Transcriptomic Changes Research Articles

Physiological and transcriptome analysis reveals the mechanism of Gymnocarpos przewalskii response to drought stress

BackgroundGymnocarpos przewalskii Bunge ex Maxim. (G. przewalskii) is an endangered xerophytic shrub that plays a crucial role as a source of forage in the Alxa Desert. However, there is limited understanding regarding the forage quality of G. przewalskii and its response to drought. This study aimed to evaluate the forage quality of G. przewalskii and investigate the physiological and transcriptomic changes in G. przewalskii response to drought stress.ResultsThe ash, fat, crude protein, lignin, crude fiber, acid detergent fiber, and neutral detergent fiber contents in G. przewalskii twigs were 10.61%, 1.85%, 5.68%, 7.08%, 21.23%, 42.16%, and 58.42%, respectively. In contrast, these ingredients in its leaves were 20.39%, 0.92%, 11.96%, 2.40%, 17.51%, 14.29% and 20.26%, respectively. Osmotic stress led to a reduction in chlorophyll levels and an increase in malondialdehyde content. Levels of hydrogen peroxide and oxygen free radicals remained relatively stable under osmotic stress. The proline content, SOD and CAT activities, and ·OH scavenging capacity were enhanced in G. przewalskii under osmotic stress. RNA-sequencing of G. przewalskii generated 44.51 Gb clean reads, which were assembled into 102,191 Unigenes and 30,809 Unigenes were successfully annotated. Comparative analysis identified 3,015 differentially expressed genes under osmotic stress. There were 2,134 and 1,739 DEGs enriched in 47 GO secondary categories and 129 KEGG pathways, respectively. 2 up-regulated DEGs were annotated to P5CS, a key enzyme in the biosynthesis of proline. 32 DEGs were annotated to various antioxidases and antioxidants. 81 DEGs were annotated to 8 plant hormone signaling pathways, in which the auxin and ABA signaling pathways exhibited dominant enrichment. 150 DEGs were annotated to 35 transcription factor families with the abundant enrichment of TF families containing WRKY, bZIP, ERF, bHLH, MYB, and NAC.ConclusionsHigh forage quality and drought stress tolerance were observed in G. przewalskii. In response to drought stress, G. przewalskii orchestrates reactive oxygen species scavenging, proline biosynthesis, and other intricate physiological processes, with substantial contributions from plant hormones and transcription factors. This study provides new insights into the forage quality and the mechanisms involved in drought adaptation of G. przewalskii, offering a foundation for its conservation and sustainable utilization.

Loss of the neuronal kinase DCLK3 leads to anxiety-like behaviour and memory deficits.

DCLK3 (Doublecortin-like kinase 3) is a kinase preferentially expressed in neurons. Dclk3 variants are risk factors for psychiatric disorders and brain alterations linked to age-related mild cognitive impairment, suggesting this kinase could be important to cognition. However, the physiological role of DCLK3 remains unknown. Here, we generated and characterized mice with constitutive or brain-region specific Dclk3 knockout. We found that constitutive pan-deletion of Dclk3 is associated with an anxiety phenotype in male, associated with changes in brain metabolites in absence of major neuroanatomical alterations, motor or memory deficits. Moreover, virally mediated loco-regional conditional Dclk3 knockout in the dorsal hippocampus of adult mice led to spatial memory deficits and transcriptomic changes, characterized by increased expression of α2, β1, and β2 subunits of the GABAA receptor, down-regulation of neuronal activity-regulated genes such as the immediate early genes (e.g. Egr1, Fos, Per1 and Nr4r1), and transcriptional regulator-activity enriched in polycomb-repressive complexes (PRC). These new data reveal that Dclk3 modulates behavior and cognition in association with transcriptomic changes in the hippocampus, providing direct physiological evidence that this kinase is involved in synaptic plasticity, consistent with its crucial role in neurodegenerative and psychiatric diseases.

An Endogenous Aryl Hydrocarbon Receptor Ligand Dysregulates Endothelial Functions, Transcriptome, and Phosphoproteome.

We have reported that an endogenous aryl hydrocarbon receptor (AhR) ligand, 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), inhibits functions of human umbilical vein endothelial cells (HUVECs) and induces preeclampsia (PE)-like symptoms in rats. Herein, we tested the hypothesis that ITE impairs endothelial functions via disturbing transcriptome and phosphoproteome in HUVECs. We measured AhR activity in human maternal and umbilical vein sera from PE and normotensive (NT) pregnancies. The serum-induced changes in CYP1A1/B1 mRNA (indexes of AhR activation) in HUVECs were quantified using RT-qPCR. ITE's effects on endothelial proliferation and monolayer integrity in female and male HUVECs were determined. We profiled ITE-induced changes in transcriptome and phosphoproteome in HUVECs using RNA-seq and bottom-up phosphoproteomics, respectively. After 12 hr of treatment, umbilical vein sera from PE increased CYP1A1 mRNA (1.7-fold of NT) HUVECs, which was blocked by CH223191, an AhR antagonist. ITE dose-dependently inhibited endothelial proliferation (76%-87% of control) and time-dependently reduced endothelial integrity with a maximum inhibition (~ 10%) at 40 hr. ITE induced 140 and 80 differentially expressed genes in female and male HUVECs, respectively. ITE altered phosphorylation of 92 and 105 proteins at 4 and 24 hr, respectively, in HUVECs. These ITE-dysregulated genes and phosphoproteins were enriched in biological functions and pathways are relevant to heart, liver, and kidney diseases, vascular functions, and inflammatory responses. Thus, endogenous AhR ligands may impair endothelial functions by disturbing transcriptome and phosphoproteome. These AhR ligand-dysregulated genes and phosphoproteins may be therapeutic and cell sex-specific targets for PE-induced endothelial dysfunction.

Impaired primitive erythropoiesis and defective vascular development in Trim71-KO embryos.

The transition of an embryo from gastrulation to organogenesis requires precisely coordinated changes in gene expression, but the underlying mechanisms remain unclear. The RNA-binding protein Trim71 is essential for development and serves as a potent regulator of post-transcriptional gene expression. Here, we show that global deficiency of Trim71 induces severe defects in mesoderm-derived cells at the onset of organogenesis. Murine Trim71-KO embryos displayed impaired primitive erythropoiesis, yolk sac vasculature, heart function, and circulation, explaining the embryonic lethality of these mice. Tie2 Cre Trim71 conditional knockout did not induce strong defects, showing that Trim71 expression in endothelial cells and their immediate progenitors is dispensable for embryonic survival. scRNA-seq of E7.5 global Trim71-KO embryos revealed that transcriptomic changes arise already at gastrulation, showing a strong up-regulation of the mesodermal pioneer transcription factor Eomes. We identify Eomes as a direct target of Trim71-mediated mRNA repression via the NHL domain, demonstrating a functional link between these important regulatory genes. Taken together, our data suggest that Trim71-dependent control of gene expression at gastrulation establishes a framework for proper development during organogenesis.

Transcriptome analysis reveals hypoxic response key genes and modules as well as adaptive mechanism of crucian carp (Carassius auratus) gill under hypoxic stress

Fish gill tissue is a primary organ responsive to acute oxygen deprivation or dissolved oxygen (DO) fluctuations in aquatic environments. However, the adaptive mechanism of crucian carp to hypoxic stress remains largely unknown. Here, we investigated gill physiological and transcriptomic changes of crucian carp exposed to hypoxic conditions (dissolved oxygen concentration of 0.6 ± 0.3 mg/L) for different durations (0 d, 1 d, 2d, 3d, 4 d, and 5d). Transcriptomic analysis revealed that the hypoxia group (0.6 ± 0.3 mg/L DO) exhibited a reduction in interlamellar cell mass (ILCM) on the gill filaments, compared with the control group (6.6 ± 0.3 mg/L DO). With prolonged hypoxia stress, the epithelial cells in the gill lamellae became sparse at 3 d to 5 d, and gill vacuoles were increased. A total of 3,502 differentially expressed genes (DEGs) were identified, and 3 hypoxia-specific modules were screened through differential expression analysis, weighted gene co-expression network analysis (WGCNA), and Bayesian network analysis. The apoptosis, necroptosis, efferocytosis and FoxO signaling pathways were significantly enriched based on the KEGG enrichment pathway analysis. The VEGF pathway genes are significantly expressed, enhancing the generation of microvessels in the gill filaments, and improving the capacity to carry oxygen, thus enabling the crucian carp to adapt to hypoxia stress. Hypoxia activated glycolysis, enhanced anaerobic metabolism, promoted β-oxidation of fatty acids, providing energy and maintaining normal physiological metabolism, eventually improving antioxidant and immune capabilities in crucian carp. In summary, this study reveals the molecular mechanism by which crucian carp adapt to hypoxic stress. Our findings provide valuable references for promoting the healthy aquaculture of hypoxic-sensitive fish and breeding hypoxia-tolerant fish varieties.

Better Transcriptomic Stability and Broader Transcriptomic Thermal Response Range Drive the Greater Thermal Tolerance in a Global Invasive Turtle Relative to Native Turtle.

Greater thermal tolerance of invasive species benefits their survival and spread under extreme climate events, especially under global warming. Revealing the mechanisms underlying the interspecific differences in thermal tolerance between invasive and native species can help understand the invasion process and predict potential invaders. Here, we link the changes in global transcriptomics and antioxidant defense at multiple temperatures with the differences in thermal limits in the juveniles of a successful globally invasive turtle, Trachemys scripta elegans, and a native turtle in China, Mauremys reevesii. The two species show different thermal tolerances and have co-existed in habitats with the risk of overheating. The majority of the transcriptional response to thermal stress is conserved in the two turtle species, including protein folding or DNA damage responses activated under relatively moderate thermal stress and regulation of the cell cycle and apoptosis during severe thermal stress. Greater thermal tolerance of T. scripta elegans can be associated with a more stable global transcriptome during thermal stress, except for necessary stress responses, and a broader thermal range of continuous up-regulation of the core mechanisms promoting survival under thermal stress, mainly protein folding and negative regulation of apoptosis. Under extreme hot conditions, the opposite change trends of genes involved in survival mechanisms during thermal stress between invasive and native turtles can be due to differences in energy turnover. The present study provides insights into the mechanisms of physiological differences between invasive and native species given global transcriptional changes and helps understand successful invasion and predict potential invasive species.

Integrated transcriptomic and metabolomic analyses reveal critical gene regulatory network in response to drought stress in Dendrobium nobile Lindl

BackgroundDendrobium nobile Lindl belongs to the genus Dendrobium of the orchid family and is a valuable herbal medicine. Drought stress severely affects the growth of D. nobile Lindl; however, the specific regulatory mechanisms have not yet been elucidated.ResultsIn the present study, we conducted a combined transcriptome and metabolome analysis of D. nobile Lindl stems under different drought stress conditions. Global transcriptomic changes were detected in Dendrobium under different drought stress conditions. KEGG enrichment analysis showed that the DEGs were enriched in plant hormone signal transduction; cutin, suberin, and wax biosynthesis; starch and sucrose metabolism; and the biosynthesis of various plant secondary metabolites. The differentially abundant metabolites (DAMs) detected using STEM analysis were enriched in pathways associated with glucosinolate biosynthesis and cyanoamino acid metabolism. We constructed a regulatory network for the drought tolerance of Dendrobium by weighted gene co-expression analysis.ConclusionsThe results showed that arginine and proline metabolism, glucosinolate biosynthesis and tyrosine metabolism pathways participated in regulating drought stress in D. nobile Lindl. Our study provides a theoretical basis for studying the drought resistance mechanisms in Dendrobium.

Conventional and antibody-enhanced DENV infection of human macrophages induces differential immunotranscriptomic profiles.

Dengue virus (DENV) is a mosquito-borne flavivirus which coexists as four genetically and immunologically distinct serotypes (DENV-1 to -4). In secondary heterologous DENV infection, pre-existing immunity is believed to contribute to severe disease through antibody-dependent enhancement (ADE). Although the elevated pathology observed in ADE conditions has been described, the cell-intrinsic mechanisms governing this process remain unclear. Using single-cell RNA sequencing (scRNAseq), we investigated the transcriptomic profiles of human monocyte-derived macrophages infected by DENV-2 in ADE compared to conventional infection conditions. Unsupervised analysis of scRNAseq data enabled the identification of two distinct cell populations in a heterogeneous cell culture, likely representing infected and bystander/uninfected cells. Differential gene expression and ingenuity pathway analyses revealed a number of significantly upregulated and downregulated genes and gene networks between cells infected by ADE compared to conventional infection. Specifically, these pathways indicated mechanisms such as suppressed interferon signaling and inflammatory chemokine transcription in cells infected via ADE. Further analysis revealed that transcriptomic changes were independent of viral RNA within infected cells, suggesting that the observed changes are reflective of cell-intrinsic responses and not simply a function of per-cell viral burden. The interpreted "bystander" cell population also demonstrated distinct profiles in ADE conditions, indicating an immunologically activated phenotype enriched for the expression of gene networks involved with protein translation, cytokine production, and antigen presentation. Together, these findings support the concept that DENV infection via ADE induces a qualitatively different transcriptomic response in infected cells, contributing to our understanding of ADE as a mechanistic driver of disease and pathogenesis.IMPORTANCEDengue virus (DENV) is a mosquito-borne human pathogen with a significant and growing global health burden. Although correlates of severe dengue disease are poorly understood, pre-existing immunity to DENV has been associated with severe disease risk and known to contribute to an alternative route of viral entry termed antibody-dependent enhancement (ADE). Using single-cell RNA sequencing, we identified distinct transcriptomic processes involved in antibody-mediated DENV entry compared to conventional receptor-mediated entry. These data provide meaningful insight into the discrete processes contributing to DENV pathogenesis in ADE conditions.

Mediator kinase inhibition drives myometrial stem cell differentiation and the uterine fibroid phenotype through super-enhancer reprogramming.

Uterine fibroids (UFs) are the most common non-cutaneous tumors in women worldwide. UFs arise from genetic alterations in myometrial stem cells (MM SCs) that trigger their transformation into tumor-initiating cells (UF SCs). Mutations in the RNA polymerase II Mediator subunit MED12 are dominant drivers of UFs, accounting for 70% of these clinically significant lesions. Biochemically, UF driver mutations in MED12 disrupt CDK8/19 kinase activity in Mediator, but how Mediator kinase disruption triggers MM SC transformation remains unknown. Here, we show that pharmacologic inhibition of CDK8/19 in MM SCs removes a barrier to myogenic differentiation down an altered pathway characterized by molecular phenotypes characteristic of UFs, including oncogenic growth and extracellular matrix (ECM) production. These perturbations appear to be induced by transcriptomic changes, arising in part through epigenomic alteration and super-enhancer reprogramming, that broadly recapitulate those found in MED12-mutant UFs. Altogether, these findings provide new insights concerning the biological role of CDK8/19 in MM SC biology and UF formation. KEY MESSAGES: Mediator kinase inhibition in myometrial stem cells (MM SCs) induces spontaneous differentiation. Transcriptional changes upon Mediator kinase inhibition recapitulate those of MED12 mutant uterine fibroids (UFs). Such transcriptional changes are partially mediated by super-enhancer reprogramming. Mediator kinase functions to enforce cell states and its loss induces cellular plasticity.

Abstract TP367: Role of STAT1 and type I interferon signaling in ischemic preconditioning-induced microglial responses

Background: Ischemic preconditioning (IPC) is a robust protective phenomenon in which a brief ischemic exposure confers tolerance against a subsequent prolonged ischemic challenge. Elucidating the mechanisms of IPC is a critical challenge in the stroke field. Innate immune pathways, particularly type I interferon (IFN) signaling, in microglia are critical in establishing protection in both grey and white matter. Our previous studies demonstrated that type I IFN receptor (IFNAR1) in microglia is required for both IPC-induced axonal protection and interferon stimulated gene (ISG) expression. We also showed that exposure of primary microglia to either IFN-beta or ischemia/reperfusion-like conditions results in phosphorylation of STAT1, a key signaling kinase downstream of IFNAR1. Therefore, we hypothesized that STAT1 is a critical mediator of the microglial response to IPC. Here we report the impact of systemic STAT1 knock-out on microglial responses after IPC. Methods: We performed a transient (15 min) middle cerebral artery occlusion on 4 wild-type (WT) and 4 Stat1 -/- mice and collected tissue 72 hours later. We processed tissue for single nucleus RNA-sequencing using 10X Genomics kits. We captured ~10,000 nuclei per sample and sequenced at a depth of 25,000 reads. We performed comparative analysis between ipsilateral and contralateral hemispheres to identify focal changes in microglial subpopulations and gene expression due to IPC. We performed immunofluorescent microscopy and quantitative stereology to validate IPC-induced changes in microglial number/morphology and expression of ISG protein products such as IFITM3. Results: We describe global in vivo transcriptomic changes in microglial subpopulations after IPC and found that STAT1-deficiency alters both the distribution of microglial subpopulations and cluster-specific transcriptional profiles. We identify and define an interferon responsive microglial cluster induced by IPC that is dependent on STAT1. We also provide data demonstrating how ischemia/reperfusion and innate immune signaling affect STAT1 phosphorylation signaling dynamics in microglia in vitro. We show that phosphorylation is dependent on both Toll-like receptor 4 (TLR4) and IFNAR1. Conclusions: Our results support a central role for STAT1 in mediating microglial phenotype in vivo following IPC. Our findings indicate that STAT1 is an important regulator of microglial type I IFN signaling in the context of ischemia.

Transcriptomic changes and mitochondrial toxicity in response to acute and repeat dose treatment with brequinar in human liver and kidney in vitro models.

The potent dihydroorotate dehydrogenase (DHODH) inhibitor brequinar has been investigated as an anticancer, immunosuppressive, and antiviral pharmaceutical agent. However, its toxicity is still poorly understood. We investigated the cellular responses of primary human hepatocytes (PHH) and telomerase-immortalised human renal proximal tubular epithelial cells (RPTEC/TERT1) after a single 24-h exposure up to 100 μM brequinar. Additionally, RPTEC/TERT1 cells underwent repeated daily exposure for five consecutive days at 0.3, 3, and 20 μM. Transcriptomic analysis revealed that PHH were less sensitive to brequinar treatment than RPTEC/TERT1 cells. Upregulation of various phase I and II drug-metabolising enzymes, particularly Cytochrome P450 (CYP) 1 A and 3 A enzymes, in PHH suggests potential detoxification. Furthermore, brequinar exposure led to a significant upregulation of several stress response pathways in PHH and RPTEC/TERT1 cells, including the unfolded protein response, Nrf2, p53, and inflammatory responses. RPTEC/TERT1 cells exhibited greater sensitivity to brequinar at 0.3 μM with repeated exposure compared to a single exposure. Furthermore, brequinar could impair the mitochondrial respiration of RPTEC/TERT1 cells after 24 h. This study provides new insights into the differential responses of PHH and RPTEC/TERT1 cells in response to brequinar exposure and highlights the biological relevance of implementing repeated dosing regimens in in vitro studies.

Transcriptomic changes during induction of tetrasporogenesis in the red seaweed Asparagopsis armata

Transcriptomic changes during induction of tetrasporogenesis in the red seaweed Asparagopsis armata

Single-cell RNA sequencing reveals tissue-specific transcriptomic changes induced by perfluorooctanesulfonic acid (PFOS) in larval zebrafish (Danio rerio)

Single-cell RNA sequencing reveals tissue-specific transcriptomic changes induced by perfluorooctanesulfonic acid (PFOS) in larval zebrafish (Danio rerio)

Neonatal exposure to morphine results in prolonged pain hypersensitivity during adolescence, driven by gut microbial dysbiosis and gut-brain axis-mediated inflammation.

Opioids, such as morphine, are used in the Neonatal Intensive Care Unit (NICU) for pain relief in neonates. However, the available evidence concerning the benefits and harms of opioid therapy in neonates remains limited. While previous studies have reported that neonatal morphine exposure (NME) results in long-term heightened pain sensitivity, the underlying mechanisms are not well understood. This study proposes that dysbiosis of the gut microbiome contributes to pain hypersensitivity following NME. Using an adolescent female murine model, pain sensitivity was evaluated using tail flick and hot plate assays for thermal pain and the Von Frey assay for mechanical pain. Gut microbiome composition was assessed using 16 s rRNA sequencing, while transcriptomic changes in midbrain samples were investigated using bulk RNA-sequencing. NME induced prolonged hypersensitivity to thermal and mechanical pain in adolescence, accompanied by persistent gut microbial dysbiosis and sustained systemic inflammation, characterized by elevated circulating cytokine levels (e.g., IL-1α, IL-12p70, IFN-γ, IL-10). Transplantation of the microbiome from NME adolescents recapitulated pain hypersensitivity in naïve adolescent mice, while neonatal probiotic intervention with Bifidobacterium infantis (B. infantis) reversed the hypersensitivity by preventing gut dysbiosis and associated systemic inflammation. Furthermore, transcriptomic analysis of the midbrain tissues revealed that NME upregulated several genes and key signaling pathways, including those related to immune activation and excitatory signaling, which were notably mitigated by neonatal B. infantis administration. Together, these findings highlight the critical role of the gut-brain axis in modulating pain sensitivity and suggest that targeting the gut microbiome offers a promising therapeutic strategy for managing neurobiological disorders following early opioid exposure.

Transcriptomic landscapes of STING-mediated DNA-sensing reveal cellular response heterogeneity.

Transfection of plasmid DNA (pDNA) encoding target genes is a routine tool in gene function studies and therapeutic applications. However, nucleic acid-sensing-mediated innate immune responses influence multiple intracellular signaling pathways. The stimulator of interferon genes (STING) is a crucial adapter protein for DNA sensors in mammalian cells. In this study, we explored the molecular mechanisms underlying DNA sensing by investigating the relationship between mRNA and protein expression levels and the STING pathway using single-cell analysis. We observed that reporter gene expression was dose-nonlinear after transfection of pDNA in cells with intact DNA-sensing pathways. Moreover, blocking the STING pathway in THP-1 cells significantly downregulated innate immune responses, upregulated exogenous gene expression, and mitigated the effects of innate immune responses on cell and gene function, but did not affect the proportion of reporter protein-positive cells. We elucidated the mechanisms of DNA sensing-induced innate immune response and cell death by analyzing heterozygous cellular responses to DNA transfection and transcriptome changes in positive cells. These findings suggest that the regulation of STING-mediated nucleic acid-sensing pathways is crucial for the accuracy of gene function studies and could enhance the efficacy of gene therapy.

Abstract TP343: Co-Expression Modules In The Periphreal Blood Transcriptome Following Subarachnoid Hemorrhage Associate With 90-Day Outcome

Background: Subarachnoid Hemorrhage (SAH) accounts for 2-7% of strokes and has high mortality and morbidity. We sought to identify peripheral blood transcriptome changes in the acute SAH phase that associated with 90-day outcome, as measured by the modified Rankin Scale (mRS), to gain insights about potential mechanisms contributing to long term outcome. Methods: We sequenced the peripheral blood transcriptome of SAH patients within 3 days post ictus and stratified the patients into patients with Good (mRS≤2, n=37) and Poor (mRS≥3, n=23) outcomes at 90-day follow up. We generated the co-expression networks using the Weighted Gene Co-Expression Network Analysis (WGCNA) package to determine modules (groups of co-expressed genes) associated with 90-day SAH outcome. The outcome-significant modules (p<0.05) were further analyzed for their biological relevance using pathway analysis. Results: We identified two outcome-significant modules, the Pink module and the Purple module. The Pink module was enriched (corrected p<0.05) in Monocyte- and Granulocyte-specific genes, while the Purple module and its hubs were enriched in Monocyte-specific genes. The hub genes are the most interconnected genes in each module, which are therefore potential master regulators. The Pink module was enriched (corrected p<0.05) in Neutrophil Degranulation, an inflammatory pathway which was predicted to be activated in patients with worse outcome, in Histone Modification Signaling, which is involved in epigenetic control of gene expression, and in SUMOylation of transcriptional cofactors, which confers post-transcriptional modifications of these cofactors. The Purple module was enriched in 45 canonical biological pathways, including numerous inflammatory pathways predicted to be activated in SAH patients with worse outcomes, such as Neutrophil Degranulation, Phagosome Formation, IL-8 Signaling, and Macrophage Classical Activation Signaling. Conclusions: Early peripheral blood changes in the transcriptome architecture following SAH are associated with long-term outcome. The identified genes and networks may guide the search for potential biomarkers of outcome and novel treatment targets.

A Head-Specific Transcriptomic Study Reveals Key Regulatory Pathways for Winter Diapause in the Mosquito Culex pipiens.

The primary vector of the West Nile virus, Culex pipiens, undergoes reproductive dormancy during the adverse winter season. While our current understanding has mainly focused on cellular signals and phenotypic shifts occurring at a global scale during diapause, information on tissue-specific transcriptomic changes remains limited. This knowledge gap is a major challenge in interpreting the regulatory mechanisms at the tissue level. To address this, the present work utilized RNA-seq technology to investigate the transcriptional changes in the head that house the brain and crucial endocrinal organs such as corpora allata. We obtained RNA samples from the heads of diapausing and nondiapausing female mosquitoes at two specific time intervals, ZT0 and ZT16, and then subjected them to sequencing. Our results revealed differences in differentially expressed genes between diapause and non-diapause at ZT0 and ZT16, highlighting the phenotypic and diel variations in gene expression. We also selected twelve genes associated with the diapause phenotype and examined the transcript abundance at six different time points over 24 h. qRT-PCR analysis showed similar up- and downregulation of transcripts between the diapause and nondiapause phenotypes thus validating the results of RNA-seq. In summary, our study identified new genes with phenotypic and diel differentiation in their expression, potentially linking photoperiod to seasonal reproductive dormancy in insects. The newly presented information will significantly advance our understanding of head-specific genes crucial for insect diapause.

A Chromosome-Level Genome Sequence Reveals Regulation of Salt Stress Response in Mesembryanthemum crystallinum.

Salt stress disturbs plant growth and photosynthesis due to its toxicity. The ice plant Mesembryanthemum crystallinum is a highly salt-tolerant facultative crassulacean acid metabolism (CAM) plant. However, the genetic basis of the salt tolerance mechanisms in ice plants remains unclear. In this study, we constructed a chromosome-level whole-genome sequence of the ice plant and performed transcriptome analysis of the effects of salt treatment on the leaves. After 24-hour 500 mM NaCl treatment to the roots, 1100 and 1394 genes, including CAM pathway, glycolysis, and inositol metabolism, were up- and down-regulated in the leaves, respectively. Salt treatment also influenced the abscisic acid (ABA) signaling components, including genes from the PYRABACTIN RESISTANCE1 (PYR1) family and the PROTEIN PHOSPHATASE 2CA (PP2CA) family. We detected the induction of the genes encoding various ion transporters after salt treatment. The expression of most v-ATPase subunits is induced, leading to vacuolar acidification, which facilitates sodium ion sequestration in the vacuoles. Additionally, some genes encoding metal ion transporters, including the genes from the ZIP family and NRAMP family, were induced by salt treatment, accompanied by the accumulation of iron, zinc, and copper ions in the leaves. Cis-motif enrichment analysis revealed that ABRE-like motifs and MYB-binding-like motifs were enriched in the upstream sequences of genes that were either up-regulated or down-regulated by salt. In conclusion, this study highlights how salt treatment induces drastic and rapid transcriptomic changes and unveils the ice plant's genomic foundation. Our resources provide further insights into the regulation of salt tolerance in the ice plants.

Effects of low-salt stress on biological characteristics and transcriptomic profiles of Vibrio parahaemolyticus.

Studies have proved that halophilic Vibrio parahaemolyticus is widely detected in freshwater environments (salinity <0.5%). However, the growth and colonization of V. parahaemolyticus in low-salt environments remain unclear. This study was envisaged to assess the effects of low-salt stress on the growth, motility and biofilm formation of V. parahaemolyticus and the transcriptomic changes that the bacterium responds to such stress. The results indicated that low salt concentrations supported the growth (allowing growth to proceed, though at a lower speed) of V. parahaemolyticus, prolonged the lag time (LT), and decreased the maximum specific growth rate (μmax) of V. parahaemolyticus. Additionally, this low salinity inhibited its motility and enhanced its biofilm formation capacity. Notably, the growth of V. parahaemolyticus on both freshwater and marine-cultured Litopenaeus vannamei exhibited a similar trend, suggesting that V. parahaemolyticus might have adapted to thrive in freshwater food. Furthermore, the reasons for the support of V. parahaemolyticus growth in 0.25% NaCl was analyzed by transcriptome sequencing (RNA-seq). RNA-seq revealed that V. parahaemolyticus can improve resistance to adverse environments by reducing energy consumption and enhancing oxidative stress resistance to adapt to a low-salt environment. This study revealed that the freshwater environment supported the growth of V. parahaemolyticus and its influence on the growth of V. parahaemolyticus, providing valuable theoretical support for risk assessment.

Local Inflammation Precedes Diaphragm Wasting and Fibrotic Remodelling in a Mouse Model of Pancreatic Cancer.

Cancer cachexia represents a debilitating muscle wasting condition that is highly prevalent in gastrointestinal cancers, including pancreatic ductal adenocarcinoma (PDAC). Cachexia is estimated to contribute to ~30% of cancer-related deaths, with deterioration of respiratory muscles suspected to be a key contributor to cachexia-associated morbidity and mortality. In recent studies, we identified fibrotic remodelling of respiratory accessory muscles as a key feature of human PDAC cachexia. To gain insight into mechanisms driving respiratory muscle wasting and fibrotic remodelling in response to PDAC, we conducted temporal histological and transcriptomic analyses on diaphragm muscles harvested from mice-bearing orthotopic murine pancreatic (KPC) tumours at time points reflective of precachexia (D8 and D10), mild-moderate cachexia (D12 and D14) and advanced cachexia (endpoint). During the precachexia phase, diaphragms showed significant leukocyte infiltration (+3-fold to +13-fold; D8-endpoint vs. Sham, p < 0.05) and transcriptomic enrichment of inflammatory processes associated with tissue injury that remained increased through endpoint. Diaphragm inflammation was followed by increases in PDGFR-ɑ+ fibroadipogenic progenitors (+2.5 to +3.8-fold; D10-endpoint vs. Sham, p < 0.05), fibre atrophy (-16% to -24%, D12 to endpoint vs. Sham, p < 0.05), ECM expansion (+1.5 to +1.8-fold; D14-endpoint vs. Sham, p < 0.05), collagen accumulation (+3.8-fold; endpoint vs. Sham, p = 0.0013) and reductions in breathing frequency (-55%, p = 0.0074) and diaphragm excursion (-43%, p = 0.0006). These biological processes were supported by changes in the diaphragm transcriptome. Ingenuity pathway analysis predicted factors involved in inflammatory responses to tissue injury, including TGF-β1, angiotensin and PDGF BB, as top upstream regulators activated in diaphragms prior to and throughout cachexia progression, while PGC-1α and the insulin receptor were among the top upstream regulators predicted to be suppressed. The transcriptomic dataset further revealed progressive disturbances to networks involved in lipid, glucose and oxidative metabolism, activation of the unfolded protein response and neuromuscular junction remodelling associated with denervation. In summary, our data support leukocyte infiltration and expansion of PDGFRα mesenchymal progenitors as early events that precede wasting and fibrotic remodelling of the diaphragm in response to PDAC that may also underlie metabolic disturbances, weakness and respiratory complications.