Probes for INS

PDGFR-β-expressing cells of the kidneys are considered as a relevant site of erythropoietin (EPO) production. The origin of these cells, their contribution to renal EPO production, and if PDGFR-β-positive cells in other organs are also capable to express EPO are less clear. We addressed these questions in mice, in which hypoxia-inducible transcription factors were stabilized in PDGFR-β+ cells by inducible deletion of the von Hippel-Lindau (Vhl) protein. Vhl deletion led to a 600-fold increase of plasma EPO concentration, 170-fold increase of renal EPO messenger RNA (mRNA) levels, and an increase of hematocrit values up to 70 %. Intrarenal localization of EPO-expressing cells coincided with the zonal heterogeneity and distribution of cells expressing PDGFR-β. Amongst a variety of extrarenal organs only adrenal glands showed significant EPO mRNA expression after Vhl deletion in PDGFR-β+ cells. EPO mRNA, plasma EPO, and hematocrit fell to subnormal values if HIF-2α, but not HIF-1α, was deleted either alone or in combination with Vhl in PDGFR-β+ cells. Treatment of mice with a prolyl-hydroxylase inhibitor caused an increase of EPO mRNA abundance and plasma EPO concentrations in wild-type mice and in mice lacking HIF-1α in PDGFR-β+ cells but exerted no effect in mice lacking HIF-2α in PDGFR-β+ cells. These findings suggest that PDGFR-β+ cells are the only relevant site of EPO expression in the kidney and that HIF-2 is the essential transcription factor triggering EPO expression therein. Moreover, our findings suggest that PDGFR-β+cells elaborating EPO might arise from the metanephric mesenchyme, rather than from the neural crest.

Forkhead box A2 (FOXA2) is a pioneer transcription factor involved in organ development, function, and cancer. In the uterus, FOXA2 is essential for pregnancy and expressed specifically in the glands of the endometrium. Loss of FOXA2 function occurs during development of endometrial cancer in humans. The current study describes the development of a mouse model for conditional expression of mouse FOXA2. Using a system consisting of a minigene located at the Rosa26 locus, we generated a CAG-S-mFOXA2 allele in embryonic stem cells and subsequently in mice; before activation, the minigene is silent because of a floxed stop cassette inserted between the promoter and the transgene. To validate functionality, mice with the CAG-S-mFOXA2 allele were crossed with progesterone receptor (Pgr)-Cre mice and lactotransferrin (Ltf)-iCre mice that express Cre in the immature and adult uterus, respectively. In immature Pgr-Cre-CAG-S-mFoxa2 mice, FOXA2 protein was expressed in the luminal epithelium (LE), glandular epithelium (GE), stroma, and inner layer of the myometrium. Interestingly, FOXA2 protein was not observed in most of the LE of uteri from adult Pgr-Cre-CAG-S-mFoxa2 mice, although FOXA2 was maintained in the stroma, GE, and myometrium. The adult Pgr-Cre-CAG-S-mFoxa2 females were completely infertile. In contrast, Ltf-iCre-CAG-S-mFoxa2 mice were fertile with no detectable histological differences in the uterus. The adult uterus of Pgr-Cre-CAG-S-mFoxa2 mice was smaller, contained few endometrial glands, and displayed areas of partially stratified LE and GE. This transgenic mouse line is a valuable resource to elucidating and exploring FOXA2 function.

Aberrantly expressed G protein-coupled receptors in tumors are considered as potential therapeutic targets. We analyzed the expressions of receptors of gonadotropin-releasing hormone (GNRHR), luteinizing hormone/chorionic gonadotropin (LHCGR) and follicle-stimulating hormone (FSHR) in human adrenocortical carcinomas and assessed their response to GnRH antagonist therapy. We further studied the effects of the GnRH antagonist cetrorelix acetate (CTX) on cultured adrenocortical tumor (ACT) cells (mouse Cα1 and Y-1, and human H295R), and in vivo in transgenic mice (SV40 T-antigen expression under inhibin α promoter) bearing Lhcgr and Gnrhr in ACT. Both models were treated with control (CT), CTX, human chorionic gonadotropin (hCG) or CTX+hCG, and their growth and transcriptional changes were analyzed. In situ hybridization and qPCR analysis of human adrenocortical carcinomas (n = 11-13) showed expression of GNRHR in 54/73%, LHCGR in 77/100% and FSHR in 0%, respectively. CTX treatment in vitro decreased cell viability and proliferation, and increased caspase 3/7 activity in all treated cells. In vivo, CTX and CTX+hCG (but not hCG alone) decreased ACT weights and serum LH and progesterone concentrations. CTX treatment downregulated the tumor markers Lhcgr and Gata4. Upregulated genes included Grb10, Rerg, Nfatc and Gnas, all recently found to be abundantly expressed in healthy adrenal vs ACT. Our data suggest that CTX treatment may improve the therapy of human adrenocortical carcinomas by direct action on GNRHR-positive cancer cells inducing apoptosis and/or reducing gonadotropin release, directing tumor cells towards a healthy adrenal gene expression profile.

Abstract BACKGROUND: Intestinal absorption of bile acids is mediated by the apical sodium-dependent bile acid transporter (ASBT). Fecal bile acid dysmetabolism has been reported in dogs with chronic inflammatory enteropathy (CIE). OBJECTIVE: Characterization of ASBT distribution along the intestinal tract of control dogs and comparison to dogs with CIE. ANIMALS: Twenty-four dogs with CIE and 11 control dogs. METHODS: The ASBT mRNA and protein expression were assessed using RNA in situ hybridization and immunohistochemistry, respectively. The concentrations of fecal bile acids were measured by gas chromatography-mass spectrometry. The fecal microbiota dysbiosis index was assessed with a quantitative polymerase chain reaction panel. RESULTS: In control dogs, ASBT mRNA expression was observed in enterocytes in all analyzed intestinal segments, with highest expression in the ileum. The ASBT protein expression was restricted to enterocytes in the ileum, cecum, and colon. Dogs with CIE had significantly decreased expression of ASBT protein in the ileum (P = .001), which was negatively correlated with histopathological score (ρ = -0.40; Pcorr = .049). Additionally, dogs with CIE had a significantly increased percentage of primary bile acids in feces compared to controls (P = .04). The fecal dysbiosis index was significantly higher in dogs with CIE than in control dogs (P = .01). CONCLUSIONS AND CLINICAL IMPORTANCE: These findings indicate that ileal protein expression of ASBT is downregulated in dogs with CIE. This change may be linked to the inflammatory process, intestinal dysbiosis, and fecal bile acid dysmetabolism observed in these patients.

The Hyperpolarization activated, cyclic nucleotide gated (HCN) channel is a candidate mediator of neuroendocrine influence over detrusor tonus during filling. In other tissues, HCN loss with aging is linked to declines in rhythmicity and function. We hypothesized that HCN has an age-sensitive expression profile and functional role in adrenergic bladder relaxation. HCN was examined in bladders from young (2–6 months) and old (18–24 months) C57BL/6 female mice, using qRT-PCR, RNAScope, and Western blots. Isometric tension studies were conducted using bladder strips from young wild-type (YWT), old wild-type (OWT), and young HCN1 knock-out (YKO) female mice to test the role HCN in effects of β-adrenergic stimulation. Hcn1 is the dominant HCN isoform RNA in the mouse bladder wall, and is diminished with age. Location of Hcn RNA within the mouse bladder wall is isoform-specific, with HCN1 limited to the detrusor layer. Passively-tensioned YWT bladder strips are relaxed by isoproterenol in the presence of HCN function, where OWT strips are relaxed only in the presence of HCN blockade. HCN has an age-specific expression and function in adrenergic detrusor relaxation in mouse bladder strips.

BACKGROUND:

Cassava brown streak disease (CBSD) has a viral aetiology and is caused by viruses belonging to the genus Ipomovirus (family Potyviridae), Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Molecular and serological methods are available for detection, discrimination and quantification of cassava brown streak viruses (CBSVs) in infected plants. However, precise determination of the viral RNA localization in infected host tissues is still not possible pending appropriate methods.

RESULTS:

We have developed an in situ hybridization (ISH) assay based on RNAscope™ technology that allows the sensitive detection and localization of CBSV RNA in plant tissues. The method was initially developed in the experimental host Nicotiana rustica and was then further adapted to cassava. Highly sensitive and specific detection of CBSV RNA was achieved without background and hybridization signals in sections prepared from non-infected tissues. The tissue tropism of CBSV RNAs appeared different between N. rustica and cassava.

CONCLUSIONS:

This study provides a robust method for CBSV detection in the experimental host and in cassava. The protocol will be used to study CBSV tropism in various cassava genotypes, as well as CBSVs/cassava interactions in single and mixed infections.

Zika virus (ZIKV) infection of pregnant women can cause congenital malformations including microcephaly, which has focused global attention on this emerging pathogen1. In addition to transmission by mosquitoes, ZIKV can be detected in the seminal fluid of affected males for extended periods of time and transmitted sexually2. Here, using a mouse-adapted African ZIKV strain (Dakar 41519), we evaluated the consequences of infection in the male reproductive tract of mice. We observed persistence of ZIKV, but not the closely related Dengue virus (DENV), in the testis and epididymis of male mice, and this was associated with tissue injury that caused diminished testosterone and inhibin B levels, and oligospermia. ZIKV preferentially infected spermatogonia, primary spermatocytes, and Sertoli cells in the testis, resulting in cell death and destruction of the seminiferous tubules. Less damage was observed with a contemporary Asian ZIKV strain (H/PF/2013), in part because this virus replicates less efficiently in mice. The extent to which these observations in mice translate to humans remains unclear, but longitudinal studies of sperm function and viability in ZIKV-infected humans seem warranted.

High-grade serous ovarian cancers (HGSOC) have been subdivided into molecular subtypes. The mesenchymal HGSOC subgroup, defined by stromal-related gene signatures, is invariably associated with poor patient survival. We demonstrate that stroma exerts a key function in mesenchymal HGSOC. We highlight stromal heterogeneity in HGSOC by identifying four subsets of carcinoma-associated fibroblasts (CAF-S1-4). Mesenchymal HGSOC show high content in CAF-S1 fibroblasts, which exhibit immunosuppressive functions by increasing attraction, survival, and differentiation of CD25+FOXP3+ T lymphocytes. The beta isoform of the CXCL12 chemokine (CXCL12β) specifically accumulates in the immunosuppressive CAF-S1 subset through a miR-141/200a dependent-mechanism. Moreover, CXCL12β expression in CAF-S1 cells plays a crucial role in CAF-S1 immunosuppressive activity and is a reliable prognosis factor in HGSOC, in contrast to CXCL12α. Thus, our data highlight the differential regulation of the CXCL12α and CXCL12β isoforms in HGSOC, and reveal a CXCL12β-associated stromal heterogeneity and immunosuppressive environment in mesenchymal HGSOC.

The hypocretin/orexin neuropeptide system coordinates the regulation of various physiological processes. Our previous study reported that a reduction in the expression of pleomorphic adenoma gene‑like 1 (Plagl1), which encodes a C2H2 zinc‑finger transcription factor, occurs in hypocretin neuron‑ablated transgenic mice, suggesting that PLAGL1 is co‑expressed in hypocretin neurons and regulates hypocretin transcription. The present study examined whether canonical prepro‑hypocretin transcription is functionally modulated by PLAGL1. Double immunostaining indicated that the majority of hypocretin neurons were positive for PLAGL1 immunoreactivity in the nucleus. Notably, PLAGL1 immunoreactivity in hypocretin neurons was altered in response to several conditions affecting hypocretin function. An uneven localization of PLAGL1 was detected in the nuclei of hypocretin neurons following sleep deprivation. Chromatin immunoprecipitation revealed that endogenous PLAGL1 may bind to a putative PLAGL1‑binding site in the proximal region of the hypocretin gene, in the murine hypothalamus. In addition, electroporation of the PLAGL1 expression vector into the fetal hypothalamus promoted hypothalamic hypocretin transcription. These results suggested that PLAGL1 may regulate hypothalamic hypocretin transcription.