Serum Copper Concentrations, Effect Modifiers and Blood Pressure: Insights from NHANES 2011-2014.

(1) Background: Epidemiological studies on the relationship between serum copper and hypertension are contradictory. We assessed the relationship between serum copper and blood pressure among adults in the United States. (2) Methods: We divided hypertension into two categories: treated hypertension and untreated hypertension. Linear or logistic regression analysis was applied to investigate the association between serum copper concentrations and blood pressure levels. (3) Results: As compared to quartile 1, the odds ratios (ORs) for untreated hypertension in quartiles 2, 3, and 4 were 1.02 (0.74-1.42), 1.23 (0.88-1.72), and 1.08 (0.74-1.58), respectively, in multivariable analysis (all p > 0.05). In non-hypertension, as compared with quartile 1, the ß (95% CI) of systolic blood pressure for quartiles 2, 3, and 4 was -0.92 (-2.07-0.23), -0.05 (-1.30-1.20), and -0.48 (-1.83-0.88), respectively, in multivariable analysis (all p > 0.05). As compared to quartile 1, the ORs for treated hypertension in quartiles 2, 3, and 4 were 1.36 (0.88-2.10), 1.35 (0.87-2.09), and 1.56 (0.98-2.47), respectively, upon multivariable analysis including antihypertensive medication use as a covariate (all p > 0.05). Furthermore, 1SD increase in serum copper was non-significantly associated with 1.16 (0.97-1.37)-fold increased risk of hypertension in multivariable analysis (p = 0.096). (4) Conclusion: In the present study, we discovered that the serum copper concentration was not related with hypertension or blood pressure levels. Antihypertensive drug use may distort the correlation between copper and blood pressure levels. Information on antihypertensive drug use may be taken into account when identifying new risk factors for hypertension.

Lasiopodomys fuscus as an important intermediate host for Echinococcus multilocularis: isolation and phylogenetic identification of the parasite.

BACKGROUND: Echinococcus multilocularis causes alveolar echinococcosis (AE) and is widely prevalent in Qinghai Province, China, where a number of different species have been identified as hosts. However, limited information is available on the Qinghai vole (Lasiopodomys fuscus), which is hyper endemic to Qinghai Province and may represent a potential intermediate host of E. multilocularis. Thus, L. fuscus could contribute to the endemicity of AE in the area. METHODS: Fifty Qinghai voles were captured from Jigzhi County in Qinghai Province for the clinical identification of E. multilocularis infection via anatomical examination. Hydatid fluid was collected from vesicles of the livers in suspected voles and subjected to a microscopic examination and PCR assay based on the barcoding gene of cox 1. PCR-amplified segments were sequenced for a phylogenetic analysis. E. multilocularis-infected Qinghai voles were morphologically identified and subjected to a phylogenetic analysis to confirm their identities. RESULTS: Seventeen of the 50 Qinghai voles had E. multilocularis-infection-like vesicles in their livers. Eleven out of the 17 Qinghai voles presented E. multilocularis infection, which was detected by PCR and sequencing. The phylogenetic analysis showed that all 11 positive samples belonged to the E. multilocularis Asian genotype. A morphological identification and phylogenetic analysis of the E. multilocularis-infected Qinghai voles confirmed that all captured animals were L. fuscus. CONCLUSIONS: L. fuscus can be infected with E. multilocularis and plays a potential role in the life cycle and epidemiology of E. multilocularis in the Qinghai-Tibetan Plateau of China.

Efficient production of a recombinant Venerupis philippinarum defensin (VpDef) in Pichia pastoris and characterization of its antibacterial activity and stability.

VpDef is a novel defensin isolated from the clam Venerupis philippinarum. Previously it was expressed in Escherichia coli; however, the E. coli-derived recombinant VpDef did not show effective antimicrobial activity against Staphyloccocus aureus or the Gram-negative bacteria tested. As such, the goal of this study was to design, express, and purify a recombinant VpDef (rVpDef) in Pichia pastoris and to determine its antibacterial potency and stability. A 6.9 KDa rVpDef was successfully expressed as a secreted peptide in P. pastoris, and the amount of rVpDef accumulation was shown to reach as high as approximate 60 µg per 1 ml of culture medium only after an initial optimization was performed. The purified rVpDef demonstrated a broad antibacterial spectrum and was active against six typical common bacteria, both Gram-positive and Gram-negative. A minimal inhibition concentration of as low as 50 µg/ml was observed for rVpDef against the growth of E. coli O157 (ATCC 35150). Moreover, rVpDef was tolerant to temperature shock and proteinase digestion and maintained a high stability over a relatively broad pH range. In addition, rVpDef had a low hemolytic activity against rabbit erythrocytes. Taken together, this study demonstrated that rVpDef could be produced in a large-scale manner in P. pastoris and has a good antibacterial activity and suitable stability. This is the first report on heterologous expression of a biologically active VpDef in P. pastoris, supporting its use for both research and application purposes.

Different strategies for preparation of non-tagged rV270 protein and its efficacy against Yersinia pestis challenge.

OBJECTIVE: LcrV is an important component for the development of a subunit vaccine against plague. To reduce immunosuppressive activity of LcrV, a recombinant LcrV variant lacking amino acids 271 to 326 (rV270) was prepared by different methods in this study. METHODS: A new strategy that produced non-tagged or authentic rV270 protein was designed by insertion of rV270-thrombin-hexahistidine fusion gene into the vector pET24a, or by insertion of hexahistidine-enterokinase-rV270 or hexahistitine-factor Xa-rV270 fusion gene into the vector pET32a. After Co(2+) affinity chromatography, a purification strategy was developed by cleavage of His tag on column, following Sephacryl S-200HR column filtration chromatography. RESULTS: Removal of His tag by thrombin, enterokinase and factor Xa displayed a yield of 99.5%, 32.4% and 15.3%, respectively. Following Sephacryl S-200HR column filtration chromatography, above 97% purity of rV270 protein was obtained. Purified rV270 that was adsorbed to 25% (v/v) Al(OH)3 adjuvant in phosphate-buffered saline (PBS) induced very high titers of antibody to rV270 in BALB/c mice and protected them (100% survival) against subcutaneous challenge with 106 CFU of Y. pestis virulent strain 141. CONCLUSION: The completely authentic rV270 protein can be prepared by using enterokinase or factor Xa, but they exhibited extremely low cleavage activity to the corresponding recognition site. Thrombin cleavage is an efficient strategy to prepare non-tagged rV270 protein and can be easily operated in a large scale due to its relatively low cost and high cleavage efficacy. The recombinant rV270 can be used as a key component to develop a subunit vaccine of plague.

[Evaluation of immunization protection efficacy of plague subunit vaccine].

OBJECTIVE: To evaluate the protective efficacy of plague subunit vaccine, BALB/c mice, guinea pigs and rabbits were used in this study. METHODS: Groups of mice (10 per group), guinea pigs (14 per group) and rabbits (6 per group) were immunized with F1 + rV270 vaccine, EV76 vaccine and alum adjuvant by intramuscular route, respectively. Serum antibody titres of mice, guinea pigs and rabbits were determined by ELISA and the immunized animals were challenged with 10(6) CFU of Y. pestis strain 141 at the 8th week after the primary immunization. RESULTS: The immunized mice, guinea pigs or rabbits with subunit vaccine developed anti-F1 IgG titre of 41 587.3 +/- 2.1, 11 543.7 +/- 2.1 or 522.4 +/- 22.4 and elicited statistical anti-F1 IgG titre difference among them (F = 17.58, P < 0.01). The immunized mice, guinea pigs or rabbits with subunit vaccine had anti-rV270 IgG titre of 15 748.7 +/- 1.6, 12.6 +/- 1.4 or 1648.0 +/- 5.0 and induced statistical anti-rV270 IgG titre difference among them (F value was 16.34, P < 0.01). There was significant anti-F1 IgG titre difference among mice, guinea pigs and rabbits immunized with EV76 vaccine that developed anti-F1 IgG titre of 913.4 +/- 4.5, 937.0 +/- 2.0 or 342.0 +/- 12.0 (F = 23.67, P < 0.01), whereas the immunized mice, guinea pigs and rabbits with EV76 vaccine developed anti-rV270 IgG titre of 12.0 +/- 1.0, 447.0 +/- 10.0, 40.0 +/- 11.0 and there was no anti-rV270 IgG titre difference between them (F = 2.20, P = 0.1314). The immunized mice with subunit vaccine developed significantly higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 30.57 and 19.04, respectively, P < 0.01), and there were no anti-F1 IgG titre differences between the immunized guinea pigs and rabbits (q = 0.04, P = 0.8485). The immunized mice with subunit vaccine developed significantly higher anti-rV270 IgG titres than immunized guinea pigs and rabbits (q value was 27.10 and 19.49, respectively, P < 0.01), and there were no anti-rV270 IgG titre differences between the immunized guinea pigs and rabbits with the subunit vaccine (q = 0.25, P = 0.6187). The immunized mice with EV76 elicited higher anti-F1 IgG titres than immunized guinea pigs and rabbits (q value was 40.67 and 29.10, respectively, P < 0.01), whereas there was no difference of F1 IgG titer between immunized guinea pigs and rabbits (q = 0.06, P = 0.8098). The immunized mice, guinea pigs and rabbits with subunit vaccine provided 100% (10/10), 86% (12/14) and 100% (5/5) protection against 10(6) CFU Y. pestis of challenge, respectively. The immunized mice, guinea pigs and rabbits with EV76 vaccine gave 100% (6/6), 93% (13/14) and 100% (6/6) protection against 10(6) CFU Y. pestis of challenge respectively. CONCLUSION: BALB/c mice is the best small animal model for valuation of protective efficacy of plague subunit vaccine. The guinea pigs showed a high individual variation for this purpose. The rabbits can be used as an alternative model for evaluating plague subunit vaccine.

[A molecular epidemiological study on human plague fulminant epidemic in Qinghai, 2004].

OBJECTIVE: To study the epidemiology of genotyping Yersinia pestis isolated in the fulminant epidemics of human plague in Qinghai province in 2004. METHODS: Primer pairs targeting the twenty-three different identified regions (DFRs) were designed to detect the presence or deletion of each DFR in 13 strains of Yersinia pestis isolated from the fulminant epidemic of human plague in Qinghai province in 2004. RESULTS: There were 4 genomovars, i.e. Genomovar 8, 10, 15 and 16 in the 13 strains of Yersinia pestis identified. The genomovar of all the strains of Yersinia pestis isolated from Nangqian county was Genomovar 10. Among the two strains of Yersinia pestis isolated from Wulan county, the genomovar of one strain was Genomovar 8 and the other was Genomovar 10. The genomovars of all the strains of Yersinia pestis isolated from Qilian, Qumalai and Chengduo county belonged to Genomovar 16. CONCLUSION: It was demonstrated that the genotyping of Yersinia pestis appeared to be a powerful tool for investigating human plague epidemics.

[Study on an epidemic of human lung plaque in Nangqian county, Qinghai province].

OBJECTIVE: To study an epidemic of human lung plague fulminant from September to October, 2004 in Nangqian county, Qinghai province. METHODS: Cases were diagnosed through data from epidemiological, clinical, bacteriological, serological and autopsy studies. RESULTS: 14 patients were identified, ending up with 6 deaths and 8 cured. The first case was diagnosed as primary pesticemia late progressed to lung plague. 4 cases were transformed from pesticemia out of 13, leaving the 9 cases as primary lung plague. Situation was under complete control through routinely handling the plague focus. CONCLUSION: The first case was bitten by the infected fleas which parasitized the marmota preyed on a dog but later these fleas were brought into the tent by the dog. The others cases were infected through droplets or dust. Programs on monitoring and controling the amount of marmotas and fleas should to be strengthened to prevent the epidemics of plague in the area.