Use of recombinant lentivirus pseudotyped with vesicular stomatitis virus glycoprotein G for efficient generation of human anti-cancer chimeric T cells by transduction of human peripheral blood lymphocytes in vitro

BACKGROUND: Genetic redirection of lymphocytes that have been genetically engineered to recognize antigens other than those originally programmed in their germlines is a potentially powerful tool for immunotherapy of cancers and potentially also of persistent viral infections. The basis for this procedure is that both cancers and some viruses have developed strikingly similar mechanisms of evading attacks by host immune mechanisms. To redirect human peripheral blood lymphocytes (PBLs) with a chimeric T cell receptor (chTCR) so that they recognize a new target requires a high degree of transfection efficiency, a process that is regarded as technically demanding. RESULTS: Infection with a retroviral vector carrying a chTCR cassette was shown to transduce 100% of rapidly dividing murine T cells but typically, only ~10% of PBLs could be infected with the same vector. In contrast with other retroviruses, lentiviruses integrate their genomes into non-dividing cells. To increase host cell range, vesicular stomatitis virus G protein was pseudotyped with a lentivirus vector, which resulted in ~100% PBL transduction efficiency. Signaling of PBLs bearing chimeric receptors was shown by specific proliferation on exposure to cells expressing cognate ligand. Further, T-bodies against CEA showed a startling abilty to cause regression of maligant colon tumors in a nude mouse model of human cancer. CONCLUSION: A lentivirus/VSV pseudotyped virus, which does not require replicating cells for integration of its genome, efficiently transduced a high proportion of human PBLs with chTCRs against CEA. PBLs transduced by infection with a lentivirus/VSV pseudotyped vector were able to proliferate specifically in vitro on exposure to CEA-expressing cells and further they had a startling therapeutic effect in a mouse model of human colon cancer

Ribosomal DNA sequence heterogeneity reflects intraspecies phylogenies and predicts genome structure in two contrasting yeast species

The ribosomal RNA encapsulates a wealth of evolutionary information, including genetic variation that can be used to discriminate between organisms at a wide range of taxonomic levels. For example, the prokaryotic 16S rDNA sequence is very widely used both in phylogenetic studies and as a marker in metagenomic surveys and the internal transcribed spacer region, frequently used in plant phylogenetics, is now recognized as a fungal DNA barcode. However, this widespread use does not escape criticism, principally due to issues such as difficulties in classification of paralogous versus orthologous rDNA units and intragenomic variation, both of which may be significant barriers to accurate phylogenetic inference. We recently analyzed data sets from the Saccharomyces Genome Resequencing Project, characterizing rDNA sequence variation within multiple strains of the baker's yeast Saccharomyces cerevisiae and its nearest wild relative Saccharomyces paradoxus in unprecedented detail. Notably, both species possess single locus rDNA systems. Here, we use these new variation datasets to assess whether a more detailed characterization of the rDNA locus can alleviate the second of these phylogenetic issues, sequence heterogeneity, while controlling for the first. We demonstrate that a strong phylogenetic signal exists within both datasets and illustrate how they can be used, with existing methodology, to estimate intraspecies phylogenies of yeast strains consistent with those derived from whole-genome approaches. We also describe the use of partial Single Nucleotide Polymorphisms, a type of sequence variation found only in repetitive genomic regions, in identifying key evolutionary features such as genome hybridization events and show their consistency with whole-genome Structure analyses. We conclude that our approach can transform rDNA sequence heterogeneity from a problem to a useful source of evolutionary information, enabling the estimation of highly accurate phylogenies of closely related organisms, and discuss how it could be extended to future studies of multilocus rDNA systems. [concerted evolution; genome hydridisation; phylogenetic analysis; ribosomal DNA; whole genome sequencing; yeast]

LotuS2: an ultrafast and highly accurate tool for amplicon sequencing analysis

Background: Amplicon sequencing is an established and cost-efficient method for profiling microbiomes. However, many available tools to process this data require both bioinformatics skills and high computational power to process big datasets. Furthermore, there are only few tools that allow for long read amplicon data analysis. To bridge this gap, we developed the LotuS2 (less OTU scripts 2) pipeline, enabling user-friendly, resource friendly, and versatile analysis of raw amplicon sequences.Results: In LotuS2, six different sequence clustering algorithms as well as extensive pre- and post-processing options allow for flexible data analysis by both experts, where parameters can be fully adjusted, and novices, where defaults are provided for different scenarios.We benchmarked three independent gut and soil datasets, where LotuS2 was on average 29 times faster compared to other pipelines, yet could better reproduce the alpha- and beta-diversity of technical replicate samples. Further benchmarking a mock community with known taxon composition showed that, compared to the other pipelines, LotuS2 recovered a higher fraction of correctly identified taxa and a higher fraction of reads assigned to true taxa (48% and 57% at species; 83% and 98% at genus level, respectively). At ASV/OTU level, precision and F-score were highest for LotuS2, as was the fraction of correctly reported 16S sequences.Conclusion: LotuS2 is a lightweight and user-friendly pipeline that is fast, precise, and streamlined, using extensive pre- and post-ASV/OTU clustering steps to further increase data quality. High data usage rates and reliability enable high-throughput microbiome analysis in minutes

Characterising the vertical separation of shale-gas source rocks and aquifers across England and Wales (UK)

Shale gas is considered by many to have the potential to provide the UK with greater energy security, economic growth and jobs. However, development of a shale gas industry is highly contentious due to environmental concerns including the risk of groundwater pollution. Evidence suggests that the vertical separation between exploited shale units and aquifers is an important factor in the risk to groundwater from shale gas exploitation. A methodology is presented to assess the vertical separation between different pairs of aquifers and shales that are present across England and Wales. The application of the method is then demonstrated for two of these pairs—the Cretaceous Chalk Group aquifer and the Upper Jurassic Kimmeridge Clay Formation, and the Triassic sandstone aquifer and the Carboniferous Bowland Shale Formation. Challenges in defining what might be considered criteria for ‘safe separation’ between a shale gas formation and an overlying aquifer are discussed, in particular with respect to uncertainties in geological properties, aquifer extents and determination of socially acceptable risk levels. Modelled vertical separations suggest that the risk of aquifer contamination from shale exploration will vary greatly between shale–aquifer pairs and between regions and this will need to be considered carefully as part of the risk assessment and management for any shale gas development

Repetitive sequence variation and dynamics in the ribosomal DNA array of Saccharomyces cerevisiae as revealed by whole-genome resequencing

Ribosomal DNA (rDNA) plays a key role in ribosome biogenesis, encoding genes for the structural RNA components of this important cellular organelle. These genes are vital for efficient functioning of the cellular protein synthesis machinery and as such are highly conserved and normally present in high copy numbers. In the baker's yeast Saccharomyces cerevisiae, there are more than 100 rDNA repeats located at a single locus on chromosome XII. Stability and sequence homogeneity of the rDNA array is essential for function, and this is achieved primarily by the mechanism of gene conversion. Detecting variation within these arrays is extremely problematic due to their large size and repetitive structure. In an attempt to address this, we have analyzed over 35 Mbp of rDNA sequence obtained from whole-genome shotgun sequencing (WGSS) of 34 strains of S. cerevisiae. Contrary to expectation, we find significant rDNA sequence variation exists within individual genomes. Many of the detected polymorphisms are not fully resolved. For this type of sequence variation, we introduce the term partial single nucleotide polymorphism, or pSNP. Comparative analysis of the complete data set reveals that different S. cerevisiae genomes possess different patterns of rDNA polymorphism, with much of the variation located within the rapidly evolving nontranscribed intergenic spacer (IGS) region. Furthermore, we find that strains known to have either structured or mosaic/hybrid genomes can be distinguished from one another based on rDNA pSNP number, indicating that pSNP dynamics may provide a reliable new measure of genome origin and stability

A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose.

Microbial consortia are a promising alternative to monocultures of genetically modified microorganisms for complex biotransformations. We developed a versatile consortium-based strategy for the direct conversion of lignocellulose to short-chain fatty acids, which included the funneling of the lignocellulosic carbohydrates to lactate as a central intermediate in engineered food chains. A spatial niche enabled in situ cellulolytic enzyme production by an aerobic fungus next to facultative anaerobic lactic acid bacteria and the product-forming anaerobes. Clostridium tyrobutyricum, Veillonella criceti, or Megasphaera elsdenii were integrated into the lactate platform to produce 196 kilograms of butyric acid per metric ton of beechwood. The lactate platform demonstrates the benefits of mixed cultures, such as their modularity and their ability to convert complex substrates into valuable biochemicals

CAPG is required for Ebola virus infection by controlling virus egress from infected cells

The replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors involved in actin nucleation, such as Rac1 and Arp2/3, were shown important in both steps. However, downstream of nucleation, many other cell factors are needed to control actin dynamics. How these regulate EBOV infection remains largely unclear. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown of CAPG specifically inhibited viral infectivity and yield of infectious particles. Cell-based mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split-green fluorescent protein reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells

Improving students’ ability at writing narrative text by using outline technique at the first grade of Mal Uin Su Medan in academic year 2016/2017

This research was aimed to know the students’ ability at writing narrative text by using outline technique. The research was conducted by using classroom action research. The subjects of this study were 36 of the tenth grade students of MAL UIN SU Medan in academic year 2016/2017. In doing this research, the technique of analyzing data was applied by using qualitative and quantitative approach. The qualitative data was taken from interview, observation, diary notes and documentation. The quantitative data was taken from the test. The result of the data analyzing showed that there was an improving on the students writing narrative text by using outline technique from each cycle. It was showed from mean of pre test was 51.41 and the mean of the students’ score for the post test I was 71.86, and the mean of the students’ score for post test II was 75.33. And based on interview, observation sheet, diary notes result and documentation showed that the students’ response at writing narrative text by using outline technique was good. It was found that teaching writing narrative text by using outline technique could improve the students’ ability

Heritability and genome-wide analyses of problematic peer relationships during childhood and adolescence

Peer behaviour plays an important role in the development of social adjustment, though little is known about its genetic architecture. We conducted a twin study combined with a genome-wide complex trait analysis (GCTA) and a genome-wide screen to characterise genetic influences on problematic peer behaviour during childhood and adolescence. This included a series of longitudinal measures (parent-reported Strengths-and-Difficulties Questionnaire) from a UK population-based birth-cohort (ALSPAC, 4–17 years), and a UK twin sample (TEDS, 4–11 years). Longitudinal twin analysis (TEDS; N ≤ 7,366 twin pairs) showed that peer problems in childhood are heritable (4–11 years, 0.60 < twin-h 2 ≤ 0.71) but genetically heterogeneous from age to age (4–11 years, twin-r g = 0.30). GCTA (ALSPAC: N ≤ 5,608, TEDS: N ≤ 2,691) provided furthermore little support for the contribution of measured common genetic variants during childhood (4–12 years, 0.02<GCTA−h2Meta ≤ 0.11) though these influences become stronger in adolescence (13–17 years, 0.14<GCTA−h2ALSPAC ≤ 0.27). A subsequent cross-sectional genome-wide screen in ALSPAC (N ≤ 6,000) focussed on peer problems with the highest GCTA-heritability (10, 13 and 17 years, 0.0002 < GCTA-P ≤ 0.03). Single variant signals (P ≤ 10−5) were followed up in TEDS (N ≤ 2835, 9 and 11 years) and, in search for autism quantitative trait loci, explored within two autism samples (AGRE: N Pedigrees = 793; ACC: N Cases = 1,453/N Controls = 7,070). There was, however, no evidence for association in TEDS and little evidence for an overlap with the autistic continuum. In summary, our findings suggest that problematic peer relationships are heritable but genetically complex and heterogeneous from age to age, with an increase in common measurable genetic variation during adolescence

Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution

Actively maintained close appositions, or contact sites, between organelle membranes, enable the efficient transfer of biomolecules between the various cellular compartments. Several such sites have been described together with their tethering machinery. Despite these advances we are still far from a comprehensive understanding of the function and regulation of most contact sites. To systematically characterize the proteome of contact sites and support the discovery of new tethers and functional molecules, we established a high throughput screening approach in Saccharomyces cerevisiae based on co-localization imaging. We imaged split fluorescence reporters for six different contact sites, two of which have never been studied before, on the background of 1165 strains expressing a mCherry-tagged yeast protein that have a cellular punctate distribution (a hallmark of contact sites). By scoring both co-localization events and effects on reporter size and abundance, we discovered over 100 new potential contact site residents and effectors in yeast. Focusing on several of the newly identified residents, we identified one set of hits as previously unrecognized homologs to Vps13 and Atg2. These proteins share their lipid transport domain, thus expanding this family of lipid transporters. Analysis of another candidate, Ypr097w, which we now call Lec1 (Lipid-droplet Ergosterol Cortex 1), revealed that this previously uncharacterized protein dynamically shifts between lipid droplets and the cell cortex, and plays a role in regulation of ergosterol distribution in the cell