Receptor Expression Research Articles

Olfactory Receptors and Tumorigenesis: Implications for Diagnosis and Targeted Therapy.

Olfactory receptors (ORs) are a class of G protein-coupled receptors (GPCR) widely distributed in olfactory sensory neurons and various non-olfactory tissues, serving significant physiological and pathological functions in the human body. Increasing evidence reveals the heightened expression of olfactory receptors in tumorous tissues and cells alongside normal tissues. Olfactory receptors have demonstrated influence over tumor cell proliferation and metastasis, establishing a close relationship with tumor initiation and progression. This review highlights the specific molecular actions and signaling pathways of olfactory receptors in the development of human tumors. The potential for precise tumor diagnosis and targeted therapy through therapeutic targeting of olfactory receptors as an adjunct anticancer treatment strategy is being considered.

Macroanatomy, Histomorphometry, and Androgen Receptor Expression in the Epididymis of Kacang Goats Aged 4, 8, and 12 Months

The epididymis is a crucial component of the goat's reproductive structure. The epididymis is responsible for the transportation, concentration, and maturation of sperm. This study aimed to examine the differences in the structure of macro and microanatomy, tissue composition, and the existence of androgen receptors in the epididymis of Kacang goats aged 4, 8, and 12 months. The assessment of macroscopic organ growth was done immediately after sampling, whereas microscopic measurements were carried out following histological preparations using hematoxylin-eosin (HE) and immunohistochemical (IHC) procedures. The results on the macroscopic anatomy of the epididymis indicated a significant association between age with the width of the caput dexter and sinister and the circumference of the right cauda. However, no significant relationship was found between age with the corpus length and the left cauda's circumference. Significant variations were observed in the diameter and concentration of the agglutinated spermatozoa in the lumen during histomorphometry of the epididymis in three age groups of Kacang goats. There were no statistically significant variations in the expression of androgen receptors among the three age groups. This study showed that the correlation coefficient test reveals a positive relationship between age and the caput width and corpus length dimensions, indicating that these measurements tend to grow as age increases. On the other hand, the diameter of the agglutinated spermatozoa in the epididymal lumen exhibits significant variations between the ages of 4 months, with the ages of 8 months and 12 months, suggesting that the sperm becomes fully matured by the age of 8 months.

Immune modulation and epigenetic therapies for enhanced outcome of treatment in triple-negative breast cancer

Triple-negative breast cancer (TNBC) is a subtype of breast cancer characterized by the absence of human epidermal growth factor receptor 2, estrogen receptor, and progesterone receptor expression. While traditional TNBC treatment primarily relies on systemic chemotherapy, epigenetic modification has an important role in immune evasion and tumor progression. Recent classifications of TNBC into “hot” and “cold” tumors, based on immune activity and mutation burden, have revealed that poor response to immune checkpoint inhibitors (ICIs) in these tumors is due to low tumor-infiltrating lymphocytes and the presence of immunosuppressive cells. Emerging treatments such as ICIs and poly(ADP-ribose) polymerase inhibitors offer promising alternatives. The tumor microenvironment (TME) shapes immune responses in TNBC, and a limited subset of patients has shown encouraging responses to ICIs in treating TNBC at both early and advanced stages. However, combination therapies face challenges, including medication interactions and side effects. Epigenetic modulators, such as histone deacetylase and DNA methyltransferase inhibitors, also show promise in reversing epigenetic changes by enhancing anti-tumor immunity in TNBC. The frequent expression of indoleamine 2,3-dioxygenase 1 (IDO1) in TNBC, which catalyzes the conversion of tryptophan to kynurenine, contributes to immune suppression in TNBC patients. High IDO1 levels impair the tumoricidal activity of natural killer cells and correlate with poor responses to anti-PD-L1 therapy, which can be improved using IDO1 inhibitors. Targeting myeloid-derived suppressor cells, regulatory T-cells, and tumor-associated macrophage, along with utilizing epigenetic mechanisms, show promise in modulating the immunosuppressive TME in TNBC. Combining these approaches with standard therapies, along with biomarker-guided patient selection, can enhance treatment efficacy. Clinical trials and preclinical models are essential for optimizing these strategies. This study emphasizes the importance of understanding the mechanisms of modulation, tumor-immune interactions, and the potential of epigenetic therapies in improving treatment outcomes in TNBC.

Correlation of Androgen Receptor Expression With Ki67 Proliferative Index and Other Clinicopathological Characteristics in Invasive Mammary Carcinomas

Correlation of Androgen Receptor Expression With Ki67 Proliferative Index and Other Clinicopathological Characteristics in Invasive Mammary Carcinomas

An in vitro approach reveals molecular mechanisms underlying endocrine disruptor-induced epimutagenesis.

Endocrine disrupting chemicals (EDCs) such as bisphenol S (BPS) are xenobiotic compounds that can disrupt endocrine signaling due to steric similarities to endogenous hormones. EDCs have been shown to induce disruptions in normal epigenetic programming (epimutations) and differentially expressed genes (DEGs) that predispose disease states. Most interestingly, the prevalence of epimutations following exposure to many EDCs persists over multiple generations. Many studies have described direct and prolonged effects of EDC exposure in animal models, but many questions remain about molecular mechanisms by which EDC-induced epimutations are introduced or subsequently propagated, whether there are cell type-specific susceptibilities to the same EDC, and whether this correlates with differential expression of relevant hormone receptors. We exposed cultured pluripotent (iPS), somatic (Sertoli and granulosa), and primordial germ cell-like (PGCLC) cells to BPS and found that differential incidences of BPS-induced epimutations and DEGs correlated with differential expression of relevant hormone receptors inducing epimutations near relevant hormone response elements in somatic and pluripotent, but not germ cell types. Most interestingly, we found that when iPS cells were exposed to BPS and then induced to differentiate into PGCLCs, the prevalence of epimutations and DEGs was largely retained, however, >90% of the specific epimutations and DEGs were replaced by novel epimutations and DEGs. These results suggest a unique mechanism by which an EDC-induced epimutated state may be propagated transgenerationally.

Anti-Müllerian hormone signaling in the ovary involves stromal fibroblasts: a study in humans and mice provides novel insights into the role of ovarian stroma.

What is the involvement of ovarian stroma in the anti-Müllerian hormone (AMH) signaling pathway and which stromal cells are involved? Mouse and human ovaries show high expression of AMH receptor II (AMHR2) in the stromal fibroblasts surrounding the follicles and activation of the post-AMHR2 pathway by recombinant AMH was evidenced by increased phosphorylation of SMAD1,5 and 9, increased expression AMHR2 and upregulation of αSMA, suggesting fibroblast activation to initiate myofibroblast differentiation. AMH secreted by small growing follicles, regulates ovarian activity. It suppresses initial primordial follicle (PMF) recruitment and FSH-dependent growth. AMH signal transduction is mediated by AMHR2, activating intracellular SMAD proteins and other signaling cascades to induce target-gene expression. Although AMHR2 expression has been reported within the follicle unit, there is evidence suggesting it may be identified in the stroma as well. Fresh murine ovaries were extracted from BALB/c mice (6 weeks old; n = 12 and 21 days old; n = 56). Frozen-thawed ovarian fragments were obtained from 10 women, aged 18-35, who had undergone ovarian tissue cryopreservation and donated frozen ovarian tissue for research. Murine (6 weeks old) and human donor ovaries were immunostained for AMHR2 and Collagen 1α/αSMA/VCAM1, with additional vimentin staining in mice. Murine (21 days old) and human donor ovaries were used for fibroblast isolation and subsequent 7-day cultures. Prior to assessing AMH effects on isolated fibroblast culture, purity validation tests were implemented to ensure the absence of epithelial, immune, endothel, granulosa, and theca ovarian cell populations. The fibroblast culture's homogeneity was validated by RT-qPCR and western-blot assays, confirming negativity for E-cadherin, CD31, aromatase, CYP17A1, and positivity for αSMA and vimentin. Fibroblasts were then subjected to rAMH treatment in vitro (200 ng/ml) for 0-72h, with an additional time point of 96 h for human samples, followed by RT-qPCR, western blot, and immunocytochemistry (ICC) for AMHR2 expression. AMHR2 post-receptor signaling was examined by pSMAD1,5,9 levels via western blot. Activated fibroblast marker, αSMA, was assessed via western blot and ICC. Immunostaining of mouse and human ovarian tissue showed that stromal cells around follicles at all developmental stages exhibit high AMHR2 expression, while granulosa cells of growing follicles show considerably lower levels. The majority of these AMHR2-positive stromal cells were identified as fibroblasts (Collagen1α in mice and human; vimentin in mice). RT-qPCR, western blot, and immunostaining were performed on cultured mouse and human fibroblasts, confirming that they consisted of a pure fibroblast population (αSMA/vimentin positive and negative for other cell-type markers). A total of 99.81% (average 28.94 ± 1.34 cells/field in mice) and 100% (average 19.20 ± 1.39 cells/field in human samples) of these fibroblasts expressed AMHR2 (ICC). rAMH treated cultured fibroblasts showed increased pSMAD1,5 and 9 levels, demonstrating the effects of AMH on its downstream signaling pathway. pSMAD1,5 and 9 expression increased, as detected by western blot: 1.92-fold in mice (48 h, P = 0.026) and 2.37-fold in human samples (48 h, P = 0.0002). In addition, rAMH treatment increased AMHR2 protein expression, as observed in ICC (human): a 2.57-fold upregulation of AMHR2 Mean Fluorescence Intensity (MFI) (96 h, P = 0.00036), and western blot, showing a 4.2-fold time-dependent increase (48 h, P = 0.026) in mice and 2.4-fold change (48 h, P = 0.0003) in human donors. Exposure to rAMH affected AMHR2 transcription upregulation, with a 6.48-fold change (72 h, P = 0.0137) in mice and a 7.87-fold change (72 h, P < 0.0001) in humans. rAMH treatment induced fibroblast activation (αSMA positive), demonstrating the dynamic effects of AMH on fibroblast behavior. αSMA expression elevation was detected in ICC with a 2.28-fold MFI increase in humans (96 h, P = 0.000067), and in western blot with a 5.12-fold increase in mice (48 h, P = 0.0345) and a 2.69-fold increase in humans (48 h, P ≤ 0.0001). Activated AMHR2-positive stained fibroblast fractions were solely located around growing follicles, in both human and mice. In addition, a small population of AMHR2-positive stained theca cells (VCAM1 positive) was observed. N/A. Ex vivo, fibroblast gene expression might be changed by adhesion to the tissue-culture plate. Nevertheless, cultured fibroblasts (with and without rAMH) are subjected to the same conditions. Observations or significant differences can therefore be considered reliable. In addition, the presented effect of rAMH on fibroblasts is not directly linked to the known inhibitory effect of AMH on follicle activation. Clarifying the populations of AMH-responsive cells in the ovary provides a foundation for further investigation of the complex AMH signaling across the ovary. The composition of AMH-releasing and -responsive cells can shed light on the communication network between follicles and their environment, which may elucidate the mechanisms behind the AMH inhibitory effect on PMF activation. This work was financially supported by grants from the Kahn Foundation. There are no competing interests in this study. N/A.

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.

Molecular characterization and stage-dependent gene expression of gonadotropin receptors in Pacific bluefin tuna, Thunnus orientalis, ovarian follicles

To understand the physiological mechanisms by which pituitary-derived gonadotropins (Gths), follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) regulate asynchronous oocyte development, we investigated the function and expression of Fsh and Lh receptors (Fshr and Lhr, respectively) in Pacific bluefin tuna (PBT, Thunnus orientalis). As a first, we cloned the full-length cDNAs encoding PBT Fshr and Lhr. Recombinant PBT Fsh and Lh single-chain proteins were produced in abundance using stable CHO-DG44 cell lines and were subsequently purified from the culture medium, culminating in their yields being 87.0 and 88.2%, respectively. An in vitro reporter assay using homologous recombinant Gths revealed that PBT Fshr and Lhr responded strongly to their corresponding ligands in a dose-dependent manner, with no cross-activation over a wide range of concentrations. Moreover, quantitative expression analysis of Fshr and Lhr at the follicle level showed that fshr gene expression was highly upregulated in the ovarian follicles through vitellogenesis, while lhr expression was significantly upregulated and peaked in fully vitellogenic ovarian follicles. These findings suggest that asynchronous-type oocyte development is primarily attributed to the differential function and expression of Gthrs, rather than the ligand, in PBT.

Stimulus duration encoding occurs early in the moth olfactory pathway

Pheromones convey rich ethological information and guide insects’ search behavior. Insects navigating in turbulent environments are tasked with the challenge of coding the temporal structure of an odor plume, obliging recognition of the onset and offset of whiffs of odor. The coding mechanisms that shape odor offset recognition remain elusive. We designed a device to deliver sharp pheromone pulses and simultaneously measured the response dynamics from pheromone-tuned olfactory receptor neurons (ORNs) in male moths and Drosophila. We show that concentration-invariant stimulus duration encoding is implemented in moth ORNs by spike frequency adaptation at two time scales. A linear-nonlinear model fully captures the underlying neural computations and offers an insight into their biophysical mechanisms. Drosophila use pheromone cis-vaccenyl acetate (cVA) only for very short distance communication and are not faced with the need to encode the statistics of the cVA plume. Their cVA-sensitive ORNs are indeed unable to encode odor-off events. Expression of moth pheromone receptors in Drosophila cVA-sensitive ORNs indicates that stimulus-offset coding is receptor independent. In moth ORNs, stimulus-offset coding breaks down for short ( < 200 ms) whiffs. This physiological constraint matches the behavioral latency of switching from the upwind surge to crosswind cast flight upon losing contact with the pheromone.

Androgen Receptor Mediates Dopamine Agonist Resistance by Regulating Intracellular reactive oxygen species (ROS) in Prolactin-secreting Pituitary Adenoma.

Dopamine agonists (DA) are the first-line treatment for patients with prolactin-secreting pituitary adenoma (PRL adenoma). However, a subset of individuals exhibits poor responses, known as DA resistance. Previous studies have reported that DA resistance is more prevalent in male patients. This study aims to investigate the relationship between androgen receptor (AR) expression and DA resistance, as well as to explore underlying mechanisms of AR-mediated DA resistance. Our results demonstrated that patients with higher AR expression exhibit greater resistance to DA in our cohort of DA-resistant PRL adenoma. Furthermore, AR was found to be involved in cell proliferation, PRL secretion, and resistance to BRC both in vitro and in vivo. Mechanistically, we demonstrated that intracellular ROS function as upstream mediators of apoptosis and ferroptosis following BRC treatment. As a ligand-dependent transcription factor, AR could translocate to the nucleus and transcriptionally promote NRF2 expression, which regulates intracellular ROS levels, thereby enhancing cell viability, and conferring DA resistance to PA cells. Finally, AR targeting agents were used to inhibit AR signaling, downregulate NRF2 transcription, and sensitize PA cells to BRC treatment. Conclusion and innovation: We demonstrated that AR plays a crucial role in mediating DA resistance in PRL-adenoma. Mechanistically, AR promotes cell proliferation and PRL secretion and confers drug resistance by transcriptionally regulating NRF2 expression to maintain redox homeostasis in PA cells. Finally, combining AR targeting agents with BRC shows promise as a therapeutic strategy for treating PRL adenomas.

Seasonal heterochrony of reproductive development and gene expression in a polymorphic salamander.

Life cycle evolution includes ecological transitions and shifts in the timing of somatic and reproductive development (heterochrony). However, heterochronic changes can be tissue-specific, ultimately leading to the differential diversification of traits. Salamanders exhibit alternative life cycle polymorphisms involving either an aquatic to terrestrial metamorphosis (biphasic) or retention of aquatic larval traits into adulthood (paedomorphic). In this study, we used gene expression and histology to evaluate how life cycle evolution impacts temporal reproductive patterns in males of a polymorphic salamander. We found that heterochrony shifts the distribution of androgen signaling in the integument, which is correlated with significant differences in seasonal reproductive gland development and pheromone gene expression. In the testes, androgen receptor (ar) expression does not significantly vary between morphs or across seasons. We found significant differences in the onset of spermatogenesis, but by peak breeding season the testes were the same with respect to both histology and gene expression. This study provides an example of how seasonal heterochronic shifts in tissue-specific ar gene expression can disparately impact seasonal development and expression patterns across tissues, providing a potential mechanism for differential diversification of reproductive traits.

Ursolic Acid Regulates Immune Balance, Modulates Gut Microbial Metabolism, and Improves Liver Health in Mice

Ursolic acid (UA) has demonstrated significant immunomodulatory and hepatoprotective effects; however, the underlying mechanisms remain unclear. This study aims to analyze the impact of UA on the gut microbiome, metabolome, and liver transcriptome, investigate UA’s role in maintaining gut immune homeostasis and liver health, and evaluate the potential contributions of gut microbes and their metabolites to these beneficial effects. Our findings indicate that UA enhances immune balance in the jejunum, fortifies intestinal barrier function, and promotes overall gut health. UA modulates the intestinal microbiota and its metabolic processes, notably increasing the abundance of beneficial bacteria such as Odoribacter and Parabacteroides, along with their metabolites, including ornithine and lactucin. Additionally, UA inhibits the expression of interleukin-1 receptor 1 (IL1R1) and calcium (Ca2+) voltage-gated channel auxiliary subunit beta 2 (CACNB2) while enhancing the synthesis pathways of retinol and ascorbic acid, thereby exerting a protective influence on liver function. In summary, UA enhances intestinal immune homeostasis and promotes liver health, with these advantageous effects potentially mediated by beneficial bacteria (Odoribacter and Parabacteroides) and their metabolites (ornithine and lactucin).

Enhanced Gene Expression of Toll-Like Receptors 3, 7, and 9 in Thalassemia Patients: Implications for Inflammation and Potential Therapeutic Targets

Enhanced Gene Expression of Toll-Like Receptors 3, 7, and 9 in Thalassemia Patients: Implications for Inflammation and Potential Therapeutic Targets

Investigating the IgM and IgG B Cell Receptor Repertoires and Expression of Ultralong Complementarity Determining Region 3 in Colostrum and Blood from Holstein-Friesian Cows at Calving

In cattle, colostral maternal immunoglobulins and lymphocytes transfer across the neonate’s intestinal epithelium to provide protection against pathogens. This study aimed to compare repertoires of B cell populations in blood and colostrum in cows for the first time, with an emphasis on ultralong complementarity determining region 3 (CDR3, ≥40 amino acids). Blood mononuclear cells (BMCs, n= 7) and colostral cells (n = 7) were isolated from Holstein-Friesian dairy cows. Magnetic-activated cell sorting was used to capture IgM and IgG B cells from BMCs. Colostral cells were harvested by centrifugation. RNA was extracted and cDNA was produced; IgM and IgG transcripts were amplified using polymerase chain reactions. Amplicons were sequenced using the Nanopore Native barcoding kit 24 V14 and MinION with R10.4 flow cells. In colostrum, there was a significantly greater percentage of IgM B cells with ultralong CDR3s (8.09% ± 1.73 standard error of the mean) compared to blood (4.22% ± 0.70, p = 0.05). There was a significantly greater percentage of IgG B cells in colostrum with ultralong CDR3s (12.98% ± 1.98) compared to blood (6.61% ± 1.11, p = 0.05). A higher percentage of IgM and IgG B cells with ultralong CDR3s in colostrum may be indicative of a potential role in protecting the neonate.

B-255 Identification of the protein expression of Androgen Receptor variants using Targeted proteomics in clinical Castration Resistant Prostate Cancer models

Abstract Background Castration Resistant Prostate Cancer (CRPC) is a treatment resistant form of prostate cancer (PCa). Currently, there is not a way to identify which patients will develop this resistance before full blown CRPC develops. Therefore, all PCa treatment approaches are similar yet this results in tumor regression in some cases and progression in others. Targeting the androgen receptor (AR) is still the main focus of current therapies even in CRPC. Emergence of AR splice variants (AR-Vs) after initial treatment is thought to be one of the primary mechanisms of resistance. AR-Vs lack the ligand binding domain rendering Androgen Deprivation Therapy (ADT) ineffective in tumors expressing these variants. Recent work has identified the DNA and RNA species of ARVs in CRPC but investigation into whether the protein is translated are unknown. One exception is the approval of an antibody test that detects a specific AR-v, AR-v7, from the blood of PC patients and predicts ADT response. However, there are instances where a patient may not express ARv7 and are still resistant to ADT. This may suggest that other AR-Vs, not currently detected at the protein level, are important predictors for ADT response. Methods The primary goal of this work is to expand the protein identification of known and unknown AR-Vs that may predict response to ADT using targeted liquid chromatography tandem mass spectrometry (LC-MS/MS). We developed the AR-V targeted LC-MS/MS assay by first designing AR splice variants from unique splice regions via RNA-guided sequences translated to amino acid sequences. The AR-variants full amino acid sequences were then in-silico trypsin digested using Expasy software tool that generated tryptic peptides. Blast analysis of the AR-V peptides was performed where no overlap with other human proteins was observed. These AR-V peptide sequences were commercially synthesized along with heavy isotopically analogs to be used as internal standards in the LC-MS/MS method. The method examined 9 peptides and included 2 to 3 MRM transitions per peptide and calibration curves were constructed using unlabeled peptides. Specimens examined consisted of a panel of PCa cell lines (n= 6), CRPC patient derived xenografts tumors (PDX) (n=4) and a negative control cell line (no androgen receptor expression), Cos-1, which served with a dual function and was used as matrix for peptide MRM optimization experiments. Linearity and sensitivity for each peptide included in the method was examined using calibration curves. Results The targeted MRM approached was successfully achieved for the quantification of 9 peptides from 8 proteins in 6 PCa cell lines and 4 PDX tumor specimens. Out of the 8 proteins evaluated, we observed 3 known targets (AR-exon1, AR-V7 and AR-V12) and 4 novel AR-Vs in 22Rv1 PCa cell lines. Additionally, CRPC PDX were assessed where 3 novel AR-Vs were detected. Conclusions These preliminary results using a LC-MS/MS platform show promising identification and quantification of novel AR-Vs that have not been measured before at the protein level. Subsequent analysis of larger cohorts will further elucidate AR-Vs role in PCa treatment resistance mechanisms and possibly assist in future treatment approaches.

Orexins mitigate obesity-associated dysfunctions in mice.

Obesity is a chronic disease that affects more than 400 million adults with severe comorbidities. The search for new treatments to reduce its negative consequences is necessary. Orexins are hypothalamic neuropeptides involved in various physiological processes related to obesity. The aim of this study was to investigate the consequences of chronic orexin-A treatment in mouse models. Female wild-type C57BL/6 mice that were obesity-prone or obesity-resistant and mice that were deficient for orexin receptors were fed with a high-fat diet. Glucose tolerance, indirect calorimetry, expression of brain neuropeptides and receptors, microglial activation, and microbiota were determined to evaluate the role of orexins on metabolic flexibility. Orexin-A reduces weight gain in obesity-prone mice. This reduction is associated with a decrease in body fat, food intake, steatosis, and insulin resistance, as well as alterations of intestinal microbiota composition. A decreased expression of orexin receptors and neuropeptides involved in food intake was also observed in the hypothalamus. Our data support the notion that orexin receptor signaling is involved in different aspects of energy metabolism and can mitigate several dysfunctions associated with obesity, suggesting that orexin receptors can represent new targets for obesity treatment.

The expression level of VEGFR2 regulates mechanotransduction, tumor growth and metastasis of high grade serous ovarian cancer cells

Recent data shows that alterations in the expression and/or activation of the vascular endothelial growth factor receptor 2 (VEGFR2) in high grade serous ovarian cancer (HGSOC) modulate tumor progression. However, controversial results have been obtained, showing that in some cases VEGFR2 inhibition can promote tumorigenesis and metastasis. Thus, it is urgent to better define the role of the VEGF/VEGFR2 system to understand/predict the effects of its inhibitors administered as anti-angiogenic in HGSOC. Here, we modulated the expression levels of VEGFR2 and analyzed the effects in two cellular models of HGSOC. VEGFR2 silencing (or its pharmacological inhibition) promote the growth and invasive potential of OVCAR3 cells in vitro and in vivo. Consistent with this, the low levels of VEGFR2 in OV7 cells are associated with more pronounced proliferative and motile phenotypes when compared to OVCAR3 cells, and VEGFR2 overexpression in OV7 cells inhibits cell growth. In vitro data confirmed that VEGFR2 silencing in OVCAR3 cells favors the acquisition of an invasive phenotype by loosening cell-ECM contacts, reducing the size and the signaling of focal adhesion contacts (FAs). This is translated into a reduced FAK activity at FAs, ECM-dependent alterations of mechanical forces through FAs and YAP nuclear translocation. Together, the data show that low expression, silencing or inhibition of VEGFR2 in HGSOC cells alter mechanotransduction and lead to the acquisition of a pro-proliferative/invasive phenotype which explains the need for a more cautious use of anti-VEGFR2 drugs in ovarian cancer.

Spatially restricted ecto-5'-nucleotidase expression promotes the growth of uterine leiomyomas by modulating Akt activity.

Found in as many as 80% of women, uterine leiomyomas are a frequent cause of abnormal uterine bleeding, pelvic pain, and infertility. Despite their significant clinical impact, the mechanisms responsible for driving leiomyoma growth remain poorly understood. After obtaining IRB permission, expression of ecto-5'-nucleotidase (NT5E, CD73) was assessed in matched specimens of myometrium and leiomyoma by real-time qPCR, Western blot, and immunohistochemistry (IHC). Adenosine concentrations were measured by enzyme-linked assay. Primary cultures were used to assess the impact of adenosine and/or adenosine receptor agonists on proliferation, apoptosis, and patterns of intracellular signaling invitro. When compared to matched specimens of healthy myometrium, uterine leiomyomas were characterized by reduced CD73 expression. Largely limited to thin-walled vascular structures and the pseudocapsule of leiomyomas despite diffuse myometrial distribution. Restricted intra-tumoral CD73 expression was accompanied by decreased levels of intra-tumoral adenosine. Invitro, incubation of primary leiomyoma cultures with adenosine or its hydrolysis-resistant analog 2-chloro-adenosine (2-CL-AD) inhibited proliferation, induced apoptosis, and reduced proportion of myocytes in S- and G2-M phases of the cell cycle. Decreased proliferation was accompanied by reduced expression of phospho-Akt, phospho-Cdk2-Tyr15, and phospho-Histone H3. Enforced expression of the A2B adenosine receptor (ADORA2B) and ADORA2B-selective agonists similarly suppressed proliferation and inhibited Akt phosphorylation. Collectively, these observations broadly implicate CD73 and reducedextracellular concentrations ofadenosine as key regulators of leiomyoma growth and potentiallyidentify novel strategies for clinically managing these common tumors.

Detection of SARS-CoV-2 binding receptors and miscellaneous targets as well as mucosal surface area of the human lacrimal drainage system

Our aim was to evaluate a potential role for the lacrimal drainage system (LDS) as a portal of entry and conduit for SARS-CoV-2 in human infection. We also investigate the mucosal surface area. The relatively long tear contact time in a closed system raises the possibility that this pathway may contribute to the initiation of systemic infection. We looked for expression of ACE2, the main receptor for SARS-CoV-2, as well as cofactors such as TMPRSS2 and other enzymes such as cathepsinB, CD147, elastase1, furin, neuropilin1, neuropilin2, TMPRSS11D and trypsin which also play a role in SARS-CoV-2 infection, in this system. Human tissue samples of the draining tear ducts from body donors were analyzed by RT-PCR, Western blot and immunohistochemistry. It is not known whether the respective body donors were Sars-Cov-2 positive at any time; they were negative when they entered the institute. Besides, the draining LDS of body donors were measured to determine the mucosal surface in the lacrimal system. The expression of the main receptor studied, ACE2, cofactors such as TMPRSS2 and other enzymes such as cathepsinB, CD147, elastase1, furin, neuropilin1, neuropilin2, TMPRSS11D and trypsin were all detected at the gene and protein level. The average mucosal surface area of the lacrimal sac and nasolacrimal duct was calculated to be 110mm2. The results show the presence of all analyzed receptors in the efferent LDS. With an average tear passage time of 3min and a relatively large mucosal surface area, the LDS could therefore be considered as a portal of entry and conduit for SARS-CoV-2. In addition, it represents a surface that should be taken into consideration in the administration of topically applied medication to the ocular surface.

Targeted immune cell therapy for hepatocellular carcinoma using expanded liver mononuclear cell-derived natural killer cells

Natural killer (NK) cells are a promising cellular therapy for T cell-refractory cancers but are frequently deficient or dysfunctional in patients with hepatocellular carcinoma (HCC). In the present study, we explored a novel therapy for HCC using NK cells derived from donor liver graft perfusate. These liver-derived NK cells, named LMNC-NK cells, are more abundant in liver mononuclear cells (LMNCs) than in peripheral blood mononuclear cells (PBMCs) from the same donor. We developed a method to expand LMNC-NK cells by 33.8±54.4-fold, enhancing their cytotoxic properties and cytokine production, including granzyme B, CD107a, TNF-α, and IFN-γ. These cells also showed an increased expression of cytotoxicity receptors. An RNA-seq analysis revealed considerable differences in gene expression between LMNC-NK and PBMC-NK cells, with 453 genes upregulated and 449 downregulated in LMNC-NK cells. These genes are involved in the mitogen-activated protein kinase cascade and cell differentiation, explaining the increased activity of LMNC-NK cells. Quantitative reverse transcription polymerase chain reaction confirmed the significant upregulation of TLR6, KIT, MMP14, IRF8, TCF7, FCERIG, LEF1, NLRp3, and IL16 in LMNC-NK cells. LMNC-NK cells effectively eliminated HepG-2-Luc cells in vitro, and in an orthotopic murine model of HCC, they exhibited a potent anti-tumor effect, outperforming PBMC-NK cells. The expression of the activation marker CD69+ in LMNC-NK cells was also significantly higher among tumor-infiltrating lymphocytes compared to PBMC-NK cells. Our research suggests that the adoptive transfer of LMNC-NK cells could be a promising treatment for HCC, offering a novel and effective source of NK cells with superior cytotoxic functions.