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The effects of genetic variation on complex traits act mainly through changes in gene regulation. Although many genetic variants have been linked to target genes in cis, the trans-regulatory cascade mediating their effects remains largely uncharacterized. Mapping trans-regulators based on natural genetic variation has been challenging due to small effects, but experimental perturbations offer a complementary approach. Using CRISPR, we knocked out 84 genes in primary CD4+ T cells, targeting inborn error of immunity (IEI) disease transcription factors (TFs) and TFs without immune disease association. We developed a novel gene network inference method called linear latent causal Bayes (LLCB) to estimate the network from perturbation data and observed 211 regulatory connections between genes. We characterized programs affected by the TFs, which we associated with immune genome-wide association study (GWAS) genes, finding that JAK-STAT family members are regulated by KMT2A, an epigenetic regulator. These analyses reveal the trans-regulatory cascades linking GWAS genes to signaling pathways.

Genome-wide association studies typically evaluate the autosomes and sometimes the X Chromosome, but seldom consider the Y or mitochondrial (MT) Chromosomes. We genotyped the Y and MT Chromosomes in heterogeneous stock (HS) rats (Rattus norvegicus), an outbred population created from 8 inbred strains. We identified 8 distinct Y and 4 distinct MT Chromosomes among the 8 founders. However, only 2 types of each nonrecombinant chromosome were observed in our modern HS rat population (generations 81-97). Despite the relatively large sample size, there were virtually no significant associations for behavioral, physiological, metabolome, or microbiome traits after correcting for multiple comparisons. However, both Y and MT Chromosomes were strongly associated with the expression of a few genes located on those chromosomes, which provided a positive control. Our results suggest that within modern HS rats there are no Y and MT Chromosomes differences that strongly influence behavioral or physiological traits. These results do not address other ancestral Y and MT Chromosomes that do not appear in modern HS rats, nor do they address effects that may exist in other rat populations, or in other species.

Two correspondences raised concerns or comments about our analyses regarding exaggerated false positives found by differential expression (DE) methods. Here, we discuss the points they raise and explain why we agree or disagree with these points. We add new analysis to confirm that the Wilcoxon rank-sum test remains the most robust method compared to the other five DE methods (DESeq2, edgeR, limma-voom, dearseq, and NOISeq) in two-condition DE analyses after considering normalization and winsorization, the data preprocessing steps discussed in the two correspondences.

Increasingly, scRNA-Seq studies explore cell populations across different samples and the effect of sample heterogeneity on organisms phenotype. However, relatively few bioinformatic methods have been developed which adequately address the variation between samples for such population-level analyses. We propose a framework for representing the entire single-cell profile of a sample, which we call a GloScope representation. We implement GloScope on scRNA-Seq datasets from study designs ranging from 12 to over 300 samples and demonstrate how GloScope allows researchers to perform essential bioinformatic tasks at the sample-level, in particular visualization and quality control assessment.

The cuticle constitutes the outermost defensive barrier of most land plants. It comprises a polymeric matrix-cutin, surrounded by soluble waxes. Moreover, the cuticle constitutes the first line of defense against pathogen invasion, while also protecting the plant from many abiotic stresses. Aliphatic monomers in cutin have been suggested to act as immune elicitors in plants. This study analyses the potential of cutin oligomers to activate rapid signaling outputs reminiscent of pattern-triggered immunity in the model plant Arabidopsis. Cutin oligomeric mixtures led to Ca2+ influx and mitogen-activated protein kinase activation. Comparable responses were measured for cutin, which was also able to induce a reactive oxygen species burst. Furthermore, cutin oligomer treatment resulted in a unique transcriptional reprogramming profile, having many archetypal features of pattern-triggered immunity. Targeted spectroscopic and spectrometric analyses of the cutin oligomers suggest that the elicitor compounds consist mostly of two up to three 10,16-dihydroxyhexadecanoic acid monomers linked together through ester bonds. This study demonstrates that cutin breakdown products can act as inducers of early plant immune responses. Further investigation is needed to understand how cutin breakdowns are perceived and to explore their potential use in agriculture.

Modification of lignin in feedstocks via genetic engineering aims to reduce biomass recalcitrance to facilitate efficient conversion processes. These improvements can be achieved by expressing exogenous enzymes that interfere with native biosynthetic pathways responsible for the production of the lignin precursors. In planta expression of a bacterial 3-dehydroshikimate dehydratase in poplar trees reduced lignin content and altered the monomer composition, which enabled higher yields of sugars after cell wall polysaccharide hydrolysis. Understanding how plants respond to such genetic modifications at the transcriptional and metabolic levels is needed to facilitate further improvement and field deployment. In this work, we acquired fundamental knowledge on lignin-modified poplar expressing 3-dehydroshikimate dehydratase using RNA-seq and metabolomics. The data clearly demonstrate that changes in gene expression and metabolite abundance can occur in a strict spatiotemporal fashion, revealing tissue-specific responses in the xylem, phloem, or periderm. In the poplar line that exhibited the strongest reduction in lignin, we found that 3% of the transcripts had altered expression levels and ~19% of the detected metabolites had differential abundance in the xylem from older stems. The changes affected predominantly the shikimate and phenylpropanoid pathways as well as secondary cell wall metabolism, and resulted in significant accumulation of hydroxybenzoates derived from protocatechuate and salicylate.

BACKGROUND: RNA sequencing (RNA-Seq) offers profound insights into the complex transcriptomes of diverse biological systems. However, standard differential expression analysis pipelines based on DESeq2 and edgeR encounter challenges when applied to the immediate early transcriptomes of Chlamydia spp., obligate intracellular bacteria. These challenges arise from their reliance on assumptions that do not hold in scenarios characterized by extensive transcriptomic activation and limited repression. RESULTS: Standard analyses using unique chlamydial RNA-Seq reads alone identify nearly 300 upregulated and about 300 downregulated genes, significantly deviating from actual RNA-Seq read trends. By incorporating both chlamydial and host reads or adjusting for total sequencing depth, the revised normalization methods each detected over 700 upregulated genes and 30 or fewer downregulated genes, closely aligned with observed RNA-Seq data. Further validation through qRT-PCR analysis confirmed the effectiveness of these adjusted approaches in capturing the true extent of transcriptomic activation during the immediate early phase of chlamydial infection. CONCLUSIONS: This study highlights the limitations of standard RNA-Seq analysis tools in scenarios with extensive transcriptomic activation, such as in Chlamydia spp. during early infection. Our revised normalization methods, incorporating host reads or total sequencing depth, provide a more accurate representation of gene expression dynamics. These approaches may inform similar adjustments in other systems with unbalanced gene expression dynamics, enhancing the accuracy of transcriptomic analysis.

Merging diverse single-cell RNA sequencing (scRNA-seq) data from numerous experiments, laboratories and technologies can uncover important biological insights. Nonetheless, integrating scRNA-seq data encounters special challenges when the datasets are composed of diverse cell type compositions. Scanorama offers a robust solution for improving the quality and interpretation of heterogeneous scRNA-seq data by effectively merging information from diverse sources. Scanorama is designed to address the technical variation introduced by differences in sample preparation, sequencing depth and experimental batches that can confound the analysis of multiple scRNA-seq datasets. Here we provide a detailed protocol for using Scanorama within a Scanpy-based single-cell analysis workflow coupled with Google Colaboratory, a cloud-based free Jupyter notebook environment service. The protocol involves Scanorama integration, a process that typically spans 0.5-3 h. Scanorama integration requires a basic understanding of cellular biology, transcriptomic technologies and bioinformatics. Our protocol and new Scanorama-Colaboratory resource should make scRNA-seq integration more widely accessible to researchers.

Conventional type 1 dendritic cells (cDC1s) are critical for innate sensing of cancer, yet they are scarce in the tumor microenvironment (TME). Here, we present a protocol to identify and isolate cDC1 subsets from murine implantable tumors for subsequent transcriptomic profiling using a flow sorting-based strategy. We describe steps for cell culture of mouse tumors, tumoral growth, dissociation and isolation of tumoral cells, extracellular staining, and cell sorting. We then detail procedures for RNA isolation, mRNA library preparation, and sequencing. For complete details on the use and execution of this protocol, please refer to Papadas et al.1.

Single-gene missense mutations remain challenging to interpret. Here, we deploy scalable functional screening by sequencing (SEUSS), a Perturb-seq method, to generate mutations at protein interfaces of RUNX1 and quantify their effect on activities of downstream cellular programs. We evaluate single-cell RNA profiles of 115 mutations in myelogenous leukemia cells and categorize them into three functionally distinct groups, wild-type (WT)-like, loss-of-function (LoF)-like, and hypomorphic, that we validate in orthogonal assays. LoF-like variants dominate the DNA-binding site and are recurrent in cancer; however, recurrence alone does not predict functional impact. Hypomorphic variants share characteristics with LoF-like but favor protein interactions, promoting gene expression indicative of nerve growth factor (NGF) response and cytokine recruitment of neutrophils. Accessible DNA near differentially expressed genes frequently contains RUNX1-binding motifs. Finally, we reclassify 16 variants of uncertain significance and train a classifier to predict 103 more. Our work demonstrates the potential of targeting protein interactions to better define the landscape of phenotypes reachable by missense mutations.

The Long-read RNA-Seq Genome Annotation Assessment Project Consortium was formed to evaluate the effectiveness of long-read approaches for transcriptome analysis. Using different protocols and sequencing platforms, the consortium generated over 427 million long-read sequences from complementary DNA and direct RNA datasets, encompassing human, mouse and manatee species. Developers utilized these data to address challenges in transcript isoform detection, quantification and de novo transcript detection. The study revealed that libraries with longer, more accurate sequences produce more accurate transcripts than those with increased read depth, whereas greater read depth improved quantification accuracy. In well-annotated genomes, tools based on reference sequences demonstrated the best performance. Incorporating additional orthogonal data and replicate samples is advised when aiming to detect rare and novel transcripts or using reference-free approaches. This collaborative study offers a benchmark for current practices and provides direction for future method development in transcriptome analysis.

BACKGROUND: Sex-differential biology may contribute to the consistently male-biased prevalence of autism spectrum disorder (ASD). Gene expression differences between males and females in the brain can indicate possible molecular and cellular mechanisms involved, although transcriptomic sex differences during human prenatal cortical development have been incompletely characterized, primarily due to small sample sizes. METHODS: We performed a meta-analysis of sex-differential expression and co-expression network analysis in 2 independent bulk RNA sequencing datasets generated from cortex of 273 prenatal donors without known neuropsychiatric disorders. To assess the intersection between neurotypical sex differences and neuropsychiatric disorder biology, we tested for enrichment of ASD-associated risk genes and expression changes, neuropsychiatric disorder risk genes, and cell type markers within identified sex-differentially expressed genes (sex-DEGs) and sex-differential co-expression modules. RESULTS: We identified 101 significant sex-DEGs, including Y-chromosome genes, genes impacted by X-chromosome inactivation, and autosomal genes. Known ASD risk genes, implicated by either common or rare variants, did not preferentially overlap with sex-DEGs. We identified 1 male-specific co-expression module enriched for immune signaling that is unique to 1 input dataset. CONCLUSIONS: Sex-differential gene expression is limited in prenatal human cortex tissue, although meta-analysis of large datasets allows for the identification of sex-DEGs, including autosomal genes that encode proteins involved in neural development. Lack of sex-DEG overlap with ASD risk genes in the prenatal cortex suggests that sex-differential modulation of ASD symptoms may occur in other brain regions, at other developmental stages, or in specific cell types, or may involve mechanisms that act downstream from mutation-carrying genes.

BackgroundThe neuronal and gliaI populations within the brain are tightly interwoven, making isolation and study of large populations of a single cell type from brain tissue a major technical challenge. Concurrently, cell-type specific extracellular vesicles (EVs) hold enormous diagnostic and therapeutic potential in neurodegenerative disorders including Alzheimer's disease (AD).New methodPostmortem AD cortical samples were thawed and gently dissociated. Following filtration, myelin and red blood cell removal, cell pellets were immunolabeled with fluorescent antibodies and analyzed by flow cytometry. The cell pellet supernatant was applied to a triple sucrose cushion for brain EV isolation.ResultsNeuronal, astrocyte and microglial cell populations were identified. Cell integrity was demonstrated using calcein AM, which is retained by cells with esterase activity and an intact membrane. For some experiments cell pellets were fixed, permeabilized, and immunolabeled for cell-specific markers. Characterization of brain small EV fractions showed the expected size, depletion of EV negative markers, and enrichment in positive and cell-type specific markers.Comparison with existing methods and conclusionsWe optimized and integrated established protocols, aiming to maximize information obtained from each human autopsy brain sample. The uniqueness of our method lies in its capability to isolate cells and EVs from a single cryopreserved brain sample. Our results not only demonstrate the feasibility of isolating specific brain cell subpopulations for RNA-seq but also validate these subpopulations at the protein level. The accelerated study of EVs from human samples is crucial for a better understanding of their contribution to neuron/glial crosstalk and disease progression.

Splicing factor 3b subunit 1 (SF3B1) is the largest subunit and core component of the spliceosome. Inhibition of SF3B1 was associated with an increase in broad intron retention (IR) on most transcripts, suggesting that IR can be used as a marker of spliceosome inhibition in chronic lymphocytic leukemia (CLL) cells. Furthermore, we separately analyzed exonic and intronic mapped reads on annotated RNA-sequencing transcripts obtained from B cells (n = 98 CLL patients) and healthy volunteers (n = 9). We measured intron/exon ratio to use that as a surrogate for alternative RNA splicing (ARS) and found that 66% of CLL-B cell transcripts had significant IR elevation compared with normal B cells (NBCs) and that correlated with mRNA downregulation and low expression levels. Transcripts with the highest IR levels belonged to biological pathways associated with gene expression and RNA splicing. A >2-fold increase of active pSF3B1 was observed in CLL-B cells compared with NBCs. Additionally, when the CLL-B cells were treated with macrolides (pladienolide-B), a significant decrease in pSF3B1, but not total SF3B1 protein, was observed. These findings suggest that IR/ARS is increased in CLL, which is associated with SF3B1 phosphorylation and susceptibility to SF3B1 inhibitors. These data provide additional support to the relevance of ARS in carcinogenesis and evidence of pSF3B1 participation in this process.

The molecular organization of the human neocortex historically has been studied in the context of its histological layers. However, emerging spatial transcriptomic technologies have enabled unbiased identification of transcriptionally defined spatial domains that move beyond classic cytoarchitecture. We used the Visium spatial gene expression platform to generate a data-driven molecular neuroanatomical atlas across the anterior-posterior axis of the human dorsolateral prefrontal cortex. Integration with paired single-nucleus RNA-sequencing data revealed distinct cell type compositions and cell-cell interactions across spatial domains. Using PsychENCODE and publicly available data, we mapped the enrichment of cell types and genes associated with neuropsychiatric disorders to discrete spatial domains.

BACKGROUND: Single-cell transcriptome sequencing (scRNA-Seq) has allowed new types of investigations at unprecedented levels of resolution. Among the primary goals of scRNA-Seq is the classification of cells into distinct types. Many approaches build on existing clustering literature to develop tools specific to single-cell. However, almost all of these methods rely on heuristics or user-supplied parameters to control the number of clusters. This affects both the resolution of the clusters within the original dataset as well as their replicability across datasets. While many recommendations exist, in general, there is little assurance that any given set of parameters will represent an optimal choice in the trade-off between cluster resolution and replicability. For instance, another set of parameters may result in more clusters that are also more replicable. RESULTS: Here, we propose Dune, a new method for optimizing the trade-off between the resolution of the clusters and their replicability. Our method takes as input a set of clustering results-or partitions-on a single dataset and iteratively merges clusters within each partitions in order to maximize their concordance between partitions. As demonstrated on multiple datasets from different platforms, Dune outperforms existing techniques, that rely on hierarchical merging for reducing the number of clusters, in terms of replicability of the resultant merged clusters as well as concordance with ground truth. Dune is available as an R package on Bioconductor: https://www.bioconductor.org/packages/release/bioc/html/Dune.html . CONCLUSIONS: Cluster refinement by Dune helps improve the robustness of any clustering analysis and reduces the reliance on tuning parameters. This method provides an objective approach for borrowing information across multiple clusterings to generate replicable clusters most likely to represent common biological features across multiple datasets.

Dorsal interneurons (dIs) in the spinal cord encode the perception of touch, pain, heat, itchiness and proprioception. Previous studies using genetic strategies in animal models have revealed important insights into dI development, but the molecular details of how dIs arise as distinct populations of neurons remain incomplete. We have developed a resource to investigate dI fate specification by combining a single-cell RNA-Seq atlas of mouse embryonic stem cell-derived dIs with pseudotime analyses. To validate this in silico resource as a useful tool, we used it to first identify genes that are candidates for directing the transition states that lead to distinct dI lineage trajectories, and then validated them using in situ hybridization analyses in the developing mouse spinal cord in vivo. We have also identified an endpoint of the dI5 lineage trajectory and found that dIs become more transcriptionally homogeneous during terminal differentiation. This study introduces a valuable tool for further discovery about the timing of gene expression during dI differentiation and demonstrates its utility in clarifying dI lineage relationships.

An organizational feature of neural circuits is the specificity of synaptic connections. A striking example is the direction-selective (DS) circuit of the retina. There are multiple subtypes of DS retinal ganglion cells (DSGCs) that prefer motion along one of four preferred directions. This computation is mediated by selective wiring of a single inhibitory interneuron, the starburst amacrine cell (SAC), with each DSGC subtype preferentially receiving input from a subset of SAC processes. We hypothesize that the molecular basis of this wiring is mediated in part by unique expression profiles of DSGC subtypes. To test this, we first performed paired recordings from isolated mouse retinas of both sexes to determine that postnatal day 10 (P10) represents the age at which asymmetric synapses form. Second, we performed RNA sequencing and differential expression analysis on isolated P10 ON-OFF DSGCs tuned for either nasal or ventral motion and identified candidates which may promote direction-specific wiring. We then used a conditional knock-out strategy to test the role of one candidate, the secreted synaptic organizer cerebellin-4 (Cbln4), in the development of DS tuning. Using two-photon calcium imaging, we observed a small deficit in directional tuning among ventral-preferring DSGCs lacking Cbln4, though whole-cell voltage-clamp recordings did not identify a significant change in inhibitory inputs. This suggests that Cbln4 does not function primarily via a cell-autonomous mechanism to instruct wiring of DS circuits. Nevertheless, our transcriptomic analysis identified unique candidate factors for gaining insights into the molecular mechanisms that instruct wiring specificity in the DS circuit.

In droplet-based single-cell and single-nucleus RNA-seq assays, systematic contamination of ambient RNA molecules biases the quantification of gene expression levels. Existing methods correct the contamination for all genes globally. However, there lacks specific evaluation of correction efficacy for varying contamination levels. Here, we show that DecontX and CellBender under-correct highly contaminating genes, while SoupX and scAR over-correct lowly/non-contaminating genes. Here, we develop scCDC as the first method to detect the contamination-causing genes and only correct expression levels of these genes, some of which are cell-type markers. Compared with existing decontamination methods, scCDC excels in decontaminating highly contaminating genes while avoiding over-correction of other genes.

BackgroundAdvancing age is a major risk factor for respiratory viral infections. The infections are often prolonged and difficult to resolve resulting hospitalizations and mortality. The recent COVID-19 pandemic has highlighted this as elderly subjects have emerged as vulnerable populations that display increased susceptibility and severity to SARS-CoV-2. There is an urgent need to identify the probable mechanisms underlying this to protect against future outbreaks of such nature. Innate immunity is the first line of defense against viruses and its decline impacts downstream immune responses. This is because dendritic cells (DCs) and macrophages are key cellular elements of the innate immune system that can sense and respond to viruses by producing inflammatory mediators and priming CD4 and CD8 T-cell responses.ResultsWe investigated the changes in innate immune responses to SARS-CoV-2 as a function of age. Our results using human PBMCs from aged, middle-aged, and young subjects indicate that the activation of DCs and monocytes in response to SARS-CoV-2 is compromised with age. The impairment is most apparent in pDCs where both aged and middle-aged display reduced responses. The secretion of IL-29 that confers protection against respiratory viruses is also decreased in both aged and middle-aged subjects. In contrast, inflammatory mediators associated with severe COVID-19 including CXCL-8, TREM-1 are increased with age. This is also apparent in the gene expression data where pathways related host defense display an age dependent decrease with a concomitant increase in inflammatory pathways. Not only are the inflammatory pathways and mediators increased after stimulation with SARS-CoV-2 but also at homeostasis. In keeping with reduced DC activation, the induction of cytotoxic CD8 T cells is also impaired in aged subjects. However, the CD8 T cells from aged subjects display increased baseline activation in accordance with the enhanced baseline inflammation.ConclusionsOur results demonstrate a decline in protective anti-viral immune responses and increase in damaging inflammatory responses with age indicating that dysregulated innate immune responses play a significant role in the increased susceptibility of aged subjects to COVID-19. Furthermore, the dysregulation in immune responses develops early on as middle-aged demonstrate several of these changes.

Genome-wide association studies (GWASs) have uncovered susceptibility loci associated with psychiatric disorders such as bipolar disorder (BP) and schizophrenia (SCZ). However, most of these loci are in non-coding regions of the genome, and the causal mechanisms of the link between genetic variation and disease risk is unknown. Expression quantitative trait locus (eQTL) analysis of bulk tissue is a common approach used for deciphering underlying mechanisms, although this can obscure cell-type-specific signals and thus mask trait-relevant mechanisms. Although single-cell sequencing can be prohibitively expensive in large cohorts, computationally inferred cell-type proportions and cell-type gene expression estimates have the potential to overcome these problems and advance mechanistic studies. Using bulk RNA-seq from 1,730 samples derived from whole blood in a cohort ascertained from individuals with BP and SCZ, this study estimated cell-type proportions and their relation with disease status and medication. For each cell type, we found between 2,875 and 4,629 eGenes (genes with an associated eQTL), including 1,211 that are not found on the basis of bulk expression alone. We performed a colocalization test between cell-type eQTLs and various traits and identified hundreds of associations that occur between cell-type eQTLs and GWASs but that are not detected in bulk eQTLs. Finally, we investigated the effects of lithium use on the regulation of cell-type expression loci and found examples of genes that are differentially regulated according to lithium use. Our study suggests that applying computational methods to large bulk RNA-seq datasets of non-brain tissue can identify disease-relevant, cell-type-specific biology of psychiatric disorders and psychiatric medication.

Crown gall disease (Agrobacterium tumefaciens), crown/root rot disease (Phytophthora spp.), root lesion disease (Pratylenchus vulnus) and tree vigor are key traits affecting the productivity and quality of walnuts in California. Unchallenged hybrid rootstocks were analyzed by RNA-seq to examine pre-formed factors affecting these traits. Enrichment analysis of the differentially expressed genes revealed that the increased expression of cell wall biogenesis-related genes plays a key role in susceptibility to A. tumefaciens, susceptibility to Phytophthora spp. and increased vigor. Analysis of the predicted subcellular loci of the encoded proteins revealed that many gene products associated with vigor and susceptibility were targeted to the plasma membrane and extracellular space, connecting these traits to sustaining barrier function. We observed that RNA processing and splicing, along with predicted nuclear targeting, were associated with resistance to A. tumefaciens, resistance to Phytophthora spp. and low vigor. Four genes within the J. microcarpa QTL region for resistance to A. tumefaciens and Phytophthora spp. were represented among our transcripts, with two of the genes being differentially expressed in association with resistance to A. tumefaciens and decreased vigor. No differential expression related to Phytophthora spp. or P. vulnus resistance was observed in this region. Additionally, the J. microcarpa haplotype expressed more transcripts associated with resistance to A. tumefaciens, Phytophthora spp. and low vigor, but not P. vulnus, than the J. regia haplotype. We also report unique and shared hormone and defense responses associated with each trait. This research suggests a link between cell wall biogenesis, vigor and critical root diseases of walnut.

IntroductionThe domestic cat (Felis catus) is a valued companion animal and a model for virally induced cancers and immunodeficiencies. However, species-specific limitations such as a scarcity of immune cell markers constrain our ability to resolve immune cell subsets at sufficient detail. The goal of this study was to characterize circulating feline T cells and other leukocytes based on their transcriptomic landscape and T-cell receptor repertoire using single cell RNA-sequencing.MethodsPeripheral blood from 4 healthy cats was enriched for T cells by flow cytometry cell sorting using a mouse anti-feline CD5 monoclonal antibody. Libraries for whole transcriptome, αβ T cell receptor transcripts and γδ T cell receptor transcripts were constructed using the 10x Genomics Chromium Next GEM Single Cell 5' reagent kit and the Chromium Single Cell V(D)J Enrichment Kit with custom reverse primers for the feline orthologs.ResultsUnsupervised clustering of whole transcriptome data revealed 7 major cell populations - T cells, neutrophils, monocytic cells, B cells, plasmacytoid dendritic cells, mast cells and platelets. Sub cluster analysis of T cells resolved naive (CD4+ and CD8+), CD4+ effector T cells, CD8+ cytotoxic T cells and γδ T cells. Cross species analysis revealed a high conservation of T cell subsets along an effector gradient with equitable representation of veterinary species (horse, dog, pig) and humans with the cat. Our V(D)J repertoire analysis identified a subset of CD8+ cytotoxic T cells with skewed TRA and TRB gene usage, conserved TRA and TRB junctional motifs, restricted TRA/TRB pairing and reduced diversity in TRG junctional length. We also identified a public γδ T cell subset with invariant TRD and TRG chains and a CD4+ TEM-like phenotype. Among monocytic cells, we resolved three clusters of classical monocytes with polarization into pro- and anti-inflammatory phenotypes in addition to a cluster of conventional dendritic cells. Lastly, our neutrophil sub clustering revealed a larger mature neutrophil cluster and a smaller exhausted/activated cluster.DiscussionOur study is the first to characterize subsets of circulating T cells utilizing an integrative approach of single cell RNA-sequencing, V(D)J repertoire analysis and cross species analysis. In addition, we characterize the transcriptome of several myeloid cell subsets and demonstrate immune cell relatedness across different species.

Rheumatoid arthritis (RA) management leans toward achieving remission or low disease activity. In this study, we conducted single-cell RNA sequencing (scRNA-Seq) of peripheral blood mononuclear cells (PBMCs) from 36 individuals (18 patients with RA and 18 matched controls, accounting for age, sex, race, and ethnicity), to identify disease-relevant cell subsets and cell type-specific signatures associated with disease activity. Our analysis revealed 18 distinct PBMC subsets, including an IFN-induced transmembrane 3-overexpressing (IFITM3-overexpressing) IFN-activated monocyte subset. We observed an increase in CD4+ T effector memory cells in patients with moderate-high disease activity (DAS28-CRP ≥ 3.2) and a decrease in nonclassical monocytes in patients with low disease activity or remission (DAS28-CRP < 3.2). Pseudobulk analysis by cell type identified 168 differentially expressed genes between RA and matched controls, with a downregulation of proinflammatory genes in the γδ T cell subset, alteration of genes associated with RA predisposition in the IFN-activated subset, and nonclassical monocytes. Additionally, we identified a gene signature associated with moderate-high disease activity, characterized by upregulation of proinflammatory genes such as TNF, JUN, EGR1, IFIT2, MAFB, and G0S2 and downregulation of genes including HLA-DQB1, HLA-DRB5, and TNFSF13B. Notably, cell-cell communication analysis revealed an upregulation of signaling pathways, including VISTA, in both moderate-high and remission-low disease activity contexts. Our findings provide valuable insights into the systemic cellular and molecular mechanisms underlying RA disease activity.

IntroductionMaternal intervillous monocytes (MIMs) and fetal Hofbauer cells (HBCs) are myeloid-derived immune cells at the maternal-fetal interface. Maternal reproductive history is associated with differential risk of pregnancy complications. The molecular phenotypes and roles of these distinct monocyte/macrophage populations and the influence of gravidity on these phenotypes has not been systematically investigated.MethodsHere, we used RNA sequencing to study the transcriptional profiles of MIMs and HBCs in normal term pregnancies.ResultsOur analyses revealed distinct transcriptomes of MIMs and HBCs. Genes involved in differentiation and cell organization pathways were more highly expressed in MIMs vs. HBCs. In contrast, HBCs had higher expression of genes involved in inflammatory responses and cell surface receptor signaling. Maternal gravidity influenced monocyte programming, as expression of pro-inflammatory molecules was significantly higher in MIMs from multigravidae compared to primigravidae. In HBCs, multigravidae displayed enrichment of gene pathways involved in cell-cell signaling and differentiation.DiscussionOur results demonstrated that MIMs and HBCs have highly divergent transcriptional signatures, reflecting their distinct origins, locations, functions, and roles in inflammatory responses. Furthermore, maternal gravidity influences the gene signatures of MIMs and HBCs, potentially modulating the interplay between tolerance and trained immunity. The phenomenon of reproductive immune memory may play a novel role in the differential susceptibility of primigravidae to pregnancy complications.

Dermestes frischii Kugelann, 1792 is a storage pest worldwide, and is important for estimating the postmortem interval in forensic entomology. However, because of the lack of transcriptome and genome resources, population genetics and biological control studies on D. frischii have been hindered. Here, single‐molecule real‐time sequencing and next‐generation sequencing were combined to generate the full‐length transcriptome of the five developmental stages of D. frischii, namely egg, young larva, mature larva, pupa and adult. A total of 41,665 full‐length non‐chimeric sequences and 59,385 non‐redundant transcripts were generated, of which 42,756 were annotated in public databases. Using the weighted gene co‐expression network analysis, gene co‐expression modules related to the five developmental stages were constructed and screened, and the genes in these modules were subjected to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The expression patterns of the differentially expressed genes (DEGs) related to olfaction and insect hormone biosynthesis were also explored. Transcription of most odorant binding proteins was up‐regulated in the adult stage, suggesting they are important for foraging in adults. Many genes encoding for the ecdysone‐inducible protein were up‐regulated in the pupal stage, may be mainly responsible for the tissue remodelling of metamorphosis. The results of the quantitative real‐time polymerase chain reaction (qRT‐PCR) were consistent with the RNA‐seq results. This is the first full‐length transcriptome sequencing of dermestids, and the data obtained here are vital for understanding the stage‐specific development and olfactory system of D. frischii, providing valuable resources for storage pest and forensic research. [ABSTRACT FROM AUTHOR]

SUMMARY Phytosulfokine (PSK) is a plant growth‐promoting peptide hormone that is perceived by its cell surface receptors PSKR1 and PSKR2 in Arabidopsis. Plants lacking the PSK receptors show phenotypes consistent with PSK signaling repressing some plant defenses. To gain further insight into the PSK signaling mechanism, comprehensive transcriptional profiling of Arabidopsis treated with PSK was performed, and the effects of PSK treatment on plant defense readouts were monitored. Our study indicates that PSK's major effect is to downregulate defense‐related genes; it has a more modest effect on the induction of growth‐related genes. WRKY transcription factors (TFs) emerged as key regulators of PSK‐responsive genes, sharing commonality with a pathogen‐associated molecular pattern (PAMP) responses, flagellin 22 (flg22), but exhibiting opposite regulatory directions. These PSK‐induced transcriptional changes were accompanied by biochemical and physiological changes that reduced PAMP responses, notably mitogen‐activated protein kinase (MPK) phosphorylation (previously implicated in WRKY activation) and the cell wall modification of callose deposition. Comparison with previous studies using other growth stimuli (the sulfated plant peptide containing sulfated tyrosine [PSY] and Pseudomonas simiae strain WCS417) also reveals WRKY TFs' overrepresentations in these pathways, suggesting a possible shared mechanism involving WRKY TFs for plant growth–defense trade‐off. [ABSTRACT FROM AUTHOR]

Background: South China carp (Cyprinus carpio rubrofuscus), which is an economically important species, is traditionally cocultured with rice. Our previous study indicated that approximately 10–30% of these fish experienced growth arrest, severely impacting production. However, the molecular mechanism underlying growth inhibition in south China carp is currently unknown. Results: In this study, we compared the transcriptomes of the livers, muscles and intestines of carp in the fast-growing and slow-growing groups. We identified 2182, 2355 and 916 differentially expressed genes (DEGs), respectively. In the slow-growing group, the oxidative phosphorylation pathway was significantly upregulated in the liver. Transmission electron microscopy (TEM) confirmed mitochondrial damage in the liver, which was characterized by broken cristae and heterogeneous matrix. Additionally, analysis of antioxidant enzyme and transaminase activity also revealed that the livers in slow-growing individuals were unhealthy. In muscle tissue, the mitophagy and autophagy pathways were significantly dysregulated. Consequently, manifestations of mitochondrial damage and sparse myofilaments were clearly observed in slow-growing south China carp via TEM. Furthermore, pathways that regulate cell proliferation and migration, including the ECM receptor and focal adhesion, were significantly enriched in the intestine. Morphological examination revealed that the villus height and muscular layer height in the slow-growing group were significantly shorter than those in the fast-growing group, suggesting decreased intestinal cell motility. Overall, our study elucidated mitochondrial damage in the liver and muscle and detected morphological changes in intestinal villi. Conclusions: In summary, our results help elucidate the genetic architecture related to growth arrest in south China carp and provide a basis for further research on the growth of teleosts. [ABSTRACT FROM AUTHOR]

Background: Dorper sheep are celebrated for their fast maturation and superior meat quality, with some shedding their wool each spring. Wool shedding occurs naturally due to the hair follicle (HF) cycle, but its regulatory mechanisms remain unclear and need further investigation. Results: In this study, shedding and non-shedding sheep were selected from the same Dorper flock. Skin samples were collected in September of the first year and January and March of the following years. RNA sequencing was performed on these samples. Principal component analysis (PCA) was used to assess the results. A total of 2536 differentially expressed genes (DEGs) were identified. Using a clustering heatmap and fuzzy clustering analysis three distinct gene expression patterns were identified: A pattern (high expression in anagen), T1 pattern, and T2 pattern (high expression in telogen). For each pattern, differentially expressed genes (DEGs) were analyzed through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. Combining this with pathway expression analysis, six A-pattern and fourteen T-pattern pathways linked to telogen-anagen transition in the HF cycle were identified. Networks of key pathways were then constructed. Additionally, key genes were identified in the telogen-anagen transition, including one A-pattern gene and seven T-pattern (T1, 1; T2, 6) genes, using the Maximal Clique Centrality (MCC) tool in Cytoscape. Predicted transcription factors (TFs) involved in key pathways, such as LEF and STAT5B, were identified. Finally, RNA-seq results were confirmed by RT-qPCR. Conclusion: This study highlights critical genes and pathways in the telogen-anagen transition, and transcriptome sequencing along with bioinformatics analysis provides new insights into the regulatory mechanisms of the HF cycle and development. [ABSTRACT FROM AUTHOR]

Environmental factors such as altitude may significantly influence both the morphological trait and accumulation of metabolite products in plant. In present study, gene expression profiles of Z. planispinum var. Dintanensisin grown at different altitude (A1:1054 m.a.s.l., A2:821 m.a.s.l., A3:645 m.a.s.l.) were generated by using RNA-Seq. Four main medicinal ingredients of peels were determined, including the total acylamide, total flavonoids, total alkaloids and volatile oil content. The results showed that a total of 10,703 differentially expressed genes (DEGs) were identified among the three samples (A1, A2, A3), of these, 315 DEGs were putative transcription factors (TFs) belonging to 74 TF families. In particular, some candidate genes related to accumulation of main medicinal ingredients have been screened through combining Kmeans cluster and KEGG enrichment analysis. Besides, the total flavonoid accumulation exhibited a positive correlation with increasing altitude, suggesting that higher altitudes favor metabolite accumulation. Notably, seven genes and one MYB TF involved in the biosynthesis of the main medicinal ingredients were confirmed through RT-qPCR analysis The data obtained in this study offers a global survey of gene expression patterns of Z. planispinum var. Dintanensis grown in rocky desertification control areas, which support for further research on the high-altitude adaptation mechanism and identification of peels with higher medicinal value. [ABSTRACT FROM AUTHOR]

De novo assembly of transcriptomes from species without reference genome remains a common problem in functional genomics. While methods and algorithms for transcriptome assembly are continually being developed and published, the quality of de novo assemblies using short reads depends on the complexity of the transcriptome and is limited by several types of errors. One problem to overcome is the research gap regarding the best method to use in each study to obtain high-quality de novo assembly. Currently, there are no established protocols for solving the assembly problem considering the transcriptome complexity. In addition, the accuracy of quality metrics used to evaluate assemblies remains unclear. In this study, we investigate and discuss how different variables accounting for the complexity of RNA-Seq data influence assembly results independently of the software used. For this purpose, we simulated transcriptomic short-read sequence datasets from high-quality full-length predicted transcript models with varying degrees of complexity. Subsequently, we conducted de novo assemblies using different assembly programs, and compared and classified the results using both reference-dependent and independent metrics. These metrics were assessed both individually and combined through multivariate analysis. The degree of alternative splicing and the fragment size of the paired-end reads were identified as the variables with the greatest influence on the assembly results. Moreover, read length and fragment size had different influences on the reconstruction of longer and shorter transcripts. These results underscore the importance of understanding the composition of the transcriptome under study, and making experimental design decisions related to the need to work with reads and fragments of different sizes. In addition, the choice of assembly software will positively impact the final assembly outcome. This selection will affect the completeness of represented genes and assembled isoforms, as well as contribute to error reduction. [ABSTRACT FROM AUTHOR]

Macrobrachium nipponense is a commercially important freshwater species of prawn that is widely distributed across Asian countries. In order to investigate the molecular mechanisms of growth in M. nipponense, and to provide a foundation for molecular breeding, we used genome-wide association analysis (GWAS) and transcriptomic analysis to screen polymorphisms and genes related to growth traits. We recorded the growth traits of 100 adult M. nipponense at the same growth stage, and each individual genotype was evaluated by whole genome resequencing. GWAS of growth traits detected 12 growth-related single-nucleotide polymorphisms (SNPs) and eight growth-related genes from 49 chromosomes. Of the 100 individuals, we sampled muscle tissue from a total of 18 female and male M. nipponense exhibiting large differences in growth rate for RNA-seq. Transcriptome analysis revealed a total of 27,996 unigenes; of these, 33 and 60 differentially expressed genes were identified from males and females, respectively. Of these, 12 genes associated with energy metabolism and cytoskeletal pathways were identified as growth-related genes. Notably, genes from the actin family and the ubiquitin C-terminal hydrolase 2 (UCH2) gene were identified by both GWAS and transcriptomic analysis. Two growth-related SNPs, S40_12327385 and S40_12327391, were found to be mapped to the ACTB gene. The ACTA1 gene, also from the actin family, was up-regulated in fast-growing males and females, while the ACT57B was down-regulated. In addition, the growth associated SNP S7_35313774 was located in the UCH2 gene; transcriptomics analysis revealed that the UCH2 gene was up-regulated in female individuals exhibiting high growth rates. Overall, our results provided a set of markers and candidate genes related to the growth of M. nipponense. These findings could facilitate the breeding management of this species and help us to further understand the genetic mechanisms of growth in crustaceans. [ABSTRACT FROM AUTHOR]

Introduction: Ischemic post-conditioning (I-post C) is a recognized therapeutic strategy for lung ischemia/reperfusion injury (LIRI). However, the specific mechanisms underlying the lung protection conferred by I-post C remain unclear. This study aimed to investigate the protective mechanisms and potential molecular regulatory networks of I-post C on lung tissue. Methods: Transcriptome analysis was performed on rat lung tissues obtained from Sham, ischemia-reperfusion (IR), and I-post C groups using RNA-seq to identify differentially expressed genes (DEGs). Subsequently, gene ontology (GO) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and gene set enrichment analysis (GSEA) were conducted to elucidate significantly enriched pathways in the IR and I-post C groups. Additionally, protein-protein interaction (PPI) network analysis was carried out to examine associations among the DEGs. Pathological changes in lung tissues were assessed using hematoxylin-eosin (H&E) staining. The expression levels of CXCL1 and CXCL6 in the IR and I-post C groups were evaluated through immunofluorescence and Western blotting. Results: Our results showed that I-post C significantly attenuated both pulmonary edema and inflammatory cell infiltration. Transcriptome analysis identified 38 DEGs in the I-post C group compared to the IR group, comprising 21 upregulated and 17 downregulated genes. Among these, seven inflammation-related DEGs exhibited co-expression patterns with the Sham and IR groups, with notable downregulation of C xcl1 and C xcl6. GO analysis primarily linked these DEGs to neutrophil activation, chemotaxis, cytokine activity, and CCR chemokine receptor binding. KEGG analysis revealed enriched pathways, including the IL-17, TNF, and NF-κB signaling pathways. GSEA indicated downregulation of neutrophil chemotaxis and the IL-17 signaling pathway, correlating with reduced expression of C xcl1 and C xcl6. Validation of Cxcl1 and Cxcl6 mRNA expression via immunofluorescence and Western blotting supported the RNA-seq findings. Furthermore, a PPI network was constructed to elucidate interactions among the 29 DEGs. Conclusions: Through RNA-Seq analysis, we concluded that I-post C may reduce inflammation and suppress the IL-17 signaling pathway, thereby protecting against lung damage caused by LIRI, potentially involving neutrophil extracellular traps. [ABSTRACT FROM AUTHOR]

Background: Fusion transcripts (FTs), generated by the fusion of genes at the DNA level or RNA-level splicing events significantly contribute to transcriptome diversity. FTs are usually considered unique features of neoplasia and serve as biomarkers and therapeutic targets for multiple cancers. The latest findings show the presence of FTs in normal human physiology. Several discrete reports mentioned the presence of fusion transcripts in planta, has important roles in stress responses, morphological alterations, or traits (e.g. seed size, etc.). Results: In this study, we identified 169,197 fusion transcripts in 2795 transcriptome datasets of Arabidopsis thaliana, Cicer arietinum, and Oryza sativa by using a combination of tools, and confirmed the translational activity of 150 fusion transcripts through proteomic datasets. Analysis of the FT junction sequences and their association with epigenetic factors, as revealed by ChIP-Seq datasets, demonstrated an organised process of fusion formation at the DNA level. We investigated the possible impact of three-dimensional chromatin conformation on intra-chromosomal fusion events by leveraging the Hi-C datasets with the incidence of fusion transcripts. We further utilised the long-read RNA-Seq datasets to validate the most reoccurring fusion transcripts in each plant species followed by further authentication through RT-PCR and Sanger sequencing. Conclusions: Our findings suggest that a significant portion of fusion events may be attributed to alternative splicing during transcription, accounting for numerous fusion events without a proportional increase in the number of RNA pairs. Even non-nuclear DNA transcripts from mitochondria and chloroplasts can participate in intra- and inter-chromosomal fusion formation. Genes in close spatial proximity are more prone to undergoing fusion formation, especially in intra-chromosomal FTs. Most of the fusion transcripts may not undergo translation and serve as long non-coding RNAs. The low validation rate of FTs in plants indicated that the fusion transcripts are expressed at very low levels, like in the case of humans. FTs often originate from parental genes involved in essential biological processes, suggesting their relevance across diverse tissues and stress conditions. This study presents a comprehensive repository of fusion transcripts, offering valuable insights into their roles in vital physiological processes and stress responses. [ABSTRACT FROM AUTHOR]

Introduction: Sexual dimorphism in Muscovy ducks results in substantial differences in muscle development potential between males and females, leading to significant variations in growth rates and body weights throughout their development. Methods: This study aimed to investigate the regulatory mechanisms underlying the differences in muscle development between genders in black Muscovy ducks, we analyzed the phenotypic characteristics and transcriptome profiles of breast muscles in male and female black Muscovy ducks at different developmental stages (postnatal days 28, 42, and 70). Results: In the analysis of tissue physical morphology, the results showed that females exhibit larger myofiber diameters and lower myofiber densities compared to males at postnatal day 42 (p < 0.05). The difference becomes more pronounced by day 70, however, no significant difference was observed at postnatal day 28. Transcriptome analysis identified a total of 1,118 unique differentially expressed genes (DEGs) across the various comparison groups. In different growth and development stages of black Muscovy ducks, the DEGs like MYLK4 , KIT , CD36 , ATP2A1 were significantly associated with myofiber hypertrophy, and key pathways such as AMPK signaling pathway, focal adhesion, and ECM-receptor interactions have been found to be closely associated with muscle size and hypertrophy. In the breast muscles of different sexes black Muscovy ducks, the DEGs such as TPM2 , HNRNPK , VCP , ATP2A2 , and ANKRD1 may be the reason for the difference in breast muscle size between male and female ducks. Furthermore, key pathways, including the cGMP-PKG signaling pathway, calcium signaling pathway, and hypertrophic cardiomyopathy are also involved in regulating the developmental potential differences in muscle between male and female ducks. Discussion: This study reveals the molecular mechanism regulating the muscle development in male and female black Muscovy ducks at different growth stages, and provides valuable insights into the specific genes responsible for muscle development, laying a theoretical foundation for enhancing the genetic quality of duck meat. [ABSTRACT FROM AUTHOR]

One of the fundamental aspects of genomic research is the identification of differentially expressed (DE) genes between two conditions. In the past decade, numerous DE analysis tools have been developed, employing various normalization methods and statistical modelling approaches. In this article, we introduce DElite , an R package that leverages the capabilities of four state-of-the-art DE tools: edgeR, limma, DESeq2, and dearseq. DElite returns the outputs of the four tools with a single command line, thus providing a simplified way for non-expert users to perform DE analysis. Furthermore, DElite provides a statistically combined output of the four tools, and in vitro validations support the improved performance of these combination approaches for the detection of DE genes in small datasets. Finally, DElite offers comprehensive and well-documented plots and tables at each stage of the analysis, thus facilitating result interpretation. Although DElite has been designed with the intention of being accessible to users without extensive expertise in bioinformatics or statistics, the underlying code is open source and structured in such a way that it can be customized by advanced users to meet their specific requirements. DElite is freely available for download from https://gitlab.com/soc-fogg-cro-aviano/DElite. [ABSTRACT FROM AUTHOR]

Enterovirus 71 (EV71) is a significant causative agent of hand, foot and mouth disease (HFMD). However, the precise mechanism by which EV71 infection leads to alterations in the immune response remain elusive. To address this knowledge gap, we conducted a study where we introduced the Inner Mongolia EV71 C33λ strain into Vero cells, derived from African green monkey kidney cells. Subsequently, we performed RNA sequencing (RNA-Seq) to investigate the changes in the transcriptome of these infected Vero cells. Our primary objective was to establish a foundational understanding that could inform future research on EV71-associated immune factors. In our study, we identified a total of 942 differentially expressed genes (DEGs) in Vero cells infected with Enterovirus 71 (EV71), with 568 gene exhibiting increased expression and 374 gene showing decreased expression. To elucidate the functional implications of these DEGs, we conducted a comprehensive functional enrichment analysis using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. This analysis revealed three genes that were significantly upregulated, which we subsequently validated using reverse transcription polymerase chain reaction technology (RT-qPCR). The RT-qPCR results were in strong agreement with our RNA-Seq data, confirming the reliability of our findings. This study represents the pioneering RNA-Seq analysis that delves into the cellular response of Vero cells to EV71 infection. Our results not only provide a foundational understanding of the molecular changes induced by EV71 but also offer crucial insights into the mechanisms by which EV71 modulates the host immune system. These insights are pivotal for future research endeavors aimed at developing effective therapeutic strategies against EV71 and related pathogens, as well as for understanding the broader implications of viral infections on host immunity. [ABSTRACT FROM AUTHOR]

The GRAS transcription factor (TF) is a plant-specific regulator that plays a critical role in plant growth, development and response to various abiotic stresses. While the functions of many GRAS TFs have been extensively studied in numerous model plants with fully sequenced genomes, the GRAS TFs in white clover (Trifolium repens L.) remain elusive despite the sequencing of its genome. To bridge this knowledge gap, homologs of GRAS TFs from Arabidopsis were employed and subjected to a genome-wide blast against white clover proteins. This analysis identified and characterized 102 putative GRAS genes, designated as TrGRAS, based on the presence of GRAS domains in positive blast hits. Concurrently, an exploration of their gene structures, classification, evolutionary relationships and conservative motifs provided insights into the distinctive features of TrGRAS in white clover. Furthermore, gene duplication analysis revealed an expansion of TrGRAS genes in the PAT1 and LISCL subfamilies, indicating functional diversity within the white clover genome. Lastly, RNA-seq and quantitative reverse transcription-PCR (qRT-PCR) experiments confirmed their higher expression during the early or intermittent responses to cold stress, suggesting their potential and crucial roles in determining the cold tolerances of white clover. The analysis of TrGRAS genes, coupled with expression studies, contributes to the functional characterization of TrGRAS genes in white clover. This knowledge is pivotal for informing genetic improvement strategies in future breeding efforts aimed at enhancing cold tolerance in white clover. [ABSTRACT FROM AUTHOR]

SUMMARY: Volvox carteri is an excellent system for investigating the origins of cell differentiation because it possesses just two cell types, reproductive gonidia and motile somatic cells, which evolved relatively recently. The somatic phenotype depends on the regA gene, which represses cell growth and reproduction, preventing cells expressing it from growing large enough to become gonidia. regA encodes a putative transcription factor and was generated in an undifferentiated ancestor of V. carteri through duplication of a progenitor gene whose ortholog in V. carteri is named rlsD. Here we analyze the function of rlsD through knockdown, overexpression, and RNA‐seq experiments, to gain clues into the function of a member of an understudied putative transcription factor family and to obtain insight into the origins of cell differentiation in the volvocine algae. rlsD knockdown was lethal, while rlsD overexpression dramatically reduced gonidial growth. rlsD overexpression led to differential expression of approximately one‐fourth of the genome, with repressed genes biased for those typically overexpressed in gonidia relative to somatic cells, and upregulated genes biased toward expression in soma, where regA expression is high. Notably, rlsD overexpression affects accumulation of transcripts for genes/Pfam domains involved in ribosome biogenesis, photosynthetic light harvesting, and sulfate generation, functions related to organismal growth, and responses to resource availability. We also found that in the wild type, rlsD expression is induced by light deprivation. These findings are consistent with the idea that cell differentiation in V. carteri evolved when a resource‐responsive, growth‐regulating gene was amplified, and a resulting gene duplicate was co‐opted to repress growth in a constitutive, spatial context. Significance Statement: Little is known about the function of VARL (Volvocine Algal RegA‐Like) family transcription factors beyond RegA. Moreover, the relationship (if any) between function across VARL gene products and how cell differentiation arose in the green alga Volvox carteri has only begun to be examined. Here we provide additional evidence that the evolution of cell differentiation in V. carteri involved rewiring of a pathway that regulates cell and organismal growth in response to resource limitation. [ABSTRACT FROM AUTHOR]

Human satellitess(HSats) are pericentric, tandemly repeating satellite DNA sequences in the human genome. While silent in normal cells, a subset of HSat2 noncoding RNA is expressed and accumulates in the nucleus of cancer cells. We developed a FISH-based approach for identification of the distribution of three subfamilies of HSat2 (A1, A2, B) sequences on individual human chromosomes. Further, using the HSat subfamily annotations in the T2T completed centromere satellite (CenSat) sequence, we isolated, defined and mapped differentially expressed sequence variants of nuclear-restricted HSat2 and HSat3 RNA from cancer cell lines and heat-shocked cells. We identified chromosome-specific and subfamily-specific expression of HSat2 and HSat3 and established a computational pipeline for differential expression analysis of tandemly repeated satellite sequences. Results suggest the differential expression of chromosome-specific HSat2 arrays in the human genome may underlie their accumulation in cancer cells and that specific HSat3 loci are upregulated upon heat shock. [ABSTRACT FROM AUTHOR]

Folsomia candida is a tiny soil-living arthropod belonging to the Collembola, which is an outgroup to Insecta. It resembles insects as having a pair of antennae and three pairs of thorax legs, while it also possesses three abdominal appendages: a ventral tube located in the first abdominal segment (A1), a retinaculum in A3, and a furca in A4. Collembolan Ubx and AbdA specify abdominal appendages, but they are unable to repress appendage marker gene Dll. The genetic basis of collembolan appendage formation and the mechanisms by which Ubx and AbdA regulate Dll transcription and appendage development remains unknown. In this study, we analysed the developmental transcriptomes of F. candida and identified candidate appendage formation genes, including Ubx (FcUbx). The expression data revealed the dominance of Dll over Ubx during the embryonic 3.5 and 4.5 days, suggesting that Ubx is deficient in suppressing Dll at early appendage formation stages. Furthermore, via electrophoretic mobility shift assays and dual luciferase assays, we found that the binding and repression capacity of FcUbx on Drosophila Dll resembles those of the longest isoform of Drosophila Ubx (DmUbx_Ib), while the regulatory mechanism of the C-terminus of FcUbx on Dll repression is similar to that of the crustacean Artemia franciscana Ubx (AfUbx), demonstrating that the function of collembolan Ubx is intermediate between that of Insecta and Crustacea. In summary, our study provides novel insights into collembolan appendage formation and sheds light on the functional evolution of Ubx. Additionally, we propose a model that collembolan Ubx regulates abdominal segments in a context-specific manner. [ABSTRACT FROM AUTHOR]

Environmental stresses are responsible for limiting soybean yield. To mitigate the impacts generated by water deficit, molecular biology tools are being used to develop genetically modified plants. Previous studies showed that two independent events (B1 and B3) of soybean transgenic plants expressing a Solanum nigrum osmotin (SnOLP) had an increment in drought tolerance. The present study aims to investigate the modulated pathways that results in the drought tolerance promoted by osmotin overexpression in soybean. Transgenic and non-transgenic (NT) plants in the vegetative stage were submitted to water deficit by irrigation suppression for seven days. Control plants were kept irrigated. Physiological variables were monitored and confirmed that the transgenic plants present better performance when compared to the NT plants. The total RNA extracted from leaves was sequenced and data was normalized by DESeq2. A total of 2044 and 1505 differentially expressed genes (DEGs) were identified in B1 and B3 events, respectively. Regarding the B1 event, 769 genes were upregulated and 1275 downregulated. For B3, 541 genes were upregulated and 964 genes were downregulated. Excluding common differentially expressed genes (DEGs) between transgenic and non-transgenic (NT) plants yielded 395 upregulated and 234 downregulated genes, which were shared by B1 and B3 events. The metabolic pathways and gene ontology categories identified are known to be involved in plant responses to drought. Hormonal, photosynthetic, carbohydrate and amino acid metabolism, reactive oxygen species, and post-translational modifications pathways were significantly modulated in transgenic plants. Altogether, the results suggest that osmotin promotes tolerance through an increment in the plant responses elicited by drought. [ABSTRACT FROM AUTHOR]

Endoplasmic reticulum (ER) stress is a cellular condition induced by environmental stressors. Transcriptomic approaches reveal the response of plants to ER stress and provide a broadened view. Tomato plants were treated with tunicamycin, and total RNA isolated from the tomato leaves was sequenced on an RNA-sequencing platform to be analyzed for differential expressions. A total of 856 differentially expressed genes (DEGs) were discovered in tunicamycin-treated tomato leaves. Upregulation of ER stress marker gene expressions was detected after 2h, while a prolonged ER stress downregulated most of the protein-coding genes. Pathways such as: response to stress, jasmonic acid mediated signaling, signal transduction regulation, plant hormone signal transduction, and protein processing in ER were enriched. Prolonged stress mostly resulted in reduced transcript levels, possibly related to a mechanism functioning to lower the load of nascent transcripts in the ER. Our findings emphasize the key role of JA-regulated signaling in tomato ER stress responses due to the differential expressions of the pathway components, along with other plant hormones and signaling-related or regulatory genes. ER stress being a common response to many stressors in plants, transcriptome data obtained here will provide for further studies of understanding plant stress tolerance or generating stress-resistant tomato plants. [ABSTRACT FROM AUTHOR]

Auxin and gibberellin significantly influence plant pistil development, but their specific roles in the formation of the three pistils trait in wheat are still unclear. To investigate the effects of auxin and gibberellin on the three pistils trait in wheat, we measured the contents of auxin and gibberellin in young spikes from three pistils wheat (TP and CM28TP) and single pistil wheat (CM28) at three stages. We also performed transcriptome sequencing. A total of 14 different auxins and 7 different gibberellins were detected from all samples, among which the contents of IAA-Glc and GA19 showed significant differences among different samples. The transcriptome sequencing yielded over 1.28 billion clean reads and 3.2 million transcripts. A total of 2,018 and 6,163 differentially expressed genes (DEGs) were identified from the comparisons of CM28 vs CM28TP and CM28 vs TP, respectively. Through the correlation analysis between auxin, gibberellin, and DEGs, 4 ARF genes, 3 AMI genes, and 3 DELLA genes were identified. These results suggest that IAA-Glc, GA19, and the 10 key genes were crucial factors in the formation of three pistils trait. Further analysis showed that ARF expression level and GA contents were negatively correlated with three pistils trait, while DELLA expression level and auxin contents were positively correlated. This study suggests that auxin and gibberellin signal transduction have an antagonistic relationship during wheat flower development, influencing the formation of the three pistils. This discovery helps further understanding of the mechanism behind the formation of the three pistils trait and provides theoretical support for the cultivation of high-yield wheat. [Display omitted] • IAA-Glc, a storage forms of IAA, accumulates in wheat's three pistils formation. • Ten key genes were identified by correlation analysis of IAA, GA content & DEGs. • ARF and DELLA family genes express differently in wheat's three pistils formation. • Auxin and gibberellin may collaboratively influence the formation of three pistils. [ABSTRACT FROM AUTHOR]

The global prevalence of iron deficiency-induced "hidden hunger" highlights a critical health concern, underscoring the pressing need to improve iron nutrition through safe and efficient means, such as increasing iron intake from plant-based foods. Yellow Stripe-Like (YSL) genes play a crucial role in long-distance iron transport between source and sink tissues in plants. Here, we report on the analysis of YSL family genes in the common bean (Phaseolus vulgaris L.), an iron-rich legume crop. We identified 9 YSL genes in the common bean genome using BLAST and HMM methods. Gene duplication analysis revealed that PvYSL7a and PvYSL7b originated through tandem duplication events. Structural analysis noted an absence of conservative motifs in PvYSL3b and PvYSL7a, which led to distinct predicted 3D protein structures. Leveraging publicly available RNA-seq data from developing bean pods, the expression patterns of PvYSL genes alongside pod and seed development were analyzed. Notably, PvYSL7a and PvYSL7b, as well as PvYSL1a and PvYSL1b, exhibited diverged expression patterns in seeds, signifying their functional divergence in this tissue. Moreover, PvYSL3a and PvYSL3b exhibited divergent expression patterns in both pod walls and seeds during pod development, underscoring their distinct roles in facilitating iron transportation between pods and seeds. This study provides valuable insights into the gene regulatory basis of iron accumulation in bean pods and seeds. [ABSTRACT FROM AUTHOR]

Plexus injury results in lifelong suffering from flaccid paralysis, sensory loss, and intractable pain. For this clinical problem, regenerative medicine concepts set high expectations. However, it is largely unknown how dorsal root ganglia (DRG) are affected by accidental deafferentation. Here, we phenotyped DRG of a clinically and MRI-characterised cohort of 13 patients with plexus injury. Avulsed DRG were collected during reconstructive nerve surgery. For control, we used DRG from forensic autopsy. The cellular composition of the DRG was analysed in histopathological slices with multicolour high-resolution immunohistochemistry, tile microscopy, and deep-learning-based bioimage analysis. We then sequenced the bulk RNA of corresponding DRG slices. In about half of the patients we found loss of the typical DRG units consisting of neurones and satellite glial cells. The DRG cells were replaced by mesodermal/connective tissue. In the remaining patients, the cellular units were well preserved. Preoperative plexus MRI neurography was not able to distinguish the two types. Patients with 'neuronal preservation' had less maximum pain than patients with 'neuronal loss'. Arm function improved after nerve reconstruction, but severe pain persisted. Transcriptome analysis of preserved DRGs revealed expression of subtype-specific sensory neurone marker genes, but downregulation of neuronal attributes. Furthermore, they showed signs of ongoing inflammation and connective tissue remodelling. Patients with plexus injury separate into two groups with either neuronal preservation or neuronal loss. The former could benefit from anti-inflammatory therapy. For the latter, studies should explore mechanisms of neuronal loss especially for regenerative approaches. DRKS00017266. [ABSTRACT FROM AUTHOR]

Rice production is severely hampered by drought stress, which causes enormous economic losses. The issue of global climate change is gaining importance, and hence development of rice genotypes tolerant to drought stress is becoming more critical. To address this issue, backcross inbred lines (BILs) developed were subjected to drought stress, and their molecular mechanism was studied. The drought‐tolerant parent Apo and drought‐susceptible, high‐yielding IR64 along with two BILs, namely, CB 229 (qDTY2.2 + qDTY3.1 + qDTY8.1) and CB 193‐3 (qDTY3.1 + qDTY8.1) were tested in a greenhouse for their response to drought. In this study, CB 229 showed better performance under water stress irrigated conditions; it was on par with IR64. Drought‐responsive transcriptome profiling was carried out in both the parents and the superior BIL CB 229 through the RNA‐Seq approach. About 3050 differentially expressed genes (DEGs) (2021 upregulated and 1029 downregulated) were detected in tolerant BIL CB 229 in drought stress. Most of the DEGs were involved in carbohydrate metabolism and the formation of cell walls, as well as genes associated with metabolite adaptability, ROS homeostasis and post‐transcriptional regulation. Genes such as chaperone protein, senescence‐induced receptor‐like serine, mannose‐6‐phosphate isomerase, aquaporin and heat shock proteins (LOC_Os02g26840, LOC_Os02g25720, LOC_Os07g35570, LOC_Os01g64970, etc.) were upregulated in the tolerant Apo and CB 229. It was observed that the BIL CB 229 yielded higher grains than both parents under moisture stress. Ninety‐four genes in the quantitative trait loci (QTLs) region were found to be differentially regulated in CB 229, Apo and IR64. Out of 94, nine genes co‐localized within the QTL qDTY2.2, 12 genes within qDTY3.1 and four genes within qDTY8.1 were differentially upregulated in CB 229 and downregulated in the susceptible genotype. The study revealed that the QTLs qDTY2.2, qDTY3.1 and qDTY8.1 are found to have complementary effects and offer enhanced levels of tolerance against drought due to complementation. Additionally, this analysis discovered new DEGs that may be involved in functions related to drought tolerance but lack function annotations. Future research can focus on a few of the significant DEGs found in this study. Taken together, our findings provide insight into drought tolerance mechanisms and could assist the development of novel strategies for improving drought tolerance in rice. [ABSTRACT FROM AUTHOR]

Summary: Arsenic poses a global threat to living organisms, compromising crop security and yield. Limited understanding of the transcriptional network integrating arsenic‐tolerance mechanisms with plant developmental responses hinders the development of strategies against this toxic metalloid.Here, we conducted a high‐throughput yeast one‐hybrid assay using as baits the promoter region from the arsenic‐inducible genes ARQ1 and ASK18 from Arabidopsis thaliana, coupled with a transcriptomic analysis, to uncover novel transcriptional regulators of the arsenic response.We identified the GLABRA2 (GL2) transcription factor as a novel regulator of arsenic tolerance, revealing a wider regulatory role beyond its established function as a repressor of root hair formation. Furthermore, we found that ANTHOCYANINLESS2 (ANL2), a GL2 subfamily member, acts redundantly with this transcription factor in the regulation of arsenic signaling. Both transcription factors act as repressors of arsenic response. gl2 and anl2 mutants exhibit enhanced tolerance and reduced arsenic accumulation. Transcriptional analysis in the gl2 mutant unveils potential regulators of arsenic tolerance.These findings highlight GL2 and ANL2 as novel integrators of the arsenic response with developmental outcomes, offering insights for developing safer crops with reduced arsenic content and increased tolerance to this hazardous metalloid. [ABSTRACT FROM AUTHOR]