Allogeneic Stem Cells Alter Gene Expression and Improve Healing of Distal Limb Wounds in Horses.

Distal extremity wounds are a significant clinical problem in horses and humans and may benefit from mesenchymal stem cell (MSC) therapy. This study evaluated the effects of direct wound treatment with allogeneic stem cells, in terms of gross, histologic, and transcriptional features of healing. Three full-thickness cutaneous wounds were created on each distal forelimb in six healthy horses, for a total of six wounds per horse. Umbilical cord-blood derived equine MSCs were applied to each wound 1 day after wound creation, in one of four forms: (a) normoxic- or (b) hypoxic-preconditioned cells injected into wound margins, or (c) normoxic- or (d) hypoxic-preconditioned cells embedded in an autologous fibrin gel and applied topically to the wound bed. Controls were one blank (saline) injected wound and one blank fibrin gel-treated wound per horse. Data were collected weekly for 6 weeks and included wound surface area, thermography, gene expression, and histologic scoring. Results indicated that MSC treatment by either delivery method was safe and improved histologic outcomes and wound area. Hypoxic-preconditioning did not offer an advantage. MSC treatment by injection resulted in statistically significant increases in transforming growth factor beta and cyclooxygenase-2 expression at week 1. Histologically, significantly more MSC-treated wounds were categorized as pro-healing than pro-inflammatory. Wound area was significantly affected by treatment: MSC-injected wounds were consistently smaller than gel-treated or control wounds. In conclusion, MSC therapy shows promise for distal extremity wounds in horses, particularly when applied by direct injection into the wound margin. Stem Cells Translational Medicine 2018;7:98-108

Rapid Tumor Induction in Zebrafish by TALEN-Mediated Somatic Inactivation of the Retinoblastoma1 Tumor Suppressor rb1

Investigating the in vivo role of tumor suppressor genes in cancer is technically challenging due to their essential requirement during early animal development. To address this bottleneck, we generated genetic mosaic adult zebrafish using TALEN genome editing and demonstrate somatic inactivation of the tumor suppressor retinoblastoma1 (rb1) induces tumorigenesis at high frequency. 11–33% of 1-cell stage embryos injected with TALEN mRNAs targeting rb1 exon 2 or 3 develop tumors beginning as early as 3.5 months of age. Lesions predominantly arise in the brain and show features of neuroectodermal-like and glial-like tumors. Mutant allele analysis is consistent with tumor initiation due to somatic inactivation of rb1, revealing a conserved role for rb1 in tumor suppression across vertebrates. In contrast to genetic mosaics, heterozygous rb1−/+ adults show no evidence of neoplasia, while homozygous mutant rb1−/− are larval lethal. This is the first demonstration that somatic inactivation of a tumor suppressor causes cancer in zebrafish, and highlights the utility of site-specific nucleases to create genetic mosaic zebrafish for tumor suppressor gene discovery. Somatic inactivation with site-directed nucleases in zebrafish presents a rapid and scalable strategy to study tumor suppressor gene function in cancer

The Effects of Atmospheric Dispersion on High-Resolution Solar Spectroscopy

We investigate the effects of atmospheric dispersion on observations of the Sun at the ever-higher spatial resolutions afforded by increased apertures and improved techniques. The problems induced by atmospheric refraction are particularly significant for solar physics because the Sun is often best observed at low elevations, and the effect of the image displacement is not merely a loss of efficiency, but the mixing of information originating from different points on the solar surface. We calculate the magnitude of the atmospheric dispersion for the Sun during the year and examine the problems produced by this dispersion in both spectrographic and filter observations. We describe an observing technique for scanning spectrograph observations that minimizes the effects of the atmospheric dispersion while maintaining a regular scanning geometry. Such an approach could be useful for the new class of high-resolution solar spectrographs, such as SPINOR, POLIS, TRIPPEL, and ViSP

Novel selective β1-adrenoceptor antagonists for concomitant cardiovascular and respiratory disease

β-Blockers reduce mortality and improve symptoms in people with heart disease. However, current clinically available β-blockers have poor selectivity for the cardiac β1-adrenoceptor (AR) over the lung β2-AR. Unwanted β2-blockade risks causing life-threatening bronchospasm and a reduction in the efficacy of β2-agonist emergency rescue therapy. Thus current life-prolonging β-blockers are contraindicated in people with both heart disease and asthma. Here we describe NDD-713 and NDD-825, novel highly β1-selective neutral antagonists with good pharmaceutical properties that can potentially overcome this limitation. Radioligand binding studies and functional assays using human receptors expressed in CHO cells demonstrate that NDD-713 and NDD-825 have nanomolar β1-AR affinity, greater than 500-fold β1-AR vs β2-AR selectivity and no agonism. Studies in conscious rats demonstrated that they are orally bioavailable and cause pronounced β1-mediated reduction of heart rate while showing no effect on β2-mediated hindquarters vasodilatation. The compounds also have good disposition properties and show no adverse toxicological effects. They potentially offer a truly cardioselective β-blocker therapy for the large number of people with heart and respiratory, or peripheral vascular comorbidities

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

BACKGROUND:Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival. METHODOLOGY AND PRINCIPAL FINDINGS:Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease. CONCLUSIONS:S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers

R132H IDH1 sensitizes glioma to the antiproliferative and cytotoxic effects of BET inhibition

IntroductionMutations in isocitrate dehydrogenase 1/2 (IDHmut) identify a subset of gliomas that exhibit epigenetic dysregulation via aberrant DNA methylation. These tumors are ultimately fatal and lack effective therapeutic strategies. Considering the epigenetic dysregulation of IDHmut gliomas, we hypothesized that epigenetic-targeting drugs may yield therapeutic benefits in gliomas bearing IDHmut. One set of targets includes the bromodomain and extraterminal (BET) family of transcriptional coactivators.MethodsWe used TCGA data from glioma patients to determine whether BET proteins affect patient survival differently based on IDH status. Follow-up experiments using a set of IDH wildtype/mutant glioma cultures, as well as an IDH wildtype glioblastoma cell line expressing exogenous R132H IDH1, focused on cell health assays to investigate whether IDHmut was associated with increased sensitivity to the BET inhibitor JQ1. Immunoblots were used to evaluate the molecular response to JQ1 in these cultures.ResultsWe identified that high BRD4 expression associated with decreased survival only in IDHmut glioma patients. Cell viability analysis showed that IDHmut sensitized glioma cells to delayed cytotoxicity (10 days) in response to JQ1. Early effects of JQ1 (3 days) were primarily antiproliferative, with IDHmut glioma exhibiting a modest increase in sensitivity. Finally, exogenous R132H IDH1 expression in a resistant IDH wildtype cell line recapitulated the JQ1-mediated delayed cytotoxicity seen in our endogenous IDHmut glioma cells.ConclusionOverall, these data suggest that BRD4 enhances malignancy primarily in gliomas bearing IDHmut and is associated with greater sensitivity to BET inhibition. The finding that BET inhibition primarily exhibits delayed cytotoxicity may be overlooked in conventional short endpoint dose-response assays. Follow-up mechanistic and animal studies will help address the translational potential of these findings

IDH1 mutant glioma is preferentially sensitive to the HDAC inhibitor panobinostat

IntroductionA large subset of diffusely infiltrative gliomas contains a gain-of-function mutation in isocitrate dehydrogenase 1 or 2 (IDH1/2mut) which produces 2-hydroxglutarate, an inhibitor of α-ketoglutarate-dependent DNA demethylases, thereby inducing widespread DNA and histone methylation. Because histone deacetylase (HDAC) enzymes are localized to methylated chromatin via methyl-binding domain proteins, IDH1/2mut gliomas may be more dependent on HDAC activity, and therefore may be more sensitive to HDAC inhibitors.MethodsSix cultured patient-derived glioma cell lines, IDH1wt (n = 3) and IDH1mut (n = 3), were treated with an FDA-approved HDAC inhibitor, panobinostat. Cellular cytotoxicity and proliferation assays were conducted by flow cytometry. Histone modifications and cell signaling pathways were assessed using immunoblot and/or ELISA.ResultsIDH1mut gliomas exhibited marked upregulation of genes associated with the HDAC activity. Glioma cell cultures bearing IDH1mut were significantly more sensitive to the cytotoxic and antiproliferative effects of panobinostat, compared to IDH1wt glioma cells. Panobinostat caused a greater increase in acetylation of the histone residues H3K14, H3K18, and H3K27 in IDH1mut glioma cells. Another HDAC inhibitor, valproic acid, was also more effective against IDH1mut glioma cells.ConclusionThese data suggest that IDH1mut gliomas may be preferentially sensitive to HDAC inhibitors. Further, IDH1mut glioma cultures showed enhanced accumulation of acetylated histone residues in response to panobinostat treatment, suggesting a direct epigenetic mechanism for this sensitivity. This provides a rationale for further exploration of HDAC inhibitors against IDH1mut gliomas

Fatal balamuthosis in a Siberian tiger and a literature review of detection options for free-living amoebic infections in animals

Free-living amoebae are rare causes of morbidity and mortality in humans and animals around the globe. Because the route of exposure and clinical progression of disease caused by different species of amoebae may vary in people and animals, determining the species of amoeba present is important. We describe here a fatal infection by the free-living amoeba Balamuthia mandrillaris in a Siberian tiger (Panthera tigris altaica). The 17-y-old patient had a rapid clinical decline after a peracute onset of severe lethargy, dull mentation, and anorexia. Autopsy did not identify a cause of death. Histology revealed inflammation associated with amoebic trophozoites in the brain, lungs, and iris of one eye. These amoebae were confirmed to be B. mandrillaris based on a PCR assay and sequencing. Although there are subtle morphologic differences between cyst stages of Acanthamoeba spp., B. mandrillaris, and Naegleria fowleri when present and identified on routine staining, other modalities, including PCR, immunofluorescence, electron microscopy, and immunohistochemistry, are typically utilized to confirm the pathogen involved in these cases. We review the reports of balamuthosis in animals