SPEED-MODE cell line development (CLD): Reducing Chinese hamster ovary (CHO) CLD timelines via earlier suspension adaptation and maximizing time spent in the exponential growth phase.

Chinese hamster ovary (CHO) cells are the preferred system for expression of therapeutic proteins and the majority of all biotherapeutics are being expressed by these cell lines. CHO expression systems are readily scalable, resistant to human adventitious agents, and have desirable post-translational modifications, such as glycosylation. Regardless, drug development as a whole is a very costly, complicated, and time-consuming process. Therefore, any improvements that result in reducing timelines are valuable and can provide patients with life-saving drugs earlier. Here we report an effective method (termed SPEED-MODE, herein) to speed up the Cell line Development (CLD) process in a targeted integration (TI) CHO CLD system. Our findings show that (1) earlier single cell cloning (SCC) of transfection pools, (2) speeding up initial titer screening turnaround time, (3) starting suspension adaptation of cultures sooner, and (4) maximizing the time CHO cultures spend in the exponential growth phase can reduce CLD timelines from ~4 to ~3 months. Interestingly, SPEED-MODE timelines closely match the theoretical minimum timeline for CHO CLD assuming that CHO cell division is the rate limiting factor. Clones obtained from SPEED-MODE CLD yielded comparable titer and product quality to those obtained via a standard CLD process. Hence, SPEED-MODE CLD is advantageous for manufacturing biotherapeutics in an industrial setting as it can significantly reduce CLD timelines without compromising titer or product quality.

Branched-chain amino acid levels are inversely associated with incident and prevalent chronic kidney disease in people with type 2 diabetes.

AIM: To investigate the association of plasma metabolites with incident and prevalent chronic kidney disease (CKD) in people with type 2 diabetes and establish whether this association is causal. MATERIALS AND METHODS: The Hoorn Diabetes Care System cohort is a large prospective cohort consisting of individuals with type 2 diabetes from the northwest part of the Netherlands. In this cohort we assessed the association of baseline plasma levels of 172 metabolites with incident (Ntotal = 462/Ncase = 81) and prevalent (Ntotal = 1247/Ncase = 120) CKD using logistic regression. Additionally, replication in the UK Biobank, body mass index (BMI) mediation and causality of the association with Mendelian randomization was performed. RESULTS: Elevated levels of total and individual branched-chain amino acids (BCAAs)-valine, leucine and isoleucine-were associated with an increased risk of incident CKD, but with reduced odds of prevalent CKD, where BMI was identified as an effect modifier. The observed inverse effects were replicated in the UK Biobank. Mendelian randomization analysis did not provide evidence for a causal relationship between BCAAs and prevalent CKD. CONCLUSIONS: Our study shows the intricate relationship between plasma BCAA levels and CKD in individuals with type 2 diabetes. While an association exists, its manifestation varies based on disease status and BMI, with no definitive evidence supporting a causal link between BCAAs and prevalent CKD.

Taken to the extreme: Transformational leadership, psychological capital, and follower health outcomes in extreme contexts.

The purpose of this study is to examine the boundary conditions of transformational leadership, follower psychological capital, and their effects on follower mental health outcomes. Specifically, we utilize archival, multi-wave data from a military sample to examine whether the negative relationship between transformational leadership and adverse follower stress outcomes increases as the context shifts from a relatively safe environment to one in which follower lives are at risk. Additionally, psychological capital, a constellation of personal psychological resources, is also assessed to account for individual buffers against extreme stressors. Findings from the current study suggest that the negative relationship between transformational leadership and follower stress increases significantly when the context shifts to a high-risk, mortality-salient environment.

Clarifying the association between adverse childhood experiences and postdeployment posttraumatic stress disorder symptom severity: A meta-analysis and large-sample investigation.

We report on two studies designed to shed light on the association between adverse childhood experiences (ACEs) and posttraumatic stress disorder (PTSD) symptom severity in military personnel. In particular, we examined the evidence for both additive and multiplicative associations between ACEs and combat exposure in predicting PTSD symptom severity. Study 1 was a meta-analysis of 50 samples (N > 50,000), and we found evidence for a moderate linear association between ACEs and PTSD symptom severity, ρ = .24. We also found that ACEs explained substantial variance in PTSD symptom severity after controlling for combat exposure, ΔR2 = .048. In Study 2, which is preregistered, we relied on a large sample of combat-deployed U.S. soldiers (N > 6,000) to examine evidence of a multiplicative association between ACEs and combat exposure in predicting PTSD symptom severity. In line with theoretical arguments that individuals who have experienced childhood trauma are more vulnerable to subsequent trauma exposure, we found a weak but meaningful interaction effect, ΔR2 = .00, p < .001, between ACEs and deployment-related traumatic events in the prediction of PTSD symptom severity. Implications for clinical applications and future research are discussed.

Longitudinal association of electrocardiogram abnormalities with major adverse cardiac events in people with Type 2 diabetes: the Hoorn Diabetes Care System cohort.

AIMS: To investigate the association of (changes in) electrocardiogram (ECG) abnormalities with incident major adverse cardiac events (MACE) in people with Type 2 diabetes (T2D) without pre-existing cardiovascular disease (CVD). METHODS AND RESULTS: A prospective longitudinal study of 11 993 people with T2D without known CVD from the Hoorn Diabetes Care System cohort. Annually repeated measurements (1998-2018), included cardiovascular risk factors, over 70 000 ECG, and self-reported cardiovascular events. ECG abnormalities were classified according to the Minnesota Classification as prolonged PR duration, prolonged QRS duration, left QRS-axis, QS pattern, ST-segment/T-wave abnormalities, or tall R-wave. The association of ECG abnormalities with MACEs was assessed using time-dependent Cox-regression models, adjusted for time-varying cardiovascular risk factors, and medication use [hazard ratios (HRs) with 95% confidence intervals (CIs)]. During a median follow-up of 6.6 (IQR, 3.1-10.7) years, 5445 (45.4%) of the participants had an ECG abnormality (prevalent or incident) at any of the median 6 (IQR, 3-10) annual ECG recordings, and 905 people (7.5%) had a MACE (529 coronary heart disease (CHD), 250 heart failure (HF), and 126 sudden cardiac arrest (SCA)). After adjustment, most ECG abnormalities were associated with HF: prolonged QRS duration [HR, 4.01 (95% CI, 2.67-6.03)], QS pattern [2.68 (0.85-8.49)], ST-segment/T-wave abnormalities [4.26 (2.67-6.80)], and tall R-wave [2.23 (1.33-3.76)]. Only QS pattern [2.69 (1.20-6.03)] and ST-segment/T-wave abnormalities [2.11 (1.48-3.02)] were associated with CHD. These associations were robust across age, sex, hypertension, or estimated CVD risk subgroups. CONCLUSION: In people with T2D without pre-existing CVD, ECG abnormalities related to decelerated conduction, ischaemia, and hypertrophy are predominantly early signs of emerging HF, while only abnormalities related to ischaemic disorders are signs of CHD.

In this cohort study of 11 993 people with Type 2 diabetes (T2D) that were still free of cardiovascular disease (CVD), the people with electrocardiogram (ECG) abnormalities were up to four times as likely to experience heart failure and up to twice as likely to experience a heart attack, regardless of their age, sex, blood pressure, or estimated risk of CVD. • Most ECG abnormalities are related to a higher risk of heart failure, but only ECG abnormalities that indicate reduced oxygen supply to the heart are related to a higher risk of a heart attack. • Periodical ECG examinations can help detect developing heart disease in an early stage for all people with T2D still free of CVD.

Longitudinal association of premature atrial contractions with atrial fibrillation and brain ischemia in people with type 2 diabetes: The Hoorn Diabetes Care System cohort.

Background: Premature atrial contractions (PACs) are potential markers for imminent onset of both atrial fibrillation (AF) and brain ischemia (BI; transient ischemic attack [TIA] or ischemic stroke). We investigated the association of PACs with incident AF and BI events separately, and of incident AF with BI events in people with type 2 diabetes (T2D) without pre-existing AF or cerebrovascular disease. Methods: A prospective longitudinal study of 12,242 people with T2D without known AF or cerebrovascular disease from the Hoorn Diabetes Care System cohort. Annual measurements (1998-2018) included cardiovascular risk factors, over 85,000 ECGs, and self-reported cardiovascular events. We assessed the association of PACs with incident AF and BI events and of incident AF with BI events using time-dependent Cox-regression models, adjusted for time-varying cardiovascular risk factors and medication use (Hazard Ratios with 95%CIs). Results: The baseline mean age was 62.2 ± 11.9 years. During a median follow-up of 7.0 (IQR 3.4-11.0) years, 1031 (8.4 %) participants had PACs, and 566 (4.6 %) had incident AF at any of the median 6 (IQR 3-10) annual ECG recordings. BI events occurred in 517 (4.2 %) people (304 TIAs, 213 ischemic strokes). After adjustment, PACs were associated with incident AF (Hazard Ratio, 1.96 (95%CI, 1.53-2.50)), but not with overall BI events (1.09 (0.76-1.56)), or with TIA (0.91 (0.57-1.46)) or ischemic stroke (1.50 (0.88-2.54)) separately. AF was not associated with BI events (0.95 (0.55-1.63)). Conclusions: In people with T2D without a history of AF or BI events, PACs are associated with a two-fold increased risk of incident AF.

Discovery of predictors of sudden cardiac arrest in diabetes: rationale and outline of the RESCUED (REcognition of Sudden Cardiac arrest vUlnErability in Diabetes) project.

INTRODUCTION: Early recognition of individuals with increased risk of sudden cardiac arrest (SCA) remains challenging. SCA research so far has used data from cardiologist care, but missed most SCA victims, since they were only in general practitioner (GP) care prior to SCA. Studying individuals with type 2 diabetes (T2D) in GP care may help solve this problem, as they have increased risk for SCA, and rich clinical datasets, since they regularly visit their GP for check-up measurements. This information can be further enriched with extensive genetic and metabolic information. AIM: To describe the study protocol of the REcognition of Sudden Cardiac arrest vUlnErability in Diabetes (RESCUED) project, which aims at identifying clinical, genetic and metabolic factors contributing to SCA risk in individuals with T2D, and to develop a prognostic model for the risk of SCA. METHODS: The RESCUED project combines data from dedicated SCA and T2D cohorts, and GP data, from the same region in the Netherlands. Clinical data, genetic data (common and rare variant analysis) and metabolic data (metabolomics) will be analysed (using classical analysis techniques and machine learning methods) and combined into a prognostic model for risk of SCA. CONCLUSION: The RESCUED project is designed to increase our ability at early recognition of elevated SCA risk through an innovative strategy of focusing on GP data and a multidimensional methodology including clinical, genetic and metabolic analyses.

Prevalence of ECG abnormalities in people with type 2 diabetes: The Hoorn Diabetes Care System cohort.

AIMS: The American Diabetes Association, and the joint European Society of Cardiology and European Association for the Study of Diabetes guidelines recommend a resting ECG in people with type 2 diabetes with hypertension or suspected cardiovascular disease (CVD). However, knowledge on the prevalence of ECG abnormalities is incomplete. We aimed to analyse the prevalence of ECG abnormalities and their cross-sectional associations with cardiovascular risk factors in people with type 2 diabetes. METHODS: We used data of the Diabetes Care System cohort obtained in 2018. ECG abnormalities were defined using the Minnesota Classification and categorised into types of abnormalities. The prevalence was calculated for the total population (n = 8068) and the subgroup of people without a history of CVD (n = 6494). Logistic regression models were used to asses cross-sectional associations. RESULTS: Approximately one-third of the total population had minor (16.0%) or major (13.1%) ECG abnormalities. Of the participants without a CVD history, approximately one-quarter had minor (14.9%) or major (9.1%) ECG abnormalities, and for those with hypertension or very high CVD risk, the prevalence was 27.5% and 39.6%, respectively. ECG abnormalities were significantly and consistently associated with established CVD risk factors. CONCLUSIONS: Resting ECG abnormalities are common in all people with type 2 diabetes (29.1%), including those without a history of CVD (24.0%), and their prevalence is related to traditional cardiovascular risk factors such as older age, male sex, hypertension, lower HDL cholesterol, higher BMI, and smoking behaviour.

An anti-apoptotic HEK293 cell line provides a robust and high titer platform for transient protein expression in bioreactors.

HEK293 transient expression systems are used to quickly generate proteins for research and pre-clinical studies. With the aim of engineering a high-producing host that grows and transfects robustly in bioreactors, we deleted the pro-apoptotic genes Bax and Bak in an HEK293 cell line. The HEK293 Bax Bak double knock-out (HEK293 DKO) cell line exhibited resistance to apoptosis and shear stress. HEK293 DKO cells sourced from 2 L seed train bioreactors were most productive when a pH setpoint of 7.0, a narrow pH deadband of ±0.03, and a DO setpoint of 30% were used. HEK293 DKO seed train cells cultivated for up to 60 days in a 35 L bioreactor showed similar productivities to cells cultivated in shake flasks. To optimize HEK293 DKO transfection cultures, we first evaluated different pH and agitation parameters in ambr15 microbioreactors before scaling up to 10 L wavebag bioreactors. In ambr15 microbioreactors with a pH setpoint of 7.0, a wide pH deadband of ±0.3, and an agitation of 630 rpm, HEK293 DKO transient cultures yielded antibody titers up to 650 mg/L in 7 days. The optimal ambr15 conditions prompted us to operate the 10 L wavebag transfection without direct pH control to mimic the wide pH deadband ranges. The HEK293 DKO transfection process produces high titers at all scales tested. Combined, our optimized HEK293 DKO 35 L bioreactor seed train and 10 L high titer transient processes support efficient, large-scale recombinant protein production for research studies.

High Throughput Transfection of HEK293 Cells for Transient Protein Production.

Transient transfection of mammalian cells is used in the biotechnology industry to quickly supply recombinant protein for research and large molecule drug development. Here, we describe a method for high throughput transient transfection of Human Embryonic Kidney 293 (HEK293) cells in 30 mL tubespins using polyethylenimine (PEI) as a transfection reagent. An automated liquid handler can be used to perform pipetting steps for transfecting batches of 96 tubespins, and septa in the tubespin caps allow for rapid processing without decapping. The addition of valproic acid (VPA) to transfection cultures enhances recombinant protein production. The thawing and passaging operations for HEK293 cultures to source the transient transfections are also described.

Systems Perspective of Amazon Mechanical Turk for Organizational Research: Review and Recommendations.

Amazon Mechanical Turk (MTurk) is becoming a prevalent source of quick and cost effective data for organizational research, but there are questions about the appropriateness of the platform for organizational research. To answer these questions, we conducted an integrative review based on 75 papers evaluating the MTurk platform and 250 MTurk samples used in organizational research. This integrative review provides four contributions: (1) we analyze the trends associated with the use of MTurk samples in organizational research; (2) we develop a systems perspective (recruitment system, selection system, and work management system) to synthesize and organize the key factors influencing data collected on MTurk that may affect generalizability and data quality; (3) within each factor, we also use available MTurk samples from the organizational literature to analyze key issues (e.g., sample characteristics, use of attention checks, payment); and (4) based on our review, we provide specific recommendations and a checklist for data reporting in order to improve data transparency and enable further research on this issue.

Much ado about grit: A meta-analytic synthesis of the grit literature.

Grit has been presented as a higher order personality trait that is highly predictive of both success and performance and distinct from other traits such as conscientiousness. This paper provides a meta-analytic review of the grit literature with a particular focus on the structure of grit and the relation between grit and performance, retention, conscientiousness, cognitive ability, and demographic variables. Our results based on 584 effect sizes from 88 independent samples representing 66,807 individuals indicate that the higher order structure of grit is not confirmed, that grit is only moderately correlated with performance and retention, and that grit is very strongly correlated with conscientiousness. We also find that the perseverance of effort facet has significantly stronger criterion validities than the consistency of interest facet and that perseverance of effort explains variance in academic performance even after controlling for conscientiousness. In aggregate our results suggest that interventions designed to enhance grit may only have weak effects on performance and success, that the construct validity of grit is in question, and that the primary utility of the grit construct may lie in the perseverance facet. (PsycINFO Database Record

Assessing the Utility of Compound Trait Estimates of Narrow Personality Traits.

It has been argued that approximations of narrow traits can be made through linear combinations of broad traits such as the Big Five personality traits. Indeed, Hough and Ones ( 2001 ) used a qualitative analysis of scale content to arrive at a taxonomy of how Big Five traits might be combined to approximate various narrow traits. However, the utility of such compound trait approximations has yet to be established beyond specific cases such as integrity and customer service orientation. Using data from the Eugene-Springfield Community Sample (Goldberg, 2008 ), we explore the ability of linear composites of scores on Big Five traits to approximate scores on 127 narrow trait measures from 5 well-known non-Big-Five omnibus measures of personality. Our findings indicate that individuals' standing on more than 30 narrow traits can be well estimated from 3 different types of linear composites of scores on Big Five traits without a substantial sacrifice in criterion validity. We discuss theoretical accounts for why such relationships exist as well as the theoretical and practical implications of these findings for researchers and practitioners.

When Friends' and Society's Expectations Collide: A Longitudinal Study of Moral Decision-Making and Personality across College.

Early adulthood is a developmentally important time period, with many novel life events needing to be traversed for the first time. Despite this important transition period, few studies examine the development of moral decision-making processes during this critical life stage. In the present study, college students completed moral decision-making measures during their freshman and senior years of college. Results indicate that, across four years, moral decision-making demonstrates considerable rank-order stability as well as change, such that people become more likely to help a friend relative to following societal rules. To help understand the mechanisms driving changes in moral decision-making processes, we examined their joint development with personality traits, a known correlate that changes during early adulthood in the direction of greater maturity. We found little evidence that personality and moral decision-making developmental processes are related. In sum, findings indicate that while moral decision-making processes are relatively stable across a four-year period, changes do occur which are likely independent of developmental processes driving personality trait change.

Optimization and automation of an end-to-end high throughput microscale transient protein production process.

High throughput protein production from transient transfection of mammalian cells is used in multiple facets of research and development studies. Commonly used formats for these high number expressions are 12-, 24- and 96-well plates at various volumes. However there are no published examples of a 96-deep well plate microscale (1,000 µL) suspension process for mammalian transient expression. For this reason, we aimed to determine the optimal operating conditions for a high producing, microscale HEK293 transient system. We evaluated the hydrodynamic flow and measured the oxygen transfer rate (OTR) and transient protein expression for 96-deep well plates of different well geometries filled at 600-1,000 µL working volumes and agitated at various speeds and orbital diameters. Ultimately, a round well-round bottom (RR) 96-deep well plate with a working volume of 1,000 µL agitated at 1,000 RPM and a 3 mm orbital diameter yielded the highest and most consistent total transient protein production. As plate cultures are subject to evaporation, water loss from different plate seals was measured to identify an optimal plate sealing method. Finally, to enable higher capacity protein production, both expression and purification processes were automated. Functionality of this end-to-end automation workflow was demonstrated with the generation of high levels of human IgG1 antibodies (≥360 µg/mL) with reproducible productivity, product quality and ≥78% purification recovery.

Development of a semi-automated high throughput transient transfection system.

Transient transfection of mammalian cells provides a rapid method of producing protein for research purposes. Combining the transient transfection protein expression system with new automation technologies developed for the biotechnology industry would enable a high throughput protein production platform that could be utilized to generate a variety of different proteins in a short amount of time. These proteins could be used for an assortment of studies including proof of concept, antibody development, and biological structure and function. Here we describe such a platform: a semi-automated process for PEI-mediated transient protein production in tubespins at a throughput of 96 transfections at a time using a Biomek FX(P) liquid handling system. In one batch, 96 different proteins can be produced in milligram amounts by PEI transfection of HEK293 cells cultured in 50 mL tubespins. Methods were developed for the liquid handling system to automate the different processes associated with transient transfections such as initial cell seeding, DNA:PEI complex activation and DNA:PEI complex addition to the cells. Increasing DNA:PEI complex incubation time resulted in lower protein expression. To minimize protein production variability, the methods were further optimized to achieve consistent cell seeding, control the DNA:PEI incubation time and prevent cross-contamination among different tubespins. This semi-automated transfection process was applied to express 520 variants of a human IgG1 (hu IgG1) antibody.

Reciprocal relationship between proactive personality and work characteristics: a latent change score approach.

Previous proactivity research has predominantly assumed that proactive personality generates positive environmental changes in the workplace. Grounded in recent research on personality development from a broad interactionist theoretical approach, the present article investigates whether work characteristics, including job demands, job control, social support from supervisors and coworkers, and organizational constraints, change proactive personality over time and, more important, reciprocal relationships between proactive personality and work characteristics. Latent change score analyses based on longitudinal data collected in 3 waves across 3 years show that job demands and job control have positive lagged effects on increases in proactive personality. In addition, proactive personality exerts beneficial lagged effects on increases in job demands, job control, and supervisory support, and on decreases in organizational constraints. Dynamic reciprocal relationships are observed between proactive personality with job demands and job control. The revealed corresponsive change relationships between proactive personality and work characteristics contribute to the proactive personality literature by illuminating more nuanced interplays between the agentic person and work characteristics, and also have important practical implications for organizations and employees.

An evaluation of the consequences of using short measures of the Big Five personality traits.

Researchers often use very abbreviated (e.g., 1-item, 2-item) measures of personality traits due to their convenience and ease of use as well as the belief that such measures can adequately capture an individual's personality. Using data from 2 samples (N = 437 employees, N = 355 college students), we show that this practice, particularly the use of single-item measures, can lead researchers to substantially underestimate the role that personality traits play in influencing important behaviors and thereby overestimate the role played by new constructs. That is, the use of very short measures of personality may substantially increase both the Type 1 and Type 2 error rates. We argue that even slightly longer measures can substantially increase the validity of research findings without significant inconvenience to the researcher or research participants.

Perceiver effects as projective tests: what your perceptions of others say about you.

In 3 studies, we document various properties of perceiver effects--or how an individual generally tends to describe other people in a population. First, we document that perceiver effects have consistent relationships with dispositional characteristics of the perceiver, ranging from self-reported personality traits and academic performance to well-being and measures of personality disorders, to how liked the person is by peers. Second, we document that the covariation in perceiver effects among trait dimensions can be adequately captured by a single factor consisting of how positively others are seen across a wide range of traits (e.g., how nice, interesting, trustworthy, happy, and stable others are generally seen). Third, we estimate the 1-year stability of perceiver effects and show that individual differences in the typical perception of others have a level of stability comparable to that of personality traits. The results provide compelling evidence that how individuals generally perceive others is a stable individual difference that reveals much about the perceiver's own personality.

Mechanisms of health: Education and health-related behaviours partially mediate the relationship between conscientiousness and self-reported physical health.

The personality trait of conscientiousness is an important predictor of health and longevity. The present research examined how conscientiousness, in combination with educational attainment and health-related behaviours, predicted self-reported physical health across adulthood. These relations were investigated in two studies, one using a large, representative sample of Illinois residents (N = 617) and the other using a community sample with a multi-method assessment of conscientiousness (N = 274). Across both studies, structural path analyses provided evidence for a model wherein conscientiousness predicted health, in part, through its relationship to both educational attainment and health-related behaviours. The findings suggest conscientiousness predicts health through a diverse set of mechanisms including, but not limited to, educational attainment and health-related behaviours.