Base-resolution detection of N4-methylcytosine in genomic DNA using 4mC-Tet-assisted-bisulfite- sequencing

Restriction-modification (R-M) systems pose a major barrier to DNA transformation and genetic engineering of bacterial species. Systematic identification of DNA methylation in R-M systems, including N⁶-methyladenine (6mA), 5-methylcytosine (5mC) and N⁴-methylcytosine (4mC), will enable strategies to make these species genetically tractable. Although single-molecule, real time (SMRT) sequencing technology is capable of detecting 4mC directly for any bacterial species regardless of whether an assembled genome exists or not, it is not as scalable to profiling hundreds to thousands of samples compared with the commonly used next-generation sequencing technologies. Here, we present 4mC-Tet-assisted bisulfite-sequencing (4mC-TAB-seq), a next-generation sequencing method that rapidly and cost efficiently reveals the genome-wide locations of 4mC for bacterial species with an available assembled reference genome. In 4mC-TAB-seq, both cytosines and 5mCs are read out as thymines, whereas only 4mCs are read out as cytosines, revealing their specific positions throughout the genome. We applied 4mC-TAB-seq to study the methylation of a member of the hyperthermophilc genus, Caldicellulosiruptor, in which 4mC-related restriction is a major barrier to DNA transformation from other species. In combination with MethylC-seq, both 4mC- and 5mC-containing motifs are identified which can assist in rapid and efficient genetic engineering of these bacteria in the future.     

Randomised controlled trial of an occupational therapy intervention to increase outdoor mobility after stroke

Objective To evaluate an occupational therapy intervention to improve outdoor mobility after stroke.Design Randomised controlled trial.Setting General practice registers, social services departments, a primary care rehabilitation service, and a geriatric day hospital.Participants 168 community dwelling people with a clinical diagnosis of stroke in previous 36 months: 86 were allocated to the intervention group and 82 to the control group.Interventions Leaflets describing local transport services for disabled people (control group) and leaflets with assessment and up to seven intervention sessions by an occupational therapist (intervention group).Main outcome measures Responses to postal questionnaires at four and 10 months: primary outcome measure was response to whether participant got out of the house as much as he or she would like, and secondary outcome measures were response to how many journeys outdoors had been made in the past month and scores on the Nottingham extended activities of daily living scale, Nottingham leisure questionnaire, and general health questionnaire.Results Participants in the treatment group were more likely to get out of the house as often as they wanted at both four months (relative risk 1.72, 95% confidence interval 1.25 to 2.37) and 10 months (1.74, 1.24 to 2.44). The treatment group reported more journeys outdoors in the month before assessment at both four months (median 37 in intervention group, 14 in control group: P < 0.01) and 10 months (median 42 in intervention group, 14 in control group: P < 0.01). At four months the mobility scores on the Nottingham extended activities of daily living scale were significantly higher in the intervention group, but there were no significant differences in the other secondary outcomes. No significant differences were observed in these measures at 10 months.Conclusion A targeted occupational therapy intervention at home increases outdoor mobility in people after stroke.     

IRAK-4 inhibitors. Part II: a structure-based assessment of imidazo[1,2-a]pyridine binding

A potent IRAK-4 inhibitor was identified through routine project cross screening. The binding mode was inferred using a combination of in silico docking into an IRAK-4 homology model, surrogate crystal structure analysis and chemical analogue SAR.     

Locating interaction sites on proteins: the crystal structure of thermolysin soaked in 2% to 100% isopropanol

Multiple-solvent crystal structure determination (MSCS) allows the position and orientation of bound solvent fragments to be identified by determining the structure of protein crystals soaked in organic solvents. We have extended this technique by the determination of high-resolution crystal structures of thermolysin (TLN), generated from crystals soaked in 2% to 100% isopropanol. The procedure causes only minor changes to the conformation of the protein, and an increasing number of isopropanol interaction sites could be identified as the solvent concentration is increased. Isopropanol occupies all four of the main subsites in the active site, although this was only observed at very high concentrations of isopropanol for three of the four subsites. Analysis of the isopropanol positions shows little correlation with interaction energy computed using a molecular mechanics force field, but the experimentally determined positions of isopropanol are consistent with the structures of known protein-ligand complexes of TLN.     

Avian fruit preferences across a Puerto Rican forested landscape: pattern consistency and implications for seed removal

Avian fruit consumption may ensure plant reproductive success when frugivores show consistent preference patterns and effectively remove and disperse seeds. In this study we examined avian fruit preferences and their seed-removal services at five study sites in north-central Puerto Rico. At each site, we documented the diet of seven common fruit-eating avian species from February to September 1998. Using foraging observations and area-based estimates of fruit abundance, we examined preference patterns of birds. We found that 7 out of 68 fleshy-fruited plant species were responsible for most of the fruit diet of birds. Seventeen plant species were preferred and four of them were repeatedly preferred across several study sites and times by at least one avian species. Preferred plant species comprised a small percentage of fleshy fruits at each site (<15% in four out of five study sites), but showed extended phenology patterns. The quantity of seeds removed by frugivore species was not strictly related to preferences. Some frugivores showing no preference could effectively remove more seeds from plants at some locations than species exhibiting constancy in their patterns of preference. Only two frugivores, Euphonia musica and Vireo altiloquous, removed most of the seeds of plants for which they exhibited repeated preference across the landscape. Preference patterns, particularly those exhibiting consistency in space and time for plant species having prolonged fruiting periods, may have important mechanistic consequences for the persistence, succession, and regeneration of tropical plant communities.     

Recent introduction of an allodapine bee into Fiji: A new model system for understanding biological invasions by pollinators

Morphology‐based studies have suggested a very depauperate bee fauna for islands in the South West Pacific, and recent genetic studies since have indicated an even smaller endemic fauna with many bee species in this region resulting from human‐aided dispersal. These introduced species have the potential to both disrupt native pollinator suites as well as augment crop pollination, but for most species the timings of introduction are unknown. We examined the distribution and nesting biology of the long‐tongued bee Braunsapis puangensis that was first recorded from Fiji in 2007. This bee has now become widespread in Fiji and both its local abundance and geographical range are likely to increase dramatically. The impacts of this invasion are potentially enormous for agriculture and native ecosystems, but they also provide opportunities for understanding how social insect species adapt to new environments. We outline the major issues associated with this recent invasion and argue that a long‐term monitoring study is needed.     

Characterization of Metabolites during Biodegradation of Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (RDX) with Municipal Anaerobic Sludge

The biodegradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in liquid cultures with municipal anaerobic sludge showed that at least two degradation routes were involved in the disappearance of the cyclic nitramine. In one route, RDX was reduced to give the familiar nitroso derivatives hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX) and hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX). In the second route, two novel metabolites, methylenedinitramine [(O₂NNH)₂CH₂] and bis(hydroxymethyl)nitramine [(HOCH₂)₂NNO₂], formed and were presumed to be ring cleavage products produced by enzymatic hydrolysis of the inner C—N bonds of RDX. None of the above metabolites accumulated in the system, and they disappeared to produce nitrous oxide (N₂O) as a nitrogen-containing end product and formaldehyde (HCHO), methanol (MeOH), and formic acid (HCOOH) that in turn disappeared to produce CH₄ and CO₂ as carbon-containing end products.