A meningococcal factor H binding protein mutant that eliminates factor H binding enhances protective antibody responses to vaccination

Certain pathogens recruit host complement inhibitors such as factor H (fH) to evade the immune system. Microbial complement inhibitor-binding molecules can be promising vaccine targets by eliciting Abs that neutralize this microbial defense mechanism. One such Ag, meningococcal factor H-binding protein (fHbp), was used in clinical trials before the protein was discovered to bind fH. The potential effect of fH binding on vaccine immunogenicity had not been assessed in experimental animals because fHbp binds human fH specifically. In this study, we developed a human fH transgenic mouse model. Transgenic mice immunized with fHbp vaccine had 4- to 8-fold lower serum bactericidal Ab responses than those of control mice whose native fH did not bind the vaccine. In contrast, Ab responses were unimpaired in transgenic mice immunized with a control meningococcal group C polysaccharide-protein conjugate vaccine. In transgenic mice, immunization with an fH nonbinding mutant of fHbp elicited Abs with higher bactericidal activity than that of fHbp vaccination itself. Abs elicited by the mutant fHbp more effectively blocked fH binding to wild-type fHbp than Abs elicited by fHbp that bound fH. Thus, a mutant fHbp vaccine that does not bind fH but that retains immunogenicity is predicted to be superior in humans to an fHbp vaccine that binds human fH. In the case of mutant fHbp vaccination, the resultant Ab responses may be directed more at epitopes in or near the fH binding site, which result in greater complement-mediated serum bactericidal activity; these epitopes may be obscured when human fH is bound to the wild-type fHbp vaccine.     

Pig transgenesis by Sleeping Beauty DNA transposition

Modelling of human disease in genetically engineered pigs provides unique possibilities in biomedical research and in studies of disease intervention. Establishment of methodologies that allow efficient gene insertion by non-viral gene carriers is an important step towards development of new disease models. In this report, we present transgenic pigs created by Sleeping Beauty DNA transposition in primary porcine fibroblasts in combination with somatic cell nuclear transfer by handmade cloning. Göttingen minipigs expressing green fluorescent protein are produced by transgenesis with DNA transposon vectors carrying the transgene driven by the human ubiquitin C promoter. These animals carry multiple copies (from 8 to 13) of the transgene and show systemic transgene expression. Transgene-expressing pigs carry both transposase-catalyzed insertions and at least one copy of randomly inserted plasmid DNA. Our findings illustrate critical issues related to DNA transposon-directed transgenesis, including coincidental plasmid insertion and relatively low Sleeping Beauty transposition activity in porcine fibroblasts, but also provide a platform for future development of porcine disease models using the Sleeping Beauty gene insertion technology.     

A conserved cysteine motif is critical for rice ceramide kinase activity and function

Background

Ceramide kinase (CERK) is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare) and investigate the effects of ceramides on rice cell viability.

Principal Findings

OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg²⁺ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK''s substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing.

Conclusions

OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.     

TNFα-induced up-regulation of miR-155 inhibits adipogenesis by down-regulating early adipogenic transcription factors

Tumor necrosis factor α (TNFα) is known to inhibit adipogenesis, but the molecular mechanism of this inhibition remains elusive. In the present study, we found that TNFα-induced inhibition of adipogenesis mainly occurs when 3T3-L1 preadipocytes are treated with TNFα within 2 h induction of adipogenesis. We revealed that TNFα treatment results in the up-regulation of miR-155 through the NFκB pathway in 3T3-L1 cells. This overexpression of miR-155 may suppress the expression of C/EBPβ and CREB by directly targeting their 3′ untranslated regions (3′ UTRs). Importantly, anti-miR-155 reduces the TNFα-induced inhibition of adipogenesis, whereas exogenous expression of mir-155 inhibits adipogenesis. Taken together, these findings reveal a novel role for TNFα in the regulation of anti-adipogenic miRNAs.     

Comparison of HIV-, HBV-, HCV- and co-infection prevalence between Chinese and Burmese intravenous drug users of the China-Myanmar border region

Background

Co-infection with HIV and HCV and/or HBV is highly prevalent in intravenous drug users (IDUs). Because of the proximity to the “Golden Triangle”, HIV prevalence among the IDUs is very high in the China-Myanmar border region. However, there are few studies about co-infection with HIV and HCV and/or HBV, especially in the region that belongs to Myanmar.

Methods

721 IDUs, including 403 Chinese and 318 Burmese, were investigated for their HIV, hepatitis B virus (HBV), and hepatitis C virus (HCV) serological status. Statistical analysis was performed to evaluate the differences of the epidemic situation between the Chinese IDUs and the Burmese IDUs.

Results

Among the Chinese IDUs and the Burmese IDUs, HCV infection was the most prevalent (69.0% vs 48.1%, P<0.001), followed by HBV (51.6% vs 43.1%, P<0.05) and HIV (33.7% vs 27.0%, P>0.05). Besides, there were more HIV-HBV co-infected IDUs (20.1% vs 11.3%, P<0.005), and HIV-HCV co-infected IDUs (31.8% vs 23.9%, P<0.05) in China than in Myanmar, as well as HIV-HBV-HCV triple infection (19.1% vs 10.4%, P<0.005).

Conclusion

Co-infection with HIV and HCV and/or HBV is highly prevalent among the IDUs in the China-Myanmar border region. The HIV epidemic appears to be in a downward trend, compared with previous reports. However, all infections were more prevalent among the Chinese IDUs than among the Burmese.     

Improvement of a Potential Anthrax Therapeutic by Computational Protein Design

Past anthrax attacks in the United States have highlighted the need for improved measures against bioweapons. The virulence of anthrax stems from the shielding properties of the Bacillus anthracis poly-γ-d-glutamic acid capsule. In the presence of excess CapD, a B. anthracis γ-glutamyl transpeptidase, the protective capsule is degraded, and the immune system can successfully combat infection. Although CapD shows promise as a next generation protein therapeutic against anthrax, improvements in production, stability, and therapeutic formulation are needed. In this study, we addressed several of these problems through computational protein engineering techniques. We show that circular permutation of CapD improved production properties and dramatically increased kinetic thermostability. At 45 °C, CapD was completely inactive after 5 min, but circularly permuted CapD remained almost entirely active after 30 min. In addition, we identify an amino acid substitution that dramatically decreased transpeptidation activity but not hydrolysis. Subsequently, we show that this mutant had a diminished capsule degradation activity, suggesting that CapD catalyzes capsule degradation through a transpeptidation reaction with endogenous amino acids and peptides in serum rather than hydrolysis.     

A model for random genetic damage directing selection of diploid or aneuploid tumours

Objectives: To test whether genetic instability may determine whether tumours become aneuploid or diploid. Materials and methods: We have identified genes needed for cell survival or replication by combining Affymetrix gene expression array data from 12 experimental cell lines with in silico GEO+GNF and expO databases. Specific loss of heterozygosis (LOHs), chromosomal abnormalities (called derivative chromosomes) and numbers of normal homologues were identified by SNP and SKY analyses. Random gene losses were calculated under the assumption that bi‐allelic MMR gene inactivation causes a 20‐fold increase in rate of gene loss. Results: There were ～1.23 × 10⁴ genes widely dispersed throughout the genome and possibly expressed by all cells for survival or proliferation, many of these genes performed housekeeping functions. Conservation of the genes may explain the complete haploid genomes found for 15 different cell types and derivative chromosomes selectively retained in aneuploid cancer cell lines after LOH formations, and normal homologue losses. Loss of cell survival/replication genes was calculated to be higher in colon stem cells of carriers of MMR gene mutations than carriers of APC gene mutations. Conclusion: Random loss of cell survival/replication genes was calculated to be low enough for colon stem cells with APC gene mutations to ‘select’ LOH and derivative chromosome combinations favouring tumour cell proliferation. However, cell survival/replication gene loss was calculated to be too high for colonic stem cells lacking MMR genes to survive chromosomal instability, explaining why MMR mutations only produce tumours with diploid chromosome cells.     

Enzymatic placement of 6-O-sulfo groups in heparan sulfate

Heparan sulfate is a highly sulfated polysaccharide that exhibits important physiological and pathological functions. The glucosamine residue of heparan sulfate can carry sulfo groups at the 2-N, 3-O, and 6-O positions, leading to diverse polysaccharide structures. 6-O-Sulfation at the glucosamine residue contributes to a wide range of biological functions. Here, we report a method for controlling the positioning of 6-O-sulfo groups in oligosaccharides. This was achieved by synthesizing oligosaccharide backbones from a disaccharide building block utilizing glycosyltransferases followed by modifications using heparan sulfate N-sulfotransferase and 6-O-sulfotransferases. This method offers a viable approach for preparing heparan sulfate oligosaccharides with precisely located 6-O-sulfo groups.     

BMI-1 autoantibody as a new potential biomarker for cervical carcinoma

BMI-1 is overexpressed in a variety of cancers, which can elicit an immune response leading to the induction of autoantibodies. However, BMI-1 autoantibody as a biomarker has seldom been studied with the exception of nasopharyngeal carcinoma. Whether BMI-1 autoantibodies can be used as a biomarker for cervical carcinoma is unclear. In this study,BMI-1 proteins were isolated by screening of a T7 phage cDNA library from mixed cervical carcinoma tissues. We analyzed BMI-1 autoantibody levels in serum samples from 67 patients with cervical carcinoma and 65 controls using ELISA and immunoblot. BMI-1 mRNA or protein levels were over-expressed in cervical carcinoma cell lines. Immunoblot results exhibited increased BMI-1 autoantibody levels in patient sera compared to normal sera. Additionally, the results for antibody affinity assay showed that there was no difference between cervical polyps and normal sera of BMI-1 autoantibody levels, but it was significantly greater in patient sera than that in normal controls (patient 0.827±0.043 and normal 0.445±0.023; P<0.001). What''s more, the levels of BMI-1 autoantibody increased significantly at stage I (0.672±0.019) compared to normal sera (P<0.001), and levels of BMI-1 autoantibodies were increased gradually during the tumor progression (stage I 0.672±0.019; stage II 0.775 ±0.019; stage III 0.890 ±0.027; stage IV 1.043±0.041), which were significantly correlated with disease progression of cervical carcer (P<0.001). Statistical analyses using logistic regression and receiver operating characteristics (ROC) curves indicated that the BMI-1 autoantibody level can be used as a biomarker for cervical carcinoma (sensitivity 0.78 and specificity 0.76; AUC = 0.922). In conclusion, measuring BMI-1 autoantibody levels of patients with cervical cancer could have clinical prognostic value as well as a non-tissue specific biomarker for neoplasms expressing BMI-1.     

Nitric oxide production is associated with response to brown planthopper infestation in rice

Nilaparvata lugens Stål, the brown planthopper (BPH), is one of the most destructive phloem-feeding insects of rice (Oryza sativa L.) throughout Asia. Here, we show that BPH feeding increases the level of endogenous nitric oxide (NO) in the leaf and sheath tissue of both resistant and susceptible rice cultivars. However, in the roots, the NO level increased in the resistant cultivar, but decreased in the susceptible one. A burst of NO production occurred in the sheath within 1 h of infestation with BPH. The production of NO in response to BPH feeding appears to be dependent primarily on the activity of nitric oxide synthase. The application of exogenous NO reduced plant water loss by its effect on both stomatal opening and root architecture. It also stimulated the expression of certain drought stress-related genes, reduced plant height and delayed leaf senescence. Over the short term, NO supplementation reduced the seedling mortality caused by BPH feeding. This suggests that NO signaling plays a role in the rice tolerance response to BPH feeding.