Decoding the rice genome

Rice cultivation is one of the most important agricultural activities on earth, with nearly 90% of it being produced in Asia. It belongs to the family of crops that includes wheat, maize and barley, and it supplies more than 50% of calories consumed by the world population. Its immense economic value and a relatively small genome size makes it a focal point for scientific investigations, so much so that four whole genome sequence drafts with varying qualities have been generated by both public and privately funded ventures. The availability of a complete and high-quality map-based sequence has provided the opportunity to study genome organization and evolution. Most importantly, the order and identity of 37,544 genes of rice have been unraveled. The sequence provides the required ingredients for functional genomics and molecular breeding programs aimed at unraveling intricate cellular processes and improving rice productivity.     

Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1s-1. The Km values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 microM and 308.28 microM, respectively whereas the Vmax and kcat values for the enzyme are 0.02074 micromoles/min/ml and 0.003 s-1, respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme.     

Evolution and Taxonomic Classification of Alphapapillomavirus 7 Complete Genomes: HPV18, HPV39, HPV45, HPV59, HPV68 and HPV70

Background

The species Alphapapillomavirus 7 (alpha-7) contains human papillomavirus genotypes that account for 15% of invasive cervical cancers and are disproportionately associated with adenocarcinoma of the cervix. Complete genome analyses enable identification and nomenclature of variant lineages and sublineages.

Methods

The URR/E6 region was sequenced to screen for novel variants of HPV18, 39, 45, 59, 68, 70, 85 and 97 from 1147 cervical samples obtained from multiple geographic regions that had previously been shown to contain an alpha-7 HPV isolate. To study viral heterogeneity, the complete 8 kb genome of 128 isolates, including 109 sequenced for this analysis, were annotated and analyzed. Viral evolution was characterized by constructing phylogenic trees using maximum-likelihood and Bayesian algorithms. Global and pairwise alignments were used to calculate total and ORF/region nucleotide differences; lineages and sublineages were assigned using an alphanumeric system. The prototype genome was assigned to the A lineage or A1 sublineage.

Results

The genomic diversity of alpha-7 HPV types ranged from 1.1% to 6.7% nucleotide sequence differences; the extent of genome-genome pairwise intratype heterogeneity was 1.1% for HPV39, 1.3% for HPV59, 1.5% for HPV45, 1.6% for HPV70, 2.1% for HPV18, and 6.7% for HPV68. ME180 (previously a subtype of HPV68) was designated as the representative genome for HPV68 sublineage C1. Each ORF/region differed in sequence diversity, from most variable to least variable: noncoding region 1 (NCR1) / noncoding region 2 (NCR2) > upstream regulatory region (URR) > E6 / E7 > E2 / L2 > E1 / L1.

Conclusions

These data provide estimates of the maximum viral genomic heterogeneity of alpha-7 HPV type variants. The proposed taxonomic system facilitates the comparison of variants across epidemiological and molecular studies. Sequence diversity, geographic distribution and phylogenetic topology of this clinically important group of HPVs suggest an independent evolutionary history for each type.     

Genome analysis and genetic enhancement of tomato

The Solanaceae is an important family of vegetable crops, ornamentals and medicinal plants. Tomato has served as a model member of this family largely because of its enriched cytogenetic, genetic, as well as physical, maps. Mapping has helped in cloning several genes of importance such as Pto, responsible for resistance against bacterial speck disease, Mi-1.2 for resistance against nematodes, and fw2.2 QTL for fruit weight. A high-throughput genome-sequencing program has been initiated by an international consortium of 10 countries. Since heterochromatin has been found to be concentrated near centromeres, the consortium is focusing on sequencing only the gene-rich euchromatic region. Genomes of the members of Solanaceae show a significant degree of synteny, suggesting that the tomato genome sequence would help in the cloning of genes for important traits from other Solanaceae members as well. ESTs from a large number of cDNA libraries have been sequenced, and microarray chips, in conjunction with wide array of ripening mutants, have contributed immensely to the understanding of the fruit-ripening phenomenon. Work on the analysis of the tomato proteome has also been initiated. Transgenic tomato plants with improved abiotic stress tolerance, disease resistance and insect resistance, have been developed. Attempts have also been made to develop tomato as a bioreactor for various pharmaceutical proteins. However, control of fruit quality and ripening remains an active and challenging area of research. Such efforts should pave the way to improve not only tomato, but also other solanaceous crops.     

Immunization with Hexon Modified Adenoviral Vectors Integrated with gp83 Epitope Provides Protection against Trypanosoma cruzi Infection

Background

Trypanosoma cruzi is the causative agent of Chagas disease. Chagas disease is an endemic infection that affects over 8 million people throughout Latin America and now has become a global challenge. The current pharmacological treatment of patients is unsuccessful in most cases, highly toxic, and no vaccines are available. The results of inadequate treatment could lead to heart failure resulting in death. Therefore, a vaccine that elicits neutralizing antibodies mediated by cell-mediated immune responses and protection against Chagas disease is necessary.

Methodology/Principal Findings

The “antigen capsid-incorporation” strategy is based upon the display of the T. cruzi epitope as an integral component of the adenovirus'' capsid rather than an encoded transgene. This strategy is predicted to induce a robust humoral immune response to the presented antigen, similar to the response provoked by native Ad capsid proteins. The antigen chosen was T. cruzi gp83, a ligand that is used by T. cruzi to attach to host cells to initiate infection. The gp83 epitope, recognized by the neutralizing MAb 4A4, along with His₆ were incorporated into the Ad serotype 5 (Ad5) vector to generate the vector Ad5-HVR1-gp83-18 (Ad5-gp83). This vector was evaluated by molecular and immunological analyses. Vectors were injected to elicit immune responses against gp83 in mouse models. Our findings indicate that mice immunized with the vector Ad5-gp83 and challenged with a lethal dose of T. cruzi trypomastigotes confer strong immunoprotection with significant reduction in parasitemia levels, increased survival rate and induction of neutralizing antibodies.

Conclusions/Significance

This data demonstrates that immunization with adenovirus containing capsid-incorporated T. cruzi antigen elicits a significant anti-gp83-specific response in two different mouse models, and protection against T. cruzi infection by eliciting neutralizing antibodies mediated by cell-mediated immune responses, as evidenced by the production of several Ig isotypes. Taken together, these novel results show that the recombinant Ad5 presenting T. cruzi gp83 antigen is a useful candidate for the development of a vaccine against Chagas disease.     

Weight Loss in Nonalcoholic Fatty Liver Disease Patients in an Ambulatory Care Setting Is Largely Unsuccessful but Correlates with Frequency of Clinic Visits

Background and Aims

Nonalcoholic fatty liver disease (NALFD) is a leading cause of liver disease. Weight loss improves clinical features of NAFLD; however, maintenance of weight loss outside of investigational protocols is poor. The goals of this study were to characterize patterns and clinical predictors of long-term weight loss in ambulatory patients with NAFLD.

Methods

We retrospectively reviewed 924 non-cirrhotic patients with NAFLD presenting to a liver clinic from May 1^st 2007 to April 30^th 2013. Overweight and obese patients were counseled on lifestyle modifications for weight loss as per USPSTF guidelines. The primary outcome was percent weight change between the first and last recorded visits: % weight change  =  (weight^initial – weight^final)/(weight^initial). Baseline BMI and percent BMI change were secondary measures. Predictors of weight loss were determined using logistic regression.

Results

The mean baseline BMI was 33.3±6.6 kg/m², and the mean follow-up duration was 17.3±17.6 months. Most patients with NAFLD were in either overweight (26.1%) or class I obesity (30.5%) categories at baseline, while the prevalence of underweight and class III obesity was lower (0.2% and 15.4%, respectively). Overall, there was no change in mean weight or BMI during the follow-up period, and only 183 patients (19.8%) lost at least 5% body weight during the follow up period. Independent predictors of weight loss included number of clinic visits and baseline BMI, and patients with higher baseline BMI required more clinic visits to lose weight.

Conclusions

Weight loss is largely unsuccessful in NAFLD patients in the ambulatory care setting. Frequent clinical encounters are associated with weight reduction, especially among individuals with high baseline BMI. Future studies are required to define effective weight loss strategies in NAFLD patients.