Entry of Vibrio cincinnatiensis into viable but nonculturable state and its resuscitation

Aims:  The aim was to characterize the viable but nonculturable (VBNC) state of Vibrio cincinnatiensis and its resuscitation.
Methods and Results:  Vibrio cincinnatiensis VIB287 was cultured in sterilized seawater microcosms at 4°C. Plate counts, direct viable counts and total counts were used. A large population of the V. cincinnatiensis became nonculturable after approx. 50 day at 4°C. Electron microscopy revealed that the VBNC cells changed from rod to coccoid and decreased in size. Resuscitation of VBNC cells was achieved by temperature upshift in nutrition of yeast extract and peptone by addition of catalase or compound vitamin B. The VBNC and resuscitative cells were intraperitoneally injected into zebra fish separately. No death was observed in the group inoculated with the VBNC cells.
Conclusions:  Vibrio cincinnatiensis VIB287 could enter VBNC state in adverse environments. Resuscitation of VBNC cells occurred by addition of compound vitamin B or catalase to VBNC cells containing nutrient. The resuscitative cells might retain their pathogenicity.
Significance and Impact of the Study:  The study confirmed that V. cincinnatiensis could enter into VBNC state in seawater at low temperature and resuscitated. The resuscitative cells retained their pathogenicity, which may be important in future studies of ecology of V. cincinnatiensis .     

Dependence of alignment direction on magnitude of strain in esophageal smooth muscle cells

The response of cells in vitro to mechanical forces has been the subject of much research using devices to exert controlled mechanical stimulation on cultured cells or isolated tissue. In this study, esophageal smooth muscle cells were seeded on flexible polyurethane membranes to form a confluent cell layer. The cells were then subjected to uniform cyclic stretch of varying magnitudes at a frequency of approximately five cycles per minute in a custom made mechatronic bioreactor, providing similar strains experienced in the in vivo mechanical environment of the esophagus. The results show that the orientation response is dependent on the magnitude of cyclic stretch applied. Smooth muscle cells showed parallel alignment to the force direction at low cyclic strains (2%) compared to the hill‐valley morphology of static controls. At higher strains (5% and 10% magnitude), the cells exhibited a consistent alignment perpendicular to the strain. To our knowledge, this is the first time that the alignment direction's dependence on strain magnitude has been demonstrated. MTS analysis indicated that cell metabolism was reduced when mechanical strain was applied, and proliferation was inhibited by mechanical strain. Protein expression indicates a decrease in smooth muscle α‐actin, indicative of changes in cell phenotype, an increase in vimentin, which is associated with increased cell motility, and an increase in desmin, indicating differentiation in stimulated cells. Biotechnol. Bioeng. 2009;102: 1703–1711. © 2008 Wiley Periodicals, Inc.     

Biodegradation of p-nitrophenol by Pseudomonas aeruginosa HS-D38 and analysis of metabolites with HPLC–ESI/MS

Pseudomonas aeruginosa strain HS-D38 was capable of mineralizing p-nitrophenol (PNP) as the sole source of carbon, nitrogen and energy. Degradation of 200 mg L^?1 PNP was examined in different media including: (i) MSM (mineral salts medium, no carbon and nitrogen source); (ii) addition of 1% ammonium chloride as additional nitrogen source (ANM); and (iii) addition of 1% glucose as a carbon source (ACM). Complete degradation of 200 mg L^?1 PNP was achieved in 12 h in MSM. Additional ammonium chloride accelerated the PNP degradation, but additional glucose inhibited this process. This strain metabolized as high concentration as 300 and 500 mg L^?1 of PNP in 14 h and 24 h, respectively, in MSM. The degradation was accompanied by release of stoichiometric amount of nitrate from PNP. During the bacterial growth on PNP, hydroquinone and 1,2,4-benzenetriol were observed as the key degradation intermediates by using a combination of techniques, including HPLC–DAD and LC–ESI/MS compared with the authentic standards. These results indicated that PNP was degraded via a hydroquinone pathway.     

Fed-batch fermentation of Tuber melanosporum for the hyperproduction of mycelia and bioactive Tuber polysaccharides

For the first time, a fed-batch fermentation process of Tuber melanosporum was developed for the efficient production of bioactive mycelia and Tuber polysaccharides. Each 1.67 g/L of peptone and 8.33 g/L of yeast extract were added on day 3, 6, and 9, respectively, and sucrose was fed to maintain its concentration around 35–5 g/L when its residual level decreased to 10–5 g/L. Then, the maximal biomass, the production of extracellular polysaccharides (EPS) and intracellular polysaccharides (IPS) reached 53.72 ± 2.57 g DW/L, 7.09 ± 0.62 and 4.43 ± 0.21 g/L, respectively. Compared with the batch culture conducted in the enriched medium, the biomass, the production of EPS and IPS were enhanced by 55.8%, 222.3% and 103.2%, respectively. Not only the cell density but also the production of EPS and IPS were the highest ever reported in truffle fermentation, and the biomass was also the highest as ever reported in mushroom fermentation.     

Genetic Structure of Daphnia galeata Populations in Eastern China

This study presents the first examination of the genetic structure of Daphnia longispina complex populations in Eastern China. Only one species, D. galeata, was present across the eight investigated lakes; as identified by taxon assignment using allelic variation at 15 microsatellite loci. Three genetically differentiated D. galeata subgroups emerged independent of the type of statistical analysis applied. Thus, Bayesian clustering, discriminant analysis based on results from factorial correspondence analysis, and UPGMA clustering consistently showed that populations from two neighbouring lakes were genetically separated from a mixture of genotypes found in other lakes, which formed another two subgroups. Clonal diversity was high in all D. galeata populations, and most samples showed no deviation from Hardy-Weinberg equilibrium, indicating that clonal selection had little effect on the genetic diversity. Overall, populations did not cluster by geographical origin. Further studies will show if the observed pattern can be explained by natural colonization processes or by recent anthropogenic impact on predominantly artificial lakes.     

Thermal Characteristics of Hyperaccumulator and Fate of Heavy Metals during Thermal Treatment of Sedum plumbizincicola

Thermal treatment is one of the most promising disposal techniques for heavy metal- (HM)-enriched hyperaccumulators. However, the thermal characteristics and fate of HMs during thermal treatment of hyperaccumulator biomass need to be known in detail. A horizontal tube furnace was used to analyze the disposal process of hyperaccumulator biomass derived from a phyto-extracted field in which the soil was moderately contaminated with heavy metals. Different operational conditions regarding temperature and gas composition were tested. A thermo-dynamic analysis by advanced system for process engineering was performed to predict HM speciation during thermal disposal and SEM-EDS, XRD and sequential chemical extraction were used to characterize the heavy metals. The recovery of Zn, Pb and Cd in bottom ash decreased with increasing temperature but recovery increased in the fly ash. Recovery of Zn, Pb and Cd fluctuated with increasing air flow rate and the metal recovery rates were higher in the fly ash than the bottom ash. Most Cl, S, Fe, Al and SiO₂ were found as alkali oxides, SO₂, Fe₂(SO₄)₃, iron oxide, Ca₃Al₂O₆, K₂SiO₃ and SiO₂ instead of reacting with HMs. Thus, the HMs were found to occur as the pure metals and their oxides during the combustion process and as the sulfides during the reducing process.     

A Phosphorylation-Related Variant ADD1-rs4963 Modifies the Risk of Colorectal Cancer

It is well-established that abnormal protein phosphorylation could play an essential role in tumorgenesis by disrupting a variety of physiological processes such as cell growth, signal transduction and cell motility. Moreover, increasing numbers of phosphorylation-related variants have been identified in association with cancers. ADD1 (α-adducin), a versatile protein expressed ubiquitously in eukaryotes, exerts an important influence on membrane cytoskeleton, cell proliferation and cell-cell communication. Recently, a missense variant at the codon of ADD1’s phosphorylation site, rs4963 (Ser586Cys), was reported to modify the risk of non-cardia gastric cancer. To explore the role of ADD1-rs4963 in colorectal cancer (CRC), we conducted a case-control study with a total of 1054 CRC cases and 1128 matched controls in a Chinese population. After adjustment for variables including age, gender, smoking and drinking, it was demonstrated that this variant significantly conferred susceptibility to CRC (G versus C: OR = 1.16, 95% CI = 1.03–1.31, P = 0.016; CG versus CC: OR = 1.25, 95% CI = 1.02–1.55, P = 0.036; GG versus CC: OR = 1.35, 95% CI = 1.06–1.72, P = 0.015). We further investigated the interaction of ADD1-rs4963 with smoking or drinking exposure, but found no significant result. This study is the first report of an association between ADD1 and CRC risk, promoting our knowledge of the genetics of CRC.     

miR-186 and 326 Predict the Prognosis of Pancreatic Ductal Adenocarcinoma and Affect the Proliferation and Migration of Cancer Cells

MicroRNAs can function as key tumor suppressors or oncogenes and act as biomarkers for cancer diagnosis or prognosis. Although high-throughput assays have revealed many miRNA biomarkers for pancreatic ductal adenocarcinoma (PDAC), only a few have been validated in independent populations or investigated for functional significance in PDAC pathogenesis. In this study, we correlated the expression of 36 potentially prognostic miRNAs within PDAC tissue with clinico-pathological features and survival in 151 Chinese patients. We then analyzed the functional roles and target genes of two miRNAs in PDAC development. We found that high expression of miR-186 and miR-326 predict poor and improved survival, respectively. miR-186 was over-expressed in PDAC patients compared with controls, especially in patients with large tumors (>2 cm), lymph node metastasis, or short-term survival (< 24 months). In contrast, miR-326 was down-regulated in patients compared with controls and displayed relatively increased expression in the patients with long-term survival or without venous invasion. Functional experiments revealed that PDAC cell proliferation and migration was decreased following inhibition and enhanced following over-expression of miR-186. In contrast, it was enhanced following inhibition and decreased after over-expression of miR-326. A luciferase assay indicated that miR-186 can bind directly to the 3′-UTR of NR5A2 to repress gene expression. These findings suggest that miR-186 over-expression contributes to the invasive potential of PDAC, likely via suppression of NR5A2, thereby leading to a poor prognosis; high miR-326 expression prolongs survival likely via the decreasing invasive potential of PDAC cells. These two miRNAs can be used as markers for clinical diagnosis and prognosis, and they represent therapeutic targets for PDAC.     

The Cold-Inducible RNA-Binding Protein (CIRP) Level in Peripheral Blood Predicts Sepsis Outcome

Objectives

Sepsis is a lethal and complex clinical syndrome caused by infection or suspected infection. Cold-inducible RNA-binding protein (CIRP) is a widely distributed cold-shock protein that plays a proinflammatory role in sepsis and that may induce organ damage. However, clinical studies regarding the use of CIRP for the prognostic evaluation of sepsis are lacking. The purpose of this research was to investigate the prognostic significance of peripheral blood concentrations of CIRP in sepsis. Sepsis was assessed using several common measures, including the Acute Physiology and Chronic Health Evaluation II (APACHE II) score; the Sepsis-related Organ Failure Assessment (SOFA) score; the lactate, serum creatinine, and procalcitonin (PCT) levels; the white blood cell (WBC) count; and the neutrophil ratio (N%).

Design

Sixty-nine adult patients with sepsis were enrolled in this study. According to the mortality data from the hospital, 38 patients were survivors, and 31 were nonsurvivors. The plasma levels of the biomarkers were measured and the APACHE II and SOFA scores were calculated within 24 hours of patient enrollment into our study. The CIRP level was measured via ELISA.

Results

The plasma level of CIRP was significantly higher in the nonsurvivors than in the survivors (median (IQR) 4.99 (2.37–30.17) ng/mL and 1.68 (1.41–13.90) ng/mL, respectively; p = 0.013). The correlations of the CIRP level with the APACHE II score (r = 0.248, p = 0.040, n = 69), the SOFA score (r = 0.323, p = 0.007, n = 69), the serum creatinine level (r = 0.316, p = 0.008, n = 69), and the PCT level (r = 0.282, p = 0.019, n = 69) were significant. Receiver operator characteristic (ROC) curve analysis showed that the area under the ROC curve (AUC) for the CIRP level was 0.674 (p = 0.013). According to Cox proportional hazards models, the CIRP level independently predicts sepsis mortality. When the CIRP level in the peripheral blood increased by 10 ng/mL, the mortality risk increased by 1.05-fold (p = 0.012). Thus, the CIRP level reflects the degree of renal injury but does not predict the severity of sepsis or organ damage.

Conclusion

An elevated plasma concentration of CIRP was significantly associated with poor prognosis among patients with sepsis. Therefore, CIRP is a potential predictor of sepsis prognosis.