Receptor mRNA Research Articles

A Novel Approach for In Vitro Testing and Hazard Evaluation of Nanoformulated RyR2-Targeting siRNA Drugs Using Human PBMCs

Nucleic acid (NA)-based drugs are promising therapeutics agents. Beyond efficacy, addressing safety concerns—particularly those specific to this class of drugs—is crucial. Here, we propose an in vitro approach to screen for potential adverse off-target effects of NA-based drugs. Human peripheral blood mononuclear cells (PBMCs), purified from buffy coats of healthy donors, were used to investigate the ability of NA-drugs to trigger toxicity pathways and inappropriate immune stimulation. PBMCs were selected for their ability to represent potential human responses, given their likelihood of interacting with administered drugs. As proof of concept, a small interfering RNA (siRNA) targeting Ryanodine Receptor mRNA (RyR2) identified by the Italian National Center for Gene Therapy and Drugs based on RNA Technology as a potential therapeutic target for dominant catecholaminergic polymorphic ventricular tachycardia, was selected. This compound and its scramble were formulated within a calcium phosphate nanoparticle-based delivery system. Positive controls for four toxicity pathways were identified through literature review, each associated with a specific type of cellular stress: oxidative stress (tert-butyl hydroperoxide), mitochondrial stress (rotenone), endoplasmic reticulum stress (thapsigargin), and autophagy (rapamycin). These controls were used to define specific mRNA signatures triggered in PBMCs, which were subsequently used as indicators of off-target effects. To assess immune activation, the release of pro-inflammatory cytokines (interleukin-6, interleukin-8, tumor necrosis factor-α, and interferon-γ) was measured 24 h after exposure. The proposed approach provides a rapid and effective screening method for identifying potential unintended effects in a relevant human model, which also allows to address gender effects and variability in responses.

Acute heat stress regulates estradiol synthesis in ovine ovarian granulosa cells through the SREBPs/MVK–LHR pathway

The adverse effects of heat stress on reproductive performance of sheep are becoming increasingly severe. Previous research has revealed that heat stress decreases both cholesterol and estradiol content; however, regulation of estradiol by cholesterol and its regulatory mechanism under heat stress are unclear. Mevalonate kinase (MVK), a key cholesterol synthesis pathway enzyme, binds to the luteinizing hormone receptor (LHR; a key gene regulating hormone synthesis) mRNA. In this study, ovine ovarian granulosa cells (GCs) were used in an in vitro model. To elucidate the underlying molecular mechanism, immunofluorescence, quantitative reverse transcription polymerase chain reaction, western blotting, enzyme-linked immunosorbent assay, and an RNA electrophoretic mobility shift assay (REMSA) were used to investigate whether the decrease in cholesterol caused by acute heat stress resulted in a decrease in estradiol synthesis. Acute heat stress reduced the cholesterol content in ovine ovarian GCs, which transactivated the cholesterol synthesis pathway corresponding to the gene expression of sterol regulatory element-binding protein (SREBP-1A), SREBP-2, and MVK. Upregulated MVK increased the MVK–LHR mRNA complex, which caused LHR mRNA decay and downregulation, further leading to the downregulation of CYP19A1 and a decrease in estradiol. The cholesterol synthesis inhibitor, PF-429242, alleviated the decrease in estradiol synthesis caused by acute heat stress. Overall, acute heat stress caused a decrease in total cholesterol, which transactivated the expression of cholesterol synthesis genes, such as SREBP-1A, SREBP2, and MVK, increasing the MVK–LHR complex, downregulating LHR expression, and further decreasing estradiol.

Peripheral inflammation enhances opioid-induced gastrointestinal motility inhibition via up-regulating spinal mu opioid receptor.

Opioids are potent analgesics in clinical pain management but exert variable analgesia in different pain types. Opioid-induced constipation is a common side effect of opioid therapy, and whether opioids induce different gastrointestinal motility inhibitions in different pain types is unknown. In this study, we evaluated the antinociceptive effects and inhibition of upper gastrointestinal transit and colonic bead expulsion of morphine, DAMGO, and Deltorphin in mouse CFA chronic inflammatory pain, SNI chronic neuropathic pain, and carrageenan chronic inflammatory pain models. Furthermore, quantitative PCR and immunofluorescence were used to investigate the mechanisms underlying the altered inhibition. Results showed that intrathecal administration of morphine, DAMGO, and Deltorphin produced higher antinociceptive effects in the CFA and carrageenan groups than in the SNI group. Upper gastrointestinal transit inhibition was significantly enhanced in the carrageenan group by morphine and DAMGO; colonic bead expulsion inhibition was also enhanced in the CFA and carrageenan groups by morphine and DAMGO, but not in Deltorphin treatment. Additionally, mu (MOR) opioid receptor mRNA and MOR-expressing cell density in the lumbar spinal cord of CFA and carrageenan mice were increased, whereas delta opioid receptor expression remained unchanged in these groups. Finally, the pharmacological blockade of MOR completely prevented the enhanced upper gastrointestinal transit inhibition in the carrageenan group by morphine and DAMGO. Altogether, our results indicate that gastrointestinal motility inhibition induced by MOR agonists can be enhanced with upregulated spinal MOR expression in chronic inflammatory pain.

Evaluation of Reference Gene Stability for Investigations of Intracellular Signalling in Human Cancer and Non-Malignant Mesenchymal Stromal Cells.

Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful tool for analysing target gene expression in biological samples. To achieve reliable results by RT-qPCR, the most stable reference genes must be selected for proper data normalisation, particularly when comparing cells of different types. We aimed to choose the least variable candidate reference genes among eight housekeeping genes tested within a set of human cancer cell lines (HeLa, MCF-7, SK-UT-1B, A549, A431, SK-BR-3), as well as four lines of normal, non-malignant mesenchymal stromal cells (MSCs) of different origins. The reference gene stability was evaluated using four algorithms (BestKeeper, NormFinder, geNorm and the comparative ΔCt method) and ranked with the RefFinder web-based tool. We found increased variability in the housekeeping genes' expression in the cancer cell lines compared to that in normal MSCs. POP4 and GAPDH were identified as the most suitable reference genes in cancer cells, while 18S and B2M were the most suitable in MSCs. POP4 and EIF2B1 were shown to be the least variable genes when analysing normal and cancer cell lines together. Epidermal growth factor receptor (EGFR) mRNA relative expression was normalised by the three most stable or three least stable reference genes to demonstrate the reliability of reference genes validation. We analysed and selected stable reference genes for RT-qPCR analysis in the wide panel of cancer cell lines and MSCs. The study provides a reliable tool for future research concerning the expression of genes involved in various intracellular signalling pathways and emphasises the need for careful selection of suitable references before analysing target gene expression.

Regulation of abscisic acid receptor mRNA stability: Involvement of microRNA5628 in PYL6 transcript decay.

Phytohormone signaling is fine-tuned by regulatory feedback loops. The phytohormone abscisic acid (ABA) plays key roles in plant development and abiotic stress tolerance. PYRABACTIN RESISTENCE 1/PYR1-LIKE/REGULATORY COMPONENT OF ABA RECEPTOR (PYR/PYL/RCAR) receptors sense ABA, and in turn, ABA represses their expression. Conversely, ABA induces expression of Type 2C PROTEIN PHOSPHATASES (PP2C) genes, which negatively regulate the ABA signaling pathway. This regulatory feedback scheme is likely important for modulating ABA signaling. Here, we provide insight into the mechanisms underlying the ABA-induced repression of PYR/PYL/RCAR expression in Arabidopsis (Arabidopsis thaliana). ABA time course analyses revealed strong and sustained repression of PYR/PYL/RCARs, suggesting that receptor gene regulation is an important step in resetting the ABA signaling pathway. Cordycepin-induced transcription inhibition showed that PYL1/4/5/6 mRNA destabilization is involved in the ABA-induced repression of these genes. Furthermore, genetic evidence indicated that decapping may play a role in PYL4/5/6 mRNA decay. We also provide evidence that the Arabidopsis-specific microRNA5628 (miR5628), which is transiently induced by the ABA core signaling pathway, guides PYL6 transcript cleavage in response to ABA. After cleavage, the resulting 5'- and 3'-cleaved fragments of PYL6 mRNA may be degraded by the XRN4 exoribonuclease. miR5628 is an evolutionary novelty that may enhance PYL6 mRNA degradation, along with decapping and XRN4 activity. Thus, regulating the stability of PYR/PYL/RCAR transcripts maintains ABA signaling homeostasis.

Influence of oxytocin level and oxytocin receptor gene expression on the status and severity of autism spectrum disorder in a group of Egyptian children

Background The use of oxytocin (OXT) as a biomarker of autism spectrum disorder (ASD) and its role in the pathophysiology of ASD is still controversial and not straightforward. Objective To evaluate, the association of ASD risk with OXT concentration and the expression of its receptor (OXTR) mRNA in a group of children with autism, in addition, to investigating the influence of OXT and OXTR on the status and severity of the disorder in the study ASD group. Patients and methods This study included 30 children diagnosed with ASD, representing the cases group, and a comparable 30 neurotypical children with matched age and sex as a control group. After detailed history taking, pedigree construction, and clinical examination to exclude recognizable syndromes, OXT level and OXTR gene expression were assessed in all included children. Results and conclusion The OXT level in autistic cases was significantly lower than in controls (P=0.0001). Also, the fold change of OXTR was significantly lower than controls (P=0.01). Receiver operating characteristic curve analysis showed that at a cut-off point of less than 0.2806, OXTR showed reasonable diagnostic performance with sensitivity, specificity, positive predictive value, and negative predictive value of 53.33, 78.2, 72.7, and 63.2, respectively (P=0.0067). At a cut-off point of less than or equal to 119 pg/ml, the OXT level showed good diagnostic performance with sensitivity (86.67%), specificity (76.67%), positive predictive value (78.8%), and negative predictive value (85.2%) (P<0.0001). Our results proved the potential role of OXT plasma measurements and gene expression in diagnosing ASD at optimal cut-off values. These results support the use of OXT as a biomarker for ASD and also as a possible therapeutic option to improve the social behavior of ASD children.

Replace part of regenerative rice silage feed with whole cottonseed enhances growth performance and nutrient digestibility via regulating gH/IGF axis in beef cattle

This work was designed to assess the impacts of whole cottonseed (WCs) on growth performance, nutrient digestion, GH /IGF-1 axis, and meat quality in beef cattle. Twenty-four healthy male simmental cattle (18 months old and 370.5 ± 12.5 kg) were arbitrarily classified into two groups (n=12 per group): Control group (without WCs) and WCs replaced group (part of regenerative rice silage was replaced with WCs). The dry matter intake (DMI) was declined(P < 0.01), while the average daily gain (ADG) was increased(P < 0.05), therefore the feed conversion ratio (FCR) was lower (P < 0.05) in WCs replaced group. Beef cattle consuming WCs had a higher digestibility of total gastrointestinal nutrients (P < 0.01 or P < 0.05). The growth hormone receptor (GHR), insulin-like growth factor 1 (IGF-1), and insulin-like growth factor 1 receptor (IGF-1R) mRNA amounts were all enhanced (P < 0.05) in liver, longissimus dorsi muscle, and semitendinosus muscle of beef cattle after feeding WCs relative to control group. The mRNA amounts of phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR), and ribosomal protein S6 kinase beta-1 (S6K1) and the protein phosphorylation levels of Akt、mTOR, and S6K1 were all markedly enhanced (P < 0.05 or P < 0.01) in liver, longissimus dorsi muscle, and semitendinosus muscle for WCs replacement. The shear force and cooking loss values having somewhat raised(P < 0.05) for WCs replacement. Altogether, WCs improves the growth performance and nutrient digestibility via regulating GH/IGF axis in beef cattle, and has negative effects on meat quality.

Basal forebrain-lateral habenula inputs and control of impulsive behavior.

Deficits in impulse control are observed in several neurocognitive disorders, including attention deficit hyperactivity (ADHD), substance use disorders (SUDs), and those following traumatic brain injury (TBI). Understanding brain circuits and mechanisms contributing to impulsive behavior may aid in identifying therapeutic interventions. We previously reported that intact lateral habenula (LHb) function is necessary to limit impulsivity defined by impaired response inhibition in rats. Here, we examine the involvement of a synaptic input to the LHb on response inhibition using cellular, circuit, and behavioral approaches. Retrograde fluorogold tracing identified basal forebrain (BF) inputs to LHb, primarily arising from ventral pallidum and nucleus accumbens shell (VP/NAcs). Glutamic acid decarboxylase and cannabinoid CB1 receptor (CB1R) mRNAs colocalized with fluorogold, suggesting a cannabinoid modulated GABAergic pathway. Optogenetic activation of these axons strongly inhibited LHb neuron action potentials and GABA release was tonically suppressed by an endogenous cannabinoid in vitro. Behavioral experiments showed that response inhibition during signaled reward omission was impaired when VP/NAcs inputs to LHb were optogenetically stimulated, whereas inhibition of this pathway did not alter LHb control of impulsivity. Systemic injection with the psychotropic phytocannabinoid, Δ9-tetrahydrocannabinol (Δ9-THC), also increased impulsivity in male, and not female rats, and this was blocked by LHb CB1R antagonism. However, as optogenetic VP/NAcs pathway inhibition did not alter impulse control, we conclude that the pro-impulsive effects of Δ9-THC likely do not occur via inhibition of this afferent. These results identify an inhibitory LHb afferent that is controlled by CB1Rs that can regulate impulsive behavior.

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.

Adenosine relaxes vagina smooth muscle through the cyclic guanosine monophosphate- and cyclic guanosine monophosphate-dependent pathways.

In males, adenosine (ADO) is known to relax penile smooth muscles, although its role in the vagina is not yet fully elucidated. This study investigated the effect of ADO on vagina smooth muscle activity, using a validated female Sprague-Dawley rat model. Contractility studies, using noradrenaline-precontracted vaginal strips, tested the effects of ADORA1/3 antagonists and ADORA2A/2B antagonists and agonists. Increasing doses of ADO were tested after in vivo or in vitro treatment with Nω-nitro-L-arginine-methyl-ester hydrochloride (L-NAME) or with guanylate or adenylate cyclase inhibitors. Immunopositivity for ADORA2A and ADORA2B was assessed, and messenger RNA (mRNA) analysis was performed. Cyclic ADO monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) were quantified both in rat vagina smooth muscle cells (rvSMCs) and in vaginal tissues with increasing doses of ADO. Demonstrating ADO's role in the relaxing/contractile mechanism in distal vagina smooth muscle. All ADO receptors mRNAs were expressed in vaginal tissue, with a prevalent content of ADORA2B. A high expression of genes regulating ADO catabolism (ADK) and de novo synthesis (NT5E) was found. In vaginal strips, ADO induced relaxation with IC50 = 144.7μM and a flat pseudo-Hill coefficient value = -0.42, indicating an activity on heterogeneous receptors. Blocking ADORA1/3 shifted ADO response to the left and with a steeper slope. ADORA2A/2B agonists showed a higher potency than ADO in inducing relaxation. Immunolocalization confirmed the presence of ADORA2A/2B in vaginal musculature, in the blood vessels endothelium, and in the epithelium. ADO stimulation of vagina tissues induced a significant increase in cAMP and cGMP contents. Experiments on rvSMCs confirmed that ADO time- and dose-dependently stimulated cAMP production in these cells. However, ADORA2A/2B antagonists, although reducing the ADO-induced relaxation, did not completely block it. A similar inhibition was obtained by blocking adenylate cyclase. Overall, these findings suggest that ADO relaxation involves other pathways, eg, nitric oxide (NO)/cGMP. Accordingly, blocking NO formation through L-NAME substantially blunted ADO responsiveness, as it does the block of cGMP formation through 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. Simultaneous incubation with cGMP and cAMP blockers completely inhibited ADO responsiveness. The study highlights ADO's role in regulating vaginal smooth muscle activity, suggesting its potential effect on the vagina. This is the first study on ADO in the vagina, although the results are preliminary and limited to the rat model. These results show that ADO acts as a vaginal relaxing modulator through selective activation of receptors involving not only cAMP but also cGMP.

Antidepressant effects of SY-2476: A caffeine derivative’s role in A1/A2A gene expression modulation in corticosterone-induced depressed rats

Depression is a pervasive mood disorder that continues to challenge researchers and clinicians worldwide. Caffeine and its derivatives have been studied for their neuroprotective and antidepressant effect. Current study aimed to explore the potential antidepressant effect of a caffeine derivative, Sy-2476 [4-(1, 3, 7-trimethyl-2, 6-dioxo-2, 3, 6, 7-tetrahydro-1H-purin-8-yl) benzo nitrile], in corticosterone-induced rat model of depression. Depression-like behaviour in rats was induced by administering 20 mg/kg hydrocortisone s.c for 21 days. Behavioural studies evaluated the potential antidepressant effect of caffeine derivative Sy-2476, its effect on cortisol levels, modulation of A1/A2A receptors mRNA expression and antioxidant assays. Treatment of rats with Sy-2476 exhibited robust antidepressant-like effects in corticosterone-exposed rats by increasing sucrose preference (p = 0.0002) while reducing immobility time (p = 0.0118) in the forced swim test. Sy-2476 also reduced lipid peroxidation and increased the level of antioxidant enzymes, including glutathione, catalase, and superoxide dismutase. Moreover, Sy-2476 significantly lowered cortisol levels (p = 0.0019) and up-regulated mRNA expression of A1 (p = 0.0001) and A2A receptors (p = 0.0016) compared to the corticosterone-only treated group. In conclusion, Sy-2476 showed an antidepressant effect primarily by suppressing serum cortisol levels, modulating the expression of adenosine receptors, and exhibiting antioxidant properties.

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.

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 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.

Deciphering Folate Receptor alphaGene Expression and mRNA Signatures in Ovarian Cancer: Implications for Precision Therapies.

Antibody-drug conjugates targeting folate receptor alpha (FRα) are a promising treatment for platinum-resistant ovarian cancer (OC) with high FRα expression. Challenges persist in accurately assessing FRα expression levels. Our study aimed to better elucidate FRα gene expression and identify mRNA signatures in OC. We pooled OC gene expression data from 16 public datasets, encompassing 1832 OC and 30 normal ovarian tissues. Additional data included DNA copy number and methylation data from TCGA and protein data from 363 cancer cell lines from the Broad Institute Cancer Cell Line Encyclopedia. FOLR1 mRNA expression was significantly correlated with protein expression in pan-cancer cell lines and ovarian cancer cell lines. FOLR1 expression was higher in OC samples than in normal ovarian tissues (OR = 3.88, p = 6.97 × 10-12). Patients with high FOLR1 expression were more likely to be diagnosed with serous histology, FIGO stage III-IV, and high-grade tumors; however, nearly similar percentages of patients with low FOLR1 expression were also diagnosed with these features. FOLR1 mRNA expression was not correlated with platinum sensitivity or complete surgery, nor with prognosis. However, we identified a 187-gene signature associated with high FOLR1 expression that was significantly associated with improved survival (HR = 0.71, p = 1.18 × 10-6), independently from clinicopathological features. We identified a gene expression signature correlated to high FRα expression and OC prognosis, which may be used to refine therapeutic strategies targeting FRα in OC. These findings warrant validation in larger cohorts.

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.

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.

Distribution of progesterone receptors and the membrane component of progesterone receptor in various organs and tissues of male and female rats

Progesterone regulates reproductive processes and affects many functions of various non-reproductive organs. Its effects in mammals and humans are mediated by nuclear (nPRs) and membrane progesterone receptors (mPRs). The action of progesterone through different types of receptors may differ significantly and has tissue specific features. The expression of known types and subtypes of progesterone receptors in the tissues of male and female rats has been studied fragmentarily. The purpose of our work was to study the expression of five mPRs genes, as well as the nPRs gene and the membrane component of the progesterone receptor PGRMC I in the reproductive organs and in 17 non-reproductive tissues of male and female rats using reverse transcription followed by real-time PCR. In this study, it was shown that a high level of nPRs gene expression in rats is found not only in reproductive organs of females (uterus, ovary, mammary glands), but also in seminal vesicles of males, in the brain and trachea of both sexes, in blood vessels, and in the pancreas of females. The highest level of expression of mPRs genes of all subtypes was found in the testes, while expression of the gene encoding nPRs was practically undetectable in them. Expression of genes encoding mPRs was also detected in the liver and spleen of male and female rats, while expression of the gene encoding nPRs was at background levels. Virtually no expression of nPRs, mPRs, and membrane component of progesterone receptor (PGRMC I) genes was detected in muscle, and its level was very low in the heart in animals of both sexes. We found sex-specific differentiation of nuclear and membrane receptor mRNA levels in rats in non-reproductive tissues, characterized by a predominance of nPRs transcripts and three subtypes of mPRs (α, β, δ) in females and two subtypes of mPRs (γ, ε) in males. Data on the presence of progesterone receptors in tissues not involved in reproduction confirm the effect of progesterone on these organs. High levels of mRNA for various progesterone receptors in the tissues of male rats, such as the pancreas, lungs, kidney, and trachea, indicate an important physiological role of progestins not only in females, but also in males, which is still poorly understood. The work also discusses the known functions of progesterone receptors in the tissues studied.

Interaction between corticotropin-releasing factor, orexin, and dynorphin in the infralimbic cortex may mediate exacerbated alcohol-seeking behavior

A major challenge for the treatment of alcohol use disorder (AUD) is relapse to alcohol use, even after protracted periods of self-imposed abstinence. Stress significantly contributes to the chronic relapsing nature of AUD, given its long-lasting ability to elicit intense craving and precipitate relapse. As individuals transition to alcohol dependence, compensatory allostatic mechanisms result in insults to hypothalamic-pituitary-adrenal axis function, mediated by corticotropin-releasing factor (CRF), which is subsequently hypothesized to alter brain reward pathways, influence affect, elicit craving, and ultimately perpetuate problematic drinking and relapse vulnerability. Orexin (OX; also called hypocretin) plays a well-established role in regulating diverse physiological processes, including stress, and has been shown to interact with CRF. Interestingly, most hypothalamic cells that express Ox mRNA also express Pdyn mRNA. Both dynorphin and OX are located in the same synaptic vesicles, and they are co-released. The infralimbic cortex (IL) of the medial prefrontal cortex (mPFC) has emerged as being directly involved in the compulsive nature of alcohol consumption during dependence. The IL is a CRF-rich region that receives OX projections from the hypothalamus and where OX receptor mRNA has been detected. Although not thoroughly understood, anatomical and behavioral pharmacology data suggest that CRF, OX, and dynorphin may interact, particularly in the IL, and that functional interactions between these three systems in the IL may be critical for the etiology and pervasiveness of compulsive alcohol seeking in dependent subjects that may render them vulnerable to relapse. The present review presents evidence of the role of the IL in AUD and discusses functional interactions between CRF, OX, and dynorphin in this structure and how they are related to exacerbated alcohol drinking and seeking.