NGM282 for treatment of non-alcoholic steatohepatitis: a multicentre, randomised, double-blind, placebo-controlled, phase 2 trial.

BACKGROUND: Non-alcoholic steatohepatitis is a chronic liver disease characterised by the presence of hepatic steatosis, inflammation, and hepatocellular injury, for which no Food and Drug Administration (FDA)-approved treatment exists. FGF19 is a hormone that regulates bile acid synthesis and glucose homoeostasis. We aimed to assess the safety and efficacy of NGM282, an engineered FGF19 analogue, for the treatment of non-alcoholic steatohepatitis. METHODS: In this randomised, double-blind, placebo-controlled, phase 2 study, we recruited patients aged 18-75 years with biopsy-confirmed non-alcoholic steatohepatitis as defined by the non-alcoholic steatohepatitis clinical research network histological scoring system, from hospitals and gastroenterology and liver clinics in Australia and the USA. Key eligibility criteria included a non-alcoholic fatty liver disease activity score of 4 or higher, stage 1-3 fibrosis, and at least 8% liver fat content. Patients were randomly assigned (1:1:1) via a web-based system and stratified by diabetic status to receive either 3 mg or 6 mg subcutaneous NGM282 or placebo. The primary endpoint was the absolute change from baseline to week 12 in liver fat content. Responders were patients who achieved a 5% or larger reduction in absolute liver fat content as measured by MRI-proton density fat fraction. Efficacy analysis was by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT02443116. FINDINGS: Between July 14, 2015, and Aug 30, 2016, 166 patients were screened across 18 sites in Australia and the USA. 82 patients were randomly assigned to receive 3 mg NGM282 (n=27), 6 mg NGM282 (n=28), or placebo (n=27). At 12 weeks, 20 (74%) patients in the 3 mg dose group and 22 (79%) in the 6 mg dose group achieved at least a 5% reduction in absolute liver fat content from baseline (relative risk 10·0 [95% CI 2·6-38·7] vs 11·4 [3·0-43·8], respectively; p<0·0001 for both comparisons) versus two (7%) in the placebo group. Overall, 76 (93%) of 82 patients experienced at least one adverse event, most of which were grade 1 (55 [67%]), and only five (6%) were grade 3 or worse. The most commonly (≥10%) reported adverse events were injection site reactions (28 [34%]), diarrhoea (27 [33%]), abdominal pain (15 [18%]), and nausea (14 [17%]). These adverse events were reported more frequently in the NGM282 groups compared with the placebo group. No life-threatening events or patient deaths occurred during the study. INTERPRETATION: NGM282 produced rapid and significant reductions in liver fat content with an acceptable safety profile in patients with non-alcoholic steatohepatitis. Further study of NGM282 is warranted in this patient population. FUNDING: NGM Biopharmaceuticals.

Redox heterogeneity of subsurface waters in the Mesoproterozoic ocean.

A substantial body of evidence suggests that subsurface water masses in mid-Proterozoic marine basins were commonly anoxic, either euxinic (sulfidic) or ferruginous (free ferrous iron). To further document redox variations during this interval, a multiproxy geochemical and paleobiological investigation was conducted on the approximately 1000-m-thick Mesoproterozoic (Lower Riphean) Arlan Member of the Kaltasy Formation, central Russia. Iron speciation geochemistry, supported by organic geochemistry, redox-sensitive trace element abundances, and pyrite sulfur isotope values, indicates that basinal calcareous shales of the Arlan Member were deposited beneath an oxygenated water column, and consistent with this interpretation, eukaryotic microfossils are abundant in basinal facies. The Rhenium-Osmium (Re-Os) systematics of the Arlan shales yield depositional ages of 1414±40 and 1427±43 Ma for two horizons near the base of the succession, consistent with previously proposed correlations. The presence of free oxygen in a basinal environment adds an important end member to Proterozoic redox heterogeneity, requiring an explanation in light of previous data from time-equivalent basins. Very low total organic carbon contents in the Arlan Member are perhaps the key--oxic deep waters are more likely (under any level of atmospheric O2) in oligotrophic systems with low export production. Documentation of a full range of redox heterogeneity in subsurface waters and the existence of local redox controls indicate that no single stratigraphic section or basin can adequately capture both the mean redox profile of Proterozoic oceans and its variance at any given point in time.

Cigarette smoke induces genetic instability in airway epithelial cells by suppressing FANCD2 expression.

Chromosomal abnormalities are commonly found in bronchogenic carcinoma cells, but the molecular causes of chromosomal instability (CIN) and their relationship to cigarette smoke has not been defined. Because the Fanconi anaemia (FA)/BRCA pathway is essential for maintenance of chromosomal stability, we tested the hypothesis that cigarette smoke suppresses that activity of this pathway. Here, we show that cigarette smoke condensate (CSC) inhibited translation of FANCD2 mRNA (but not FANCC or FANCG) in normal airway epithelial cells and that this suppression of FANCD2 expression was sufficient to induce both genetic instability and programmed cell death in the exposed cell population. Cigarette smoke condensate also suppressed FANCD2 function and induced CIN in bronchogenic carcinoma cells, but these cells were resistant to CSC-induced apoptosis relative to normal airway epithelial cells. We, therefore, suggest that CSC exerts pressure on airway epithelial cells that results in selection and emergence of genetically unstable somatic mutant clones that may have lost the capacity to effectively execute an apoptotic programme. Carcinogen-mediated suppression of FANCD2 gene expression provides a plausible molecular mechanism for CIN in bronchogenic carcinogenesis.

Is infliximab safe to use while breastfeeding?

Inflammatory bowel disease (IBD) often affects women around the age of conception and pregnancy. Most drugs used to treat IBD are safe in pregnancy, but physicians must consider the clinical implications of certain treatment regimens in young, fertile females. We report an informative case of a pregnant patient with IBD who underwent treatment with infliximab during her pregnancy and while nursing her infant. Serum and breast milk infliximab levels were monitored throughout this time period. This case report suggests that targeted monoclonal antibodies and other biologic agents can be used with caution in pregnant and breastfeeding patients.

New advances in evaluation and management of patients with polycystic liver disease.

Adult polycystic liver disease (APLD) is an autosomal dominant condition most commonly associated with polycystic kidney disease. However, over the last decade it has come to be recognized that APLD is a genetically heterogeneous disorder involving derangements on at least three different chromosomes. Mutations involving chromosomes 16 and 4 accounting for autosomal dominant polycystic kidney disease (ADPKD) type 1 and type 2 have been well described as have their gene products, polycystin-1 and polycystin-2. These have since been joined by a more recently recognized mutation in the short arm of chromosome 19 thought to be responsible for a much rarer form of autosomal dominant polycystic liver disease without any associated renal involvement. Despite the sometimes impressive physical and radiologic findings, only a minority of patients will progress to advanced liver disease or develop complications as a result of massive hepatomegaly. In these patients, medical management alone has proved ineffectual. Therefore, in the symptomatic APLD patient, surgical therapy remains the mainstay of therapy and includes cyst aspiration and sclerosis, fenestration with and without hepatic resection and orthotopic liver transplantation. The surgical literature on treatment of APLD, to include outcome measurements and complication rates are summarized. Additionally, we review other potential organ involvement and resultant complications.

Safety of band ligator use in the small bowel and the colon.

BACKGROUND: Endoscopic band ligation for bleeding small-bowel vascular lesions has been reported as safe and efficacious based on small case series. There have been several other published case reports of band ligators used for bleeding lesions, usually Dieulafoy's lesions, in the stomach, the proximal small bowel, and the colon. In addition, this method has been used for postpolypectomy bleeding stalks. There has never been a critical look at the anatomic consequences of banding in the thinner sections of bowel. METHOD: The purpose of this study is to define the anatomic and histologic consequences of applying band ligator devices to the small and the large bowel. Fresh surgical specimens, both large and small bowel, that were excised because of neoplastic lesions were transported to our endoscopy unit where one end of the intact bowel was sutured shut. A standard upper endoscope was passed via the open end, and the bowel was closed tightly with rubber band ties. The bowel then was insufflated, and band ligators were applied to unaffected mucosa by using a standard technique. Photodocumentation from inside and outside the bowel was obtained. Some of the band polyps were cut above the band, and some were cut below the band in the fresh state. Some were fixed in formalin and examined microscopically. Histologic sectioning occurred at the level of the bands. RESULTS: The results were striking in that there were large holes (1 cm) in the fresh ileum specimen. There was gross serosal entrapment manifested by visible puckers on the outer surfaces of the specimens, especially in the small bowel and the right colon. The left colon, anatomically thicker, was less affected. The histologic evaluation revealed inclusion by the band ligator of the muscularis propria and serosa on the small bowel, the muscularis propria in the right colon, and the submucosa in the left colon. CONCLUSIONS: Based on these findings, we conclude that band ligator devices are not safe in the small bowel and the right colon but probably are safe in the thicker left colon.

Intragranular targeting of syncollin, but not a syncollinGFP chimera, inhibits regulated insulin exocytosis in pancreatic beta-cells.

Several proteins play a role in the mechanism of insulin exocytosis. However, these 'exocytotic proteins' have yet to account for the regulated aspect of insulin exocytosis, and other factors are involved. In pancreatic exocrine cells, the intralumenal zymogen granule protein, syncollin, is required for efficient regulated exocytosis, but it is not known whether intragranular peptides similarly influence regulated insulin exocytosis. Here, this issue has been addressed using expression of syncollin and a syncollin-green fluorescent protein (syncollinGFP) chimera in rat islet beta-cells as experimental tools. Syncollin is not normally expressed in beta-cells but adenoviral-mediated expression of both syncollin and syncollinGFP indicated that these were specifically targeted to the lumen of beta-granules. Syncollin expression in isolated rat islets had no effect on basal insulin secretion but significantly inhibited regulated insulin secretion stimulated by glucose (16.7 mM), glucagon-like peptide-1 (GLP-1) (10 nM) and glyburide (5 microM). Consistent with specific localization of syncollin to beta-granules, constitutive secretion was unchanged by syncollin expression in rat islets. Syncollin-mediated inhibition of insulin secretion was not due to inadequate insulin production. Moreover, secretagogue-induced increases in cytosolic intracellular Ca2+, which is a prerequisite for triggering insulin exocytosis, were unaffected in syncollin-expressing islets. Therefore, syncollin was most likely acting downstream of secondary signals at the level of insulin exocytosis. Thus, syncollin expression in beta-cells has highlighted the importance of intralumenal beta-granule peptide factors playing a role in the control of insulin exocytosis. In contrast to syncollin, syncollinGFP had no effect on insulin secretion, underlining its usefulness as a 'fluorescent tag' to track beta-granule transport and exocytosis in real time.

Acute behavioral and physiological effects of modafinil in drug abusers.

Modafinil, a novel stimulant, is effective in the treatment of excessive daytime sleepiness associated with narcolepsy. It is biochemically and pharmacologically distinct from prototypical stimulants such as D-amphetamine, cocaine, and methylphenidate. The present experiment was designed to assess the acute behavioral effects of oral modafinil, cocaine, and placebo in participants (n=9) with recent histories of cocaine use (i.e. positive urine for cocaine or benzoylecgonine during the initial screening interview). Drug effects were assessed with a battery of self-reported drug-effect questionnaires, performance measures, and physiological indices. Cocaine, but not modafinil, produced stimulant-like self-reported drug effects (e.g. increased ratings of High and Stimulated). Modafinil and cocaine dose-dependently increased heart rate and blood pressure. The results of the present study suggest that modafinil has minimal abuse potential, but should be viewed cautiously because of the relatively small sample size. Future studies should further characterize the abuse potential of modafinil using other behavioral arrangements, such as drug discrimination or drug self-administration. A full characterization of the abuse potential of modafinil will become important as the use of this drug increases.

Retrograde facilitation of memory by triazolam: effects on automatic processes.

RATIONALE: Retrieval processes have been implicated as a potential mechanism by which benzodiazepines can produce retrograde memory facilitation. OBJECTIVES: This study tested the degree to which benzodiazepine-induced retrograde facilitation of memory was due to an enhancement of automatic retrieval processes. METHODS: Forty healthy adults were randomly assigned to one of three dose conditions (double-blind), under which they received 0.0 mg (placebo), 0.125 mg, or 0.25 mg of the short-acting benzodiazepine triazolam (Halcion). Subjects studied a list of words just prior to dose administration. One hour after dose administration, subjects performed a word-stem completion task which tested their retrieval of the studied words. A process-dissociation procedure was used to estimate the degree to which retrieval was under the influence of memory processes that were automatic (i.e., unintentional) versus controlled (i.e., intentional). RESULTS: Subjects who received active doses of triazolam displayed a greater probability of using studied words as stem completions. Estimates of memory processes showed a greater influence of automatic influences during memory retrieval under triazolam doses. CONCLUSIONS: The findings indicate that retrograde memory facilitation following benzodiazepine administration does not necessarily reflect an improved ability to intentionally retrieve information but could instead reflect increased responsiveness to cues that automatically elicit retrieval of pre-drug information.

The low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor is a receptor for connective tissue growth factor.

Connective tissue growth factor (CTGF) expression is regulated by transforming growth factor-beta (TGF-beta) and strong up-regulation occurs during wound healing; in situ hybridization data indicate that there are high levels of CTGF expression in fibrotic lesions. Recently the binding parameters of CTGF to both high and lower affinity cell surface binding components have been characterized. Affinity cross-linking and SDS-polyacrylamide gel electrophoresis analysis demonstrated the binding of CTGF to a cell surface protein with a mass of approximately 620 kDa. We report here the purification of this protein by affinity chromatography on CTGF coupled to Sepharose and sequence information obtained by mass spectroscopy. The binding protein was identified as the multiligand receptor, low density lipoprotein receptor-related protein/alpha2-macroglobulin receptor (LRP). The identification of LRP as a receptor for CTGF was validated by several studies: 1) binding competition with many ligands that bind to LRP, including receptor-associated protein; 2) immunoprecipitation of CTGF-receptor complex with LRP antibodies; and 3) cells that are genetically deficient for LRP were unable to bind CTGF. Last, CTGF is rapidly internalized and degraded and this process is LRP-dependent. In summary, our data indicate that LRP is a receptor for CTGF, and may play an important role in mediating CTGF biology.

Factors affecting leakage of trapped solutes from phospholipid vesicles during thermotropic phase transitions.

Liposomes are commonly used as models for chilling and freezing damage, with leakage of water-soluble contents from the aqueous interior as the most frequently used measurement of damage. In order to achieve an understanding of the mechanism of the leakage, we have conducted a study of the factors that influence the leakage from liposomes during phase transitions. While such investigations have appeared sporadically in the literature, a detailed study has not been undertaken previously, despite the fact that liposomes are widely used as models for stress injury. Thus, we suggest that these findings will be of general interest in the cryobiology community. We now report that the following variables affected leakage from liposomes during chilling: (i) increasing the rate of cooling and warming resulted in decreased leakage; (ii) maximal leakage occurred at the measured phase transition temperature; (iii) addition of defect-forming additives such as a second phospholipid or a surfactant increased leakage from the liposomes during the phase transition but not above or below that temperature; (iv) small unilamellar vesicles leaked much more rapidly than large unilamellar vesicles; and (v) increasing the pH of the external buffer decreased leakage of carboxyfluorescein, an effect that is probably particular to ionizable solutes.

Arp2/3 complex and actin dynamics are required for actin-based mitochondrial motility in yeast.

The Arp2/3 complex is implicated in actin polymerization-driven movement of Listeria monocytogenes. Here, we find that Arp2p and Arc15p, two subunits of this complex, show tight, actin-independent association with isolated yeast mitochondria. Arp2p colocalizes with mitochondria. Consistent with this result, we detect Arp2p-dependent formation of actin clouds around mitochondria in intact yeast. Cells bearing mutations in ARP2 or ARC15 genes show decreased velocities of mitochondrial movement, loss of all directed movement and defects in mitochondrial morphology. Finally, we observe a decrease in the velocity and extent of mitochondrial movement in yeast in which actin dynamics are reduced but actin cytoskeletal structure is intact. These results support the idea that the movement of mitochondria in yeast is actin polymerization driven and that this movement requires Arp2/3 complex.

Triazolam impairs inhibitory control of behavior in humans.

This study tested the effects of the sedative-hypnotic drug triazolam (Halcion) on the ability to inhibit behavior in humans. Thirty adults practiced a stop-signal task that measured their ability to inhibit and activate behavioral responses on a choice reaction time task. Equal numbers of participants (i.e., n = 10) then received either 0.25 mg, 0.125 mg, or 0 mg (placebo) of triazolam under double-blind conditions and performed the task intermittently over a 3-hr period. In accord with the hypothesis, triazolam reduced response inhibitions and increased the time required to inhibit a response. The drug also slowed the activation of responses. The findings contribute to the understanding of the basic behavioral mechanisms by which sedative-hypnotic drugs can produce states of behavioral disinhibition in some individuals.

Proteomics of rat liver Golgi complex: minor proteins are identified through sequential fractionation.

The discovery of novel proteins resident to the Golgi complex will fuel our future studies of Golgi structure/function and provide justification for proteomic analysis of this organelle. Our approach to Golgi proteomics was to first isolate and characterize the intact organelle free of proteins in transit by use of tissue pretreated with cycloheximide. Then the stacked Golgi fraction was fractionated into biochemically defined subfractions: Triton X-114 insoluble, aqueous, and detergent phases. The aqueous and detergent phases were further fractionated by anion-exchange column chromatography. In addition, radiolabeled cytosol was incubated with stacked Golgi fractions containing proteins in transit, and the proteins bound to the Golgi stacks in an energy-dependent manner were characterized. All fractions were analyzed by two-dimensional (2-D) gel electrophoresis and identification numbers were given to 588 unique 2-D spots. Tandem mass spectrometry was used to analyze 93 of the most abundant 2-D spots taken from preparative Triton X-114 insoluble, aqueous and detergent phase 2-D gels. Fifty-one known and 22 unknown proteins were identified. This study represents the first installment in the mammalian Golgi proteome database. Our data suggest that cell fractionation followed by biochemical dissection of specific classes of molecules provides a significant advantage for the identification of low abundance proteins in organelles.

Two-hybrid system for characterization of protein-protein interactions in E. coli.

The yeast two-hybrid system has been used to characterize many protein-protein interactions. A two-hybrid system for E. coli was constructed in which one hybrid protein bound to a specific DNA site recruits another to an adjacent DNA binding site. The first hybrid comprises a test protein, the bait, fused to a chimeric protein containing the 434 repressor DNA binding domain. In the second hybrid, a second test protein, the prey, is fused downstream of a chimeric protein with the DNA binding specificity of the lambda repressor. Reporters were designed to express cat and lacZ under the control of a low-affinity lambda operator. At low expression levels, lambda repressor hybrids weakly repress the reporter genes. A high-affinity operator recognized by 434 repressor was placed nearby, in a position that does not yield repression by 434 repressor alone. If the test proteins interact, the 434 hybrid bound to the 434 operator stabilizes the binding of the lambda repressor hybrid to the lambda operator, causing increased repression of the reporter genes. Reconstruction experiments with the fos and jun leucine zippers detected protein-protein interactions between either homodimeric or heterodimeric leucine zippers.

Heart Messages: a tailored message intervention for improving heart failure outcomes.

Heart failure is a major health problem in the United States leading to high rates of mortality and morbidity and impaired quality of life. Assisting patients to improve compliance with their self-care regimen, including medications, dietary sodium restrictions, and self-monitoring (daily weights, edema assessment), may improve these poor outcomes. This article describes the development and initial evaluation of Heart Messages, a theory-based, tailored message intervention to improve compliance with the self-care regimen recommended for patients with heart failure. The project involved four phases, each of which is described in this article. The Heart Messages tailored message intervention program is available in both printed and Web-based formats. In a pilot study and clinical evaluation project, the program was found to be useful for patient education and feasible for implementation. Larger randomized trials are now warranted to evaluate the effectiveness of the intervention in improving compliance with the self-care regimen and thereby improving outcomes among patients with heart failure.

Comparison of the solution conformation and dynamics of antifreeze glycoproteins from Antarctic fish.

The (1)H- and (13)C-NMR spectra of antifreeze glycoprotein fractions 1-5 from Antarctic cod have been assigned, and the dynamics have been measured using (13)C relaxation at two temperatures. The chemical shifts and absence of non-sequential (1)H-(1)H NOEs are inconsistent with a folded, compact structure. (13)C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1-5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555-1563). The infrared (IR) spectra of AFGP8 and AFGP1-5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1-5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible o, psi angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops.