Immunolocalization of estrogen receptor beta in the epididymis of mature and immature pigs.

A growing body of evidence suggests a role of estrogens in the male reproduction via their specific estrogen receptors (ERalpha/ERbeta). Estrogen receptor distribution along the genital tract tissues has been described in different species, but it is unknown in the pig. Therefore, the aim of the present study was to localize ERbeta in the epididymis of mature and immature pigs (aged 2 and 18 months, respectively). Immunohistochemistry was carried out on paraffin-embedded tissues using a mouse anti-human monoclonal IgG against ERbeta as the primary antibody, and a goat anti-mouse biotinylated IgG as the secondary antibody. Avidin-biotin-peroxidase complex was then applied followed by diaminobenzidine. In immature pigs, the epithelial cells from the caput, corpus and cauda epididymis showed no or very weak immunoreactivity for ERbeta, whereas they were all strongly immmunoreactive in mature pigs. A various intensity of immunostaining from weak to strong in the smooth muscle cells as well as in the connective tissue cells were detected in the epididymis of both, young and adult pigs. This is the first report on the cellular localization of ERbeta protein in porcine epidydimis. The present study demonstrated that (1) irrespectively of the epididymal region, the epithelial cells of caput, corpus and cauda epididymis of mature pigs revealed a strong immunoreactivity for ERbeta, and (2) ERbeta expression in the epididymal epithelium is regulated by puberty. Finally, although the biological activity of ERbeta has not yet been established, the results of the present study suggest its involvement in estrogen modulation of pig epididymal function

The FXR agonist obeticholic acid inhibits the cancerogenic potential of human cholangiocarcinoma

Cholangiocarcinoma (CCA) is an aggressive cancer with high resistance to chemotherapeutics. CCA is enriched in cancer stem cells, which correlate with aggressiveness and prognosis. FXR, a member of the metabolic nuclear receptor family, is markedly down-regulated in human CCA. Our aim was to evaluate, in primary cultures of human intrahepatic CCA (iCCA), the effects of the FXR agonist obeticholic acid (OCA), a semisynthetic bile acid derivative, on their cancerogenic potential. Primary human iCCA cell cultures were prepared from surgical specimens of mucinous or mixed iCCA subtypes. Increasing concentrations (0–2.5 μM) of OCA were added to culture media and, after 3–10 days, effects on proliferation (MTS assay, cell population doubling time), apoptosis (annexin V-FITC/propidium iodide), cell migration and invasion (wound healing response and Matrigel invasion assay), and cancerogenic potential (spheroid formation, clonogenic assay, colony formation capacity) were evaluated. Results: FXR gene expression was downregulated (RT-qPCR) in iCCA cells vs normal human biliary tree stem cells (p < 0.05) and in mucinous iCCA vs mixed iCCA cells (p < 0.05) but was upregulated by addition of OCA. OCA significantly (p < 0.05) inhibited proliferation of both mucinous and mixed iCCA cells, starting at a concentration as low as 0.05 μM. Also, CDCA (but not UDCA) inhibited cell proliferation, although to a much lower extent than OCA, consistent with its different affinity for FXR. OCA significantly induced apoptosis of both iCCA subtypes and decreased their in vitro cancerogenic potential, as evaluated by impairment of colony and spheroid formation capacity and delayed wound healing and Matrigel invasion. In general, these effects were more evident in mixed than mucinous iCCA cells. When tested together with Gemcitabine and Cisplatin, OCA potentiated the anti-proliferative and pro-apoptotic effects of these chemotherapeutics, but mainly in mixed iCCA cells. OCA abolished the capacity of both mucinous and mixed iCCA cells to form colonies when administered together with Gemcitabine and Cisplatin. In subcutaneous xenografts of mixed iCCA cells, OCA alone or combined with Gemcitabine or Cisplatin markedly reduced the tumor size after 5 weeks of treatment by inducing necrosis of tumor mass and inhibiting cell proliferation. In conclusion, FXR is down-regulated in iCCA cells, and its activation by OCA results in anti-cancerogenic effects against mucinous and mixed iCCA cells, both in vitro and in vivo. The effects of OCA predominated in mixed iCCA cells, consistent with the lower aggressiveness and the higher FXR expression in this CCA subtype. These results, showing the FXR-mediated capacity of OCA to inhibit cholangiocarcinogenesis, represent the basis for testing OCA in clinical trials of CCA patients

Histamine stimulates the proliferation of small and large cholangiocytes by activation of both IP3/Ca2+ and cAMP-dependent signaling mechanisms

Although large cholangiocytes exert their functions by activation of cyclic adenosine 3',5'-monophosphate (cAMP), Ca(2+)-dependent signaling regulates the function of small cholangiocytes. Histamine interacts with four receptors, H1-H4HRs. H1HR acts by Gαq activating IP(3)/Ca(2+), whereas H2HR activates Gα(s) stimulating cAMP. We hypothesize that histamine increases biliary growth by activating H1HR on small and H2HR on large cholangiocytes. The expression of H1-H4HRs was evaluated in liver sections, isolated and cultured (normal rat intrahepatic cholangiocyte culture (NRIC)) cholangiocytes. In vivo, normal rats were treated with histamine or H1-H4HR agonists for 1 week. We evaluated: (1) intrahepatic bile duct mass (IBDM); (2) the effects of histamine, H1HR or H2HR agonists on NRIC proliferation, IP(3) and cAMP levels and PKCα and protein kinase A (PKA) phosphorylation; and (3) PKCα silencing on H1HR-stimulated NRIC proliferation. Small and large cholangiocytes express H1-H4HRs. Histamine and the H1HR agonist increased small IBDM, whereas histamine and the H2HR agonist increased large IBDM. H1HR agonists stimulated IP(3) levels, as well as PKCα phosphorylation and NRIC proliferation, whereas H2HR agonists increased cAMP levels, as well as PKA phosphorylation and NRIC proliferation. The H1HR agonist did not increase proliferation in PKCα siRNA-transfected NRICs. The activation of differential signaling mechanisms targeting small and large cholangiocytes is important for repopulation of the biliary epithelium during pathologies affecting different-sized bile ducts

Application of the MEGNO technique to the dynamics of Jovian irregular satellites

We apply the MEGNO (Mean Exponential Growth of Nearby Orbits) technique to the dynamics of Jovian irregular satellites. We demonstrate the efficiency of applying the MEGNO indicator to generate a mapping of relevant phase-space regions occupied by observed jovian irregular satellites. The construction of MEGNO maps of the Jovian phase-space region within its Hill-sphere is addressed and the obtained results are compared with previous studies regarding the dynamical stability of irregular satellites. Since this is the first time the MEGNO technique is applied to study the dynamics of irregular satellites we provide a review of the MEGNO theory. We consider the elliptic restricted three-body problem in which Jupiter is orbited by a massless test satellite subject to solar gravitational perturbations. The equations of motion of the system are integrated numerically and the MEGNO indicator computed from the systems variational equations. An unprecedented large set of initial conditions are studied to generate the MEGNO maps. The chaotic nature of initial conditions are demonstrated by studying a quasi-periodic orbit and a chaotic orbit. As a result we establish the existence of several high-order mean-motion resonances detected for retrograde orbits along with other interesting dynamical features. The computed MEGNO maps allows to qualitatively differentiate between chaotic and quasi-periodic regions of the irregular satellite phase-space given only a relatively short integration time. By comparing with previous published results we can establish a correlation between chaotic regions and corresponding regions of orbital instability.Comment: 15 pages, 13 figures, 2 tables, submitted to MNRA

CoRoT light curves of RR Lyrae stars. CoRoT 101128793: long-term changes in the Blazhko effect and excitation of additional modes

The CoRoT (Convection, Rotation and planetary Transits) space mission provides a valuable opportunity to monitor stars with uninterrupted time sampling for up to 150 days at a time. The study of RR Lyrae stars, performed in the framework of the Additional Programmes belonging to the exoplanetary field, will particularly benefit from such dense, long-duration monitoring. The Blazhko effect in RR Lyrae stars is a long-standing, unsolved problem of stellar astrophysics. We used the CoRoT data of the new RR Lyrae variable CoRoT 101128793 (f0=2.119 c/d, P=0.4719296 d) to provide us with more detailed observational facts to understand the physical process behind the phenomenon. The CoRoT data were corrected for one jump and the long-term drift. We applied different period-finding techniques to the corrected timeseries to investigate amplitude and phase modulation. We detected 79 frequencies in the light curve of CoRoT 101128793. They have been identified as the main frequency f0, and its harmonics, two independent terms, the terms related to the Blazhko frequency, and several combination terms. A Blazhko frequency fB=0.056 c/d and a triplet structure around the fundamental radial mode and harmonics were detected, as well as a long-term variability of the Blazhko modulation. Indeed, the amplitude of the main oscillation is decreasing along the CoRoT survey. The Blazhko modulation is one of the smallest observed in RR Lyrae stars. Moreover, the additional modes f1=3.630 and f2=3.159 c/d are detected. Taking its ratio with the fundamental radial mode into account, the term f1 could be the identified as the second radial overtone. Detecting of these modes in horizontal branch stars is a new result obtained by CoRoT.Comment: 13 pages, 2 figures, 2 long tables. Accepted for publication in A&

The Italian Registry for Primary Immunodeficiencies (Italian Primary Immunodeficiency Network; IPINet): Twenty Years of Experience (1999&#8211;2019)

Primary immunodeficiencies (PIDs) are heterogeneous disorders, characterized by variable clinical and immunological features. National PID registries offer useful insights on the epidemiology, diagnosis, and natural history of these disorders. In 1999, the Italian network for primary immunodeficiencies (IPINet) was established. We report on data collected from the IPINet registry after 20\ua0years of activity. A total of 3352 pediatric and adult patients affected with PIDs are registered in the database. In Italy, a regional distribution trend of PID diagnosis was observed. Based on the updated IUIS classification of 2019, PID distribution in Italy showed that predominantly antibody deficiencies account for the majority of cases (63%), followed by combined immunodeficiencies with associated or syndromic features (22.5%). The overall age at diagnosis was younger for male patients. The minimal prevalence of PIDs in Italy resulted in 5.1 per 100.000 habitants. Mortality was similar to other European registries (4.2%). Immunoglobulin replacement treatment was prescribed to less than one third of the patient cohort. Collectively, this is the first comprehensive description of the PID epidemiology in Italy

Circulating miR-26b-5p and miR-451a as diagnostic biomarkers in medullary thyroid carcinoma patients

Purpose/methodsThe determination of tumour biomarkers is paramount to advancing personalized medicine, more so in rare tumours like medullary thyroid carcinoma (MTC), whose diagnosis is still challenging. The aim of this study was to identify non-invasive circulating biomarkers in MTC. To achieve this goal, paired MTC tissue and plasma extracellular vesicle samples were collected from multiple centres and microRNA (miRNA) expression levels were evaluated.ResultsThe samples from a discovery cohort of 23 MTC patients were analysed using miRNA arrays. Lasso logistic regression analysis resulted in the identification of a set of circulating miRNAs as diagnostic biomarkers. Among them, miR-26b-5p and miR-451a, were highly expressed and their expression decreased during follow-up in disease-free patients in the discovery cohort. Circulating miR-26b-5p and miR-451a were validated using droplet digital PCR in a second independent cohort of 12 MTC patients.ConclusionThis study allowed the identification and validation of a signature of two circulating miRNAs, miR-26b-5p and miR-451a, in two independent cohorts reporting a significant diagnostic performance for MTC. The results of this study offer advancements in molecular diagnosis of MTC proposing a novel non-invasive tool to use in precision medicine

Post-assembly modification of kinetically metastable Fe(II)2L3 triple helicates.

We report the covalent post-assembly modification of kinetically metastable amine-bearing Fe(II)2L3 triple helicates via acylation and azidation. Covalent modification of the metastable helicates prevented their reorganization to the thermodynamically favored Fe(II)4L4 tetrahedral cages, thus trapping the system at the non-equilibrium helicate structure. This functionalization strategy also conveniently provides access to a higher-order tris(porphyrinatoruthenium)-helicate complex that would be difficult to prepare by de novo ligand synthesis.This work was supported by the UK Engineering and Physical Sciences Research Council (EPSRC). D.A.R. acknowledges the Gates Cambridge Trust for Ph.D. (Gates Cambridge Scholarship) and conference funding.This is the final published version. It first appeared at http://pubs.acs.org/doi/abs/10.1021/ja5042397