An Optimized, Chemically Regulated Gene Expression System for Chlamydomonas

BACKGROUND: Chlamydomonas reinhardtii is a model system for algal and cell biology and is used for biotechnological applications, such as molecular farming or biological hydrogen production. The Chlamydomonas metal-responsive CYC6 promoter is repressed by copper and induced by nickel ions. However, induction by nickel is weak in some strains, poorly reversible by chelating agents like EDTA, and causes, at high concentrations, toxicity side effects on Chlamydomonas growth. Removal of these bottlenecks will encourage the wide use of this promoter as a chemically regulated gene expression system. METHODOLOGY: Using a codon-optimized Renilla luciferase as a reporter gene, we explored several strategies to improve the strength and reversibility of CYC6 promoter induction. Use of the first intron of the RBCS2 gene or of a modified TAP medium increases the strength of CYC6 induction up to 20-fold. In the modified medium, induction is also obtained after addition of specific copper chelators, like TETA. At low concentrations (up to 10 microM) TETA is a more efficient inducer than Ni, which becomes a very efficient inducer at higher concentrations (50 microM). Neither TETA nor Ni show toxicity effects at the concentrations used. Unlike induction by Ni, induction by TETA is completely reversible by micromolar copper concentrations, thus resulting in a transient "wave" in luciferase activity, which can be repeated in subsequent growth cycles. CONCLUSIONS: We have worked out a chemically regulated gene expression system that can be finely tuned to produce temporally controlled "waves" in gene expression. The use of cassettes containing the CYC6 promoter, and of modified growth media, is a reliable and economically sustainable system for the temporally controlled expression of foreign genes in Chlamydomonas

Big data and data repurposing – using existing data to answer new questions in vascular dementia research

Introduction: Traditional approaches to clinical research have, as yet, failed to provide effective treatments for vascular dementia (VaD). Novel approaches to collation and synthesis of data may allow for time and cost efficient hypothesis generating and testing. These approaches may have particular utility in helping us understand and treat a complex condition such as VaD. Methods: We present an overview of new uses for existing data to progress VaD research. The overview is the result of consultation with various stakeholders, focused literature review and learning from the group’s experience of successful approaches to data repurposing. In particular, we benefitted from the expert discussion and input of delegates at the 9th International Congress on Vascular Dementia (Ljubljana, 16-18th October 2015). Results: We agreed on key areas that could be of relevance to VaD research: systematic review of existing studies; individual patient level analyses of existing trials and cohorts and linking electronic health record data to other datasets. We illustrated each theme with a case-study of an existing project that has utilised this approach. Conclusions: There are many opportunities for the VaD research community to make better use of existing data. The volume of potentially available data is increasing and the opportunities for using these resources to progress the VaD research agenda are exciting. Of course, these approaches come with inherent limitations and biases, as bigger datasets are not necessarily better datasets and maintaining rigour and critical analysis will be key to optimising data use

Assessing pooled BAC and whole genome shotgun strategies for assembly of complex genomes

<p>Abstract</p> <p>Background</p> <p>We investigate if pooling BAC clones and sequencing the pools can provide for more accurate assembly of genome sequences than the "whole genome shotgun" (WGS) approach. Furthermore, we quantify this accuracy increase. We compare the pooled BAC and WGS approaches using <it>in silico </it>simulations. Standard measures of assembly quality focus on assembly size and fragmentation, which are desirable for large whole genome assemblies. We propose additional measures enabling easy and visual comparison of assembly quality, such as rearrangements and redundant sequence content, relative to the known target sequence.</p> <p>Results</p> <p>The best assembly quality scores were obtained using 454 coverage of 15× linear and 5× paired (3kb insert size) reads (15L-5P) on <it>Arabidopsis</it>. This regime gave similarly good results on four additional plant genomes of very different GC and repeat contents. BAC pooling improved assembly scores over WGS assembly, coverage and redundancy scores improving the most.</p> <p>Conclusions</p> <p>BAC pooling works better than WGS, however, both require a physical map to order the scaffolds. Pool sizes up to 12Mbp work well, suggesting this pooling density to be effective in medium-scale re-sequencing applications such as targeted sequencing of QTL intervals for candidate gene discovery. Assuming the current Roche/454 Titanium sequencing limitations, a 12 Mbp region could be re-sequenced with a full plate of linear reads and a half plate of paired-end reads, yielding 15L-5P coverage after read pre-processing. Our simulation suggests that massively over-sequencing may not improve accuracy. Our scoring measures can be used generally to evaluate and compare results of simulated genome assemblies.</p

Predicting discharge to institutional long-term care after stroke: a systematic review &amp; meta-analysis

Background/Objectives: Stroke is a leading cause of disability worldwide, and a significant proportion of stroke survivors require long-term institutional care. Understanding who cannot be discharged home is important for health and social care planning. Our aim was to establish predictive factors for discharge to institutional care after hospitalization for stroke. Design: We registered and conducted a systematic review and meta-analysis (PROSPERO: CRD42015023497) of observational studies. We searched MEDLINE, EMBASE, and CINAHL Plus to February 2017. Quantitative synthesis was performed where data allowed. Setting: Acute and rehabilitation hospitals. Participants: Adults hospitalized for stroke who were newly admitted directly to long-term institutional care at the time of hospital discharge. Measurements: Factors associated with new institutionalization. Results: From 10,420 records, we included 18 studies (n = 32,139 participants). The studies were heterogeneous and conducted in Europe, North America, and East Asia. Eight studies were at high risk of selection bias. The proportion of those surviving to discharge who were newly discharged to long-term care varied from 7% to 39% (median 17%, interquartile range 12%), and the model of care received in the long-term care setting was not defined. Older age and greater stroke severity had a consistently positive association with the need for long-term care admission. Individuals who had a severe stroke were 26 times as likely to be admitted to long-term care than those who had a minor stroke. Individuals aged 65 and older had a risk of stroke that was three times as great as that of younger individuals. Potentially modifiable factors were rarely examined. Conclusion: Age and stroke severity are important predictors of institutional long-term care admission directly from the hospital after an acute stroke. Potentially modifiable factors should be the target of future research. Stroke outcome studies should report discharge destination, defining the model of care provided in the long-term care setting

454 sequencing of pooled BAC clones on chromosome 3H of barley

<p>Abstract</p> <p>Background</p> <p>Genome sequencing of barley has been delayed due to its large genome size (ca. 5,000Mbp). Among the fast sequencing systems, 454 liquid phase pyrosequencing provides the longest reads and is the most promising method for BAC clones. Here we report the results of pooled sequencing of BAC clones selected with ESTs genetically mapped to chromosome 3H.</p> <p>Results</p> <p>We sequenced pooled barley BAC clones using a 454 parallel genome sequencer. A PCR screening system based on primer sets derived from genetically mapped ESTs on chromosome 3H was used for clone selection in a BAC library developed from cultivar "Haruna Nijo". The DNA samples of 10 or 20 BAC clones were pooled and used for shotgun library development. The homology between contig sequences generated in each pooled library and mapped EST sequences was studied. The number of contigs assigned on chromosome 3H was 372. Their lengths ranged from 1,230 bp to 58,322 bp with an average 14,891 bp. Of these contigs, 240 showed homology and colinearity with the genome sequence of rice chromosome 1. A contig annotation browser supplemented with query search by unique sequence or genetic map position was developed. The identified contigs can be annotated with barley cDNAs and reference sequences on the browser. Homology analysis of these contigs with rice genes indicated that 1,239 rice genes can be assigned to barley contigs by the simple comparison of sequence lengths in both species. Of these genes, 492 are assigned to rice chromosome 1.</p> <p>Conclusions</p> <p>We demonstrate the efficiency of sequencing gene rich regions from barley chromosome 3H, with special reference to syntenic relationships with rice chromosome 1.</p

The Cassava Genome: Current Progress, Future Directions

The starchy swollen roots of cassava provide an essential food source for nearly a billion people, as well as possibilities for bioenergy, yet improvements to nutritional content and resistance to threatening diseases are currently impeded. A 454-based whole genome shotgun sequence has been assembled, which covers 69% of the predicted genome size and 96% of protein-coding gene space, with genome finishing underway. The predicted 30,666 genes and 3,485 alternate splice forms are supported by 1.4 M expressed sequence tags (ESTs). Maps based on simple sequence repeat (SSR)-, and EST-derived single nucleotide polymorphisms (SNPs) already exist. Thanks to the genome sequence, a high-density linkage map is currently being developed from a cross between two diverse cassava cultivars: one susceptible to cassava brown streak disease; the other resistant. An efficient genotyping-by-sequencing (GBS) approach is being developed to catalog SNPs both within the mapping population and among diverse African farmer-preferred varieties of cassava. These resources will accelerate marker-assisted breeding programs, allowing improvements in disease-resistance and nutrition, and will help us understand the genetic basis for disease resistance

A mixed methods study of hope, transitions, and quality of life in family caregivers of persons with Alzheimer's disease

<p>Abstract</p> <p>Background</p> <p>Several research studies have reported the poor quality of life of family caregivers of persons with Alzheimer's disease (AD). However, factors that influence their quality of life have not been clearly defined. The purpose of this study was to examine factors associated with the quality of life of these caregivers such as demographic variables, their transition experience, and hope. A secondary aim was to explore the transition experience of family caregivers of persons with AD.</p> <p>Methods</p> <p>A cross-sectional triangulation data transformation model mixed method design (Quant +Qual) was utilized to address the purpose of the study. Eighty family caregivers of persons with AD completed a survey with quantitative measures [demographic variables, Herth Hope Index (HHI-hope), World Health Organization Quality of Life -BREF (WHOQOL-BREF)] and a qualitative survey about their transitions experience. The qualitative data (transition open ended- survey) was converted to quantitative data using content analysis. Variables significant at the p < 0.10 level in the univariate analysis were entered in the multivariate generalized linear model used to determine significant factors associated with quality of life.</p> <p>Results</p> <p>Subjects with higher hope scores (p < 0.0001) (Factor 1: temporality and future-cognitive-temporary dimension of hope) and who dealt with their transitions by actively seeking out knowledge and assistance (p = 0.02) had higher overall quality of life scores. HHI scores were associated with overall quality of life and for each of the four quality of life domains (physical psychosocial, relationships, and environment).</p> <p>Conclusions</p> <p>Hope played a significant role in the subjects' perceptions of overall quality of life as well as the 4 quality of life domains. This underscores the need to develop ways to foster hope in family caregivers. Moreover, the active engagement of families in seeking information and help, as a way to deal with their transitions, suggests encouraging this engagement is important. The findings of this study also suggest many directions for future research, such as increasing our understanding of the processes of transitions for this population.</p

Interleukin-15 Treatment Induces Weight Loss Independent of Lymphocytes

Obesity is a chronic inflammatory condition characterized by activation and infiltration of proinflammatory immune cells and a dysregulated production of proinflammatory cytokines. While known as a key regulator of immune natural killer (NK) cell function and development, we have recently demonstrated that reduced expression of the cytokine Interleukin-15 (IL-15) is closely linked with increased body weight and adiposity in mice and humans. Previously, we and others have shown that obese individuals have lower circulating levels of IL-15 and NK cells. Lean IL-15 overexpressing (IL-15 tg) mice had an accumulation in adipose NK cells compared to wildtype and NK cell deficient obese IL-15−/− mice. Since IL-15 induces weight loss in IL-15−/− and diet induced obese mice and has effects on various lymphocytes, the aim of this paper was to determine if lymphocytes, particularly NK cells, play a role in IL-15 mediated weight loss. Acute IL-15 treatment resulted in an increased accumulation of NK, NKT, and CD3+ T cells in adipose tissue of B6 mice. Mice depleted of NK and NKT cells had similar weight loss comparable to controls treated with IL-15. Finally, IL-15 treatment induces significant weight loss in lymphocyte deficient RAG2−/−γc−/− mice independent of food intake. Fat pad cross-sections show decreased pad size with cytokine treatment is due to adipocyte shrinkage. These results clearly suggest that IL-15 mediates weight loss independent of lymphocytes

A pilot study for channel catfish whole genome sequencing and de novo assembly

<p>Abstract</p> <p>Background</p> <p>Recent advances in next-generation sequencing technologies have drastically increased throughput and significantly reduced sequencing costs. However, the average read lengths in next-generation sequencing technologies are short as compared with that of traditional Sanger sequencing. The short sequence reads pose great challenges for <it>de novo </it>sequence assembly. As a pilot project for whole genome sequencing of the catfish genome, here we attempt to determine the proper sequence coverage, the proper software for assembly, and various parameters used for the assembly of a BAC physical map contig spanning approximately a million of base pairs.</p> <p>Results</p> <p>A combination of low sequence coverage of 454 and Illumina sequencing appeared to provide effective assembly as reflected by a high N50 value. Using 454 sequencing alone, a sequencing depth of 18 X was sufficient to obtain the good quality assembly, whereas a 70 X Illumina appeared to be sufficient for a good quality assembly. Additional sequencing coverage after 18 X of 454 or after 70 X of Illumina sequencing does not provide significant improvement of the assembly. Considering the cost of sequencing, a 2 X 454 sequencing, when coupled to 70 X Illumina sequencing, provided an assembly of reasonably good quality. With several software tested, Newbler with a seed length of 16 and ABySS with a K-value of 60 appear to be appropriate for the assembly of 454 reads alone and Illumina paired-end reads alone, respectively. Using both 454 and Illumina paired-end reads, a hybrid assembly strategy using Newbler for initial 454 sequence assembly, Velvet for initial Illumina sequence assembly, followed by a second step assembly using MIRA provided the best assembly of the physical map contig, resulting in 193 contigs with a N50 value of 13,123 bp.</p> <p>Conclusions</p> <p>A hybrid sequencing strategy using low sequencing depth of 454 and high sequencing depth of Illumina provided the good quality assembly with high N50 value and relatively low cost. A combination of Newbler, Velvet, and MIRA can be used to assemble the 454 sequence reads and the Illumina reads effectively. The assembled sequence can serve as a resource for comparative genome analysis. Additional long reads using the third generation sequencing platforms are needed to sequence through repetitive genome regions that should further enhance the sequence assembly.</p

Understanding atmospheric organic aerosols via factor analysis of aerosol mass spectrometry: a review

Organic species are an important but poorly characterized constituent of airborne particulate matter. A quantitative understanding of the organic fraction of particles (organic aerosol, OA) is necessary to reduce some of the largest uncertainties that confound the assessment of the radiative forcing of climate and air quality management policies. In recent years, aerosol mass spectrometry has been increasingly relied upon for highly time-resolved characterization of OA chemistry and for elucidation of aerosol sources and lifecycle processes. Aerodyne aerosol mass spectrometers (AMS) are particularly widely used, because of their ability to quantitatively characterize the size-resolved composition of submicron particles (PM1). AMS report the bulk composition and temporal variations of OA in the form of ensemble mass spectra (MS) acquired over short time intervals. Because each MS represents the linear superposition of the spectra of individual components weighed by their concentrations, multivariate factor analysis of the MS matrix has proved effective at retrieving OA factors that offer a quantitative and simplified description of the thousands of individual organic species. The sum of the factors accounts for nearly 100% of the OA mass and each individual factor typically corresponds to a large group of OA constituents with similar chemical composition and temporal behavior that are characteristic of different sources and/or atmospheric processes. The application of this technique in aerosol mass spectrometry has grown rapidly in the last six years. Here we review multivariate factor analysis techniques applied to AMS and other aerosol mass spectrometers, and summarize key findings from field observations. Results that provide valuable information about aerosol sources and, in particular, secondary OA evolution on regional and global scales are highlighted. Advanced methods, for example a-priori constraints on factor mass spectra and the application of factor analysis to combined aerosol and gas phase data are discussed. Integrated analysis of worldwide OA factors is used to present a holistic regional and global description of OA. Finally, different ways in which OA factors can constrain global and regional models are discussed