In cells, biosynthetic machinery coordinates protein synthesis and folding to optimize efficiency and minimize off-pathway outcomes. However, it has been difficult to delineate experimentally the ...mechanisms responsible. Using fluorescence resonance energy transfer, we studied cotranslational folding of the first nucleotide-binding domain from the cystic fibrosis transmembrane conductance regulator. During synthesis, folding occurred discretely via sequential compaction of N-terminal, α-helical, and α/β-core subdomains. Moreover, the timing of these events was critical; premature α-subdomain folding prevented subsequent core formation. This process was facilitated by modulating intrinsic folding propensity in three distinct ways: delaying α-subdomain compaction, facilitating β-strand intercalation, and optimizing translation kinetics via codon usage. Thus, de novo folding is translationally tuned by an integrated cellular response that shapes the cotranslational folding landscape at critical stages of synthesis.
Extracorporeal cardiopulmonaryresuscitation (ECPR) is emerging as a viable rescue strategy for refractory out-of-hospital cardiac arrest. In the U.S., limited training of emergency medicine providers ...is a barrier to widespread implementation.
Test the hypothesis that emergency medicine physicians and nurses can acquire and retain the skills to rapidly and safely initiate ECPR using high-fidelity simulation.
Prospective interventional study.
U.S. tertiary academic medical center.
Emergency medicine physicians and nurses with no prior ECPR/ECMO experience.
Teams of three physicians and three nurses underwent a two-day ECPR training course including didactics, hands-on training, and simulation. Teams were videotaped initiating ECPR in a high-fidelity simulation scenario before and after simulation training. The primary outcome was the proportion of simulations in which full ECPR support was achieved within 30 min of patient arrival.
Five teams completed the entire study. Full ECPR support was achieved within 30 min of patient arrival in 11/15, 15/15, and 15/15 attempts at baseline (B), post-testing (PT) and 3-month post-testing (3-PT), respectively (p = 0.06). Intervals (mean ± sd) required to achieve full ECPR support at B, PT, and 3-PT were 25.8±5.3, 17.2±4.6, and 19.2±1.9 min respectively (p < 0.05 for B vs. PT and 3-PT).
High fidelity simulation training is effective in preparing emergency medicine physicians and nurses to rapidly and safely initiate ECPR in a simulated cardiac arrest scenario, and should be considered when implementing an ED-based ECPR program.
Gadolinium (Gd) based contrast agents (GBCAs) in magnetic resonance imaging (MRI) are used in daily clinical practice and appear safe in most patients; however, nephrogenic systemic fibrosis (NSF) is ...a recently recognized severe complication associated with GBCAs. It affects primarily patients with renal disease, such as stage 4 or 5 chronic kidney disease (CKD; glomerular filtration rate <30 ml/min per 1.73 m2), acute kidney injury, or kidney and liver transplant recipients with kidney dysfunction. Contrast-enhanced MRI and computed tomography (CT) scans provide important clinical information and influence patient management. An alternative contrast agent is needed to obtain adequate imaging results while avoiding the risk of NSF in this vulnerable patient group. One potential alternative is ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles, which provide enhancement characteristics similar to GBCAs. We review our experience in approximately 150 patients on the potential benefits of the USPIOs ferumoxtran-10 and ferumoxytol. We focus on central nervous system (CNS) MRI but also review imaging of other vascular beds. Safety studies, including USPIO administration (ferumoxytol) as iron supplement therapy in CKD patients on and not on dialysis, suggest that decreased kidney function does not alter the safety profile. We conclude that for both CNS MR imaging and MR angiography, USPIO agents like ferumoxytol are a viable option for patients at risk for NSF.
Classifying indolent prostate cancer represents a significant clinical challenge. We investigated whether integrating data from different omic platforms could identify a biomarker panel with improved ...performance compared to individual platforms alone. DNA methylation, transcripts, protein and glycosylation biomarkers were assessed in a single cohort of patients treated by radical prostatectomy. Novel multiblock statistical data integration approaches were used to deal with missing data and modelled via stepwise multinomial logistic regression, or LASSO. After applying leave‐one‐out cross‐validation to each model, the probabilistic predictions of disease type for each individual panel were aggregated to improve prediction accuracy using all available information for a given patient. Through assessment of three performance parameters of area under the curve (AUC) values, calibration and decision curve analysis, the study identified an integrated biomarker panel which predicts disease type with a high level of accuracy, with Multi AUC value of 0.91 (0.89, 0.94) and Ordinal C‐Index (ORC) value of 0.94 (0.91, 0.96), which was significantly improved compared to the values for the clinical panel alone of 0.67 (0.62, 0.72) Multi AUC and 0.72 (0.67, 0.78) ORC. Biomarker integration across different omic platforms significantly improves prediction accuracy. We provide a novel multiplatform approach for the analysis, determination and performance assessment of novel panels which can be applied to other diseases. With further refinement and validation, this panel could form a tool to help inform appropriate treatment strategies impacting on patient outcome in early stage prostate cancer.
In this study, we built a novel statistical model across multiple omic platforms to predict indolent and aggressive prostate cancer. We demonstrate using ROC, calibration and decision curves that our combined biomarker panel significantly improves on the prediction of indolent disease compared to current clinical features. This will inform appropriate treatment strategies impacting on patient outcomes in early stage prostate cancer.
Ionic liquids have been considered for their potential applications within the nuclear fuel cycle. If ionic liquids are to be successful in their application as solvents for highly radioactive ...materials in any future process, there will be a requirement for them to be robust to high radiation doses. A preliminary assessment of the radiation stability of 1,3-dialkylimidazolium cation based ionic liquids containing nitrate and chloride anions has been performed. The results of radiolysis studies are reported, in which the samples were exposed to alpha radiation from a tandem Van der Graaff generator, beta radiation from a linear accelerator and gamma radiation from cobalt 60 sources. These results suggest that their stability is similar to that of benzene and that they are much more stable than mixtures of tributylphosphate and odourless kerosene under similar irradiation conditions. The radiolysis of 1,3-dialkylimidazolium cation based ionic liquids reflects their combination of the properties of a salt, an alkane and an aromatic. They appear to be relatively radiation resistant and there is certainly no major decomposition of the organic component.
Transmembrane topology of polytopic membrane proteins (PMPs) is established in the endoplasmic reticulum (ER) by the ribosome Sec61-translocon complex (RTC) through iterative cycles of translocation ...initiation and termination. It remains unknown, however, whether tertiary folding of transmembrane domains begins after the nascent polypeptide integrates into the lipid bilayer or within a proteinaceous environment proximal to translocon components. To address this question, we used cysteine scanning mutagenesis to monitor aqueous accessibility of stalled translation intermediates to determine when, during biogenesis, hydrophilic peptide loops of the aquaporin-4 (AQP4) water channel are delivered to cytosolic and lumenal compartments. Results showed that following ribosome docking on the ER membrane, the nascent polypeptide was shielded from the cytosol as it emerged from the ribosome exit tunnel. Extracellular loops followed a well defined path through the ribosome, the ribosome translocon junction, the Sec61-translocon pore, and into the ER lumen coincident with chain elongation. In contrast, intracellular loops (ICLs) and C-terminalresidues exited the ribosome into a cytosolically shielded environment and remained inaccessible to both cytosolic and lumenal compartments until translation was terminated. Shielding of ICL1 and ICL2, but not the C terminus, became resistant to maneuvers that disrupt electrostatic ribosome interactions. Thus, the early folding landscape of polytopic proteins is shaped by a spatially restricted environment localized within the assembled ribosome translocon complex.
Background: Mechanisms that guide membrane protein folding in the endoplasmic reticulum membrane remain unresolved.
Results: During aquaporin-4 synthesis, extracellular peptides loops enter the endoplasmic reticulum lumen sequentially, whereas delivery of cytosolic loops is actively delayed.
Conclusion: The assembled ribosome translocon complex (RTC) shields large regions of the protein from the cytosol throughout synthesis.
Significance: Early membrane protein folding occurs in a proteinaceous environment provided by the RTC.
Oral pre-exposure prophylaxis has been introduced in more than 70 countries, including many in sub-Saharan Africa, but women experience considerable barriers to daily pill-taking, such as stigma, ...judgement, and the fear of violence. Safe and effective long-acting agents for HIV prevention are needed for women. We aimed to evaluate the safety and efficacy of injectable cabotegravir compared with daily oral tenofovir diphosphate plus emtricitabine (TDF-FTC) for HIV prevention in HIV-uninfected women.
HPTN 084 was a phase 3, randomised, double-blind, double-dummy, active-controlled, superiority trial in 20 clinical research sites in seven countries in sub-Saharan Africa. Participants were eligible for enrolment if they were assigned female sex at birth, were aged 18–45 years, reported at least two episodes of vaginal intercourse in the previous 30 days, were at risk of HIV infection based on an HIV risk score, and agreed to use a long-acting reversible contraceptive method. Participants were randomly assigned (1:1) to either active cabotegravir with TDF-FTC placebo (cabotegravir group) or active TDF-FTC with cabotegravir placebo (TDF-FTC group). Study staff and participants were masked to study group allocation, with the exception of the site pharmacist who was responsible for study product preparation. Participants were prescribed 5 weeks of daily oral product followed by intramuscular injections every 8 weeks after an initial 4-week interval load, alongside daily oral pills. Participants who discontinued injections were offered open-label daily TDF-FTC for 48 weeks. The primary endpoints of the study were incident HIV infection in the intention-to-treat population, and clinical and laboratory events that were grade 2 or higher in all women who had received at least one dose of study product. This study is registered with ClinicalTrials.gov, NCT03164564.
From Nov 27, 2017, to Nov 4, 2020, we enrolled 3224 participants (1614 in the cabotegravir group and 1610 in the TDF-FTC group). Median age was 25 years (IQR 22–30); 1755 (54·7%) of 3209 had two or more partners in the preceding month. 40 incident infections were observed over 3898 person-years (HIV incidence 1·0% 95% CI 0·73–1·40); four in the cabotegravir group (HIV incidence 0·2 cases per 100 person-years 0·06–0·52) and 36 in the TDF-FTC group (1·85 cases per 100 person-years 1·3–2·57; hazard ratio 0·12 0·05–0·31; p<0·0001; risk difference –1·6% –1·0% to –2·3%. In a random subset of 405 TDF-FTC participants, 812 (42·1%) of 1929 plasma samples had tenofovir concentrations consistent with daily use. Injection coverage was 93% of the total number of person-years. Adverse event rates were similar across both groups, apart from injection site reactions, which were more frequent in the cabotegravir group than in the TDF-FTC group (577 38·0% of 1519 vs 162 10·7% of 1516) but did not result in injection discontinuation. Confirmed pregnancy incidence was 1·3 per 100 person-years (0·9–1·7); no congenital birth anomalies were reported.
Although both products for HIV prevention were generally safe, well tolerated, and effective, cabotegravir was superior to TDF-FTC in preventing HIV infection in women.
National Institute of Allergy and Infectious Diseases, ViiV Healthcare, and the Bill & Melinda Gates Foundation. Additional support was provided through the National Institute of Mental Health, the National Institute on Drug Abuse, and the Eunice Kennedy Shriver National Institute of Child Health and Human Development. ViiV Healthcare and Gilead Sciences provided pharmaceutical support.
In vitro methods for CFTR biogenesis Matsumura, Yoshihiro; Rooney, LeeAnn; Skach, William R
Methods in molecular biology (Clifton, N.J.),
2011, Volume:
741
Journal Article
Cell-free expression systems provide unique tools for understanding CFTR biogenesis because they reconstitute the cellular folding environment and are readily amenable to biochemical and ...pharmacological manipulation. The most common system for this purpose is rabbit reticulocyte lysate (RRL), supplemented with either canine pancreatic microsomes or semi-permeabilized cells, which has yielded important insights into the folding of CFTR and its individual domains. A common problem in such studies, however, is that biogenesis of large proteins such as CFTR is often inefficient due to low translation processivity, ribosome stalling, and/or premature termination. The first part of this chapter therefore describes parameters that affect in vitro translation of CFTR in RRL. We have found that CFTR expression is uniquely dependent upon 5'- and 3'-untranslated regions (UTRs) of the mRNA. Full-length CFTR expression can be markedly increased using mRNA lacking a 5'-cap analog (G(5')ppp(5')G), whereas the reverse usually holds for smaller proteins and individual CFTR domains. In the context of the full-length mRNA, translation was further stimulated by the presence of a long 3'-UTR. The second part of this chapter describes CFTR translation in lysates derived from cultured mammalian cells including human bronchial epithelial cells. Unfortunately, mammalian cell-derived lysates showed limited ability to sustain full-length CFTR synthesis. However, they provide a unique opportunity to examine specific CFTR domains (i.e., nucleotide-binding domain 1 and transmembrane domain 1) under conditions that more closely resemble the native folding environment.
... the United States was in a housing boom. Because of the unprecedented need for building materials, domestic drywall manufacturers were unable to keep up with the demand for drywall.
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