Biology subjects

Murphy, D.

Publications and source records attributed to Murphy, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

inSequio: A Programmable 3D CAD Application for Designing DNA Nanostructures

DNA nanotechnology is evolving rapidly, paralleling the historic trajectory of the 1970s electronics industry. However, current DNA nanostructure (DN) design software limits users to either manual design with minimal automation or a constrained range of automated designs. inS[e]quio Design Studio, developed by Parabon(R) NanoLabs, bridges this gap as a programmable 3D computer-aided design (CAD) application, integrating a domain-specific graphical editor with a Python API for versatile DN design. Developed in C++ for Windows(R) and Macintosh(R) systems, inS[e]quio features a user-friendly GUI with extensive CAD tools, capable of managing complex designs and offloading computational tasks to the cloud. It supports various DNA design formats, PDB molecule integration, residue modifications, and includes preloaded designs and thorough documentation. With its combination of features, inS[e]quio enables a code-centric design (CCD) approach, enhancing DN construction with improved precision, scalability, and efficiency. This approach is elucidated through a streptavidin barrel cage designed via Python notebook and a spheroid origami case study. Marking a significant advance in DN design automation, inS[e]quio, the first fully programmable 3D CAD tool for DN design, enables both manual and programmatic 3D editing. This fusion of features establishes inS[e]quio as a transformative tool, poised to significantly enhance designer productivity and expand the scope of possible designs. Extended AbstractAdvances in DNA nanotechnology have positioned the field at a juncture reminiscent of the pivotal growth phase of the electronics industry in the 1970s. The evolution of software for designing DNA nanostructures (DNs) is following a similar historical trajectory and dozens of software packages have been developed for creating them. Existing software options, however, require users to choose between manual design with minimal automation support or selecting from a limited set of designs, typically wireframe, that can be generated from a high-level structural description. Here, we introduce the inS[e]quio Design Studio, a programmable 3D computer-aided design (CAD) application that effectively bridges this gap. By integrating a domain-specific, freeform graphical editor with a Python application programming interface (API), inS[e]quio provides a comprehensive and extensible platform for designing complex nucleic acid (NA) nanostructures. The inS[e]quio desktop application, developed in C++, runs on Windows(R) and Macintosh(R) operating systems. Its graphical user interface (GUI) features multiple synchronized view panels and a diverse set of CAD and NA-specific editing tools. Its optimized graphics pipeline enables editing of designs with >2M nucleotides, and it includes an integrated service infrastructure for offloading heavy computations to cloud servers. The software also supports import and export of various DNA design file formats, integration of arbitrary PDB molecules, and specification of residue modifications. Additionally, it includes preloaded sample designs, scripts, and comprehensive documentation. Parabon has used evolving versions of inS[e]quio for over a decade to design a variety of proprietary DNs and have now transitioned it into a commercially available product. This paper summarizes inS[e]quios features, discusses its strengths and limitations, and outlines planned enhancements. Although freeform 3D design is well supported in inS[e]quio, the integration of its CAD environment with its API facilitates a code-centric design (CCD) approach for DN construction that offers notable productivity advantages over traditional methods, including enhanced precision, scalability, and efficiency. Here we describe CCD, outline its benefits and demonstrate its use through a well-documented Python notebook, included with the product, which generates a sample design within the inS[e]quio application. A spheroid origami created using CCD is also presented. As the first commercial fully programmable 3D CAD application specifically created for DN design, the release of inS[e]quio represents a milestone in the field of DN design automation. It introduces a new dimension to the discipline by enabling both manual and programmatic 3D editing, thereby facilitating an innovative CCD approach. The availability of extensive documentation and technical support enables designers to efficiently adopt and utilize these capabilities. This combination of features establishes inS[e]quio as a noteworthy addition to the tools available for DN design, with the potential to significantly increase designer productivity and broaden the scope of designs that can be developed by practitioners of all skill levels. Windows and Mac versions of the inS[e]quio desktop application are available for download at https://parabon.com/insequio. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=119 SRC="FIGDIR/small/586810v1_ufig1.gif" ALT="Figure 1"> View larger version (64K): org.highwire.dtl.DTLVardef@16a2714org.highwire.dtl.DTLVardef@2bc610org.highwire.dtl.DTLVardef@1d852e2org.highwire.dtl.DTLVardef@129b3fa_HPS_FORMAT_FIGEXP M_FIG C_FIG An illustration of the inS[e]quio Design Studio desktop application interoperating with a Python Jupyter notebook and molecular dynamics (MD) simulation tools to support an iterative code-centric design (CCD) process. The design cycle includes (a) programmatic and/or manual creation of objects in the inS[e]quio editors; (b) visual inspection and manipulation of objects via user interface; (c) in silico evaluation of designs via MD simulation using native or external tools; repeating a-c as necessary; and (d) procurement of strands and synthesis of DNA nanostructures (DNs).

bioengineering↗

bioRxiv2024

Deciphering the History of ERK Activity from Fixed-Cell Immunofluorescence Measurements

The Ras/ERK pathway drives cell proliferation and other oncogenic behaviors, and quantifying its activity in situ is of high interest in cancer diagnosis and therapy. Pathway activation is often assayed by measuring phosphorylated ERK. However, this form of measurement overlooks dynamic aspects of signaling that can only be observed over time. In this study, we combine a live, single-cell ERK biosensor approach with multiplexed immunofluorescence staining of downstream target proteins to ask how well immunostaining captures the dynamic history of ERK activity. Combining linear regression, machine learning, and differential equation models, we develop an interpretive framework for immunostains, in which Fra-1 and pRb levels imply long term activation of ERK signaling, while Egr-1 and c-Myc indicate recent activation. We show that this framework can distinguish different classes of ERK dynamics within a heterogeneous population, providing a tool for annotating ERK dynamics within fixed tissues.

molecular biology↗

bioRxiv2024

Spatiotemporal Clusters of ERK Activity Coordinate Cytokine-induced Inflammatory Responses in Human Airway Epithelial Cells

RATIONALESpatially coordinated ERK signaling events ("SPREADs") transmit radially from a central point to adjacent cells via secreted ligands for EGFR and other receptors. SPREADs maintain homeostasis in non-pulmonary epithelia, but it is unknown whether they play a role in the airway epithelium or are dysregulated in inflammatory disease. OBJECTIVES(1) To characterize spatiotemporal ERK activity in response to pro-inflammatory ligands, and (2) to assess pharmacological and metabolic regulation of cytokine-mediated SPREADs. METHODSSPREADs were measured by live-cell ERK biosensors in human bronchial epithelial cell lines (HBE1 and 16HBE) and primary human bronchial epithelial (pHBE) cells, in both submerged and biphasic Air-Liquid Interface (ALI) culture conditions (i.e., differentiated cells). Cells were exposed to pro-inflammatory cytokines relevant to asthma and chronic obstructive pulmonary disease (COPD), and to pharmacological treatments (gefitinib, tocilizumab, hydrocortisone) and metabolic modulators (insulin, 2-deoxyglucose) to probe the airway epithelial mechanisms of SPREADs. Phospho-STAT3 immunofluorescence was used to measure localized inflammatory responses to IL-6. RESULTSPro-inflammatory cytokines significantly increased the frequency of SPREADs. Notably, differentiated pHBE cells display increased SPREAD frequency that coincides with airway epithelial barrier breakdown. SPREADs correlate with IL-6 peptide secretion and localized pSTAT3. Hydrocortisone, inhibitors of receptor signaling, and suppression of metabolic function decreased SPREAD occurrence. CONCLUSIONSPro-inflammatory cytokines modulate SPREADs in human airway epithelial cells via both secreted EGFR and IL6R ligands. SPREADs correlate with changes in epithelial barrier permeability, implying a role for spatiotemporal ERK signaling in barrier homeostasis and dysfunction during inflammation. The involvement of SPREADs in airway inflammation suggests a novel signaling mechanism that could be exploited clinically to supplement corticosteroid treatment for asthma and COPD. Brief SummaryCombining live-cell ERK biosensors with multiple human airway epithelial models, we demonstrate that pro-inflammatory cytokines cause spatiotemporally organized ERK signaling events called "SPREADs", correlating with conditions that disrupt epithelial barrier function. Additionally, common anti-inflammatory treatments tocilizumab, gefitinib, and hydrocortisone suppress cytokine-induced SPREADs. These findings suggest that localized ERK signaling coordinates the innate immune response via spatially restricted cytokine release and regulation of airway barrier permeability.

cell biology↗

bioRxiv2023

Systematic mapping and modeling of 3D enhancer-promoter interactions in early mouse embryonic lineages reveal regulatory principles that determine the levels and cell-type specificity of gene expression

Mammalian embryogenesis commences with two pivotal and binary cell fate decisions that give rise to three essential lineages, the trophectoderm (TE), the epiblast (EPI) and the primitive endoderm (PrE). Although key signaling pathways and transcription factors that control these early embryonic decisions have been identified, the non-coding regulatory elements via which transcriptional regulators enact these fates remain understudied. To address this gap, we have characterized, at a genome-wide scale, enhancer activity and 3D connectivity in embryo-derived stem cell lines that represent each of the early developmental fates. We observed extensive enhancer remodeling and fine-scale 3D chromatin rewiring among the three lineages, which strongly associate with transcriptional changes, although there are distinct groups of genes that are irresponsive to topological changes. In each lineage, a high degree of connectivity or "hubness" positively correlates with levels of gene expression and enriches for cell-type specific and essential genes. Genes within 3D hubs also show a significantly stronger probability of coregulation across lineages, compared to genes in linear proximity or within the same contact domains. By incorporating 3D chromatin features, we build a novel predictive model for transcriptional regulation (3D-HiChAT), which outperformed models that use only 1D promoter or proximal variables in predicting levels and cell-type specificity of gene expression. Using 3D-HiChAT, we performed genome-wide in silico perturbations to nominate candidate functional enhancers and hubs in each cell lineage, and with CRISPRi experiments we validated several novel enhancers that control expression of one or more genes in their respective lineages. Our study comprehensively identifies 3D regulatory hubs associated with the earliest mammalian lineages and describes their relationship to gene expression and cell identity, providing a framework to understand lineage-specific transcriptional behaviors. HIGHLIGHTS- Cell lines representing early embryonic lineages undergo drastic enhancer remodeling and fine-scale 3D chromatin reorganization - Highly interacting 3D hubs strongly enrich for highly expressed, cell-type specific and essential genes - 3D chromatin features greatly improve prediction of cell-type specific gene expression compared to 1D promoter features - In silico and experimental perturbations identify novel enhancers regulating the expression of two or more genes in early embryonic lineages

molecular biology↗

bioRxiv2023

Single-cell analysis of bidirectional reprogramming between early embryonic states reveals mechanisms of differential lineage plasticities

Two distinct fates, pluripotent epiblast (EPI) and primitive (extra-embryonic) endoderm (PrE), arise from common progenitor cells, the inner cell mass (ICM), in mammalian embryos. To study how these sister identities are forged, we leveraged embryonic (ES) and eXtraembryonic ENdoderm (XEN) stem cells - in vitro counterparts of the EPI and PrE. Bidirectional reprogramming between ES and XEN coupled with single-cell RNA and ATAC-seq analyses uncovered distinct rates, efficiencies and trajectories of state conversions, identifying drivers and roadblocks of reciprocal conversions. While GATA4-mediated ES-to-iXEN conversion was rapid and nearly deterministic, OCT4, KLF4 and SOX2-induced XEN-to-iPS reprogramming progressed with diminished efficiency and kinetics. The dominant PrE transcriptional program, safeguarded by Gata4, and globally elevated chromatin accessibility of EPI underscored the differential plasticities of the two states. Mapping in vitro trajectories to embryos revealed reprogramming in either direction tracked along, and toggled between, EPI and PrE in vivo states without transitioning through the ICM.

developmental biology↗

bioRxiv2023

Dynamic network-guided CRISPRi screen reveals CTCF loop-constrained nonlinear enhancer-gene regulatory activity in cell state transitions

Comprehensive enhancer discovery is challenging because most enhancers, especially those affected in complex diseases, have weak effects on gene expression. Our network modeling revealed that nonlinear enhancer-gene regulation during cell state transitions can be leveraged to improve the sensitivity of enhancer discovery. Utilizing hESC definitive endoderm differentiation as a dynamic transition system, we conducted a mid-transition CRISPRi-based enhancer screen. The screen discovered a comprehensive set of enhancers (4 to 9 per locus) for each of the core endoderm lineage-specifying transcription factors, and many enhancers had strong effects mid-transition but weak effects post-transition. Through integrating enhancer activity measurements and three-dimensional enhancer-promoter interaction information, we were able to develop a CTCF loop-constrained Interaction Activity (CIA) model that can better predict functional enhancers compared to models that rely on Hi-C-based enhancer-promoter contact frequency. Our study provides generalizable strategies for sensitive and more comprehensive enhancer discovery in both normal and pathological cell state transitions.

genetics↗

bioRxiv2022

Chromatin regulators in the TBX1 network confer risk for conotruncal heart defects in 22q11.2DS and sporadic congenital heart disease

BackgroundCongenital heart disease (CHD) affecting the conotruncal region of the heart, occur in half of patients with 22q11.2 deletion syndrome. This syndrome is a rare disorder with relative genetic homogeneity that can facilitate identification of genetic modifiers. Haploinsufficiency of TBX1, mapped to the 22q11.2 region, encoding a T-box transcription factor, is one of the main genes for the etiology of the syndrome. We suggest that genetic modifiers of CHD in patients with 22q11.2 deletion syndrome may be in the TBX1 gene network. MethodsTo identify genetic modifiers of 22q11.2 deletion syndrome, we analyzed whole genome sequence of subjects with 22q11.2DS, of which 456 were cases with conotruncal heart defects and 537 were controls with normal cardiac structures. We retained the most damaging rare coding variants and examined 19 functional gene sets for association that were weighted upon expression of genes in cardiac progenitor cells in mouse embryos identified by RNA-sequencing. ResultsWe identified rare damaging coding variants in chromatin regulatory genes as modifiers of conotruncal heart defects in 22q11.2DS. Chromatin genes with recurrent damaging variants include EP400, KAT6A, KMT2C, KMT2D, NSD1, CHD7 and PHF21A. In total, we identified 37 chromatin regulatory genes, that may increase risk for conotruncal heart defects in 8.5% of 22q11.2 deletion syndrome cases. Many of these genes were identified as risk factors for sporadic CHD in the general population increasing the likelihood that these genes are medically important contributors for CHD. These genes are co-expressed in cardiac progenitor cells with TBX1, suggesting that they may be in the same genetic network. Some of the genes identified, such as KAT6A, KMT2C, CHD7 and EZH2, have been previously shown to genetically interact with TBX1 in mouse models, providing mechanistic validation of these genes found. ConclusionsOur findings indicate that disturbance of chromatin regulatory genes impact a TBX1 gene network serving as genetic modifiers of 22q11.2 deletion syndrome. Since some of these chromatin regulatory genes were found in individuals with sporadic CHD, we suggest that there are shared mechanisms involving the TBX1 gene network in the etiology of CHD.

genetics↗

bioRxiv2022

Clinical, socio-demographic, and parental correlates of early autism traits

BackgroundAutism traits emerge between the ages of 1 and 2. It is not known if experiences which increase the likelihood of childhood autism are related to early trait emergence, or if other exposures are more important. Identifying factors linked to toddler autism traits in the general population may improve our understanding of the mechanisms underlying atypical neurodevelopment. MethodsClinical, socio-demographic, and parental information was collected at birth from 536 toddlers in London, UK (gestational age at birth, sex, maternal body mass index, age, parental education level, parental first language, parental history of neurodevelopmental disorders) and at 18 months (parent cohabiting status, two measures of social deprivation, three measures of maternal parenting style, and a measure of maternal postnatal depression). General neurodevelopment was assessed with the Bayley Scales of Infant and Toddler Development, 3rd Edition (BSID-III), and autism traits were assessed using the Quantitative Checklist for Autism in Toddlers (Q-CHAT). Multivariable models were used to identify associations between variables and Q-CHAT. A model including BSID-III was used to identify factors associated with Q-CHAT independent of general neurodevelopment. Models were also evaluated addressing variable collinearity with principal component analysis (PCA). ResultsA multivariable model explained 20% of Q-CHAT variance, with four individually significant variables (two measures of parenting style and two measures of socio-economic deprivation). After adding general neurodevelopment into the model 36% of Q-CHAT variance was explained, with three individually significant variables (two measures of parenting style and one measure of language development). After addressing variable collinearity with PCA, parenting style and social deprivation were positively correlated with Q-CHAT score via a single principal component, independently of general neurodevelopment. Neither sex nor family history of autism were associated with Q-CHAT score. LimitationsThe Q-CHAT is parent rated and is therefore a subjective opinion rather than a clinical assessment. We measured Q-CHAT at a single timepoint, and to date no participant has been followed up in later childhood, so we are focused purely on emerging traits rather than clinical autism diagnoses. ConclusionsAutism traits are common at age 18 months, and greater emergence is specifically related to exposure to early life adversity.

neuroscience↗

bioRxiv2022

Hypothalamic extraretinal photoreceptor Opsin3 regulates water balance, body temperature and motor activity

Opsin 3 (Opn3) is a light-sensitive protein present throughout the mammalian brain, although its functions in this organ are currently unknown. Since Opn3 encoded mRNA increases in the supraoptic nucleus (SON) of the hypothalamus in response to hyperosmotic stimuli, we have explored the role of Opn3 in this nucleus. By in situ hybridization, we have demonstrated that Opn3 is expressed in neurones expressing both arginine vasopressin and oxytocin in the rat SON, suggesting that OPN3 functions in both neuronal types. Gene profiling analysis by RNA-seq suggested that neuropeptide production is impaired when knocking down Opn3 in the rat SON, as confirmed by quantitative PCR and immunolabelling. Knocking down Opn3 in the rat SON altered physiological parameters including water intake, body temperature and motor activity. Altogether the data indicates that Opn3 in the SON is involved in the regulation of a number of neuropeptides and other proteins that participate in water homeostasis, body temperature and motor activity.

neuroscience↗

bioRxiv2022

Multiomic deep delve of synthesis and secretion processes in a model peptidergic system

The cell bodies of hypothalamic magnocellular neurones are densely packed in the hypothalamic supraoptic nucleus (SON) whereas their axons project to the anatomically discrete posterior pituitary gland. We have taken advantage of this unique anatomical structure to establish proteome and phosphoproteome dynamics in neuronal cell bodies and axonal terminals in response to physiological stimulation. We have found that proteome and phosphoproteome responses are very different between somatic and axonal neuronal compartments, indicating the need of each cell domain to differentially adapt. In particular, changes in the phosphoproteome in the cell body are involved in the reorganisation of the cytoskeleton and in axonal terminals the regulation of synaptic and secretory processes. We have identified that prohormone precursors including vasopressin and oxytocin are phosphorylated in axonal terminals and become hyperphosphorylated following stimulation. By multi-omic integration of transcriptome and proteomic data we identify changes to proteins present in afferent inputs to this nucleus. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/494122v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@adc36org.highwire.dtl.DTLVardef@ff7d0corg.highwire.dtl.DTLVardef@16abd16org.highwire.dtl.DTLVardef@a41e3d_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2022

Patterns of connectome variability in autism across five functional activation tasks. Fndings from the LEAP project

BackgroundAutism spectrum disorder (autism) is a complex neurodevelopmental condition with pronounced behavioural, cognitive, and neural heterogeneities across individuals. Here, our goal was to characterise heterogeneity in autism by identifying patterns of neural diversity as reflected in BOLD fMRI in the way individuals with autism engage with a varied array of cognitive tasks. MethodsAll analyses were based on the EU-AIMS/AIMS-2-TRIALS multisite Longitudinal European Autism Project (LEAP) with participants with autism and typically developing controls (TD) between 6 and 30 years of age. We employed a novel task-potency approach which combines the unique aspects of both resting-state fMRI and task-fMRI to quantify task-induced variations in the functional connectome. Normative modelling was used to map atypicality of features on an individual basis with respect to their distribution in neurotypical control participants. We applied robust out-of-sample canonical correlation analysis (CCA) to relate connectome data to behavioural data. ResultsDeviation from the normative ranges of global functional connectivity was greater for individuals with autism compared to TD in each fMRI task paradigm (all tasks p<0.001). The similarity across individuals of the deviation pattern was significantly increased in autistic relative to TD individuals (p<0.002). The CCA identified significant and robust brainbehavior covariation between functional connectivity atypicality and autism-related behavioral features. ConclusionsIndividuals with autism engage with tasks in a globally atypical way, but the particular spatial pattern of this atypicality is nevertheless similar across tasks. Atypicalities in the tasks originate mostly from prefrontal cortex and default mode network regions, but also speech and auditory networks. We show, moving forward, sophisticated modeling methods such as task-potency and normative modeling will prove key to unravelling complex heterogeneous conditions like autism.

neuroscience↗

bioRxiv2021

Excitatory/inhibitory imbalance in autism: the role of glutamate and GABA gene-sets in symptoms and cortical brain structure

BackgroundThe excitatory/inhibitory (E/I) imbalance hypothesis posits that an imbalance between excitatory (glutamatergic) and inhibitory (GABAergic) mechanisms underlies the behavioral characteristics of autism spectrum disorder (autism). However, how E/I imbalance arises and how it may differ across autism symptomatology and brain regions is not well understood. MethodsWe used innovative analysis methods - combining competitive gene-set analysis and gene-expression profiles in relation to cortical thickness (CT)- to investigate the relationship between genetic variance, brain structure and autism symptomatology of participants from the EU-AIMS LEAP cohort (autism=360, male/female=259/101; neurotypical control participants=279, male/female=178/101) aged 6 to 30 years. Competitive gene-set analysis investigated associations between glutamatergic and GABAergic signaling pathway gene-sets and clinical measures, and CT. Additionally, we investigated expression profiles of the genes within those sets throughout the brain and how those profiles relate to differences in CT between autistic and neurotypical control participants in the same regions. ResultsThe glutamate gene-set was associated with all autism symptom severity scores on the Autism Diagnostic Observation Schedule-2 (ADOS-2) and the Autism Diagnostic Interview-Revised (ADI-R) within the autistic group, while the GABA set was associated with sensory processing measures (using the SSP subscales) across all participants. Brain regions with greater gene expression of both glutamate and GABA genes showed greater differences in CT between autistic and neurotypical control participants. ConclusionsOur results suggest crucial roles for glutamate and GABA genes in autism symptomatology as well as CT, where GABA is more strongly associated with sensory processing and glutamate more with autism symptom severity.

neuroscience↗

bioRxiv2021

Broad-scale variation in human genetic diversity levels is predicted by purifying selection on coding and non-coding elements

Analyses of genetic variation in many taxa have established that neutral genetic diversity is shaped by natural selection at linked sites. Whether the mode of selection is primarily the fixation of strongly beneficial alleles (selective sweeps) or purifying selection on deleterious mutations (background selection) remains unknown, however. We address this question in humans by fitting a model of the joint effects of selective sweeps and background selection to autosomal polymorphism data from the 1000 Genomes Project. After controlling for variation in mutation rates along the genome, a model of background selection alone explains [~]60% of the variance in diversity levels at the megabase scale. Adding the effects of selective sweeps driven by adaptive substitutions to the model does not improve the fit, and when both modes of selection are considered jointly, selective sweeps are estimated to have had little or no effect on linked neutral diversity. The regions under purifying selection are best predicted by phylogenetic conservation, with [~]80% of the deleterious mutations affecting neutral diversity occurring in non-exonic regions. Thus, background selection is the dominant mode of linked selection in humans, with marked effects on diversity levels throughout autosomes.

evolutionary biology↗

bioRxiv2020

Host Specific SARS-CoV-2 Mutations: Insertion of the Phenylalanine in the NSP6 Linked to the United Kingdome and Premature Termination of the ORF-8 Associated with the European and the United States of America Derived Samples.

The coronavirus belongs to the order Nidovirales, which is known for the longest RNA genome virus. The polymerase enzyme of SARS-CoV-2 has proofreading functions, but still, the RNA viruses have a higher mutation rate than DNA viruses. The mutations in the viral genome provide a replication advantage in any population/geographical location and that may have profound consequences in the outcome and pathogenesis, diagnosis and patient management of the viral infection. In the present study, we have analysed full-length SARS-CoV-2 genome sequences, derived from symptomatic/asymptomatic COVID-19 patients from all six continents to investigate the common mutations globally. Our results revealed that SARS-CoV-2 is mutating independently, we identified total 313 mutations and some (21 mutations) of them are prevailing over time irrespective of geographical location. Another important finding, we are reporting here is, the mutation rate of the virus varies in different geographical locations suggesting the virus is adapting different strategies in the infected populations, having different genetic backgrounds across the globe. We have identified 11085TTT insertion (insertion of the Phenylalanine in NSP6 at position 38) mutation, which is mainly linked to the UK derived SARS-CoV-2 samples, we have also discovered non-sense mutation in ORF-8 after 17 amino acid is linked to the European and the USA derived SARS-CoV-2 samples.

genomics↗

bioRxiv2020

Biallelic mutation of CLRN2 causes non-syndromic hearing loss in humans

Deafness, the most frequent sensory deficit in humans, is extremely heterogenous with hundreds of genes probably involved. Clinical and genetic analyses of an extended consanguineous family with pre-lingual, moderate-to-profound autosomal recessive sensorineural hearing loss, allowed us to identify CLRN2, encoding a tetraspan protein as a new deafness gene. Homozygosity mapping followed by exome sequencing identified a 15.2 Mb locus on chromosome 4p15.32p15.1 containing a missense pathogenic variant in CLRN2 (c.494C>A, NM_001079827.2) segregating with the disease. Using in vitro RNA splicing analysis, we show that the CLRN2 c.494C>A mutation leads to two events: 1) the substitution of a highly conserved threonine (uncharged amino acid) to lysine (charged amino acid) at position 165, p.(Thr165Lys), and 2) aberrant splicing, with the retention of intron 2 resulting in a stop codon after 26 additional amino acids, p.(Gly146Lysfs*26). Expression studies and phenotyping of newly produced zebrafish and mouse models deficient for clarin 2 further confirm that clarin 2, expressed in the inner ear hair cells, is essential for normal organization and maintenance of the auditory hair bundles, and for hearing function. Together, our findings identify CLRN2 as a new deafness gene, which will impact future diagnosis and treatment for deaf patients.

genetics↗

bioRxiv2020

PP1 promotes cyclin B destruction and the metaphase-anaphase transition by dephosphorylating CDC20

Ubiquitin-dependent proteolysis of cyclin B and securin initiates sister chromatid segregation and anaphase. The anaphase promoting complex/cyclosome (APC/C) and its co-activator CDC20 form the main ubiquitin E3 ligase for these proteins. APC/CCDC20 is regulated by CDK1-cyclin B and counteracting PP1 and PP2A family phosphatases through modulation of both activating and inhibitory phosphorylations. Here we report that PP1 promotes cyclin B destruction at the onset of anaphase by removing specific inhibitory phosphorylation in the N-terminus of CDC20. Depletion or chemical inhibition of PP1 stabilises cyclin B and results in a pronounced delay at the metaphase-to-anaphase transition after chromosome alignment. This requirement for PP1 is lost in cells expressing CDK1-phosphorylation defective CDC206A mutants. These CDC206A cells show a normal spindle checkpoint response, but once all chromosomes have aligned rapidly degrade cyclin B and enter into anaphase in the absence of PP1 activity. PP1 therefore facilitates the metaphase-to-anaphase by promoting APC/CCDC20-dependent destruction of cyclin B in human cells.

cell biology↗

bioRxiv2019

Antiviral signalling in human IPSC-derived neurons recapitulates neurodevelopmental disorder phenotypes

Maternal immune activation increases the risk of neurodevelopmental disorders. Elevated cytokines, such as interferon-gamma (IFN{gamma}), in offsprings brains play a central role. IFN{gamma} activates an antiviral cellular state, limiting viral entry and replication. In addition, IFN{gamma} has been implicated in brain development. Here, we hypothesise that IFN{gamma}-induced antiviral signalling contributes to molecular and cellular phenotypes associated with neurodevelopmental disorders. We find that transient IFN{gamma} treatment of neural progenitors derived from human induced pluripotent stem cells (hIPSCs) persistently increases neurite outgrowth, phenocopying hIPSC-neurons from autistic individuals. IFN{gamma} upregulates antiviral PML bodies and MHC class I (MHCI) genes, which persists through neuronal differentiation. Critically, IFN{gamma}-induced neurite outgrowth requires both PML and MHCI. We also find that IFN{gamma} disproportionately alters expression of autism and schizophrenia risk genes, suggesting convergence between these genetic and environmental risk factors. Together, these data indicate that IFN{gamma}-induced antiviral signalling may contribute to neurodevelopmental disorder aetiology.

neuroscience↗

bioRxiv2019

Do direct nose-to-brain pathways underlie intranasal oxytocin-induced changes in regional cerebral blood flow in humans?

Could nose-to-brain pathways mediate the effects of peptides such as oxytocin (OT) on brain physiology when delivered intranasally? We address this question by contrasting two methods of intranasal administration (a standard nasal spray, and a nebulizer expected to improve OT deposition in nasal areas putatively involved in direct nose-to-brain transport) to intravenous administration in terms of effects on regional cerebral blood flow during two hours post-dosing. We demonstrate that OT-induced decreases in amygdala perfusion, a key hub of the OT central circuitry, are explained entirely by OT increases in systemic circulation following both intranasal and intravenous OT administration. Yet we also provide robust evidence confirming the validity of the intranasal route to target specific brain regions. Our work has important translational implications and demonstrates the need to carefully consider the method of administration in our efforts to engage specific central oxytocinergic targets for the treatment of neuropsychiatric disorders.

neuroscience↗