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Singh, M.

Publications and source records attributed to Singh, M..

At least 109 records · Page 6Linked to original sources
bioRxiv2023

KAT6A mutations in Arboleda-Tham syndrome drive epigenetic regulation of posterior HOXC cluster

Arboleda-Tham Syndrome (ARTHS) is a rare genetic disorder caused by heterozygous, de novo truncating mutations in Lysine(K) acetyltransferase 6A (KAT6A). ARTHS is clinically heterogeneous and characterized by several common features including intellectual disability, developmental and speech delay, hypotonia and affects multiple organ systems. KAT6A is highly expressed in early development and plays a key role in cell-type specific differentiation. KAT6A is the enzymatic core of a histone-acetylation protein complex, however the direct histone targets and gene regulatory effects remain unknown. In this study, we use ARTHS patient (n=8) and control (n=14) dermal fibroblasts and perform comprehensive profiling of the epigenome and transcriptome caused by KAT6A mutations. We identified differential chromatin accessibility within the promoter or gene body of 23%(14/60) of genes that were differentially expressed between ARTHS and controls. Within fibroblasts, we show a distinct set of genes from the posterior HOXC gene cluster (HOXC10, HOXC11, HOXC-AS3, HOXC-AS2, HOTAIR) that are overexpressed in ARTHS and are transcription factors critical for early development body segment patterning. The genomic loci harboring HOXC genes are epigenetically regulated with increased chromatin accessibility, high levels of H3K23ac, and increased gene-body DNA methylation compared to controls, all of which are consistent with transcriptomic overexpression. Finally, we used unbiased proteomic mass spectrometry and identified two new histone post-translational modifications (PTMs) that are disrupted in ARTHS: H2A and H3K56 acetylation. Our multi-omics assays have identified novel histone and gene regulatory roles of KAT6A in a large group of ARTHS patients harboring diverse pathogenic mutations. This work provides insight into the role of KAT6A on the epigenomic regulation in somatic cell types.

genomics↗
bioRxiv2023

Aberrant tissue stiffness impairs neural tube development in Mthfd1l mutant mouse embryos

Neurulation is a highly synchronized biomechanical process leading to the formation of the brain and spinal cord, and its failure leads to neural tube defects (NTDs). Although we are rapidly learning the genetic mechanisms underlying NTDs, the biomechanical aspects are largely unknown. To understand the correlation between NTDs and tissue stiffness during neural tube closure (NTC), we imaged an NTD murine model using optical coherence tomography (OCT), Brillouin microscopy, and confocal fluorescence microscopy. Here, we associate structural information from OCT with local stiffness from the Brillouin signal of embryos undergoing neurulation. The stiffness of neuroepithelial tissues in Mthfd1l null embryos was significantly lower compared to that of wild-type embryos, while exogenous formate supplementation improved tissue stiffness and gross embryonic morphology in both nullizygous and heterozygous embryos. Our results demonstrate the significance of proper tissue stiffness for normal NTC and pave the way for future studies on the mechanobiology of normal and abnormal embryonic development.

developmental biology↗
bioRxiv2023

Disruption of Fuz in mouse embryos generates hypoplastic hindbrain development and reduced cranial nerve ganglia.

The formation of the brain and spinal cord is initiated in the earliest stages of mammalian pregnancy in a highly organized process known as neurulation. Convergent and extension movements transforms a flat sheet of ectodermal cells into a narrow and elongated line of neuroepithelia, while a major source of Sonic Hedgehog signaling from the notochord induces the overlying neuroepithelial cells to form two apposed neural folds. Afterward, neural tube closure occurs by synchronized coordination of the surface ectoderm and adjacent neuroepithelial walls at specific axial regions known as neuropores. Environmental or genetic interferences can impair neurulation resulting in neural tube defects. The Fuz gene encodes a subunit of the CPLANE complex, which is a macromolecular planar polarity effector required for ciliogenesis. Ablation of Fuz in mouse embryos results in exencephaly and spina bifida, including dysmorphic craniofacial structures due to defective cilia formation and impaired Sonic Hedgehog signaling. In this work, we demonstrate that knocking Fuz out during embryonic mouse development results in a hypoplastic hindbrain phenotype, displaying abnormal rhombomeres with reduced length and width. This phenotype is associated with persistent loss of ventral neuroepithelial stiffness, in a notochord adjacent area at the level of the rhombomere 5, preceding the development of exencephaly in Fuz ablated mutants. The formation of cranial and paravertebral ganglia is also impaired in these embryos, indicating that Fuz has a critical function sustaining normal neural tube development and neuronal differentiation. O_FIG O_LINKSMALLFIG WIDTH=153 HEIGHT=200 SRC="FIGDIR/small/552068v1_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@1c1d63forg.highwire.dtl.DTLVardef@d8e5e0org.highwire.dtl.DTLVardef@1dbf248org.highwire.dtl.DTLVardef@ff6969_HPS_FORMAT_FIGEXP M_FIG C_FIG SIGNIFICANCE STATEMENTNeural tube defects (NTDs) are a common cause of disability in children, representing the second most common congenital structural malformation in humans following only congenital cardiovascular malformations. NTDs affect approximately 1 to 2 pregnancies per 1000 births every year worldwide, when the mechanical forces folding the neural plate fails to close at specific neuropores located anteriorly (cranial) or posteriorly (caudal) along the neural tube, in a process known as neurulation, which happens throughout the third and fourth weeks of human pregnancy.

developmental biology↗
bioRxiv2023

Elucidation of the essentiality of lumazine synthase (RibH) for Mycobacterium tuberculosis survival and discovery of potent inhibitors for enhanced antimycobacterial therapy

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate the indispensable role of lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, in the survival of Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We show that genetic and functional ablation of RibH in M. tb cannot be compensated by exogenous supply of Riboflavin, or co-factors Flavin Adenine Dinucleotide (FAD) or Flavin Mononucleotide (FMN). Capitalizing on the essentiality of RibH, we employ a high-throughput molecular docking approach to screen [~]600,000 compounds and identify inhibitors of RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 inhibitors that exhibit potent antimycobacterial activity. These compounds effectively also eradicate intracellular M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these 3 compounds synergistically enhance the bactericidal effect of first-line anti-TB drugs, Isoniazid and Rifampicin. Corroborating with the in silico predicted high docking scores along with favorable ADME and toxicity profiles, all 3 compounds demonstrate exceptional binding affinity towards purified lumazine synthase enzyme in vitro, and display an acceptable safety profile in mammalian cells, with a high selective index. By providing mechanistic evidence for the essentiality of RibH in M. tb survival, and discovering potent RibH inhibitors with outstanding antimycobacterial activity, our study contributes to the development of superior TB treatment strategies and advances the global fight against this devastating disease.

microbiology↗
bioRxiv2023

Second messenger c-di-AMP regulates multiple antibiotic sensitivity pathways in Mycobacterium smegmatis by discrete mechanisms

One of the debilitating causes of high mortality in the case of tuberculosis and other bacterial infections is the resistance development against standard drugs. There are limited studies so far to describe how a bacterial second messenger molecule can directly participate in distinctive antibiotic tolerance characteristics of a cell in a mechanism-dependent manner. Here we show that intracellular cyclic di-AMP (c-di-AMP) concentration can modulate drug sensitivity of Mycobacterium smegmatis by directly interacting with either a protein effector or with the 5-UTR regions in mRNA of the genes and thus causing transcriptional downregulation of important genes in the pathways. We studied four antibiotics with different mechanisms of action: rifampicin, ciprofloxacin, erythromycin, and tobramycin and subsequently found that the level of drug sensitivity of the bacteria is directly proportional to the c-di-AMP concentration inside the cell. Further, we unraveled the underlying molecular mechanisms to delineate the specific genes and pathways regulated by c-di-AMP and hence result in differential drug sensitivity in M. smegmatis. Thus, our findings of c-di-AMP messenger controlling drug resistance phenotypes of mycobacteria against four different classes of antibiotics is a unique observation that will contribute to scientific advancement in the field. One Sentence SummaryHere we describe how second messenger c-di-AMP modulates the antibiotic sensitivity and resistance profile of M. smegmatis by diverse mechanisms.

microbiology↗
bioRxiv2023

Global transcriptome analysis reveals Salmonella Typhimurium employs the nitrate-dependent anaerobic pathway to combat bile stress

Salmonella Typhimurium is an enteric pathogen that is highly tolerant to bile. Next-generation mRNA sequencing was performed to analyse the stress and adaptive responses of S. Typhimurium to bile. We identified the cellular pathways affected during bile stress in wild type (WT) and a mutant lacking cspE ({Delta}cspE), which plays an essential role in protection from bile stress. We observed transcriptional upregulation of several genes involved in nitrate metabolism, in response to bile stress. These genes were also differentially expressed between the bile-resilient WT and the bile-sensitive {Delta}cspE strain. To understand the role of nitrate metabolism in bile stress response, we generated a strain lacking fnr ({Delta}fnr), which is the global regulator of nitrate metabolism in S. Typhimurium. fnr was highly induced in the bile treated WT strain but not in the {Delta}cspE strain. Notably, the {Delta}fnr strain was susceptible to bile-mediated killing. Our studies revealed a new role for fnr in mediating the bile stress response. In addition, a strain lacking arcA ({Delta}arcA), a two-component system response regulator involved in anaerobic metabolism, also showed a marked reduction in growth in presence of bile. This corroborated the significance of anaerobic metabolism in S. Typhimurium bile tolerance. Importantly, overexpression of fnr and arcA lowered reactive oxygen species and significantly enhanced the survival of the bile-sensitive {Delta}cspE strain. We also observed that S. Typhimurium pre-treated with nitrate displayed better growth in the presence of bile. Together, these results demonstrate that nitrate-dependent anaerobic metabolism promotes adaptation of S. Typhimurium to bile. ImportanceSalmonella Typhimurium, as an enteric pathogen, manifests an extreme example of bile tolerance. This study describes the diverse metabolic changes at the level of transcriptome in S. Typhimurium exposed to bile. We identified the differential expression of several genes involved in anaerobic metabolism between bile-tolerant WT and bile-sensitive {Delta}cspE strains. Two major regulators of anaerobic metabolism, fnr and arcA, support the growth of S. Typhimurium in bile. Our results highlight that, in presence of bile, S. Typhimurium activates genes involved in anaerobic metabolism, specifically nitrate metabolism, that improves survival of bacteria during bile stress.

microbiology↗
bioRxiv2023

The axonal localization of Dual Leucine Zipper Kinase defines its protein-turnover

Dual Leucine Zipper Kinase (DLK) mediates multiple neuronal stress responses, and its expression levels are constantly suppressed to prevent excessive stress signaling. We found that Wallenda (Wnd), the Drosophila ortholog of DLK, is highly enriched in the axon terminals of Drosophila sensory neurons in vivo and that this subcellular localization is necessary for Highwire-mediated Wnd protein turnover under normal conditions. Our structure-function analysis found that Wnd palmitoylation is essential for its axon terminal localization. Palmitoylation-defective Wnd accumulated in neuronal cell bodies, exhibited dramatically increased protein expression levels, and triggered excessive neuronal stress responses. Defective intracellular transport is implicated in neurodegenerative conditions. Comprehensive dominant-negative Rab protein screening identified Rab11 as an essential factor for Wnd localization in axon terminals. Consequently, Rab11 loss-of-function increased the protein levels of Wnd and induced neuronal stress responses. Inhibiting Wnd activity significantly ameliorated neuronal loss and c-Jun N-terminal kinase signaling triggered by Rab11 loss-of-function. Taken together, these suggest that DLK proteins are constantly transported to axon terminals for protein turnover and a failure of such transport can lead to neuronal loss. Our study demonstrates how subcellular protein localization is coupled to protein turnover for neuronal stress signaling. HighlightsO_LIWnd is highly enriched in axon terminals. C_LIO_LIWnd protein turnover by Hiw is restricted in the axon terminals. C_LIO_LIProtein palmitoylation of Wnd and Rab11 activity is essential for Wnd axonal localization. C_LIO_LIRab11 mutations and defective Wnd palmitoylation impair Wnd protein turnover leading to increased Wnd protein levels and neuronal loss. C_LIO_LIInhibiting Wnd activity mitigates neuronal stress response caused by Rab11 loss-of-function. C_LI

neuroscience↗
bioRxiv2023

SifA SUMOylation governs Salmonella intracellular survival via modulation of lysosomal function

Gastroenteritis causing pathogen Salmonella Typhimurium (S. Tm) during its infection in host cells thrives in a vacuolated compartment, Salmonella Containing Vacuole (SCV), which sequentially acquires host endosomal and lysosomal markers. Long tubular structures, called as Salmonella induced filaments (SIFs), are known to be required for SCVs nutrient acquisition, membrane maintenance and stability. A tightly coordinated interactions involving prominent effector SifA and various host adapters PLEKHM1, PLEKHM2 and Rab GTPases govern SCV integrity and SIF formation. Here, we report for the first time, the functional regulation of SifA is modulated by its SUMOylation at lysine 11. S. Tm expressing lysine 11 mutant SifA (SifAK11R) is defective in intracellular proliferation due to compromised SIF formation and enhanced lysosomal acidification. Furthermore, murine competitive index experiments reveal defective in vivo proliferation and weakened virulence of SifAK11R mutant. Concisely, our results demonstrate that SUMO deficient SifA mutant nearly behaves like a SifA knockout strain which impacts PLEKHM2-M6PR mediated lysosomal acidification pathway. Thus, our results bring forth a novel S. Tm-host crosstalk mechanism involving host mediated effector SUMOylation critical for pathogenicity.

microbiology↗
bioRxiv2023

Single cell transcriptomics reveals early photoreceptor trajectories and a cancer-predisposed cone precursor state

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin, yet the developmental basis for their distinct behaviors is poorly understood. Here, we used deep full-length single-cell RNA-sequencing (scRNA-seq) to distinguish post-mitotic cone and rod developmental states and identify cone-specific features that contribute to retinoblastomagenesis. The analyses revealed nascent, immediately post-mitotic cone- and rod precursor populations characterized by higher THRB or NRL regulon activities, immature and maturing cone and rod precursors with concurrent cone- and rod-related gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Cell-state-specific gene expression features inferred based on full-length scRNA-seq were consistent with past single nucleus 3 RNA-seq analyses. Beyond the cell state characterizations, full-length scRNAseq revealed that both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL and THRB isoforms and prematurely terminated THRB transcripts. Moreover, early L/M cone precursors exhibited successive expression of several lncRNAs along with MYCN, which composed the seventh most L/M-cone-specific regulon, and SYK, which was implicated in the cone precursors proliferative response to RB1 loss. These findings reveal previously unresolved photoreceptor precursor states and suggest a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation. Impact StatementFeatures acquired by early post-mitotic retinal cells underlie the distinct behaviors of rods and the cone cells of origin of retinoblastoma tumors.

developmental biology↗
bioRxiv2023

An associative transcriptomics study on rice bean (Vigna umbellata) provides new insights into genetic basis and candidate genes governing flowering, maturity and seed weight

Rice bean is an underrated legume with significant potential to support food and nutritional security worldwide, being a rich source of proteins, minerals, and essential fatty acids. Therefore, we considered three pivotal production traits of rice bean; flowering, maturity and seed weight, to identify associated candidate genes. One-hundred diverse genotypes out of 1800 evaluated rice bean accessions from the Indian National Genebank were considered for phenotypic data collection and genotyping by transcriptome sequencing approach. Association analysis involving various GWAS models was conducted to identify significant marker-trait associations. The results revealed association of 82 markers on 48 transcripts for flowering, 26 markers on 22 transcripts for maturity and 22 markers on 21 transcripts for seed weight. The annotation of associated transcripts unraveled the functional genes related to the considered traits. Among the significant candidate genes identified, HSC80, P-II PsbX, phospholipid-transporting-ATPase-9, pectin-acetylesterase-8 and E3-ubiquitin-protein-ligase-RHG1A were found associated with flowering. Further, associations of WRKY1 and DEAD-box-RH27 with seed weight, PIF3 and pentatricopeptide-repeat-containing-gene with maturity & seed weight and aldo-keto-reductase with flowering & maturity have been revealed. The present investigation provides insights into the genetic mechanisms governing economically-essential traits like flowering, maturity and seed weight that can be potentially utilized for rice bean improvement. HighlightsThe present investigation is the first associative transcriptomics approach implemented on Vigna umbellata. Based on the marker-trait association analysis, candidate genes for flowering, maturity and seed weight traits are reported.

bioinformatics↗
bioRxiv2023

PNMA2 forms non-enveloped virus-like capsids that trigger paraneoplastic neurological syndrome

The paraneoplastic Ma antigen (PNMA) genes are associated with cancer-induced paraneoplastic syndromes that present with neurological symptoms and autoantibody production. How PNMA proteins trigger a severe autoimmune disease is unclear. PNMA genes are predominately expressed in the central nervous system with little known functions but are ectopically expressed in some tumors. Here, we show that PNMA2 is derived from a Ty3 retrotransposon that encodes a protein which forms virus-like capsids released from cells as non-enveloped particles. Recombinant PNMA2 capsids injected into mice induce a robust autoimmune reaction with significant generation of autoantibodies that preferentially bind external "spike" PNMA2 capsid epitopes, while capsid-assembly-defective PNMA2 protein is not immunogenic. PNMA2 autoantibodies present in cerebrospinal fluid of patients with anti-Ma2 paraneoplastic neurologic disease show similar preferential binding to PNMA2 "spike" capsid epitopes. These observations suggest that PNMA2 capsids released from tumors trigger an autoimmune response that underlies Ma2 paraneoplastic neurological syndrome.

neuroscience↗
bioRxiv2023

Transcriptional networks predating cognition-associated pyramidal lineages are restructured by erythropoietin

Recombinant human erythropoietin (rhEPO) has potent procognitive effects, hematopoiesis-independent, but underlying mechanisms and physiological role of brain-expressed EPO have remained obscure. Here, we provide encyclopedic transcriptional hippocampal profiling of mice treated with rhEPO. Based on [~]108,000 single nuclei, we unmask multiple pyramidal lineages with their comprehensive molecular signatures. By temporal profiling and gene regulatory analysis, we build a developmental trajectory of CA1 pyramidal neurons derived from multiple predecessor lineages and elucidate gene regulatory networks underlying their fate determination. With EPO as tool, we discover novel populations of newly differentiating pyramidal neurons, overpopulating to [~]200% upon rhEPO with upregulation of genes crucial for neurodifferentiation, dendrite growth, synaptogenesis, memory formation, and cognition. Using a Cre-based approach to visually distinguish pre-existing from newly formed pyramidal neurons for patch-clamp recordings, we learn that rhEPO treatment differentially affects excitatory and inhibitory inputs. Our findings provide mechanistic insight into how EPO modulates neuronal functions and networks.

neuroscience↗
bioRxiv2023

Functional characterization of Salmonella Typhimurium encoded YciF, a domain of unknown function (DUF892) family protein, and its role in protection during bile and oxidative stress

YciF (STM14_2092) is a member of domain of unknown function (DUF892) family. It is an uncharacterized protein, involved in stress responses in Salmonella Typhimurium. In this study, we investigated the significance of YciF and its DUF892 domain during bile and oxidative stress responses of S. Typhimurium. Purified wild type YciF forms higher order oligomers, binds to iron and displays ferroxidase activity. Studies on the site-specific mutants revealed that the ferroxidase activity of YciF is dependent on the two metal binding sites present within the DUF892 domain. Transcriptional analysis displayed that the {Delta}cspE strain, which has compromised expression of YciF, encounters iron toxicity due to dysregulation of iron homeostasis in presence of bile. Utilizing this observation, we demonstrate that the bile mediated iron toxicity in {Delta}cspE causes lethality, primarily through the generation of reactive oxygen species (ROS). Expression of wild type YciF, but not the three mutants of the DUF892 domain, in {Delta}cspE alleviate ROS in presence of bile. Our results establish the role of YciF as a ferroxidase that can sequester excess iron in the cellular milieu to counter ROS-associated cell death. In fact, pre-treatment with an iron chelator mitigates the hypersensitivity of {Delta}cspE to bile. ImportanceThe DUF892 domain has a wide taxonomic distribution encompassing several bacterial pathogens. This domain belongs to the ferritin-like superfamily; however, it has not been biochemically and functionally characterized. This is the first report of characterization of a member of this family. In this study, we demonstrate that S. Typhimurium YciF is an iron binding protein with ferroxidase activity, which is dependent on the metal binding sites present within the DUF892 domain. It combats iron toxicity and oxidative damage caused due to exposure to bile. The functional characterization of YciF delineates the significance of the DUF892 domain in bacteria. In addition, our studies on S. Typhimurium bile stress response divulged the importance of comprehensive iron homeostasis in bacteria in presence of bactericidal compounds that tend to generate ROS irrespective of their primary targets.

microbiology↗
bioRxiv2022

Truncating ASXL1 mutations in Bohring-Opitz Syndrome dysregulate canonical and non-canonical Wnt Signaling

ASXL1 (Additional sex combs-like 1) plays key roles in epigenetic regulation of early developmental gene expression. De novo truncating mutations in ASXL1 cause Bohring-Opitz syndrome (BOS, OMIM #605039), a rare neurodevelopmental condition characterized by severe intellectual disabilities, characteristic facial features, hypertrichosis, increased risk of Wilms tumor, and variable congenital anomalies including heart defects and severe skeletal defects giving rise to a typical BOS posture. These BOS-causing ASXL1 variants are also high-prevalence somatic driver mutations in acute myeloid leukemia (AML). We use primary cells from BOS individuals (n = 18) and controls (n = 49) to dissect gene regulatory changes caused by ASXL1 mutations using comprehensive multi-omics assays for chromatin accessibility (ATAC-seq), DNA methylation, histone methylation binding, and transcriptome in peripheral blood and skin fibroblasts. Our data shows that regardless of cell type, ASXL1 mutations drive strong cross-tissue effects that disrupt multiple layers of the epigenome. The data showed a broad activation of canonical Wnt signaling at the transcriptional and protein levels and upregulation of VANGL2, a planar cell polarity pathway protein that acts through non-canonical Wnt signaling to direct tissue patterning and cell migration. This multi-omics approach identifies the core impact of ASXL1 mutations and therapeutic targets for BOS and myeloid leukemias. Brief summaryGermline ASXL1 mutations that cause Bohring Optiz syndrome disrupt the epigenome and dysregulate gene expression resulting in activation of canonical and non-canonical Wnt signaling pathways.

genetics↗
bioRxiv2022

Secreted ADAMTS-like 2 promotes myoblast differentiation by potentiating Wnt signaling

The formation of multinucleated contractile myofibers from muscle stem cells during myogenesis is indispensable for skeletal muscle formation. Myogenesis is governed by myogenic regulatory transcription factors, including MYOD. However, very few MYOD- regulated effector proteins were shown to be sufficient to promote myogenesis. Here, we identified an unexpected role for the secreted matricellular protein ADAMTS-like 2 (ADAMTSL2) as a rheostat for Wnt signaling during myogenesis downstream of MYOD. ADAMTSL2 expression was induced during myoblast differentiation and ADAMTSL2 was required for myoblast differentiation. ADAMTSL2 ablation in myogenic precursor cells resulted in aberrant muscle architecture in vivo. The pro-myogenic ADAMTSL2 function was dependent on WNT ligands. Mechanistically, ADAMTSL2 potentiated WNT signaling by binding to WNT ligands and WNT receptors. Finally, we identified a WNT-binding ADAMTSL2 peptide that was sufficient to promote myogenesis. Since ADAMTSL2 was previously described as a negative regulator of TGF{beta} signaling in fibroblasts, ADAMTSL2 now emerges as a signaling node that could integrate and fine-tune WNT, TGF{beta} and potentially other signaling pathways within the dynamic microenvironment of differentiating myoblasts during skeletal muscle development and regeneration.

cell biology↗
bioRxiv2022

Comparative analysis of single-cell and single-nucleus RNA-sequencing in a rabbit model of retinal detachment-related proliferative vitreoretinopathy.

Structured AbstractO_ST_ABSPurposeC_ST_ABSProliferative vitreoretinopathy (PVR) is the most common cause of failure of retinal reattachment surgery and the molecular changes leading to this aberrant wound healing process is currently unknown. We aimed to study PVR pathogenesis using single-cell transcriptomics to dissect cellular heterogeneity in a rabbit PVR model. MethodsPVR was induced unilaterally in Dutch Belted rabbits. At different timepoints following PVR induction, retinas were dissociated into either cells or nuclei suspension and processed for single-cell or single-nucleus RNA sequencing (scRNA-seq or snRNA-seq). ResultsscRNA-Seq and snRNA-Seq were conducted on retinas at 4 hours and 14 days after disease induction. While the capture rate of unique molecular identifiers (UMI) and genes were greater in scRNA-seq samples, overall gene expression profiles of individual cell types were highly correlated between scRNA-seq and snRNA-seq. A major disparity between the two sequencing modalities is the cell type capture rate, however, with glial cell types over-represented in scRNA-seq, and inner retinal neurons were enriched by snRNA-seq. Furthermore, fibrotic Muller glia were over-represented in snRNA-seq samples, while reactive Muller glia were in scRNA-seq samples. Trajectory analyses were similar between the two methods, allowing for the combined analysis of the scRNA-seq and snRNA-seq datasets. ConclusionsThese findings highlight limitations of both scRNA-seq and snRNA-seq analysis and imply that use of both techniques can more accurately identify transcriptional networks critical for aberrant fibrogenesis in PVR.

neuroscience↗
bioRxiv2022

Vector-clustering Multiple Sequence Alignment: Aligning into the twilight zone of protein sequence similarity with protein language models

Multiple sequence alignment is a critical step in the study of protein sequence and function. Typically, multiple sequence alignment algorithms progressively align pairs of sequences and combine these alignments with the aid of a guide tree. These alignment algorithms use scoring systems based on substitution matrices to measure amino-acid similarities. While successful, standard methods struggle on sets of proteins with low sequence identity - the so-called twilight zone of protein alignment. For these difficult cases, another source of information is needed. Protein language models are a powerful new approach that leverage massive sequence datasets to produce high-dimensional contextual embeddings for each amino acid in a sequence. These embeddings have been shown to reflect physicochemical and higher-order structural and functional attributes of amino acids within proteins. Here, we present a novel approach to multiple sequence alignment, based on clustering and ordering amino acid contextual embeddings. Our method for aligning semantically consistent groups of proteins circumvents the need for many standard components of multiple sequence alignment algorithms, avoiding initial guide tree construction, intermediate pairwise alignments, gap penalties, and substitution matrices. The added information from contextual embeddings leads to higher accuracy alignments for structurally similar proteins with low amino-acid similarity. We anticipate that protein language models will become a fundamental component of the next generation of algorithms for generating MSAs. Software availability: https://github.com/clairemcwhite/vcmsa

bioinformatics↗