Transcript Levels Of Genes Research Articles

Transcriptomics-Based Study of Immune Genes Associated with Subclinical Mastitis in Bactrian Camels

The significant increase in demand for camel milk has led to a rapid increase in the number of Bactrian camels. However, the widespread occurrence of mastitis significantly impacts the development of the Bactrian camel milk industry and poses a public health risk. Despite this, there is a lack of research on the transcriptional response, immune response pathways, and changes in core genes of Bactrian camels with subclinical mastitis. This study aimed to reveal the changes in immune-related response pathways and gene transcription levels in Bactrian camels with subclinical mastitis by analyzing the blood transcriptional response after the occurrence of subclinical mastitis in natural conditions. This study focused on 7-year-old Bactrian camels and collected 2 mL of blood from the camels that tested positive with a 4-peak California Mastitis Test (CMT) and those that tested negative with a 3-peak CMT. RNA sequencing (RNA-Seq) technology was used to analyze gene expression in the blood samples. Gene expression was verified using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Overall, 1722 differentially expressed genes were sequenced in the blood samples of CMT-positive and CMT-negative Bactrian camels, including 1061 upregulated and 661 downregulated genes. After conducting gene ontology functional enrichment, 453 differentially expressed genes were identified. We also discovered pathways such as immune response, the G-protein-coupled receptor signaling pathway, and internal signal transmission. Kyoto Encyclopedia of Genes and Genomes (KEGG) signaling pathway enrichment detected 668 differentially expressed genes annotated onto 309 metabolic pathways, with significantly enriched immune pathways including cytokine–cytokine receptor interaction, complex and coalescence cascades, natural killer cell-mediated cytotoxicity, and T helper type 17 cell differentiation, among others. Through a STRING protein interaction database and cytoscape analysis, it was found that core differentially expressed genes related to immunity included IL10, CCL5, IL1B, OSM, TNFRSF1B, IL7, and CCR3, among others. The RT-qPCR results for six randomly selected core differentially expressed genes showed that the RT-qPCR expression pattern was consistent with the RNA Seq results. The immune-related genes in Bactrian camels affected by subclinical mastitis are primarily concentrated in the immune response and the cytokine–cytokine receptor interaction pathway. Given the importance of these pathways and the connections among related genes, the immune genes within these pathways may play a crucial role in the pathogenesis of subclinical mastitis in Bactrian camels. This study provides a valuable reference for investigating the immune regulatory mechanisms of subclinical mastitis in Bactrian camels.

Functional characterization and mechanism of the multidrug resistance transport potein YoeA in Bacillus subtilis.

Transport proteins are essential for bacterial resistance to antibiotics and toxins, but their mechanisms remain poorly understood in Bacillus subtilis. In the present study, overexpression of yoeA enhanced resistance to various antibiotics, with its expression induced by these antibiotics, especially penicillin and plipastatin. The ΔyoeA strain exhibited significant growth inhibition at 100μg/mL of plipastatin, while as high as 10,000μg/mL of iturin/surfactin are required to achieve comparable inhibition, suggesting a higher sensitivity of ΔyoeA to plipastatin. The transcript level of yoeA gene was increased 2.71-fold in response to plipastatin, significantly higher than the levels induced by surfactin and iturin. The ethidium bromide (EtBr) efflux activity of YoeA was inhibited by carbonyl cyanide chlorophenylhydrazone (CCCP) and enhanced by Na+. Molecular modeling studies revealed that cation-π interactions of Na+ with Y287 and Y434 residues in the C-terminal domain of YoeA contribute to its ion channel function, and Cu2+ can form coordination bonds with the N atoms of H278 and H421 residues on the C-terminal surface of YoeA, promoting plipastatin efflux in a dose-dependent manner. The present study characterized the main factors influencing YoeA's efflux activity, revealed its transport mechanism, and provided new insights into enhancing antimicrobial peptide production and controlling bacterial resistance.

Effects of exopolysaccharides form Lactobacillus plantarum KX041 on high fat diet-induced gut microbiota and inflammatory obesity.

Lactobacillus plantarum KX041 is a probiotics obtained from early screening, which can relieve inflammation and enrich the anti-obesity intestinal flora, and produce high yield of exopolysaccharides (EPS). The extraction, structure and physicochemical properties of EPS have been completed earlier. However, whether the functional activity of L. plantarum KX041 is related to the EPS is still unclear. In this study, combined in vivo and in vitro tests explored the effects of EPS on inflammatory obesity. In vitro tests, EPS relieased inflammation by inhibiting TLR4/p-NFκB, and inhibited adipocyte differentiation and reduced the transcriptional level of inflammation and lipid-synthesis genes (especially CD36 and peroxisome proliferators-activated receptors-γ PPARγ). In vivo tests demonstrated that EPS could alleviate weight gain, white adipocyte expansion, organ damage, insulin resistance, hyperlipidemia, and inflammatory response caused by high fat diet. Combined with in vitro tests, the transcription level of inflammation and lipid-synthesis genes in adipose tissue identified that IL-1β and PPARγ were the key genes for the effect of EPS on inflammatory obesity. Moreover, EPS increased the abundance of Akkermansia and Lachnospiraceae_NK4A136_group, and intestinal levels of isobutyrate, which is one of the key factors in alleviating inflammatory obesity. These insights may inform the development of functionalized dietary interventions in obesity.

Estrogen and glucocorticoid promote the lactoferrin synthesis and secretion ability of bovine mammary epithelial cells through ER and GR signaling pathways.

Lactoferrin (LF) is an innate immunity glycoprotein with antibacterial, anti-inflammatory, antiviral, anti-tumor, and autoantibody activity-enhancing properties. Steroid hormones are essential for development and lactation in the dairy cow mammary gland, and act through binding to receptors that drive gene transcription. However, it remains unclear whether steroid hormone receptors play roles in LF synthesis in bovine mammary epithelial cells (BMECs). In this study, we investigated the direct effects of estrogen and glucocorticoid on LF synthesis and secretion by BMECs. The results show that treatment of BMECs with estrogen (17-β-estradiol, E2) and glucocorticoid (hydrocortisone) significantly promoted cell proliferation. Furthermore, E2 or hydrocortisone increased the expression levels of estrogen receptor (ER) and glucocorticoid receptor (GR), and stimulated the synthesis and secretion of LF in BMECs. Treatment of BMECs with various inhibitors (fulvestrant, mifepristone, and pimozide) decreased LF gene transcript and LF protein levels. It was concluded that fulvestrant and mifepristone inhibit LF transcription and translation via inhibiting ER and GR, respectively. Our data indicate that E2 and hydrocortisone regulate LF protein synthesis through the ER and GR signaling pathways. These results provide new information about the regulation of the synthesis of functional proteins in milk.

Metabolic fuel selection in rainbow trout: Coping with Intralipid infusion.

The impact of hyperlipidemia on fuel selection has never been investigated in fish. This study quantifies how Intralipid administration affects: (i) in vivo mobilization of lipids (lipolytic rate: Ra glycerol) and carbohydrates (hepatic glucose production: Ra glucose) in rainbow trout, and (ii) key proteins involved in the regulation of fuel metabolism that could explain changes in glycerol and glucose kinetics. Results show that Intralipid triples lipolytic rate (from 2.5 ± 0.5 to 7.8 ± 1.1 µmol glycerol kg-1 min-1) and inhibits glucose production by 36% (from 7.3 ± 0.9 to 4.7 ± 0.4 µmol kg-1 min-1). The stimulation of lipolysis is probably driven by lipase activation (gene expression of hormone sensitive lipase increases in muscle), or by mass action effect. Such a strong lipolytic response is quite surprising because baseline Ra glycerol is already particularly high in fish, and well known for its stability under a variety of stresses that have important effects in mammals. The reduction in trout Ra glucose is likely caused by a large decrease in glycogen mobilization because hepatic gluconeogenic pathway capacity may rise as a consequence of increases in gluconeogenesis gene transcript levels. In contrast to humans, which maintain steady glucose production in response to Intralipid infusion, rainbow trout appear to overcompensate increased gluconeogenic capacity with a disproportionately large inhibition of glycogen breakdown. Overall, these Intralipid-driven changes in glycerol and glucose kinetics allow fish to decrease their reliance on carbohydrates and amino acids by replacing them, in part, with fatty acids as metabolic fuels.

Corneal stromal cells from patients with keratoconus exhibit alterations in the ESCRT-dependent machinery responsible for multivesicular body formation.

Previous studies have reported that exosomes produced by corneal stromal cells from keratoconus patients exhibit a molecular content distinct from those produced by cells from healthy donors. This study investigates differences in the expression of ESCRT components, regarded as the most critical mechanism in exosome biogenesis. The study included analysis of transcription levels of system-encoding genes using qRT-PCR reactions, as well as semiquantitative protein determination through immunocytochemistry. Of the 34 molecules analyzed, mRNA downregulation was observed in 8 in pathological cells. In keratoconus, genes encoding STAM2 from the ESCRT-0 complex and VPS37A, VPS37C, VPS37D and UBAP1 from the ESCRT-I complex were found to be underexpressed, although VPS37D could not be confirmed at the protein level. Additionally, two other expression alterations affected the ESCRT-III complex, involving the core protein CHMP4C and the regulatory protein CHMP1B. Finally, deregulation of the ubiquitin-specific peptidase UBPY was observed. Most changes identified in this study affected specific isoforms, which could suggest functional diversification and differences in cargo recognition in the context of pathology. Altogether, these findings suggest that the previously reported alteration in the molecular content of exosomes produced by stromal cells in keratoconus may be, at least partially, due to disruptions in the exosome synthesis machinery.

Enhanced methanol-xylose co-utilization strategy in Komagataella phaffii.

Bio-manufacturing based on non-food carbon sources is conducive to alleviating the global food crisis and greenhouse effect. However, the mechanism of the utilization of methanol and xylose in Komagataella phaffii based on endogenous metabolic pathways has not been fully explored. In this study, transcriptomics revealed a positive correlation between methanol metabolic efficiency and the transcription level of genes related to xylose metabolism and phosphate metabolism. By providing sufficient phosphate to the strain, the methanol utilization rate of the Komagataella phaffii GA01 strain was improved, and the final biomass reached 7.5g DCW/L. Metabolomics further confirmed that methanol could effectively activate xylose metabolism of the strain, and the consumption rates of methanol and xylose of the Komagataella phaffii GA01 strain could reach 3.87g/L/d and 1.83g/L/d, which were 34% and 357.5% higher than that of the wild-type strain, respectively. This study further promotes the application of methanol and xylose in microbial fermentation.

Ethylene-Mediated Production and Emission of Limonene Influence Brown Planthopper Preference for Rice Plants.

Volatile organic compounds (VOCs) play a key role in plant communication with other organisms in the natural environment. However, the regulatory role of the phytohormone ethylene in volatile production in plants remains unclear. In this study, we demonstrated that the application of an ethylene precursor and amplification of ethylene signaling make rice plants more attractive to brown planthopper (BPH) females for feeding and oviposition. A combination of transcriptome and VOCs analyses indicated that overexpression of OsEIL1, a master transcription factor in the ethylene pathway, influences the transcript levels of several terpene synthase genes as well as the production of volatile terpenes. Further investigation revealed that the expression of the limonene synthase gene, OsTPS19, was down-regulated in OsEIL1-overexpressing rice plants, leading to a decrease in limonene production and release. Genetic analysis confirmed the essential role of limonene in the OsEIL1-mediated attractiveness of rice plants to BPH. Our findings provide new perspectives for understanding the role of ethylene signaling in volatile-mediated plant-insect interactions.

P0066 Enhanced transcription of human endogenous retroviruses and downregulation of TRIM28 in patients with Crohn’s disease and ulcerative colitis

Abstract Background Human endogenous retroviruses (HERVs) represent 8% of the human genome. They originate from ancestral infections of germinal cells. HERVs are no longer infectious, but some retroviral sequences can be activated, and their aberrant expressions have been implicated in a number of diseases, including chronic inflammatory and autoimmune disorders. HERV transcription is regulated by TRIM28 and SETDB1, which are directly involved in epigenetic processes and in the modulation of the immune response. HERVs and TRIM28/SETDB1 expressions have not been investigated in patients affected by inflammatory bowel disease (IBD). Methods We assessed, through a PCR real-time Taqman amplification assay, the transcription levels of pol genes of HERV-H, -K, and -W families, of env genes of syncytin 1 (SYN1), SYN2, and HERV-W, as well as of TRIM28 and SETDB1 in whole blood of 48 patients with Crohn’s disease (CD), 20 with ulcerative colitis (UC) and in age-matched 104 healthy controls (HC). Results The transcriptional levels of HERV-H-pol (p=0.0003) and HERV-K-pol (p=0.001) were significantly higher in IBD patients as compared with HC, while no significant differences between the two groups were found for the remaining HERVs (Figure 1). The median mRNA levels of HERV-H-pol in CD was 1.53 (1.22–2.02), in UC was 1.56 (0.96–1.99), in HC was 1.24 (0.63–1.63); median HERV-K-pol in CD was 1.27 (1.00–1.76), in UC was 1.41 (0.99–1.85), in HC was 0.97 (0.71–1.34). The mRNA levels of TRIM28 were significantly downregulated in IBD patients (median mRNA levels of TRIM28 in CD = 0.73, 0.56–0.92, in UC = 0.76, 0.60–0.92, in HC = 1.01, 0.79–1.25, p < 0.001), while the SETDB1 levels were preserved (p = 0.95) (Figure 2). Conclusion Over-expressions of HERVs and impaired transcription of TRM28 in patients affected by CD or UC suggest that they might be main actors in the pathophysiology of IBD and open the way to innovative targeted interventions.

The Role of Naphthaleneacetic Acid and 1-Methylcyclopropene in Preventing Preharvest Berry Dropping in Vitis vinifera L.

Fruit dropping represents a concern in many fruit species, including Vitis vinifera L. This research investigated the role of two plant growth regulators (PGRs), naphthaleneacetic acid (NAA) and 1-methylcyclopropene (1-MCP), in mitigating preharvest berry dropping (PHBD) through affecting ethylene (ET) and auxin (AUX) metabolism and interactions, key hormones involved in abscission. The experiment was carried out on cv. Dolcetto, with PGR treatments applied at 43, 53, and 90 days after anthesis (DAA) for NAA and at 56 DAA for 1-MCP. Berry dropping incidence, yield parameters, and transcript levels of genes related to ET and AUX pathways were analyzed, including VIT_212s0059g01380, VIT_211s0016g02380, VIT_207s0005g00820, VIT_216s0013g00980, VIT_203s0091g00310, and VIT_207s0104g00800. Both NAA and 1-MCP significantly reduced PHBD, with NAA achieving a 92% reduction and 1-MCP an 82% reduction compared to control vines. Transcript analysis revealed differential gene expression patterns, indicating that NAA affects the ET biosynthesis pathway, while 1-MCP interferes with ET receptor signaling. The results suggest that both PGRs effectively reduced berry dropping, providing a basis for integrated crop management strategies to mitigate PHBD in grapevine cultivars susceptible to this physiological disorder.

Cancer-Specific Activation of the Vesicular Stomatitis Virus Matrix by Survivin Promoter in Breast Cancer Cells.

Oncolytic viral-based therapy and specific gene expression by promoters are modern targeted oncotherapy approaches that have gained significant attention in recent years. In this study, both strategies were combined by designing cancer-specific activation of vesicular stomatitis virus matrix expression under the survivin promoter. The matrix sequence was cloned downstream of the survivin promoter (pM). After transfecting MCF-7 cells with pM, cell proliferation and apoptosis induction were assessed. Additionally, the transcript levels of matrix and apoptosis-related genes in response to pM was assessed. The proliferation of MCF-7 cells was significantly reduced by the constructed matrix-expressing plasmid at 48 and 72 h post-transfection (p < 0.05). Enhanced matrix expression resulted in the down-regulation of MMP-9, TP53, and NF-kB, while simultaneously up-regulating Bax transcripts. Evaluating the effect of pM vector on apoptosis induction revealed a significant increase in the MCF-7 cells compared to untreated cells (p < 0.05). The absence of significant matrix gene expression in HDF cells, relative to MCF-7 cells, further underscores the specific function of the Survivin promoter in cancer cells. These findings suggest that the matrix may have various biological functions in a diverse set of non-apoptotic pathways. Further research on the association of the matrix with other genes could provide insights into the biomedical significance and future perspectives of the matrix in cancer gene therapy.

Mitochondrial Gene Expression of Three Different Dragonflies Under the Stress of Chlorpyrifos.

Chlorpyrifos (CPF) is an organophosphate insecticide that is extensively utilized globally due to its effectiveness against over 200 pest species. CPF exhibits its toxicity primarily through the inhibition of the acetylcholinesterase (AChE) enzyme, while mitochondrial damage and dysfunction have also been observed. The present study quantified the transcript levels of mitochondria protein-coding genes (mtPCGs) using quantitative real-time polymerase chain reaction (RT-qPCR) in samples of larvae of three dragonfly species (A. parthenope, E. elegans, and G. confluens) under different levels of CPF stress. By exposing larvae from uncontaminated populations to 0.05 μg/L CPF for 24 h, the transcript levels of seven mtPCGs in A. parthenope were significantly increased (p < 0.05) by 1.89 ± 0.42-fold for COI, 4.30 ± 0.24-fold for COIII, 5.94 ± 0.17-fold for ND1, 4.69 ± 0.56-fold for ND2, 3.44 ± 0.48-fold for ND4, 2.19 ± 0.53-fold for ND4L, and 5.05 ± 0.36-fold for Cytb, respectively. In E. elegans, the transcript levels of ND1, ND2, and ND4 increased by 1.23 ± 0.15, 1.48 ± 0.31, and 1.98 ± 0.25-fold, respectively (p < 0.05). In G. confluens, the transcript levels of COI, COIII, and ND4 increased by 1.56 ± 0.13, 1.50 ± 0.26, and 3.74 ± 0.40-fold, respectively (p < 0.01). It was demonstrated that the transcript levels of different mtPCGs showed significant up-regulation in the three different dragonfly larvae under CPF stress in the absence of mortality. ND4 was significantly increased in all three species, indicating that it is an important target gene. The present study underscores the response of mitochondrial gene expression in larvae of three different species in response to CPF pollutants, indicating that pesticide influences can potentially alter mitochondrial gene expression and potentially act as a method for assessing aquatic ecosystem health.

Transcriptome analysis of novel B16 melanoma metastatic variants generated by serial intracarotid artery injection

The incidence of brain metastases (BrM) in patients with metastatic melanoma is reported to be 30–50% and constitutes the third most frequent BrM after breast and renal cancers. Treatment strategies including surgical resection, stereotactic radiation, and immunotherapy have improved clinical response rates and overall survival, but the changes that occur in circulating melanoma cells to promote invasion of the brain are not fully understood. To investigate brain tropism, we generated new variants of the B16 mouse melanoma model by serially passaging B16 cells through the brain of immune competent syngeneic C57BL/6 mice. Cells were injected into the right carotid artery and recovered from the brain after the mice had reached the study endpoint due to tumor burden, then expanded in vitro and reinjected. We compared the transcriptomes of 4th generation B16 cell populations from separate lineages with the founder B16-F0 cells. Gene set enrichment analysis (GSEA) of differentially expressed protein coding genes revealed that cells isolated from the brain as well as from the lung and meninges expressed higher levels of genes associated with an epithelial to mesenchymal transition (EMT), upregulation of the KRAS signaling pathway, and a metastasis aggressiveness gene signature associated with poor survival in melanoma patients. Principal component analysis of differentially expressed genes showed that 4th generation melanoma cells isolated from the brain, lung and meninges from one lineage were distinct from those of the other three lineages. Among the differentially expressed genes, transcript levels of several genes, including Itgb2, Rftn2, and Kcnn4, were significantly higher in all cell populations that comprised this lineage compared with all cell populations from the other three lineages. In conclusion, we have derived an aggressive, highly brain metastatic B16 variant associated with leptomeningeal disease by serially passaging cells in vivo.

FRET analysis of the unwrapping of nucleosomal DNA containing a sequence characteristic of the + 1 nucleosome

Sequence-dependent mechanical properties of DNA could play essential roles in nuclear processes by affecting histone-DNA interactions. Previously, we found that the DNA entry site of the first nucleosomes from the transcription start site (+ 1 nucleosome) in budding yeast enriches AA/TT steps, but not the exit site, and the biased presence of AA/TT in the entry site was associated with the transcription levels of yeast genes. Because AA/TT is a rigid dinucleotide step, we considered that AA/TT causes DNA unwrapping. However, our previous MNase-seq experiments with reconstituted nucleosomes left some doubt regarding this interpretation, owing to its high exonuclease activity. Furthermore, MNase cleavage did not provide direct evidence of its structural state. In this study, Förster resonance energy transfer (FRET) measurements were used to investigate salt-induced conformational changes in nucleosomal DNA containing AA/TT repeats at the entry site. We observed that the AA/TT region wrapped around the histone core was as likely as other DNA sequences at physiological salt concentrations. However, it unwrapped at a lower salt concentration, indicating weaker electrostatic interactions with the histone core. Ethidium-induced nucleosome disruption assay showed that the intercalator had greater access to DNA with AA/TT at the entry site. Taken together, these results suggest that AA/TT at the entry sites induces DNA unwrapping from the histone core on the promoter side, which may promote transcriptional activation in response to the approach of transcription-related proteins.

Convergent pairs of highly transcribed genes restrict chromatin looping in Dictyostelium discoideum.

Dictyostelium discoideum is a unicellular slime mold, developing into a multicellular fruiting body upon starvation. Development is accompanied by large-scale shifts in gene expression program, but underlying features of chromatin spatial organization remain unknown. Here, we report that the Dictyostelium 3D genome is organized into positionally conserved, largely consecutive, non-hierarchical and weakly insulated loops at the onset of multicellular development. The transcription level within the loop interior tends to be higher than in adjacent regions. Loop interiors frequently contain functionally linked genes and genes which coherently change expression level during development. Loop anchors are predominantly positioned by the genes in convergent orientation. Results of polymer simulations and Hi-C-based observations suggest that the loop profile may arise from the interplay between transcription and extrusion-driven chromatin folding. In this scenario, a convergent gene pair serves as a bidirectional extrusion barrier or a 'diode' that controls passage of the cohesin extruder by relative transcription level of paired genes.

Post-transcriptional cross- and auto-regulation buffer expression of the human RNA helicases DDX3X and DDX3Y.

The Y-linked gene DDX3Y and its X-linked homolog DDX3X survived the evolution of the human sex chromosomes from ordinary autosomes. DDX3X encodes a multifunctional RNA helicase, with mutations causing developmental disorders and cancers. We find that, among X-linked genes with surviving Y homologs, DDX3X is extraordinarily dosage sensitive. Studying cells of individuals with sex chromosome aneuploidy, we observe that when the number of Y Chromosomes increases, DDX3X transcript levels fall; conversely, when the number of X Chromosomes increases, DDX3Y transcript levels fall. In 46,XY cells, CRISPRi knockdown of either DDX3X or DDX3Y causes transcript levels of the homologous gene to rise. In 46,XX cells, chemical inhibition of DDX3X protein activity elicits an increase in DDX3X transcript levels. Thus, perturbation of either DDX3X or DDX3Y expression is buffered: by negative cross-regulation of DDX3X and DDX3Y in 46,XY cells and by negative auto-regulation of DDX3X in 46,XX cells. DDX3X-DDX3Y cross-regulation is mediated through mRNA destabilization-as shown by metabolic labeling of newly transcribed RNA-and buffers total levels of DDX3X and DDX3Y protein in human cells. We infer that post-transcriptional auto-regulation of the ancestral (autosomal) DDX3X gene transmuted into auto- and cross-regulation of DDX3X and DDX3Y as these sex-linked genes evolved from ordinary alleles of their autosomal precursor.

The Involvement of Glutamate-mGluR5 Signaling in the Development of Vulvar Hypersensitivity.

Provoked vulvodynia (PV) is the leading cause of vulvar pain and dyspareunia. The etiology of PV is multifactorial and remains poorly understood. PV is associated with a history of repeated vulvar inflammation and is often accompanied by sensory neuromodulation as a result of activation of the metabotropic glutamate receptor 5 (mGluR5) in the sensory nerve terminals. Therefore, this study aims to examine the role of glutamate-mGluR5 signaling during the initial inflammatory phase in chronic vulvar pain development in an animal model of PV.Thermal and mechanical vulvar sensitivity was assessed for three weeks following zymosan vulvar challenges. Anxiety-like behavior and locomotor activity were assessed at the end of the experiment. To investigate the role of glutamate mGluR5, the MTEP (mGluR5 antagonist) was injected into the vulva during vulvar inflammation. On the other hand, glutamate or CHPG (mGluR5 agonist) were injected in order to examine the effects of mGluR5 activation. RT-PCR was performed to assess changes in the transcription of genes related to neuroinflammation, neuromodulation, and neuroplasticity in the spinal cord (L6-S3). Zymosan-induced inflammation resulted in a significant thermal and mechanical vulvar hypersensitivity that persisted for over a month after the zymosan injection. However, local treatment with MTEP enhanced the vulvar mechanical and thermal hypersensitivity. On the other hand, activation of the mGluR5 via injection of glutamate or CHPG into the vulva leads to long-lasting vulvar mechanical and thermal hypersensitivity. The activation of the glutamate pathway was found to be accompanied by an increase in the transcription level of genes related to neuroinflammation and neuroplasticity in the sacral spine region. The present findings indicate that vulvar hypersensitivity is mediated by mGluR5 activation during inflammation. Hence, modulation of the mGluR5 pathway during the critical period of inflammation contributes to preventing chronic vulvar pain development. Conversely, activation of the mGluR5 pathway leads to long-lasting mechanical and thermal hypersensitivity.

ENTPD1, NT5E, ADORA2A, FOXP3 and RORγ gene expression in peripheral blood of patients with ulcerative colitis

Development of ulcerative colitis (UC) is accompanied by activation of the purinergic signaling pathway and an increased level of extracellular adenosine. Adenosine is involved in regulating the balance of pro-inflammatory T-helper type 17 (Th17) cells and immunosuppressive regulatory T-cells (Treg), Their disturbance is believed to be one of the main immune factors causing this disease. However, expression of genes encoding Treg cell and Th17 transcription factors (FOXP3 and RORγ, respectively) and genes encoding members of the CD39/CD73/A2AR signaling pathway (ENTPD1, NT5E, ADORA2A) in peripheral blood has not been studied enough. The purpose of our study was to evaluate the expression levels of ENTPD1, NT5E, ADORA2A, FOXP3 and RORγ genes in ulcerative colitis (UC). Thirty-eight patients were examined including 20 patients diagnosed with UC (15 patients on a basic therapy with 5-ASA derivatives, UC1 group); 5 patients treated with Prednisone (group UC2), and 18 apparently healthy people. Total RNA was isolated from peripheral blood leukocytes (PBLCs). The levels of gene transcripts were studied by real-time PCR technique. The ENTPD1 gene mRNA content in UC patients on a basic therapy with 5-ASA derivatives was higher than in healthy people (p = 0.0045). The levels of NT5E and ADORA2A transcripts in the PBLCs of patients in the UC1 group were higher than those of patients in the UC2 group (p = 0.0486 and p = 0.0289, respectively) and healthy individuals (p = 0.0007 and p &lt; 0.001, respectively). The mRNA content of FOXP3 gene in PBLCs of the UC1 group of patients was higher than in conditionally healthy individuals (p = 0.0093). The level of RORγ gene expression in PBLCs of the patients from UC1 and UC2 groups was higher than in healthy individuals (p = 0.0005). Increased levels of FOXP3 and RORγ gene expression (the transcription factors of Treg and Th17 cells, respectively), were observed in PBLCs of UC patients on baseline therapy, whereas expression levels of these genes in prednisolone-treated UC patients did not differ from controls. Correlations between the expression levels of some genes under study were revealed in control persons and in the UC1 group.

Behavioural responses to acute warming precede critical shifts in the cellular and physiological thermal stress responses in a salmonid fish (brook trout, Salvelinus fontinalis).

From a conservation perspective, it is important to identify when sub-lethal temperatures begin to adversely impact an organism. However, it is unclear whether, during acute exposures, sub-lethal cellular thresholds occur at similar temperatures to other physiological or behavioural changes, or at temperatures associated with common physiological endpoints measured in fishes to estimate thermal tolerance. To test this, we estimated temperature preference (15.1±1.1°C) using a shuttle box, agitation temperature (22.0±1.4°C) as the point where a fish exhibits a behavioural avoidance response and the CTmax (28.2±0.4°C) as the upper thermal limit for 1 yr old brook trout (Salvelinus fontinalis) acclimated to 10°C. We then acutely exposed a different subset of fish to the mean temperatures associated with the pre-determined physiological endpoints and sampled tissues when they reached the target temperature or after 60 min of recovery at 10°C for transcriptomic analysis. We used qPCR to estimate mRNA transcript levels of genes associated with heat shock proteins, oxidative stress, apoptosis, and inducible transcription factors. A major shift in the transcriptome response occurred once the agitation temperature was reached, which may identify a possible link between the cellular stress response and the behavioural avoidance response.

Enhancement of FK520 production in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 by overexpressing the regulatory gene fkbR2.

Ascomycin (FK520) is a 23-membered macrolide antibiotic primarily produced by the Streptomyces hygroscopicus var. ascomyceticus. Structurally similar to tacrolimus and rapamycin, it serves as an effective immunosuppressant widely used in the treatment of rejection reactions after organ transplantation and certain autoimmune diseases. Currently, FK520 is mainly produced through microbial fermentation, but its yield remains low. Since the gene fkbR2 is a regulatory gene within the FK520 biosynthesis gene cluster that has not been studied, this paper focuses on the overexpression of the gene fkbR2 in Streptomyces hygroscopicus var. ascomyceticus ATCC 14891 (WT). By constructing a strain with overexpressed fkbR2 gene, we initially obtained a high-yield strain R2-17 through shake flask fermentation, with a 28% increase in yield compared to WT. In the process of further improving the stability of the high-yield strain, this paper defines two indices: high-yield index and stability index. After two consecutive rounds of natural breeding, strain R2-17 achieved a high-yield index of 100% and a stability index of 80%. Finally, the high-yield strain R2-17-3-10 was successfully screened, and the yield was increased by 34% compared with the strain WT, reaching 686.47mg/L. A comparative analysis between the high-yield strain R2-17-3-10 and the original strain WT revealed differences in fermentation process parameters such as FK520 synthesis rate, pH, bacterial growth, glycerol consumption rate, ammonia nitrogen level, and ammonium ion concentration. In addition, the transcription levels of genes involved in the synthesis of precursors 4,5-dihydroxycyclohex-1-enecarboxylic acid (fkbO), ethylmalonyl-CoA (fkbE, fkbU, fkbS), and pipecolic acid (fkbL), as well as pathway-specific regulatory genes (fkbN, fkbR1), were significantly increased at different time points in the high-yield strain R2-17-3-10. EMSAs analysis showed that the FkbR2 protein could not bind to the promoter region of above genes. This suggests that the gene fkbR2 may enhance the supply of FK520 synthetic precursors by indirectly regulating the transcription levels of these genes, thereby promoting an increase in FK520 production. These results demonstrate that modifying genes within the biosynthetic gene clusters of natural products can be successfully applied to increase the yields of industrially and clinically important compounds. However, it is found that fkbR2 gene is a regulatory gene that has not been fully studied, and it is worth further studying its regulatory mechanism.