bluuebunny/crossref_metadata_2025 huggingface dataset:
Interpretable and predictive models based on high-dimensional data in ecology and evolution
Authors: Jahner Joshua P. , Buerkle C. Alex , Gannon Dustin G. , Grames Eliza M. , McFarlane S. Eryn , Siefert Andrew , Bell Katherine L. , DeLeo Victoria L. , Forister Matthew L. , Harrison Joshua G. , Laughlin Daniel C. , Patterson Amy C. , Powers Breanna F. , Werner Chhaya M. , Oleksy Isabella A.
Published: March 2024

The proliferation of high-dimensional data in ecology and evolutionary biology raise the promise of statistical and machine learning models that are highly predictive and interpretable. However, high-dimensional data are commonly burdened with an inherent trade-off: in-sample prediction of outcomes will improve as additional predictors are included in the model, but this may come at the cost of poor predictive accuracy and limited generalizability for future or unsampled observations (out-of-sample prediction). To confront this problem of overfitting, sparse models can focus on key predictors by correctly placing low weight on unimportant variables. We competed nine methods to quantify their performance in variable selection and prediction using simulated data with different sample sizes, numbers of predictors, and strengths of effects. Overfitting was typical for many methods and simulation scenarios. Despite this, in-sample and out-of-sample prediction converged on the true predictive target for simulations with more observations, larger causal effects, and fewer predictors. Accurate variable selection to support process-based understanding will be unattainable for many realistic sampling schemes in ecology and evolution. We use our analyses to characterize data attributes for which statistical learning is possible, and illustrate how some sparse methods can achieve predictive accuracy while mitigating and learning the extent of overfitting.
Single-animal, single-tube RNA extraction for comparison of relative transcript levels via qRT-PCR in the tardigradeHypsibius exemplaris
Authors: Kirk Molly J. , Xu Chaoming , Paules Jonathan , Rothman Joel H.
Published: March 2024

The tardigradeHypsibius exemplarisis an emerging model organism renowned for its ability to survive environmental extremes. To explore the molecular mechanisms and genetic basis of such extremotolerance, many studies rely on RNA-sequencing (RNA-seq), which can be performed on populations ranging from large cohorts to individual animals. Reverse Transcription Polymerase Chain Reaction (RT-PCR) and RNA interference (RNAi) are subsequently used to confirm RNA-seq findings and assess the genetic requirements for candidate genes, respectively. Such studies require an efficient, accurate, and affordable method for RNA extraction and measurement of relative transcript levels by quantitative RT-PCR (qRT-PCR). This work presents an efficient single-tardigrade, single-tube RNA extraction method (STST) that not only reliably isolates RNA from individual tardigrades but also reduces the required time and cost for each extraction. This RNA extraction method yields quantities of cDNA that can be used to amplify and detect multiple transcripts by quantitative PCR (qRT-PCR). The method is validated by analyzing dynamic changes in the expression of genes encoding two heat-shock-regulated proteins, Heat-Shock Protein 70 β2 (HSP70 β2) and Heat-Shock Protein 90α (HSP90α), making it possible to assess their relative expression levels in heat-exposed individuals using qRT-PCR. STST effectively complements existing bulk and single tardigrade RNA extraction methods, permitting rapid and affordable examination of individual tardigrade transcriptional levels by qRT-PCR.

This work presents a rapid RNA extraction and transcript level comparison method for analyzing gene expression in the tardigradeHypsibius exemplaris.Using physical lysis, this high-throughput method requires a single tardigrade as the starting material and results in robust production of cDNA for quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR).
A transcription factor (TF) inference method that broadly measures TF activity and identifies mechanistically distinct TF networks
Authors: Jones Taylor , Sigauke Rutendo F. , Sanford Lynn , Taatjes Dylan J. , Allen Mary A. , Dowell Robin D.
Published: March 2024

TF profiler is a method of inferring transcription factor regulatory activity, i.e. when a TF is present and actively regulating transcription, directly directly from nascent sequencing assays such as PRO-seq and GRO-seq. Transcription factors orchestrate transcription and play a critical role in cellular maintenance, identity and response to external stimuli. While ChIP assays have measured DNA localization, they fall short of identifying when and where transcription factors are actively regulating transcription. Our method, on the other hand, uses RNA polymerase activity to infer TF activity across hundreds of data sets and transcription factors. Based on these classifications we identify three distinct classes of transcription factors: ubiquitous factors that play roles in cellular homeostasis, driving basal gene programs across tissues and cell types, tissue specific factors that act almost exclusively at enhancers and are themselves regulated at transcription, and stimulus responsive TFs which are regulated post-transcriptionally but act predominantly at enhancers. TF profiler is broadly applicable, providing regulatory insights on any PRO-seq sample for any transcription factor with a known binding motif.
Transcriptomic analyses in the gametophyte of Dryopteris affinis: apomixis and more
Authors: Ojosnegros Sara , Alvarez Jose Manuel , Gagliardini Valeria , Quintanilla Luis G. , Grossniklaus Ueli , Fernandez Helena
Published: March 2024

The gametophyte of the fern Dryopteris affinis ssp. affinis represents a good model to explore the molecular basis of vegetative and reproductive development, as well as stress responses. Specifically, this fern reproduces asexually by apogamy, a peculiar case of apomixis whereby a sporophyte forms directly from a gametophytic cell without fertilization. Using an RNA-sequencing approach, we have previously annotated more than six thousand transcripts. Here, we selected one hundred of the inferred proteins that seemed particularly interesting for a detailed study of their potential functions, protein-protein interactions, and molecular phylogenies. As expected, a plethora of proteins associated with gametogenesis and embryogenesis in angiosperms, such as FERONIA (FER) and CHROMATING REMODELING 11 (CHR11) were identified, and more than a dozen candidates potentially involved in apomixis, such as ARGONAUTE4 (AGO4), AGO9, and AGO10, BABY BOOM (BBM), FASCIATED STEM4 (FAS4), FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MATERNAL EFFECT EMBRYO ARREST29 (MEE29). In addition, proteins involved in the response to biotic and abiotic stresses were widely represented, as shown by the enrichment of heat-shock proteins. Using the String platform, studying interactomes revealed that most of the protein-protein interactions were predicted based on experimental, database, and text mining datasets, with MULTICOPY SUPPRESSOR OF IRA4 (MSI4) showing the highest number of 16 interactions. Lastly, some proteins were studied from a phylogenetic point of view, comparing the alignments with respect to more distantly or closely related plant groups, identifying AGO1 as the evolutionarily most similar to that other ferns and the most distant to the predicted common ancestor. This work sets the stage for future functional characterizations in relation to gametophyte development including apomictic reproduction.
Identification of novel genes in cattle (Bos taurus) and biological insights into their function in embryo development
Authors: Schettini Gustavo P , Morozyuk Michael , Biase Fernando H
Published: March 2024

Appropriate regulation of genes expressed in oocytes and embryos is essential for acquisition of developmental competence in mammals. Here, we hypothesized that several genes expressed in oocytes and pre-implantation embryos remain unknown. Our goal was to reconstruct the transcriptome of oocytes (germinal vesicle and metaphase II) and pre-implantation cattle embryos (blastocysts) using short-read and long-read sequences to identify putative new genes. We identified 274,342 transcript sequences, and 3,033 of those transcripts do not match a gene present in an annotation, thus are potential new genes. Notably, 63.67% (1,931/3,033) of potential novel genes exhibited coding potential. Also noteworthy, 97.92% of the putative novel genes overlapped annotation with transposable elements. Comparative analysis of transcript abundance identified that 1,840 novel genes (recently added to the annotation) or potential new genes were differentially expressed between developmental stages (FDR<0.01). We also determined that 522 novel or potential new genes (448 and 34 respectively) were upregulated at eight-cell embryos compared to oocytes (FDR<0.01). In eight-cell embryos, 102 novel or putative new genes were co-expressed (|r|>0.85, P<1x10-8) with several genes annotated with gene ontology processes related to pluripotency maintenance and embryo development. CRISPR-Cas9 genome editing confirmed that the disruption of one of the novel genes highly expressed in eight-cell embryos reduced blastocyst development (ENSBTAG00000068261, P=1.55x10-7). In conclusion, our results revealed several putative new genes that need careful annotation. Many of the putative new genes have dynamic regulation during pre-implantation development and are important components of gene regulatory networks involved in pluripotency and blastocyst formation.
Benchmarking reveals superiority of deep learning variant callers on bacterial nanopore sequence data
Authors: Hall Michael B. , Wick Ryan R. , Judd Louise M. , Nguyen An N. T. , Steinig Eike J. , Xie Ouli , Davies Mark R. , Seemann Torsten , Stinear Timothy P. , Coin Lachlan J. M.
Published: March 2024

Variant calling is fundamental in bacterial genomics, underpinning the identification of disease transmission clusters, the construction of phylogenetic trees, and antimicrobial resistance prediction. This study presents a comprehensive benchmarking of SNP and indel variant calling accuracy across 14 diverse bacterial species using Oxford Nanopore Technologies (ONT) and Illumina sequencing. We generate gold standard reference genomes and project variations from closely-related strains onto them, creating biologically realistic distributions of SNPs and indels.

Our results demonstrate that ONT variant calls from deep learning-based tools delivered higher SNP and indel accuracy than traditional methods and Illumina, with Clair3 providing the most accurate results overall. We investigate the causes of missed and false calls, highlighting the limitations inherent in short reads and discover that ONT’s traditional limitations with homopolymer-induced indel errors are absent with high-accuracy basecalling models and deep learning-based variant calls. Furthermore, our findings on the impact of read depth on variant calling offer valuable insights for sequencing projects with limited resources, showing that 10x depth is sufficient to achieve variant calls that match or exceed Illumina.

In conclusion, our research highlights the superior accuracy of deep learning tools in SNP and indel detection with ONT sequencing, challenging the primacy of short-read sequencing. The reduction of systematic errors and the ability to attain high accuracy at lower read depths enhance the viability of ONT for widespread use in clinical and public health bacterial genomics.
Eicosapentaenoic acid induces an anti-inflammatory transcriptomic landscape in T cells implicating a pathway independent of triglyceride lowering in cardiovascular risk reduction
Authors: Reilly Nathalie A. , Dekkers Koen F. , Molenaar Jeroen , Arumugam Sinthuja , Kuipers Thomas B. , Ariyurek Yavuz , Hoeksema Marten A. , Jukema J. Wouter , Heijmans Bastiaan T.
Published: March 2024

A twice-daily dose of highly purified eicosapentaenoic acid (EPA) reduces the risk of atherosclerotic cardiovascular disease among patients with high triglycerides and either known cardiovascular disease or those at high risk for developing it. However, the process by which EPA exerts its beneficial effects remains poorly understood. Here, we show that EPA can induce an anti-inflammatory transcriptional profile in non-activated CD4+T cells. We find that EPA-exposed CD4+T cells downregulate immune response related genes, such asHLA-DRA, CD69, andIL2RA, while upregulating genes involved in oxidative stress prevention, such asNQO1. Furthermore, transcription footprint analysis based on ATAC-sequencing reveals downregulation of GATA3 and PU.1, key transcription factors in TH2 and TH9 differentiation, and upregulation of REV-ERB, an antagonist of TH17 differentiation. By in parallel examining T cell responses to oleic acid, a monounsaturated fatty acid, and palmitic acid, a saturated fatty acid, we find that both the intensity of the transcriptomic response and the involvement of anti-inflammatory pathways is highly specific for EPA. Thus, EPA can induce an anti-inflammatory transcriptomic landscape in CD4+T cells, a process that may contribute to the unexpectedly strong beneficial effects of EPA on the risk of atherosclerotic cardiovascular disease in clinical trials.
Variability and correlations among vital rates and their influence on population growth in mule and black-tailed deer
Authors: Ruprecht Joel , Forrester Tavis D. , Clark Darren A. , Wisdom Michael J. , Smith Joshua B. , Levi Taal
Published: March 2024

To reverse observed range-wide population declines, managers of mule and black- tailed deer (Odocoileus hemionus) require information on the vital rates and life stages that are most influential to population growth for which to target management actions. We conducted a range-wide literature review and used hierarchical models to provide biological descriptions of mule and black-tailed deer vital rates, their variability, and how they correlate with one another. We then used matrix models and life-stage simulation analysis to determine the individual vital rates that contributed most to lambda, i.e., annual population growth rate. Adult female survival was the vital rate with the greatest ability to predict lambda and explained 62% of the variation in population growth. While annual juvenile survival explained 44% of the variation in lambda, summer and winter juvenile survival by themselves were far less explanatory than adult female survival. Winter fawn:doe ratios, a metric often collected by management agencies, only explained 10% of the variation in lambda. When adult female survival was 0.84, our simulations estimated an equal probability that a population would increase versus decrease, and correspondingly, the estimate of lambda was 1.0 with a 95% credible interval of 0.88–1.14. Simulations suggested that populations with adult survival rates less than 70% would always decline, but as survival increased beyond this value lambda increased linearly and never plateaued. In contrast, the probability of observing a stable lambda plateaued when annual juvenile survival reached approximately 0.5. The mean lambda calculated from all simulated values within the observed range of vital rate values across the species’ geographical distribution was 0.975, and in 61% of the simulations lambda was < 1. After 20 years, we estimated that this distribution of lambda values would cause populations to decrease in 92% of instances with a mean decrease of 44%. Our results align with the observed declines in mule deer populations throughout their range over recent decades and suggest that these trends will continue until management can improve survival of adult females.
Impact of Chemical Dynamics of Commercial PURE Systems on Malachite Green Aptamer Fluorescence
Authors: Jurado Zoila , Murray Richard M.
Published: March 2024

The malachite green aptamer (MGapt) is known for its utility in RNA measurementin vivoand lysate-based cell-free protein systems. However, MGapt fluorescence dynamics do not accurately reflect mRNA concentration. Our study finds that MGapt fluorescence is unstable in commercial PURE systems. We discovered that the chemical composition of the cell-free reaction strongly influences MGapt fluorescence, which leads to inaccurate RNA calculations. Specific to the commercial system, we posit that MGapt fluorescence is significantly affected by the system’s chemical properties, governed notably by the presence of dithiothreitol (DTT). We propose a model that, on average, accurately predicts MGapt measurement within a 10% margin, leveraging DTT concentration as a critical factor. This model sheds light on the complex dynamics of MGapt in cell-free systems and underscores the importance of considering environmental factors in RNA measurements using aptamers.
N-acetyltransferase 10-mediated mRNA N4-acetylation is Essential for the Translational Regulation During Oocyte Meiotic Maturation in Mice
Authors: Chen Lu , Liu Shao-Yuan , Su Rui-Bao , Wu Yu-Ke , Wang Wen-Jing , Wu Xuan , Zhang Song-Ying , Qiao Jie , Sha Qian-Qian , Fan Heng-Yu
Published: March 2024

Mammalian oocyte maturation is driven by the strict translational regulation of maternal mRNAs stored in the cytoplasm. However, the function and mechanism of post-transcriptional chemical modifications, especially the newly identified N4-acetylcytidine (ac4C) modification catalyzed by N-acetyltransferase 10 (NAT10), are unknown. In this study, we developed a low-input ac4C sequencing technology, ac4C LACE-seq, and mapped 8241 ac4C peaks at the whole-transcriptome level using 50 mouse oocytes at the germinal vesicle stage. We profiled the mRNA landscapes of NAT10-interactions and ac4C modifications. The NAT10-interacted and ac4C-modified transcripts are associated with high translation efficiency in oocytes. Oocyte-specificNat10knockout wiped out ac4C signals in oocytes and caused severe defects in meiotic maturation and female infertility. ac4C LACE-seq results indicated thatNat10deletion led to a failure of ac4C deposition on mRNAs encoding key maternal factors, such as MSY2, ZAR1, BTG4, and cyclin B1, which regulate transcriptome stability and maternal-to-zygotic transition.Nat10-deleted oocytes showed decreased mRNA translation efficiency during meiotic maturation, partially due to the direct inhibition of ac4C sites on specific transcripts. In summary, we developed a low-input, high-sensitivity mRNA ac4C profiling approach and highlighted the important physiological function of ac4C in the precise regulation of oocyte meiotic maturation by enhancing translation efficiency.
Genome-wide CRISPRa screens nominate modulators of CAR T cell survival within distinct tumor cytokine milieus
Authors: Curtis Benjamin C , Spurrell Cailyn H , Flint Lindsay , Lande Aalton M , Leonardi Marissa , Rosser James M , Valdez Ardizon , Murren Nat , Chai Tiffanie , Fitzgerald Michael , Martinez-Reyes Jasmin , Saxby Christopher P , Oda Shannon K , Jensen Michael CV
Published: March 2024

Chimeric Antigen Receptor (CAR) T cell therapy has revolutionized the treatment of B cell malignancies and translating this success to other cancers remains an ongoing clinical objective. Next-generation T cell products in development aim to genetically modulate many facets of cell behavior, for which gene-nominating platforms provide a useful framework for prioritization. Among competing screening approaches, CRISPR activation (CRISPRa) technology permits gain-of-function (GoF) gene surveys at genome-wide scale, but routine implementation in primary T cells has been stymied by high cell requirements (∼107- 108) and abbreviated activity. Here, we describe a novel cell manufacturing schema using an all-in-one transposon-based gene delivery system coupled with CAR-restricted cell expansion to generate yields (109) of primary T cells bearing CAR and CRISPRa transgenes that are well above the threshold needed for genome-scale screening. CRISPRa activity is sustained via the inclusion of divergent, duplicate Elongation Factor 1α core/human T-cell leukemia virus (EF1α-HTLV) hybrid promoters; while guide RNA representation is preserved through late lentiviral transduction, thus preventing bottlenecking and premature candidate pruning. CRISPRa-CAR T cells manufactured via this pipeline retain potent on-target gene-overexpression (>85% target+) across varied cell subsets (e.g. Tim-3+Lag3+or serial-challenge) and timescales (>14 days). When deployed to survival-based genome-wide selection landscapes, CRISPRa-CAR pools nominate known and novel endogenous genes capable of enhancing CD8+CAR T survival in cytokine-rich (e.g.MYC, FUT6, IRF4, GSE1) and cytokine-depleted (e.g.CSF2RB,STAT6,IRF4,GSE1) settings of tumor challenge. This system will have broad utility for therapy-enhancing gene discovery.
GNN codon adjacency regulates protein translation
Authors: Sun Joyce , Hwang Pete , Sakkas Eric D. , Zhou Yancheng , Perez Luis , Dave Ishani , Kwon Jack B. , McMahon Audrey E. , Wichman Mia , Raval Mitsu , Scopino Kristen , Krizanc Daniel , Thayer Kelly M. , Weir Michael P.
Published: March 2024

The central dogma treats the ribosome as a molecular machine that reads one mRNA codon at a time as it adds each amino acid to its growing peptide chain. However, this and previous studies suggest that ribosomes actually perceive pairs of adjacent codons as they take three-nucleotide steps along the mRNA. We examined GNN codons which we find are surprisingly overrepresented in eukaryote protein-coding open reading frames (ORFs), especially immediately after NNU codons. Ribosome profiling experiments in yeast revealed that ribosomes with NNU at their aminoacyl (A) site have particularly elevated densities when NNU is immediately followed (3') by a GNN codon, indicating slower mRNA threading of the NNU codon from the ribosome's A to peptidyl (P) sites. Moreover, if the assessment was limited to ribosomes that have only recently arrived at the next codon, by examining 21-nucleotide ribosome footprints (21-nt RFPs), elevated densities were observed for multiple codon classes when followed by GNN. This striking translation slowdown at adjacent 5'-NNN GNN codon pairs is likely mediated in part by the ribosome's CAR surface which acts as an extension of the A-site tRNA anticodon during ribosome translocation and interacts through hydrogen bonding and pi stacking with the GNN codon. The functional consequences of 5'-NNN GNN codon adjacency are expected to influence the evolution of protein coding sequences.
Cell culture differentiation and proliferation conditions influence thein vitroregeneration of the human airway epithelium
Authors: Redman Elisa , Fierville Morgane , Cavard Amélie , Plaisant Magali , Arguel Marie-Jeanne , Ruiz Garcia Sandra , McAndrew Eamon M , Girard-Riboulleau Cédric , Lebrigand Kevin , Magnone Virginie , Ponzio Gilles , Gras Delphine , Chanez Pascal , Abelanet Sophie , Barbry Pascal , Marcet Brice , Zaragosi Laure-Emmanuelle
Published: March 2024

The human airway mucociliary epithelium can be recapitulatedin vitrousing primary cells cultured in an Air-Liquid Interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist between media used for ALI-cultured human airway epithelial cells, our study aimed to evaluate the impact of several media (BEGMTM, PneumaCultTM, “Half&Half” and “Clancy”) on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCultTM-ALI and Half&Half, stronger EGF signaling from basal cells in BEGMTM-ALI, differential expression of the SARS-CoV-2 entry factorACE2, and distinct secretome transcripts depending on media used. We also established that proliferation in PneumaCultTM-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will help to choose the most relevant medium according to the processes to be investigated such as cilia, mucus biology or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.
Selectivity and evolution of Aqp10 in solute permeability influenced by pore molecular weight
Authors: Nagashima Ayumi , Ushio Kazutaka , Nishihara Hidenori , Akimoto Jin , Kato Akira , Furuta Tadaomi
Published: March 2024

Aqp10 is an aquaglyceroporin that transports not only water but also uncharged low-molecular-weight compounds. We previously demonstrated the evolution of solute permeability in Aqp10 paralogs and showed that the urea and boric acid permeabilities of Aqp10.2 were much weaker than those of Aqp10.1 and plesiomorphic Aqp10s. However, the molecular mechanism responsible for the weak permeability of Aqp10.2 to urea and boric acid remains unclear. Herein, we present a novel hypothesis that explains the solute selectivity of Aqp10. We deduced the ancestral sequences of Aqp10.1 and Aqp10.2 paralogs via molecular phylogenetic analysis. Constructed structural models of these sequences revealed that both the well known amino acid site at position 3 and the sum of molecular weights of the four amino acid sites in the ar/R region were important for the formation of the Aqp10 selectivity filter. Site-directed mutagenesis revealed that a decrease in the sum of the molecular weights of the four amino acid sites enhanced the Aqp10 permeability to urea and boric acid. Based on this, we proposed a model in which the presence of two or more bulky amino acids in the ar/R region, which increases the sum of the molecular weights of amino acids in the ar/R region, was essential for the formation of a filter that limited urea and boric acid transport. Our results outline the molecular mechanism by which Aqp10.2 acquired a selectivity filter during evolution and provide structural insights into the narrowly tuned filter responsible for the solute selectivity of aquaglyceroporins.

Significance Statement

In aquaglyceroporins, particularly Aqp10s, the urea and boric acid permeabilities of Aqp10.2 paralogs are much weaker than those of plesiomorphic Aqp10s. Here, we deduced the ancestral sequences of Aqp10.1 and Aqp10.2 via molecular phylogenetic analysis. Structural models of these sequences revealed that the sum of the four amino acid site molecular weights in the ar/R region, if more than one is bulky, contributed to the selectivity filter formation in the pore region. Using site-directed mutagenesis, reduction in the molecular weight of one bulky amino acid residue in the ar/R region restricted urea and boric acid permeability. Therefore, Aqp10.2 acquired a selectivity filter during evolution, and structural differences in this selectivity filter are responsible for the variable solute permeability of aquaglyceroporins.
Growth parameters of the clupeidsLimnothrissa miodonandStolothrissa tanganicaein northern Lake Tanganyika (Bujumbura sub-basin)
Authors: N’sibula Mulimbwa T. , Gordó-Vilaseca Cesc , Milec Leona J. M. , Patel Deepti M. , Alidor Kankonda Busanga , Micha Jean-Claude , Sarvala Jouko , Raeymaekers Joost A. M.
Published: March 2024

The analysis of growth parameters in fish stocks, such as the asymptotic length (L∞) and the curvature parameter (K), is crucial for the estimation of production rates and total mortality rates in fisheries management. Here, we estimated the growth parameters of the clupeidsLimnothrissa miodonandStolothrissa tanganicaein the northern part of Lake Tanganyika (Bujumbura sub-basin). Both species are important targets of pelagic fisheries, but the available estimates are based on scarce size frequency data, and rarely on size as a function of age data. We provide new estimates of L∞and K based on size as a function of age data, with age being inferred from otolith weights. Furthermore, we reanalyze several existing length frequency datasets using advanced statistical procedures to test if growth parameters estimated by length frequency analysis are consistent with those obtained from size as a function of age. We found that length frequency analysis consistently underestimates L∞and overestimates K as compared to length-at-age data. We recommend that length-at-age data are collected at a much broader spatial and temporal scale than currently available to advance our understanding of the growth dynamics of these two sardine species. This will improve the assessment of stock productivity of Lake Tanganyika’s pelagic fisheries and the conservation of these economically important fish species.
Biomimetic fractal topography enhances podocyte maturation in vitro
Authors: Liu Chuan , Aggarwal Praful , Wagner Karl T. , Landau Shira S. , Cui Teng , Song Xin , Shamaei Laleh , Rafatian Naimeh , Zhao Yimu , Rodriguez-Ramirez Sonia , Morton Keith , Virlee Elizabeth , Li Chen Yu , Bannerman Dawn , Pascual-Gil Simon , Okhovatian Sargol , Radisic Anastasia , Clotet-Freixas Sergi , Veres Teodor , Sadrzadeh Mohtada , Filleter Tobin , Broeckel Ulrich , Konvalinka Ana , Radisic Milica
Published: March 2024

Cells and tissues in their native environment are organized into intricate fractal structures, which are rarely recapitulated in their culturein vitro. The extent to which fractal patterns that resemble complex topography in vivo influence cell maturation, and the cellular responses to such shape stimulation remain inadequately elucidated. Yet, the application of fractal cues (topographical stimulation via self-similar patterns) as an external input may offer a much-needed solution to the challenge of improving the differentiated cell phenotypein vitro. Here, we established fractality in podocytes, branching highly differentiated kidney cells, and glomerulus structure. Biomimetic fractal patterns derived from glomerular histology were used to generate topographical (2.5-D) substrates for cell culture. Podocytes grown on fractal topography were found to express higher levels of functional markers and exhibit enhanced cell polarity. To track morphological complexities of differentiated podocytes, we employed a fluorescent labelling assay where labelled individual cells are tracked within otherwise optically silent confluent cell monolayer to reveal cell-cell interdigitation. RNAseq analysis suggests enhanced ECM deposition and remodeling in podocytes grown on fractal topography compared to flat surface or non-fractal microcurvature, mediated by YAP signaling. The incorporation of fractal topography into standard tissue culture well plates as demonstrated here may serve as a user-friendly bioengineered platform for high-fidelity cell culture.
High-frequency extracellular spiking in electrically-active cancer cells
Authors: Melikov Rustamzhon , De Angelis Francesco , Moreddu Rosalia
Published: March 2024

Microelectrode matrices have been extensively used in the past 30 years as a reliable platform to record the extracellular activity of cells traditionally regarded to as excitable, i.e. brain cells and muscle cells. Meanwhile, fundamental biology studies on cancer cells since the 1970s reveal that they exhibit altered functionalities of ion channels, membrane potentials, metabolism, and communication mechanisms when compared to their healthy counterparts. In this work, we present for the first time the presence of extracellular voltage spikes occurring at high frequencies (0.1-3.5 kHz) in breast cancer cells, resembling the ones observed in excitable cells, and the possibility to record them with 30 µm TiN microelectrode matrices. These preliminary findings may open a new path for exploration in a variety of research fields, enabling the access to bioelectrical dynamics in cancer cells and cell networks, targeting bioelectricity as a tool in anticancer drug development and cancer diagnostics, as well as provide a market expansion for companies commercializing microelectrodes and microelectrode recording systems.
Neuronal activity promotes axonal node-like clustering prior to myelination and remyelination in the central nervous system
Authors: Ronzano Rémi , Perrot Clément , Mazuir Elisa , Thetiot Melina , Aigrot Marie-Stéphane , Stheneur Paul , Lejeune François-Xavier , Stankoff Bruno , Lubetzki Catherine , Sol-Foulon Nathalie , Desmazières Anne
Published: March 2024

Nodes of Ranvier ensure the fast saltatory conduction along myelinated axons, through their enrichment in voltage-gated sodium and potassium channels. We and others have shown that node-like cluster assembly can occur before myelination. In multiple sclerosis, demyelination is associated with node of Ranvier disassembly, but node-like reassembly can occur prior to remyelination. Given the crucial role of neuronal activity in inducing (re)myelination, we asked whether neuronal activity could regulate node-like clustering.

We show that node-like clustering is promoted by neuronal activity and decreased when excitatory glutamatergic receptors are inhibited. Altering glutamatergic neurotransmission leads to the downregulation of Nav1.1 expression, which we show to be critical for node-like clustering. Neuronal activity also promotes node-like clustering in remyelination. As node-like clusters modulate conduction velocity and myelination initiation along axons, we propose that activity-dependent node-like clustering could modulate neuronal network establishment, as well as myelination regulation and patterning during development, plasticity and repair.
Promoter and Gene-Body RNA-Polymerase II co-exist in partial demixed condensates
Authors: Changiarath Arya , Flores-Solis David , Michels Jasper J. , Rodriguez Rosa Herrera , Hanson Sonya M. , Schmid Friederike , Zweckstetter Markus , Padeken Jan , Stelzl Lukas S.
Published: March 2024

In cells, transcription is tightly regulated on multiple layers. The condensation of the transcription machinery into distinct phases is hypothesised to spatio-temporally fine tune RNA polymerase II behaviour during two key stages, transcription initiation and the elongation of the nascent RNA transcripts. However, it has remained unclear whether these phases would mix when present at the same time or remain distinct chemical environments; either as multi-phase condensates or by forming entirely separate condensates. Here we combine particle-based multi-scale simulations and experiments in the model organismC. elegansto characterise the biophysical properties of RNA polymerase II condensates. Both simulations and the in vivo work describe a lower critical solution temperature (LCST) behaviour of RNA Polymerase II, with condensates dissolving at lower temperatures whereas higher temperatures promote condensate stability. Importantly this gradual change in temperature correlates with an incremental transcriptional response to temperature, but is largely uncoupled from the classical stress response. The LCST behaviour of CTD also highlights that these condensates are physio-chemically distinct from heterochromatin condensates. Expanding the simulations we model how the degree of phosphorylation of the disordered C-terminal domain of RNA polymerase II (CTD), which is characteristic for each step of transcription, controls demixing of CTD and pCTD in line with phase separation experiments. We show that the two phases putatively underpinning the initiation of transcription and transcription elongation constitute distinct chemical environments and are in agreement with RNA polymerase II condensates observed inC. elegansembryos by super resolution microscopy. Our analysis reveals how depending on its post-translational modifications and its interaction partners a single protein can adopt multiple morphologies and how partially engulfed condensates promote the selective recruitment of additional factors to the different phases.
Spectral waveform analysis dissociates human cortical alpha rhythms
Authors: Giehl Janet , Siegel Markus
Published: March 2024

The non-sinusoidal waveform of neuronal oscillations reflects the physiological properties of underlying circuit interactions and may serve as an informative biomarker of healthy and diseased human brain function. However, little is known about which brain rhythms can be dissociated based on their waveform and methods to comprehensively characterize waveforms are missing. Here, we introduce a novel spectral waveform analysis (SWA) that provides a complete waveform description, is noise-resistant, and allows to reconstruct time-domain waveforms. We applied this framework to human magnetoencephalography (MEG) recordings during rest and identified several distinct and previously unknown cortical alpha waveforms that were temporally stable and specific for individual subjects. Our findings suggest at least four distinct alpha rhythms in human sensorimotor, occipital, temporal, and parietal cortex. SWA provides a powerful new framework to characterize the waveform of neural oscillations in the healthy and diseased human brain.
Naturalistic Object Representations Depend on Distance and Size Cues
Authors: Fairchild Grant T. , Holler Desiree E. , Fabbri Sara , Gomez Michael A. , Walsh-Snow Jacqueline C.
Published: March 2024

Egocentric distance and real-world size are important cues for object perception and action. Nevertheless, most studies of human vision rely on two-dimensional pictorial stimuli that convey ambiguous distance and size information. Here, we use fMRI to test whether pictures are represented differently in the human brain from real, tangible objects that convey unambiguous distance and size cues. Participants directly viewed stimuli in two display formats (real objects and matched printed pictures of those objects) presented at different egocentric distances (near and far). We measured the effects of format and distance on fMRI response amplitudes and response patterns. We found that fMRI response amplitudes in the lateral occipital and posterior parietal cortices were stronger overall for real objects than for pictures. In these areas and many others, including regions involved in action guidance, responses to real objects were stronger for near vs. far stimuli, whereas distance had little effect on responses to pictures—suggesting that distance determines relevance to action for real objects, but not for pictures. Although stimulus distance especially influenced response patterns in dorsal areas that operate in the service of visually guided action, distance also modulated representations in ventral cortex, where object responses are thought to remain invariant across contextual changes. We observed object size representations for both stimulus formats in ventral cortex but predominantly only for real objects in dorsal cortex. Together, these results demonstrate that whether brain responses reflect physical object characteristics depends on whether the experimental stimuli convey unambiguous information about those characteristics.

Significance Statement

Classic frameworks of vision attribute perception of inherent object characteristics, such as size, to the ventral visual pathway, and processing of spatial characteristics relevant to action, such as distance, to the dorsal visual pathway. However, these frameworks are based on studies that used projected images of objects whose actual size and distance from the observer were ambiguous. Here, we find that when object size and distance information in the stimulus is less ambiguous, these characteristics are widely represented in both visual pathways. Our results provide valuable new insights into the brain representations of objects and their various physical attributes in the context of naturalistic vision.
Diclofenac-eugenol modulation of Kv7 and TRPV1 ion channels
Authors: Rivera-Ruedas Adrián , Medina-Vilchis Alejandro Rafael , Romero-Tovar Juan José , Cristóbal-Mondragón Gema R. , De la Rosa Victor
Published: March 2024

Pharmacological targeting of ion channels represents a crucial avenue for pain management. Among these, the Kv7 family of ion channels plays a significant role controlling neuronal excitability and the generation and propagation of pain-related nerve impulses, thus mitigating excessive electrical signaling and curtailing the exaggerated transmission of pain signals. Pain management strategies often involve a multimodal approach, combining various medications with distinct mechanisms of action to achieve optimal outcomes. Eugenol possesses a spectrum of biological activities, including analgesic and anti-inflammatory properties. When used in conjunction with the anti-inflammatory drug diclofenac, eugenol demonstrates enhanced analgesic efficacy in animal models. We investigated the effects of diclofenac and eugenol on Kv7 and TRPV1 ion channels, both agents act as Kv7 activators, whereas diclofenac inhibits the TRPV1 current, and eugenol reduce the capsaicin-activated current presumably competing for the same binding site. Eugenol shows a time dependent biphasic effect on acid-activated TRPV1 current, first activating and then a slow decay of the current. When eugenol and diclofenac are used together, they limit the extent of depolarization of cells expressing Kv7 and TRPV1. Our results shed light on the combined effectiveness of eugenol and diclofenac in the treatment of acute pain.
Mechanistic principles of hydrogen evolution in the membrane-bound hydrogenase
Authors: Sirohiwal Abhishek , Gamiz-Hernandez Ana P. , Kaila Ville R. I.
Published: March 2024

The membrane-bound hydrogenase (Mbh) fromPyrococcus furiosusis an archaeal member of the Complex I superfamily. It catalyzes the reduction of protons to H2gas powered by a [NiFe] active site and transduces the free energy into proton pumping and Na+/H+-exchange across the membrane. Despite recent structural advances (1–4), the mechanistic principles of H2catalysis and ion transport in Mbh remain elusive. Here we probe how the redox chemistry drives the proton reduction to H2and how the catalysis couples to conformational dynamics in the membrane domain of Mbh. By combining large-scale quantum chemical density functional theory (DFT) and correlatedab initiowave function methods with atomistic molecular dynamics simulations, we show that the proton transfer reactions required for the catalysis are gated by electric field effects that direct the protons by water-mediated reactions from Glu21Ltowards the [NiFe] site, or alternatively along the nearby His75Lpathway that also becomes energetically feasible in certain reaction steps. These local proton-coupled electron transfer (PCET) reactions induce conformational changes around the active site that provide a key coupling element via conserved loop structures to the ion transport activity. We find that H2forms in a heterolytic proton reduction step, with spin crossovers tuning the energetics along key reaction steps. On a general level, our work showcases the role of electric fields in enzyme catalysis, and how these effects are employed by the [NiFe] active site of Mbh to drive the PCET reactions and ion transport.

Significance statement

Hydrogen (H2) serves as a crucial solar fuel in renewable energy systems that can be efficiently produced by microbial hydrogenases. Here we probe the elusive mechanistic principles underlying the H2production in the ancient membrane-bound hydrogenase (Mbh) from the thermophilic archaeonPyrococcus furiosus. Distinct from other hydrogenases, Mbh not only produces H2, but it couples this activity with ion transport across a membrane that powers the archaeal energy metabolism. Our study elucidates key mechanistic principles underlying H2production and shed light on energy transducing enzymes that led to the evolution of modern mitochondrial respiratory enzymes.
Deep learning based genomic breeding of pest-resistant grapevine
Authors: Gan Yu , Liu Zhenya , Zhang Fan , Xu Qi , Wang Xu , Xue Hui , Su Xiangnian , Ma Wenqi , Long Qiming , Ma Anqi , Huang Guizhou , Liu Wenwen , Xu Xiaodong , Sun Lei , Zhang Yingchun , Liu Yuting , Fang Xinyue , Li Chaochao , Yang Xuanwen , Wei Pengcheng , Fan Xiucai , Zhang Chuan , Zhang Pengpai , Liu Chonghuai , Zhang Zhiwu , Huang Sanwen , Wang Yiwen , Liu Zhongjie , Zhou Yongfeng
Published: March 2024

Crop pests have profoundly deleterious effects on crop yield and food security. However, conventional pest control depends heavily on the utilization of insecticides, which develops strong pesticide resistance and concerns of food safety. Crop and their wild relatives display diverse levels of pest resistance, indicating the feasibility for breeding of pest-resistant crop varieties. In this study, we integrate deep learning (DL)/machine learning (ML) algorithms, plant phenomics and whole genome sequencing (WGS) data to conduct genomic selection (GS) of pest-resistance in grapevine. We employ deep convolutional neural networks (DCNN) to accurately calculate the severity of damage by pests on grape leaves, which achieves a classification accuracy of 95.3% (Visual Geometry Group 16, VGG16, for binary trait) and a correlation coefficient of 0.94 in regression analysis (DCNN with Pest Damage Score, DCNN-PDS, for continuous trait). We apply DL models to predict and integrate phenotype (both binary and continuous) along with WGS data from 231 grape accessions, conducting Genome-Wide Association Studies (GWAS). This analysis detects a total of 69 QTLs, encompassing 139 candidate genes involved in pathways associated with pest resistance, including jasmonic acid (JA), salicylic acid (SA), ethylene, and other related pathways. Furthermore, through the combination with transcriptome data, we identify specific pest-resistant genes, such asACA12andCRK3, which play distinct roles in resisting herbivore attacks. Machine learning-based GS demonstrates a high accuracy (95.7%) and a strong correlation (0.90) in predicting the leaf area damaged by pests as binary and continuous traits in grapevine, respectively. In general, our study highlights the power of DL/ML in plant phenomics and GS, facilitating genomic breeding of pest-resistant grapevine.
Alternative silencing states of Transposable Elements in Arabidopsis
Authors: Hure Valentin , Piron-Prunier Florence , Yehouessi Tamara , Vitte Clémentine , Kornienko Aleksandra E. , Adam Gabrielle , Nordborg Magnus , Déléris Angélique
Published: March 2024

The DNA methylation/H3K9me2 and Polycomb-group proteins (PcG)-H3K27me3 pathways have long been considered mutually exclusive and specific to TEs and genes, respectively. However, H3K27me3 can be recruited to many TEs in the absence of DNA methylation machinery and sometimes also co-occur with DNA methylation. In this study, we show that TEs can also be solely targeted by H3K27me3 in wild-type Arabidopsis plants. These H3K27me3-marked TEs not only comprise degenerate relics but also seemingly intact copies that display the epigenetic features of responsive PcG target genes as well as an active H3K27me3 regulation. We also show that H3K27me3 can be deposited on newly inserted transgenic TE sequences in a TE-specific manner indicating that silencing is determined incis. Finally, comparison of Arabidopsis natural accessions reveals the existence of a category of TEs - which we refer to as “bifrons” - that are marked by DNA methylation or H3K27me3 depending on the ecotype. This variation can be linked to intrinsic TE features and totrans- acting factors, and reveals a change in epigenetic status across TE lifespan. Our study sheds light on an alternative mode of TE silencing associated with H3K27me3 instead of DNA methylation in flowering plants. It also suggests dynamic switching between the two epigenetic marks at the species level, a new paradigm that might extend to other multicellular eukaryotes.