Stress Stimuli Research Articles

Exploring the possibility of a proprietary black cumin oil extract as a Dual Orexin Receptor Antagonist in restoring stress-sleep balance on stress-induced sleep deprived animals

BackgroundOrexins act as a molecular switch for the release of cortisol/corticosterone in response to a stress stimulus and hence to regulate sleep/wake cycle. Orexin agonism during the day promotes wakefulness, and Dual Orexin Receptor Antagonists (DORA) can promote sleep signals by enhancing melatonin, which is an inhibitor of orexin. It was reported that a proprietary black cumin (Nigella sativa) oil (BCO-5) alleviated stress and improved sleep quality. The present study investigated the mechanism of action of BCO-5 using stress-induced and sleep-deprived model of rats. MethodsAdult Sprague Dawley rats (n = 24) were randomised into 4 groups (Group I – Sham; Group II – Stress-induced group; Group III – BCO-5 treated normal animals; Group IV – Stress + BCO-5 (20 mg/kg b. wt.) for 14 days and monitored the behaviour and biochemical markers. ResultsThe co-supplementation of BCO-5 significantly decreased the body weight, locomotor activity, rearing and grooming frequencies among Group IV animals significantly compared to Group II. The observed behaviour was also correlated with the significant decrease in orexin, corticosterone and c-fos expression levels, while an increase was observed in melatonin concentration. ConclusionOur results support the plausible role of BCO-5 as a DORA to manage stress and improve sleep.

Revisiting p38 Mitogen-Activated Protein Kinases (MAPK) in Inflammatory Arthritis: A Narrative of the Emergence of MAPK-Activated Protein Kinase Inhibitors (MK2i).

The p38 mitogen-activated protein kinase (p38-MAPK) is a crucial signaling pathway closely involved in several physiological and cellular functions, including cell cycle, apoptosis, gene expression, and responses to stress stimuli. It also plays a central role in inflammation and immunity. Owing to disparate p38-MAPK functions, it has thus far formed an elusive drug target with failed clinical trials in inflammatory diseases due to challenges including hepatotoxicity, cardiac toxicity, lack of efficacy, and tachyphylaxis, which is a brief initial improvement with rapid disease rebound. To overcome these limitations, downstream antagonism of the p38 pathway with a MAPK-activated protein kinase (MAPKAPK, also known as MK2) blockade has demonstrated the potential to abrogate inflammation without the prior recognized toxicities. Such MK2 inhibition (MK2i) is associated with robust suppression of key pro-inflammatory cytokines, including TNFα and IL-6 and others in experimental systems and in vitro. Considering this recent evidence regarding MK2i in inflammatory arthritis, we revisit the p38-MAPK pathway and discuss the literature encompassing the challenges of p38 inhibitors with a focus on this pathway. We then highlight how novel MK2i strategies, although encouraging in the pre-clinical arena, may either show evidence for efficacy or the lack of efficacy in emergent human trials data from different disease settings.

The Current State of Research on Sirtuin-Mediated Autophagy in Cardiovascular Diseases.

Sirtuins belong to the class III histone deacetylases and possess nicotinamide adenine dinucleotide-dependent deacetylase activity. They are involved in the regulation of multiple signaling pathways implicated in cardiovascular diseases. Autophagy is a crucial adaptive cellular response to stress stimuli. Mounting evidence suggests a strong correlation between Sirtuins and autophagy, potentially involving cross-regulation and crosstalk. Sirtuin-mediated autophagy plays a crucial regulatory role in some cardiovascular diseases, including atherosclerosis, ischemia/reperfusion injury, hypertension, heart failure, diabetic cardiomyopathy, and drug-induced myocardial damage. In this context, we summarize the research advancements pertaining to various Sirtuins involved in autophagy and the molecular mechanisms regulating autophagy. We also elucidate the biological function of Sirtuins across diverse cardiovascular diseases and further discuss the development of novel drugs that regulate Sirtuin-mediated autophagy.

Novel Therapeutic Avenues for Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a complicated, heterogeneous genetic condition that causes left ventricular hypertrophy, fibrosis, hypercontractility, and decreased compliance. Despite the advances made over the past 3decades in understanding the molecular and cellular mechanisms aggravating HCM, the relationship between pathophysiological stress stimuli and distinctive myocyte growth profiles is still imprecise. Currently, mavacamten, a selective and reversible inhibitor of cardiac myosin ATPase, is the only drug approved by the US FDA for the treatment of HCM. Thus, there is an unmet need for developing novel disease-specific therapeutic approaches. This article provides an overview of emerging therapeutic targets for the treatment of HCM based on various molecular pathways and novel developments that are hopefully soon to enter the clinical study. These newly discovered targets include the dual specificity tyrosine-phosphorylation-regulated kinase 1B, the absence of the melanoma 1 inflammasome, the leucine-rich repeat kinase 2 enzyme, and the cluster of differentiation 147.

The S100 calcium-binding protein A6 plays a crucial role in hepatic steatosis by mediating lipophagy.

S100 calcium-binding protein A6 (S100A6) is a calcium-binding protein that is involved in a variety of cellular processes, such as proliferation, apoptosis, and the cellular response to various stress stimuli. However, its role in NAFLD and associated metabolic diseases remains uncertain. In this study, we revealed a new function and mechanism of S100A6 in NAFLD. S100A6 expression was upregulated in human and mouse livers with hepatic steatosis, and the depletion of hepatic S100A6 remarkably inhibited lipid accumulation, insulin resistance, inflammation, and obesity in a high-fat, high-cholesterol (HFHC) diet-induced murine hepatic steatosis model. In vitro mechanistic investigations showed that the depletion of S100A6 in hepatocytes restored lipophagy, suggesting S100A6 inhibition could alleviate HFHC-induced NAFLD. Moreover, S100A6 liver-specific ablation mediated by AAV9 alleviated NAFLD in obese mice. Our study demonstrates that S100A6 functions as a positive regulator of NAFLD, targeting the S100A6-lipophagy axis may be a promising treatment option for NAFLD and associated metabolic diseases.

The Beneficial and Adverse Effects of Autophagic Response to Caloric Restriction and Fasting.

Each cell is equipped with a conserved housekeeping mechanism, known as autophagy, to recycle exhausted materials and dispose of injured organelles via lysosomal degradation. Autophagy is an early-stage cellular response to stress stimuli in both physiological and pathological situations. It is thought that the promotion of autophagy flux prevents host cells from subsequent injuries by removing damaged organelles and misfolded proteins. As a correlate, the modulation of autophagy is suggested as a therapeutic approach in diverse pathological conditions. Accumulated evidence suggests that intermittent fasting or calorie restriction can lead to the induction of adaptive autophagy and increase longevity of eukaryotic cells. However, prolonged calorie restriction with excessive autophagy response is harmful and can stimulate a type II autophagic cell death. Despite the existence of a close relationship between calorie deprivation and autophagic response in different cell types, the precise molecular mechanisms associated with this phenomenon remain unclear. Here, we aimed to highlight the possible effects of prolonged and short-term calorie restriction on autophagic response and cell homeostasis.

A mathematical representation of the reactive scope model

Researchers have long sought to understand and predict an animal’s response to stressful stimuli. Since the introduction of the concept of homeostasis, a variety of model frameworks have been proposed to describe what is necessary for an animal to remain within this stable physiological state and the ramifications of leaving it. Romero et al. (Horm Behav 55(3):375–389, 2009) introduced the reactive scope model to provide a novel conceptual framework for the stress response that assumes an animal’s ability to tolerate a stressful stimulus may degrade over time in response to the stimulus. We provide a mathematical formulation for the reactive scope model using a system of ordinary differential equations and show that this model is capable of recreating existing experimental data. We also provide an experimental method that may be used to verify the model as well as several potential additions to the model. If future experimentation provides the necessary data to estimate the model’s parameters, the model presented here may be used to make quantitative predictions about physiological mediator levels during a stress response and predict the onset of homeostatic overload.

Bone Marrow Microenvironment Involvement in t-MN: Focus on Mesenchymal Stem Cells.

Therapy-related myeloid neoplasms (t-MN) are a late complication of cytotoxic therapy (CT) used in the treatment of both malignant and non-malignant diseases. Historically, t-MN has been considered to be a direct consequence of DNA damage induced in normal hematopoietic stem or progenitor cells (HSPC) by CT. However, we now know that treatment-induced mutations in HSC are not the only players involved in t-MN development, but additional factors may contribute to the onset of t-MN. One of the known drivers involved in this field is the bone marrow microenvironment (BMM) and, in particular, bone marrow mesenchymal stem cells (BM-MSC), whose role in t-MN pathogenesis is the topic of this mini-review. BM-MSCs, physiologically, support HSC maintenance, self-renewal, and differentiation through hematopoietic-stromal interactions and the production of cytokines. In addition, BM-MSCs maintain the stability of the BM immune microenvironment and reduce the damage caused to HSC by stress stimuli. In the t-MN context, chemo/radiotherapy may induce damage to the BM-MSC and likewise alter BM-MSC functions by promoting pro-inflammatory response, clonal selection and/or the production of factors that may favor malignant hematopoiesis. Over the last decade, it has been shown that BM-MSC isolated from patients with de novo and therapy-related MN exhibit decreased proliferative and clonogenic capacity, altered morphology, increased senescence, defective osteogenic differentiation potential, impaired immune-regulatory properties, and reduced ability to support HSC growth and differentiation, as compared to normal BM-MSC. Although the understanding of the genetic and gene expression profile associated with ex vivo-expanded t-MN-MSCs remains limited and debatable, its potential role in prognostic and therapeutic terms is acting as a flywheel of attraction for many researchers.

Correlation between preoperative frailty and postoperative delirium in elderly patients undergoing hip arthroplasty.

Postoperative delirium (POD) refers to acute brain dysfunction occurring within 7 days after operation or before discharge. Frailty refers to the state that the body's physiological reserve is insufficient, so that the compensative capacity to endogenous and exogenous stress stimuli decreases. The purpose of this study is to explore the association of preoperative frailty (PF) with POD in elderly patients undergoing hip arthroplasty. Totally 228 elderly patients (age ≥ 65 years) who received elective hip arthroplasty in the Ningbo No. 6 Hospital between December 2021 and June 2022 were enrolled. One day before surgery, the frailty phenotype scale was adopted for evaluation of patients' frailty. On the 1st-3rd day after operation, the confusion assessment method was adopted for evaluation of delirium, and the patients were grouped into a POD group and non-POD group. Logistic regression was conducted to analyze the correlation between PF and POD. Among the patients, the incidence of PF was 30.70% (70/228), and the incidence of delirium within 3 days after operation was 25.88% (59/228). According to binary logistic regression analysis, PF, age, hypertension, diabetes mellitus, and preoperative sleep disorder were independent risk factors for POD in elderly patients undergoing hip arthroplasty (all P < .05). PF is a crucial risk factor for POD in elderly patients undergoing hip arthroplasty.

Acute Stress Modulates Social Approach and Social Maintenance in Adult Zebrafish.

Stress alters social functioning in a complex manner. An important variable determining the final effects of stress is stressor intensity. However, the precise relationship between stressor intensity and social behavior is not well understood. Here, we investigate the effects of varying acute stressor intensity exposure on social behavior using adult zebrafish. We first establish a novel test using adult zebrafish that allows distinguishing fish's drive to approach a social cue and its ability to engage and maintain social interaction within the same behavioral paradigm. Next, we combined this test with a new method to deliver an acute stress stimulus of varying intensities. Our results show that both social approach and social maintenance are reduced in adult zebrafish on acute stress exposure in an intensity-dependent manner. Interestingly, lower stress intensity reduces social maintenance without affecting the social approach, while a higher stress level is required to alter social approach. These results provide evidence for a direct correlation between acute stressor intensity and social functioning and suggest that distinct steps in social behavior are modulated differentially by the acute stress level.

Physiological Stress Responses in Cattle Used in the Spanish Rodeo.

Certain events can cause distress in cattle. In Spain, there is a sport similar to rodeo called persecution and takedown, in which calves are harassed and knocked down by riders. In this study, the physiological stress response of calves (n = 260) is assessed by measuring hormonal physiological parameters. Salivary samples were collected from Salers (n = 110) and Lidia (n = 150) calves before, during, and after the persecution and takedown event. The hormones epinephrine, cortisol, serotonin, and dopamine were determined in saliva samples using enzyme-immunoassay techniques. The results obtained revealed that epinephrine and cortisol levels increased during the event in Salers calves, with a significant increase (p < 0.05) in the case of epinephrine, although after the event, these values returned to their initial state. Therefore, this sport supposes an assumable punctual stressor stimulus for the animal. In contrast, in Lidia calves, cortisol and epinephrine levels decreased, with a significant decrease (p < 0.05) in the case of cortisol, which may be related to the temperament of this breed and facing a stressful situation in a different manner. This is confirmed by serotonin and dopamine levels that were altered in Lidia calves with respect to the other group studied. In conclusion, the sport of persecution and takedown produces a physiological response of adaptive stress assumable for the animals.

Assessing brain function in stressed healthy individuals following the use of a combination of green tea, Rhodiola, magnesium, and B vitamins: an fMRI study.

This randomized, controlled, single-blinded trial assessed the effect of magnesium (Mg)-Teadiola (Mg, vitamins B6, B9, B12, Rhodiola, and green tea/L-theanine) versus placebo on the brain response to stressful thermal stimulus in chronically stressed, but otherwise healthy subjects. Impacts on stress-related quality-of-life parameters (depression, anxiety, sleep, and perception of pain) were also explored. The study recruited a total of 40 adults (20 per group), suffering from stress for more than 1 month and scaling ≥14 points on the Depression Anxiety Stress Scale (DASS)-42 questionnaire at the time of inclusion. Individuals received oral Mg-Teadiola or placebo for 28 days (D). fMRI analysis was used to visualize the interplay between stress and pain cerebral matrices, using thermal stress model, at baseline (D0) and after D28. Based on blood-oxygen-level-dependent (BOLD) signal variations during the stress stimulation (before pain perception), a significantly increased activation between D0 and D28 was observed for left and right frontal area (p = 0.001 and p = 0.002, respectively), left and right anterior cingulate cortex (ACC) (p = 0.035 and p = 0.04, respectively), and left and right insula (p = 0.034 and p = 0.0402, respectively) in Mg-Teadiola versus placebo group. During thermal pain stimulation, a significantly diminished activation of the pain matrix was observed between D0 and D28, for left and right prefrontal area (both p = 0.001), left and right insula (p = 0.008 and p = 0.019, respectively), and left and right ventral striatum (both p = 0.001) was observed in Mg-Teadiola versus placebo group. These results reinforce the clinical observations, showing a perceived benefit of Mg-Teadiola on several parameters. After 1 month of treatment, DASS-42 stress score significantly decreased in Mg-Teadiola group [effect size (ES) -0.46 (-0.91; -0.01), p = 0.048]. Similar reductions were observed on D14 (p = 0.011) and D56 (p = 0.008). Sensitivity to cold also improved from D0 to D28 for Mg-Teadiola versus placebo [ES 0.47 (0.02; 0.92) p = 0.042]. Supplementation with Mg-Teadiola reduced stress on D28 in chronically stressed but otherwise healthy individuals and modulated the stress and pain cerebral matrices during stressful thermal stimulus.

Oxidative stress-induced by different stressors alters kidney tissue antioxidant markers and levels of creatinine and urea: the fate of renal membrane integrity

The cellular integrity of the kidney in homeostatic regulation has constantly been compromised by oxidative stress following exposure to varying nature of stressor present within the environment. The objective of the work was to evaluate the renal effect of the different stressor stimuli applied. Twenty-four adult female rats weighing averagely 160–200 g and within the ages of 12–14 weeks were used for experiment-1, while 12 offspring were utilized for experiment-2. Three stress models namely; restraint, mirror chamber and cat intruder stressors were used. Tissues were isolated from the animal and homogenized for tissue antioxidant assay. Serum was collected for assays of urea and creatinine for the kidney function test using ELISA. Data collected were analyzed for Mean ± SEM using One Way ANOVA. The present study revealed that exposure of rats to different stressors reduced relative kidney weights but did not significantly alter serum creatinine concentration in the Wistar rats, although the concentrations were slightly increased compared to controls. Urea concentration was significantly (p < 0.05) increased in rats exposed to restraint and intruder stressors. Exposure to a mirror chamber stressor did not significantly alter urea concentration. Offspring from parents of stressed female rats exhibited a significant (p < 0.05) increase in serum urea level, minimal increase in serum creatinine levels. GSH and GST levels showed no significant difference when compared to control group, whereas, GPx were significantly (p < 0.05) decreased irrespective of the stressor applied. SOD activity were significantly (p < 0.05) reduced in the group exposed to restraint or cat intruder stressor. CAT activities were significantly (p < 0.05) reduced in the rats exposed to restraint or cat intruder stressor. In all, the different stress model altered the antioxidant capacity of the kidney tissues. Exposure of rats to a stressful condition of the different nature of stressor has the tendency of compromising the functional integrity of the kidney, thus, with the potency of complicating female renal function.

Mechanical stress and inflammation have opposite effects on Wnt signaling in human chondrocytes.

Dysregulation of Wingless and Int-1 (Wnt) signaling has been strongly associated with development and progression of osteoarthritis (OA). Here, we set out to investigate the independent effects of either mechanical stress (MS) or inflammation on Wnt signaling in human neocartilage pellets, and to relate this Wnt signaling to OA pathophysiology. OA synovium-conditioned media (OAS-CM) was collected after incubating synovium from human end-stage OA joints for 24 h in medium. Cytokine levels in the OAS-CM were determined with a multiplex immunoassay (Luminex). Human neocartilage pellets were exposed to 20% MS, 2% OAS-CM or 1 ng/mL Interleukin-1β(IL-1β). Effects on expression levels of Wnt signaling members were determined by reverse transcription-quantitative polymerase chain reaction. Additionally, the expression of these members in articular cartilage from human OA joints was analyzed in association with joint space narrowing (JSN) and osteophyte scores. Protein levels of IL-1β, IL-6, IL-8, IL-10, tumor necrosis factor α, and granulocyte-macrophage colony-stimulating factor positively correlated with each other. MS increased noncanonical WNT5A and FOS expression. In contrast, these genes were downregulated upon stimulation with OAS-CM or IL-1β. Furthermore, Wnt inhibitors DKK1 and FRZB decreased in response to OAS-CM or IL-1β exposure. Finally, expression of WNT5A in OA articular cartilage was associated with increased JSN scores, but not osteophyte scores. Our results demonstrate that MS and inflammatory stimuli have opposite effects on canonical and noncanonical Wnt signaling in human neocartilage. Considering the extent to which MS and inflammation contribute to OA in individual patients, we hypothesize that targeting specific Wnt pathways offers a more effective, individualized approach.

Effects of mindfulness-based stress reduction meditation on the emotional reaction to affective pictures assessed by electrodermal activity

ObjectiveIn recent years the psychophysiological benefits of Mindfulness meditation on emotional processing have drawn great interest in scientific research. Currently, the effects of this meditation practice on stress, anxiety and well-being have been mostly evaluated using self-reporting questionnaires, which lead to a quite subjective assessment. This study assesses the effect of Mindfulness practice on the reaction to emotionally charged visual stimuli through Electrodermal Activity (EDA) data. MethodsTwenty-five healthy volunteers, without any previous experience of meditation techniques completed a 12-week Mindfulness-Based Stress Reduction (MBSR) course. EDA and psychological measures were collected longitudinally in 4 scheduled sessions. Statistical analysis was performed to find changes in the most relevant EDA parameters throughout the 4 sessions of data collection. ResultsWe found an increase in response latency, and a decrease in amplitude, area, number of specific responses, and skin conductance level along Mindfulness training. Both outcomes might suggest a reduction in the reactivity to the presented stimuli and an improvement in the emotional well-being of the practitioners. Furthermore, this study showed preliminary evidence that women improve more their attitude towards stressful stimuli than man, after the mindfulness practice.The statistical analysis also showed a correlation between the main EDA parameters and the scores reported by each participant in the depression, anxiety, and stress scale (DASS) questionnaire. Conclusion and SignificanceThis study contributed to a more objective evaluation of the physiological changes observed during Mindfulness practice, and so to understand the underlying mechanisms that explain the benefits of meditation training.

Aerobic Exercise Improves Depressive-like Behavior in CUMS-Induced Rats via the SIRT3/ROS/NLRP3 Signaling Pathway.

This study aimed to investigate the effect of exercise on depressive-like behavior induced by chronic unpredictable mild stress (CUMS) in rats and to explore the role of the SIRT3/ROS/NLRP3 signaling pathway in this process. Twenty-nine male 8-week-old Sprague Dawley rats were divided into a control group (CON) (nine rats) and a model group (twenty rats). Thirteen chronic stress stimuli were randomly applied once or twice per day for 35 days to induce depression in the model group rats. After the model was established, the model group rats were randomly divided into the CUMS group (CUMS) and the aerobic exercise + CUMS group (EX + CUMS). The EX + CUMS group received 8 weeks of aerobic exercise intervention for 6 days per week. Behavioral assessments were performed using the sucrose preference test and forced swimming test. The expression of SIRT3, NLRP3, IL-1β, and IL-18 in the hippocampus was detected using RT-PCR. The ROS level in the hippocampus was detected using immunofluorescence. The protein levels of SIRT3 and NLRP3 in the hippocampus were detected using western blotting. The protein levels of IL-1β and IL-18 in the hippocampus were measured using ELISA. After 5 weeks of chronic stress stimuli, the hippocampal function of rats in the CUMS model group was impaired, and their sucrose preference was reduced, while their forced swimming time was prolonged. The expression of SIRT3 decreased, ROS increased, and the expression of NLRP3 and the levels of IL-1β and IL-18 increased. Aerobic exercise increased the sucrose preference of rats, shortened their immobility time, increased the expression of SIRT3, and reduced the levels of ROS, NLRP3, IL-1β, and IL-18. Exercise can improve the depressive behavior of CUMS model rats, and its mechanism may be related to the upregulation of SIRT3 in the hippocampus, which plays an anti-inflammatory role.

Glucocorticoids and Their Receptor Isoforms: Roles in Female Reproduction, Pregnancy, and Foetal Development.

Alterations in the hypothalamic-pituitary-adrenal (HPA) axis and associated changes in circulating levels of glucocorticoids are integral to an organism's response to stressful stimuli. Glucocorticoids acting via glucocorticoid receptors (GRs) play a role in fertility, reproduction, placental function, and foetal development. GRs are ubiquitously expressed throughout the female reproductive system and regulate normal reproductive function. Stress-induced glucocorticoids have been shown to inhibit reproduction and affect female gonadal function by suppressing the hypothalamic-pituitary-gonadal (HPG) axis at each level. Furthermore, during pregnancy, a mother's exposure to prenatal stress or external glucocorticoids can result in long-lasting alterations to the foetal HPA and neuroendocrine function. Several GR isoforms generated via alternative splicing or translation initiation from the GR gene have been identified in the mammalian ovary and uterus. The GR isoforms identified include the splice variants, GRα and GRβ, and GRγ and GR-P. Glucocorticoids can exert both stimulatory and inhibitory effects and both pro- and anti-inflammatory functions in the ovary, in vitro. In the placenta, thirteen GR isoforms have been identified in humans, guinea pigs, sheep, rats, and mice, indicating they are conserved across species and may be important in mediating a differential response to stress. Distinctive responses to glucocorticoids, differential birth outcomes in pregnancy complications, and sex-based variations in the response to stress could all potentially be dependent on a particular GR expression pattern. This comprehensive review provides an overview of the structure and function of the GR in relation to female fertility and reproduction and discusses the changes in the GR and glucocorticoid signalling during pregnancy. To generate this overview, an extensive non-systematic literature search was conducted across multiple databases, including PubMed, Web of Science, and Google Scholar, with a focus on original research articles, meta-analyses, and previous review papers addressing the subject. This review integrates the current understanding of GR variants and their roles in glucocorticoid signalling, reproduction, placental function, and foetal growth.

Abstract P1134: RNA Elongation Mediates Rapid Induction Of Gene Expression During Endothelial-to-mesenchymal Transition

Endothelial-to-mesenchymal transition (EndMT) is triggered by stress stimuli and characterized by the conversion of endothelial cells (EC) into mesenchymal cells. Stress stimuli such as hypoxia or inflammation depend on rapid gene expression mediated by RNA elongation. RNA elongation is controlled by the super elongation complex (SEC) through transient pausing of RNA Polymerase II (RNAPII). We aimed to analyze the regulatory role of RNA elongation on rapid gene expression during the onset of EndMT. In an in vitro EndMT model, SEC scaffolds AFF1 and AFF4 were upregulated compared to controls. SEC inhibition using chemical compounds reduced the protein levels of AFF1 and AFF4. EndMT was reduced by SEC inhibition, whereas overexpression of AFF4 increased EndMT as measured by mesenchymal marker gene expression. Rapid gene expression depends on the release of paused RNAPII at promoter sites. ChIP-sequencing revealed a reduction of RNAPII occupancy at promoter sites in EndMT compared to controls, suggesting the release of RNAPII during EndMT. Transcriptome-wide RNA elongation rates were increased as early as 10 min after induction of EndMT, as determined by analysis of nascent 4sU-labeled RNA. SEC inhibition reduced global RNA elongation rates, suggesting a regulatory contribution of RNA elongation in EndMT. Among the genes that were immediately upregulated after the induction of EndMT was the SPARC mRNA. RNA initiation and elongation of SPARC mRNA were increased 10 min after EndMT induction, which was reduced by SEC inhibition. At 72 h after EndMT induction, inhibition of SEC decreased SPARC mRNA, protein concentration and SPARC delivery to the supernatant. Silencing of SPARC reduced the angiogenic potential of EC and enhanced EndMT induction, which was attenuated by treatment with recombinant SPARC. Treatment with recombinant SPARC also improved endothelial impedance under EndMT conditions. Spatial transcriptomics revealed increased SPARC expression in brachiocephalic artery plaques from LDLR-/- mice. In conclusion, we observed increased RNA elongation rates associated with reduced RNAPII pausing during EndMT that mediate rapid gene expression of target genes such as SPARC. Our results suggest that RNA elongation plays a regulatory role in EndMT.

Arabidopsis cell suspension culture and RNA sequencing reveal regulatory networks underlying plant-programmed cell death.

Programmed cell death (PCD) facilitates selective, genetically controlled elimination of redundant, damaged, or infected cells. In plants, PCD is often an essential component of normal development and can mediate responses to abiotic and biotic stress stimuli. However, studying the transcriptional regulation of PCD is hindered by difficulties in sampling small groups of dying cells that are often buried within the bulk of living plant tissue. We addressed this challenge by using RNA sequencing and Arabidopsis thaliana suspension cells, a model system that allows precise monitoring of PCD rates. The use of three PCD-inducing treatments (salicylic acid, heat, and critical dilution), in combination with three cell death modulators (3-methyladenine, lanthanum chloride, and conditioned medium), enabled isolation of candidate core- and stimuli-specific PCD genes, inference of underlying regulatory networks and identification of putative transcriptional regulators of PCD in plants. This analysis underscored a disturbance of the cell cycle and mitochondrial retrograde signaling, and repression of pro-survival stress responses, as key elements of the PCD-associated transcriptional signature. Further, phenotyping of Arabidopsis T-DNA insertion mutants in selected candidate genes validated the potential of generated resources to identify novel genes involved in plant PCD pathways and/or stress tolerance.

Functional characterization of porcine nucleophosmin (NPM1) gene in promoting the replication of Japanese encephalitis virus and induction of inflammatory cytokines

Nucleophosmin (NPM1) is a multifunctional nucleolar protein that plays a role in cell cycle control, tumorigenesis, induction of the inflammatory cytokine, virus replication, as well as the cellular responses to a variety of stress stimuli. However, its physiological functions in pigs have not been well understood. Here, we cloned the porcine NPM1 (porNPM1) gene and analyzed the functions of the porNPM1 protein in pigs. The full-length porNPM1 gene encoded a 294-amino acid protein with 94.5%–99.3% sequence identity to its orthologues in mammals and was extensively expressed in various pig tissues at the mRNA level. The porNPM1 primarily localizes in the nucleus of ST cells, while it translocates from the nucleus to nucleoplasm upon UV irradiation or H2O2 treatment. Notably, JEV infection blocked the translocation of porNPM1 from the nucleolus to the nucleoplasm. Furthermore, porNPM1 interacted with the JEV C protein and facilitated JEV replication in ST cells. The overexpression and knockdown of porNPM1 respectively enhanced or impaired JEV replication, suggesting the important role of porNPM1 in JEV replication. Additionally, the purified ectodomain of porNPM1 induced the production of inflammatory cytokines (TNF-α, IL-6, and IL-8). Together, these data demonstrated that porNPM1 is involved in cellular stress stimuli, JEV replication, and induction of inflammatory cytokines.