Biology subjects

Sharma, S.

Publications and source records attributed to Sharma, S..

At least 289 records · Page 16Linked to original sources

bioRxiv2022

Identifying Optimal Neuroinflammation Treatment Using Nanoligomer Discovery Engine

Acute activation of innate immune response in the brain, or neuroinflammation, protects this vital organ from a range of external pathogens and promotes healing after traumatic brain injury. However, chronic neuroinflammation leads to the activation of immune cells like microglia and astrocytes causes damage to the nervous tissue, and is causally linked to a range of neurodegenerative diseases such as Alzheimers diseases (AD), Multiple Sclerosis (MS), Parkinsons diseases (PD), and many others. While neuroinflammation is a key target for a range of neuropathological diseases, there is a lack of effective countermeasures to tackle it, and existing experimental therapies require fairly invasive intracerebral and intrathecal delivery due to difficulty associated with the therapeutic crossover between the blood-brain barrier (BBB), making such treatments impractical to treat neuroinflammation long-term. Here, we present the development of an optimal neurotherapeutic using our Nanoligomer discovery engine, by screening downregulation of several proinflammatory cytokines (e.g., Interleukin-1{beta} or IL-1{beta}, tumor necrosis factor-alpha or TNF-, TNF receptor 1 or TNFR1, Interleukin 6 or IL-6), inflammasomes (e.g., NLRP1), key transcription factors (e.g., nuclear factor kappa-B or NF-{kappa}{beta}) and their combinations, as upstream regulators and canonical pathway targets, to identify and validate the best-in-class treatment. Using our high-throughput drug discovery, target validation, and lead molecule identification via a bioinformatics and AI-based ranking method to design sequence-specific peptide molecules to up-or down-regulate gene expression of the targeted gene at will, we used our discovery engine to perturb and identify most effective upstream regulators and canonical pathways for therapeutic intervention to reverse neuroinflammation. The lead neurotherapeutic was a combination of Nanoligomers targeted to NF-{kappa}{beta} (SB.201.17D.8_ NF-{kappa}{beta}1) and TNFR1 (SB.201.18D.6_TNFR1), which were identified using in vitro cell-based screening in donor-derived human astrocytes, and further validated in vivo using a mouse model of lipopolysaccharide (LPS)-induced neuroinflammation. The combination treatment SB_NI_111 was delivered without any special formulation using a simple intraperitoneal injection of low-dose (5mg/kg) and was found to significantly suppress the expression of LPS-induced neuroinflammation in mouse hippocampus. These results point to the broader applicability of this approach towards the development of therapies for chronic neuroinflammation-linked neurodegenerative diseases, sleep countermeasures, and others, and the potential for further investigation of the lead neurotherapeutic molecule as reversible gene therapy.

neuroscience↗

bioRxiv2022

Structures of human Malic Enzyme 3

Malic enzymes (ME1, ME2, and ME3) are involved in cellular energy regulation, redox homeostasis, and biosynthetic processes, through the production of pyruvate and reducing agent NAD(P)H. Recent studies have implicated the third and least well-characterized isoform, mitochondrial NADP+-dependent malic enzyme 3 (ME3), as a therapeutic target for pancreatic cancers. Here, we utilized an integrated structure approach to capture the structures of ME3 in various ligand binding states. ME3 exists as a stable tetramer and its dynamic Domain C is critical for activity. Catalytic assay results reveal that ME3 is a non-allosteric enzyme and does not require modulators for activity while structural analysis suggests that the inner stability of ME3 domain A relative to ME2 disables allostery in ME3. With structural information available for all three malic enzymes, the foundation has been laid to understand the structural and biochemical differences of these enzymes and could aid in the development of specific malic enzyme small molecule drugs.

molecular biology↗

bioRxiv2022

Transcriptional activation of Jun and Fos members of the AP-1 complex is a conserved signature of immune aging that contributes to inflammaging

Diverse mouse strains have different health and life spans, mimicking the diversity among humans. To capture conserved aging signatures, we studied long-lived C57BL/6J and short-lived NZO/HILtJ mouse strains by profiling transcriptomes and epigenomes of immune cells from peripheral blood and the spleen from young and old mice. Transcriptional activation of the AP-1 transcription factor complex, particularly Fos, Junb, and Jun genes, was the most significant and conserved aging signature across tissues and strains. ATAC-seq data analyses showed that the chromatin around these genes was more accessible with age and there were significantly more binding sites for these TFs with age across all studied tissues, targeting pro-inflammatory molecules including Il6. Age-related increases in binding sites of Jun/Fos factors were also conserved in human peripheral blood ATAC-seq data. Single-cell RNA-seq data from the mouse aging cell atlas Tabula Muris Senis showed that the expression of these genes increased with age in B, T, NK cells, and macrophages, with macrophages from old mice expressing these molecules more abundantly than other cells. Functional data showed that upon myeloid cell activation via poly(I:C), the levels of c-JUN protein and its binding activity increased more significantly in spleen cells from old mice compared to cells from young mice. In addition, upon activation, old cells produced more IL6 compared to young cells. In sum, we showed that the aging-related transcriptional activation of Jun/Fos members of the AP-1 complex is conserved across immune tissues and long- and short-living mouse strains, possibly contributing to increased inflammation with age.

systems biology↗

bioRxiv2022

Screening Avian Pathogens in Eggs from Commercial Hatcheries in Nepal- an Effective Poultry Disease Surveillance Tool

BackgroundCommercial hatcheries play an important role in the overall poultry value chain-providing small to large poultry farmers with day old chicks. Any outbreak in such hatcheries can spread diseases to other farms. Regular screening of major avian pathogens, along with strict bio-security measures, can prevent spread of diseases in hatcheries. Newcastle Disease Virus (NDV), Infectious Bronchitis Virus (IBV), Mycoplasma gallisepticum (MG), Mycoplasma synoviae (MS), Infectious Bursal Disease Virus (IBDV) and Influenza A Virus (IAV) are among the most prevalent poultry diseases which can be detected in egg albumin. MethodWe retrospectively (August 2020-August 2021, except October 2020) analyzed diagnostic results for six selected avian pathogens (NDV, IBV, MS, MG, IBDV and IAV) on eggs (n=4343) received from eleven major commercial poultry hatcheries located in the five adjoining districts of Kathmandu, Nepal. Albumin from 10% randomly selected eggs from each hatchery were tested for the six avian pathogens using multiplex PCR. ResultMajority (7/11, 64%) of the poultry hatcheries had at least one of the six pathogens present. We detected at least one avian pathogen in nine out of eleven months (82%) of screening. Except for IBDV, we found one or more of the other major avian pathogens-Influenza A (IAV) (n=4 times) and Mycoplasma gallisepticum (MG) (n=4 times) were detected the most, followed by Newcastle Virus (NDV) (n=3 times). Infectious bronchitis virus (IBV) were detected twice, and Mycoplasma synoviae (MS) was detected once. ConclusionIn a resource strapped country like Nepal, poultry disease outbreak investigation in particular and surveillance in general are challenging. Meanwhile, poultry production is highly impacted by disease outbreaks often triggered by poor bio-security and lack of pathogen screening practices. Our molecular screening tests have picked up major poultry pathogens present throughout the year in eggs collected from hatcheries. Influenza A was detected at 4 different incidences throughout the year, which is of concern to both human and animal health. Quick systematic screening of eggs at key distribution points (hatcheries) for major avian pathogens is an effective surveillance tool for early disease detection and containment of outbreaks.

microbiology↗

bioRxiv2022

Fiber Diameter and Architecture Direct Three-Dimensional Assembly of Pericytes into Spheroids

Due to their physiological relevance, multicellular 3D spheroids are actively replacing standard 2D monolayer cultures. How spheroids are formed through the assembly of individual cells in natural fibrous environments that include a mix of diameters and architectures in vivo remains unknown. Here, we demonstrate that the spontaneous assembly of human vasa vasorum-derived pericytes in 3D spheroids depends on the fiber diameter and network architecture. A parallel arrangement of suspended fibers of all tested diameters (200, 500, and 800 nm) leads to the formation of spheroids, while on crosshatch networks, spheroid assembly on larger diameters is absent. The design of fibrous networks of a mix of diameters and architectures leads to the patterning of spheroids in desired locations. Fiber remodeling in parallel arrangements serves as force sensors providing mechanical insights into the assembly dynamics of spheroids and subsequent cell sprouting from spheroids. Translocation and merger of spheroids occur predominantly on parallel fiber networks, while on crosshatch networks, a cellular exchange is observed between spheroids connected with remodeled fibers. Rho kinase inhibition by Y27632 and subsequent wash-off leads to spheroid disintegration and reassembly, thus, highlighting the role of cell contractility in the assembly and integrity of 3D spheroids. Overall, using extracellular mimicking fiber networks of varying diameters and architectures, we report new insights into the 3D dynamics of spheroids which may inform pericytes role in vasculogenesis, and (patho)physiological angiogenesis

bioengineering↗

bioRxiv2022

Comprehensive analysis using DNA metabarcoding, PCR, and HPLC unveils the adulteration in Brahmi herbal products

BackgroundThe herbal products market is expanding and creating a bottleneck for raw materials. Hence, economically motivated adulteration has a high prevalence. DNA barcoding and species-specific PCR assays are now revolutionising the molecular identification of herbal products and are included in a number of pharmacopoeias for the identification of raw materials. High-throughput sequencing with barcoding advances toward metabarcoding, which enables the identification of unintentionally or intentionally unlabelled plant material present in herbal products. Brahmi is one of the most commercially significant and nootropic botanicals, with great controversy over the terms "Brahmi" being used to describe both Bacopa monneri (BM) and Centella asiatica (CA) species. PurposeThis study evaluates DNA-based methods for Brahmi herbal products with the traditional HPLC-based analytical approach in order to assess their effectiveness. MethodsWe employed a species-specific PCR assay, DNA metabarcoding using rbcL minibarcode, and HPLC to detect the presence of the Brahmi (either BM or CA) in eighteen market samples. All the methods have been validated using in-house blended formulations. ResultsComprehensive analysis of all three methods revealed the presence of 22.2%, 55.6%, and 50.0% of Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively, in Brahmi market formulations, whereas blended formulations only exhibited targeted plant species with all three methods. ConclusionSpecies-specific PCR can be used as a cost-effective and rapid method to detect the presence of the Brahmi, while in high-throughput methods, DNA metabarcoding can be used to detect the presence of widespread adulterated botanicals, and further, bioactive compounds could be detected by HPLC. These results emphasise the need for quality control of the marketed Brahmi herbal products as well as the implementation of all methodologies in accordance with fit for purpose.

molecular biology↗

bioRxiv2022

Alpha-B-Crystallin overexpression is sufficient to promote tumorigenesis and metastasis in mice

B-Crystallin is a heat shock chaperone protein which binds to misfolded proteins to prevent their aggregation. It is overexpressed in a wide-variety of cancers. Previous studies using human cancer cell lines and human xenograft models have reported tumor suppressor or tumor promoter (oncogene) roles for B-Crystallin depending on cellular context and environmental conditions. To determine the causal relationship between CRYAB overexpression and cancer, we generated a Cryab overexpression knock-in mouse model. This model revealed that constitutive overexpression of Cryab results in the formation of a variety of lethal spontaneous primary and metastatic tumors in mice. In vivo, the overexpression of Cryab correlated with the upregulation of epithelial-to-mesenchymal (EMT) markers, angiogenesis and some oncogenic proteins including Basigin. In vitro, using E1A/Ras transformed mouse embryonic fibroblasts (MEFs), we observed that the overexpression of Cryab led to the promotion of cell survival via upregulation of Akt signaling and downregulation of pro-apoptotic pathway mediator JNK, with subsequent attenuation of apoptosis as assessed by cleaved caspase-3. Overall, through the generation and characterization of Cryab overexpression model, we provide evidence supporting the role of B-Crystallin as an oncogene, where its upregulation is sufficient to induce tumors, promote cell survival and inhibit apoptosis.

cancer biology↗

bioRxiv2022

Room-temperature serial synchrotron crystallography of apo PTP1B

Room-temperature X-ray crystallography provides unique insights into protein conformational heterogeneity, but a common hurdle is obtaining sufficiently large protein crystals. Serial synchrotron crystallography (SSX) helps address this hurdle by allowing the use of many medium- to small-sized crystals. We have used a recently introduced serial sample support chip system to obtain the first SSX structure of a human phosphatase, specifically Protein Tyrosine Phosphatase 1B (PTP1B) in the unliganded (apo) state. In previous apo room-temperature structures, the active site and allosteric sites adopted alternate conformations, including open and closed conformations for the active-site WPD loop and for a distal allosteric site. By contrast, in our SSX structure, the active site is best fit with a single conformation, but the distal allosteric site is best fit with alternate conformations. This observation argues for additional nuance in interpreting the nature of allosteric coupling in this protein. Overall, our results illustrate the promise of serial methods for room-temperature crystallography, as well as future avant-garde crystallography experiments, for PTP1B and other proteins.

biophysics↗

bioRxiv2022

Viability Discrimination of Bacterial Microbiomes in Home Kitchen Dish Sponges via Propidium Monoazide Treatment

Dish sponges are known to support the survival and growth of human bacterial pathogens yet are commonly used by consumers to wash dishes and clean kitchen surfaces. Exposure to foodborne pathogens via sponge use may lead to foodborne illness, which is of particular concern among susceptible populations. Limitations associated with culture-independent or - dependent methods for bacterial community characterization have challenged the complete assessment of foodborne pathogen exposure risk presented by sponges. In this study, the bacterial microbiomes of five dish sponges were characterized to evaluate the presence of viable bacterial foodborne pathogens using propidium monoazide treatment, which is a novel approach in this medium. Total and viable sponge microbiomes were subsequently metataxonomically evaluated via targeted 16S rRNA sequencing. Select pathogen viability was confirmed using targeted selective enrichment. The comparison of total and viable sponge microbiome beta diversity indicated that sponge taxonomic abundance profiles did not vary significantly according to PMA treatment. The numbers of unique bacterial species (p-value = 0.0465) and unique bacterial foodborne pathogens (p-value = 0.0102) identified were significantly lower after PMA-treatment. A total of 20 unique bacterial foodborne pathogens were detected among sponge microbiomes regardless of PMA treatment. Three to six unique viable foodborne pathogens were identified in each sponge. Escherichia coli and Staphylococcus aureus were identified in all five viable sponge microbiomes evaluated. Viable E. coli were recovered from two of five sponges via targeted selective enrichment. These findings suggest that most sponge-associated bacterial communities may be viable and contain multiple viable bacterial foodborne pathogens. ImportanceBacterial pathogens may exist in the domestic kitchen environment, threatening both sanitation levels and the health of residents. Dish sponges are commonly used kitchen tools that can harbor foodborne pathogens as they present adequate conditions for the survival and growth of bacteria. Using a contaminated sponge may lead to foodborne illness through direct contact with pathogens or via cross-contamination with food or other surfaces. Although bacterial foodborne pathogens have been identified in sponges, previous limitations in methodology have prevented the complete understanding of sponge microbiomes. This study used a novel application of a chemical reagent coupled with targeted amplicon sequencing to identify sponge communities and differentiate between viable and non-viable bacteria. Insights into dish sponge microbiomes and potential risks of bacterial foodborne pathogen exposure can inform food safety education programs to aid in the prevention of home-acquired foodborne illness and cross-contamination events.

microbiology↗

bioRxiv2022

Targeting MAPAKAP2(MK2) to combat inflammation by avoiding the differential regulation of anti-inflammatory genes by p38 MAPK inhibitors

p38 mitogen-activated protein kinase (p38 MAPK) plays an important role in the key cellular processes related to inflammation. Several small molecule inhibitors of p38 MAPK therefore have been evaluated for their anti-inflammatory potential and progressed from early discovery to late phase clinical trials. Most of these efforts however have failed due to severe toxicity concerns. Since p38 MAPK has several downstream substrates, inhibition of p38 MAPK, therefore, leads to the modulation of all its substrates, resulting into a dis-balance of pro- and anti-inflammatory response and multiple toxicity concerns. Targeting p38MAPK MAPKAPK2 (MK2), one of the downstream substrates of p38 MAPK directly, is expected to be a better anti-inflammatory approach without having any toxicity concerns. In this manuscript, we are reporting biological data of representative MK2 inhibitor to validate its anti-inflammatory potential and a comparison of p38 MAPK and MK2 inhibitors in cell based assays to understand their relative toxicities.

pharmacology and toxicology↗

bioRxiv2022

Generalising uncertainty improves accuracy and safety of deep learning analytics applied to oncology

Trust and transparency are critical for deploying deep learning (DL) models into the clinic. DL application poses generalisation obstacles since training/development datasets often have different data distributions to clinical/production datasets that can lead to incorrect predictions with underestimated uncertainty. To investigate this pitfall, we benchmarked one pointwise and three approximate Bayesian DL models used to predict cancer of unknown primary with three independent RNA-seq datasets covering 10,968 samples across 57 primary cancer types. Our results highlight simple and scalable Bayesian DL significantly improves the generalisation of uncertainty estimation (e.g., p-value = 0.0013 for calibration). Moreover, we demonstrate Bayesian DL substantially improves accuracy under data distributional shifts when utilising uncertainty thresholding by designing a prototypical metric that evaluates the expected (accuracy) loss when deploying models from development to production, which we call the Area between Development and Production curve (ADP). In summary, Bayesian DL is a hopeful avenue of research for generalising uncertainty, which improves performance, transparency, and therefore safety of DL models for deployment in real-world.

bioinformatics↗

bioRxiv2022

Targeting Acid Ceramidase Ameliorates Fibrosis in Mouse Models of Nonalcoholic Steatohepatitis

1)Nonalcoholic fatty liver disease (NAFLD) is a common cause of liver disease worldwide, and is characterized by the accumulation of fat in the liver. Nonalcoholic steatohepatitis (NASH), an advanced form of NAFLD, is a leading cause of liver transplantation. Fibrosis is the histologic feature most associated with liver-related morbidity and mortality in patients with NASH, and treatment options remain limited. In previous studies, we discovered that acid ceramidase (aCDase) is a potent antifibrotic target using human hepatic stellate cells (HSCs) and models of hepatic fibrogenesis. Using two dietary mouse models, we demonstrate that depletion of aCDase in HSC reduces fibrosis without worsening metabolic features of NASH, including steatosis, inflammation, and insulin resistance. Consistently, pharmacologic inhibition of aCDase ameliorates fibrosis but does not alter metabolic parameters. The findings suggest that targeting aCDase is a viable therapeutic option to reduce fibrosis in patients with NASH.

systems biology↗

bioRxiv2022

Integration of synthetic microbial consortia based bioprocessing with pyrolysis for efficient conversion of cellulose to valuables

Improved technologies are needed for sustainable conversion of cellulosic waste to valuable products. Here we demonstrate the successful integration of a synthetic microbial consortium (SynCONS) based consolidated bioprocessing with pyrolysis to produce commodity chemicals from cellulose. Promising microbial partners were rationally identified from 7626 organisms via comparative metabolic mapping which led to establishing two promising SynCONS with abilities to convert cellulose to ethanol and lactate in bioreactors. The partners in the two SynCONS were a) the mesophilic fungus Trichoderma reesei grown sequentially with the thermophilic bacterium Parageobacillus thermoglucosidasius NCIMB 11955 (TrPt) and b) a thermophilic bacterium Thermobifida fusca grown together with Parageobacillus thermoglucosidasius NCIMB 11955 (TfPt). TrPt sequential bioprocessing resulted in 39% (g/g) cellulose consumption with product yields up to 9.3% g/g (ethanol + lactate). The TfPt co-cultures demonstrated a cellulose consumption of 30% (g/g) and combined yields of ethanol and lactic acid up to 23.7% g/g of consumed cellulose. The total product yields were further enhanced (51% g/g cellulose) when commercially available cellulases were used in place of T. fusca. Furthermore, when the metabolically engineered ethanol-producing strain of P. thermoglucosidasius TM242 (TfPt242) was substituted in the thermophilic TfPt co-culture consortium, ethanol yields were substantially higher (32.7% g/g of consumed cellulose). Finally, subjecting the residual cellulose and microbial biomass to pyrolysis resulted in carbon material with physicochemical properties similar to commercially available activated carbon as analysed using Scanning Electron Microscopy, X-Ray Diffraction and Raman spectroscopy. Overall, the integration of this synthetic microbial consortia-based bioprocessing strategy with pyrolysis demonstrated a promising strategy for conversion of waste cellulose to chemicals, biofuels, and industrial carbon potentially suitable for several industrial applications.

bioengineering↗

bioRxiv2022

Estimating the causal relationship between smoking and abdominal adiposity using Mendelian randomization

Background and AimsSmokers tend to have a lower body weight than non-smokers, but also more abdominal fat. It remains unclear whether the relationship between smoking and abdominal obesity is causal. Previous Mendelian randomization studies have investigated this relationship by relying on a single genetic variant for smoking heaviness. This approach is sensitive to pleiotropic effects and may produce imprecise causal estimates. We aimed to assess causality between smoking and abdominal obesity using multiple genetic instruments. MethodsWe used GWAS results for smoking initiation (n=1,232,091), lifetime smoking (n=462,690) and smoking heaviness (n=337,334) as exposure traits, and waist-hip ratio (WHR) and waist and hip circumferences (WC and HC) (n up to 697,734), with and without adjustment for body mass index (adjBMI), as outcome traits. We implemented Mendelian randomization using the CAUSE and LHC-MR methods that instrument smoking using genome-wide data. ResultsBoth CAUSE and LHC-MR indicated a positive causal effect of smoking initiation on WHR (0.13 [95%CI 0.10, 0.16] and 0.49 [0.41, 0.57], respectively) and WHRadjBMI (0.07 [0.03, 0.10] and 0.31 [0.26, 0.37]). Similarly, they indicated a positive causal effect of lifetime smoking on WHR (0.35 [0.29, 0.41] and 0.44 [0.38, 0.51]) and WHRadjBMI (0.18 [0.13, 0.24] and 0.26 [0.20, 0.31]). In follow-up analyses, smoking particularly increased visceral fat. There was no evidence of a mediating role by cortisol or sex hormones. ConclusionsSmoking initiation and higher lifetime smoking may lead to abdominal fat distribution. The increase in abdominal fat due to smoking was characterized by an increase in visceral fat. Thus, efforts to prevent and cease smoking can have the added benefit of reducing abdominal fat.

genomics↗

bioRxiv2022

Anti-COVID-19 Activity of FDA Approved Drugs through RNA G-quadruplex Binding

The COVID-19 pandemic caused by SARS-CoV-2 has caused millions of infections and deaths worldwide. Limited treatment options and the threat from emerging variants underline the need for novel and widely accessible therapeutics. G-quadruplexes (G4s) are nucleic acid secondary structures known to affect many cellular processes including viral replication and transcription. We identified heretofore not reported G4s with remarkably low mutation frequency across >5 million SARS-CoV-2 genomes. The G4 structure was targeted using FDA-approved drugs that can bind G4s - Chlorpromazine (CPZ) and Prochlorperazine (PCZ). We found significant inhibition in lung pathology and lung viral load of SARS-CoV-2 challenged hamsters when treated with CPZ, PCZ that was comparable to the widely used antiviral drug Remdesivir. In support, in vitro G4 binding, inhibition of reverse transcription from RNA isolated from COVID-infected humans, and attenuated viral replication and infectivity in Vero cell cultures were clear in case of both CPZ/PCZ. Apart from the wide accessibility of CPZ/PCZ, targeting relatively invariant nucleic acid structures poses an attractive strategy against fast mutating viruses like SARS-CoV-2.

molecular biology↗

bioRxiv2022

Universal functions of prion candidates across all three domains of life suggest a primeval role of protein self-templating.

Amyloid-based prions have simple structures, a wide phylogenetic distribution, and a plethora of functions in contemporary organisms, suggesting they may be an ancient phenomenon. However, this hypothesis has yet to be addressed with a systematic, computational, and experimental approach. Here we present a framework to help guide future experimental verification of candidate prions with conserved functions in order to understand their role in the early stages of evolution and potentially in the origins of life. We identified candidate prions in all high-quality proteomes available in UniProt computationally, assessed their phylogenomic distributions, and analyzed candidate-prion functional annotations. Of the 27,980,560 proteins scanned, 228,561 were identified as candidate prions ([~]0.82%). Among these candidates, there were 84 Gene Ontology (GO) terms conserved across the 3 domains of life. We found that candidate prions with a possible role in adaptation were particularly well-represented within this group. We discuss unifying features of candidate prions to elucidate the primeval roles of prions and their associated functions. Candidate prions annotated as transcription factors, DNA binding, and kinases are particularly well suited to generating diverse responses to changes in their environment and could allow for adaptation and population expansion into more diverse environments. We hypothesized that these functions could be evolutionarily ancient, even if individual prion domains themselves are not evolutionarily conserved. Candidate prions annotated with these universally-occurring functions potentially represent the oldest extant prions on Earth and are therefore excellent experimental targets.

bioinformatics↗

bioRxiv2022

Transcriptional signature pattern in black, blue and purple wheat and impact on seed pigmentation and other associated features: Comparative transcriptomics, genomics and metabolite profiling

Anthocyanin biosynthesis in plants is complex, especially in a polyploid monocot wheat plant. Using whole-genome sequencing, transcriptomics, and LC-MS/MS, we investigated anthocyanin production in pigmented (black, blue, and purple) wheat seeds. According to differential gene expression profiling, 2AS-MYC, 7DL-MYB, WD40 regulatory genes controls purple pericarp coloration, 4DL-MYC, 2AS-MYC, 7DL-MYB, WD40 controls blue aleurone coloration, and 4DL-MYC, 7DL-MYB, WD40 controls black aleurone colour. We believe that at least one MYC and MYB isoform is sufficient to regulate the anthocyanin synthesis in pericarp or aleurone. Based upon the reduced expressions of the genes belonging to the 4D, SSR molecular marker mapping, variant calling using genome sequencing and IGV browser gene structure visualization, it was inferred that the advanced black and blue wheat lines were substitution lines (4E{4D}), with very small recombinations. Pericarp anthocyanin profiling is controlled by a mutation in chromosome 2AS of purple wheat, and environmental variations more influence pigmented pericarp trait. The expression patterns of anthocyanin structural and other genes varied in different colored wheat, corroborating differences in agronomical metrics.

plant biology↗

bioRxiv2022

Identification of a novel deFADding activity in 5' to 3' exoribonucleases

Identification of metabolite caps including FAD on the 5 end of RNA has uncovered a previously unforeseen intersection between cellular metabolism and gene expression. To understand the function of FAD caps in cellular physiology, we characterised the proteins interacting with FAD caps in budding yeast. Here we demonstrate that highly conserved 5-3 exoribonucleases, Xrn1 and Rat1, physically interact with the RNA 5 FAD cap and both possess FAD cap decapping (deFADding) activity and subsequently degrade the resulting RNA. Xrn1 deFADding activity was also evident in human cells indicating its evolutionary conservation. Furthermore, we report that the recently identified bacterial 5-3 exoribonuclease RNase AM also possesses deFADding activity that can degrade FAD-capped RNAs in vitro and in E. coli cells. To gain a molecular understanding of the deFADding reaction, an RNase AM crystal structure with three manganese ions coordinated by a sulfate molecule and the active site amino acids was generated that provided details underlying hydrolysis of the FAD cap. Our findings reveal a general propensity for 5-3 exoribonucleases to hydrolyse and degrade RNAs with 5 end noncanonical caps in addition to their well characterized 5 monophosphate RNA substrates indicating an evolutionarily conserved intrinsic property of 5-3 exoribonucleases.

molecular biology↗