Biology subjects

Shrestha, S.

Publications and source records attributed to Shrestha, S..

83 records · Page 5Linked to original sources

bioRxiv2019

In situ miRNA delivery from a hydrogel promotes osteogenesis of encapsulated mesenchymal stromal cells

AO_SCPLOWBSTRACTC_SCPLOWHydrogels have many properties that emulate biological tissues and are therefore attractive candidates for use in tissue engineering. In particular the encapsulation and subsequent differentiation of mesenchymal stem/stromal cells (MSCs) is a strategy that holds great promise for the repair and regeneration of bone and cartilage. However, MSCs are well-known for their sensitivity to mechanical cues, particularly substrate stiffness, and so the inherent softness of hydrogels is poorly matched to the mechanical cues that drive efficient osteogenesis. This limits the success of bone tissue engineering using MSCs encapsulated in a hydrogel. One approach to overcome this limitation is to harness mechanotransductive signalling pathways and override the signals cells receive from their environment. Previous reports have shown that the mechanosensitive miRNAs, miR-100-5p and miR-143-3p can enhance MSC osteogenesis, but this required a complex multi-step procedure to transfect, encapsulate and differentiate the cells. In this study, we develop and characterise a facile system for in situ transfection of MSCs encapsulated within a light-crosslinkable gelatin-PEG hydrogel. Comparing the influence of different transfection agents and hydrogel compositions, we determine the factors affecting transfection agent release and MSC transfection, showing that it is possible to transfect MSCs with miRNAs in situ. We then compare the efficacy of both pretransfection and in situ transfection on the osteogenic capacity of hydrogel-encapsulated MSCs, demonstrating superior mineralisation and osteogenic gene expression for in situ transfected samples. Our platform therefore demonstrates a simple, one-pot system for delivery of pro-osteogenic miRNAs and in situ transfection that is able to enhance MSC osteogenic potential without the need of multi-step transfection procedures, thus demonstrating significant promise for bone tissue engineering.

bioengineering↗

bioRxiv2019

Single-Cell, Human Sperm Transcriptomes and Variants from Fathers of Autistic and Healthy Children

The human sperm is one of the smallest cells in the body, but also one of the most important, as it serves as the entire paternal genetic contribution to a child. This is especially relevant for diseases such as Autism Spectrum Disorders (ASD), which have been correlated with advance paternal age. Historically, most studies of sperm have focused on the assessment of a bulk sperm, wherein millions of individual sperm are present and only high-frequency variants can be detected. Using 10X Chromium single cell sequencing technology, we have assessed the RNA from >65,000 single sperm cells across 6 donors (scsperm-RNA-seq), including two of whom have autistic children and four that do not. Using multiple RNA-seq methods for differential expression and variant analysis, we found clusters of sperm mutations in each donor that are indicative of the sperm being produced by different stem cell pools. Moreover, by comparing the two groups, we have found expression changes that can separate out the two sets of donors. Finally, through our novel variant calling from single-cell RNA-seq methods, we have shown that we can detect mutation rates in sperm from ASD donors that is distinct from the controls, highlighting this method as a new means to characterize ASD risk.

genomics↗

bioRxiv2019

Pluripotent Stem Cells Can Be Isolated from Human Peripheral Nerves after in vitro BMP-2 Stimulation

We have recently identified a population of cells within the peripheral nerves of adult mice that can respond to BMP-2 exposure or physical injury to rapidly proliferate. More importantly, these cells exhibited embryonic differentiation potentials that could be induced into osteoblastic and endothelial cells in vitro. The current study examined human nerve specimens to compare and characterize the cells after BMP-2 stimulation. Fresh pieces of human nerve tissue were minced and treated with either BMP-2 (750ng/ml) or vehicle for 12 hours at 37{degrees}C, before digested in 0.2% collagenase and 0.05% trypsin-EDTA. Isolated cells were cultured in restrictive stem cell medium. Significantly more cells were obtained from the nerve pieces with BMP-2 treatment in comparison with the non-treated controls. Cell colonies were starting to form at day 3. Expressions of the 4 transcription factors Klf4, c-Myc, Sox2 and Oct4 were confirmed at both transcriptional and translational levels. The cells can be maintained in the stem cell culture medium for at least 6 weeks without changing morphologies. When the cells were switched to fibroblast growth medium, dispersed spindle-shaped cells were noted and became fibroblast activated protein- (FAP) positive following immunocytochemistry staining. The data suggested that human peripheral nerve tissue also contain a population of cells that can respond to BMP-2 and express all four transcription factors KLF4, Sox2, cMyc, and Oct4. These cells are capable to differentiate into FAP-positive fibroblasts. It is proposed that these cells are possibly at the core of a previously unknown natural mechanism for healing injury.

cell biology↗

bioRxiv2019

Neutrophil-derived extracellular vesicles: proinflammatory trails and anti-inflammatory microvesicles

Extracellular vesicles (EVs) are membrane-derived vesicles that mediate intercellular communications. Neutrophils produce different subtypes of EVs during inflammatory responses. Neutrophil-derived trails (NDTRs) are generated by neutrophils migrating toward inflammatory foci, whereas neutrophil-derived microvesicles (NDMVs) are thought to be generated by neutrophils that have arrived at the inflammatory foci. However, the physical and functional characteristics of neutrophil-derived EVs are incompletely understood. In this study, we investigated the similarities and differences between neutrophil-derived EV subtypes. Neutrophil-derived EVs shared similar characteristics regarding stimulators, generation mechanisms, and surface marker expression. Both neutrophil-derived EV subtypes exhibited similar functions, such as direct bactericidal activity and induction of monocyte chemotaxis via MCP-1. However, NDTR generation was dependent on the integrin signaling, while NDMV generation was dependent on the PI3K pathway. The CD16 expression level differentiated the neutrophil-derived EV subtypes. Interestingly, both subtypes showed different patterns of miRNA expression and were easily phagocytosed by monocytes. NDTRs induced M1 macrophage polarization, whereas NDMVs induced M2 macrophage polarization. Moreover, NDTRs but not NDMVs exerted protective effects against sepsis-induced lethality in a murine sepsis model and pathological changes in a murine chronic colitis model. These results suggest a new insight into neutrophil-derived EV subtypes: proinflammatory NDTRs and anti-inflammatory NDMVs.\n\nKey pointsO_LINeutrophil-derived trails are proinflammatory extracellular vesicles that induce M1 macrophage polarization and protect against inflammation\nC_LIO_LINeutrophil-derived microvesicles are anti-inflammatory extracellular vesicles that induce M2 macrophage polarization\nC_LI

immunology↗

bioRxiv2019

An evolutionary-conserved redox regulatory mechanism in human Ser/Thr protein kinases

Reactive oxygen species (ROS) are products of oxygen metabolism, but are also recognized as endogenous physiological mediators of cellular signaling. Eukaryotic protein kinase (ePK) regulation occurs through reversible phosphorylation events in the flexible activation segment. In this study, we demonstrate that the catalytic phosphotransferase output from the mitotic Ser/Thr kinase Aurora A is also controlled by cysteine (Cys) oxidation. Reversible regulation occurs by direct modification of a conserved residue (Cys 290), which lies adjacent to Thr 288, the activating site of phosphorylation. Strikingly, redox modulation of the Cys 290-equivalent in other ePKs is predicted to be an underappreciated regulatory mechanism, since ~100 human Ser/Thr kinases possess a Cys at this position in the conserved activation loop. Using real-time enzyme assays, we confirm that the presence of the equivalent Cys residue is prognostic for redox-sensitivity amongst a cohort of human CAMK, AGC and AGC-like kinases, including AKT, AMPK, CAMK1, MAPKAP-K2/3 and SIK1-3. Our findings demonstrate that dominant Cys-based redox-switching in the activation segment represents an evolutionary-conserved mode of regulation for a significant subset of the human kinome. This finding has important implications for understanding physiological and pathological signaling responses to ROS, and emphasises the importance of multivalent activation segment regulation in ePKs.\n\nONE-SENTENCE SUMMARYThe catalytic activity of Ser/Thr kinases is regulated through a conserved Cys-based redox mechanism.

biochemistry↗

bioRxiv2019

High-throughput phenotyping with deep learning gives insight into the genetic architecture of flowering time in wheat

BackgroundPrecise measurement of plant traits with precision and speed on large populations has emerged as a critical bottleneck in connecting genotype to phenotype in genetics and breeding. This bottleneck limits advancements in understanding plant genomes and the development of improved, high-yielding crop varieties. ResultsHere we demonstrate the application of deep learning on proximal imaging from a mobile field vehicle to directly score plant morphology and developmental stages in wheat under field conditions. We developed and trained a convolutional neural network with image datasets labeled from expert visual scores and used this breeder-trained network to directly score wheat morphology and developmental stages. For both morphological (awned) and phenological (flowering time) traits, we demonstrate high heritability and extremely high accuracy against the ground-truth values from visual scoring. Using the traits scored by the network, we tested genotype-to-phenotype association using the deep learning phenotypes and uncovered novel epistatic interactions for flowering time. Enabled by the time-series high-throughput phenotyping, we describe a new phenotype as the rate of flowering and show heritable genetic control. ConclusionsWe demonstrated a field-based high-throughput phenotyping approach using deep learning that can directly score morphological and developmental phenotypes in genetic populations. Most powerfully, the deep learning approach presented here gives a conceptual advancement in high-throughput plant phenotyping as it can potentially score any trait in any plant species through leveraging expert knowledge from breeders, geneticist, pathologists and physiologists.

genetics↗

bioRxiv2019

Prevalence and risk factors of postpartum depression among women living in urban slums of Dhaka, Bangladesh

Postpartum depression (PPD) is a serious pubic health concern and known to have the adverse effects on mothers perinatal wellbeing; and childs physical and codnitive development. There were limited literatures on PPD in Bangladesh, especially in urban slum context. The aim of this study was to assess the burden and risk factors of PPD among the urban slum women. A cross-sectional study was conducted between November-December 2017 in three urban slums on 376 women within first 12 months of postpartum. A validated Bangla version of Edinburgh Postnatal Depression Scale was used to measure the depression status. Respondents socio-economic characteristics and other risk factors were collected with structured validated questionaire by trained interviewers. Unadjusted Prevalence Ratio (PR) and Adjusted Prevalence Ratio (APR) were estimated with Generalized Linear Model(GLM) and Generalized Estimating Equation (GEE) respectively to identify the risk factors of PPD. The prevalence of PPD was 39.4% within first 12 months following the child birth. Job involvement after child delivery (APR=1.9, 95% CI= 1.1, 3.3), job loss due to pregnancy (APR=1.5, 95% CI= 1.0, 2.1), perinatal and postnatal death (APR=1.4, 95% CI= 0.97, 2.0), unintended pregnancy (APR=1.8, 95% CI= 1.3, 2.5), management of delivery cost by borrowing, selling or mortgaging assets (APR=1.3, 95% CI= 0.94, 1.9), depressive symptom during pregnancy (APR=2.5, 95% CI= 1.7, 3.8) and intimate partner violence (APR=2.0, 95% CI= 1.2, 3.3), were identified as risk factors. PPD was not associated with poverty, mother in law and any child related factors. The burden of postpartum depression is high in the urban slum of Bangladesh. Maternal mental health services should be integrated with existing maternal health services. Research is required for the innovation of effective, low cost and culturally appropriate PPD case management and preventive intervention in urban slum of Bangladesh.

epidemiology↗

bioRxiv2018

Uncovering human transcription factor interactions associated with genetic variants, novel DNA motifs, and repetitive elements using enhanced yeast one-hybrid assays

Identifying transcription factor (TF) binding to noncoding variants, uncharacterized DNA motifs, and repetitive genomic elements has been technically and computationally challenging. Current experimental methods, such as chromatin immunoprecipitation, generally test one TF at a time, and computational motif algorithms often lead to false positive and negative predictions. To address these limitations, we developed two approaches based on enhanced yeast one-hybrid assays. The first approach interrogates the binding of >1,000 human TFs to repetitive DNA elements, while the second evaluates TF binding to single nucleotide variants, short insertions and deletions (indels), and novel DNA motifs. Using the first approach, we detected the binding of 75 TFs, including several nuclear hormone receptors and ETS factors, to the highly repetitive Alu elements. Using the second approach, we identified cancer-associated changes in TF binding, including gain of interactions involving ETS TFs and loss of interactions involving KLF TFs to different mutations in the TERT promoter, and gain of a MYB interaction with an 18 bp indel in the TAL1 super-enhancer. Additionally, we identified the TFs that bind to three uncharacterized DNA motifs identified in DNase footprinting assays. We anticipate that these approaches will expand our capabilities to study genetic variation and under-characterized genomic regions.

genomics↗

bioRxiv2018

Diagnostic host gene signature to accurately distinguish enteric fever from other febrile diseases

Misdiagnosis of enteric fever is a major global health problem resulting in patient mismanagement, antimicrobial misuse and inaccurate disease burden estimates. Applying a machine-learning algorithm to host gene expression profiles, we identified a diagnostic signature which could accurately distinguish culture-confirmed enteric fever cases from other febrile illnesses (AUROC<95%). Applying this signature to a culture-negative suspected enteric fever cohort in Nepal identified a further 12.6% as likely true cases. Our analysis highlights the power of data-driven approaches to identify host-response patterns for the diagnosis of febrile illnesses. Expression signatures were validated using qPCR highlighting their utility as PCR-based diagnostic for use in endemic settings.

molecular biology↗

bioRxiv2018

Repurposing covalent EGFR/HER2 inhibitors for on-target degradation of human Tribbles 2 (TRIB2) pseudokinase

ONE SENTENCE SUMMARYA Tribbles 2 pseudokinase small molecule screen led to the identification of known EGFR/HER2 inhibitors that alter the stability of TRIB2 in vitro and lead to rapid on-target degradation of TRIB2 in human cancer cells.\n\nSHORT ABSTRACTTribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, including the AKT signaling module. Substantial evidence demonstrates that TRIB2 dysregulation is important in multiple human tumors. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine rich region and interacts with a peptide motif in its own C-terminal tail. We demonstrate that TRIB2 is a target for previously described small molecule protein kinase inhibitors, which were originally designed to inhibit the catalytic domain of EGFR/HER2 tyrosine kinases. Using thermal-shift assays and drug repurposing, we classify ligands that stabilize or destabilize the TRIB2 pseudokinase domain. TRIB2 destabilizing agents, including the clinical inhibitor afatinib, lead to rapid and on-target TRIB2 protein degradation in tumor cells, eliciting tractable effects on cell signaling and survival. Our data identifies leads for further development of TRIB2-degrading drugs and highlights compound-induced TRIB2 downregulation, which might be mechanistically relevant for other catalytically-deficient (pseudo)kinases targeted by small molecules.\n\nFULL ABSTRACTA major challenge associated with biochemical and cellular analysis of pseudokinases is the lack of target-validated small molecule ligands with which to probe molecular function. Human Tribbles 2 (TRIB2) is a cancer-associated pseudokinase with a diverse interactome, which includes the canonical AKT signaling module. There is substantial evidence that human TRIB2 is a therapeutic target in both solid tumors and blood cancers. The non-canonical TRIB2 pseudokinase domain contains a unique cysteine-rich region and interacts with a peptide motif in its own C-terminal tail, which was previously shown to drive interaction with cellular E3 ubiquitin ligases. In this study we demonstrate that TRIB2 is a target for previously described small molecule protein kinase inhibitors, which were originally designed to inhibit the canonical catalytic domain of the tyrosine kinases EGFR/HER2. Using a thermal-shift assay, we discovered TRIB2 ligands within the Published Kinase Inhibitor Set (PKIS), and employed a drug repurposing approach to classify compounds that either stabilize or destabilize TRIB2 in vitro. Remarkably, TRIB2 destabilizing agents, including the clinical covalent drug afatinib, lead to rapid and on-target TRIB2 degradation in human cells, eliciting tractable effects on signaling and survival. Our data reveal the first drug-leads for development of TRIB2-degrading ligands, which will also be invaluable for unravelling the cellular mechanisms of TRIB2-based signaling. Our study highlights that small molecule-induced protein downregulation through drug off-targets might be relevant for other inhibitors that serendipitously target pseudokinases.

biochemistry↗

bioRxiv2018

Laboratory and Molecular Surveillance of Paediatric Typhoidal Salmonella in Nepal: Antimicrobial Resistance and Implications for Vaccine Policy

BackgroundChildren are substantially affected by enteric fever in most settings with a high burden of the disease, which could be due to immune naivety, or enhanced risk of exposure to the pathogen. Although Nepal is a high burden setting for enteric fever, the bacterial population structure and transmission dynamics are poorly delineated in young children, the proposed target group for immunization programs.\n\nMethodsBlood culture surveillance amongst children aged 2 months to 15 years of age was conducted at Patan Hospital between 2008 and 2016. A total of 198 S. Typhi and 66 S. Paratyphi A isolated from children treated in both inpatient and outpatient settings were subjected to whole genome sequencing and antimicrobial susceptibility testing. Demographic and clinical data were also collected from the inpatients. The resulting data were used to place these paediatric Nepali isolates into a worldwide context, based on their phylogeny and carriage of molecular determinants of antimicrobial resistance (AMR).\n\nResultsChildren aged [≤]4 years made up >40% of the inpatient population. The majority of isolates (78 %) were S. Typhi, comprising several distinct genotypes but dominated by 4.3.1 (H58). Several distinct S. Typhi genotypes were identified, but the globally disseminated S. Typhi clade 4.3.1 (H58) dominated. The majority of isolates (86%) were insusceptible to fluoroquinolones. This was mainly associated with S. Typhi H58 Lineage II and S. Paratyphi A; non-susceptible strains from these two genotypes accounted for 50% and 25% of all enteric fever cases. Multi-drug resistance (MDR) was rare (3.5% of S. Typhi, 0 S. Paratyphi A) and restricted to chromosomal insertions of AMR genes in H58 lineage I strains. Comparison to global data sets showed the local S. Typhi and S. Paratyphi A strains had close genetic relatives in other South Asian countries, indicating regional strain circulation.\n\nConclusionsThese data indicate that enteric fever in Nepal continues to be a major public health issue with ongoing inter- and intra-country transmission, and highlights the need for regional coordination of intervention strategies. The absence of a S. Paratyphi A vaccine is cause for concern, given its prevalence as an enteric fever agent in this setting, and the large proportion of isolates displaying fluoroquinolone resistance. This study also highlights an urgent need for routine laboratory and molecular surveillance to monitor the epidemiology of enteric fever and evolution of antimicrobial resistance within the bacterial population as a means to facilitate public health interventions in prevention and control of this febrile illness.

molecular biology↗