Heat shock and cold sensitivity – The Hindu

DNA is a linear chain of nucleotides, portions of which are faithfully transcribed into linear messenger RNA. The message in this RNA is translated into strings of amino acids - proteins. Proteins need to take a precise three-dimensional shape to become functional entities. This protein folding does not happen all by itself, at least most of the time. A special bunch of proteins called molecular chaperones assist in correctly folding the protein.

The idea of chaperones may sound quaint and Victorian, but in biological systems they play crucial roles. After the new protein chain has been shaped correctly, chaperones move on. Or else the new chain is eliminated. Without chaperones, newly synthesised proteins would soon become a tangled mess of insoluble aggregates, hindering cellular processes.

Many molecular chaperones belong to the class of heat shock proteins (or stress-response proteins). This is because whenever an organism is subjected to elevated temperatures a heat shock proteins in the system begin to lose their native shapes, and chaperones are produced in large quantities to restore order.

Chaperones are needed under physiological conditions too, for normal cellular function.

Misfolding of proteins can cause a number of diseases. Alpha-synuclein protein, present in neurons, is wrongly folded in Parkinson's disease. Brains of Alzheimer's patients have plaques formed from aggregates of amyloid beta-peptide. This accumulation of amyloid fibrils is toxic, leading to widespread destruction of neurons a 'neurodegenerative disorder. Aberrant folding of crystallins of the eye lens leads to cataract. In the eye lens, an abundant subset of proteins called alpha-crystallins themselves serve as chaperones a single R116G mutation in human alpha crystallin is responsible for autosomal dominant congenital cataract.

Major chaperones in humans include HSP70, HSC70 and HSP90: the numbers express the size of the proteins in kilodaltons. In normal cells 1%2% of all proteins present are heat shock proteins. This number rises threefold during stressful conditions.

HSP70 is induced by heat, whereas HSC70 is always present at high levels in normal cells. HSC70 appears to be more like a molecular thermometer, with an ability to sense cold temperatures. This knowledge comes from the study of an intriguing set of disorders, exemplified by Familial Cold Autoinflammatory Syndrome (FCAS). Symptoms of these disorders include rashes on the skin, pain in joints and fever. Periodic episodes may last from a few hours to a few weeks. These episodes begin early in life, the trigger being exposure to cold, or a stress such as fatigue. The confusing set of symptoms shown in this rare disorder make diagnosis difficult it often takes ten years from first clinical presentation to a confirmed diagnosis.

The first family with confirmed FCAS in India was reported only in August this year. Sagar Bhattad and colleagues, at the Aster CMI Hospital in Bengaluru, traced the genetic underpinnings of FCAS in a four-year old boy who frequently suffered from winter rashes. It turns out that several family members, including his paternal great-grandfather, had similar symptoms. This was published in Indian Journal of Pediatrics, August 2021, 88(8):834.

Addressing the role of HSC70 in sensing low temperatures, the group of Ghanshyam Swarup at the Centre for Cellular and Molecular Biology has worked out a framework for the triggering of autoinflammatory conditions This was published in The FEBS Journal, 2021; doi:10.1111/febs.16203. Disorders related to cold sensitivity are caused by mutations in proteins that regulate inflammation. At normal body temperatures, HSC70 is able to coax these mutated proteins to fold correctly and thus function normally. In cold conditions, however, the HSC70 molecule is itself slightly altered in its shape and is not able to unerringly interact with the mutated regulators of inflammation. This leads to a pathological state with symptoms such as chills, joint pains and rich red skin rashes setting in within two hours.

Cancer cells divide at break-neck pace, and heat shock proteins are very important in maintaining the stressful cancerous state. An overabundance of heat shock proteins in cancer cells is an indicator of a poor prognosis. Cancerous cells accumulate mutations in proteins that would normally suppress tumours. HSP70 and HSP90 play the roles of villains, as they continue to fold the mutated proteins, thus allowing tumour progression. In the laboratory, inhibitors of HSP90 have shown much promise as anti-cancer agents. However, no inhibitor has yet been approved for human use, as the levels required for these to be effective are too toxic for your body.

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Heat shock and cold sensitivity - The Hindu

Sun is teaching COVID-19 researchers how to use high-performance AMD computers – William & Mary News

by Joseph McClain | December 8, 2021

Large segments of the worlds research community refocused in early 2020 in response to the growing COVID-19 pandemic.

Biochemists, epidemiologists, molecular biologists, geneticists and other specialists began working on various ways to model, track and attack the novel coronavirus using the most sophisticated scientific techniques and methods. Those techniques have become increasingly computationally intensive in nature, requiring high-performance computing not always available to the COVID research community.

The multinational semiconductor company Advanced Micro Devices AMD stepped into the fight against COVID-19 by supporting 23 organizations in addressing issues such as vaccine development, genetic sequencing and modeling of the outbreak. AMD provides researchers with high-performance computing devices, especially graphics processing units, supported by the AMD Radeon Open Compute (ROCm) platform.

AMD brought in a set of instructors, including William & Marys Yifan Sun, to make sure the researchers get the maximum benefit of the combined 12 petaflops of computing capacity. Sun and his fellow instructors are holding a series of online lectures and office hours to get the COVID researchers up to speed on graphics processing unit (GPU) computing and, in particular, the computing language needed.

Even before COVID, we were writing a book designed for the HIP program language, which is what you need to work with AMD GPUs, said Sun, an assistant professor in William & Marys Department of Computer Science. The book is not done yet, but almost there. So, the AMD people put together experts in component computing with experts in research for a 10-week session.

Sun said he has 33 people taking his classes. Its an international group, with representatives from research institutions in the U.S., Italy, Germany, the U.K., India, France and Canada.

Theyre assistant professors, researchers, Ph.D. students, he added. Five of them are from AMD; I think theyre want to take the course to get a better understanding about how to program AMD GPUs and how to improve GPU program performance.

The researchers are not novices to high performance computing, so Sun says he and the other instructors were able to hit the ground running, introducing the HIP language in the early weeks.

Then, later on, we dive into performance tuning, invoking libraries and other tools, he explained. This is a hyper-focused community. They care about performance. They care about how fast they can get a result.

The researchers develop their algorithms themselves. Sun said many of the attendees are interested in applying high-performance computing to tasks such as protein folding modeling, as well as gene sequencing and alignment. GPU-based computing shines in such computation-heavy chores, he added.

Sun explained that traditional computer architecture is based on the CPU the central processing unit. A CPU can have numerous cores, or brains, and CPU cores can number into the tens, or hundreds. To link all of those cores, CPU servers communicate from rack-to-rack of servers.

So the communications between CPU cores are intrinsically very slow, Sun explained. But in this initiative, were using GPU devices. GPUs are highly parallel, and there are thousands of cores in a single GPU.

The GPU architecture is much faster, he said, because of the parallel nature and assembly of more cores on a single device. No need for slow fabrics such as an Ethernet cable to communicate computer to computer. Sun said the result is much quicker computation for researchers working to combat the virus causing a global pandemic.

For common algorithms, GPUs can be hundreds to thousand times faster than CPUs. For more specific algorithms, like gene alignment, I would say at least tens of times faster, Sun said. You could think about it reducing a computation assignment from 10 days to one day. Thats a lot of speed-up.

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Structural dynamics in the evolution of a bilobed protein scaffold – pnas.org

Significance

Proteins conduct numerous complex biological functions by use of tailored structural dynamics. The molecular details of how these emerged from ancestral peptides remains mysterious. How does nature utilize the same repertoire of folds to diversify function? To shed light on this, we analyzed bilobed proteins with a common structural core, which is spread throughout the tree of life and is involved in diverse biological functions such as transcription, enzymatic catalysis, membrane transport, and signaling. We show here that the structural dynamics of the structural core differentiate predominantly via terminal additions during a long-period evolution. This diversifies substrate specificity and, ultimately, biological function.

Novel biophysical tools allow the structural dynamics of proteins and the regulation of such dynamics by binding partners to be explored in unprecedented detail. Although this has provided critical insights into protein function, the means by which structural dynamics direct protein evolution remain poorly understood. Here, we investigated how proteins with a bilobed structure, composed of two related domains from the periplasmic-binding proteinlike II domain family, have undergone divergent evolution, leading to adaptation of their structural dynamics. We performed a structural analysis on 600 bilobed proteins with a common primordial structural core, which we complemented with biophysical studies to explore the structural dynamics of selected examples by single-molecule Frster resonance energy transfer and HydrogenDeuterium exchange mass spectrometry. We show that evolutionary modifications of the structural core, largely at its termini, enable distinct structural dynamics, allowing the diversification of these proteins into transcription factors, enzymes, and extracytoplasmic transport-related proteins. Structural embellishments of the core created interdomain interactions that stabilized structural states, reshaping the active site geometry, and ultimately altered substrate specificity. Our findings reveal an as-yet-unrecognized mechanism for the emergence of functional promiscuity during long periods of evolution and are applicable to a large number of domain architectures.

Proteins drive and maintain all fundamental cellular processes (1) by interactions with small molecules and/or other biopolymers. Important mechanistic information on proteins are accessible via structural analysis of their functional cycle (2). While classical approaches rely on the interpretation of static structure snapshots, the visualization of structural dynamics (i.e., to follow the interconversion of distinct structural states at high spatial and temporal resolution) (37) has been recognized as an essential complement. The folding funnel model (8), rooted in the free-energy landscape theory (911), has by now become a widely accepted way to describe the ensemble of such states (1214).

Distinct structural states can originate from local flexibility (i.e., bond vibrations, and side-chain rotations [Fig. 1A, Tier-2 dynamics]), changes in secondary structure (Fig. 1A, Tier-1 dynamics) or large-scale domain motions (Fig. 1A, Tier-0 dynamics). The free-energy landscape of a protein defines the lifetime of its structural states, ranging from nanoseconds (local flexibility) to seconds (large-scale motions). Transitions between the states are referred to as structural changes and are induced by interactions with ligands, posttranslational modifications (e.g., phosphorylation), or chemical events such as nucleotide hydrolysis. The coupling of the latter to structural changes enables proteins to perform a diverse range of functions.

Energetic funnel, structure, and evolution of the cherry-core. (A) One-dimensional cross-section of a hypothetical protein energy landscape adapted from Kern and coworkers (12) according to the Tier description and definitions introduced by Ansari and coworkers (15). A structural state is defined as the lowest point of a well on the energy surface. The populations of the Tier-0 states, closed and open, are defined as Boltzmann distributions, and their relative probabilities (pC, pO) are determined in this paper by smFRET, which follows large-scale domain motions. Tier-1 states describe local and fast structural fluctuations (e.g., changes in secondary structure elements like loop motions or loss of secondary structure). Tier-1 dynamics were probed in this study by HDX-MS. Rapid and localized Tier-2 dynamics (e.g., side-chain rotations) were not considered here but can be monitored via molecular dynamics (MD) simulations. Changes in the chemical environment (i.e., absence or presence of a ligand) modify the energy landscape via a bias for one of the two states. Typically, thermodynamic parameters such as the free energy difference of Tier-0 wells (GOC) can be determined by ITC. (B) Structure of a representative bilobed protein, the substrate binding domain 2 (SBD2) of the ATP-binding cassette (ABC) amino acid transporter GlnPQ from Lactococcus lactis (PDB: 4KR5). (C) Summary of the structure-based phylogenetic tree with schematic representations of the different structural classes (class A through G) highlighting their termini. Complete sequence and structure-based phylogenetic trees are provided in SI Appendix, Fig. S1 and Datasets S1 and S2. We used the following notation: Secondary structure elements that are common between the different classes were assigned a number identifier (e.g., helix H4 in classes F and G), whereas the unique elements have a letter followed by a number identifier (e.g., HD1, first unique helix of class D proteins). An asterisk (*) marks a structural subclass (shown in detail in Dataset S3). Schematics are based on the crystal structures of the selected proteins. (D) Topology of bilobed proteins depicting the consensus of secondary structure elements: strands (s) or helices (H) belonging to the Domains (D1, D2), hinge-forming -strands (H1, H2), and C-tails of all seven classes (A through G) are shown. Revised alignments of bilobed proteins are shown in Dataset S3. The secondary structure elements forming the consensus cherry-core structure are depicted on the Top row.

Tier-0 dynamics were observed and characterized in various settings (e.g., in motor proteins, in which they are used in propelling movement along filaments) (16), in the transport of molecules or biopolymers across biological membranes (1721), or in the activity of proteins that perform mechanical work (22). Tier-1 dynamics drive the actions of various signaling proteins for transmission of signals (2325). Structural and biochemical data indicate that enzymes also show varying degrees of structural dynamics (26), although it is not well understood what precise role this plays for catalytic activity. The current belief is that extensive structural dynamics in enzymes are not necessarily required for catalysis (27) but rather enact in regulation. For instance, many protein kinases exploit Tier-0 dynamics to generate active or inactive structural states (28). Tier-2 dynamics have been shown to be important for the evolution of enzymatic function (29). In addition to domain motions occurring within a structure, protein oligomerization and the possible quaternary dynamics can also be relevant for function (30). A well-characterized example is the allostery of hemoglobin that occurs on the transition between two distinct quaternary states (relaxed and tense) (31, 32). All this highlights that multi-Tier dynamics of proteins occur on various time- and length-scales (Fig. 1A) and are often the basis for function. It is, however, not yet well understood how structural dynamics are optimized during evolution to tailor protein function.

Analysis of protein sequences and structures has provided important insights into the evolution of protein function (33). A powerful approach is to assign the domain components of proteins to families and superfamilies on the basis of sequence alone (Pfam database for the protein family) (34) or in combination with structural information (class architecture topology homologous superfamily, CATH and structural classification of proteins, SCOP databases) (35, 36). The CATH and SCOP databases combined have identified 3,000 domain superfamilies comprising >50 million domains that account for 70% of the domains in completed genomes (37). More recently, the database ECOD (evolutionary classification of protein domains), groups domains by considering their evolutionary relationships (38). In ECOD, 760,000 domains have been assigned to 3,700 homologous groups (39). The most highly populated domain superfamilies/homologous groups are universal to all kingdoms of life (40, 41). The prevalence of proteins with multidomain architectures and the recurrent appearance of the same domain in nonhomologous proteins suggests that functional domains are reused when creating new proteins and functions (42, 43). Analyses of selected domain groups (44, 45), and more-recent large-scale investigations (4648) have shown that domains in such groups generally share a common structural core (40 to 50% of the domain) that is highly conserved, even for relatives separated by billions of years. To achieve functional promiscuity of such a structural core, it has been proposed that larger structural embellishments (secondary structure elements or even entire domains) need to be added to the core for altered biochemical function (49). Such fusions occur frequently at the N or C termini (40), which has led to the belief that structural elements act as Lego bricks (47). Those are recombined in various ways for new functions to emerge during evolution (42, 49).

Much less is known on the role played by structural dynamics in the evolution of protein function (5053). A recent model suggests that the native state of an evolved protein is the most abundant state of all possible structural states, which was selected for a specific function (54, 55). The avant-garde evolvability theory proposes the existence of a highly promiscuous primordial protein structure. It is assumed that for the emergence of the native state, the ability to evolve (evolvability) was traded for ligand-functional specificity (56, 57). These observations were experimentally verified (58) and agree with the proposal that changes in structural dynamics serve as a mechanism for the evolution of specialist enzymes from promiscuous generalists (59), though discordant examples are also observed in evolution (60). Furthermore, recent studies indicate that this evolvability theory can explain the short-period evolution (e.g., variation of enzyme local flexibility [variation of Tier-1/2 dynamics] to acquire new functions) particularly well (5456, 61). Remarkably, this short-period evolution can be faithfully reproduced invitro using directed evolutionary approaches based on consecutive rounds of single point mutations (54, 61). Ancestral protein reconstruction has also been useful in elucidating the role of structural dynamics in the emergence of specialized amino acid binding proteins from a promiscuous ancestor (62). However, it still remains unclear how, during longer periods of evolution, a primordial core structure evolves to modulate or diverge Tier-0 and/or quaternary dynamics, generating completely new functionalities.

Here, we test this aspect of the evolvability theory for proteins separated by long evolutionary periods during which Tier-0 or quaternary dynamics were introduced to an existing protein core structure. For this, we identified proteins with a bilobed domain structure with a high degree of plasticity and neutrality (29, 55), having 1) a conserved core structure, 2) large sequence diversity and related functional divergence, and 3) occurrence in all kingdoms of life. The selected structural core is composed of two Rossmann-like domains, which are believed to be among the most ancient architectures (63) and that are connected by a single -sheet (Fig. 1B). We analyzed 600 proteins with this structural core that contain two related domains from the periplasmic-binding protein (PBP)like II domain homologs (ECOD: X-, H- and T-groups PBP-like II). We show here that different members of these homologous proteins diverged with respect to domains or secondary structure elements, predominantly at their termini (Fig. 1 C and D). Using a combination of structural analysis and biophysical investigations, we demonstrate that such structural embellishments confer multi-Tier structural dynamics that diversify the function of the core structure to yield transcription factors, enzymes, or extracytoplasmic transport-related proteins. To understand both the mechanistic distinction of these proteins and the evolutionary trajectories, we used single-molecule Frster resonance energy transfer (smFRET) (64) and HydrogenDeuterium Exchange Mass Spectrometry (HDX-MS) (65). smFRET allows to monitor Tier-0 dynamics with a temporal resolution down to microseconds and subnanometer spatial resolution at the single-molecule level, even for highly heterogeneous structural ensembles (12, 6668). HDX-MS complements smFRET, as it can probe Tier-1 dynamics throughout the structure that occur within the structural states identified by smFRET (Fig. 1A) (69). Using this combination of techniques, we show how the C-terminal extensions modulate Tier-0 dynamics in the structural core in a manner specific to their three-dimensional orientation. This arrangement also dictates specific geometrical criteria, crucial for establishing specific ligand interactions, all of which we detail in this work. On the other hand, we reveal the means by which N-terminal domain additions enable oligomerization to provide distinct quaternary dynamics in LysR-type transcriptional regulators (LTTRs). The remarkable modularity of these proteins permits us to confirm and expand the evolvability theory for the primordial core structure during a long-period evolution, which is largely facilitated, seemingly, by genetic recombination events.

In this study, our focus was on proteins composed of two globular lobes (bilobed), each of three layers (//), articulated around a central -sheet hinge (Fig. 1B). This focus meant that not all multidomain architectures harboring the PBP-like II domains were included. To investigate the structural dynamics of the selected proteins, we constructed phylogenetic trees based on both sequence and structural information (Fig. 1C). This analysis indicated that the proteins evolved from a common ancestor that diversified into seven distinct structural classes A to G (Fig. 1 C and D), members of which are found throughout all kingdoms of life, in which some are even present in viruses (SI Appendix, Fig. S1A and Datasets S1 and S2). They have a consensus structure (Fig. 1B, Fig. 1 D, Top, and Dataset S3), which we have dubbed the cherry-core (hereafter CC, and proteins harboring this core, cherry-core proteins, CCPs) because of the bilobed structures resemblance to a cherry (SI Appendix, Fig. S2A), that contains two PBP-like II domains (70). According to ECOD database, the two domains of CCPs belong to the same X-, H-, and T-groups (PBP-like II; Dataset S3), supporting their common ancestry (38). In the consensus structure (Fig. 1 C and D), domains D1 and D2 adopt a face-to-face mirror-fashioned geometry with the active site, which is typically a ligand-binding site, located at their interface (SI Appendix, Fig. S2B).

Most of the selected proteins have distinct segments linked N-terminally to the CC (Fig. 1 C and D). Class A proteins have the addition of a LysR winged helix-turn-helix (HTH)type DNA-binding domain (ECOD: X-, H-group: HTH, T-group: Winged; Fig. 1C). This element has been shown to be responsible for oligomerization and binding to promoter DNA (7173). Most proteins of classes B through D, and F and G contain N-terminal localization signals for export via the general secretion (Sec) or the twin-arginine translocation (Tat) pathway (7477). The presence of these signal peptides was obtained from UniProtKB (78) and verified manually by inspecting all 600 protein sequences with PRED-TAT, prediction of twin-arginine and secretory signal peptides (79). Class E proteins are predominantly cytosolic and lack an N-terminal signal peptide.

In addition, classes B through G have distinct C-terminal structural embellishments, hereafter termed C-tails (Fig. 1D). For example, Helical-tail 1 (H1) is common to all classes except C, and importantly, it has a similar placement in the three-dimensional architecture of the proteins. Other C-tails are unique to a specific class, such as the Helical-tails G (HG1 and 2), present only in class G proteins.

The ligand specificity and function of the CCPs, as documented in UniProtKB (78), were found to correlate with the assigned structural class (SI Appendix, Fig. S1A). Class A proteins are bacterial transcription factors of the LTTR family. Class E proteins are predominantly eukaryotic single-turnover enzymes. The majority of the remainder (class B through D, F, and G) are found in prokaryotes and associate with the translocator domains of ABC transporters or with the membrane-embedded domains of chemoreceptors, in which they mediate unidirectional solute transport and signal transduction, respectively.

To investigate what role the distinct C-tails of these proteins might have played in structural dynamics and the evolution of new functions, we examined CCPs for which high-resolution structures of unliganded (apo) and liganded (holo) states were available (Fig. 2 and SI Appendix, Table S1). Interestingly, class A and E proteins, in most cases, exhibit nearly identical apo and holo structures. They also display the widest variety of substrates with little chemical structure similarity (SI Appendix, Fig. S3). For most members of the other classes, D1 and D2 of the CC undergo a rigid body rotation of varying degrees (Fig. 2 and SI Appendix, Table S1). For the solute-binding proteins, this mode of substrate binding has been termed the Venus-Fly Trap mechanism (80). These proteins recognize ligands with a specific pharmacophore (81): amino acids, ethanolamines, phosphonates, ironphosphate complexes, and carbohydrates are recognized by classes B, C, D, F, and G, respectively (SI Appendix, Fig. S3). Another striking difference is that proteins in classes B through G are monomeric, whereas those in class A are oligomeric (see Fig. 4C).

Structures of CCPs highlighting their C-tails. Structures (Top) and schematics (Bottom) of the identified structural classes (A through G). The apo and holo structures of the indicated CCPs are superimposed to highlight the role of the C-tail. The domain to which the two structures were superimposed is represented in gray (D1 in A through E and D2 in F and G), whereas the one that is displaced relative to the C-tail in the open state is represented in green. Arrows and schematics indicate domain displacement (SI Appendix, Table S1). Interaction contact maps are presented in SI Appendix, Table S2. The most prominent contacts between the indicated secondary structure elements of the C-tail and D1 (F, G) or D2 (B through E) that stabilize the open state are indicated by a yellow dashed line. The PDB codes and protein names are indicated.

To investigate structural dynamics of the CCPs, we first used smFRET (12, 6668) to probe Tier-0 dynamics at near-physiological conditions in aqueous buffer at room temperature. One representative protein from each class was investigated: the effector binding domain (EBD) of CynR, representing the CC of full-length CynR (72) (class A), SBD2 (82) (class B), OpuAC (83) (class C), PhnD (84) (class D), CmpA (85) (class E), FbpA (86) (class F), and MalE (87) (class G). For these experiments, D1 and D2 were stochastically labeled with donor and acceptor fluorophores via cysteines that were substituted for nonconserved and surface-exposed residues, one in each domain (Fig. 3A). Fluorophore labeling was performed by maleimidethiol conjugation (22, 88). Labeling positions were selected based on the crystal structures to show large changes in separation between the apo and holo states. smFRET was performed by confocal microscopy with alternating laser excitation (ALEX) (66).

Monitoring structural changes and ligand binding in CCPs using smFRET and ITC. (A) Schematic of the experimental strategy to monitor structural states by FRET efficiency via stochastic labeling of D1 and D2 with donor and acceptor fluorophores. (BH) Solution-based apparent FRET efficiency histograms in the absence (Top) or presence (Bottom) of saturating ligand concentrations for the indicated proteins. Gray bars are experimental data, and solid line is the fit. Centre position of Gaussian fits are given in SI Appendix, Table S5. (I and J) Binding isotherms of the calorimetric titration of azide (I) and calcium carbonate (J) to CynR (I) or CmpA (J), respectively, with the indicated thermodynamic parameters. For the apo condition of FbpA (E), the sample was treated extensively with citrate, as Iron (III) and the synergistic anion carbonate required for high-affinity binding to FbpA are removed efficiently by citrate treatment at low pH (92). According to high-resolution structural data (93), both Ca2+ and CO3 are present in the CmpA binding cleft. Indeed, we observed heat release upon titration of Ca2+ to a CO3 bound CmpA. Data points represent the heat of reaction per injection, and the line is the fit.

As predicted by our structural analysis (Fig. 2 and SI Appendix, Table S1) and in line with our previous observations (89), the FRET efficiency histograms and fitted distributions (Fig. 3 BF) shifted toward higher FRET efficiency (E) values upon addition of saturating concentrations of ligand, for SBD2, OpuAC, PhnD, FbpA, and MalE. This indicates that in the apo state, the donor and acceptor dyes are further apart (Fig. 3 BF, Upper, low FRET) compared to the holo state (Fig. 3 BF, Lower, high FRET). Thus, our data suggest that ligand binding drives Tier-0 dynamics in these CCPs.

In contrast, the distributions of class A and E proteins [i.e., CynR (90) and CmpA (85), respectively] (Fig. 3 G and H), were virtually identical in the absence or presence of saturating ligand concentrations. Ligand binding was confirmed via isothermal titration calorimetry (ITC) showing binding affinities of both proteins in the micromolar range (Fig. 3 I and J). Thus, we conclude that the CC of class A and E proteins lack Tier-0 dynamics on the probed reaction coordinates for the selected FRETdistance pairs in contrast to the other structural classes.

Interestingly, this observation is in line with the known biological function. Class E proteins are predominantly single-turnover enzymes (SI Appendix, Fig. S1A), for which the rigidity of their active site is a prerequisite for catalysis (SI Appendix, Fig. S4A) (91). As previously suggested by us and others, Tier-0 dynamics in periplasmic-binding proteins (classes B, C, D, F, and G) are utilized in the regulation of transport in ABC importers (Discussion). A remaining question is, however, how ligand binding in class A proteins triggers transcriptional processes without major structural changes related to ligand binding.

The DNA-binding domain of class A proteins typically comprises a 58-aa HTH motif. This is followed by a 20-aa-long helix that provides a dimerization interface and a connecting loop that links the DNA-binding domain to the CC (Fig. 4A). The CC acts as the tetramerization interface within the full-length CCP or the dimerization interface within the EBD (Fig. 4B and SI Appendix, Fig. S4 BE). Indeed, by using size exclusion chromatography with multi angle light scattering (SEC-MALS) we observed that (full-length) CynR is tetrameric, whereas its CC is dimeric (Fig. 4C).

CynR tertiary and quaternary assemblies probed by MALS and HDX-MS. (A) Crystal structure of the CynR CC (PDB: 2HXR) with colored secondary structure elements discussed in the text that are critical for its quaternary dynamics (Top). A homology model of full-length CynR obtained from the SWISS-MODEL server with CbnR (PDB: 1IZ1) as a template (Bottom). The HTH domain is colored green, and the loop connecting it to the CC splitpea. W64 at the tip of the dimerization helix is indicated with an arrow. (B, Top) Schematic representation of the CC of CynR using the same color-coding as in A. (Middle) Schematic representation of the CynR homology model, with one of the protomers in the compact and the other in the extended configuration. The CC of the CynR protomers self-associate to form dimers or, for the full-length protein, tetramers. For clarity, two of the protomers have been omitted. (Bottom) The tetrameric assembly formed by the self-association of the dimerization helices and the CC. Of the two interacting protomers, one is present in the compact configuration, whereas the other is extended. The two protomers with the compact configuration are shown at the top of the plane, whereas those that are extended are at the bottom. (C) SEC-MALS analysis of full-length and CC of CynR (3 M). Ultraviolet (UV) traces of the chromatograms were superimposed on the measured mass (black cycles). (D) Structural dynamics of full-length CynR in the absence of ligand and DNA by HDX-MS. Deuterium uptake values are reported for the incubation times in deuterated buffer and expressed relative to the fully deuterated control. These values are mapped onto the CynR homology model (as in A), using the indicated color gradient. Proline residues as well as the first residue of each peptide were excluded from mapping, as they do not contribute to the observed D-uptake. (E) Scatter plot visualization of the statistical analysis of D-uptake differences between the apo and holo states of full-length CynR (Upper) and the CC of CynR (Bottom). Three statistical criteria were used (SI Appendix, Material and Method), as described previously (98). Statistically significant differences would appear as black spheres (indicating that D-uptake > 2SD for a specific peptide), lying outside the 99% confidence threshold (1-P 0.99; indicated on y-axis) and outside the 4 pooled average SD cut-off (indicated on x-axis; value given on the right).

To determine whether ligand binding to CynR resulted in structural changes that were not detectable along our selected reaction coordinate or that were too small or fast for smFRET (Fig. 3G), we performed HDX-MS. In contrast to smFRET, which reports on a single distance along a single reaction coordinate, HDX-MS probes structural dynamics at nearresidue level resolution, providing global insights into Tier-1 dynamics. HDX-MS detects the exchange of hydrogens with deuterium at solvent-accessible and nonhydrogen-bonded backbone amides (94, 95). Hydrogens involved in stabilizing the secondary, tertiary, or quaternary structure of a protein via hydrogen bonds are exchanged more slowly through structural transitions that disrupt these bonds. Deuterium incorporation into the protein can then be determined, following proteolysis, by MS. The mass difference between hydrogen (1H) and deuterium (2H) results in a mass shift between nondeuterated and deuterated peptides that is a measure of the number of exchanged hydrogens (65, 96, 97).

For such investigations, the CC of CynR or full-length CynR were isotopically labeled (pD, 7.4; 25C) for different time periods (10 to 105 sec) either in free or DNA-bound states, and the peptic fragments were identified by MS (Fig. 4D and Dataset S4). For each peptide, the fraction of deuterium uptake relative to the maximum determined deuterium incorporation was calculated (Dataset S4). The data reveal a rigid character of the CC of CynR, whereas the dimerization helix and the rest of the DNA-binding domain turned out to be more flexible (Fig. 4D). To identify the regions in which pronounced structural changes were induced by azide binding, we performed comparative HDX-MS. For this, we determined the difference in deuterium uptake (D) for each peptide between different conditions (e.g., CynR apo versus holo states). Observed differences would indicate a decrease (protection) or an increase (deprotection) of deuterium uptake upon azide binding (Dataset S4). No statistically significant change in the deuterium uptake was observed (Fig. 4E). From this, we can conclude that no detectable change in structural dynamics is induced by the ligand in the CC of CynR in its free or DNA-bound form (Fig. 4E and Dataset S4). This includes Tier-0 dynamics (detected by smFRET and HDX-MS) and Tier-1 or quaternary dynamics (both detected by HDX-MS).

From our results on the selected CCPs, only those with an asymmetric C-tail display Tier-0 dynamics (Figs. 2 and 3). To investigate how the C-tails introduced Tier-0 dynamics to the CC, we compared crystal structures of the apo and holo states of the CCPs to identify interactions between their C-tails and the CC. Fig. 2 summarizes the results of these comparisons. Interestingly, we found that the holo structures are similar for all CCPs, but the apo states are class specific. Contact mapping of the interactions between the CC and C-tail using the protein interaction calculator web-server (99) showed that the number and characteristics of the interactions depends on the structural class (Fig. 2 and SI Appendix, Table S2). Strikingly, these interactions stabilize predominantly the open state of the CC, the only exception being for classes A (C-tail-less) and E. In the former cases, stabilization is associated with an asymmetrical placement of the C-tail with respect to D1 and D2. In contrast, the C-tail of class E (predominantly H3 and HE1; Fig. 2) is placed symmetrically around D1 and D2 and thus cannot provide the required structural asymmetry needed to create a stable open state. The interactions of the C-tail to stabilize the open state involve the consensus CC-helices of D1 and D2 (Fig. 2 and SI Appendix, Table S2). In classes B, C, and D, such interactions involve D2, whereas D1 is contacted in classes F and G. These asymmetrical interactions create active sites with distinct geometries (Fig. 2 and SI Appendix, Fig. S2).

In alternative phylogenetic trees, based on the protein sequence using either D1/D2-domains or the C-tail, the clustering remains similar (SI Appendix, Fig. S1 BD and Datasets S1 and S2), indicating that D1/D2 (CC domains) and the C-tail of a specific class coevolved to be part of the same polypeptide. This is in line with the role of the C-tail to interact with one specific domain, D1 or D2, to stabilize the open structural state.

To confirm the role of the C-tail interactions with D1 or D2 for stabilization of the open state, we manipulated the relevant ones (Fig. 2 and SI Appendix, Table S2) in SBD2, SBD1, and MalE (Fig. 5 and SI Appendix, Figs. S5 and S6). The impact was tested via assessing structural states and ligand-binding affinities and monitored in smFRET experiments. Test cases were selected from class B and G, as these CCP classes are only remotely related by the first major clade in the evolutionary trees (Fig. 1C), and their open state is stabilized by distinct helices within the C-tails (Fig. 2), contacting either D2 (class B) or D1 (class G).

Experimental verification of the role of C-tail interactions in stabilizing the open conformation of SBD2 and MalE by smFRET and HDX-MS. (A) Dotted rectangles (Left) on the SBD2 (in A, PDB: 4KR5) structure highlights the critical contact region between the C-tail and the CC that stabilize the open state. Zoom in of rectangle regions (Middle and Right) depicting interactions between the C-tail helix H1 and D2 in the indicated apo (open) or holo (closed) states. Distances () between L480 and P419 are shown as black dotted lines. (B and C) Solution-based apparent FRET efficiency histograms of SBD2 (B) and SBD2 (L480A) (C) at different conditions as indicated. (D) Fraction of the closed state (high-FRET state) of SBD2 and the indicated derivative as a function of glutamine concentration. (E) As in panel (A) for the MalE (PDB: 1OMP) structure with the indicated secondary structure elements and critical contacts. (F and G) Solution-based apparent FRET efficiency histograms of MalE (F) and MalE (M321K) (G) at different conditions as indicated. (H) Fraction of the closed state (high-FRET state) of MalE and its derivatives as a function of maltose concentration. (I) Maltose release from MalE (M321K) over time determined by solution-based smFRET (reference detailed values in SI Appendix, Fig. S6). Data points (panels D and H) and gray bars (panels B, C, F, G, and I) are the experimental data, and the solid line is the fit. (J) Map of regions in MalE structure that show statistically significant increase in deuterium uptake caused by M321K (numerical values and complete statistical analysis presented in Dataset S5). n = 3. (K and L) Deuterium uptake for the indicated MalE C-tail helices. SDs (SD) are shown in the deuterium uptake plots.

In SBD2, a hydrophobic interaction between L480 in the C-tail and P419 in D2 was weakened by substitution of L480 with alanine (L480A; Fig. 5A and SI Appendix, Table S2). The mutation resulted in the appearance of a subpopulation of molecules (20%) that were in the closed state in the absence of glutamine (Fig. 5 C versus B). To rule out the possibility that this might have been due to an artifact introduced by the choice of fluorophores, a second pair was tested, which showed a comparable result (SI Appendix, Fig. S5 A and B). We also examined whether residual endogenous ligand might account for this subpopulation by performing smFRET measurements on diluted samples, but these experiments displayed subpopulations of a similar size (SI Appendix, Fig. S5C). A closed-unliganded conformation was also observed for SBD2 previously but with a much lower abundance (1%). Detection of this small subpopulation required the use of confocal scanning microscopy (89), as populations <5% cannot be detected reliably with ALEX spectroscopy. We also observed small differences in the mean E values for apo and holo states of SBD2 (L480A) as compared to SBD2, suggesting that the structural landscape had been altered (SI Appendix, Fig. S5B). Destabilizing the open state is in line with the 10-fold increase in glutamine-binding affinity of SBD2 (L480A) as compared to SBD2 (Kd of 209 64 nM and 1,990 130 nM, respectively; Fig. 5D).

Determination of the dissociation constant (Kd) by smFRET measurements reports on the stability of the open state via Kd(1+exp()), where =1/kbT, kb is the Boltzmann constant, T is the absolute temperature, and =GC-GO is the conformational (free) energy difference, where GC and GO are the (free) energies of the closed and open structural states without ligand bound, respectively (100). Thus, destabilizing the open state will decrease Kd and vice versa. From the percentages of open and closed in the absence of ligand, we obtain =1.4 and =4.4kBT for SBD2 (89) and SBD2 (L480A) (Fig. 5 BD and SI Appendix, Fig. S5 AC), respectively. Based on these results, the Kd value would be expected to decrease by 16 folds due to the L480A mutation, in close agreement with the estimated 10-fold difference. Similar trials on SBD1 did not show this trend (SI Appendix, Fig. S5 DF), as the mutation had no impact on the structural dynamics of SBD1 (I249A). The absence of a subpopulation of molecules in the closed state at apoprotein conditions (alike SBD2) is based on the inability to evaluate all stabilizing open state interactions (SI Appendix, Table S2, compare SBD1 versus SBD2) so as to abolish the relevant ones. This is likely due to the fact that H1 residues of SBD1 (SI Appendix, Fig. S5G) participating in interprotomer contacts arose from crystallographic conditions (SI Appendix, Fig. S5H).

For MalE, we constructed the derivatives MalE (M321A) (101) and MalE (M321K), with weakened interactions between the C-tail HG1 and D1, by disrupting the hydrophobic interaction of M321 with Y90 and F92 and the aromatic sulfur interactions with Y90 (Fig. 5E and SI Appendix, Table S2). For MalE (M321A), the stabilizing interactions of M321 are partially abolished. This resulted in an eightfold increase of maltose affinity (Kd from 2,400 400 nM in MalE to 300 50 nM in MalE [M321A] [Fig. 5H and SI Appendix, Fig. S6]). An even-stronger effect was observed for MalE (M321K) with an affinity enhanced by 3,000-fold (Kd of 0.81 0.15 nM; Fig. 5 G versus F and Fig. 5H). Shilton and coworkers have also proposed the hydrophobic interactions of M321 as being important structural determinants of the open state (101), affecting the affinity of MalE for maltose, in agreement with our results.

We next compared the lifetime of the closed, maltose-bound conformation of MalE (M321K) with that of MalE. Addition of 10 nM maltose allowed MalE (M321K) to occupy the closed state exclusively (SI Appendix, Fig. S6H). A total 20 M unlabeled MalE (M321K) protein was subsequently added to scavenge maltose, which is stochastically released from the labeled protein. In a time-course experiment, the decrease in the population of closed, maltose-bound MalE (M321K) was then followed as a function of time (102) (Fig. 5I and SI Appendix, Fig. S6H). From these experiments, we established that the lifetime of the closed, maltose-bound conformation of MalE (M321K) was 122 12 s. This value was 2,500-fold higher than the value determined for MalE (0.048 0.010 s; SI Appendix, Fig. S6E). This result is consistent with the observed increase affinity of the derivatives for maltose.

In the above-described MalE derivatives, the structural basis for the destabilization of the open state could not be addressed mechanistically. Our smFRET experiments report on Tier-0 dynamics that affect the proteins tertiary structure (Fig. 1A). Considering that we observed differences in the Tier-0 dynamics, as a consequence of C-tail-D1/D2 destabilization, only for SBD2, we performed comparative HDX-MS (Dataset S5) to monitor changes in the secondary structure of MalE in comparison to MalE (M321K). For this, we determined the difference in deuterium uptake (D) for each peptide between MalE and MalE (M321K) in their apo states (Fig. 5J). Remarkably, comparative HDX-MS indicates that the differences between MalE and MalE (M321K) are localized almost exclusively at the C-tail and specifically in regions interacting with D1 (Dataset S5 and Fig. 5J). The D-uptake of the C-tail helices H2/HG1 that are critical for stabilizing the open state (Fig. 5E and SI Appendix, Table S2) denotes that their rigidity was significantly reduced in the MalE derivative (Fig. 5 K and L). As might be expected, reduced rigidity occurred also in a region within the CC containing Y90 and F92. Notably, the same regions become allosterically destabilized in MalE upon maltose binding (Dataset S5). These results support the idea that the mutation leads to a weaker interaction between the C-tail and the CC resulting in a destabilization of the open state, because these C-tail elements and the region containing contact residues were found to be more flexible and solvent exposed.

Taken together, we postulate that ligands attenuate the stability of the open state in the CCPs, adopting multiple allosteric models for signal propagation (103): either via Tier-0 (SBD2 [Fig. 5 C and D]) or Tier1/2 (MalE [Fig. 5 G, H, K, and L]) dynamics.

Common (structural) origin represents the hallmark of Darwinian evolution. Homology or descent from a common ancestor is often deduced from similarities in protein sequences or better from structures, as the latter are more conserved during evolution (104). However, similar structures can originate from divergent, convergent or parallel evolution (105). The most common tricks nature uses to vary a protein domain are the following: -strand invasion/withdrawal, insertions/deletions/substitutions of secondary structure elements, domain flip/swaps, and circular permutations (106, 107). The currently established evolvability theory relies on investigations involving a fixed-length polypeptide chain by observing the effects of sequence variations, accomplished primarily by directed evolutionary approaches or by investigating closely related functional homologs. The functional promiscuity originating from structural variability is altered by the few amino acid modifications that can yield alterations of local structural fluctuations. For this reason, this theory can well explain protein evolution during short time periods.

In this study, we focused on the analysis of structures that have diverged over longer evolutionary periods. We analyzed a group of 600 proteins that share a core structure with the same topology of secondary structure elements giving rise to identical three-dimensional structures. The structure was predominantly varied by terminal embellishments and exhibits detectable sequence identity, used for constructing the sequence-based phylogenetic trees (SI Appendix, Fig. S1). The identified proteins likely emerged from divergent long-term evolution from a common ancestor, which spreads throughout the tree of life. This common ancestor is seemingly represented by the consensus core structure (CC; SI Appendix, Fig. S2) and encountered within the type-II class of PBPs (70, 108). As proposed previously (70), the CC derived possibly from a gene duplication of a PBP-like II domain (ECOD; Dataset S3) connected by a -sheet (Fig. 6A).

Model for the evolution of the cherry-core and hypothetical energetic funnels. (A) A gene duplication of a PBP-like II domain gave rise to the CC, being composed of a unique closed structural state, represented by a single well in the energetic funnel. The function of the CC was uniquely binding. Extant proteins acquired different extensions that altered their localization and dynamics and by that their function and specificity. (B) An N-terminal signal peptide and an asymmetrical C-terminal tail generated extracytoplasmic proteins evolving an additional open state. The different flavors of open states conferred distinct substrate specificities. The two states of CCPs, predominantly, signal substrate transport by their association to the membrane-embedded translocator domains of (ABC) transporters. (C) A symmetrical C-tail rigidified the closed state and yielded primarily single-turnover enzymes. (D) The N-terminal domain addition of a flexible dimerization helix and a DNA-binding motif (HTH-type) conferred distinct oligomeric assemblies with different quaternary dynamics, yielding transcription factors. (E) The different flavors of open states, conferred distinct substrate specificities. For details, refer to SI Appendix, Fig. S8.

When the CC is fused N-terminally to a signal peptide and C-terminally to an asymmetric C-tail, the CC acts as an extracytoplasmic monomeric protein that associates with the translocator domains of ABC transporters or with chemoreceptors (SI Appendix, Fig. S1 and Fig. 6B). The two structural states are apo-open versus holo-closed and originate from Tier-0 dynamics, which are critical determinants of their biological function: the membrane-embedded partners can discriminate between open versus closed states to activate or inactivate a biological process (88, 109112), such as solute transport. These structural transitions rely on the generation of an open state of the CC, accomplished by the C-tail, through its asymmetric interactions with either of the domains of the CC (D1 or D2; Fig. 2). Evidently, the open state is stabilized by such enthalpic contributions and destabilized by mutations or ligand binding that increase the flexibility of the interacting regions (Fig. 5J) (the C-tail elements with either D2 [classes B, C, and D] or D1 [classes F and G] [Fig. 2]). Seemingly, entropic contributions (protein conformational entropy) bias the structural equilibrium toward the closed state. Given the fact that the holo is the closed state driven by ligand binding (89), we anticipate that the interactions of the ligand with the CC cleft allosterically induce order-to-disorder transitions to alter the structural equilibrium. Depending on the placement of the asymmetric C-tails in the three-dimensional space, we identified five different flavors of open states, establishing distinct geometries of active sites; all resembling a triangle distinctly oriented in space (Figs. 2 and 6E). We verified that each active site geometry can recognize a specific chemical structure (SI Appendix, Fig. S3), in full agreement with ligand binding triggering closing.

In contrast to the C-tail, the presence of the N-terminal signal peptide does not affect the Tier-0 states of MalE (MalE versus proMalE; SI Appendix, Table S5). However, to render protein trafficking to extracytoplasmic locations possible, the presence of the signal peptide is known to delay MalE folding (113, 114). By that, preproteins are allowed to be secreted by the protein translocase (115).

The evolvability theory of Tawfik and coworkers (29, 57) explains the functional promiscuity encountered within the different structural classes. We selected two closely related proteins in our phylogenetic tree in class B (Fig. 1C) to illustrate this (Dataset S2, marked red asterisk). SBD1 (Protein Data Bank [PDB]: 4LA9) mediates the unidirectional transport of two substrates (glutamine and asparagine), whereas SBD2 (PDB: 4KR5) transports one of them (glutamine), though it captures it with higher affinity (88). SBD1 can be transformed to bind glutamine with higher affinity like SBD2 by mutating three amino acids (82). Clearly, functional promiscuity is traded seemingly with ligand specificity (62). However, only the modularity notion introduced in this study can explain a greater ligand-functional promiscuity. Class B proteins recognizing and mediating amino acid transport (SI Appendix, Fig. S1) would gain tertiary structural variability by acquiring four more helical elements at their C termini (H2, HG1, H4, and HG2; Fig. 1 C and D) (Fig. 6). This would potentially allow to switch them to class G to bind and mediate transport of carbohydrates (SI Appendix, Fig. S1A). According to another evolutionary trajectory, class B proteins, by acquiring helices H3, HE1, and HE2, could divert to class E and switch from transport-related proteins to enzymes (Fig. 6) by restricting their tertiary structural variability to a unique closed state. An extreme case of functional divergence has been experimentally verified in a recent study that restored the evolutionary history of the enzyme cyclohexadienyl dehydratase (116), a class B member according to our classification. The reconstructed ancestor of this enzyme was a highly promiscuous, transport-related protein possessing the open-unliganded and the closed-liganded structural states and was able to bind four different cationic amino acids. On the other side, cyclohexadienyl dehydratase binds a single substrate and forms a unique closed state for its catalytic function, since it is believed that structural sampling represents a constrain for catalytic activities (117, 118). Our study can now explain the means by which the addition of modular elements to a conserved core structure diversify its function (i.e., by modulating its structural landscape).

When the CC is combined C-terminally to a symmetric C-tail during evolution (Fig. 6C), the CC either operates as an enzyme or is associated with ABC transporters (SI Appendix, Fig. S1). Those proteins are present in a unique (apo and holo) closed state (Fig. 2), as asymmetrical interactions are an essential prerequisite for open state formation. Interestingly, these ABC transporterassociated proteins are both interacting with the actual translocator from the extracytoplasmic side (like the classes B, C, D, F, and G; described in the previous paragraph and in such a case also possess an N-terminal signal peptide) but are also tethered covalently to the translocator ATPase motor domains (93). As the switching (open to closed) behaviorto activate the transport cycleis missing (88, 89, 109), the noncanonical arrangement of these ABC transporters could only trigger transport activation using a yet-uncharacterized mechanism. On the other hand, the lack of Tier-0 dynamics observed in the single-turnover enzymes is conforming to their enzymatic mechanism demanding an extremely rigid active site (SI Appendix, Fig. S4A) (91). Evidently, the rigidity required for the chemistry is so dramatic that the flexibility of a noncovalently (to the polypeptide chain) bound histidine to the cleft would render the reaction unproductive.

Lastly, the CC bearing an N-terminal domain harboring the HTH-type DNA-binding domain but no C-tail (Fig. 6D) yields transcription factors of the LTTR family. Apparently, in such a case, Tier-0 dynamics of the CC are not required for function (Fig. 3G). The rigidity of the CC (Fig. 4D) is evidently required for the stability of the quaternary assemblies, as those have large cavities and holes (SI Appendix, Fig. S7) (119124).

Our data and analysis gave insights into the steps taken during evolution to shape the CC into functional ligand receptorassociated proteins (classes B, C, D, F, and G) but also one-turn enzymes (class E). Yet, how the ligand-driven signal propagates within the LTTR family, such as CynR, remains largely elusive.

The CC of CynR constitutes only one part of its structure (i.e., the sensory EBD) of a full-length transcription factor of the LTTR family with an additional winged HTH-type DNA-binding domain (Fig. 4 A and B). Although only little direct experimental evidence is available, it has been proposed that changes in the tetrameric assembly of LysR-type transcription factors can be induced by ligand binding (72, 125). These quaternary dynamics were suggested to activate transcription by a transition from a bent DNA (transcription OFF) to an unbent state (transcription ON) (72, 125). Our smFRET and HDX results (Figs. 3G and 4E), however, did not reveal any detectable changes of the CC upon azide binding. This suggests that ligand-driven structural changes within the CC protomers are minor and might thus be very hard to detect.

To gain insights into potential quaternary dynamics, we analyzed the available high-resolution structures of the oligomeric assemblies belonging to the LysR transcription factors (119124) to identify the evolutionary relevant ones by Evolutionary ProteinProtein Interface Classifier (EPIC) (126, 127). Subsequently, we modeled the CynR sequence after these structures in the presence or absence of DNA (SI Appendix, Fig. S7). The structural basis of the distinct tetrameric assemblies is the positioning of the dimerization helix with respect to the CC, dictated by the connecting loop (Fig. 4 A and B). In the extreme case that an additional helix (RD1-CH5; Dataset S3) is linked to and displaces the connecting loop, an octameric assembly is obtained (SI Appendix, Fig. S7H). To shed light on this, we monitored intrinsic Trp fluorescence during thermal melting (SI Appendix, Table S3). From the two tryptophans of CynR, one present in the dimerization helix (64 aa) and the other on D1 (274 aa) of the CC (Fig. 4 A and B), only the former one contributes to a fluorescent signal (SI Appendix, Table S3). Under DNA-free apo conditions, CynR displays a Tm(app) 55C that is significantly destabilized (6C) after binding to DNA. Addition of azide at free or DNA-bound CynR causes a ligand-characteristic signature throughout the Trp-temperature spectrums, giving rise to a secondary Tm(app) at 27C in both cases (SI Appendix, Table S3).

To understand the signal propagation originating from ligand binding, we inspected the available structural information (SI Appendix, Fig. S7). In agreement with the experimental findings (Figs. 3G and 4E), our structural analysis indicated that D1 and D2 motions are not required (SI Appendix, Table S4) for the oligomeric assemblies of the same transcription factor (OxyR) to differ dramatically in order to trigger structural changes on DNA (SI Appendix, Fig. S7 EG). What varies between those assemblies is the orderdisorder of specific secondary structure elements within D2 of the CC (S4 till 5; Dataset S3). We anticipate that such changes in the flexibility of secondary structure elements are induced by ligand binding, like in the case of MalE (Fig. 5 JL).

We conclude that signal propagation in CCPs comprising large D1/D2 rearrangements (Fig. 2; classes B, C, D, F, and G) driven by the C-tail/D1 and D2 interactions involves bending/unbending of the spring-like hinge (108). On the other side, propagation in class A is initiated by small/localized rearrangements of secondary structure element within the CC somehow transmitted to the N-terminal HTH domain leading to global quaternary structural changes (SI Appendix, Figs. S7 and S8). Additional analysis regarding the molecular mechanisms of LTTR-transcriptional regulators will be the subject of future studies.

Fig. 6 summarizes our proposed evolutionary path taken by the CC proteins over long time periods. The terminal modules impact and finetune the multi-Tier structural dynamics as can be described by the folding funnel model (8). The CCPs with asymmetric C-tails displaying Tier-0 dynamics (i.e., open and closed states) have the two characteristic wells in the funnel separated by a large energetic barrier (Fig. 6B and SI Appendix, Fig. S8C). For that reason, such proteins are found predominantly in the lowestenergetic level open state (apo energetic funnel; SI Appendix, Fig. S8C), with infrequent transitions to the closed one (Fig. 5B). Only in the SBD1/2 proteins (class B), 1% occurrence of a closed state has been experimentally observed (89). By destabilizing the open state of SBD2, we obtained a 20% occurrence of a closed state (Fig. 5C). As in the holo state energetic funnel, the lowest energy state is the closed one (SI Appendix, Fig. S8C); addition of the ligand shifts the equilibrium toward the closed state. Since the dissociation constant (KD) derives from the difference between the lowest energetic levels of the apo and holo funnels (SI Appendix, Fig. S8), destabilization of the C-tail (Fig. 5 D and H) that directs the open state of the apo funnel at a higher energetic level leads to an increased affinity.

Alternatively, CCPs with a symmetric C-tail (Fig. 6C) have a single main energetic well, defining the unique closed structural state both in the apo or holo funnels (SI Appendix, Fig. S8D).

The CCPs with N-terminal domain additions (Fig. 6D) have multiple energetic funnels corresponding to oligomerization states (SI Appendix, Fig. S8E). The energetic funnel of such CCPs is having multiple minima corresponding to the many different arrangements of the flexible N-terminal domain with respect to the CC (SI Appendix, Fig. S8E). Self-association (oligomer formation) and/or binding to their partners/ligands deepens specific wells that are required for function.

We anticipate the energetic funnel of the primordial consensus CC to be extremely rugged, with small energetic barriers between the wells, with a single well somewhat deeper (corresponding to the closed state), thus allowing sampling of multiple structural states (SI Appendix, Fig. S8A). Such a structural variability would lead to increased substrate promiscuity, however, exploiting extremely weak interactions (i.e., low binding affinities; SI Appendix, Fig. S8B).

Clearly, the structural promiscuity achieved by the modularity introduced in this study, expands the protein evolvability theory and establishes the notion that in order to comprehend protein evolution, it is essential to decode the energetic funnel of structural homologs. Structural elements or even domains added alike Lego bricks to a structural core, being the Lego Board, trigger distinct evolutionary trajectories.

Detailed materials and methods are included in SI Appendix, Material and Method. All proteins were expressed in Escherichia coli cells (BL21 DE3 or BL21 pLysS DE3) and grew in Luria Bertani or Terrific Broth media. Purification was based on affinity [Ni-NTA (Qiagen)], anion exchange [Q Sepharose (GE Healthcare)], and/or size-exclusion [Hi Load 26/60 Superdex 200 (GE Healthcare)] chromatography. Samples used for analysis (ITC, smFRET, HDX-MS, etc.) were single monodisperse peaks. Phylogenetic, structural analysis, alignments, and protein visualization were accomplished by widely accepted procedures via freely available software or servers: Structure similarity search-PDB webserver (128, 129), PDBeFold (130), Dali server (131), multidimensional QR factorization of multiple sequence and structure alignments (132)/visual molecular dynamics 1.9.2 software package (133), Protein blast (134), DynDom domain motion server (135, 136), Protein Interaction calculator webserver (99), EPIC server (126, 127), ECOD database (38), ConSurf-DB server (137, 138), SWISS-MODEL server (139), and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrdinger, LLC). smFRET experiments were performed with a custom-made confocal ALEX microscope that has been previously described in the literature (22, 89) and HDX-MS by a nanoACQUITY Ultra Performance Liquid Chromatography System with HDX technology (Waters, United Kingdom). Data and statistical analyses were performed by established procedures described in detail in the literature.

This work was financed by an Netherlands Organization for Scientific Research (NWO Veni grant 722.012.012 to G.G.), an ERC Starting Grant (European Research Council StG No. 638536, SM-IMPORT to T.C.), Deutsche Forschungsgemeinschaft within GRK2062 (project C03 to T.C.), SFB863 (project A10 to M.Z. and A13 to T.C.), Research Foundation Flanders (FWO CARBS #G0C6814N to G.G. and A.E.). G.G. also acknowledges a fellowship from the European Molecular Biology Organization (EMBO long-term fellowship ALF 47-2012 to G.G.) and financial support by the Zernike Institute for Advanced Materials and the Rega foundation. Y.A.M. was supported by the Indonesia Endowment Fund for Education (Lembaga Pengelola Dana Pendidikan, Republik Indonesia, LPDP RI PhD scholarship). G.G. and Y.A.M. acknowledge financial support from the IMBB-FORTH (start-up grant to G.G.). N.Z. acknowledges an Alexander von Humboldt postdoctoral fellowship. N.E. acknowledges a fellowship from the Marie Sklodowska Curie Action (MSCA SoE FWO (195872). R.X. was supported by the Chinese Scholarship Council grant. T.C. was supported by the German Academic Exchange Service, Center of Nanoscience Munich, LMU excellent and the Center for Integrated Protein Science Munich. We thank Eitan Lerner for useful comments and critical reading of the manuscript, Monique Wiertsema for help with smFRET experiments, and Florence Husada for support with ITC experiments.

Author contributions: G.G., Y.A.M., M.d.B., and T.C. designed research; G.G., Y.A.M., M.d.B., D.A.G., A.T., K.T., N.Z., R.X., Y.S., and S.K. performed research; G.G., Y.A.M., M.d.B., D.A.G., K.T., R.X., Y.S., M.Z., A.D., C.P., and A.E. contributed new reagents/analytic tools; G.G., Y.A.M., M.d.B., D.A.G., N.Z., A.T., N.E., and T.C. analyzed data; and G.G., Y.A.M., D.A.G., and T.C. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

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The Best Science and Tech Breakthroughs of 2021 – Nerdist

Scientists and engineers explored new frontiers in every technological category in 2021. Advances in everything from spaceflight to microrobotics to artificial intelligence abounded, offering a glimpse of a world in which humanity is a multiplanet species. As well as one physiologically connected to intelligent machines. Below are the best science and tech breakthroughs of 2021, in our humble opinion, which may change when we get our Neuralink brain implants.

Although SpaceX had several spectacular failures trying to fly and land its prototype Starship rocket, that just made the first successful attempt (below) all the sweeter. According to SpaceX, the company plans to use Starships to send people to the Moon and Mars. The complete Starship system, once it comes online, will be an astounding 394 feet tall.

While seeing rovers roll around on Mars can feel commonplace, mobility breakthroughs on the Red Planet are beginning to happen. Below is video of the first-ever (mini) helicopter flight on Mars, which occurred on April 19. The flight, while short, was exceptional thanks not only to the helicopters long journey to Mars in the Perseverance rover, but also the planets super-thin atmosphere.

In July of this year, Googles DeepMind subsidiary announced it had solved a grand challenge in biology known as the protein folding problem. Using its cutting-edge AI, AlphaFold, DeepMind released the structures of 350,000 proteins. And noted that the tech will eventually be able to help identify and cure diseases.

Engineers the world over have been working on ways to shrink robots. Emblematic of the efforts from this year are microflier robots that can float on the wind. While the microfliers themselves will reportedly record things like changes in climate and the spread of disease, we cant help but experience foreboding Black Mirror vibes.

As their name implies, brain organoids, or cerebral organoids, are very much like tiny human brains; a fact that makes scientists giving them eye balls in August of this year all the wilder. The eyed organoids, while somewhat disturbing, will hopefully help to cure congenital retinal disorders and even personalize drug testing. And help to raise some important issues for bioethics as well, we imagine.

Smart clothes that can sense and record all of your movements, as well as give you posture suggestions, are now here thanks to MIT. While not wholly new, MITs smart clothes are unique because they consist of simple, knitted conductive yarn, and are amenable to mass production. As well as collecting large amounts of data from their users for robot training.

Finally on the list is Neuralinks breakthrough demonstration of a monkey telepathically playing Pong. Or, in this context, MindPong. Neuralink was able to pull off the feat by plunging 1,024 ultra-thin electrodes into a Macaques brain. (Banana smoothies were essential as well.) The company says that, in the near-term, the tech could help paralyzed people surf the net and express themselves artistically. Merging with superintelligent AI is also apparently not off the table for this rapidly moving decade.

Feature image: Neuralink/Cell Stem Cell/NASA

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The Best Science and Tech Breakthroughs of 2021 - Nerdist

Doing This in the Bathroom Can Reduce Your Dementia Risk Best Life – Best Life

As you age, your dementia risk increases rapidly. In fact, according to the Alzheimer's Association, your risk of Alzheimer's doubles every five years after the age of 65. However, there are some things you can do to lower your risk of developing cognitive impairmentincluding one you may be able to do in your very own bathroom. Experts say that by doing this one thing roughly three times per week, you can slash your odds of dementia in half over a 20 year period. Read on to find out which habit may stave off dementia, and how to do it safely.

RELATED:This Heartburn Medication Raises Your Dementia Risk 44 Percent, Study Says.

According to a 2020 study published in the journal Preventive Medicine Reports, "repeated heat exposure like sauna bathing" seems to be beneficial in preventing dementia development. The cohort study, which was conducted in Finland, utilized surveys and existing medical records from 13,994 middle-aged men and women who had not previously been diagnosed with dementia.

When the team compared the health data from those who took a sauna between nine and 12 times per month with that of those who did so four or fewer times per month, they found that those who regularly took a sauna had lower dementia risk. "During the first 20 years of follow-up, the dementia risk of those reporting 912 sauna baths per month (i.e., approximately three per week) was less than a half of the risk of those who had sauna baths only 04 times per month," the team wrote. "The reduction in the dementia risk was attenuated during the follow-up, but the decrease of the risk was still evident after nearly 40 years. Accordingly, a sauna bathing frequency of three times per week may be associated with a reduced risk of dementia," they added, noting that further research is required to verify the benefits.

RELATED:This Could Be Your First Sign of Dementia Years Before Diagnosis, Study Says.

Because sauna bathing is considered commonplace in Finland, nearly all of the study participants practiced the habit, and did so on average 6.03 times per month. These sessions typically lasted under 15 minutes at a temperature below 100 degrees Celsius (212 degrees Fahrenheit), the researchers wrote.

They found that "a straight stay in heat for five to 14 minutes per heat session vs. less than 5 minutes was suggestively related to a reduced risk [of dementia]. The most favorable sauna temperature for dementia protection was 8099 degrees Celsius [176-210 degrees Fahrenheit]," they wrote.

Wondering why a sauna would lower your dementia risk? The Finnish team said that while more research is needed on the matter, there are several possible answers related to "physiological, metabolic, and cellular changes which may affect brain function."

In particular, they say that a sudden elevation in temperature causes heat shock, which leads to the creation of something called "heat shock proteins." The researchers explained that these "are important regulators in normal cell functions and have an essential role in guarding and controlling protein formation. Because disturbances of protein construction and folding are central to the development of neurological diseases, heat shock proteins may be important in maintaining protein homeostasis in the brain."

Additionally, saunas may improve vascular and cardiovascular function, which increases cerebral blood flow and lowers inflammation. "It is possible that some of the effects of sauna in the brain are conveyed via reduced inflammation," the team wrote.

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Though the researchers found a lower risk of dementia in those who took a sauna on average three times per week, they also observed that those who did so in extremely hot temperatures were at significantly elevated risk of the brain disease. "Sauna heat which is too high may not be good for the brain. The dementia risk of those bathing in sauna temperatures higher than 100 degrees Celsius [212 degrees Fahrenheit] doubled compared to those bathing at temperatures lower than 80 degrees Celsius [176 degrees Fahrenheit] during the first twenty years of follow-up," the team warned.

However, most saunas in the U.S.like the one you may have at your gym, or, if you're lucky, in your homeare heated to temperatures between 150 and 195 degrees Fahrenheit. They typically include a thermometer and temperature controls. Be sure to check the sauna's settings before entering, and limit your time inside to no more than 15 minutes.

RELATED:If You Do This in Your Sleep, Get Checked for Dementia, Says Mayo Clinic.

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Doing This in the Bathroom Can Reduce Your Dementia Risk Best Life - Best Life

The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting – DocWire News

This article was originally published here

Nat Commun. 2021 Dec 10;12(1):7193. doi: 10.1038/s41467-021-27431-0.

ABSTRACT

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses, including SARS-CoV-2. It allows production of essential viral, structural and replicative enzymes that are encoded in an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshift elements and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the SARS-CoV-2 frameshift element and the host proteome. We reveal that the short isoform of the zinc-finger antiviral protein (ZAP-S) is a direct regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and inhibits viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and interferes with the folding of the frameshift RNA element. Together, these data identify ZAP-S as a host-encoded inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

PMID:34893599 | DOI:10.1038/s41467-021-27431-0

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The short isoform of the host antiviral protein ZAP acts as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting - DocWire News

The roadmap to an effective AI assurance ecosystem – extended version – GOV.UK

Introduction

This extended Roadmap is designed to complement the CDEIs Roadmap to an effective AI assurance ecosystem, which sets out the key steps, and the roles and responsibilities required to develop an effective, mature AI assurance ecosystem.

Where the short version of the roadmap is designed to be accessible as a quick-read for decision makers, this extended version incorporates further research and examples to provide a more detailed picture of the ecosystem and necessary steps forward.

Additionally, chapter 1 of this extended roadmap offers further context on the AI assurance process, delivering AI assurance, the role of AI assurance in broader AI governance and the roles and responsibilities for AI assurance. Chapter 3 discusses some of the ongoing tensions and challenges in a mature AI assurance ecosystem.

This extended roadmap will be valuable for readers interested in finding out more information about how to build an effective, mature assurance ecosystem for AI.

Data-driven technologies, such as artificial intelligence (AI), have the potential to bring about significant benefits for our economy and society. AI systems offer the opportunity to make existing processes faster and more effective, and in some sectors offer new tools for decision-making, analysis and operations.

AI is being harnessed across the economy, helping businesses to improve their day-to-day operations, such as achieving more efficient and adaptable supply chain management. AI has also enabled researchers to make a huge leap forward in solving one of biologys greatest challenges, the protein folding problem. This breakthrough could vastly accelerate efforts to understand the building blocks of cells, and could improve and speed up drug discovery. AI presents game changing opportunities in other sectors too, through the potential for operating an efficient and resilient green energy grid, as well as helping tackle misinformation on social media platforms.

However, AI systems also introduce risks that need to be managed. The autonomous, complex and scalable nature of AI systems (in particular, machine learning) pose risks beyond that of regular software. These features pose fundamental challenges to our existing methods for assessing and mitigating the risks of using digital technologies.

The autonomous nature of AI systems makes it difficult to assign accountability to individuals if harms occur; the complexity of AI systems often prevents users or affected individuals from explaining or understanding the link between a systems output or decision and its causes, providing further challenges to assigning accountability; and the scalability of AI makes it particularly difficult to define legitimate values and governance frameworks for a systems operation e.g. across social contexts or national jurisdictions.

As these technologies are more widely adopted, there is an increasing need for a range of actors to check that these tools are functioning as expected and demonstrate this to others. Without being able to assess the trustworthiness of an AI system against agreed criteria, buyers or users of AI systems will struggle to trust them to operate effectively, as intended. Furthermore they will have limited means of preventing or mitigating potential harms if a system is not in fact trustworthy.

Assurance as a service draws originally from the accounting profession, but has since been adapted to cover many areas such as cyber security and quality management. In these areas, mature ecosystems of assurance products and services enable people to understand whether systems are trustworthy. These products and services include: process and technical standards; repeatable audits; certification schemes; advisory and training services. For example, in financial accounting, auditing services provided by independent accountancy firms enable an assurance user to have confidence in the trustworthiness of the financial information presented by a company.

AI assurance services have the potential to play a distinctive and important role within AI governance. Its not enough to set out standards and rules about how we expect AI systems to be used. It is also important that we have trustworthy information about whether they are following those rules.

Assurance is important for assessing efficacy, for example through performance testing; addressing compliance with rules and regulations, for example performing an impact assessment to comply with data protection regulation; and also for assessing more open ended risks. In the latter category, rules and regulations cannot be relied upon to ensure that a system is trustworthy, more individual judgement is required. For example, assessing whether an individual decision made by an AI system is fair in a specific context.

By ensuring both trust in and the trustworthiness of AI systems, AI assurance will play an important enabling role in the development and deployment of AI, unlocking both the economic and social benefits of AI systems. Consumer trust in AI systems is crucial to widespread adoption, and trustworthiness is essential if systems are going to perform as expected and therefore bring the benefits we want without causing unexpected harm.

An effective AI assurance ecosystem is needed to coordinate appropriate responsibilities, assurance services, standards and regulations to ensure that those who need to trust AI have the sort of evidence they need to justify that trust. In other industries, we have seen healthy assurance ecosystems develop alongside professional services to support businesses from traditional accounting, to cybersecurity services. Encouraging a similar ecosystem to develop around AI in the UK would be a crucial boon to the economy.

For example, the UKs cyber security industry employed 43,000 full-time workers, and contributed nearly 4bn to the UK economy in 2019. More recently, research commissioned by the Open Data Institute (ODI) on the nascent but buoyant data assurance market found that 890 data assurance firms are now working in the UK with 30,000 staff. The research carried out by Frontier Economics and glass.ai noted that 58% of these firms incorporated in the last 10 years. Following this trend, AI assurance is likely to become a significant economic activity in its own right. AI assurance is an area in which the UK, with particular strengths in legal and professional services, has the potential to excel.

The roadmap provides a vision of what a mature ecosystem for AI assurance might look like in the UK and how the UK can achieve this vision. It builds on the CDEIs analysis of the current state of the AI assurance ecosystem and examples of other mature assurance ecosystems.

The first section of the roadmap looks at the role of AI assurance in ensuring trusted and trustworthy AI. We set out how assurance engagements can build justified trust in AI systems, drawing on insights from more mature assurance ecosystems, from product safety through to quality management and cyber security. We illustrate the structure of assurance engagements and highlight assurance tools relevant to AI and their applications for ensuring trusted and trustworthy AI systems. In the latter half of this section we zoom out to consider the role of assurance within the broader AI governance landscape and highlight the responsibilities of different actors for demonstrating trustworthiness and their needs for building trust in AI.

The second section sets out how an AI assurance ecosystem needs to develop to support responsible innovation and identifies six priority areas:

We set out the current state of the ecosystem and highlight the actions needed in each of these areas to achieve a vision for an effective, mature AI assurance ecosystem. Following this, we discuss the ongoing tensions that will need to be managed, as well as the promise and limits of assurance. We conclude by outlining the role that the CDEI will play in helping deliver this mature AI assurance ecosystem.

We have combined multiple research methods to build the evidence and analysis presented in this roadmap. We carried out literature and landscape reviews of the AI assurance ecosystem to ground our initial thinking and performed further desk research on a comparative analysis of mature assurance ecosystems. Based on this evidence, we drew on multidisciplinary research methods to build our analysis of AI assurance tools and the broader ecosystem.

Our desk-based research is supported by expert engagement, through workshops, interviews and discussions with a diverse range of expert researchers and practitioners. We have also drawn on practical experience from assurance pilot projects with organisations adopting or considering deploying AI systems, across both private sector organisations (in partnership with researchers from University College London), along with the CDEIs work with public sector organisations across recruitment, policing and defence.

Building and maintaining trust is crucial to realising the benefits of AI systems. If organisations dont trust AI systems, they will be less willing to adopt these technologies because they dont have the confidence that an AI system will actually work or benefit them. They might not adopt for fear of facing reputational damage and public backlash. Without trust, consumers will also be cautious about using data-driven technologies, as well as sharing the data that is needed to build them.

The difficulty is, however, that these stakeholders often have limited information, or lack the appropriate specialist knowledge to check and verify others claims to understand whether AI systems are actually deserving of their trust.

This is where assurance is important. Being assured is about having confidence or trust in something, for example a system or process, documentation, a product or an organisation. Assurance engagements require providing evidence - often via a trusted independent third party - to show that the AI system being assured is reliable and trustworthy.

The distinction between trust and trustworthiness is important here: when we talk about trustworthiness, we mean whether something is deserving of peoples trust. On the other hand, when we talk about trust, we mean whether something is actually trusted by someone. Someone might trust something, even if it is not in fact trustworthy.

A successful relationship built on justified trust requires both trust and trustworthiness:

Trust without trustworthiness = misplaced trust. If we trust technology or the organisations deploying a technology when they are not in fact trustworthy, we incur potential risks by misplacing our trust.

Trustworthy but not trusted = (unjustified) mistrust. If we fail to trust a technology or organisation which is in fact trustworthy, we incur the opportunity costs of not using good technology.

Fulfilling both of these requirements produces justified trust.

There are two key problems which organisations must overcome to build justified trust:

An information problem: Organisations need to reliably and consistently evaluate whether an AI system is trustworthy to provide the evidence base for whether or not people should trust it.

A communication problem: Organisations need to communicate their evidence to other assurance users and translate this evidence at the appropriate level of complexity so that they can direct their trust or distrust accordingly.

The value of assurance is overcoming both of these problems to enable justified trust.

Assurance requires measuring and evaluating a variety of information to show that the AI system being assured is reliable and trustworthy. This includes how these systems perform, how they are governed and managed, whether they are compliant with standards and regulations, and whether they will reliably operate as intended. Assurance provides the evidence required to demonstrate that a system is trustworthy.

Assurance engagements rely on clear metrics and standards against which organisations can communicate that their systems are effective, reliable and ethical. Assurance engagements therefore provide a process for (1) making and assessing verifiable claims to which organisations can be held accountable and (2) for communicating these claims to the relevant actors so that they can build justified trust, where a system is deserving of their trust.

This challenge of assessing the trustworthiness of systems, processes and organisations to build justified trust is not unique to AI. Across different mature assurance ecosystems, we can see how different assurance models have been developed and deployed to respond to different types of risks that arise in different environments. For example: from risks around professional integrity, qualifications and expertise in legal practice; to assuring operational safety and performance risks in safety critical industries, such as aviation or medicine.

The requirements for a robust assurance process are most clearly laid out in the accounting profession, although we see very similar characteristics in a range of mature assurance ecosystems.

In the accounting profession, the 5 elements of assurance are specified as:

The accounting model is helpful for thinking about the structure that AI assurance engagements need to take. The five elements help to ensure that information about the trustworthiness of different aspects of AI systems is reliably evaluated and communicated.

While this roadmap draws on the formal definitions developed by the accounting profession, similar roles, responsibilities and institutions for standard setting, assessment and verification are present across the range of assurance ecosystems - from cybersecurity to product safety - providing transferable assurance approaches.

Within these common elements, there is also variation in the use of different assurance models across mature ecosystems. Some rely on direct performance testing, while others rely on reviewing processes or ensuring that accountable people have thought about the right issues at the right time. In each case, the need to assure different subject matters has led to variation in the development and use of specific assurance models, to achieve the same ends.

In this section, we will build on our analysis of AI assurance and how it can help to build justified trust in AI systems, by briefly explaining some of the mechanisms that can be used to deliver AI assurance. We will explore where they are useful for assuring different types of subject matter that are relevant to the trustworthiness of AI systems.

A more detailed exploration of AI assurance mechanisms and how they apply to different subject matter in AI assurance is included in our AI assurance guide.

There are multiple approaches to delivering assurance. The spectrum of assurance techniques offer different processes for providing assurance, enabling assurance users to have justified trust in a range of subject matters relevant to the trustworthiness of AI systems.

On one end of this spectrum, impact assessments are designed to account for uncertainty, ambiguity and the unobservability of potential future harms. Impact assessments require expertise and subjective judgement to account for these factors, but they enable standardised processes for qualitatively assessing potential impacts. Assurance can be provided against these processes and the mitigation strategies put in place to deal with potential adverse impacts.

At the other end of this spectrum, formal verification is appropriate for assessing trustworthiness for subject matters which can be measured objectively and with a high degree of certainty. It is ineffective if the subject matter is ambiguous, subjective or uncertain. For example, formal guarantees of fairness cannot be provided for an AI systems outputs.

AI assurance services are a distinctive and important aspect of broader AI governance. AI governance covers all the means by which the development, use, outputs and impacts of AI can be shaped, influenced and controlled, whether by government or by those who design, develop, deploy, buy or use these technologies. AI governance includes regulation, but also tools like assurance, standards and statements of principles and practice.

Regulation, standards and other statements of principles and practice set out criteria for how AI systems should be developed and used. Alongside this, AI assurance provides the infrastructure for checking, assessment and verification, to provide reliable information about whether organisations are following these criteria.

An AI assurance ecosystem can offer an agile regulatory market of assurance services, consisting of both for-profit and not-for-profit services. This regulatory market can support regulators as well as standards development bodies and other responsible AI authorities to ensure trustworthy AI development and deployment while enabling industry to innovate at pace and manage risk.

AI assurance services will play a crucial role in a regulatory environment by providing a toolbox of mechanisms and processes to monitor regulatory compliance, as well as the development of common practice beyond statutory requirements to which organisations can be held accountable.

Compliance with regulation

AI assurance mechanisms facilitate the implementation of regulation and the monitoring of regulatory compliance in the following ways: implementing and elaborating rules for the use of AI systems in specific circumstances; translating rules into practical forms useful for end users and evaluating alternative models of implementation; and providing technical expertise and capacity to assess regulatory compliance across the system lifecycle.

Assurance mechanisms are also important in the international regulatory context. Assurance mechanisms can be used to facilitate assessment against designated technical standards that can provide a presumption of conformity with essential legal requirements. The presumption of conformity can enable interoperability between different regulatory regimes, to facilitate trade. For example, the EUs AI act states that compliance with standardsshould be a means for providers to demonstrate conformity with the requirements of this Regulation.

Managing risk and building trust

Assurance services also enable stakeholders to manage risk and build trust by ensuring compliance with standards, norms and principles of responsible innovation, alongside or as an alternative to more formal regulatory compliance. Assurance tools can be effective as post compliance tools where they can draw on alternative, commonly recognised sources of authority. These might include industry codes of conduct, standards, impact assessment frameworks, ethical guidelines, public values, organisational values or preferences stated by the end users.

Post-compliance assurance is particularly useful in the AI context where the complexity of AI systems can make it very challenging to craft meaningful regulation for them. Assurance services can offer means to assess, evaluate and assign responsibility for AI systems impacts, risks and performance without the need to encode explicit, scientific understandings in law.

Effective AI assurance will rely on a variety of actors with different roles and responsibilities for evaluating and communicating the trustworthiness of AI systems. In the diagram below we have categorised four important groups of actors who will need to play a role in the AI assurance ecosystem: the AI supply chain, AI assurance service providers, independent research and oversight, and supporting structures for AI assurance. The efforts of different actors in this space are both interdependent and complimentary. Building a mature assurance ecosystem will therefore require an active and coordinated effort.

The actors specified in the diagram are not meant to be exhaustive, but represent the key roles in the emerging AI assurance ecosystem. For example, Business to Business to Consumer (B2B2C) deployment models can greatly increase the complexity of assurance relationships in the real-world, where the chain of deployment between AI developers and end consumers can go through multiple client layers.

Similarly, it is important to note that while the primary role of the supporting structures in developing an AI assurance ecosystem is to set out the requirements for trustworthy AI through regulation, standards or guidance, these actors can also play other roles in the assurance ecosystem. For example, regulators also provide assurance services via advisory, audit and certification functions e.g. the Information Commissioners Offices (ICO) investigation and assurance teams assess the compliance of organisations using AI. Government and other public sector organisations also play the executive role when procuring and deploying AI systems.

These actors play a number of interdependent roles within an assurance ecosystem. The table below illustrates each actors role in demonstrating the trustworthiness of AI systems and their own requirements for building trust in AI systems.

This section sets out a vision for a mature AI assurance ecosystem, and the practical steps that can be taken to make this vision a reality. We have based this vision on our assessment of the current state of the AI assurance ecosystem, as well as comparison with more mature ecosystems in other domains.

An effective AI assurance ecosystem matters for the development of AI. Without it, we risk either trust without trustworthiness, where risky, unsafe or inappropriately used AI systems are deployed, leading to real world harm to people, property, and society. Alternatively, the prospect of these harms could lead to unjustified mistrust in AI systems, where organisations hesitate to deploy AI systems even where they could deliver significant benefit. Worse still, we risk both of these happening simultaneously.

An effective AI assurance ecosystem will rely on accommodating the perspectives of multiple stakeholders who have different concerns about AI systems and their use, different incentives to respond to those concerns, and different skills, tools and expertise for assurance. This coordination task is particularly challenging for AI, as it is a general purpose group of technologies that can be applied in many domains. Delivering meaningful assurance requires understanding not only the technical details of AI systems, but also relies on subject matter expertise and knowledge of the context in which these systems are used.

The current ecosystem contains the right ingredients for success, but is highly fragmented and needs to mature in a number of different ways to become fully effective. Responsibilities for assurance need to be distributed appropriately between actors, the right standards need to be developed and the right skills are needed throughout the ecosystem.

The market for AI assurance is already starting to grow, but action is needed to shape this ecosystem into an effective one that can respond to the full spectrum of risks and compliance issues presented by AI systems. To distribute responsibilities effectively and develop the skills and supporting structures needed for assurance, we have identified six key areas for development. These are:

In the following sections, we will outline the current state of the AI assurance ecosystem with regard to these six areas and compare this with our vision for a mature future ecosystem. We highlight the roles of different actors and outline important next steps for building towards a mature AI assurance ecosystem.

Early demand for AI assurance has been driven primarily by the reputational concerns of actors in the AI supply chain, along with proactive efforts by AI developers to build AI responsibly. However, pressure on organisations to take accountability for their use of AI is now coming from a number of directions. Public awareness of issues related to AI assurance (especially bias) is growing in response to high profile failures. We are also seeing increasing interest from regulators, higher customer expectations, and concerns about where liability for harms will sit. The development community is being proactive in this space, managing risks as part of the responsible AI movement, however we need others in the ecosystem to better recognise assurance needs.

Increased interest and higher consumer expectations mean that organisations will need to demand more evidence from their suppliers and their internal teams to demonstrate that the systems they use are safe and otherwise trustworthy.

Organisations developing and deploying AI systems already have to respond to existing regulations including data protection law, equality law and sector specific regulations. As existing motivations to regulate AI appear, organisations will need to anticipate future regulation to remain competitive. This will include both UK sector-based regulation and for organisations exporting products, non-UK developments such as the EU AI regulations and the Canadian AIA.

Regulators are starting to demand evidence that AI systems being deployed are safe and otherwise trustworthy, with some regulators starting to set out assurable recommendations and guidelines for the use of AI systems.

In a mature assurance ecosystem, assurance demand is driven by:

Organisations desire to know that their systems or processes are effective and functioning as intended.

The need for organisations to earn and keep the trust of their customers and staff, by demonstrating the trustworthiness of the AI systems they deploy. This will partly need to happen proactively but will also be driven by commercial pressures.

An awareness of and a duty to address real material risks to the organisation and wider society.

The need to comply with, and demonstrate compliance with regulations and legal obligations.

Demonstrating trustworthiness to the wider public, competing on the basis of public trust.

The importance of these drivers will vary by sector. For example, in safety-critical industries the duty to address material risks and build consumer trust will be stronger in driving assurance demand, compared to low risk industries. In most industries where AI is being adopted, the primary driver for assurance services will be gaining the confidence that their systems will actually work effectively, as they intend them to.

To start to respond to these demands, organisations building or deploying AI systems should be developing a clear understanding of the concrete risks and concerns that arise. Regulators and professional bodies have an important supporting role here in setting out guidance to inform industry about key concerns and drive effective demand for assurance. When these concerns have been identified, organisations need to think about the sorts of evidence that is needed to understand, manage and mitigate these risks, to provide assurance to other actors in the ecosystem.

In a mature AI assurance ecosystem, those accountable for the use of AI systems will demand and receive evidence that these systems are fit-for-purpose. Organisations developing, procuring or using AI systems should be aware of the risks, governance requirements and performance outcomes that they are accountable for, and provide assurance accordingly. Organisations that are aware of their accountabilities for risks will be better placed to demand the right sort of assurance services to address these risks. As well as setting accountabilities, regulation and standards will play an important role in structuring incentives for assurance i.e. setting assurance requirements and criteria to incentivise effective demand.

The drivers discussed above will inevitably increase the demand for AI assurance, but we still need to take care to ensure that the demand is focused on services that add real value. Many current AI assurance services are focused primarily on aspects of risk and performance that are most salient to an organisations reputation. This creates risks of deception and ethics washing, where actors in the supply chain can selectively commision or perform assurance services primarily to benefit their reputation, rather than address the underlying drivers of trustworthiness.

This risk of ethics washing relates to an incentive problem. The economic incentives of actors within the AI supply chain come into conflict with incentives to provide reliable, trustworthy assurance. Incentive problems in the AI supply chain currently prevent demand for AI assurance from satisfactorily ensuring AI systems are trustworthy, and misalign demand for AI assurance with broader societal benefit. Avoiding this risk will require a combination of ensuring that assurance services are valuable and attractive for organisations, but also that assurance requirements whether regulatory or non-regulatory are clearly defined across the spectrum of relevant risks, and organisations are held to account on this basis.

Demand is also constrained by challenges with skills and accountability within the AI supply chain. In many organisations there is a lack of awareness about: the types of risks and different aspects of systems and development processes that need to be assured for AI systems to be trustworthy, and appropriate assurance approaches for assessing trustworthiness across these different areas. There is also a lack of knowledge and coordination across the supply chain around who is accountable for assurance across different areas. Clearer understanding of accountabilities is required to drive demand for assurance.

As demand for AI assurance grows, a market for assurance services needs to develop in response to limitations in skills and competing incentives that actors in the AI supply chain, government and regulators are not well placed to overcome.

Organisations in the AI supply chain will increasingly demand evidence that systems are trustworthy and compliant as they become aware of their own accountabilities for developing, procuring and deploying AI systems.

However, actors in the AI supply chain wont have the expertise required to provide assurance in all of these areas. In some cases building specialist in-house capacity to serve these needs will make sense. For example, in the finance industry, model risk management is a crucial in-house function. In other areas, building specialist in-house capacity will be difficult and will likely not be an efficient way to distribute skills and resources for providing assurance services.

The business interests of actors in the AI supply chain means that without independent verification, in many cases first and second party assurance will be insufficient to build justified trust. Assurance users will be unable to have confidence that the assurance provided by the first party faithfully reflects the trustworthiness of the AI system.

Therefore, as demand for AI assurance grows, a market for external assurance providers will need to grow to meet this demand. This market for independent assurance services should include a dynamic mix of small and large providers offering a variety of services to suit a variety of needs.

A market of AI-specific assurance services has started to emerge, with a range of companies including established professional services firms, research institutions and specialised start-ups beginning to offer assurance services. There is a more established market of services addressing data protection issues, with a relatively new but growing sector of services addressing the fairness and robustness of AI systems. More novel services are also emerging to enable effective assurance, such as testbeds to promote the responsible development of autonomous vehicles. Similarly, the Maritime Autonomy Surface Testbed enables the testing of autonomous maritime systems for verification and proof of concept.

Not all AI assurance will be new though. In some use-cases and sectors, existing assurance mechanisms will need to evolve to adapt to AI. For example, routes such as conformity assessment, audit and certification used in safety assurance mechanisms will inevitably need to be updated to consider AI issues. Regulators in safety critical industries are leading the way here. The Medicines and Healthcare Products Regulatory Agency (MHRA) is committed to developing the worlds leading regulatory system for the regulation of Software as a Medical Device (SaMD) including AI.

The ICO has also begun to develop a number of initiatives to ensure that AI systems are developed and used in a trustworthy manner. The ICO has produced an AI Auditing Framework alongside Draft Guidance, which is designed to complement their guidance on Explaining decisions made with AI, produced in collaboration with the Alan Turing Institute. In September 2021, Healthily, the creator of an AI-based smart symptom checker submitted the first AI explainability statement to the ICO. ForHumanity, a US led non-profit, has submitted a draft UK GDPR Certification scheme for accreditation by the ICO and UK Accreditation Body (UKAS).

There are a range of toolkits and techniques for assuring AI emerging. However, the AI assurance market is currently fragmented and at a nascent stage. We are now in a window of opportunity to shape how this market emerges. This will involve a concerted effort across the ecosystem, in both the public and private sectors, to ensure that AI assurance services can meet the UKs objectives for ethical innovation.

Assuring AI systems requires a mix of different skills. Data scientists will be needed to provide formal verification and performance testing, audit professionals will be required to assess organisations compliance with regulations, and risk management experts will be required to assess risks and develop mitigation processes.

Given the range of skills, it is perhaps unlikely that demand will be met entirely by multi-skilled individuals; multi-disciplinary teams bringing a diverse range of expertise will be needed. A diverse market of assurance providers needs to be supported to ensure the right specialist skills are available. The UKs National AI Strategy has begun to set out initiatives to help develop, validate and deploy trustworthy AI, including building AI and data science skills through skills bootcamps. It will be important for the UK to develop both general AI skills and the specialist skills in assurance to do this well.

In addition to independent assurance providers, there needs to be a balance of skills for assurance across different roles in the ecosystem. For example, actors within the AI supply chain will require a baseline level of skills for assurance to be able to identify risks to provide or procure assurance services effectively. This balance of skills should reflect the complexity of different assurance processes, the need for independence, and the role of expert judgement in building justified trust.

Supporting an effective balance of skills for assurance, and more broadly enabling a trustworthy market of independent assurance providers, will rely on the development of two key supporting structures. Standards (both regulatory and technical) are needed to set shared reference points for assurance engagements enabling agreement between assurance users and independent providers. Secondly, professionalisation will be important in developing the skills and best-practice for AI assurance across the ecosystem. Professionalisation could involve a range of complementary options, from university or vocational courses to more formal accreditation services.

The next section will outline the role of standards in enabling independent assurance services to succeed as part of a mature assurance ecosystem. After exploring the role of standards, the following section will expand on the possible options for developing an AI assurance profession.

Standards are crucial enablers for AI assurance. Across a whole host of industries, the purpose of a standard is to provide a reliable basis for people to share the same expectations about a product, process, system or service.

Without commonly accepted standards to set a shared reference point, a disconnect between the values and opinions of different actors can prevent assurance from building justified trust. For example, an assurance user might disagree with the views of an assurance provider about the appropriate scope of an impact assessment, or how to measure the level of accuracy of a system. As well as enabling independent assurance, commonly understood standards will also support the scalability and viability of self-assessment and assurance more generally across the ecosystem.

There are a range of different types of standards that can be used to support AI assurance, including technical, regulatory and professional standards. The rest of this section will specifically focus on the importance of Global technical standards for AI assurance. Global technical standards set out good practice that can be consistently applied to ensure that products, processes and services perform as intended safely and efficiently. They are generally voluntary and developed through an industry-led process in global standards developing organisations, based on the principles of consensus, openness, and transparency, and benefiting from global technical expertise and best practice. As a priority, independent assurance requires the development of commonly understood technical standards which are built on consensus.

Read more here:
The roadmap to an effective AI assurance ecosystem - extended version - GOV.UK

Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor – pnas.org

Significance

This study reveals a mechanism for effector perception by a plant NLR immune receptor that contains an integrated domain (ID) that mimics an authentic effector target. The Arabidopsis immune receptors RRS1 and RPS4 detect the Pseudomonas syringae pv. pisi secreted effector AvrRps4 via a WRKY ID in RRS1. We used structural biology to reveal the mechanisms of AvrRps4CWRKY interaction and demonstrated that this binding is essential for effector recognition in planta. Our analysis revealed features of the WRKY ID that mediate perception of structurally distinct effectors from different bacterial pathogens. These insights could enable engineering NLRs with novel recognition specificities, and enhance our understanding of how effectors interact with host proteins to promote virulence.

Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the 2-3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4CRRS1WRKY interactions invitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKYW-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.

Plants coevolve with their pathogens, resulting in extensive genetic variation in host immune receptor and pathogen virulence factor (effector) repertoires (1). To enable host colonization, pathogenic microbes deliver effector proteins into host cells that suppress host immune responses and elevate host susceptibility by manipulating host physiology (2, 3). Plants have evolved surveillance mechanisms to detect and then activate defenses that combat pathogens, and detect host-translocated effectors via nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)containing receptors (NLRs) (4). NLR genes are highly diverse, showing both copy-number and presence/absence of polymorphisms, and different alleles can exhibit distinct effector recognition specificities (5, 6). As described by the gene-for-gene model, plant NLRs usually recognize a single effector (7). However, NLRs capable of responding to multiple effectors are known (5, 8, 9).

NLRs typically contain an N-terminal Toll/interleukin-1 receptor (TIR) or coiled-coil (CC) domain, a central NBD (NB-ARC [NBD shared with APAF-1, various R proteins, and CED-4]), and a C-terminal LRR domain (6). In addition to these canonical domains, some NLRs have evolved to carry integrated domains that mimic effector virulence targets and facilitate immune activation by directly binding effectors (1015). Interestingly, integrated domain-containing NLRs (NLR-IDs) usually function with a paired helper NLR, which is required for immune signaling (10, 16).

The Arabidopsis NLR pair RRS1-R/RPS4 is a particularly interesting NLR-ID/NLR pair that confers resistance to bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum, and also to a fungal pathogen (Colletotrichum higginsianum) where the effector is unknown (1720). RRS1-R contains an integrated WRKY domain near its C terminus (RRS1WRKY), which interacts with two structurally distinct type III secreted bacterial effectors, AvrRps4 from P. syringae pv. pisi and PopP2 from R. solanacearum (13, 14, 21, 22). The RRS1WRKY domain may mimic the DNA-binding domain of WRKY transcription factors (TFs), the putative virulence targets of AvrRps4 and PopP2, to enable immune perception of these effectors (13). Two alleles of RRS1 have been identified that differ in the length of the C-terminal extension after the WRKY domain (SI Appendix, Fig. S1). RRS1-R, from the accession Ws-2, has a 101amino acid C-terminal extension beyond the end of the WRKY domain, and can perceive AvrRps4 and PopP2, while RRS1-S from Col-0, which perceives AvrRps4 but not PopP2, is likely a derived allele with a premature stop codon, and has only an 18amino acid C-terminal extension (23). Most Arabidopsis ecotypes also carry a paralogous and genetically linked RRS1B/RPS4B NLR pair, which only perceives AvrRps4 (24). RRS1B/RPS4B share a similar domain architecture with RRS1/RPS4, including 60% sequence identity in the integrated WRKY domain.

AvrRps4 is proteolytically processed in planta to produce a 133amino acid N-terminal fragment (AvrRps4N) and an 88amino acid C-terminal fragment (AvrRps4C) (25, 26). Previous studies have highlighted the role of AvrRps4C in triggering RRS1/RPS4-dependent immune responses (25, 26). AvrRps4N has been reported to potentiate immune signaling from AvrRps4C (27, 28). PopP2 is sequence and structurally distinct from AvrRps4 and has an acetyltransferase activity that is likely related to its role in virulence. The structural basis of PopP2 perception by RRS1WRKY has been determined (29), but how RRS1WRKY binds AvrRps4C and whether this is via a shared or different interface to PopP2 is unknown.

Here, we determined the structural basis of AvrRps4C recognition by the integrated WRKY ID of RRS1. The recognition of AvrRps4C is mediated by the 2-3 segment of RRS1WRKY, the same region used to bind PopP2. This segment interacts with surface-exposed acidic residues of AvrRps4C. Structure-informed mutagenesis at the AvrRps4CRRS1WRKY interface identifies AvrRps4 residues required for proteinprotein interactions invitro and in planta and AvrRps4 perception and immune responses. Residues mediating the interaction of AvrRps4C and RRS1WRKY are conserved in both the RRS1BWRKY and the DNA-binding domain of WRKY TFs, and AvrRps4C mutants that prevent interaction with RRS1WRKY also disrupt binding to AtWRKY41. This supports the hypothesis that the RRS1WRKY mimics host WRKY TFs through a shared effector-binding mechanism. We also show that AvrRps4C prevents the interaction of RRS1WRKY and AtWRKY41 with W-box DNA, most likely via steric blocking, at the same WRKY domain site acetylated by PopP2.

To investigate how AvrRps4C interacts with the RRS1WRKY domain, constructs comprising residues 134 to 221 of AvrRps4C (the in planta processed C-terminal fragment) and residues 1194 to 1273 of RRS1-R (corresponding to the RRS1WRKY domain) were separately expressed in Escherichia coli and proteins were purified via a combination of immobilized metal-affinity chromatography (IMAC) via 6His tags and gel filtration (Superdex 75 26/60 and Superdex S75 16/60) (see SI Appendix, Materials and Methods for full details). We qualitatively assessed the interaction of purified AvrRps4C with RRS1WRKY using analytical gel filtration chromatography. Individually, the proteins displayed well-separated elution profiles. RRS1WRKY eluted at a volume (Ve) of 14.9 mL and AvrRps4C eluted at a Ve of 12.1 mL (Fig. 1A). Following incubation of a 1:1 molar ratio of the proteins, we observed a new elution peak with an earlier Ve of 11.8 mL, and a lack of absorption peaks for the separate proteins (Fig. 1A). This demonstrates complex formation invitro and suggests a 1:1 stoichiometry of the AvrRps4CRRS1WRKY complex.

AvrRps4C interacts with the WRKY domain of RRS1 invitro. (A) Analytical gel filtration traces (using a Superdex 75 10/300 column) for AvrRps4C alone (gold), RRS1WRKY alone (green), and AvrRps4C with RRS1WRKY (blue) with sodium dodecyl sulfatepolyacrylamide gels of relevant fractions. An equimolar ratio of AvrRps4C and RRS1WRKY was used for the analysis. AvrRps4C runs as a dimer invitro. Poor absorbance for AvrRps4C at 280 nm is due to its low molar extinction coefficient. (B) ITC titrations of AvrRps4C with RRS1WRKY. (B, Upper) Raw processed thermogram after baseline correction and noise removal. (B, Lower) The experimental binding isotherm obtained for the interaction of AvrRps4C and RRS1WRKY together with the global fitted curves (displayed in red) were obtained from three independent experiments using AFFINImeter software (61). Kd and binding stoichiometry (N) were derived from fitting to a 1:1 binding model.

We then determined the binding affinities of the interaction using isothermal titration calorimetry (ITC). Titration of AvrRps4C into a solution of RRS1WRKY resulted in an exothermic binding isotherm with a fitted dissociation equilibrium constant (Kd) of 0.103 M (Fig. 1B) and stoichiometry of 1:1. The thermodynamic parameters of the interaction are given in SI Appendix, Table S1. As RRS1WRKY may be a mimic of WRKY TFs, we explored the binding kinetics of AvrRps4C with AtWRKY41 and AtWRKY70 by ITC [previous reports have shown that AvrRps4 interacts with these proteins in yeast two-hybrid assay and by in planta coimmunoprecipitation (13, 30)]. We chose AtWRKY41 for further study as this protein expressed and purified stably from E. coli. AvrRps4C interacted with AtWRKY41 with a Kd of 0.02 M, and with similar thermodynamic parameters as RRS1WRKY (SI Appendix, Fig. S2 and Table S1).

To reveal the molecular basis of the AvrRps4C and RRS1WRKY interaction, we coexpressed the proteins in E. coli, purified the complex, and obtained crystals that diffracted with 2.65- resolution at the Diamond Light Source (SI Appendix, Materials and Methods). The crystal structure of the AvrRps4CRRS1WRKY complex was solved by molecular replacement using the structure of RRS1WRKY (from the PopP2RRS1WRKY complex, Protein Data Bank [PDB] ID code 5W3X) and AvrRps4C (PDB ID code 4B6X) as models (SI Appendix, Materials and Methods). X-ray data collection, refinement, and validation statistics are shown in SI Appendix, Table S2.

The structure comprises a 1:1 complex of AvrRps4C and RRS1WRKY (Fig. 2A), which supports the 1:1 binding model in ITC. Overall, AvrRps4C adopts the same antiparallel -helical CC structure in both free [PDB ID code 4B6X (26)] and complexed forms, with an rmsd of 0.66 over 59 C atoms (SI Appendix, Fig. S3A). Also, RRS1WRKY adopts a conventional WRKY domain fold [rmsd of 2.03 over 61 C atoms compared with AtWRKY1, PDB ID code 2AYD (31)] comprising a four-stranded antiparallel -sheet (2 to 5) stabilized by a zinc ion (C2H2 type). Comparison of RRS1WRKY in the AvrRps4CRRS1WRKY and PopP2RRS1WRKY complex (PDB ID code 5W3X) structures reveals high conformational similarity, with an rmsd of 1.81 over 64 C atoms. The characteristic WRKY sequence signature motif WRKYGQK maps to the 2-strand of RRS1WRKY and is directly involved in contacting AvrRps4C (Fig. 2B and SI Appendix, Figs. S3A and S4). The same surface, including the 2-3 strands of RRS1WRKY, forms contacts with PopP2 in the PopP2RRS1WRKY complex (29) (SI Appendix, Fig. S4), and mutants at this surface showed it to be essential for PopP2 recognition.

Structure of the AvrRps4CRRS1WRKY complex. (A) Electrostatic surface representation of AvrRps4C in the AvrRps4CRRS1WRKY crystal structure displaying a prominent negative patch in AvrRps4 at the interacting interface. (B) Schematic representation of AvrRps4CRRS1WRKY, highlighting interfacing residues. AvrRps4C is shown in gold cartoon and RRS1WRKY is shown in green with surface-exposed side chains as sticks. (C) Close-up view of the interactions of AvrRps4C with the 2-3 segment of RRS1WRKY. Hydrogen bonds are shown as dashed lines, and water molecules are depicted as red spheres. The Zn2+ ion is also displayed.

The total interface area buried in the AvrRps4CRRS1WRKY complex is 591.8 2, encompassing 12.3% (589.7 2) and 11.9% (593.9 2) of the total accessible surface areas of the effector and integrated domain, respectively [as calculated by PDBePISA (32); full details are given in SI Appendix, Table S3]. The binding interface between AvrRps4C and RRS1WRKY is largely formed by residues from the 2-3 strand of RRS1WRKY, which present a positive surface patch that interacts with acidic residues on the surface of AvrRps4C (Fig. 2A, SI Appendix, Fig. S3B, and Movie S1). The interaction between the 2-segment of RRS1WRKY, which harbors the WRKYGQK motif, and AvrRps4C includes hydrogen bonds and/or salt-bridge interactions involving Tyr1218 and Lys1221 of RRS1WRKY and AvrRps4 Glu175, Glu187, and Asn171. Notably, the side chain of RRS1WRKY Lys1221 protrudes into an acidic cleft on the surface of AvrRps4C to contact the side chains of both AvrRps4 Glu175 and Glu187 (Fig. 2 B and C and Movie S1). The OH atom of RRS1WRKY Tyr1218 forms a hydrogen bond with the ND2 atom of AvrRps4 Asn171 (Fig. 2 B and C). Additional intermolecular contacts are formed by the 2-3 loop of RRS1WRKY involving the backbone carbonyl oxygen and nitrogen of Asp1222, which form hydrogen bonds with the side chains of AvrRps4 Asn190 and Gln194. The complex between AvrRps4C and RRS1WRKY is further stabilized by the 3-strand of RRS1WRKY that forms hydrogen bonds and salt-bridge interactions via side chains of RRS1WRKY Arg1230, Tyr1232, and Arg1234 to AvrRps4 Glu175 and Asp164 (Fig. 2 B and C). A detailed interaction summary is provided in SI Appendix, Table S4.

To evaluate the contribution of residues at the AvrRps4CRRS1WRKY interface to complex formation invitro, we generated six structure-guided mutants in AvrRps4C (native amino acid to Ala) and tested the effect on protein interactions by ITC. Each AvrRps4C mutant was purified from E. coli under the same conditions as for the wild-type protein, and proper folding was evaluated by circular dichroism (CD) spectroscopy (SI Appendix, Fig. S5). ITC titrations were carried out as for the wild-type interactions. Individual ITC isotherms are shown in Fig. 3, and the thermodynamic parameters of the interactions are shown in SI Appendix, Table S1. We found that mutating AvrRps4 residues Asp164 (D164A), Glu175 (E175A), Glu187 (E187A), and double mutant Glu175/Glu187 (EE/AA) essentially abolished complex formation invitro (Fig. 3). Mutations in residues Asn171 (N171A) and Gln194 (Q194A) retained binding to RRS1WRKY, with N171A displaying wild-type levels and Q194A showing an approximately sevenfold reduction in affinity. Besides structure-guided mutants, we also tested binding of an AvrRps4 quadruple mutant, carrying mutations in the N-terminal KRVY motif (KRVY/AAAA) [previously identified to be essential for the virulence activity and perception of AvrRps4 (25)], with RRS1WRKY. Unlike most interface mutants, the AvrRps4C KRVY/AAAA mutant retained wild typelike binding affinity with RRS1WRKY (Fig. 3).

Structure-guided mutants of AvrRps4C at the AvrRps4CRRS1WRKY interface disrupt interaction with RRS1WRKY invitro. ITC titrations of wild-type AvrRps4C and mutants with RRS1WRKY. (Upper) Raw processed thermograms after baseline correction and noise removal. (Lower) Experimental binding isotherms obtained for the interaction of AvrRps4C wild type and mutants with RRS1WRKY together with the global fitted curves (displayed in red) obtained from three independent experiments using AFFINImeter software (61). Kd was derived from fitting to a 1:1 binding model. N.B., nonbinding.

Since AvrRps4C binds RRS1WRKY and AtWRKY41 with similar affinity (SI Appendix, Fig. S2), we tested the impact of the AvrRps4C EE/AA double mutant on the binding to AtWRKY41. We found that this mutant also abolishes interaction with AtWRKY41, suggesting the same AvrRps4-binding interface is shared with different WRKY proteins (SI Appendix, Fig. S2).

To validate the biological relevance of the AvrRps4CRRS1WRKY interface observed in the crystal structure, we tested the effect of the AvrRps4C mutants above on RRS1-R/RPS4mediated immunity by monitoring the cell-death response in N. tabacum. Agrobacterium-mediated transient expression of wild-type AvrRps4 triggers a hypersensitive cell-death response (HR) 5 d post infiltration when coexpressed with RRS1-R/RPS4 (Fig. 4A). The previously characterized inactive AvrRps4 KRVY/AAAA mutant (25, 26) was used as a negative control. We found that AvrRps4 mutations at positions D164, E175, and E187 and the double mutant E175/E187 prevented RRS1-R/RPS4dependent cell-death responses, consistent with their loss of binding to RRS1WRKY invitro (Fig. 4A). Interestingly, the N171A mutation, which retained its binding to RRS1WRKY invitro, displayed wild typelike cell deathinducing activity, and Q194A with an approximately sevenfold reduction in RRS1WRKY affinity consistently exhibited a weaker cell-death response. Expression of all mutants was confirmed by immunoblotting (Fig. 4B). In addition to RRS1-R/RPS4, we also explored the effect of AvrRps4 structure-based mutations on RRS1-S/RPS4dependent cell death in N. tabacum (SI Appendix, Figs. S1 and S6A). We found that AvrRps4 variants elicited similar immune responses when transiently coexpressed with RRS1-S/RPS4 or RRS1-R/RPS4.

Structure-guided mutants of AvrRps4 at the AvrRps4CRRS1WRKY interface compromise RRS1-R/RPS4mediated cell-death responses and invivo binding in Nicotiana. (A) Representative leaf images showing RRS1-R/RPS4mediated cell-death response to wild-type structure-guided mutants of AvrRps4. Agroinfiltration assays were performed in 4- to 5-wk-old N. tabacum leaves, and cell death was assessed at 4 d post infiltration. The experiment was repeated three times with similar results. (B) Coimmunoprecipitation (co-IP) of RRS1-RWRKY+83 (6His/3FLAG-tagged) with AvrRps4 and variants (4myc-tagged) in N. benthamiana. Blots show protein accumulations in total protein extracts (input) and immunoprecipitates obtained with anti-FLAG magnetic beads when probed with appropriate antisera. Empty vector was used as a control. The experiment was repeated at least three times, with similar results.

To determine whether loss of RRS1-R/RPS4mediated HR in transient assays correlates with the loss of AvrRps4 binding to RRS1WRKY invivo as well as invitro, we performed coimmunoprecipitation assays using full-length C-terminal 4myc-tagged AvrRps4 constructs and C-terminal 6His/3FLAG-tagged constructs of RRS1-RWRKY+83 (equivalent to RRS1-D5/6R as defined in ref. 23). Wild-type AvrRps4 associates with RRS1-RWRKY+83 in its in planta processed form (Fig. 4B). Consistent with the cell-death phenotype and invitro binding data, no association between AvrRps4 mutants D164A, E175A, E187A, or EE/AA and RRS1WRKY+83 was detected (Fig. 4B). Further, we observed wild-type levels of association of AvrRps4 N171A with RRS1WRKY+83, while AvrRps4 Q194A appeared to coimmunoprecipitate weakly. The AvrRps4 KRVY/AAAA mutant displayed wild typelike binding affinity toward RRS1WRKY+83, as observed previously (26).

Next, we investigated the impact of AvrRps4 structure-guided mutations on the activation of RRS1-R/RPS4dependent immune responses using HR assays in Arabidopsis. Constructs carrying full-length AvrRps4 wild type and mutants, flanked by a 126-bp native AvrRps4 promoter, were delivered into plant cells by infiltration using the Pf0-EtHAn (Pseudomonas fluorescens effector-to-host analyzer, hence Pf0) system (33). HR assays used Arabidopsis ecotype Ws-2 (encoding RRS1-R/RPS4 and RPS4B/RRS1B) and Ws-2 rrs1-1/rps4-21/rps4b-1 (RRS1-R/RPS4/RPS4B triple-knockout) lines and were scored at 20 h post infiltration. Pf0 carrying wild-type AvrRps4 triggered HR in Ws-2, but not in Ws-2 rrs1-1/rps4-21/rps4b-1, as previously reported (13, 26). AvrRps4 KRVY/AAAA, an HR inactive mutant, was used as a negative control (26). The structure-guided mutants AvrRps4 D164A, E175A, E187A, and EE/AA all showed a complete loss of HR in Ws-2, with AvrRps4 Q194A showing a weaker HR and N171A showing a wild typelike phenotype (Fig. 5A). None of the AvrRps4 variants triggered HR in Ws-2 rrs1-1/rps4-21/rps4b-1 (Fig. 5A).

Structure-guided mutants of AvrRps4 compromise RRS1-R/RPS4dependent recognition specificities and restriction of bacterial growth in Arabidopsis. (A) HR assay in different Arabidopsis accessions using P. fluorescens Pf0-1 secreting AvrRps4 wild type and structure-guided mutants. Constructs were delivered to the Arabidopsis Ws-2 and rrs1-1/rps4-21/rps4b-1 knockout background and HR was recorded 20 h post infiltration. Fraction refers to the number of leaves showing HR of 12 randomly inoculated leaves. This experiment was repeated at least three times with similar results. (B) In planta bacterial growth assays of Pto DC3000 secreting AvrRps4 wild type and mutant constructs. Bacterial suspensions with OD600 = 0.001 were pressure-infiltrated into the leaves of 4- to 5-wk-old Arabidopsis plants. Values are plotted from three independent experiments (denoted in different colors). Statistical significance of the values was calculated by one-way ANOVA followed by post hoc Tukey honestly significant difference analysis. Letters above the data points denote significant differences (P < 0.05). A detailed statistical summary can be found in SI Appendix, Table S5. CFU, colony forming unit.

In addition to Ws-2, we also performed a parallel set of experiments in Arabidopsis ecotype Col-0 (which encodes the RRS1-S allele) and the Col-0 rrs1-3/rrs1b-1 (RRS1-S/RRS1B double-knockout) line. Overall, we observed a weaker HR toward AvrRps4 wild type and mutants in Col-0 in comparison with Ws-2. Nevertheless, a similar pattern of HR phenotypes was observed in Col-0 compared with Ws-2, and none of the AvrRps4 variants triggered HR in the Col-0 rrs1-3/rrs1b-1 line (SI Appendix, Fig. S6B). The pattern of HR phenotypes conferred by the AvrRps4 interface mutants further validates the AvrRps4CRRS1WRKY structure and the role of these residues in recognition of AvrRps4 by the RRS1/RPS4 receptor pair.

Having demonstrated the role of AvrRps4 interface residues in effector-triggered HR in Arabidopsis, we next investigated their effects on bacterial growth. We performed bacterial growth assays on Arabidopsis ecotypes Ws-2, Col-0, Ws-2 rrs1-1/rps4-21/rps4b-1, and Col-0 rrs1-3/rrs1b-1 using the P. syringae pv. tomato (Pto) DC3000 strain carrying AvrRps4 wild type or structure-based mutants. Since both the single mutants AvrRps4 E175A and E187A displayed the same impaired HR as the double AvrRps4 EE/AA mutant in previous assays, we focused on AvrRps4 EE/AA only for this assay. Bacterial growth was scored at 3 d post infection. Pto DC3000 carrying wild-type AvrRps4 displayed reduced growth on Ws-2 when compared with the mutant background (Ws-2 rrs1-1/rps4-21/rps4b-1), presumably due to the activation of RRS1-R/RPS4dependent immunity (Fig. 5B). The effector mutants AvrRps4 D164A, EE/AA, and KRVY/AAAA, which displayed a complete loss of HR in Ws-2, showed a severe or complete lack of restriction of bacterial growth in Ws-2 (Fig. 5B). Pto DC3000:AvrRps4 Q194A and Pto DC3000:AvrRps4 N171A showed reduced bacterial growth (but not full restriction) when compared with wild-type AvrRps4, even though they displayed a similar cell-death phenotype in N. tabacum (albeit weaker for AvrRps4 Q194A) and HR in Arabidopsis (Figs. 4A and 5A). All the Pto DC3000:AvrRps4 variants tested displayed indistinguishable bacterial growth in the RRS1-R/RPS4 loss-of-function line (Fig. 5B). Finally, all the Pto DC3000:AvrRps4 variants displayed similar bacterial growth profiles in the Col-0 and Col-0 rrs1-3/rrs1b-1 line when compared with Ws-2 and Ws-2 rrs1-1/rps4-21/rps4b-1 (SI Appendix, Fig. S6C).

In addition to RRS1/RPS4, the RRS1B/RPS4B pair can confer recognition of AvrRps4 in Arabidopsis (24). Sequence alignment revealed an overall 60% amino acid identity of the integrated WRKY domains from RRS1 and RRS1B, with the WRKYGQK motif and all residues interfacing with AvrRps4C conserved (SI Appendix, Fig. S7). To explore AvrRps4 recognition by RRS1B/RPS4B, we performed ITC titrations of RRS1BWRKY with wild-type AvrRps4C invitro. We found that RRS1BWRKY binds to AvrRps4C three times more weakly than RRS1WRKY (SI Appendix, Fig. S7), possibly due to subtle changes imposed by residues outside the direct binding interface. When comparing the binding kinetics with the strength of immune responses in planta, we observed a weaker RRS1B/RPS4B-dependent HR to AvrRps4 compared with RRS1/RPS4. Nonetheless, both NLR pairs displayed a similar profile of immune responses toward the AvrRps4 structure-guided mutants in transient cell-death assays and in Arabidopsis HR assays (SI Appendix, Fig. S7).

To regulate gene expression, WRKY TFs bind to specific W-box DNA motifs in the promoters of their target genes (3436). Intriguingly, the majority of the AvrRps4-interacting residues are conserved within the DNA-binding domain of WRKY TFs (SI Appendix, Fig. S4 and Movie S2) and are indispensable for DNA binding (34). To test if AvrRps4 interferes with the W-box DNA-binding activity of RRS1WRKY and AtWRKY41, we preincubated increasing concentrations of AvrRps4C and the AvrRps4C EE/AA mutant (as a negative control) and studied their effect on the DNA-binding capacity of RRS1WRKY and AtWRKY41 using both electrophoretic mobility-shift assay (EMSA) and surface plasmon resonance (SPR)based assays. We found that the interaction of RRS1WRKY and AtWRKY41 with W-box DNA was reduced after preincubation with increasing concentrations of AvrRps4C but not the AvrRps4C EE/AA mutant (Fig. 6 and SI Appendix, Figs. S8S10), revealing that AvrRps4C interferes with WRKY binding to W-box DNA.

AvrRps4 interferes with W-box DNA binding by the RRS1WRKY domain. (A) EMSA of DNA binding by RRS1WRKY following preincubation of increasing concentrations of AvrRps4C or AvrRps4C EE/AA mutant. Bovine serum albumin (BSA) was used as a negative control for W-box DNA binding. Scrambled DNA was used as a negative control to test the specificity of RRS1WRKY to W-box DNA. The experiment was repeated three times with similar results. (B) An SPR ReDCaT assay was performed using W-box and scrambled DNA (as a negative control). Percentage of normalized response (% Rmax) of RRS1WRKY binding to W-box DNA and scrambled DNA (denoted by an asterisk) immobilized on a ReDCaT SPR chip. Titrations were performed following preincubation of 2 M RRS1WRKY with increasing concentrations of AvrRps4C wild type and AvrRps4C EE/AA mutant. The experiment was performed in eight replicates (each dot represents one replicate).

Despite recent advances, structural knowledge of how diverse integrated domains in plant NLRs perceive pathogen effectors is limited. Here, we investigated how the integrated WRKY domain of the Arabidopsis NLR RRS1 binds to the Pseudomonas effector AvrRps4, and how this underpins RRS1/RPS4-dependent immunity in planta. Further, through this work, we gained insights into interfaces in the RRS1WRKY domain that are crucial for perception of two structurally unrelated effectors from distinct bacterial pathogens, which may have implications for NLR integrated domain engineering.

Transcriptional reprogramming upon NLR activation is well-established as an early immune response in plants (3739), and direct interactions between NLRs and TFs have been reported (4044). WRKY TFs are important molecular players in the regulation of plant growth and development and abiotic and biotic stresses (35, 36, 45). Typically, WRKY TFs target genes by binding W-box DNA in promoters, via a signature amino acid motif, WRKYGQK, to either promote or repress transcription (34, 4648). As WRKY TFs play an important role in plant immunity, it is unsurprising that they are often found as integrated domains in NLR immune receptors (49), supporting the hypothesis that pathogen effectors enhance virulence by targeting WRKY TFs. Therefore, understanding how effectors bind to WRKY integrated domains may inform how effector/WRKY binding promotes disease. The structure of the AvrRps4CRRS1WRKY complex reveals that the effector directly interacts with the DNA-binding WRKYGQK motif, likely rendering it unavailable for binding to DNA (SI Appendix, Fig. S4 and Movie S2). AvrRps4C binds to AtWRKY41 with similar thermodynamic parameters to RRS1WRKY, and interface mutants that prevent AvrRps4C interaction with RRS1WRKY prevent interaction with AtWRKY41, supporting the hypothesis that AvrRps4C binds different WRKY proteins via a similar interface. WRKY TFs bind W-box DNA sequences in the promoters of their target genes. We used EMSAs and SPR assays to observe how the interaction of AvrRps4C with RRS1WRKY or AtWRKY41 affects the binding of these proteins to a generic W-box DNA sequence. Preincubation of RRS1WRKY and AtWRKY41 with increasing amounts of AvrRps4C reduced DNA-binding activity, whereas preincubation with AvrRps4 EE/AA showed no significant difference (Fig. 6 and SI Appendix, Figs. S8S10). WRKY domain residues interacting with AvrRps4C are well-conserved in these TFs (SI Appendix, Fig. S11), suggesting that AvrRps4 could interfere with and sterically block DNA binding of multiple WRKY TFs, thus promoting virulence. In addition to WRKY TFs, a recent publication suggests AvrRps4 can interact with BTS (nucleus-located Fe sensor BRUTUS) domains to affect pathogen colonization (50). Understanding whether these functions are related requires further investigation.

Comparing the AvrRps4CRRS1WRKY structure with that of PopP2RRS1WRKY (29) reveals an overlapping binding site for the effectors, primarily mediated by the 2-3 segment of the WRKY domain (SI Appendix, Fig. S4 and Movie S2). The second lysine of the WRKYGQK motif, Lys1221, is acetylated by PopP2, abolishing the affinity of the WRKY domain for W-box DNA (13, 14, 29). Intriguingly, this acetylation event also abolished the association of AvrRps4C with RRS1WRKY (13), highlighting the important role of this interface in mediating the association of RRS1WRKY with both effectors. It also highlights the likely shared role of these effectors in preventing interaction of WRKY domains with DNA as their virulence activity, either via enzymatic modification or steric blocking.

Studies with the NLR pair Pik from rice have shown that the strength of effector binding to integrated domains invitro can correlate with immune responses in planta (5153). Of the AvrRps4 mutants we tested to validate the RRS1WRKY interface, all except N171A and Q194A prevented binding invitro (by ITC) and in planta (by coimmunoprecipitation), and these did not give cell death in Nicotiana species when coexpressed with either RRS1-R/RPS4 or RRS1-S/RPS4. Further, they did not give HR or restrict bacterial growth in Arabidopsis Ws-2 or Col-0 ecotypes (except for a partial restriction of bacterial growth for the D164A mutation in the Col-0 background). The N171A mutant retained the same level of binding as wild type invitro, and displayed the same in planta phenotypes, although restriction of bacterial growth in Arabidopsis was reduced compared with wild type in both Ws-2 and Col-0 ecotypes. Finally, the Q194A mutant showed a reduced binding invitro (approximately sevenfold compared with wild type) but maintained an HR in Arabidopsis as well as displaying a restriction of bacterial growth in Arabidopsis, albeit reduced compared with wild type. Interestingly, this mutant consistently showed a qualitative reduction in the intensity of cell death in Nicotiana. Taken together, these AvrRps4 mutations validate the complex with RRS1WRKY in that they prevent interaction invitro and in planta, but they are not sufficient to determine whether strength of binding invitro can directly correlate with in planta phenotypes. Further studies, including additional mutants, will be required to study this in the RRS1/RPS4 system.

Structural studies of singleton NLRs have shown that interactions between effectors and multiple domains within an NLR can be essential for activation (5457). It is yet to be established whether this is also the case for effector perception involving paired NLRs with integrated domains, although the rice blast pathogen effector AVR-Pia immunoprecipitates with its sensor NLR Pia-2 (RGA5) when the integrated HMA domain has been deleted. However, this interaction does not promote immune responses in planta (58). Although unresolved in the structure of AvrRps4C alone, or in complex with RRS1WRKY, the N-terminal KRVY motif is known to be required for both the virulence activity of the effector and its perception by RRS1/RPS4 (25, 26). Here, we verified that the quadruple mutant AvrRps4 KRVY/AAAA retains interaction with RRS1WRKY at wild-type levels invitro and invivo, but did not trigger RRS1/RPS4-dependent responses in our in planta assays. This suggests that while binding of AvrRps4 to the RRS1WRKY domain is essential for immune activation, an additional interaction mediated by the N-terminal region of the effector to a region of RRS1 and/or RPS4 outside this domain is also required for initiation of defense. Further studies are required to determine how additional receptor domains outside of integrated domains in NLR-IDs contribute to receptor function.

The Arabidopsis NLR pair RRS1B/RPS4B perceives AvrRps4, but not PopP2 (24). Phylogenetically, the RRS1 WRKY belongs to group III of the WRKY superfamily, whereas RRS1BWRKY is grouped into group IIe (14, 24, 48). Here we found that AvrRps4C binds RRS1BWRKY with threefold lower affinity and RRS1B/RPS4B shows a similar pattern of recognition specificity in planta but with reduced phenotypes compared with RRS1/RPS4. A full investigation addressing why AvrRps4 shows differential interaction strength and phenotypes between RRS1 and RRS1B is beyond the scope of this work, but will be a direction for future research.

The unique ability of RRS1/RPS4 to perceive two effectors that differ both in sequence and structure, via the same integrated domain, highlights the potential for engineering of sensor NLRs to recognize diverse effectors. Recently, the range of rice blast pathogen effectors recognized by the integrated HMA domain of Pia-2 (RGA5) has been expanded by molecular engineering (58). However, this expanded recognition was toward structurally related effectors and may not be via a shared interface. Further, although cell-death responses were observed in Nicotiana benthamiana, the engineered NLR was not able to deliver an expanded disease resistance profile in transgenic rice. This suggests we still require a better understanding of how NLR-IDs interact with effectors, and their partner helper NLRs, to enable bespoke engineering of disease resistance.

For invitro studies, the gene fragments of AvrRps4C (Gly134 to Gln221), RRS1WRKY (Ser1194 to Thr1273), RRS1BWRKY (Asn1164 to Thr1241), and AtWRKY41 (Thr125 to Ile204) were cloned in various pOPIN expression vectors using an in-fusion cloning strategy as described in SI Appendix, Materials and Methods.

For transient assays in N. tabacum and N. benthamiana, domesticated genomic fragments encoding RRS1-R, RRS1-S, RRS1B, RPS4, and RPS4B were cloned into binary vector pICSL86977 under a 35S (CaMV) promoter with a C-terminal 6His/3FLAG tag using the Golden Gate assembly method as described (23). Similar cloning techniques were used to generate constructs expressing RRS1WRKY+83. Full-length AvrRps4 (P. syringae pv. pisi) was PCR-amplified from published constructs (13, 23, 26) and assembled with a C-terminal 4myc tag in binary vector pICSL86977 under the control of the 35S (CaMV) promoter using the Golden Gate assembly method. DNA encoding each mutation was synthesized and cloned into pICSL86977 as described above.

For HR and bacterial growth assays in Arabidopsis, full-length AvrRps4 and variants were cloned into a Golden Gatecompatible pEDV3 vector with a C-terminal 4myc tag.

Plasmids expressing the in planta processed C-terminal fragment of AvrRps4 (AvrRps4C) and integrated WRKY domain of RRS1 (RRS1WRKY) were expressed in E. coli SHuffle cells. The proteins were purified via IMAC followed by size-exclusion chromatography. Purified fractions were pooled and concentrated to 15 mg/mL and used for further studies. Detailed procedures are provided in SI Appendix, Materials and Methods.

Crystals of the AvrRps4CRRS1WRKY complex were obtained from a 1:1 solution of 15 mg/mL protein with 0.8 M potassium sodium tartrate tetrahydrate, 0.1 M sodium Hepes (pH 7.5). Diffraction data were collected at the Diamond Light Source on the i03 beamline and processed in the P61/522 space group. The structure was determined by molecular replacement using the model of a monomer of AvrRps4C (PDB ID code 4B6X) and the RRS1WRKY from the PopP2RRS1WRKY complex (PDB ID code 5W3X) as the search model. Further details are provided in SI Appendix, Materials and Methods. X-ray data collection and refinement statistics are summarized in SI Appendix, Table S2.

AvrRps4CRRS1WRKY complex formation invitro was studied using analytical gel filtration chromatography and ITC. The effect of structure-guided mutations on the AvrRps4CRRS1WRKY interaction invitro was investigated using ITC as described in SI Appendix, Materials and Methods.

Agrobacterium-mediated transient cell-death assays were performed in N. tabacum and coimmunoprecipitation assays were performed in N. benthamiana. Detailed information concerning plant materials, growth conditions, plasmid construction, and immunoblotting are provided in SI Appendix, Materials and Methods.

Bacterial strains P. fluorescens Pf0-EtHAn and Pto DC3000 were used for HR or in planta bacterial growth assays, respectively. The Arabidopsis accessions Ws-2 and Col-0 were used as wild type for all the assays in this study. Further details about plant materials, growth conditions, plasmid construction and mobilization, pathogen infection assays, and bacterial growth assays are provided in SI Appendix, Materials and Methods.

Complementary single-stranded DNA fragments encoding the W-box DNA sequence (forward strand: 5-CGCCTTTGACCAGCGC-3) were synthesized by IDT. EMSAs were performed using a Cy3-labeled W-box DNA probe in a reaction buffer containing 10 mM TrisCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, and 5% glycerol as described in SI Appendix, Materials and Methods.

Complementary single-stranded DNA fragments encoding the W-box DNA sequence were synthesized by IDT. For SPR assays, the forward strand encoded the W-box DNA sequence (5-CGCCTTTGACCAGCGC-3) while the complementary reverse strand added an extra 20-bp ReDCaT sequence (5-CCTACCCTACGTCCTCCTGC-3) to complement the linker DNA added to the SA chip. The double-stranded DNA was then diluted to a working concentration of 1 M. SPR measurements were performed at 25C using the reusable DNA capture technique (ReDCaT) as described (59, 60) and using a Biacore 8K System (Cytiva). Further details are provided in SI Appendix, Materials and Methods.

All study data are included in the article and/or supporting information.

This work was supported by the European Research Council (Proposal 669926, ImmunityByPairDesign); the UK Research and Innovation (UKRI) Biotechnology and Biological Sciences Research Council (BBSRC) Norwich Research Park Biosciences Doctoral Training Partnership, UK (Grant BB/M011216/1); the UKRI BBSRC, UK (Grants BB/P012574 and BBS/E/J/000PR9795); and the BBSRC Future Leader Fellowship (Grant BB/R012172/1). We thank Julia Mundy and Professor David Lawson from the John Innes Centre (JIC) Biophysical Analysis and X-Ray Crystallography platform for their support with CD spectroscopy, protein crystallization, and X-ray data collection; Andrew Davies and Phil Robinson from JIC Scientific Photography for their help with leaf imaging; and Dr. Tung Lee for advice on EMSAs. We also thank Dr. Kee Hoon Sohn for helpful suggestions for triparental mating and other members of the M.J.B. and J.D.G.J. laboratories for discussions.

Author contributions: N.M., H.B., P.D., J.D.G.J., and M.J.B. designed research; N.M. and D.G. performed research; H.B., P.D., and C.E.M.S. contributed new reagents/analytic tools; N.M., A.R.B., C.E.M.S., and M.J.B. analyzed data; and N.M., J.D.G.J., and M.J.B. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2113996118/-/DCSupplemental.

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Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor - pnas.org

In breakthrough, DeepMind’s AI has cracked two mathematical problems that have stumped experts for decades – Times Now

Representational image.  |  Photo Credit: iStock Images

DeepMind's AI is probably best known for cracking the popular strategy game Go, but in the last few years, machine learning has proved extremely valuable in an array of applications like protein-folding and deep intuition.

Now, for the first time, the technology has been used to identify mathematical connections that have eluded researchers for decades. Teaming up with mathematicians, DeepMind's AI sought to tackle two distinct problems one in the study of symmetries and the other in knot theory.

I was very struck at just how useful the machine-learning tools could be as a guide for intuition, said Marc Lackenby, one of the mathematicians from the University of Oxford who participated in the study. I was not expecting to have some of my preconceptions turned on their head.

The study of math may turn a lot of people off but, at its core, it facilitates a greater human understanding of the fundamental properties that govern our universe. It's only through painstaking work in the area of pure mathematics that we now have revolutionary technologies like airplanes and computers.

Mathematicians try to spot patterns in large datasets which they then seek to formulate conjectures out of. These conjectures are then reviewed and tested by their peers in various hypothetical cases and, if they hold up, turn into theorems.

But the amount of data now available is impossible for any human to process. And this is where machine learning comes in. Machine learning can discover patterns much quicker than humans insights that can then guide new mathematical ideas.

Take the theory of knots for example. At a superficial level, knots describe how a piece of string or rope is entangled. But at a much deeper level, they revolve around key mathematical principles that can be applied in the realm of quantum computing.

Algebra, geometry, and quantum theory all share unique perspectives on these objects and a long-standing mystery is how these different branches relate: for example, what does the geometry of the knot tell us about the algebra? wrote the researchers.

The team of researchers created a machine learning model to probe these connections and one particular trick called saliency maps proved immensely valuable. The ML model was able to spot specific geometrical properties known as a 'signature' that researchers could then use to formulate a conjecture.

In another instance, DeepMind teamed up with mathematicians to probe a problem in symmetries one that scientists have traditionally studied using charts or graphs. But as more data is incorporated, these charts inevitably grow dauntingly large, making it nearly impossible for a human to comprehend. But DeepMind's AI discovered numerous interesting patterns that, the researchers, think could guide mathematicians toward a proof.

I was just blown away by how powerful this stuff is, said Dr Geordie Williamson from the University of Sydney. I think I spent basically a year in the darkness just feeling the computer knew something that I didn't.

DeepMind has been consistently proving that the applications of machine learning extend well beyond just games, and the latest breakthrough is another testament to the technology's growing value in solving some of humanity's most complex problems. But ultimately, due to its inherent probabilistic nature, it needs to be accompanied by human intuition and rigour. Nevertheless, the man-machine combination, the researchers believe, could inspire other scientists to incorporate AI into their own research.

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In breakthrough, DeepMind's AI has cracked two mathematical problems that have stumped experts for decades - Times Now

Rumbleverse, a pro wrestling battle royale, announced at TGAs 2021 – Polygon

The next take on the battle royale genre is Rumbleverse, a 40-player brawler royale from Extinction maker Iron Galaxy. Revealed Thursday night during the Game Awards, Rumbleverse is a city full of zany-costumed pro rasslers, elbow-dropping and pile-driving one another until the last is standing.

And yes, the characters are rasslers, not wrestlers. The day that we were first jamming on the concept, Chelsea [Blasko] our co-CEO, she just goes, We should do rasslin!, said Adam Boyes, Iron Galaxys other co-CEO since 2016. Just like that, right? And then we just started, like, What would happen in a world where a wrestling match could break out anywhere on planet Earth?

Rumbleverse, to be published by Epic Games for PlayStation 4, PlayStation 5, Windows PC, Xbox One, and Xbox Series X, borrows the battle royale conventions of parachuting into a map, scarfing up loot and power-ups, and moving to stay within a steadily closing area. But because there are no guns except the ones attached to your arms, says lead designer Adam Hart Iron Galaxys developers hope the fighting will more engaging and more entertaining than the fast-twitch, shoot-or-be-shot immediacy of Fortnite or PUBG Battlegrounds.

When you see somebody in this game, theyre not a threat to you just because youve seen them, Hart said. You can kind of watch them fight or have a, you know, emote conversation with them across rooftops. Of course, at some point theyll start throwing down. While sneak attacks are possible, normally this is mutually initiated combat.

Iron Galaxy figures most events will take between 12 and 15 minutes to crown a winner. The ring shrinks tighter and more quickly to compensate for inactivity, Hart said, to force combat on anyone who is avoiding a fight. The games map, Grapital City, is quite large but more importantly, it has a lot of verticality. It packs a lot of visual appeal into the fights, and of course, supplies a lot more force to moves landed from way higher than the top rope.

I saw Hart, whose fighter was kitted out in a tuxedo under a karate gi, with a full-head cat mask (customization, of course, is very important here) pull out an old-school belly-to-back suplex on a clown (another devs costume), landing it from what looked like the observation deck of the Empire State Building.

How often do you guys imitate Jim Ross, by the way? I asked.

Every day! Boyes laughed. For the past couple of years! Hart added.

Eliminations are a simple case of draining another players health bar, which can be replenished or buffed by the power-ups strewn about Grapital City. Hart guzzled down protein powder, for example, and picked up weightlifting magazines to get himself in shape. Theres also plenty of roasted poultry, the international sign of video game health since Castlevania and Gauntlet. Its available from a drive-thru window for Squatch Chicken: The Home of Slow-Squatted Chicken.

[The cook] has all the chicken on a squat rack, and he just dips it into the fire, Hart explained.

Other pick-ups supply perks or modify one of three core attributes arms (power), core (health), and legs (stamina). Players can work up a match-to-match character build that emphasizes certain areas of their wrestling prowess. Harts character, in the playthrough, went 3-5-2, for example, with his core being the top attribute. But a max-stamina fighter could do comparable damage per second with a flurry of lesser attacks and I saw plenty of chained, juggling strikes that fighting game fans will recognize.

One thing we found out is that a lot of people that are crazy-good platform players, like Mario, Crash, Spyro fans, became the best players, very quickly, Boyes said. So its as much, I think, about how you move around the world as it is your offensive integrity.

Melee weapons are also available, like baseball bats and the de-rigueur folding chairs. But as Hart pointed out, anything a player can hold can also be knocked from their hands (and used) weapon or power-up. That means you can also improvise throwing attacks with the can of whey.

Grapital Citys environment also presents tactical possibilities for players, too. No one can swim, so if the circle is closing around an area with water, players can get rung out quickly even if theyre at maximum health. Being outside the circle doesnt inflict damage, as it does in Fortnite, but it does start a 10-second countdown, akin to disqualification matches in real pro rasslin. (Not that pro rasslin is real. Just, you know, real-life.)

Other wrestling tropes include a perk that revives a player after theyve been counted out (that is, when their health has been fully drained), much like the scripted reversals and comebacks in epic-length wrestling matches.

What I love about this is, I see someone, and I see a series of choices for me, Boyes summarized. Do I have enough stuff that Ive picked up? Do I feel like my stats are good enough? Do I have that one move I need? Can I sneak up behind him? Can I sort of stalk them? Or can I just run away, get health, and come back.

I dont want to say its casual, but because theres so much depth to the combat, it makes it less pressure, Boyes said, but more, you know, just more fun to experiment and try new things.

Rumbleverse kicks off a First Look gameplay event on Friday, Dec. 10, available to a limited number of players (the games official website has registration information and more details). Iron Galaxy expects to launch Rumbleverse in early access on the Epic Games Store on Feb. 8, 2022, as well as on PlayStation 4, PlayStation 5, Xbox One, and Xbox Series X through those consoles marketplaces. Rumbleverse will support cross-platform play and progression, Iron Galaxy said.

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Rumbleverse, a pro wrestling battle royale, announced at TGAs 2021 - Polygon

Letters to the editor – December 12, 2021 – Times of Malta

Japanese longevity

The feature about ikigai and Japanese longevity (in particular Okinawa, December 5) is most interesting and useful. Ikigai is essentially about maintaining a positive mental attitude, physical and mental activity and avoiding overeating.

Japan remains at the top of human longevity league while Sardinia is said to be home to the highest percentage of European centenarians. A recent British study of this Sardinian phenomenon claims these long-lived individuals live in hilltop villages, are active most of the day, do not make much use of cars, their diet is mainly vegetables and goat cheese based, and are free from chronic disputes and anger.

Japanese longevity is not just related to ikigai but also to their traditional diet of fish, vegetables, green tea and no animal meats and dairy produce. Several decades ago, US medical statistics identified that, whereas Japanese living in Japan had low rates of heart disease, breast and prostate cancer, compared to the US, Japanese residents in America acquired similar disease patterns to other Americans within two generations. This suggested the diet in Japan was an important factor in Japan residents longevity.

Two important dietary factors contributing to Japanese longevity are thought to be fish and a fermented soya product. Fish, particularly from cold waters, is rich in omega-3 fat, which has anti-inflammatory and anti-coagulant properties besides lowering blood triglycerides levels (the worst factor in cholesterol tests). Fermented soya, which the Japanese call natto, has blood clot loosening properties.

The dietary combination of fish and natto would, therefore, be expected to be just as an effective (if not superior and safer) alternative to aspirin and cholesterol-lowering pharmaceuticals (statins). In the West, if one is not eating fish on a daily basis, one can replicate this Japanese dietary pattern with pure fish oil (marine omega-3) and nattokinase capsules.

Nattokinase is natto in capsule form and, if not available locally, can be purchased online from European suppliers.

In the 1970s and 1980s, US laboratory animal studies and a combined US and Chinese university field study in China produced evidence incriminating excessive animal-derived foods as the main promoter of cancer.

The traditional Japanese diet, containing little or no animal-derived food, probably also contributes to their longevity by lowering cancer risk. Furthermore, soya beans (and all beans and lentils) contain substances which lower breast and prostate cancer risk.

ALBERT CILIA-VINCENTI former European Medicines Agency scientific delegate, Attard

In the run-up to the Christmas season, or holiday festivities, if you will, a shadow has been cast that temporarily diminished the sparkle of led lights and Christmas cheer. The European Commissioner for Equality, Helena Dalli sought to issue some sugar-coated equality guidelines which were, fortunately, withdrawn following scathing criticism from various quarters within the European Union.

This move coming from Dalli takes me to revisit one of my favourite movies, The Nightmare Before Christmas. In Tim Burtons stop-motion animated masterpiece, the grotesque but charismatic character Jack Skellington naively tries to fuse Halloween with Christmas, going so far as to send his minions to capture Father Christmas and replacing presents with Halloween versions, which shock and terrify children and parents.

After realising his folly, Jack the Pumpkin King sets things right by reversing his actions and restores Christmas to its normal state.

Dalli has, likewise, attempted to distort the meaning of Christmas and its symbolism to suit her vision of equality but retracted her steps because of the negative backlash. However, while Skellingtons motivations may have been comical and well-intentioned, those of the commissioner could be different.

I distinctly recall, a few years back, the first draft of the Equality Bill, issued when Dalli was a minister for equality in Malta, which included a rather sinister definition of pregnancy: the state of a person who has within the ovary or womb an implanted embryo, which gradually becomes developed in the latter receptacle.

After the social partners protested against this mad scientist definition of pregnancy, which, underhandedly, attempted to separate the mother from the child, the definition was later changed to a more humane woman with child. Yet, the attempt to strip the concept of a pregnancy of any human element was evident, understood and exposed.

More recently, Dalli bragged about how she deceived the electorate by disguising the true intentions of the Labour Party electoral manifesto through the use of obscure terminology. It seems to me that Dalli harbours opinions to which she is perfectly entitled but would go to any lengths to see these ideas imposed on the rest of society, even by stealth and Macchiavellian tactics.

The proposed guidelines by the European Commission also tried to dissuade the use of names like Mary because of their Christian connotations, under the guise of promoting multiculturalism. Why my mothers name, which Leonard Bernstein in the classic West Side Story describes as all the beautiful sounds of the world in a single word, should have the effect of brandishing a crucifix to a vampire on some people eludes me.

Multiculturalism should be all-embracing. If Frank Zappa chose to name his daughter Moon Unit, I love his music no less, though I still prefer the name Mary to Moon Unit.

The EU has to grapple with striking a balance between its historical and cultural roots and a rapidly changing sociocultural environment. Yet, there is no need to resort to a sledgehammer approach to accept the new by obscuring the history and traditions that unite the countries within the Union.

The branding of the Union its flag is also an affirmation of predominantly Christian culture and values, even if the Union and member states are secular. The blue background and yellow stars are a direct reference to the biblical Mary, not Moon Unit.

In time, this may be challenged by the likes of Commissioner Dalli. Who knows, in future, we may remove the 12 yellow stars that can represent the apostles, the zodiac or the 12 labours of Hercules and replace them with a deconstructed foetus as a symbol of equality!

JOSEPH FARRUGIA Attard

Letters to the editor should be sent to editor@timesofmalta.com. Please include your full name, address and ID card number. The editor may disclose personal information to any person or entity seeking legal action on the basis of a published letter.

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Letters to the editor - December 12, 2021 - Times of Malta

What was the Mossad director thinking? – Haaretz

Here is what Mossad director David Barnea said: Iran will never have nuclear weapons not in the coming years, and not ever. This is my pledge; this is the Mossads pledge.

I couldnt believe my ears. I wanted to see it with my own eyes. A video of this official event shows the president lighting Hanukkah candles, the prime minister spouting historical nonsense and the Mossad director spouting clichs. But the sentence quoted above nothing. Its disappeared.

Presumably it was left on the cutting room floor in the hopes it would be forgotten. Nevertheless, theres no doubt the Mossad chief said what he did, and his comments deserve to be discussed.

First, the Mossad director needs to grasp a painful truth he doesnt decide anything. Not whether Iran will have nuclear weapons, and not whether Iran wont have nuclear weapons. Because what can you do, the Mossad director only belongs to the servant class, not the decision-making class. And like all service providers (including secret services), the Mossad director is authorized to do only what hes told to do.

Heres an incident from the past that proves the point. Some decades ago, the heads of the secret services were ordered to assist the (evil) secret services of Irans then-ruler by providing equipment and training. And of course, they obeyed.

And thats what will happen in the future as well. If any Mossad director, including the current one, is again asked to help an Iranian tyrant (perhaps because of his support for the settlements or something like that), he too will obey. After all, thats the essence of his job obedience. Perhaps not blind obedience, but definitely squinting obedience.

Moving on, the Mossad director also declared that his pledge was forever. Thats a bit presumptuous, isnt it? Especially coming from someone whose term will end in another five and a half years at most.

His lifespan to everyones great regret, of course also isnt eternal. In just another 55 years, hell turn 120. And that, as everyone knows, is the upper limit God set for human longevity, according to Jewish tradition.

But its still permissible to hope that the world will go on for a few years after that. Therefore, the Mossad directors personal pledge that Iran will never have a nuclear bomb is about as coherent and impressive as a personal pledge by his Iranian counterpart that Israel will never have a nuclear bomb.

Moreover, the Mossad is undoubtedly an efficient assassination enterprise, and assassinations are very good for the ego, morale and the enthusiastic media. But they dont produce any real benefits. The Mossad also has a successful dirty tricks department that knows how to plant viruses in electric razors, disrupt the timetables of cable cars and even cause centrifuges to spin out of control. This department, too, is good at tactical annoyances but fails to produce any strategic benefits.

By contrast, the Mossad and its ilk around the world have been consistent failures for many years at anything connected to actual intelligence and strategic analysis. As proof, consider the collapse of the Soviet bloc, the fall of the Berlin Wall, the Yom Kippur War, the first intifada, the Iraq war, the attack on the Twin Towers, the war in Afghanistan and more and more and more resounding, embarrassing strategic failures that have escaped my gaze. But the top-secret operations are all really terrific.

Therefore, the Mossad director should try to enhance the functioning of his modesty gland and practice lowering his nose in front of a mirror. That might help him later on.

In his defense, I will say one thing only. His remarks were so arrogant, conceited, aggressive and inflated that together they create a frighteningly accurate reflection of Israel in 2021 arrogant, conceited, aggressive and inflated. And for that, he has my thanks.

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What was the Mossad director thinking? - Haaretz

Why ‘Sunny’ is the greatest sitcom of all time – The Bona Venture

BY TUCKER REILLY, MANAGING EDITOR

Last week, the FX sitcom Its Always Sunny in Philadelphia premiered its 15th season, officially marking it as the longest running live-action sitcom in US history. The secret to the series longevity lies in its strong cast of characters, clever dialogue and constant boundary-pushing humor, which has allowed Sunny to garner an ever-increasing cult audience over nearly two decades. Additionally, the show has continued to produce quality content throughout its entire run, not suffering from the late season fatigue of many other sitcoms. With its unique comedic genius and historical precedent, Its Always Sunny should be considered the greatest American sitcom of all time.

Like many sitcoms, Sunny is based on a simple premise: five friends run a bar in Philadelphia, concocting schemes and settling scores with the local population. Unlike its thematic predecessor Cheers however, Sunny endeavors from the start to prove that its core cast are the absolute worst people imaginable. In the second season alone, our core five make a fake terrorist threat video, get addicted to crack cocaine, solicit bribes from local politicians and start a deadly Vietnamese gambling ring in the basement, among other crimes. The genius of Sunny lies in this darkness, in showing the viewer each characters rock bottom, then revealing that rock bottom is actually a cliff. Often the world around them acts as a universal straight man to the gangs antics, although Sunny does occasionally relish in finding someone even crazier than its own characters.

One of the defining features of Sunnys longevity is the shows acceptance of change, in contrast to the sitcom norm of status quo. When Dennis (Glenn Howerton) and Dee (Kaitlin Olson) get addicted to crack cocaine or suffer heart attacks (on separate occasions), they remain encumbered with these problems, although the culmination of their sins make each but a single aspect of chaos within the overall storm. Frank Reynolds (Danny DeVito) enters the series as a disciplined, business-savvy father figure to the group, who gradually devolves into a near-animalistic train wreck of a human being over time. Characters have children, gain and lose dramatic amounts of weight, suffer permanent afflictions and occasionally die. Sunny understands that its characters are not a mere collection of gimmicks, however, and molds its comedy around the irreplaceable actors themselves.

Its Always Sunny is an inherently character-driven comedy that mercilessly lambasts and pities its characters in equal measure. Many episodes of the show function like car wrecks, locking viewers into the sordid spectacle, while acknowledging the human flaws at each characters center. And more often than not, there is something profound there: we are not these people, yet we understand where their fears, desires and insecurities come from before they are turned up to 11. We want to see them win or better themselves but crave the inevitable implosion of their despicable actions. The snowball rolls on, keeping us firmly stuck within an ever-expanding web of degeneracy.

It is also important to emphasize the exceptional talent brought to Its Always Sunny by the cast and writing staff, who happen to be one in the same. The series core group of Rob McElhenney (the shows creator), Charlie Day, Kaitlin Olson, Glenn Howerton and Danny DeVito have built larger-than-life characters around their own strengths and eccentricities, sharply refined over time. The casts familiarity, mixed with Sunnys trademark rapid-fire dialogue, have allowed them to make nearly every scenario comedically valuable, while pushing the boundaries of the show to new heights. A key factor in this development has been creative freedom: due to the shows relatively low profile on FX, McElhenney and co. have been allowed to stretch the series into strange new directions largely without restriction. While some creative decisions such as the shows ironic use of blackface have been poorly received, Sunny has never lacked nuance in its satire. Its Always Sunny is a unique and terrible creation developed by a singular set of people, consistently sustaining itself for over 15 years (even though we all know seasons five through nine are the best, of course). It deserves, without a doubt, a seat at the table as the greatest American sitcom of all time.

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Why 'Sunny' is the greatest sitcom of all time - The Bona Venture

Kevin Holland Calls Out Conor McGregor Over Weight Gain – Heavy.com

Getty ImagesUFC star Conor McGregor.

Conor McGregor showed off his bulky new look this week and Kevin Holland isnt too sure that the UFC stars gains are all-natural.

Holland is never scared to make a joke or speak his mind and took to Instagram with a message about McGregors new look.

Conor vitro Belfort McGregor, Holland wrote, appearing to reference former UFC champ Vitor Belfort, who has been known for using testosterone replacement therapy.

Holland used the same photo McGregor posted earlier in the week, hinting his gains were from eating at his restaurant, The Black Forge.

Before a feed at The Black Forge. After a feed at The Black Forge,McGregor wrote in a tweet. Quality Irish produce. All across the board. Unmatched!

McGregor is coming off a broken leg suffered while fighting Dustin Poirier at UFC 264 and has been on the mend, obviously putting in work. He mentioned previously on social media that he was tipping the scales around 190 pounds a big shift for McGregor who has fought as low as 145 pounds in his career.

Holland is a meme master and also poked fun at McGregor following his loss to Poirier with an edited interview of his interview with Joe Rogan.

In case you missed [Conor McGregors] first words after the fight he was really letting go after the fight, Holland wrote.

After catching some heat from the McGregor army he apologized in the comments.

Dam I forgot the Conor fans would murder me. I sorry, Holland wrote.

Whether theres a belt on the line or not, a McGregor fight brings the eyeballs and cash. And while the Irishman could sit back on his piles of cash, hes still hungry to get in the octagon.

Hes chomping at the bit to get back in there and compete again, White said of McGregor. Its going to be a while before he can put some torque on that leg. April, for him to start training again in April is probably about right.

White said theres no question about McGregors commitment and he wants to get back to winning fights. Hes won just one fight since 2018, which came against Donald Cerrone.

He obviously still has the desire to compete, White said. You dont ever hear Conor going, You know what, man? I just dont have the desire for this anymore. You now what I mean? Yes, hes obviously very rich, and hes training like Rocky in Rocky III, but he still has the desire to compete and the desire to fight, and well see what happens.

There are a few routes McGregor could go with his next fight and his longtime head coach John Kavanagh noted some of those during Mondays The MMA Hour.

Ill be honest, the Nate Diaz trilogy is very, very tempting, Kavanagh said. Its a fight that gives me nightmares. The man doesnt stop coming forward, whether its three rounds or five rounds, but its an intriguing fight. So that one is definitely very interesting. But also the Tony Ferguson one it never happened. It was talked about a lot. And Tony still has, I believe, a lot to offer the game. Hes a very unorthodox striker, grappler. I think the buildup would be fun for the fans. So any of those kind of legacy guys.

It might be a while before we see McGregor back in the octagon but his next fight result will dictate quite a bit about how the rest of his career goes.

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Kevin Holland Calls Out Conor McGregor Over Weight Gain - Heavy.com

Crain’s Health Pulse is your source for actionable, exclusive and inside news on the health care industry. – Crain’s Chicago Business

IS LAB SPACE BOOM TOO SCATTERED? Locally born life sciences companies no longer have to worry about a lack of high-quality lab space forcing them to leave town as they grow, thanks to developers racing to build it during the COVID-19 pandemic. Where those companies decide to go in the Chicago area is a separate issue, and one that is shaping the next chapter of the city's evolution into a biotech and pharmaceutical research hub.

While more mature life sciences markets like Boston, Raleigh-Durham and San Diego feature centralized clusters of startups, well-established companies and lab buildings all close to academic institutions that churn out research, Chicago's growing biotech landscape thus far has been anything but concentrated. New lab space in the area has been scattered by geographically separated universities and where real estate developers want to build, fragmenting the momentum among city and suburban nodes that all show promise, but have little connective tissue.

The research that seeds many of the life sciences companies grown in Chicago, meanwhile, has historically been done closer to Northwestern in Evanston, University of Chicago in Hyde Park and the hospital systems in the Illinois Medical District on the city's Near West Side. But instead of building more lab space on or near those campuses, developers have been jockeying to create what they hope will emerge as the nucleus for local life sciences startups in places like Fulton Market or Lincoln Yards. READ MORE.

MILLS CLAN'S POTENTIAL $29 BILLION WINDFALL, AND THE FATE OF MEDLINE:As the Mills family weighs what to do with an enormous pile of cash from selling a 79% stake in Northfield-based Medline, managers are charged with steering a suddenly debt-laden medical-supply firm through an industry besieged by supply issues.

Investors dont seem too concerned. Septembers offering of more than $14 billion in junk bonds and leveraged loans to finance the deal was met withenthusiasm.The company also raised an additional $2.1 billion of commercial mortgage bonds.

In Medlines favor, the family executivesCEO Charlie, his cousin Andy Mills and their in-law, Jim Abramswill remain in their roles and the family will keep a significant stake. The Mills family is rolling $3.5 billion of their equity into the business, allowing them to retain 21% of Medline, according to bond documents seen by Bloomberg. READ MORE.

TITLE FIGHT PITS 'PHYSICIAN ASSISTANTS' AGAINST DOCTORS:Seeking greater respect for their profession, physician assistants are pushing to rebrand themselves as physician associates. Their national group, formally replaced assistant with associate in its name in May, transforming into the American Academy of Physician Associates. The group wants state legislatures and regulatory bodies to legally enshrine the name change in statutes and rules.

Rechristening the P.A. name has spiked the blood pressures of physicians, Kaiser Health News reports.

They complain that some patients will wrongly assume a physician associate is a junior doctor, much as an attorney who has not yet made partner is an associate. The head of the Chicago-based American Medical Associationwarnedthat the change will undoubtedly confuse patients and is clearly an attempt to advance their pursuit toward independent practice. The American Osteopathic Association, also based in Chicago,accusedthe P.A.s and other nonphysician clinicians of trying to obfuscate their credentials through title misappropriation.

But the P.A. group says theres no ulterior motive in altering their name.

Changing the title is really just to address that misperception that we only assist, said Jennifer Orozco, president of the Alexandria Virginia-based P.A. association and an administrator at Rush University Medical Center. It wont change what we do.

ALS THERAPY SHOULD TARGET BRAIN, NORTHWESTERN STUDY SAYS: Flipping the notion that ALS starts in the spine, Northwestern Medicine scientists said in a study published Thursday that the brain is indeed a target for treating ALS, Northwestern said in a statement.

The study in the Nature journal, Gene Therapy, shows the degeneration of brain motor neurons is not merely a byproduct of the spinal motor neuron degeneration, as had been previously thought, the statement said.

We have discovered that the brain degenerates early in diseases like ALS, sends us warning signals and shows defects very early in the disease, lead study author Hande Ozdinler, an associate professor of neurology at Northwestern University Feinberg School of Medicine said in the statement. Therefore, we need to repair the brain motor neurons if we want long-term and effective treatment strategies. The brain is important in ALS.

HEALTH CARE EQUITY FIRM FORMS VETERINARY REIT: Chicago-based Thurston Group, a private-equity firm focused on investments in health care service companies, said in a statement its formed a Real Estate Investment Trust, or REIT, dedicated to serving veterinary property owners. For the National Veterinary REIT, Thurston is partnering with real estate investors, AMO Partners, which is experienced in the veterinary business, the statement said.

"Partnering with AMO is the perfect fit for National Veterinary REIT because it lets us replicate our health care REIT success in a very similar, yet underserved space: the veterinary market. We have a proven track record of bringing wealth creation tools to clinicians. AMO's experience and depth of understanding will bring the same opportunities to an entirely new group of caring yet underserved providers," Thurston Group Chairman and CEO Patrick J. Haynes III said in the statement.

Thurston has a similar REIT strategy in the dental arena, where it has over 70 properties in 12 states, the statement said.

GTCR'S CORZA MEDICAL BUYS KATENA PRODUCTS: Chicago-based private-equity firm GTCR said in a statement that its medical device portfolio company Corza Medical has acquired Katena Products, Inc., a medical device company focused on ophthalmic surgery products. The acquisition of Katena expands Corza's portfolio of surgical products and adds significant scale, product breadth and infrastructure to Corza's ophthalmic surgery segment, the statement said. Terms were not disclosed.

CLARUS SELLING SECURITIES IN PRIVATE PLACEMENT: Northbrook-based Clarus Therapeutics Holdings, which develops androgen and metabolic therapies for men and women, said in a statement that it has entered into a definitive agreement to sell securities in a private placement with a leading health care investor.

Clarus said it intends to use the net proceeds from gross proceeds of about $15 million, "to support growth initiatives for its near-term commercial objectives for its oral testosterone replacement product, Jatenzo," the statement said.

Pursuant to the terms of the securities purchase agreement, Clarus said it will issue to the investor 3,024,194 units at a price of $4.96 per unit. The securities are being sold in a private placement and have not been registered under the Securities Act of 1933, as amended, Clarus said in the statement.

VILLAGEMD ADDS ANOTHER MICHIGAN PRACTICE: Chicago-based VillageMD said in a statement that Envision Medical Group, with 12 locations across three Michigan counties, will begin practicing as Village Medical starting Nov. 17. The practices are located in Metro Detroit. Village Medical also has two practices in Westland, Mich.

JOIN US FOR CRAIN'S HOSPITAL CEO BREAKFAST:Hear how Chicago-area hospitals have been weathering the storm and what's on the horizon at Crain's Hospital CEO Breakfast on Dec. 14.Register now for the in-person event.

PEOPLE ON THE MOVE

Mike Ferson and Michelle Werr have been promoted to the roles of managing director at HealthScape Advisors.

Ferson has extensive experience supporting health plans, systems and physician groups in solving complex strategic and operational challenges. Werr serves as a key advisor to health plans, providers and private-equity firms as they navigate the health care market, particularly in government programs.

Kimberly Bors, senior vice president and chief human resources officer at Dover Corporation has been appointed to the American Heart Associations Midwest Board of Directors for a two-year term.

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Crain's Health Pulse is your source for actionable, exclusive and inside news on the health care industry. - Crain's Chicago Business

Understanding the Immune Response to Nanomedicine Pharmaceuticals – AZoNano

The existence of microbial antigens and other impurities mistakenly introduced during the development and purification of bionanopharmaceutical devices can stimulate the innate immune system, as described in a paper published in the journal Molecules.

An immediate but largely non-specific local immune reaction including both biochemical and molecular components initiates the body's first "innate" defense against foreign armies.

Trained immunity is anon-specific, T-cell self-sufficient innate immunity that relies primarily on macrophage activation and pro-inflammatory cytokine secretion for long-term functional reconfiguration of the innate immune cell response instead of the epigenetic hybridization required by innate and adaptive immunity.

Due tothe high financial and social expenses of medicine development, research, and approval, it iscritical that any prospective product "failure" is not caused by the accidental inclusion of innate immunity modulating impurities IIMIs

Activated phagocytes produce simultaneously stimulatory as well as inhibitory cytokines in the influence of IIMIs to stimulate and control the immune response.

Chemokines are the most diversified family of cytokines, with roles ranging from cell migratory regulation (e.g., recruiting and activation of local neutrophils and basophils to the infection site) through embryogenesis, innate and adaptive body's immune function and structure, and cancer metastasis.

In most cases, cytokine-driven immunostimulation is beneficial, such as when it is activated by adjuvants to boost vaccine effectiveness.

Immunological stimulation that is unanticipated or uncontrolled, particularly in the presence of therapeutic substances, causes unwanted cellular immune responses and antibody formation in reaction to the medicinal product.

Immunotoxicity is defined as "any unfavorable effect on the structure or function of the immune mechanism, or other systems influenced by the same biologic mediatorsas a result of immune response malfunction."

It is further divided into three categories based on the intensity of the response: non-specific immunostimulation, uncontrolled hypersensitivity that causes tissue injury, and immunosuppression.

Impurities in drug products trigger innate cellular responses and produce biomarkers for bioassay detection and Quantification. Currently, only -glucans and endotoxins can be detected and quantified directly using specialized assays. The remaining population of impurities must instead be detected and quantified indirectly using downstream biomarkers (e.g., proteins, peptides, and nucleic acids) and immune cell activation as hallmarks of contamination. Image Credit:Holley, C., and Dobrovolskaia, M.

When compared to classically formulated variants of such prescription medications, the use of nanotechnology is becoming a popular method for reducing drug immunotoxicity whilst also improving medicinal solubility, biodistribution, and cell-specific distribution. However, several nanocarriers have been shown to have immunomodulatory properties.

For example, RNA nanoparticles have been found to increase inflammation by inducing pro-inflammatory cytokine release. The raw materials used to make nanoplatforms can have a variety of immunological impacts, either as a result of contamination or because of the chemical features of the material.

Certain nanomaterials, including lipid-based nanocarriers and carbon nanotubes, are immunostimulatory, causing cytokine production and inflammation.

The rabbit pyrogen test (RPT) became the bioassay used to identify microbial contamination. It detects pyrogens, as well as any contaminants that causea histamine reaction, chills, fever, and other inflammation side effects.

As the rabbit pyrogen test identifies all pyrogens, it has a high level of unpredictability, is costly, and requiressignificant animal usage for tests.

As issues with beta-glucan and endotoxin identification in nanoformulations arise from excipient-, carrier-, or drug-mediated external interference, sources of interferences and techniques to overcome them have been discovered. Here, direct detection methods are often utilized.

For an efficient test, a suitable biomarkercan be any chemical with a beneficial attribute, such as a mechanical by-product, that can be measured or assessed, either direct or indirect, and utilized as an indication of anormal biological, pathological, or pharmacological condition.

Recent experimentshave placed focus on thein vitro and in vivo effects of IIMIsbecause as the long-term objective of these investigations is to prevent human immunotoxicity and probable immunogenicity.

These biological tests detect immune cell growth and proliferation or measure quantities of released innate immunity biomarkers,which may help to prime immune cells and contribute to immunogenicity.

The FDA's mandated panel of IIMIs for measurement should be broadened to include a far larger range of impurities, such as microbial antigens that may trigger additional innate immune pathways, popular manufacturing leachates and solvents, and hazardous chemicals needed to keep host cells alive.

The utilization of a single high-throughput platform designed to detect a large panel of indicators from the same class (proteins, small molecules, or nucleic acids) simultaneously, such as multiplex MS, ELISAs, or genomic arrays, should be used to standardize data across trials and laboratories. Broader nanoassortment of cytokines can be applied to make the data more complete.

Continue reading: Why Nanotoxicology Should be the First Step Towards a Nanotechnology Future.

Holley, C., and Dobrovolskaia, M. (2021). Innate Immunity Modulating Impurities and the Immunotoxicity of Nanobiotechnology-Based Drug Products. Molecules 26(23). Available at:https://www.mdpi.com/1420-3049/26/23/7308

Disclaimer: The views expressed here are those of the author expressed in their private capacity and do not necessarily represent the views of AZoM.com Limited T/A AZoNetwork the owner and operator of this website. This disclaimer forms part of the Terms and conditions of use of this website.

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Understanding the Immune Response to Nanomedicine Pharmaceuticals - AZoNano

"Hawkeye" reminds us why Jeremy Renner’s archer is the least Avenger – Salon

The opening episode of "Hawkeye" meets up with the mostly retired heroic archer operating in the uncharacteristic role of the powerless witness. Clint Barton is off the clock, having brought his kids to see a Broadway show during a holiday trip to New York City. The musical in question, "Rogers," is a ludicrous ode to Captain America highlighted by the 2012 intergalactic invasion that the Avengers thwarted.

In that showstopper tune the Avengers' super-powered members are center stage as the actor playing Steve sings their praises, and Black Widow's double ridiculously high-kicks her way across the stage. But it takes a minute to notice whether Hawkeye is being portrayed, too. He is, but at first he blends into the background.

RELATED: "Black Widow" is a triumph for Marvel fans, yet that's what makes it so infuriating

Lining up all the heroes in the "Avengers" pantheon, Hawkeye is the one most people could probably take or leave. He's also the hero whom dedicated nutballs could persuade themselves they could actually become with enough archery training, protein powder, push-ups, "low T" supplements, yams, and inspirational speeches from Joe Rogan.

An unenhanced marksman who never misses his targets and is an expert in hand-to-hand combat, Clint is a problematic fave. While he's saved the world a few times, he outright murdered a slew of criminals in his guise as the Ronin when half the universe wasn't looking.

Renner hasn't done such a bang-up job of winning widespread endearment either. Infuriating headlines like "Jeremy Renner won't stop calling Black Widow a 'slut'" or "Jeremy Renner says it's 'not my job' to help female costars get equal pay" keep popping up in answer to questions of, "Remind me, why don't we like Jeremy Renner?" The good news there is that thisproves how little time most of us spend thinking about Jeremy Renner.

Actors are not the characters they play. We know this. But some are happy to court the public's association them with the most popular of their fictional personas. Chris Evans, the actor behind the first Captain America, happily parted from Marvel while continuing to embrace the hero's nice guy reputation. It's good for his brand.

Renner approaches the whole hero business with a resounding "meh," which is his prerogative. Along the way he's been dogged by ugly allegations surrounding a custody battle with his ex-wife Sonni Pacheco, which he's either refuted or refused to comment upon, and still doesn't leave the most flattering impression.

Neither have we been trained to expect much from his solo efforts, like his very basic acid-washed jeans rock or his starring role as an organized crime "fixer" in the Paramount+ series "Mayor of Kingstown," a 2003-era brood fest that somehow time-traveled to 2021.

What does all this have to do with "Hawkeye"? Simple it explains why a show named for the least of the world's mightiest superheroes works best as an introduction to the wonderful Hailee Steinfeld and her character Kate Bishop, set up via this series to inherit Hawkeye's mantle.

That makes "Hawkeye" the latest in a trend of properties named for or associated with alpha males being reforged as a female fighter's story.In the same way that Netflix's "Master of the Universe" animated series was not, in fact, about He-Man and "Mad Max: Fury Road" stars Charlize Theron's post-apocalyptic Amazon Furiosa, "Hawkeye" introduces Steinfeld's heroine as a wealthy young woman with a vigilante's soul.

This wasn't Kate's plan, to be clear. Danger comes into her house in the present day, the same way it dropped out of the clouds in 2012 and decimated New York, where she witnesses some of the battle and Hawkeye's derring-do from the blown-out window of her family's luxury midtown apartment. She returns from her university's holiday break having destroyed a landmark while showing off her archery skills and discovers her mother Eleanor (Vera Farmiga) has become romantically involved with a snake-like jerk (Tony Dalton of "Better Call Saul," smartly here).

Kate can sense something's not right about the guy, confirmed when she tails him to a black market auction offering an array of contraband for the 1%. She gets no such hit off Clint, which she wouldn't since she's the latest in a line of women who make Hawkeye a better man.

This directly contradicts what much of the public knows or thinks it knows about Renner, a man who couldn't make it through presenting a Golden Globe without ogling and commenting upon his partner Jennifer Lopez's cleavage. Whether acting as a force lawful good or "taking out the trash" as an antihero, Clint Barton's charisma is increased by his proximity to one or both of the women in his life who could vouch for him, like his wife Laura (played by Linda Cardellini), or have, as best friend Natasha Romanoff (Scarlett Johansson) often did.

Kate continues this trend by informing Clint that he's her hero, something he views as more of a nuisance than a responsibility. Her Christmas wish is for him to help her become the superhero she's trained her whole life to be. His is to wrap up the inconvenient public resurrection of his shameful past in time to get home in time to carve the Christmas roast with his own daughter.

This erector set of a premise is made to charm, as if Marvel is asking both Renner and the public to lighten up a little by placing him in inside a Christmas-themed buddy action romp with the star of "Dickinson," a superb performer.

Want a daily wrap-up of all the news and commentary Salon has to offer? Subscribe to our morning newsletter, Crash Course.

Steinfeld deserves a better introduction than this piece of tinsel which, in its first two episodes, never transcends the rating of fine. Nevertheless, her presence and performance elevate a story into which Hawkeye has glaring downward in disappointment before eventually figuring out how to have fun again.

This scenario makes one wonder if Marvel'snoticed that when Florence Pugh's Yelena Belova received orders to assassinate Hawkeye in the "Black Widow" post-credits sequence, more than a few people were into the idea and may have been rooting for Yelena's success. (Pugh is confirmed to appear in "Hawkeye.")

To be fair, Renner and Hawkeye have their fans, otherwise he would not be toplining this show. His (likely Dockers-wearing, American pick-up driving, dog-loving) constituency still loves him for his excellent performance in "The Hurt Locker" and, remembering how well he and Taylor Sheridan worked together in 2017's "Wind River," are committed to seeing whether "Mayor of Kingstown" goes anywhere.

Next to that gloom "Hawkeye" is a sugar plum hit teasing us with the prospect that Kate can somehow help Clint shed his Grinch persona and help him to turn around what she calls his branding problem. "People want sincerity," she says while championing the cause of wearing one's heart on one's sleeve.

The man called Hawkeye may have to be reminded of that, but the audience already knows that to be true about Renner himself. He trades in an image of being rough, unvarnished and real, a guy who told Men's Health in a recent profile that he'd rather collect and restore decommissioned fire trucks than slap his name on a vanity tequila brand. "So f**k you, Ryan Reynolds or George Clooney or whoever," he tells his interviewer. "I'll come put out the fire on your agave farm."

Good one, as long as you forget Renner's role in the Great Jeremy Renner Vanity App Debacle of 2019. We very well might by the end of the six-episode run of "Hawkeye," which could shape up to be the most forgettable Marvel Cinematic Universe property since "Iron Man 3" and make Renner's Hawkeye fade into the curtains yet again. But he's an Avenger of a bygone era. As long as Renner doesn't dim Steinfeld's light we'll be fine to see him step back to let the next generation shine.

New episodes of "Hawkeye" premiere Wednesdays on Disney+.

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"Hawkeye" reminds us why Jeremy Renner's archer is the least Avenger - Salon

Jain vegetarianism – Wikipedia

Set of religion-based dietary rules

Jain vegetarianism is practiced by the followers of Jain culture and philosophy. It is one of the most rigorous forms of spiritually motivated diet on the Indian subcontinent and beyond. The Jain cuisine is completely lacto-vegetarian and also excludes root and underground vegetables such as potato, garlic, onion etc, to prevent injuring small insects and microorganisms; and also to prevent the entire plant getting uprooted and killed. It is practised by Jain ascetics and lay Jains.

Jain objections to the eating of meat, fish and eggs are based on the principle of non-violence (ahimsa, figuratively "non-injuring"). Every act by which a person directly or indirectly supports killing or injury is seen as act of violence (himsa), which creates harmful reaction karma. The aim of ahimsa is to prevent the accumulation of such karma. The extent to which this intention is put into effect varies greatly among Hindus, Buddhists and Jains. Jains believe nonviolence is the most essential religious duty for everyone (ahins paramo dharma, a statement often inscribed on Jain temples). It is an indispensable condition for liberation from the cycle of reincarnation,[7] which is the ultimate goal of all Jain activities. Jains share this goal with Hindus and Buddhists, but their approach is particularly rigorous and comprehensive. Their scrupulous and thorough way of applying nonviolence to everyday activities, and especially to food, shapes their entire lives and is the most significant hallmark of Jain identity. A side effect of this strict discipline is the exercise of asceticism, which is strongly encouraged in Jainism for lay people as well as for monks and nuns. Out of the five types of living beings, a householder is forbidden to kill, or destroy, intentionally, all except the lowest (the one sensed, such as vegetables, herbs, cereals, etc., which are endowed with only the sense of touch).

For Jains, vegetarianism is mandatory. In the Jain context, Vegetarianism excludes all animal products except dairy products. Food is restricted to that originating from plants, since plants have only one sense ('ekindriya') and are the least developed form of life, and dairy products. Food that contains even the smallest particles of the bodies of dead animals or eggs is unacceptable. Some Jain scholars and activists support veganism, as they believe the modern commercialised production of dairy products involves violence against farm animals.[18][19][20] In ancient times, dairy animals were well cared for and not killed.[21] According to Jain texts, a rvaka (householder) should not consume the four maha-vigai (the four perversions) - wine, flesh, butter and honey; and the five udumbara fruits (the five udumbara trees are Gular, Anjeera, Banyan, Peepal, and Pakar, all belonging to the fig class). Lastly, Jains should not consume any foods or drinks that have animal products or animal flesh. A common misconception is that Jains cannot eat animal-shaped foods or products. As long as the foods do not contain animal products or animal flesh, animal shaped foods can be consumed without the fear of committing a sin.[23]

Jains go out of their way so as not to hurt even small insects and other tiny animals, because they believe that harm caused by carelessness is as reprehensible as harm caused by deliberate action.[28][29][30] Hence they take great pains to make sure that no minuscule animals are injured by the preparation of their meals and in the process of eating and drinking.

Traditionally Jains have been prohibited from drinking unfiltered water. In the past, when stepwells were used for the water source, the cloth used for filtering was reversed, and some filtered water poured over it to return the organisms to the original body of water. This practice of jivani or bilchavani is no longer possible because of the use of pipes for water supply. Modern Jains may also filter tap water in the traditional fashion and a few continue to follow the filtering process even with commercial mineral or bottled drinking water.

Jains make considerable efforts not to injure plants in everyday life as far as possible. Jains only accept such violence in as much as it is indispensable for human survival, and there are special instructions for preventing unnecessary violence against plants. Strict Jains do not eat root vegetables such as potatoes, onions, roots and tubers as they are considered ananthkay.[23] Ananthkay means one body, but containing infinite lives. A root vegetable such as potato, though from the looks of it is one article, is said to contain infinite lives in it. Also, tiny life forms are injured when the plant is pulled up and because the bulb is seen as a living being, as it is able to sprout. Also, consumption of most root vegetables involves uprooting and killing the entire plant, whereas consumption of most terrestrial vegetables does not kill the plant (it lives on after plucking the vegetables or it was seasonally supposed to wither away anyway). Green vegetables and fruits contain uncountable, but not infinite, lives. Dry beans, lentils, cereals, nuts and seeds contain a countable number of lives and their consumption results in the least destruction of life.

Mushrooms, fungi and yeasts are forbidden because they grow in unhygienic environments and may harbour other life forms.[citation needed]

Honey is forbidden, as its collection would amount to violence against the bees.[41]

Jain texts declare that a rvaka (householder) should not cook or eat at night. According to Acharya Amritchandra's Purushartha Siddhyupaya:

And, how can one who eats food without the light of the sun, albeit a lamp may have been lighted, avoid his of minute beings which get into food?

Strict Jains do not consume food that has been stored overnight, as it possesses a higher concentration of micro-organisms (for example, bacteria, yeast etc.) as compared to food prepared and consumed the same day. Hence, they do not consume yoghurt or dhokla and idli batter unless they have been freshly set on the same day.

During certain days of the month and on important religious days such as Paryushana and 'Ayambil', strict Jains avoid eating green leafy vegetables along with the usual restrictions on root vegetables.

Jains do not consume fermented foods (beer, wine and other alcohols) to avoid killing of a large number of microorganisms associated with the fermenting process.[44] According to Pururthasiddhyupya:

Wine deludes the mind and a deluded person tends to forget piety; the person who forgets piety commits his without hesitation.

The vegetarian cuisines of some regions of the Indian subcontinent have been strongly influenced by Jainism. These include

In India, vegetarian food is considered appropriate for everyone for all occasions. This makes vegetarian restaurants quite popular. Many vegetarian restaurants and Mishtanna sweet-shops for example, the Ghantewala sweets of Delhi[47] and Jamna Mithya in Sagar are run by Jains.

Some restaurants in India serve Jain versions of vegetarian dishes that leave out carrots, potatoes, onions and garlic. A few airlines serve Jain vegetarian dishes[48][49] upon prior request.

When Mahavira revived and reorganized the Jain community in the 6th century BCE, ahimsa was already an established, strictly observed rule. Parshvanatha, a tirthankara whom modern Western historians consider a historical figure, lived in about the 8th century BCE and founded a community to which Mahaviras parents belonged.[56] Parshvanathas followers vowed to observe ahimsa; this obligation was part of their caujjama dhamma (Fourfold Restraint).[58]

In the times of Mahavira and in the following centuries, Jains criticized Buddhists and followers of the Vedic religion or Hindus for negligence and inconsistency in the implementation of ahimsa. In particular, they strongly objected to the Vedic tradition of animal sacrifice with subsequent meat-eating, and to hunting.

According to the famous Tamil classic, Tirukkua, which is also considered a Jain work by some scholars:

If the world did not purchase and consume meat, no one would slaughter and offer meat for sale. (Kural 256)

Some BrahminsKashmiri Pandits and Bengali Brahminshave traditionally eaten meat (primarily seafood). However, in regions with strong Jain influence such as Rajasthan and Gujarat, or strong Jain influence in the past such as Karnataka and Tamil Nadu, Brahmins are strict vegetarians. Bal Gangadhar Tilak has described Jainism as the originator of ahimsa. He wrote in a letter:

In ancient times, innumerable animals were butchered in sacrifices. Evidence in support of this is found in various poetic compositions such as the Meghaduta. But the credit for the disappearance of this terrible massacre from the Brahminical religion goes to Jainism.[67]

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9 Reasons To Reject Vegetarianism – Listverse

Lets be honest: eating meat is an objectively bad idea. Its expensive, has been linked to cancer and causes devastating crises in the developing world.

SEE ALSO: 10 Surprising Facts About Vegetarians

Yet, for all the rational arguments against it, some of us just cant give our carnivorous habits up. Show us a cross-section of our disease-ridden gut and well show you a juicy steak just begging to be eaten. Show us a slaughterhouse and well ask for a knife and fork. It may sound callous, but well only give up our bacon when you pry it from our cold, dead handsand heres why:

Our Bodies Are Designed For Meat

Thanks to the miracles of evolution, we humans can survive just fine on a meat-free diet. But that doesnt mean were natural vegetarians. Far from it: as far back as 2003, scientists had established our ancestors were eating meat up to 2.5 million years ago. In other words, that juicy slab of barbecue isnt some icon of modern decadence; its part of our traditional diet, and there are plenty of other clues too. First, our bodies lack most of the equipment youd associate with herbivores. For instance, we dont have four stomachs, any ability to break down cellulose, or the sort of complex intestinal tracts most leaf-eaters possess. Second, our teeth are obviously designed to handle both meat and non-meat diets. And a good job too, because

From a strictly logical perspective, there are a number of oddities about us humans. For starters, our brains seemingly shouldnt be this big. If you look across most primate species, brain size increases with body size: humans are noticeable outliers. Then theres the added complexity of our brains, which are so stuffed full of neurons theyre likely capable of holding more individual thoughts than there are stars in the universe. So what makes us so special? Well, according to one 2011 study, its our appetite for meat.

Seriously: researchers from Spain identified signs of malnutrition in a childs skull dating from 1.5 million years ago, consistent with a meat-deficient diet. Whats interesting about this is it suggests we were so used to eating meat back then our brains couldnt develop without ita theory supported by other evidence that links primate brain complexity to the number of calories consumed per day. Since we didnt begin cooking our food until long after our brains went supernova, the only likely candidate for our calorific diets is meat. Meaning were only capable of making logical choices like vegetarianism because we originally ate other animals.

Other Primates Eat Meat

One argument often put forward for going vegetarian is that humans are the only primates to eat meat. Ergo, it must be unnatural: like using the internet to moan about steakhouses. But guess what? Its not just untrue; its about as scientific as punching biology in the face.

Back in 1960, Jane Goodall observed chimps hunting and eating other animals in the wild. In the years since, its been shown that certain chimp communities eat as much as one ton of meat annually. In other words, theyre less indulging occasional cravings than they are taking part in the chimpanzee equivalent of Man V. Food. Not only that, but they apparently use the slaughtered meat to gain a reproductive and political advantage over one another. So, to recap: our evolutionary cousins love a good steak so much; theyll literally whore themselves out to get it.

Meat Can Be Sustainable

One of the big reasons for giving up meat is the devastating environmental impact of shipping, say, a chunk of dead cow halfway across the world. So if youre into environmentalism, dropping meat should be a no-brainer, right?

Not quite. While our current model of shipping is about as environmentally-friendly as a forest fire, it doesnt have to be this way. See, livestockmanaged properlycan be used to do a lot of stuff that would otherwise require a heck-load of fossil fuel. For example, grazing animals can help cycle nutrients and aid in land management: while also requiring little in the way of chemicals and pesticides to grow to an edible size. Not only that, but a lone cow slaughtered on a small farm can feed its owners for ages, which is why we got into agriculture in the first place. So its not meat itself which is the issue, so much as our current supply chain.

Damage to the Environment

In our modern age, its taken as read that eating meat is a bigger planet killer than chowing down on tofu. But thats not always the case. For example, compare organically reared animals with industrially produced tofu. The quantities of land needed are greater, the treatment and harvesting of the soya involves more fossil fuels, and the end product often has to be shipped great distances if you live somewhere like Britainwhere the climate is really, really bad for growing meat substitutes. Simply put: that tasteless tofu burger youre forcing down to preserve our planets future may actually be more atmosphere-frying than the delicious hunk of beef being eaten by that smug bastard across the table from you.

It May Reduce Aggression

There are certain psychological traits among humans that seem so obvious we shouldnt need a study to prove them. One is that exposure to weapons triggers violence. Another is that meat-eaters are more aggressive than vegetarians. However, a group of scientists decided to look into the meat/aggression issue anywayand what they found turns common sense on its head.

By exposing men to pictures of red meat then placing them in a position of power over another subject, researchers discovered that thinking about steak might actually reduce aggression in humans. No-ones really got any idea whybeyond hazily linking it to evolutionbut the conclusion seems valid. So, while we may imagine a rabid steak-eater to be more violence-prone than a guy who lives off soy beans and lentils; the opposite may well be true.

It Doesnt Have to Harm Animals

Of course, one of the big arguments against eating meat is that its cruel. However you look at it, cramming a bunch of chickens together in a cage and feeding them until theyre too fat to stand isnt a particularly pleasant thing to do. Even if you give the animal the best life possible, theres no getting around the fact youre killing a sentient creature for no better reason than dinner. So its easy to see why some people just flat-out refuse to eat meat.

Only thats about to change. Thanks to Dutch scientist Willem van Eelen, were now at the stage where we can grow burgers in a lab. Slow down and read that again: were now so advanced as a species we can grow a hunk of cow in a lab without ever actually involving a living cow. Currently, the technology is too expensive for mass-productionthe first lab-grown burger cost $300,000 to make and tasted only reasonably good. But were conceivably only a decade or two away from a world where steak, sausages, bacon and even veal cutlets can be created without harming a single animal.

It Could Save the Planet

Go for a walk in the countryside and chances areunless you live near a National Parkthat the natural landscape youre seeing is nothing like how nature intended it. For thousands of years, animals belonging to our ancestors grazed dense natural forests to destruction, resulting in the great big open spaces we now associate with being outdoors. And while it may seem kinda sad, this slow-motion deforestation is actually just what we need. See, if the country ever gets its act together and decides to go green, were gonna need as much open space for wind farms and solar panels as we can get. Know the most eco-friendly way for maintaining such places? Yep: grazing livestock. This isnt just me speculating either, British eco warrior Simon Fairlie famously argued that rearing livestock is essential for increasing biodiversity and creating a truly-sustainable world. And what do we ultimately do with all this necessary livestock? Thats right: we eat it.

OKI admit this isnt much of a point. But lets be honest: a huge amount of the vegetarian v. carnivore internet war comes down to this simple fact. For all we talk about protein and write long list articles defending our choices, most of us meat-eaters just basically like the taste. Does that make us callous, immoral people? Well, maybe kinda. But we live in a world thats an ethical minefieldevery single day we log onto computers manufactured by tax-dodging multinationals using sweatshop labor; wear clothes made by virtual slaves in third world countries; give a big chunk of our paychecks to a sociopathic government; and generally reap the rewards of living in a nation subsidized by the unethical treatment of most of the rest of the planet. If eating a hunk of bacon each day is what it takes to get me through this headache-inducing liberal guilt-trip, then so be it.

Morris M. is Listverse's official news human, trawling the depths of the media so you don't have to. He avoids Facebook and Twitter like the plague.

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9 Reasons To Reject Vegetarianism - Listverse

Opinion: Gujarat is a vegetarian state only in the minds of its self-chosen custodians – Scroll.in

Ane ardho dozen eenda aapjo, (and half a dozen eggs please), I say to the owner of a provision store near my house in Ahmedabad. I am given six eggs packed discretely in a newspaper bag. It feels like buying sanitary napkins, the exchange is so minimal in words. A fast-food joint right next to this provision store sells mock chicken lollipops. Okay, let me explain. They are made of potatoes but look like chicken lollipops, and ditto the case with kebabs and other items that provide to a vegetarian foodie some comfortable alternative to non-vegetarian food. I mean theres no dearth of such surrogate food in India, is there?

In my community of Sindhis, those who begin to follow the Radhaswami satsang eat a specific version of nutria-nugget subzi that is meant to taste like mutton but is called Radaswami baadji. This kind of proxy-fulfilment is evident even in mocktails and fruit beer and wine bars that are not wine bars.

My point is that we (from the state) and others (not from the state) may continue to be surprised, mortified even by how the topic of vegetarianism and the prohibitive rules against meat sales and consumption keeps coming up in Gujarat. However, this matter is not comprehensible through contexts of citizenship and freedom of choice. It can be contested in courts on those grounds, but in order to understand the nature of this pathology, we need to see what happens in Gujarat, ordinarily.

The two examples I began with involve two different forms of visuality. The sight of real eggs is an anathema to most customers who are not there to buy eggs, hence the eggs must be hidden from their view lest they stop coming to that store. The sight of mock lollipops and kebabs is a visual reminder of the prohibited items without the guilt of having eaten them. These negotiations characterise everyday life in Gujarat.

The sight of meat would be a reminder to the upper-caste Hindu and Jain Gujarati of the prohibited parts in the psyche which they have managed to cleanse out through a range of strategies. Among the first is not to give a home to a meat-eating family in a colony or neighbourhood. In the 1980s my family had a tough time finding a house because Sindhis are known as non-vegetarian, ergo, Muslim-like.

It is a different matter that many Sindhis in Gujarat have now taken to vegetarianism. The second strategy has been to keep communities (read Muslims) away from sight so that neither their presence nor their lifestyle reminds the Hindu Gujaratis of what and who they do not want to see. And this collapsing between the what and who also needs to be understood.

Gujarat takes the principle of you are what you eat quite literally. The third strategy is to make the life of non-vegetarian restaurants precarious so that while some survive, a large number become suddenly vegetarian and South Indian or Punjabi restaurants doing paneer dishes. The fourth strategy is not even to socialise with, or visit homes of families that eat meat, and should you do visit, do not accept any food from them.

Now some of these forms of abstinence may be common across vegetarian communities in India. However, in Gujarat, they all exist without resistance from within. All this may lead us to assume Gujarat is a vegetarian state and while figures of meat-eating population vary from 40% to 60 % the truth is that it is not a fully vegetarian state except in the mind of its self-chosen custodians.

Amrita Shah has persuasively argued in her recent piece for Moneycontrol that vegetarianism is Gujarats commonsense or mythicised face and not its lived reality. There is certain rhetoric by which this myth-making has taken place. The Gujarat State Gazetteer of 1989 generalises vegetarianism by outsourcing it to western style.

Non-vegetarianism in Gujarat is common but made to feel illegitimate. Meat selling and consumption happens in Gujarat, but it must be away from the sight and sensory world of the upper-castes. To ask for a restriction on displaying meat is to go just one step further in the existing scheme of things.

It is to say not only that I must not see, but I must also avoid that rare and accidental occasion of stumbling upon its sight if I am in that part of the city. In other words, should Ahmedabad or Vadodara be my city, it must be on my terms and whom I represent. The recent ban on non-veg food stalls is not only a violation of freedom to eat but also the prerogative to see and smell only what I wish to as a Hindu or Jain citizen of the state.

Rita Kothari is the author of several books, most notable among which are The Burden of Refuge and Unbordered Memories. She teaches at Ashoka University.

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Opinion: Gujarat is a vegetarian state only in the minds of its self-chosen custodians - Scroll.in