A One Pot, One Step, Precision Cloning Method with High Throughput Capability

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible, but have the drawback of leaving recombination site sequences in the final construct, adding an extra 8 to 13 amino acids to the expressed protein. We have devised a simple and rapid subcloning strategy to transfer any DNA fragment of interest from an entry clone into an expression vector, without this shortcoming. The strategy is based on the use of type IIs restriction enzymes, which cut outside of their recognition sequence. With proper design of the cleavage sites, two fragments cut by type IIs restriction enzymes can be ligated into a product lacking the original restriction site. Based on this property, a cloning strategy called ‘Golden Gate’ cloning was devised that allows to obtain in one tube and one step close to one hundred percent correct recombinant plasmids after just a 5 minute restriction-ligation. This method is therefore as efficient as currently used recombination-based cloning technologies but yields recombinant plasmids that do not contain unwanted sequences in the final construct, thus providing precision for this fundamental process of genetic manipulation

The development and preliminary psychometric properties of two positive psychology outcome measures for people with dementia: the PPOM and the EID-Q.

Background: Positive psychology research in dementia care has largely been confined to the qualitative literature because of the lack of robust outcome measures. The aim of this study was to develop positive psychology outcome measures for people with dementia. Methods: Two measures were each developed in four stages. Firstly, literature reviews were conducted to identify and operationalise salient positive psychology themes in the qualitative literature and to examine existing measures of positive psychology. Secondly, themes were discussed within a qualitative study to add content validity for identified concepts (n = 17). Thirdly, draft measures were submitted to a panel of experts for feedback (n = 6). Finally, measures were used in a small-scale pilot study (n = 33) to establish psychometric properties. Results: Salient positive psychology themes were identified as hope, resilience, a sense of independence and social engagement. Existing measures of hope and resilience were adapted to form the Positive Psychology Outcome Measure (PPOM). Due to the inter-relatedness of independence and engagement for people with dementia, 28 items were developed for a new scale of Engagement and Independence in Dementia Questionnaire (EID-Q) following extensive qualitative work. Both measures demonstrated acceptable internal consistency (α = .849 and α = .907 respectively) and convergent validity. Conclusions: Two new positive psychology outcome measures were developed using a robust four-stage procedure. Preliminary psychometric data was adequate and the measures were easy to use, and acceptable for people with dementia

The peculiar debris disk of HD 111520 as resolved by the Gemini Planet Imager

This is the author accepted manuscript. The final version is available from American Astronomical Society / IOP Publishing via the DOI in this record.Using the Gemini Planet Imager, we have resolved the circumstellar debris disk around HD 111520 at a projected range of ∼30-100 AU in both total and polarized H-band intensity. The disk is seen edge-on at a position angle of 165° along the spine of emission. A slight inclination and asymmetric warp are covariant and alter the interpretation of the observed disk emission. We employ three point-spread function subtraction methods to reduce the stellar glare and instrumental artifacts to confirm that there is a roughly 2:1 brightness asymmetry between the NW and SE extension. This specific feature makes HD 111520 the most extreme example of asymmetric debris disks observed in scattered light among similar highly inclined systems, such as HD 15115 and HD 106906. We further identify a tentative localized brightness enhancement and scale height enhancement associated with the disk at ∼40 AU away from the star on the SE extension. We also find that the fractional polarization rises from 10% to 40% from 0.″5 to 0.″8 from the star. The combination of large brightness asymmetry and symmetric polarization fraction leads us to believe that an azimuthal dust density variation is causing the observed asymmetry.Z.H.D. and B.C.M. acknowledge a Discovery Grant and Accelerator Supplement from the Natural Science and Engineering Research Council of Canada. Supported by NSF grants AST-0909188, AST-1313718 (J.R.G., J.J.W., P.G.K.), AST-141378 (G.D., M.F.), and AST-1411868 (K.F., J.L.P., A.R., K.W.D.). Supported by NASA grants NNX15AD95G/NEXSS, NNX14AJ80G, and NNX11AD21G (J.R.G., J.J.W., P.G.K.)

The peculiar debris disk of HD 111520 as resolved by the Gemini Planet Imager

This is the author accepted manuscript. The final version is available from American Astronomical Society / IOP Publishing via the DOI in this record.Using the Gemini Planet Imager, we have resolved the circumstellar debris disk around HD 111520 at a projected range of ∼30-100 AU in both total and polarized H-band intensity. The disk is seen edge-on at a position angle of 165° along the spine of emission. A slight inclination and asymmetric warp are covariant and alter the interpretation of the observed disk emission. We employ three point-spread function subtraction methods to reduce the stellar glare and instrumental artifacts to confirm that there is a roughly 2:1 brightness asymmetry between the NW and SE extension. This specific feature makes HD 111520 the most extreme example of asymmetric debris disks observed in scattered light among similar highly inclined systems, such as HD 15115 and HD 106906. We further identify a tentative localized brightness enhancement and scale height enhancement associated with the disk at ∼40 AU away from the star on the SE extension. We also find that the fractional polarization rises from 10% to 40% from 0.″5 to 0.″8 from the star. The combination of large brightness asymmetry and symmetric polarization fraction leads us to believe that an azimuthal dust density variation is causing the observed asymmetry.Z.H.D. and B.C.M. acknowledge a Discovery Grant and Accelerator Supplement from the Natural Science and Engineering Research Council of Canada. Supported by NSF grants AST-0909188, AST-1313718 (J.R.G., J.J.W., P.G.K.), AST-141378 (G.D., M.F.), and AST-1411868 (K.F., J.L.P., A.R., K.W.D.). Supported by NASA grants NNX15AD95G/NEXSS, NNX14AJ80G, and NNX11AD21G (J.R.G., J.J.W., P.G.K.)

Effects of an Infectious Fungus, Batrachochytrium dendrobatidis, on Amphibian Predator-Prey Interactions

The effects of parasites and pathogens on host behaviors may be particularly important in predator-prey contexts, since few animal behaviors are more crucial for ensuring immediate survival than the avoidance of lethal predators in nature. We examined the effects of an emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis, on anti-predator behaviors of tadpoles of four frog species. We also investigated whether amphibian predators consumed infected prey, and whether B. dendrobatidis caused differences in predation rates among prey in laboratory feeding trials. We found differences in anti-predator behaviors among larvae of four amphibian species, and show that infected tadpoles of one species (Anaxyrus boreas) were more active and sought refuge more frequently when exposed to predator chemical cues. Salamander predators consumed infected and uninfected tadpoles of three other prey species at similar rates in feeding trials, and predation risk among prey was unaffected by B. dendrobatidis. Collectively, our results show that even sub-lethal exposure to B. dendrobatidis can alter fundamental anti-predator behaviors in some amphibian prey species, and suggest the unexplored possibility that indiscriminate predation between infected and uninfected prey (i.e., non-selective predation) could increase the prevalence of this widely distributed pathogen in amphibian populations. Because one of the most prominent types of predators in many amphibian systems is salamanders, and because salamanders are susceptible to B. dendrobatidis, our work suggests the importance of considering host susceptibility and behavioral changes that could arise from infection in both predators and prey

The crucial role of particle surface reactivity in respirable quartz-induced reactive oxygen/nitrogen species formation and APE/Ref-1 induction in rat lung

Persistent inflammation and associated excessive oxidative stress have been crucially implicated in quartz-induced pulmonary diseases, including fibrosis and cancer. We have investigated the significance of the particle surface reactivity of respirable quartz dust in relation to the in vivo generation of reactive oxygen and nitrogen species (ROS/RNS) and the associated induction of oxidative stress responses in the lung. Therefore, rats were intratracheally instilled with 2 mg quartz (DQ12) or quartz whose surface was modified by either polyvinylpyridine-N-oxide (PVNO) or aluminium lactate (AL). Seven days after instillation, the bronchoalveolar lavage fluid (BALF) was analysed for markers of inflammation (total/differential cell counts), levels of pulmonary oxidants (H(2)O(2), nitrite), antioxidant status (trolox equivalent antioxidant capacity), as well as for markers of lung tissue damage, e.g. total protein, lactate dehydrogenase and alkaline phosphatase. Lung homogenates as well as sections were investigated regarding the induction of the oxidative DNA-lesion/oxidative stress marker 8-hydroxy-2'-deoxyguanosine (8-OHdG) using HPLC/ECD analysis and immunohistochemistry, respectively. Homogenates and sections were also investigated for the expression of the bifunctional apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) by Western blotting and immunohistochemistry. Significantly increased levels of H(2)O(2 )and nitrite were observed in rats treated with non-coated quartz, when compared to rats that were treated with either saline or the surface-modified quartz preparations. In the BALF, there was a strong correlation between the number of macrophages and ROS, as well as total cells and RNS. Although enhanced oxidant generation in non-coated DQ12-treated rats was paralleled with an increased total antioxidant capacity in the BALF, these animals also showed significantly enhanced lung tissue damage. Remarkably however, elevated ROS levels were not associated with an increase in 8-OHdG, whereas the lung tissue expression of APE/Ref-1 protein was clearly up-regulated. The present data provide further in vivo evidence for the crucial role of particle surface properties in quartz dust-induced ROS/RNS generation by recruited inflammatory phagocytes. Our results also demonstrate that quartz dust can fail to show steady-state enhanced oxidative DNA damage in the respiratory tract, in conditions were it elicits a marked and persistent inflammation with associated generation of ROS/RNS, and indicate that this may relate to compensatory induction of APE/Ref-1 mediated base excision repair

BMP signaling components in embryonic transcriptomes of the hover fly Episyrphus balteatus (Syrphidae)

<p>Abstract</p> <p>Background</p> <p>In animals, signaling of Bone Morphogenetic Proteins (BMPs) is essential for dorsoventral (DV) patterning of the embryo, but how BMP signaling evolved with changes in embryonic DV differentiation is largely unclear. Based on the extensive knowledge of BMP signaling in <it>Drosophila melanogaster</it>, the morphological diversity of extraembryonic tissues in different fly species provides a comparative system to address this question. The closest relatives of <it>D. melanogaster </it>with clearly distinct DV differentiation are hover flies (Diptera: Syrphidae). The syrphid <it>Episyrphus balteatus </it>is a commercial bio-agent against aphids and has been established as a model organism for developmental studies and chemical ecology. The dorsal blastoderm of <it>E. balteatus </it>gives rise to two extraembryonic tissues (serosa and amnion), whereas in <it>D. melanogaster</it>, the dorsal blastoderm differentiates into a single extraembryonic epithelium (amnioserosa). Recent studies indicate that several BMP signaling components of <it>D. melanogaster</it>, including the BMP ligand Screw (Scw) and other extracellular regulators, evolved in the dipteran lineage through gene duplication and functional divergence. These findings raise the question of whether the complement of BMP signaling components changed with the origin of the amnioserosa.</p> <p>Results</p> <p>To search for BMP signaling components in <it>E. balteatus</it>, we generated and analyzed transcriptomes of freshly laid eggs (0-30 minutes) and late blastoderm to early germband extension stages (3-6 hours) using Roche/454 sequencing. We identified putative <it>E. balteatus </it>orthologues of 43% of all annotated <it>D. melanogaster </it>genes, including the genes of all BMP ligands and other BMP signaling components.</p> <p>Conclusion</p> <p>The diversification of several BMP signaling components in the dipteran linage of <it>D. melanogaster </it>preceded the origin of the amnioserosa.</p> <p>[Transcriptome sequence data from this study have been deposited at the NCBI Sequence Read Archive (SRP005289); individually assembled sequences have been deposited at GenBank (<ext-link ext-link-id="JN006969" ext-link-type="gen">JN006969</ext-link>-<ext-link ext-link-id="JN006986" ext-link-type="gen">JN006986</ext-link>).]</p

Transcriptomic Analysis of Human Retinal Detachment Reveals Both Inflammatory Response and Photoreceptor Death

Background Retinal detachment often leads to a severe and permanent loss of vision and its therapeutic management remains to this day exclusively surgical. We have used surgical specimens to perform a differential analysis of the transcriptome of human retinal tissues following detachment in order to identify new potential pharmacological targets that could be used in combination with surgery to further improve final outcome. Methodology/Principal Findings Statistical analysis reveals major involvement of the immune response in the disease. Interestingly, using a novel approach relying on coordinated expression, the interindividual variation was monitored to unravel a second crucial aspect of the pathological process: the death of photoreceptor cells. Within the genes identified, the expression of the major histocompatibility complex I gene HLA-C enables diagnosis of the disease, while PKD2L1 and SLCO4A1 -which are both down-regulated- act synergistically to provide an estimate of the duration of the retinal detachment process. Our analysis thus reveals the two complementary cellular and molecular aspects linked to retinal detachment: an immune response and the degeneration of photoreceptor cells. We also reveal that the human specimens have a higher clinical value as compared to artificial models that point to IL6 and oxidative stress, not implicated in the surgical specimens studied here. Conclusions/Significance This systematic analysis confirmed the occurrence of both neurodegeneration and inflammation during retinal detachment, and further identifies precisely the modification of expression of the different genes implicated in these two phenomena. Our data henceforth give a new insight into the disease process and provide a rationale for therapeutic strategies aimed at limiting inflammation and photoreceptor damage associated with retinal detachment and, in turn, improving visual prognosis after retinal surgery

The impact of viral mutations on recognition by SARS-CoV-2 specific T cells.

We identify amino acid variants within dominant SARS-CoV-2 T cell epitopes by interrogating global sequence data. Several variants within nucleocapsid and ORF3a epitopes have arisen independently in multiple lineages and result in loss of recognition by epitope-specific T cells assessed by IFN-γ and cytotoxic killing assays. Complete loss of T cell responsiveness was seen due to Q213K in the A∗01:01-restricted CD8+ ORF3a epitope FTSDYYQLY207-215; due to P13L, P13S, and P13T in the B∗27:05-restricted CD8+ nucleocapsid epitope QRNAPRITF9-17; and due to T362I and P365S in the A∗03:01/A∗11:01-restricted CD8+ nucleocapsid epitope KTFPPTEPK361-369. CD8+ T cell lines unable to recognize variant epitopes have diverse T cell receptor repertoires. These data demonstrate the potential for T cell evasion and highlight the need for ongoing surveillance for variants capable of escaping T cell as well as humoral immunity.This work is supported by the UK Medical Research Council (MRC); Chinese Academy of Medical Sciences(CAMS) Innovation Fund for Medical Sciences (CIFMS), China; National Institute for Health Research (NIHR)Oxford Biomedical Research Centre, and UK Researchand Innovation (UKRI)/NIHR through the UK Coro-navirus Immunology Consortium (UK-CIC). Sequencing of SARS-CoV-2 samples and collation of data wasundertaken by the COG-UK CONSORTIUM. COG-UK is supported by funding from the Medical ResearchCouncil (MRC) part of UK Research & Innovation (UKRI),the National Institute of Health Research (NIHR),and Genome Research Limited, operating as the Wellcome Sanger Institute. T.I.d.S. is supported by a Well-come Trust Intermediate Clinical Fellowship (110058/Z/15/Z). L.T. is supported by the Wellcome Trust(grant number 205228/Z/16/Z) and by theUniversity of Liverpool Centre for Excellence in Infectious DiseaseResearch (CEIDR). S.D. is funded by an NIHR GlobalResearch Professorship (NIHR300791). L.T. and S.C.M.are also supported by the U.S. Food and Drug Administration Medical Countermeasures Initiative contract75F40120C00085 and the National Institute for Health Research Health Protection Research Unit (HPRU) inEmerging and Zoonotic Infections (NIHR200907) at University of Liverpool inpartnership with Public HealthEngland (PHE), in collaboration with Liverpool School of Tropical Medicine and the University of Oxford.L.T. is based at the University of Liverpool. M.D.P. is funded by the NIHR Sheffield Biomedical ResearchCentre (BRC – IS-BRC-1215-20017). ISARIC4C is supported by the MRC (grant no MC_PC_19059). J.C.K.is a Wellcome Investigator (WT204969/Z/16/Z) and supported by NIHR Oxford Biomedical Research Centreand CIFMS. The views expressed are those of the authors and not necessarily those of the NIHR or MRC