CAS agar diffusion assay for the measurement of siderophores in biological fluids

We developed a simple and universal method, by modifying the universal CAS (Chrome azurol S) assay, measuring siderophores in various biological fluids. We named the assay as CAS agar diffusion (CASAD) assay. CAS plate devoid of nutrients was prepared by using Bacto-agar (1.5%, w/v) as a matrix. Holes with 5-mm-diameter were punched on the CAS agar plate. Each hole was added by 35 microl of the test fluids containing Desferal that was twofold serially diluted. After incubating at 37 degrees C or room temperature for 4-8 h, the size of orange haloes formed around the holes was measured. The size of orange haloes correlated well with the concentration of Desferal in all the biological fluids tested in this study. CASAD assay showed consistent results in wide pH range from 5 to 9. Addition of iron to the test fluids containing Desferal decreased the size of orange haloes in a dose-dependent manner, which suggests that the CASAD assay detects only iron non-bound siderophore. These results suggest that CASAD assay would serve as a simple, stable, and highly reproducible test for screening and quantitative siderophore analysis in biological fluids.     

Frequent Users of Hospital Emergency Departments in Korea Characterized by Claims Data from the National Health Insurance: A Cross Sectional Study

The Korean National Health Insurance, which provides universal coverage for the entire Korean population, is now facing financial instability. Frequent emergency department (ED) users may represent a medically vulnerable population who could benefit from interventions that both improve care and lower costs. To understand the nature of frequent ED users in Korea, we analyzed claims data from a population-based national representative sample. We performed both bivariate and multivariable analyses to investigate the association between patient characteristics and frequent ED use (4+ ED visits in a year) using claims data of a 1% random sample of the Korean population, collected in 2009. Among 156,246 total ED users, 4,835 (3.1%) were frequent ED users. These patients accounted for 14% of 209,326 total ED visits and 17.2% of $76,253,784 total medical expenses generated from all ED visits in the 1% data sample. Frequent ED users tended to be older, male, and of lower socio-economic status compared with occasional ED users (p < 0.001 for each). Moreover, frequent ED users had longer stays in the hospital when admitted, higher probability of undergoing an operative procedure, and increased mortality. Among 8,425 primary diagnoses, alcohol-related complaints and schizophrenia showed the strongest positive correlation with the number of ED visits. Among the frequent ED users, mortality and annual outpatient department visits were significantly lower in the alcohol-related patient subgroup compared with other frequent ED users; furthermore, the rate was even lower than that for non-frequent ED users. Our findings suggest that expanding mental health and alcohol treatment programs may be a reasonable strategy to decrease the dependence of these patients on the ED.     

Use of Pseudomonas putida EstA as an Anchoring Motif for Display of a Periplasmic Enzyme on the Surface of Escherichia coli

The functional expression of proteins on the surface of bacteria has proven important for numerous biotechnological applications. In this report, we investigated the N-terminal fusion display of the periplasmic enzyme β-lactamase (Bla) on the surface of Escherichia coli by using the translocator domain of the Pseudomonas putida outer membrane esterase (EstA), which is a member of the lipolytic autotransporter enzymes. To find out the transport function of a C-terminal domain of EstA, we generated a set of Bla-EstA fusion proteins containing N-terminally truncated derivatives of the EstA C-terminal domain. The surface exposure of the Bla moiety was verified by whole-cell immunoblots, protease accessibility, and fluorescence-activated cell sorting. The investigation of growth kinetics and host cell viability showed that the presence of the EstA translocator domain in the outer membrane neither inhibits cell growth nor affects cell viability. Furthermore, the surface-exposed Bla moiety was shown to be enzymatically active. These results demonstrate for the first time that the translocator domain of a lipolytic autotransporter enzyme is an effective anchoring motif for the functional display of heterologous passenger protein on the surface of E. coli. This investigation also provides a possible topological model of the EstA translocator domain, which might serve as a basis for the construction of fusion proteins containing heterologous passenger domains.     

Lipase-catalyzed synthesis of lysophosphatidic acid in a solvent free system

Summary The direct, lipase-catalyzed esterifications of glycerol-3-phosphate in an organic solvent system and in a solvent free system were carried out. In a solvent free system only, LPA synthesis could be achieved within the acceptable reaction time. Open reaction system was preferable to closed reaction system for LPA synthesis. Yield of LPA isolated by silica gel column chromatography was 32.3%.     

Vibrio vulnificus has the transmembrane transcription activator ToxRS stimulating the expression of the hemolysin gene vvhA

In an attempt to dissect the virulence regulatory mechanism in Vibrio vulnificus, we tried to identify the V. cholerae transmembrane virulence regulator toxRS (toxRS(Vc)) homologs in V. vulnificus. By comparing the sequences of toxRS of V. cholerae and V. parahaemolyticus (toxRS(Vp)), we designed a degenerate primer set targeting well-conserved sequences. Using the PCR product as an authentic probe for Southern blot hybridization, a 1.6-kb BglII-HindIII fragment and a 1.2-kb HindIII fragment containing two complete open reading frames and one partial open reading frame attributable to toxR(Vv), toxS(Vv), and htpG(Vv) were cloned. ToxR(Vv) shared 55.0 and 63.0% sequence homology with ToxR(Vc) and ToxR(Vp), respectively. ToxS(Vv) was 71.5 and 65.7% homologous to ToxS(Vc) and ToxS(Vp), respectively. The amino acid sequences of ToxRS(Vv) showed transmembrane and activity domains similar to those observed in ToxRS(Vc) and ToxRS(Vp). Western blot analysis proved the expression of ToxR(Vv) in V. vulnificus. ToxRS(Vv) enhanced, in an Escherichia coli background, the expression of the V. vulnificus hemolysin gene (vvhA) fivefold. ToxRS(Vv) also activated the ToxR(Vc)-regulated ctx promoter incorporated into an E. coli chromosome. A toxR(Vv) null mutation decreased hemolysin production. The defect in hemolysin production could be complemented by a plasmid harboring the wild-type gene. The toxR(Vv) mutation also showed a reversed outer membrane protein expression profile in comparison to the isogenic wild-type strain. These results demonstrate that ToxR(Vv) may regulate the virulence expression of V. vulnificus.     

Growth Inhibitors of Lettuce Seedlings From Bacillus cereus EJ-121

The ethyl acetate extract of the Bacillus sp. EJ-121 culture broth exhibited growth inhibitory activity on a lettuce (Lactuca sativa L.) seedlings assay. Bacillus sp. EJ-121 was identified as Bacillus cereus by the morphological characteristic and nucleotide sequence of the 16S rDNA. The bioassay-guided fractionation of the ethyl acetate extract led to the isolation of two compounds. Their structures were deduced by spectroscopic methods and determined as sodium vanillate (1) and 2-aminobenzoic acid (2). Both compounds 1 and 2 inhibited more than 90% of root length at 50 ppm (0.26 and 0.36 mM, respectively) while they had a limited effect on shoot growth at the same concentration level. Roots and shoots of lettuce seedlings showed severe deterioration at 100 ppm. In order to study the fundamental structure–activity relationship, several structurally related benzoic acid derivatives were also assayed. The existence of a polar carboxyl moiety seemed to be responsible for the stronger activity.     

Lipocalin 2 (Lcn2) interferes with iron uptake by Brucella abortus and dampens immunoregulation during infection of RAW 264.7 macrophages

Lipocalin 2 (Lcn2) is an important innate immunity component against bacterial pathogens. In this study, we report that Lcn2 is induced by Brucella (B.) abortus infection and significantly contributes to the restriction of intracellular survival of Brucella in macrophages. We found that Lcn2 prevented iron uptake by B. abortus through two distinct mechanisms. First, Lcn2 is secreted to capture bacterial siderophore(s) and abrogate iron import by Brucella. Second, Lcn2 decreases the intracellular iron levels during Brucella infection, which probably deprives the invading Brucella of the iron source needed for growth. Suppression of Lcn2 signalling resulted in a marked induction of anti‐inflammatory cytokine, interleukin 10, which was shown to play a major role in Lcn2‐induced antibrucella immunity. Similarly, interleukin 6 was also found to be increased when Lcn2 signalling is abrogated; however, this induction was thought to be an alternative pathway that rescues the cell from infection when the effective Lnc2 pathway is repressed. Furthermore, Lcn2 deficiency also caused a marked decrease in brucellacidal effectors, such as reactive oxygen species and nitric oxide but not the phagolysosome fusion. Taken together, our results indicate that Lcn2 is required for the efficient restriction of intracellular B. abortus growth that is through limiting iron acquisition and shifting cells to pro‐inflammatory brucellacidal activity in murine macrophages.