Biology subjects

Sharma, R.

Publications and source records attributed to Sharma, R..

At least 163 records · Page 9Linked to original sources

bioRxiv2022

Optimization of the Illumina COVIDSeq™ protocol for decentralized, cost-effective genomic surveillance

A decentralized surveillance system to identify local outbreaks and monitor SARS-CoV-2 Variants of Concern is one of the primary strategies for the pandemics containment. Although next-generation sequencing (NGS) is a gold standard for genomic surveillance and variant discovery, the technology is still cost-prohibitive for decentralized sequencing, particularly in small independent labs with limited resources. We have optimized the Illumina COVID-seq protocol to reduce cost without compromising accuracy. 90% of genomic coverage was achieved for 142/153 samples analyzed in this study. The lineage was correctly assigned to all samples (152/153) except for one. This modified protocol can help laboratories with constrained resources contribute to decentralized SARS-CoV-2 surveillance in the post-vaccination era.

genomics↗

bioRxiv2022

Flexible IrOx Neural Electrode for Mouse Vagus Nerve Stimulation

Vagus nerve stimulation (VNS) is being actively explored as a treatment for multiple conditions as part of bioelectronic medicine research. Reliable and safe VNS in mouse models is a critical need for understanding mechanisms of these. We report on the development and evaluation of a microfabricated cuff electrode (MouseFlex) constructed of polyimide (PI) and with iridium oxide (IrOx) electrodes that is thermoformed to 86 {micro}m {+/-} 12 {micro}m radius to interface the mouse cervical vagus nerve (r {approx} 50 {micro}m). Innovative bench-top methods were used to evaluated the stimulation stability and electrochemical properties of electrodes. Our aggressive stimulation stability (Stim-Stab) test utilized 1 billion pulses at a 1000 Hz with a current density of 6.28 A/cm2 (1.51 mC/cm2/phase) to evaluate electrode lifetimes, and all electrodes remained functional. We also investigated the effects of thermoforming on their impedance, charge storage capacity (CSC), and charge injection capacity (CIC). The modest changes in electrochemical properties indicate that the thermoforming process was well tolerated. Thermoformed electrode safety and efficacy were evaluated in-vivo by performing acute VNS in mice and monitoring their heart and respiration rate as biomarkers. Their electrochemical properties were also measured before, during and after VNS. Bradycardia and bradypnea were reliably induced at stimulation currents of 100 to 200 {micro}A, well below the in-vivo CIC of ~1250 {micro}A (~0.5 mC/cm2), supporting their safety and efficacy. The electrode impedance increased and CIC decreased during in-vivo use, but largely reversed these changes in in-vitro testing after enzymatic cleaning, supporting their tolerance for surgical use.

bioengineering↗

bioRxiv2022

In-vitro activity of Cefiderocol against clinical isolates of meropenem-resistant Klebsiella pneumoniae from India.

BackgroundCefiderocol (FDC), a novel siderophore drug, is active against gram negative bacteria producing carbapenemases, including metallo-beta-lactamases. ObjectiveTo compare the in-vitro activity of FDC with ceftazidime-avibactam (CZA), CZA/ aztreonam (AT) combination and colistin (CST), in clinical isolates of meropenem-resistant (MER-R) Klebsiella pneumoniae. MethodsFrom 2052 clinical specimens submitted for culture testing, 245 K. pneumoniae isolates were recovered within a six month period in 2021. 103 non-duplicate, non-outbreak, MER-R (MIC >4 g/ml) strains were included in the study. Identification and susceptibility was performed using VITEK-2 (bioMerieux). 10 meropenem-susceptible isolates served as controls. For FDC, BMD was performed after in-house standardisation. Disc diffusion (Liofilchem, Italy) and broth microdilution (BMD; ComASP, STC, Liofilchem, Italy) were used for susceptibility testing of CZA and CST respectively. Synergy testing for CZA and aztreonam (AT) was performed using disk approximation method. CLSI breakpoints were used for interpretation of results. ResultsFor FDC, MIC50 and MIC90 was 2 g/ml and 8 g/ml, respectively. 80% isolates were susceptible to FDC. 26.2% isolates were susceptible to CZA, synergy testing with CZA/ AT was positive for 74 (72%) of the isolates. 89.3% had intermediate susceptibility to CST. Nine (8.7%) were susceptible only to FDC. ConclusionFDC is active in-vitro against MER-R K. pneumoniae > CZA/AT> CZA > CST, as observed in this study, applying CLSI criteria. Clinico-microbiological studies should be performed for assessing clinical efficacy of this novel drug in this region with high prevalence of carbapenem resistance among gram-negative organisms.

microbiology↗

bioRxiv2022

Chromosome 9p21.3 Coordinates Cell Intrinsic and Extrinsic Tumor Suppression

Somatic chromosomal deletions are prevalent in cancer, yet their functional contributions remain ill-defined. Among the most prominent of these events are deletions of chromosome 9p21.3, which disable a cell intrinsic barrier to tumorigenesis by eliminating the CDKN2A/B tumor suppressor genes. However, half of 9p21.3 deletions encompass a cluster of 16 type I interferons (IFNs) whose co-deletions have not been functionally characterized. To dissect how 9p21.3 and other genomic deletions impact cancer, we developed MACHETE (Molecular Alteration of Chromosomes with Engineered Tandem Elements), a genome engineering strategy that enables flexible modeling of megabase-sized deletions. Generation of 9p21.3-syntenic deletions in a mouse model of pancreatic cancer revealed that concomitant loss of Cdkn2a/b and the IFN cluster led to immune evasion and metastasis compared to Cdkn2a/b-only deletions. Mechanistically, IFN co-deletion disrupted type I IFN signaling, altered antigen-presenting cells, and facilitated escape from CD8+ T cell surveillance in a cell extrinsic manner requiring loss of interferon epsilon (Ifne). Our results establish co-deletions of the IFN cluster as a pervasive route to tumor immune evasion and metastasis, revealing how deletions can disable physically linked cell intrinsic and extrinsic tumor suppression. Our study establishes a framework to dissect the functions of genomic deletions in cancer and beyond.

cancer biology↗

bioRxiv2022

Deep-Worm-Tracker: Deep Learning Methods for Accurate Detection and Tracking for Behavioral Studies in C. elegans

Accurate detection and tracking of model organisms such as C. elegans worms remains a fundamental task in behavioral studies. Traditional Machine Learning (ML) and Computer Vision (CV) methods produce poor detection results and suffer from repeated ID switches during tracking under occlusions and noisy backgrounds. Using Deep Learning (DL) methods, the task of animal tracking from video recordings, like those in camera trap experiments, has become much more viable. The large amount of data generated in ethological studies, makes such models suitable for real world scenarios in the wild. We propose Deep-Worm-Tracker, an end to end DL model, which is a combination of You Only Look Once (YOLOv5) object detection model and Strong Simple Online Real Time Tracking (Strong SORT) tracking backbone that is highly accurate and provides tracking results in real time inference speeds. Present literature has few solutions to track animals under occlusions and even fewer publicly available large scale animal re-ID datasets. Thus, we also provide a worm re-ID dataset to minimize worm ID switches, which, to the best of our knowledge, is first-of-its-kind for C. elegans. We are able to track worms at a mean Average Precision (mAP@0.5) > 98% within just 9 minutes of training time with inference speeds of 9-15 ms for worm detection and on average 27 ms for worm tracking. Our tracking results show that Deep-Worm-Tracker is well suited for ethological studies involving C. elegans.

animal behavior and cognition↗

bioRxiv2022

Reduced γ-glutamyl hydrolase activity likely contributes to high folate levels in Periyakulam-1 tomato

Tomato cultivars show wide variation in nutraceutical folate in ripe fruits, yet the loci regulating folate levels in fruits remain unexplored. To decipher regulatory points, we compared two contrasting tomato cultivars: Periyakulam-1 (PKM-1) with high folate and Arka Vikas (AV) with low folate. The progression of ripening in PKM-1 was nearly similar to AV but had substantially lower ethylene emission. In parallel, the levels of phytohormones salicylic acid, ABA, and jasmonic acid were substantially lower than AV. The fruits of PKM-1 were also metabolically distinct from AV, with upregulation of several amino acids. Consistent with higher {degrees}Brix levels, the red ripe fruits also showed upregulation of sugars and sugar-derived metabolites. In parallel with higher folate, PKM-1 fruits also had higher carotenoid levels, especially lycopene and {beta}-carotene. Transcript levels of genes encoding folate biosynthesis did not show a perceptible difference in relative expression. The proteome analysis showed upregulation of carotenoid sequestration and folate metabolism-related proteins in PKM-1. The deglutamylation pathway mediated by {gamma}-glutamyl hydrolase (GGH) was substantially reduced in PKM-1 at the red-ripe stage. The red-ripe fruits had reduced transcript levels of GGHs and lower GGH activity than AV. Conversely, the percent polyglutamylation of folate was much higher in PKM-1. Our analysis indicates the regulation of GGH activity as a potential target to elevate folate levels in tomato fruits.

plant biology↗

bioRxiv2022

Oxytocin receptor is not required for social attachment in prairie voles

Prairie voles are among a small group of mammals that display long-term social attachment between mating partners. Many pharmacological studies show that signaling via the oxytocin receptor (OxtR) is critical for the display of social monogamy in these animals. We used CRISPR-mutagenesis to independently generate three different OxtR null mutant prairie vole lines. OxtR mutants displayed social attachment such that males and females showed a behavioral preference for their mating partners over a stranger of the opposite sex when assayed using different paradigms. Mothers lacking OxtR delivered viable pups, and parents displayed care of their young and raised them to the weanling stage. Together, our studies unexpectedly reveal that OxtR-mediated signaling is genetically dispensable for social attachment, parturition, and parental behavior.

animal behavior and cognition↗

bioRxiv2022

C-terminal disulfide bond in SUN2 regulate dynamic remodeling of LINC complexesat the nuclear envelope

The LINC complex tethers the cell nucleus to the cytoskeleton to regulate mechanical forces during cell migration, differentiation, and diseases. The function of LINC complexes relies on the interaction between highly conserved SUN and KASH proteins that form higher-order assemblies capable of load bearing. These structural details have emerged from in vitro assembled LINC complexes however, the principles of in vivo assembly remain obscure. Here, we report a conformation-specific SUN2 antibody as a tool to visualize LINC complex dynamics in situ. Using imaging, biochemical and cellular methods, we find that conserved cysteines in SUN2 undergo KASH dependent inter- and intra- molecular disulfide bond rearrangements. Disruption of the SUN2 terminal disulfide bond compromises SUN2 localization, turnover, LINC complex assembly as well as cytoskeletal organization and cell migration. Moreover, using pharmacological and genetic interventions, we identify components of the ER lumen as SUN2 cysteines redox state regulators. Overall, we provide evidence for SUN2 disulfide bond rearrangement as a physiologically relevant structural modification that regulates LINC complex functions.

cell biology↗

bioRxiv2022

The autism-associated gene SYNGAP1 regulates human cortical neurogenesis

Autism spectrum disorder (ASD) is a genetically heterogeneous disorder linked with rare, inherited and de novo mutations occurring in two main functional gene categories: gene expression regulation and synaptic function1. Accumulating evidence points to dysregulation in cortical neurogenesis as a convergent mechanism in ASD pathophysiology2-8. While asynchronous development has been identified as a shared feature among ASD-risk genes in the category of gene expression regulation, it remains unknown whether this phenotype is also associated with ASD-risk genes in the synaptic function category. Here we show for the first time the expression of the synaptic Ras GTP-ase activating protein 1 (SYNGAP1), one of the top ASD risk genes9, in human cortical progenitors (hCPs). Interestingly, we found that multiple components of the postsynaptic density (PSD) of excitatory synapses, of which SYNGAP1 is one of the most abundant components 10,11, are enriched in the proteome of hCPs. Specifically, we discover that SYNGAP1 is expressed within the apical domain of human radial glia cells (hRGCs) where it lines the wall of the developing cortical ventricular zone colocalizing with the tight junction-associated protein and MAGUK family member TJP1. In a cortical organoid model of SYNGAP1 haploinsufficiency, we show dysregulated cytoskeletal dynamics that impair the scaffolding and division plane of hRGCs, resulting in disrupted lamination of the cortical plate and accelerated maturation of cortical projection neurons. Overall, the discovery of the expression and function of SYNGAP1 in cortical progenitor cells reframes our understanding of the pathophysiology of SYNGAP1-related disorders and, more broadly, underscores the importance of dissecting the role of synaptic genes associated with neurodevelopmental disorders in distinct cell types across developmental stages.

developmental biology↗

bioRxiv2022

An auto-inhibited state of protein kinase G and implications for selective activation

Cyclic GMP-dependent protein kinases (PKGs) are key mediators of the nitric oxide/cGMP signaling pathway that regulates biological functions as diverse as smooth muscle contraction, cardiac function, and axon guidance. Campaigns targeting nitric oxide synthases and cyclic nucleotide phosphodiesterases in this signaling axis suggest that understanding how cGMP differentially triggers mammalian PKG isoforms could lead to new therapeutics that inhibit or activate PKGs. Alternate splicing of PRKG1 transcripts confers distinct leucine zippers, linkers, and auto-inhibitory pseudo-substrate sequences to PKG I and I{beta} that result in isoform-specific activation properties, but the mechanism of enzyme auto-inhibition and its alleviation by cGMP is still not well understood. Here we present a crystal structure of PKG I{beta} in which the auto-inhibitory sequence and the cyclic nucleotide binding domains are bound to the catalytic domain, providing a snapshot of the auto-inhibited state. Specific contacts between the PKG I{beta} auto-inhibitory sequence and the enzyme active site help explain isoform-specific activation constants and the effects of phosphorylation in the linker. We also present a crystal structure of a PKG I cyclic nucleotide binding domain with an activating mutation linked to Thoracic Aortic Aneurysms and Dissections. Similarity of this structure to wild type cGMP-bound domains and differences with the auto-inhibited enzyme provide a mechanistic basis for constitutive activation. We show that PKG I{beta} auto-inhibition is mediated by contacts within each monomer of the native full-length dimeric protein, and using the available structural and biochemical data we develop a model for the regulation and activation of PKGs.

biochemistry↗

bioRxiv2022

Upregulation of Somatostatin Receptor Type 2 in a Receptor-Deficient In Vivo Pancreatic Neuroendocrine Tumor Model Improves Tumor Response to Targeted 177Lu-DOTATATE

PurposeThe goal of this study was to test whether histone deacetylase inhibitors (HDACis) restore somatostatin receptor type 2 (SSTR2) expression in models of high-grade pancreatic neuroendocrine tumors (PNETs), thereby facilitating effective treatment with 177Lu-DOTATATE therapy. MethodsTo assess tumor grade correlation with SSTR2 expression, we assessed human SSTR2 promoter methylation and expression levels in 96 NIH patient samples and merged the GSE149395 and GSE117852 datasets. We used three NET cell lines (QGP-1, BON-1, GOT-1) characterized by variable SSTR2 expression profiles for functional in vitro studies using HDACis. Finally, the QGP-1 xenograft mouse model, with low basal SSTR2 expression, was used to analyze the therapeutic efficacy of combined HDACi and 177Lu-DOTATATE therapies. ResultsHuman PNET SSTR2 promoter methylation showed a significant positive correlation with higher tumor grades (P = 0.000014). We also found a significant negative correlation (P < 0.0001) between SSTR2 promoter methylation and SSTR2 expression in three NET cell lines. In vitro, SSTR2 expression increased significantly in BON-1 and QGP-1 cells at 48 and 72 hours in a dose-dependent fashion using two different HDACis, valproic acid and CI-994. In vivo studies demonstrated a significant increase in 177Lu-DOTATATE tumor uptake in QGP-1-engrafted mice after 10 days of CI-994 pretreatment (P = 0.0175). Treatment with 177Lu-DOTATATE reduced tumor size in mice pretreated with CI-994 compared to 177Lu-DOTATATE alone (at 15 days, P = 0.0028). ConclusionHDACis increase SSTR2 surface expression in models of high-grade, SSTR2-deficient PNETs. This approach has the potential to improve tumor response to targeted therapy with 177 Lu-DOTATATE in patients with receptor-negative, metastatic PNETs. Translational Relevance StatementPancreatic neuroendocrine tumors (PNETs) express high levels of somatostatin receptor type 2 (SSTR2), a unique target for both tumor imaging and therapy. Unfortunately, high-grade PNETs lose SSTR2 surface expression and thus become ineligible for SSTR2-targeted 177Lu-DOTATATE peptide receptor radionuclide therapy (PRRT). Restoring SSTR2 expression through the reversal of inhibitory epigenetic gene silencing mechanisms has the potential for improving tumor responsiveness to PRRT. We demonstrate that histone deacetylase inhibitors (HDACis) upregulate SSTR2 surface expression in three NET cell lines in vitro. In an in vivo PNET xenograft model with low basal SSTR2 expression, our studies validate a significantly higher tumor uptake of SSTR2-targeted 177Lu-DOTATATE in animals pretreated with HDACis compared to controls. Furthermore, we show that this higher tumor uptake results in significant anti-tumor response when compared to standard PRRT alone. Our preclinical results thus provide a rationale for utilizing HDACi pretreatment to improve targeted radionuclide therapy in patients with SSTR2-negative, metastatic PNETs.

molecular biology↗

bioRxiv2022

Whole-Genome Profiling of Ethyl Methanesulfonate Mutagenesis in Tomato

The induced mutations accelerate crop improvement by providing novel disease resistance and yield alleles. However, the alleles with no perceptible phenotype but having an altered function remain hidden in mutagenized plants. The whole-genome sequencing (WGS) of mutagenized individuals uncovers the complete spectrum of mutations in the genome. We sequenced 132 doubly ethyl methanesulfonate (EMS)-mutagenized lines of tomato and detected ca. 41 million SNPs and 5.5 million short-INDELs. We found a very high average density of mutations 1/3.05 Kb compared to other species. About 97% of the genome had mutations, including the genes, promoters, UTRs, and introns. More than 1/3rd of genes in the mutagenized population had one or more deleterious mutations predicted by SIFT. Nearly 1/4th of deleterious genes mapped on tomato metabolic pathways modulating multiple pathway steps. Contrary to the reported GC>AT transition bias for EMS, we found EMS also produced nearly equal AT>GC transitions. Comparing mutation frequency among synonymous codons revealed that the most preferred codon is least mutagenic towards EMS. The reduction in carotenoids in {zeta}-carotene isomerase mutant fruits and chloroplasts relocation loss in phototropin1 mutant validated the mutation discovery pipeline. Our study makes a large repertoire of mutations accessible to genetic studies and the breeding of tomato.

plant biology↗

bioRxiv2022

Lhx2 in germ cells suppresses endothelial cell migration in the developing ovary

LIM-homeobox genes play multiple roles in developmental processes, but their roles in gonad development are not completely understood. Herein, we report that Lhx2, Ils2, Lmx1a, and Lmx1b are expressed in a sexually dimorphic manner in mouse, rat, and human gonads during sex determination. Amongst these, Lhx2 has female biased expression in the developing gonads of species with environmental and genetic modes of sex determination. Single-cell RNAseq analysis revealed that Lhx2 is exclusively expressed in the germ cells of the developing mouse ovaries. To elucidate the roles of Lhx2 in the germ cells, we analyzed the phenotypes of Lhx2 knockout XX gonads. While the gonads developed appropriately in Lhx2 knockout mice and the somatic cells were correctly specified in the developing ovaries, transcriptome analysis revealed enrichment of genes in the angiogenesis pathway. There was an elevated expression of several pro-angiogenic factors in the Lhx2 knockout ovaries. The elevated expression of pro-angiogenic factors was associated with an increase in numbers of endothelial cells in the Lhx2-/-ovaries at E13.5. Gonad recombination assays revealed that the increased numbers of endothelial cells in the XX gonads in absence of Lhx2 was due to ectopic migration of endothelial cells in a cell non-autonomous manner. We also found that, there was increased expression of several endothelial cell-enriched male-biased genes in Lhx2 knockout ovaries. Also, in absence of Lhx2, the migrated endothelial cells formed an angiogenic network similar to that of the wild type testis, although the coelomic blood vessel did not form. Together, our results suggest that Lhx2 in the germ cells is required to suppress vascularization in the developing ovary. These results suggest a need to explore the roles of germ cells in the control of vascularization in developing gonads. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/483280v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@59ca47org.highwire.dtl.DTLVardef@ef193eorg.highwire.dtl.DTLVardef@162651corg.highwire.dtl.DTLVardef@dc3d0f_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightO_LIMultiple LIM-HD genes are expressed in developing gonads during the window of sex determination with Lhx2 having female dominating expression in an evolutionarily conserved manner C_LIO_LILhx2 is expressed in the germ cells of developing mouse ovaries C_LIO_LILoss of Lhx2 in the developing ovaries alters the expression of genes involved in various pathways including angiogenesis C_LIO_LILhx2 in germ cells suppress endothelial cell migration in the developing ovaries C_LI

developmental biology↗

bioRxiv2022

Dissociable default-mode subnetworks subserve childhood attention and cognitive flexibility: evidence from deep learning and stereotaxic electroencephalography

BackgroundCognitive flexibility encompasses the ability to efficiently shift focus and forms a critical component of goal-directed attention. The neural substrates of this process are incompletely understood in part due to difficulties in sampling the involved circuitry. MethodsStereotactic intracranial recordings that permit direct resolution of local-field potentials from otherwise inaccessible structures were employed to study moment-to-moment attentional activity in children with epilepsy during the performance of an attentional set-shifting task. A combined deep learning and model-agnostic feature explanation approach was used to analyze these data and decode attentionally-relevant neural features. Connectomic profiling of highly predictive attentional nodes was further employed to examine task-related engagement of large-scale functional networks. ResultsThrough this approach, we show that beta/gamma power within executive control, salience, and default mode networks accurately predicts single-trial attentional performance. Connectomic profiling reveals that key attentional nodes exclusively recruit dorsal default mode subsystems during attentional shifts. ConclusionsThe identification of distinct substreams within the default mode system supports a key role for this network in cognitive flexibility and attention in children. Furthermore, convergence of our results onto consistent functional networks despite significant inter-subject variability in electrode implantations supports a broader role for deep learning applied to intracranial electrodes in the study of human attention. FundingNo funds supported this specific investigation. Awards and grants supporting authors include: Canadian Institutes of Health Research (CIHR) Vanier Scholarship (NMW, HY); CIHR Frederick Banting and Charles Best Canada Graduate Scholarship Doctoral Award (SMW); CIHR Canada Graduate Scholarship Masters Award (ONA); and a CIHR project grant (GMI).

neuroscience↗

bioRxiv2022

Development of duplex real time PCR for quick detection of Cryptosporidia in goats

Cryptosporidium spp. is the most important foodborne and waterborne pathogens and the leading cause of mortality from foodborne and waterborne gastrointestinal disease. In neonates of domestic animals it is associated with consistent diarrhoea and dehydration. Cryptosporidium infection begins with the ingestion of sporulated oocytes disseminated by carrier animals that consistently contaminate the environment. Many diagnostic tests are available including microscopy, antigen trap ELISA, but none of the diagnostic tests available currently cannot differentiate between active and passive infection in the host. In the current study, to address this challenge an mRNA based duplex TaqMan(R) probe PCR (dRT-qPCR) was developed to target the Cryptosporidium oocyst wall protein (COWP) gene and 18ssu rRNA gene in a single tube that can detect metabolically active Cryptosporidial oocysts. The mRNA transcripts are the direct indicator of any actively replicating cell and it will help decipher the active stages of its lifecycle in host. This diagnostic assay was standardized by computing transcript copy number-based limit of detection. For COWP and 188ssu rRNA genes the limit of detection was 7.08x1004 and 5.95x1005 respectively. During active infections the oocyst wall protein will be active and so its COWP gene transcripts will act as marker for active infection. While transcripts for 18SSU rRNA are constitutively expressing in Cryptosporidial life cycle. This current diagnostic assay will be a quantitative marker that will help assess active stages of Cryptosporidium infection in neonates. The disease dynamics will help better understand to formulate the control strategies and contain infection among the healthy animals. ImportanceCryptosporidiosis is an important neonatal disease affecting goats causing diarrhoea, dehydration and stunted growth. For diagnosing this condition, many diagnostic tests are available including microscopy, immunological tests, but none of the diagnostic tests available currently can differentiate between active and passive infection in the host. The mRNA transcripts are the direct indicator of any actively replicating cell and especially in intracellular parasites it will help decipher the infective stages of a lifecycle in the host, and hence the test was developed in a reverse transcriptional format in a duplex mode. The currently developed diagnostic assay for cryptosporidiosis was evaluated for sensitivity using Limit of detection (LOD). This diagnostic test will act as a quantitative marker to aid in detecting active stages of Cryptosporidium infection in neonatal goats and will eventually lead to better control strategies for managing cryptosporidial infections in the future.

molecular biology↗

bioRxiv2022

Confirming Multiplex Q-PCR Use in COVID-19 with Next Generation Sequencing: Strategies for Epidemiological Advantage

Rapid classification and tracking of emerging SARS-CoV-2 variants are critical for understanding the transmission dynamics and developing strategies for interrupting the transmission chain. Next-Generation Sequencing (NGS) is an exceptional tool for whole-genome analysis and deciphering new mutations. The technique has been instrumental in identifying the Variants of Concern and tracking this pandemic. However, NGS remains expensive and time-consuming for large-scale monitoring of COVID-19. This study analyzed a total of 78 de-identified samples that screened positive for SARS-CoV-2 from two timeframes, August 2020 and July 2021. All 78 samples were classified into WHO lineages by whole genome sequencing then compared with two commercially available Q-PCR assays for spike protein mutation(s). The data showed good concordance with Q-PCR and NGS analysis for specific SARS-COV-2 lineages and characteristic mutations. Deployment of Q-PCR testing to detect known SARS-COV-2 variants may be extremely beneficial. These assays are quick and cost-effective, thus can be implemented as an alternative to sequencing for screening known mutations of SARS-COV-2 for clinical and epidemiological interest. The findings support the great potential for Q-PCR to be an effective strategy offering several COVID-19 epidemiological advantages.

genomics↗

bioRxiv2022

Call count surveys as a tool to monitor habitats occupied by Cheer Pheasants (Catreus wallichii) in Shimla, Himachal Pradesh

The vulnerable Cheer Pheasant (Catreus wallichii) is found in the western Himalayas. It prefers successional grasslands which are maintained by residents near villages. Therefore, it is prone to anthropogenic pressures which have led to local extinctions and a decline in population. We conducted five call count surveys at dusk and dawn from 15 April - 15 May 2020 in grasslands of Dharbhog panchayat, Shimla, Himachal Pradesh to assess presence of Cheer Pheasants in different habitat types. We used frequency of occurrence (number of encounters / number of visits) to determine presence or absence of Cheer Pheasant in an area of 10.5 km2 with ten established call count stations. We encountered Cheer Pheasants at six call count stations and the frequency of occurrence of the species ranged from 0 - 0.60. This survey provides an index of occupancy of the species and can be used to assess the change in habitat occupied by the species with time. The local forest department can also use these results to regulate over grazing and burning of grasslands during the breeding season in areas where we recorded the species.

ecology↗

bioRxiv2022

Triple-helix potential of the mouse genome

Certain DNA sequences, including mirror-symmetric polypyrimidine/polypurine runs, are capable of folding into a triple-helix-containing non-B-form DNA structure called H-DNA. Such H-DNA-forming sequences are frequent in many eukaryotic genomes, including in mammals, and multiple lines of evidence indicate that these motifs are mutagenic and can impinge on DNA replication, transcription, and other aspects of genome function. In this study, we show that the triplex-forming potential of H-DNA motifs in the mouse genome can be evaluated using S1-sequencing (S1-seq), which uses the single-stranded DNA (ssDNA)-specific nuclease S1 to generate deep-sequencing libraries that report on the position of ssDNA throughout the genome. When S1-seq was applied to genomic DNA isolated from mouse testis cells and splenic B cells, we observed prominent clusters of S1-seq reads that appeared to be independent of endogenous double-strand breaks, that coincided with H-DNA motifs, and that correlated strongly with the triplex-forming potential of the motifs. Fine-scale patterns of S1-seq reads, including a pronounced strand asymmetry in favor of centrally-positioned reads on the pyrimidine-containing strand, suggested that this S1-seq signal is specific for one of the four possible isomers of H-DNA (H-y5). By leveraging the abundance and complexity of naturally occurring H-DNA motifs across the mouse genome, we further defined how polypyrimidine repeat length and the presence of repeat-interrupting substitutions modify the structure of H-DNA. This study provides a new approach for studying DNA secondary structure genome wide at high spatial resolution.

molecular biology↗