Lysine specific demethylase 1 inhibits sodium arsenite activation of HSCs by regulating SESN2/AMPK/ULK1 signaling pathway activity.

Lysine specific demethylase 1 (LSD1) is a histone demethylase that specifically catalyzes the demethylation of histone H3K4 (H3K4me1/2) and regulates gene expression. In addition, it can mediate the process of autophagy through its demethylase activity. Sestrin2 (SESN2) is a stress-induced protein and a positive regulator of autophagy. In NaAsO2-induced mouse fibrotic livers and activated hepatic stellate cells (HSCs), LSD1 expression is decreased, SESN2 expression is increased, and autophagy levels are also increased. Overexpression of LSD1 and silencing of SESN2 decreased the level of autophagy and attenuated the activation of HSCs induced by NaAsO2. LSD1 promoted SESN2 gene transcription by increasing H3K4me1/2 in the SESN2 promoter region. 3-methyladenine (3-MA) and chloroquine were used to inhibit autophagy of HSCs, and the degree of activation was also alleviated. Taken together, LSD1 positively regulates SESN2 by increasing H3K4me1/2 enrichment in the SESN2 promoter region, which in turn increases the level of autophagy and promotes the activation of HSCs. Our results may provide new evidence for the importance of LSD1 in the process of autophagy and activation of HSCs induced by arsenic poisoning. Increasing the expression and activity of LSD1 is expected to be an effective way to reverse the autophagy and activation of HSCs induced by arsenic poisoning.

Dose selection for aztreonam-avibactam, including adjustments for renal impairment, for Phase IIa and Phase III evaluation.

PURPOSE: A series of iterative population pharmacokinetic (PK) modeling and probability of target attainment (PTA) analyses based on emerging data supported dose selection for aztreonam-avibactam, an investigational combination antibiotic for serious Gram-negative bacterial infections. METHODS: Two iterations of PK models built from avibactam data in infected patients and aztreonam data in healthy subjects with "patient-like" assumptions were used in joint PTA analyses (primary target: aztreonam 60% fT > 8 mg/L, avibactam 50% fT > 2.5 mg/L) exploring patient variability, infusion durations, and adjustments for moderate (estimated creatinine clearance [CrCL] > 30 to ≤ 50 mL/min) and severe renal impairment (> 15 to ≤ 30 mL/min). Achievement of > 90% joint PTA and the impact of differential renal clearance were considerations in dose selection. RESULTS: Iteration 1 simulations for Phase I/IIa dose selection/modification demonstrated that 3-h and continuous infusions provide comparable PTA; avibactam dose drives joint PTA within clinically relevant exposure targets; and loading doses support more rapid joint target attainment. An aztreonam/avibactam 500/137 mg 30-min loading dose and 1500/410 mg 3-h maintenance infusions q6h were selected for further evaluation. Iteration 2 simulations using expanded PK models supported an alteration to the regimen (500/167 mg loading; 1500/500 mg q6h maintenance 3-h infusions for CrCL > 50 mL/min) and selection of doses for renal impairment for Phase IIa/III clinical studies. CONCLUSION: A loading dose plus 3-h maintenance infusions of aztreonam-avibactam in a 3:1 fixed ratio q6h optimizes joint PTA. These analyses supported dose selection for the aztreonam-avibactam Phase III clinical program. CLINICAL TRIAL REGISTRATION: NCT01689207; NCT02655419; NCT03329092; NCT03580044.

Suberoylanilide hydroxamic acid upregulates reticulophagy receptor expression and promotes cell death in hepatocellular carcinoma cells.

BACKGROUND: Hepatocellular carcinoma (HCC) is a common clinical condition with a poor prognosis and few effective treatment options. Potent anticancer agents for treating HCC must be identified. Epigenetics plays an essential role in HCC tumorigenesis. Suberoylanilide hydroxamic acid (SAHA), the most common histone deacetylase inhibitor agent, triggers many forms of cell death in HCC. However, the underlying mechanism of action remains unclear. Family with sequence similarity 134 member B (FAM134B)-induced reticulophagy, a selective autophagic pathway, participates in the decision of cell fate and exhibits anticancer activity. This study focused on the relationship between FAM134B-induced reticulophagy and SAHA-mediated cell death. AIM: To elucidate potential roles and underlying molecular mechanisms of reticulophagy in SAHA-induced HCC cell death. METHODS: The viability, apoptosis, cell cycle, migration, and invasion of SAHA-treated Huh7 and MHCC97L cells were measured. Proteins related to the reticulophagy pathway, mitochondria-endoplasmic reticulum (ER) contact sites, intrinsic mitochondrial apoptosis, and histone acetylation were quantified using western blotting. ER and lysosome colocalization, and mitochondrial Ca2+ levels were characterized via confocal microscopy. The level of cell death was evaluated through Hoechst 33342 staining and propidium iodide colocalization. Chromatin immunoprecipitation was used to verify histone H4 lysine-16 acetylation in the FAM134B promoter region. RESULTS: After SAHA treatment, the proliferation of Huh7 and MHCC97L cells was significantly inhibited, and the migration and invasion abilities were greatly blocked in vitro. This promoted apoptosis and caused G1 phase cells to increase in a concentration-dependent manner. Following treatment with SAHA, ER-phagy was activated, thereby triggering autophagy-mediated cell death of HCC cells in vitro. Western blotting and chromatin immunoprecipitation assays confirmed that SAHA regulated FAM134B expression by enhancing the histone H4 lysine-16 acetylation in the FAM134B promoter region. Further, SAHA disturbed the Ca2+ homeostasis and upregulated the level of autocrine motility factor receptor and proteins related to mitochondria-endoplasmic reticulum contact sites in HCC cells. Additionally, SAHA decreased the mitochondrial membrane potential levels, thereby accelerating the activation of the reticulophagy-mediated mitochondrial apoptosis pathway and promoting HCC cell death in vitro. CONCLUSION: SAHA stimulates FAM134B-mediated ER-phagy to synergistically enhance the mitochondrial apoptotic pathway, thereby enhancing HCC cell death.

Organizational trust, usability, and inclusivity are key implementation facilitators for a proposed assets-based mobile health intervention.

Assets-based interventions can address child health disparities by connecting families to existing community resources. Community collaboration when designing interventions may identify barriers and facilitators to implementation. The objective of this study was to identify crucial implementation considerations during the design phase of an asset-based intervention to address disparities in childhood obesity, Assets for Health. We conducted focus groups and semi-structured interviews with caregivers of children (<18 years) (N = 17) and representatives of community-based organizations (CBOs) which serve children and families (N = 20). Focus group and interview guides were developed based on constructs from the Consolidated Framework for Implementation Research. Data were analyzed using rapid qualitative analysis and matrices were used to identify common themes within and across groups of community members. Desired intervention characteristics included an easy-to-use list of community programs that could be filtered based on caregiver preferences and local community health workers to promote trust and engagement among Black and Hispanic/Latino families. Most community members felt an intervention with these characteristics could be advantageous versus existing alternatives. Key outer setting characteristics which were barriers to family engagement included families' financial insecurity and lack of access to transportation. The CBO implementation climate was supportive but there was concern that the intervention could increase staff workload beyond current capacity. Assessment of implementation determinants during the intervention design phase revealed important considerations for intervention development. Effective implementation of Assets for Health may depend on app design and usability, fostering organizational trust and minimizing the costs and staff workload of caregivers and CBOs, respectively.

The purpose of our work was to design a program to connect families with children to existing health-promoting resources in their communities (i.e., group exercise, food pantries, community gardens). We specifically wanted to capture the needs and preferences of parents with children and community-based organizations and determine the possible barriers to creating this program. Based on prior community listening sessions, the program, called Assets for Health, would consist of a mobile app which lists community resources and a community health worker to help connect families to these resources. We presented the idea for Assets for Health to a diverse group of parents and community-based organizations using focus groups and interviews to carefully capture their thoughts. We then analyzed what was said. This work showed that parents were struggling to find community programs that fit their needs and thought a program like Assets for Health could be helpful. Also organizations were struggling to show families that they could be trusted and that all families were welcome.

[Dithiothreitol promotes apoptosis in rat hepatocytes by inhibiting family with sequence similarity 134 member B (FAM134B)-mediated endoplasmic reticulophagy].

Objective To investigate the effect of family with sequence similarity 134 member B (FAM134B)-mediated endoplasmic reticulophagy on apoptosis of hepatocytes induced by endoplasmic reticulum stress (ERS) and identify its potential regulatory mechanism. Methods BRL-3A cells were treated with 0, 0.5, 1.0, 2.0, 4.0, 6.0 mmol/L dithiothreitol (DTT) for 48 hours. The effect of DTT treatment on the proliferation and apoptosis was analyzed using real time cellular dynamic analysis (RTCA) and flow cytometry. The level of proteins related to ERS, endoplasmic reticulophagy, mitochondria-endoplasmic reticulum contact sites (MERCs), and mitochondrial apoptosis pathway were determined using Western blot analysis. Co-localization of ER and lysosomes were detected using ER and lysosomal fluorescence probes. A Ca2+ fluorescence probe was used to detect the level of Ca2+ in mitochondria. Results DTT treatment significantly inhibited cell proliferation and promoted apoptosis in hepatocytes. The levels of proteins related to ERS and endoplasmic reticulophagy, MERCs and the mitochondrial apoptosis pathway significantly increased in BRL-3A cells treated with DTT. DTT treatment decreased the ER-lysosome co-localization and enhanced the fluorescence intensity of Ca2+ in mitochondria. Conclusion DTT aggravates hepatocyte apoptosis by inhibiting FAM134B-mediated endoplasmic reticulophagy and enhancing the level of mitochondrial Ca2+.

Roles of C/EBP-homologous protein and histone H3 lysine 4 methylation in arsenic-induced mitochondrial apoptosis in hepatocytes.

C/EBP-homologous protein (CHOP) and histone H3 lysine 4 (H3K4) methylation have been verified to be correlated with apoptosis, whereas their biological function in arsenic-induced hepatocyte apoptosis through the mitochondrial pathway is still unclear. This study aimed to explore the specific regulatory mechanism of CHOP and H3K4me1/2 in arsenic-induced mitochondrial apoptosis in hepatocytes. Apoptosis and proliferation results showed arsenic promoted apoptosis and inhibited cell growth in BRL-3A cells. Meanwhile, arsenic treatment significantly upregulated the 78-kDa glucose-regulated protein (GRP78), CHOP, su(var)-3-9,enhancer-of-zeste,trithorax (SET) domain containing 7/9 (SET7/9), H3K4me1/2, BIM and BAX expression, while markedly downregulated lysine-specific histone demethylase 1 (LSD1) and BCL2 expression. After down-regulating CHOP, LSD1, and (su(var)-3-9,enhancer-of-zeste,trithorax) domain-containing protein 7/9 (SET7/9) in BRL-3A cells by siRNA, silencing CHOP and SET7/9 notably attenuated the pro-apoptotic and anti-proliferative effects of arsenic treatment on BRL-3A cells, which was reversed after inhibiting LSD1. In addition, our results suggested that knockdown of CHOP altered the expression of mitochondrial-associated proteins BCL2 and BIM, whereas knockdown of LSD1 and SET7/8 regulated the level of H3K4me1/2 modification and BAX protein. Coupled with chromatin immunoprecipitation results, we found that the level of CHOP in the promoter regions of BCL2 and BIM was significantly increased in BRL-3A cells exposed to 30 µmol/L NaAsO2 for 24 h, whereas the levels of H3K4me1/2 in the promoter regions of BAX were unchanged. Collectively, these data indicated that arsenic triggered the mitochondrial pathway to induce hepatocyte apoptosis by up-regulating the levels of CHOP and H3K4me1/2.

Family with sequence similarity 134 member B-mediated reticulophagy ameliorates hepatocyte apoptosis induced by dithiothreitol.

BACKGROUND: Endoplasmic reticulum (ER) stress-related hepatocyte apoptosis is responsible for multiple hepatic diseases. Previous studies have revealed that endoplasmic reticulophagy (ER-phagy) promotes the selective clearance of damaged ER fragments during ER stress, playing a crucial role in maintaining ER homeostasis and inhibiting apoptosis. Family with sequence similarity 134 member B (FAM134B) is a receptor involved in ER-phagy that can form a complex with calnexin (CNX) and microtubule-associated protein 1 light chain 3 (LC3). The complex can mediate the selective isolation of ER fragments to attenuate hepatocyte apoptosis. However, the precise regulatory mechanisms remain unclear. AIM: To elucidate the effect of FAM134B-mediated ER-phagy on ER stress-induced apoptosis in buffalo rat liver 3A (BRL-3A) rat hepatocytes and the potential regulatory mechanisms. METHODS: ER stress-related hepatocyte apoptosis was induced using dithiothreitol (DTT). Proteins related to ER stress and autophagy were measured with western blotting. Protein complex interactions with FAM134B were isolated by co-immunoprecipitation. ER-phagy was evaluated in immunofluorescence experiments. Cell cycle distribution and apoptosis were measured by flow cytometry. Mitochondrial Ca2+ levels were evaluated by the co-localization of intracellular Ca2+-tracker and Mito-tracker. The small interfering RNA against FAM134B was used to knockdown FAM134B in BRL-3A cells. RESULTS: ER stress-related and autophagy-related proteins in BRL-3A cells were elevated by both short and long-term DTT treatment. Furthermore, co-immunoprecipitation confirmed an interaction between FAM134B, CNX, FAM134B, and LC3 in BRL-3A cells. Immunofluorescence assays revealed that autolysosomes significantly decreased following short-term DTT treatment, but increased after long-term treatment. Mitochondrial Ca2+ levels and apoptotic rates were dramatically elevated, and more cells were arrested in the G1 stage after short-term DTT treatment; however, these decreased 48 h later. Moreover, FAM134B downregulation accelerated mitochondrial apoptotic pathway activation and aggravated hepatocyte apoptosis under ER stress. CONCLUSION: FAM134B-mediated ER-phagy attenuates hepatocyte apoptosis by suppressing the mitochondrial apoptotic pathway. Our findings provide new evidence highlighting the importance of FAM134B-mediated ER-phagy in attenuating hepatocyte apoptosis.

[Rat serum containing oxymatrine inhibits the proliferation of LX2 human hepatic stellate cells induced by sodium arsenite via blocking PI3K/AKT pathway].

Objective To investigate the effect of rat serum containing oxymatrine (OM) on the activation of LX2 human hepatic stellate cells induced by sodium arsenite and its mechanism. Methods SD rats were gavaged with 100 mg/kg OM or equal volume of normal saline to prepare OM-containing serum and blank serum. LO2 human embryonic liver cell line was treated with 100 µmol/L sodium arsenite for 24 hours, and then the supernatant was collected. LX2 cells were incubated with the mixture of the supernatant and normal medium at the ratio of 1:4 for 24 hours to establish the cell model of indirect arsenic exposure. Blank serum group (160 mL/L blank serum), indirect arsenic exposure group (160 mL/L blank serum with arsenic exposure), low-dose OM-containing serum group (80 mL/L blank serum and 80 mL/L OM-containing serum with arsenic exposure), high-dose OM-containing serum group (160 mL/L medicated serum with arsenic exposure) were set up. MTT assay and flow cytometry were used to detect cell proliferation and cell cycle, respectively. Western blot analysis was performed to detect the protein expressions of α-SMA, Bcl2, BAX, cyclin D1, PI3K, and phospho-AKT (p-AKT) in LX2 cells. Results After indirect arsenic treatment, the proliferation rate of LX2 cells increased, the proportion of G1 phase decreased, the proportion of apoptosis decreased, the expression of α-SMA, PI3K, p-AKT, cyclin D1, Bcl2 were significantly up-regulated, and the expression of BAX decreased. After OM-containing serum treatment, the proportion of cells in G1 phase increased, the proportion of apoptosis increased, the expression of BAX protein increased significantly, and the expression of other proteins were significantly down-regulated, especially in the high-dose group. Conclusion OM-containing serum can effectively inhibit the proliferation of LX2 hepatic stellate cells induced by arsenite and promote their apoptosis, which may be related to the blocking of PI3K/AKT signaling pathway.

[Endoplasmic reticulum stress in hepatic stellate cells induced by tunicamycin promotes apoptosis and cell cycle arrest].

Objective To investigate the effect of endoplasmic reticulum stress (ERS) induced by tunicamycin on proliferation, activation, and apoptosis of HSC-T6 rat hepatic stellate cells and its possible mechanism. Methods With the expression level of glucose regulated protein 78 (GRP78) as an indicator to explore the optimal concentration and time, a cell model of tunicamycin-induced ERS in HSC-T6 cells was established. HSC-T6 cells were randomized into control group, treatment group with 1 mL/L of dimethyl sulfoxide (DMSO), and treatment group with 1 µg/mL of tunicamycin, and the cells were treated for 12 h. MTT assay was used to detect cell proliferation, flow cytometry to detect apoptosis and cell cycle, and Western blot to detect the protein expressions of α-smooth muscle actin (α-SMA), C/EBP cAMP homologous protein (CHOP), caspase-12, and cyclin D1. Results The optimal dose of tunicamycin to induce ERS in HSC-T6 cells was 1 µg/mL and the optimal time was 12 hours. Compared with the control group and treatment group with DMSO, the treatment group with 1 µg/mL of tunicamycin had no significant change in cell proliferation, but the expression of α-SMA was up-regulated with the apoptosis increased, the proportion of G1 phase cells was significantly increased and that of S phase cells decreased, the ERS induced apoptosis related signal proteins CHOP and caspase-12 were significantly up-regulated, and the expression of cyclin D1 was significantly down-regulated. Conclusion Tunicamycin treatment of HSC-T6 cells for 12 hours induces significant ERS and activation of the cells. The insignificant change in the number of cells during the activation may be related to the increased apoptosis and the cell cycle arrest induced by the activation of the GRP78/CHOP/caspase-12 pathway.

Roles of SET7/9 and LSD1 in the Pathogenesis of Arsenic-induced Hepatocyte Apoptosis.

BACKGROUND AND AIMS: Multiple regulatory mechanisms play an important role in arsenic-induced liver injury. To investigate whether histone H3 lysine 4 (H3K4) methyltransferase (SET7/9) and histone H3K4 demethyltransferase (LSD1/KDM1A) can regulate endoplasmic reticulum stress (ERS)-related apoptosis by modulating the changes of H3K4 methylations in liver cells treated with arsenic. METHODS: Apoptosis, proliferation and cell cycles were quantified by flow cytometry and real-time cell analyzer. The expression of ERS- and epigenetic-related proteins was detected by Western blot analysis. The antisense SET7/9 expression vector and the overexpressed LSD1 plasmid were used for transient transfection of LO2 cells. The effects of NaAsO2 on the methylation of H3 in the promoter regions of 78 kDa glucose-regulated protein, activating transcription factor 4 and C/EBP-homologous protein were evaluated by chromatin immunoprecipitation assay. RESULTS: The protein expression of LSD1 (1.25±0.08 vs. 1.77±0.08, p=0.02) was markedly decreased by treatment with 100 µM NaAsO2, whereas the SET7/9 (0.68±0.05 vs. 1.10±0.13, p=0.002) expression level was notably increased, which resulted in increased H3K4me1/2 (0.93±0.64, 1.19±0.22 vs. 0.71±0.13, 0.84±0.13, p=0.03 and p=0.003). After silencing SET7/9 and overexpressing LSD1 by transfection, apoptosis rate (in percentage: 3.26±0.34 vs. 7.04±0.42, 4.80±0.32 vs. 7.52±0.38, p=0.004 and p=0.02) was significantly decreased and proliferation rate was notably increased, which is reversed after inhibiting LSD1 (in percentage: 9.31±0.40 vs. 7.52±0.38, p=0.03). Furthermore, the methylation levels of H3 in the promoter regions of GRP78 (20.80±2.40 vs. 11.75±2.47, 20.46±2.23 vs. 14.37±0.91, p=0.03 and p=0.01) and CHOP (48.67±4.04 vs. 16.67±7.02, 59.33±4.51 vs. 20.67±3.06, p=0.004 and p=0.001) were significantly increased in LO2 cells exposed to 100 µM NaAsO2 for 24 h. CONCLUSIONS: Histone methyltransferase SET7/9 and histone demethyltransferase LSD1 jointly regulate the changes of H3K4me1/me2 levels in arsenic-induced apoptosis. NaAsO2 induces apoptosis in LO2 cells by activating the ERS-mediated apoptotic signaling pathway, at least partially by enhancing the methylation of H3 on the promoter regions of ERS-associated genes, including GRP78 and CHOP.

Resequencing of Microbial Isolates: A Lab Module to Introduce Novices to Command-Line Bioinformatics.

Familiarity with genome-scale data and the bioinformatic skills to analyze it have become essential for understanding and advancing modern biology and human health, yet many undergraduate biology majors are never exposed to hands-on bioinformatics. This paper presents a module that introduces students to applied bioinformatic analysis within the context of a research-based microbiology lab course. One of the most commonly used genomic analyses in biology is resequencing: determining the sequence of DNA bases in a derived strain of some organism, and comparing it to the known ancestral genome of that organism to better understand the phenotypic differences between them. Many existing CUREs - Course Based Undergraduate Research Experiences - evolve or select new strains of bacteria and compare them phenotypically to ancestral strains. This paper covers standardized strategies and procedures, accessible to undergraduates, for preparing and analyzing microbial whole-genome resequencing data to examine the genotypic differences between such strains. Wet-lab protocols and computational tutorials are provided, along with additional guidelines for educators, providing instructors without a next-generation sequencing or bioinformatics background the necessary information to incorporate whole-genome sequencing and command-line analysis into their class. This module introduces novice students to running software at the command-line, giving them exposure and familiarity with the types of tools that make up the vast majority of open-source scientific software used in contemporary biology. Completion of the module improves student attitudes toward computing, which may make them more likely to pursue further bioinformatics study.

Population Pharmacokinetics of Tofacitinib in Patients With Moderate to Severe Ulcerative Colitis.

Tofacitinib is an oral, small molecule Janus kinase inhibitor for the treatment of ulcerative colitis (UC). We characterized tofacitinib pharmacokinetics in patients with moderate to severe UC, and the effects of covariates on variability in pharmacokinetic parameter estimates. Data were pooled from 1 8-week phase 2 and 2 8-week phase 3 induction studies, and a 52-week phase 3 maintenance study (N = 1096). Population pharmacokinetic analysis was conducted using nonlinear mixed-effects modeling. Potential predictors of apparent oral clearance (CL/F) and volume of distribution (V/F) were evaluated. The PK was described by a 1-compartment model parameterized in terms of CL/F (26.3 L/hour [h]) and V/F (115.8 L), with first-order absorption (Ka ; 9.85 h-1 ) and lag time (0.236 h). The derived elimination half-life was approximately 3.05 h. In the final model, baseline creatinine clearance, sex, and race (Asian vs non-Asian) were significant covariates for CL/F; significant covariates for V/F were age, sex, and body weight; baseline albumin and baseline Mayo score were not significant covariates. CL/F between-patient variability was estimated at 22%. Tofacitinib exposure did not change significantly over the duration of induction/maintenance treatment in patients with UC. Although statistically significant covariate effects on CL/F and V/F were observed, the magnitude of the effects are not clinically significant. Therefore, dose adjustment/restrictions for age, body weight, sex, race, or baseline disease severity are not required during tofacitinib treatment. ClinicalTrials.gov numbers: NCT00787202, NCT01465763, NCT01458951, NCT01458574.

IOX1 protects from TGF-ß induced fibrosis in LX-2 cells via the regulation of extracellular matrix protein expression.

The aim of the present study was to investigate the effect of the histone H3K9 demethylase inhibitor, IOX1, on the mechanism of hepatic fibrosis in TGF-ß-induced human hepatic stellate LX-2 cells. Cellular proliferation, apoptosis, histone H3K9 dimethylation (H3K9me2), protein expression of extracellular matrix (ECM)-related proteins α-smooth muscle actin (SMA), type I collagen (Col I), MMP-1 and TIMP-1 were measured. H3K9me2 levels in the promoter region of ECM-related genes were detected by real-time cell analysis (RTCA), flow cytometry, western blotting and chromatin immunoprecipitation (ChIP) in LX-2 cells. IOX1 significantly inhibited cell proliferation and the IC50 of IOX1 was 100 µM in cells treated with IOX1 for 48 h. IOX1 significantly induced apoptosis in LX-2 cells in a concentration-dependent manner. In addition, different concentration of IOX1 increased the level of H3K9me2 and downregulated the expression of α-SMA, Col I, MMP-1 and TIMP-1 in TGF-ß-induced LX-2 cells. ChIP measurements indicated that H3K9me2 levels in the promotor region of the corresponding genes were increased in TGF-ß-induced LX-2 cells. IOX1 may elevate H3K9me2 in the promotor region of Col I, MMP-1, and TIMP-1 genes to regulate α-SMA, Col I, MMP-1 and TIMP-1 protein expression to induce cell apoptosis, inhibit LX-2 cell proliferation and oppose hepatic fibrotic activity.

Fabrication of diverse carbon forms and their reversed applications in hexane/water separation.

Melamine foam is an important material in production and life. A series of porous carbon foams were obtained through a simple carbonization process of melamine foam at different temperatures. The carbon foams obtained at the carbonization temperature of 400 and 600 °C reveal a hydrophobic and even super-hydrophobic property (water contact angle larger than 150°) with a hexane adsorption much larger than that of melamine foam. However, the carbon foam obtained at the carbonization temperature of 800 °C reveals a super-hydrophilic property (water contact angle smaller than 5°) due to its severest shrinkage during the carbonization process. Interestingly, this series of carbon foams have an excellent performance in oil adsorption. However, the carbon membranes derived from the 800 °C carbon foam reveals oleophobicity under water (the adsorbed water at the surface was extremely important), which allows the penetration of water and blocks the infiltration of hexane at the same time. These different carbon forms have reversed applications in hexane/water separation.

Calpain-2 activity promotes aberrant endoplasmic reticulum stress-related apoptosis in hepatocytes.

BACKGROUND: Calpain-2 is a Ca2+-dependent cysteine protease, and high calpain-2 activity can enhance apoptosis mediated by multiple triggers. AIM: To investigate whether calpain-2 can modulate aberrant endoplasmic reticulum (ER) stress-related apoptosis in rat hepatocyte BRL-3A cells. METHODS: BRL-3A cells were treated with varying doses of dithiothreitol (DTT), and their viability and apoptosis were quantified by 3-[4, 5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2-H-tetrazolium bromide and flow cytometry. The expression of ER stress- and apoptosis-related proteins was detected by Western blot analysis. The protease activity of calpain-2 was determined using a fluorescent substrate, N-succinyl-Leu-Leu-Val-Tyr-AMC. Intracellular Ca2+ content, and ER and calpain-2 co-localization were characterized by fluorescent microscopy. The impact of calpain-2 silencing by specific small interfering RNA on caspase-12 activation and apoptosis of BRL-3A cells was quantified. RESULTS: DTT exhibited dose-dependent cytotoxicity against BRL-3A cells and treatment with 2 mmol/L DTT triggered BRL-3A cell apoptosis. DTT treatment significantly upregulated 78 kDa glucose-regulated protein, activating transcription factor 4, C/EBP-homologous protein expression by >2-fold, and enhanced PRKR-like ER kinase phosphorylation, caspase-12 and caspase-3 cleavage in BRL-3A cells in a trend of time-dependence. DTT treatment also significantly increased intracellular Ca2+ content, calpain-2 expression, and activity by >2-fold in BRL-3A cells. Furthermore, immunofluorescence revealed that DTT treatment promoted the ER accumulation of calpain-2. Moreover, calpain-2 silencing to decrease calpain-2 expression by 85% significantly mitigated DTT-enhanced calpain-2 expression, caspase-12 cleavage, and apoptosis in BRL-3A cells. CONCLUSION: The data indicated that Ca2+-dependent calpain-2 activity promoted the aberrant ER stress-related apoptosis of rat hepatocytes by activating caspase-12 in the ER.

Population Analysis of Anidulafungin in Infants to Older Adults With Confirmed or Suspected Invasive Candidiasis.

In a pooled population analysis, we investigated the pharmacokinetics of i.v. anidulafungin in four studies across a full range of adult and pediatric ages in patients with confirmed, suspected, or at high risk of invasive candidiasis (IC). Relationships between anidulafungin exposure and key efficacy end points (global response of success and all-cause mortality) and safety end points (all-cause hepatic or gastrointestinal adverse events) in all patients and separately in pediatric patients and the appropriate dosing regimen for IC treatment in pediatric patients were evaluated. Pediatric patients received a 3.0 mg/kg (maximum 200 mg) i.v. loading dose and 1.5 mg/kg (maximum 100 mg) daily thereafter. Adults received a 200 mg i.v. loading dose and 100 mg daily thereafter. Estimated systemic anidulafungin exposures were similar across age groups (neonates to adults) at the weight-based doses studied in pediatric patients. No clear associations were identified between anidulafungin exposure and efficacy or safety end points.

Blueberry Attenuates Liver Fibrosis, Protects Intestinal Epithelial Barrier, and Maintains Gut Microbiota Homeostasis.

Objective: Recently, blueberry has been identified as a candidate for the treatment of liver fibrosis. Given the role of gut-liver axis in liver fibrosis and the importance of the gut microbiota homeostasis to the maintenance of the intestinal epithelial barrier, this study aimed to investigate whether blueberry could attenuate liver fibrosis and protect the intestinal epithelial barrier by maintaining the homeostasis of the gut microbiota. Method: A CCl4-induced rat liver fibrosis model was used to detect the roles of blueberry in liver fibrosis and intestinal epithelial barrier. The liver weight and body weight were measured, the liver function was monitored by ALT and AST activity, protein and mRNA were determined by western blot and RT-qPCR, and the gut microbiome was detected by Miseq. Results: The results showed that blueberry could reduce the rate of liver weight/body weight gain (p < 0.05), ALT (p < 0.01) and AST (p < 0.05) activity, and the expression of collagen I (p < 0.01), collagen IV (p < 0.01), and α-SMA (p < 0.01) expression in CCl4-induced rat liver. CCl4 impaired the intestinal epithelial barrier and decreased the expression of the tight junction protein. Blueberry restored the intestinal epithelial barrier and increased the expression of the tight junction protein. The gut microbiota homeostasis was impaired by CCl4, but after treatment with blueberry, the intestinal flora returned to normal. Conclusion: Blueberry attenuated liver fibrosis, protected intestinal epithelial barrier, and maintained the homeostasis of the gut microbiota in a CCl4-induced injury rat model.

Suberoylanilide hydroxamic acid upregulates histone acetylation and activates endoplasmic reticulum stress to induce apoptosis in HepG2 liver cancer cells.

Suberoylanilide hydroxamic acid (SAHA) is a histone deacetylase inhibitor that has demonstrated clinical activity against various solid tumors. The aim of the present study was to explore the effects of SAHA on the apoptosis of HepG2 liver cancer cells, as well as the potential mechanisms involved in histone acetylation and endoplasmic reticulum (ER) stress. HepG2 cells were treated with various doses of SAHA (0, 1, 6 and 12 µM), and apoptosis was measured by flow cytometry. The levels of ER stress-associated molecules, including 78 kDa glucose-regulated protein (GRP78), PRKR-like endoplasmic reticulum kinase (PERK), phosphorylated (p)-PERK, activating transcription factor 4 (ATF4) and C/EBP-homologous protein (CHOP), were quantitated by western blot analysis and reverse transcription-quantitative PCR assay. The expression levels of acetylated histone H4 (acH4, acH4 lysine (K)5 and acH4K12) were detected by western blot analysis. The effects of SAHA on the acetylation of H4 in the promoter regions of GRP78, ATF4 and CHOP were evaluated by chromatin immunoprecipitation assays. Following treatment with higher doses of SAHA (6 and 12 µM) for 48 h, the proliferation of HepG2 cells was significantly suppressed. SAHA induced dose-dependent apoptosis and increased both protein and mRNA expression levels of GRP78, ATF4 and CHOP in HepG2 cells. The protein expression of PERK was markedly decreased by treatment with SAHA, whereas the p-PERK expression level was notably increased, which resulted in increased p-PERK/PERK ratio. Furthermore, the acetylation levels of H4 in the promoter regions of GRP78, ATF4 and CHOP were significantly increased in HepG2 cells exposed to 6 µM SAHA for 36 h. Thus, SAHA induces apoptosis in HepG2 cells by activating the ER stress-mediated apoptotic signaling pathway, at least partially by enhancing the acetylation of histone H4 on the promoter regions of ER-stress associated genes, including GRP78, ATF4 and CHOP.

Population pharmacokinetics of tofacitinib in patients with psoriatic arthritis
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OBJECTIVE: Tofacitinib is an oral Janus kinase inhibitor for the treatment of psoriatic arthritis (PsA). This analysis characterized the pharmacokinetics (PK) of tofacitinib in adult patients with active PsA and evaluated the impact of covariates (baseline characteristics) on the disposition of tofacitinib. MATERIALS AND METHODS: Data were pooled from two phase 3 studies of tofacitinib of up to 12 months' duration in patients with active PsA (OPAL Broaden (NCT01877668); OPAL Beyond (NCT01882439)). This analysis included 650 tofacitinib-treated patients with 3,252 tofacitinib plasma concentration measurements. Tofacitinib PK was described using a one-compartment disposition model parameterized in terms of apparent oral clearance (CL/F) and apparent volume of distribution (V/F) with first-order absorption rate (Ka) and a lag time. Covariates evaluated were baseline age, baseline body weight, sex, race, ethnicity, baseline creatinine clearance (BCCL), and baseline C-reactive protein. RESULTS: The estimates (95% confidence interval) of PK model parameters of a reference patient were CL/F: 20.4 (18.6, 21.8) L/h; V/F: 110 (108, 113) L; and Ka: 13.8 (12.1, 16.6)/h. Among the covariates, only BCCL led to clinically relevant changes in exposure; however, this was consistent with the known contribution of renal excretion to the total clearance of tofacitinib (~ 30%). CONCLUSION: Tofacitinib did not require dose modification or restrictions for age, body weight, sex, race, ethnicity, or baseline disease severity in patients with active PsA based on the magnitude of exposure relative to a reference patient. Dosing adjustments for renal impairment were derived from a separate phase 1 study.