Biosynthesis and structure of the Burkholderia cenocepacia K56-2 lipopolysaccharide core oligosaccharide:truncation of the core oligosaccharide leads to increased binding and sensitivity to polymyxin B

Burkholderia cenocepacia is an opportunistic pathogen that displays a remarkably high resistance to antimicrobial peptides. We hypothesize that high resistance to antimicrobial peptides in these bacteria is because of the barrier properties of the outer membrane. Here we report the identification of genes for the biosynthesis of the core oligosaccharide (OS) moiety of the B. cenocepacia lipopolysaccharide. We constructed a panel of isogenic mutants with truncated core OS that facilitated functional gene assignments and the elucidation of the core OS structure in the prototypic strain K56-2. The core OS structure consists of three heptoses in the inner core region, 3-deoxy-d-manno-octulosonic acid, d-glycero-d-talo-octulosonic acid, and 4-amino-4-deoxy-l-arabinose linked to d-glycero-d-talo-octulosonic acid. Also, glucose is linked to heptose I, whereas heptose II carries a second glucose and a terminal heptose, which is the site of attachment of the O antigen. We established that the level of core truncation in the mutants was proportional to their increased in vitro sensitivity to polymyxin B (PmB). Binding assays using fluorescent 5-dimethylaminonaphthalene-1-sulfonyl-labeled PmB demonstrated a correlation between sensitivity and increased binding of PmB to intact cells. Also, the mutant producing a heptoseless core OS did not survive in macrophages as compared with the parental K56-2 strain. Together, our results demonstrate that a complete core OS is required for full PmB resistance in B. cenocepacia and that resistance is due, at least in part, to the ability of B. cenocepacia to prevent binding of the peptide to the bacterial cell envelope.

Diphenylamine increases cloacin DF13 sensitivity in avian septicemic strains of Escherichia coli

Thirteen avian septicemic isolates of Escherichia coli were examined for the presence of the aerobactin iron transport system. All of the strains possessed a functional aerobactin system and hybridization experiments showed that the aerobactin genes were located on ColV-type plasmids in all cases. The expression of the aerobactin receptor IutA was also studied by determining the bacterial susceptibility to the bacteriocin cloacin DF13. Twelve of the 13 isolates were cloacin-resistant but became sensitive to this bacteriocin upon treatment with diphenylamine which caused a reduction in the amount of O-side chain lipopolysaccharide.

Moxifloxacin sensitivity of respiratory pathogens in the United Kingdom

The in vitro activity of moxifloxacin and comparator agents against respiratory isolates from a range of geographically distinct centres around the United Kingdom was investigated in the following study. Clinical isolates of Streptococcus pneumoniae (n = 257), Haemophilus influenzae (n = 399) and Moraxella catarrhalis (n = 253) were obtained between March 1998 and April 1999 from nine centres in the United Kingdom. Sensitivity was determined by testing each isolate for its minimum inhibitory concentration (MIC) by agar dilution. Against Streptococcus pneumoniae moxifloxacin and grepafloxacin were the most active (MIC90 = 0.25 mg/l). Trovafloxacin and sparfloxacin were the next most active (MIC90 = 0.5 mg/l) followed by levofloxacin and ciprofloxacin. MIC90 values of the six fluoroquinolones versus H. influenzae ranged from ciprofloxacin > levofloxacin. Against M. catarrhalis the lowest MIC90 was that of grepafloxacin at 0.0625 mg/l followed by moxifloxacin, sparfloxacin, levofloxacin and ciprofloxacin. Trovafloxacin demonstrated the highest MIC90 at 0.5 mg/l. These results demonstrate that moxifloxacin has superior in vitro activity against respiratory tract pathogens than any other comparator quinolones available for clinical use.

Assessing the sensitivity and specificity of fish community indicators to management action

We assessed ten trophodynamic indicators of ecosystem status for their sensitivity and specificity to fishing management using a size-resolved multispecies fish community model. The responses of indicators to fishing depended on effort and the size selectivity (sigmoid or Gaussian) of fishing mortality. The highest specificity against sigmoid (trawl-like) size selection was seen from inverse fishing pressure and the large fish indicator, but for Gaussian size selection, the large species indicator was most specific. Biomass, mean trophic level of the community and of the catch, and fishing in balance had the lowest specificity against both size selectivities. Length-based indicators weighted by biomass, rather than abundance, were more sensitive and specific to fishing pressure. Most indicators showed a greater response to sigmoid than Gaussian size selection. Indicators were generally more sensitive at low levels of effort because of nonlinear sensitivity in trophic cascades to fishing mortality. No single indicator emerged as superior in all respects, so given available data, multiple complementary indicators are recommended for community monitoring in the ecosystem approach to fisheries management.

Clinicopathological features of homologous recombination-deficient epithelial ovarian cancers: Sensitivity to PARP inhibitors, platinum, and survival

Up to 50% of epithelial ovarian cancers (EOC) display defects in the homologous recombination (HR) pathway. We sought to determine the ramifications of the homologous recombination-deficient (HRD) status on the clinicopathologic features, chemotherapy response, and survival outcomes of patients with EOCs. HR status was determined in primary cultures from ascitic fluid in 50 chemotherapy-naïve patients by a functional RAD51 immunofluorescence assay and correlated with in vitro sensitivity to the PARP inhibitor (PARPi), rucaparib. All patients went on to receive platinum-based chemotherapy; platinum sensitivity, tumor progression, and overall survival were compared prospectively in HR-competent versus HRD patients. Compared with HR-competent patients, the HRD group was predominantly serous with a higher median CA125 at presentation. HRD was associated with higher ex vivo PARPi sensitivity and clinical platinum sensitivity. Median follow-up duration was 14 months; patients in the HRD group had lower tumor progression rates at 6 months, lower overall/disease-specific death rates at 12 months, and higher median survival. We therefore suggest that HRD as predicted by a functional RAD51 assay correlates with in vitro PARPi sensitivity, clinical platinum sensitivity, and improved survival outcome.

Conformational Change of Mitochondrial Complex I Increases ROS Sensitivity During Ischemia

Aims: Myocardial ischemia/reperfusion (I/R) is associated with mitochondrial dysfunction and subsequent cardiomyocyte death. The generation of excessive quantities of reactive oxygen species (ROS) and resultant damage to mitochondrial enzymes is considered an important mechanism underlying reperfusion injury. Mitochondrial complex I can exist in two interconvertible states: active (A) and deactive or dormant (D). We have studied the active/deactive (A/D) equilibrium in several tissues under ischemic conditions in vivo and investigated the sensitivity of both forms of the heart enzyme to ROS.

Results: We found that in the heart, t½ of complex I deactivation during ischemia was 10?min, and that reperfusion resulted in the return of A/D equilibrium to its initial level. The rate of superoxide generation by complex I was higher in ischemic samples where content of the D-form was higher. Only the D-form was susceptible to inhibition by H2O2 or superoxide, whereas turnover-dependent activation of the enzyme resulted in formation of the A-form, which was much less sensitive to ROS. The mitochondrial-encoded subunit ND3, most likely responsible for the sensitivity of the D-form to ROS, was identified by redox difference gel electrophoresis.

Innovation: A combined in vivo and biochemical approach suggests that sensitivity of the mitochondrial system to ROS during myocardial I/R can be significantly affected by the conformational state of complex I, which may therefore represent a new therapeutic target in this setting.

Conclusion: The presented data suggest that transition of complex I into the D-form in the absence of oxygen may represent a key event in promoting cardiac injury during I/R.

Predicting IQ of biologically "at risk" children from age 3 to school entry:sensitivity and specificity of the Stanford-Binet Intelligence Scale IV

Predictive validity of the Stanford-Binet Intelligence Scale Fourth Edition (S-B IV) from age 3 years to ages 4-5 years was evaluated with biologically "at risk" children without major sensory or motor impairments (n = 236). Using the standard scoring, children with full scale IQ <or = 84 on the Wechsler Preschool and Primary Scale of Intelligence at age 4-5 years were poorly identified (sensitivity 54%) from the composite S-B IV score at age 3. However, sensitivity improved greatly to 78% by including as a predictor the number of subtests the child was actually able to perform at age 3 years. Measures from the Home Screening Questionnaire and ratings of mother-child interaction further improved sensitivity to 83%. The standard method for calculating the composite score on the S-B IV excludes subtests with a raw score of 0, which overestimates cognitive functioning in young biologically high risk children. Accuracy of early identification was improved significantly by considering the number of subtests the child did not perform at age 3 years.

Pain sensitivity and temperament in extremely low-birth-weight premature toddlers and preterm and full-term controls

High-technology medical care of extremely low-birth-weight (ELBW) infants (<1001 g) involves repeated medical interventions which are potentially painful and may later affect reaction to pain. At 18 months corrected age (CCA), we examined parent ratings of pain sensitivity and how pain sensitivity ratings related to child temperament and parenting style in 2 groups of ELBW children (49 with a birth weight of 480-800 g and 75 with a birth weight of 801-1000 g) and 2 control groups (42 heavier preterm (1500-2499 g) and 29 full-birth-weight (FBW) children (> 2500 g). Both groups of ELBW toddlers were rated by parents as significantly lower in pain sensitivity compared with both control groups. The relationships between child temperament and pain sensitivity rating varied systematically across the groups. Temperament was strongly related to rated pain sensitivity in the FBW group, moderately related in the heavier preterm and ELBW 801-1000 g groups, and not related in the lowest birth-weight group (<801 g). Parental style did not mediate ratings of pain sensitivity. The results suggest that parents perceive differences in pain behavior of ELBW toddlers compared with heavier preterm and FBW toddlers, especially for those less than 801 g. Longitudinal research into the development of pain behavior for infants who experience lengthy hospitalization is warranted.

High sensitivity (1 ppm) hydrogen detection using an unconventional Pd/n-InP Schottky device

Hydrogen is detected using a Pd/n-InP Schottky diode in which the elongated, very thin Pd electrode is of greater resistance than the underlying semiconductor substrate. Four-probe measurements of the device resistance, as a function of hydrogen concentration, are made by contacting only the Pd electrode, with a sensitivity of 1 ppm being achieved. On hydrogen exposure the device resistance drops from an initial high value, characteristic of the Pd electrode alone, to a lower value due to a hydrogen-induced lowering of the Schottky barrier that opens up the InP substrate as a parallel current carrying channel.