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Discovery of the missing UDP-glycosyltransferase for ginsenoside Rg2 and Re biosynthesis

Previously, we systematically characterized a series of UGTs involved in the biosynthesis of ginsenosides and completely resolved the biosynthetic pathway of Rh1 and Rg121. The downstream pathway from Rh1 and Rg1 to Rg2 and Re is speculated to be catalyzed by an unknown UGT enzyme (Fig.1). Because all previous characterized UGTs responsible for the sugar elongation of ginsenosides belong to the UGT94 family30, we thus focused our efforts on screening UGT candidates belong to this family.

Bluish green arrows represent glycosylation steps using UDP-glucose as a sugar donor, blue arrows represent the biosynthetic pathway of UDP-rhamnose and glycosylation steps using UDP-rhamnose as a sugar donor. Multi-step conversions were presented as multi arrows. Bluish green marked genes represented previous reported genes and thebule marked gene represented the identified onein this study. PPD protopanaxadiol, PPT protopanaxatriol.

We identified 665 UGTs with >350 amino acids residues (typical plant UGTs length) from the P. ginseng transcriptome that were predicted to have a conserved Plant Secondary Products Glycosyltransferase (PSPG) box. These could be clustered into 187 OTUs with a 95% cutoff. Twenty-two OTUs and their representative UGTs which possessed >40% amino acid identity to the previous identified UGT94 family UGT member, PgUGT94Q230 were recognized as belong to the UGT94 family. Through gene co-expression analysis of these UGT candidates, previously characterized P450s, and UGTs involved in PPT-type ginsenoside biosynthesis from P. ginseng transcriptome data, a UGT94 gene named PgURT94 was identified. The expression pattern of PgURT94 was strongly correlated with PgDDS, CYP716A47, CYP716A53v2, PgUGT71A53, and PgUGT71A54, which are involved in Rh1 and Rg1 biosynthesis (Fig.2a). Besides, PgURT94 was highly expressed in the root hairs of P. ginseng, which is consistent with the distribution of Re in this tissue (Supplementary Table1). Thus, we speculated that PgURT94 is most likely to be involved in the biosynthesis of Rg2 and Re.

a Heat-map analysis of the relative abundance of PgURT94 expression, along with PgDDS, CYP716A47, CYP716A53v2, and PgUGT71A53 in different parts of P. ginseng. b HPLC analysis of the in vitro reaction products catalyzed by PgURT94 crude enzyme using Rh1 as sugar acceptor and UDP-rhamnose as sugar donor. c HPLC analysis of the in vitro reaction products catalyzed by PgURT94 crude enzyme using Rg1 as sugar acceptor and UDP-rhamnose as sugar donor. Crude enzymes of E. coli strain harboring pET28a empty vector were used as a negative control for above assays and authentic ginsenoside samples Rh1, Rg1, Rg1, and Re were monitored as standards.

To verify this hypothesis, we firstly cloned this gene from callus of P. ginseng. The PgURT94 gene has an open reading frame (ORF) of 1404bp, encoding a protein of 467 amino acids. PgURT94 protein has a sequence identify of 46.1%, 47.1%, and 44.8% with PgUGT94Q2 (UGT catalyzing C3-O-Glc glucosylation of PPD-type ginsenosides), PgUGT94Q3 (UGT catalyzing C6-O-Glc glucosylation of PPT-type ginsenosides), and PgUGT94Q6 (UGT catalyzing C20-O-Glc glucosylation of PPD and PPT-type ginsenosides), respectively30. For the enzymatic activity test, PgURT94 was initially expressed in E. coli (Supplementary Fig.1) and the crude enzymes from E. coli expressing PgURT94 were incubated with Rh1 and Rg1 as substrates, and UDP-Rha as a sugar donor. The reaction products were subjected to HPLC analysis and results indicated a product was generated in the reaction extract from PgURT94 and Rh1 incubations, which had the same retention time as the Rg2 standard. This compound was not detected in the control reaction with Rh1 and crude enzyme of E. coli strain harboring empty pET28a vector (Fig.2b). A product was also observed in the reaction extract from PgURT94 using Rg1 as a substrate, and was monitored along with the Re standard (Fig.2c). The structures of these two newly produced compounds were confirmed to be Rg2 and Re, respectively, by HPLC/electrospray ionization mass spectrometry (ESIMS) (Supplementary Fig.2) and NMR (Supplementary Fig.3).

To test the sugar donor specificity of PgURT94, in vitro enzymatic assays were performed by using UDP-glucose as a sugar donor and incubating PgURT94 with Rh1 and Rg1, respectively. To ensure accuracy of the assay, a previously reported UGT (PgUGT94Q3) which could catalyze the glycosylation modification of Rh1 and Rg1 using UDP-glucose as a sugar donor30, was used as a positive control. TLC and HPLC analyses of the reaction extracts revealed that while production of glycosylated products Rf and C20-O-Glc-Rf could be detected by PgUGT94Q3 as expected, no products were detected by PgURT94, indicating that PgURT94 could not use UDP-glucose as a sugar donor (Supplementary Fig.4). These results demonstrated that PgURT94 is a specific rhamnosylation UGT.

Through functional characterization of PgURT94, the complete biosynthetic pathway of Rg2 became clear: PgUGT71A54 catalyzes the C6-OH glycosylation of PPT to form Rh1, and PgURT94 then transfers a rhamnose moiety to the C6-O-Glc of Rh1 to produce Rg2 (Fig.1). To achieve de novo biosynthesis of Rg2 in yeast, codon-optimized PgUGT71A54 and PgURT94 (hereafter referred to as synPgURT94), under the control of two strong constitutive promoters respectively, were introduced into the chromosome of strain PPT-10, a PPT-producing chassis constructed in our previous work23. Since S. cerevisiae lacks the native UDP-Rha biosynthetic pathway, AtRHM2 from A. thaliana, which catalyze the formation of UDP-Rha from UDP-glucose, was also expressed in PPT-1031,32. The resulting strain, called Rg2-01, produced 36.8mg/L Rg2 according to the analysis of metabolites in subsequent flask fermentations (Fig.3a, b).

a HPLC analyses of Rg2, Rh1, PPT and PPD production in yeast strains Rg2-01, Rg2-02, Rg2-03, and Rg2-04. The PPT chassis strain PPT-10 was used as a control. Mixed samples of Rf, Rh1, Rg2, PPT and PPD were monitored as standards. b Quantitative analysis of Rg2 and its related intermediates Rh1, PPT, and PPD in yeast strains Rg2-01, Rg2-02, Rg2-03, and Rg2-04. Genetic modification of each strain was drawn under the column, + represent the strain possess the corresponding engineering, while represent the corresponding engineering are missing the strain. All data represent the mean of n=3 biologically independent samples and error bars show standard deviation.

The total triterpenoid production (PPD+PPT+Rh1+Rg2) in strain Rg2-01 decreased sharply compared to that of the parent strain PPT-10 (Fig.3b). Since the triterpenoid biosynthetic pathway is a highly NADPH-consuming pathway and the biosynthesis of UDP-Rha from UDP-Glc by AtRHM2 in the Rg2-producing strain Rg2-01 is also an NADPH-dependent pathway33, we thus focused our attention on the NADPH consumption. For PPT biosynthesis, the precursor pathway from acetyl-CoA to 2, 3-oxidosuqalene requires three NADPH molecules (the formation of mevalonate from 3-hydroxy-3-methylglutaryl-CoA need two NADPH molecules34 and the formation of 2, 3-oxidosqalene from squalene need one NADPH molecule35). Besides, two P450s that catalyze the formation of PPT from DM also consume two NADPH molecules12,13 (Supplementary Fig.5). Therefore, the NADPH supply is of great importance for PPT production. We speculate that the introduced of NADPH-dependent UDP-Rha synthase AtRHM2 may further increase NADPH consumption and caused a reduction in total triterpenoid production in strain Rg2-01 (Supplementary Table6).

To address this issue, we utilized a NADPH-independent UDP-Rha synthase to alleviate the NADPH limitation. Several studies demonstrated that an engineered RHM enzyme (VvRHM-NRS) formed by fusing a bifunctional UDP-4-keto-6-deoxy-d-glucose 3,5-epimerase (NRS)/UDP-4-keto-rhamnose 4-keto-reductase (ER) from A. thaliana to the N-terminal of a Vitis vinifera UDP-Rha synthase VvRHM can be a self-sufficient NADPH-independent enzyme for UDP-Rha synthesis36,37. VvRHM-NRS and synPgURT94 were then introduced into PPT-10 to construct strain Rg2-02. Metabolite analysis of strain Rg2-02 indicated that Rg2 production increase to 66.4mg/L, which is approximately 1.8-fold the amount of Rg2-01 (Fig.3b). No significant reduction in total triterpenoid production was observed compared to the parent strain, PPT-10. These results clearly demonstrate that utilization of a NADPH-independent enzyme for UDP-Rha synthesis could rescue the triterpenoid reduction resulting from insufficient NADPH supply.

During screening the single clones of the construction of strain Rg2-02, a clone, hereafter designated as strain Rg2-03, was found to produce Rg2 with a production of 107.5mg/L, which is significantly higher than Rg2-02. The synPgURT94 gene of Rg2-03 was then amplified and sequenced; a missense T-to-A mutation of the 163th nucleotide was found, which resulted in a leucine to methionine mutation at the 55th amino acid. To explore whether this amino acid mutation contributed to the enhanced production of Rg2 in strain Rg2-03, we expressed the synPgURT94 mutant (synPgURT94m1) in E. coli and performed an in vitro enzymatic activity assay. Using Rh1 as a substrate, the conversion ratio of Rh1 to Rg2 by synPgURT94 and synPgURT94m1 was 70.6% and 92.4%, respectively, which demonstrated a great improvement in catalysis efficiency by synPgURT94m1 (Fig.4a, c, Supplementary Table7). The improved catalytic performance of synPgURT94m1 may explain the increased Rg2 production of strain Rg2-03. The catalytic activity of synPgURT94m1 towards Rg1, to produce Re, was also assessed; significant enhancement of catalytic activity was also observed (Fig.4b, d, Supplementary Table7). Therefore, this mutant was used for Re-cell factory construction.

a, b HPLC analyses of the in vitro reaction products catalyzed by synPgURT94 and synPgURT94m1 crude enzymes using Rh1 (a) and Rg1 (b) as thesugar acceptor and UDP-rhamnose as thesugar donor. Crude enzymes of E. coli strain harboring pET28a empty vector were used as a negative control and authentic ginsenoside samples Rh1, Rg1, Rg1 and Re were monitored as standards. c, d Quantitative analysis of the conversion ratio catalyzed by synPgURT94 and synPgURT94m1, from Rh1 to Rg2 (c) and from Rg1 to Re (d). All data represent the mean of n=3 biologically independent samples and error bars show standard deviation.

Since Rh1 did not accumulate in any of the engineered Rg2-producing strains, the conversion of PPT to Rh1 may be a limiting step in Rg2 production. To address this, we introduced an additional copy of PgUGT71A54 into strain Rg2-03 to create Rg2-04. As expected, the production of Rg2 by Rg2-04 reached 147.1mg/L, representing a 40% improvement compared to Rg2-03 (Fig.3, Supplementary Table6). By combining all engineering strategies, the production of Rg2 increased 4.0-fold from Rg2-01 to Rg2-04. However, in our final strain, there was still more than 263.3mg/L of PPT accumulated and no Rh1 was detected (Supplementary Table6). Therefore, the conversion of PPT into Rh1 remains a major bottleneck for Rg2 production. We believe that the production of Rg2 could be further improved by addressing this limiting step in the future.

A Rg1-producing yeast cell factory, Rg1-02, was constructed previously by inserting PgUGT71A53 and PgUGT71A54 respectively into the single-copy YORW22 and multi-copy delta DNA sites. The production level of Rg1 was 111.45mg/L in shake flasks and 1.95g/L in fed-batch fermentations23. For Re production in yeast, VvRHM-NRS and synPgURT94m1, under the control of strong constitutive promoters, were introduced into Rg1-02 to generate strain Re-01 (Fig.1). Subsequent shaken flask fermentation tests detected Re at a production level of 215mg/L (Fig.5b, Supplementary Fig.6).

a HPLC analyses of Rg1, Re, F1, PPT, CK, DMG, and PPD produced in yeast strain Re-01. Mixed samples of ginsenosides were monitored as standards. The Rg1 chassis strain Rg1-02 was used as a negative control. b Quantitative analysis of Re and its related intermediate Rg1 in engineered yeast strains. All data represent the mean of n=3 biologically independent samples and error bars show standard deviation.

Re production in strain Re-01 was much higher than Rg2 production in strain Rg2-04, despite the fact that Re has a more complicated biosynthetic pathway. The Re content is also much higher than that of Rg2 in Panax plants, including P. ginseng and P. notoginseng1,2. To test whether this phenomenon is determined by some intrinsic factors or just a coincidence, we then examined the difference between the two yeast strains. Re-01 and Rg2-04 share the same PPT-producing background strain and downstream rhamnosylation pathway (Fig.1), thus PgUGT71A53 and PgUGT71A54 may contribute to different production between the two strains.

Previous enzyme kinetics assays indicated that the kcat/Km value of PgUGT71A53 for PPT is higher than that of PgUGT71A54 by more than 400-fold23. Thus, the poor PPT-catalyzing efficiency of PgUGT71A54 in strain Rg2-04 may severely limit the formation of Rh1 and lead to the lower production of Rg2. Accordingly, the high efficiency of PgUGT71A53 towards PPT ensured the rapid conversion of PPT into F1 which can be subsequently converted into Rg1 by PgUGT71A54. To assess the catalytic performance of PgUGT71A54 towards F1, enzyme kinetics assays were performed. Although the kcat/Km value of PgUGT71A54 towards PPT is low (2.58102mM1s1), its kcat/Km value towards F1 is much higher (7.87101mM1s1) (Supplementary Table8). We also observed that PgUGT71A53 could also catalyze the conversion of F1 to Rg1, with a kcat/Km value of 7.40102mM1s1. Thus, the elevated activity of PgUGT71A54 and PgUGT71A53 towards F1 enabled highly efficient conversion of F1 into Rg1 in the Re-01 strain. These results demonstrated that the elaborate coordination of PgUGT71A53 and PgUGT71A54 in the pathway precisely regulated the production of downstream products. The enzyme characteristic of UGTs might determine the contents of Re and Rg2 in Panax plants, as well as that ofour engineered yeast strains.

With the above engineering efforts, we obtained two yeast strains (Rg2-04 and Re-01) that could in turn produce ginsenosides Rg2 and Re directly from glucose, respectively. However, the production titer in shaken flasks remained at relatively low levels. We previously reported a series of successful examples of the promotion of ginsenosides through fed-batch fermentations in bioreactors, including Rh2, CK, Rg1, NgR1, and NgR2, all of which reached the gram-per-liter scale after optimization of the fed-batch fermentation conditions (Supplementary Table9). To achieve higher production of Rg2 and Re, we performed high-density fed-batch fermentation of Rg2-04 and Re-01 using a 1.3-L parallel bioreactor system. Since Rg2-04 and Re-01 have the same strain background (strain PPT-10) to previous constructed NgR1- and NgR2-producing strains, the fermentation control parameters were set as reported previously23. Fresh medium was fed into the fermenter at approximately 24h and the cell biomass of both strains continuously increased after feeding. For strain Rg2-04, the OD600 continuously increased to a maximum of 330.4 at 108h and remained unchanged until 120h. Re-01 reached a growth plateau at 96h (OD600=456.8) and exhibited a slight decrease at 108h (Fig.6 and Supplementary Table6). Unexpectedly, the final cell biomass of Re-01 is more than 37.2% higher than that of Rg2-04, although the mechanism underlying this phenomenon is unclear.

a Time course analysis of cell growth and triterpenoid production of strain Rg2-04. b Time course analysis of cell growth and triterpenoid production of strain Re-01. PPD protopanaxadiol, PPT protopanaxatriol, DMG 20SO--(D-glucosyl)-dammarenediol-II.

The final production amount of Rg2 by Rg2-04 was 1.3g/L, which represents an 8.9-fold increase over shaken flask production. Other important ginsenosides precursors, including PPD and PPT, could also be detected in the fermentation broth, with titers of 2.2 and 3.0g/L, respectively (Fig.6, Supplementary Table6). The final production level of Re by Re-01 reached 3.6g/L, which represents a 16.6-fold increase over shaken flasks. Many important ginsenoside precursors, such as PPD, DMG (20SO--(D-glucosyl)-dammarenediol-II), CK, PPT, F1, and Rg1, could also be detected in the fermentation broth, with titers of 1.0, 0.5, 0.7, 0.2, 0.1, and 0.2g/L, respectively (Fig.6 and Supplementary Table6). The accumulation of numerous triterpenoid precursors in both strains indicated that some rate-limiting steps remained in the engineered strains, and there is great potential for even higher Rg2 and Re production levels if these obstacles can be overcome. Since the physicochemical properties of these ginsenoside precursors vary significantly, it will not be difficult to separate and purify them from the fermentation broth. Thus, engineered strains Rg2-04 and Re-01 may also be useful for the preparation of these valuable ginsenosides.

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Pathway elucidation of bioactive rhamnosylated ginsenosides in Panax ginseng and their de novo high-level production by engineered Saccharomyces...

Novel CRISPR solution leverages Eclipse Platform to enable a faster path to therapeutic development

REDWOOD CITY, Calif., August 02, 2022--(BUSINESS WIRE)--Synthego, the genome engineering company, today announced the launch of Engineered Cell Libraries, a novel offering that further enables access to CRISPR by providing arrayed CRISPR-edited cells for direct use in functional screening assays. The innovative solution leverages Synthegos Eclipse Platform. This high-throughput cell engineering platform delivers cell-based models for disease research by providing highly predictable CRISPR-engineered cells at scale through the integration of engineering, bioinformatics, and proprietary science. Synthegos Engineered Cell Libraries provide unparalleled speed, scalability, and efficiency to accelerate the drug discovery process by enabling a faster path between experimental design and execution.

As the newest addition to Synthego's Engineered Cells product line, the offering is a custom arrayed library of multi-guide knockout cell pools (immortalized or iPSC) delivered ready-made to researchers, efficiently enabling them to bypass the need for equipment and reagent sourcing, transfection, generation of an extensive CRISPR library, and hiring and training of staff. This allows researchers to overcome common hurdles associated with CRISPR-based target discovery and focus specifically on the science.

"Arrayed screening approaches are more sensitive, compatible with a broader assortment of downstream assays, and can yield data that is more readily interpretable than the more commonly used pooled approach but are underutilized due to lack of infrastructure and limited bandwidth," said Travis Maures, Synthegos Chief Technology Officer. "With Engineered Cell Libraries, Synthego continues to provide transformative solutions for accelerated drug discovery which ultimately can bring a wider range of therapeutics to market faster."

With Engineered Cell Libraries, scientists specify the human or mouse cell type (Immortalized or iPSC available at launch) and gene targets they desire to knockout to generate a custom "Knockout Cell Library." The cells are then edited on Synthegos Eclipse Platform, which handles guide design, cell line optimization, editing through transfection, and assessment of editing efficiencies, so cells are ready to screen upon arrival.

Engineered Cell Libraries on the Eclipse Platform additionally benefit customers with:

Scalability and flexibility - Engineered Cell Libraries allow researchers to maximize their screening power and identify more targets earlier in the screening process.

Predictability and transparency - Synthegos multi-guide technology achieves reliably high knockout efficiencies so researchers can confidently proceed with their screens.

Cell engineering expertise and support - Synthego uses its expertise to bring automation innovation that provides greater consistency in outcomes and scalability.

Synthegos Eclipse Platform and Engineered Cell Libraries enable a wide range of applications in research and development across various disease areas and research disciplines such as oncology and neurology. Engineered Cell Libraries were employed in a recent study that used genetic screening to identify host factors that either facilitate or inhibit infection by SARS-CoV-2 and that could potentially be targeted with existing drugs that have been approved for other indications.

"We were able to quickly combine our proteomic expertise with Synthego's genome engineering capabilities in a matter of weeks," said Nevan J. Krogan, Director, Quantitative Biosciences Institute, University of California, San Francisco. "Normally, work such as this would take many years. We were able to quickly pinpoint which human genes are important for infection, and that allowed us to jump to which ones if we were able to drug them, could have a positive pharmacological effect on SARS-CoV-2 infection. That whole pipeline allowed us to identify several potential drug candidates, several of which we're still looking at."

Ultimately, Synthegos goal is to enable scientists to spend less time thinking about method development and more time running their functional assays. The addition of Engineered Cell Libraries is driving impact in biopharma research and development. For more information about Engineered Cell Libraries and Synthegos Eclipse Platform, visit synthego.com

ABOUT SYNTHEGO

Synthego was founded to revolutionize genome engineering technology, helping translate genomics into the clinic and ultimately making engineered biological therapies accessible to all patients. The company leverages machine learning, automation, and gene editing to build platforms for science at scale. With its foundations in engineering disciplines, the companys platforms vertically integrate proprietary hardware, software, bioinformatics, chemistries, and molecular biology to advance both basic research and therapeutic development programs. With its technologies cited in more than a thousand peer-reviewed publications and utilized by thousands of commercial and academic researchers and therapeutic drug developers, Synthego is at the forefront of innovation enabling the next generation of medicines by delivering genome editing at an unprecedented scale.

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Synthego Launches Engineered Cell Libraries to Validate Targets with Speed and Accelerate Drug Discovery - Yahoo Finance

Renewed efforts are being made to cover up the origin of Wuhan virus.

Bengaluru: As a defeat by the Democratic Party in the mid-term elections in the United States this year appears to be a certainty, and as possibility increases of the Republicans coming to control the US House of Representatives, the prospects of a Congressional hearing/investigation into the origin of the Wuhan virus, also known as Covid-19, are a given. To head off a possible Congressional probe, the cast of US characters involved in the research and development of this genetically engineered virus that has killed millions all over the globe, is back to obfuscating the source of the virusChinas Wuhan Institute of Virology (WIV)by calling it a natural virus in a bid to whitewash their own and the Xi Jinping regimes culpability. This is apparent from the publishing of two articles on 26 July 2022, in the journal Science. Both these articles have multiple authors under Dr Kristian G. Andersens guidance in the US. Andersen is known for infamously switching from suspecting Covid-19 to be genetically engineered, to trying to prove that it naturally jumped from wild bats to humans. These two latest studies further prove that Covid-19 originated in Wuhan, but do not prove the zoonotic (jumping from animals to humans) origin of the virus that the authors are at pains to prove, based on numerous assumptions. Co-conspirators in the diversion of large US research funds tothe WIV in China ensured the suppression of facts and orchestrated the publication of misleading scientific correspondence in the early days of the pandemic in 2020 to prove that the virus had a natural origin in the Wuhan wet market. This concerted disinformation campaign has been given life once again by some virologists who fear a prospective ban on high risk virus research, an exposure of their role in research like genetic engineering and gain of function, and are more concerned about their own funding than on the safety and welfare of humanity.A MISLEADING STUDY: One of these two articles, an 18-page write-up by 18 authors is titled, The Hunan seafood wholesale market in Wuhan was the early epicentre of the Covid-19 pandemic. It starts with the premise that understanding how Covid-19 virus emerged in 2019 is critical to prevent zoonotic outbreaks. Hence the article predetermines that the virus was natural in origin. The article provides the geographical distribution of the early suspected Covid-19 cases around the wet market in Wuhan, based on a flawed and biased sample. Their spatial distribution maps highlight the wet market location and ignore the Wuhan CDC (the agency that monitored the outbreak initially) just 280 metres away, leave alone the WIV 12 kilometres away. The Wuhan CDC had hosted experimental wild animals including bats collected from Hubei and Zhejiang provinces. The writers of the article found many early cases that had no direct links with the market. They found susceptible mammals such as racoon dogs for sale, but were unable to identify an intermediate host. They conceded that there is insufficient evidence to define upstream events, and exact circumstances remain obscure. They still concluded that our analyses indicate that the emergence of SARS-CoV-2 occurred via the live wildlife trade in China, and show that the Huanan market was the epicentre of the COVID-19 pandemic. It is to be noted that the epicentre of an outbreak would be a crowded place near the source of the virus, and not necessarily the source itself.THE OTHER MISLEADING STUDY: The other article, a 15-page study by 29 authors from the same institutions, and titled, Molecular epidemiology of multiple zoonotic origins of SARS-CoV2 examines the strains of the virus found in the early stages of the outbreak in Wuhan. They mention two virus lineages A and B and propose multiple cross species transmissionsof lineage B virus to humans around 18 November 2019 and later of lineage A within a few weeks. It is simple logic that a cross species transmission that did not occur in centuries of existence of wet markets in China is most unlikely to occur multiple times in quick succession. Their claim papers over the well-known fact that gain of function research produces multiple strains. Who should know this better than these virologists? They speculate about racoon dogs and other mammals being the intermediate hosts, but their numerous errors suggest that animals and their samples may have been contaminated by infected humans. Their conclusion also ignores the fact that the only bats in Wuhan existed in the Wuhan labs and not in the wet market.LAY MEDIA AND PUBLIC MISLED: Newspaper and network news journalists and ombudsman have always had a tough time understanding technical jargon and making sense of scientific claims. It is worse when leading experts publish scientific articles with dubious claims. Ideally the results of a scientific study should be explained rationally and should lead to a logical conclusion. It should not be reverse engineered to achieve a predetermined conclusion. Sometimes, as with these two studies the elaborate data and statistical analysis seems authentic but the authors jump to a conclusion that is not justified. An article by Laura Ungar on 27 July 2020 in Associated Press based on these two studies in Science and titled New studies bolster theory coronavirus emerged from the wild quotes Dr Kristian G. Andersen as saying, Have we disproven the lab leak theory? No, we have not, but I think whats really important here is there are possible scenarios and there are plausible scenarios and its really important to understand that possible does not mean equally likely. This article was prominently republished by many leading Indian newspapers with the headline eventually evolving to an emphatic Covid did originate in Wuhan market, say 2 studies. Tragically, this will now be accepted as gospel truth by many in academia, intelligence, political and administrative circles.SUMMARY OF EVENTS LEADING TO COVID-19 ORIGIN: After leaks even from the safest of western virology laboratories and outcry about creation of deadly Chimera viruses by virologists hoping to profit on vaccines for novel human viruses; this risky virus research with technology, equipment and facilities was outsourced to China. Chinese researchers were trained in gain of function and genetic engineering techniques, funded and hand held by well-connected senior US virologists. Western collaboration enabled Chinese researchers to clandestinely or otherwise collect deadly viruses existing in the wild in various parts of the world and steal samples from western laboratories.THE ORIGINAL COVER-UP: My article of 6 June 2021, in The Sunday Guardian, titled, International scientists covered up the lab origin of Covid-19 details the original cover-up. Here is a brief recap of the original cover-up from that article: On 1 Feb 2021, within hours of the researchers from IIT New Delhi submitting their findings online on bioRxiv, alarm bells rang around the world. Dr Kristian G. Andersen of Scripps Research Institute emailed Dr Fauci: Some of the features look engineered, inconsistent with expectations from evolutionary theory. Following this a concerted suppression of findings, including of the New Delhi group was done by vested interests. On 19 February 2020, a group of 27 senior virologists from the US, Australia, Germany, Spain, UK, Netherlands, Italy, Malaysia, Hong Kong including Peter Daszak, president of the EcoHealth Alliance, that was funding WIV, published in Lancet a Statement in support of the scientists, public health professionals, and medical professionals of China combatting COVID-19. In a correspondence published on 17 March 2020 in Nature titled The proximal origin of SARS-CoV-2, Kristian G. Andersen, who on 1 February had emailed Dr Fauci, now turned contrarian and with four other researchers argued that Our analyses clearly show that SARS-CoV-2 is not a laboratory construct or a purposefully manipulated virus. On 26 March 2020, Dr Francis Collins supported Dr Andersens analysis on the NIV directors blog: next time you come across something about COVID-19 online that disturbs or puzzles you, I suggest going to FEMAs new Coronavirus Rumor Control web site. It will help to distinguish rumours from facts.The motto of these compromised researchers is: If you cant convince them, confuse them.Dr P.S. Venkatesh Rao is Consultant Endocrine, Breast & Laparoscopic Surgeon, Bengaluru.

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China's apologists again try to cover up Wuhan lab leak of Covid-19 - The Sunday Guardian Live - The Sunday Guardian

History was made in the Bihar Assembly when, heedless of the fact that nobody heard a word of what he said, Chief Minister Jagannath Mishra, went through all the motions to secure passage of the controversial bill to deal with newspapers publishing grossly indecent or scurrilous matter. The Opposition kept shouting slogans while tearing copies of the bill and the books of reporters recordings of the proceedings.

One major result of Prime Minister Indira Gandhis visit to Washington is the agreement to set up a blue ribbon panel for scientific co-operation between the two countries. Mrs Gandhi listed the following in the many rewarding areas in which US and Indian Science could cooperate: Biomass production and the application of tissue culture and genetic engineering; biomedical research to control leprosy, tuberculosis and waterborne diseases and; materials research to reduce energy consumption and costs.

A disciple of the late Mahatma Gandhi, Manibhai Bhimbai Desai, 62, was awarded the 1982 Roman Magsaysay Award for public service for his work with Indias poor, the award foundation announced.

New racial skirmishing in southern Sri Lanka has killed one more person and led authorities to continue a dusk-to-dawn curfew for the third day. The clash between Sinhalese, who dominate the island nation, and minority muslims in the southern coastal town of Galle, 120 km south of Colombo, has killed three people including the latest casualty. Sri Lanka declared a nation-wide emergency.

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August 1, 1982, Forty Years Ago: Bill To Curb Press - The Indian Express

High-fat diet (HFD) feeding in mice promotes induction of aldose reductase (AR) activity, expression, and senescence of adipocytes in subcutaneous adipose tissue (scAT), according to a new study inObesity. The researchers, headed by a New York University Grossman School of Medicine team, suggest that their findings could point to potential new strategies for treating obesity. Our data demonstrate that aldose reductase gene expression increases in scAT of obese humans and mice, and that an inhibitor of aldose reductase attenuates weight gain, reduces adipocyte senescence, and promotes lipolysis in HFD-fed mice, said Ravichandran Ramasamy, PhD, professor, diabetes research program, department of medicine, NYU Grossman School of Medicine. These data pave the way for testing these inhibitors as therapeutic adjuncts in treating patients with obesity.

Ramasamy is corresponding author of the teams study, which is titled, Aldose reductase promotes diet-induced obesity via induction of senescence in subcutaneous adipose tissue, and in which they concluded, These data unveil new opportunities to target these pathways to combat obesity.

Senescence is a state of permanent proliferative cell cycle arrest, the authors noted, which occurs in cells exposed to a variety of stresses. In HFD-induced models of obesity, various cell types, including adipocytes, immune cells, neuronal cells, and osteoprogenitors, have been shown to undergo senescence, they further commented. Although adipose tissue senescence has been observed in humans with obesity and in mouse models of obesity, the pathways driving this process are unclear.

In their newly reported study, Ramasamy and colleagues identified a novel role for the enzyme aldose reductase (akr1b3 in mice, and AKR1B1 in humans) in response to high-fat feeding in mice. Their studies showed that AR promotes induction of senescence in subcutaneous inguinal white adipose tissue in wild-type (WT) mice fed a high-fat diet.

To determine whether the AR pathway affected obesity in HFD-fed mice, and to establish its relationship to senescence and lipolysis, WT mice were fed an HFD for 11 weeks, followed by three weeks of continued HFD, plus treatment using either the AR inhibitor (ARI) zopolrestat (zop) or vehicle (potassium bicarbonate buffer used to dissolve zop). All the animals were male, had free access to water and food, and were subjected to 12-hour light/dark cycles. For a study in human subjects, researchers obtained cDNA samples of subcutaneous fat from fasted lean and fasted subjects with obesity.

The study results showed that in animals fed an HFD there was a significant increase in AR and the senescence marker Cdkn2a expression in all tissues, when compared with results in mice fed normal chow. Glycerol, non-esterified free fatty acid (NEFA), and triglycerides measured from plasma after a four-hour fast revealed significantly higher NEFA levels in HFD-fed mice when compared with the chow-fed mice. Analysis of cDNA samples from human subjects with obesity showed also increased expression of AR and senescence marker.

Increased expression of aldose reductase and senescence in the adipose tissue of humans and mice with obesity were demonstrated, the team wrote. In the animals given the aldose reductase inhibitor, zop, however, the collective data suggested thatthe treatment enhanced lipolysis and attenuated HFD-induced weight gain and scAT senescence.

Separately, the team carried out an analysis of Akr1b3 knockdown mice (Akr1b3-/- animals) that were fed an HFD. The resulting data also indicated that Akr1b3-/- HFD-fed mice are protected from HFD-induced obesity and that protection is linked to attenuation of senescence markers and enhanced adrenergic pathway-driven lipolysis in scAT, the authors noted. Genetic deletion of Akr1b3 or pharmacological blockade of AKRIB3 with zopolrestat reduced HFD-induced obesity, attenuated markers of adipose tissue senescence, and increased lipolysis.

They concluded, AKR1B1/Akr1b3 modulation of senescence in subcutaneous adipose tissue contributes to aberrant metabolic responses to high-fat feeding Our data demonstrate that AR gene expression increases in subcutaneous adipose tissue of humans and mice with obesity and that an inhibitor of AR attenuates weight gain, reduces senescence, and promotes lipolysis in HFD-fed mice; these data pave the way for the testing of these inhibitors as therapeutic adjuncts in treating patients with obesity.

AdventHealth senior vice president and CSO Steven R. Smith, MD, an expert in this areathough not linked to the reported researchfurther commented, This is an important discovery, and the data is compelling. Today, we dont have good medicines that target dysfunctional adipose tissue. Im very excited by this work; these findings should compel scientists to find drugs that impact this novel pathway and could be used to treat both obesity and diabetes.

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Enzyme that Promotes Diet-Induced Obesity Could Point to Inhibitor Therapy - Genetic Engineering & Biotechnology News

Gene and cell therapies are an emerging therapy class. Here, GEN talks to Nneka Onwudiwe, PharmaD, PhD, the PRO/PE Regulatory Review Officer for the FDA, about current and future challenges for manufacturing regulation.

GEN: What do you feel are the main areas where the gene and cell therapy industry are working to meet regulatory requirements?

Onwudiwe: The manufacturing processes [in these industries] are in their infancy, so a lot of validation is needed. [This is the case,] for example, with starting materials, especially if a manufacturer is starting with a small batch and is scaling up ready for commercialization.

Even when a product makes it to clinic, there are lots of other issues that need to be considered. For example, how do you store it within a critical range of temperatures? And how do you ensure safe delivery to clinical sites?

GEN: What do you think is the biggest challenge facing manufacturers in this sector?

Onwudiwe: Capacity constraints is the biggest challenge. If regulations require certain manufacturing processes or additional testing, that becomes a bottleneck if the capacity isnt thereas the manufacturer hasnt anticipated it.

GEN: How would you recommend manufacturers respond?

Onwudiwe: Its important for companies to engage with the health authority at an early stage because the last thing a manufacturer wants to find out is that either their manufacturing process is not in compliance or theres some issue with sourcing raw materials.

The FDA has put out several guidances around cell and gene therapy, but theyre not prescriptive. And, so, manufacturers still need to engage [with the agency] because there may be nuances around their molecule or development process. As these therapies are in their infancy (20 years in this space is still new) then there are things we are still learning. Manufacturers need to have adequate resources to anticipate challenges and a designated team of regulatory professionals who really understand the regulatory pathway for their molecule and can engage with the FDA at each step.

GEN: Where do you see future challenges arising?

Onwudiwe: I think theres a shift, not only in the biotechnology industry, but across other industries too in terms of sourcing much-needed raw materials [due to global supply chain issues]. Thats where I see the challenge in the next couple of years. And that will remain a challenge even if manufacturers have a good understanding of the manufacturing process for their molecule and its been validated by the FDA.

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Outlining the Latest Regulatory Trends in Advanced Therapies, Like Cell and Gene Therapy - Genetic Engineering & Biotechnology News