Cis-regulatory elements (CREs, e.g., promoters and enhancers) regulate gene expression, and variants within CREs can modulate disease risk. Next-generation sequencing has enabled the rapid generation ...of genomic data that predict the locations of CREs, but a bottleneck lies in functionally interpreting these data. To address this issue, massively parallel reporter assays (MPRAs) have emerged, in which barcoded reporter libraries are introduced into cells, and the resulting barcoded transcripts are quantified by next-generation sequencing. Thus far, MPRAs have been largely restricted to assaying short CREs in a limited repertoire of cultured cell types. Here, we present two advances that extend the biological relevance and applicability of MPRAs. First, we adapt exome capture technology to instead capture candidate CREs, thereby tiling across the targeted regions and markedly increasing the length of CREs that can be readily assayed. Second, we package the library into adeno-associated virus (AAV), thereby allowing delivery to target organs in vivo. As a proof of concept, we introduce a capture library of about 46,000 constructs, corresponding to roughly 3500 DNase I hypersensitive (DHS) sites, into the mouse retina by ex vivo plasmid electroporation and into the mouse cerebral cortex by in vivo AAV injection. We demonstrate tissue-specific cis-regulatory activity of DHSs and provide examples of high-resolution truncation mutation analysis for multiplex parsing of CREs. Our approach should enable massively parallel functional analysis of a wide range of CREs in any organ or species that can be infected by AAV, such as nonhuman primates and human stem cell-derived organoids.
CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a ...scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.
Outcomes after exacerbations of chronic obstructive pulmonary disease (COPD) requiring acute noninvasive ventilation (NIV) are poor and there are few treatments to prevent hospital readmission and ...death.
To investigate the effect of home NIV plus oxygen on time to readmission or death in patients with persistent hypercapnia after an acute COPD exacerbation.
A randomized clinical trial of patients with persistent hypercapnia (Paco2 >53 mm Hg) 2 weeks to 4 weeks after resolution of respiratory acidemia, who were recruited from 13 UK centers between 2010 and 2015. Exclusion criteria included obesity (body mass index BMI >35), obstructive sleep apnea syndrome, or other causes of respiratory failure. Of 2021 patients screened, 124 were eligible.
There were 59 patients randomized to home oxygen alone (median oxygen flow rate, 1.0 L/min interquartile range {IQR}, 0.5-2.0 L/min) and 57 patients to home oxygen plus home NIV (median oxygen flow rate, 1.0 L/min IQR, 0.5-1.5 L/min). The median home ventilator settings were an inspiratory positive airway pressure of 24 (IQR, 22-26) cm H2O, an expiratory positive airway pressure of 4 (IQR, 4-5) cm H2O, and a backup rate of 14 (IQR, 14-16) breaths/minute.
Time to readmission or death within 12 months adjusted for the number of previous COPD admissions, previous use of long-term oxygen, age, and BMI.
A total of 116 patients (mean SD age of 67 10 years, 53% female, mean BMI of 21.6 IQR, 18.2-26.1, mean SD forced expiratory volume in the first second of expiration of 0.6 L 0.2 L, and mean SD Paco2 while breathing room air of 59 7 mm Hg) were randomized. Sixty-four patients (28 in home oxygen alone and 36 in home oxygen plus home NIV) completed the 12-month study period. The median time to readmission or death was 4.3 months (IQR, 1.3-13.8 months) in the home oxygen plus home NIV group vs 1.4 months (IQR, 0.5-3.9 months) in the home oxygen alone group, adjusted hazard ratio of 0.49 (95% CI, 0.31-0.77; P = .002). The 12-month risk of readmission or death was 63.4% in the home oxygen plus home NIV group vs 80.4% in the home oxygen alone group, absolute risk reduction of 17.0% (95% CI, 0.1%-34.0%). At 12 months, 16 patients had died in the home oxygen plus home NIV group vs 19 in the home oxygen alone group.
Among patients with persistent hypercapnia following an acute exacerbation of COPD, adding home noninvasive ventilation to home oxygen therapy prolonged the time to readmission or death within 12 months.
clinicaltrials.gov Identifier: NCT00990132.
The laboratory mouse is the workhorse of immunology, used as a model of mammalian immune function, but how well immune responses of laboratory mice reflect those of free-living animals is unknown. ...Here we comprehensively characterize serological, cellular and functional immune parameters of wild mice and compare them with laboratory mice, finding that wild mouse cellular immune systems are, comparatively, in a highly activated (primed) state. Associations between immune parameters and infection suggest that high level pathogen exposure drives this activation. Moreover, wild mice have a population of highly activated myeloid cells not present in laboratory mice. By contrast, in vitro cytokine responses to pathogen-associated ligands are generally lower in cells from wild mice, probably reflecting the importance of maintaining immune homeostasis in the face of intense antigenic challenge in the wild. These data provide a comprehensive basis for validating (or not) laboratory mice as a useful and relevant immunological model system.
Mobilities and lifetimes of photogenerated charge carriers are core properties of photovoltaic materials and can both be characterized by contactless terahertz or microwave measurements. Here, the ...expertise from fifteen laboratories is combined to quantitatively model the current‐voltage characteristics of a solar cell from such measurements. To this end, the impact of measurement conditions, alternate interpretations, and experimental inter‐laboratory variations are discussed using a (Cs,FA,MA)Pb(I,Br)3 halide perovskite thin‐film as a case study. At 1 sun equivalent excitation, neither transport nor recombination is significantly affected by exciton formation or trapping. Terahertz, microwave, and photoluminescence transients for the neat material yield consistent effective lifetimes implying a resistance‐free JV‐curve with a potential power conversion efficiency of 24.6 %. For grainsizes above ≈20 nm, intra‐grain charge transport is characterized by terahertz sum mobilities of ≈32 cm2 V−1 s−1. Drift‐diffusion simulations indicate that these intra‐grain mobilities can slightly reduce the fill factor of perovskite solar cells to 0.82, in accordance with the best‐realized devices in the literature. Beyond perovskites, this work can guide a highly predictive characterization of any emerging semiconductor for photovoltaic or photoelectrochemical energy conversion. A best practice for the interpretation of terahertz and microwave measurements on photovoltaic materials is presented.
Bringing together the expertise from fifteen laboratories the current‐voltage characteristics of a solar cell are modeled using contactless terahertz and microwave measurements. To this end, the impact of measurement conditions, alternate interpretations, and experimental inter‐laboratory variations are discussed. For a neat (Cs,FA,MA)Pb(I,Br)3 thin film, the implied resistance‐free JV‐curve and the fill factor losses by its finite mobility are revealed.
Background. Initial management of community-acquired pneumonia (CAP) has been a Centers for Medicare and Medicaid Services performance measure for a decade. We hypothesized that an intervention ...directed at management of CAP that assesses areas not covered by the performance measures—treatment duration and antimicrobial selection after additional microbiology data are available—would further improve CAP management. Methods. We performed a single-center, prospective study to compare management of adult inpatients with presumed CAP before (from 1 January 2008 through 31 March 2008) and after (from 1 February 2010 through 10 May 2010) an intervention consisting of education and prospective feedback to teams regarding antibiotic choice and duration. The primary outcome measure was duration of antibiotic therapy in the 2 periods. Results. There were 62 patients in the preintervention period and 65 patients in the intervention period. The duration of antibiotic therapy decreased from a median of 10 to 7 days (P < .001), with 148 fewer days of antibiotic therapy. The median lengths of stay were similar in the 2 groups (4 vs 5 days). A causative pathogen was identified less frequently during the intervention period (14% vs 34%); however, antibiotics were more frequently narrowed or modified on the basis of susceptibility results during the intervention period (67% vs 19%). Fewer patients received duplicate therapy within 24 hours in the intervention period (90% vs 55%). Conclusions. The duration of therapy for CAP was excessive at our institution and was decreased with a stewardship intervention. Confirmatory studies at other institutions are needed; efforts to assess and reduce duration of therapy for CAP should be strongly considered.
Despite known low sensitivity, culture of periprosthetic tissue specimens on agars and in broths is routine. Culture of periprosthetic tissue samples in blood culture bottles (BCBs) is potentially ...more convenient, but it has been evaluated in a limited way and has not been widely adopted. The aim of this study was to compare the sensitivity and specificity of inoculation of periprosthetic tissue specimens into blood culture bottles with standard agar and thioglycolate broth culture, applying Bayesian latent class modeling (LCM) in addition to applying the Infectious Diseases Society of America (IDSA) criteria for prosthetic joint infection. This prospective cohort study was conducted over a 9-month period (August 2013 to April 2014) at the Mayo Clinic, Rochester, MN, and included all consecutive patients undergoing revision arthroplasty. Overall, 369 subjects were studied; 117 (32%) met IDSA criteria for prosthetic joint infection, and 82% had late chronic infection. Applying LCM, inoculation of tissues into BCBs was associated with a 47% improvement in sensitivity compared to the sensitivity of conventional agar and broth cultures (92.1 versus 62.6%, respectively); this magnitude of change was similar when IDSA criteria were applied (60.7 versus 44.4%, respectively; P = 0.003). The time to microorganism detection was shorter with BCBs than with standard media (P < 0.0001), with aerobic and anaerobic BCBs yielding positive results within a median of 21 and 23 h, respectively. Results of our study demonstrate that the semiautomated method of periprosthetic tissue culture in blood culture bottles is more sensitive than and as specific as agar and thioglycolate broth cultures and yields results faster.
Prosthetic joint infections are a devastating complication of arthroplasty surgery. Despite this, current microbiological techniques to detect and diagnose infections are imperfect. This study examined a new approach to diagnosing infections, through the inoculation of tissue samples from around the prosthetic joint into blood culture bottles. This study demonstrated that, compared to current laboratory practices, this new technique increased the detection of infection. These findings are important for patient care to allow timely and accurate diagnosis of infection.
•Experimentally measured falling-film evaporation heat transfer coefficients.•Effects of temperature, flow rate, tube spacing, tube row, and heat flux discussed.•Newly observed heat transfer ...coefficient trend that is not seen in round tubes.•New trend due to film breakdown quantified and supported by force balance.
An experimental study on falling-film evaporation on horizontal rectangular tubes, the external geometry of extruded flat, microchannel tubes, was conducted. Previous falling-film evaporation studies have focused on horizontal and vertical round tubes, and the usage of microchannel tubes provides a promising new heat exchanger design option. The test sections used in this study have external dimensions of 203 × 1.42 × 27.4 mm (length × width × height) and were tested with water as the refrigerant. Heat transfer coefficients were measured for saturation temperatures from 10 to 30 °C, test section spacings from 5 to 15 mm, heat fluxes from 10 to 20 kW m−2, and film Reynolds numbers of 48 to 544. This paper, Part I, reports the experimental methodology and the results, while in the companion paper, Part II, models for predicting heat transfer are presented.
Summary Background Malignant pleural mesothelioma incidence continues to rise, with few available evidence-based therapeutic options. Results of previous non-randomised studies suggested that ...video-assisted thoracoscopic partial pleurectomy (VAT-PP) might improve symptom control and survival. We aimed to compare efficacy in terms of overall survival, and cost, of VAT-PP and talc pleurodesis in patients with malignant pleural mesothelioma. Methods We undertook an open-label, parallel-group, randomised, controlled trial in patients aged 18 years or older with any subtype of confirmed or suspected mesothelioma with pleural effusion, recruited from 12 hospitals in the UK. Eligible patients were randomly assigned (1:1) to either VAT-PP or talc pleurodesis by computer-generated random numbers, stratified by European Organisation for Research and Treatment of Cancer risk category (high vs low). The primary outcome was overall survival at 1 year, analysed by intention to treat (all patients randomly assigned to a treatment group with a final diagnosis of mesothelioma). This trial is registered with ClinicalTrials.gov , number NCT00821860. Findings Between Oct 24, 2003, and Jan 24, 2012, we randomly assigned 196 patients, of whom 175 (88 assigned to talc pleurodesis, 87 assigned to VAT-PP) had confirmed mesothelioma. Overall survival at 1 year was 52% (95% CI 41–62) in the VAT-PP group and 57% (46–66) in the talc pleurodesis group (hazard ratio 1·04 95% CI 0·76–1·42; p=0·81). Surgical complications were significantly more common after VAT-PP than after talc pleurodesis, occurring in 24 (31%) of 78 patients who completed VAT-PP versus ten (14%) of 73 patients who completed talc pleurodesis (p=0·019), as were respiratory complications (19 24% vs 11 15%; p=0·22) and air-leak beyond 10 days (five 6% vs one 1%; p=0·21), although not significantly so. Median hospital stay was longer at 7 days (IQR 5–11) in patients who received VAT-PP compared with 3 days (2–5) for those who received talc pleurodesis (p<0·0001). Interpretation VAT-PP is not recommended to improve overall survival in patients with pleural effusion due to malignant pleural mesothelioma, and talc pleurodesis might be preferable considering the fewer complications and shorter hospital stay associated with this treatment. Funding BUPA Foundation.
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