Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Determinants of serum concentrations of organochlorine compounds in Swedish pregnant women: a cross-sectional study

BACKGROUND: We performed a cross-sectional study of associations between personal characteristics and lipid-adjusted serum concentrations of certain PCB congeners and chlorinated pesticides/metabolites among 323 pregnant primiparous women from Uppsala County (age 18–41 years) sampled 1996–1999. METHODS: Extensive personal interviews and questionnaires about personal characteristics were performed both during and after pregnancy. Concentrations of organochlorine compounds in serum lipids in late pregnancy were analysed by gas chromatography. Associations between personal characteristics and serum levels of organochlorine compounds were analysed by multiple linear regression. RESULTS: Participation rate was 82% (325 of 395 women). Serum concentrations of PCB congeners IUPAC no. 28, 52, 101, 105 and 167, and o, p'-DDT and -DDE, p, p'-DDT and -DDD, oxychlordane, and γ- and α-HCH were in many cases below the limit of quantification (LOQ). No statistical analysis of associations with personal characteristics could be performed for these substances. Concentrations of PCB congeners IUPAC no. 118, 138, 153, 156 and 180, HCB, β-HCH, trans-nonachlor and p, p'-DDE increased with increased age and were highest in women sampled early during the 4 year study period. This shows that older women and women sampled early in the study had experienced the highest life-time exposure levels, probably mainly during childhood and adolescence. The importance of early exposures was supported by lower PCB concentrations and higher β-HCH and p, p'-DDE concentrations among women born in non-Nordic countries. Moreover, serum concentrations of certain PCBs and pesticide/metabolites were positively associated with consumption of fatty fish during adolescence, and concentrations of CB 156, CB 180 and p, p'-DDE increased significantly with number of months women had been breast-fed during infancy. Short-term changes in bodily constitution may, however, also influence serum concentrations, as suggested by negative associations between concentrations of organochlorine compounds and BMI before pregnancy and weight change during pregnancy. CONCLUSION: Although some of the associations could be caused by unknown personal characteristics confounding the results, our findings suggest that exposures to organochlorine compounds during childhood and adolescence influence the body burdens of the compounds during pregnancy

Bringing Back a Healthy Buzz? Invertebrate Parasites and Reintroductions:A Case Study in Bumblebees

Reintroductions can play a key role in the conservation of endangered species. Parasites may impact reintroductions, both positively and negatively, but few case studies of how to manage parasites during reintroductions exist. Bumblebees are in decline at regional and global scales, and reintroductions can be used to re-establish extinct local populations. Here we report on how the risks associated with parasites are being managed in an ongoing reintroduction of the short-haired bumblebee, Bombus subterraneus, to the UK. Disease risk analysis was conducted and disease risk management plans constructed to design a capture-quarantine-release system that minimised the impacts on both the bumblebees and on their natural parasites. Given that bumblebee parasites are (i) generalists, (ii) geographically ubiquitous, and (iii) show evidence of local adaptation, the disease risk management plan was designed to limit the co-introduction of parasites from the source population in Sweden to the destination site in the UK. Results suggest that this process at best eliminated, or at least severely curtailed the co-introduction of parasites, and ongoing updates of the plan enabled minimization of impacts on natural host-parasite dynamics in the Swedish source population. This study suggests that methods designed for reintroductions of vertebrate species can be successfully applied to invertebrates. Future reintroductions of invertebrates where the parasite fauna is less well known should take advantage of next-generation barcoding and multiple survey years prior to the start of reintroductions, to develop comprehensive disease risk management plans

Peaks and Troughs of Three-Dimensional Vestibulo-ocular Reflex in Humans

The three-dimensional vestibulo-ocular reflex (3D VOR) ideally generates compensatory ocular rotations not only with a magnitude equal and opposite to the head rotation but also about an axis that is collinear with the head rotation axis. Vestibulo-ocular responses only partially fulfill this ideal behavior. Because animal studies have shown that vestibular stimulation about particular axes may lead to suboptimal compensatory responses, we investigated in healthy subjects the peaks and troughs in 3D VOR stabilization in terms of gain and alignment of the 3D vestibulo-ocular response. Six healthy upright sitting subjects underwent whole body small amplitude sinusoidal and constant acceleration transients delivered by a six-degree-of-freedom motion platform. Subjects were oscillated about the vertical axis and about axes in the horizontal plane varying between roll and pitch at increments of 22.5° in azimuth. Transients were delivered in yaw, roll, and pitch and in the vertical canal planes. Eye movements were recorded in with 3D search coils. Eye coil signals were converted to rotation vectors, from which we calculated gain and misalignment. During horizontal axis stimulation, systematic deviations were found. In the light, misalignment of the 3D VOR had a maximum misalignment at about 45°. These deviations in misalignment can be explained by vector summation of the eye rotation components with a low gain for torsion and high gain for vertical. In the dark and in response to transients, gain of all components had lower values. Misalignment in darkness and for transients had different peaks and troughs than in the light: its minimum was during pitch axis stimulation and its maximum during roll axis stimulation. We show that the relatively large misalignment for roll in darkness is due to a horizontal eye movement component that is only present in darkness. In combination with the relatively low torsion gain, this horizontal component has a relative large effect on the alignment of the eye rotation axis with respect to the head rotation axis

The assessment of the quality of reporting of meta-analyses in diagnostic research: a systematic review

<p>Abstract</p> <p>Background</p> <p>Over the last decade there have been a number of guidelines published, aimed at improving the quality of reporting in published studies and reviews. In systematic reviews this may be measured by their compliance with the PRISMA statement. This review aims to evaluate the quality of reporting in published meta-analyses of diagnostic tests, using the PRISMA statement and establish whether there has been a measurable improvement over time.</p> <p>Methods</p> <p>Eight databases were searched for reviews published prior to 31^stDecember 2008. Studies were selected if they evaluated a diagnostic test, measured performance, searched two or more databases, stated the search terms and inclusion criteria, and used a statistical method to summarise a test's performance. Data were extracted on the review characteristics and items of the PRISMA statement. To measure the change in the quality of reporting over time, PRISMA items for two periods of equal duration were compared.</p> <p>Results</p> <p>Compliance with the PRISMA statement was generally poor: none of the reviews completely adhered to all 27 checklist items. Of the 236 meta-analyses included following selection: only 2(1%) reported the study protocol; 59(25%) reported the searches used; 76(32%) reported the results of a risk of bias assessment; and 82(35%) reported the abstract as a structured summary. Only 11 studies were published before 2000. Thus, the impact of QUOROM on the quality of reporting was not evaluated. However, the periods 2001-2004 and 2005-2008 (covering 93% of studies) were compared using relative risks (RR). There was an increase in the proportion of reviews reporting on five PRISMA items: eligibility criteria (RR 1.13, 95% CI 1.00 - 1.27); risk of bias across studies (methods) (RR 1.81, 95% CI 1.34 - 2.44); study selection results (RR 1.48, 95% CI 1.05 - 2.09); results of individual studies (RR 1.37, 95% CI 1.09 - 1.72); risk of bias across studies (results) (RR 1.65, 95% CI 1.20 - 2.25).</p> <p>Conclusion</p> <p>Although there has been an improvement in the quality of meta-analyses in diagnostic research, there are still many deficiencies in the reporting which future reviewers need to address if readers are to trust the validity of the reported findings.</p

TRY plant trait database - enhanced coverage and open access

Plant traits-the morphological, anatomical, physiological, biochemical and phenological characteristics of plants-determine how plants respond to environmental factors, affect other trophic levels, and influence ecosystem properties and their benefits and detriments to people. Plant trait data thus represent the basis for a vast area of research spanning from evolutionary biology, community and functional ecology, to biodiversity conservation, ecosystem and landscape management, restoration, biogeography and earth system modelling. Since its foundation in 2007, the TRY database of plant traits has grown continuously. It now provides unprecedented data coverage under an open access data policy and is the main plant trait database used by the research community worldwide. Increasingly, the TRY database also supports new frontiers of trait-based plant research, including the identification of data gaps and the subsequent mobilization or measurement of new data. To support this development, in this article we evaluate the extent of the trait data compiled in TRY and analyse emerging patterns of data coverage and representativeness. Best species coverage is achieved for categorical traits-almost complete coverage for 'plant growth form'. However, most traits relevant for ecology and vegetation modelling are characterized by continuous intraspecific variation and trait-environmental relationships. These traits have to be measured on individual plants in their respective environment. Despite unprecedented data coverage, we observe a humbling lack of completeness and representativeness of these continuous traits in many aspects. We, therefore, conclude that reducing data gaps and biases in the TRY database remains a key challenge and requires a coordinated approach to data mobilization and trait measurements. This can only be achieved in collaboration with other initiatives