Direct unfolding of RuvA-HJ complex at the single-molecule level

The repair of double-stranded DNA breaks via homologous recombination involves a four-way cross-strand intermediate known as Holliday junction (HJ), which is recognized, processed, and resolved by a specific set of proteins. RuvA, a prokaryotic HJ-binding protein, is known to stabilize the square-planar conformation of the HJ, which is otherwise a short-lived intermediate. Despite much progress being made regarding the molecular mechanism of RuvA-HJ interactions, the mechanochemical aspect of this protein-HJ complex is yet to be investigated. Here, we employed an optical-tweezers-based, single-molecule manipulation assay to detect the formation of RuvA-HJ complex and determined its mechanical and thermodynamic properties in a manner that would be impossible with traditional ensemble techniques. We found that the binding of RuvA increases the unfolding force (F_unfold) of the HJ by ～2-fold. Compared with the ΔG_unfold of the HJ alone (54 ± 13 kcal/mol), the increased free energy of the RuvA-HJ complex (101 ± 20 kcal/mol) demonstrates that the RuvA protein stabilizes HJs. Interestingly, the protein remains bound to the mechanically melted HJ, facilitating its refolding at an unusually high force when the stretched DNA molecule is relaxed. These results suggest that the RuvA protein not only stabilizes the HJs but also induces refolding of the HJs. The single-molecule platform that we employed here for studying the RuvA-HJ interaction is broadly applicable to study other HJ-binding proteins involved in the critical DNA repair process.     

Assessment of tissue allograft safety monitoring with administrative healthcare databases: a pilot project using Medicare data

Assess whether Medicare data are useful for monitoring tissue allograft safety and utilization. We used health care claims (billing) data from 2007 for 35 million fee-for-service Medicare beneficiaries, a predominantly elderly population. Using search terms for transplant-related procedures, we generated lists of ICD-9-CM and CPT^® codes and assessed the frequency of selected allograft procedures. Step 1 used inpatient data and ICD-9-CM procedure codes. Step 2 added non-institutional provider (e.g., physician) claims, outpatient institutional claims, and CPT codes. We assembled preliminary lists of diagnosis codes for infections after selected allograft procedures. Many ICD-9-CM codes were ambiguous as to whether the procedure involved an allograft. Among 1.3 million persons with a procedure ascertained using the list of ICD-9-CM codes, only 1,886 claims clearly involved an allograft. CPT codes enabled better ascertainment of some allograft procedures (over 17,000 persons had corneal transplants and over 2,700 had allograft skin transplants). For spinal fusion procedures, CPT codes improved specificity for allografts; of nearly 100,000 patients with ICD-9-CM codes for spinal fusions, more than 34,000 had CPT codes indicating allograft use. Monitoring infrequent events (infections) after infrequent exposures (tissue allografts) requires large study populations. A strength of the large Medicare databases is the substantial number of certain allograft procedures. Limitations include lack of clinical detail and donor information. Medicare data can potentially augment passive reporting systems and may be useful for monitoring tissue allograft safety and utilization where codes clearly identify allograft use and coding algorithms can effectively screen for infections.     

Rising Midlife Obesity Will Worsen Future Prevalence of Dementia

Background

Midlife body weight status has been found to affect late life dementia outcomes. A cohort projections model was developed to assess the impact of midlife body mass index (BMI) profile on dementia in older Australians.

Methods

A baseline projection using age-sex specific dementia prevalence rates was constructed and the results of scenarios that took account of midlife BMI were compared with those from population ageing only.

Results

This modelling predicts that if the rising trend in midlife obesity and declining trend in midlife normal weight in Australia are to be taken into account in projecting future numbers of Australians with dementia then the number of people aged 65 or more years with dementia, by 2050, would be 14% higher than that expected from demographic ageing only. If midlife obesity prevalence was decreased to 20% and normal weight increased to 40% over the period of 2015–2025, then dementia cases among persons aged 65–69 years would be lower by about 10% in 2050 compared with the “doing nothing to stop current trends in obesity” projection.

Conclusion

The rising tide of obesity in Australian adults will increase the dementia epidemic expected in future years.     

Draft Genome Comparison of Representatives of the Three Dominant Genotype Groups of Dairy Bacillus licheniformis Strains

The spore-forming bacterium Bacillus licheniformis is a common contaminant of milk and milk products. Strains of this species isolated from dairy products can be differentiated into three major groups, namely, G, F1, and F2, using random amplification of polymorphic DNA (RAPD) analysis; however, little is known about the genomic differences between these groups and the identity of the fragments that make up their RAPD profiles. In this work we obtained high-quality draft genomes of representative strains from each of the three RAPD groups (designated strain G-1, strain F1-1, and strain F2-1) and compared them to each other and to B. licheniformis ATCC 14580 and Bacillus subtilis 168. Whole-genome comparison and multilocus sequence typing revealed that strain G-1 contains significant sequence variability and belongs to a lineage distinct from the group F strains. Strain G-1 was found to contain genes coding for a type I restriction modification system, urease production, and bacitracin synthesis, as well as the 8-kbp plasmid pFL7, and these genes were not present in strains F1-1 and F2-1. In agreement with this, all isolates of group G, but no group F isolates, were found to possess urease activity and antimicrobial activity against Micrococcus. Identification of RAPD band sequences revealed that differences in the RAPD profiles were due to differences in gene lengths, 3′ ends of predicted primer binding sites, or gene presence or absence. This work provides a greater understanding of the phylogenetic and phenotypic differences observed within the B. licheniformis species.     

Bio-transformation of artemisinin using soil microbe: Direct C-acetoxylation of artemisinin at C-9 by Penicillium simplissimum

Potent antimalarial compound artemisinin, 1 was bio-transformed to C-9 acetoxy artemisinin, 2 using soil microbe Penicillium simplissimum along with C-9 hydroxy derivative 3. The products were characterized using high field NMR and MS–MS data. The absolute stereochemistry of the newly generated chiral centers has been ascertained by COSY and 1D NOESY experiments. This is the first Letter of direct C-acetoxylation of artemisinin using microbial strains.     

Cross-Protective Effect of Antisense Oligonucleotide Developed Against the Common 3′ NCR of Influenza A Virus Genome

The influenza A virus (IAV) has eight segmented single-stranded RNA genome containing a common and evolutionarily conserved non-coding region (NCRs) at 5′ and 3′ ends that are important for the virus replication. In this study, we designed an antisense oligonucleotide against the 3′ NCR of vital segments of the IAV genome to inhibit its replication. The results demonstrated that the co-transfection of Madine Darby Canine Kidney (MDCK) cells with the antisense oligonucleotide and the plasmids encoding the viral genes led to the down-regulation of the viral gene expression. The designed antisense molecules reduced the cytopathic effect caused by A/PR/8/34 (H1N1), A/Udorn/307/72 (H3N2), and A/New Caledonia/20/99 (H1N1) strains of IAV for almost 48 h. Furthermore, the intra-venous delivery of this oligonucleotide significantly reduced the viral titers in the lungs of infected mice and protected the mice from lethal effects of all the strains of influenza virus. The study demonstrated that the antisense oligonucleotide designed against the NCR region inhibits the expression of the viral genome. The decrease of the cytopathic effect in the MDCK cells and increase in survival of mice confirmed the reduction of virus multiplication and pathogenesis in the presence of antisense oligonucleotide. Thus, we demonstrate that a single antisense oligonucleotide is capable of providing protection against more than one strains of the IAV.     

Cucurbitacin B Suppresses Hyperglycemia Associated with a High Sugar Diet and Promotes Sleep in Drosophila melanogaster

Secondary metabolites enable plants to protect themselves from herbivorous insects. Among these, cucurbitacin B (cuc-B) is a bitter-tasting compound with promising pharmacological potential. Dietary exposure to cuc-B lowered the hemolymph glucose levels of Drosophila melanogaster fed with a high carbohydrate diet, which is homologous to high blood glucose in humans, and its effect was comparable to that of metformin, a well-known glucose-lowering drug. Furthermore, cuc-B reduced tissue sugar levels and glycogen levels, as well as triacylglycerol levels. Our results thus highlight the potential applicability of this compound to treat chronic metabolic diseases such as diabetes and obesity. Additionally, we analyzed sleep quality and taste-associative memory enhancement after cuc-B and metformin treatment. Both supplements increased nighttime bout length and metformin increased memory consolidation. Therefore, discarded shell of Cucurbitaceae could be processed into health supplements.     

Stable nuclear transformation of rhodophyte species Porphyridium purpureum: advanced molecular tools and an optimized method

Photosynthesis Research - A mutated phytoene desaturase (pds) gene, pds-L504R, conferring resistance to the herbicide norflurazon has been reported as a dominant selectable marker for the genetic...     

Biosynthesis and characterization of a new copolymer, poly(3-hydroxyvalerate-co-5-hydroxydecenoate), from Pseudomonas aeruginosa

Using a new isolate of Pseudomonas aeruginosa, we obtained 7 g cell dry wt (CDW/l) using 5 % (w/v) glucose. Crude polyhydroxyalkanoates were obtained at 14.6 % of CDW. FTIR and NMR analysis confirmed that this was a new co-polymer: 3-hydroxyvalerate-co-5-hydroxydecenoate. Differential scanning calorimetry analysis showed two different melting temperatures of the copolymer and also indicated the glass transition temperature to be 4 °C. The polydispersity index of the polymer was 1.059.