The deubiquitinating enzyme BAP1 is a tumor suppressor, among others involved in cholangiocarcinoma. BAP1 has many proposed molecular targets, while its Drosophila homolog is known to deubiquitinate histone H2AK119. We introduce BAP1 loss-of-function by CRISPR/Cas9 in normal human cholangiocyte organoids. We find that BAP1 controls the expression of junctional and cytoskeleton components by regulating chromatin accessibility. Consequently, we observe loss of multiple epithelial characteristics while motility increases. Importantly, restoring the catalytic activity of BAP1 in the nucleus rescues these cellular and molecular changes. We engineer human liver organoids to combine four common cholangiocarcinoma mutations (TP53, PTEN, SMAD4, and NF1). In this genetic background, BAP1 loss results in acquisition of malignant features upon xenotransplantation. Thus, control of epithelial identity through the regulation of chromatin accessibility appears to be a key aspect of BAP1's tumor suppressor function. Organoid technology combined with CRISPR/Cas9 provides an experimental platform for mechanistic studies of cancer gene function in a human context.

Hypobaric hypoxia (HH), an environmental condition of high altitude encountered by mountaineers, miners, and observatory, rural health, border patrol, and rural education workers, jeopardizes normal physiologic functions in humans. The present study was conducted to evaluate the effects of intermittent HH (IHH; equivalent to 4600 m above mean sea level) on oxidative stress and the protective role of dietary ascorbic acid on rat testis and epididymis. Ten-week-old male Wistar rats were assigned to 1 of 6 groups: 1) normobaric (Nx), 2) Nx + physiologic solution (Nx + PS), 3) Nx + ascorbic acid (Nx + AA), 4) IHH, 5) IHH + PS, or 6) IHH + AA. Animals subjected to IHH were exposed for 96 hours followed by normobaric conditions for 96 hours for a total of 32 days. The control groups (2 and 5) were injected with doses of PS, and the treated groups (3 and 6) were injected with doses of AA (10 mg 6kg 21 body weight) at an interval of 96 hours. Rats were sacrificed on day 32 after initiation of the protocol. The testis and epididymis were collected to determine the activity and expression of glutathione reductase and the levels of lipid peroxide formation. An epididymal sperm count was also performed in each animal. The results of this study revealed that IHH induced lipid peroxidation, a reduction in glutathione reductase activity in testis and epididymis, and a significant decrease in epididymal sperm count. Treatment with AA prevented these changes. In conclusion, AA was capable of decreasing oxidative stress in testis and epididymis under IHH. This protection by AA of the IHH-induced lipid peroxidation can be explained in part by the preservation of glutathione reductase activity in these organs.

IntroductionAlthough the prognostic value of persistent hyperlactatemia in septic shock is unequivocal, its physiological determinants are controversial. Particularly, the role of impaired hepatic clearance has been underestimated and is only considered relevant in patients with liver ischemia or cirrhosis. Our objectives were to establish whether endotoxemia impairs whole body net lactate clearance, and to explore a potential role for total liver hypoperfusion during the early phase of septic shock.MethodsAfter anesthesia, 12 sheep were subjected to hemodynamic/perfusion monitoring including hepatic and portal catheterization, and a hepatic ultrasound flow probe. After stabilization (point A), sheep were alternatively assigned to lipopolysaccharide (LPS) (5 mcg/kg bolus followed by 4 mcg/kg/h) or sham for a three-hour study period. After 60 minutes of shock, animals were fluid resuscitated to normalize mean arterial pressure. Repeated series of measurements were performed immediately after fluid resuscitation (point B), and one (point C) and two hours later (point D). Monitoring included systemic and regional hemodynamics, blood gases and lactate measurements, and ex-vivo hepatic mitochondrial respiration at point D. Parallel exogenous lactate and sorbitol clearances were performed at points B and D. Both groups included an intravenous bolus followed by serial blood sampling to draw a curve using the least squares method.ResultsSignificant hyperlactatemia was already present in LPS as compared to sham animals at point B (4.7 (3.1 to 6.7) versus 1.8 (1.5 to 3.7) mmol/L), increasing to 10.2 (7.8 to 12.3) mmol/L at point D. A significant increase in portal and hepatic lactate levels in LPS animals was also observed. No within-group difference in hepatic DO2, VO2 or O2 extraction, total hepatic blood flow (point D: 915 (773 to 1,046) versus 655 (593 to 1,175) ml/min), mitochondrial respiration, liver enzymes or sorbitol clearance was found. However, there was a highly significant decrease in lactate clearance in LPS animals (point B: 46 (30 to 180) versus 1,212 (743 to 2,116) ml/min, P <0.01; point D: 113 (65 to 322) versus 944 (363 to 1,235) ml/min, P <0.01).ConclusionsEndotoxemia induces an early and severe impairment in lactate clearance that is not related to total liver hypoperfusion.Electronic supplementary materialThe online version of this article (doi:10.1186/s13054-015-0928-3) contains supplementary material, which is available to authorized users.

Abstract. Chronic hypobaric hypoxia (CHH) induces a decrease in sperm output and spermatogenesis in male rats. The mechanisms that underlie these changes in testicular function are unknown and could involve changes in the hypophysis-gonad axis. We have tested the hypothesis that changes take place in the endocrine status (FSH, follicle stimulating hormone; LH, luteinizing hormone; testosterone) of rats subjected to CHH. Male Wistar rats were maintained under normobaric or hypobaric conditions (428 torr, 4,600 m). On days 0, 5, 15 and 30 post-exposure, 12 rats were anesthetized, their body weights were measured and blood samples were collected. The testicles were fixed in 4% formaldehyde and processed for histological analysis. In this time course, the FSH levels rose by day 5 post-exposure. On subsequent days, the FSH levels decreased in rats subjected to CHH with a tendency to remain higher than the normoxic group. The LH plasma levels decreased in rats exposed to CHH. Consistent with the decrease in LH levels, the plasma testosterone level decreased significantly after 30 days of CHH exposure. Integrated analysis of hormonal changes in rats subjected to CHH and the body dehydration that occurs in HH allows us to conclude that the effects of CHH on spermatogenesis may be partially related to changes in the hypophysis-gonad hormonal axis. Key words: Fertility, High altitude, Hypobaric hypoxia, Testicular function, Testis (J. Reprod. Dev. 54: [18][19][20][21] 2008) hronic hypobaric hypoxia (CHH) is experienced by an increasing number of sea level natives exposed to high altitude because of tourism, border patrol, mining or rural health and education activities [1]. It has been suggested that hypobaric hypoxia reduces fertility in humans. Nonetheless, epidemiological studies of high and low altitude populations have not been able to verify this proposal [2]. Based on the hormonal changes observed in men and rats at high altitude, some authors have proposed that CHH affects the hypothalamic/gonad axis (e.g. [3][4][5]). However, the results in the literature have been controversial. Nelson et al. reported that the plasma levels of GH, LH, FSH and TSH of hypoxic rats did not differ from the control values [3]. These results are in agreement with the reported absence of changes in the testosterone levels of rats exposed to hypoxia compared with sea level controls published by Gonzales et al. [5]. Instead, Sawhaney et al. [4] reported a decrease in the LH and testosterone levels in men exposed to CHH. Exposure of male rats to chronic hypobaric hypoxia and intermittent hypobaric hypoxia induced evident changes in testicular morphology [6][7][8][9], strong metabolic stress and loss of spermatogenic cells [8]. Local changes observed in testicles exposed to hypoxia include neovascularization and an increase in temperature. Thus, local changes that could lead to the observed effects on spermatogenesis do occur. However, it is highly likely that these local changes are also accompanied by changes in the hypophysis-gon...

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Abstract. The presence of cells positive for cytokeratins or prostate-specific antigen (PSA) in bone marrow aspirates (BMAs) has been used to indicate the presence of micrometastasis. The aim of this prospective study of prostate cancer patients was to determine the presence of prostate cells in blood and BMAs and to compare them with bone marrow biopsy touch prep samples. The results indicated that there was a satisfactory concordance between circulating prostate cells (CPCs) in blood and disseminated tumor cells (DTCs) in BMAs for all Gleason scores (κ>0.50). However, neither were concordant with the presence of prostate cells in bone marrow biopsies except for high-grade tumors, Gleason 8 and 9. Phenotypic characteristics of CPCs and DTCs were identical (κ>0.9) but were different than cells detected in bone marrow biopsies (κ<0.2). The expression of matrix metalloproteinase-2 (MMP-2) in bone marrow biopsies was positively associated with the Gleason score (trend Chi-squared <0.05) and may explain the differences between the presence of DTCs and the presence of prostate cells in bone marrow biopsies. If the presence of DTCs was used to indicate micrometastatic disease, 20% of patients would be misclassified compared to micrometastasis defined as patients with a positive biopsy. This may have clinical implications for patients with low-grade tumors.

HIGHLIGHTS c-Abl is a TFEB regulator that mediates its tyr phosphorylation c-Abl inhibition promotes TFEB activity independently of mTORC1 c-Abl inhibition reduces cholesterol accumulation in NPC1 models