Monthly Archives: September 2023

From there it has only expanded further. In 2021,JLL foundthat the number of proptech start-ups had tripled over the previous decade. The first six months of 2021 had seen a record $9.7 billion in funding activity for the sector, propelled by the pandemic.

While the surge continued through to 2022, funds have tailed off in 2023, withPitchBook data recording US$2.2bnin proptech venture capital deals globally to May. Nonetheless, confidence in the sector remains high.

There are now technological solutions for almost every aspect of real estate functions, including investment management, design and construction, building and facility operations and portfolio management, Wang says.

However, with over 500 companies globally currently developing AI-powered services relevant to real estate, AI is set to wield the biggest influence. In fact, the total capital raised to fund AI-powered Proptech reached $4 billion globally in 2022 twice that raised in 2021, according to venture capital and private equity research firm PitchBook.

The early adopters are already reaping financial benefits.

For example, U.K. investment firm Royal London Asset Management has used JLLs AI- powered Hank technologies to improve its heating, ventilation and air conditioning (HVAC) operations and overall energy efficiency in its 11,600 square meter (125,000 square feet) Birmingham office building. Since the start of 2023 it has achieved a 708% return on investment, saved 59% in energy output, and reduced its carbon emissions by 500 metric tons per year.

AI technology is also used to analyse data from the building management system to understand the performance of the equipment in that building, identifying clearly what might need to be changed or maintained, says Carolyn Trickett, Growth Principal at JLL Spark Global Ventures, the corporate venture arm of JLL and part of the JLL Technologies division focused on investing in strategically relevant technology to optimize the needs of JLLs clients.

Another strength of AI is its ability to rapidly analyze large amounts of data, even from different data streams, something that can more efficiently inform management decisions.

Take the work of VergeSense, spearheaded by JLL Spark, which uses optical sensor technology combined with AI to offer insights about how buildings are used.

Which desks are used most frequently? How often are meeting rooms used? What times do people arrive and leave? Trickett says. In a post COVID-19 world with an increase in hybrid working, this can help analyse the space needs of a company.

AI can also work 24/7, and generative AI, which uses advanced algorithms to generate content like the answer to customer queries, can be trained to respond to out-of-hours queries, increasing satisfaction from existing tenants, or the potential conversation rate of prospective ones.

While AI technology and its capabilities are still in their infancy, there are some legitimate concerns that any early adopter needs to consider.

The regulation landscape for AI isnt moving quite as fast as the technology, Wang says, So you should consider potential risks in cybersecurity or data security when considering implementation.

She adds: We are at a critical point for businesses to start thinking about the pain points AI technology might be able to help you with and how you might need to refine your processes or technology to realise the most value from it.

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AI and the evolution of proptech - JLL

In the world of firearms, specific to open and concealed handgun carry, the holster is often an unsung hero, silently safeguarding both the firearm and the wearer.

Over time, the holster has undergone a remarkable journey of transformation, evolving from simple leather sheaths to advanced handgun-retention systems that cater to the demands of military, law enforcement and civilians alike. The origin, history and development of retention holsters are a testament of shooter ingenuity and the ever-evolving landscape of firearm technology.

Humble Origins

The roots of the holster can be traced back to ancient civilizations, where rudimentary forms of sheaths or scabbards were used to carry the likes of swords and knives. These early holsters were typically crafted from leather or fabric, providing a simple means of accessibility by carrying a weapon close at hand.

As firearms emerged, so did the need for holsters designed to accommodate these new tools. The earliest pistol holsters were often crude leather pouches, providing little more than a convenient way to carry a firearm. Security, ergonomics and retention were not priorities for these early designs.

The mid-19th century saw the advent of revolvers, which introduced new challenges and opportunities for holster design. Holsters became more specialized to accommodate these wheel guns. However, the primary focus remained on accessibility rather than retention. Most holsters of this era were open-top designs, allowing quick access to the firearm but offering minimal security.

It wasn't until the late 19th century that the first attempts at retention were made. The "Texas" or "Mexican" style holster featured a strap that secured the firearm in the holster, offering a rudimentary form of retention. While not as sophisticated as modern retention systems, it was at least a step in the right direction.

Holsters for Semi-Automatics

The early 20th century brought semi-automatic pistols into prominence. With the introduction of these firearms, holster design once again had to adapt. The first holsters for semi-automatic pistols were similar to their revolver counterparts, open-top and offering minimal retention.

However, World War I served as a catalyst for change. The need for secure holsters became apparent in the trenches, where the chaos of battle required soldiers to keep their firearms firmly in place. The flap holster, with a top cover secured by a button or strap, became the standard issue for many military forces. It was an early forerunner of modern retention holsters, ensuring that the firearm stayed put even in tumultuous situations.

Military and Police Innovations

The mid-20th century witnessed significant advancements in holster technology, largely driven by law enforcement and military needs. The need for quick and reliable access to firearms, combined with enhanced retention, became paramount. This led to the development of new materials such as Kydex (originally produced in 1965 by Rohm and Haas, having been originally designed for use in aircraft interiors), and practical innovations such as thumb-break holsters, featuring a strap or snap that could be quickly released with a downward motion of the thumb.

The 1970s marked a pivotal moment in holster evolution with the introduction of the Safariland ALS (Automatic Locking System). This groundbreaking design incorporated a rotating hood that locked into place when the firearm was holstered, adding a significant level of security. The ALS retention holster (and with the latter development and production of the GLS (Grip Lock System) and the QLS (Quick Lock System)), set a new standard for retention holsters, balancing accessibility and safety.

Today, retention holsters have reached unprecedented levels of sophistication. They cater to a wide range of users, from law enforcement officers requiring a higher level of security to concealed carriers who need quick access while ensuring their weapon stays secure. Some of the more common modern retention mechanisms include:

The evolution of retention holsters is a testament to the dynamic nature of the firearm industry. From simple leather sheaths to sophisticated automatic locking systems, holsters have come a long way in providing both accessibility and security.

Whether carried by military personnel, law enforcement officers, or concealed weapons carriers, modern retention holsters are designed to cater to a diverse range of needs, ensuring that the firearm is readily accessible when needed and securely retained when not in use. As technology continues to advance, we can only imagine what the future holds for this indispensable accessory to those carrying a sidearm.

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Holster Evolution | An Official Journal Of The NRA - Shooting Illustrated

Phylogenetic properties of Deg/ENaC channels from metazoans and non-metazoans

To better understand the relationships of T. adhaerens TadNaC channels to other Deg/ENaC channels, including those from the fellow placozoan Hoiliungia hongkongensis, we used CLuster ANalysis of Sequences (CLANS)27 on a set of 1074 Deg/ENaC channel protein sequences extracted from high-quality gene datasets of representative species spanning the major animal groupings, followed by phylogenetic inference. We tested a range of P value cut-offs for the CLANS analysis (i.e., 1E10, 1E20, 1E30, 1E40, and 1E50), finding in all cases that the sequences formed one major cluster comprised of two inter-connected sub-clusters (Fig.1, Supplementary Data3 to 7). One of these sub-clusters contained the chordate ASIC and BASIC channels, along with the T. adhaerens channels TadNaC1 to 9 and TadNaC11 (and corresponding H. hongkongensis homologs), and the other the chordate ENaC channels with the singleton placozoan homologs TadNaC10 and HhoNaC10. Our analysis is altogether consistent with a previous study19, both also finding the peptide-gated FaNaC and WaNaC channels from lophotrochozoans to associate with the ENaC sub-cluster, and the peptide-gated HyNaC channels from Hydra magnipapillata to associate with the ASIC/BASIC sub-cluster.

Nodes correspond to individual channel sequences and are colored by taxon as indicated by the legend. Edges correspond to BLAST connections with P-values<1E30. The general locations of the chordate ASIC and ENaC channels, the cnidarian HyNaC and NeNaC channels, the lophotrochozoan FaNaC and WaNaC channels, and the C. elegans ACD channels are indicated. Singletons and non-connected clusters with less than five sequences are masked but available in the corresponding CLANS file (Supplementary Data5).

Comparing the various CLANS analyses at different thresholds, we found that decreasing the P value from 1E20 to 1E30 caused numerous sequences to no longer associate with the main cluster, including a large group of ctenophore sequences (Fig.1, Supplementary Data4 and 5). Decreasing it further to 1E40 caused a large group of D. melanogaster PPK channels to no longer associate with the ENaC sub-cluster, and a set of C. elegans ACD channels to lose their relatively strong connectivity with the ASIC sub-cluster (Fig.1), instead forming a single connection with the ENaC sub-cluster (Supplementary Data6). We therefore selected a P value cut-off of 1E30 to isolate a central cluster of sequences for phylogenetic inference, reasoning that this cut-off struck a balance between strategically removing divergent and/or truncated sequences that would interfere with phylogenetic analysis, while being permissive enough to include most PPK channels. In agreement, a previous study that employed a similar CLANS pre-filtering approach prior to phylogenetic analysis but with a P value of 1E50 excluded the D. melanogaster PPK channels19. In our analysis, pre-filtering the sequences at 1E30 resulted in the removal of 200 sequences, which in addition to the noted cluster of ctenophore channels, included numerous singletons and smaller clusters from platyhelminths and cnidarians (Fig.1). Lastly, our clustering analysis revealed that several Deg/ENaC homologs present in the gene data for unicellular eukaryotic species from the clades Heterokonta (i.e., from the SAR supergroup, for Stramenopila, Alveolata, and Rhizaria) and Filasterea, clustered the ASIC and ENaC sub-clusters (Fig.1), corroborating a report that Deg/ENaC channels are present outside of animals, in select unicellular organisms28.

A maximum likelihood phylogeny inferred from the aligned protein sequences, rooted on the Deg/ENaC channel homologs from the unicellular filasterea-related species Tunicaraptor unikontum, reveals strong phylogenetic support for two distinct clades, termed Clades A and B, corresponding to the ASIC and ENaC sub-clusters (Fig.2), which is consistent with another recent phylogenetic analysis8. In both analyses, most TadNaC channels fall within Clade A (TadNaC1 to 9 and 11), forming a sister relationship with chordate BASIC channels. Instead, the singleton channel TadNaC10, along with its orthologue from fellow placozoan Hoilungia hongkongensis, falls within Clade B. Our analysis also identifies several groups of uncharacterized channels that are positioned between the TadNaC and BASIC channels in Clade A, with representatives from chordates (i.e., cephalochordate and urochordate), ambulacrarians (i.e., echinoderm and hemichordate), and lophotrochozoans (i.e., annelid and brachiopod). Our tree also expands the group of C. elegans channels that form a sister relationship with BASIC channels by including the channels ACD-1, ACD-5, and FLR-1, which notably, resemble TadNaC6 and BASIC channels in being inhibited/blocked by external protons13,14,24,29, and ACD-2 which is proton-activated14. Between T. adhaerens and H. hongkongensis, most Clade A Deg/ENaC channel sequences exhibit one-to-one orthology, except for TadNaC4, 6, and 7, for which H. hongkongensis only bears the single homolog, HhoNaC4/6/7. Also consistent with previous reports10,25, ASIC channels within our phylogenetic tree form two distinct subgroups, Groups A and B (not to be confused with Clades A and B), with chordates (vertebrates, urochordates, and cephalochordates) possessing only Group A orthologues, cephalochordates also possessing Group B orthologues, and ambulacrarians and lophotrochozoans only possessing Group B orthologues. Together, these various described channels form a well-supported subclade within Clade A (i.e., subclade I), which is distinct from subclade II which bears representatives from a broad range of bilaterian and non-bilaterian animals. This includes a clade of C. elegans channels bearing ACD-1, ACD-5, and FLR-1, which resemble TadNaC6 and BASIC channels in being inhibited/blocked by external protons13,14,24,29, and ACD-2 which is proton-activated14, and a large clade of arthropod channels including the D. melanogaster PPK channels, of which PPK1 is also proton-activated16. In addition, Clade A subclade II includes two groups of cnidarian channels, one bearing the neuropeptide-gated HyNaC channels from Hydra magnipapillata21 and the proton-activated channel NeNaC2 from Nematostella vectensis8, and several distinct groups of uncharacterized channels from protostomes, ambulacrarians, ctenophores, and poriferans.

The tree was generated with the program IQ-TREE 2 with the best-fit model WAG+F+G4 and rooted with the filasterean-related Deg/ENaC channel homologs. Node support values are for 100 standard bootstrap replicates (green). The asterisks and labels (pink) indicate single channels or clades bearing Deg/ENaC channels that have been characterized as proton-activated.

Clade B similarly subdivides into two major subclades, with subclade I bearing the chordate ENaC channels and the C.elegans mechanosensory channels MEC-4 and MEC-10 and the proton-activated channel ASIC-18,14. Also within subclade I are the placozoan channels TadNaC10 and HhoNaC10, along with a diversity of uncharacterized channels from cephalochordates, ambulacrarians, and protostomes, and a clade of cnidarian channels which includes the N. vectensis proton-activated channel NeNaC148. Clade B subclade II contains a large group of protostome channels which includes the peptide-gated FaNaC and WaNaC channels from annelids and molluscs9, and several uncharacterized representatives from ambulacrarians, cephalochordates, and protostomes (i.e., lophotrochozoans and ecdysozoans including a large clade of channels from Centroides sculpturatus). Lastly, a set of cnidarian channels and Deg/ENaC homologs from the unicellular heterokont Cafeteria roenbergensis form a sister clade relationship with all other Clade B channels.

Altogether, our combined CLANS and phylogenetic analysis provide strong evidence that most TadNaC channels, including the previously described TadNaC6 and the currently described TadNaC2, are phylogenetically closer to BASIC channels than ASIC channels. Furthermore, our analysis corroborates the existence of two major groups of metazoan channels8,19, identifies numerous groups of uncharacterized channels with phylogenetic proximity to channels with known properties, and provides phylogenetic evidence for the existence of Deg/ENaC channels outside of Metazoa.

Previously, we found that the T. adhaerens Deg/ENaC channel TadNaC6 conducts constitutive Na+ leak currents in vitro that are blocked by external protons and Ca2+ ions24 (Fig.3a). Here, we set out to characterize the in vitro properties of a second T. adhaerens Deg/ENaC channel, TadNaC2. Whole-cell patch clamp recording of Chinese Hamster Ovary (CHO)-K1 cells transfected with the TadNaC2 cDNA revealed robust inward macroscopic cation currents elicited by perfusing a pH 5 solution over the recorded cells. No such currents were evident in untransfected cells, but we did observe a small endogenous inward current in these cells that became activated by solutions with a pH of 4 or lower (Fig.3a). For comparison, we also transfected mouse ASIC1a (mASIC1a) which has been extensively studied in vitro, observing robust inward currents at pH 5 with a noticeably faster desensitization than TadNaC2. TadNaC2 whole-cell currents were quite large in amplitude, reaching upwards of 5000 picoamperes (Fig.3b), despite the cDNA not being codon optimized as was required for efficient expression of the cnidarian HyNaC channels in mammalian cells30.

a Sample whole-cell currents recorded for the previously characterized Trichoplax Deg/ENaC sodium leak channel TadNaC6 that is blocked by extracellular protons24, a newly identified endogenous current in CHO-K1 cells that becomes activated upon perfusion of strongly acidic solutions below pH 4.0, and large, prominent proton-activated currents conducted by the in vitro expressed Trichoplax TadNaC2 and the mouse ASIC1a (mASIC1a) channels. b Plot of average peak inward current amplitude (in picoamps or pA) for currents shown in (a) standard deviation. Orange symbols denote values for individual cells/recordings.

Next, we sought to compare the general properties of TadNaC2 and mASIC1a proton-activated currents. Perfusion of external solutions of various pH revealed that TadNaC2 begins activating at pH 5.5, with current kinetics that accelerate from a slow onset non-desensitizing current at pH 5.5, to a faster transient and partially desensitizing current at pH 4.0 (Fig.4a). These currents are markedly different from those of mASIC1a, which began activating at the more basic pH of 6.7, with much faster activation and desensitization evident across all tested values of pH. Notably, the TadNaC2 currents appear biphasic, particularly upon activation with a pH 4.5 solution, with a fast/early transient component followed by a slower/late sustained component. Doseresponse curves generated from these experiments revealed that TadNaC2 is considerably less sensitive to external protons than mASIC1a (Fig.4b), with a pH50 of 5.10.1 vs. 6.70.1, and a Hill coefficient (nH) value of only 1.70.4 vs. 8.42.7. Notably, these values for the mASIC1a channel are closely in line with those reported for the human ASIC1a channel recorded in Xenopus oocytes25,26. Together, the lower pH50 and nH values observed for TadNaC2 indicate a lower binding affinity and reduced cooperativity for extracellular proton binding, more inline with the sensitivity reported for the rat ASIC2a channel31,32,33.

a Sample recordings of TadNaC2 currents (top) and mouse ASIC1a currents (mASIC1a, bottom) elicited by perfusion of solutions with decreasing pH. b pH doseresponse curves for TadNaC2 (n=1417) and mASIC1a (n=713) revealing a right shifted pH50 for the Trichoplax channel relative to mASIC1a, and a smaller Hill coefficient (nH). The values observed for mASIC1a are consistent with previous reports26. c Sample sequential TadNaC2 currents exhibiting rundown or tachyphylaxis similar to mASIC1a. d Plot of average normalized current amplitude standard deviation through successive sweeps for TadNaC2 (i.e., early and late currents at pH 4.5, n=6, and peak current at pH 5.5, n=56) and mASIC1a (peak current at pH 5.5, n=8), revealing decaying amplitudes for all conditions that are statistically indistinguishable from each other (i.e., p>0.05 for one-way ANOVAs comparing raw normalized values for each condition at each pulse). The asterisks indicate statistically significant p values (i.e., <0.05) for pairwise post hoc Tukey tests after one-way ANOVAs of each set of pulses for each condition (TadNaC2 pH 4.5 early: p=1.0E4, F=7.6; TadNaC2 pH 4.5 late: p=2.4E3, F=4.8; TadNaC2 pH 5.5: p=4.3E7, F=14.7; mASIC1 pH 5.5: p=1.4E14, F=38.2). e Sample current recordings for TadNaC2 and mASIC1a before (black traces) and after (red traces) perfusion of 3mM amiloride, revealing a nearly complete block for mASIC1a (at pH 5.5) and only ~50% block for TadNaC2 (pH 4.5). f Plot of average percent block of inward current standard deviation for TadNaC2 (n=8) and mASIC1a (n=7) before and after perfusion of 3mM amiloride. Individual replicates are included as gray circles. B+T indicates the total decay in average current for a successive sweep, which includes the effects of drug block (B) and tachyphylaxis (T), while B indicates the isolated component of drug block alone, obtained by subtracting the average decline in amplitude caused by tachyphylaxis. Denoted p values are for post hoc Tukeys tests after one-way ANOVA (p=1.7E11, F=56.1). g Sample sequential TadNaC2 currents elicited by perfusion of pH 4.5 solutions bearing increasing concentrations of amiloride. h Average amiloride doseresponse curve (n=9) revealing a more pronounced decline in normalized peak inward current with increasing concentration of amiloride, compared to the rundown observed in the absence of drug attributable to tachyphylaxis.

In early experiments, we found that TadNaC2 currents exhibit a non-recovering decay in amplitude upon repeated activation. For example, applying paired 30s pulses of pH 4.5 solution separated by neutral pH wash steps of either 40 or 80s resulted in similar decreases in amplitude of 55.516.5% with a 40s interval vs. 50.749.2% with an 80s interval. Since doubling the interpulse interval from 40 to 80s did not diminish the current decay amplitude, the observed process is not likely due to incomplete recovery from fast/acute desensitization. This feature of TadNaC2 thus resembles the rodent ASIC1a channel which undergoes slow desensitization or tachyphylaxis, a unique process not observed for ASIC2 and ASIC3 proposed to involve a prolonged inactivated state that is distinct from acute desensitization34,35. To better characterize this property of TadNaC2, we employed an experimental paradigm similar to one used previously to study tachyphylaxis of rat ASIC1a in Xenopus oocytes34. Specifically, we applied six 15-second pulses of pH 4.5 or 5.5 solutions over recorded cells expressing TadNaC2 or mouse ASIC1a, separated by 55-second interpulse intervals. Consistent with observations in oocytes, mouse ASIC1a peak currents decayed upon repeated activation at pH 5.5 (Fig.4c), decreasing to 49.49.5% of their original value after 6 pulses (Fig.4d). Similarly, peak TadNaC2 currents declined to 57.920.5% at pH 4.5, and 43.516.4% at pH 5.5, while the late/sustained component of the TadNaC2 current at pH 4.5 declined to 59.522.9%. Analysis of the average data revealed that although the decline in current amplitude for each condition relative to the first pulse was statistically significant, the degree and rate of decline between the different channels and conditions was not.

Next, we tested the sensitivity of TadNaC2 to the general Deg/ENaC channel blocker amiloride, having previously found that the T. adhaerens TadNaC6 channel was potently activated by this drug24, a rare feature also reported for ASIC3 channels36,37. Application of 3mM amiloride almost completely blocked mASIC1, but only partially blocked TadNaC2, altering the current waveform such that the fast early current component was no longer evident (Fig.4e). Given that TadNaC2 and mASIC1 currents, respectively, decay by 19.111.6% and 17.38.3% upon successive activation, we reasoned that a component of the attenuated current amplitude in these experiments was attributable to tachyphylaxis. Subtracting the effect of tachyphylaxis to isolate the amiloride block of both channels reduced the decrease in average peak inward current from 90.34.1% down to 73.04.1% for mASIC1a, and from 60.59.5% to only 41.49.6% for TadNaC2 (Fig.4f). To better characterize amiloride block of TadNaC2, we applied increasing concentrations of the drug while activating at pH 4.5, revealing a continuing decline in peak current amplitude coupled with a marked reduction in the fast transient current with amiloride concentrations greater than 1mM (Fig.4g). Although a component of the decline in current amplitude is likely due to the tachyphylaxis-like property of TadNaC2, there is a marked difference in the current waveforms, in that tachyphylaxis does not markedly alter the macroscopic current waveform (Fig.4c), while high concentrations of amiloride alter the kinetics of the macroscopic current such that the fast transient current is considerably inhibited (Fig.4g). Although these observations suggest that amiloride has a more potent effect on the early compared to the late current, more detailed studies will be required to characterize this phenomenon. Analysis of the decline in peak current as a function of amiloride concentration reveals a more pronounced decay in amplitude compared to tachyphylaxis with amiloride concentrations greater than 100M, with an IC50 of 52.029.6M attributable to the combined effect of amiloride plus tachyphylaxis (Fig.4h).

TadNaC2 resembles mammalian ASIC3 in conducting biphasic macroscopic currents comprised of an early current that activates and desensitizes quickly, followed by a late current that activates and desensitizes more slowly38. These two components of the TadNaC2 current become even more distinguishable at pH 3.5, where two separate peaks can be observed (Fig.5a). We thus wondered whether these two components of the macroscopic current exhibit differences in their ion selectivity. To test this, we employed the bi-ionic reversal potential technique by perfusing different monovalent cations over recorded cells (Li+, Na+, and K+), while maintaining equimolar Na+ in the internal recording solution, and measuring changes in current reversal potential (voltage where currents reverse from inward to outward) when external permeating ions are altered39. This technique allows quantification of permeability ratios of desired cations relative to Na+ (pX+/pNa+, where X+ is the external cation). Recording TadNaC2 currents at different fixed voltages at pH 4.5, with 150mM Na+ on each side of the cell membrane, produced slowly activating currents that lacked a fast transient component (Fig.5b). As expected, these currents reversed from inward (negative) to outward (positive) near zero millivolts (i.e., 0.870.87mV; Fig.5c). Replacement of extracellular Na+ with an equal concentration of Li+, which has a smaller ionic radius than Na+, produced similar currents that reversed near 0mV and lacked a transient component (2.510.72mV), indicating that TadNaC2 is equally permeable to Na+ and Li+. Notably, all our previous recordings made using standard salines with external Na+ and internal K+ or Cs+ ions produced biphasic currents at pH values below 5.5, unlike currents observed under the bi-ionic conditions of Na+In/Na+Out and Na+In/Li+Out. Thus, it appears that the kinetics of the macroscopic current can differ depending on the types of permeating ions present across the cell membrane, an interesting observation that will require deeper analysis in future studies.

a Sample current recordings of the TadNaC2 channel at pH 4 and 3.5, revealing a biphasic current with a fast transient component (i.e., early current), and a slower, sustained (late current) component. The biphasic current becomes more evident at pH 3.5. b Sample proton-activated TadNaC2 currents recorded at different voltages (voltage protocol on top), under bi-ionic conditions of equimolar intracellular Na+ and extracellular Na+ (Na+ ext.) or K+ (K+ ext.). The star and square symbols denote regions of the currents that were measured to determine reversal potentials. c Plot of average reversal potential data ( standard deviation) for the bi-ionic reversal potential experiments, revealing a leftward shift for both the early and late current components of bimodal currents in the presence of external K+ (n=67) compared to external Na+ (n=7) and Li+ (n=4). d Box plot of average reversal potential data, showing statistically significant differences for both the early and late currents when extracellular Na+ was replaced with K+. The denoted p values are from Tukey post hoc tests after one-way ANOVA (p<1E30, F=2569). e Na+/K+ permeability ratios calculated using the bi-ionic reversal potential data, revealing that the late current exhibits higher Na+ selectivity compared to the early current (p value is for a two-sample t-test). f Sample sequential TadNaC2 currents elicited by pH 4.5 solutions bearing increasing Ca2+ concentrations. g Average Ca2+ doseresponse curve (n=12) revealing a similar decline in normalized peak inward current with increasing Ca2+ concentration compared to tachyphylaxis (in constant 2mM Ca2+). h Sample sequential paired currents elicited by pH 4.5 solutions bearing either 0mM or 10mM Ca2+ ions. i Plot of average percent block of peak inward current (e.g., 1 - P2/P1 from (h) 100%) after switching from 0mM Ca2+ to either 0mM or 10mM Ca2+(n=13 and 10, respectively). The denoted p value is for a two-sample T-test.

Instead, replacement of extracellular Na+ with equimolar K+ (i.e., Na+In/K+Out) produced canonical biphasic currents with a fast transient component and a late sustained component (Fig.5b). The occurrence of these two clearly delineated current components allowed us to measure reversal potentials for each, revealing that although both exhibit a negative shift in voltage compared to bi-ionic sodium, the late current exhibited a more marked hyperpolarizing shift compared to the early current (i.e., 60.422.30 vs. 49.011.62mV, respectively; Fig.5c). A box plot of the reversal potential data for the different bi-ionic measurements, coupled with ANOVA and post hoc tests (Fig.5d), substantiates the negative shift in reversal potentials for both the late and early currents in the presence of external K+, reflecting a general preference of TadNaC2 for Na+ over K+ ions. Furthermore, the more pronounced shift in reversal potential for the late vs. the early current indicates that ion selectivity changes over the course of the biphasic current, such that the early current is less selective for Na+ over K+ compared to the late current (pNa+/pK+ permeability ratios of 7.30.5 and 11.01.1, respectively; Fig.5e).

Next, we sought to determine whether external Ca2+ ions can block inward Na+ currents through TadNaC2. Perfusion of a pH 4.5 external solution containing 140mM Na+ and increasing concentrations of Ca2+ revealed a sequential decline in current amplitude (Fig.5f), which however was not statistically different from that attributed to tachyphylaxis (Fig.5g). Nonetheless, 10mM Ca2+ appeared to cause a downward inflection in the doseresponse curve (Fig.5g), suggesting that at this higher concentration, Ca2+ is able to mildly block TadNaC2. We therefore designed a paired pulse experiment aimed at distinguishing the decline in current caused by tachyphylaxis, from that caused by 10mM Ca2+ block. Specifically, we applied paired pulses of pH 4.5 solutions containing either 0mM of 10mM Ca2+ over recorded cells (Fig.5h), and quantified the decline in peak current amplitude of the second pulse relative to the first (Fig.5i). When both pulses lacked external Ca2+, the peak current amplitude declined by 13.24.6%, while addition of 10mM Ca2+ to the second pulse resulted in a decline of 27.65.2%). Thus, 10mM Ca2+ exerts a low-affinity block of the TadNaC2 current of roughly 14.4%.

Deg/ENaC channels like ASIC channels are homo- and/or hetero-trimeric in nature, with each separate subunit forming a ball in hand tertiary structure comprised of wrist, palm, thumb, finger, knuckle, and -ball regions (Fig.6a). Cumulative efforts have uncovered several core molecular determinants for proton activation of ASIC channels, namely a critical histidine residue in the wrist region (H73 in mASIC1), and a lysine in the palm region (K211) situated at the extracellular interface between subunits (Fig.6a)25,40,41,42,43. A protein alignment of several regions bearing these and other determinants for proton-activation of ASIC channels, including the group A ASIC channels from mice (i.e., ASIC1 to 4), selected group A and B channels from Branchiostoma belcheri25, and the singleton group B channel from Lingula anatina10 reveals near complete conservation of the H73 and K211 residues (Fig.6b). The only exception are the proton-insensitive ASIC2b splice variant which lacks H7344, and ASIC4 which is also proton-insensitive and lacks K21145. Indeed, except for the zebrafish ASIC1 homolog zASIC1.110, all functional group A and B ASIC channels that have been experimentally characterized in vitro bear a conserved H73 residue and most bear a K211 equivalent. The mouse ASIC5/BASIC channel, which falls in a separate clade from ASIC channels (Fig.2) and is not activated by protons, lacks both the H73 and K211 residues. These residues are also absent in other Deg/ENaC channels that are sensitive to external protons in vitro, including TadNaC6, the proton-inhibited channel from T. adhaerens24, and the proton-activated channels TadNaC2, human ENaC-6, C. elegans ACD-2, DEL-9, and ASIC-114, D. melanogaster Pickpocket116, and NeNaC2 from the sea anemone Nematostella vectensis8. However, TadNaC2, as well as the mouse ASIC4 and BASIC channels, possess a cationic residue just one amino acid upstream of the K211 position (i.e., R201 in TadNaC2). TadNaC2 and its Hoilungia hongkongensis orthologue HhoNaC2 also possess a conserved lysine one position downstream (K203 in TadNaC2). Furthermore, all of the ASIC channels except for the non-functional ASIC4 isotype bear a conserved aromatic residue 2 positions upstream of H73 (i.e., Y71). Y71 forms an aromatic bridge with a conserved tryptophan (W287) in mouse ASIC1a, shown to be important for coupling conformation changes in the extracellular domain with gating of the pore helices46. This aromatic residue is absent in all included non-ASIC channels except for TadNaC2 and HhoNaC2 which bear phenylalanine and tyrosine residues at this position, respectively (i.e., F70 in TadNaC2), as well as a tryptophan corresponding to W287 (i.e., W276 in TadNaC2). Also notable is that TadNaC2 possesses several protonatable amino acids that are in proximity of the ASIC H73 position, with aspartate and glutamate residues at positions 75 and 77, and a histidine at position 80 that aligns with residues in the palm region placing it proximal to the noted R201 and K203 residues.

a Ribbon diagrams of the chick ASIC1 homotrimeric channel crystal structure (left, PDB number 6VTK), and the AlphaFold-predicted tertiary structure of the mouse ASIC1a subunit (right). The three separate subunits of the homotrimeric channel are colored in red, white, and gray, and the colored circles denote the carbon atoms of critical residues corresponding to the back-colored residues of the mouse ASIC1a channel in the protein alignment shown in (b) (i.e., atoms in blue are within the acid pocket, pink are within the wrist, green are within the palm, and purple are within the finger). The dashed boxes denote structural regions of the single mASIC1a subunit structure bearing these same critical residues. b Protein sequence alignment of the acid pocket (enclosed by blue dashed boxes), wrist (pink), palm (green), and finger (purple) regions of select proton-activated Deg/ENaC channels from cnidarians and bilaterians with TadNaC2, TadNaC6, HhoNaC2,and HhoNaC4/6/7 channels from the placozoans Trichoplax adhaerens and Hoilungia hongkongensis. Residues that are back-colored in black represent conserved residues for proton activation of ASIC channels, while those back-colored red denote key residues that render the ASIC2b splice variant insensitive to external protons. Residues that are back-colored in gray denote protonatable amino acids in TadNaC2 within these key structural regions, some of which are conserved in cnidarian and bilaterian homologs, while those back-colored in brown denote cationic residues in TadNaC2 that flank the critical K211 residue of ASIC channels, also found in several other channels. Notable is the complete conservation of the critical residues H73 and K211 in all included ASIC channels, and their absence in most non-ASIC proton-activated channels including TadNaC2. c Homology model of the homotrimeric TadNaC2 channel structure (left), and AlphaFold-predicted structure of the single subunit, with a similar annotation as described for (a). d Left panels: Close-up view of the acid pocket region of mASIC1a (top) and TadNaC2 (bottom) within corresponding AlphaFold-predicted structures. The six rendered residues in the TadNaC2 channel correspond to residues that align with the six acid pocket residues in mASIC1a as depicted in (a). Right panels: Surface rendering of the acid pocket region of mASIC1a (top) and TadNaC2 (bottom) reveals a stark difference in the electrostatic potential between the two channel subunits. e Close-up view of the wrist and palm regions of mASIC1a and TadNaC2. Apparent in the wrist region is the absence of a critical H73 proton-sensing residue in TadNaC2, but conservation of the aromatic amino acids F70 and W276, which in mASIC1a (i.e., Y71 and W278) form an aromatic bridge critical for channel gating. Instead, TadNaC2 bears a putative proton-sensing amino acid (H80) at the opposite end of a strand that projects from the first transmembrane helix in wrist region (TMH1) to the palm domain, placing it near the residuesR201 and K203 that flank the critical K211 residue of mASIC1a. f Close-up view of the finger and acid pocket regions, with rendered amino acids corresponding to the positions in the ASIC2b splice variant that make the channel insensitive to protons. Also labeled are the equivalent acid pocket residues, and the predicted 1 to 3 helices in the finger region.

Another region associated with proton activation (and desensitization) of ASIC channels is the acid pocket, comprised of a cluster of four acidic amino acids located between the finger and thumb regions of the subunit monomer, and another pair in the palm region close to K211 (Fig.6a, b). In the trimeric channel, the four acidic residues from one subunit and two from an adjacent subunit combine to form a namesake pocket-like structure where protons are thought to bind and affect channel conformation and gating26. Of note, mutation of these glutamate/aspartate residues does not completely disrupt proton activation, and instead, these appear to be more important for channel desensitization26. Accordingly, the group B ASIC channels from B. belcheri and L. anatina lack most glutamate/aspartate residues in the acid pocket (Fig.6b), while they are largely conserved among the group A channels. Furthermore, the two TadNaC channels, as well as the various other non-ASIC Deg/ENaC channels included in the alignment lack most if not all acidic residues at equivalent positions of the acid pocket.

A third region of interest with respect to proton activation is the finger region, where a motif of four amino acids distinguishes the proton-sensitive ASIC2a mRNA splice variant from the insensitive ASIC2b variant (Fig.6b). Specifically, ASIC2a bears a motif of TTN-XXX-H and is proton-activatable, while ASIC2b bears an SKG-XXX-Y motif and is not47. Moreover, introducing the SKG and Y elements of the ASIC2b motif into ASIC2a, together but not separately, abrogates proton activation, and insertion of the finger region of ASIC2a into ASIC1 causes a marked reduction in proton sensitivity making the latter less sensitive to protons similar to theASIC2a channel48. Notably, a histidine residue within the finger region of ASIC2a (H109) is conserved among many of the included Deg/ENaC channels including TadNaC2 (Fig.6b). However, its functional significance remains unclear, with one mutation study reporting no effect on ASIC2a activation42, and a subsequent study reporting a contribution but not a requirement47 In this region, TadNaC2 also bears two protonatable glutamate residues (E104 and E105).

To better infer how the structure of TadNaC2 compares to the well-studied structures of ASIC channels, we generated a homology model of the homotrimeric channel using the crystal structure of chick ASIC1 as a template (Fig.6c; left panel)49. We also predicted the tertiary structures of the monomeric mouse ASIC1 and TadNaC2 channel subunits with AlphaFold (Fig.6a, c; right panels)50. Labeling the carbon atoms of the Y71, W287, H79, K211, acid pocket, and ASIC2 finger motif equivalents in the homotrimeric structure of the chick ASIC1 channel (Fig.6a), and the F70, W276, H80, R201, K203, D413 (single acid pocket residue), and finger motif equivalents in the model of the TadNaC2 homotrimer (Fig.6c), illustrates the general absence of acid pocket residues in TadNaC2. Also evident are the noted differences in the wrist region, where TadNaC2 lacks an H73 equivalent, and in the palm, where the residues H80, R201, and K203 in TadNaC2 are arranged in a triangular cluster at the interface between subunits, in asimilar position as the K211 residue in ASIC channels. Furthermore, the aromatic residues F70 and W276 residues in TadNaC2 are in proximity to each other, suggesting that like Y71 and W287 in ASIC1, these can form hydrophobic interactions.

The predicted structures of the mASIC1 and TadNaC2 monomers also highlight key differences and similarities. First, whereas the cluster of four acid pocket residues in mASIC1 (D237, E238, D345, and D349) are arranged in a tight cluster, the equivalent residues in TadNaC2 are not (S228, P229, L332, and S336) (Fig.6d, left panels). Rendering the electrostatic potential on the surface of the two channel subunits also illustrates a stark difference in the acid pocket region, with the acidic residues of mASIC1a contributing to a highly electronegative surface, while those in TadNaC2 contribute to a surface that is slightly positive and hence unlikely to attract and bind H+ ions (Fig.6d, right panels). In the wrist region, the W276 sidechain at the base of the thumb of TadNaC2 is in a similar position as theW287 sidechainin mASIC1, situatedbetween the Y71 and H73 equivalent residues F70 and threonine 72 (Fig.6e). Furthermore, both the SKN-XXX-H and SEE-XXX-H finger motifs of mASIC1 and TadNaC2 are within a short loop and adjacent descending alpha helix, consistent with the 1 helix identified in the crystal structure of the chick ASIC1a finger region7. However, this helix is predicted to be two helical rotations longer in TadNaC2, with a short loop connecting it to the downstream 2 helix that is also longer than its predicted counterpart in mASIC1 by one rotation (Fig.6f). Lastly, it is notable that the finger regions of the two channels are positioned above the divergent acid pocket, suggesting that any structural alterations taking place in the finger region would be differentially transferred to the thumb and pore regions that mediate channelgating.

Despite lacking key deterministic residues for proton activation, the similar predicted structure of TadNaC2 compared to mASIC1a prompted us to examine whether corresponding structural regions in the placozoan channel bear unique or conserved elements involved in channel gating. Thus, we performed site-directed mutagenesis on selected aromatic or protonatable residues in the wrist region (F70, D75, and E77), protonatable residues in the finger region (E104, E105, and H109), and protonatable or cationic residues in the palm region (H80, R201, and K203) (Fig.6a). To assess changes in H+ sensitivity and gating properties at different pH, we tested each mutant with a series of perfused solutions of various pH to generate doseresponse curves of recorded macroscopic currents (Fig.7ac; plots of individual variants with replicates provided in Supplementary Fig.1).

ac Average pH doseresponse curves standard deviation for wild-type (wt) TadNaC2 and variants bearing amino acid substitutions within the wrist (a), finger (b), and palm (c) regions. d, e Plot of average standard deviation pH50 (d) and normalized peak current amplitude (e) for wild-type (wt) and various point mutated TadNaC2 channels. Red-colored asterisks denote p value thresholds for two-sample t-tests comparing wild-type to mutant values. f Sample whole-cell currents of wild-type TadNaC2 and select mutant variants.

In the wrist region of rat ASIC1a, mutation of the Y71 aromatic residue to a histidine imposes a ~70% reduction in elicited current amplitude, while mutation to alanine completely disrupts activation by protons46. In contrast, analogous mutations of the F70 residue in TadNaC2 had negligible effects on the pH50 (Fig.7d, e), and no effect on average peak inward current amplitude at pH 4.0 compared to the wild-type channel (Fig.7e). Thus, this residue in TadNaC2 most likely does not form a functionally analogous aromatic interaction with the conserved W276 residue in the thumb region, akin to the Y71-W287 interaction in ASIC1 channels. As noted, TadNaC2 bears two protonatable residues in the wrist region (D75 and E77), within a predicted strand that in ASIC channels projects from the H73 residue in the wrist towards the K211 residue in the palm (Fig.6a, b). The E77 residue in TadNaC2 aligns with D78 in ASIC1a and ASIC2a, which when mutated to asparagine in the rat channels disrupts proton activation41,43. In contrast, alanine substitution of E77 in TadNaC2 had no noticeable effect, while mutation of the D75 residue two positions upstream caused a marked increase in the pH50 (Fig.7d, e). Furthermore, neither the D75A nor the E77A mutation caused a change in maximal peak current amplitude (Fig.7f), or in the overall shape of macroscopic currents (Fig.7f). Overall, TadNaC2 appears different from ASIC channels in lacking homologous molecular determinants in the wrist region that areinvolved in proton gating.

In the finger region, single mutations of E104A and E105A had no effect on pH50 or peak current amplitude (Fig.7b, d, e). However, mutation of both together caused a moderate decrease in both metrics, and altered the macroscopic current waveform by diminishing the fast/early component (Fig.7f). A more dramatic effect occurred for the single mutation H109A, which in addition to reducing maximal peak current amplitude (Fig.7e), produced a biphasic macroscopic current and a complete loss of the early current component at pH 4.5 and 4.0 (Fig.7b, f; Supplementary Fig.1h). Interestingly, mutation of the acid pocket residue D345 in mouse ASIC1a (Fig.6b), which is close to the predicted finger region of TadNaC2 where H109 resides (Fig.6f), also imposes biphasic sensitivity to pH, attributed to the loss of one of two separate proton binding sites involved in channel activation (the other being in the palm domain)51. However, the biphasic effect is much more severe for the TadNaC2 H109A mutation, where instead of plateauing between pH 5.0 and 4.0 like mAIC1a, the current amplitude first decreases from pH 5.0 to 4.5, then increases again from pH 4.5 to 4.0 (Fig.7b, f, Supplementary Fig.1h). This atypical feature precluded accurate fitting of the doseresponse data with either standard or biphasic doseresponse curves, since both serve to model strictly incremental processes (i.e., R2 values of 0.64 and 0.68, respectively). Nonetheless, imposing a standard doseresponse curve over the data revealed reduced sensitivity to protons compared to the wild-type channel, with an average pH50 of 4.70.2 vs. 5.20.1 (Fig.7d). Instead, fitting the data with a bimodal doseresponse curve produced a pH50-1 value of 5.50.3 and a pH50-2 value of 4.50.3, both of which are statistically different from wild type (i.e., P values for two-sample t-Tests 0.005 and 0.0005, respectively). However, since the H109A variant shows diminished sensitivity to protons at the threshold pH of 5.5 (Supplementary Fig.1h), this mutant channel is not likely more sensitive to protons at threshold pH values, but rather, has an overestimated the pH50-1 value caused by the poor curve fit. Of note, while macroscopic current amplitudes varied considerably for the wild-type channel, the pH50 values were less variable (Fig.7d, e). Furthermore, we found no correlation between current amplitude and pH50 for the wild-type channel, altogetherindicating that observed differences in pH sensitivity for the H109A mutant and other channels variants was not due to altered current amplitudes. It is alsonotable that the transient current observed at pH 5.0 desensitized more rapidly compared to the wild-type channel, while at more acidic conditions the transient current was absent leaving only a slowly activating sustained current that increased in amplitude from pH 4.5 to 4.0 (Fig.7f). The most severe of all mutations tested was a triple mutation E104A/E105A/H109A, which produced a channel with very weak activation at pH 5.5 and 4.5, completely lacking transient/early currents at all tested pH (Fig.7f). This resulted in the most significant change in proton gating with an average pH50 of 4.40.1 (Fig.7d, e). Altogether, it appears as though the H109 residue, together with E104 and E105, plays an important role in the proton gating of TadNaC2.

In ASIC1a, deletion of the K211 palm residue results in a strong decrease in proton sensitivity, while mutation to glutamate causes a more moderate effect25. In TadNaC2, mutation of the two cationic residues that flank the K211 position, R201 and K203, produced an increase in proton sensitivity with respective pH50 values of 5.30.1 and 5.50.0 (Fig.7c, d). Notably, the R201A mutation also altered the macroscopic current waveform such that the amplitude difference between the early and late components was greater at pH 5.0 and 4.5 compared to wild-type, but not at pH 4.0 (Supplementary Fig.2). Instead, the K203E mutation disrupted the early current such that it only became evident at very acidic pH values (Fig.7f). Deletion of this same residue (K203), to emulate K211 variants of ASIC channels, resulted in an inability to detect currents even with very acidic pH. Alanine substitution of the unique protonatable H80 residue in the palm region, which is proximal to R201 and K203 in our predicted structures (Fig.6), caused the doseresponse data to become more variable, and the pH sensitivity to become biphasic similar to the H109A mutation (Fig.7c, f; Supplementary Fig.1j). Furthermore, and like the K203E mutation, the H80A mutation strongly disrupted the transient current, which was only evident under very acidic pH conditions (Fig.7f). Fitting a standard doseresponse curve over the data revealed a decrease in pH sensitivity compared to wild-type (i.e., pH50=4.70.3; R2 for global fit = 0.91). Instead, a biphasic curve fit produced a pH50-1 value that was not statistically different from wild-type (pH50-1=5.10.7), but a pH50-2 value that was considerably lower (4.30.3; P value for two-sample t-Test 0.0005; R2 for global fit = 0.96). Of note, mutation of a glutamate residue in mouse ASIC1a, just two amino acids upstream of H80 in our protein alignment and in a similar region of the palm domain (Fig.6b, e), also imposed a biphasic sensitivity to pH51. Altogether, these observations indicate that the H80 residue also plays a role in the proton gating of TadNaC2. Furthermore, the R201 and K203 residues also contribute to TadNaC2 gating, however, their mutation did not produce a rightward shift in the pH doseresponse curve as it did for the analogous K211 residue in ASIC channels25, indicating key functional differences. Finally, all tested mutations in the palm region caused a significant decrease in maximal current amplitude (Fig.7e), most extreme for the K203 variant which was either completely non-functional, not trafficking to the cell membrane, or both.

Next, we wanted to determine whether the noted decrease in current amplitude caused by select mutations was due to reduced functionality or a reduction in channel protein expression. Hence, we N-terminally tagged the wild-type channel with enhanced green fluorescent protein (EGFP), as well as the mutants H80A, H109A, and K203 that, respectively, caused moderate, strong, and severe effects on current amplitude. This permitted inference of the total channel protein levels in transfected CHO-K1 cells via EGFP fluorescence quantification, relative to a co-transfected blue fluorescent protein from the empty vector pIRES2-EBFP. Of note, we tested whether tagging the wild-type TadNaC2 channel with EGFP disrupted its function, finding it to conduct proton-activated currents that were visually indistinguishable from the untagged channel (Supplementary Fig.3). Fluorescence micrographs of transfected cells reveal a noticeable decrease in EGFP fluorescence of all three mutant channels relative to wild type (Fig.8a), with respective normalized average integrated density values of 675%, 434%, and 577% for the H80A, H109A, and K203 mutants (Fig.8b). Average integrated density measurements for the co-transfected EBFP were statistically indistinguishable for all transfections, indicating that the differences in EGFP fluorescence were not due to differences in transfection efficiency. Thus, all three of the tested mutations cause a decrease in total protein expression in vitro.

a Representative fluorescence micrographs of CHO-K1 cells co-transfected with pEGFP-TadNaC2 fusion vector (left panels) and an empty pIRES2-EBFP vector (right panels). b Plot of percent average integrated density standard deviation, quantifying the emitted fluorescence of pEGFP-TadNaC2 wild type (wt) and mutant channels, normalized to the average integrated density of wild-type TadNaC2 (n=3 for each transfection condition). EBFP fluorescence was also quantified to determine transfection efficiency. Cyan-colored asterisks denote p value thresholds for Tukey post hoc means comparisons of fluorescence signals between wild-type and mutant channels after one-way ANOVAs (EGFP: p=5.6E11, F=73.6; EBFP: not significant). c Top panel: Western blot of select EGFP-tagged TadNaC2 channel variants in CHO-K1 cell lysates using anti-GFP polyclonal antibodies, comparing total channel protein content (T) with membrane/surface expressed channel protein content (S) for each variant. Bottom panel: Western blot of the lower half of the membrane used in the top panel, using anti-GAPDH (top bands) and anti-EBFP (bottom bands), polyclonal antibodies. d Quantified band intensity (mean gray area) of TadNaC2 bands in (c), relative to the wild type EGFP-TadNaC2 total protein band, revealing decreased total and surface protein expression of TadNaC2 channels bearing mutations, and a near complete absence of membrane expressed variants harboring a K203 deletion, consistent with our inability to record current for this channel in vitro. Bands for each channel variant were also normalized to the intensity of EBFP present in corresponding total protein lanes.

Using a cell surface biotinylation strategy, we also wanted to characterize the effect of mutations on total protein and membrane expressed protein levels in transfected cells. A Western blot probed with anti-EGFP antibodies revealed a marked reduction in both total protein and membrane expressed (surface) protein levels of mutant TadNaC channels relative to wild-type (Fig.8c). Measurements of the mean gray value of the different bands on the blot reveals similar reductions in total protein levels for all three mutants, and notably, extreme reduction of membrane expressed K203 (Fig.8d). Altogether, this data is consistent with our current amplitude measurements and inability to record currents for the K203 variant of TadNaC2.

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Function and phylogeny support the independent evolution of an ... - Nature.com

Scientists have confirmed that human brains are naturally wired to perform advanced calculations, much like a high-powered computer, to make sense of the world through a process known as Bayesian inference.

In a study published in the journal Nature Communications, researchers from the University of Sydney, University of Queensland and University of Cambridge developed a specific mathematical model that closely matches how human brains work when it comes to reading vision. The model contained everything needed to carry out Bayesian inference.

Bayesian inference is a statistical method that combines prior knowledge with new evidence to make intelligent guesswork. For example, if you know what a dog looks like and you see a furry animal with four legs, you might use your prior knowledge to guess it's a dog.

This inherent capability enables people to interpret the environment with extraordinary precision and speed, unlike machines that can be bested by simple CAPTCHA security measures when prompted to identify fire hydrants in a panel of images.

The study's senior investigator Dr Reuben Rideaux, from the University of Sydney's School of Psychology, said: "Despite the conceptual appeal and explanatory power of the Bayesian approach, how the brain calculates probabilities is largely mysterious."

"Our new study sheds light on this mystery. We discovered that the basic structure and connections within our brain's visual system are set up in a way that allows it to perform Bayesian inference on the sensory data it receives.

"What makes this finding significant is the confirmation that our brains have an inherent design that allows this advanced form of processing, enabling us to interpret our surroundings more effectively."

The study's findings not only confirm existing theories about the brain's use of Bayesian-like inference but open doors to new research and innovation, where the brain's natural ability for Bayesian inference can be harnessed for practical applications that benefit society.

"Our research, while primarily focussed on visual perception, holds broader implications across the spectrum of neuroscience and psychology," Dr Rideaux said.

"By understanding the fundamental mechanisms that the brain uses to process and interpret sensory data, we can pave the way for advancements in fields ranging from artificial intelligence, where mimicking such brain functions can revolutionise machine learning, to clinical neurology, potentially offering new strategies for therapeutic interventions in the future."

The research team, led by Dr William Harrison, made the discovery by recording brain activity from volunteers while they passively viewed displays, engineered to elicit specific neural signals related to visual processing. They then devised mathematical models to compare a spectrum of competing hypotheses about how the human brain perceives vision.

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Evolution wired human brains to act like supercomputers - Science Daily

Commercial UAV Expo features a wide variety of different use cases for which drones can be used in commercial settings, ranging from deliveries to public safety to surveying, and much more. Among the industries which held a significant presence at this years show was the energy and utilities industry, who use UAVs to perform safe and efficient inspections of their assets. There were many companies who offer hardware and software for these verticals on the exhibit floor, and on the second day of the conference there was an entire session dedicated to these workflows and how the space has changed in recent years.

That session was broken into two halves, the first of which featured four presentations from professionals working for some of the biggest infrastructure companies across the United States. Each presenter highlighted a case study showing how drones are being used in their space and some of the barriers that they are still crossing in what is still the early days of the usage.

This portion of the session began with Rohit Gohil of Ameren, who spoke about the companys infrared inspections being done with UAVs. He discussed why drones are particularly useful for this non-contact data acquisition, allowing for sensors to get closer than more traditional methods, and thus collect more accurate information about the assets. After him, Teena Deering from TeeDeeUAS, LLC and San Diego Gas & Electric spoke about her experience with the public trying to shoot down drones inspecting assets in their neighborhoods. She spoke to how professionals should respond to these scenarios and gave tips on working with local and federal law enforcement after such an event occurred.

We also heard from Jake Lahmann from Valmont Industries, one of the largest utility asset owners in the United States. He spoke to how the companys drone operations have grown over the years, and what steps theyve taken to ensure effective scaling of that work. Finally, Stanley McHann of SparkMeter spoke similarly to building up drone divisions for inspections and talked about the importance of finding the right equipment and ensuring you are getting the most ROI from your workflows.

Following a short break, the session resumed with a four-person panel moderated by Cynthia Huang, CEO of ACSL, Inc. She was joined on stage by the following industry experts for a discussion about what challenges they are still facing in utility inspection space and what they see for the future of drones in these workflows:

The panel discussion began with a nod from Huang about how much this industry has grown even over the last half-decade I remember back in 2018 when I first got involved with this wonderful industry, a lot of the programs that I was working with at the time were starting out about 10, 15, 20 drones. And nowadays, it's very common to talk to large utilities like the ones you're about to hear from who have collectively hundreds or even 1000s of drones but also an acknowledgement that there is still room to grow. She asked the panel what hurdles they needed to overcome in order for the space to take that next step.

Broadly speaking, there was agreement that one of the major hurdles simply revolves around providing more education to other parts of the organization. Drone programs are mostly known about organizations, but the specifics were a bit of a mystery. The panel called for better education throughout companies about why these programs exist and what they add to the bottomline for organizations of all sizes.

Beyond that, there was also discussion about honing in on what the drones do best, and acknowledging that they arent a solution for every problem. There are, in other words, still use cases for which other sensing technology could work better. Robie summed this up nicely, saying, I think where were going next is a bit more specialized, really dialing in on those use cases and seeing where we can make the biggest impact.

The conversation also touched on what the next steps are for the growing space. There were a few threads of this conversation, one of which revolved around the software used to process data collected via UAVs. On this front, the running themes of the conversation largely came in three buckets: Flexibility, proof of worth, and standardization. On the first point, Scott said, We need flexibility. A lot of time, manufacturers go out and they try to build the workflow for us, and a lot of times that creates challenges for us because we all appear to probably have different workflows across the board.

Piece also talked about the growing presence of artificial intelligence and automation, acknowledging the power of these tools but showing skepticism of whether or not full automation is realistic. That said, he and Robie agreed that these companies need to be able to prove that their solutions work and are viable for their businesses rather than just preaching the broad concepts and buzzwords.

Finally, Spurlock touched on the need within the industry to create a standardized file type. In these workflows, massive amounts of data are collected and there are different solutions out there for downsizing the data in a way that makes it ingestible. He said, We only want to ingest the stuff thats useful to us, because otherwise were going to be in paralysis. How do we manage the amount of data that were capturing now, much less when we have these automated flights taken off of the system?

This was only a small portion of the presentations and discussions that took place on the stage at Commercial UAV Expo, but broadly speaking the big theme was how much pride all of these professionals take in their work, which was something Robie spoke to in closing the session.

He said, Everybody up here that works for utility companies are certainly very proud of what they do and what they bring to their community. Be cognizant of that when youre a third-party vendor and approaching the utility companies in the seriousness with which we take our jobs. Its not just the earned paycheck, and its not just to get a contract for the third-party vendors. It's something that we all take pride in.

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The evolution of UAV implementation in utility inspection workflows - Commercial UAV News

NEW YORK, Sept. 18, 2023 /PRNewswire/ --TheSemiconductor Foundry Marketis projected to have a remarkable growth, with a Compound Annual Growth Rate (CAGR) of 7.74% between 2022 and 2027. This expansion is expected to result in a substantial increase in market size, amounting to an impressive USD 42.78 billion. A comprehensive analysis of the market reveals that the increasing demand for IoT, growing demand for 14-nm/16-nm FinFET technology, and the evolution of 3D printing are the primary driving forces behind this robust growth. Download a free sample report here

The Semiconductor Foundry Market report, covering the period from 2023 to 2027, encompasses an extensive examination of market segmentation, including type (pure-play foundry and IDMs), application (communications, PCs/desktop, consumers, automotive, and others), and geography (North America, APAC, Europe, South America, and Middle East and Africa).Additionally, the report provides a detailed analysis of key drivers, trends, challenges, and historical market data spanning from 2017 to 2021.

The growth of 3D printing technology is driving various industries, including medical, aerospace, and automotive.It has the potential to revolutionize the foundry market, with companies like Taiwan Semiconductor Manufacturing Co. Ltd. and Intel exploring its use in semiconductor production. While it may not replace traditional manufacturing entirely, 3D printing is increasingly being used to extend the life of ageing tools in 200-mm manufacturing, offering a cost-effective solution. This technology is particularly well-suited for foundry needing high-value, low-volume production. Overall, 3D printing is expected to play a significant role in these sectors during the forecast period. For more details, get the free sample report now

An emerging trend in semiconductor foundry is the increasing use of advanced analytics, particularly for harnessing big data.These foundry incorporate sensors and data-collecting devices in their tools, enabling the extraction of valuable data for improved decision-making. This data-driven approach helps foundry identify new business models and strategies, adding extra value to the company. As a result, these developments are expected to drive the global market in the foreseeable future.

One of the challenges affecting semiconductor foundry market growth is the unpredictable fluctuations in demand, driven by products like mobile devices, computers, and consumer electronics.This industry's cyclic nature, with periods of rapid growth followed by retrenchment, leads to high volatility. Factors like excess production capacity and intense competition contribute to sharp price drops for semiconductor chips. These fluctuations can result in inventory issues during low and high-demand periods, ultimately hindering semiconductor foundry market growth.

North America is expected to contribute 40% to global semiconductor foundry market growth due to its high sales and concentration of leading semiconductor companies.Government initiatives in the US and strong demand for advanced semiconductor foundry drive this growth. Despite setbacks from COVID-19, the region is rebounding in 2021, with potential for further expansion through new product launches and industry strategies.

Companies in the semiconductor foundry market are deploying various strategies, including strategic alliances, partnerships, mergers and acquisitions, geographical expansion, and product/service launches, to strengthen their market presence. Notable market players include:

For complete insights of the Semiconductor Foundry Market, buy the full report now

Related reports

Semiconductor Market: The semiconductor market sizeis estimated to grow at aCAGR of 5.09%between 2022 and 2027. Themarket size is forecast to increase byUSD168.3 billion.

Front End of the Line Semiconductor Equipment Market: The front end of the line semiconductor equipment market has the potential to grow by $ 5.89 bn during 2021-2025, and the market's growth momentum will accelerate at a CAGR of 4%.

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Technavio is a leading global technology research and advisory company. Their research and analysis focus on emerging market trends and provide actionable insights to help businesses identify market opportunities and develop effective strategies to optimize their market positions.

With over 500 specialized analysts, Technavio's report library consists of more than 17,000 reports and counting, covering 800 technologies, spanning across 50 countries. Their client base consists of enterprises of all sizes, including more than 100 Fortune 500 companies. This growing client base relies on Technavio's comprehensive coverage, extensive research, and actionable market insights to identify opportunities in existing and potential markets and assess their competitive positions within changing market scenarios.

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Semiconductor Foundry Market - 2022 to 2027 | The evolution of 3D ... - Longview News-Journal

The two-day event witnessed an overwhelming response from the attendees, reaffirming the strength of the newspaper publishing business in India. The event featured a diverse range of sessions and exhibitions that celebrated innovation, sustainability, and excellence in print.

During the conference opening remarks, Jacob Mathew, Former President, WAN-IFRA & Managing Editor, Malayala Manorama, India said, We have been able to keep our print business alive thanks to the resilient business strategies of news publishers. This conference gives us the opportunity to learn from some of the best business practices as they share their experiences.

He addressed the various challenges that the news industry has been facing today and encouraged the media industry to enhance its use of the latest technologies, such as artificial intelligence.

The keynote address by Mylene Sylvestre, Publishing Director, Guardian News & Media Ltd, who joined the audience live from London, reflected on how print still plays a crucial role for The Guardian, and offered valuable perspectives on the coexistence of print and digital media in todays media landscape.

The following panel discussion on the Print business trends and current demands of the industry in India was moderated by Vinodini Sukumar, Managing Director, Team One Advertising Pvt Ltd.

The panellists included:

The panel discussed various initiatives and strategies to attract younger audiences and explained how these changes led to increased profitability for businesses. The panellists acknowledged the growing trend of younger audiences avoiding the news. They collectively agreed that the print medium can remain relevant in India through publisher resilience and active engagement with advertisers and audiences.

During the day, eminent speakers from various prestigious news publications discussed the role of renewable energy, sustainability and Industry 4.0 in print operations.

The Indian Printers Summit featured two parallel tracks of conference: the Printing Summit and the Advertising Summit, providing attendees with comprehensive insights into the industrys dynamics.

The Best in Print Asia 2023 Awards were presented at the end of the first day of the event. The awards recognise the newspapers across Asia who excel in printing the newspaper as per ISO 12647-3 and WAN-IFRA quality standards.

The awards were presented in two categories newspapers with a circulation of less than 100,000 copies and newspapers with circulation of more than 100,000 copies. The winners emerged from the Times of India, United Printing & Publishing, Abu Dhabi, Anandabazar Patrika, Malayala Manorama, and The Hindu.

Earlier in the day, WIZONE, WAN-IFRAs new online marketplace was launched.

The Day 2 of the summit started with a keynote by Laya Menon, Executive Vice President & Business Head South, Lodestar UM (IPG Mediabrands) and she presented insights into understanding media solutions from the advertisers eyes.

Emmy DSilva, Engineering & Newsprint Consultant, received the audiences standing ovation for his insightful newsprint session. The session addressed the reasons behind newsprint cost fluctuations in India and elsewhere.

The summit concluded with a newspaper distribution and circulation session featuring M Rajagopalan Nair, Vice President Circulation, and Cinu Mathews, Chief General Manager Circulation, both from Malayala Manorama. They shared the remarkable circulation success story of the Kerala market and how they ensured newspaper distribution even during the challenging COVID-19 pandemic.

The concluding panel included insights from:

The panellists expressed their views on the potential benefits of publishers collaborating and sharing distribution infrastructure to enhance operational efficiencies.

During the end of the summit, Magdoom Mohamed, Managing Director, WAN-IFRA South Asia, said, This year, we saw higher engagement levels, one of the finest. I appreciate the curiosity to learn when we come back after a year.

Throughout the two-day conference, various industry leaders and insiders shared their expertise and experiences on print readership, production, circulation and distribution, and print augmenting the growth of other mediums.

Contact and Inquiries

For further inquiries, pleasecontact: Magdoom Mohamed, Managing Director, WAN-IFRA South Asia, Chennai, India (magdoom.mohamed@wan-ifra.org),Thai Anban, Business Development Manager, WAN-IFRA South Asia, Chennai, India (thai.anban@wan-ifra.org).

WAN-IFRA is the global organisation of the worlds press, comprising 3,000 news publishers and technology companies and 60 national publishers associations representing 18,000 publications in 120 countries. With a mission to protect the rights of journalists and publishers around the world to operate independent media, WAN-IFRA provides its members with expertise and services to innovate and prosper in a digital world and perform their crucial role in society. wan-ifra.org

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Embrace evolution: Print augments the growth of new media - WAN-IFRA

The multibillion-dollar question right now is whether the Search Generative Experience (SGE) will blow up Googles Search business model. Based on early testing, Google CEO Sundar Pichai is confident that wont happen.

Its important to us to connect users with whats out on the web, and we are working deeply to make sure that continues to work well, Pichai told Steven Levy in a Q&A published on Wired.

Why we care. Google may be confident that the SGE experiment will continue to send people to websites, but were still finding it hard to trust Google at this point. They wont reveal any real data to us about whether AI answers are driving clicks to websites, click-through rate data, or advertising performance data on what we are calling CHERPs, or Chat Engine Results Pages. And we have no way to track this in Google Search Console, Google Ads or Google Analytics.

Ads vs. organic. Levy pointed out to Pichai that the AI-generated answers are different from a list of links, which could further add confusion about whether an SGE answer is sponsored or organic.

Helping people find information. Pichai pointed out that even though the way Google presents information has evolved greatly in 25 years but ultimately people are searching for information.

Like desktop to mobile? Pichai pointed out that people asked similar questions as Google shifted its focus from desktop to making everything mobile-first.

The search community was also extremely worried about Googles featured snippets stealing traffic. When Bard launched in February, there were no links. And SGE only finally added links to websites Aug. 30 after months of pressure by search marketers and publishers.

Bing burn. Did the new Bing, powered by ChatGPT, make Google dance. Pichai essentially dissed their efforts, comparing it to Alexa and Siri. Pichai also claimed:

Could that time in late 2022 coincide with OpenAI launching ChatGPT in late November? Generative AI exploded on the scene and supposedly triggered a code red within Google, leading to the return of Google founders Larry Page and Sergey Brin.

Pichai was also asked about the antitrust trial, but had little to say beyond throwing around the word innovation.

You can read the full interview here: Sundar Pichai on Googles AI, Microsofts AI, OpenAI, and Did We Mention AI?

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Google CEO on SGE and Search evolution: 'We'll get it right' - Search Engine Land

From humble beginnings to a performance car icon, the Honda Civic has helped to shape the very foundations of the auto industry, with an influence that now spans over half a century.

Originally, the Honda Civic was launched as an exciting alternative to lazy, miserable, and generally uninspiring American-made offerings in the 1970s. From there on out, popularity exploded as Honda continued to bring value to their dedicated customers.

While the model never set out to break records, it did eventually end up doing just that, with the legendary Honda Civic Type-R. Join us as we venture back over the last eleven generations of Civic, discovering just how this hatchback ended up as the global icon we know and love today.

Information has been sourced from Honda, Ultimate Specs, and Edmunds, to bring you the history of the Honda Civic, from 1972 to today.

The first Honda Civic featured a transversely mounted 52 horsepower 1.2-liter engine, which offered not only considerably better economy than American peers in the day, but also a lively, enjoyable drive; optimistic economy cars were few and far between in the 1970s, and as such, the earliest Honda Civics were almost without any real competition.

By the mid-1970s, engineers at Honda had developed new systems within the Civic's engine that meant catalytic converters weren't needed, unlike anything else on offer at the time. This economy car had struck a chord with many Americans, announced as the EPA's most efficient car of 1977, just as Honda cracked the 100,000th sale in America.

As the 1970s progressed, subtle styling tweaks were introduced, such as larger safety bumpers front and rear, blacked-out front grills, and relocated indicators. In the top trim level, the now-1.5-liter Civic produced a hearty 63 horsepower.

Related: Honda Is Set To Unleash A Powerful New Type-R GT Race Car

For the second generation, the diminutive Civic grew in length by 2 inches, which saw the wheelbase swell up to 88.6 inches. Two powertrains were on offer, with both 1.3 and 1.5-liter engines available, most with 4-speed gearboxes, although the range-topping DX trims afforded a 5-speed. A 2-speed automatic gearbox was also available.

While it was clearly no Rolls-Royce, Honda did begin to introduce luxurious upgrades for the GL trim, such as a tachometer and clock, radial tires, plus a rear window wash and wiper.

Soon after, it was time to celebrate, as for the final year of this second generation (1983), Honda dropped the aforementioned GL model and introduced the S. This sporting hatch was equipped with firmer suspension, a rear stabilizer bar, and 13" 165/70 Michelin tires.

As 1984 dawned, so did the third-generation Honda Civic, which was now 5 inches longer than the previous model, with sedan and wagon models now sharing the same basic platform as the Accord and Prelude. The 1.5-liter unit was now standard across the range, apart from the base model 3-door hatch, which still made do with the old 1.3-liter engine.

Such was the success of Civic in America that production was moved to a facility in Ohio in 1986. Here, the first Civic Si was developed, alongside an all-wheel-drive Civic wagon.

Just 4 short years later, Honda dropped the fourth-generation Civic. Larger and more powerful still, this latest fourth generation did away with many of the sharp creases and jagged lines that so defined the previous generations.

Creature comforts such as power locks, windows, and mirrors were also introduced in the higher trim levels, in addition to intermittent wipers. Competition was fierce against the likes of Europe's Volkswagen Fox, but the Civic had enough of a fan base to continue battling it out into the 1990s.

The fifth generation was a global success and set the Civic up with so many of the bases that we still recognize today. The base model CX made do with a 70 horsepower 1.5-liter unit, whereas the VX gifted owners the use of a 92 horsepower 1.5-liter with variable valve timing, dubbed VTEC-E, which was optimized for fuel savings. Further up the food chain, the DX offered a 102 horsepower 1.5-liter engine, and the flagship Si sported a 125 horsepower VTEC engine.

In addition to the Del-Sol, the Civic coupe debuted in 1993, further broadening the customer base of this now-global icon. Airbags are now featured across the range too, Honda always has a keen eye on safety, in addition to value and economy.

Perhaps one of the more instantly recognizable Civics, the 1996 sixth-generation continued building upon the huge success of previous models. More tech was introduced, now in an aptly named 'value package', which bundled A/C, a CD player, an automatic transmission, and other sought-after options into a suitably priced bundle.

The ever-popular VTEC dual-overhead-cam B16 unit appeared for the first time in the Civic Si coupe, producing 160 horsepower, clearly defining the Civic as a sporting model. This engine could rev up to 8,500 rpm and enabled the coupe to hit 60 MPH from a standstill in just a shade over 7 seconds.

1997 also gave the world the first-ever Civic Type-R, the EK9, which yielded blistering results from an 182-horsepower VTEC unit. Sadly, the EK9 was never offered in America, although the seed for a true performance Civic was planted, and an icon was born.

Related: Here Are The Best And Worst Honda Civic Models Ever Made

The seventh and eighth generation Civics saw modern technology integrated with the humble Honda, with MP3 compatibility and a plethora of new driving aids added across the range. In addition, safety was continually improved, with greater focus on NCAP safety results, and additional airbags fitted as standard.

Honda got adventurous with the styling, in particular with the eighth generation, which also saw leather seats added as an option for the first time in the Civic's history.

The ninth-generation Civic saw more conservative styling, a step back in terms of interior quality and, according to period reviews, lost some of its charm on the road too. Fortunately, Honda acted quickly to rectify this, and in 2013 stiffer suspension springs and beefed-up anti-roll bars helped to deliver the characterful handling once more.

The tenth generation was introduced in 2016, with a range of fizzy engines, including a 174 horsepower turbocharged 1.5-liter inline-4 and 158 horsepower 2.0-liter inline-4, both of which would please driving enthusiasts, with stick-shift options available through the model run.

Just one year later, Honda re-launched an Si model, which featured an enigmatic 205 horsepower version of the previously mentioned 1.5-liter turbo engine, and the all-important stick-shift six-speed transmission.

Apart from the 1997 Acura Integra Type-R, America has otherwise been denied a true sporting Honda, having had to make do with Si models for decades. The tenth-generation Civic Type-R changed all that, finally bringing a 306 horsepower, wide-bodied hot hatchback to dealerships, coated in splitters, spoilers, and an awesome triple exhaust pipe.

Way more than just a marketing gimmick, the Type-R set the lap record around the Nrburgring for a front-wheel-drive production car, setting a time of 7:43:80; that's almost 3.5 seconds quicker than the Golf GTI Clubsport.

Related: 10 Most Reliable Honda Civic Models To Buy Used

We are onto the eleventh generation of Honda Civic now, and Honda has continued to impress with this relatively new model, only introduced in 2022.

Ever looking forward, the Civic comes standard with a smart 7.0" touchscreen display, which sits proudly in the center of the dashboard. As you would expect, there's also a plethora of driver and safety aids, in addition to the more interesting infotainment features, such as a wireless charging pad, a BOSE premium surround sound system, and satellite navigation.

Keen to prove their sporting credentials, Honda once again offers consumers the choice of an Si and a Type-R with this latest Civic, both of which set new standards in performance; here's how they compare against each other.

Model

2023 Civic Si

2023 Civic Type R

0 to 60 MPH

6.6 seconds

4.9 seconds

Top Speed

137 MPH

169 MPH

Curb Weight

2,952 lbs

3,188 lbs

Power

200 horsepower

315 horsepower

Torque

192 lb-ft

310 lb-ft

Layout

Front-engine, front-wheel-drive

Front-engine, front-wheel-drive

Starting Price

$28,800

$43,990

There's a lot of speculation about what the future will look like for the Honda Civic. Many think Honda will continue to build upon the heritage of this gas-powered hatchback, whereas they could just as easily take a new direction with it like so many manufacturers have done with their long-standing models.

One thing is for sure, the legacy of the Honda Civic will continue either way, having shaped the auto industry for the better over the last five decades.

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The Honda Civic's Evolution In Photos, From 1972 To Today - HotCars

Even when in Nashville for business, Childers sometimes looks like he is ready to go walk the land at his farm. Work boots, Wrangler jeans, and a feed store cap. Some country stars pay stylists lots of money to dress them similarly, but they cant quite pull off the look; they dont know how to move realistically in clothes made for climbing hills and slipping between strands of barbed wire. But Childers has been doing such all of his life. One morning when we go to a coffee shop in Nashville for breakfast, he is wearing a sharply cut linen jacket, a crisp button-down, chinos, and a pair of brown leather Sabahs. Somehow he pulls off wearing these Turkish espadrilles, too, mostly because he seems comfortable in his own skin. There is no pretense about Childers. He is not easily defined.

Nor is the sound of his new album, Rustin in the Rain, which was released September 8. The title track of the seven-song record includes barn-burners that allow Childers incredibly tight band, The Food Stamps, plenty of room to show off their talents, as well as precise ballads that let Childers use his full vocal range, including on the lead single, In Your Love. After a euphonious piano intro, the song opens with Childers at the top of his vocal game, singing about a cosmic love before the song continues with a ghostly synthesizer effect. By the time he sings Honey, I will stand my ground, the listener is hooked on this extraordinary ballad that is bound to become a wedding staple.

I first heard the song on a long drive with Childers as we ambled over the glowing green Kentucky hills on an afternoon in early spring. We had been friends for a couple years but mutual admirers of each others work before that. He read my first novel, Clays Quilt, when he was in high school and has said it helped him better appreciate his heritage; hed later write an introduction for a reissue of the book. I have loved his music since the very beginning of his career. His wife, singer-songwriter Senora May, was one of my students at Berea College and used to bring me CDs shed burn of him picking and singing at home.

To me, In Your Love announced a fuller, richer sound. I knew right away the song would take his career in a whole new direction, but I was stunned when he asked me if Id be interested in writing a treatment for the video. Childers had only three suggestions: that Casey Campbell, an actor and musician who has appeared in all of his videos, show up in some way; that there be a scene that includes a mule; and, best of all, that it would be a gay love story. Childers has always been as close as brothers with a cousin who is gay, and he told me that he wanted his cousin to be able to see himself in a country music video.

I collaborated with my husband, Jason Kyle Howard, on the story of two coal miners who not only fall in love but also leave the toxic masculinity and industrial confines of the mines to live out an agrarian existence where they can control their own lives. I also wrote the treatment and served as the creative director on the shoot, orchestrated by director Bryan Schlam and starring Colton Haynes and James Scully, two gay actors who brought tremendous chemistry to the roles.

The video struck a nerve, racking up more than 1 million views in the first day. (As of this writing, it has more than 7.8 million global views.) The song has amassed 30 million combined streams across all platforms globally andbecame the highest-charting single of Childers career.

Childers knew he was making a statement, but most importantly, he wanted to make a piece of art.

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