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Background & Aims: Primary biliary cholangitis (PBC) is a chronic cholangiopathy characterised by immuno-mediated injury of interlobular bile ducts leading to intrahepatic cholestasis and progressive liver fibrosis. PBC histology is characterised by portal inflammation, progressive fibrosis, ductopenia, and the appearance of the so-called ductular reaction. The aim of the present study was to investigate the pathogenetic relevance of ductular reaction in PBC. Methods: Liver biopsies were collected from naïve people with PBC (N = 87). Clinical–serological parameters were obtained at diagnosis and after 1 year of ursodeoxycholic acid (UDCA) treatment. Histological staging was performed on all slides according to multiple scoring systems and criteria for PBC. Liver samples were obtained from Mdr2−/− mice treated with or without UDCA. Samples were processed for histology, immunohistochemistry, and immunofluorescence. Results: Ductular reaction in people with PBC correlated with the disease stage and liver fibrosis, but not with disease activity; an extensive ductular reaction correlated with serum alkaline phosphatase levels at diagnosis, response to UDCA, and individuals’ estimated survival, independently from other histological parameters, including disease stage. In people with PBC, reactive ductules were associated with the establishment of junctions with bile canaliculi and with fibrogenetic cell activation. Consistently, in a mouse model of intrahepatic cholestasis, UDCA treatment was effective in reducing ductular reaction and fibrosis and increasing ductular–canalicular junctions. Conclusions: Extensive ductular reaction outlines a severe histologic phenotype in PBC and is associated with an inadequate therapy response and a worse estimated prognosis. Lay summary: In people affected by primary biliary cholangitis (PBC), the histological appearance of extensive ductular reaction identifies individuals at risk of progressive fibrosis. Ductular reaction at dia

Background and Aims: Serotonin (5HT) is a neuroendocrine hormone synthetized in the central nervous system (CNS) as well as enterochromaffin cells of the gastrointestinal tract. Tryptophan hydroxylase (TPH1) and monoamine oxidase (MAO-A) are the key enzymes for the synthesis and catabolism of 5HT, respectively. Previous studies demonstrated that 5-hydroxytryptamine receptor (5HTR)1A/1B receptor agonists inhibit biliary hyperplasia in bile-duct ligated (BDL) rats, whereas 5HTR2B receptor antagonists attenuate liver fibrosis (LF) in mice. Our aim was to evaluate the role of 5HTR2A/2B/2C agonists/antagonists in cholestatic models. Approach and Results: While in vivo studies were performed in BDL rats and the multidrug resistance gene 2 knockout (Mdr2–/–) mouse model of PSC, in vitro studies were performed in cell lines of cholangiocytes and hepatic stellate cells (HSCs). 5HTR2A/2B/2C and MAO-A/TPH1 are expressed in cholangiocytes and HSCs from BDL rats and Mdr2–/–- mice. Ductular reaction, LF, as well as the mRNA expression of proinflammatory genes increased in normal, BDL rats, and Mdr2–/–- mice following treatment 5HTR2A/2B/2C agonists, but decreased when BDL rats and Mdr2–/– mice were treated with 5HTR2A/2B/2C antagonists compared to BDL rats and Mdr2–/– mice, respectively. 5HT levels increase in Mdr2–/– mice and in PSC human patients compared to their controls and decrease in serum of Mdr2–/– mice treated with 5HTR2A/2B/2C antagonists compared to untreated Mdr2–/– mice. In vitro, cell lines of murine cholangiocytes and human HSCs express 5HTR2A/2B/2C and MAO-A/TPH1; treatment of these cell lines with 5HTR2A/2B/2C antagonists or TPH1 inhibitor decreased 5HT levels as well as expression of fibrosis and inflammation genes compared to controls. Conclusions: Modulation of the TPH1/MAO-A/5HT/5HTR2A/2B/2C axis may represent a therapeutic approach for management of cholangiopathies, including PSC.

Melatonin, a neuroendocrine hormone synthesized by the pineal gland and cholangiocytes, decreases biliary hyperplasia and liver fibrosis during cholestasis-induced biliary injury via melatonin-dependent autocrine signaling through increased biliary arylalkylamine N-acetyltransferase (AANAT) expression and melatonin secretion, downregulation of miR-200b and specific circadian clock genes. Melatonin synthesis is decreased by pinealectomy (PINX) or chronic exposure to light. We evaluated the effect of PINX or prolonged light exposure on melatonin-dependent modulation of biliary damage/ductular reaction/liver fibrosis. Studies were performed in male rats with/without BDL for 1 week with 12:12 h dark/light cycles, continuous light or after 1 week of PINX. The expression of AANAT and melatonin levels in serum and cholangiocyte supernatant were increased in BDL rats, while decreased in BDL rats following PINX or continuous light exposure. BDL-induced increase in serum chemistry, ductular reaction, liver fibrosis, inflammation, angiogenesis and ROS generation were significantly enhanced by PINX or light exposure. Concomitant with enhanced liver fibrosis, we observed increased biliary senescence and enhanced clock genes and miR-200b expression in total liver and cholangiocytes. In vitro, the expression of AANAT, clock genes and miR-200b was increased in PSC human cholangiocyte cell lines (hPSCL). The proliferation and activation of HHStecs (human hepatic stellate cell lines) were increased after stimulating with BDL cholangiocyte supernatant and further enhanced when stimulated with BDL rats following PINX or continuous light exposure cholangiocyte supernatant via intracellular ROS generation. Conclusion: Melatonin plays an important role in the protection of liver against cholestasis-induced damage and ductular reaction.

We have investigated trifluoroacetic acid (TFA) formation from emissions of HFO-1234yf (CF3CFH2), its dry and wet deposition, and rainwater concentration over India, China, and the Middle East with GEOS-Chem and WRF-Chem models. We estimated the TFA deposition and rainwater concentrations between 2020 and 2040 for four previously published HFO-1234yf emission scenarios to bound the possible levels of TFA. We evaluated the capability of GEOS-Chem to capture the wet deposition process by comparing calculated sulfate in rainwater with observations. Our calculated TFA amounts over the USA, Europe, and China were comparable to those previously reported when normalized to the same emission. A significant proportion of TFA was found to be deposited outside the emission regions. The mean and the extremes of TFA rainwater concentrations calculated for the four emission scenarios from GEOS-Chem and WRF-Chem were orders of magnitude below the no observable effect concentration. The ecological and human health impacts now, and the continued use of HFO-1234yf in India, China, and the Middle East, are estimated to be insignificant based on the current understanding, as summarized by Neale et al. (2021).

We have investigated trifluoroacetic acid (TFA) formation from emissions of HFO-1234yf (CF3CFH2), its dry and wet deposition, and rainwater concentration over India, China, and the Middle East with GEOS-Chem and WRF-Chem models. We estimated the TFA deposition and rainwater concentrations between 2020 and 2040 for four previously published HFO-1234yf emission scenarios to bound the possible levels of TFA. We evaluated the capability of GEOS-Chem to capture the wet deposition process by comparing calculated sulfate in rainwater with observations. Our calculated TFA amounts over the USA, Europe, and China were comparable to those previously reported when normalized to the same emission. A significant proportion of TFA was found to be deposited outside the emission regions. The mean and the extremes of TFA rainwater concentrations calculated for the four emission scenarios from GEOS-Chem and WRF-Chem were orders of magnitude below the no observable effect concentration. The ecological and human health impacts now, and the continued use of HFO-1234yf in India, China, and the Middle East, are estimated to be insignificant based on the current understanding, as summarized by Neale et al. (2021).

We have investigated trifluoroacetic acid (TFA) formation from emissions of HFO-1234yf (CF3CFH2), its dry and wet deposition, and rainwater concentration over India, China, and the Middle East with GEOS-Chem and WRF-Chem models. We estimated the TFA deposition and rainwater concentrations between 2020 and 2040 for four previously published HFO-1234yf emission scenarios to bound the possible levels of TFA. We evaluated the capability of GEOS-Chem to capture the wet deposition process by comparing calculated sulfate in rainwater with observations. Our calculated TFA amounts over the USA, Europe, and China were comparable to those previously reported when normalized to the same emission. A significant proportion of TFA was found to be deposited outside the emission regions. The mean and the extremes of TFA rainwater concentrations calculated for the four emission scenarios from GEOS-Chem and WRF-Chem were orders of magnitude below the no observable effect concentration. The ecological and human health impacts now, and the continued use of HFO-1234yf in India, China, and the Middle East, are estimated to be insignificant based on the current understanding, as summarized by Neale et al. (2021).

Multi-year droughts in Mediterranean climates may shift the water balance, that is, the partitioning rule of precipitation across runoff, evapotranspiration, and subsurface storage. Mechanisms causing these shifts remain largely unknown and are not well represented in hydrologic models. Focusing on measurements from the headwaters of California s Feather River, we found that also in these mixed rain snow Mediterranean basins a lower fraction of precipitation was partitioned to runoff during multi-year droughts compared to non-drought years. This shift in the precipitation runoff relationship was larger in the surfacerunoff-dominated than subsurface-flow-dominated headwaters (39% vs. 18% decline of runoff, respectively, for a representative precipitation amount). The predictive skill of the Precipitation Runoff Modeling System (PRMS) hydrologic model in these basins decreased during droughts, with evapotranspiration (ET) being the only water-balance component besides runoff for which the drop in predictive skill during drought vs. non-drought years was statistically significant. In particular, the model underestimated the response time required by ET to adjust to interannual climate variability, which we define as climate elasticity of ET. Differences between simulated and data-driven estimates of ET were well correlated with accompanying data-driven estimates of changes in sub-surface storage (1S, r D 0:78). This correlation points to shifts in precipitation runoff relationships being evidence of a hysteretic response of the water budget to climate elasticity of ET during and after multi-year droughts. This hysteresis is caused by carryover storage offsetting precipitation deficit during the initial drought period, followed by vegetation mortality when storage is depleted and subsequent post-drought vegetation expansion. Our results point to a general improvement in hydrologic predictions across drought and recovery cycles by including the climate elasticity of ET a

Multi-year droughts in Mediterranean climates may shift the water balance, that is, the partitioning rule of precipitation across runoff, evapotranspiration, and subsurface storage. Mechanisms causing these shifts remain largely unknown and are not well represented in hydrologic models. Focusing on measurements from the headwaters of California s Feather River, we found that also in these mixed rain snow Mediterranean basins a lower fraction of precipitation was partitioned to runoff during multi-year droughts compared to non-drought years. This shift in the precipitation runoff relationship was larger in the surfacerunoff-dominated than subsurface-flow-dominated headwaters (39% vs. 18% decline of runoff, respectively, for a representative precipitation amount). The predictive skill of the Precipitation Runoff Modeling System (PRMS) hydrologic model in these basins decreased during droughts, with evapotranspiration (ET) being the only water-balance component besides runoff for which the drop in predictive skill during drought vs. non-drought years was statistically significant. In particular, the model underestimated the response time required by ET to adjust to interannual climate variability, which we define as climate elasticity of ET. Differences between simulated and data-driven estimates of ET were well correlated with accompanying data-driven estimates of changes in sub-surface storage (1S, r D 0:78). This correlation points to shifts in precipitation runoff relationships being evidence of a hysteretic response of the water budget to climate elasticity of ET during and after multi-year droughts. This hysteresis is caused by carryover storage offsetting precipitation deficit during the initial drought period, followed by vegetation mortality when storage is depleted and subsequent post-drought vegetation expansion. Our results point to a general improvement in hydrologic predictions across drought and recovery cycles by including the climate elasticity of ET a

Hepatic fibrosis occurs during the progression of primary sclerosing cholangitis (PSC) and is characterized by accumulation of extracellular matrix proteins. Proliferating cholangiocytes and activated hepatic stellate cells (HSCs) participate in the promotion of liver fibrosis during cholestasis. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone synthesized by hypothalamic neurons and the biliary epithelium and exerts its biological effects on cholangiocytes by interaction with the receptor subtype (GnRHR1) expressed by cholangiocytes and HSCs. Previously, we demonstrated that administration of GnRH to normal rats increased intrahepatic biliary mass (IBDM) and hepatic fibrosis. Also, miR-200b is associated with the progression of hepatic fibrosis; however, the role of the GnRH/GnRHR1/miR-200b axis in the development of hepatic fibrosis in PSC is unknown. Herein, using the mouse model of PSC (multidrug resistance gene 2 knockout), the hepatic knockdown of GnRH decreased IBDM and liver fibrosis. In vivo and in vitro administration of GnRH increased the expression of miR-200b and fibrosis markers. The GnRH/GnRHR1 axis and miR-200b were up-regulated in human PSC samples. Cetrorelix, a GnRHR1 antagonist, inhibited the expression of fibrotic genes in vitro and decreased IBDM and hepatic fibrosis in vivo. Inhibition of miR-200b decreased the expression of fibrosis genes in vitro in cholangiocyte and HSC lines. Targeting the GnRH/GnRHR1/miR-200b axis may be key for the management of hepatic fibrosis during the progression of PSC.

Hepatic fibrosis occurs during the progression of primary sclerosing cholangitis (PSC) and is characterized by accumulation of extracellular matrix proteins. Proliferating cholangiocytes and activated hepatic stellate cells (HSCs) participate in the promotion of liver fibrosis during cholestasis. Gonadotropin-releasing hormone (GnRH) is a trophic peptide hormone synthesized by hypothalamic neurons and the biliary epithelium and exerts its biological effects on cholangiocytes by interaction with the receptor subtype (GnRHR1) expressed by cholangiocytes and HSCs. Previously, we demonstrated that administration of GnRH to normal rats increased intrahepatic biliary mass (IBDM) and hepatic fibrosis. Also, miR-200b is associated with the progression of hepatic fibrosis; however, the role of the GnRH/GnRHR1/miR-200b axis in the development of hepatic fibrosis in PSC is unknown. Herein, using the mouse model of PSC (multidrug resistance gene 2 knockout), the hepatic knockdown of GnRH decreased IBDM and liver fibrosis. In vivo and in vitro administration of GnRH increased the expression of miR-200b and fibrosis markers. The GnRH/GnRHR1 axis and miR-200b were up-regulated in human PSC samples. Cetrorelix, a GnRHR1 antagonist, inhibited the expression of fibrotic genes in vitro and decreased IBDM and hepatic fibrosis in vivo. Inhibition of miR-200b decreased the expression of fibrosis genes in vitro in cholangiocyte and HSC lines. Targeting the GnRH/GnRHR1/miR-200b axis may be key for the management of hepatic fibrosis during the progression of PSC.

Fibroblast growth factor receptor 2 (FGFR2) might have an important role in the pathogenesis and biology of cholangiocarcinoma (CCA). We examined FGFR expression in CCA tumor specimens obtained from patients and CCA cell lines, and then determined the effects of the novel FGFR inhibitor, derazantinib (DZB; formally, ARQ 087), which is currently in clinical phase 2 trials for intrahepatic CCA. DZB inhibited the growth of CCA cell lines in a dose-dependent manner, and extracellular signal-regulated kinase 1/2 and AKT. It also activated apoptotic and cell growth arrest signaling. DZB reduced the in vitro invasiveness and the expression of key epithelial-mesenchymal transition genes. The in vitro data correlated with the expression of FGFRs in human CCA specimens by immunohistochemistry (FGFR1, 30% positive; and FGFR2, 65% positive) and the CCA cell lines assayed by Western blot analysis. These correlated in vitro studies suggest that FGFR may play an important role in the pathogenesis and biology of CCA. Our findings support the notion that FGFR inhibitors, like DZB, should be further evaluated at the clinical stage as targeted therapy for CCA treatment.

Detecting underground voids, such as old mine workings, solution cavities in karst terrain, or unknown tunnels such as illicit cross-border tunnels, is a challenging problem for geophysics and an important concern for geotechnical design, public safety, and domestic security. Seismic surface-wave-based detection methods have become increasingly popular for detecting relatively shallow and small targets; however, the theoretical limitations of these methods have thus far remained unclear. We use a suite of 3D numerical simulations inspired by a tunnel detection experiment carried out at the Black Diamond Mines Regional Preserve (BDM) in northern California. The geophysical anomalies predicted by our numerical simulations at BDM agree with field observations, and our estimates for the location of the primary tunnel target agree with historical records in the area. Using our calibrated numerical model, we perform a parametric study to determine the effect of tunnel size, depth of burial, filling material, and source characteristics to determine the range over which surface-wave backscattering and attenuation-based methods are effective. In addition, we perform a regression analysis to determine a relationship for the maximum depth at which a tunnel may be detected via these approaches, given the target diameter, wavelength of interest, and the signal-to-noise ratio.

We strategically placed spatially distributed sensors to provide representative measures of changes in snowpack and subsurface water storage, plus the fluxes affecting these stores, in a set of nested headwater catchments. The high temporal frequency and distributed coverage make the resulting data appropriate for process studies of snow accumulation and melt, infiltration, evapotranspiration, catchment water balance, (bio)geochemistry, and other critical-zone processes. We present 8 years of hourly snow-depth, soil-moisture, and soil-temperature data, as well as 14 years of quarter-hourly streamflow and meteorological data that detail water-balance processes at Providence Creek, the upper part of which is at the current 50 % rain versus snow transition of the southern Sierra Nevada, California. Providence Creek is the long-term study cooperatively run by the Southern Sierra Critical Zone Observatory (SSCZO) and the USDA Forest Service Pacific Southwest Research Station's Kings River Experimental Watersheds (KREW). The 4.6 km2 montane Providence Creek catchment spans the current lower rain–snow transition elevation of 1500–2100 m. Two meteorological stations bracket the high and low elevations of the catchment, measuring air temperature, relative humidity, solar radiation, precipitation, wind speed and direction, and snow depth, and at the higher station, snow water equivalent. Paired flumes at three subcatchments and a V-notch weir at the integrating catchment measure quarter-hourly streamflow. Measurements of meteorological and streamflow data began in 2002. Between 2008 and 2010, 50 sensor nodes were added to measure distributed snow depth, air temperature, soil temperature, and soil moisture within the top 1 m below the surface. These sensor nodes were installed to capture the lateral differences of aspect and canopy coverage. Data are available at hourly and daily intervals by water year (1 October–30 September) in nonproprietary formats from online data reposi

We strategically placed spatially distributed sensors to provide representative measures of changes in snowpack and subsurface water storage, plus the fluxes affecting these stores, in a set of nested headwater catchments. The high temporal frequency and distributed coverage make the resulting data appropriate for process studies of snow accumulation and melt, infiltration, evapotranspiration, catchment water balance, (bio)geochemistry, and other critical-zone processes. We present 8 years of hourly snow-depth, soil-moisture, and soil-temperature data, as well as 14 years of quarter-hourly streamflow and meteorological data that detail water-balance processes at Providence Creek, the upper part of which is at the current 50 % rain versus snow transition of the southern Sierra Nevada, California. Providence Creek is the long-term study cooperatively run by the Southern Sierra Critical Zone Observatory (SSCZO) and the USDA Forest Service Pacific Southwest Research Station's Kings River Experimental Watersheds (KREW). The 4.6 km2 montane Providence Creek catchment spans the current lower rain–snow transition elevation of 1500–2100 m. Two meteorological stations bracket the high and low elevations of the catchment, measuring air temperature, relative humidity, solar radiation, precipitation, wind speed and direction, and snow depth, and at the higher station, snow water equivalent. Paired flumes at three subcatchments and a V-notch weir at the integrating catchment measure quarter-hourly streamflow. Measurements of meteorological and streamflow data began in 2002. Between 2008 and 2010, 50 sensor nodes were added to measure distributed snow depth, air temperature, soil temperature, and soil moisture within the top 1 m below the surface. These sensor nodes were installed to capture the lateral differences of aspect and canopy coverage. Data are available at hourly and daily intervals by water year (1 October–30 September) in nonproprietary formats from online data reposi

We strategically placed spatially distributed sensors to provide representative measures of changes in snowpack and subsurface water storage, plus the fluxes affecting these stores, in a set of nested headwater catchments. The high temporal frequency and distributed coverage make the resulting data appropriate for process studies of snow accumulation and melt, infiltration, evapotranspiration, catchment water balance, (bio)geochemistry, and other critical-zone processes. We present 8 years of hourly snow-depth, soil-moisture, and soil-temperature data, as well as 14 years of quarter-hourly streamflow and meteorological data that detail water-balance processes at Providence Creek, the upper part of which is at the current 50 % rain versus snow transition of the southern Sierra Nevada, California. Providence Creek is the long-term study cooperatively run by the Southern Sierra Critical Zone Observatory (SSCZO) and the USDA Forest Service Pacific Southwest Research Station's Kings River Experimental Watersheds (KREW). The 4.6 km2 montane Providence Creek catchment spans the current lower rain–snow transition elevation of 1500–2100 m. Two meteorological stations bracket the high and low elevations of the catchment, measuring air temperature, relative humidity, solar radiation, precipitation, wind speed and direction, and snow depth, and at the higher station, snow water equivalent. Paired flumes at three subcatchments and a V-notch weir at the integrating catchment measure quarter-hourly streamflow. Measurements of meteorological and streamflow data began in 2002. Between 2008 and 2010, 50 sensor nodes were added to measure distributed snow depth, air temperature, soil temperature, and soil moisture within the top 1 m below the surface. These sensor nodes were installed to capture the lateral differences of aspect and canopy coverage. Data are available at hourly and daily intervals by water year (1 October–30 September) in nonproprietary formats from online data reposi

Detecting underground voids, such as old mine workings, solution cavities in karst terrain, or unknown tunnels such as illicit cross-border tunnels, is a challenging problem for geophysics and an important concern for geotechnical design, public safety, and domestic security. Seismic surface-wave-based detection methods have become increasingly popular for detecting relatively shallow and small targets; however, the theoretical limitations of these methods have thus far remained unclear. We use a suite of 3D numerical simulations inspired by a tunnel detection experiment carried out at the Black Diamond Mines Regional Preserve (BDM) in northern California. The geophysical anomalies predicted by our numerical simulations at BDM agree with field observations, and our estimates for the location of the primary tunnel target agree with historical records in the area. Using our calibrated numerical model, we perform a parametric study to determine the effect of tunnel size, depth of burial, filling material, and source characteristics to determine the range over which surface-wave backscattering and attenuation-based methods are effective. In addition, we perform a regression analysis to determine a relationship for the maximum depth at which a tunnel may be detected via these approaches, given the target diameter, wavelength of interest, and the signal-to-noise ratio.

Cancer cells activate telomere maintenance mechanisms (TMMs) to bypass replicative senescence and achieve immortality by either upregulating telomerase or promoting homology-directed repair (HDR) at chromosome ends to maintain telomere length, the latter being referred to as ALT (Alternative Lengthening of Telomeres). In yeast telomerase mutants, the HDR-based repair of telomeres leads to the generation of 'survivors’ that escape senescence and divide indefinitely. So far, yeast has proven to provide an accurate model to study the generation and maintenance of telomeres via HDR. Recently, it has been established that up-regulation of the lncRNA, TERRA (telomeric repeat-containing RNA), is a novel hallmark of ALT cells. Moreover, RNA-DNA hybrids are thought to trigger HDR at telomeres in ALT cells to maintain telomere length and function. Here we show that, also in established yeast type II survivors, TERRA levels are increased in an analogous manner to human ALT cells. The elevated TERRA levels are independent of yeast-specific subtelomeric structures, i.e. the presence or absence of Y’ repetitive elements. Furthermore, we show that RNase H1 overexpression, which degrades the RNA moiety in RNA-DNA hybrids, impairs the growth of yeast survivors. We suggest that even in terms of TERRA regulation, yeast survivors serve as an accurate model that recapitulates many key features of human ALT cells

Cholangiocarcinoma (CCA) is a devastating liver tumour arising from malignant transformation of bile duct epithelial cells. Cancer stem cells (CSC) are a subset of tumour cells endowed with stem-like properties, which play a role in tumour initiation, recurrence and metastasis. In appropriate conditions, CSC form 3D spheres (SPH), which retain stem-like tumour-initiating features. Here, we found different expression of iron proteins indicating increased iron content, oxidative stress and higher expression of CSC markers in CCA-SPH compared to tumour cells growing as monolayers. Exposure to the iron chelator desferrioxamine decreased SPH forming efficiency and the expression of CSC markers and stem-like genes, whereas iron had an opposite effect. Microarray profiles in CCA samples (n = 104) showed decreased H ferritin, hepcidin and ferroportin expression in tumours respect to surrounding liver, whereas transferrin receptor was up-regulated. Moreover, we found a trend toward poorer outcome in CCA patients with elevated expression of ferritin and hepcidin, two major proteins of iron metabolism. These findings, which represent the first evidence of a role for iron in the stem cell compartment as a novel metabolic factor involved in CCA growth, may have implications for a better therapeutic approach.

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24–dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.

Menin (MEN1) is a tumor-suppressor protein in neuroendocrine tissue. Therefore, we tested the novel hypothesis that menin regulates cholangiocarcinoma proliferation. Menin and miR-24 expression levels were measured in the following intrahepatic and extrahepatic cholangiocarcinoma (CCA) cell lines, Mz-ChA-1, TFK-1, SG231, CCLP, HuCCT-1, and HuH-28, as well as the nonmalignant human intrahepatic biliary line, H69. miR-24 miRNA and menin protein levels were manipulated in vitro in Mz-ChA-1 cell lines. Markers of proliferation and angiogenesis (Ki-67, vascular endothelial growth factors A/C, vascular endothelial growth factor receptors 2/3, angiopoietin 1/2, and angiopoietin receptors 1/2) were evaluated. Mz-ChA-1 cells were injected into the flanks of nude mice and treated with miR-24 inhibitor or inhibitor scramble. Menin expression was decreased in advanced CCA specimens, whereas miR-24 expression was increased in CCA. Menin overexpression decreased proliferation, angiogenesis, migration, and invasion. Inhibition of miR-24 increased menin protein expression while decreasing proliferation, angiogenesis, migration, and invasion. miR-24 was shown to negatively regulate menin expression by luciferase assay. Tumor burden and expression of proliferative and angiogenic markers was decreased in the miR-24 inhibited tumor group compared to controls. Interestingly, treated tumors were more fibrotic than the control group. miR-24–dependent expression of menin may be important in the regulation of nonmalignant and CCA proliferation and may be an additional therapeutic tool for managing CCA progression.

Cholangiocarcinoma (CCA) is a devastating liver tumour arising from malignant transformation of bile duct epithelial cells. Cancer stem cells (CSC) are a subset of tumour cells endowed with stem-like properties, which play a role in tumour initiation, recurrence and metastasis. In appropriate conditions, CSC form 3D spheres (SPH), which retain stem-like tumour-initiating features. Here, we found different expression of iron proteins indicating increased iron content, oxidative stress and higher expression of CSC markers in CCA-SPH compared to tumour cells growing as monolayers. Exposure to the iron chelator desferrioxamine decreased SPH forming efficiency and the expression of CSC markers and stem-like genes, whereas iron had an opposite effect. Microarray profiles in CCA samples (n = 104) showed decreased H ferritin, hepcidin and ferroportin expression in tumours respect to surrounding liver, whereas transferrin receptor was up-regulated. Moreover, we found a trend toward poorer outcome in CCA patients with elevated expression of ferritin and hepcidin, two major proteins of iron metabolism. These findings, which represent the first evidence of a role for iron in the stem cell compartment as a novel metabolic factor involved in CCA growth, may have implications for a better therapeutic approach.

Substance P (SP) is involved in the proliferation of cholangiocytes in bile duct–ligated (BDL) mice and human cholangiocarcinoma growth by interacting with the neurokinin-1 receptor (NK-1R). To identify whether SP regulates liver fibrosis during cholestasis, wild-type or NK-1R knockout (NK-1R–/–) mice that received BDL or sham surgery and multidrug resistance protein 2 knockout (Mdr2–/–) mice treated with either an NK-1R antagonist (L-733,060) or saline were used. Additionally, wild-type mice were treated with SP or saline intraperitoneally. In vivo, there was increased expression of tachykinin precursor 1 (coding SP) and NK-1R in both BDL and Mdr2–/–mice compared to wild-type mice. Expression of tachykinin precursor 1 and NK-1R was significantly higher in liver samples from primary sclerosing cholangitis patients compared to healthy controls. Knockout of NK-1R decreased BDL-induced liver fibrosis, and treatment with L-733,060 resulted in decreased liver fibrosis in Mdr2–/–mice, which was shown by decreased sirius red staining, fibrosis gene and protein expression, and reduced transforming growth factor-Î21 levels in serum and cholangiocyte supernatants. Furthermore, we observed that reduced liver fibrosis in NK-1R–/–mice with BDL surgery or Mdr2–/–mice treated with L-733,060 was associated with enhanced cellular senescence of hepatic stellate cells and decreased senescence of cholangiocytes. In vitro, L-733,060 inhibited SP-induced expression of fibrotic genes in hepatic stellate cells and cholangiocytes; treatment with L-733,060 partially reversed the SP-induced decrease of senescence gene expression in cultured hepatic stellate cells and the SP-induced increase of senescence-related gene expression in cultured cholangiocytes. Conclusion: Collectively, our results demonstrate the regulatory effects of the SP/NK-1R axis on liver fibrosis through changes in cellular senescence during cholestatic liver injury. (Hepatology 2017

Melatonin therapy or prolonged exposure to complete darkness reduces biliary hyperplasia and liver fibrosis in bile-duct–ligated (BDL) rats; however, no information exists in primary sclerosing cholangitis (PSC). Thus, we aimed to determine the therapeutic effects of prolonged dark therapy or melatonin administration on hepatic fibrosis in the multidrug resistance gene 2–knockout (Mdr2-/-) mouse model of PSC. Melatonin levels, biliary mass, liver fibrosis, angiogenesis and miR-200b expression were evaluated in wild-type and Mdr2-/- mice exposed to darkness or melatonin treatment or in male patients with PSC and healthy controls. Mdr2-/- mice were also treated with miR-200b inhibitor or control before evaluating biliary mass, liver fibrosis, and angiogenesis. After overexpression of arylalkylamine N-acetyltransferase (AANAT; the enzyme regulating melatonin synthesis) or inhibition of miR-200b in cholangiocytes and hepatic stellate cells in vitro, we evaluated angiogenesis and fibrosis gene expression. After exposure to darkness or administration of melatonin, Mdr2-/- mice show elevated serum melatonin levels and inhibition of biliary mass, along with reduction of liver fibrosis and angiogenesis. MicroRNA PCR analysis demonstrated that miR-200b expression increased in Mdr2-/- mice and patients with PSC compared with controls and decreased in Mdr2-/- mice subjected to dark exposure or melatonin treatment. Inhibition of miR-200b in Mdr2-/- ablates biliary proliferation, liver fibrosis, and angiogenesis. In vitro, overexpression of AANAT or inhibition of miR-200b in cholangiocytes and hepatic stellate cells decreased the expression of miR-200b, angiogenesis, and fibrosis genes. Dark therapy or targeting melatonin/miR-200b axis may be important in the management of biliary damage and liver fibrosis in cholangiopathies including PSC.

Biliary-committed progenitor cells (small mouse cholangiocytes; SMCCs) from small bile ducts are more resistant to hepatobiliary injury than large mouse cholangiocytes (LGCCs) from large bile ducts. The definitive endoderm marker, forkhead box A2 (FoxA2), is the key transcriptional factor that regulates cell differentiation and tissue regeneration. Our aim was to characterize the translational role of FoxA2 during cholestatic liver injury. Messenger RNA expression in SMCCs and LGCCs was assessed by polymerase chain reaction (PCR) array analysis. Liver tissues and hepatic stellate cells (HSCs) from primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) patients were tested by real-time PCR for methylation, senescence, and fibrosis markers. Bile duct ligation (BDL) and multidrug resistance protein 2 (MDR2) knockout mice (MDR2-/-) were used as animal models of cholestatic liver injury with or without healthy transplanted large or small cholangiocytes. We demonstrated that FoxA2 was notably enhanced in murine liver progenitor cells and SMCCs and was silenced in human PSC and PBC liver tissues relative to respective controls that are correlated with the epigenetic methylation enzymes, DNA methyltransferase (DNMT) 1 and DNMT3B. Serum alanine aminotransferase and aspartate aminotransferase levels in nonobese diabetic/severe combined immunodeficiency mice engrafted with SMCCs post-BDL showed significant changes compared to vehicle-treated mice, along with improved liver fibrosis. Enhanced expression of FoxA2 was observed in BDL mouse liver after SMCC cell therapy. Furthermore, activation of fibrosis signaling pathways were observed in BDL/MDR2-/- mouse liver as well as in isolated HSCs by laser capture microdissection, and these signals were recovered along with reduced hepatic senescence and enhanced hepatic stellate cellular senescence after SMCC engraft. Conclusion: The definitive endoderm marker and the positive regulator of biliary development, FoxA

Background & Aims A therapeutically challenging subset of cells, termed cancer stem cells (CSCs) are responsible for cholangiocarcinoma (CCA) clinical severity. Presence of tumor-associated macrophages (TAMs) has prognostic significance in CCA and other malignancies. Thus, we hypothesized that CSCs may actively shape their tumor-supportive immune niche. Methods CCA cells were cultured in 3D conditions to generate spheres. CCA sphere analysis of in vivo tumorigenic-engraftment in immune-deficient mice and molecular characterization was performed. The in vitro and in vivo effect of CCA spheres on macrophage precursors was tested after culturing healthy donor cluster of differentiation (CD)14+ with CCA-sphere conditioned medium. Results CCA spheres engrafted in 100% of transplanted mice and revealed a significant 20.3-fold increase in tumor-initiating fraction (p = 0.0011) and a sustained tumorigenic potential through diverse xenograft-generations. Moreover, CCA spheres were highly enriched for CSC, liver cancer and embryonic stem cell markers both at gene and protein levels. Next, fluorescence-activated cell sorting analysis showed that in the presence of CCA sphere conditioned medium, CD14+ macrophages expressed key markers (CD68, CD115, human leukocyte antigen-D related, CD206) indicating that CCA sphere conditioned medium was a strong macrophage-activator. Gene expression profile of CCA sphere activated macrophages revealed unique molecular TAM-like features confirmed by high invasion capacity. Also, freshly isolated macrophages from CCA resections recapitulated a similar molecular phenotype of in vitro-educated macrophages. Consistent with invasive features, the largest CD163+ set was found in the tumor front of human CCA specimens (n = 23) and correlated with a high level of serum cancer antigen 19.9 (n = 17). Among mediators released by CCA spheres, only interleukin (IL)13, IL34 and osteoactivin were detected and further confirmed in CCA patient sera (n = 12). S

Next-generation hydrologic science and monitoring requires real-time, spatially distributed measurements of key variables including: soil moisture, air/soil temperature, snow depth, and air relative humidity. The SierraNet project provides these measurements by deploying low-power mesh networks across the California Sierra Nevada. This demo presents a replica of the end-to-end SierraNet monitoring system deployed in the Southern Sierra. This system is a highly reliable, low-power turn-key solution for environmental monitoring.

Next-generation hydrologic science and monitoring requires real-time, spatially distributed measurements of key variables including: soil moisture, air/soil temperature, snow depth, and air relative humidity. The SierraNet project provides these measurements by deploying low-power mesh networks across the California Sierra Nevada. This demo presents a replica of the end-to-end SierraNet monitoring system deployed in the Southern Sierra. This system is a highly reliable, low-power turn-key solution for environmental monitoring.

Recent genome-wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified associations with genetic variation at both HLA and non-HLA loci; however, much of heritable HL susceptibility remains unexplained. Here we perform a meta-analysis of three HL GWAS totaling 1,816 cases and 7,877 controls followed by replication in an independent set of 1,281 cases and 3,218 controls to find novel risk loci. We identify a novel variant at 19p13.3 associated with HL (rs1860661; odds ratio (OR) = 0.81, 95% confidence interval (95% CI) = 0.76-0.86, P-combined 3.5 x 10(-10)), located in intron 2 of TCF3 (also known as E2A), a regulator of B-and T-cell lineage commitment known to be involved in HL pathogenesis. This meta-analysis also notes associations between previously published loci at 2p16, 5q31, 6p31, 8q24 and 10p14 and HL subtypes. We conclude that our data suggest a link between the 19p13.3 locus, including TCF3, and HL risk.

Recent genome-wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified associations with genetic variation at both HLA and non-HLA loci; however, much of heritable HL susceptibility remains unexplained. Here we perform a meta-analysis of three HL GWAS totaling 1,816 cases and 7,877 controls followed by replication in an independent set of 1,281 cases and 3,218 controls to find novel risk loci. We identify a novel variant at 19p13.3 associated with HL (rs1860661; odds ratio (OR)=0.81, 95% confidence interval (95% CI)=0.76-0.86, P combined =3.5 × 10 10), located in intron 2 of TCF3 (also known as E2A), a regulator of B-and T-cell lineage commitment known to be involved in HL pathogenesis. This meta-analysis also notes associations between previously published loci at 2p16, 5q31, 6p31, 8q24 and 10p14 and HL subtypes. We conclude that our data suggest a link between the 19p13.3 locus, including TCF3, and HL risk.

Background: Genome-wide association studies (GWAS) have identified numerous loci influencing cross-sectional lung function, but less is known about genes influencing longitudinal change in lung function. Methods: We performed GWAS of the rate of change in forced expiratory volume in the first second (FEV1) in 14 longitudinal, population-based cohort studies comprising 27,249 adults of European ancestry using linear mixed effects model and combined cohort-specific results using fixed effect meta-analysis to identify novel genetic loci associated with longitudinal change in lung function. Gene expression analyses were subsequently performed for identified genetic loci. As a secondary aim, we estimated the mean rate of decline in FEV1 by smoking pattern, irrespective of genotypes, across these 14 studies using meta-analysis. Results: The overall meta-analysis produced suggestive evidence for association at the novel IL16/STARD5/TMC3 locus on chromosome 15 (P = 5.71 x 10(-7)). In addition, meta-analysis using the five cohorts with >= 3 FEV1 measurements per participant identified the novel ME3 locus on chromosome 11 (P = 2.18 x 10(-8)) at genome-wide significance. Neither locus was associated with FEV1 decline in two additional cohort studies. We confirmed gene expression of IL16, STARD5, and ME3 in multiple lung tissues. Publicly available microarray data confirmed differential expression of all three genes in lung samples from COPD patients compared with controls. Irrespective of genotypes, the combined estimate for FEV1 decline was 26.9, 29.2 and 35.7 mL/year in never, former, and persistent smokers, respectively. Conclusions: In this large-scale GWAS, we identified two novel genetic loci in association with the rate of change in FEV1 that harbor candidate genes with biologically plausible functional links to lung function.

Background & Aims Proliferating cholangiocytes secrete and respond to neuroendocrine hormones, including secretin. We investigated whether secretin secreted by S cells and cholangiocytes stimulates biliary proliferation in mice. Methods Cholestasis was induced in secretin knockout (Sct-/-) and wild-type (control) mice by bile duct ligation (BDL). At days 3 and 7 after BDL, control and Sct-/- mice received tail-vein injections of morpholinos against microRNA 125b or let7a. One week later, liver tissues and cholangiocytes were collected. Immunohistochemical, immunoblot, luciferase reporter, and real-time polymerase chain reaction assays were performed. Intrahepatic bile duct mass (IBDM) and proliferation were measured. Secretin secretion was measured in conditioned media from cholangiocytes and S cells and in serum and bile. Results Secretin secretion was increased in supernatants from cholangiocytes and S cells and in serum and bile after BDL in control mice. BDL Sct-/- mice had lower IBDM, reduced proliferation, and reduced production of vascular endothelial growth factor (VEGF) A and nerve growth factor (NGF) compared with BDL control. BDL and control mice given morpholinos against microRNA 125b or let7a had increased IBDM. Livers of mice given morpholinos against microRNA 125b had increased expression of VEGFA, and those treated with morpholinos against microRNA let7a had increased expression of NGF. Secretin regulated VEGF and NGF expression that negatively correlated with microRNA 125b and let7a levels in liver tissue. Conclusions After liver injury, secretin produced by cholangiocytes and S cells reduces microRNA 125b and let7a levels, resulting in up-regulation of VEGF and NGF. Modulation of cholangiocyte expression of secretin could be a therapeutic approach for biliary diseases. © 2014 by the AGA Institute.

Recent genome-wide association studies (GWAS) of Hodgkin lymphoma (HL) have identified associations with genetic variation at both HLA and non-HLA loci; however, much of heritable HL susceptibility remains unexplained. Here we perform a meta-analysis of three HL GWAS totaling 1,816 cases and 7,877 controls followed by replication in an independent set of 1,281 cases and 3,218 controls to find novel risk loci. We identify a novel variant at 19p13.3 associated with HL (rs1860661; odds ratio (OR)=0.81, 95% confidence interval (95% CI)=0.76-0.86, P combined =3.5 × 10 10), located in intron 2 of TCF3 (also known as E2A), a regulator of B-and T-cell lineage commitment known to be involved in HL pathogenesis. This meta-analysis also notes associations between previously published loci at 2p16, 5q31, 6p31, 8q24 and 10p14 and HL subtypes. We conclude that our data suggest a link between the 19p13.3 locus, including TCF3, and HL risk.

The etiology of Hodgkin lymphoma (HL) remains incompletely characterized. Studies of the association between smoking and HL have yielded ambiguous results, possibly due to differences between HL subtypes.

The etiology of Hodgkin lymphoma (HL) remains incompletely characterized. Studies of the association between smoking and HL have yielded ambiguous results, possibly due to differences between HL subtypes.

Genome-wide association studies have identified numerous genetic loci for spirometic measures of pulmonary function, forced expiratory volume in one second (FEV1), and its ratio to forced vital capacity (FEV1/FVC). Given that cigarette smoking adversely affects pulmonary function, we conducted genome-wide joint meta-analyses (JMA) of single nucleotide polymorphism (SNP) and SNP-by-smoking (ever-smoking or pack-years) associations on FEV1 and FEV1/FVC across 19 studies (total N = 50,047). We identified three novel loci not previously associated with pulmonary function. SNPs in or near DNER (smallest P-JMA = 5.00 x 10(-11)), HLA-DQB1 and HLA-DQA2 (smallest P-JMA = 4.35 x 10(-9)), and KCNJ2 and SOX9 (smallest P-JMA = 1.28 x 10(-8)) were associated with FEV1/FVC or FEV1 in meta-analysis models including SNP main effects, smoking main effects, and SNP-by-smoking (ever-smoking or pack-years) interaction. The HLA region has been widely implicated for autoimmune and lung phenotypes, unlike the other novel loci, which have not been widely implicated. We evaluated DNER, KCNJ2, and SOX9 and found them to be expressed in human lung tissue. DNER and SOX9 further showed evidence of differential expression in human airway epithelium in smokers compared to non-smokers. Our findings demonstrated that joint testing of SNP and SNP-by-environment interaction identified novel loci associated with complex traits that are missed when considering only the genetic main effects.

Cholangiocarcinoma (CCA) is a devastating biliary cancer. Melatonin is synthesized in the pineal gland and peripheral organs from serotonin by two enzymes, serotonin N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT). Cholangiocytes secrete neuroendocrine factors, including serotonin-regulating CCA growth by autocrine mechanisms. Melatonin exerts its effects by interaction with melatonin receptor type 1A/1B (MT1/MT2) receptors. We propose that 1) in CCA, there is decreased expression of AANAT and ASMT and secretion of melatonin, changes that stimulate CCA growth; and 2) in vitro overexpression of AANAT decreases CCA growth. We evaluated the 1) expression of AANAT, ASMT, melatonin, and MT1/MT2 in human nonmalignant and CCA lines and control and CCA biopsy samples; 2) melatonin levels in nonmalignant and CCA lines, and bile and serum from controls and patients with intrahepatic CCA; 3) effect of melatonin on the growth and expression of AANAT/ASMT and MT1/MT2 in CCA lines implanted into nude mice; and 4) effect of AANAT overexpression on the proliferation, apoptosis, and expression of MT1/MT2 in Mz-ChA-1 cells. The expression of AANAT, ASMT, and melatonin decreased, whereas MT1/MT2 expression increased in CCA lines and biopsy samples. Melatonin secretion decreased in the supernatant of CCA lines and bile of CCA patients. Melatonin decreased xenograft CCA tumor growth in nude mice by increased AANAT/ ASMT and melatonin, along with reduced MT1/MT2 expression. Overexpression of AANAT in Mz-ChA-1 cells inhibited proliferation and MT1/ MT2 expression and increased apoptosis. There is dysregulation of the AANAT/ASMT/melatonin ¡ melatonin receptor axis in CCA, which inhibited melatonin secretion and subsequently enhanced CCA growth. © 2011 the American Physiological Society

During gametogenesis and pre-implantation development, the mammalian epigenome is reprogrammed to establish pluripotency in the epiblast. Here we show that the histone 3 lysine 4 (H3K4) methyltransferase, MLL2, controls most of the promoter-specific chromatin modification, H3K4me3, during oogenesis and early development. Using conditional knockout mutagenesis and a hypomorph model, we show that Mll2 deficiency in oocytes results in anovulation and oocyte death, with increased transcription of p53, apoptotic factors, and Iap elements. MLL2 is required for (1) bulk H3K4me3 but not H3K4me1, indicating that MLL2 controls most promoters but monomethylation is regulated by a different H3K4 methyltransferase; (2) the global transcriptional silencing that preceeds resumption of meiosis but not for the concomitant nuclear reorganization into the surrounded nucleolus (SN) chromatin configuration; (3) oocyte survival; and (4) normal zygotic genome activation. These results reveal that MLL2 is autonomously required in oocytes for fertility and imply that MLL2 contributes to the epigenetic reprogramming that takes place before fertilization. We propose that once this task has been accomplished, MLL2 is not required until gastrulation and that other methyltransferases are responsible for bulk H3K4me3, thereby revealing an unexpected epigenetic control switch amongst the H3K4 methyltransferases during development.