The molecular pathogenesis of renal cell carcinoma (RCC) is poorly understood. Whole-genome and exome sequencing followed by innovative tumorgraft analyses (to accurately determine mutant allele ratios) identified several putative two-hit tumor suppressor genes including BAP1. BAP1, a nuclear deubiquitinase, is inactivated in 15% of clear-cell RCCs. BAP1 cofractionates with and binds to HCF-1 in tumorgrafts. Mutations disrupting the HCF-1 binding motif impair BAP1-mediated suppression of cell proliferation, but not H2AK119ub1 deubiquitination. BAP1 loss sensitizes RCC cells in vitro to genotoxic stress. Interestingly, BAP1 and PBRM1 mutations anticorrelate in tumors (P=3×10−5), and combined loss of BAP1 and PBRM1 in a few RCCs was associated with rhabdoid features (q=0.0007). BAP1 and PBRM1 regulate seemingly different gene expression programs, and BAP1 loss was associated with high tumor grade (q=0.0005). Our results establish the foundation for an integrated pathological and molecular genetic classification of RCC, paving the way for subtype-specific treatments exploiting genetic vulnerabilities.

BackgroundPreterm birth is the second leading cause of death in children under the age of five years worldwide, but the etiology of many cases remains enigmatic. The dogma that the fetus resides in a sterile environment is being challenged by recent findings and the question has arisen whether microbes that colonize the fetus may be related to preterm birth. It has been posited that meconium reflects the in-utero microbial environment. In this study, correlations between fetal intestinal bacteria from meconium and gestational age were examined in order to suggest underlying mechanisms that may contribute to preterm birth.MethodsMeconium from 52 infants ranging in gestational age from 23 to 41 weeks was collected, the DNA extracted, and 16S rRNA analysis performed. Resulting taxa of microbes were correlated to clinical variables and also compared to previous studies of amniotic fluid and other human microbiome niches.FindingsIncreased detection of bacterial 16S rRNA in meconium of infants of <33 weeks gestational age was observed. Approximately 61·1% of reads sequenced were classified to genera that have been reported in amniotic fluid. Gestational age had the largest influence on microbial community structure (R = 0·161; p = 0·029), while mode of delivery (C-section versus vaginal delivery) had an effect as well (R = 0·100; p = 0·044). Enterobacter, Enterococcus, Lactobacillus, Photorhabdus, and Tannerella, were negatively correlated with gestational age and have been reported to incite inflammatory responses, suggesting a causative role in premature birth.InterpretationThis provides the first evidence to support the hypothesis that the fetal intestinal microbiome derived from swallowed amniotic fluid may be involved in the inflammatory response that leads to premature birth.

OBJECTIVETo evaluate the value of fasting plasma glucose (FPG) value in the first prenatal visit to diagnose gestational diabetes mellitus (GDM).RESEARCH DESIGN AND METHODSMedical records of 17,186 pregnant women attending prenatal clinics in 13 hospitals in China, including the Peking University First Hospital (PUFH), were examined. Patients with pre-GDM were excluded; data for FPG at the first prenatal visit and one-step GDM screening with 75-g oral glucose tolerance test (OGTT) performed between 24 and 28 weeks of gestation were collected and analyzed.RESULTSThe median ± SD FPG value was 4.58 ± 0.437. FPG decreased with increasing gestational age. FPG level at the first prenatal visit was strongly correlated with GDM diagnosed at 24–28 gestational weeks (χ2 = 959.3, P < 0.001). The incidences of GDM were 37.0, 52.7, and 66.2%, respectively, for women with FPG at the first prenatal visit between 5.10 and 5.59, 5.60 and 6.09, and 6.10–6.99 mmol/L. The data of PUFH were not statistically different from other hospitals.CONCLUSIONSPregnant women (6.10 ≤ FPG < 7.00 mmol/L) should be considered and treated as GDM to improve outcomes; for women with FPG between 5.10 and 6.09 mmol/L, nutrition and exercise advice should be provided. An OGTT should be performed at 24–28 weeks to confirm or rule out GDM. Based on our data, we cannot support an FPG value ≥5.10 mmol/L at the first prenatal visit as the criterion for diagnosis of GDM.

BACKGROUND & AIMS Identification of intestinal stem cells (ISCs) has relied heavily on the use of transgenic reporters in mice, but this approach is limited by mosaic expression patterns and difficult to directly apply to human tissues. We sought to identify reliable surface markers of ISCs and establish a robust functional assay to characterize ISCs from mouse and human tissues. METHODS We used immunohistochemistry, real-time reverse-transcription polymerase chain reaction, and fluorescence-activated cell sorting (FACS) to analyze intestinal epithelial cells isolated from mouse and human intestinal tissues. We compared different combinations of surface markers among ISCs isolated based on expression of Lgr5–green fluorescent protein. We developed a culture protocol to facilitate the identification of functional ISCs from mice and then tested the assay with human intestinal crypts and putative ISCs. RESULTS CD44+CD24loCD166+ cells, isolated by FACS from mouse small intestine and colon, expressed high levels of stem cell–associated genes. Transit-amplifying cells and progenitor cells were then excluded based on expression of GRP78 or c-Kit. CD44+CD24loCD166+ GRP78lo/− putative stem cells from mouse small intestine included Lgr5-GFPhi and Lgr5-GFPmed/lo cells. Incubation of these cells with the GSK inhibitor CHIR99021 and the E-cadherin stabilizer Thiazovivin resulted in colony formation by 25% to 30% of single-sorted ISCs. CONCLUSIONS We developed a culture protocol to identify putative ISCs from mouse and human tissues based on cell surface markers. CD44+CD24loCD166+, GRP78lo/−, and c-Kit− facilitated identification of putative stem cells from the mouse small intestine and colon, respectively. CD44+CD24−/loCD166+ also identified putative human ISCs. These findings will facilitate functional studies of mouse and human ISCs.

The intestinal crypt-niche interaction is thought to be essential to the function, maintenance, and proliferation of progenitor stem cells found at the bases of intestinal crypts. These stem cells are constantly renewing the intestinal epithelium by sending differentiated cells from the base of the crypts of Lieberkühn to the villus tips where they slough off into the intestinal lumen. The intestinal niche consists of various cell types, extracellular matrix, and growth factors and surrounds the intestinal progenitor cells. There have recently been advances in the understanding of the interactions that regulate the behavior of the intestinal epithelium and there is great interest in methods for isolating and expanding viable intestinal epithelium. However, there is no method to maintain primary human small intestinal epithelium in culture over a prolonged period of time. Similarly no method has been published that describes isolation and support of human intestinal epithelium in an in vivo model. We describe a technique to isolate and maintain human small intestinal epithelium in vitro from surgical specimens. We also describe a novel method to maintain human intestinal epithelium subcutaneously in a mouse model for a prolonged period of time. Our methods require various growth factors and the intimate interaction between intestinal sub-epithelial myofibroblasts (ISEMFs) and the intestinal epithelial cells to support the epithelial in vitro and in vivo growth. Absence of these myofibroblasts precluded successful maintenance of epithelial cell formation and proliferation beyond just a few days, even in the presence of supportive growth factors. We believe that the methods described here can be used to explore the molecular basis of human intestinal stem cell support, maintenance, and growth.