Biology subjects

Singh, A. K.

Publications and source records attributed to Singh, A. K..

At least 91 records · Page 5Linked to original sources

bioRxiv2022

Dynamic single-cell RNA sequencing reveals BCG vaccination curtails SARS-CoV-2 induced disease severity and lung inflammation

COVID-19 continues to exact a toll on human health despite the availability of several vaccines. Bacillus Calmette Guerin (BCG) has been shown to confer heterologous immune protection against viral infections including COVID-19 and has been proposed as vaccine against SARS-CoV-2 (SCV2). Here we tested intravenous BCG vaccination against COVID-19 using the golden Syrian hamster model together with immune profiling and single cell RNA sequencing (scRNAseq). We observed that BCG reduced both lung SCV2 viral load and bronchopneumonia. This was accompanied by an increase in lung alveolar macrophages, a reversal of SCV2-mediated T cell lymphopenia, and reduced lung granulocytes. Single cell transcriptome profiling showed that BCG uniquely recruits immunoglobulin-producing plasma cells to the lung suggesting accelerated antibody production. BCG vaccination also recruited elevated levels of Th1, Th17, Treg, CTLs, and Tmem cells, and differentially expressed gene (DEG) analysis showed a transcriptional shift away from exhaustion markers and towards antigen presentation and repair. Similarly, BCG enhanced lung recruitment of alveolar macrophages and reduced key interstitial macrophage subsets, with both cell-types also showing reduced IFN-associated gene expression. Our observations indicate that BCG vaccination protects against SCV2 immunopathology by promoting early lung immunoglobulin production and immunotolerizing transcriptional patterns among key myeloid and lymphoid populations.

immunology↗

bioRxiv2022

Cryo-EM structures of Doravirine and Rilpivirine with HIV-1 Reverse Transcriptase/DNA Aptamer: Nonnucleoside Inhibitor Resistance by E138K and M184I Mutations

Structures trapping a verity of functional and conformational states of HIV-1 reverse transcriptase (RT) have been determined by X-ray crystallography. These structures have played important roles in understanding the mechanisms of catalysis, inhibition and drug resistance, and in driving drug design. However, structures of several desired complexes of RT could not be obtained even after many crystallization or crystal soaking experiments. The ternary complexes of doravirine and rilpivirine with RT/DNA are such examples. Structural study of HIV-1 RT by single-particle cryo-EM has been challenging due to the enzymes relatively smaller size and higher flexibility. We optimized a protocol for rapid structure determination of RT complexes by cryo-EM and determined six structures of wild-type and E138K/M184I mutant RT/DNA in complexes with the nonnucleoside inhibitors rilpivirine, doravirine, and nevirapine. RT/DNA/rilpivirine and RT/DNA/doravirine complexes have structural differences between them and from the representative conformation of RT/DNA/nevirapine (or efavirenz); the primer grip in the RT/DNA/doravirine and the YMDD motif in the RT/DNA/rilpivirine complexes have large shifts. The DNA primer 3-end in the doravirine-bound structure is positioned at the active site, but the complex is in a non-productive state. In the mutant RT/DNA/rilpivirine structure, I184 is stacked with the DNA such that their relative positioning can influence rilpivirine in the pocket. Simultaneously, E138K mutation widens the NNRTI-binding pocket entrance, potentially contributing to a faster rate of rilpivirine dissociation by E138K/M184I mutant RT, as reported by earlier kinetic studies. These structural differences have implications for drug design and for understanding molecular mechanisms of drug resistance.

biophysics↗

bioRxiv2022

Rice Pan-genome Array (RPGA): an efficient genotyping solution for pan-genome-based accelerated crop improvement in rice

The advent of the pan-genome era has unraveled previously unknown genetic variation existing within diverse crop plants including rice. This untapped genetic variation is believed to account for a major portion of phenotypic variation existing in crop plants and might be responsible for missing heritability. However, the use of conventional single reference-guided genotyping often fails to capture large portion of this genetic variation leading to a reference bias. This makes it difficult to identify and utilize novel population/cultivar-specific genes for crop improvement. To overcome this challenge, we developed a rice pan-genome genotyping array (RPGA) includes 80K genome-wide SNPs which provides simple, user-friendly and cost-effective solution for rapid pan-genome-based genotyping in rice. The GWAS conducted using RPGA-SNP genotyping data of a rice diversity panel detected total of 42 loci, including previously known as well as novel genomic loci regulating grain size/weight traits in rice. Eight of the identified trait-associated loci (dispensable loci) could not be detected with conventional single reference genome-based GWAS and found to be missing from the commonly used Nipponbare reference genome. WD repeat-containing PROTEIN 12 gene underlying one of such dispensable locus on chromosome 7 (qLWR7) along with few other non-dispensable loci was subsequently detected using high-resolution QTL mapping confirming authenticity of RPGA-led GWAS. This demonstrates the potential of RPGA-based genotyping to overcome reference bias. Besides GWAS, the application of RPGA-based genotyping for natural allelic diversity and population structure analysis, seed purity and hybridity testing, ultra-high-density genetic map construction and chromosome level genome assembly, and marker-assisted foreground/background selection was successfully demonstrated. Based on these salient outcomes, a web application (http://www.rpgaweb.com) was also developed to provide easy to use platform for imputation of RPGA-based genotyping data using 3K Rice Reference Panel and subsequent GWAS in order to drive genetic improvement of rice.

genomics↗

bioRxiv2022

Outer membrane protein A (OmpA) deficient Salmonella Typhimurium displays enhanced susceptibility towards β-lactam antibiotics: third-generation cephalosporins (ceftazidime) and carbapenems (meropenem)

The invasive non-typhoidal serovar of Salmonella enterica, namely Salmonella Typhimurium ST313, causes bloodstream infection in sub-Saharan Africa. Like other bacterial pathogens, the development of antimicrobial resistance is a severe problem in curing non-typhoidal Salmonella infection. In this work, we have investigated the role of four prominent outer membrane porins of S. Typhimurium, namely OmpA, OmpC, OmpD, and OmpF, in resistance against broad-spectrum {beta}-lactam antibiotics-ceftazidime and meropenem. We found that deleting OmpA from Salmonella makes the bacteria susceptible to {beta}-lactam drugs. The MIC for both the antibiotics reduced significantly for STM{Delta} ompA compared to the wild-type and the ompA complemented strains. Despite the presence of antibiotics, the uninterrupted growth of STM{Delta} ompC,{Delta} ompD, and{Delta} ompF endorsed the dispensability of these three porins in antimicrobial resistance. The {beta}-lactam antibiotics caused massive depolarization in the outer membrane of the bacteria in the absence of OmpA. We have proved that none of the extracellular loops but the complete structure of perfectly folded OmpA is required by the bacteria for developing antimicrobial resistance. Our data revealed that STM{Delta} ompA consumed more antibiotics than the wild-type and the complemented strain, resulting in severe damage of the bacterial outer membrane and subsequent killing of the pathogen by antibiotic-mediated oxidative stress. Upon deleting ompA, the steady decrease in the relative proportion of antibiotic-resistant persisters and the clearance of the STM{Delta} ompA from the liver and spleen of C57BL/6 mice upon treatment with ceftazidime proved the role of OmpA in rendering protection against {beta}-lactam antibiotics.

microbiology↗

bioRxiv2022

Persistence of parental age effect on somatic mutation rates across generations in Arabidopsis.

In the model plant Arabidopsis thaliana, parental age is known to affect somatic mutation rates in their immediate progeny and here we show that this age dependent effect persists across successive generations. Using a set of detector lines carrying the mutated uidA gene, we examined if a particular parental age maintained across five consecutive generations affected the rates of base substitution (BSR), intrachromosomal recombination (ICR), frameshift mutation (FS), and transposition. The frequency of functional GUS reversions were assessed in seedlings as a function of identical/different parental ages across generations. In the context of a fixed parental age, BCR/ICR rates were unaffected in the first three generations, then dropped significantly in the 4th and increased in most instances in the 5th generation. On the other hand, with advancing parental ages, BSR/ICR rates remained high in the first two/three generations, with a striking resemblance in the pattern of mutation rates. We adopted a novel approach of identifying and tagging flowers pollinated on a particular day, thereby avoiding biases due to potential emasculation induced stress responses. Our results suggest a time component in counting the number of generations a plant has passed through self-fertilization at a particular age in determining the somatic mutation rates.

developmental biology↗

bioRxiv2022

Presence of sodium taurocholate co-transporting polypeptide and Hepatitis B replication marker on placenta: Another home for the virus

BackgroundThe role of sodium taurocholate co-transporting polypeptide (NTCP), in facilitating the binding of Hepatitis B virus (HBV) on surface of hepatocytes is well documented. Expression of NTCP in extra hepatic cells may make these cells susceptible to HBV infection and support cellular proliferation akin to hepatocytes. Placental replication of HBV is not well explored. In this study we have assessed the expression of NTCP and HBV replication markers (HBeAg, HBcAg, and HBV DNA) in placental cells, to investigate if these cells act as host for HBV. MethodsFourty one HBsAg+ve pregnant women along with 10 healthy controls were enrolled after obtaining informed consent. The HBV DNA in placenta was detected by qPCR using primers for X and core ORF. Expression of NTCP in placenta was analyzed by qRT-PCR and further investigated by immunohistochemistry (IHC) along with HBV replication biomarkers, HBeAg, and HBcAg. ResultsHBsAg positive subjects were divided in two groups on the basis of viral load [High Viral Load (HVL) Group; viral load [≥] 2000IU/ml, Low Viral Load (LVL) Group; viral load <2000IU/ml] according to INASL guidelines 2018. HBV infected females showed increased expression of NTCP in trophoblasts of placenta compared to control group (HVL 3.69{+/-}0.13 Vs Control 1.74{+/-}0.15, p=0.0117). Furthermore, significant difference in NTCP expression was also observed between HVL and LVL group (HVL 3.69{+/-}0.13 Vs LVL 1.98{+/-}0.17, p=0.022) and positively correlated with the maternal HBV DNA load. Membranous and/or cytoplasmic immunostaining of NTCP, and cytoplasmic staining of HBeAg and HBcAg in trophoblasts along with presence of HBV DNA indicated that trophoblasts are not only susceptible to HBV infection but may also be a site for viral replication. ConclusionsThis is the pioneer study, which demonstrates expression of NTCP on placenta which may facilitate the entry of HBV. Furthermore, the study establishes the presence of HBeAg in placenta of patients without circulating HBeAg, indicating these cells may act as replication host/reservoir. This pioneering finding hints at the possibility of exploring the potential of NTCP blocking strategies in preventing vertical transmission of HBV.

microbiology↗

bioRxiv2021

miRNA mediated downregulation of non-muscle Cyclase associated protein 1 is required for myogenic differentiation

Myoblast fusion is crucial for the formation, growth and regeneration of healthy skeletal muscle, but the molecular mechanisms that govern fusion and myofiber formation remain poorly understood. Here we report that Cyclase-associated protein 1 (Cap1), a regulator of actin dynamics, plays a critical role in cytoskeletal remodeling during myoblast fusion and formation of myotubes. Cap1 mRNA and protein are expressed in murine C2C12 and human LHCN-M2 myoblasts, but its abundance decreases during myogenic differentiation. Perturbing the temporally controlled expression of Cap1 by overexpression or Crispr-Cas9 mediated knockout impaired actin rearrangement, myoblast alignment, expression of profusion molecules, differentiation into multinucleated myotubes and myosin heavy chain expression. Endogenous Cap1 expression is posttranscriptionally downregulated during differentiation by canonical myomiRs miR-1, miR-133 and miR-206, which have conserved binding sites in the 3 UTR of the Cap1 mRNA. Deletion of the endogenous 3 UTR in C2C12 cells phenocopies overexpression of Cap1 by inhibiting myotube formation. Our findings implicate Cap1 and its myomiR-mediated downregulation in the myoblast fusion process and the generation of skeletal muscle.

cell biology↗

bioRxiv2021

Acute silencing uncovers multiple forms of activity-dependent neuronal survival in the mature entorhinal cortex

Neurodegenerative diseases are characterized by selective vulnerability of distinct cell populations; however, the cause for this specificity remains elusive. Many circuits that degenerate in disease are shaped by neural activity during development, raising the possibility that mechanisms governing early cell loss may be misused when activity is compromised in the mature brain. Here we show that electrical activity and synaptic transmission are both required for neuronal survival in the adult entorhinal cortex, but these silencing methods trigger distinct means of degeneration in the same neuronal population. Competition between active and inactive cells drives axonal disintegration caused by synaptic inhibition, but not axon retraction due to electrical suppression. These findings suggest that activity-dependence may persist in some areas of the adult brain long after developmental critical periods have closed. We speculate that lifelong plasticity required to support memory may render entorhinal neurons vulnerable to prolonged activity changes in disease.

neuroscience↗

bioRxiv2021

Multi-layer Regulation of Rubisco in Response to Altered Carbon Status in Synechococcus elongatus PCC 7942

Photosynthetic organisms possess a variety of mechanisms to achieve balance between absorbed light (source) and the capacity to metabolically utilize or dissipate this energy (sink). While regulatory processes that detect changes in metabolic status/balance are relatively well-studied in plants, analogous pathways remain poorly characterized in photosynthetic microbes. Herein, we explore systemic changes that result from alterations in carbon availability in the model cyanobacterium Synechococcus elongatus PCC 7942 by taking advantage of an engineered strain where influx/efflux of a central carbon metabolite, sucrose, can be regulated experimentally. We observe that induction of a high-flux sucrose export pathway leads to depletion of internal carbon storage pools (glycogen), and concurrent increases in photosynthetic parameters. Further, a proteome-wide analysis and fluorescence reporter-based analysis revealed that upregulated factors following the activation of the metabolic sink are strongly concentrated on ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) and axillary modules involved in Rubisco maturation. Carboxysome number and Rubisco activity also increase following engagement of sucrose secretion. Conversely, reversing the flux of sucrose by feeding exogenous sucrose heterologously results in increased glycogen pools, decreased Rubisco abundance, decreased photosystem II quantum efficiency, and carboxysome reorganization. Our data suggest that Rubisco activity and organization are key outputs connected to regulatory pathways involved in metabolic balancing in cyanobacteria. One sentence summaryRubisco activity and organization are key outputs involve in source-sink balancing in cyanobacteria.

microbiology↗

bioRxiv2021

KMT2D Loss Promotes Head and Neck Squamous Cell Carcinoma Through Enhancer Reprogramming and Modulation of Immune Microenvironment

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide, with 5-year survival of [~]50%. Genomic profiling studies have identified important somatic mutations in this disease which presents an opportunity for precision medicine. We demonstrate that KMT2D, a histone methyltransferase harbors somatic mutations in [~]17% of HNSCC and is associated with 2-year recurrence in TCGA data. Consistent with algorithmic prediction of bring a driver tumor-suppressor event, its loss results in larger oral tumors in immune-proficient orthotopic models. Mechanistically, we find that KMT2D knockdown or KMT2D mutation causes loss of H3K4me1-marked enhancers harboring IRF7/9 binding sites, which is known to regulate interferon signaling. Indeed, KMT2D loss in human and murine cell lines deregulated transcriptional levels of cytokine expression and impacted numerous immune signaling pathways, including interferon signaling. Consistently, Kmt2d knockdown in murine tumors exhibited decrease in IFN{gamma}-producing effector T cells and an increase in T-cells with an exhausted phenotype. Epistasis experiments showed that exogenous treatment with IFN{gamma} abrogated the increased tumor growth in Kmt2d-deficient oral tumors. Together, these results support the role of KMT2D as a tumor suppressor in HNSCC that regulates the tumor microenvironment by modulating H3K4me1-marked enhancers controlling interferon signaling.

cancer biology↗

bioRxiv2021

ZRC3308 monoclonal antibody cocktail shows protective efficacy in Syrian hamsters against SARS-CoV-2 infection

We have developed a monoclonal antibody (mAb) cocktail (ZRC-3308) comprising of ZRC3308-A7 and ZRC3308-B10 in the ratio 1:1 for COVID-19 treatment. The mAbs were designed to have reduced immune effector functions and increased circulation half-life. mAbs showed good binding affinities to non-competing epitopes on RBD of SARS-CoV-2 spike protein and were found neutralizing SARS-CoV-2 variants B.1, B.1.1.7, B.1.351, B.1.617.2 and B.1.617.2 AY.1 in vitro. The mAb cocktail demonstrated effective prophylactic and therapeutic activity against SARS-CoV-2 infection in Syrian hamsters. The antibody cocktail appears to be a promising candidate for the prophylactic use and for therapy in early COVID-19 cases which have not progressed to severe disease.

microbiology↗

bioRxiv2021

EP300 (p300) mediated histone butyrylation is critical for adipogenesis

ObjectiveThe master epigenetic enzyme EP300 (p300) besides having lysine acetyltransferase activity can also catalyse other acylation modifications (propionylation, butyrylation, crotonylation etc.), the physiological implications of which are yet to be established fully. We hypothesized that p300 catalysed histone butyrylation may have a causal relationship with adipogenesis and the consequent obesity. MethodsHistone butyrylation pattern was investigated in 3T3L1 cells upon adipogenesis by immunoblotting and chromatin immunoprecipitation experiments. A small molecule modulator that could specifically inhibit p300 catalysed butyrylation without affecting its canonical acetyltransferase activity was screened from a series of compounds and then administered in differentiating 3T3L1 adipocytes as well as high fat diet-induced and genetically obese mice to validate the importance of butyrylation in adipogenesis. ResultsHistone butyrylation was increased upon adipogenesis both globally and locally in the promoters of pro-adipogenic genes along with an upregulation in the expression of acyl CoA generating enzyme Acss2, knockdown of which led to reduced butyrylation. Treatment of differentiating 3T3L1 cells with the p300 specific butyrylation inhibitor LTK-14A led to abrogation of adipogenesis with reduced expression of pro-adipogenic genes and inhibition of H4K5 butyrylation. LTK-14A administration could also attenuate weight gain in both mice models of obesity by preventing adipocyte hypertrophy via H4K5 butyrylation inhibition. ConclusionOur results indicate that p300 catalysed histone butyrylation may have a causal relationship with the process of adipogenesis. Site specific inhibition of butyrylation could lead to adipogenesis repression and hence this epigenetic modification could be targeted for obesity treatment. HighlightsO_LIHistone butyrylation has been established as a new epigenetic signature in the context of adipogenesis. C_LIO_LITo the best of our knowledge, this is the first report of a selective inhibitor of p300 catalysed histone acylation (butyrylation) without affecting its canonical acetyltransferase activity. C_LIO_LISpecific inhibition of H4K5 butyrylation could be a possible mechanism for inhibiting adipogenesis and hepatic steatosis leading to better control of obesity. C_LIO_LILTK-14A class of molecule could be developed as anti-obesity therapeutics. C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=195 HEIGHT=200 SRC="FIGDIR/small/454641v3_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1f29b35org.highwire.dtl.DTLVardef@abc5fcorg.highwire.dtl.DTLVardef@1534388org.highwire.dtl.DTLVardef@176d706_HPS_FORMAT_FIGEXP M_FIG Proposed model for the role of p300-mediated histone butyrylation in adipogenesis: In pre-adipocytes, there exists a basal level of histone acetylation while butyrylation is present to a much lesser extent owing to low stoichiometric levels of butyryl CoA. Induction of adipogenesis causes a simultaneous upregulation of histone acetylation and butyrylation marks leading to increased rate of adipogenesis and concomitant transcriptional activation of pro-adipogeneic genes. Onset of obesity in mice, either due to excess energy intake through high fat diet consumption or increased de novo synthesis of fatty acids due to leptin receptor gene mutation leading to hyperphagic behavior, is accompanied by adipocyte hyperplasia and hypertrophy. Both the organs of adipose tissue and liver were found to have enhanced levels of H4K5 butyrylation during obesity. LTK-14A, a butyrylation specific inhibitor could efficiently prevent the processes of adipogenesis and adipocyte hypertrophy due to inhibition of H4K5 butyrylation in these organs. Thus the compound could attenuate weight gain by selective inhibition of butyrylation without affecting acetylation, thereby highlighting the importance of histone butyrylation in adipogenesis and obesity. C_FIG

molecular biology↗

bioRxiv2021

Active enhancers strengthen TAD insulation by bRNA mediated CTCF enrichment at the TAD boundaries

Vertebrate genomes are partitioned into Topologically Associating Domains (TADs), which are typically bound by head-to-head pairs of CTCF binding sites. Transcription at TAD boundaries correlates with better insulation, however, it is not known whether the boundary transcripts themselves contribute to boundary function. Here we characterize boundary-associated RNAs genome-wide, focusing on the disease-relevant INK4a/ARF TAD. Using CTCF site deletions and boundary-associated RNA knockdowns, we observe that boundary-associated RNAs facilitate recruitment and clustering of CTCF at TAD borders. The resulting CTCF enrichment enhances TAD insulation, enhancer:promoter interactions and TAD gene expression. Importantly, knockdown of boundary-associated RNAs results in loss of boundary insulation function. Using enhancer deletions and CRISPRi of promoters we show that active TAD enhancers but not promoters induce boundary-associated RNA transcription, thus defining a novel class of regulatory enhancer RNAs.

genomics↗

bioRxiv2021

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - Targeting P-pocket by fragment screening

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography and identified binding of two to P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drugresistance mutations. Analogs of a fragment were synthesized of which two showed noticeable RT inhibition. An engineered RT/DNA aptamer complex trapped the transient P-pocket in solution. Structures of the RT/DNA complex were determined with a fragment and a synthesized analog bound at P-pocket by single-particle cryo-EM. Identification of P-pocket and the devised structurebased platform provide an opportunity for designing new types of polymerase inhibitors.

biophysics↗

bioRxiv2021

Scalable and Automated CRISPR-Based Strain Engineering Using Droplet Microfluidics

We present a droplet-based microfluidic system that enables CRISPR-based gene editing and high-throughput screening on chip. The microfluidic device contains a 10 x 10 element array, each element containing sets of electrodes for two electric field actuated operations-electrowetting for merging droplets to mix reagents and electroporation for transformation. It can perform up to 100 genetic modifications in parallel, providing a scalable platform for generating the large number of engineered strains required for combinatorial optimization of genetic pathways and predictable bioengineering. We demonstrate the systems capabilities through CRISPR-based engineering of two test cases-1) disruption of the function of enzyme galactokinase (galK) in E. coli and 2) targeted engineering of glutamine synthetase gene (glnA) and blue-pigment synthetase (bpsA) enzyme to improve indigoidine production in E. coli.

synthetic biology↗

bioRxiv2021

Neuro-musculoskeletal Upper Limb in-silico asvirtual patient

Virtual patients and physiologies allow experimentation, design, and early-stage clinical trials in-silico. Virtual patient technology for human movement systems that encompasses musculoskeleton and its neural control are few and far in between. In this work, we present one such neuro-musculoskeletal upper limb in-silico model. This upper limb is both modular in architecture and generates movement as an emergent phenomenon out of a multiscale co-simulation of spinal cord neural control and musculoskeletal dynamics. It is developed on the NEUROiD movement simulation platform that enables a co-simulation of popular neural simulator NEURON and the musculoskeletal simulator OpenSim. In this work, we describe the design and development of the upper limb in a modular fashion, while reusing existing models and modules. We further characterize and demonstrate the use of this model in generating a range of commonly observed movements by means of a spatio temporal stimulation pattern delivered to the cervical spinal cord. We believe this work enables a first and small step towards an in-silico paradigms for understanding upper limb movement, disease pathology, medication, and rehabilitation.

neuroscience↗

bioRxiv2021

New cellular models of undifferentiated pleomorphic sarcoma and malignant peripheral nerve sheath tumor

Undifferentiated pleomorphic sarcoma (UPS) and malignant peripheral nerve sheath tumor (MPNST) are aggressive soft tissue sarcomas that do not respond well to current treatment modalities. The limited availability of UPS and MPNST cell lines makes it challenging to identify potential therapeutic targets in a laboratory setting. Understanding the urgent need for improved treatments for these tumors and the limited cellular models led us to generate additional cell lines to study these rare cancers further. Patient-derived tumors were used to establish 5 new UPS models, including one radiation-associated UPS--UPS060.1, UPS271.1, UPS511, UPS0103, and RIS620--and 3 new models of MPNST--MPNST007, MPNST3813E, and MPNST4970. This study examined the utility of the new cell lines as sarcoma models by assessing tumorigenic potential and mutation status for known sarcoma-related genes. All the cell lines formed colonies and migrated in vitro. The in vivo tumorigenic potential of each cell line was determined by either subcutaneous injection of cells or implantation of tumor tissue into immunocompromised mice. UPS060.1, UPS271.1, and UPS511 cells formed tumors in mice upon subcutaneous injection. UPS0103 and RIS620 tumor implants formed tumors in vivo, as did MPNST007 and MPNST3813E tumor implants. Mutation analysis of a panel of genes frequently mutated in sarcomas showed that two of the three MPNST cell lines had NF1 mutations. Two of the three MPNST cell lines had mutations in polycomb repressive complex 2 members. These new cellular models provide the scientific community with powerful tools for detailed studies of sarcomagenesis and investigate novel therapies for UPS and MPNST.

cancer biology↗

bioRxiv2021

Genome-wide chromosomal association of Upf1 is linked to Pol II transcription in Schizosaccharomyces pombe

Although the RNA helicase Upf1 has hitherto been examined mostly in relation to its cytoplasmic role in nonsense mediated mRNA decay (NMD), here we report high-throughput ChIP data indicating genome-wide association of Upf1 with active genes in Schizosaccharomyces pombe. This association is RNase sensitive and it correlates with Pol II transcription and mRNA expression levels. While changes in Pol II occupancy were detected at only some genes in a Upf1-deficient (upf1{Delta}) strain, there is an increased Ser2 Pol II signal at all highly transcribed genes examined by ChIP-qPCR. Furthermore, upf1{Delta} cells are hypersensitive to the transcription elongation inhibitor 6-azauracil and display Pol II abnormalities suggestive of Pol II hyperphosphorylation. A significant proportion of the genes associated with Upf1 in wild-type conditions are also mis-regulated in upf1{Delta}. These data envisage that by operating on the nascent transcript Upf1 might influence Pol II phosphorylation and transcription.

molecular biology↗