Structural asymmetry in the Thermus thermophilus RuvC dimer suggests a basis for sequential strand cleavages during Holliday junction resolution

Holliday junction (HJ) resolvases are structure-specific endonucleases that cleave four-way DNA junctions (HJs) generated during DNA recombination and repair. Bacterial RuvC, a prototypical HJ resolvase, functions as homodimer and nicks DNA strands precisely across the junction point. To gain insights into the mechanisms underlying symmetrical strand cleavages by RuvC, we performed crystallographic and biochemical analyses of RuvC from Thermus thermophilus (T.th. RuvC). The crystal structure of T.th. RuvC shows an overall protein fold similar to that of Escherichia coli RuvC, but T.th. RuvC has a more tightly associated dimer interface possibly reflecting its thermostability. The binding mode of a HJ-DNA substrate can be inferred from the shape/charge complementarity between the T.th. RuvC dimer and HJ-DNA, as well as positions of sulfate ions bound on the protein surface. Unexpectedly, the structure of T.th. RuvC homodimer refined at 1.28 Å resolution shows distinct asymmetry near the dimer interface, in the region harboring catalytically important aromatic residues. The observation suggests that the T.th. RuvC homodimer interconverts between two asymmetric conformations, with alternating subunits switched on for DNA strand cleavage. This model provides a structural basis for the ‘nick-counter-nick’ mechanism in HJ resolution, a mode of HJ processing shared by prokaryotic and eukaryotic HJ resolvases.     

Dynamic Change of Prohibitin2 Expression in Rat Sciatic Nerve After Crush

As a novel cell cycle inhibitor, PHB2 controls the G1/S transition in cycling cells in a complex manner. Its aberrant expression is closely related to cell carcinogenesis. While its expression and role in peripheral nervous system lesion and repair were still unknown. Here, we performed an acute sciatic nerve crush (SNC) model in adult rats to examine the dynamic changes of PHB2. Temporally, PHB2 expression was sharply decreased after sciatic nerve crush and reached a valley at day 5. Spatially, PHB2 was widely expressed in the normal sciatic nerve including axons and Schwann cells. While after injury, PHB2 expression decreased predominantly in Schwann cells. The alteration was due to the decreased expression of PHB2 in Schwann cells after SNC. PHB2 expression correlated closely with Schwann cells proliferation in sciatic nerve post injury. Furthermore, PHB2 largely localized with GAP43 in axons in the crushed segment. Collectively, we suggested that PHB2 participated in the pathological process response to sciatic nerve injury and may be associated with Schwann cells proliferation and axons regeneration.     

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines

Key message

Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS.

Abstract

Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III–VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6–orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.     

Silicon-Mediated Tomato Resistance Against Ralstonia solanacearum is Associated with Modification of Soil Microbial Community Structure and Activity

Bacterial wilt caused by Ralstonia solanacearum is a serious soil-borne disease of Solanaceae crops. In this study, the soil microbial effects of silicon-induced tomato resistance against R. solanacearum were investigated through pot experiment. The results showed that exogenous 2.0 mM Si treatment reduced the disease index of bacterial wilt by 19.18 % to 52.7 % compared with non-Si-treated plants. The uptake of Si was significantly increased in the Si-treated tomato plants, where the Si content was higher in the roots than that in the shoots. R. solanacearum inoculation resulted in a significant increase of soil urease activity and reduction of soil sucrase activity, but had no effects on soil acid phosphatase activity. Si supply significantly increased soil urease and soil acid phosphatase activity under pathogen-inoculated conditions. Compared with the non-inoculated treatment, R. solanacearum infection significantly reduced the amount of soil bacteria and actinomycetes by 52.5 % and 16.5 %, respectively, but increased the ratio of soil fungi/soil bacteria by 93.6 %. After R. solanacearum inoculation, Si amendments significantly increased the amount of soil bacteria and actinomycetes and reduced soil fungi/soil bacteria ratio by 53.6 %. The results suggested that Si amendment is an effective approach to control R. solanacearum. Moreover, Si-mediated resistance in tomato against R. solanacearum is associated with the changes of soil microorganism amount and soil enzyme activity.     

Dysbiosis Signature of Fecal Microbiota in Colorectal Cancer Patients

The human gut microbiota is a complex system that is essential to the health of the host. Increasing evidence suggests that the gut microbiota may play an important role in the pathogenesis of colorectal cancer (CRC). In this study, we used pyrosequencing of the 16S rRNA gene V3 region to characterize the fecal microbiota of 19 patients with CRC and 20 healthy control subjects. The results revealed striking differences in fecal microbial population patterns between these two groups. Partial least-squares discriminant analysis showed that 17 phylotypes closely related to Bacteroides were enriched in the gut microbiota of CRC patients, whereas nine operational taxonomic units, represented by the butyrate-producing genera Faecalibacterium and Roseburia, were significantly less abundant. A positive correlation was observed between the abundance of Bacteroides species and CRC disease status (R?=?0.462, P?=?0.046?<?0.5). In addition, 16 genera were significantly more abundant in CRC samples than in controls, including potentially pathogenic Fusobacterium and Campylobacter species at genus level. The dysbiosis of fecal microbiota, characterized by the enrichment of potential pathogens and the decrease in butyrate-producing members, may therefore represent a specific microbial signature of CRC. A greater understanding of the dynamics of the fecal microbiota may assist in the development of novel fecal microbiome-related diagnostic tools for CRC.     

Biomimetic injectable HUVEC‐adipocytes/collagen/alginate microsphere co‐cultures for adipose tissue engineering

Engineering adipose tissue that has the ability to engraft and establish a vascular supply is a laudable goal that has broad clinical relevance, particularly for tissue reconstruction. In this article, we developed novel microtissues from surface‐coated adipocyte/collagen/alginate microspheres and human umbilical vein endothelial cells (HUVECs) co‐cultures that resembled the components and structure of natural adipose tissue. Firstly, collagen/alginate hydrogel microspheres embedded with viable adipocytes were obtained to mimic fat lobules. Secondly, collagen fibrils were allowed to self‐assemble on the surface of the microspheres to mimic collagen fibrils surrounding the fat lobules in the natural adipose tissue and facilitate HUVEC attachment and co‐cultures formation. Thirdly, the channels formed by the gap among the microspheres served as the room for in vitro prevascularization and in vivo blood vessel development. The endothelial cell layer outside the microspheres was a starting point of rapid vascular ingrowth. Adipose tissue formation was analyzed for 12 weeks at 4‐week intervals by subcutaneous injection into the head of node mice. The vasculature in the regenerated tissue showed functional anastomosis with host blood vessels. Long‐term stability of volume and weight of the injection was observed, indicating that the vasculature formed within the constructs benefited the formation, maturity, and maintenance of adipose tissue. This study provides a microsurgical method for adipose regeneration and construction of biomimetic model for drug screening studies. Biotechnol. Bioeng. 2013; 110: 1430–1443. © 2012 Wiley Periodicals, Inc.     

Gene Content and Diversity of the Loci Encoding Biosynthesis of Capsular Polysaccharides of the 15 Serovar Reference Strains of Haemophilus parasuis

Haemophilus parasuis is the causative agent of Glässer''s disease, a systemic disease of pigs, and is also associated with pneumonia. H. parasuis can be classified into 15 different serovars. Here we report, from the 15 serotyping reference strains, the DNA sequences of the loci containing genes for the biosynthesis of the group 1 capsular polysaccharides, which are potential virulence factors of this bacterium. We contend that these loci contain genes for polysaccharide capsule structures, and not a lipopolysaccharide O antigen, supported by the fact that they contain genes such as wza, wzb, and wzc, which are associated with the export of polysaccharide capsules in the current capsule classification system. A conserved region at the 3′ end of the locus, containing the wza, ptp, wzs, and iscR genes, is consistent with the characteristic export region 1 of the model group 1 capsule locus. A potential serovar-specific region (region 2) has been found by comparing the predicted coding sequences (CDSs) in all 15 loci for synteny and homology. The region is unique to each reference strain with the exception of those in serovars 5 and 12, which are identical in terms of gene content. The identification and characterization of this locus among the 15 serovars is the first step in understanding the genetic, molecular, and structural bases of serovar specificity in this poorly studied but important pathogen and opens up the possibility of developing an improved molecular serotyping system, which would greatly assist diagnosis and control of Glässer''s disease.     

4-Bicyclic heteroaryl-piperidine derivatives as potent,orally bioavailable Stearoyl-CoA desaturase-1 (SCD1) inhibitors. Part 1: Urea-based analogs

A new series of urea-based, 4-bicyclic heteroaryl-piperidine derivatives as potent SCD1 inhibitors is described. The structure–activity relationships focused on bicyclic heteroarenes and aminothiazole–urea portions are discussed. A trend of dose-dependent decrease in body weight gain in diet-induced obese (DIO) mice is also demonstrated.     

TOPS-MODE model of multiplexing neuroprotective effects of drugs and experimental-theoretic study of new 1,3-rasagiline derivatives potentially useful in neurodegenerative diseases

The interest on computational techniques for the discovery of neuroprotective drugs has increased due to recent fail of important clinical trials. In fact, there is a huge amount of data accumulated in public databases like CHEMBL with respect to structurally heterogeneous series of drugs, multiple assays, drug targets, and model organisms. However, there are no reports of multi-target or multiplexing Quantitative Structure–Property Relationships (mt-QSAR/mx-QSAR) models of these multiplexing assay outcomes reported in CHEMBL for neurotoxicity/neuroprotective effects of drugs. Accordingly, in this paper we develop the first mx-QSAR model for multiplexing assays of neurotoxicity/neuroprotective effects of drugs. We used the method TOPS-MODE to calculate the structural parameters of drugs. The best model found correctly classified 4393 out of 4915 total cases in both training and validation. This is representative of overall train and validation Accuracy, Sensitivity, and Specificity values near to 90%, 98%, and 80%, respectively. This dataset includes multiplexing assay endpoints of 2217 compounds. Every one compound was assayed in at least one out of 338 assays, which involved 148 molecular or cellular targets and 35 standard type measures in 11 model organisms (including human). The second aim of this work is the exemplification of the use of the new mx-QSAR model with a practical case of study. To this end, we obtained again by organic synthesis and reported, by the first time, experimental assays of the new 1,3-rasagiline derivatives 3 different tests: assay (1) in absence of neurotoxic agents, (2) in the presence of glutamate, and (3) in the presence of H₂O₂. The higher neuroprotective effects found for each one of these assays were for the stereoisomers of compound 7: compound 7b with protection = 23.4% in assay (1) and protection = 15.2% in assay (2); and for compound 7a with protection = 46.2% in assay (3). Interestingly, almost all compounds show protection values >10% in assay (3) but not in the other 2 assays. After that, we used the mx-QSAR model to predict the more probable response of the new compounds in 559 unique pharmacological tests not carried out experimentally. The results obtained are very significant because they complement the pharmacological studies of these promising rasagiline derivatives. This work paves the way for further developments in the multi-target/multiplexing screening of large libraries of compounds potentially useful in the treatment of neurodegenerative diseases.