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BACKGROUND: Eplerenone is known to reduce time to first hospitalization for heart failure or cardiovascular death in patients with heart failure and mild symptoms. In chronic diseases such as heart failure, characterized by repeat hospitalizations, analyzing all heart failure hospitalizations, not just the first, should give a more complete picture of treatment benefits. METHODS AND RESULTS: The Eplerenone in Mild Patients Hospitalization and SurvIval Study in Heart Failure (EMPHASIS-HF) trial compared eplerenone with placebo in 2737 patients with mild heart failure, followed for a median 2.08 years (interquartile range, 1.08-3.10 years). Data were collected on all hospitalizations, with a focus on those due to heart failure. Heart failure hospitalization rates in the eplerenone and placebo groups were 10.70 and 16.99 per 100 patient-years, respectively. Allowing for skewness in the frequency of hospitalizations by using the negative binomial generalized linear model, the rate ratio (eplerenone versus placebo) was 0.53 (95% confidence interval, 0.42-0.66; P<0.0001). A plot of cumulative hospitalization rates over time revealed that most of the reduced risk on eplerenone occurred in the first year of follow-up. Several baseline variables strongly predicted the risk of hospitalization. More complex statistical methods, adjusting for mortality (as informative censoring), made a negligible difference in these findings. CONCLUSIONS: Eplerenone markedly reduces the risk of heart failure hospitalizations in patients with heart failure and mild symptoms to a greater extent than is captured by only studying the time to first hospitalization. Future clinical trials in heart failure would gain from incorporating repeat hospitalizations into their primary evaluation of treatment effects. CLINICAL TRIAL REGISTRATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT00232180. [ABSTRACT FROM AUTHOR]

Background: Patients who present with only simple or complex partial seizures have a poorly documented prognosis. Treatment may be advocated to prevent future secondary generalised seizures, reduce the frequency of further simple or complex partial seizures or a combination of both.Methods: A full statistical analysis on 1334 patients was carried out. The outcomes measured were post-randomisation times to first seizure of any type and first tonic-clonic seizure. Methodology was adopted that accounted for individuals' underlying pre-randomisation seizure counts and allowed for the possibility that there may be a proportion of the sample that will not experience post-randomisation seizure recurrence.Results: 103 subjects randomised to the MESS (Multicentre Study of Early Epilepsy and Single Seizures) study had only partial seizures at randomisation. Only 17 of these had a tonic-clonic seizure during follow-up. The presence of an abnormal EEG at randomisation influenced this risk: an estimated 23% of those with EEG abnormality were at risk of tonic-clonic seizures during follow-up compared with 16% of those with a normal EEG. The group did, however, continue to have partial seizures during follow-up, and modelling showed that the impact of treatment on these seizures was significantly less than the effects of treatment on the frequency of tonic-clonic seizures in those patients with such pre-randomisation seizures.Conclusion: Patients presenting with a history of only partial seizures are at low risk of subsequent tonic-clonic seizures in the period of time to which therapeutic decisions are relevant. The effects of the antiepileptic drugs used in the MESS study are greater for tonic-clonic seizures than they are for partial seizures. [ABSTRACT FROM AUTHOR]

Bermudagrass ( Cynodon dactylon L.) stockpiling and cool-season annual pastures can extend grazing seasons in cow-calf operations and reduce winter feeding costs, but less is known about how these practices interact and their effect on producer profitability. Data from a completely randomized-design experiment in South-Central Oklahoma were collected on three grazing systems for cows and calves: bermudagrass pasture (CONTROL), stockpiled bermudagrass and interseeded cool-season pasture (SPINT), and stockpiled bermudagrass plus cropland no-till seeded with a summer cover-crop followed by cool-season annuals (SPCROP). A mixed model was used to estimate the effects of grazing system on weaning weights, total hay, and total range cubes (crude protein [CP] = 30%) fed in each system. Enterprise budgeting was used to calculate the expected net return of each system. Weaning weight did not vary between systems ( P  = 0.6940), resulting in similar revenues. Relative to other treatments, the quantity of cubes fed in the CONTROL system were significantly higher ( P  < 0.0001) while hay fed was significantly higher in the SPCROP system ( P  = 0.0036). Increased machinery costs, seed costs, and fertilization requirements in bermudagrass stockpiling, interseeding, and cropland production outweighed the cost savings associated with less feeding. Total costs were $446 ha -1 ($722 hd -1 ), $451 ha -1 ($732 hd -1 ), and $553 ha -1 ($895 ha -1 ) for the CONTROL, SPINT, and SPCROP systems, respectively. Overall, the CONTROL system was $3.13 ha -1 ($5.08 hd -1 ) and $98.91 ha -1 ($160.10 hd -1 ) more profitable than the SPINT and SPCROP systems., Competing Interests: None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science.)

Background: Overall survival is the "gold standard" endpoint in cancer clinical trials. It plays a key role in determining the clinical- and cost-effectiveness of a new intervention and whether it is recommended for use in standard of care. The assessment of overall survival usually requires trial participants to be followed up for a long period of time. In this time, they may stop receiving the trial intervention and receive subsequent anti-cancer treatments, which also aim to extend survival, during trial follow-up. This can potentially change the interpretation of overall survival in the context of the clinical trial. This review aimed to determine how overall survival has been assessed in cancer clinical trials and whether subsequent anti-cancer treatments are considered., Methods: Two searches were conducted using MEDLINE within OVID© on the 9th of November 2021. The first sought to identify papers publishing overall survival results from randomised controlled trials in eight reputable journals and the second to identify papers mentioning or considering subsequent treatments. Papers published since 2010 were included if presenting or discussing overall survival in the context of treating cancer., Results: One hundred and thirty-four papers were included. The majority of these were presenting clinical trial results (98, 73%). Of these, 45 (46%) reported overall survival as a (co-) primary endpoint. A lower proportion of papers including overall survival as a (co-) primary endpoint compared to a secondary endpoint were published in recent years. The primary analysis of overall survival varied across the papers. Fifty-nine (60%) mentioned subsequent treatments. Seven papers performed additional analysis, primarily when patients in the control arm received the experimental treatment during trial follow-up (treatment switching)., Discussion: Overall survival has steadily moved from being the primary to a secondary endpoint. However, it is still of interest with papers presenting overall survival results with the caveat of subsequent treatments, but little or no investigation into their effect. This review shows that there is a methodological gap for what researchers should do when trial participants receive anti-cancer treatment during trial follow-up. Future research will identify the stakeholder opinions, on how this methodological gap should be addressed., (© 2023. The Author(s).)

The objective of this study was to examine the effects of diet energy density on ranking for dry matter intake (DMI), residual feed intake (RFI), and greenhouse gas emissions. Forty-two mature, gestating Angus cows (600 ± 69 kg body weight [BW]; body condition score [BCS] 5.3 ± 1.1) with a wide range in DMI expected progeny difference (-1.38 to 2.91) were randomly assigned to two diet sequences; forage then concentrate (FC) or concentrate then forage (CF). The forage diet consisted of long-stem native grass hay plus protein supplement (HAY; 1.96 Mcal ME/kg DM). The concentrate diet consisted of 35% chopped grass hay and 65% concentrate feeds on a dry matter basis (MIX; 2.5 Mcal ME/kg DM). The GreenFeed Emission Monitoring system was used to determine carbon dioxide (CO2), oxygen (O2), and methane (CH4) flux. Cow performance traits, ultrasound back fat and rump fat, feed DMI, and gas flux data were analyzed in a crossover design using a mixed model including diet, period, and sequence as fixed effects and pen and cow within sequence as random effects. For all measured traits excluding DMI, there was a diet × sequence interaction (P < 0.05). The correlation between MIX and HAY DMI was 0.41 (P = 0.067) and 0.47 (P = 0.03) for FC and CF sequences, respectively. There was no relationship (P > 0.66) between HAY and MIX average daily gain (ADG), regardless of sequence. Fifty-seven percent of the variation in DMI was explained by metabolic BW, ADG, and BCS for both diets during the first period. During the second period, the same three explanatory variables accounted for 38% and 37% of the variation in DMI for MIX and HAY diets, respectively. The negative relationship between BCS and DMI was more pronounced when cows consumed the MIX diet. There was no relationship between MIX and HAY RFI, regardless of sequence (P > 0.18). During the first period, correlations for CO2, CH4, and O2 with MIX DMI were 0.69, 0.81, and 0.56 (P ≤ 0.015), respectively, and 0.76, 0.74, and 0.64 (P < 0.01) with HAY DMI. During the second period, correlations for CO2, CH4, and O2 with MIX DMI were 0.62, 0.47, and 0.56 (P ≤ 0.11), respectively. However, HAY DMI during the second period was not related to gas flux (P > 0.47). Results from this experiment indicate that feed intake of two energy-diverse diets is moderately correlated while ADG while consuming the two diets is not related. Further experimentation is necessary to determine if gas flux data can be used to predict feed intake in beef cows., (© The Author(s) 2022. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multimorbidity is a defining challenge for health systems and requires coordination of care delivery and care management. Care management is a clinical service designed to remotely engage patients between visits and after discharge in order to support self-management of chronic and emergent conditions, encourage increased use of scheduled care and address the use of unscheduled care. Care management can be provided using digital technology - digital care management. A robust methodology to assess digital care management, or any traditional or digital primary care intervention aimed at longitudinal management of multimorbidity, does not exist outside of randomized controlled trials (RCTs). RCTs are not always generalizable and are also not feasible for most healthcare organizations. We describe here a novel and pragmatic methodology for the evaluation of digital care management that is generalizable to any longitudinal intervention for multimorbidity irrespective of its mode of delivery. This methodology implements propensity matching with bootstrapping to address some of the major challenges in evaluation including identification of robust outcome measures, selection of an appropriate control population, small sample sizes with class imbalances, and limitations of RCTs. We apply this methodology to the evaluation of digital care management at a U.S. payor and demonstrate a 9% reduction in ER utilization, a 17% reduction in inpatient admissions, and a 29% increase in the utilization of preventive medicine services. From these utilization outcomes, we drive forward an estimated cost saving that is specific to a single payor's payment structure for the study time period of $641 per-member-per-month at 3 months. We compare these results to those derived from existing observational approaches, 1:1 and 1:n propensity matching, and discuss the circumstances in which our methodology has advantages over existing techniques. Whilst our methodology focuses on cost and utilization and is applied in the U.S. context, it is applicable to other outcomes such as Patient Reported Outcome Measures (PROMS) or clinical biometrics and can be used in other health system contexts where the challenge of multimorbidity is prevalent.

Background & Aims: Gut-homing lymphocytes that express the integrin α4β7 and CCR9 might contribute to development of primary sclerosing cholangitis (PSC). Vedolizumab, which blocks the integrin α4β7, is used to treat patients with inflammatory bowel diseases (IBD), but there are few data on its efficacy in patients with PSC. We investigated the effects of vedolizumab in a large international cohort of patients with PSC and IBD., Methods: We collected data from European and North American centers participating in the International PSC Study Group from patients with PSC and IBD who received at least 3 doses of vedolizumab (n = 102; median vedolizumab treatment duration, 412 days). Demographic and clinical data were collected from baseline and during the follow-up period (until liver transplantation, death, or 56 days after the final vedolizumab infusion). We analyzed overall changes in biochemical features of liver and proportions of patients with reductions in serum levels of alkaline phosphatase (ALP) of 20% or more, from baseline through last follow-up evaluation. Other endpoints included response of IBD to treatment (improved, unchanged, or worsened, judged by the treating clinician, as well as endoscopic score) and liver-related outcomes., Results: In the entire cohort, the median serum level of ALP increased from 1.54-fold the upper limit of normal at baseline to 1.64-fold the upper limit of normal at the last follow-up examination (P = .018); serum levels of transaminases and bilirubin also increased by a small amount between baseline and the last follow-up examination. Serum levels of ALP decreased by 20% or more in 21 patients (20.6%); only the presence of cirrhosis (odds ratio, 4.48; P = .019) was independently associated with this outcome. Of patients with available endoscopic data, 56.8% had a response of IBD to treatment. Liver-related events occurred in 21 patients (20.6%), including bacterial cholangitis, cirrhosis decompensation, or transplantation., Conclusions: In an analysis of patients with PSC and IBD in an international study group, we found no evidence for a biochemical response to vedolizumab, although serum level of ALP decreased by 20% or more in a subset of patients. Vedolizumab appears to be well tolerated and the overall response of IBD was the same as expected for patients without PSC., (Copyright © 2020 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Echocardiography plays an essential role in the diagnosis and assessment of cardiovascular disease. Measurements derived from echocardiography are also used to determine the severity of disease, its progression over time, and to aid in the choice of optimal therapy. It is therefore clinically important that echocardiographic measurements be reproducible, repeatable, and reliable. There are a variety of statistical tests available to assess these parameters, and in this article the authors summarize those available for use by echocardiographers to improve their clinical practice. Correlation coefficients, linear regression, Bland-Altman plots, and the coefficient of variation are explored, along with their limitations. The authors also provide an online tool for the easy calculation of these statistics in the clinical environment (www.birmingham.ac.uk/echo). Quantifying and enhancing the reproducibility of echocardiography has important potential to improve the value of echocardiography as the basis for good clinical decision-making., (Copyright © 2019 American Society of Echocardiography. Published by Elsevier Inc. All rights reserved.)

Background: The logrank test and the Cox proportional hazards model are routinely applied in the design and analysis of randomised controlled trials (RCTs) with time-to-event outcomes. Usually, sample size and power calculations assume proportional hazards (PH) of the treatment effect, i.e. the hazard ratio is constant over the entire follow-up period. If the PH assumption fails, the power of the logrank/Cox test may be reduced, sometimes severely. It is, therefore, important to understand how serious this can become in real trials, and for a proven, alternative test to be available to increase the robustness of the primary test., Methods: We performed a systematic search to identify relevant articles in four leading medical journals that publish results of phase 3 clinical trials. Altogether, 50 articles satisfied our inclusion criteria. We digitised published Kaplan-Meier curves and created approximations to the original times to event or censoring at the individual patient level. Using the reconstructed data, we tested for non-PH in all 50 trials. We compared the results from the logrank/Cox test with those from the combined test recently proposed by Royston and Parmar., Results: The PH assumption was checked and reported only in 28% of the studies. Evidence of non-PH at the 0.10 level was detected in 31% of comparisons. The Cox test of the treatment effect was significant at the 0.05 level in 49% of comparisons, and the combined test in 55%. In four of five trials with discordant results, the interpretation would have changed had the combined test been used. The degree of non-PH and the dominance of the p value for the combined test were strongly associated. Graphical investigation suggested that non-PH was mostly due to a treatment effect manifesting in an early follow-up and disappearing later., Conclusions: The evidence for non-PH is checked (and, hence, identified) in only a small minority of RCTs, but non-PH may be present in a substantial fraction of such trials. In our reanalysis of the reconstructed data from 50 trials, the combined test outperformed the Cox test overall. The combined test is a promising approach to making trial design and analysis more robust.

Rotational grazing management is commonly recommended to improve persistence of legumes interseeded into bermudagrass, but impacts on animal performance are poorly understood. Steers (n = 365, BW = 249 ± 22.9 kg) grazed mixed alfalfa (Medicago sativa L.)/bermudagrass (Cynodon dactylon [L.] Pers.) pastures (n = 10; 1.6-ha) with either continuous grazing (CONT) or rotational grazing (ROT) management over 3 yr. Initial stocking rate was set at 6.25 steers/ha. When forage allowance of CONT became limiting in mid-July each year, 2 randomly selected steers were removed from all pastures to maintain equal stocking rates in CONT and ROT. Rotational grazing pastures were divided into 8 paddocks with 3-d grazing and 21-d rest between grazing events. Initial, final, and interim (28-d) BW were collected following a 16-h removal from feed and water. Data were analyzed as a repeated measures experiment with completely random design using the mixed procedure of SAS (SAS Inst. Inc., Cary, NC) using pasture within treatment by year in the random statement. Across years, steer BW did not differ (P ≥ 0.47) at the midpoint or end of the grazing season. Daily BW gains during the early summer period tended (P = 0.10) to be 0.11 kg greater for CONT than ROT in yr 1, but did not differ (P ≥ 0.24) in yr 2 and 3. In the late summer, ADG was 0.15 kg less (P < 0.01) for CONT than ROT in yr 1, were not different (P = 0.23) in yr 2, and were 0.29 kg less (P < 0.01) for CONT than ROT in yr 3. Over the entire experimental period, ADG did not differ (P ≥ 0.67) in yr 1 or 2, but tended (P = 0.09) to be 0.10 kg greater for ROT than CONT in yr 3. Forage mass of ROT was greater (P < 0.01) than CONT throughout the grazing season. Because stocking rates were maintained at the same level, ROT pastures provided greater (P < 0.01) forage allowance during the grazing season than CONT explaining differences in ADG between treatments. Alfalfa stand percentage did not differ (P = 0.79) at the beginning of the experiment, but was less (P ≤ 0.03) for CONT than ROT at all other sampling dates. These data indicate that at equal stocking rates, ROT can maintain greater alfalfa persistence, forage nutritive quality, and forage allowance compared with continuous grazing and provided increased animal performance during the late summer when the alfalfa stand was reduced in CONT pastures.

Objectives: The influence of choice of endpoint on trial size, duration, and interpretation of results was examined in patients with heart failure who were enrolled in BEST (Beta-blocker Evaluation of Survival Trial)., Background: The choice of endpoints in heart failure trials has evolved over the past 3 decades., Methods: In the BEST trial, we used Cox regression analysis to examine the effect of bucindolol on the current standard composite of cardiovascular death or heart failure hospitalization (CVD/HFH) compared with the original primary mortality endpoint and the expanded composite that included emergency department (ED) visits. We also undertook an analysis of recurrent events primarily using the Lin, Wei, Ying, and Yang model., Results: Overall, 448 (33%) patients on placebo and 411 (30%) patients on bucindolol died (hazard ratio [HR]: 0.90; 95% confidence interval [CI]: 0.78 to 1.02; p = 0.11). A total of 730 (54%) patients experienced CVD/HFH on placebo and 624 (46%) on bucindolol (HR: 0.80; 95% CI: 0.72 to 0.89; p < 0.001). Adding ED visits increased these numbers to 768 (57%) and 668 (49%), respectively (HR: 0.81; 95% CI: 0.73 to 0.90; p < 0.001). A total of 568 (42%) patients on placebo experienced HFH compared with 476 (35%) patients on bucindolol (HR: 0.78; 95% CI: 0.69 to 0.89; p < 0.001), with a total of 1,333 and 1,124 admissions, respectively. With the same statistical assumptions, using the composite endpoint instead of all-cause mortality would have reduced the trial size by 40% and follow-up duration by 69%. The rate ratio for recurrent events (CVD/HFH) was 0.83 (95% CI: 0.73 to 0.94; p = 0.003)., Conclusions: Choice of endpoint has major implications for trial size and duration, as well as interpretation of results. The value of broader composite endpoints and inclusion of recurrent events needs further investigation. (Beta Blocker Evaluation in Survival Trial [BEST]; NCT00000560)., (Copyright © 2017 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Biosynthetic pathways provide an enzymatic route from inexpensive renewable resources to valuable metabolic products such as pharmaceuticals and plastics. Designing these pathways is challenging due to the complexities of biology. Advances in the design and construction of genetic variants has enabled billions of cells, each possessing a slightly different metabolic design, to be rapidly generated. However, our ability to measure the quality of these designs lags by several orders of magnitude. Recent research has enabled cells to report their own success in chemical production through the use of genetically encoded biosensors. A new engineering discipline is emerging around the creation and application of biosensors. Biosensors, implemented in selections and screens to identify productive cells, are paving the way for a new era of biotechnological progress., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

This paper considers the analysis of a repeat event outcome in clinical trials of chronic diseases in the context of dependent censoring (e.g. mortality). It has particular application in the context of recurrent heart failure hospitalisations in trials of heart failure. Semi-parametric joint frailty models (JFMs) simultaneously analyse recurrent heart failure hospitalisations and time to cardiovascular death, estimating distinct hazard ratios whilst individual-specific latent variables induce associations between the two processes. A simulation study was carried out to assess the suitability of the JFM versus marginal analyses of recurrent events and cardiovascular death using standard methods. Hazard ratios were consistently overestimated when marginal models were used, whilst the JFM produced good, well-estimated results. An application to the Candesartan in Heart failure: Assessment of Reduction in Mortality and morbidity programme was considered. The JFM gave unbiased estimates of treatment effects in the presence of dependent censoring. We advocate the use of the JFM for future trials that consider recurrent events as the primary outcome. © 2016 The Authors. Statistics in Medicine Published by John Wiley & Sons Ltd., (© 2016 The Authors. Statistics in Medicine Published by John Wiley & Sons Ltd.)

Summer-active (continental) and summer-dormant (Mediterranean) tall fescue morphotypes are each adapted to different environmental conditions. Endophyte presence provides plant parasitic nematode resistance, but not with all endophyte strains and cultivar combinations. This study sought to compare effects of four nematode genera on continental and Mediterranean cultivars infected with common toxic or novel endophyte strains. A 6-mon greenhouse study was conducted with continental cultivars, Kentucky 31 (common toxic) and Texoma MaxQ II (novel endophyte) and the Mediterranean cultivar Flecha MaxQ (novel endophyte). Endophyte-free plants of each cultivar were controls. Each cultivar × endophyte combination was randomly assigned to a control, low or high inoculation rate of a mixed nematode culture containing stunt nematodes (Tylenchorhynchus spp.), ring nematodes (Criconemella spp.), spiral nematodes (Helicotylenchus spp.), and lesion nematodes (Pratylenchus spp.). Endophyte infection had no effect on nematode population densities. The cultivar × endophyte interaction was significant. Population densities of stunt nematode, spiral nematode, and ring nematodes were higher for Flecha MaxQ than other cultivar × endophyte combinations. Novel endophyte infection enhances suitability of Flecha MaxQ as a nematode host.

Biotechnology is the manufacturing technology of the future. However, engineering biology is complex, and many possible genetic designs must be evaluated to find cells that produce high levels of a desired drug or chemical. Recent advances have enabled the design and construction of billions of genetic variants per day, but evaluation capacity remains limited to thousands of variants per day. Here we evaluate biological engineering through the lens of the design–build–test cycle framework and highlight the role that multiplexing has had in transforming the design and build steps. We describe a multiplexed solution to the ‘test’ step that is enabled by new research. Achieving a multiplexed test step will permit a fully multiplexed engineering cycle and boost the throughput of biobased product development by up to a millionfold.

Engineering cells to produce valuable metabolic products is hindered by the slow and laborious methods available for evaluating product concentration. Consequently, many designs go unevaluated, and the dynamics of product formation over time go unobserved. In this work, we develop a framework for observing product formation in real time without the need for sample preparation or laborious analytical methods. We use genetically encoded biosensors derived from small-molecule responsive transcription factors to provide a fluorescent readout that is proportional to the intracellular concentration of a target metabolite. Combining an appropriate biosensor with cells designed to produce a metabolic product allows us to track product formation by observing fluorescence. With individual cells exhibiting fluorescent intensities proportional to the amount of metabolite they produce, high-throughput methods can be used to rank the quality of genetic variants or production conditions. We observe production of several renewable plastic precursors with fluorescent readouts and demonstrate that higher fluorescence is indeed an indicator of higher product titer. Using fluorescence as a guide, we identify process parameters that produce 3-hydroxypropionate at 4.2 g/L, 23-fold higher than previously reported. We also report, to our knowledge, the first engineered route from glucose to acrylate, a plastic precursor with global sales of $14 billion. Finally, we monitor the production of glucarate, a replacement for environmentally damaging detergents, and muconate, a renewable precursor to polyethylene terephthalate and nylon with combined markets of $51 billion, in real time, demonstrating that our method is applicable to a wide range of molecules.

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. We engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). The ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

Characterization and standardization of inducible transcriptional regulators has transformed how scientists approach biology by allowing precise and tunable control of gene expression. Despite their utility, only a handful of well-characterized regulators exist, limiting the complexity of engineered biological systems. We apply a characterization pipeline to four genetically encoded sensors that respond to acrylate, glucarate, erythromycin and naringenin. We evaluate how the concentration of the inducing chemical relates to protein expression, how the extent of induction affects protein expression kinetics, and how the activation behavior of single cells relates to ensemble measurements. We show that activation of each sensor is orthogonal to the other sensors, and to other common inducible systems. We demonstrate independent control of three fluorescent proteins in a single cell, chemically defining eight unique transcriptional states. To demonstrate biosensor utility in metabolic engineering, we apply the glucarate biosensor to monitor product formation in a heterologous glucarate biosynthesis pathway and identify superior enzyme variants. Doubling the number of well-characterized inducible systems makes more complex synthetic biological circuits accessible. Characterizing sensors that transduce the intracellular concentration of valuable metabolites into fluorescent readouts enables high-throughput screening of biological catalysts and alleviates the primary bottleneck of the metabolic engineering design-build-test cycle., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Ivabradine, a specific heart rate lowering agent, was shown in the SHIFT study to reduce time to first hospitalization for worsening heart failure (HF) in chronic systolic HF patients and also to reduce recurrent/total hospitalizations over the study interval. We assessed the effects of adding ivabradine in patients with systolic HF on the number needed to treat (NNT) to reduce recurrent hospitalizations., Methods: The SHIFT trial included 6505 patients with symptomatic HF (NYHA II-IV), left ventricular ejection fraction ≤35% and heart rate ≥70 bpm in sinus rhythm. Patients were randomized to either ivabradine or placebo in addition to guidelines-based drug therapy. The times to first hospitalization were analyzed using a univariate Cox proportional-hazards model; the associated NNT was calculated using Kaplan-Meier estimates of the time-to-event curves at 1 year in each treatment arm. Recurrent hospitalizations were analyzed using a negative binomial and the estimated annual event rates used to calculate the associated patient-time NNTs respectively., Results: The estimated NNT (number needed to initiate treatment with ivabradine to prevent one first HF hospitalization within 1 year) was 27 (estimated hazard ratio: 0.75, P < 0.0001). For recurrent HF hospitalizations, one event would be prevented on average per 14 patient-years for any year of follow-up over the course of SHIFT (estimated rate ratio: 0.71, P < 0.0001). A key limitation of this analysis is that it did not account for a relationship between recurrent HF hospitalizations and subsequent mortality., Conclusion: In chronic systolic HF the effect of ivabradine on reducing recurrent HF hospitalizations results in a lower NNT compared to the effect on the time for first hospitalization. The effect of ivabradine on recurrent hospitalizations, in addition to first events, may be a more appropriate measure when considering the impact of a treatment with ivabradine on healthcare resource utilization.

Engineering biosynthetic pathways for chemical production requires extensive optimization of the host cellular metabolic machinery. Because it is challenging to specify a priori an optimal design, metabolic engineers often need to construct and evaluate a large number of variants of the pathway. We report a general strategy that combines targeted genome-wide mutagenesis to generate pathway variants with evolution to enrich for rare high producers. We convert the intracellular presence of the target chemical into a fitness advantage for the cell by using a sensor domain responsive to the chemical to control a reporter gene necessary for survival under selective conditions. Because artificial selection tends to amplify unproductive cheaters, we devised a negative selection scheme to eliminate cheaters while preserving library diversity. This scheme allows us to perform multiple rounds of evolution (addressing ∼10(9) cells per round) with minimal carryover of cheaters after each round. Based on candidate genes identified by flux balance analysis, we used targeted genome-wide mutagenesis to vary the expression of pathway genes involved in the production of naringenin and glucaric acid. Through up to four rounds of evolution, we increased production of naringenin and glucaric acid by 36- and 22-fold, respectively. Naringenin production (61 mg/L) from glucose was more than double the previous highest titer reported. Whole-genome sequencing of evolved strains revealed additional untargeted mutations that likely benefit production, suggesting new routes for optimization.

Objectives: This study sought to examine the effect of statin therapy hospitalizations for heart failure (HFH) in patients in the CORONA (Controlled Rosuvastatin Multinational Trial in Heart Failure) trial., Background: HFH is an important, frequently recurrent event. Conventional time-to-first event analyses do not take account repeat events. We used a number of statistical approaches to examine the effect of treatment on first and repeat HFH in the CORONA trial., Methods: In the CORONA trial, 5,011 patients ≥60 years of age with chronic New York Heart Association functional classes II to IV systolic heart failure resulting from ischemia were randomized to receive rosuvastatin or placebo. Poisson, Andersen-Gill, and negative binomial methods (NB) were used to analyze the effect of rosuvastatin on HFH, and the NB and a parametric joint frailty model (JF) were used to examine this effect while accounting for the competing risk of cardiovascular (CV) death. Rosuvastatin/placebo rate ratios were calculated, both unadjusted and adjusted., Results: A total of 1,291 patients had 1 or more HFH (750 of these had a single HFH only), and there were a total of 2,408 HFHs. The hazard ratio for the conventional time-to-first event analysis for HFH was 0.91 (95% confidence interval [CI]: 0.82 to 1.02, p = 0.105). In contrast, the NB on repeat hospitalizations gave an unadjusted RR (RR) for HFH of 0.86 (95% CI: 0.75 to 0.99, p = 0.030), adjusted 0.82 (95% CI: 0.72 to 0.92, p = 0.001), and after including CV death as the last event, adjusted RR of 0.85 (95% CI: 0.77 to 0.94, p = 0.001). The JF gave an adjusted RR of 0.82 (95% CI: 0.73 to 0.92, p = 0.001). Similar results were found in analyses of all CV hospitalizations and all-cause hospitalizations., Conclusions: When repeat events were included, rosuvastatin was shown to reduce the risk of HFH by approximately 15% to 20%, equating to approximately 76 fewer admissions per 1,000 patients treated over a median 33 months of follow-up. Including repeat events could increase the ability to detect treatment effects in heart failure trials., (Copyright © 2014 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

Aims: Heart failure is characterized by recurrent hospitalizations, but often only the first event is considered in clinical trial reports. In chronic diseases, such as heart failure, analysing all events gives a more complete picture of treatment benefit. We describe methods of analysing repeat hospitalizations, and illustrate their value in one major trial., Methods and Results: The Candesartan in Heart failure Assessment of Reduction in Mortality and morbidity (CHARM)-Preserved study compared candesartan with placebo in 3023 patients with heart failure and preserved systolic function. The heart failure hospitalization rates were 12.5 and 8.9 per 100 patient-years in the placebo and candesartan groups, respectively. The repeat hospitalizations were analysed using the Andersen-Gill, Poisson, and negative binomial methods. Death was incorporated into analyses by treating it as an additional event. The win ratio method and a method that jointly models hospitalizations and mortality were also considered. Using repeat events gave larger treatment benefits than time to first event analysis. The negative binomial method for the composite of recurrent heart failure hospitalizations and cardiovascular death gave a rate ratio of 0.75 [95% confidence interval (CI) 0.62-0.91, P = 0.003], whereas the hazard ratio for time to first heart failure hospitalization or cardiovascular death was 0.86 (95% CI 0.74-1.00, P = 0.050)., Conclusions: In patients with preserved EF, candesartan reduces the rate of admissions for worsening heart failure, to a greater extent than apparent from analysing only first hospitalizations. Recurrent events should be routinely incorporated into the analysis of future clinical trials in heart failure., (© 2013 The Authors. European Journal of Heart Failure © 2013 European Society of Cardiology.)

As the nursing profession seeks to develop evidence-based practice, nurses are being encouraged to embark on research in their institutions. Staff nurses and nurse clinicians often struggle with the process of obtaining institutional review board approval for their projects. When classes are available, the focus is frequently on informing nurses about the federal regulations for protection of human subjects. This article addresses practical strategies for addressing these regulations from the perspective of both the individual nurse and the nursing leader., (Copyright 2012, SLACK Incorporated.)

Continuous-flow centrifugation is widely utilized as the primary clarification step in the recovery of biopharmaceuticals from cell culture. However, it is a challenging operation to develop and characterize due to the lack of easy to use, small-scale, systems that can be used to model industrial processes. As a result, pilot-scale continuous centrifugation is typically employed to model large-scale systems requiring a significant amount of resources. In an effort to reduce resource requirements and create a system which is easy to construct and utilize, a capillary shear device, capable of producing energy dissipation rates equivalent to those present in the feed zones of industrial disk stack centrifuges, was developed and evaluated. When coupled to a bench-top, batch centrifuge, the capillary device reduced centrate turbidity prediction error from 37% to 4% compared to using a bench-top centrifuge alone. Laboratory-scale parameters that are analogous to those routinely varied during industrial-scale continuous centrifugation were identified and evaluated for their utility in emulating disk stack centrifuge performance. The resulting relationships enable bench-scale process modeling of continuous disk stack centrifuges using an easily constructed, scalable, capillary shear device coupled to a typical bench-top centrifuge., (Copyright © 2010 Wiley Periodicals, Inc.)

The extraction of antibodies using a polyethylene glycol (PEG)-citrate aqueous two-phase system (ATPS) was investigated. Studies using purified monoclonal antibody (mAb) identified operating ranges for successful phase formation and factors that significantly affected antibody partitioning. The separation of antibody and host cell protein (HCP) from clarified cell culture media was examined using statistical design of experiments (DOE). The partitioning of antibody was nearly complete over the entire range of the operating space examined. A model of the HCP partitioning was generated in which both NaCl and citrate concentrations were identified as significant factors. To achieve the highest purity, the partitioning of HCP from cell culture fluid into the product containing phase was minimized using a Steepest Descent algorithm. An optimal ATPS consisting of 14.0% (w/w) PEG, 8.4% (w/w) citrate, and 7.2% (w/w) NaCl at pH 7.2 resulted in a product yield of 89%, an approximate 7.6-fold reduction in HCP levels relative to the clarified cell culture fluid before extraction and an overall purity of 70%. A system consisting of 15% (w/w) PEG, 8% (w/w) citrate, and 15% (w/w) NaCl at pH 5.5 reduced product-related impurities (aggregates and low molecular product fragments) from ∼40% to less than 0.5% while achieving 95% product recovery. At the experimental conditions that were optimized in the batch mode, a scale-up model for the use of counter-current extraction technology was developed to identify potential improvements in purity and recovery that could be realized in the continuous operational mode., (Copyright © 2010 American Institute of Chemical Engineers (AIChE).)

One method of transmitting wavelet based zerotree encoded images over noisy channels is to add channel coding without altering the source coder. A second method is to reorder the embedded zerotree bitstream into packets containing a small set of wavelet coefficient trees. We consider a hybrid mixture of these two approaches and demonstrate situations in which the hybrid image coder can outperform either of the two building block methods, namely on channels that can suffer packet losses as well as statistically varying bit errors.

Conditioned suppression was examined for control (N = 8) and lead-treated (N = 8) adult rats. Treated animals were exposed daily to 10 mg/kg lead administered via the diet as lead acetate. 40 days of operant lever press training on a multiple schedule using food as reward was given. Lever press training continued for an additional 5 days but during this period subjects experienced superimposed 3 min tone presentations followed by electric shock (1.3 mA, 1.5 s duration) to a grid floor. Tone presentations were observed to be significantly more disruptive of operant steady state responding for treated subjects as compared to controls. This finding supports earlier claims that lead exposure enhances emotionality.