Team Situation Awareness, Cohesion, and Autonomic Synchrony 2: Group-level Effects and their Combined Influence on Team Performance.

Situation awareness (SA) is a mental state that is instrumental to performance of complex dynamic tasks. SA within teams is thought to be supported by favorable social conditions within the team. The present study was organized in two parts: (a) causal relationships among SA, group cohesion, and autonomic synchrony, the latter being a fundamentally nonlinear process, and (b) the combined impact of the three variables on performance in a dynamic decisions task. Experimental conditions assessed changes in task difficulty, group size, and method of obtaining SA measures. Participants were 136 undergraduates organized into 32 teams of three to five members engaged in two matches of a first-person shooter computer game. They completed self-report measures of cohesion and SA. Synchrony was determined through time series analysis of electrodermal responses using the driver-empath framework. ANOVA results showed that cohesion and SA improved over the two matches, and SA was better in smaller groups during the second match. Synchrony was stronger in larger groups. Granger regression indicated no causal or circular relationship between SA and cohesion. Synchrony had a small positive effect on cohesion during the first match. SA had a strong negative impact on synchrony early on and dissipated afterwards. The best performing teams during the first match were those that: were larger, were measured for SA without pausing the simulation, were less synchronized, showed more accurate SA, and reported stronger cohesion. The study opens new questions concerning the role of synchrony in volatile situations and the role of automated team members operating alongside humans.

Impact of dual-baculovirus infection on the Sf9 insect cell transcriptome during rAAV production using single-cell RNA-seq.

The insect cell-baculovirus expression vector system (IC-BEVS) has shown to be a powerful platform to produce complex biopharmaceutical products, such as recombinant proteins and virus-like particles. More recently, IC-BEVS has also been used as an alternative to produce recombinant adeno-associated virus (rAAV). However, little is known about the variability of insect cell populations and the potential effect of heterogeneity (e.g., stochastic infection process and differences in infection kinetics) on product titer and/or quality. In this study, transcriptomics analysis of Sf9 insect cells during the production of rAAV of serotype 2 (rAAV2) using a low multiplicity of infection, dual-baculovirus system was performed via single-cell RNA-sequencing (scRNA-seq). Before infection, the principal source of variability in Sf9 insect cells was associated with the cell cycle. Over the course of infection, an increase in transcriptional heterogeneity was detected, which was linked to the expression of baculovirus genes as well as to differences in rAAV transgenes (rep, cap and gfp) expression. Noteworthy, at 24 h post-infection, only 29.4% of cells enclosed all three necessary rAAV transgenes to produce packed rAAV2 particles, indicating limitations of the dual-baculovirus system. In addition, the trajectory analysis herein performed highlighted that biological processes such as protein folding, metabolic processes, translation, and stress response have been significantly altered upon infection. Overall, this work reports the first application of scRNA-seq to the IC-BEVS and highlights significant variations in individual cells within the population, providing insight into the rational cell and process engineering toward improved rAAV2 production in IC-BEVS.

Transcriptome analysis of Sf9 insect cells during production of recombinant Adeno-associated virus.

The insect cell-baculovirus expression vector system (IC-BEVS) has emerged as an alternative time- and cost-efficient production platform for recombinant Adeno-associated virus (AAV) for gene therapy. However, a better understanding of the underlying biological mechanisms of IC-BEVS is fundamental to further optimize this expression system toward increased product titer and quality. Here, gene expression of Sf9 insect cells producing recombinant AAV through a dual baculovirus expression system, with low multiplicity of infection (MOI), was profiled by RNA-seq. An 8-fold increase in reads mapping to either baculovirus or AAV transgene sequences was observed between 24 and 48 h post-infection (hpi), confirming a take-over of the host cell transcriptome by the baculovirus. A total of 336 and 4784 genes were identified as differentially expressed at 24 hpi (vs non-infected cells) and at 48 hpi (vs. infected cells at 24 hpi), respectively, including dronc, birc5/iap5, and prp1. Functional annotation found biological processes such as cell cycle, cell growth, protein folding, and cellular amino acid metabolic processes enriched along infection. This work uncovers transcriptional changes in Sf9 in response to baculovirus infection, which provide new insights into cell and/or metabolic engineering targets that can be leveraged for rational bioprocess engineering of IC-BEVS for AAV production.

Team Situation Awareness, Cohesion, and Autonomic Synchrony.

OBJECTIVE: This study evaluated the causal relationships among situation awareness (SA), cohesion, and autonomic synchrony (SE) within teams. SA is often a team effort and should be more accurate in better-functioning teams. BACKGROUND: Cohesive teams perform better overall, although the relationship appears reciprocal; the relationship to SA has not been considered previously. SE is a collective neurocognitive activity that has been connected to team coordination, communication, and performance in some circumstances. METHOD: In this experiment, 71 undergraduates, organized into 16 teams, played two matches of a first-person shooter computer game and completed self-report measures of cohesion and SA. SE was determined through time series analysis of electrodermal responses using the driver-empath framework. RESULTS: Empaths and those who came from more synchronized teams reported less cohesion in the team. Granger causality regression showed reciprocal relations among SA, SE, and cohesion that were both positive and negative after controlling for match difficulty. CONCLUSION: The cohesion-SA relationship is similar to the reciprocal cohesion-performance relationship. SE plays an important and independent role in both the social and cognitive aspects of team behavior. It is possible, furthermore, that individuals who are more attuned to their co-workers reported a more accurate, and less obliging, social situation. APPLICATION: Results are applicable to situations requiring teamwork in a dynamic environment.

Tracing production instability in a clonally derived CHO cell line using single-cell transcriptomics.

A variety of mechanisms including transcriptional silencing, gene copy loss, and increased susceptibility to cellular stress have been associated with a sudden or gradual loss of monoclonal antibody (mAb) production in Chinese hamster ovary (CHO) cell lines. In this study, we utilized single-cell RNA-seq (scRNA-seq) to study a clonally derived CHO cell line that underwent production instability leading to a dramatic reduction of the levels of mAb produced. From the scRNA-seq data, we identified subclusters associated with variations in the mAb transgenes and observed that heavy chain gene expression was significantly lower than that of the light chain across the population. Using trajectory inference, the evolution of the cell line was reconstructed and was found to correlate with a reduction in heavy and light chain gene expression. Genes encoding for proteins involved in the response to oxidative stress and apoptosis were found to increase in expression as cells progressed along the trajectory. Future studies of CHO cell lines using this technology have the potential to dramatically enhance our understanding of the characteristics underpinning efficient manufacturing performance as well as product quality.

Subphysiological temperature induces pervasive alternative splicing in Chinese hamster ovary cells.

RNA sequencing (RNASeq) has been widely used to associate alterations in Chinese hamster ovary (CHO) cell gene expression with bioprocess phenotypes; however, alternative messenger RNA (mRNA) splicing, has thus far, received little attention. In this study, we utilized RNASeq for transcriptomic analysis of a monoclonal antibody (mAb) producing CHO K1 cell line subjected to a temperature shift. More than 2,465 instances of differential splicing were observed 24 hr after the reduction of cell culture temperature. A total of 1,197 of these alternative splicing events were identified in genes where no changes in abundance were detected by standard differential expression analysis. Ten examples of alternative splicing were selected for independent validation using quantitative polymerase chain reaction in the mAb-producing CHO K1 cell line used for RNASeq and a further two CHO K1 cell lines. This analysis provided evidence that exon skipping and mutually exclusive splicing events occur in genes linked to the cellular response to changes in temperature and mitochondrial function. While further work is required to determine the impact of these changes in mRNA sequence on cellular phenotype, this study demonstrates that alternative splicing analysis can be utilized to gain a deeper understanding of post-transcriptional regulation in CHO cells during biopharmaceutical production.

Validation of the systematic scoring of immunohistochemically stained tumour tissue microarrays using QuPath digital image analysis.

AIMS: Output from biomarker studies involving immunohistochemistry applied to tissue microarrays (TMA) is limited by the lack of an efficient and reproducible scoring methodology. In this study, we examine the functionality and reproducibility of biomarker scoring using the new, open-source, digital image analysis software, QuPath. METHODS AND RESULTS: Three different reviewers, with varying experience of digital pathology and image analysis, applied an agreed QuPath scoring methodology to CD3 and p53 immunohistochemically stained TMAs from a colon cancer cohort (n = 661). Manual assessment was conducted by one reviewer for CD3. Survival analyses were conducted and intra- and interobserver reproducibility assessed. Median raw scores differed significantly between reviewers, but this had little impact on subsequent analyses. Lower CD3 scores were detected in cases who died from colorectal cancer compared to control cases, and this finding was significant for all three reviewers (P-value range = 0.002-0.02). Higher median p53 scores were generated among cases who died from colorectal cancer compared with controls (P-value range = 0.04-0.12). The ability to dichomotise cases into high versus low expression of CD3 and p53 showed excellent agreement between all three reviewers (kappa score range = 0.82-0.93). All three reviewers produced dichotomised expression scores that resulted in very similar hazard ratios for colorectal cancer-specific survival for each biomarker. Results from manual and QuPath methods of CD3 scoring were comparable, but QuPath scoring revealed stronger prognostic stratification. CONCLUSIONS: Scoring of immunohistochemically stained tumour TMAs using QuPath is functional and reproducible, even among users of limited experience of digital pathology images, and more accurate than manual scoring.