Conversion of bovine pancreatic DNase I to a repair endonuclease with a high selectivity for abasic sites.

Bovine pancreatic deoxyribonuclease I (DNase I) is a nuclease of relatively low specificity which interacts with DNA in the minor groove. No contacts are made between the protein and the major groove of the nucleic acid. DNase I is structurally homologous to exonuclease III, a DNA-repair enzyme with multiple activities. One of the main differences between the two enzymes is the presence of an additional alpha-helix in exonuclease III, in a position suggestive of interaction with the major groove of DNA. Recombinant DNA techniques have been used to add 14 amino acids, comprising the 10 amino acids of the exonuclease III alpha-helix flanked by a glycine rich region, to DNase I. The polypeptide has been inserted after serine 174, an amino acid on the surface of DNase I corresponding to the location of the extra alpha-helix in exonuclease III. The recombinant protein, DNase-exohelix, has been purified and its catalytic activities towards DNA investigated. The recombinant protein demonstrated a high selectivity for endonucleolytic cleavage at abasic sites in DNA, a property of exonuclease III but not native DNase I. Thus the insertion of 14 amino acids at Ser174, converts DNase I to an exonuclease III-like enzyme with DNA-repair properties.     

Active coping toward predatory stress is associated with lower corticosterone and progesterone plasma levels and decreased methylation in the medial amygdala vasopressin system

An active coping style displayed under stress – which involves proactive investigatory responses toward environmental threats – has been associated with reduced vulnerability to psychiatric illness. However, the neurobiological determinants of coping styles are not well understood. When rats are exposed to a naturalistic stressor (cat fur) in a group, some individuals in the group show robust active investigation of the stimulus while others show a passive response involving retreat, immobility and close aggregation with conspecifics. Here we explored endocrine and epigenetic correlates of these contrasting coping styles. Male Wistar rats (n = 48) were exposed to cat fur in groups of 4 and the passive and active responders were identified and assessed for endocrine and epigenetic differences. Three days after the final cat fur exposure, active responders had substantially lower plasma levels of corticosterone and progesterone than passive responders. Plasma and testicular testosterone levels did not differ between active and passive responders. Active responders had markedly less methylation of the AVP CGCG promoter region located at base 4970 in the posterodorsal region of the medial amygdala but did not differ in the methylation status of the CCGG sequence located at base 2243. This is in agreement with prior research suggesting that AVP and progesterone act in opposition within the medial amygdala to modulate stress-related behaviors. The present study reports striking endocrine and epigenetic differences between active and passive responders, providing insight into potential systems involved in the manifestation of differing coping styles.     

Generation of tactile maps for artificial skin

Prior research has shown that representations of retinal surfaces can be learned from the intrinsic structure of visual sensory data in neural simulations, in robots, as well as by animals. Furthermore, representations of cochlear (frequency) surfaces can be learned from auditory data in neural simulations. Advances in hardware technology have allowed the development of artificial skin for robots, realising a new sensory modality which differs in important respects from vision and audition in its sensorimotor characteristics. This provides an opportunity to further investigate ordered sensory map formation using computational tools. We show that it is possible to learn representations of non-trivial tactile surfaces, which require topologically and geometrically involved three-dimensional embeddings. Our method automatically constructs a somatotopic map corresponding to the configuration of tactile sensors on a rigid body, using only intrinsic properties of the tactile data. The additional complexities involved in processing the tactile modality require the development of a novel multi-dimensional scaling algorithm. This algorithm, ANISOMAP, extends previous methods and outperforms them, producing high-quality reconstructions of tactile surfaces in both simulation and hardware tests. In addition, the reconstruction turns out to be robust to unanticipated hardware failure.     

The structure of Bacillus subtilis RecU Holliday junction resolvase and its role in substrate selection and sequence-specific cleavage

We have determined the structure of the enzyme RecU from Bacillus subtilis, that is the general Holliday junction resolving enzyme in Gram-positive bacteria. The enzyme fold reveals a striking similarity to a class of resolvase enzymes found in archaeal sources and members of the type II restriction endonuclease family to which they are related. The structure confirms the presence of active sites formed around clusters of acidic residues that we have also shown to bind divalent cations. Mutagenesis data presented here support the key role of certain residues. The RecU structure suggests a basis for Holliday junction selectivity and suggests how sequence-specific cleavage might be achieved. Models for a resolvase-DNA complex address how the enzyme might organize junctions into an approximately 4-fold symmetric form.     

Probiotic therapy for the prevention and treatment of Clostridium difficile-associated diarrhea: a systematic review

BackgroundThe recent increase in the number and severity of cases of nosocomial Clostridium difficile-associated diarrhea (CDAD) has prompted interest in the use of probiotics for the prevention and treatment of this disease. We performed a systematic review of randomized controlled trials to assess the effectiveness of probiotic therapy.MethodsWe searched the PubMed, EMBASE, INAHTA, HEN and Cochrane Collaboration databases to identify trials in which the prevention or treatment of CDAD with probiotic therapy was the primary or secondary outcome. We extracted data on the number of patients randomly assigned to receive probiotic or placebo, the number of patients with CDAD, the type of probiotic, criteria for diagnosing CDAD, persistence of infection after treatment, compliance and adverse effects.ResultsWe identified 4 eligible studies in which prevention (n = 1) or treatment (n = 3) of CDAD was the primary outcome. The benefit of probiotic therapy seen in 2 of the studies was restricted to subgroups characterized by severe CDAD and increased use of vancomycin. The remaining 2 studies were too methodologically flawed for us to draw meaningful conclusions. We also identified 4 trials in which prevention of antibiotic-associated diarrhea with probiotics was the primary outcome and prevention of CDAD a secondary outcome. These studies were limited primarily by too few CDAD cases and provided no evidence of effective prophylaxis. Overall, heterogeneity in choice and dose of probiotic and in criteria for diagnosing CDAD makes it difficult to synthesize information from the 8 studies.InterpretationStudies conducted to date provide insufficient evidence for the routine clinical use of probiotics to prevent or treat CDAD. Better designed and larger studies are needed.Both the incidence and severity of Clostridium difficile-associated diarrhea (CDAD) have increased in hospitals across North America.^1,2,3,4 Recent outbreaks in Montreal and Sherbrooke resulted in a 4-fold increase in the number of CDAD cases as well as an increase in the number of attributable deaths.^1,5,6,7 CDAD is a debilitating and costly illness, particularly among patients with recurring episodes. Probiotics, or naturally occurring “good bacteria,” have been suggested as a means of both preventing and treating the disease.^8,9,10,11 The disturbance of normal gastrointestinal flora, particularly after antibiotic use, is believed to predispose patients to colonization by C. difficile;² probiotics, by delivering bacteria to the gastrointestinal tract, are believed to restore equilibrium in the altered gastrointestinal flora and thus protect against colonization.⁸ Probiotics that have been proposed for prevention and treatment of antibiotic-associated diarrhea and CDAD include various bacteria (Bifidobacterium, Lactobacillus GG, L. rhamnosus, L. casei, L. plantarum 299v, Enteroccus faecium [SF68]) and yeasts (Saccharomyces boulardii, S. cerevisiae). They are commonly available as lyophilized capsules or in the form of a fermented drink. We performed a systematic review of randomized controlled trials of probiotic therapy for the prevention and treatment of CDAD.