Description: Genetic structure can be altered by chemical mutagenesis, which is a common method applied in molecular biology and genetics. Second-generation sequencing provides a platform to reveal base alterations occurring in the whole genome due to mutagenesis. A model legume, Lotus japonicus ecotype Miyakojima , was chemically mutated with alkylating ethyl methanesulfonate (EMS) for the scanning of DNA lesions throughout the genome. Using second-generation sequencing, two individually mutated third-generation progeny (M3, named AM and AS) were sequenced and analyzed to identify single nucleotide polymorphisms and reveal the effects of EMS on nucleotide sequences in these mutant genomes. Single-nucleotide polymorphisms were found in every 208 kb (AS) and 202 kb (AM) with a bias mutation of G/C-to-A/T changes at low percentage. Most mutations were intergenic. The mutation spectrum of the genomes was comparable in their individual chromosomes; however, each mutated genome has unique alterations, which are useful to identify causal mutations for their phenotypic changes. The data obtained demonstrate that whole genomic sequencing is applicable as a high-throughput tool to investigate genomic changes due to mutagenesis. The identification of these single-point mutations will facilitate the identification of phenotypically causative mutations in EMS-mutated germplasm.

Description: Background and Purpose— The National Institutes of Health policy calls for the inclusion of under-represented groups, such as women and minorities, in clinical research. Poor minority recruitment and retention in stroke clinical trials remain a significant challenge limiting safety and efficacy in a general population. Previous research examines participant barriers to clinical trial involvement, but little is known about the investigator perspective. This study addresses this gap and examines researcher-reported barriers and best practices of minority involvement in stroke clinical trials. Methods— Quantitative and qualitative methods, including surveys, focus groups, and key informant interviews were used. Results— In a survey of 93 prominent stroke researchers, 43 (51.2%; 70% response rate) respondents reported proactively setting recruitment goals for minority inclusion, 29 respondents (36.3%) reported requiring cultural competency staff training, and 44 respondents (51.2%) reported using community consultation about trial design. Focus groups and key informant interviews highlighted structural and institutional challenges to recruitment of minorities, including mistrust of the research/medical enterprise, poor communication, and lack of understanding of clinical trials. Researcher-identified best practices included using standardized project management procedures and protocols (eg, realistic budgeting to support challenges in recruitment, such as travel/parking reimbursement for participants), research staff cultural competency and communication training, and developing and fostering community partnerships that guide the research process. Conclusions— This study’s formative evaluation contributes a new dimension to the literature as it highlights researcher-reported barriers and best practices for enhancing participation of minority populations into stroke clinical trials.

Description: Background: This paper reports the development and testing of a construct measuring parental fire safety behaviours for planning escape from a house fire. Methods: Latent variable modelling of data on parental-reported fire safety behaviours and plans for escaping from a house fire and multivariable logistic regression to quantify the association between groups defined by the latent variable modelling and parental-report of having a plan for escaping from a house fire. Data comes from 1112 participants in a cluster randomised controlled trial set in children’s centres in 4 study centres in the UK. Results: A two class model provided the best fit to the data, combining responses to five fire safety planning behaviours. The first group (‘more behaviours for escaping from a house fire’) comprised 86 % of participants who were most likely to have a torch, be aware of how their smoke alarm sounds, to have external door and window keys accessible, and exits clear. The second group (‘fewer behaviours for escaping from a house fire’) comprised 14 % of participants who were less likely to report these five behaviours. After adjusting for potential confounders, participants allocated to the ‘more behaviours for escaping from a house fire group were 2.5 times more likely to report having an escape plan (OR 2.48; 95 % CI 1.59–3.86) than those in the “fewer behaviours for escaping from a house fire” group. Conclusions: Multiple fire safety behaviour questions can be combined into a single binary summary measure of fire safety behaviours for escaping from a house fire. Our findings will be useful to future studies wishing to use a single measure of fire safety planning behaviour as measures of outcome or exposure.Trial registration number: NCT 01452191. Date of registration 13/10/2011

Description: Klebsiella pneumoniae is now recognized as an urgent threat to human health because of the emergence of multidrug-resistant strains associated with hospital outbreaks and hypervirulent strains associated with severe community-acquired infections. K. pneumoniae is ubiquitous in the environment and can colonize and infect both plants and animals. However, little is...

Description: Human T-cell lymphotropic virus type 1 (HTLV-1) and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of auxiliary protein-encoding open reading frames (ORFs) in HTLV-3, the latest HTLV to be discovered, is unknown. Simian T-cell lymphotropic virus type 3 (STLV-3) is almost identical to HTLV-3. Given the lack of HTLV-3-infected cell lines, we took advantage of STLV-3-infected cells and of an STLV-3 molecular clone to search for the presence of auxiliary transcripts. Using reverse transcriptase PCR (RT-PCR), we first uncovered the presence of three unknown viral mRNAs encoding putative proteins of 5, 8, and 9 kDa and confirmed the presence of the previously reported RorfII transcript. The existence of these viral mRNAs was confirmed by using splice site-specific RT-PCR with ex vivo samples. We showed that p5 is distributed throughout the cell and does not colocalize with a specific organelle. The p9 localization is similar to that of HTLV-1 p12 and induced a strong decrease in the calreticulin signal, similarly to HTLV-1 p12. Although p8, RorfII, and Rex-3 share an N-terminal sequence that is predicted to contain a nucleolar localization signal (NoLS), only p8 is found in the nucleolus. The p8 location in the nucleolus is linked to a bipartite NoLS. p8 and, to a lesser extent, p9 repressed viral expression but did not alter Rex-3-dependent mRNA export. Using a transformation assay, we finally showed that none of the STLV-3 auxiliary proteins had the ability to induce colony formation, while both Tax-3 and antisense protein of HTLV-3 (APH-3) promoted cellular transformation. Altogether, these results complete the characterization of the newly described primate T-lymphotropic virus type 3 (PTLV-3). IMPORTANCE Together with their simian counterparts, HTLVs form the primate T-lymphotropic viruses. HTLVs arose from interspecies transmission between nonhuman primates and humans. HTLV-1 and HTLV-2 encode auxiliary proteins that play important roles in viral replication, viral latency, and immune escape. The presence of ORFs encoding auxiliary proteins in HTLV-3 or STLV-3 genomes was unknown. Using in silico analyses, ex vivo samples, or in vitro experiments, we have uncovered the presence of 3 previously unknown viral mRNAs encoding putative proteins and confirmed the presence of a previously reported viral transcript. We characterized the intracellular localization of the four proteins. We showed that two of these proteins repress viral expression but that none of them have the ability to induce colony formation. However, both Tax and the antisense protein APH-3 promote cell transformation. Our results allowed us to characterize 4 new retroviral proteins for the first time.

Description: Many electrochemical devices are formulated on conjugated polymer films. A model is presented for electrogenerated chemiluminescence in such systems. The resulting diffusion equation for ion transport has a standard Fickian form, but with a highly nonlinear diffusion coefficient. The asymptotic analysis of the equation involves both intermediate layers and logarithmic matching. The asymptotic results show a sharp Fickian front, and compare favourably with numerical simulations and experimental results. Additional results are also presented for the solution beyond saturation, as well as in the more realistic case of polar coordinates.

Description: Klebsiella pneumoniae clonal group (CG) 258, comprising sequence types (STs) 258, 11, and closely related variants, is associated with dissemination of the K. pneumoniae carbapenemase (KPC). Hospital outbreaks of KPC CG258 infections have been observed globally and are very difficult to treat. As a consequence, there is renewed interest in alternative infection control measures such as vaccines and phage or depolymerase treatments targeting the K. pneumoniae polysaccharide capsule. To date, 78 immunologically distinct capsule variants have been described in K. pneumoniae. Previous investigations of ST258 and a small number of closely related strains suggested that capsular variation was limited within this clone; only two distinct ST258 capsule polysaccharide synthesis ( cps ) loci have been identified, both acquired through large-scale recombination events (〉50 kb). In contrast to previous studies, we report a comparative genomic analysis of the broader K. pneumoniae CG258 ( n = 39). We identified 11 different cps loci within CG258, indicating that capsular switching is actually common within the complex. We observed several insertion sequences (IS) within the cps loci, and show further intraclone diversification of two cps loci through IS activity. Our data also indicate that several large-scale recombination events have shaped the genomes of CG258, and that definition of the complex should be broadened to include ST395 (also reported to harbor KPC). As only the second report of extensive intraclonal cps variation among Gram-negative bacterial species, our findings alter our understanding of the evolution of these organisms and have key implications for the design of control measures targeting K. pneumoniae capsules.

Description: A series of three crosslink occultation experiments have been acquired between the Mars Odyssey and Mars Reconnaissance Orbiter spacecraft to probe the Martian atmosphere i\n 2007. While crosslink occultations between Earth orbiting satellites have been used to profile the Earth's atmosphere and ionosphere since 1995, this represents the first demonstration of crosslink occultation measurements at another planet. These measurements leverage the proximity link telecommunication payloads on each orbiter, which are nominally used to provide relay communication and navigation services to Mars landers and rovers. Analysis of the observed Doppler shift on each crosslink measurement reveals a clear signature of the Martian atmosphere, primarily the ionosphere. Inversion of the observed Doppler data yields vertical profiles of the Martian refractivity and electron density. The electron density profiles show the presence of two layers with peak densities and peak heights that are consistent with empirical model results. Our study demonstrates the feasibility and future potential of the crosslink radio occultation technique in the exploration of planetary atmospheres.

Description: Dymeclin is a Golgi-associated protein whose deficiency causes Dyggve–Melchior–Clausen syndrome (DMC, MIM #223800), a rare recessively inherited spondyloepimetaphyseal dysplasia consistently associated with postnatal microcephaly and intellectual disability. While the skeletal phenotype of DMC patients has been extensively described, very little is known about their cerebral anomalies, which result in brain growth defects and cognitive dysfunction. We used Dymeclin -deficient mice to determine the cause of microcephaly and to identify defective mechanisms at the cellular level. Brain weight and volume were reduced in all mutant mice from postnatal day 5 onward. Mutant mice displayed a narrowing of the frontal cortex, although cortical layers were normally organized. Interestingly, the corpus callosum was markedly thinner, a characteristic we also identified in DMC patients. Consistent with this, the myelin sheath was thinner, less compact and not properly rolled, while the number of mature oligodendrocytes and their ability to produce myelin basic protein were significantly decreased. Finally, cortical neurons from mutant mice and primary fibroblasts from DMC patients displayed substantially delayed endoplasmic reticulum to Golgi trafficking, which could be fully rescued upon Dymeclin re-expression. These findings indicate that Dymeclin is crucial for proper myelination and anterograde neuronal trafficking, two processes that are highly active during postnatal brain maturation.