The ability to program new modes of catalysis into proteins would allow the development of enzyme families with functions beyond those found in nature. To this end, genetic code expansion methodology ...holds particular promise, as it allows the site-selective introduction of new functional elements into proteins as noncanonical amino acid side chains
. Here we exploit an expanded genetic code to develop a photoenzyme that operates by means of triplet energy transfer (EnT) catalysis, a versatile mode of reactivity in organic synthesis that is not accessible to biocatalysis at present
. Installation of a genetically encoded photosensitizer into the beta-propeller scaffold of DA_20_00 (ref.
) converts a de novo Diels-Alderase into a photoenzyme for 2+2 cycloadditions (EnT1.0). Subsequent development and implementation of a platform for photoenzyme evolution afforded an efficient and enantioselective enzyme (EnT1.3, up to 99% enantiomeric excess (e.e.)) that can promote intramolecular and bimolecular cycloadditions, including transformations that have proved challenging to achieve selectively with small-molecule catalysts. EnT1.3 performs >300 turnovers and, in contrast to small-molecule photocatalysts, can operate effectively under aerobic conditions and at ambient temperatures. An X-ray crystal structure of an EnT1.3-product complex shows how multiple functional components work in synergy to promote efficient and selective photocatalysis. This study opens up a wealth of new excited-state chemistry in protein active sites and establishes the framework for developing a new generation of enantioselective photocatalysts.
Purpose
Selection of an open or minimally invasive approach to total mesorectal excision (TME) is generally based on surgeon preference and an intuitive assessment of patient characteristics but ...there consensus on criteria to predict surgical difficulty. Pelvimetry has been used to predict the difficult surgical pelvis, typically using only bony landmarks. This study aimed to assess the relationship between pelvic soft tissue measurements on preoperative MRI and surgical difficulty.
Methods
Preoperative MRIs for patients undergoing laparoscopic rectal resection in the Australasian Laparoscopic Cancer of the Rectum Trial (ALaCaRT) were retrospectively reviewed by two blinded surgeons and pelvimetric variables measured. Pelvimetric variables were analyzed for predictors of successful resection of the rectal cancer, defined by clear circumferential and distal resection margins and completeness of TME.
Results
There was no association between successful surgery and any measurement of distance, area, or ratio. However, the was a strong association between the primary outcome and the estimated total pelvic volume on adjusted logistic regression analysis (OR = 0.99,
P
= 0.01). For each cubic centimeter increase in the pelvic volume, there was a 1% decrease in the odds of successful laparoscopic rectal cancer surgery. Intuitive prediction of unsuccessful surgery was correct in 43% of cases, and correlation between surgeons was poor (ICC = 0.18).
Conclusions
A surgeon’s intuitive assessment of the difficult pelvis, based on visible MRI assessment, is not a reliable predictor of successful laparoscopic surgery. Further assessment of pelvic volume may provide an objective method of defining the difficult surgical pelvis.
Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created ...knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.
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•CRISPRi knockdown of essential genes enables discovery of direct antibiotic targets•An essential gene network reveals functional connections between core processes•A cell morphology screen identifies essential genes intimately tied to cell shape•A majority of essential genes show morphological defects as a terminal phenotype
A systematic analysis of all essential genes in Bacillus subtilis using a CRISPR-based knockdown approach established the essential gene network, identified modes of action for antibiotics, and discerned fundamental underpinnings of growth and morphological characteristics of cells.
With the recent rise of teleophthalmology due to coronavirus disease, health care needs accurate and reliable methods of checking visual acuity remotely. The visual acuity as measured by the GoCheck ...Kids application was compared with that of the Amblyopia Treatment Study (ATS) and the authors' clinic protocol.
This was a prospective, comparison of visual acuity assessment methods.
Established patients (3-18 years of age) in the practice of a single pediatric ophthalmologist were eligible. Visual acuity was measured 1) by GoCheck Kids mobile application, by the patient's family member; 2) by HOTV-ATS, by study personnel; and 3) by regular clinic protocol, by an ophthalmic technician. To assess agreement between measurement of acuity, intraclass correlations with 95% confidence intervals (CI) were computed.
A total of 53 children participated. The mean differences between GoCheck Kids and HOTV-ATS acuities (0.094) were significantly different (P < .001). The intraclass correlation coefficient (ICC) was 0.55 (95% CI: 0.40-0.68). The mean differences between GoCheck Kids and chart acuities (0.010) were not significantly different (P = .319; ICC: 0.59; 95% CI: 0.45-0.71). The mean differences between HOTV-ATS and chart acuities (0.084) were significantly different (P < .001; ICC: 0.66; 95% CI: 0.53-0.76). The percentages of eyes with visual acuity measured by GoCheck Kids within 1 line of the HOTV-ATS and chart acuity were 65.3% and 86.7%, respectively.
GoCheck Kids as checked by a family member provided a modest correlation of visual acuity compared to the chart screen and a fair correlation of visual acuity compared to HOTV-Amblyopia Treatment Study protocol, although most were within 1 line.
•Teleophthalmology is part of our practices due to coronavirus disease (COVID-19).•Visual acuity can be checked at home using paper charts or devices.•GoCheck Kids has a modest correlation of visual acuity compared to the chart screen.•GoCheck Kids has a fair correlation of visual acuity compared to HOTV-ATS.
Introduction/Aims
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative illness with great unmet patient need. We aimed to evaluate whether mesenchymal stem cells induced to secrete high ...levels of neurotrophic factors (MSC‐NTF), a novel autologous cell‐therapy capable of targeting multiple pathways, could safely slow ALS disease progression.
Methods
This randomized, double‐blind, placebo‐controlled study enrolled ALS participants meeting revised El Escorial criteria, revised ALS Functional Rating Scale (ALSFRS‐R) ≥25 (screening) and ≥3 ALSFRS‐R points decline prior to randomization. Participants received three treatments of MSC‐NTF or placebo intrathecally. The primary endpoint evaluated efficacy of MSC‐NTF through a responder analysis and safety. A change in disease progression post‐treatment of ≥1.25 points/mo defines a clinical response. A pre‐specified analysis leveraged baseline ALSFRS‐R of 35 as a subgroup threshold.
Results
Overall, MSC‐NTF treatment was well tolerated; there were no safety concerns. Thirty‐three percent of MSC‐NTF and 28% of placebo participants met clinical response criteria at 28 wk (odds ratio OR = 1.33, P = .45); thus, the primary endpoint was not met. A pre‐specified analysis of participants with baseline ALSFRS‐R ≥ 35 (n = 58) showed a clinical response rate at 28 wk of 35% MSC‐NTF and 16% placebo (OR = 2.6, P = .29). Significant improvements in cerebrospinal biomarkers of neuroinflammation, neurodegeneration, and neurotrophic factor support were observed with MSC‐NTF, with placebo unchanged.
Discussion
The study did not reach statistical significance on the primary endpoint. However, a pre‐specified subgroup suggests that MSC‐NTF participants with less severe disease may have retained more function compared to placebo. Given the unmet patient need, the results of this trial warrant further investigation.
Choosing optimal outcome measures maximizes statistical power, accelerates discovery and improves reliability in early-phase trials. We devised and evaluated a modification to a pragmatic measure of ...oxygenation function, the Formula: see text ratio. Because of the ceiling effect in oxyhaemoglobin saturation, Formula: see text ratio ceases to reflect pulmonary oxygenation function at high Formula: see text values. We found that the correlation of Formula: see text with the reference standard (Formula: see text/Formula: see text ratio) improves substantially when excluding Formula: see text and refer to this measure as Formula: see text. Using observational data from 39,765 hospitalised COVID-19 patients, we demonstrate that Formula: see text is predictive of mortality, and compare the sample sizes required for trials using four different outcome measures. We show that a significant difference in outcome could be detected with the smallest sample size using Formula: see text. We demonstrate that Formula: see text is an effective intermediate outcome measure in COVID-19. It is a non-invasive measurement, representative of disease severity and provides greater statistical power.
IMPORTANCE: Statins affect several mechanisms underlying acute kidney injury (AKI). OBJECTIVE: To test the hypothesis that short-term high-dose perioperative atorvastatin would reduce AKI following ...cardiac surgery. DESIGN, SETTING, AND PARTICIPANTS: Double-blinded, placebo-controlled, randomized clinical trial of adult cardiac surgery patients conducted from November 2009 to October 2014 at Vanderbilt University Medical Center. INTERVENTIONS: Patients naive to statin treatment (n = 199) were randomly assigned 80 mg of atorvastatin the day before surgery, 40 mg of atorvastatin the morning of surgery, and 40 mg of atorvastatin daily following surgery (n = 102) or matching placebo (n = 97). Patients already taking a statin prior to study enrollment (n = 416) continued taking the preenrollment statin until the day of surgery, were randomly assigned 80 mg of atorvastatin the morning of surgery and 40 mg of atorvastatin the morning after (n = 206) or matching placebo (n = 210), and resumed taking the previously prescribed statin on postoperative day 2. MAIN OUTCOMES AND MEASURES: Acute kidney injury defined as an increase of 0.3 mg/dL in serum creatinine concentration within 48 hours of surgery (Acute Kidney Injury Network criteria). RESULTS: The data and safety monitoring board recommended stopping the group naive to statin treatment due to increased AKI among these participants with chronic kidney disease (estimated glomerular filtration rate <60 mL/min/1.73 m2) receiving atorvastatin. The board later recommended stopping for futility after 615 participants (median age, 67 years; 188 30.6% were women; 202 32.8% had diabetes) completed the study. Among all participants (n = 615), AKI occurred in 64 of 308 (20.8%) in the atorvastatin group vs 60 of 307 (19.5%) in the placebo group (relative risk RR, 1.06 95% CI, 0.78 to 1.46; P = .75). Among patients naive to statin treatment (n = 199), AKI occurred in 22 of 102 (21.6%) in the atorvastatin group vs 13 of 97 (13.4%) in the placebo group (RR, 1.61 0.86 to 3.01; P = .15) and serum creatinine concentration increased by a median of 0.11 mg/dL (10th-90th percentile, −0.11 to 0.56 mg/dL) in the atorvastatin group vs by a median of 0.05 mg/dL (10th-90th percentile, −0.12 to 0.33 mg/dL) in the placebo group (mean difference, 0.08 mg/dL 95% CI, 0.01 to 0.15 mg/dL; P = .007). Among patients already taking a statin (n = 416), AKI occurred in 42 of 206 (20.4%) in the atorvastatin group vs 47 of 210 (22.4%) in the placebo group (RR, 0.91 0.63 to 1.32; P = .63). CONCLUSIONS AND RELEVANCE: Among patients undergoing cardiac surgery, high-dose perioperative atorvastatin treatment compared with placebo did not reduce the risk of AKI overall, among patients naive to treatment with statins, or in patients already taking a statin. These results do not support the initiation of statin therapy to prevent AKI following cardiac surgery. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00791648
The Largest M Dwarf Flares from ASAS-SN Schmidt, Sarah J.; Shappee, Benjamin J.; van Saders, Jennifer L. ...
The Astrophysical journal,
05/2019, Letnik:
876, Številka:
2
Journal Article
Recenzirano
Odprti dostop
The All-sky Automated Survey for Supernovae (ASAS-SN) is the only project in existence to scan the entire sky in optical light approximately every day, reaching a depth of g ∼ 18 mag. Over the course ...of its first 4 yr of transient alerts (2013-2016), ASAS-SN observed 53 events classified as likely M dwarf flares. We present follow-up photometry and spectroscopy of all 53 candidates, confirming flare events on 47 M dwarfs, one K dwarf, and one L dwarf. The remaining four objects include a previously identified T Tauri star, a young star with outbursts, and two objects too faint to confirm. A detailed examination of the 49 flare star light curves revealed an additional six flares on five stars, resulting in a total of 55 flares on 49 objects ranging in V-band contrast from ΔV = −1 to −10.2 mag. Using an empirical flare model to estimate the unobserved portions of the flare light curve, we obtain lower limits on the V-band energy emitted during each flare, spanning -35, which are among the most energetic flares detected on M dwarfs. The ASAS-SN M dwarf flare stars show a higher fraction of H emission, as well as stronger H emission, compared to M dwarfs selected without reference to activity, consistent with belonging to a population of more magnetically active stars. We also examined the distribution of tangential velocities, finding that the ASAS-SN flaring M dwarfs are likely to be members of the thin disk and are neither particularly young nor old.
Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to ...measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry (MS)-based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a green fluorescent protein (GFP)-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose-dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by MS and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high-throughput applications of variables that affect aspartate levels.
The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, ...sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.
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