Receptor mRNA Expression Research Articles

Lactobacillus johnsonii GLJ001 prevents DSS-induced colitis in mice by inhibiting M1 macrophage polarization via gut microbiota-SCFAs axis

Inflammatory Bowel Disease (IBD) is increasing worldwide and has become a global emergent disease. Probiotics have been reported to be effective in relieving colitis. Previous studies found ripened Pu-erh tea (RPT) promoted gut microbiota resilience against dextran sulfate sodium (DSS)-induced colitis in mice by increasing relative abundance of Lactobacillus. However, whether and how it alleviated DSS-induced colitis in mice need to be explored. Here, we screened a probiotic Lactobacillus johnsonii GLJ001 from feces of ripened Pu-erh tea (RPT)-administrated mice. In this study, L. johnsonii GLJ001 attenuated symptoms of DSS-induced colitis in mice, including weight loss, increased disease activity index (DAI), colon shortening and colon tissue damage, as well as high expression of inflammatory cytokines and disturbances of intestine barrier function. Furthermore, abundances of short-chain fatty acids (SCFAs)-producing bacteria (i.e. Clostridium cluster IV and XIVa, Lachnospiracea_incertae_sedis and Ruminococcus) were enhanced in the cecum of mice treated with L. johnsonii GLJ001, accompanying by an increase of SCFAs. It was also found that SCFAs inhibited mRNA expression of M1 macrophage markers (Inos and CD86), inflammatory cytokines (TNF-α and Il-1β) and SCFAs receptors (Gpr41 and Gpr43) induced by lipopolysaccharide (LPS) and interferon-γ (IFN-γ) in THP-1 cell line. Collectively, L. johnsonii GLJ001 prevented DSS-induced colitis in mice by inhibiting M1 macrophage polarization via gut microbiota-SCFAs axis, and can be administered for management of colitis.

Differential effects of leptin on energy metabolism in murine cell models of metastatic triple negative breast cancer

BackgroundLeptin, an energy balance regulator secreted by adipocytes, increases metastatic potential of breast cancer cells. The impact on cancer cell metabolism remains unclear given that most studies of leptin and breast cancer cell metabolism utilize supraphysiological glucose concentrations.MethodsUsing two murine models of metastatic triple-negative breast cancer (TNBC) differing in genetic alterations (4T1: p53 and Pik3ca mutations; metM-Wntlung: increased Wnt signaling) and cultured in physiological (5 mM) glucose media, we tested the hypothesis that leptin increases migration of metastatic breast cancer cells through regulation of glucose metabolism.ResultsOur results showed that leptin treatment, compared with vehicle, increased cell migration in each cell line, with decreased leptin receptor (Ob-R) mRNA expression in 4T1, but not metM-Wntlung, cells. AMP-activated protein kinase (AMPK) was activated in 4T1 with leptin treatment but decreased in metM-Wntlung. Leptin decreased fatty acid synthase (Fasn) and carnitine palmitoyltransferase 1a (Cpt1a) mRNA expression in 4T1 cells but increased their expression in metM-Wntlung cells. Fatty acid oxidation was not necessary for leptin-induced migration in either cell line. Leptin increased palmitate synthesis from glucose in metM-Wntlung, but not 4T1 cells. Moreover, although leptin increased glucose transporter 1 (Glut1) mRNA expression in both cell lines and inhibition of glycolysis blocked leptin-induced migration in metM-Wntlung, but not 4T1 cells.ConclusionTaken together, these results demonstrate that at physiological glucose concentrations, leptin increases migration of 4T1 and metM-Wntlung cells via shared and distinct effects on energy metabolism, suggesting that the type of TNBC genetic alteration plays a role in differential metabolic regulation of leptin-induced migration.

Dysregulated HPA axis during postnatal developmental stages in the BTBR T+ Itpr3tf/J mouse: A model of autism spectrum disorder.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder. Some children with ASD show enhanced cortisol response to stress. BTBR T+ Itpr3tf/J (BTBR) mice, an ASD model, display behavior consistent with the three diagnostic categories of ASD and exhibit an exaggerated response to stress in adulthood. However, it remains unclear how basal corticosterone levels change and how the hypothalamic-pituitary-adrenal axis responds to stress during the early life stages in BTBR mice. In this study, we found that basal corticosterone levels showed characteristic changes, peaking at weaning during postnatal development in both BTBR and control C57BL/6J (B6J) mice. Furthermore, we observed higher corticosterone and corticotropin-releasing hormone levels in BTBR mice than in B6J mice following acute stress exposure during weaning; however, adrenocorticotropic hormone levels were lower in BTBR mice. Glucocorticoid receptor mRNA expression levels in the hippocampus and lateral septum after stress were higher in BTBR mice than in B6J mice. This study documented changes in corticosterone levels at baseline during postnatal development in mice and showed that BTBR mice exhibited disrupted stress responses at weaning.

Opposite-sex pairing alters social interaction-induced GCaMP and dopamine activity in the insular cortex of male prairie voles.

The prairie vole ( Microtus ochrogaster ) is a monogamous rodent species which displays selective social behaviors to conspecifics after establishing a pair bonded relationship, specifically partner-directed affiliation and stranger-directed aggression. This social selectivity relies on the ability of an individual to respond appropriately to a social context and requires salience detection and valence assignment. The anterior insular cortex (aIC) has been implicated in stimulus processing and categorization across a variety of contexts and is well-situated to integrate environmental stimuli and internal affective states to modulate complex goal-directed behaviors and social decision-making. Surprisingly, the contribution of the aIC to the expression of pair bond-induced social selectivity in prairie voles has been drastically understudied. Here we examined whether neural activity and gene expression in the aIC change in response to opposite-sex pairing and/or as a function of pairing length in male prairie voles. Opposite-sex pairing was characterized by changes to calcium and dopamine (DA) transients in the aIC that corresponded with the display of social selectivity across pair bond maturation. Furthermore, D1 and D2 receptor mRNA expression was significantly higher in males after 48 hrs of cohabitation with a female partner compared to same-sex housed males, and D2 mRNA remained significantly higher in males with a female partner compared to same-sex housed males after a week of cohabitation. Together, these results implicate a role for DA and its receptors in the aIC across the transition from early-to late-phase pair bonding.

Regulatory Effects of Copper on Ghrelin Secretion in Rat Fundic Glands.

Copper (Cu) is an effective additive in feed for promoting growth. Growth dan axis comprising growth hormone (GH), somatostatin (SS) and GH-releasing hormone (GHRH), with ghrelin regulating their release. The growth-promoting effects of Cu are closely related to ghrelin, but the specific mechanism behind the relationship remains unknown. We investigated the adjustment of ghrelin synthesis and secretion by Cu. Sprague-Dawley rats were fed basal diets with an addition of 0, 120 or 240 mg/kg Cu sulfate for 28 day to establish a growth-promoting model. Signalling molecules relevant to ghrelin synthesis and secretion were detected and mechanistically explored using enzyme-linked immunosorbent assay, quantitative reverse-transcription polymerase chain reaction and Western blot analysis. The 120 mg/kg supplement improved growth performance; significantly increased the serum levels of ghrelin, ghrelin O-acyltransferase (GOAT), acylated ghrelin (AG), GH, and reactive oxygen species (ROS) and decreased those of SS; significantly increased the mRNA and protein expression of ghrelin, GOAT, ghrelin receptor (GHS-R1α), and activator protein 1 (AP-1); increased the phosphorylation ratio of JNK and p38 MAPK; and inhibited the mRNA and protein expression of SS and SS receptor subtype 2 (SSTR2) in gastric fundic gland tissues. Thus, Cu may affect gastric ghrelin synthesis at the transcriptional level by activating the JNK/p38 MAPK pathway through increased ROS levels and regulating the activation of the downstream redox-sensitive transcription factor AP-1. SS plays a crucial determinant role in ghrelin regulation via intragastric Cu. Cu promotes GOAT activity and ghrelin secretion by inhibiting SS secretion, affecting AG levels, and promoting ghrelin acylation through ghrelin/GOAT/GHS-R1α system, modulating ghrelin secretion.

The impact of long-term isolation on anxiety, depressive-like and social behavior in aging Wistar-Kyoto (WKY) and spontaneously hypertensive (SHR) male rats

Aging is a complex process associated with multimorbidity. Hypertension, one of widespread states, is among main causes of age-related alterations in behavior, emotionality and sociability. We studied the effects of long-term isolated housing on anxiety, depressive-like and social behavior as well as changes in the adrenocortical and sympathetic systems in the aging normotensive Wistar Kyoto (WKY) and spontaneously hypertensive rats (SHR). Ten-month-old male rats of both strains were subjected to 90-day isolated or group housing. Surprisingly, social isolation induced only mild effect on anxiety without influencing other affective-related behaviors. No effects of isolated housing on sociability or social novelty preferences were revealed. Despite the adrenal gland hypertrophy in the SHRs, corticosterone levels remained stable within the period of isolation but the expression of nuclear glucocorticoid receptor (Nr3c1) mRNA in the adrenals was lower in the SHR as compared to WKY rats. Pre-existing hypertension, associated with SHR genotype, did not significantly contribute to the effects of social isolation. The data suggest that the aged WKY and SHR rats are relatively resilient to chronic social stress associated with isolated housing.

Transgenic rat with ubiquitous expression of angiotensin-(1-7)-producing fusion protein: a new tool to study the role of protective arm of the renin-angiotensin system in the pathophysiology of cardio-renal diseases.

The aim of the present study was to assess systemic circulatory and tissue activities of both the classical arm and of the alternative arm of the renin-angiotensin system (RAS) in a new transgenic rat line (TG7371) that expresses angiotensin-(1-7) (ANG 1-7)-producing fusion protein; the results were compared with the activities measured in control transgene-negative Hannover Sprague-Dawley (HanSD) rats. Plasma and tissue concentrations of angiotensin II (ANG II) and ANG 1-7, and kidney mRNA expressions of receptors responsible for biological actions of ANG II and ANG 1-7 [i.e. ANG II type 1 and type 2 (AT1 and AT2) and Mas receptors] were assessed in TG7371 transgene-positive and in HanSD rats. We found that male TG7371 transgene-positive rats exhibited significantly elevated plasma, kidney, heart and lung ANG 1-7 concentrations as compared with control male HanSD rats; by contrast, there was no significant difference in ANG II concentrations and no significant differences in mRNA expression of AT1, AT2 and Mas receptors. In addition, we found that in male TG7371 transgene-positive rats blood pressure was lower than in male HanSD rats. These data indicate that the balance between the classical arm and the alternative arm of the RAS was in male TGR7371 transgene-positive rats markedly shifted in favor of the latter. In conclusion, TG7371 transgene-positive rats represent a new powerful tool to study the long-term role of the alternative arm of the RAS in the pathophysiology and potentially in the treatment of cardio-renal diseases.

Dietary bile acids supplementation protects against Salmonella Typhimurium infection via improving intestinal mucosal barrier and gut microbiota composition in broilers

BackgroundSalmonella Typhimurium (S. Typhimurium) is a common pathogenic microorganism and poses a threat to the efficiency of poultry farms. As signaling molecules regulating the interaction between the host and gut microbiota, bile acids (BAs) play a protective role in maintaining gut homeostasis. However, the antibacterial effect of BAs on Salmonella infection in broilers has remained unexplored. Therefore, the aim of this study was to investigate the potential role of feeding BAs in protecting against S. Typhimurium infection in broilers.MethodsA total of 144 1-day-old Arbor Acres male broilers were randomly assigned to 4 groups, including non-challenged birds fed a basal diet (CON), S. Typhimurium-challenged birds (ST), S. Typhimurium-challenged birds treated with 0.15 g/kg antibiotic after infection (ST-ANT), and S. Typhimurium-challenged birds fed a basal diet supplemented with 350 mg/kg of BAs (ST-BA).ResultsBAs supplementation ameliorated weight loss induced by S. Typhimurium infection and reduced the colonization of Salmonella in the liver and small intestine in broilers (P < 0.05). Compared to the ST group, broilers in ST-BA group had a higher ileal mucosal thickness and villus height, and BAs also ameliorated the increase of diamine oxidase (DAO) level in serum (P < 0.05). It was observed that the mucus layer thickness and the number of villous and cryptic goblet cells (GCs) were increased in the ST-BA group, consistent with the upregulation of MUC2 gene expression in the ileal mucosa (P < 0.05). Moreover, the mRNA expressions of Toll-like receptor 5 (TLR5), Toll-like receptor 4 (TLR4), and interleukin 1 beta (IL1b) were downregulated in the ileum by BAs treatment (P < 0.05). 16S rDNA sequencing analysis revealed that, compared to ST group, BAs ameliorated the decreases in Bacteroidota, Bacteroidaceae and Bacteroides abundances, which were negatively correlated with serum DAO activity, and the increases in Campylobacterota, Campylobacteraceae and Campylobacter abundances, which were negatively correlated with body weight but positively correlated with serum D-lactic acid (D-LA) levels (P < 0.05).ConclusionsDietary BAs supplementation strengthens the intestinal mucosal barrier and reverses dysbiosis of gut microbiota, which eventually relieves the damage to the intestinal barrier and weight loss induced by S. Typhimurium infection in broilers.

Screening of bile salt hydrolase-producing lactic acid bacteria and evaluation of cholesterol-lowering activity in vitro

Hypercholesterolemia is a great threat to humans. Bile salt hydrolase (BSH)-producing lactic acid bacteria (LAB) may alleviate it, but current screening methods are inadequate. This study aimed to establish a more comprehensive in vitro method to screen BSH-producing LAB with greater potential for anti-hypercholesterolemia. Forty-one LAB were initially isolated from infant feces and 37 strains could produce BSH. Strains with BSH over 2 U/mg for sodium glycodeoxycholate and sodium taurodeoxycholate were used to analyse cholesterol degradation rate, hydrophobicity, self-aggregation, gastric fluid tolerance, and intestinal fluid tolerance. Based on the results of these indicators, Lactococcus lactis Y17, L. rhamnosus Q2, and L. fermentum Q11 with the top scores were considered anti-hypercholesterolemia candidates using a principal component analysis evaluation. Antioxidant and Caco-2 adhesion assays showed that these three strains exhibited excellent antioxidant ability and Caco-2 adhesion ability. Their total antioxidant capacity was above 0.99 μmol/L, while the Caco-2 adhesion rate was higher than 8.22%. Safety assessments, via antibiotic susceptibility and hemolysis tests, confirmed their safety. In the HepG2 cells assay, the total cholesterol content of the Y17, Q2, and Q11 groups was reduced by 0.525 mmol/L, 0.426 mmol/L, and 0.581 mmol/L, which verified the potential of the three LAB in anti-hypercholesterolemia. In addition, they could enhance cholesterol uptake and bile acid synthesis in HepG2 cells by upregulating the mRNA expression of bile acid synthase (CYP7A1 and CYP8B1) and cholesterol uptake receptors (NPC1L1 and LDLR). The results showed that the three LAB could be developed into anti-hypercholesterolemia foods with beneficial properties.

Exosomes derived from Umbilical cord mesenchymal stem cell promote hair regrowth in C57BL6 mice through upregulation of the RAS/ERK signaling pathway.

Androgenetic alopecia is one of the common types of hair loss and has become a medical and social problem due to its increasingly young onset. Existing therapies, although effective, have serious side effects and therefore better treatments need to be sought. The aim of this study was to evaluate the efficacy of umbilical cord mesenchymal stem cell-derived exosomes in the treatment of androgenetic alopecia and to investigate the mechanism of exosome regulation of hair growth. First, we randomly divided 20 C57BL/6J mice into blank group, model group, positive control group and exosomal hydrogel group, and mice were treated with hair removal on the back. The mice were injected intraperitoneally with dihydrotestosterone solution except for the blank group. At the end of the experiment, new hairs were collected and the differences in length, diameter and number of hair follicles were compared among the groups; the histopathological changes of hair follicles were observed by HE staining; the expression of androgen receptor mRNA and protein in skin tissues were compared; and the skin tissues were analyzed by real-time PCR, western blotting, immunofluorescence staining and transcriptome sequencing. Finally, the results of transcriptome sequencing experiments were verified by real-time PCR, western blotting and other techniques for the corresponding genes and proteins. Compared with the blank group, mice in the model group had shorter hair length and reduced hair diameter, and pathological observation showed that the total number of hair follicles was significantly reduced and the hair follicles were miniaturized; compared with the model group, mice in the positive control and exosome groups had longer hair length, larger hair diameter and more hair follicles; the androgen receptor mRNA content and protein expression in the skin tissue of mice in the model group were significantly higher than those in the blank group, and the protein expression in the exosome gel group was lower than that in the model group. Similarly, compared with the model group, the expression of stemness-related proteins K15 and CD200 in the skin tissues of mice in the exosome group increased, and the expression of PCNA, a protein related to cell proliferation, increased. The KEGG data showed that the differential genes were mainly enriched in the RAS/ERK pathway. In this study, we demonstrated the therapeutic effect of umbilical cord MSC-derived exosomes on androgenetic alopecia and verified that exosomes regulate hair follicle stem cell stemness through the RAS/ERK pathway to promote hair proliferation and thus hair growth in mice with androgenetic alopecia, providing a potential therapeutic strategy for androgenetic alopecia.

ERα status of invasive ductal breast carcinoma as a result of regulatory interactions between lysine deacetylases KAT6A and KAT6B

Breast cancer (BC) is the leading cause of death among cancer patients worldwide. In 2020, almost 12% of all cancers were diagnosed with BC. Therefore, it is important to search for new potential markers of cancer progression that could be helpful in cancer diagnostics and successful anti-cancer therapies. In this study, we investigated the potential role of the lysine acetyltransferases KAT6A and KAT6B in the outcome of patients with invasive breast carcinoma. The expression profiles of KAT6A/B in 495 cases of IDC and 38 cases of mastopathy (FBD) were examined by immunohistochemistry. KAT6A/B expression was also determined in the breast cancer cell lines MCF-7, BT-474, SK-BR-3, T47D, MDA-MB-231, and MDA-MB-231/BO2, as well as in the human epithelial mammary gland cell line hTERT-HME1 – ME16C, both at the mRNA and protein level. Statistical analysis of the results showed that the nuclear expression of KAT6A/B correlates with the estrogen receptor status: KAT6ANUC vs. ER r = 0.2373 and KAT6BNUC vs. ER r = 0.1496. Statistical analysis clearly showed that KAT6A cytoplasmic and nuclear expression levels were significantly higher in IDC samples than in FBD samples (IRS 5.297 ± 2.884 vs. 2.004 ± 1.072, p < 0.0001; IRS 5.133 ± 4.221 vs. 0.1665 ± 0.4024, p < 0.0001, respectively). Moreover, we noticed strong correlations between ER and PR status and the nuclear expression of KAT6A and KAT6B (nucKAT6A vs. ER, p = 0.0048; nucKAT6A vs. PR p = 0.0416; nucKAT6B vs. ER p = 0.0306; nucKAT6B vs. PR p = 0.0213). Significantly higher KAT6A and KAT6B expression was found in the ER-positive cell lines T-47D and BT-474, whereas significantly lower expression was observed in the triple-negative cell lines MDA-MB-231 and MDA-MB-231/BO2. The outcomes of small interfering RNA (siRNA)-mediated suppression of KAT6A/B genes revealed that within estrogen receptor (ER) positive and negative cell lines, MCF-7 and MDA-MB-231, attenuation of KAT6A led to concurrent attenuation of KAT6A, whereas suppression of KAT6B resulted in simultaneous attenuation of KAT6A. Furthermore, inhibition of KAT6A/B genes resulted in a reduction in estrogen receptor (ER) mRNA and protein expression levels in MCF-7 and MDA-MMB-231 cell lines. Based on our findings, the lysine acetyltransferases KAT6A and KAT6B may be involved in the progression of invasive ductal breast cancer. Further research on other types of cancer may show that KAT6A and KAT6B could serve as diagnostic and prognostic markers for these types of malignancies.

Anti-EGFR antibody immunotoxins improve cytotoxic effects in the salivary gland cancer A253 cell line

ObjectiveSalivary gland cancers account for a relatively small proportion, accounting for 3–5 %, among head and neck cancers. The range of antibody drugs available for head and neck cancers, including salivary gland cancers, has been expanding in recent years. However, reports indicated insufficient clinical efficacy despite high target expression. Our research has been investigating methods to improve the cytotoxic effects of antibody drugs in head and neck cancers, using immunotoxins (ITs), which are antibodies conjugated with toxins. Our studies on squamous cell carcinoma, which constitutes the majority of head and neck cancers, indicated that promoting endosomal escape of ITs improves their cytotoxic effects. Therefore, the current study aimed to confirm the antitumor effects of IT-Cetuximab (Cmab), which is cetuximab conjugated with a toxin, targeting salivary gland cancers—a type of cancer with limited effective treatment options aside from surgery. MethodsWe confirmed protein and mRNA expression of epidermal growth factor receptor (EGFR) in the human salivary duct carcinoma cell line, A253. We analyzed the cytotoxicity of saporin-conjugated anti-EGFR antibody (IT-Cmab). ResultsEGFR expression in A253 was comparably higher than in epidermoid carcinoma cell line A431 which highly expresses EGFR. Cmab alone exhibited no cytotoxic effects, but IT-Cmab demonstrated concentration-dependent cytotoxic effects in A253. We revealed the promising cytotoxic effects of IT-Cmab in salivary gland cancer cells. ConclusionWe first demonstrate the cytotoxic effects of IT in salivary gland cancers in vitro. The IT we used, cetuximab toxin conjugated antibody may be a remarkable treatment option for salivary gland cancers.

Female specific interactions of serotonin and testosterone in the rostral ventromedial medulla after activity-induced muscle pain

Classical preclinical studies show that serotonin (5-HT) injected into the rostral ventromedial medulla (RVM) produces analgesia that is blocked by 5-HT2 receptor antagonists. One key modulator of 5-HT activity is the serotonin transporter (SERT) which reduces serotonergic signaling through reuptake into the presynaptic terminal. In the activity-induced muscle pain model, females show widespread pain and increased SERT expression in the RVM whereas males show localized pain and no changes in SERT expression. Since prior studies show testosterone protects from the development of widespread pain, and females have widespread pain in the activity-induced pain model, we hypothesized that testosterone modulates serotonin signaling to enhance analgesia in female mice with widespread pain. We showed that testosterone reduced the enhanced SERT protein expression and increased 5-HT2A receptor mRNA expression in the RVM normally observed in the activity-induced pain model in females, but not males. Inhibition of SERT in the RVM was analgesic in both female and male mice; this analgesia was blocked by co-administration of 5-HT2A antagonist. Next, using in situ hybridization, we demonstrated co-expression of SERT, 5-HT2A receptor, and androgen receptor mRNA in cells within the RVM in female mice. Lastly, activation of androgen receptors using dihydrotestosterone reduced hyperalgesia in female mice. These data therefore show for the first time expression of androgen receptors in the RVM in female mice, that activation of androgen receptors reduces nociceptive behaviors, and endogenous testosterone modulates SERT and 5-HT2 receptor expression. Thus, we show a sex-specific role for how testosterone modulates analgesia in female mice. PerspectiveThis article presents novel mechanisms testosterone’s protection against muscle pain in female mice showing modulation of the serotonin system in the rostral ventromedial medulla. Understanding the relationship between testosterone and serotonin could lead to better treatment of individuals with muscle pain.

Chronic olanzapine treatment leads to increased opioid receptor expression and changes in feeding regulating neurons in the female rat hypothalamus

Opioid receptor antagonists have shown increasing promise as an adjunct therapy to psychotropic medication. The goal is to reduce the weight gain and metabolic adverse effects that are associated with certain second-generation antipsychotics, such as olanzapine and clozapine. In this study, female rats were treated for 4 weeks with a long-acting injectable formulation of olanzapine to assess effects on hypothalamic feeding regulation. Using quantitative spatial in situ hybridization and receptor autoradiography, expression levels of the mu, kappa and delta opioid receptors were defined in the five hypothalamic areas: paraventricular nucleus (PVN), arcuate nucleus (ARC), ventromedial nucleus (VMN), dorsomedial nucleus (DMN) and lateral hypothalamus (LH). In addition, hypothalamic neuron number and size were estimated using the unbiased optical fractionator and spatial rotator methods. Hyperphagia was observed after only 24 hours of olanzapine treatment, with continued weight gain throughout the duration of the study. In contrast, the observed food intake reversed to control levels after 2 weeks of olanzapine treatment. Chronic olanzapine treatment increased expression of kappa opioid receptor mRNA and receptor availability in the PVN, as well as increased mu opioid receptor availability in the PVN, ARC and VMN. These changes were accompanied by fewer anorexigenic proopiomelanocortin-expressing neurons of the ARC and corticotropin-releasing hormone expressing neurons of the PVN. This study links olanzapine-driven metabolic effects to increased opioid receptor expression in the hypothalamus, thus providing a rationale for the positive effects of using opioid receptor antagonists to relieve olanzapine adverse effects.

Ammonia Exposure-Induced Immunological Damage in Chicken Lymphoid Organs via TLR-7/MYD88/NF-κB Signaling Pathway and NLRP3 Inflammasome Activation.

Ammonia (NH3) is a hazardous gas that pollutes the environment and causes irritation. Its harmful effects on chickens, including its impact on their immune system, have previously been observed. However, the mechanism by which NH3 exposure causes immune system disorders in chickens remains unclear. The bursa of Fabricius (BF) and thymus are the two main lymphoid organs responsible for the proliferation, differentiation, and selection of B- and T-lymphocytes, both critical for the innate immune response of the host. In this study, we investigated the mechanism of NH3-induced immune dysregulation in broiler chickens. Transmission electron microscopy (TEM) revealed swollen mitochondria and breakage of the large crista lining, membrane deformation, chromatin condensation, increased vacuolation, and blood vessel spasms in the NH3-exposed BF and thymus tissues. Immunofluorescent analysis showed clustering of CD4+ and CD8+ cells, indicating an active immune response to NH3 exposure. Furthermore, NH3 exposure enhanced the mRNA expressions of Toll-like receptor 7 (TLR-7), myeloid differentiation primary response 88 (MYD88), and nuclear factor-kappa B (NF-κB), along with their proteins, and led to activation of the TLR-7/MyD88/NF-κB signaling pathway and NLRP3 inflammasome in chicken thymus tissues. Both mRNA and protein levels of key inflammation-related genes and proteins were upregulated in the NH3-treated group, highlighting a robust inflammatory response due to NH3 exposure. The specific findings of significant structural damage to key lymphoid organs and activation of inflammatory pathways in broiler chickens upon NH3 exposure can provide guidance for future, targeted therapies to improve poultry health.

Glycine Transporter 1 Inhibitors Minimize the Analgesic Tolerance to Morphine.

Opioid analgesic tolerance (OAT), among other central side effects, limits opioids' indispensable clinical use for managing chronic pain. Therefore, there is an existing unmet medical need to prevent OAT. Extrasynaptic N-methyl D-aspartate receptors (NMDARs) containing GluN2B subunit blockers delay OAT, indicating the involvement of glutamate in OAT. Glycine acts as a co-agonist on NMDARs, and glycine transporters (GlyTs), particularly GlyT-1 inhibitors, could affect the NMDAR pathways related to OAT. Chronic subcutaneous treatments with morphine and NFPS, a GlyT-1 inhibitor, reduced morphine antinociceptive tolerance (MAT) in the rat tail-flick assay, a thermal pain model. In spinal tissues of rats treated with a morphine-NFPS combination, NFPS alone, or vehicle-comparable changes in µ-opioid receptor activation, protein and mRNA expressions were seen. Yet, no changes were observed in GluN2B mRNA levels. An increase was observed in glycine and glutamate contents of cerebrospinal fluids from animals treated with a morphine-NFPS combination and morphine, respectively. Finally, GlyT-1 inhibitors are likely to delay MAT by mechanisms relying on NMDARs functioning rather than an increase in opioid efficacy. This study, to the best of our knowledge, shows for the first time the impact of GlyT-1 inhibitors on MAT. Nevertheless, future studies are required to decipher the exact mechanisms.

ROR1 MRNA EXPRESSION IN GLIOMA - A NOVEL PROGNOSTIC BIOMARKER

Abstract AIMS Glioblastoma is the most common and aggressive primary brain tumour in adults. Despite maximal surgical resection followed by concomitant chemo-/radiotherapy, the three-year survival remains less than 5%. Receptor tyrosine kinase-like orphan receptor 1 (ROR1) overexpression is associated with poor prognosis in several cancers. ROR1 therapeutics are in Phase I/II clinical trials making it an exciting novel target with translational potential to explore for glioblastoma treatment. The aim of this study was to examine the association between ROR1 mRNA expression and overall survival (OS), by applying the WHO 2021 classification of transcriptomic glioma datasets. METHOD Clinical, histological, and molecular data were extracted from four independent glioma datasets (TCGA, CGGA, Rembrandt, and Gravendeel) via the GlioVis portal and analysed using the WHO 2021 classification. Only confirmed cases of astrocytoma, oligodendroglioma, or glioblastoma, and ROR1 mRNA expression data were included (2,216 cases). Kaplan-Meier survival curves were produced for OS with low/high grade glioma versus ROR1 mRNA expression. Log-rank (Mantel-Cox) multivariate regression was applied to analyse the effect of selected covariates on OS and ROR1 mRNA expression. RESULTS We observed a significant decrease in median OS in patients with high-ROR1 mRNA expression when compared to the low-ROR1 mRNA expression cohort (20 and 70 months, respectively). High-grade gliomas represented the highest ROR1 mRNA expression across the consortium, resulting in a poor median OS of 16 months. CONCLUSION This study represents the first report of an association between OS and the oncogenic role of ROR1 in glioma, a targetable cell surface marker for novel putative treatment in glioblastoma.

Influence of N- and/or P-restriction on bone metabolism in young goats.

Ruminants can recycle nitrogen (N) and phosphorus (P), which are essential for vital body processes. Reduced N- and P-intake in ruminants is desirable for economic and ecologic reasons. Simultaneous modulation of mineral homoeostasis and bone metabolism occurs in young goats. This study aimed to investigate potential effects of dietary N- and/or P-restriction on molecular changes in bone metabolism. The twenty-eight young male goats were fed a control diet, an N-reduced diet, a P-reduced diet or a combined N- and P-reduced diet for 6-8 weeks. The N-restricted goats had lower plasma Ca concentration and higher plasma osteocalcin (OC) and CrossLaps concentrations. The P-restricted goats had reduced plasma inorganic phosphate (Pi) concentrations and increased plasma Ca concentrations. Due to the initiation of a signalling pathway that inhibits the fibroblast growth factor 23 (FGF23) expression, this was lower with P-restriction. Consequently, lower Pi concentrations were the main factor influencing the reduction in FGF23. The changes in mineral homoeostasis associated with P-restriction led to a reduction in OC, bone mineral content and mineral density. Simultaneously, bone resorption potentially increased with P-restriction as indicated by an increased receptor activator of NF-κB ligand/osteoprotegerin (OPG) ratio and an increase in OPG mRNA expression. Additionally, the increased mRNA expression of the calcitonin receptor during P-restriction points to a higher number of osteoclasts. This study demonstrates an impairment of bone remodelling processes in young goats by N- or P-restriction. With P-restriction, bone mineralisation rate was potentially reduced and bone quality impaired, while with N-restriction, bone remodelling increased.

Characterization of human placental fetal vessels in gestational diabetes mellitus.

Gestational diabetes mellitus is one of the most common complications during pregnancy. Its prevalence is rapidly increasing worldwide. Gestational diabetes mellitus is leading to an elevated risk for the development of endothelial dysfunction and cardiovascular diseases both in the mother and the child in later life. The underlying pathophysiological mechanisms are not well-understood. Therefore, we aimed to characterize the endothelial function in fetal placental vessels from mothers with gestational diabetes mellitus. In this study, we distinguished between insulin-treated and diet-controlled gestational diabetes mothers and compared them to a normoglycemic control group. The clinical data confirmed pre-conceptional overweight as a risk factor in women with insulin-treated gestational diabetes mellitus. The insulin-treated gestational diabetes group was also characterized by a recent family history of diabetes compared to mothers of the control or diet-controlled gestational diabetes group. Analyses of blood serum from umbilical cords suggested a reduced fetal insulin metabolism in the insulin-treated gestational diabetes group. Vascular function analysis in fetal placental vessels revealed an altered substance P-induced vasorelaxation in vessels from patients with insulin-dependent gestational diabetes. Inhibition of nitric oxide synthase affected only fetal vessel segments from the control group or diet-controlled gestational diabetes group, but not from insulin-dependent gestational diabetes. Finally, we found a significantly decreased substance P receptor (TACR1) mRNA expression in fetal vessel segments from patients with insulin-treated gestational diabetes. In conclusion, we provide evidence that different pathophysiological mechanisms might be responsible for the development of insulin-treated versus diet-controlled gestational diabetes. Only in fetal vessels from patients with insulin-treated gestational diabetes were we able to detect an endothelial dysfunction and a reduced fetal insulin conversion. This provides novel insights into the pathophysiology of the subtypes of gestational diabetes.

Maternal stress and the early embryonic microenvironment: investigating long-term cortisol effects on bovine oviductal epithelial cells using air–liquid interface culture

The oviduct epithelium is the initial maternal contact site for embryos after fertilization, offering the microenvironment before implantation. This early gestation period is particularly sensitive to stress, which can cause reduced fertility and reproductive disorders in mammals. Nevertheless, the local impact of elevated stress hormones on the oviduct epithelium has received limited attention to date, except for a few reports on polyovulatory species like mice and pigs. In this study, we focused on the effects of chronic maternal stress on cattle, given its association with infertility issues in this monoovulatory species. Bovine oviduct epithelial cells (BOEC) differentiated at the air–liquid interface (ALI) were stimulated with 250 nmol/L cortisol for 1 or 3 weeks. Subsequently, they were assessed for morphology, bioelectrical properties, and gene expression related to oviduct function, glucocorticoid pathway, cortisol metabolism, inflammation, and apoptosis. Results revealed adverse effects of cortisol on epithelium structure, featured by deciliation, vacuole formation, and multilayering. Additionally, cortisol exposure led to an increase in transepithelial potential difference, downregulated mRNA expression of the major glucocorticoid receptor (NR3C1), upregulated the expression of cortisol-responsive genes (FKBP5, TSC22D3), and significant downregulation of oviductal glycoprotein 1 (OVGP1) and steroid receptors PGR and ESR1. The systematic comparison to a similar experiment previously performed by us in porcine oviduct epithelial cells, indicated that bovine cultures were more susceptible to elevated cortisol levels than porcine. The distinct responses between both species are likely linked to their divergence in the cortisol-induced expression changes of HSD11B2, an enzyme controlling the cellular capacity to metabolise cortisol. These findings provide insights into the species-specific reactions and reproductive consequences triggered by maternal stress.