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Objective:To investigate the cytopathic effect of amino acids 86-175 of rotavirus non-structural protein 4 (NSP4 86-175) on rat cardiomyocytes and the possible mechanism. Methods:Rat H9C2 cardiomyocytes were treated with NSP4 86-175 protein. Changes in the growth and morphology of the cells were observed. The activity of LDH in the cell culture medium was detected. Fluo-3AM was used to label intracellular free calcium ions and the concentrations of calcium ions in rat cardiomyocytes with and without NSP4 86-175 treatment were detected by confocal laser microscopy. The expression of Bax, Bcl-2, caspase-3, 78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP) and caspase-12 at mRNA level was detected by real-time PCR. The expression of caspase-3, caspase-8, caspase-9, GRP78, CHOP and caspase-12 at protein level was detected by Western blot. Results:Normal cardiomyocytes showed a typical myoblast-like morphology, presenting as spindle-shaped cells with clear boundaries. Obvious cytopathic effect, vacuolar degeneration, shriveled and rounded cells, and cell fragmentation were observed after the treatment with purified NSP4 86-175 protein. The activity of LDH in cell culture medium was enhanced by NSP4 86-175 protein. NSP4 86-175 protein also enhanced the fluorescence of the calcium ions in rat cardiomyocytes, promoted cell apoptosis, up-regulated the expression of apoptotic factors including caspase-3, caspase-8, caspase-9 and Bax-2, and increased the expression of classical markers of endoplasmic reticulum stress such as GRP78, CHOP and caspase-12. Conclusions:NSP4 86-175 had a cytopathic effect on rat cardiomyocytes, which might be related to the induced intracellular calcium overload, endoplasmic reticulum stress, apoptosis and necrosis. These results might be used as theoretical reference for further study on rotavirus infection and myocardial injury.
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Objective:To construct a mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 with hfq gene deletion and to analyze its biological characteristics. Methods:The hfq gene of NTUH-K2044 was knocked out by homologous recombination technology to construct △ hfq mutant strain. Its biological characteristics including growth rate, environmental stress tolerance, biofilm formation, capsular polysaccharide synthesis, resistance to neutrophil phagocytosis and lethality to Galleria mellonella larvae were analyzed by comparing with the wild-type strain using phenotypic experiments. Results:The △ hfq mutant strain of hypervirulent Klebsiella pneumoniae NTHU-K2044 was successfully constructed. Phenotypic experiments showed that the △ hfq mutant strain had significantly slower growth rate, smaller colonies and decreased hypermucoviscosity. Its growth was significantly inhibited under different environmental stress conditions such as pH9, pH5.5, 0.7 mmol/L SDS, 5% NaCl, 0.1% H 2O 2 and high temperature of 50℃. In terms of virulence and pathogenicity, the △ hfq mutant strain showed decreased ability to form biofilm and capsule, significantly down-regulated expression of magA and rmpA genes required for capsule synthesis, lower survival rate in the neutrophil bactericidal test and obviously reduced lethality to Galleria mellonella larvae. Conclusions:As a RNA chaperone, Hfq protein could participate in post-transcriptional regulation and play an important role in regulating the physiology, environmental adaptability and virulence of hypervirulent Klebsiella pneumoniae. This study provided reference for further study on hypervirulent Klebsiella pneumoniae.
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Objective To explore the value of Sysmex UF-1000i urinary sediment analyzer in diagnosis of urinary tract infection (UTI) and clarify the influence caused by different specimen on UF-1000i diagnosing UTI.Methods Totally 466 specimens examined bacterial culture and routine urinalysis were collected from patients suspected of UTI during July and August,2015 (samples of group A).148 specimens during late March and early April were gathered to implement a urine culture and then the rest of urine were detected byUF -1000i urinary sediment analyzer instantly(samples of group B).Bacteria and leukocyte counts were gathered and then receiver operating characteristic (ROC) carve was drawn regarding thegold standard as bacterial culture by SPSS18.0.Next,the threshold values of bacteria and leukocyte counts for diagnosis of UTI were found out.Meanwhile,itssensitivity,specificity,positive/negative predictive value,false positive/false negative value,and diagnostic accuracy were calculated.Results The cut off values to samples of group A were101.7 bacteria/μl and 18.8WBC/μl respectively and to samples of group B were 98.7 bacteria/μl and 11.1WBC/μl respectively.The area of Bacteria and leukocyte counts under ROC carve was 0.604 and 0.661 to samples of group A and 0.941 and 0.848 to samples Of group B.To samples of group B,combined Bacteria and leukocyte counts for UTI,the optimum sensitivity was 82.4％,specificity was 92.1％,positive predictive value was 77.8％,negative predictive value was 93.8％,false positive rate was 7.9％,false negative rate was 17.6％,and accuracy was 89.9％.Bacterial culture was reduced by 70.9％.Conclusion UF-1000i urine sediment analyzers have the value of early screening value and help to diagnose UTI.Urine that was sterilely collected and examined within two hours can make the value of UF-1000i maximized.
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Objective To investigate the cytopathic effect of amino acid residues 86 to 175 of rotavirus nonstructural protein 4 (NSP486-175) on rat neurons and to analyze the underlying mechanism.MethodsPrimary cultured rat neurons were treated with NSP486-175 and the morphological changes induced in rat neurons were observed.Lactate dehydrogenase (LDH) activity in the culture supernatant of NSP486-175 treated-neurons was measured.Laser scanning confocal microscope was used to detect the concentration of intracellular Ca2+ labeled with Fluo-3 AM.Results Exogenous addition of NSP486-175 induced obvious cytopathic effect on rat neurons.The LDH activity in the culture supernatant of treated-neurons was stronger than that of the control group.The intensity and the distribution of fluorescence in neurons were altered after stimulation with NSP486-175.Conclusion NSP486-175 can induce the damage of rat neurons, which may be related to its role in increasing the concentration of intracellular Ca2+.This study may provide certain theoretical basis for understanding extra-intestinal spread and pathogenesis of rotavirus infection.