Biology subjects

Vargas, J.

Publications and source records attributed to Vargas, J..

At least 19 recordsLinked to original sources

bioRxiv2026

SSUplex: fast, both-strand extraction and origin-sorting of small-subunit rRNA for environmental DNA metabarcoding

Ribosomal RNA metabarcoding sits at the center of how we characterize microbial and eukaryotic communities in environmental samples, and long-read sequencing has made full-length small-subunit (SSU; 16S/18S) profiling routine. The broadly conserved primers that make rRNA such a convenient marker are also its liability: by design they co-amplify organellar (mitochondrial, chloroplast) and cross-domain SSU alongside the intended target. Left unsorted before taxonomic assignment, these passengers are systematically misclassified, and the error propagates straight into estimates of community composition and diversity. Reads must therefore be detected, extracted, and sorted by origin before they ever reach a classifier. We present SSUplex, an open-source tool that detects SSU rRNA, assigns each read to one of five origins (bacteria, archaea, eukaryota, mitochondria, chloroplast), and extracts the SSU region for downstream classification. SSUplex reimplements the extraction-and-origin logic of the widely used Metaxa2 in the Rust programming language, scans both strands, and ships as a single dependency-light binary suited to long-read (Oxford Nanopore, PacBio HiFi) and short-read data. Benchmarked against Metaxa2 on public data, SSUplex reproduces Metaxa2 origin calls on full-length reads (96.8% concordance) and matches its extraction speed on small inputs, then pulls away to run up to [~]3.4x faster with [~]35% lower peak memory at 200,000 reads, the per-sample scale a long-read amplicon run typically reaches. We are candid about a genuine, measured trade-off in the origin-ranking statistic, and we pinpoint the bacteria-versus-mitochondria boundary as the methods one intrinsically lower-confidence edge. For the now-common workflow in which origin-sorted reads are handed to a dedicated classifier rather than classified in place, SSUplex is a fast, reproducible, embeddable stand-in for Metaxa2s extraction role. Source code and a benchmark harness that regenerates every result from public data are available under the MIT license at https://github.com/ayobi/ssuplex.

bioinformatics↗

bioRxiv2026

scLASER: a robust framework for simulating and detecting time-dependent single-cell dynamics in longitudinal studies

Longitudinal single-cell clinical studies enable tracking within-individual cellular dynamics, but methods for modeling temporal phenotypic changes and estimating power remain limited. We present scLASER, a framework detecting time-dependent cellular neighborhood dynamics and simulating longitudinal single-cell datasets for power estimation. Across benchmark experiments, scLASER shows consistently higher sensitivity than traditional cluster--based approaches, with particularly pronounced gains in rare cell types and non-linear temporal patterns. Applications to inflammatory bowel disease (95,813 cells, 38 patients) reveal treatment-responsive NOTCH3+ stromal trajectories with high cell type discrimination (AUC > 0.92), while analysis of COVID-19 data (188,181 cells, 84 patients) identifies three distinct axes of T cell activity (cytotoxic effector, NK immunoreceptor signaling, and interferon-stimulated gene programs) over disease progression. scLASER enables robust longitudinal single-cell analysis and optimization of study design. TeaserA new framework detects time-dependent cellular dynamics and enables power estimation for longitudinal clinical single-cell studies.

bioinformatics↗

bioRxiv2026

SPP1hi macrophages in fibrin niches promote hyperplastic tissue remodeling in rheumatoid arthritis synovium

In chronic inflammatory diseases, maladaptive tissue remodelling is driven by a complex interplay of resident cells, immune filtrates and the extracellular matrix. In the autoimmune disorder rheumatoid arthritis (RA), synovial tissue undergoes assive expansion to form an invasive pannus that drives the erosion of cartilage and bone. The mechanisms mediating this ggressive growth are incompletely defined. Using spatial transcriptomics profiling of patient tissue, we detected an bundance of proliferating fibroblasts near the synovial tissue lining surface and adjacent to SPP1hi macrophages. Notably, ese synovial lining regions were also distinctly marked by deposits of the clot-forming protein fibrin. While the SPP1hi acrophages phenotypically resemble pro-fibrotic macrophages that drive lung and liver fibrosis, these niches were devoid f the dense highly ordered collagen that marks fibrosis. Functionally, we found that SPP1hi macrophages degrade and hagocytose fibrin matrices and promote fibroblast proliferation. As fibrin provides transient matrices for de novo tissue eneration in the context of wound healing, these data support a model of hyperplastic tissue outgrowth involving SPP1hi acrophages, fibroblasts and fibrin matrices adhered to the exterior synovial tissue surface. While current RA therapies rimarily aim to dampen pro-inflammatory responses, our findings provide rationale for targeting pro-generative pathways nd SPP1hi macrophages. Once Sentence SummarySPP1hi macrophages in RA synovial fibrin deposits promote tissue hyperplasia.

immunology↗

bioRxiv2025

ETS1 Orchestrates a Hybrid EMT Program Driving in vivo Metastasis and Immune Evasion

Transcriptional Intratumoral heterogeneity (ITH) is a hallmark of aggressive cancers, yet how transcriptional ITH programs drive tumor metastasis and immune evasion in upper aerodigestive squamous cell carcinoma (UASCC) remains unclear. Through single-cell RNA sequencing analysis of UASCC cells and patient tumors, we uncovered a hybrid epithelial mesenchymal transition (hEMT) ITH program linked to metastatic dissemination. The transcription factor ETS1 was identified as a master regulator of the hEMT program, directly activating pro-metastatic genes and promoting distant spread in vivo. Unexpectedly, ETS1 also orchestrated an immune-cold tumor microenvironment by transcriptionally activating both STAT1 and PD-L1 (CD274) genes, suppressing T lymphocyte infiltration, and elevating immune checkpoint molecules. Clinically, ETS1-high tumors strongly correlated with poor survival and resistance to immune checkpoint blockade across multiple cohorts. Leveraging drug screens, we discovered that ETS1-high cancers are vulnerable to HSP90 inhibitors (e.g., Alvespimycin), which suppress ETS1 by disrupting HIF1A-mediated transcriptional activation. Together, our work reveals ETS1 as a dual driver of tumor distal metastasis and immune evasion in UASCC, while nominating HSP90 inhibition as a tailored treatment strategy for ETS1-driven tumors. These findings provide a roadmap for targeting aggressive ITH subsets and overcoming immunotherapy resistance.

cancer biology↗

bioRxiv2025

Structural determinants of rotavirus proteolytic activation

The infectivity of rotavirus (RV), the leading cause of childhood diarrhea, hinges on the activation of viral particles through the proteolysis of the spike protein by trypsin-like proteases in the host intestinal lumen. Despite comprehensive structural characterization of the virus particle, the structural rationale behind the necessity of trypsin digestion of the VP4 protein for infectivity remains poorly understood. In this study, using cryo-electron microscopy (cryo-EM) and advanced image processing techniques, we compared uncleaved and cleaved RV virions and found that the conformation of the non-proteolyzed spike is constrained by the position of loops that surround its structure, linking the lectin domains of the spike head to its body. The proteolysis of these loops removes this structural constraint, thereby enabling the spike to undergo the necessary conformational changes required for cell membrane penetration. Thus, these loops function as regulatory elements to ensure that the spike protein is activated precisely when and where it is needed to facilitate a successful infection.

microbiology↗

bioRxiv2024

Regulatory Roles of Long Non-Coding RNAs in Arterial Stiffness and Hypertension: Insights from Two African American Studies

BackgroundArterial stiffness, commonly assessed via pulse wave velocity (PWV), is marked by reduced arterial elasticity and serves as a significant risk factor for cardiovascular disease and an early indicator of hypertension. This study investigated the regulatory roles of long non-coding RNAs (lncRNAs) in modulating mRNAs associated with arterial stiffness and hypertension, with a particular focus on African Americans, a population disproportionately impacted by hypertension. MethodsWe utilized whole-blood transcriptome sequencing data from two African American (AA) cohorts with high hypertension prevalence: the GENE-FORECAST study (436 subjects) and the MH-GRID study (179 subjects). Our objectives were to: (1) identify lncRNAs and mRNAs differentially expressed (DE) between the upper and lower tertiles of PWV, (2) determine DE lncRNAs associated with the expression levels of each DE mRNA, and (3) link the lncRNA-modulated mRNAs to hypertension across both datasets. ResultsDifferential expression analysis revealed 1,035 DE mRNAs and 31 DE lncRNAs between upper and lower PWV groups. Then lncRNA-mRNA pairs significantly associated were identified, involving 31 unique lncRNAs and 1,034 unique mRNAs. Finally, 22 of the lncRNA-modulated mRNAs initially linked to PWV were found associated with hypertension, in both datasets. Interestingly, 30 lncRNAs were linked to the expression of UCP2 (Uncoupling Protein 2), a gene implicated in oxidative stress and endothelial function. ConclusionsOur findings underscore the significant roles of lncRNAs in regulating gene expression associated with arterial stiffness and hypertension. The differential expression of UCP2 in relation to PWV and hypertension, along with its potential regulation by lncRNAs, offers valuable insights into the molecular mechanisms underlying arterial stiffness and its connection with hypertension.

genomics↗

bioRxiv2023

Uncover spatially informed shared variations for single-cell spatial transcriptomics with STew

MotivationThe recent spatial transcriptomics (ST) technologies have enabled characterization of gene expression patterns and spatial information, advancing our understanding of cell lineages within diseased tissues. Several analytical approaches have been proposed for ST data, but effectively utilizing spatial information to unveil the shared variation with gene expression remains a challenge. ResultsWe introduce STew, a Spatial Transcriptomic multi-viEW representative learning method, to jointly analyze spatial information and gene expression in a scalable manner, followed by a data-driven statistical framework to measure the goodness of model fit. Through benchmarking using Human DLPFC data with true manual annotations, STew achieved superior performance in both clustering accuracy and continuity of identified spatial domains compared with other methods. STew is also robust to generate consistent results insensitive to model parameters, including sparsity constraints. We next applied STew to various ST data acquired from 10x Visium and Slide-seqV2, encompassing samples from both mouse and human brain, which revealed spatially informed cell type clusters. We further identified a pro-inflammatory fibroblast spatial niche using ST data from psoriatic skins. Hence, STew is a generalized method to identify both spatially informed clusters and disease-relevant niches in complex tissues. AvailabilitySource code and the R software tool STew are available from github.com/fanzhanglab/STew. Contactfan.3.zhang@cuanschutz.edu Supplementary informationSupplementary data are provided.

bioinformatics↗

bioRxiv2022

Cryo-EM map anisotropy can be attenuated by map post-processing and a new method for its estimation

One of the most important challenges in cryogenic electron microscopy (cryo-EM) is the substantial number of samples that exhibit preferred orientations, which leads to an uneven coverage of the projection sphere. As a result, the overall quality of the reconstructed maps can be severely affected, as manifested by the presence of anisotropy in the map resolution. Several methods have been proposed to measure the directional resolution of maps in tandem with experimental protocols to address the problem of preferential orientations in cryo-EM. Following these works, in this manuscript we identified one potential limitation that may affect most of the existing methods and we proposed an alternative approach to evaluate the presence of preferential orientations in cryo-EM reconstructions. In addition, we also showed that some of the most recently proposed cryo-EM map post-processing algorithms can attenuate map anisotropy, thus offering alternative visualization opportunities for cases affected by moderate levels of preferential orientations.

bioinformatics↗

bioRxiv2022

Multivalent interactions essential for lentiviral integrase function

A multimer of retroviral integrase (IN) synapses viral DNA ends within a stable intasome nucleoprotein complex for integration into a host cell genome. Reconstitution of the intasome from the maedi-visna virus (MVV), an ovine lentivirus, revealed a large assembly containing sixteen IN subunits (1). Herein, we report cryo-EM structures of the lentiviral intasome prior to engagement of target DNA and following strand transfer, refined at 3.4 and 3.5 [A] resolution, respectively. The structures elucidate details of the protein-protein and protein-DNA interfaces involved in lentiviral intasome formation. We show that the homomeric interfaces involved in IN hexadecamer formation and the -helical configuration of the linker connecting the C-terminal and catalytic core domains are critical for MVV IN strand transfer activity in vitro and for virus infectivity. Single-molecule microscopy in conjunction with photobleaching revealed that the MVV intasome can bind a variable number, up to sixteen molecules, of the lentivirus-specific host factor LEDGF/p75. Concordantly, ablation of endogenous LEDGF/p75 resulted in gross redistribution of MVV integration sites in human and ovine cells. Our data confirm the importance of the expanded architecture observed in cryo-EM studies of lentiviral intasomes and suggest that this organization underlies multivalent interactions with chromatin for integration targeting to active genes.

biochemistry↗

bioRxiv2020

IFN-{lambda}4 may contribute to HCV persistence by increasing ER stress and enhancing IRF1 signaling

Chronic hepatitis C virus (HCV) infection and cirrhosis are major risk factors for developing hepatocellular carcinoma (HCC). Genetic polymorphisms in the IFNL3/IFNL4 locus have been associated both with impaired clearance of HCV and protection from liver fibrosis, an early stage of cirrhosis. Here, we aimed to address the genetic and functional relationships between IFNL3/IFNL4 polymorphisms, HCV-related cirrhosis, and HCC risk. We evaluated associations between IFNL4 genotype, defined as the presence of rs368234815-dG or rs12979860-T alleles, with cirrhosis and HCC risk in patients with chronic HCV - 2,931 from Taiwan and 3,566 from Japan. We detected associations between IFNL4 genotype and decreased risk of cirrhosis (OR=0.66, 95%CI=0.46-0.93, P=0.018, in Taiwan), but increased risk of HCC (OR=1.28, 95%CI=1.07-1.52, P=0.0058, in Japan). In-vitro, IFN-{lambda}4 expression increased ER stress, and enhanced positive regulation of IFN responses via IRF1 induction, which mediated antiproliferative effects in hepatic cells. Our data present novel IFN-{lambda}4-associated pathways that may be contributing to HCV persistence and development of HCC.

cancer biology↗

bioRxiv2020

Interferons and viruses induce a novel primate-specific isoform dACE2 and not the SARS-CoV-2 receptor ACE2

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes COVID-19, utilizes angiotensin-converting enzyme 2 (ACE2) for entry into target cells. ACE2 has been proposed as an interferon-stimulated gene (ISG). Thus, interferon-induced variability in ACE2 expression levels could be important for susceptibility to COVID-19 or its outcomes. Here, we report the discovery of a novel, primate-specific isoform of ACE2, which we designate as deltaACE2 (dACE2). We demonstrate that dACE2, but not ACE2, is an ISG. In vitro, dACE2, which lacks 356 N-terminal amino acids, was non-functional in binding the SARS-CoV-2 spike protein and as a carboxypeptidase. Our results reconcile current knowledge on ACE2 expression and suggest that the ISG-type induction of dACE2 in IFN-high conditions created by treatments, inflammatory tumor microenvironment, or viral co-infections is unlikely to affect the cellular entry of SARS-CoV-2 and promote infection.

genetics↗

bioRxiv2020

DeepEMhacer: a deep learning solution for cryo-EM volume post-processing

Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on B-factor correction, suffer from limitations. For instance, they ignore the heterogeneity in the map local quality that reconstructions tend to exhibit. Aiming to overcome these problems, we present DeepEMhancer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental maps and maps sharpened using their respective atomic models, DeepEMhancer has learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhancer was evaluated on a testing set of 20 different experimental maps, showing its ability to obtain much cleaner and more detailed versions of the experimental maps. Additionally, we illustrated the benefits of DeepEMhancer on the structure of the SARS-CoV-2 RNA polymerase.

bioinformatics↗

bioRxiv2020

Local computational methods to improve the interpretability and analysis of cryo-EM maps

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, over-sharpening due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.e. ab initio modelling. In this work, we propose 1) approaches to enhance high-resolution features of cryo-EM maps, while preventing map distortions and 2) methods to obtain local B-factors and electron density occupancy maps. These algorithms have as common link the use of the spiral phase transformation and are called LocSpiral, LocBSharpen, LocBFactor and LocOccupancy. Our results, which include improved maps of recent SARS-CoV-2 structures, show that our methods can improve the interpretability and analysis of obtained reconstructions.

biophysics↗

bioRxiv2020

Alternative Conformations and Motions Adopted by 30S Ribosomal Subunits Visualized by Cryo-Electron Microscopy.

It is only after recent advances in cryo-electron microscopy that is now possible to describe at high resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between the "active" and "inactive" conformations. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved. Here we used cryo-electron microscopy to obtain the structure of the inactive conformation of the 30S subunit to 3.6[A] resolution and study its motions. In the inactive conformation, three nucleotides at the 3 end of the 16S rRNA cause the region of helix 44 forming the decoding center to adopt an unlatched conformation and the 3 end of the 16S rRNA positions similarly to the mRNA during translation. Incubation of inactive 30S subunits at 42 {degrees}C reverts these structural changes. The position adopted by helix 44 dictates the most prominent motions of the 30S subunit. We found that extended exposures to low magnesium concentrations induces unfolding of large rRNA structural domains. The air-water interface to which ribosome subuints are exposed during sample preparation also peel off some ribosomal proteins. Overall this study provides new insights about the conformational space explored by the 30S ribosomal subunit when the ribosomal particles are free in solution.

molecular biology↗

bioRxiv2020

The connector between the kinase and RNase domains of IRE1α transmits the conformational change that underlies ER stress-induced activation.

The unfolded protein response (UPR) plays an evolutionarily conserved role in homeostasis, and its dysregulation often leads to human disease, including diabetes and cancer. IRE1 is a major transducer that conveys endoplasmic reticulum (ER) stress to biochemical signals, yet major gaps persist in our understanding of how the detection of stress is converted to one of several molecular outcomes. It is known that upon sensing unfolded proteins via its ER luminal domain, IRE1 dimerizes and oligomerizes (often visualized as clustering), and then trans-autophosphorylates. The IRE1 kinase activity is required for activation of its RNase effector domain and for clustering of IRE1. It is not yet clear if IRE1 clustering is a platform for the RNase activity, or if the two represent distinct biological functions. Here, we uncover a previously unrecognized role for helix K between IRE1 kinase and RNase domains in conveying critical conformational changes. Using mutants within this inter-domain helix, we show for the first time that: 1) distinct substitutions (specifically, of Leu827) selectively affect oligomerization, RNase activity, and, unexpectedly, the kinase activity of IRE1; 2) RNase activation can be uncoupled from IRE1 oligomerization, and phosphorylation of S729 marks the former but not the latter; 3) The nature of residue 827 determines the conformation that the IRE1 protein adopts, leading to different patterns of biochemical activities. In summary, this work reveals a previously unappreciated role for the inter-domain helix as a pivotal conduit for attaining the stress-responsive conformation of IRE1.

biochemistry↗

bioRxiv2019

The inner junction complex of the cilia is an interaction hub that involves tubulin post-translational modifications

Microtubules are cytoskeletal structures involved in structural support, microtubule-based transport and the organization of organelles in the cells. The building blocks of the microtubule, the - and {beta}-tubulin heterodimers, polymerize into protofilaments, that associate laterally to form the hollow microtubule. There exists a specific type of microtubule structures in the cilia, termed doublet microtubules, where high stability is required for ciliary beating and function. The doublet microtubule, consisting of a complete A-tubule and a partial B-tubule maintains its stability through unique interactions at its outer and inner junctions, where the A- and B-tubules meet.\n\nUsing cryo-electron microscopy, we present the answer to the long-standing question regarding the identities, localizations and structures of the Chlamydomonas doublet microtubule inner junction proteins. Using a combination of sequence bioinformatics and mass spectrometry, we identified two new inner junction proteins, FAP276 and FAP106, and an inner junction associated protein FAP126. We show that inner junction proteins PACRG and FAP20, together with FAP52, previously unidentified FAP276, FAP106 and FAP126, form an interaction hub at the inner junction, which involves tubulin sites for post-translational modifications. We further compare the Chlamydomonas and Tetrahymena doublet microtubule structures to understand the common and species-specific features of the inner junction.

biophysics↗

bioRxiv2019

Tubulin Lattice in Cilia is in a Stressed Form Regulated by Microtubule Inner Proteins

Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around the cell, are composed of radially bundled doublet microtubules. The doublet microtubule is composed of a 13-protofilament A-tubule, a partial 10-protofilament B-tubule and microtubule inner proteins (MIPs) inside the tubulin lattice. In this study, we present the near-atomic resolution map of the Tetrahymena doublet microtubules. The map demonstrates that the network of microtubule inner proteins is weaving into the tubulin lattice, forming an inner sheath of proteins. In addition, we also obtain the tubulin lattice structure with missing MIPs by Sarkosyl treatment. In this structure, the tubulin lattice showed significant longitudinal compaction and lateral angle changes between protofilaments. These results are evidence that the binding of MIPs directly affects and stabilizes the tubulin lattice. It is also suggested that the doublet microtubule is an intrinsically stressed filament and this stress could be exploited in the regulation of ciliary waveforms.

biophysics↗

bioRxiv2019

Role of Era in Assembly and Homeostasis of the Ribosomal Small Subunit

To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, cryo-electron microscopy and in-cell footprinting. Our combined approach allowed for visualization of the small subunit as it assembled and revealed that with the exception of key helices in the platform domain, all other 16S rRNA domains were able to fold even in the absence of Era. Notably, the maturing particles did not stall while waiting for the platform domain to mature and instead re-routed their folding pathway to enable concerted maturation of other structural motifs spanning multiple rRNA domains. We also found that binding of Era to the mature 30S subunit destabilized helix 44 and the decoding center preventing binding of YjeQ, another assembly factor. This work establishes Eras role in ribosome assembly and suggests new roles in maintaining ribosome homeostasis.

molecular biology↗