Molecular characterization of the PhoPQ-PmrD-PmrAB mediated pathway regulating polymyxin B resistance in Klebsiella pneumoniae CG43

<p>Abstract</p> <p>Background</p> <p>The cationic peptide antibiotic polymyxin has recently been reevaluated in the treatment of severe infections caused by gram negative bacteria.</p> <p>Methods</p> <p>In this study, the genetic determinants for capsular polysaccharide level and lipopolysaccharide modification involved in polymyxin B resistance of the opportunistic pathogen <it>Klebsiella pneumoniae </it>were characterized. The expressional control of the genes responsible for the resistance was assessed by a LacZ reporter system. The PmrD connector-mediated regulation for the expression of <it>pmr </it>genes involved in polymyxin B resistance was also demonstrated by DNA EMSA, two-hybrid analysis and <it>in vitro </it>phosphor-transfer assay.</p> <p>Results</p> <p>Deletion of the <it>rcsB</it>, which encoded an activator for the production of capsular polysaccharide, had a minor effect on <it>K. pneumoniae </it>resistance to polymyxin B. On the other hand, deletion of <it>ugd </it>or <it>pmrF </it>gene resulted in a drastic reduction of the resistance. The polymyxin B resistance was shown to be regulated by the two-component response regulators PhoP and PmrA at low magnesium and high iron, respectively. Similar to the control identified in <it>Salmonella</it>, expression of <it>pmrD </it>in <it>K. pneumoniae </it>was dependent on PhoP, the activated PmrD would then bind to PmrA to prolong the phosphorylation state of the PmrA, and eventually turn on the expression of <it>pmr </it>for the resistance to polymyxin B.</p> <p>Conclusions</p> <p>The study reports a role of the capsular polysaccharide level and the <it>pmr </it>genes for <it>K. pneumoniae </it>resistance to polymyxin B. The PmrD connector-mediated pathway in governing the regulation of <it>pmr </it>expression was demonstrated. In comparison to the <it>pmr </it>regulation in <it>Salmonella</it>, PhoP in <it>K. pneumoniae </it>plays a major regulatory role in polymyxin B resistance.</p

Improving Antigenicity of the Recombinant Hepatitis C Virus Core Protein via Random Mutagenesis

In order to enhance the sensitivity of diagnosis, a recombinant clone containing domain I of HCV core (amino acid residues 1 to 123) was subjected to random mutagenesis. Five mutants with higher sensitivity were obtained by colony screening of 616 mutants using reverse ELISA. Sequence analysis of these mutants revealed alterations focusing on W84, P95, P110, or V129. The inclusion bodies of these recombinant proteins overexpressed in E. coli BL21(DE3) were subsequently dissolved using 6 M urea and then refolded by stepwise dialysis. Compared to the unfolded wild-type antigen, the refolded M3b antigen (W84S, P110S and V129L) exhibited an increase of 66% antigenicity with binding capacity of 0.96 and affinity of 113 μM−1. Moreover, the 33% decrease of the production demand suggests that M3b is a potential substitute for anti-HCV antibody detection

Novel variant Pro143Ala in HTRA2 contributes to Parkinson’s disease by inducing hyperphosphorylation of HTRA2 protein in mitochondria

Mutations in the gene encoding the mitochondrial protein high temperature requirement A2 (HTRA2) are inconsistently associated with a risk of Parkinson’s disease (PD). We assessed the presence of HTRA2 mutations among patients with PD and performed functional assay of identified mutations or variants. Among the total 1,373 subjects, the entire HTRA2 coding region was sequenced in 113 early-onset PD (EOPD), 20 familial PD patients and 150 control subjects. An additional 390 sporadic late-onset PD patients and 700 controls were subsequently screened to validate possible mutations found in the first set. We identified two novel heterozygous variants, c.427C > G (Pro143Ala) and c.906 +3 G > A, in 2 (1.5%) EOPD patients. The missense variant, Pro143Ala, was also observed in one late-onset PD patient but was absent in total 850 control subjects (relative risk 2.3, 95% CI 1.5–2.8, P = 0.04). Expressing Pro143Ala variant of HTRA2 in primary dopaminergic neurons causes neurite degeneration. Following exposure to rotenone, the ultra-structural mitochondrial abnormality, the percentage of mitochondrial dysfunction and apoptosis in cells carrying the HTRA2 Pro143Ala variant was significantly higher than wild-type cells. Mechanistically, protein level of phosphorylated HTRA2 was increased in cells carrying the Pro143Ala variant, suggesting Pro143Ala variant promotes HTRA2 phosphorylation with resultant mitochondrial dysfunction. Our results support a biologically relevant role of HTRA2 in PD susceptibility in Taiwanese. Further large-scale association studies are warranted to confirm the role of HTRA2 Pro143Ala variant in the risk of PD

Isokinetic eccentric exercise can induce skeletal muscle injury within the physiologic excursion of muscle-tendon unit: a rabbit model

BACKGROUND AND PURPOSE: Intensive eccentric exercise can cause muscle damage. We simulated an animal model of isokinetic eccentric exercise by repetitively stretching stimulated triceps surae muscle-tendon units to determine if such exercise affects the mechanical properties of the unit within its physiologic excursion. METHODS: Biomechanical parameters of the muscle-tendon unit were monitored during isokinetic eccentric loading in 12 rabbits. In each animal, one limb (control group) was stretched until failure. The other limb (study group) was first subjected to isokinetic and eccentric cyclic loading at the rate of 10.0 cm/min to 112% (group I) or 120% (group II) of its initial length for 1 hour and then stretched to failure. Load-deformation curves and biomechanical parameters were compared between the study and control groups. RESULTS: When the muscle-tendon unit received eccentric cyclic loading to 112%, changes in all biomechanical parameters – except for the slope of the load-deformation curve – were not significant. In contrast, most parameters, including the slope of the load-deformation curve, peak load, deformation at peak load, total energy absorption, and energy absorption before peak load, significantly decreased after isokinetic eccentric cyclic loading to 120%. CONCLUSION: We found a threshold for eccentrically induced injury of the rabbit triceps surae muscle at between 12% and 20% strain, which is within the physiologic excursion of the muscle-tendon units. Our study provided evidence that eccentric exercise may induce changes in the biomechanical properties of skeletal muscles, even within the physiologic range of the excursion of the muscle-tendon unit

The prevalence of ocular diseases in primary and junior high school students on Orchid Island

AbstractObjectiveTo assess the prevalence of refractive error and ocular diseases in primary and junior high school students on Orchid Island.Materials and MethodsThis is a cross-sectional study of all students in the primary and junior high schools on Orchid Island conducted within 1 week in 2008. Each student received a visual acuity examination without correction with the Landolt-C chart. An experienced ophthalmologist performed associated assessments through retinoscopy, slit lamp, and fundoscopy.ResultsOf the 403 student residents, 260 were primary school students (139 boys and 121 girls) and 143 were junior high school students (74 boys and 69 girls). Visual acuity in two eyes was < 0.1, in 14 eyes was between 0.1 and 0.3, in 34 eyes was between 0.4 and 0.7, in 225 eyes was between 0.8 and 1.0, and in 531 eyes was between 1.2 and 2.0. Myopia was found in 21 students (21/403, 5.21%; 9 primary school students and 12 junior high school students). Four students (4/403, 0.99%) had amblyopia, of whom two had anisometropia (unilateral high hyperopia), one had high astigmatism in both eyes, and the other had unilateral esotropia. Lens dislocation was found in one student (0.25%) with Marfan syndrome. Retinal vasculitis and optic atrophy were found in one student (0.25%) with systemic lupus erythematosus.ConclusionBecause it is a small, isolated island, Orchid Island still has a unique traditional culture and life style. Therefore the prevalence of myopia in primary school and junior high school students on Orchid Island is low, and 94% of all the students had uncorrected visual acuity above 0.8

Antimicrobial Drug Resistance in Pathogens Causing Nosocomial Infections at a University Hospital in Taiwan, 1981-1999

To determine the distribution and antimicrobial drug resistance in bacterial pathogens causing nosocomial infections, surveillance data on nosocomial infections documented from 1981 to 1999 at National Taiwan University Hospital were analyzed. During this period, 35,580 bacterial pathogens causing nosocomial infections were identified. Candida species increased considerably, ranking first by 1999 in the incidence of pathogens causing all nosocomial infections, followed by Staphylococcus aureus and Pseudomonas aeruginosa. Candida species also increased in importance as bloodstream infection isolates, from 1.0% in 1981-1986 to 16.2% in 1999. The most frequent isolates from urinary tract infections were Candida species (23.6%), followed by Escherichia coli (18.6%) and P. aeruginosa (11.0%). P. aeruginosa remained the most frequent isolates for respiratory tract and surgical site infections in the past 13 years. A remarkable increase in incidence was found in methicillin-resistant S. aureus (from 4.3% in 1981-1986 to 58.9% in 1993-1998), cefotaxime-resistant E. coli (from 0% in 1981-1986 to 6.1% in 1993-1998), and cefotaxime-resistant Klebsiella pneumoniae (from 4.0% in 1981-1986 to 25.8% in 1993-1998). Etiologic shifts in nosocomial infections and an upsurge of antimicrobial resistance among these pathogens, particularly those isolated from intensive care units, are impressive and alarming

Detection of SARS-associated Coronavirus in Throat Wash and Saliva in Early Diagnosis

Early detection of SARS-CoV in throat wash and saliva suggests that these specimens are ideal for SARS diagnosis

Modernização e ressignificação: as contradições na formação do espaço urbano oeirense (1900-1945).

A presente dissertação visa a analisar a produção do espaço urbano oeirense e suas eventuais contradições compreendendo o período de 1900 a 1945, atentando para a ideia de crise atribuída à perda do status de capital e à introdução de alguns signos do moderno, tomados pelas elites como forma de ressignificação da cidade. Portanto, neste propósito, constitui-se um projeto de modernização urbana ilusória e de exclusão, pois apenas o núcleo central urbano foi merecedor dessa ação. Enquanto isso, o subúrbio estava fora de qualquer tentativa de melhoramento, embora fantasmagoricamente o discurso de modernização para todos fosse divulgado de modo homogêneo entre o grupo político e os letrados locais. Buscamos ainda identificar o ressentimento presente no imaginário local e suas formas reativas, como também as relações do cotidiano dos citadinos. No entanto, esta política de reforma urbana, além de ser destinadas a obras que priorizavam a cultura moderna e o embelezamento urbano, imprimiu um conjunto de normas e condutas, inclusive higiênicas aos moradores da área central. Muitas das quais pretendiam afastar sujeiras e doenças epidêmicas, mas, nesse meio tempo, as zonas suburbana e rural eram consumidas por doenças como a febre tifoide e a malária, sem nenhuma intervenção pública no sentido de preveni-las. Por fim, nesta pesquisa, apoiamo-nos em diversas fontes, sendo elas documentais, fotográficas, orais e bibliográficas. Buscamos encontrar nelas as evidências do passado local, que nos possibilitaram compreender o cotidiano da sociedade oeirense.This dissertation aims to examine the first attempts to modernize in Oeiras-PI, the time frame of 1900-1945, including the idea of seeking crisis attributed to this city by the intellectuals and the local political elite after the transfer of capital to these featuring the "decadent", generalizing the effect of the political crisis and some material loss. Which in turn, not elapsed in its entirety by the loss of capital status, but by their own economic conditions of the state and the municipality, however, not noted by this group insisted that the tirade of "crisis." It also aims to understand the transformations in everyday life oeirense from some achievements, such as theater, traveling cinema, newspaper production, and strengthening the local economy caused by the extraction of latex maniçoba in the early years of the twentieth century. Only after 1930 the political and intellectual discourse points to the advent of local progress, which now has the following material and symbolic transformations: a) urban sanitation; b) construction of the town hall, with modern architecture to the city standards; c) Installation of electric light; d) construction of the airfield with weekly landing; e) film and square. The arrival of these elements of modernity in a society characterized by parochial habits causes changes in sociability and sensitivities both within the elite and among the popular. The sources used in this work are based on surveys of official documents, oral, pictures, newspapers and local literature