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Results for “molecular biology”

Search indexed bioRxiv preprints in genomics, neuroscience, cell biology and bioinformatics. Read source abstracts and check manuscript versions; preprints are not peer reviewed.

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bioRxiv2022

Proteomics Characteristics Reveal the Risk of T1 Colorectal Cancer Metastasis to Lymph Nodes

BackgroundThe presence of lymph node metastasis (LNM) affects treatment strategy decisions in T1NxM0 colorectal cancer (CRC), but the currently used clinicopathological-based risk stratification cannot predict LNM accurately. In this study, we established a classifier for predicting LNM in T1 CRC. MethodsWe detected proteins in formalin-fixed paraffin-embedded (FFPE) tumor samples from 143 LNM-negative and 78 LNM-positive patients with T1 CRC and revealed changes in molecular and biological pathways by label-free LC-MS/MS. An effective prediction model was built and validated in a training cohort (N=132) and two validation cohorts (VC1, N=42; VC2, N=47) by machine learning. We further built a simplified classifier with 9 proteins. The expression patterns of 13 proteins were confirmed by immunohistochemistry, and the IHC score of 5 proteins were used to build a IHC predict model. ResultPatients with or without LNM have different molecular signatures. The 55-proteins prediction model achieved an impressive AUC of 1.00 in the training cohort, 0.96 in VC1 and 0.93 in VC2. The 9-protein classifier achieved an AUC of 0.824, and the calibration plot was excellent. We found that 5 biomarkers could predict LNM by the IHC score, with an AUC of 0.825. RHOT2 silence significantly enhanced migration and invasion of colon cancer cells. ConclusionsOur study explored the mechanism of metastasis in T1 CRC and can be used to facilitate the individualized prediction of LNM in patients with T1 CRC, which may provide a guidance for clinical practice in T1 CRC.

cancer biology↗

bioRxiv2024

LotOfCells: data visualization and statistics of single cell metadata

Single-cell sequencing unveils a treasure trove into the biological and molecular characteristics of samples. Yet, within this flood of data, the challenge to draw meaningful conclusions sometimes can be time consuming and a tortuous process. Here we introduce LotOfCells: a simple R package designed to explore the intricate landscape of phenotypic data within single-cell studies. Normally, we are interested in visualizing and measuring if the differences in the proportion of number of cells across various covariates is significant or biologically relevant. As an example, one of the most common questions is the proportion of different cell types across conditions in our experiment, or the cluster composition before and after treatment (e.g.: difference in cell type proportions between wild type and mutant). LotOfCells helps with the interpretation and visualization of meta-data of these recurrent scenarios, including the test of proportion changes across multiple ordered stages. Additionally, it computes a symmetric divergence score to measure global deregulation of cell proportions due to a condition. Code repositoryR package, manual and relevant examples can be accessed on the GitHub repository: https://github.com/OscarGVelasco/LotOfCells

bioinformatics↗

bioRxiv2020

Identification of specific biomarkers and pathways in the synovial tissues of patients with osteoarthritis in comparison to rheumatoid arthritis

Osteoarthritis and rheumatoid arthritis are two common arthritis with different pathogenesis. Here, we explore the difference of genes and biological pathways in human synovial fibroblasts by using a bioinformatics method to clarify their potential pathogenesis. The GSE7669 dataset was originally produced by using an Affymetrix Human Genome U95 platform. We used the KEGG and GO analysis to identify the functional categories and pathways. Our results suggested that biological adhesion and cell adhesion are the main signaling pathways in osteoarthritis in comparison to rheumatoid arthritis. Furthermore, Albumin, MAPK3, PTPRC, COL1A1, and CXCL12 may be key genes in osteoarthritis. Therefore, our study provides potential targets for the specific and accurate therapy of osteoarthritis.

molecular biology↗

bioRxiv2022

Inhibited KdpFABC resides in an E1 off-cycle state

KdpFABC is a high-affinity prokaryotic K+ uptake system that forms a functional chimera between a channel-like subunit (KdpA) and a P-type ATPase (KdpB). At high K+ levels, KdpFABC needs to be inhibited to prevent excessive K+ accumulation to the point of toxicity. This is achieved by a phosphorylation of the serine residue in the TGES162 motif in the A domain of the pump subunit KdpB (KdpBS162-P). Here, we explore the structural basis of inhibition by KdpBS162 phosphorylation by determining the conformational landscape of KdpFABC under inhibiting and non-inhibiting conditions. Under turnover conditions, we identified a new inhibited KdpFABC conformation that we termed E1-P tight, which is not part of the canonical Post-Albers transport cycle of P-type ATPases. It likely represents the biochemically described stalled E1-P state adopted by KdpFABC upon KdpBS162 phosphorylation. The E1-P tight state exhibits a compact fold of the three cytoplasmic domains and is likely adopted when the transition from high-energy E1-P states to E2-P states is unsuccessful. This study represents a structural characterization of a biologically relevant off-cycle state in the P-type ATPase family and supports the emerging discussion of P-type ATPase regulation by such conformations.

molecular biology↗

bioRxiv2023

A palmitoyl transferase chemical genetic system to map ZDHHC-specific S-acylation

The 23 human ZDHHC S-acyltransferases catalyze long-chain S-acylation at cysteine residues across an extensive network of hundreds of proteins important for normal physiology or dysregulated in disease. Here we present a technology platform to directly map the protein substrates of a specific ZDHHC for the first time at the whole proteome level, in intact cells. Structure-guided engineering of paired ZDHHC hole mutants and bumped chemically tagged fatty acid probes enabled probe transfer to specific protein substrates with excellent selectivity over wild type ZDHHCs. Chemical genetic systems were exemplified for five ZDHHCs (3, 7, 11, 15 and 20), and applied to generate the first de novo ZDHHC substrate profiles, identifying >300 unique and shared substrates across multiple cell lines and S-acylation sites for novel functionally diverse substrates. We expect that this powerful and versatile platform will open a new window on S-acylation biology for a wide range of models and organisms.

molecular biology↗

bioRxiv2024

Effective lowering of α-synuclein expression by targeting G-quadruplex structures within the SNCA genome

Alpha-synuclein, encoded by the SNCA gene, is a pivotal protein implicated in the pathogenesis of synucleinopathies, including Parkinsons disease. Current approaches for modulating alpha-synuclein levels involve antisense nucleotides, siRNAs, and small molecules targeting SNCAs 5-UTR mRNA. Here, we propose a groundbreaking strategy targeting G-quadruplex structures to effectively modulate SNCA gene expression and lowering alpha-synuclein amount. Novel G-quadruplex sequences, identified on the SNCA genes transcription starting site and 5-UTR of SNCA mRNAs, were experimentally confirmed for their stability through biophysical assays and in vitro experiments on human genomic DNA. Biological validation in differentiated SH-SY5Y cells revealed that well-known G-quadruplex ligands remarkably stabilized these structures, inducing the modulation of SNCA mRNAs expression, and the effective decrease in alpha-synuclein amount. Besides, a novel peptide nucleic acid conjugate, designed to selectively disrupt of G-quadruplex within the SNCA gene promoter, caused a promising lowering of both SNCA mRNA and alpha-synuclein protein. Altogether our findings highlight G-quadruplexes key role as intriguing biological targets in achieving a notable and successful reduction in alpha-synuclein expression, pointing to a novel approach against synucleinopathies.

molecular biology↗

bioRxiv2024

Multisite Assembly of Gateway Induced Clones (MAGIC): a flexible cloning toolbox withdiverse applications in vertebrate model systems.

Here we present the Multisite Assembly of Gateway Induced Clones (MAGIC) system, which harnesses site-specific recombination-based cloning via Gateway technology for rapid, modular assembly of between 1 and 3 "Entry" vector components, all into a fourth, standard high copy "Destination" plasmid backbone. The MAGIC toolkit spans a range of in vitro and in vivo uses, from directing tunable gene expression, to driving simultaneous expression of microRNAs and fluorescent reporters, to enabling site-specific recombinase-dependent gene expression. All MAGIC system components are directly compatible with existing multisite gateway Tol2 systems currently used in zebrafish, as well as existing eukaryotic cell culture expression Destination plasmids, and available mammalian lentiviral and adenoviral Destination vectors, allowing rapid cross-species experimentation. Moreover, herein we describe novel vectors with flanking piggyBac transposon elements for stable genomic integration in vitro or in vivo when used with piggyBac transposase. Collectively, the MAGIC system facilitates transgenesis in cultured mammalian cells, electroporated mouse and chick embryos, as well as in injected zebrafish embryos, enabling the rapid generation of innovative DNA constructs for biological research due to a shared, common plasmid platform.

molecular biology↗

bioRxiv2025

An integrated DNA interactome and transcriptome profiling reveals a PU.1/enhancer RNA-mediated Feed-forward Regulatory Loop Regulating monocyte/macrophage development and innate immune functions

High expression of the myeloid master ETS transcription factor PU.1 drives the development of monocyte/macrophage (Mono/M{Phi}), a crucial cellular component of the innate immune system. Disruptions in normal expression patterns of PU.1 are linked to a variety myeloid malignancy and immune diseases. It is evidenced that PU.1 binds to and modulates enhancers of several myeloid genes. While noncoding RNAs transcribed from noncoding genes at the enhancers are increasingly reported to be involved in enhancer regulation, the crosstalk between PU.1 and noncoding RNAs in enhancer-mediated myeloid gene regulation in Mono/M{Phi} differentiation and immune response has not been systematically investigated. In this study, we interrogated the PU.1-mediated transcriptome and cistrome with our comprehensive collection of putative and verified enhancers. Among a repertoire of noncoding genes present at PU.1-bound enhancers, we discovered that PU.1 acts as a potent transcription factor inducer of the noncoding RNA LOUP, which we previously identified as an RNA inducer of PU.1. The genomic region within the LOUP locus occupied by PU.1 is characterized by the epigenetic features of a myeloid-specific super-enhancer. Targeted disruption of the PU.1-binding motifs resulted in the downregulation of LOUP promoter activity. Depletion of LOUP reduced the expression of Mono/M{Phi} cell markers as well as the transcriptional program associated with Mono/M{Phi} differentiation Mono/M{Phi} innate defense mechanisms, including phagocytosis, antimicrobial activity, and chemoattractant cytokine production. LOUP induces Mono/M{Phi} phagocytic activities. Collectively, our findings indicate that PU.1 and enhancer RNA LOUP are biomolecular components of an unidentified feed-forward loop that promotes their mutual expression, contributing to Mono/M{Phi} differentiation and innate immune functions. The identification of the PU.1/LOUP regulatory circuit provides valuable insights into the mechanisms underlying cell-type and gene-specific enhancer activity and Mono/M{Phi} biology, as well as significant implications for advancing our understanding of immune diseases and myeloid malignancies.

molecular biology↗

bioRxiv2025

Liver angiocrine myeloid-derived growth factor protects against endothelial dysfunction in pulmonary arterial hypertension

Myeloid-derived growth factor (MYDGF) is a hepatic angiokine with protective effects in systemic vascular beds, but its role in pulmonary arterial hypertension (PAH) is unknown. We hypothesized that hepatic MYDGF deficiency contributes to pulmonary endothelial activation in PAH and that recombinant MYDGF could rescue endothelial injury. In the Sugen-hypoxia (SuHx) rat model, hepatic MYDGF expression was decreased, while pulmonary vascular cell adhesion molecule-1 (VCAM-1) expression was increased. Human hepatic sinusoidal endothelial cells exposed to pro-inflammatory macrophage conditioned media downregulated MYDGF, and recombinant MYDGF restored pulmonary artery endothelial cell resistance to inflammatory activation via MAP4K4-NF{kappa}B signaling. In the Brown University PHiNE PAH cohort (n=41 PAH, n=27 controls), plasma proteomics demonstrated increased MYDGF in PAH patients compared with controls, but MYDGF levels declined with worsening liver stiffness and correlated with higher pulmonary vascular resistance. In the independent Servetus PAH cohort (n=117), higher plasma MYDGF was associated with mortality and right ventricular dilation. Together, these findings demonstrate hepatic MYDGF deficiency in experimental PAH, tissue specificity of endothelial MYDGF to the liver, and MYDGFs potential to mitigate pulmonary endothelial inflammation. However, human data suggest a paradoxical association of elevated circulating MYDGF with adverse outcomes, underscoring the complex biology of angiogenic growth factors in PAH. MYDGF may represent a novel hepatic angiokine linking systemic inflammation, liver dysfunction, and pulmonary vascular disease.

molecular biology↗

bioRxiv2025

Automatic colocalization of high resolution MALDI MSI and Raman imaging applied to cardiac tissue of Fabry disease mouse models

Understanding early molecular changes in biological tissues is crucial for diagnosing pathological and genetic diseases and for elucidating their underlying mechanisms. However, localized molecular alterations of low molecular-weight compounds are not inferred from conventional staining or genetic methods. Here, we established a multimodal imaging approach that integrates Raman spectroscopy and atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry imaging (AP-MALDI MSI): two complementary, label-free techniques enabling molecular profiling of a broad spectrum of biomolecules from one single tissue section. This method was applied to detect Gb3 accumulation in heart tissue of murine models of Fabry disease, including mice deficient in -galactosidase A (GLA) activity (GLA knock-out) and transgenic mice with a GLA knock-out and an upregulation of globotriaosylceramides (Gb3) synthase. With AP-MALDI MSI we were able to discern the heterogenous expression of Gb3 lipoforms with down to 5 {micro}m pixel size and reveal the significantly increased Gb3 content in mice containing a GLA knock-out combined with human Gb3 synthase overexpression compared to GLA knock-out and wild type samples. By employing Raman microscopy with a pixel size of 2 {micro}m, we were able to contextualize the physiological alterations in cardiac tissue by identifying components associated with nuclei, tissue, collagen, and lipids for the same three genotypes. An automated co-localization algorithm aligned Raman and AP-MALDI-MSI data from the same tissue section with (5.1 {+/-} 1.6) {micro}m precision, enabling overlay at 5 {micro}m and 2 {micro}m resolution. The method resolved heterogeneous Gb3 distributions and distinct lipid species in cardiac mouse tissue.

molecular biology↗

bioRxiv2019

Solving the first novel protein structure by 3D micro-crystal electron diffraction

Micro-crystal electron diffraction (MicroED) has recently shown potential for structural biology. It enables studying biomolecules from micron-sized 3D crystals that are too small to be studied by conventional X-ray crystallography. However, to the best of our knowledge, MicroED has only been applied to re-determine protein structures that had already been solved previously by X-ray diffraction. Here we present the first unknown protein structure - an R2lox enzyme - solved using MicroED. The structure was phased by molecular replacement using a search model of 35% sequence identity. The resulting electrostatic scattering potential map at 3.0 [A] resolution was of sufficient quality to allow accurate model building and refinement. Our results demonstrate that MicroED has the potential to become a widely applicable tool for revealing novel insights into protein structure and function, opening up new opportunities for structural biologists.

molecular biology↗

bioRxiv2026

Muscleblind-like proteins dimerize by forming disulfide bonds to regulate alternative splicing and pathogenic RNA foci formation

Muscleblind-like (MBNL) RNA-binding proteins (RBPs) possess modular domains that mediate regulation of alternative splicing and RNA localization. Myotonic Dystrophy Type 1 is a CTG repeat expansion disorder where MBNL is sequestered into intranuclear RNA foci, impairing its function. Previous studies found that MBNL self-associates through its exon 7, but the nature of this interaction is not well understood. We identified a cysteine in MBNL1 exon 7 that enables dimerization through formation of an intermolecular disulfide bond. We likewise demonstrate that MBNL2 dimerizes by forming disulfide bonds between multiple cysteines in its carboxy-terminus. Nucleocytoplasmic fractionation revealed a greater proportion of MBNL1 dimer in the nucleus, suggesting a nuclear function for the MBNL1 dimer. We investigated a connection between MBNL1 dimerization and MBNL1-mediated regulation of alternative splicing. To accomplish this, we mutated the MBNL1 cysteine in question to alanine (C325A) and performed RNAseq. We uncovered novel splicing events sensitive to MBNL1 dimerization. We also found that MBNL1 C325A, when co-expressed with expanded CTG repeats, produces smaller, more numerous foci, suggesting a role for the MBNL1 dimer in maintaining foci integrity. These results provide insight into biological and pathological mechanisms of MBNL1 dimerization and suggest other RBPs might similarly dimerize to regulate function. GRAPHICAL ABSTRACT

molecular biology↗

bioRxiv2020

Regulated control of gene therapies with a drug induced switch

To date, gene therapies for human application rely on engineered promoters that cannot be finely controlled. Here, we report a universal switch element that allows precise control for gene silencing or gene replacement after exposure to a small molecule. Importantly, these small molecule inducers are in human use, are orally bioavailable when given to animals or humans, and can reach both peripheral tissues and the brain. Moreover, the switch system (Xon) does not require the co-expression of any regulatory proteins. Using Xon, translation of desired elements for gene knockdown or gene replacement occurs after a single oral dose, and expression levels can be controlled by drug dose or in waves with repeat drug intake. This universal switch can provide temporal control of gene editing machinery and gene addition cassettes that can be adapted to cell biology applications and animal studies. Additionally, due to the oral bioavailability and safety of the drugs employed, the Xon switch provides an unprecedented opportunity to refine gene therapies for more appropriate human application.

molecular biology↗

bioRxiv2025

Defying expectations: sex-biased telomere dynamics and extended lifespan in the tropical bat species, Molossus molossus.

Telomeres are key biomarkers of cellular ageing, yet their dynamics remain poorly studied in tropical and short-lived bat species. Here, we present the first investigation of telomere length across age in Molossus molossus, a tropical bat historically categorised as the shortest-lived bat on record. Through a multi-year mark-recapture study in Gamboa, Panama, we sampled 492 individuals (n = 317 females, 175 males) and documented a female M. molossus surviving to at least 13 years of age, more than doubling the previously reported maximum lifespan of 5.6 years. Across the population, relative telomere length (rTL) showed no overall significant decline with age. No evidence was found for sex-specific rates of telomere attrition. Rather these results suggest that males and females follow parallel age-related telomere trajectories, with any sex differences primarily reflecting differences in mean telomere length rather than ageing dynamics. Overall, the findings here challenge previous assumptions about the lifespan and ageing biology of M. molossus. They demonstrate that telomere maintenance is not limited to temperate bats, show that sex differences in telomere biology are subtle and species-specific, and reinforce the value of long-term field studies for understanding ageing processes in the wild.

molecular biology↗

bioRxiv2024

Parameters that influence bipartite reporter system expression in C. elegans

The development of bipartite reporter systems in C. elegans has lagged by more than a decade behind its adoption in Drosophila, the other invertebrate model commonly used to dissect biological mechanisms. Here, we characterize many parameters that influence expression in recently developed C. elegans bipartite systems. We examine how DNA binding site number and spacing influence expression and characterize how these expression parameters vary in distinct tissue types. Furthermore, we examine how both basal promoters and 3 UTR influence the specificity and level of expression. These studies provide both a framework for the rational design of driver and reporter transgenes as well as molecular and genetic tools for the creation, characterization, and optimization of bipartite system components for expression in other cell types.

molecular biology↗

bioRxiv2021

Selection and structural characterisation of anti-TREM2 scFvs that reduce levels of shed ectodomain

Single point mutations in TREM2, a receptor expressed by microglia in the brain, are associated with an increased risk of neurodegeneration including Alzheimers disease. Numerous studies support a role for TREM2 in sensing damaging stimuli and triggering signalling cascades necessary for neuroprotection. Despite its significant role, ligands and regulators of TREM2 activation, and the mechanisms governing TREM2-dependent responses and its cleavage from the membrane, remain poorly characterised. Here, we present phage display generated scFv antibody binders to human TREM2 ectodomain. Cocrystal structures revealed the binding of two scFvs to an epitope on the globular TREM2 domain distal to the putative ligand-binding site. Enhanced functional activity was observed for oligomeric scFv species which inhibited the production of soluble TREM2 in a HEK293 cell model. We hope that detailed characterisation of their epitopes and properties will facilitate the use of these renewable binders as structural and functional biology tools for TREM2 research.

molecular biology↗

bioRxiv2021

Melbournevirus-encoded histone doublets are recruited to virus particles and form destabilized nucleosome-like structures

The organization of genomic DNA into defined nucleosomes has long been viewed as a hallmark of eukaryotes. This paradigm has been challenged by the identification of minimalist histones in archaea, and more recently by the discovery of genes that encode fused remote homologs of the four eukaryotic histones in Marseilleviridae, a subfamily of giant viruses that infect amoebae. We demonstrate that viral doublet histones localize to the cytoplasmic viral factories after virus infection, and ultimately to mature virions. CryoEM structures of viral nucleosome-like particles show strong similarities to eukaryotic nucleosomes, despite the limited sequence identify. The unique connectors that link the histone chains contribute to the observed instability of viral nucleosomes, and some histone tails assume structural roles. Our results further expand the range of organisms that have nucleosomes and suggest a specialized function of histones in the biology of these unusual viruses. One Sentence SummarySome large DNA viruses encode fused histone doublets that are targeted to viral factories and assemble into open nucleosome-like structures.

molecular biology↗

bioRxiv2022

A synergistic activation of RARb and RARg nuclear receptors restores cell-types specialization during stem cells differentiation by hijacking RARa-controlled program

How cells respond to different external cues to develop along defined cell lineages to form complex tissues is a major question in systems biology. Here, we investigated the potential of retinoic acid receptor (RARs)-selective synthetic agonists to activate the gene-regulatory programs driving cell specialization during nervous tissue formation from P19 stem cells. Specifically, we found that the synergistic activation of the RAR{beta} and RAR{gamma} by selective ligands (BMS641 or BMS961) induces cell maturation to specialized neuronal subtypes, as well as to astrocytes and oligodendrocyte precursors. Using RAR istoype knockout lines exposed to RAR-specific agonists, interrogated by global transcriptome landscaping and in silico modeling of transcription regulatory signal propagation, revealed major RAR-driven gene programs essential for optimal neuronal cell specialization, and hijacked by the synergistic activation of the RAR{beta} and RAR{gamma} receptors. Overall, this study provides a systems biology view of the gene programs accounting for the previously observed redundancy between RAR receptors, paving the way towards their potential use for directing cell specialization during nervous tissue formation.

molecular biology↗