A Hippo and Fibroblast Growth Factor Receptor Autocrine Pathway in Cholangiocarcinoma

Herein, we have identified cross-talk between the Hippo and fibroblast growth factor receptor (FGFR) oncogenic signaling pathways in cholangiocarcinoma (CCA). Yes-associated protein (YAP) nuclear localization and up-regulation of canonical target genes was observed in CCA cell lines and a patient-derived xenograft (PDX). Expression of FGFR1, -2, and -4 was identified in human CCA cell lines, driven, in part, by YAP coactivation of TBX5. In turn, FGFR signaling in a cell line with minimal basal YAP expression induced its cellular protein expression and nuclear localization. Treatment of YAP-positive CCA cell lines with BGJ398, a pan-FGFR inhibitor, resulted in a decrease in YAP activation. FGFR activation of YAP appears to be driven largely by FGF5 activation of FGFR2, as siRNA silencing of this ligand or receptor, respectively, inhibited YAP nuclear localization. BGJ398 treatment of YAP-expressing cells induced cell death due to Mcl-1 depletion. In a YAP-associated mouse model of CCA, expression of FGFR 1, 2, and 4 was also significantly increased. Accordingly, BGJ398 treatment was tumor-suppressive in this model and in a YAP-positive PDX model. These preclinical data suggest not only that the YAP and Hippo signaling pathways culminate in an Mcl-1-regulated tumor survival pathway but also that nuclear YAP expression may be a biomarker to employ in FGFR-directed therapy.     

Cryopreserved CD90+ cells obtained from mobilized peripheral blood in sheep: a new source of mesenchymal stem cells for preclinical applications

Mobilized peripheral blood (MPB) bone marrow cells possess the potential to differentiate into a variety of mesenchymal tissue types and offer a source of easy access for obtaining stem cells for the development of experimental models with applications in tissue engineering. In the present work, we aimed to isolate by magnetic activated cell sorting CD90+ cells from MPB by means of the administration of Granulocyte-Colony Stimulating Factor and to evaluate cell proliferation capacity, after thawing of the in vitro culture of this population of mesenchymal stem cells (MSCs) in sheep. We obtained a median of 8.2 ± 0.6 million of CD90+ cells from the 20-mL MPB sample. After thawing, at day 15 under in vitro culture, the mean CD90+ cells determined by flow cytometry was 92.92 ± 1.29 % and cell duplication time determined by crystal violet staining was 47.59 h. This study describes for the first time the isolation, characterization, and post-in vitro culture thawing of CD90+ MSCs from mobilized peripheral blood in sheep. This population can be considered as a source of MSCs for experimental models in tissue engineering research.     

Hb Santa Clara (beta 97His-->Asn), a human haemoglobin variant: functional characterization and structure modelling

This study examines the functional and structural effects of amino acid substitution at alpha(1)beta(2) interface of Hb Santa Clara (beta 97His-->Asn). We have characterized the variation by a combination of electrospray ionisation mass spectrometry and DNA sequence analysis followed by oxygen-binding experiments. Functional studies outlined an increased oxygen affinity, reduced effect of organic phosphates and a reduced Bohr effect with respect to HbA. In view of the primary role of this interface in the cooperative quaternary transition from the T to R conformational state, a theoretical three-dimensional model of Hb Santa Clara was generated. Structural investigations suggest that replacement of Asn for His beta 97 results in a significant stabilization of the high affinity R-state of the haemoglobin molecule with respect to the low affinity T-state. The role of beta FG4 position has been further examined by computational models of known beta FG4 variants, namely Hb Malm? (beta 97His-->Gln), Hb Wood (beta 97His-->Leu), Hb Nagoya (beta 97His-->Pro) and Hb Moriguchi (beta 97His-->Tyr). These findings demonstrate that, among the various residues at the alpha(1)beta(2) (and alpha(2)beta(1)) intersubunit interface, His beta FG4 contributes significantly to the quaternary constraints that are responsible for the low oxygen affinity of human deoxyhaemoglobin.     

Developmentally Regulated Expression of Persyn, a Member of the Synuclein Family, in Skin

Synucleins constitute a group of unique, evolutionarily conserved proteins that are expressed predominantly in neurons of the central and peripheral nervous system. Although the normal cellular functions of synucleins are not clear, these proteins have been implicated in various neurodegenerative conditions in humans. We found that persyn, a recently characterized member of the synuclein family, is expressed not only in the nervous system but also in the stratum granulosum of the epidermis of neonatal and adult mice. This finding together with our recent observations that persyn influences neurofilament network integrity in sensory neurons raises the possibility that persyn in skin could be involved in modulation of the keratin network.     

Up-regulated P21CIP1 expression is part of the regulation quantitatively controlling serum deprivation-induced apoptosis

Among the many genes which have been suggested to be required by the molecular mechanism dictating apoptotic death, some have been shown to function as pacemakers to pave the way for cells either to live or to die. Previously we have shown that immediate early gene expressions associated with the G₁ phase of cell cycle traverse are candidates for this function. Here we report that the well-known key regulator for halting cell cycling at the G₁/S border, the p21 protein known as WAF1, Cip1, Pic1, or Sdi1, is also involved in the execution of cells' suicidal death. p21 up-regulation is seen in quiescent mouse 3T3 fibroblasts stimulated to die by serum deprivation, at both message and protein levels, evidenced by increased protein presence in its targeted functional site, the nucleus. In addition, we show that this up-regulation of p21 is functionally related to the operational efficiency of the apoptotic process, in that when cells are stably transfected with an antisense construct to repress the endogenous p21-protein level, death is delayed. Quantitative protection from apoptosis with antisense p21 transfection is relatively proportional to the repressed level of this protein in the cells. Taken together, our results suggest that the apoptosis-dependent additional increase of p21 beyond the base level, seen in serum-deprived quiescent cells, may be involved in the molecular events precipitating a rapid program of cell demise, and that repression of this increase may obstruct the operation of this program and postpone the eventual death. J. Cell. Biochem. 64:434–446. © 1997 Wiley-Liss, Inc.     

Enfermedad de Erdheim-Chester: primer caso pediátrico reportado en Colombia

The Erdheim-Chester’s disease is extremely rare in children. We present the case of a 12-year-old girl with histological and radiological diagnosis of this disease and mutation of the BRAF gene, who developed multisystemic compromise requiring treatment with dabrafenib. We identified 22 reports of this condition among children worldwide and this is the second pediatric case in Latin America. Diagnostic imaging is critical to confirm Erdheim-Chester disease and for the surgical planning of the biopsy. Additionally, we identified the parasellar dark sign, which has previously been reported on lymphocytic hypophysitis.This report contributes to the current practice as it shows the clinical presentation and the diagnostic workout of this disease in pediatrics.     

The localization of a heterologous displayed antigen in the baculovirus-budded virion determines the type and strength of induced adaptive immune response

In the search of strategies of presentation of heterologous antigens to elicit humoral or cellular immune responses that modulate and properly potentiate each type of response, researchers have been studying baculovirus (BV) as vaccine vectors with promising results. For some years, several research groups explored different antigen presentation approaches using the BV AcNPV by expressing polypeptides on the surface of budded virions or by de novo synthesis of heterologous antigens by transduction of mammalian cells. In the case of expression on the surface of budded virions, for example, researchers have expressed polypeptides in peplomers as GP64 glycoprotein fusions or distributed throughout the entire surface by fusions to portions of the G protein of vesicular stomatitis virus, VSV. Recently, our group developed the strategy of cross-presentation of antigens by fusions of GP64 to the capsid protein VP39 (capsid display) for the generation of cytotoxic responses. While the different strategies showed to be effective in raising immune responses, the individuality of each analysis makes difficult the comparison of the results. Here, by comparing the different strategies, we show that localization of the model antigen ovalbumin (OVA) strongly determined the quality and intensity of the adaptive response to the heterologous antigen. Furthermore, surface display favored humoral responses, whereas capsid display favored cytotoxic responses. Finally, capsid display showed a much more efficient strategy to activate CD8-mediated responses than transduction. The incorporation of adjuvants in baculovirus formulations dramatically diminished the immunostimulatory properties of baculovirus.     

Macroinvertebrate community traits and nitrate removal in stream sediments

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Hypoxia enhances antibody‐dependent dengue virus infection

Dengue virus (DENV) has been found to replicate in lymphoid organs such as the lymph nodes, spleen, and liver in post‐mortem analysis. These organs are known to have low oxygen levels (~0.5–4.5% O₂) due to the vascular anatomy. However, how physiologically low levels of oxygen affect DENV infection via hypoxia‐induced changes in the immune response remains unknown. Here, we show that monocytes adapted to 3% O₂ show greater susceptibility to antibody‐dependent enhancement of DENV infection. Low oxygen level induces HIF1α‐dependent upregulation of fragment crystallizable gamma receptor IIA (FcγRIIA) as well as HIF1α‐independent alterations in membrane ether lipid concentrations. The increased FcγRIIA expression operates synergistically with altered membrane composition, possibly through increase membrane fluidity, to increase uptake of DENV immune complexes for enhanced infection. Our findings thus indicate that the increased viral burden associated with secondary DENV infection is antibody‐dependent but hypoxia‐induced and suggest a role for targeting hypoxia‐induced factors for anti‐dengue therapy.     

A recombinant iron transport protein from Bordetella pertussis confers protection against Bordetella parapertussis

Whooping cough, which is caused by Bordetella pertussis and B. parapertussis, is a reemerging disease. New protective antigens are needed to improve the efficacy of current vaccines against both species. Using proteomic tools, it was here found that B. parapertussis expresses a homolog of AfuA, a previously reported new vaccine candidate against B. pertussis . It was found that this homolog, named AfuA_Bpp, is expressed during B. parapertussis infection, exposed on the surface of the bacteria and recognized by specific antibodies induced by the recombinant AfuA cloned from B. pertussis (rAfuA). Importantly, the presence of the O‐antigen, a molecule that has been found to shield surface antigens on B. parapertussis , showed no influence on antibody recognition of AfuA_Bpp on the bacterial surface. The present study further showed that antibodies induced by immunization with the recombinant protein were able to opsonize B. parapertussis and promote bacterial uptake by neutrophils. Finally, it was shown that this antigen confers protection against B. parapertussis infection in a mouse model. Altogether, these results indicate that AfuA is a good vaccine candidate for acellular vaccines protective against both causative agents of whooping cough.