bluuebunny/crossref_metadata_2025 huggingface dataset:
The regulatory grammar of human promoters uncovered by MPRA-trained deep learning
Authors: Barbadilla-Martínez Lucía , Klaassen Noud , Franceschini-Santos Vinícius H. , Breda Jérémie , Hernandez-Quiles Miguel , van Lieshout Tijs , Urzua Traslaviña Carlos G. , Yücel Hatice , Luong Boi Minh Chau , Hermana-Garcia-Agullo Celia , Gregoricchio Sebastian , Zwart Wilbert , Voest Emile , Franke Lude , Vermeulen Michiel , de Ridder Jeroen , van Steensel Bas
Published: July 2024

One of the major challenges in genomics is to build computational models that accurately predict genome-wide gene expression from the sequences of regulatory elements. At the heart of gene regulation are promoters, yet their regulatory logic is still incompletely understood. Here, we report PARM, a cell-type specific deep learning model trained on specially designed massively parallel reporter assays that query human promoter sequences. PARM requires ∼1,000 times less computational power than state-of-the-art technology, and reliably predicts autonomous promoter activity throughout the genome from DNA sequence alone, in multiple cell types. PARM can even design purely synthetic strong promoters. We leveraged PARM to systematically identify binding sites of transcription factors (TFs) that are likely to contribute to the activity of each natural human promoter. We uncovered and experimentally confirmed striking positional preferences of TFs that differ between activating and repressive regulatory functions, as well as a complex grammar of motif-motif interactions. For example, many, but not all, TFs act as repressors when their binding motif is located near or just downstream of the transcription start site. Our approach lays the foundation towards a deep understanding of the regulation of human promoters by TFs.

Highlights

Causality-trained deep learning model PARM captures regulatory grammar of human promoters

PARM is highly economical, both experimentally and computationally

Transcription factors have different preferred positions for their regulatory activity

Many (but not all) transcription factors act as repressors when binding downstream of transcription start sites
Visual analysis of spatial transcriptomics data with RedeViz
Authors: Wang Dehe , Ren Xianwen
Published: July 2024

Spatial transcriptomics (ST) technologies are powerful tools to illustrate the spatial hierarchy and heterogeneity of tissues with the lens of multiplexed gene readouts. However, ST technologies generate sequence data rather than images, preventing intuitive examination of the cellular contexture of tissues. Moreover, the inherent sparsity of ST data caused by molecular crowdedness and sequencing dropouts poses great challenges to accurate and clear visualization. In this study, we introduce RedeViz, a toolkit crafted for enhancing and visualizing subcellular-resolution ST data. RedeViz applies a pixel-level enhancement strategy, visualizes ST data in automatic or customized manners, and can display the cellular and genic spatial patterns with effects akin to HE staining. Strict evaluations confirm that RedeViz fits a wide range of ST platforms, including Xenium, Visium HD, MERFISH, CosMx, Stereoseq, as well as spatial proteomic platforms like CODEX. The impressive performance of RedeViz across various scales from cell-, tissue-, organ-, to organism-levels brings us a universal “What You See Is What You Get” framework for visual analysis of ST data.
A latitudinal gradient of reference genomes
Authors: Linck Ethan B. , Cadena Carlos Daniel
Published: July 2024

Global inequality rooted in legacies of colonialism and uneven development can lead to systematic biases in scientific knowledge. In ecology and evolutionary biology, findings, funding and research effort are disproportionately concentrated at high latitudes while biological diversity is concentrated at low latitudes. This discrepancy may have a particular influence in fields like phylogeography, molecular ecology and conservation genetics, where the rise of genomics has increased the cost and technical expertise required to apply state-of-the-art methods. Here we ask whether a fundamental biogeographic pattern—the latitudinal gradient of species richness in tetrapods—is reflected in available reference genomes, an important data resource for various applications of molecular tools for biodiversity research and conservation. We also ask whether sequencing approaches differ between the Global South and Global North, reviewing the last five years of conservation genetics research in four leading journals. We find that extant reference genomes are scarce relative to species richness at low latitudes, and that reduced-representation and whole-genome sequencing are disproportionately applied to taxa in the Global North. We conclude with recommendations to close this gap and improve international collaborations in biodiversity genomics.
Changes in the functional diversity and abundance of ectomycorrhizal fungi are decoupled from water uptake patterns in European beech forests
Authors: Rodríguez-Uña Asun , Moreno-Mateos David , Matesanz Silvia , Wingate Lisa , Barbeta Adrià , Porras Javier , Gimeno Teresa E.
Published: July 2024

Temperate forests on their warm and dry distribution limit are expected to be most vulnerable to reductions in water availability. This prediction is mostly based on studies assessing single forest functions, mainly growth. Water and nutrient cycling are functions that rely on tree roots and their symbiotic association with ectomycorrhizal (ECM) fungi. Trees can compensate for seasonal reductions in water availability by shifting root water-uptake (RWU) towards deeper soil layers, but ECM fungi dwell in the upper soil, thus suffering from desiccation and compromising nutrient uptake. We hypothesised that drier sites should depict larger seasonal shifts in RWU, but at the expense of lower diversity and colonization of fine roots by ECM fungi. We selected three beech (Fagus sylvatica) forests in their warm distribution limit with contrasting geographic locations and mean annual precipitation: northern Atlantic (2500mm), intermediate transitional (1150mm) and southern Mediterranean (780mm). We collected soil, stem and root samples in spring (wet) and summer (dry) to quantify fine-root density and colonization by ECM fungi, to infer RWU from isotopic composition of plant and soil water, and to characterize ECM fungal diversity through DNA-metabarcoding. High moisture in the upper soil benefited the ECM community, but higher diversity and fine-root colonization by ECM fungi in the upper soil did not imply larger contributions of this soil layer to RWU. The prevailing climate and local abiotic conditions determined how ECM communities structured, more than seasonal variability. Across sites, ECM communities differed in their functional diversity: ECM fungi with long hyphae, more vulnerable to water scarcity, dominated at the site with the highest water availability. Our results suggest that transient reductions in soil water availability might not compromise RWU but could be detrimental for maintaining ECM-mediated nutrient uptake in beech forests experiencing longer and more severe drought periods under current climate change.
Interleukin-33 from oligodendrocytes sustains effector differentiation of tissue-resident CD8+ T cells and is a druggable target in CNS autoimmune disease
Authors: Fonta Nicolas , Page Nicolas , Klimek Bogna , Piccinno Margot , Di Liberto Giovanni , Lemeille Sylvain , Kreutzfeldt Mario , Kastner Anna Lena , Ertuna Yusuf I. , Vincenti Ilena , Wagner Ingrid , Pinschewer Daniel D. , Merkler Doron
Published: July 2024

In chronic inflammatory disorders of the central nervous system (CNS), tissue-resident self-reactive T cells perpetuate disease. The specific tissue factors governing the persistence and continuous differentiation of these cells remain undefined but could represent attractive therapeutic targets.

In a model of chronic CNS autoimmunity, we find that oligodendrocyte-derived interleukin-33 (IL-33), an alarmin, is key for locally regulating the pathogenicity of self-reactive CD8+ T cells. The selective ablation of IL-33 from neo-self-antigen-expressing oligodendrocytes mitigates CNS disease. In this context, fewer self-reactive CD8+ T cells persist in the inflamed CNS, and the remaining cells are largely locked into a stem-like precursor program, failing to form TCF-1loweffector progeny. Importantly, interventional IL-33 blockade by locally administered somatic gene therapy reduces T cell infiltrates and improves the disease course.

Our study identifies oligodendrocyte-derived IL-33 as a druggable tissue factor regulating the differentiation and survival of self-reactive CD8+ T cell in the inflamed CNS. This finding introduces tissue factors as a novel category of immune targets for treating chronic CNS autoimmune diseases.
Spleen Tyrosine Kinase (SYK) negatively regulates ITAM-mediated human NK cell signaling and CD19-CAR NK cell efficacy
Authors: Millan Alberto J. , Allain Vincent , Nayak Indrani , Aguilar Oscar A. , Arakawa-Hoyt Janice S. , Ureno Gabriella , Rothrock Allison Grace , Shemesh Avishai , Eyquem Justin , Das Jayajit , Lanier Lewis L.
Published: July 2024

NK cells express activating receptors that signal through ITAM-bearing adapter proteins. The phosphorylation of each ITAM creates binding sites for SYK and ZAP70 protein tyrosine kinases to propagate downstream signaling including the induction of Ca2+influx. While all immature and mature human NK cells co-express SYK and ZAP70, clonally driven memory or adaptive NK cells can methylateSYKgenes and signaling is mediated exclusively using ZAP70. Here, we examined the role of SYK and ZAP70 in a clonal human NK cell line KHYG1 by CRISPR-based deletion using a combination of experiments and mechanistic computational modeling. Elimination ofSYKresulted in more robust Ca++influx after cross-linking of the CD16 and NKp30 receptors and enhanced phosphorylation of downstream proteins, whereasZAP70deletion diminished these responses. By contrast,ZAP70depletion increased proliferation of the NK cells. As immature T cells express both SYK and ZAP70 but mature T cells often express only ZAP70, we transduced the human Jurkat cell line with SYK and found that expression of SYK increased proliferation but diminished TCR-induced Ca2+flux and activation. We performed transcriptional analysis of the matched sets of variant Jurkat and KHYG1 cells and observed profound alterations caused by SYK expression. As depletion ofSYKin NK cells increased their activation, primary human NK cells were transduced with a CD19-targeting CAR and were CRISPR edited to ablateSYKorZAP70. Deletion ofSYKresulted in more robust cytotoxic activity and cytokine production, providing a new therapeutic strategy of NK cell engineering for cancer immunotherapy.
A novel mechanism of microbial attachment: the flagellar pump ofGiardia lamblia
Authors: Picou Theodore J. , Luo Haibei , Polackwich Robert J. , Gabilondo Beatriz Burrola , McAllister Ryan G. , Gagnon David A. , Powers Thomas R. , Elmendorf Heidi G. , Urbach Jeffrey S.
Published: July 2024

The ability of microbes to attach to biological and inert substrates is a necessary prerequisite for colonization of new habitats. In contrast to well-characterized mechanisms that rely on specific or non-specific chemical interactions between microbe and substrate, we describe here an effective hydrodynamic mechanism of attachment that relies on fluid flow generated by the microbe. The microbeGiardia lamblia, a flagellated protozoan parasite, naturally attaches to the microvilliated surface of the small intestine but is also capable of attaching indiscriminately to a wide range of natural and artificial substrates. By tracking fluorescent quantum dots, we demonstrate a persistent flow between the parasite and substrate generated by a pair ofGiardiaflagella. Using both experimental measures and computational modeling, we show that the negative pressure generated by this fluid flow is sufficient to generate the previously measured force of attachment. We further show that this dynamically-generated negative pressure allowsGiardiato attach to both solid and porous surfaces, thereby meeting the real-world demands of attachment to the microvilliated surface of intestinal cells. These findings provide experimental support for a hydrodynamic model of attachment that may be shared by other ciliated and flagellated microbes.
Roles of P-body factors inCandida albicansfilamentation and stress response
Authors: Tosiano Melissa A. , Lanni Frederick , Mitchell Aaron P. , McManus C. Joel
Published: July 2024

Hyphal growth is strongly associated with virulence in the human fungal pathogenCandida albicans. While hyphal transcriptional networks have been the subject of intense study, relatively little is known about post-transcriptional regulation. Previous work reported that P-Body (PB) factors Dhh1 and Edc3 were required forC. albicansvirulence and filamentation, suggesting an essential role for post-transcriptional regulation of these processes. However, the molecular roles of these factors have not been determined. To further study the function of PB factors in filamentation, we generated homozygous deletions ofDHH1andEDC3in diverse prototrophic clinical strains using transient CRISPR-Cas9. HomozygousDHH1deletion strongly impaired growth, altered filamentation, and exhibited unusual colony morphology in response to heat stress in five strain backgrounds. Using RNA-seq, we foundDHH1deletion disrupts the regulation of thousands of genes under both yeast and hyphal growth conditions in SC5314 and P57055. This included upregulation of many stress response genes in the absence of external stress, similar to deletion of theS. cerevisiae DHH1homolog. In contrast, we foundEDC3was not required for heat tolerance or filamentation in diverse strains. These results support a model in whichDHH1, but notEDC3, represses hyphal stress response transcripts in yeast and remodels the transcriptome during filamentation. Our work supports distinct requirements for specific mRNA decay factors, bolstering evidence for post-transcriptional regulation of filamentation inC. albicans.

Author summary

In the dimorphic fungal pathogenC. albicans, the hyphal phenotype strongly correlates with pathogenicity. While transcriptional control of hyphal growth has been extensively studied, comparatively little is known about post-transcriptional regulation of this significant morphological shift. PB factors are associated with mRNA decay and translational repression. Here we investigate the roles of two PB factors in growth, filamentation, and gene expression. Although deletion of PB factorEDC3did not impact growth or filamentation,dhh1Δ/Δhad greatly impaired growth and heat tolerance as well as unusual hyphal phenotypes in multiple clinical isolate strains. Additionally, we found that the transcriptomes of SC5314 and P57055dhh1Δ/Δyeast and hyphae were highly dysregulated. The extensive transcriptomic impacts of the absence of Dhh1 correlated with our phenotypic findings. Stress-associated genes were induced under non-stress conditions and the filamentation response was blunted under physiologically relevantin vitroconditions. We demonstrate that mRNA decay factors play distinct roles in regulatingC. albicansmorphology and that Dhh1 contributes to environmentally appropriate expression of the stress response and hyphal growth.
Cytokinin acts as a systemic signal coordinating reproductive effort in Arabidopsis
Authors: Volkava Darya , Bukovcakova Sona Valuchova , Mikulkova Pavlina , Pecinkova Jana , Skalak Jan , Hejatko Jan , Riha Karel
Published: July 2024

The duration of the flowering phase is a key adaptive trait related to reproductive effort and plant longevity. In plants with indeterminate inflorescences, the number of flowers produced is determined by the activity of the inflorescence meristem. However, the integration of inflorescence meristem activity with the physiological and developmental status of a plant is not well understood. We combined quantitative imaging of the inflorescence meristem with the manipulation of cytokinin levels and distribution to investigate how meristem activity is regulated. Our data support the hypothesis that emerging fruits drain the acropetal flow of cytokinin away from the meristem inducing its proliferative arrest. We further show that the duration of flowering and cytokinin signaling in the inflorescence meristem is influenced by nitrate availability. Taken together, these data suggest that cytokinin functions as a systemic signal that integrates information about ongoing reproductive effort and plant nutrient status to control flower production.
E. colido not count single molecules
Authors: Mattingly Henry H , Kamino Keita , Kottou Rafaela , Ong Jude , Emonet Thierry , Machta Benjamin B
Published: July 2024

Organisms must perform sensory-motor behaviors to survive. What bounds or constraints limit behavioral performance? Previously, we found that the gradient-climbing speed of a chemotaxing Escherichia coli is near a bound set by the limited information they acquire from their chemical environments. Here we ask what limits their sensory accuracy. Past theoretical analyses have shown that the stochasticity of single molecule arrivals sets a fundamental limit on the precision of chemical sensing. Although it has been argued that bacteria approach this limit, direct evidence is lacking. Here, using information theory and quantitative experiments, we find that E. coli's chemosensing is not limited by the physics of particle counting. First, we derive the physical limit on the behaviorally-relevant information that any sensor can get about a changing chemical concentration, assuming that every molecule arriving at the sensor is recorded. Then, we derive and measure how much information E. coli's signaling pathway encodes during chemotaxis. We find that E. coli encode two orders of magnitude less information than an ideal sensor limited only by shot noise in particle arrivals. These results strongly suggest that constraints other than particle arrival noise limit E. coli's sensory fidelity.
The impact of physical activity on physical performance, mitochondrial bioenergetics, ROS production and calcium handling across the human adult lifespan
Authors: Cefis Marina , Marcangeli Vincent , Hammad Rami , Granet Jordan , Leduc-Gaudet Jean-Philippe , Gaudreau Pierrette , Trumpff Caroline , Huang Hannah , Picard Martin , Aubertin-Leheudre Mylène , Bélanger Marc , Robitaille Richard , Morais José A. , Gouspillou Gilles
Published: July 2024

Aging-related muscle atrophy and weakness contribute to loss of mobility, falls and disability. Mitochondrial dysfunction is widely considered a key contributing mechanism to muscle aging. However, mounting evidence position physical activity as a confounding factor, making unclear whether muscle mitochondria accumulate bona fide defects with aging. To disentangle aging from physical activity-related mitochondrial adaptations, we functionally profiled skeletal muscle mitochondria in 51 inactive and 88 active men aged 20-93. Physical activity status conferred partial protection against age-related decline in physical performance. A trend for reduced muscle mitochondrial respiration with aging was observed in inactive but not in active participants, indicating that agingper sedoes not alter mitochondrial respiratory capacity. Mitochondrial reactive oxygen species (ROS) production was unaffected by aging and active participants displayed higher ROS production. In contrast, mitochondrial calcium retention capacity decreased with aging regardless of physical activity status and correlated with muscle mass, performance and the stress-responsive metabokine GDF15. Targeting mitochondrial calcium handling may hold promise for treating aging-related muscle impairments.
Regulation of Kv1.2 redox-sensitive gating by the transmembrane lectin LMAN2
Authors: Lamothe Shawn M. , Das Damayantee , Wong Anson A. , Hao Yubin , Maguire Aislinn D. , Kerr Bradley J. , Baronas Victoria A. , Kurata Harley T.
Published: July 2024

Kv1.2 potassium channels influence excitability and action potential propagation in the nervous system. Unlike closely-related Kv1 channels, Kv1.2 exhibits highly variable voltage-dependence of gating, attributed to regulation by unidentified extrinsic factors. Variable Kv1.2 gating is strongly influenced by the extracellular redox potential, and we demonstrate that Kv1.2 currents in dorsal root ganglion sensory neurons exhibit similar variability and redox sensitivity as observed when the channel is heterologously expressed in cell lines. We used a functional screening approach to test the effects of candidate regulatory proteins on Kv1.2 gating, using patch clamp electrophysiology. Among 52 candidate genes tested, we observed that co-expression with the transmembrane lectin LMAN2 led to a pronounced gating shift of Kv1.2 activation to depolarized voltages in CHO and L(tk-) cell lines, caused by deceleration of activation kinetics. Overexpression of LMAN2 promoted a slow gating mode of Kv1.2 that mimics the functional outcomes of extracellular reducing conditions, and enhanced sensitivity to extracellular reducing agents. In contrast, shRNA-mediated knockdown of endogenous LMAN2 in cell lines reduced Kv1.2 redox sensitivity and gating variability. Kv1.2 sensitivity to LMAN2 is abolished by mutation of neighboring residues F251 and T252 in the intracellular S2-S3 linker, and these also abolish redox-dependent modulation of Kv1.2, suggesting that LMAN2 is an important contributor to the mechanism of redox sensitivity. In conclusion, we identified LMAN2 as a candidate regulatory protein that influences redox-dependent modulation of Kv1.2, and clarified the structural elements of the channel that are required for sensitivity.
AGEing of Collagen: The Effects of Glycation on Collagen’s Stability, Mechanics and Assembly
Authors: Sloseris Daniel , Forde Nancy R.
Published: July 2024

Advanced Glycation End Products (AGEs) are the end result of the irreversible, non-enzymatic glycation of proteins by reducing sugars. These chemical modifications accumulate with age and have been associated with various age-related and diabetic complications. AGEs predominantly accumulate on proteins with slow turnover rates, of which collagen is a prime example. Glycation has been associated with tissue stiffening and reduced collagen fibril remodelling. In this study, we investigate the effects of glycation on the stability of type I collagen, its molecular-level mechanics and its ability to perform its physiological role of self-assembly. Collagen AGEing is inducedin vitroby incubation with ribose. We confirm and assess glycation using fluorescence measurements and changes in collagen’s electrophoretic mobility. Susceptibility to trypsin digestion and circular dichroism (CD) spectroscopy are used to probe changes in collagen’s triple helical stability, revealing decreased stability due to glycation. Atomic Force Microscopy (AFM) imaging is used to quantify how AGEing affects collagen flexibility, where we find molecular-scale stiffening. Finally we use microscopy to show that glycated collagen molecules are unable to self-assemble into fibrils. These findings shed light on the molecular mechanisms underlying AGE-induced tissue changes, offering insight into how glycation modifies protein structure and stability.
TPM4 condensates glycolytic enzymes to fuel actin reorganization under hyperosmotic stress
Authors: Yang Wenzhong , Wang Yuan , Liu Geyao , Wang Yan , Wu Congying
Published: July 2024

Actin homeostasis is fundamental for cell structure and consumes a large portion of cellular ATP. It has been documented in the literature that certain glycolytic enzymes can interact with actin, indicating an intricate interplay between the cytoskeleton and cellular metabolism. Here we report that hyperosmotic stress triggers actin severing and subsequent phase separation of the actin-binding protein TPM4. TPM4 condensates glycolytic enzymes such as HK2, PFKM, and PKM2, and adhere to and wrap around actin filaments. Notably, the condensates of TPM4 and glycolytic enzymes are enriched of NADH and ATP, suggestive of their functional importance in cell metabolism. At cellular level, actin filaments assembly is enhanced upon hyperosmotic stress and TPM4 condensation, while depletion of TPM4 impaired osmolarity-induced actin reorganization. At tissue level, co-localized condensates of TPM4 and glycolytic enzymes are observed in renal tissues subjected to hyperosmotic stress. Together, our findings suggest that stress-induced actin perturbation may act on TPM4 to organize glycolytic hubs that tether energy production to cytoskeletal reorganization.

Graphical Abstract
eIF1 and eIF5 dynamically control translation start site fidelity
Authors: Grosely Rosslyn , Alvarado Carlos , Ivanov Ivaylo P. , Nicholson Oliver B. , Puglisi Joseph D. , Dever Thomas E. , Lapointe Christopher P.
Published: July 2024

Translation initiation defines the identity of a synthesized protein through selection of a translation start site on a messenger RNA. This process is essential to well-controlled protein synthesis, modulated by stress responses, and dysregulated in many human diseases. The eukaryotic initiation factors eIF1 and eIF5 interact with the initiator methionyl-tRNAiMeton the 40S ribosomal subunit to coordinate start site selection. Here, using single-molecule analysis of in vitro reconstituted human initiation combined with translation assays in cells, we examine eIF1 and eIF5 function. During translation initiation on a panel of RNAs, we monitored both proteins directly and in real time using single-molecule fluorescence. As expected, eIF1 loaded onto mRNAs as a component of the 43S initiation complex. Rapid (∼ 2 s) eIF1 departure required a translation start site and was delayed by alternative start sites and a longer 5’ untranslated region (5’UTR). After its initial departure, eIF1 rapidly and transiently sampled initiation complexes, with more prolonged sampling events on alternative start sites. By contrast, eIF5 only transiently bound initiation complexes late in initiation immediately prior to association of eIF5B, which allowed joining of the 60S ribosomal subunit. eIF5 association required the presence of a translation start site and was inhibited and destabilized by alternative start sites. Using both knockdown and overexpression experiments in human cells, we validated that eIF1 and eIF5 have opposing roles during initiation. Collectively, our findings demonstrate how multiple eIF1 and eIF5 binding events control start-site selection fidelity throughout initiation, which is tuned in response to changes in the levels of both proteins.
Defining a Muscle Stem Cell matrisome signature: from transcriptome data to extracellular matrix niche topology
Authors: Guillon Emilie , Hisoilat Bacar , Sasaki Takako , Mourikis Philippos , Ruggiero Florence
Published: July 2024

Although intensively investigated, the regulation of skeletal muscle stem cells (MuSCs) by their niche remains an open question. The extracellular matrix (ECM) components of the niche represent a dynamic microenvironment that undoubtedly participates in MuSCs behavior. We used bioinformatics analysis of transcriptomic data to define the matrisome profile of skeletal muscle resident cells, comprising genes encoding ECM and ECM-associated proteins. We identified quiescent MuSCs as key ECM producers of the niche, notably through the expression of specific basement membrane genes asCol19a1andLama3and regulators of ECM assembly,Thsd4 and Aebp1. Unexpectedly, quiescent MuSCs also expressed matrisome neurogenesis-related genes. Immunofluorescence staining of selected ECM components showed their organization in isolated murine myofiber bundles. Upon activation, MuSCs strikingly downregulated the niche-related ECM genes and instead expressed genes involved in basement membrane disruption and matrisome genes linked to cell motility. This study identified distinct matrisome signatures of quiescent and activated MuSCs that are consistent with their function in homeostasis and repair of damaged skeletal muscle.
MDRepo – an open environment for data warehousing and knowledge discovery from molecular dynamics simulations
Authors: Roy Amitava , Ward Ethan , Choi Illyoung , Cosi Michele , Edgin Tony , Hughes Travis S. , Islam Md. Shafayet , Khan Asif M. , Kolekar Aakash , Rayl Mariah , Robinson Isaac , Sarando Paul , Skidmore Edwin , Swetnam Tyson L. , Wall Mariah , Xu Zhuoyun , Yung Michelle L. , Merchant Nirav , Wheeler Travis J.
Published: July 2024

Background

Molecular Dynamics (MD) simulation of biomolecules provides important insights into conformational changes and dynamic behavior, revealing critical information about folding and interactions with other molecules. This enables advances in drug discovery and the design of therapeutic interventions. The collection of simulations stored in computers across the world holds immense potential to serve as training data for future Machine Learning models that will transform the prediction of structure, dynamics, drug interactions, and more.

A need

Ideally, there should exist an open access repository that enables scientists to submit and store their MD simulations of proteins and protein-drug interactions, and to find, retrieve, analyze, and visualize simulations produced by others. However, despite the ubiquity of MD simulation in structural biology, no such repository exists; as a result, simulations are instead stored in scattered locations without uniform metadata or access protocols.

A solution

Here, we introduce MDRepo, a robust infrastructure that supports a relatively simple process for standardized community contribution of simulations, activates common downstream analyses on stored data, and enables search, retrieval, and visualization of contributed data. MDRepo is built on top of the open-source CyVerse research cyberinfrastructure, and is capable of storing petabytes of simulations, while providing high bandwidth upload and download capabilities and laying a foundation for cloud-based access to its stored data.
ABCA1 and apoA-I dependent 12-hydroxyeicosatetraenoic acid efflux regulates macrophage inflammatory signaling
Authors: Harsch Brian A. , Borkowski Kamil , Walker Rachel E. , Pedersen Theresa L. , Newman John W. , Shearer Gregory C.
Published: July 2024

Aberrant high-density lipoprotein (HDL) function is implicated in inflammation-associated pathologies. While HDL ABCA1-mediated reverse cholesterol and phospholipid transport are well described, the movement of pro-/anti-inflammatory lipids has not been explored. HDL phospholipids are the largest reservoir of circulating arachidonic acid-derived oxylipins. Endotoxin-stimulation activates inflammatory cells leading to hydroxyeicosatetraenoic acid (HETE) production, oxylipins which are involved in inflammatory response coordination. Active signaling in the non-esterified (NE) pool is terminated by sequestration of HETEs as esterified (Es) forms and degradation. We speculate that an ABCA1-apoA-I-dependent efflux of HETEs from stimulated cells could regulate intracellular HETE availability. Here we test this hypothesis both in vitro and in vivo. In endotoxin-stimulated RAW-264.7 macrophages preloaded with d8-arachidonic acid we use compartmental tracer modeling to characterize the formation of HETEs, and their efflux into HDL. We found that in response to endotoxin: I) Cellular NE 12-HETE is positively associated with MCP-1 secretion (p<0.001); II) HETE transfer from NE to Es pools is ABCA1-depedent (p<0.001); III) Cellular Es HETEs are transported into media when both apoA-I and ABCA1 are present (p<0.001); IV) The stimulated efflux of HETEs >> arachidonate (p<0.001). Finally, in endotoxin challenged humans (n=17), we demonstrate that intravenous lipopolysaccharide (0.6 ng/kg body weight) resulted in accumulation of 12-HETE in HDL over a 168-hour follow-up. Therefore, HDL can suppress inflammatory responses in macrophages by regulating intracellular HETE content in an apoA-I/ABCA1 dependent manner. The described mechanism may apply to other oxylipins and explain anti-inflammatory properties of HDL. This newly defined HDL property opens new doors for the study of lipoprotein interactions in metabolic diseases.
Maturation of Human Intestinal Epithelial Cell Layers Fortifies the Apical Surface againstSalmonellaAttack
Authors: van Rijn Jorik M. , Lopes Ana C. C. , Ceylan Merve , Eriksson Jens , Bergholtz Alexandra , Ntokaki Angelika , Hammar Rebekkah , Sundbom Magnus , Skogar Martin , Graf Wilhelm , Webb Dominic-Luc , Hellström Per M. , Artursson Per , Pelaseyed Thaher , Di Martino Maria Letizia , Sellin Mikael E.
Published: July 2024

The active invasion of intestinal epithelial cells (IECs) represents a key event in the infection cycle of many gut pathogens. Studies of howSalmonella entericaTyphimurium (S.Tm) bacteria enter transformed cell lines have shaped the paradigm for swift type-three-secretion-system-1 (TTSS-1)-driven IEC invasion, fueled by expansive membrane ruffles. However, comparative studies suggest that non-transformed IECs in the intact gut context comprise a much more challenging target for the attack. The molecular and cellular features that explain these discrepancies remain undefined. By live-cell imaging in human enteroid-and colonoid-derived IEC layers, we demonstrate that the maturation state of gut epithelia dramatically impacts permissiveness toS.Tm invasion. IEC layers kept under immature progenitor-cell-promoting conditions are permissive to the bacterial invasion, whereas maturation towards an enterocyte/colonocyte fate reduces the frequency ofS.Tm-induced epithelial entry structures, and lowers the invasion efficiency by up to tenfold. This phenotypic shift during IEC maturation couples to an altered expression of actin regulatory proteins implicated in the invasion process, and an increased dependence on theS.Tm TTSS-1 effector SipA for successful entry. In addition, IEC maturation involves upregulation of cell surface mucins, e.g. MUC13, and shifts in glycocalyx composition, as revealed by multiple lectin stainings. Enzymatic treatment of the apical surface with the StcE mucinase converts maturing IEC layers back to theS.Tm-invasion-permissive state of their immature counterparts. Taken together, these results showcase how the maturation state of human IECs dictates the susceptibility to invasion by a prototype enterobacterium.
Long-term single-molecule tracking in living cells using weak-affinity protein labeling
Authors: Catapano Claudia , Dietz Marina S. , Kompa Julian , Jang Soohyen , Freund Petra , Johnsson Kai , Heilemann Mike
Published: July 2024

Single-particle tracking (SPT) has become a powerful tool to monitor the dynamics of membrane proteins in living cells. However, permanent labeling strategies for SPT suffer from photobleaching as a major limitation, restricting observation times, and obstructing the study of long-term cellular processes within single living cells. Here, we use exchangeable HaloTag Ligands (xHTLs) as an easy-to-apply labeling approach for live-cell SPT and demonstrate extended observation times of individual live cells of up to 30 minutes. Using the xHTL/HT7 labeling system, we measure the ligand-induced activation kinetics of the epidermal growth factor receptor (EGFR) in single living cells. Furthermore, we generate spatial maps of receptor diffusion in cells, report non-uniform distributions of receptor activation, and the formation of spatially confined ‘hot spots’ of EGFR activation. This approach represents a general strategy to monitor protein dynamics in a functional context and for extended observation times in single living cells.
Cryptic cycling by electroactive bacterioplankton in Trout Bog Lake
Authors: Olmsted Charles N. , Gahler Mark , Roden Eric , Peterson Ben , Lazarcik James , Tran Patricia Q. , Berg Maureen , Bryant Donald A. , Goudeau Danielle , Malmstrom Rex R. , Qin Mohan , McMahon Katherine D.
Published: July 2024

The genetic potential for extracellular electron transfer (EET)-based metabolism has been shown to be a prevailing feature of humic lakes where bacterioplankton may be able to use EET to cycle dissolved organic matter (DOM) extracellularly between oxidized and reduced states, but measurable abiotic features resulting from this phenomenon have yet to be demonstrated. We observed an anoxygenic photosyntheticChlorobiumsp. bloom each summer in Trout Bog Lake in northern WI, USA. Given this bloom’s characteristics, we hypothesized that EET-based metabolisms ofChlorobiumsp. and accompanying bacteria cycle DOM between oxidized and reduced states with seasonal or diel-timescale oscillations; therefore, we anticipated this could be measured by weekly and subdaily sampling. We collected vertical profiles on these timescales using a multiparameter sonde, including oxidation-reduction potential measurement, and we assayed for inorganic electron donors. We also developed and deployed a buoy to measure electric current flow between many pairs of electrodes simultaneously. Using metagenomics analyses, we examined the EET genes and other oxidoreductases of bacteria from water column samples at select depths and from biofilms that developed on electrodes at similar depths. Our results indicate the occurrence of diel electron cycling between phototrophic oxidation (electrotrophic metabolism) and anaerobic respiration (electrogenic metabolism), likely involving DOM. We also observed a gradual seasonal increase in hypolimnion oxidation-reduction potential. These diel and seasonal patterns have implications for carbon emissions and the ecology of electroactive bacteria in lakes.

IMPORTANCE

We investigated the physical, chemical, and redox characteristics of a bog lake and electrodes hung therein to test the hypothesis that dissolved organic matter is being cycled between oxidized and reduced states by electroactive bacterioplankton powered by phototrophy. To do so we performed field-based analyses on multiple timescales using both established and novel instrumentation. We paired these analyses with recently developed bioinformatics pipelines for metagenomics data to investigate genes that enable electroactive metabolism and accompanying metabolisms. Our results are consistent with our hypothesis and yet upend some of our other expectations. Our findings have implications for understanding greenhouse gas emissions from lakes, including electroactivity as an integral part of lake metabolism throughout more of the anoxic parts of lakes and for a longer portion of the summer than expected. Our results also give a sense of what electroactivity occurs at given depths and provide a strong basis for future studies.
Surviving the Storm: Exploring the Role of Natural Transformation in Nutrition and DNA Repair of StressedDeinococcus radiodurans
Authors: Sharma Dhirendra Kumar , Soni Ishu , Gupta Gagan D. , Rajpurohit Yogendra Singh
Published: July 2024

Deinococcus radiodurans, a natural transformation (NT) enabled bacterium renowned for its exceptional radiation resistance, employs unique DNA repair and oxidative stress mitigation mechanisms as a strategic response to DNA damage. This study excavate into the intricate roles of NT machinery in the stressedD. radiodurans, focusing on the genescomEA,comEC,endA,pilTanddprA, which are instrumental in the uptake and processing of extracellular DNA (eDNA). Our data reveals that NT not only supports the nutritional needs ofD. radioduransunder stress but also have roles in DNA repair. The study findings establish that NT-specific proteins (ComEA, ComEC, and EndA) might contribute to support the nutritional requirements in unstressed and heavily DNA-damaged cells while DprA contribute differently and in a context-dependent manner to navigating through the DNA damage storm. Thus, this dual functionality of NT-specific genes is proposed to be one of factor inD. radioduransremarkable ability to survive and thrive in environments characterized by high levels of DNA-damaging agents.

Author Summary:

Deinococcus radiodurans, a bacterium known for its extraordinary radiation resistance. This study explores the roles of natural transformation (NT) machinery in the radiation-resistant bacteriumDeinococcus radiodurans, focusing on the genescomEA,comEC,endA,pilT, anddprA. These genes are crucial for the uptake and processing of extracellular DNA (eDNA) and contribute to the bacterium nutritional needs and DNA repair under stress. The findings suggest that the NT-specific proteins ComEA, ComEC, and EndA may help meet the nutritional needs of unstressed and heavily DNA-damaged cells, whereas DprA plays a distinct role that varies depending on the context in aiding cells to cope with DNA damage. The functionality of NT genes is proposed to enhanceD. radioduranssurvival in environments with high levels of DNA-damaging agents.
A conserved code for anatomy: Neurons throughout the brain embed robust signatures of their anatomical location into spike trains
Authors: Tolossa Gemechu B. , Schneider Aidan M. , Dyer Eva L. , Hengen Keith B.
Published: July 2024

Neurons in the brain are known to encode diverse information through their spiking activity, primarily reflecting external stimuli and internal states. However, whether individual neurons also embed information about their own anatomical location within their spike patterns remains largely unexplored. Here, we show that machine learning models can predict a neuron’s anatomical location across multiple brain regions and structures based solely on its spiking activity. Analyzing high-density recordings from thousands of neurons in awake, behaving mice, we demonstrate that anatomical location can be reliably decoded from neuronal activity across various stimulus conditions, including drifting gratings, naturalistic movies, and spontaneous activity. Crucially, anatomical signatures generalize across animals and even across different research laboratories, suggesting a fundamental principle of neural organization. Examination of trained classifiers reveals that anatomical information is enriched in specific interspike intervals as well as responses to stimuli. Within the visual isocortex, anatomical embedding is robust at the level of layers and primary versus secondary but does not robustly separate individual secondary structures. In contrast, structures within the hippocampus and thalamus are robustly separable based on their spike patterns. Our findings reveal a generalizable dimension of the neural code, where anatomical information is multiplexed with the encoding of external stimuli and internal states. This discovery provides new insights into the relationship between brain structure and function, with broad implications for neurodevelopment, multimodal integration, and the interpretation of large-scale neuronal recordings. Computational approximations of anatomy have potential to support in-vivo electrode localization.
Resolving a neonatal intensive care unit outbreak of methicillin-resistantStaphylococcus aureusto the SNV level using Oxford Nanopore simplex reads and HERRO error correction
Authors: Bloomfield Max , Bakker Sarah , Burton Megan , Castro M. Leticia , Dyet Kristin , Eustace Alexandra , Hutton Samantha , Macartney-Coxson Donia , Taylor William , White Rhys T.
Published: July 2024

Objectives

Our laboratory began prospective genomic surveillance for healthcare-associated organisms in 2022 using Oxford Nanopore Technologies (ONT) sequencing as a standalone platform. This has permitted the early detection of outbreaks but has been insufficient for single-nucleotide variant (SNV)-level analysis due to lower read accuracy than Illumina sequencing. This study aimed to determine whether Haplotype-aware ERRor cOrrection (HERRO) of ONT data could permit high-resolution comparison of outbreak isolates.

Methods

We used ONT simplex reads from isolates involved in a recent outbreak of methicillin-resistantStaphylococcus aureus(MRSA) in our neonatal unit. The raw sequence data were re-basecalled and adapter-trimmed using Dorado v0.7.0. The simplex reads then underwent HERRO correction. The resulting genome assemblies and phylogenies were compared with previous analyses (using Dorado v0.3.4, no HERRO correction and data generated by Illumina sequencing).

Results

Five of nine outbreak isolates were included in the analysis. The remaining four isolates had insufficient read lengths (N50 values <10,000 bp) and did not provide complete chromosome coverage after HERRO correction. The average chromosome sequencing depth for nanopore data was 147× (range: 44–220×) with an average read N50 of 12,215 bp (interquartile range (IQR): 11,439–12,711 bp). The median pairwise SNV distance between outbreak isolates from the original investigation was 51 SNVs (range: 40–68), which decreased to 3 SNVs (range: 1–15) with HERRO correction. Illumina sequencing generated a median SNV distance of 2 (range: 0–13). The resulting standalone ONT HERRO-corrected phylogeny was almost indistinguishable from the standalone Illumina-generated phylogeny.

Conclusions

The addition of HERRO correction meant isolates from this MRSA outbreak could be resolved to a level on par with Illumina sequencing. ONT data following HERRO correction represents a viable standalone option for high-resolution genomic analysis of hospital outbreaks, provided sufficient read lengths can be generated.
HIV broadly neutralizing antibody escapability drives the therapeutic efficacy of vectored immunotherapy
Authors: Galvez Nicolas M.S. , Cao Yi , Nitido Adam D. , Deal Cailin E. , Boutros Christine L. , MacDonald Scott W. , Soto Albrecht Yentli E. , Lam Evan C. , Sheehan Maegan L. , Parsons Dylan , Lin Allen Z. , Deymier Martin J. , Brady Jacqueline M. , Moon Benjamin , Bullock Christopher B. , Tanno Serah , Pegu Amarendra , Chen Xuejun , Liu Cuiping , Koup Richard A. , Mascola John R. , Vrbanac Vladimir D. , Lingwood Daniel , Balazs Alejandro B.
Published: July 2024

Broadly neutralizing antibodies (bNAbs) have shown great promise for prevention and treatment of HIV infection. Breadth of bNAb neutralization, measuredin vitroacross panels of diverse viral isolates, is often used as a predictor of clinical potential. However, recent prevention studies demonstrate that the clinical efficacy of a broad and potent bNAb (VRC01) is undermined by neutralization resistance of circulating strains. Using HIV-infected humanized mice, we find that therapeutic efficacy of bNAbs delivered as Vectored ImmunoTherapy (VIT) is a function of both the fitness cost and resistance benefit of mutations that emerge during viral escape, which we term ‘escapability’. Applying this mechanistic framework, we find that the sequence of the envelope V5-loop alters the resistance benefits of mutants that arise during escape, thereby impacting the therapeutic efficacy of VIT-mediated viral suppression. We also find that an emtricitabine-based antiretroviral drug regimen dramatically enhances the efficacy of VIT, by reducing the fitness of mutants along the escape path. Our findings demonstrate that bNAb escapability is a key determinant to consider in the rational design of antibody regimens with maximal efficacy and illustrates a tractable means of minimizing viral escape from existing bNAbs.