Regulation of Ras p21 and RalA GTPases activity by quinine in mammary epithelial cells.

Quinine, a bitter compound, can act as an agonist to activate the family of bitter taste G protein-coupled receptor family of proteins. Previous work from our laboratory has demonstrated that quinine causes activation of RalA, a Ras p21-related small G protein. Ral proteins can be activated directly or indirectly through an alternative pathway that requires Ras p21 activation resulting in the recruitment of RalGDS, a guanine nucleotide exchange factor for Ral. Using normal mammary epithelial (MCF-10A) and non-invasive mammary epithelial (MCF-7) cell lines, we investigated the effect of quinine in regulating Ras p21 and RalA activity. Results showed that in the presence of quinine, Ras p21 is activated in both MCF-10A and MCF-7 cells; however, RalA was inhibited in MCF-10A cells, and no effect was observed in the case of MCF-7 cells. MAP kinase, a downstream effector for Ras p21, was activated in both MCF-10A and MCF-7 cells. Western blot analysis confirmed the expression of RalGDS in MCF-10A cells and MCF-7 cells. The expression of RalGDS was higher in MCF-10A cells in comparison to the MCF-7 cells. Although RalGDS was detected in MCF-10A and MCF-7 cells, it did not result in RalA activation upon Ras p21 activation with quinine suggesting that the Ras p21-RalGDS-RalA pathway is not active in the MCF-10A cells. The inhibition of RalA activity in MCF-10A cells due to quinine could be as a result of a direct effect of this bitter compound on RalA. Protein modeling and ligand docking analysis demonstrated that quinine can interact with RalA through the R79 amino acid, which is located in the switch II region loop of the RalA protein. It is possible that quinine causes a conformational change that results in the inhibition of RalA activation even though RalGDS is present in the cell. More studies are needed to elucidate the mechanism(s) that regulate Ral activity in mammary epithelial cells.

Consensus statements on endoscopic ultrasound-guided tissue acquisition. Guidelines from the Asian Endoscopic Ultrasound Group.

OBJECTIVES: This consensus was developed by the Asian EUS Group (AEG), who aimed to formulate a set of practice guidelines addressing various aspects of endoscopic ultrasound-guided tissue acquisition (EUS-TA). METHODS: The AEG initiated the development of consensus statements and formed an expert panel comprising surgeons, gastroenterologists, and pathologists. Three online consensus meetings were conducted to consolidate the statements and votes. The statements were presented and discussed in the first two consensus meetings and revised according to comments. Final voting was conducted at a third consensus meeting. The Grading of Recommendations, Assessment, Development, and Evaluation system was adopted to define the strength of the recommendations and quality of evidence. RESULTS: A total of 20 clinical questions and statements regarding EUS-TA were formulated. The committee recommended that fine-needle biopsy (FNB) needles be preferred over conventional fine-needle aspiration (FNA) needles for EUS-TA of subepithelial lesions. For solid pancreatic masses, rapid on-site evaluation is not routinely recommended when FNB needles are used. For dedicated FNB needles, fork-tip and Franseen-tip needles have essentially equivalent performance. CONCLUSION: This consensus provides guidance for EUS-TA, thereby enhancing the quality of EUS-TA.

Indian Council of Medical Research consensus document on hepatocellular carcinoma.

This document aims to assist oncologists in making clinical decisions encountered while managing their patients with hepatocellular carcinoma (HCC), specific to Indian practice, based on consensus among experts. Most patients are staged by Barcelona Clinic Liver Cancer (BCLC) staging system which comprises patient performance status, Child-Pugh status, number and size of nodules, portal vein invasion and metastasis. Patients should receive multidisciplinary care. Surgical resection and transplant forms the mainstay of curative treatment. Ablative techniques are used for small tumours (<3 cm) in patients who are not candidates for surgical resection (Child B and C). Patients with advanced (HCC should be assessed on an individual basis to determine whether targeted therapy, interventional radiology procedures or best supportive care should be provided. In advanced HCC, immunotherapy, newer targeted therapies and modern radiation therapy have shown promising results. Patients should be offered regular surveillance after completion of curative resection or treatment of advanced disease.

Craniofacial skeleton of MEXICAN tetra (Astyanax mexicanus): As a bone disease model.

A small fresh water fish, the Mexican tetra (Astyanax mexicanus) is a novel animal model in evolutionary developmental biology. The existence of morphologically distinct surface and cave morphs of this species allows simultaneous comparative analysis of phenotypic changes at different life stages. The cavefish harbors many favorable constructive traits (i.e., large jaws with an increased number of teeth, neuromast cells, enlarged olfactory pits and excess storage of adipose tissues) and regressive traits (i.e., reduced eye structures and pigmentation) which are essential for cave adaptation. A wide spectrum of natural craniofacial morphologies can be observed among the different cave populations. Recently, the Mexican tetra has been identified as a human disease model. The fully sequenced genome along with modern genome editing tools has allowed researchers to generate transgenic and targeted gene knockouts with phenotypes that resemble human pathological conditions. This review will discuss the anatomy of the craniofacial skeleton of A. mexicanus with a focus on morphologically variable facial bones, jaws that house continuously replacing teeth and pharyngeal skeleton. Furthermore, the possible applications of this model animal in identifying human congenital and metabolic skeletal disorders is addressed. Developmental Dynamics 248:153-161, 2019. © 2018 Wiley Periodicals, Inc.

Carvedilol delays the progression of small oesophageal varices in patients with cirrhosis: a randomised placebo-controlled trial.

BACKGROUND AND AIMS: Carvedilol is effective in the primary prophylaxis for large oesophageal varices. We investigated its use in preventing progression of small to large oesophageal varices. METHODS: Consecutive cirrhotics with small oesophageal varices were prospectively randomised to either carvedilol (n=70) or placebo (n=70) and followed up for a minimum of 24 months. Endoscopy was done at baseline and six monthly intervals. Hepatic vein pressure gradient (HVPG) was measured at baseline and at 12 months. The primary endpoint was development of large varices. RESULTS: Baseline characteristics in two groups were comparable. The predominant aetiology of cirrhosis was non-alcoholic fatty liver disease in both the groups. The mean dose of carvedilol administered was 12±1.67 mg/day and the target heart rate achieved was 58±3 bpm. A higher proportion of patients in carvedilol group had non-progression to large varices than placebo (79.4% vs 61.4%; p=0.04); the mean time of non-progression to large varices was 20.8 months (95% CI 19.4 to 22.4) in carvedilol group and 18.7 months (95% CI 17.1 to 20.4) in placebo group (p=0.04). There was a modest reduction of HVPG at 1 year in carvedilol group (-8.64%) compared with placebo (+0.33%) (p=0.22). None of the patients in either group died of variceal bleeding or liver-related causes. No major adverse events were observed in either group. CONCLUSIONS: Carvedilol is safe and effective in delaying the progression of small to large oesophageal varices in patients with cirrhosis. TRIAL REGISTRATION NUMBER: NCT01196507; post-results.

Controlled lid-opening in Thermomyces lanuginosus lipase- An engineered switch for studying lipase function.

Here, we present a lipase mutant containing a biochemical switch allowing a controlled opening and closing of the lid independent of the environment. The closed form of the TlL mutant shows low binding to hydrophobic surfaces compared to the binding observed after activating the controlled switch inducing lid-opening. We directly show that lipid binding of this mutant is connected to an open lid conformation demonstrating the impact of the exposed amino acid residues and their participation in binding at the water-lipid interface. The switch was created by introducing two cysteine residues into the protein backbone at sites 86 and 255. The crystal structure of the mutant shows the successful formation of a disulfide bond between C86 and C255 which causes strained closure of the lid-domain. Control of enzymatic activity and binding was demonstrated on substrate emulsions and natural lipid layers. The locked form displayed low enzymatic activity (~10%) compared to wild-type. Upon release of the lock, enzymatic activity was fully restored. Only 10% binding to natural lipid substrates was observed for the locked lipase compared to wild-type, but binding was restored upon adding reducing agent. QCM-D measurements revealed a seven-fold increase in binding rate for the unlocked lipase. The TlL_locked mutant shows structural changes across the protein important for understanding the mechanism of lid-opening and closing. Our experimental results reveal sites of interest for future mutagenesis studies aimed at altering the activation mechanism of TlL and create perspectives for generating tunable lipases that activate under controlled conditions.

Membrane curvature enables N-Ras lipid anchor sorting to liquid-ordered membrane phases.

Trafficking and sorting of membrane-anchored Ras GTPases are regulated by partitioning between distinct membrane domains. Here, in vitro experiments and microscopic molecular theory reveal membrane curvature as a new modulator of N-Ras lipid anchor and palmitoyl chain partitioning. Membrane curvature was essential for enrichment in raft-like liquid-ordered phases; enrichment was driven by relief of lateral pressure upon anchor insertion and most likely affects the localization of lipidated proteins in general.

Predictors of fifty days in-hospital mortality in patients with culture negative neutrocytic ascites.

BACKGROUND: Culture negative neutrocytic ascites is a variant of spontaneous bacterial peritonitis. But there are conflicting reports regarding the mortality associated with culture negativeneutrocytic ascites. Therefore we aim to determine the predictors of mortality associated with culture negativeneutrocytic ascites in a larger sample population. METHODS: We analysed 170 patients consecutively admitted to intensive care unit with diagnosis of culture negative neutrocytic ascites. The clinical, laboratory parameters, etiology of liver cirrhosis was determined along with the scores like model for end stage liver disease, child turcotte pugh were recorded. RESULTS: The 50 day in-hospital mortality rate in culture negative neutrocytic ascites was 39.41% (n = 67). In univariate analysis, means of parameters like total leucocyte count, urea, bilirubin, alanine transaminase, aspartate transaminase, international normalized ratio, acute kidney injury, septic shock, hepatic encephalopathy and model for end stage liver disease were significantly different among survived and those who died (P value ≤0.05). Cox proportional regression model showed the hazard ratio (HR) of acute kidney injury was 2.212 (95% CI: 1.334-3.667), septic shock (HR = 1.895, 95% CI: 1.081-3.323) and model for end stage liver disease (HR = 1.054, 95% CI: 1.020-1.090). Receiver operating characteristics curve showed aspartate aminotransferase (AST) had highest area under the curve 0.761 (95% CI: 0.625-0.785). CONCLUSION: Patients with culture negative neutrocytic ascites have a mortality rate comparable to spontaneous bacterial peritonitis. aspartate aminotransferase, alanine aminotransferase (ALT), acute kidney injury (AKI), model for end stage liver disease (MELD) and septic shock are the independent predictors of 50 days in-hospital mortality in culture negative neutrocytic ascites.

Endoscopic ultrasonography-guided tissue acquisition: How to achieve excellence.

Endoscopic ultrasound (EUS)-guided tissue acquisition is a basic forte of an endosonographer. The multiple skills required to accomplish successful results include not only the puncture itself, but also proper lesion identification, correct puncture sequence, collaboration with the pathologist onsite or remotely, proper handling of the specimens, choosing one or more of cytology, cell-block, and/or tissue core preparation and, last, deciding the immunohistochemistry (IHC) panels and ancillary tests which may be needed for the current case. Error in any of these decisions may lead to incomplete or inconclusive information from the procedure, even if the aspirate is 'adequate.' In the present review, we will describe the technical aspects of EUS-guided tissue acquisition, current needles available and how to choose between them, and how to appropriately handle the specimen. We will also discuss the optimal approach to common targets including lymph nodes, pancreatic masses, pancreatic cysts, and subepithelial lesions.

Residual contamination and bioburden after reprocessing of single-use endoscopic ultrasound needles: An ex vivo study.

BACKGROUND AND AIM: Endoscopic ultrasound (EUS) aspiration needles are single-use devices. However, in many centers, because of cost-constraints, these devices are reused multiple times. We studied microbiological contamination and bioburden on reprocessed needles to evaluate whether these devices can be successfully sterilized. METHODS: We studied 10 EUS needles each of 19 G, 22 G, and 25 G in size, and five 22-G ProCore needles. After initial use, each needle was reprocessed by a standardized protocol. We used standard microbiological cultures, as well as ATP bioluminescence technique to quantify bioburden as relative light units (RLU). We defined significant soil contamination by RLU values >200. We also used extractant fluid to disrupt cell membranes in an attempt to enhance ATP detection. RESULTS: We found culture positivity in 3/34 (8.8%), and detectable bioburden on the exposed surface of 33/35 (94.3%), and inside lumen of 29 (82.9%) reprocessed FNA needles. Significant bioburden was found in three (8.6%) and two (5.7%) needles on the surface and lumen, respectively. We found that use of extractant fluid enhanced detection of bioburden. Larger (19 G) needles had higher surface contamination (P = 0.016), but there was no relation of luminal contamination with needle diameter (P = 0.138). Sheath design and presence of side bevel did not influence extent of contamination. There was significant correlation between the surface and intraluminal bioburden (P < 0.001). CONCLUSIONS: There is significant bioburden in reprocessed EUS needles; standard microbiological cultures have low sensitivity for detection of needle contamination. We have provided objective evidence for the futility of reprocessing attempts, and practice of EUS needle reuse should be discontinued.

Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules.

Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX-BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX-BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX-BARs display a restricted pattern of BAR domain-mediated dimerization, and by resolving a 2.8 Å structure of a SNX1-BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR-dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX-BARs, and the formation of high order SNX-BAR oligomers through selective 'tip-loop' interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX-BAR-decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.

Combination of granulocyte colony-stimulating factor and erythropoietin improves outcomes of patients with decompensated cirrhosis.

BACKGROUND & AIMS: Patients with decompensated cirrhosis have significantly reduced survival without liver transplantation. Granulocyte colony-stimulating factor (G-CSF) has been shown to increase survival in patients with acute-on-chronic liver failure, and erythropoietin promoted hepatic regeneration in animal studies. We performed a double-blind, randomized, placebo-controlled trial to determine whether co-administration of these growth factors improved outcomes for patients with advanced cirrhosis. METHODS: In a prospective study, consecutive patients with decompensated cirrhosis seen at the Institute of Liver and Biliary Sciences, New Delhi (from May 2011 through June 2012) were randomly assigned to groups given subcutaneous G-CSF (5 µg/kg/d) for 5 days and then every third day (12 total doses), along with subcutaneous darbopoietin α(40 mcg/wk) for 4 weeks (GDP group, n = 29), or only placebos (control group, n = 26). All patients also received standard medical therapy and were followed for 12 months. Histology was performed on liver biopsies. The primary end point was survival at 12 months. RESULTS: Baseline characteristics of patients were comparable; alcohol intake was the most common etiology of cirrhosis. A higher proportion of patients in the GDP group than controls survived until 12 months (68.6% vs 26.9%; P = .003). At 12 months, Child-Turcotte Pugh scores were reduced by 48.6% in the GDP group and 39.1% in the control group, from baseline (P = .001); Model for End Stage Liver Disease scores were reduced by 40.4% and 33%, respectively (P = .03). The need for large-volume paracentesis was significantly reduced in GDP group, compared with controls (P < .05). A lower proportion of patients in the GDP group developed septic shock (6.9%) during follow-up compared with controls (38.5%; P = .005). No major adverse events were observed in either group. CONCLUSIONS: In a single-center randomized trial, a significantly larger proportion of patients with decompensated cirrhosis given a combination of G-CSF and darbopoietin α survived for 12 months more than patients given only placebo. The combination therapy also reduced liver severity scores and sepsis to a greater extent than placebo. Clinicaltrials.gov ID: NCT01384565.

PICK1 deficiency impairs secretory vesicle biogenesis and leads to growth retardation and decreased glucose tolerance.

Secretory vesicles in endocrine cells store hormones such as growth hormone (GH) and insulin before their release into the bloodstream. The molecular mechanisms governing budding of immature secretory vesicles from the trans-Golgi network (TGN) and their subsequent maturation remain unclear. Here, we identify the lipid binding BAR (Bin/amphiphysin/Rvs) domain protein PICK1 (protein interacting with C kinase 1) as a key component early in the biogenesis of secretory vesicles in GH-producing cells. Both PICK1-deficient Drosophila and mice displayed somatic growth retardation. Growth retardation was rescued in flies by reintroducing PICK1 in neurosecretory cells producing somatotropic peptides. PICK1-deficient mice were characterized by decreased body weight and length, increased fat accumulation, impaired GH secretion, and decreased storage of GH in the pituitary. Decreased GH storage was supported by electron microscopy showing prominent reduction in secretory vesicle number. Evidence was also obtained for impaired insulin secretion associated with decreased glucose tolerance. PICK1 localized in cells to immature secretory vesicles, and the PICK1 BAR domain was shown by live imaging to associate with vesicles budding from the TGN and to possess membrane-sculpting properties in vitro. In mouse pituitary, PICK1 co-localized with the BAR domain protein ICA69, and PICK1 deficiency abolished ICA69 protein expression. In the Drosophila brain, PICK1 and ICA69 co-immunoprecipitated and showed mutually dependent expression. Finally, both in a Drosophila model of type 2 diabetes and in high-fat-diet-induced obese mice, we observed up-regulation of PICK1 mRNA expression. Our findings suggest that PICK1, together with ICA69, is critical during budding of immature secretory vesicles from the TGN and thus for vesicular storage of GH and possibly other hormones. The data link two BAR domain proteins to membrane remodeling processes in the secretory pathway of peptidergic endocrine cells and support an important role of PICK1/ICA69 in maintenance of metabolic homeostasis.

PI3K/Akt/mTOR signaling & its regulator tumour suppressor genes PTEN & LKB1 in human uterine leiomyomas.

BACKGROUND & OBJECTIVES: Despite their high occurrence and associated significant level of morbidity manifesting as spectrum of clinical symptoms, the pathogenesis of uterine leiomyomas (ULs) remains unclear. We investigated expression profile of tumour suppressor genes PTEN (phosphatase and tensin homolog deleted on chromosome ten) and LKB1 (liver kinase B1), and key signaling components of P13K (phosphatidylinositol 3-kinase)/Akt (protein kinase B)/mTOR (mammalian target of rapamycin) pathway in leiomyomas and adjacent normal myometrium in women of reproductive age, to explore the possibility of targeting this pathway for future therapeutic implications. METHODS: Real time PCR (qPCR) was used to quantify relative gene expression levels of PTEN, Akt1, Akt2, mTOR, LKB1 and VEGFA (vascular endothelial growth factor A) in leiomyoma as compared to adjacent normal myometrium. Immunohistochemistry was subsequently performed to analyze expression of PTEN, phospho-Akt, phospho-mTOR, phospho-S6, LKB1 and VEGFA in leiomyoma and adjacent normal myometrium. RESULTS: Significant upregulation of PTEN (2.52 fold; P=0.03) and LKB1 (3.93 fold; P0.01), and downregulation of VEGFA (2.95 fold; P=0.01) genes were observed in leiomyoma as compared to normal myometrium. Transcript levels of Akt1, Akt2 and mTOR did not vary significantly between leiomyoma and myometrium. An increased immunoexpression of PTEN (P=0.015) and LKB1 (P<0.001) and decreased expression of VEGFA (P=0.01) was observed in leiomyoma as compared to myometrium. Immunostaining for activated (phosphorylated) Akt, mTOR and S6 was absent or low in majority of leiomyoma and myometrium. INTERPRETATION & CONCLUSIONS: Upregulation of PTEN and LKB1 in concert with negative or low levels of activated Akt, mTOR and S6 indicates that PI3K/Akt/mTOR pathway may not play a significant role in pathogenesis of leiomyoma.

Intraductal Sonography in Patients With Portal Cavernoma Cholangiopathy.

Intraductal sonography generates high-resolution images of the entire length of the biliary tree and peribiliary tissues, including 3-dimensional dual- and muti-plane reconstructions to depict complex anatomy. Portal cavernoma cholangiopathy (previously called portal biliopathy) can have multiple etiologies of obstructive cholestasis in the same patient, which can be difficult to define even with advanced imaging techniques.We describe 2 difficult cases of portal cavernoma cholangiopathy in which intraductal sonography helped in clinical management decisions. We think that intraductal sonography should be part of the standard management algorithm for patients with portal cavernoma cholangiopathy and describe the intraductal sonographic correlates of the cholangiographic changes in this condition.

Current situation of endoscopic biliary cannulation and salvage techniques for difficult cases: Current strategies in Japan.

In the pancreatobiliary session at Endoscopic Forum Japan (EFJ) 2015, current trends of routine biliary cannulation techniques and salvage techniques for difficult biliary cannulation cases were discussed. Endoscopists from nine Japanese high-volume centers along with two overseas centers participated in the questionnaires and discussion. It was concluded that, currently, in Western countries, the wire-guided cannulation (WGC) technique is favored during initial cannulation attempts. However, the conventional technique using an endoscopic retrograde cholangiopancreatography catheter with contrast medium injection is still used as first choice at most Japanese high-volume centers. The WGC technique is used as the second choice at some institutions only. After failed biliary cannulation attempts, the initial salvage option preferred in most centers includes pancreatic guidewire placement, followed by precut techniques as the second salvage choice. Among several precut techniques, the free-hand needle knife sphincterotomy with cutting upwards from the pancreatic duct is most popular. Endoscopic ultrasonography-guided rendezvous technique is also carried out as a final salvage option at select institutions.

The Enzymatic Activity of Lipases Correlates with Polarity-Induced Conformational Changes: A Trp-Induced Quenching Fluorescence Study.

Triacylglycerol hydrolases (EC 3.1.1.3) are thought to become activated when they encounter the water-lipid interface causing a "lid" region to move and expose the catalytic site. Here, we tested this idea by looking for lid movements in Thermomyces lanuginosus lipase (TL lipase), and in variants with a mutated lid region of esterase (Esterase) and esterase/lipase (Hybrid) character. To measure lid movements, we employed the tryptophan-induced quenching (TrIQ) fluorescence method to measure how effectively a Trp residue on the lid of these mutants (at position 87 or 89) could quench a fluorescent probe (bimane) placed at nearby site 255 on the protein. To test if lid movement is induced when the enzyme detects a lower-polarity environment (such as at the water-lipid interface), we performed these studies in solvents with different dielectric constants (ε). The results show that lid movement is highly dependent on the particular lid residue composition and solvent polarity. The data suggest that in aqueous solution (ε = 80), the Esterase lid is in an "open" conformation, whereas for the TL lipase and Hybrid, the lid remains "closed". At lower solvent polarities (ε < 46), the lid region for all of the mutants is more "open". Interestingly, these behaviors mirror the structural changes thought to take place upon activation of the enzyme at the water-lipid interface. Together, these results support the idea that lipases are more active in low-polarity solvents because the lid adopts an "open" conformation and indicate that relatively small conformational changes in the lid region play a key role in the activation mechanism of these enzymes.

Interferometric Detection of Single Gold Nanoparticles Calibrated against TEM Size Distributions.

Single nanoparticle analysis: An interferometric optical approach calibrates sizes of gold nanoparticles (AuNPs) from the interference intensities by calibrating their interferometric signals against the corresponding transmission electron microscopy measurements. This method is used to investigate whether size affects the diffusion behavior of AuNPs conjugated to supported lipid bilayer membranes and to multiplex the simultaneous detection of three different AuNP labels.