Role of H- and D- MATE-Type Transporters from Multidrug Resistant Clinical Isolates of Vibrio fluvialis in Conferring Fluoroquinolone Resistance

Background: The study seeks to understand the role of efflux pumps in multidrug resistance displayed by the clinical isolates of Vibrio fluvialis, a pathogen known to cause cholera-like diarrhoea. Methodology: Two putative MATE family efflux pumps (H- and D-type) were PCR amplified from clinical isolates of V. fluvialis obtained from Kolkata, India, in 2006 and sequenced. Bioinformatic analysis of these proteins was done to predict protein structures. Subsequently, the genes were cloned and expressed in a drug hypersusceptible Escherichia coli strain KAM32 using the vector pBR322. The recombinant clones were tested for the functionality of the efflux pump proteins by MIC determination and drug transport assays using fluorimeter. Results: The sequences of the genes were found to be around 99 % identical to their counterparts in V. cholerae. Protein structure predicting servers TMHMM and I-TASSER depicted ten-twelve membrane helical structures for both type of pumps. Real time PCR showed that these genes were expressed in the native V. fluvialis isolates. In the drug transport assays, the V. fluvialis clinical isolates as well as recombinant E. coli harbouring the efflux pump genes showed the energydependent and sodium ion-dependent drug transport activity. KAM32 cells harbouring the recombinant plasmids showed elevated MIC to the fluoroquinolones, norfloxacin and ciprofloxacin but H-type pumps VCH and VFH from V. cholerae and V. fluvialis respectively, showed decreased MIC to aminoglycosides like gentamicin, kanamycin and streptomycin. Decrease i

Down-Regulation of GEP100 Causes Increase in E-Cadherin Levels and Inhibits Pancreatic Cancer Cell Invasion

AIMS: Invasion and metastasis are major reasons for pancreatic cancer death and identifying signaling molecules that are specifically used in tumor invasion is of great significance. The purpose of this study was to elucidate the role of GEP100 in pancreatic cancer cell invasion and metastasis and the corresponding molecular mechanism. METHODS: Stable cell lines with GEP100 knocked-down were established by transfecting GEP100 shRNA vector into PaTu8988 cells and selected by puromycin. qRT-PCR and Western blot were performed to detect gene expression. Matrigel-invasion assay was used to detect cancer cell invasion in vitro. Liver metastasis in vivo was determined by splenic injection of indicated cell lines followed by spleen resection. Immunofluorescence study was used to detect the intracellular localization of E-cadherin. RESULTS: We found that the expression level of GEP100 protein was closely related to the invasive ability of a panel of 6 different human pancreatic cancer cell lines. Down-regulation of GEP100 in PaTu8988 cells significantly decreased invasive activity by Matrigel invasion assay, without affecting migration, invasion and viability. The inhibited invasive activity was rescued by over-expression of GEP100 cDNA. In vivo study showed that liver metastasis was significantly decreased in the PaTu8988 cells with GEP100 stably knocked-down. In addition, an epithelial-like morphological change, mimicking a mesenchymal to epithelial transition (MET) was induced by GEP100 down-regulation. The expression of E-cadherin protein was increased 2-3 folds accompanied by its redistribution to the cell-cell contacts, while no obvious changes were observed for E-cadherin mRNA. Unexpectedly, the mRNA of Slug was increased by GEP100 knock-down. CONCLUSION: These findings provided important evidence that GEP100 plays a significant role in pancreatic cancer invasion through regulating the expression of E-cadherin and the process of MET, indicating the possibility of it becoming a potential therapeutic target against pancreatic cancer

Survey of childhood empyema in Asia: Implications for detecting the unmeasured burden of culture-negative bacterial disease

<p>Abstract</p> <p>Background</p> <p>Parapneumonic empyema continues to be a disease of significant morbidity and mortality among children despite recent advances in medical management. To date, only a limited number of studies have assessed the burden of empyema in Asia.</p> <p>Methods</p> <p>We surveyed medical records of four representative large pediatric hospitals in China, Korea, Taiwan and Vietnam using <it>ICD</it>-10 diagnostic codes to identify children <16 years of age hospitalized with empyema or pleural effusion from 1995 to 2005. We also accessed microbiology records of cultured empyema and pleural effusion specimens to describe the trends in the epidemiology and microbiology of empyema.</p> <p>Results</p> <p>During the study period, we identified 1,379 children diagnosed with empyema or pleural effusion (China, n = 461; Korea, n = 134; Taiwan, n = 119; Vietnam, n = 665). Diagnoses of pleural effusion (n = 1,074) were 3.5 times more common than of empyema (n = 305), although the relative proportions of empyema and pleural effusion noted in hospital records varied widely between the four sites, most likely because of marked differences in coding practices. Although pleural effusions were reported more often than empyema, children with empyema were more likely to have a cultured pathogen. In addition, we found that median age and gender distribution of children with these conditions were similar across the four countries. Among 1,379 empyema and pleural effusion specimens, 401 (29%) were culture positive. <it>Staphylococcus aureus </it>(n = 126) was the most common organism isolated, followed by <it>Streptococcus pneumoniae </it>(n = 83), <it>Pseudomonas aeruginosa </it>(n = 37) and <it>Klebsiella </it>(n = 35) and <it>Acinetobacter </it>species (n = 34).</p> <p>Conclusion</p> <p>The age and gender distribution of empyema and pleural effusion in children in these countries are similar to the US and Western Europe. <it>S. pneumoniae </it>was the second leading bacterial cause of empyema and pleural effusion among Asian children. The high proportion of culture-negative specimens among patients with pleural effusion or empyema suggests that culture may not be a sufficiently sensitive diagnostic method to determine etiology in the majority of cases. Future prospective studies in different countries would benefit from standardized case definitions and coding practices for empyema. In addition, more sensitive diagnostic methods would improve detection of pathogens and could result in better prevention, treatment and outcomes of this severe disease.</p

A Membrane Fusion Protein αSNAP Is a Novel Regulator of Epithelial Apical Junctions

Tight junctions (TJs) and adherens junctions (AJs) are key determinants of the structure and permeability of epithelial barriers. Although exocytic delivery to the cell surface is crucial for junctional assembly, little is known about the mechanisms controlling TJ and AJ exocytosis. This study was aimed at investigating whether a key mediator of exocytosis, soluble N-ethylmaleimide sensitive factor (NSF) attachment protein alpha (αSNAP), regulates epithelial junctions. αSNAP was enriched at apical junctions in SK-CO15 and T84 colonic epithelial cells and in normal human intestinal mucosa. siRNA-mediated knockdown of αSNAP inhibited AJ/TJ assembly and establishment of the paracellular barrier in SK-CO15 cells, which was accompanied by a significant down-regulation of p120-catenin and E-cadherin expression. A selective depletion of p120 catenin effectively disrupted AJ and TJ structure and compromised the epithelial barrier. However, overexpression of p120 catenin did not rescue the defects of junctional structure and permeability caused by αSNAP knockdown thereby suggesting the involvement of additional mechanisms. Such mechanisms did not depend on NSF functions or induction of cell death, but were associated with disruption of the Golgi complex and down-regulation of a Golgi-associated guanidine nucleotide exchange factor, GBF1. These findings suggest novel roles for αSNAP in promoting the formation of epithelial AJs and TJs by controlling Golgi-dependent expression and trafficking of junctional proteins

Using multimodal biometrics, data hiding and encryption for secure healthcare imaging system

In  this  digital  era,  images  are  the  most  vital  in-formation  carrier  used  for  healthcare  communication  and  en-tertainment.  However,  the  increasing  use  of  images  in  severalapplications also poses a risk of their unauthorised usage or mod-ification  without  proper  attribution  to  the  owner.  To  overcomethis issue while ensuring one-time password (OTP)–based systemauthentication,  this  study  designed  a  highly  secure  healthcareimaging  system  with  multimodal  biometrics,  data  hiding  andencryption in a deep learning environment. First, we segmenteda   medical   image   via   a   customised,   deep   neural   network   tolocate the lesion and non-lesion areas.  Next, the lesion part wasembedded into the non-lesion part via least significant bit (LSB)substitution and timestamp. Furthermore, the marked non-lesionand  lesion  parts  were  combined  to  generate  the  marked  image.Second,  encoded  multimodal  biometric  features,  i.e.  face  andiris,  and  a  novel  2D  chaotic  system  were  used  to  encrypt  themarked  image  before  transmission  over  the  network.  Throughsimulation  findings  on  security  and  accuracy  of  segmentationand  feature  extraction  design,  we  demonstrated  the  feasibilityand  effectiveness  of  our  proposed  secure  system,  highlightingtheir  superior  performance  compared  to  existing  techniques.</p