Global Gene Expression Research Articles

Staphylococcus aureus Stress Response to Bicarbonate Depletion.

Bicarbonate and CO2 are essential substrates for carboxylation reactions in bacterial central metabolism. In Staphylococcus aureus, the bicarbonate transporter, MpsABC (membrane potential-generating system) is the only carbon concentrating system. An mpsABC deletion mutant can hardly grow in ambient air. In this study, we investigated the changes that occur in S. aureus when it suffers from CO2/bicarbonate deficiency. Electron microscopy revealed that ΔmpsABC has a twofold thicker cell wall thickness compared to the parent strain. The mutant was also substantially inert to cell lysis induced by lysostaphin and the non-ionic surfactant Triton X-100. Mass spectrometry analysis of muropeptides revealed the incorporation of alanine into the pentaglycine interpeptide bridge, which explains the mutant's lysostaphin resistance. Flow cytometry analysis of wall teichoic acid (WTA) glycosylation patterns revealed a significantly lower α-glycosylated and higher ß-glycosylated WTA, explaining the mutant's increased resistance towards Triton X-100. Comparative transcriptome analysis showed altered gene expression profiles. Autolysin-encoding genes such as sceD, a lytic transglycosylase encoding gene, were upregulated, like in vancomycin-intermediate S. aureus mutants (VISA). Genes related to cell wall-anchored proteins, secreted proteins, transporters, and toxins were downregulated. Overall, we demonstrate that bicarbonate deficiency is a stress response that causes changes in cell wall composition and global gene expression resulting in increased resilience to cell wall lytic enzymes and detergents.

The Response of Murine Gut Microbiome in the Presence of Altered rpoS Gene of Klebsiella pneumoniae.

The murine model is invaluable for studying intricate interactions among gut microbes; hosts; and diseases. However; the impact of genetic variations in the murine microbiome; especially in disease contexts such as Klebsiella pneumoniae (Kp) infection; still needs to be explored. Kp; an opportunistic global pathogen; is becoming increasingly prevalent in regions like Asia; especially China. This study explored the role of the gut microbiota during Kp infection using mouse model; including wild-type and rpoS mutants of Kp138; KpC4; and KpE4 from human; maize; and ditch water; respectively. Under stress conditions; RpoS reconfigures global gene expression in bacteria; shifting the cells from active growth to survival mode. Our study examined notable differences in microbiome composition; finding that Lactobacillus and Klebsiella (particularly in WKp138) were the most abundant genera in mice guts at the genus level in all wild-type treated mice. In contrast; Firmicutes were predominant in the healthy control mice. Furthermore; Clostridium was the dominant genus in all mutants; mainly in ∆KpC4; and was absent in wild-type treated mice. Differential abundance analysis identified that these candidate taxa potentially influence disease progression and pathogen virulence. Functional prediction analysis showed that most bacterial groups were functionally involved in biosynthesis; precursor metabolites; degradation; energy generation; and metabolic cluster formation. These findings challenge the conventional understanding and highlight the need for nuanced interpretations in murine studies. Additionally; this study sheds light on microbiome-immune interactions in K. pneumoniae infection and proposes new potential therapeutic strategies.

A 3-component module maintains sepal flatness in Arabidopsis

As in origami, morphogenesis in living systems heavily relies on tissue curving and folding through the interplay between biochemical and biomechanical cues. By contrast, certain organs maintain their flat posture over several days. Here, we identified a pathway that is required for the maintenance of organ flatness, taking the sepal, the outermost floral organ, in Arabidopsis as a model system. Through genetic, cellular, and mechanical approaches, our results demonstrate that the global gene expression regulator VERNALIZATION INDEPENDENCE 4 (VIP4) fine-tunes the mechanical properties of sepal cell walls and maintains balanced growth on both sides of the sepals, mainly by orchestrating the distribution pattern of AUXIN RESPONSE FACTOR 3 (ARF3). vip4 mutation results in softer cell walls and faster cell growth on the adaxial sepal side, which eventually cause sepals to bend outward. Downstream of VIP4, ARF3 works through modulating auxin to downregulate pectin methylesterase VANGUARD1, resulting in decreased cell wall stiffness. Thus, our work unravels a 3-component module that relates hormonal patterns to organ curvature and actively maintains sepal flatness during its growth.

Dynamics of the thymic transcriptome at stages of acute thymic involution in Japanese Black calves with a poor prognosis

Transcriptome analysis was performed on the thymus of Japanese Black calves that were necropsied due to poor prognosis, to characterize changes associated with acute thymic involution. Gene expression profiles obtained by DNA microarray analysis of eight calf thymuses were classified into three patterns that correlated with the histopathological stage of acute thymic involution. Using principal component analysis, the first principal component of the global gene expression levels in the calf thymus was associated with the stage of acute thymic involution, suggesting that histopathological changes greatly influence the gene expression profile. Gene ontology enrichment analysis revealed that genes related to cell proliferation, wound healing, and inflammatory responses were the main contributors to the first principal component. Real-time RT-PCR showed that the thymus had lower expression of PCNA, KIFC1, and HES6, and higher expression of SYNPO2, PDGFRB, and TWIST1 during acute thymic involution. Immunohistochemistry demonstrated a decrease in the rate of Ki67-positive cells in the thymic cortex during the late stage of acute thymic involution. The rate of cleaved caspase-1-positive cells increased in the thymic cortex at an earlier stage than the increase in the rate of cleaved caspase-3-positive cells. Vimentin, which was almost absent in the non-involuted thymic cortex, appeared in the thymic cortex during acute thymic involution. These results suggest that in farmed calves with a poor prognosis, inflammatory responses and impaired thymocyte proliferation are primarily involved in acute thymic involution.

Comparative phenotypic and transcriptomic analysis reveals genotypic differences in nitrogen use efficiency in sorghum

Sorghum (Sorghumbicolor L.), a model for C4 grass and an emerging biofuel crop, is known for its robust tolerance to low input field. However, the focus on enhancing nitrogen use efficiency (NUE) in sorghum under low nitrogen (N) conditions has been limited. This study conducted hydroponic experiments and field trials with two sorghum inbred lines, contrasting in their N efficiency: the N-efficient (398B) and the N-inefficient (CS3541) inbred lines. The aim was to analyze the key factors influencing NUE by integrating phenotypic, physiological, and multi-omics approaches under N deficiency conditions. The field experiments revealed that 398B displayed superior NUE and yield performance compared to CS3541. In hydroponic experiments, the growth of 398B outperformed CS3541 following N deficiency, attributing to its higher photosynthetic and sustaining activity of N metabolism-related enzymes. Genomic and transcriptomic integration highlighted fewer genomic diversities and alterations in global gene expression in 398B, which were likely contributor to its high NUE. Additionally, co-expression network analysis suggested the involvement of key genes which impact N uptake efficiency (NUpE) and N utilization efficiency (NUtE) in both lines, such as an N transporter, Sobic.003G371000.v3.2leaf(NPF5.10) and a transcription factor, Sobic.002G202800.v3.2leaf(WRKY) in bolstering NUE under low-N stress. The findings collectively suggested that 398B achieved higher NUpE and NUtE, effectively coordinating photosynthesis and N metabolism to enhance NUE. The candidate genes regulating N uptake and utilization efficiencies could provide valuable insights for developing sorghum breeds with improved NUE, contributing to sustainable agricultural practices and bioenergy crop development.

Interplay between DOT1L and HDAC1 regulates Leishmania donovani infection in human THP-1 cells

Leishmania donovani, a protozoan parasite, causes visceral leishmaniasis. The parasite modifies the global gene expressions of the host genome, facilitating its survival within the host. Thus, the host epigenetic modulators play important roles in host-pathogen interaction and host epigenetic modification in response to infection. Previously, we had reported that the host epigenetic modulator, histone deacetylase 1 (HDAC1) expression was upregulated on Leishmania donovani infection. This upregulation led to the repression of host defensin genes in response to the infection. In this paper, we have investigated the interplay between the host DOT1L, a histone methyltransferase, and HDAC1 in response to Leishmania donovani infection. We show that the expression of DOT1L is upregulated both at transcript and protein level following infection leading to increase in H3K79me, H3K79me2, and H3K79me3 levels. ChIP experiments showed that DOT1L regulated the expression of HDAC1. Downregulation of DOT1L using siRNA resulted in decreased expression of HDAC1 and increased transcription of defensin genes and thereby, lower parasite load. In turn, HDAC1 regulates the expression of DOT1L on Leishmania donovani infection as downregulation of HDAC1 using siRNA led to reduced expression of DOT1L. Thus, during Leishmania donovani infection, an interplay between DOT1L and HDAC1 regulates the expression of these two histone modifiers leading to downregulation of defensin gene expression.

Unraveling molecular mechanistic disparities in pathogenic visceral Leishmania resistance between reptiles and mammals through comparative transcriptomic analyses

Leishmaniasis is one of the most important neglected tropical parasitic diseases, manifesting various clinical forms depending on the parasite species and the genetic background of the host. In order to elucidate the underlying mechanisms of reptilian defense against pathogenic Leishmania species and to delineate the global gene expression profile alterations during host-pathogen interaction, we established experimental animal and cell models using both heterothermic lizards (Phrynocephalus przewalskii) and homothermic mammals (BALB/c mice) infected with pathogenic Leishmania infantum (high virulence HCZ strain) and Leishmania donovani (low virulence 801 strain). Overall, the lizards didn't show any obvious clinical symptoms or immune responses in vivo. Using RNA-seq methodology, differentially expressed genes identified in the HCZ and 801-comparison groups of P. przewalskii were primarily associated with arginine biosynthesis, the MAPK signaling pathway and the PI3K-Akt signaling pathway. In contrast, higher parasite loads, exacerbated hepatic inflammatory lesions and enhanced immune responses were observed in BALB/c mice, with DEGs predominantly associated with immunological diseases, innate and adaptive immune responses. By integrating transcriptional data from reptile and mammalian hosts, we elucidated the pivotal role of amino acid metabolism and lipid metabolism in parasite control. In contrast to findings from animal experiments, Leishmania parasites effectively infected peritoneal macrophages of lizards in vitro, demonstrating a high infection rate. Furthermore, we used RT-qPCR to detect changes in cytokine expression in macrophages and found that Th1-type cytokines were significantly upregulated in lizards, facilitating the clearance of the HCZ strain 24 hours post-infection. Conversely, cytokine expression was generally suppressed in BALB/c mice, allowing immune evasion by the parasites.

ScIDPMs: Single-Cell RNA-Seq Imputation Using Diffusion Probabilistic Models.

Single-cell RNA sequencing (scRNA-seq) technology has revolutionized biological research by enabling the sequencing of mRNA in individual cells, thereby providing valuable insights into cellular gene expression and functions. However, scRNA-seq data often contain false zero values known as dropout events, which can obscure true gene expression levels and compromise downstream analysis accuracy. To address this issue, several computational approaches have been proposed for imputing missing gene expression values. Nevertheless, these methods struggle to capture dropout value distributions due to the sparsity of scRNA-seq data and complex gene expression patterns. In this study, we present a novel method called scIDPMs that utilizes conditional diffusion probabilistic models to impute scRNA-seq data. Firstly, scIDPMs identifies dropout sites based on gene expression characteristics and subsequently infers the missing values by considering available gene expression information. To effectively capture global gene expression features, scIDPMs employs a deep neural network with an attention mechanism to optimize the imputation process. We evaluated the performance of scIDPMs using simulated and real scRNA-seq datasets and compared it with ten other imputation methods. The results indicate that scIDPMs outperform other methods in restoring biologically meaningful gene expression values and improving downstream analysis.

MAP3K8 is a potential therapeutic target in airway epithelial inflammation

BackgroundWe have previously discovered clusters of sequentially negative and positive modulators of acute inflammation during cytokine stimulation in epithelial cells and identified potential targets for therapy within these clusters. MAP3K8 is a druggable kinase that we found to be a hub of a principal interaction network. We describe here the results of MAP3K8 knockdown in the A549 lung cancer cell line, the BEAS-2B epithelial cell line and normal human bronchial epithelial (NHBE) cells following IL-1β stimulation. We analysed signalling transduction and global gene expression after IL-1β stimulation with and without MAP3K8 knockdown, quantifying levels of the inflammatory cytokines IL-6, IL-8 and RANTES levels by qPCRs and/or by ELISAs. We also examined potential small molecule inhibitors for MAP3K8 in the same models.ResultsIL-1β significantly and consistently increased MAP3K8 expression after 2 h in A549, BEAS-2B and NHBE cells. Phosphorylation of MAP3K8 occurred at 20 min after IL-1β stimulation and MAP3K8 protein was degraded at 30 min. MAP3K8 knockdown significantly reduced IL-6, IL-8 levels after IL-1β stimulation and yielded a 10-fold enhancement of the anti-inflammatory effects of dexamethasone. Phosphorylation of ERK1/2 (P-ERK1/2) and phosphorylation of SAPK/JNK (P-SAPK/JNK) decreased at 30 min after IL-1β stimulation with MAP3K8 knockdown. The combination of dexamethasone and MAP3K8 knockdown resulted in greater inhibition of phosphorylated ERK1/2 and SAPK/JNK. Nineteen genes including MMP1, MMP3, MMP10, ITGB8, LAMC2 and PLAT (P corrected < 0.01 respectively) demonstrated a distinct altered temporal response to IL-1β following suppression of MAP3K8. However, putative MAP3K8 inhibitors including Tpl2-1, Tpl2-2 and GSK2222867A only showed inhibition of IL-6 and IL-8 production at a high dose.ConclusionsThese results confirm that MAP3K8 is a key mediator of the early inflammatory response and that it is a potential target in inflammatory diseases. However, current tool compounds do not effectively inhibit its effects.

Integrative analysis of the transcriptome and proteome reveals the molecular responses of tobacco to boron deficiency

BackgroundBoron (B) is an essential micronutrient for plants. Inappropriate B supply detrimentally affects the productivity of numerous crops. Understanding of the molecular responses of plants to different B supply levels would be of significance in crop improvement and cultivation practices to deal with the problem.ResultsWe conducted a comprehensive analysis of the transcriptome and proteome of tobacco seedlings to investigate the expression changes of genes/proteins in response to different B supply levels, with a particular focus on B deficiency. The global gene and protein expression profiles revealed the potential mechanisms involved in the responses of tobacco to B deficiency, including up-regulation of the NIP5;1-BORs module, complex regulation of genes/proteins related to cell wall metabolism, and up-regulation of the antioxidant machinery.ConclusionOur results demonstrated that B deficiency caused severe morphological and physiological disorders in tobacco seedlings, and revealed dynamic expression changes of tobacco genes/proteins in response to different B supply levels, especially to B deficiency, thus offering valuable insights into the molecular responses of tobacco to B deficiency.

Comparative Gene Expression Analysis of Salmonella Typhimurium DT104 in Ground Chicken Extract and Brain Heart Infusion Broth.

Salmonella enterica Typhimurium DT104 (S. Typhimurium DT104) is an important foodborne pathogen that is associated with poultry and poultry products. Currently, there is very little information on the underlying molecular mechanisms that allow DT104 to survive and propagate in poultry meat and the poultry processing environment. The current study assessed the global gene expression of DT104 in ground chicken extract (GCE) compared to brain heart infusion (BHI) medium using RNA-Seq technology. DT104 was grown to the early stationary phase (ESP), inoculated into GCE or BHI, and then re-grown to the log phase before RNA was extracted and transcripts were quantified by RNA-Seq. Gene expression for DT104 grown in GCE was then compared to that of DT104 grown in BHI for samples grown to the ESP. Growth in GCE resulted in the up-regulated expression of genes related to translation, carnitine metabolism (23-283-fold change), and cobalamin (vitamin B12) biosynthesis (14-fold change). In particular, the presence of carnitine in chicken meat, and thus, in GCE, which lacks carbohydrates, may allow Salmonella to utilize this compound as a carbon and nitrogen source. This study demonstrates that RNA-Seq data can provide a comprehensive analysis of DT104 gene expression in a food model for poultry products. This study also provides additional evidence for the importance of metabolic adaptation in the ability of S. enterica to successfully adapt to and occupy niches outside of its host and provides potential targets that could be used to develop intervention strategies to control Salmonella in poultry.

CAMP-independent DNA binding of the CRP family protein DdrI from Deinococcus radiodurans.

The cAMP receptor proteins (CRPs) play a critical role in bacterial environmental adaptation by regulating global gene expression levels via cAMP binding. Here, we report the structure of DdrI, a CRP family protein from Deinococcus radiodurans. Combined with biochemical, kinetic, and molecular dynamics simulations analyses, our results indicate that DdrI adopts a DNA-binding conformation in the absence of cAMP and can form stable complexes with the target DNA sequence of classical CRPs. Further analysis revealed that the high-affinity cAMP binding pocket of DdrI is partially filled with Tyr113-Arg55-Glu65 sidechains, mimicking the anti-cAMP-mediated allosteric transition. Moreover, the second syn-cAMP binding site of DdrI at the protein-DNA interface is more negatively charged compared to that of classical CRPs, and manganese ions can enhance its DNA binding affinity. DdrI can also bind to a target sequence that mimics another transcription factor, DdrO, suggesting potential cross-talk between these two transcription factors. These findings reveal a class of CRPs that are independent of cAMP activation and provide valuable insights into the environmental adaptation mechanisms of D. radiodurans.IMPORTANCEBacteria need to respond to environmental changes at the gene transcriptional level, which is critical for their evolution, virulence, and industrial applications. The cAMP receptor protein (CRP) of Escherichia coli (ecCRP) senses changes in intracellular cAMP levels and is a classic example of allosteric effects in textbooks. However, the structures and biochemical activities of CRPs are not generally conserved and there exist different mechanisms. In this study, we found that the proposed CRP from Deinococcus radiodurans, DdrI, exhibited DNA binding ability independent of cAMP binding and adopted an apo structure resembling the activated CRP. Manganese can enhance the DNA binding of DdrI while allowing some degree of freedom for its target sequence. These results suggest that CRPs can evolve to become a class of cAMP-independent global regulators, enabling bacteria to adapt to different environments according to their characteristics. The first-discovered CRP family member, ecCRP (or CAP) may well not be typical of the family and be very different to the ancestral CRP-family transcription factor.

Epigenetic and Molecular Alterations in Obesity: Linking CRP and DNA Methylation to Systemic Inflammation.

Obesity is marked by excessive fat accumulation in the adipose tissue, which disrupts metabolic processes and causes chronic systemic inflammation. Commonly, body mass index (BMI) is used to assess obesity-related risks, predicting potential metabolic disorders. However, for a better clustering of obese patients, we must consider molecular and epigenetic changes which may be responsible for inflammation and metabolic changes. Our study involved two groups of patients, obese and healthy donors, on which routine analysis were performed, focused on BMI, leukocytes count, and C-reactive protein (CRP) and completed with global DNA methylation and gene expression analysis for genes involved in inflammation and adipogenesis. Our results indicate that obese patients exhibited elevated leukocytes levels, along with increased BMI and CRP. The obese group revealed a global hypomethylation and upregulation of proinflammatory genes, with adipogenesis genes following the same trend of being overexpressed. The study confirms that obesity is linked to systematic inflammation and metabolic dysfunction through epigenetic and molecular alterations. The CRP was correlated with the hypomethylation status in obese patients, and this fact may contribute to a better understanding of the roles of specific genes in adipogenesis and inflammation, leading to a better personalized therapy.

Circadian transcriptome oscillations in human adipose tissue depend on napping status and link to metabolic and inflammatory pathways.

Napping is a common habit in many countries. Nevertheless, studies about the chronic effects of napping on obesity are contradictory, and the molecular link between napping and metabolic alterations has yet to be studied. We aim to identify molecular mechanisms in adipose tissue (AT) that may connect napping and abdominal obesity. In this cross-sectional study, we extracted the RNA repeatedly across 24 hours from cultured AT explants and performed RNA sequencing. Circadian rhythms were analyzed using six consecutive time points across 24 hours. We also assessed global gene expression in each group (nappers vs. non-nappers). With napping, there was an 88% decrease in the number of rhythmic genes compared to that in non-nappers, a reduction in rhythm amplitudes of 29%, and significant phase changes from a coherent unimodal acrophase in non-nappers, towards a scattered and bimodal acrophase in nappers. Those genes that lost rhythmicity with napping were mainly involved in pathways of glucose and lipid metabolism, and of the circadian clock. Additionally, we found differential global gene expression between nappers and non-nappers with 34 genes down- and 32 genes upregulated in nappers. The top upregulated gene (IER3) and top down-regulated pseudogene (VDAC2P2) in nappers have been previously shown to be involved in inflammation. These new findings have implications for our understanding of napping's relationship with obesity and metabolic disorders.

RNA editing regulates glutamatergic synapses in the frontal cortex of a molecular subtype of Amyotrophic Lateral Sclerosis

BackgroundAmyotrophic Lateral Sclerosis (ALS) is a highly heterogenous neurodegenerative disorder that primarily affects upper and lower motor neurons, affecting additional cell types and brain regions. Underlying molecular mechanisms are still elusive, in part due to disease heterogeneity. Molecular disease subtyping through integrative analyses including RNA editing profiling is a novel approach for identification of molecular networks involved in pathogenesis.MethodsWe aimed to highlight the role of RNA editing in ALS, focusing on the frontal cortex and the prevalent molecular disease subtype (ALS-Ox), previously determined by transcriptomic profile stratification. We established global RNA editing (editome) and gene expression (transcriptome) profiles in control and ALS-Ox cases, utilizing publicly available RNA-seq data (GSE153960) and an in-house analysis pipeline. Functional annotation and pathway analyses identified molecular processes affected by RNA editing alterations. Pearson correlation analyses assessed RNA editing effects on expression. Similar analyses on additional ALS-Ox and control samples (GSE124439) were performed for verification. Targeted re-sequencing and qRT-PCR analysis targeting CACNA1C, were performed using frontal cortex tissue from ALS and control samples (n = 3 samples/group).ResultsWe identified reduced global RNA editing in the frontal cortex of ALS-Ox cases. Differentially edited transcripts are enriched in synapses, particularly in the glutamatergic synapse pathway. Bioinformatic analyses on additional ALS-Ox and control RNA-seq data verified these findings. We identified increased recoding at the Q621R site in the GRIK2 transcript and determined positive correlations between RNA editing and gene expression alterations in ionotropic receptor subunits GRIA2, GRIA3 and the CACNA1C transcript, which encodes the pore forming subunit of a post-synaptic L-type calcium channel. Experimental data verified RNA editing alterations and editing-expression correlation in CACNA1C, highlighting CACNA1C as a target for further study.ConclusionsWe provide evidence on the involvement of RNA editing in the frontal cortex of an ALS molecular subtype, highlighting a modulatory role mediated though recoding and gene expression regulation on glutamatergic synapse related transcripts. We report RNA editing effects in disease-related transcripts and validated editing alterations in CACNA1C. Our study provides targets for further functional studies that could shed light in underlying disease mechanisms enabling novel therapeutic approaches.

PHOSPHATASE 2A dephosphorylates PHYTOCHROME-INTERACTING FACTOR3 to modulate photomorphogenesis in Arabidopsis.

The phytochrome (phy) family of sensory photoreceptors modulates developmental programs in response to ambient light. Phys also control gene expression in part by directly interacting with the bHLH class of transcription factors, PHYTOCHROME-INTERACTING FACTORS (PIFs), and inducing their rapid phosphorylation and degradation. Several kinases have been shown to phosphorylate PIFs and promote their degradation. However, the phosphatases that dephosphorylate PIFs are less understood. In this study, we describe 4 regulatory subunits of the Arabidopsis (Arabidopsis thaliana) protein PHOSPHATASE 2A (PP2A) family (B'α, B'β, B″α, and B″β) that interact with PIF3 in yeast 2-hybrid, in vitro and in vivo assays. The pp2ab″αβ and b″αβ/b'αβ mutants display short hypocotyls, while the overexpression of the B subunits induces longer hypocotyls compared with the wild type (WT) under red light. The light-induced degradation of PIF3 is faster in the b″αβ/b'αβ quadruple mutant compared with that in the WT. Consistently, immunoprecipitated PP2A A and B subunits directly dephosphorylate PIF3-MYC in vitro. An RNA-sequencing analysis shows that B″α and B″β alter global gene expression in response to red light. PIFs (PIF1, PIF3, PIF4, and PIF5) are epistatic to these B subunits in regulating hypocotyl elongation under red light. Collectively, these data show an essential function of PP2A in dephosphorylating PIF3 to modulate photomorphogenesis in Arabidopsis.

Visfatin (NAMPT) affects global gene expression in porcine anterior pituitary cells during the mid-luteal phase of the oestrous cycle

BackgroundThe pituitary belongs to the most important endocrine glands involved in regulating reproductive functions. The proper functioning of this gland ensures the undisturbed course of the oestrous cycle and affects the female’s reproductive potential. It is believed that visfatin, a hormone belonging to the adipokine family, may regulate reproductive functions in response to the female’s metabolic state. Herein we verified the hypothesis that suggests a modulatory effect of visfatin on the anterior pituitary transcriptome during the mid-luteal phase of the oestrous cycle.ResultsRNA-seq analysis of the porcine anterior pituitary cells revealed changes in the expression of 202 genes (95 up-regulated and 107 down-regulated in the presence of visfatin, when compared to the non-treated controls), assigned to 318 gene ontology terms. We revealed changes in the frequency of alternative splicing events (235 cases), as well as long noncoding RNA expression (79 cases) in the presence of the adipokine. The identified genes were associated, among others, with reproductive system development, epithelial cell proliferation, positive regulation of cell development, gland morphogenesis and cell chemotaxis.ConclusionsThe obtained results indicate a modulatory influence of visfatin on the regulation of the porcine transcriptome and, in consequence, pituitary physiology during the mid-luteal phase of the oestrous cycle.

Loss of Tet hydroxymethylase activity causes mouse embryonic stem cell differentiation bias and developmental defects.

The TET family is well known for active DNA demethylation and plays important roles in regulating transcription, the epigenome and development. Nevertheless, previous studies using knockdown (KD) or knockout (KO) models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles, as well as compensatory effects among TET family members, which has made the understanding of the enzymatic role of TET not accurate enough. To solve this problem, we successfully generated mice catalytically inactive for specific Tet members (Tetm/m). We observed that, compared with the reported KO mice, mutant mice exhibited distinct developmental defects, including growth retardation, sex imbalance, infertility, and perinatal lethality. Notably, Tetm/m mouse embryonic stem cells (mESCs) were successfully established but entered an impaired developmental program, demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation. Intriguingly, Tet3, traditionally considered less critical for mESCs due to its lower expression level, had a significant impact on the global hydroxymethylation, gene expression, and differentiation potential of mESCs. Notably, there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation. In summary, our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation.

Molecular mechanisms of neutron radiation dose effects on M1 generation peas

The dose effect of radiation has long been a topic of concern, but the molecular mechanism behind it is still unclear. In this study, dried pea seeds were irradiated with 252Cf fission neutron source. Through analyzing the transcriptome and proteome of M1 generation pea (Pisum sativum L.) leaves, we studied the molecular rule and mechanism of neutron dose effect. Our results showed three important rules of global gene expression in the studied dose range. The rule closely related to the neutron absorbed dose at the transcription and translation levels is: the greater the difference in neutron absorbed dose between two radiation treatment groups, the greater the difference in differential expression between the two groups and the control group. We also obtained important sensitive metabolic pathways of neutron radiation, as well as related key genes. Furthermore, the overall molecular regulation mechanism of dose effect was revealed based on the main functional items obtained. Our research results can be applied to appropriate radiation dose estimation and agricultural production practice.

Cryopreservation of mesenchymal stem/stromal cells using a DMSO-free solution is comparable to DMSO-containing cryoprotectants: results of an international multicenter PACT/BEST collaborative study

Background and AimAn essential aspect of ensuring availability and stability of mesenchymal stem/stromal cells (MSCs) products for clinical use is that these cells are cryopreserved before individual infusion into patients. Currently, cryopreservation of MSCs involves use of a cryoprotectant solution containing dimethyl sulfoxide (DMSO). However, it is recognized that DMSO may be toxic for both the patient and the MSC product. In this Production Assistance for Cellular Therapies (PACT) and Biomedical Excellence for Safer Transfusion (BEST) Collaborative study, we compared a novel DMSO-free solution with DMSO containing cryoprotectant solutions for freezing MSCs. MethodsA DMSO-free cryoprotectant solution containing sucrose, glycerol, and isoleucine (SGI) in a base of Plasmalyte A was prepared at the University of Minnesota. Cryoprotectant solutions containing 5–10% DMSO (in-house) were prepared at seven participating centers (five from USA, one each from Australia and Germany). The MSCs were isolated from bone marrow or adipose tissue and cultured ex vivo per local protocols at each center. The cells in suspension were frozen by aliquoting into vials/bags. For six out of the seven centers, the vials/bags were placed in a controlled rate freezer (one center placed them at -80°C freezer overnight) before transferring to liquid nitrogen. The cells were kept frozen for at least one week before thawing and testing. Pre- and post-thaw assessment included cell viability and recovery, immunophenotype as well as transcriptional and gene expression profiles. Linear regression, mixed effects models and two-sided t-tests were applied for statistical analysis. ResultsMSCs had an average viability of 94.3% (95% CI: 87.2–100%) before cryopreservation, decreasing by 4.5% (95% CI: 0.03–9.0%; P: 0.049) and 11.4% (95% CI: 6.9–15.8%; P< 0.001), for MSCs cryopreserved in the in-house and SGI solutions, respectively. The average recovery of viable MSCs cryopreserved in the SGI was 92.9% (95% CI: 85.7–100.0%), and it was lower by 5.6% (95% CI: 1.3–9.8%, P < 0.013) for the in-house solution. Additionally, MSCs cryopreserved in the two solutions had expected level of expressions for CD45, CD73, CD90, and CD105 with no significant difference in global gene expression profiles. ConclusionMSCs cryopreserved in a DMSO-free solution containing sucrose, glycerol, and isoleucine in a base of Plasmalyte A had slightly lower cell viability, better recovery, and comparable immunophenotype and global gene expression profiles compared to MSCs cryopreserved in DMSO containing solutions. The average viability of MSCs in the novel solution was above 80% and, thus, likely clinically acceptable. Future studies are suggested to test the post-thaw functions of MSCs cryopreserved in the novel DMSO-free solution.