Construction and eukaryotic expression of recombinant large hepatitis delta antigen

Background:

Hepatitis delta virus (HDV) is a subviral human pathogen that exploits host RNA editing activity to produce two essential forms of the sole viral protein, hepatitis delta antigen (HDAg). Editing at the amber/W site of HDV antigenomic RNA leads to the production of the large form (L-HDAg), which is required for RNA packaging.

Methods:

In this study, PCR-based site-directed mutagenesis by the overlap extension method was used to create the point mutation converting the small-HDAg (S-HDAg) stop codon to a tryptophan codon through three stages.

Results:

Sequencing confirmed the desirable mutation and integrity of the L-HDAg open reading frame. The amplicon was ligated into pcDNA3.1 and transfected to Huh7 and HEK 293 cell lines. Western blot analysis using enhanced chemiluminescence confirmed L-HDAg expression. The recombinant L-HDAg localized within the nuclei of cells as determined by immunofluorescence and confocal microscopy.

Conclusion:

Because L-HDAg requires extensive post-translational modifications, the recombinant protein expressed in a mammalian system might be fully functional and applicable as a tool in HDV molecular studies, as well as in future vaccine research.Key Words: Hepatitis Delta Virus, L-HDAg, SOEing-PCR     

Occurrence and Characterization of the Bacterial Spot Pathogen Xanthomonas euvesicatoria on Pepper in Iran

We report in this study for the first time the occurrence of bacterial spot of pepper in Iran and both phenotypic and genetic characterization of its causal agent, Xanthomonas euvesicatoria. Pepper plants grown in 15 of 30 surveyed private gardens and commercial fields were infected by the pathogen in Marand County, East Azerbaijan Province, north‐western Iran. The obtained strains of X. euvesicatoria had different amylolytic and pectolytic activities compared with those reported for this species elsewhere. Pathogenicity tests showed that strains isolated from diseased pepper are able to infect tomato, in addition to pepper. Host range of the pathogen was assessed on eight annual plant species including crops and weeds by measuring the population dynamics. The host range assessment showed that in addition to pepper and tomato, known hosts of X. euvesicatoria, the Iranian strains were able to colonize a number of new hosts such as nightshade and common bean. In contrast, none of them were able to build up their population on cowpea, eggplant, bindweed and zucchini. All X. euvesicatoria strains obtained in this study were sensitive to copper sulphate and streptomycin at concentrations higher than 20 and 50 mg/l, respectively. Phylogenetic analyses of the strains using the sequences of gyrB and hrpB genes confirmed their species as X. euvesicatoria. Given a direct commercial trade of fresh solanaceous vegetables between Iran and Turkey, it is hypothesized that the pathogen entered north‐western Iran from eastern parts of Turkey through infected plant materials. Finally, the role of prevention – based on the use of healthy planting materials and resistant and/or tolerant plant varieties – to contain the potential disease epidemics is discussed.     

Gestational age‐dependent gene expression profiling of ATP‐binding cassette transporters in the healthy human placenta

The ATP‐binding cassette (ABC) transporters control placental transfer of several nutrients, steroids, immunological factors, chemicals, and drugs at the maternal‐fetal interface. We and others have demonstrated a gestational age‐dependent expression pattern of two ABC transporters, P‐glycoprotein and breast cancer resistance protein throughout pregnancy. However, no reports have comprehensively elucidated the expression pattern of all 50 ABC proteins, comparing first trimester and term human placentae. We hypothesized that placental ABC transporters are expressed in a gestational‐age dependent manner in normal human pregnancy. Using the TaqMan^® Human ABC Transporter Array, we assessed the mRNA expression of all 50 ABC transporters in first (first trimester, n = 8) and third trimester (term, n = 12) human placentae and validated the resulting expression of selected ABC transporters using qPCR, Western blot and immunohistochemistry. A distinct gene expression profile of 30 ABC transporters was observed comparing first trimester vs. term placentae. Using individual qPCR in selected genes, we validated the increased expression of ABCA1 (P < 0.01), ABCA6 (P < 0.001), ABCA9 (P < 0.001) and ABCC3 (P < 0.001), as well as the decreased expression of ABCB11 (P < 0.001) and ABCG4 (P < 0.01) with advancing gestation. One important lipid transporter, ABCA6, was selected to correlate protein abundance and characterize tissue localization. ABCA6 exhibited increased protein expression towards term and was predominantly localized to syncytiotrophoblast cells. In conclusion, expression patterns of placental ABC transporters change as a function of gestational age. These changes are likely fundamental to a healthy pregnancy given the critical role that these transporters play in the regulation of steroidogenesis, immunological responses, and placental barrier function and integrity.     

Cross talk between energy cost and expression of Methyl Jasmonate-regulated genes: from DNA to protein

Journal of Plant Biochemistry and Biotechnology - Plant cell balances energy consumption for its different biological processes under oxidative circumstances. The aim of the present study is to...     

Predicting coral community recovery using multi‐species population dynamics models

Predicting whether, how, and to what degree communities recover from disturbance remain major challenges in ecology. To predict recovery of coral communities we applied field survey data of early recovery dynamics to a multi‐species integral projection model that captured key demographic processes driving coral population trajectories, notably density‐dependent larval recruitment. After testing model predictions against field observations, we updated the model to generate projections of future coral communities. Our results indicated that communities distributed across an island landscape followed different recovery trajectories but would reassemble to pre‐disturbed levels of coral abundance, composition, and size, thus demonstrating persistence in the provision of reef habitat and other ecosystem services. Our study indicates that coral community dynamics are predictable when accounting for the interplay between species life‐history, environmental conditions, and density‐dependence. We provide a quantitative framework for evaluating the ecological processes underlying community trajectory and characteristics important to ecosystem functioning.     

Adult index patient with Currarino syndrome due to a novel HLXB9 mutation, c.336dupG (p.P113fsX224), presenting with Hirschsprung's disease, cephalgia, and lumbodynia

BACKGROUND: The symptom triad of autosomal dominant Currarino syndrome (CS; MIM #176450) consists of anorectal malformation, a sacral bone defect, and presacral masses. Mutations in the homeoboxHLXB9 gene have already been described in a subset of sacrococcygeal anomalies characterized by partial sacral agenesis. CASE: We report a 28-year-old male patient with Currarino syndrome due to a heterozygous novel frame-shift mutation c.336dupG (p.P113fsX224) in the homeoboxHLXB9 gene. CONCLUSIONS: Molecular diagnostics may be helpful in cases of Hirschsprung's disease accompanied by other symptoms suggestive for Currarino syndrome, since it can lead to major complications such as perianal sepsis, meningitis, and malignant transformation.     

Diversity of archaea and niche preferences among putative ammonia-oxidizing Nitrososphaeria dominating across European arable soils

Archaeal communities in arable soils are dominated by Nitrososphaeria, a class within Thaumarchaeota comprising all known ammonia-oxidizing archaea (AOA). AOA are key players in the nitrogen cycle and defining their niche specialization can help predicting effects of environmental change on these communities. However, hierarchical effects of environmental filters on AOA and the delineation of niche preferences of nitrososphaerial lineages remain poorly understood. We used phylogenetic information at fine scale and machine learning approaches to identify climatic, edaphic and geomorphological drivers of Nitrososphaeria and other archaea along a 3000 km European gradient. Only limited insights into the ecology of the low-abundant archaeal classes could be inferred, but our analyses underlined the multifactorial nature of niche differentiation within Nitrososphaeria. Mean annual temperature, C:N ratio and pH were the best predictors of their diversity, evenness and distribution. Thresholds in the predictions could be defined for C:N ratio and cation exchange capacity. Furthermore, multiple, independent and recent specializations to soil pH were detected in the Nitrososphaeria phylogeny. The coexistence of widespread ecophysiological differences between closely related soil Nitrososphaeria highlights that their ecology is best studied at fine phylogenetic scale.     

Positive correlation between vitamin D receptor gene TaqI variant and gastric cancer predisposition in a sample of Iranian population

Gastric cancer is the second cause of cancer-related mortality and the fourth most common cancers worldwide. Owing to the immune modulatory effect of vitamin D in the body, the role of vitamin D receptor gene in vitamin D regulation receives a great deal of research interest. The aim of the current study was to highlight the association between two variants of TaqI and FokI in the vitamin D receptor gene and gastric cancer predisposition in a sample of South Khorasan population. The present investigation consisted of 69 patients affected with gastric cancer and 100 healthy individuals. The genomic DNA was extracted by salting out the protocol from peripheral venous blood. Genotyping of TaqI and FokI variants were performed by PCR-RFLP method. Our findings manifested that TC genotype of TaqI polymorphism was statistically significant between the case and the control groups (p = 0.002). Moreover, the frequency of TC + CC genotypes was statistically significant between the two groups (p = 0.009). Furthermore, we could not find any meaningful association between FokI variant and the participant groups. The present results declared that, in our population, TC genotype of TaqI polymorphism has an association with gastric cancer susceptibility. In addition, more investigation with greater sample sizes is needed to confirm our results.     

Induction of protective immune response to intranasal administration of influenza virus-like particles in a mouse model

Human influenza A viruses (IAVs) cause global pandemics and epidemics, which remains a nonignorable serious concern for public health worldwide. To combat the surge of viral outbreaks, new treatments are urgently needed. Here, we design a new vaccine based on virus-like particles (VLPs) and show how intranasal administration of this vaccine triggers protective immunity, which can be exploited for the development of new therapies. H1N1 VLPs were produced in baculovirus vectors and were injected into BALB/c mice by the intramuscular (IM) or intranasal (IN) route. We found that there were significantly higher inflammatory cell and lymphocyte concentrations in bronchoalveolar lavage samples and the lungs of IN immunized mice; however, the IM group had little signs of inflammatory responses. On the basis of our results, immunization with H1N1 influenza VLP elicited a strong T cell immunity in BALB/c mice. Despite T cell immunity amplification after both IN and IM vaccination methods in mice, IN-induced T cell responses were significantly more intense than IM-induced responses, and this was likely related to an increased number of both CD11b^high and CD103⁺ dendritic cells in mice lungs after IN administration of VLP. Furthermore, evaluation of interleukin-4 and interferon gamma cytokines along with several chemokine receptors showed that VLP vaccination via IN and IM routes leads to a greater CD4⁺ Th1 and Th2 response, respectively. Our findings indicated that VLPs represent a potential strategy for the development of an effective influenza vaccine; however, employing relevant routes for vaccination can be another important part of the universal influenza vaccine puzzle.