A rapid method for determining dynamic binding capacity of resins for the purification of proteins

Biolayer interferometry is a novel method for quantifying macromolecules, such as proteins, in solution. The presence of other, non-binding molecules does not interfere with quantification, which allows one to measure the concentration of the molecule of interest in a crude mixture. Here we apply this method to determining the dynamic binding capacity of affinity resins.     

Immunogenicity and safety assessment of the Cuban recombinant hepatitis B vaccine in healthy adults.

Manufactures of biotechnological/biological products (including vaccines) frequently make changes to manufacturing processes of products both during development and after approval. In our case, a non-inferiority bridging study was carried out to demonstrate that changes in the production plant facilities of Cuban recombinant hepatitis B vaccine, Heberbiovac HB, did not affect the safety and immunogenicity of the vaccine. This controlled, randomized, doubled-blinded trial included 501 volunteers, aged between 20 and 64, who were given three doses of vaccine (20 microg HBsAg/mL) at month 0, 1, and 2. Four lots were evaluated (three corresponding to the new production facilities and a control one produced in the older facilities). One month after the third dose, were observed protective levels of anti-HBsAg in 97% of the subjects that concluded the study with a geometric mean antibody titer (GMT) of 931.18 IU/L. Normal values of body mass index (BMI), the younger ages, and being a female, were significantly related to a good antibody response. The vaccine was well tolerated. Pain at the injection site was the most commonly reported symptom. We conclude that Heberbiovac HB vaccine maintains its characteristics after the modifications carried out in the production plant facilities and both, lot obtained in previous facilities and in the new ones, are comparable in terms of safety and immunogenicity.     

Alternative chaperone machinery may compensate for calreticulin/calnexin deficiency in Caenorhabditis elegans

Proper folding and maintenance of the native structure are central to protein function and are assisted by a family of proteins called chaperones. Calreticulin and calnexin are ER resident chaperones well conserved from worm to human. Calreticulin/calnexin knock-out mice exhibit a severe phenotype, whereas in Caenorhabditis elegans, calreticulin [crt-1(jh101)]- and calnexin [cnx-1(nr2009)]-null mutant worms exhibit only a mild phenotype, suggesting the possible existence of alternative chaperone machinery that can compensate for the deficiency of calreticulin and/or calnexin. In order to rapidly identify the compensatory chaperone components involved in this process, we analyzed the proteome of crt-1(jh101) mutants and [crt-1(jh101);cnx-1(nr2009)] double mutants. When grown at 20 degrees C, we found that five proteins were up-regulated and two proteins were down-regulated in crt-1(jh101) mutants; nine proteins were up-regulated and five proteins were down-regulated in [crt-1(jh101);cnx-1(nr2009)] double mutants. In addition, elevation of the cultivation temperature to 25 degrees C, which is still permissive to growth but causes specific defects in mutants, led to the identification of several additional proteins. Interestingly, the consistent increment of heat shock protein-70 family members (hsp70) together with protein disulfide isomerase (PDI) at all the examined conditions suggests the possible compensatory function imparted by hsp70 and PDI family members in the absence of calreticulin and/or calnexin.     

Proteome analysis of chicken embryonic gonads: identification of major proteins from cultured gonadal primordial germ cells

The domestic chicken (Gallus gallus) is an important model for research in developmental biology because its embryonic development occurs in ovo. To examine the mechanism of embryonic germ cell development, we constructed proteome map of gonadal primordial germ cells (gPGCs) from chicken embryonic gonads. Embryonic gonads were collected from 500 embryos at 6 days of incubation, and the gPGCs were cultured in vitro until colony formed. After 7-10 days in culture, gPGC colonies were separated from gonadal stroma cells (GSCs). Soluble extracts of cultured gPGCs were then fractionated by two-dimensional gel electrophoresis (pH 4-7). A number of protein spots, including those that displayed significant expression levels, were then identified by use of matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry and LC-MS/MS. Of the 89 gPGC spots examined, 50 yielded mass spectra that matched avian proteins found in on-line databases. Proteome map of this type will serve as an important reference for germ cell biology and transgenic research.     

LINE-1 methylation in granulocyte DNA and trihalomethane exposure is associated with bladder cancer risk

DNA methylation changes contribute to bladder carcinogenesis. Trihalomethanes (THM), a class of disinfection by-products, are associated with increased urothelial bladder cancer (UBC) risk. THM exposure in animal models produces DNA hypomethylation. We evaluated the relationship of LINE-1 5-methylcytosine levels (LINE-1%5mC) as outcome of long-term THM exposure among controls and as an effect modifier in the association between THM exposure and UBC risk. We used a case-control study of UBC conducted in Spain. We obtained personal lifetime residential THM levels and measured LINE-1%5mC by pyrosequencing in granulocyte DNA from blood samples in 548 incident cases and 559 hospital controls. Two LINE-1%5mC clusters (above and below 64%) were identified through unsupervised hierarchical cluster analysis. The association between THM levels and LINE-1%5mC was evaluated with β regression analyses and logistic regression was used to estimate odds ratios (OR) adjusting for covariables. LINE-1%5mC change between percentiles 75^th and 25^th of THM levels was 1.8% (95% confidence interval (CI): 0.1, 3.4%) among controls. THM levels above vs. below the median (26 μg/L) were associated with increased UBC risk, OR = 1.86 (95% CI: 1.25, 2.75), overall and among subjects with low levels of LINE-1%5mC (n = 975), OR = 2.14 (95% CI: 1.39, 3.30), but not associated with UBC risk among subjects’ high levels of LINE-1%5mC (n = 162), interaction P = 0.03. Results suggest a positive association between LINE-1%5mC and THM levels among controls, and LINE-1%5mC status may modify the association between UBC risk and THM exposure. Because reverse causation and chance cannot be ruled out, confirmation studies are warranted.     

Nuclear DNA content of Vitis vinifera cultivars and ploidy level analyses of somatic embryo-derived plants obtained from anther culture

Flow cytometry was employed to determine the ploidy level of Vitis vinifera L. somatic embryo-derived plants obtained from anther culture. Only one among the 41 analysed plants (2.4%) presented somaclonal variation (tetraploidy); the other plants were diploid. No significant differences (P≤0.05) were detected between diploid and parental field plants. No haploid or aneuploid plants were observed. The nuclear DNA content of nine V. vinifera cultivars was also estimated using flow cytometry. A non-significant variation was found among the cultivars, with DNA content ranging from 1.17 pg/2C (cv. ‘Tinta Barroca’ and ‘Viosinho’) to 1.26 pg/2C (cv. ‘Cabernet Sauvignon’). These results and previous studies on other Vitis species suggest that Vitis genome is stable with regard to nuclear DNA content.     

Influenza A H5N1 clade 2.3.4 virus with a different antiviral susceptibility profile replaced clade 1 virus in humans in northern Vietnam

Background

Prior to 2007, highly pathogenic avian influenza (HPAI) H5N1 viruses isolated from poultry and humans in Vietnam were consistently reported to be clade 1 viruses, susceptible to oseltamivir but resistant to amantadine. Here we describe the re-emergence of human HPAI H5N1 virus infections in Vietnam in 2007 and the characteristics of the isolated viruses.

Methods and Findings

Respiratory specimens from patients suspected to be infected with avian influenza in 2007 were screened by influenza and H5 subtype specific polymerase chain reaction. Isolated H5N1 strains were further characterized by genome sequencing and drug susceptibility testing. Eleven poultry outbreak isolates from 2007 were included in the sequence analysis. Eight patients, all of them from northern Vietnam, were diagnosed with H5N1 in 2007 and five of them died. Phylogenetic analysis of H5N1 viruses isolated from humans and poultry in 2007 showed that clade 2.3.4 H5N1 viruses replaced clade 1 viruses in northern Vietnam. Four human H5N1 strains had eight-fold reduced in-vitro susceptibility to oseltamivir as compared to clade 1 viruses. In two poultry isolates the I117V mutation was found in the neuraminidase gene, which is associated with reduced susceptibility to oseltamivir. No mutations in the M2 gene conferring amantadine resistance were found.

Conclusion

In 2007, H5N1 clade 2.3.4 viruses replaced clade 1 viruses in northern Vietnam and were susceptible to amantadine but showed reduced susceptibility to oseltamivir. Combination antiviral therapy with oseltamivir and amantadine for human cases in Vietnam is recommended.     

Rational Design of Core@shell Structured CoSx@Cu2MoS4 Hybridized MoS2/N,S‐Codoped Graphene as Advanced Electrocatalyst for Water Splitting and Zn‐Air Battery

A novel hybrid of small core@shell structured CoS_x@Cu₂MoS₄ uniformly hybridizing with a molybdenum dichalcogenide/N,S‐codoped graphene hetero‐network (CoS_x@Cu₂MoS₄‐MoS₂/NSG) is prepared by a facile route. It shows excellent performance toward the oxygen reduction reaction (ORR), oxygen evolution reaction (OER), and hydrogen evolution reaction (HER) in alkaline medium. The hybrid exhibits rapid kinetics for ORR with high electron transfer number of ≈3.97 and exciting durability superior to commercial Pt/C. It also demonstrates great potential with remarkable stability for HER and OER, requiring low overpotential of 118.1 and 351.4 mV, respectively, to reach a current density of 10 mA cm^?2. An electrolyzer based on CoS_x@Cu₂MoS₄‐MoS₂/NSG produces low cell voltage of 1.60 V and long‐term stability, surpassing a device of Pt/C + RuO₂/C. In addition, a Zn‐air battery using cathodic CoS_x@Cu₂MoS₄‐MoS₂/NSG catalyst delivers a high cell voltage of ≈1.44 V and a power density of 40 mW cm^?2 at 58 mA cm^?2, better than the state‐of‐the‐art Pt/C catalyst. These achievements are due to the rational combination of highly active core@shell CoS_x@Cu₂MoS₄ with large‐area and high‐porosity MoS₂/NSG to produce unique physicochemical properties with multi‐integrated active centers and synergistic effects. The outperformances of such catalyst suggest an advanced candidate for multielectrocatalysis applications in metal‐air batteries and hydrogen production.