A Muscle-Specific p38 MAPK/Mef2/MnSOD Pathway Regulates Stress, Motor Function, and Life Span in Drosophila

SummaryMolecular mechanisms that concordantly regulate stress, life span, and aging remain incompletely understood. Here, we demonstrate that in Drosophila, a p38 MAP kinase (p38K)/Mef2/MnSOD pathway is a coregulator of stress and life span. Hence, overexpression of p38K extends life span in a MnSOD-dependent manner, whereas inhibition of p38K causes early lethality and precipitates age-related motor dysfunction and stress sensitivity, that is rescued through muscle-restricted (but not neuronal) add-back of p38K. Additionally, mutations in p38K are associated with increased protein carbonylation and Nrf2-dependent transcription, while adversely affecting metabolic response to hypoxia. Mechanistically, p38K modulates expression of the mitochondrial MnSOD enzyme through the transcription factor Mef2, and predictably, perturbations in MnSOD modify p38K-dependent phenotypes. Thus, our results uncover a muscle-restricted p38K-Mef2-MnSOD signaling module that influences life span and stress, distinct from the insulin/JNK/FOXO pathway. We propose that potentiating p38K might be instrumental in restoring the mitochondrial detoxification machinery and combating stress-induced aging

A study on outcome of hepatic fibrosis in chronic hepatitis B patients on anti-viral therapy

Background: Hepatitis B infection is a global public health problem. Chronic hepatitis B (CHB) infection leads to complications such as cirrhosis, liver failure, and hepatocellular carcinoma. Effective antiviral therapy significantly improves the stages of fibrosis in these patients. Hence, the liver stiffness measurement using transient elastography (Fibroscan) in CHB patients undergoing treatment is important to evaluate the effectiveness of the therapy and to predict the prognosis. Aims and Objectives: To see the outcome of Hepatic Fibrosis by non-invasive measurement in CHB patients after 6 months of oral anti-viral therapy. Materials and Methods: Seventy new CHB patients are included in a prospective hospital-based study and at the end of 6 months 53 patients were analysed. Results: Our study showed a significant statistical reduction in Liver Fibrosis as well as improvement of serological and biochemical parameters in CHB patients. Conclusion: There is liver fibrosis reversal in CHB patients after 6 months of anti-viral therapy and Fibroscan helps not only as a marker for initiation of treatment depending on the degree of fibrosis but also indicates the response or progression of the disease

Genetic Characterisation of Colistin Resistant Klebsiella pneumoniae Clinical Isolates From North India

BackgroundIncreasing use of colistin has led to the world-wide emergence of mobile colistin resistant gene (mcr). The present study aimed to identify and characterise mcr and other drug-resistant genes in colistin resistant Klebsiella pneumoniae clinical isolates.MethodsTwenty-two colistin resistant K. pneumoniae were analysed for mcr and other drug-resistant genes, efflux pumps, and virulence genes, and for their biofilm forming ability. Pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing (MLST) were performed for all mcr-1 positive isolates. S1-PFGE and Southern hybridisation were performed for localisation of mcr-1 and blaNDM.ResultsNineteen colistin resistant K. pneumoniae harboured mcr-1 and 3 had mgrB disruption. All isolates harboured blaOXA-48-type and ESBL genes; eight strains (five with mcr-1 and three with mgrB disruption) co-harboured blaNDM. Efflux pumps genes AcrAB and mdtK were detected in all 22 and tol-C in 21 isolates. Virulence-related genes entB and irp-1 were detected in all 22, mrkD in 20, and fimH-1 in 18 isolates; 11 isolates were strong biofilm producers. PFGE clustered mcr-1 positive isolates into eight groups based on ≥90% similarity; MLST revealed diverse sequence types, predominant being ST-15 (n = 4) and ST-16 (n = 4). Both mcr-1 and blaNDM were localised on plasmid and chromosome; mcr-1 was present on IncFII type and blaNDM on IncFIB and IncA/C type plasmids.ConclusionsColistin resistance in K. pneumoniae was predominantly mediated by mcr-1. Co-existence of colistin, carbapenem, and other drug-resistant genes along with efflux pumps indicates towards enormous genomic plasticity in K. pneumoniae with ability to emerge as super-spreader of drug-resistance

Comparative evaluation of IS6110 PCR via conventional methods in rapid diagnosis of new and previously treated cases of extrapulmonary tuberculosis

ÖZET Yeni ve önceden tedavi edilmiş ekstrapulmoner tüberkülozlu hastaların hızlı tanısında IS6110 ile konvansiyonel yöntemlerin karşılaştırılmalı değerlendirilmesi Gelişmekte olan ülkelerde ekstrapulmoner tüberküloz (EPTB) tanısı önemli bir problemdir. EPTB'de, az sayıda basil içerme özelliği, yetersiz miktarda örnek gibi birçok sorun bulunmaktadır. Bütün bu kısıtlamalar, konvansiyonel bakteriyolojik tekniklerin EPTB tanısına düşük katkısına neden olmaktadır. Nükleik asit amplifikasyon yöntemleri, mikobakteriyel DNA'nın saptanması amacıyla geliştirilen hızlı ve duyarlı tekniklerdir. Mycobacterium tuberculosis complex'in spesifik genomunda yer alan "insertion sequence" IS6110'a ait 123bp'nin DNA fragmanı, EPTB'nin hızlı tanısı amacıyla polimeraz zincir reaksiyonu (PCR) ile çoğaltıldı. Bu çalışmada, yeni ve önceden tedavi edilmiş EPTB'li hastaların hızlı tanısında IS6110 PCR ile konvansiyonel yöntemler karşılaştırıldı. EPTB şüpheli hastalardan 450 örnek toplandı ve Mycobacteria için Zeihl Neelson (ZN) boyama ve M. tuberculosis için BACTEC kültürü yapıldı. Bütün örnekler ayrıca, M. tuberculosis complex'in insertion element IS6110'un 123bp fragmanını hedefleyen primerlerle PCR amplifikasyonu ile IS6110 için çalışıldı. Testler arasında duyarlılık bakımından anlamlı fark saptandı. Dört yüz elli örnek . Bununla birlikte, testler arasında spesifisite bakımından anlamlı fark yoktu (p> 0.05). IS6110 PCR'nin hem yeni hem de önceden tedavi edilmiş hastalarda, yayma mikroskopi ve BACTEC kültüründen daha duyarlı olduğunu bulduk. IS6110 PCR, yeni ve önceden tedavi edilmiş EPTB'li hastaların tanısında kullanışlı olabilir. Şüpheli EPTB'li hastaların tedavi kararında fayda sağlayabilir. Anahtar Kelimeler: Tüberküloz, ekstrapulmoner tüberküloz, polimeraz zincir reaksiyonu, IS6110. Yazışma Adresi (Address for Correspondence): Dr. Surya KANT, Chhatrapati Shahu Ji Maharaj Medical University UP (Erstwhile King George Medical College), LUCKNOW -INDIA e-mail: [email protected] Tuberculosis (TB) continues to be a major global public health problem. Incidence of extrapulmonary tuberculosis (EPTB) is on increasing worldwide as well as in India (1,2). EPTB compromises 20% of all TB cases in India (3). Diagnosis of EPTB in different clinical presentations has been always as challenge. Smear microscopy and culture lack of sensitivity in EPTB case and culture (solid and liquid media) also takes at least two to four weeks for grow of mycobacteria. A study has reported smear positive is around 10-37% of the patients and mycobacterial culture is positive in variable proportional 12-80% in different biological specimens (3). Studies from many laboratories around the global were using primers most commonly targeting the IS6110 insertion element (4-9). The detection of the IS6110 insertion element present in form of multiple copies to detect of Mycobacterium tuberculosis complex but not other mycobacterial species (9-11). Polymerase chain reaction (PCR) using IS6110 insertion sequences as the target, has potential to conquer limitation of conventional method and to established as rapid, sensitive technique for detecting DNA of M. tuberculosis in different clinical specimens from respiratory and non respiratory sites MATERIALS and METHODS Study Design The study was performed prospectively in a blinded manner. Clinical Specimens and Data Collection 2-5 mL of specimens was collected from 450 specimens, non-repeated specimens from suspected cases of extrapulmonary tuberculosis. The specimens were included as Lymph Node Aspirate and Cold Abscesses, Pleural fluid, C.S.F, Synovial Fluid, Ascetic Fluid, Urine, Gastric Aspirate, Pus, Bone Marrow, Wound and Pus swab and Others specimens (biopsies tissues). All specimens were kept in ice box and transported Mycobacteriology Laboratory, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India for smear examination by ZN Staining, BACTEC Culture and PCR test. All patients were signed with due informed consent of the patients from indoor and outward wards of Department of Pulmonary Medicine, Chhatrapati Shahuji Maharaj Medical University, Lucknow, India and Mycobacteriology Laboratory, Department of Microbiology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India during Jan 2009 to Dec 2010. The clinical history regarding, present and past history of antitubercular treatment (ATT); family history of tuberculosis and any other associated disease were taken in prescribed Performa. Microbiological Analysis of Extra Pulmonary Specimens Specimens was divided in to two part one part was kept at -20 for PCR till processing and another part was processed for mycobacterial smear preparation and BACTEC culture. Smears were stained with Ziehl Neelsen (ZN) method and examined for acid-fast bacilli (AFB) (21). BACTEC vials were incubated and interpreted as per Becton Dickinson (BD, Sparks, MD, USA) manual instructions (22). NAP (p-nitro-α-acetylamino-β-hydroxy propiophenone) (Becton Dickinson, Sparks, MD, USA), identification was done to differentiate M. tuberculosis form non tuberculous mycobacteria (22). A decrease or unchanged growth index (GI) in nap vial indicated presence of M. tuberculosis complex (MTBC), while an increase in GI indicated the presence of Mycobacterium other than tuberculosis (MOTT). Standard H 37 Rv strain of M. tuberculosis complex was used as positive control. Extraction of DNA Extraction of DNA was done by the CTAB (cetyl-trimethyl-ammonium bromide) -phenol chloroform extraction method (23). Specimens were centrifuged at 10.000 rpm for 10 min. The supernatant was discarded and the pellet suspended in 567 µL of TE (Tris EDTA, pH 7.4) buffer, 30 µL 10% SDS (sodium dodecyl sulfate) and 3 µL proteinase K (20 mg/mL), mixed and incubated at 37°C for 1 hour. After incubation, 100 µL of 5 M NaCl and 80 µL of high-salt CTAB buffer (containing 4 M NaCl, 1.8% CTAB was added and mixed followed by incubation at 65°C for 10 min. An approximate equal volume (0.7-0.8 µL) of chloroform-isoamyl alcohol (24.1) was added, mixed thoroughly and centrifuged for 4-5 min in a microcentrifuge at 12.000 rpm. The aqueous viscous supernatant was carefully decanted and transferred to a new tube. An equal volume of phenol: chloroform-isoamyl alcohol (1:1) was added followed by a 5 min spin at 12.000 rpm. The supernatant was separated and then mixed with 0.6 volume of isopropanol to get a precipitate. The precipitated nucleic acids were washed with 75% ethanol, dried and re-suspended in 100 µL of TE buffer. Primer and IS6110 PCR The amplification reaction was performed in a final volume of 20 µL. the reaction mixture contained 10 µL Pyrostart Fast PCR Master Mix 2X (dNTP, Taq polymerase with Mgcl 2 , Fermentas, India), 1 µL (10 pmole) of each primer, 3 µL water (nuclease free) and 5 µL of extracted DNA. The oligonucleotide primers used were IS1 and IS2, are: 5'-CCT GCG AGC GTA GGC GTC GG3' and 5' CTC GTC CAG CGC CGC TTC GG 3' respectively (SBS Gentech Co. Ltd) (24). These primers amplified a target fragment at 123 base pairs (bp) from the insertion, M. tuberculosis sequence element IS6110. The PCR amplification was done in thermal cycler (MJ Research, PTC-100, GMI, Inc, USA), which involved 40 cycles of denaturation at 94°C for 2 minute, annealing of primers at 68°C for 2 minute, and primer extension at 72°C for 1 minute. The amplified products were separated on 2% agarose gels, visualized on a UV-light transilluminator (Bangalore Genei, Bangalore, India). The presence of 123bp fragment indicate as positive test as M. tuberculosis complex. The positive controls included the DNA of H37Rv strain. Negative control included PCR grade water Statistical Analysis Data were analyzed using SPSS 15.0 (Statistical Package for the Social Sciences, Chicago, IL, USA) for Maurya AK, Kant S, Nag VL, Kushwaha RAS, Kumar M, Dhole TN. 215 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 Windows. The significance of difference was taken as significance value (p< 0.05).Sensitivity was calculated as [Tp/(Tp + Fn)] x 100; specificity was calculated as [Tn/(Tn + Fp)] x 100; Tp = total number of positives; Tn = total number of negatives; Fp = total number of false positive, Fn = total number of false negative; respectively. RESULTS Specimen's Characterization of Extrapulmonary Tuberculosis Cases During the two year study period, 470 clinical specimens were strong clinical suspicion of extrapulmonary tuberculosis were subjected from tertiary care hospitals and all mention test were performed. Out of these, 20 specimens found to be contaminated in BACTEC culture. 450 specimens of results were used in the study. Out of 450 specimens, 153 (34%) lymph node aspirate and cold abscesses, 58 (12.8%) pleural fluid, 44 (9.7%) cerebrum spinal fluid (CSF), 48 (10.7%) urine, 31(6.8%) ascetic fluid, 26 (5.8%) pus, 22 (4.9%) wound and pus swab, 16 (3.5%) gastric aspirate, 10 (2.2%) bone marrow, 10 (2.2%) synovial fluid and 30 (6.7%) others specimens (biopsies tissues). Out of 450 patients, 320 (71.1%) patients were males and 130 (28.9%) females. The mean age of all patients was 39.8 ± 16.1 years. Patients 25-44 years of age accounted for 45% of the total cases. Out of 450 cases, 328 (72.8%) were new cases and 122 (22.2%) were previously treated cases of EPTB. Detection Rate of M. tuberculosis by IS6110 PCR, BACTEC Culture and ZN Smear Microscopy According to New Cases and Previously Treated Cases All specimens were colleted from suspected case of extra pulmonary tuberculosis were found to be AFB positive were 60 (13.4%). On the basis of cases, we found that sensitivity of AFB staining on EPTB were 37 (11.2%) in new cases and 23 (18.8%) in previously treated cases. The sensitivity of AFB staining was higher in comparison to previously treated cases. Overall detection rate of M. tuberculosis by AFB Staining was 60 (13.4%). The detection of M. tuberculosis by BACTEC culture was 202 (45%). Results of BACTEC culture according to cases, 151 (46.03%) were in new cases and 51 (41.8%) were in previously treated cases. We found that sensitivity of BACTEC culture was higher in new cases. All culture isolates obtained were confirmed as mycobacteria with biochemical tests mentioned. Using IS 6110 PCR, 283 (61.8%) were positive for IS6110 PCR for M. tuberculosis. 203 (61.8%) were positive in new cases and 80 (65.5%) were positive in previously treated cases. We found that sensitivity of IS6110 PCR was higher in previously treated cases. Overall comparison of tests, IS6110 PCR was found to have much higComparative evaluation of IS6110 PCR via conventional methods in rapid diagnosis of new and previously treated cases of extrapulmonary tuberculosis 216 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 Comparison of Sensitivity of IS6110 PCR Test Via Others Conventional Tests According to New Cases and Previously Treated Cases IS6110 PCR test was found to be much more sensitive than ZN staining and BACTEC culture results individually as well as in combination are shown in 217 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 DISCUSSION Tuberculosis (TB) is a major public health dilemma in India. India is the highest TB burden country accounting for one fifth of the global incidence. Global annual incidence estimate is 9.4 million cases out of which it is estimated that 1.98 million cases are from India (26). In India, EPTB comprises 20% of all TB cases. Its prevalence in the country varies between 8.3-13.1% in different districts according to cohort analysis by Central TB Division, Ministry of Health and Family Welfare in 2002 (27,28). The diagnosis of extrapulmonary tuberculosis is till now challenging for diagnostic routine laborites. Numeric reasons are showing that, lack of adequate specimens amounts or volumes; distribute of the specimens for different diagnostic tests (histology/cytology, biochemical analysis, microbiology, and PCR), non-uniform distribution of microorganisms; paucibacillary nature of the specimens; presence of inhibitors that undermine the performance of nucleic acid amplification-based techniques; and the lack of an efficient sample processing technique universally applicable on all types of extrapulmonary samples (29). The poor performance of conventional M. tuberculosis detection techniques, based on microscopic examination of Ziehl-Neelsen stained and culture of M. tuberculosis (LJ Medium and BACTEC Radiometric culture) are still in widespread use for diagnostic purposes, still though they fail to provide the required sensitivity and specificity. The PCR test would be particularly useful in the diagnosis of EPTB where conventional microbiological techniques for M. tuberculosis are showing poor performance of sensitivity. The specificity, sensitivity and speed of PCR test in diagnosis of M. tuberculosis infection shown in this study should encourage the use of this method in routine diagnosis of EPTB. Previously studies shown the success of microscopy is highly variable from 22% to 96% and most authors rate it at round 60% (30-32). Our results shown that sensitivity of smear microscopy was 13.7% and specificity was 100%. The sensitivity of microscopy depends on the clinical presentation and more than 10.000 bacilli per milliliter are necessary for secure microscopic positivity (33). Our studies shown that conventional bacteriological technique were positive in 202 (45%) specimens, where as IS6110 PCR showed that 283 (63%) specimens were positive for M. tuberculosis. The difference was found that to be statistical significant (p< 0.05). Several studies have been reported on PCR to detect M. tuberculosis (34-39). The detection of the IS6110 insertion element present in multiple copies to detect M. tuberculosis complex, but not other mycobacterial species 218 Tüberküloz ve Toraks Dergisi 2011; 59(3): 213-220 tion and PCR results were positive but BACTEC culture was negative; these could be the presence of nonviable mycobacteria in the sample as patients were receiving antitubercular treatment. IS6110 PCR test is higher sensitivity than microscopy and the culture and could help in therapeutic decision for patients with clinical suspicion of EPTB. CONCLUSION IS6110 PCR test for DNA specific M. tuberculosis may be hopes of a rapid and accurate diagnostic test for EPTB and it will help where conventional diagnosis fails and provisional diagnosis of tuberculosis is made on the basis of clinical presentation and histology/cytology examination without evidence of AFB. IS6110 PCR may be great potential to improve the clinician vision for the early diagnosis, treatment and prevention of EPTB. ACKNOWLEDGEMEN

Production of He-4 and (4) in Pb-Pb collisions at root(NN)-N-S=2.76 TeV at the LHC

Results on the production of He-4 and (4) nuclei in Pb-Pb collisions at root(NN)-N-S = 2.76 TeV in the rapidity range vertical bar y vertical bar <1, using the ALICE detector, are presented in this paper. The rapidity densities corresponding to 0-10% central events are found to be dN/dy4(He) = (0.8 +/- 0.4 (stat) +/- 0.3 (syst)) x 10(-6) and dN/dy4 = (1.1 +/- 0.4 (stat) +/- 0.2 (syst)) x 10(-6), respectively. This is in agreement with the statistical thermal model expectation assuming the same chemical freeze-out temperature (T-chem = 156 MeV) as for light hadrons. The measured ratio of (4)/He-4 is 1.4 +/- 0.8 (stat) +/- 0.5 (syst). (C) 2018 Published by Elsevier B.V.Peer reviewe

Inappropriate fixation of an endotracheal tube causing cuff malfunction resulting in difficult extubation

Abstract We discuss a case of difficult extubation, due to inadequate deflation of the tracheal tube cuff, despite collapse of the pilot balloon, on its aspiration. This was caused by inadvertent kinking of the pilot balloon tubing due to inappropriate tape fixation of the endotracheal tube

Identi¢cation ofa newRTX-like Gene Cluster InVibrio Cholerae

A gene cluster containing two genes in tandem has been identified in Vibrio cholerae ElTor N16961. Each has more than one cadherin domain and is homologous to the RTX toxin family and was common in various V. cholerae strains. Insertional mutagenesis demonstrated that each gene has a role in Hep-2 cell rounding, hemolytic activity towards human and sheep RBCs and biofilm formation. The mutants showed reduced adherence to intestinal epithelial cells as well as reduction of in vivo colonization in suckling mice. These two genes thus code for RTX-like toxins in V. cholerae and are associated with the pathogenecity of this organism