Biology subjects

Saxena, A.

Publications and source records attributed to Saxena, A..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Glioma-Induced Alterations in Excitatory Neurons are Reversed by mTOR Inhibition

Gliomas are highly aggressive brain tumors characterized by poor prognosis and composed of diffusely infiltrating tumor cells that intermingle with non-neoplastic cells in the tumor microenvironment, including neurons. Neurons are increasingly appreciated as important reactive components of the glioma microenvironment, due to their role in causing hallmark glioma symptoms, such as cognitive deficits and seizures, as well as their potential ability to drive glioma progression. Separately, mTOR signaling has been shown to have pleiotropic effects in the brain tumor microenvironment, including regulation of neuronal hyperexcitability. However, the local cellular-level effects of mTOR inhibition on glioma-induced neuronal alterations are not well understood. Here we employed neuron-specific profiling of ribosome-bound mRNA via RiboTag, morphometric analysis of dendritic spines, and in vivo calcium imaging, along with pharmacological mTOR inhibition to investigate the impact of glioma burden and mTOR inhibition on these neuronal alterations. The RiboTag analysis of tumor-associated excitatory neurons showed a downregulation of transcripts encoding excitatory and inhibitory postsynaptic proteins and dendritic spine development, and an upregulation of transcripts encoding cytoskeletal proteins involved in dendritic spine turnover. Light and electron microscopy of tumor-associated excitatory neurons demonstrated marked decreases in dendritic spine density. In vivo two-photon calcium imaging in tumor-associated excitatory neurons revealed progressive alterations in neuronal activity, both at the population and single-neuron level, throughout tumor growth. This in vivo calcium imaging also revealed altered stimulus-evoked somatic calcium events, with changes in event rate, size, and temporal alignment to stimulus, which was most pronounced in neurons with high-tumor burden. A single acute dose of AZD8055, a combined mTORC1/2 inhibitor, reversed the glioma-induced alterations on the excitatory neurons, including the alterations in ribosome-bound transcripts, dendritic spine density, and stimulus evoked responses seen by calcium imaging. These results point to mTOR-driven pathological plasticity in neurons at the infiltrative margin of glioma - manifested by alterations in ribosome-bound mRNA, dendritic spine density, and stimulus-evoked neuronal activity. Collectively, our work identifies the pathological changes that tumor-associated excitatory neurons experience as both hyperlocal and reversible under the influence of mTOR inhibition, providing a foundation for developing therapies targeting neuronal signaling in glioma.

neuroscience↗

bioRxiv2023

Polymer-mediated oligonucleotide delivery enables construction of barcoded 3D cultures for spatial single-cell analysis

Spatial transcriptomics has been widely used to capture gene expression profiles, realised as a two-dimensional (2D) projection of RNA captured from tissue sections. Wree-dimensional (3D) cultures such as spheroids and organoids are highly promising alternatives to oversimplified and homogeneous 2D cell culture models, but existing spatial transcriptomic platforms do not currently have sufficient resolution for robust analysis of 3D cultures. We present a transfection-based method for fluorescent DNA barcoding of cell populations, and the subsequent construction of spheroidal cellular architectures using barcoded cells in a layer-by-layer approach. For the first time, changes in gene expression throughout this 3D culture architecture are interrogated using multiplex single-cell RNA sequencing in which DNA barcodes are used to encode the spatial positioning of cells. We show that transfection with fluorophore-conjugated barcode oligonucleotides enables both imaging and sequencing at single-cell resolution, providing spatial maps of gene expression and drug response. Additionally, we show that fluorophore-conjugated DNA barcodes support correlative imaging studies such as mechano-microscopy to capture information about spatially-varying mechanical heterogeneity in 3D cultures. We ability to create customised, encoded cellular assemblies is a general approach that can resolve spatial differences in gene expression in 3D cell culture models.

bioengineering↗

bioRxiv2023

Metastable Intermediates Identified in Epithelial to Mesenchymal Transition are Regulated by G-Quadruplex DNA Structures

Cancer is a heterogenous disease, with multiple cellular subpopulations present within a single tumour mass that differ genetically and morphologically, and thus respond differently to chemotherapeutics. Epithelial-to-Mesenchymal transition (EMT) has been shown to play a role in tumour heterogeneity. Single-cell sequencing is critical to identify cell-type-specific transcriptomic differences with multiplexing methods increasing experimental scope with reduced cost. Cell hashing with barcoded antibodies is commonly used to multiplex samples but is limited to samples expressing target antigens. Antigen-independent methods of barcoding cells, such as barcoded lipid-anchors, have gained traction but present substantial populations that cannot be unambiguously demultiplexed. Herein we report a multiplexed single-cell transfection-enabled cell hashing sequencing (scTECH-seq) platform, which uses antigen-independent endocytic uptake to barcode cells, resulting in efficient, uniform barcoding with high cell recovery. We apply this methodology to identify distinct metastable cell states in human mammary cells undergoing EMT and show that stabilisation of G-quadruplex DNA has the potential to inhibit EMT.

genomics↗

bioRxiv2023

Epigenetic mediated functional reprogramming of immune cells leads to HBsAg seroconversion in Hepatitis B Virus Reactivation patients

BackgroundHepatitis B virus (HBV) modulates epigenetic landscape by epigenetic regulators. HBsAg seroconversion is possible with immune activation, therefore we aimed to investigate epigenetic modulation in HBV reactivation (rHBV) for viral clearance and seroconversion. MethodsSixteen retrospectively collected rHBV patients [Seroconverters (SC, n=7, HBsAg loss and anti-HBs>10 IU/ml), non- seroconverters (NSC, n=9)], chronic hepatitis B treatment naive (nCHBV, n=7) patients and healthy controls (HC, n=7) were included in this study. Genome methylation, gene expression, plasma-cytokines, and immune cell profiling was analysed by Reduced Representation Bisulfite Sequencing (RRBS), QRT-PCR, multiplex-cytokine-bead array and flow-cytometry. ResultsrHBV patients having high HBV DNA and ALT showed epigenetic remodellers; KDM2B, NCOR2 and GATA6, immune and metabolic genes; TGF-{beta}, IL-6, IRF8, RPTOR, HK3 significantly (p<0.05) hypomethylated at specific CpG islands compared to nCHBV. TOX was hypomethylated in nCHBV suggesting immune-exhaustion. At-baseline, seroconverters showed hypomethylation of KDM2B, COX19, IRF8, TLR5 and hypermethylation of LAG3 compared to non-seroconverters. Further, in seroconverters at week-24, IL17RA, IFN-{gamma}, TGF-{beta}, and STAT5B (p<0.05) were additionally hypomethylated at specific CpG islands suggesting immune activation. Cytokine-bead analysis revealed increased IL-6 (p=0.009) and decreased LAG3 plasma levels (p=0.01) also imply on significantly differentiated HBV specific CD8, Tfh and Th1/17 cells in seroconverters at baseline and week-24. However, both nCHBV and non-seroconverters had consistent hypomethylation of LAG3 and TOX, which leads to immune exhaustion. ConclusionIn rHBV, seroconversion is driven by position specific CpG islands methylation in epigenetic remodellers, immune and metabolic genes. Immune metabolic reprograming is reflected by Th1/17 differentiation, extensive interleukin production for HBsAg seroconversion. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=116 SRC="FIGDIR/small/554133v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@48e206org.highwire.dtl.DTLVardef@ef0fa7org.highwire.dtl.DTLVardef@ecb710org.highwire.dtl.DTLVardef@e6b34e_HPS_FORMAT_FIGEXP M_FIG C_FIG Lay summaryEpigenetic landscape in nCHBV depicts exhaustion and immune dysfunction. Out of many hypermethylated CpG islands of nCHBV, few become hypomethylated in rHBV and drives immune and metabolic reprogramming. This study provides insights into the cellular and molecular basis of epigenomic programs that regulate the differentiation and activation of immune cells leading to viral clearance and seroconversion. Targeting epigenetic mechanism could be promising strategy for the treatment of nCHBV and non-seroconverters.

immunology↗

bioRxiv2023

Progressive plasticity during colorectal cancer metastasis

Metastasis is the principal cause of cancer death, yet we lack an understanding of metastatic cell states, their relationship to primary tumor states, and the mechanisms by which they transition. In a cohort of biospecimen trios from same-patient normal colon, primary and metastatic colorectal cancer, we show that while primary tumors largely adopt LGR5+ intestinal stem-like states, metastases display progressive plasticity. Loss of intestinal cell states is accompanied by reprogramming into a highly conserved fetal progenitor state, followed by non-canonical differentiation into divergent squamous and neuroendocrine-like states, which is exacerbated by chemotherapy and associated with poor patient survival. Using matched patient-derived organoids, we demonstrate that metastatic cancer cells exhibit greater cell-autonomous multilineage differentiation potential in response to microenvironment cues than their intestinal lineage-restricted primary tumor counterparts. We identify PROX1 as a stabilizer of intestinal lineage in the fetal progenitor state, whose downregulation licenses non-canonical reprogramming.

cancer biology↗

bioRxiv2023

Single cell transcriptomics reveals cell type specific features of developmentally regulated responses to lipopolysaccharide between birth and 5 years.

Human perinatal life is characterized by a period of extraordinary change during which newborns encounter abundant environmental stimuli and exposure to potential pathogens. To meet such challenges, the neonatal immune system is equipped with unique functional characteristics that adapt to changing conditions as development progresses across the early years of life, but the molecular characteristics of such adaptations remain poorly understood. The application of single cell genomics to birth cohorts provides an opportunity to investigate changes in gene expression programs elicited downstream of innate immune activation across early life at unprecedented resolution. In this study, we performed single cell RNA-sequencing of mononuclear cells collected from matched birth cord blood and 5-year peripheral blood samples following stimulation (18hrs) with two well-characterized innate stimuli; lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (Poly(I:C)). We found that the transcriptional response to LPS was constrained at birth and predominantly partitioned into classical proinflammatory gene upregulation primarily by monocytes and IFN-signaling gene upregulation by lymphocytes. Moreover, these responses featured substantial cell-to-cell communication which appeared markedly strengthened between birth and 5 years. In contrast, stimulation with Poly(I:C) induced a robust IFN-signalling response across all cell types identified at birth and 5 years. Analysis of gene regulatory networks revealed IRF1 and STAT1 were key drivers of the LPS-induced IFN-signaling response in lymphocytes with a potential developmental role for IRF7 regulation. Additionally, we observed distinct activation trajectory endpoints for monocytes derived from LPS-treated cord and 5-year blood, which was not apparent among Poly(I:C)-induced monocytes. Taken together, our findings provide new insight into the gene regulatory landscape of immune cell function between birth and 5 years and point to regulatory mechanisms relevant to future investigation of infection susceptibility in early life.

immunology↗

bioRxiv2023

Patient-derived xenografts and single-cell sequencing identifies three subtypes of tumor-reactive lymphocytes in uveal melanoma metastases

Uveal melanoma (UM) is a rare melanoma originating in the eyes uvea, with 50% of patients experiencing metastasis predominantly in the liver. In contrast to cutaneous melanoma, there is only a limited effectiveness of combined immune checkpoint therapies, and half of patients succumb to recurrent disease after two years. This study aimed to provide a path towards enhancing immunotherapy efficacy by identifying and functionally validating tumor-reactive T cells in liver metastases of patients with UM. We employed single-cell RNA sequencing of biopsies and tumor-infiltrating lymphocytes (TILs) to identify potential tumor-reactive T cells. Patient-derived xenograft (PDX) models of UM metastases were created from patients, and tumor sphere cultures were generated from these models for co-culture with autologous or MART1-specific HLA-matched allogenic TILs. Activated T cells were subjected to TCR sequencing, and the TCRs were matched to those found in single-cell sequencing data from biopsies, expanded TILs and in livers or spleens of PDX models injected with TILs. Our findings revealed that tumor-reactive T cells resided not only among activated and exhausted subsets of T cells, but also in a subset of cytotoxic effector cells. In conclusion, combining single-cell sequencing and functional analysis provides valuable insights into which T cells in UM may be useful for cell therapy amplification and marker selection.

cancer biology↗

bioRxiv2023

Metabolic alterations unravel the materno fetal immune responses with disease severity in pregnant women infected with SARS-CoV-2

BackgroundPregnancy being immune compromised state, COVID-19 disease poses high risk of premature delivery and threat to fetus. Plasma metabolome regulates immune cellular responses and we aimed to analyze the plasma secretome, metabolome and immune cells in COVID-19 positive pregnant mother and cord blood. MethodsCOVID-19 RT-PCR positive pregnant females (n=112) asymptomatic (n=82), or with mild (n=21) or moderate (n=9) disease and control healthy pregnant (n=10) females were included. Mothers blood and cord blood (n=80) was analysed for untargeted metabolome profiling and plasma cytokines by high-resolution mass spectrometry (MS) and multiplex cytokine bead array. Immune scan in mothers was done using flow cytometry. ResultsIn asymptomatic SARS-CoV-2 infection, --the amino acid metabolic pathways such as glycine, serine, L-lactate and threonine metabolism was upregulated, riboflavin and tyrosine metabolism, downregulated. In mild to moderate disease, the pyruvate and NAD+ metabolism (energy metabolic pathways) were mostly altered. In addition to raised TNF-, IFN-, IFN-{gamma}, IL-6 cytokine storm, IL-9 was increased in both mothers and neonates. Pyruvate and NAD+ metabolic pathways along with IL-9 and IFN-{gamma} had impact on non-classical monocytes, increased CD4 T cells and B cells but depleted CD8+ T cells. Cord blood mimicked the mothers metabolomic profiles by showing altered valine, leucine, isoleucine, glycine, serine, threonine in asymptomatic and NAD+ and riboflavin metabolism in mild and moderate disease subjects. ConclusionsOur results demonstrate a graduated immune-metabolomic interplay in mother and fetus in pregnant females with different degrees of severity of COVID-19 disease. IL-9 and IFN- {gamma} regulated pyruvate, lactate TCA metabolism and riboflavin metabolism with context to disease severity are hall marks of this materno-fetal metabolome. HighlightsO_LISARS-CoV-2 infection alters energy consumption metabolic pathways during pregnancy. C_LIO_LIPregnant women with mild to moderate COVID-19 show increased energy demands, and consume stored glucose by upregulating pyruvate and NAD+ metabolism. C_LIO_LIIncreased TNF- and IL-9 in mild COVID-19 disease involve TCA cycle to produce lactate and consume stored glucose by up regulating pyruvate and nicotinamide and nicotinate metabolism. C_LIO_LIWith mild to moderate disease, raised IL-9 and TNF-, decreased riboflavin pathway, exhaustion of T and B cells cause pathogenesis. C_LIO_LICord blood mimics the metabolic profile of mothers peripheral blood, SARS- CoV-2 infection reshapes immune-metabolic profiles of mother-infant dyad. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=141 HEIGHT=200 SRC="FIGDIR/small/540101v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@657cc3org.highwire.dtl.DTLVardef@e1e76forg.highwire.dtl.DTLVardef@12153f2org.highwire.dtl.DTLVardef@10e5332_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2023

Investigating racial disparities in carcinomas through TCGA transcriptomic and proteomic database

Simple Summary: Racial disparities in cancer incidence and outcome rates are prevalent in the US, with a variety of contributing factors such as socioeconomic status, differences in lifestyle and environmental exposures, and gene polymorphisms. The goal of this research was to broadly analyze public data to identify critical differences in molecular signatures and pathways between races. Additionally, to ensure the clinical translatability of our work, we analyzed the impact of these differences on patient survival. Our findings will help inform the use of novel biomarkers in clinical settings and the future development of precision therapies. Abstract:Epidemiological studies highlight a disparity in cancer incidence and outcome rates between racial groups in the United States. In our study, we investigated molecular differences among racial groups in 10 carcinoma types. We used publicly available data from The Cancer Genome Atlas to identify patterns of differential gene expression in tumors obtained from 4,112 White, Black/African American, and Asian patients. We identified race-dependent expression of numerous genes whose mRNA transcript levels were significantly correlated with patient survival. A small subset of these genes was differentially expressed in multiple carcinomas, including genes involved in cell cycle progression such as CCNB1, CCNE1, CCNE2, and FOXM1. In contrast, genes such as transcriptional factor ETS1 and apoptotic gene BAK1 were differ-entially expressed and clinically significant only in specific cancer types. Our analyses also revealed race-dependent regulation of relevant pathways. Importantly, homology directed repair and ERBB4-mediated nuclear signaling were both upregulated in Black patients compared to Whites in four carcinoma types. This large-scale pan-cancer study refines our understanding of the cancer health disparity and can help inform the use of novel biomarkers in clinical settings as well as the future development of precision therapies.

bioinformatics↗

bioRxiv2023

Low dimensionality of phenotypic space as an emergent property of coordinated teams in biological regulatory networks

Biological networks driving cell-fate decisions involve complex interactions, but they often give rise to only a few phenotypes, thus exhibiting low-dimensional dynamics. The network design principles that govern such cell-fate canalization remain unclear. Here, we investigate networks across diverse biological contexts- Epithelial-Mesenchymal Transition, Small Cell Lung Cancer, and Gonadal cell-fate determination - to reveal that the presence of two mutually antagonistic, well-coordinated teams of nodes leads to low-dimensional phenotypic space such that the first principal component (PC1) axis can capture most of the variance. Further analysis of artificial team-based networks and random counterparts of biological networks reveals that the principal component decomposition is determined by the team strength within these networks, demonstrating how the underlying network structure governs PC1 variance. The presence of low dimensionality in corresponding transcriptomic data confirms the applicability of our observations. We propose that team-based topology in biological networks are critical for generating a cell-fate canalization landscape.

systems biology↗

bioRxiv2023

Gene Expression Changes in Cultured Reactive Rat Astrocyte Models and Comparison to Device-Associated Effects in the Brain

Implanted microelectrode arrays hold immense therapeutic potential for many neurodegenerative diseases. However, a foreign body response limits long-term device performance. Recent literature supports the role of astrocytes in the response to damage to the central nervous system (CNS) and suggests that reactive astrocytes exist on a spectrum of phenotypes, from beneficial to neurotoxic. The goal of our study was to gain insight into the subtypes of reactive astrocytes responding to electrodes implanted in the brain. In this study, we tested the transcriptomic profile of two reactive astrocyte culture models (cytokine cocktail or lipopolysaccharide, LPS) utilizing RNA sequencing, which we then compared to differential gene expression surrounding devices inserted into rat motor cortex via spatial transcriptomics. We interpreted changes in the genetic expression of the culture models to that of 24 hour, 1 week and 6 week rat tissue samples at multiple distances radiating from the injury site. We found overlapping expression of up to [~]250 genes between in vitro models and in vivo effects, depending on duration of implantation. Cytokine-induced cells shared more genes in common with chronically implanted tissue ([≥]1 week) in comparison to LPS-exposed cells. We revealed localized expression of a subset of these intersecting genes (e.g., Serping1, Chi3l1, and Cyp7b1) in regions of device-encapsulating, glial fibrillary acidic protein (GFAP)-expressing astrocytes identified with immunohistochemistry. We applied a factorization approach to assess the strength of the relationship between reactivity markers and the spatial distribution of GFAP-expressing astrocytes in vivo. We also provide lists of hundreds of differentially expressed genes between reactive culture models and untreated controls, and we observed 311 shared genes between the cytokine induced model and the LPS-reaction induced control model. Our results show that comparisons of reactive astrocyte culture models with spatial transcriptomics data can reveal new biomarkers of the foreign body response to implantable neurotechnology. These comparisons also provide a strategy to assess the development of in vitro models of the tissue response to implanted electrodes.

neuroscience↗

bioRxiv2022

Whole genome error-corrected sequencing for sensitive circulating tumor DNA cancer monitoring

Circulating cell-free DNA (ccfDNA) sequencing for low-burden cancer monitoring is limited by sparsity of circulating tumor DNA (ctDNA), the abundance of genomic material within a plasma sample, and pre-analytical error rates due to library preparation, and sequencing errors. Sequencing costs have historically favored the development of deep targeted sequencing approaches for overcoming sparsity in ctDNA detection, but these techniques are limited by the abundance of ccfDNA in samples, which imposes a ceiling on the maximal depth of coverage in targeted panels. Whole genome sequencing (WGS) is an orthogonal approach to ctDNA detection that can overcome the low abundance of ccfDNA by supplanting sequencing depth with breadth, integrating signal across the entire tumor mutation landscape. However, the higher cost of WGS limits the practical depth of coverage and hinders broad adoption. Lower sequencing costs may thus allow for enhanced ctDNA cancer monitoring via WGS. We therefore applied emerging lower-cost WGS (Ultima Genomics, 1USD/Gb) to plasma samples at [~]120x coverage. Copy number and single nucleotide variation profiles were comparable between matched Ultima and Illumina datasets, and the deeper WGS coverage enabled ctDNA detection at the parts per million range. We further harnessed these lower sequencing costs to implement duplex error-corrected sequencing at the scale of the entire genome, demonstrating a [~]1,500x decrease in errors in the plasma of patient-derived xenograft mouse models, and error rates of [~]10-7 in patient plasma samples. We leveraged this highly de-noised plasma WGS to undertake cancer monitoring in the more challenging context of resectable melanoma without matched tumor sequencing. In this context, duplex-corrected WGS allowed us to harness known mutational signature patterns for disease monitoring without matched tumors, paving the way for de novo cancer monitoring.

genomics↗

bioRxiv2022

Self-assembly of cellular neighborhoods converts stochastic signaling into sustained olfactory neurogenesis

Olfactory neurogenesis occurs continuously throughout the lives of vertebrates, including in humans, and relies on the rapid, unceasing differentiation and integration of neurons into a complex multicellular network. The system-wide regulation of this intricate choreography is poorly understood; in particular, it is unclear how progenitor cells convert stochastic fluctuations in cell-cell signaling, over both space and time, into streamlined fate decisions. Here, we track single-cell level multicellular dynamics in the developing zebrafish olfactory epithelium, perturb signaling pathways with temporal specificity, and find that the continuous generation of neurons is driven by the spatially-restricted self-assembly of transient groups of progenitor cells, i.e. cellular neighborhoods. Stochastic modeling and validation of the underlying genetic circuit reveals that neighborhood self-assembly is driven by a tightly regulated bistable toggle switch between Notch signaling and the transcription factor Insulinoma-associated 1a that is responsive to inter-organ retinoic acid signaling. Newly differentiating neurons emerge from neighborhoods and, in response to brain-derived neurotrophic factor signaling, migrate across the olfactory epithelium to take up residence as apically-located, mature sensory neurons. After developmental olfactory neurogenesis is complete, inducing injury results in a robust expansion of neighborhoods, followed by neuroregeneration. Taken together, these findings provide new insights into how stochastic signaling networks spatially pattern and regulate a delicate balance between progenitors and their neuronal derivatives to drive sustained neurogenesis during both development and regeneration.

developmental biology↗

bioRxiv2022

Machine learning guided signal enrichment for ultrasensitive plasma tumor burden monitoring

In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole genome sequencing (WGS). We now introduce MRD-EDGE, a composite machine learning-guided WGS ctDNA single nucleotide variant (SNV) and copy number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGE uses deep learning and a ctDNA-specific feature space to increase SNV signal to noise enrichment in WGS by 300X compared to our previous noise suppression platform MRDetect. MRD-EDGE also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1Gb to 200Mb, thereby expanding its applicability to a wider range of solid tumors. We harness the improved performance to track changes in tumor burden in response to neoadjuvant immunotherapy in non-small cell lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal to noise enrichment in MRD-EDGE enables de novo mutation calling in melanoma without matched tumor, yielding clinically informative TF monitoring for patients on immune checkpoint inhibition.

genomics↗

bioRxiv2021

BESFA: Bioinformatics based Evolutionary, Structural & Functional Analysis of Prostrate, Placenta, Ovary, Testis, and Embryo (POTE) Paralogs

The POTE family comprises 14 paralogues and is primarily expressed in Prostrate, Placenta, Ovary, Testis, Embryo (POTE), and cancerous cells. The prospective function of the POTE protein family under physiological conditions is less understood. We systematically analyzed their cellular localization and molecular docking analysis to elucidate POTE proteins structure, function, and Adaptive Divergence. Our result discerns that group three POTE paralogs (POTEE, POTEF, POTEI, POTEJ, and POTEKP (a pseudogene)) exhibits significant variation among other members could be because of their Adaptive Divergence. Furthermore, our molecular docking studies on POTE protein revealed the highest binding affinity with NCI-approved anticancer compounds. Additionally, POTEE, POTEF, POTEI, and POTEJ were subject to an explicit molecular dynamic simulation for 50ns. MM-GBSA and other essential electrostatics were calculated that showcased that only POTEE and POTEF have absolute binding affinities with minimum energy exploitation. Thus, this studys outcomes are expected to drive cancer research to successful utilization of POTE genes family as a new biomarker, which could pave the way for the discovery of new therapies.

cancer biology↗

bioRxiv2021

Use-dependent biases primarily originate from a contaminated motor plan

Repetition of a specific movement biases subsequent actions towards the recently practiced movement, a phenomenon referred to as use-dependent learning (UDL). UDL has been attributed to shifts in the tuning of neurons in the motor cortex. However, recent studies employing a forced reaction time task, including the eLife article by Marinovic et al (2017), indicate that these biases may also arise from a contaminated motor plan, one that is biased towards the practiced direction. We advanced this line of inquiry, seeking to establish the relative contribution of execution and planning processes to UDL in a center-out reaching task in which participants were able to initiate movements of their own volition. On most trials, the target appeared at a designated "frequent" location; on other trials, the target appeared at one of six "rare" locations. In Experiment 1, participants exhibited a robust movement bias towards the frequent target when movements were self-initiated quickly, but a small movement bias when movements were self-initiated slowly - the signature of a contaminated motor plan. Strikingly, the heading angles were bimodally distributed, with one peak at the frequent target location and the other at the rare target location - a finding reinforced by a re-analysis of two widely cited studies on UDL. Notably, the latter peak was shifted in the frequently practiced direction, a signature of a motor execution bias. To eliminate the contribution of planning-related UDL, we imposed a delay between target onset and movement initiation in Experiment 2. As predicted, the heading angles became unimodally distributed around the rare target. The peak of this distribution was again shifted towards the location of the frequent target, indicative of a persistent bias in motor execution. Taken together, these results highlight two distinct components of UDL even when movements are self-initiated: First, the temporal dynamics underlying movement planning, in which a default plan is progressively overridden by a new plan, produces a pronounced motor planning bias. Second, there is a small, temporally stable bias that may reflect shifts in motor unit tuning.

neuroscience↗

bioRxiv2021

In silico study predicts a key role of RNA-binding domains 3 and 4 in nucleolin-miRNA interactions.

RNA binding proteins (RBPs) regulate many important cellular processes through their interactions with RNA molecules. RBPs are critical for post-transcriptional mechanisms keeping gene regulation in a fine equilibrium. Conversely, dysregulation of RBPs and RNA metabolism pathways is an established hallmark of tumorigenesis. Human nucleolin (NCL) is a multifunctional RBP that interacts with different types of RNA molecules, in part through its four RNA binding domains (RBDs). Particularly, NCL interacts directly with microRNAs (miRNAs) and is involved in their aberrant processing linked with many cancers, including breast cancer. Nonetheless, molecular details of the NCL-miRNA interaction remain obscure. In this study, we used an in silico approach to characterize how NCL targets miRNAs and whether this specificity is imposed by a definite RBD-interface. Here, we present structural models of NCL-RBDs and miRNAs, as well as predict scenarios of NCL- miRNA interactions generated using docking algorithms. Our study suggests a predominant role of NCL RBDs 3 and 4 (RBD3-4) in miRNA binding. We provide detailed analyses of specific motifs/residues at the NCL- substrate interface in both these RBDs and miRNAs. Finally, we propose that the evolutionary emergence of more than two RBDs in NCL in higher organisms coincides with its additional role/s in miRNA processing. Our study shows that RBD3-4 display sequence/structural determinants to specifically recognize miRNA precursor molecules. Moreover, the insights from this study can ultimately support the design of novel antineoplastic drugs aimed at regulating NCL-dependent biological pathways with a causal role in tumorigenesis. Importance/impact of the studyNucleolin is a multifunctional RNA binding protein that is often linked with many cancers. Similarly, microRNAs are often dysregulated in many cancers and linked to tumorigenesis. This study focuses on the interaction of nucleolin with microRNAs to identify previously unknown mechanistic details/specificity of these interactions. The insights from this study can ultimately support the design of novel drugs aimed at regulating NCL- dependent pathways implicated in tumorigenesis.

bioinformatics↗

bioRxiv2021

Mycobacterium abscessus HelR interacts with RNA Polymerase to confer intrinsic rifamycin resistance

Rifampicin (RIF) constitutes the frontline therapy against M. tuberculosis as well as most slow-growing non-tuberculous mycobacteria (NTM). However, RIF is completely ineffective against M. abscessus despite the absence of mutations in the rifampicin resistance determining region of Mab_rpoB. This has been attributed to the presence of an ADP-ribosyltransferase (Arr) activity that inactivates RIF. Rifabutin (RBT), a close analogue of RIF, has recently been shown to be effective against M. abscessus in vitro and in a mouse model and comprises a promising therapeutic against M. abscessus infections. Using RNA sequencing we show that exposure of M. abscessus to sublethal doses of RIF and RBT results in [~]25-fold upregulation of Mab_helR in laboratory and clinical isolates; an isogenic deletion of Mab_helR is hypersensitive to RIF and RBT, and over-expression of Mab_helR confers RIF tolerance in M. tuberculosis implying that helR constitutes a significant determinant of inducible RIF and RBT resistance. We demonstrate a preferential association of MabHelR with RNA polymerase in vivo in bacteria exposed to RIF and showed that purified MabHelR can rescue transcription inhibition in the presence of RIF in in vitro transcription assays. Furthermore, MabHelR can dissociate RNAP from RIF-stalled initiation complexes in vitro, a species we envisage accumulates upon RIF exposure. Lastly, we show that the tip of the PCh-loop of Mab_helR, in particular residues E496 and D497 that are in proximity to RIF, is critical for conferring RIF resistance without being required for RNAP dissociation from stalled complexes. This suggests that HelR may be additionally involved in displacing RIF bound to RNAP and function as an RNAP protection protein. Significance StatementBacterial RNA polymerase is a target for the potent and broad-spectrum rifamycin group of antibiotics. Mutations within rpoB and inactivation by a diverse group of enzymes constitute the most widespread mechanisms of resistance. Herein we report an unprecedented mechanism of rifamycin resistance in M. abscessus mediated by MabHelR, a putative SF1 like helicase, that involves disassembly of RIF-stalled initiation complexes, likely followed by displacement of the antibiotic, leading to RNAP recycling. The mechanism is reminiscent of the role of HflX and ribosome protection proteins in resistance to ribosome targeting antibiotics and suggests that removal of stalled macromolecular complexes and their recycling comprises a widespread but underappreciated mechanism of antibiotic resistance. Rifampicin (RIF) is pivotal in the control of M. tuberculosis infections but ineffective against M. abscessus. Identification of inducible rifamycin resistance determinants in M. abscessus is therefore particularly crucial for informing treatment strategies and development of novel therapeutic approaches.

microbiology↗