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Background: The earliest manifestation of alcohol-associated liver disease (ALD) is steatosis characterized by deposition of fat in specialized organelles called lipid droplets (LDs). While alcohol administration causes a rise in LD numbers in the hepatocytes, little is known regarding their characteristics that allow their accumulation and size to increase. The aim of the present study is to gain insights into underlying pathophysiological mechanisms by investigating the ethanol-induced changes in hepatic LD proteome as a function of LD size., Methods: Adult male Wistar rats (180-200 g BW) were fed with ethanol liquid diet for 6 weeks. At sacrifice, large-, medium-, and small-sized hepatic LD subpopulations (LD1, LD2, and LD3, respectively) were isolated and subjected to morphological and proteomic analyses., Results: Morphological analysis of LD1-LD3 fractions of ethanol-fed rats clearly demonstrated that LD1 contained larger LDs compared with LD2 and LD3 fractions. Our preliminary results from principal component analysis showed that the proteome of different-sized hepatic LD fractions was distinctly different. Proteomic data analysis identified over 2000 proteins in each LD fraction with significant alterations in protein abundance among the three LD fractions. Among the altered proteins, several were related to fat metabolism, including synthesis, incorporation of fatty acid, and lipolysis. Ingenuity pathway analysis revealed increased fatty acid synthesis, fatty acid incorporation, LD fusion, and reduced lipolysis in LD1 compared to LD3. Overall, the proteomic findings indicate that the increased level of protein that facilitates fusion of LDs combined with an increased association of negative regulators of lipolysis dictates the generation of large-sized LDs during the development of alcohol-associated hepatic steatosis., Conclusion: Several significantly altered proteins were identified in different-sized LDs isolated from livers of ethanol-fed rats. Ethanol-induced increases in specific proteins that hinder LD lipid metabolism led to the accumulation and persistence of large-sized LDs in the liver., (Published 2024. This article is a U.S. Government work and is in the public domain in the USA. Alcohol, Clinical and Experimental Research published by Wiley Periodicals LLC on behalf of Research Society on Alcohol.)

This study entailed the synthesis of Ru nanocatalyst decorated on Nb-grafted SBA-15. A Nb-grafted SBA-15 support with varying Nb contents was utilized as a support for the Ru nanoparticles. The effect of Nb grafting on the immobilized Ru nanoparticle catalyst was systematically investigated, and its catalytic performance in the synthesis of furandicarboxylic acid using 5-hydroxymethylfurfural under base-free reaction conditions was evaluated. The results indicate the increased productivity of the Ru@Nb-grafted SBA-15 catalyst with a yield exceeding 95%, representing a significant advancement in catalysis. This study also affords insights into the complex relationship between the catalytic activity and selectivity and its unique surface attributes. Moreover, acidic sites were created, and the electron density within the active sites was modulated by monomeric Nb oxide species on the SBA-15. Additionally, the role of high-electron-density Ru atoms in facilitating the efficient adsorption and activation of the reactant, resulting in enhanced catalytic efficacy, was highlighted.

Our experiments aim to determine if decreasing the amount of phosphatidylcholine (PC) relative to phosphatidylethanolamine (PE) at the lipid droplet surface changes the localization of specific lipid droplet proteins. We manipulate lipid droplet phospholipids in both a cultured mouse hepatocyte (AML12) cell line and on synthetic lipid droplets. Decreasing the PC:PE ratio increases perilipin 2, decreases DGAT2, and does not change rab18 or lanosterol synthase levels on lipid droplets. These differences may be explained by the distinct structural motifs that mediate the protein-lipid droplet interactions., Competing Interests: The authors declare that there are no conflicts of interest present., (Copyright: © 2023 by the authors.)

The liver is a major metabolic organ that performs many essential biological functions such as detoxification and the synthesis of proteins and biochemicals necessary for digestion and growth. Any disruption in normal liver function can lead to the development of more severe liver disorders. Overall, about 3 million Americans have some type of liver disease and 5.5 million people have progressive liver disease or cirrhosis, in which scar tissue replaces the healthy liver tissue. An estimated 20% to 30% of adults have excess fat in their livers, a condition called steatosis. The most common etiologies for steatosis development are (1) high caloric intake that causes non-alcoholic fatty liver disease (NAFLD) and (2) excessive alcohol consumption, which results in alcohol-associated liver disease (ALD). NAFLD is now termed "metabolic-dysfunction-associated steatotic liver disease" (MASLD), which reflects its association with the metabolic syndrome and conditions including diabetes, high blood pressure, high cholesterol and obesity. ALD represents a spectrum of liver injury that ranges from hepatic steatosis to more advanced liver pathologies, including alcoholic hepatitis (AH), alcohol-associated cirrhosis (AC) and acute AH, presenting as acute-on-chronic liver failure. The predominant liver cells, hepatocytes, comprise more than 70% of the total liver mass in human adults and are the basic metabolic cells. Mitochondria are intracellular organelles that are the principal sources of energy in hepatocytes and play a major role in oxidative metabolism and sustaining liver cell energy needs. In addition to regulating cellular energy homeostasis, mitochondria perform other key physiologic and metabolic activities, including ion homeostasis, reactive oxygen species (ROS) generation, redox signaling and participation in cell injury/death. Here, we discuss the main mechanism of mitochondrial dysfunction in chronic liver disease and some treatment strategies available for targeting mitochondria.

Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time. The gp32 protein probe displayed high sensitivity and specificity to a variety of ssDNA-dependent processes of T4 phage. Several applications of the probe are illustrated here: the solution dynamics of ssDNA-binding protein, protein-protein and protein-DNA interactions involving gp32 protein and its mode of interaction, ssDNA translocation and protein displacement activities of helicases, primer extension activity of DNA polymerase holoenzyme and nucleoprotein filament formation during DNA recombination. The assay has identified new protein-protein interactions of gp32 during T4 replication and recombination. The fluorescent probe described here can thus be used as a universal probe for monitoring in real time various ssDNA-dependent processes, which is based on a well-characterized and easy-to-express bacteriophage T4 gene 32 protein, gp32., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Author.)

Fatty liver is the earliest response to excessive ethanol consumption, which increases the susceptibility of the liver to develop advanced stage of liver disease. Our previous studies have revealed that chronic alcohol administration alters metabolic hormone levels and their functions. Of current interest to our laboratory is glucagon-like peptide 1 (GLP-1), a widely studied hormone known to reduce insulin resistance and hepatic fat accumulation in patients with metabolic-associated fatty liver disease. In this study, we examined the beneficial effects of exendin-4 (a GLP-1 receptor agonist) in an experimental rat model of ALD. Male Wistar rats were pair-fed the Lieber-DeCarli control or ethanol diet. After 4 weeks of this feeding regimen, a subset of rats in each group were intraperitoneally injected every other day with either saline or exendin-4 at a dose of 3 nmol/kg/day (total 13 doses) while still being fed their respective diet. At the end of the treatment, rats were fasted for 6 h and glucose tolerance test was conducted. The following day, the rats were euthanized, and the blood and tissue samples collected for subsequent analysis. We found that exendin-4 treatment had no significant effect on body weight gain among the experimental groups. Exendin-4-treated ethanol rats exhibited improved alcohol-induced alterations in liver/body weight and adipose/body weight ratio, serum ALT, NEFA, insulin, adiponectin and hepatic triglyceride levels. Reduction in indices of hepatic steatosis in exendin-4 treated ethanol-fed rats was attributed to improved insulin signaling and fat metabolism. These results strongly suggest that exendin-4 mitigates alcohol-associated hepatic steatosis by regulating fat metabolism., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The earliest manifestation of alcohol-associated liver disease is hepatic steatosis, which is characterized by fat accumulation in specialized organelles called lipid droplets (LDs). Our previous studies reported that alcohol consumption elevates the numbers and sizes of LDs in hepatocytes, which is attenuated by simultaneous treatment with the methyl group donor, betaine. Here, we examined changes in the hepatic lipidome with respect to LD size and dynamics in male Wistar rats fed for 6 weeks with control or ethanol-containing liquid diets that were supplemented with or without 10 mg betaine/mL. At the time of sacrifice, three hepatic LD fractions, LD1 (large droplets), LD2 (medium-sized droplets), and LD3 (small droplets) were isolated from each rat. Untargeted lipidomic analyses revealed that each LD fraction of ethanol-fed rats had higher phospholipids, cholesteryl esters, diacylglycerols, ceramides, and hexosylceramides compared with the corresponding fractions of pair-fed controls. Interestingly, the ratio of phosphatidylcholine to phosphatidylethanolamine (the two most abundant phospholipids on the LD surface) was lower in LD1 fraction compared with LD3 fraction, irrespective of treatment; however, this ratio was significantly lower in ethanol LD fractions compared with their respective control fractions. Betaine supplementation significantly attenuated the ethanol-induced lipidomic changes. These were mainly associated with the regulation of LD surface phospholipids, ceramides, and glycerolipid metabolism in different-sized LD fractions. In conclusion, our results show that ethanol-induced changes in the hepatic LD lipidome likely stabilizes larger-sized LDs during steatosis development. Furthermore, betaine supplementation could effectively reduce the size and dynamics of LDs to attenuate alcohol-associated hepatic steatosis.

Binge drinking is the most common form of excessive alcohol use. Repeated episodes of binge drinking cause multiple organ injuries, including liver damage. We previously demonstrated that chronic ethanol administration causes a decline in the intrahepatic ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This decline causes impairments in essential methylation reactions that result in alcohol-induced fatty liver (steatosis) and other features of alcohol-associated liver disease (ALD). Co-treatment with betaine during chronic ethanol feeding, normalizes hepatocellular SAM:SAH ratio and alleviates many features of liver damage including steatosis. Here, we sought to examine whether betaine treatment similarly protects against liver injury in an alcohol binge-drinking model. We hypothesized that ethanol binge with prior or simultaneous betaine administration would prevent or attenuate acute alcohol-induced liver damage. Male C57Bl/6 mice were gavaged twice, 12 h apart, with either 6 g ethanol/kg BW or with an equal volume/kg BW of 0.9% NaCl. Two separate groups of mice ( n = 5/group) were gavaged with 4 g betaine/kg BW, either 2 h before or simultaneously with the ethanol or saline gavages. All mice were sacrificed 8 h after the last gavage and serum and liver parameters were quantified. Ethanol binges caused a 50% decrease in hepatic SAM:SAH ratio and a >3-fold rise in liver triglycerides ( p ≤ 0.05). These latter changes were accompanied by elevated serum AST and ALT activities and blood alcohol concentrations (BAC) that were ∼three-times higher than the legal limit of intoxication in humans. Mice that were treated with betaine 2 h before or simultaneously with the ethanol binges exhibited similar BAC as in mice given ethanol-alone. Both betaine treatments significantly elevated hepatic SAM levels thereby normalizing the SAM:SAH ratio and attenuating hepatic steatosis and other injury parameters, compared with mice given ethanol alone. Simultaneous betaine co-administration with ethanol was more effective in preventing or attenuating liver injury than betaine given before ethanol gavage. Our findings confirm the potential therapeutic value of betaine administration in preventing liver injury after binge drinking in an animal model., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Arumugam, Chava, Perumal, Paal, Rasineni, Ganesan, Donohue, Osna and Kharbanda.)

The host defense peptides or antimicrobial peptides (AMPs) often contain short sequence of amino acids, either positive or negatively charged and express broad-spectrum antibacterial, antiviral and antifungal activity. Many researchers had reported that tryptophan, arginine and proline rich AMPs have a promising source of next-generation antibiotics. Nowadays, AMPs are used as a possible therapeutic source for future antibiotics. In the present study, the amino acid sequences of 2924 AMPs belonging to various sources rich in Tryptophan, Proline and Arginine was chosen for investigation. The AMPs were further categorized according to their source, structure and antimicrobial activities. The AMPs with tryptophan, arginine, proline residues in abundance with maximum sequence length of 20 amino acids alone was obtained. Homology modeling was performed with PEP-FOLD and the modeled structures were evaluated using RAMPAGE to identify the structural information. Further, the stability of peptide in aqueous condition was probed using molecular dynamics simulations.Communicated by Ramaswamy H. Sarma.

Helicases are molecular motors with many activities. They use the energy from ATP hydrolysis to unwind double-stranded nucleic acids while translocating on the single-stranded DNA. In addition to unwinding, many helicases are able to remove proteins from nucleic acids. Bacteriophage T4 Dda is able to displace a variety of DNA binding proteins and streptavidin bound to biotinylated oligonucleotides. We have identified a subdomain of Dda that when deleted, results in a protein variant that has nearly wild type activity for unwinding double-stranded DNA but exhibits greatly reduced streptavidin displacement activity. Interestingly, this domain has little effect on displacement of either gp32 or BamHI bound to DNA but does affect displacement of trp repressor from DNA. With this variant, we have identified residues which enhance displacement of some proteins from DNA., (© 2021 The Protein Society.)

Breast cancer is the second most common cancer that causes death among women worldwide. Incidence of breast cancer is increasing worldwide, and the age at which breast cancer develops has shifted from 50- 70 years to 30-40 years. Chemotherapy is the most commonly used effective treatment strategy to combat breast cancer. However, one of the major drawbacks is low selective site-specificity and the consequent toxic insult to normal healthy cells. The nanocarrier system is consistently utilised to minimise the various limitations involved in the conventional treatment of breast cancer. The nanocarrier based targeted drug delivery system provides better bioavailability, prolonged circulation with an effective accumulation of drugs at the tumour site either by active or passive drug targeting. Active targeting has been achieved by receptor/protein anchoring and externally guided magnetic nanocarriers, whereas passive targeting accomplished by employing the access to the tunnel via leaky tumour vasculature, utilising the tumour microenvironment, because the nanocarrier systems can reduce the toxicity to normal cells. As of now a few nanocarrier systems have been approved by FDA, and various nanoformulations are in the pipeline at the preclinical and clinical development for targeting breast cancer; among them, polymeric micelles, microemulsions, magnetic microemulsions, liposomes, dendrimers, carbon nanotubes, and magnetic Nanoparticles (NPs) are the most common. The current review highlights the active and passive targeting potential of nanocarriers in breast cancer and discusses their role in targeting breast cancer without affecting normal healthy cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Many antimicrobial peptides (AMPs) have multiple antimicrobial immunity effects. One such class of peptides is temporins. Temporins are the smallest (AMPs) found in nature and are highly active against gram-positive bacteria. Nowadays, there was a rapid increase in the availability of the 3D structure of proteins in PDB (protein data bank). The conserved residues and 3D structural conformations of temporins (AMPs) were still unknown. The present study explores the sequence analysis, alpha-helical structural conformations of temporins. The sequence of temporins was deracinated from APD3 database, the three-dimensional structure was constructed by homology modeling studies. The sequence analysis results show that the conserved residues among the peptide sequences, the maximum of the sequences are 70% alike to each other. The secondary structure prediction results revealed that 99% of temporin (AMPs) exhibited in alpha-helical form. The 3D structure speculated using RAMPAGE exposes the alpha-helical conformation in all temporins (AMPs). The phylogenetic analysis reveals the evolutionary relationships of temporins (AMPs), which are branched into seven clusters. As a result, we identified a list of potential temporin AMPs which docked to the antiviral protein (MERS-CoV), it shows good protein-peptide binding. This computational approach may serve as a good model for the rationale design of temporin based antibiotics., Competing Interests: Conflict of interestAuthors declare no conflict of interest., (© Springer Nature B.V. 2019.)

Sliding clamp proteins encircle duplex DNA and are involved in processive DNA replication and the DNA damage response. Clamp proteins are ring-shaped oligomers (dimers or trimers) and are loaded onto DNA by an ATP-dependent clamp loader complex that ruptures the interface between two adjacent subunits. Here we measured the solution dynamics of the human clamp protein, proliferating cell nuclear antigen, by monitoring the change in the fluorescence of a site-specifically labeled. To unravel the origins of clamp subunit interface stability, we carried out comprehensive comparative analysis of the interfaces of seven sliding clamps. We used computational modeling (molecular dynamic simulations and MM/GBSA binding energy decomposition analyses) to identify conserved networks of hydrophobic residues critical for clamp stability and ring-opening dynamics. The hydrophobic network is shared among clamp proteins and exhibits a "key in a keyhole" pattern where a bulky aromatic residue from one clamp subunit is anchored into a hydrophobic pocket of the opposing subunit. Bioinformatics and dynamic network analyses showed that this oligomeric latch is conserved across DNA sliding clamps from all domains of life and dictates the dynamics of clamp opening and closing., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Werner syndrome protein (WRN) is a RecQ enzyme involved in the maintenance of genome integrity. Germline loss-of-function mutations in WRN led to premature aging and predisposition to cancer. We evaluated synthetic lethal (SL) interactions between WRN and another human RecQ helicase, BLM, with DNA damage response genes in cancer cell lines. We found that WRN was SL with a DNA mismatch repair protein MutL homolog 1, loss of which is associated with high microsatellite instability (MSI-H). MSI-H cells exhibited increased double-stranded DNA breaks, altered cell cycles, and decreased viability in response to WRN knockdown, in contrast to microsatellite stable (MSS) lines, which tolerated depletion of WRN. Although WRN is the only human RecQ enzyme with a distinct exonuclease domain, only loss of helicase activity drives the MSI SL interaction. This SL interaction in MSI cancer cells positions WRN as a relevant therapeutic target in patients with MSI-H tumors., (Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Differentiating inflammatory from malignant head mass in the background of chronic calcific pancreatitis (CCP) is difficult, and there is no investigation which can reliably solve this dilemma. An accurate diagnosis is crucial as the treatment is different for the two cases and a failure to identify malignancy before surgery can be disastrous. We aimed to assess the accuracy of platelet-lymphocyte ratio (PLR) and to compare it with CA 19-9 in determining the nature of pancreatic head mass (PHM)., Materials and Methods: Eighty-three patients, who presented with CCP and PHM between 2005 and 2011, were included in the study. Patients identified to have malignancy underwent pancreaticoduodenectomy, while those deemed to have a benign lesion underwent Frey's procedure. Clinical features of both the groups were compared. CA 19-9 and PLR individually and in combination were compared in both groups. Receiver operating characteristic curves were used to analyze the predictive values of CA 19-9 and PLR individually and together., Results: Histologically, 66.3% had an inflammatory head mass and 33.7% had a malignant head mass. Significant clinical features which predicted a malignancy included the presence of a head mass in CCP of tropics, older age, jaundice, sudden worsening abdominal pain, gastric outlet obstruction, and significant weight loss. Sensitivity and specificity of CA 19-9 and PLR in diagnosing malignancy were similar (85.5 vs. 81.2 and 96.4 vs. 92.8%, respectively), on combining CA 19-9 and PLR, there was an improvement in sensitivity (94.5%)., Conclusion: PLR is at least as good as CA 19-9 as a diagnostic marker to differentiate between malignant and inflammatory head mass in CCP. When used together, PLR improves the predictive value of serum CA 19-9.

Background: Postoperative infectious complications in patients undergoing pancreatic surgery are a significant cause for morbidity and mortality. Although synbiotics have beneficial effects on human health, their clinical value in surgical patients remains unclear given a paucity of applicable clinical studies., Aim: To determine the impact of perioperative synbiotic therapy on postoperative infectious complications, morbidity and mortality in patients undergoing pancreatic surgery for chronic pancreatitis., Methods: A trial was conducted in patients with chronic calcific pancreatitis undergoing Frey's procedure. Group A received a specific synbiotic composition, 5 days prior and 10 days after the surgery. Group B received a placebo. Primary study endpoint was the occurrence of postoperative infection during the first 30 days. Secondary outcome measures were mortality, length of hospital stay, days in intensive care unit, and duration of antibiotic therapy. Using previously accrued data, with α of 0.05 and power 80%, the sample size was calculated as 35 patients for each group with a dropout rate of 10%., Results: Of the 79 patients enrolled, 75 completed the trial [group A (n = 39) and group B (n = 36)]. The incidence of postoperative infectious complications (12.8% vs 39%; P < 0.05), duration of antibiotics therapy (P < 0.05), and length of hospital stay (P < 0.05) were significantly lower in the synbiotic group., Conclusions: Synbiotics significantly reduce septic complications, hospital stay, and antibiotic requirement in patients undergoing pancreatic surgery for chronic pancreatitis. Furthermore, basic and clinical research would clarify the underlying mechanisms of their therapeutic effect and define the appropriate conditions for use.

Introduction: Obstructive jaundice as a result of bile duct tumour thrombus (BDTT) is an unusual clinical entity and an uncommon presenting feature of hepatocellular carcinoma (HCC). This study evaluates the outcome of hepatectomy for HCC with obstructive jaundice as a result of BDTT in non-cirrhotic livers., Methods: Between 1997 and 2012, out of 426 patients with HCC in non-cirrhotic livers, 39 patients with BDTT (Group I n = 39), who underwent a hepatectomy, were analysed and compared with the non-BDTT group (Group II n = 387)., Results: The demographic profile and biochemical parameters between Group I and Group II were compared; apart from the presence of jaundice at presentation and an elevated serum bilirubin, there were no significant differences. Post-operative morbidity and mortality were 11 (28.2%) and 2 (5.1%), respectively, in Group I. There were no differences between the groups with regards to the operative variables and short-term outcomes. The 1-, 3- and 5-year survival rates in Group I were 82%, 48% and 10%, respectively, with a median survival of 28.6 months and were significantly poorer than Group II (90%, 55% and 38%, respectively, with a median survival of 39.2 months)., Conclusion: The mere presence of BDTT in HCC does not indicate an advanced or inoperable lesion. When technically feasible, a formal hepatic resection is the preferred first-line treatment option in these patients., (© 2015 International Hepato-Pancreato-Biliary Association.)

Background: Psoriasis is a common and chronic T cell mediated skin disorder with varied clinico-morphological types. Most of the published studies have reported chronic plaque type psoriasis to be the commonest type., Aim: To find the incidence of palmoplantar psoriasis in a tertiary health care centre in South India., Materials and Methods: A prospective study done over a period of six months recruiting the patients attending the Dermatology out-patient department in a tertiary health care centre., Results: Incidence of palmoplantar psoriasis was found to be highest among all morphological forms of psoriasis., Conclusion: Incidence of palmoplantar psoriasis is under-expressed. It is actually ahead in the race.

Background: Prebiotics and probiotics influence all pathogenic mechanisms of bacterial translocation. Used in combination, they are called synbiotics. Postoperative infective complications in patients undergoing hepatic and pancreatic surgery lead to a significant prolongation of hospital stay and increased costs. While synbiotics are considered to have beneficial effects on human health, their clinical value in surgical patients, especially in South Asia remains unclear given a paucity of applicable clinical studies. In this study we aim to assess their clinical usefulness in patients who undergo hepatic and pancreatic surgery., Methods: A prospective monocentric randomised single blind controlled trial is being conducted in patients undergoing major pancreatic resections (Whipple procedure, distal pancreatectomy, Frey procedure) and hepatic resections. Group A received a specific synbiotic composition, 5 days prior and 10 days after the surgery. Group B received a placebo. Primary study end point was the occurrence of postoperative infection during the first 30 days. Secondary outcome measures were mortality, first bowel movement, days in intensive care unit, length of hospital stay, and duration of antibiotic therapy. Side effects of probiotics were evaluated. From previous studies we assumed that perioperative synbiotics reduce the proportion of patients with infectious complications from 50% to 12%, with α of 0.05 and power 80%, the calculated sample size was 35 patients for each group with a dropout rate of 10%., Conclusions: This study is intended at determining the impact of perioperative synbiotic therapy on postoperative infectious complications, morbidity and mortality in patients undergoing major pancreatic and hepatic surgery., Clinical Trial: The Clinical Trials Registry of India (CTRI/2013/06/003737).

Aim: To analyze the differences in outcomes and the clinical impact following pancreatoduodenectomy (PD) in patients with and without aberrant right hepatic artery (aRHA)., Methods: All patients undergoing PD between January 2008 and December 2012 were divided into two groups, one with aRHA and the other without. These groups were compared to identify differences in the intraoperative variables, the oncological clearance and the postoperative morbidity, mortality and hospital stay., Results: A total of 225 patients underwent PD, of which 43 (19.1%) patients were found to have either accessory or replaced right hepatic arteries (aRHA group). The aRHA was preserved in 79% of the patients. There was no significant difference in the intraoperative blood loss but operative time was prolonged, reflecting the complexity of the procedure [420 ± 44 (240-540) min vs 480 ± 45 (300-600) min, P < 0.05)]. There were no differences in the incidence of postoperative complications (pancreatic leak, pancreatic fistula, delayed gastric emptying and mortality) and hospital stay. Oncological clearance in the form of positive resection margins [13 (7.1%) vs 3 (6.9%)] and lymph node yield were also similar in the two groups., Conclusion: An aRHA is found in approximately one fifth of patients undergoing PD. Preservation is technically possible in most patients and can increase the operative complexity but does not negatively affect the safety or oncological outcomes of the procedure.

Centrosomal kinase Nek2 is overexpressed in different cancers, yet how it contributes toward tumorigenesis remains poorly understood. dNek2 overexpression in a Drosophila melanogaster model led to upregulation of Drosophila Wnt ortholog wingless (Wg), and alteration of cell migration markers-Rho1, Rac1 and E-cadherin (Ecad)-resulting in changes in cell shape and tissue morphogenesis. dNek2 overexpression cooperated with receptor tyrosine kinase and mitogen-activated protein kinase signaling to upregulate activated Akt, Diap1, Mmp1 and Wg protein to promote local invasion, distant seeding and metastasis. In tumor cell injection assays, dNek2 cooperated with Ras and Src signaling to promote aggressive colonization of tumors into different adult fly tissues. Inhibition of the PI3K pathway suppressed the cooperation of dNek2 with other growth pathways. Consistent with our fly studies, overexpression of human Nek2 in A549 lung adenocarcinoma and HEK293T cells led to activation of the Akt pathway and increase in β-catenin protein levels. Our computational approach identified a class of Nek2-inhibitory compounds and a novel drug-like pharmacophore that reversed the Nek2 overexpression phenotypes in flies and human cells. Our finding posits a novel role for Nek2 in promoting metastasis in addition to its currently defined role in promoting chromosomal instability. It provides a rationale for the selective advantage of centrosome amplification in cancer.

Bacteriophage T4 UvsW helicase contains both unwinding and annealing activities and displays some functional similarities to bacterial RecG and RecQ helicases. UvsW is involved in several DNA repair pathways, playing important roles in recombination-dependent DNA repair and the reorganization of stalled replication forks. The T4 single-stranded DNA (ssDNA) binding protein gp32 is a central player in nearly all DNA replication and repair processes and is thought to facilitate their coordination by recruiting and regulating the various proteins involved. Here, we show that the activities of the UvsW protein are modulated by gp32. UvsW-catalyzed unwinding of recombination intermediates such as D-loops and static X-DNA (Holliday junction mimic) to ssDNA products is enhanced by the gp32 protein. The enhancement requires the presence of the protein interaction domain of gp32 (the acidic carboxy-terminus), suggesting that a specific interaction between UvsW and gp32 is required. In the absence of this interaction, the ssDNA annealing and ATP-dependent translocation activities of UvsW are severely inhibited when gp32 coats the ssDNA lattice. However, when UvsW and gp32 do interact, UvsW is able to efficiently displace the gp32 protein from the ssDNA. This ability of UvsW to remove gp32 from ssDNA may explain its ability to enhance the strand invasion activity of the T4 recombinase (UvsX) and suggests a possible new role for UvsW in gp32-mediated DNA transactions., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Gastrointestinal stromal tumors (GISTs) have been recognized as a biologically distinctive tumor type, different from smooth muscle and neural tumors of the gastrointestinal tract (GIT). They constitute the majority of gastrointestinal mesenchymal tumors of the GIT and are known to be refractory to conventional chemotherapy or radiation. They are defined and diagnosed by the expression of a proto-oncogene protein detected by immunohistochemistry which serves as a crucial diagnostic and therapeutic target. The identification of these mutations has resulted in a better understanding of their oncogenic mechanisms. The remarkable antitumor effects of the molecular inhibitor imatinib have necessitated accurate diagnosis of GIST and their distinction from other gastrointestinal mesenchymal tumors. Both traditional and minimally invasive surgery are used to remove these tumors with minimal morbidity and excellent perioperative outcomes. The revolutionary use of specific, molecularly-targeted therapies, such as imatinib mesylate, reduces the frequency of disease recurrence when used as an adjuvant following complete resection. Neoadjuvant treatment with these agents appears to stabilize disease in the majority of patients and may reduce the extent of surgical resection required for subsequent complete tumor removal. The important interplay between the molecular genetics of GIST and responses to targeted therapeutics serves as a model for the study of targeted therapies in other solid tumors. This review summarizes our current knowledge and recent advances regarding the histogenesis, pathology, molecular biology, the basis for the novel targeted cancer therapy and current evidence based management of these unique tumors.

In most organisms, clamp loaders catalyze both the loading of sliding clamps onto DNA and their removal. How these opposing activities are regulated during assembly of the DNA polymerase holoenzyme remains unknown. By utilizing FRET to monitor protein-DNA interactions, we examined assembly of the human holoenzyme. The results indicate that assembly proceeds in a stepwise manner. The clamp loader (RFC) loads a sliding clamp (PCNA) onto a primer/template junction but remains transiently bound to the DNA. Unable to slide away, PCNA re-engages with RFC and is unloaded. In the presence of polymerase (polδ), loaded PCNA is captured from DNA-bound RFC which subsequently dissociates, leaving behind the holoenzyme. These studies suggest that the unloading activity of RFC maximizes the utilization of PCNA by inhibiting the build-up of free PCNA on DNA in the absence of polymerase and recycling limited PCNA to keep up with ongoing replication. DOI:http://dx.doi.org/10.7554/eLife.00278.001.

Background. Hemosuccus pancreaticus (HP) is a very rare and obscure cause of upper gastrointestinal bleeding. Due to its rarity, the diagnostic and therapeutic strategy for the management of this potentially life threatening problem remains undefined. The objective of our study is to highlight the challenges in the diagnosis and management of HP and to formulate a protocol to effectively and safely manage this condition. Methods. We retrospectively reviewed the records of all patients who presented with HP over the last 15 years at our institution between January 1997 and December 2011. Results. There were a total of 51 patients with a mean age of 32 years. Nineteen patients had chronic alcoholic pancreatitis; twenty-six, five, and one patient had tropical pancreatitis, acute pancreatitis, and idiopathic pancreatitis, respectively. Six patients were managed conservatively. Selective arterial embolization was attempted in 40 of 45 (89%) patients and was successful in 29 of the 40 (72.5%). 16 of 51 (31.4%) patients required surgery. Overall mortality was 7.8%. Length of followup ranged from 6 months to 15 years. Conclusions. Upper gastrointestinal bleeding in a patient with a history of chronic pancreatitis could be caused by HP. All hemodynamically stable patients with HP should undergo prompt initial angiographic evaluation, and if possible, embolization. Hemodynamically unstable patients and those following unsuccessful embolization should undergo emergency haemostatic surgery. Centralization of GI bleed services along with a multidisciplinary team approach and a well-defined management protocol is essential to reduce the mortality and morbidity of this condition.

Loading of the phage T4 sliding clamp gp45 by the gp44/62 clamp loader onto DNA to form the holoenzyme and their disassembly pathways were investigated using FRET-based single-molecule and ensemble kinetic studies. gp44/62-mediated assembly of gp45 onto the DNA involves a rate-limiting conformational rearrangement of the gp45-gp44/62-DNA complex. Single-molecule measurements revealed the intermediates in gp45 loading and their interconversion, suggesting that the assembly is not concerted but is broken down into many small kinetic steps. Two populations of the gp45-gp44/62-DNA complex are formed on the end-blocked DNA that are poised to form the holoenzyme with the polymerase. In the absence of a polymerase, the two clamp populations dissociated from the DNA along with gp44/62 with distinct rates. In the presence of polymerase, holoenzyme assembly involved the recruitment of the polymerase to the gp45-gp44/62-DNA complex mediated by the chaperoning activity of gp44/62. This transient multiprotein complex then decomposed through an ATP hydrolysis-dependent exit of gp44/62 leaving the holoenzyme on DNA. The rate of dissociation of the holoenzyme from the DNA is sensitive to whether the DNA ends are blocked, underscoring its mobility on the DNA.

Helicases that both unwind and rewind DNA have central roles in DNA repair and genetic recombination. In contrast to unwinding, DNA rewinding by helicases has proved difficult to characterize biochemically because of its thermodynamically downhill nature. Here we use single-molecule assays to mechanically destabilize a DNA molecule and follow, in real time, unwinding and rewinding by two DNA repair helicases, bacteriophage T4 UvsW and Escherichia coli RecG. We find that both enzymes are robust rewinding enzymes, which can work against opposing forces as large as 35 pN, revealing their active character. The generation of work during the rewinding reaction allows them to couple rewinding to DNA unwinding and/or protein displacement reactions central to the rescue of stalled DNA replication forks. The overall results support a general mechanism for monomeric rewinding enzymes.

The restart of a stalled replication fork is a major challenge for DNA replication. Depending on the nature of the damage, different repair processes might be triggered; one is template switching, which is a bypass of a leading-strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching. We show that the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading-strand lesion damage by a pseudostochastic process, periodically forming and migrating a four-way Holliday junction. The initiation of the repair process requires partial replisome disassembly via the departure of the replicative helicase. The results support the role of fork regression pathways in DNA repair.

Polymerase δ is widely accepted as the lagging strand replicative DNA polymerase in eukaryotic cells. It forms a replication complex in the presence of replication factor C and proliferating cell nuclear antigen to perform efficient DNA synthesis in vivo. In this study, the human lagging strand holoenzyme was reconstituted in vitro. The rate of DNA synthesis of this holoenzyme, measured with a singly primed ssM13 DNA substrate, is 4.0 ± 0.4 nucleotides. Results from adenosine 5'-(3-thiotriphosphate) tetralithium salt (ATPγS) inhibition experiments revealed the nonprocessive characteristic of the human DNA polymerase (Pol δ) holoenzyme (150 bp for one binding event), consistent with data from chase experiments with catalytically inactive mutant Pol δ(AA). The ATPase activity of replication factor C was characterized and found to be stimulated ∼10-fold in the presence of both proliferating cell nuclear antigen and DNA, but the activity was not shut down by Pol δ in accord with rapid association/dissociation of the holoenzyme to/from DNA. It is noted that high concentrations of ATP inhibit the holoenzyme DNA synthesis activity, most likely due to its inhibition of the clamp loading process.

Helicases are an important class of enzymes involved in DNA and RNA metabolism that couple the energy of ATP hydrolysis to unwind duplex DNA and RNA structures. Understanding the mechanism of helicase action is vital due to their involvement in various biological processes such as DNA replication, repair and recombination. Furthermore, the duplex DNA unwinding property of this class of enzymes is closely related to their single-stranded DNA translocation. Hence the study of its translocation properties is essential to understanding helicase activity. Here we review the methods that are employed to analyze the DNA translocation and unwinding activities of the bacteriophage T4 UvsW and Dda helicases. These methods have been successfully employed to study the functions of helicases from large superfamilies., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Fast and accurate replication of DNA is accomplished by the interactions of multiple proteins in the dynamic DNA replisome. The DNA replisome effectively coordinates the leading and lagging strand synthesis of DNA. These complex, yet elegantly organized, molecular machines have been studied extensively by kinetic and structural methods to provide an in-depth understanding of the mechanism of DNA replication. Owing to averaging of observables, unique dynamic information of the biochemical pathways and reactions is concealed in conventional ensemble methods. However, recent advances in the rapidly expanding field of single-molecule analyses to study single biomolecules offer opportunities to probe and understand the dynamic processes involved in large biomolecular complexes such as replisomes. This review will focus on the recent developments in the biochemistry and biophysics of DNA replication employing single-molecule techniques and the insights provided by these methods towards a better understanding of the intricate mechanisms of DNA replication., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

UvsW protein from bacteriophage T4 controls the transition from origin-dependent to origin-independent initiation of replication through the unwinding of R-loops bound to the T4 origins of replication. UvsW has also been implicated through genetic and biochemical experiments to play a role in DNA repair processes such as replication fork regression and Holliday junction branch migration. UvsW is capable of unwinding a wide variety of substrates, many of which contain only duplex DNA without single-stranded regions. Based on this observation, it has been suggested that UvsW is a dsDNA translocase. In this work we examine the ability of UvsW to translocate on ssDNA. Kinetic analysis indicates that the rate of ATP hydrolysis is strongly dependent on the length of the ssDNA lattice, whereas the K(M)-DNA remains relatively constant, demonstrating that UvsW translocates on ssDNA in an ATP-dependent fashion. Experiments using streptavidin blocks or poly dT sequences located at either end of the ssDNA substrate indicate that UvsW translocates in a 3' to 5' direction. Mutant competition and heparin trapping experiments reveal that UvsW is extremely processive during ATP-driven translocation with a half-life on the order of several minutes. Finally, functional assays provide evidence that UvsW is monomeric while translocating on ssDNA. The ability of UvsW to unwind DNA duplexes is likely to be mechanistically linked to its ability to processively translocate on ssDNA in a 3' to 5' unidirectional fashion.

A series of aryl and arylmethyl beta-aryl-beta-ketophosphonates have been prepared as potential beta-lactamase inhibitors. These compounds, as fast, reversible, competitive inhibitors, were most effective (micromolar K(i) values) against the class D OXA-1 beta-lactamase but had less activity against the OXA-10 enzyme. They were also quite effective against the class C beta-lactamase of Enterobacter cloacae P99 but less so against the class A TEM-2 enzyme. Reduction of the keto group to form the corresponding beta-hydroxyphosphonates led to reduced inhibitory activity. Molecular modeling, based on the OXA-1 crystal structure, suggested interaction of the aryl groups with the hydrophobic elements of the enzyme's active site and polar interaction of the keto and phosphonate groups with the active site residues Ser 115, Lys 212 and Thr 213 and with the non-conserved Ser 258. Analysis of binding free energies showed that the beta-aryl and phosphonate ester aryl groups interacted cooperatively within the OXA-1 active site. Overall, the results suggest that quite effective inhibitors of class C and some class D beta-lactamases could be designed, based on the beta-ketophosphonate platform.

A series of amidoketophosph(on)ates of general structure PhCH2OCONHCH(R)COCHR'(CH2)n(O)P(O2-)(O)R'' (R = H, CH3; R' = H, CH3; n = 0, 1; R'' = H, CH3, Et, Ph) have been prepared as a potential source of beta-lactamase inhibitors. The phosphonates (n = 0) were obtained by means of the Arbuzov reaction while most of the phosphates were achieved from reaction of phosph(or/on)ic acids with the appropriate diazoketone PhCH2OCONHCH(R)COCR'N2. The electrophilicity of the carbonyl group in the resulting phosph(on)ates was assessed by the degree of hydration in aqueous solution, determined from NMR spectra. These compounds inhibited typical class C and class D beta-lactamases, particularly the latter group, but showed no activity against class A enzymes. To enhance the carbonyl electrophilicity, an alpha-difluorinated analogue (R = H, CHR' = CF2, n = 0, R'' = Et) was also prepared, but no enhanced inhibitory activity was observed. All evidence suggested that these compounds inhibited in the carbonyl form rather than by formation of tetrahedral adducts at the beta-lactamase active site. They show promise as leads to specific class D beta-lactamase inhibitors.