Efficient targeted transgenesis of large donor DNA into multiple mouse genetic backgrounds using bacteriophage Bxb1 integrase.

The development of mouse models of human disease and synthetic biology research by targeted transgenesis of large DNA constructs represent a significant genetic engineering hurdle. We developed an efficient, precise, single-copy integration of large transgenes directly into zygotes using multiple mouse genetic backgrounds. We used in vivo Bxb1 mediated recombinase-mediated cassette exchange (RMCE) with a transgene landing pad composed of dual heterologous Bxb1 attachment (att) sites in cis, within the Gt(ROSA)26Sor safe harbor locus. RMCE of donor was achieved by microinjection of vector DNA carrying cognate attachment sites flanking the donor transgene with Bxb1-integrase mRNA. This approach achieves perfect vector-free integration of donor constructs at efficiencies \u3e 40% with up to ~ 43 kb transgenes. Coupled with a nanopore-based Cas9-targeted sequencing (nCATS), complete verification of precise insertion sequence was achieved. As a proof-of-concept we describe the development of C57BL/6J and NSG Krt18-ACE2 models for SARS-CoV2 research with verified heterozygous N1 animals within ~ 4 months. Additionally, we created a series of mice with diverse backgrounds carrying a single att site including FVB/NJ, PWK/PhJ, NOD/ShiLtJ, CAST/EiJ and DBA/2J allowing for rapid transgene insertion. Combined, this system enables predictable, rapid development with simplified characterization of precisely targeted transgenic animals across multiple genetic backgrounds

Do household living arrangements explain gender and ethnicity differences in receipt of support services? Findings from LiLACS NZ Māori and non-Māori advanced age cohorts

Services providing practical support are a key component in the spectrum of social care assisting older people to age in place. Te Puāwaitanga o Ngā Tapuwae Kia Ora Tonu/Life and Living in Advanced Age: A Cohort Study in New Zealand (LiLACS NZ), a longitudinal study of Māori and non-Māori in advanced age, aims to determine predictors of successful ageing and to understand trajectories of health and wellbeing. This paper investigates whether household living arrangements (living alone or with others) might explain previously reported gender and ethnic differences in support service utilisation. We had shown that women and non-Māori received more services than men and Māori despite better health. The results of analyses in this paper show that, as expected, poorer physical function led to increased service use. After controlling for functional status, household living arrangements (living alone) were the next strongest driver of service use. In a fully adjusted model, previously observed differences around gender and ethnicity were no longer significant predictors of support service use. However, gender and ethnicity do shape living arrangements in advanced age. Women in advanced age are more likely to live alone, consequently needing more outside support, whereas men are more likely to have a spouse/partner able to provide care. Māori are more likely to live in multigenerational households, the care available at home meaning they are less likely to qualify for formal support. This study points to a need for understanding how gender and ethnicity interact with living arrangements and suggests that inequities may not be absent when the presence of others in a household renders an older person ineligible for formal care

How much change is enough?:Evidence from a longitudinal study on depression in UK primary care

BACKGROUND: The Patient Health Questionnaire (PHQ-9), the Beck Depression Inventory (BDI-II) and the Generalised Anxiety Disorder Assessment (GAD-7) are widely used in the evaluation of interventions for depression and anxiety. The smallest reduction in depressive symptoms that matter to patients is known as the Minimum Clinically Important Difference (MCID). Little empirical study of the MCID for these scales exists. METHODS: A prospective cohort of 400 patients in UK primary care were interviewed on four occasions, 2 weeks apart. At each time point, participants completed all three questionnaires and a ‘global rating of change’ scale (GRS). MCID estimation relied on estimated changes in symptoms according to reported improvement on the GRS scale, stratified by baseline severity on the Clinical Interview Schedule (CIS-R). RESULTS: For moderate baseline severity, those who reported improvement on the GRS had a reduction of 21% (95% confidence interval (CI) −26.7 to −14.9) on the PHQ-9; 23% (95% CI −27.8 to −18.0) on the BDI-II and 26.8% (95% CI −33.5 to −20.1) on the GAD-7. The corresponding threshold scores below which participants were more likely to report improvement were −1.7, −3.5 and −1.5 points on the PHQ-9, BDI-II and GAD-7, respectively. Patients with milder symptoms require much larger reductions as percentage of their baseline to endorse improvement. CONCLUSIONS: An MCID representing 20% reduction of scores in these scales, is a useful guide for patients with moderately severe symptoms. If treatment had the same effect on patients irrespective of baseline severity, those with low symptoms are unlikely to notice a benefit. FUNDING: Funding. National Institute for Health Research

Combining mirtazapine with SSRIs or SNRIs for treatment-resistant depression: The MIR RCT

Background: Depression is usually managed in primary care and antidepressants are often the first-line treatment, but only half of those treated respond to a single antidepressant. Objectives: To investigate whether or not combining mirtazapine with serotonin–noradrenaline reuptake inhibitor (SNRI) or selective serotonin reuptake inhibitor (SSRI) antidepressants results in better patient outcomes and more efficient NHS care than SNRI or SSRI therapy alone in treatment-resistant depression (TRD). Design: The MIR trial was a two-parallel-group, multicentre, pragmatic, placebo-controlled randomized trial with allocation at the level of the individual. Setting: Participants were recruited from primary care in Bristol, Exeter, Hull/York and Manchester/Keele. Participants: Eligible participants were aged ≥ 18 years; were taking a SSRI or a SNRI antidepressant for at least 6 weeks at an adequate dose; scored ≥ 14 points on the Beck Depression Inventory-II (BDI-II); were adherent to medication; and met the International Statistical Classification of Diseases and Related Health Problems, Tenth Revision, criteria for depression. Interventions: Participants were randomised using a computer-generated code to either oral mirtazapine or a matched placebo, starting at a dose of 15 mg daily for 2 weeks and increasing to 30 mg daily for up to 12 months, in addition to their usual antidepressant. Participants, their general practitioners (GPs) and the research team were blind to the allocation. Main outcome measures: The primary outcome was depression symptoms at 12 weeks post randomization compared with baseline, measured as a continuous variable using the BDI-II. Secondary outcomes (at 12, 24 and 52 weeks) included response, remission of depression, change in anxiety symptoms, adverse events (AEs), quality of life, adherence to medication, health and social care use and cost-effectiveness. Outcomes were analysed on an intention-to-treat basis. A qualitative study explored patients’ views and experiences of managing depression and GPs’ views on prescribing a second antidepressant. Results: There were 480 patients randomised to the trial (mirtazapine and usual care, n = 241; placebo and usual care, n = 239), of whom 431 patients (89.8%) were followed up at 12 weeks. BDI-II scores at 12 weeks were lower in the mirtazapine group than the placebo group after adjustment for baseline BDI-II score and minimisation and stratification variables [difference –1.83 points, 95% confidence interval (CI) –3.92 to 0.27 points; p = 0.087]. This was smaller than the minimum clinically important difference and the CI included the null. The difference became smaller at subsequent time points (24 weeks: –0.85 points, 95% CI –3.12 to 1.43 points; 12 months: 0.17 points, 95% CI –2.13 to 2.46 points). More participants in the mirtazapine group withdrew from the trial medication, citing mild AEs (46 vs. 9 participants). Conclusions: This study did not find convincing evidence of a clinically important benefit for mirtazapine in addition to a SSRI or a SNRI antidepressant over placebo in primary care patients with TRD. There was no evidence that the addition of mirtazapine was a cost-effective use of NHS resources. GPs and patients were concerned about adding an additional antidepressant. Limitations: Voluntary unblinding for participants after the primary outcome at 12 weeks made interpretation of longer-term outcomes more difficult. Future work: Treatment-resistant depression remains an area of important, unmet need, with limited evidence of effective treatments. Promising interventions include augmentation with atypical antipsychotics and treatment using transcranial magnetic stimulation

Review of the Performance of Residential PV systems in Belgium

The main objective of this paper is to review the state of the art of residential PV systems in Belgium by the analysis of the operational data of 993 installations. For that, three main questions are posed: how much energy do they produce? What level of performance is associated to their production? Which are the key parameters that most influence their quality? This work brings answers to these questions. A middling commercial PV system, optimally oriented, produces a mean annual energy of 892 kWh/kWp. As a whole, the orientation of PV generators causes energy productions to be some 6% inferior to optimally oriented PV systems. The mean performance ratio is 78% and the mean performance index is 85%. That is to say, the energy produced by a typical PV system in Belgium is 15% inferior to the energy produced by a very high quality PV system. Finally, on average, the real power of the PV modules falls 5% below its corresponding nominal power announced on the manufacturer's datasheet. Differences between real and nominal power of up to 16% have been detected

Comparison of ADCP observations and 3D model simulations of turbulence at a tidal energy site

Field measurement of turbulence in strong tidal currents is difficult and expensive, but the tidal energy industry needs to accurately quantify turbulence for adequate resource characterisation and device design. Models that can predict such turbulence could reduce measurement costs. We compare a Regional Ocean Modelling System (ROMS) simulation with acoustic Doppler current profiler (ADCP) measurements from a highly-energetic tidal site. This comparison shows the extent to which turbulence can be quantified by ROMS, using the conventional k−ε turbulence closure model. Both model and observations covered the same time period, encompassing two spring-neap cycles. Turbulent kinetic energy (TKE) density was calculated from measurements using the variance method; turbulent dissipation, ε, was calculated using the structure function method. Measurements show that wave action dominates turbulent fluctuations in the upper half of the water column; comparing results for deeper water, however, shows very strong agreement. A best fit between ROMS and ADCP results for mean velocity yields R2=0.98; for TKE, R2 is 0.84 when strongly wave-dominated times are excluded. Dissipation agrees less well: although time series of ε are well-correlated at similar depths, ROMS estimates a greater magnitude of dissipation than is measured, by a factor of up to 4.8

Ethical considerations when conservation research involves people

Social science is becoming increasingly important in conservation, with more studies involving methodologies that collect data from and about people. Conservation science is a normative and applied discipline designed to support and inform management and practice. Poor research practice risks harming participants, researchers, and can leave negative legacies. Often, those at the forefront of field‐based research are early‐career researchers, many of whom enter their first research experience ill‐prepared for the ethical conundrums they may face. Here, we draw on our own experiences as early‐career researchers to illuminate how ethical challenges arise during conservation research that involves human participants. Specifically, we discuss ethical review procedures, conflicts of values, and power relations, and provide broad recommendations on how to navigate ethical challenges when they arise during research. We encourage greater engagement with ethical review processes and highlight the pressing need to develop ethical guidelines for conservation research that involves human participants.Output Status: Forthcoming/Available Onlin

Homozygosity for a missense mutation in the 67 kDa isoform of glutamate decarboxylase in a family with autosomal recessive spastic cerebral palsy: parallels with Stiff-Person Syndrome and other movement disorders

Background Cerebral palsy (CP) is an heterogeneous group of neurological disorders of movement and/or posture, with an estimated incidence of 1 in 1000 live births. Non-progressive forms of symmetrical, spastic CP have been identified, which show a Mendelian autosomal recessive pattern of inheritance. We recently described the mapping of a recessive spastic CP locus to a 5 cM chromosomal region located at 2q24-31.1, in rare consanguineous families. Methods Here we present data that refine this locus to a 0.5 cM region, flanked by the microsatellite markers D2S2345 and D2S326. The minimal region contains the candidate gene GAD1, which encodes a glutamate decarboxylase isoform (GAD67), involved in conversion of the amino acid and excitatory neurotransmitter glutamate to the inhibitory neurotransmitter γ-aminobutyric acid (GABA). Results A novel amino acid mis-sense mutation in GAD67 was detected, which segregated with CP in affected individuals. Conclusions This result is interesting because auto-antibodies to GAD67 and the more widely studied GAD65 homologue encoded by the GAD2 gene, are described in patients with Stiff-Person Syndrome (SPS), epilepsy, cerebellar ataxia and Batten disease. Further investigation seems merited of the possibility that variation in the GAD1 sequence, potentially affecting glutamate/GABA ratios, may underlie this form of spastic CP, given the presence of anti-GAD antibodies in SPS and the recognised excitotoxicity of glutamate in various contexts

Anthropogenic reaction parameters - the missing link between chemical intuition and the available chemical space

How do skilled synthetic chemists develop such a good intuitive expertise ? Why can we only access such a small amount of the available chemical space — both in terms of the re actions used and the chemical scaffolds we make? We argue here that these seemingly unrelated questions have a common root and are strongly interdependent . We performed a comprehensive analysis of organic reaction parameters dating back to 1771 and discove red that there are several anthropogenic factors that limit the reaction parameters and thus the scop e of synthetic chemistry. Nevertheless, many of the anthropogenic limitations such as the narrow parameter space and the opportunity of the rapid and clear feedback on the progress of reactions appear to be crucial for the acquisition of valid and reliable chemical intuition. In parallel, however, all of these same factors represent limitations for the exploration of available chemistry space and we argue th at these are thus at least partly responsible for limited access to new chemistries. We advocate, therefore, that the present anthropogenic boundaries can be expanded by a more conscious expl oration of “off - road” chemistry that would also extend the intuit ive knowledge of trained chemists