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Lentiviral vectors (LV) have become the dominant tool for stable gene transfer into lymphocytes including chimeric antigen receptor (CAR) gene delivery to T cells, a major breakthrough in cancer therapy. Yet, room for improvement remains, especially for the latest LV generations delivering genes selectively into T cell subtypes, a key requirement for in vivo CAR T cell generation. Toward improving gene transfer rates with these vectors, whole transcriptome analyses on human T lymphocytes are conducted after exposure to CAR‐encoding conventional vectors (VSV‐LV) and vectors targeted to CD8+ (CD8‐LV) or CD4+ T cells (CD4‐LV). Genes related to quiescence and antiviral restriction are found to be upregulated in CAR‐negative cells exposed to all types of LVs. Down‐modulation of various antiviral restriction factors, including the interferon‐induced transmembrane proteins (IFITMs) is achieved with rapamycin as verified by mass spectrometry (LC‐MS). Strikingly, rapamycin enhances transduction by up to 7‐fold for CD8‐LV and CD4‐LV without compromising CAR T cell activities but does not improve VSV‐LV. When administered to humanized mice, CD8‐LV results in higher rates of green fluorescent protein (GFP) gene delivery. Also in vivo CAR T cell generation is improved in kinetics and tumor control, however to a moderate extent, leaving room for improvement by optimizing the rapamycin administration schedule. The data favor multi‐omics approaches for improvements in gene delivery. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Our ability to study Chinese hamster ovary (CHO) cell biology has been revolutionised over the last decade following the development of next generation sequencing technology and publication of reference DNA sequences for CHO cells and the Chinese hamster. RNA sequencing has not only enabled the association of transcript expression with bioreactor conditions and desirable bioprocess phenotypes but played a key role in the characterisation of protein coding and small noncoding RNAs. The annotation of long noncoding RNAs, and therefore our understanding of their role in CHO cell biology, has been limited to date. In this manuscript, we use high‐resolution RNASeq data to more than double the number of annotated lncRNA transcripts for the CHO K1 genome. In addition, the utilisation of strand‐specific sequencing enabled the identification of more than 1,000 new antisense and divergent lncRNAs. The utility of monitoring lncRNA expression is demonstrated through an analysis of the transcriptomic response to a reduction of cell culture temperature and identification of simultaneous sense/antisense differential expression for the first time in CHO cells. To enable further studies of lncRNAs, the transcripts annotated in this study have been made available for the CHO cell biology community. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Bicaudal D1 (BICD1), an adaptor for the dynein‐dynactin motor complex, has been identified as a susceptibility gene in chronic obstructive pulmonary disease (COPD). Autophagy, an essential cellular homeostasis process, is defective in COPD, in which oxidative stress‐induced misfolded proteins accumulate into toxic aggregates dependent on the accumulation of the autophagic cargo receptor p62. Defective autophagy can be caused by mutations in the dynein and dynactin motor complex suggesting a possible link between BICD1 and defective autophagy in COPD. BICD1 levels were measured in peripheral lung tissue from COPD patients together with markers of autophagy and found to be increased in COPD together with autophagosomes, p62 and p62 oligomers. In vitro exposure of bronchial epithelial cells to cigarette smoke extracts (CSEs) revealed that high concentrations of CSE induced defective autophagosome maturation with accumulation of BICD1, p62 and ubiquitin‐associated p62 oligomers. This was confirmed in vivo using CS‐exposed mice. Furthermore, we identified that formation of CS‐induced p62 oligomers required an interaction with Keap1. Overexpression and ablation of BICD1 confirmed that increased BICD1 negatively regulates autophagosome maturation inducing accumulation of p62 and p62 oligomers and that it can be reversed by cardiac glycosides. We conclude that defective autophagosome maturation in COPD is caused by oxidative stress‐mediated BICD1 accumulation. [ABSTRACT FROM AUTHOR]

In this study, we report an investigation of a panel of clonally‐derived Chinese hamster ovary (CHO) cell lines exhibiting variability in the proportion of full‐length IgG4 Fc‐fusion protein produced. The recombinant protein was found to be degraded during cell culture into four shorter "clipped" species (three of the four cleavage sites occurred at arginine residues) and preliminary analyses suggested that a host cell enzyme was responsible for proteolysis. To identify the specific enzyme responsible, RNA sequencing was used to identify gene expression differences between the cell lines with a "high" and "low" clipping phenotype. From this analysis, six protease‐encoding genes were found to be significantly upregulated in those cell lines yielding the lowest proportion of full‐length IgG4 Fc‐fusion protein. Four of these protease candidates were deprioritized after examination of their cleavage site specificity. The remaining enzymes, Adam19 and Furin, were found to be capable of cleavage at arginine residues, and inhibitors for both proteases were added to cell‐free media to determine if the product degradation could be reduced. While the Adam19 inhibitor had no impact, Furin inhibitor I (specific for the proprotein convertase family of enzymes) was found to result in a 33–39% increase in complete IgG4 Fc‐fusion protein when compared with untreated samples. [ABSTRACT FROM AUTHOR]

Early detection, clinical management and disease recurrence monitoring are critical areas in cancer treatment in which specific biomarker panels are likely to be very important in each of these key areas. We have previously demonstrated that levels of alpha-2-heremans-schmid-glycoprotein (AHSG), complement component C3 (C3), clusterin (CLI), haptoglobin (HP) and serum amyloid A (SAA) are significantly altered in serum from patients with squamous cell carcinoma of the lung. Here, we report the abundance levels for these proteins in serum samples from patients with advanced breast cancer, colorectal cancer (CRC) and lung cancer compared to healthy controls (age and gender matched) using commercially available enzyme-linked immunosorbent assay kits. Logistic regression (LR) models were fitted to the resulting data, and the classification ability of the proteins was evaluated using receiver-operating characteristic curve and leave-one-out cross-validation (LOOCV). The most accurate individual candidate biomarkers were C3 for breast cancer [area under the curve (AUC) = 0.89, LOOCV = 73%], CLI for CRC (AUC = 0.98, LOOCV = 90%), HP for small cell lung carcinoma (AUC = 0.97, LOOCV = 88%), C3 for lung adenocarcinoma (AUC = 0.94, LOOCV = 89%) and HP for squamous cell carcinoma of the lung (AUC = 0.94, LOOCV = 87%). The best dual combination of biomarkers using LR analysis were found to be AHSG + C3 (AUC = 0.91, LOOCV = 83%) for breast cancer, CLI + HP (AUC = 0.98, LOOCV = 92%) for CRC, C3 + SAA (AUC = 0.97, LOOCV = 91%) for small cell lung carcinoma and HP + SAA for both adenocarcinoma (AUC = 0.98, LOOCV = 96%) and squamous cell carcinoma of the lung (AUC = 0.98, LOOCV = 84%). The high AUC values reported here indicated that these candidate biomarkers have the potential to discriminate accurately between control and cancer groups both individually and in combination with other proteins. [ABSTRACT FROM AUTHOR]

Recently released sequence information on Chinese hamster ovary (CHO) cells promises to not only facilitate our understanding of these industrially important cell factories through direct analysis of the sequence, but also to enhance existing methodologies and allow new tools to be developed. In this article we demonstrate the utilization of CHO specific sequence information to improve mass spectrometry (MS) based proteomic identification. The use of various CHO specific databases enabled the identification of 282 additional proteins, thus increasing the total number of identified proteins by 40-50%, depending on the sample source and methods used. In addition, a considerable portion of those proteins that were identified previously based on inter-species sequence homology were now identified by a larger number of peptides matched, thus increasing the confidence of identification. The new sequence information offers improved interpretation of proteomic analyses and will, in the years to come, prove vital to unraveling the CHO proteome. Biotechnol. Bioeng. 2012; 109:1386-1394. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

In 1992, the police service of England and Wales adopted PEACE as a framework for interviewing suspects. This study was to evaluate the impact of PEACE interview training, workplace supervision and the presence of a legal advisor on the performance of police officers' interviewing suspects. One hundred seventy-four real-life interviews with suspects were obtained from six police forces in England and Wales. Officers trained and untrained in PEACE, and from police forces that did or did not have an interview supervision policy conducted the interviews. Interviews in this sample were generally of average standard. Whilst PEACE-trained officers conducted longer interviews, there were no other statistical differences dependent on training. Nor were there any statistical differences dependent on the presence of a legal advisor. A workplace supervision policy was found to be related to performance, particularly during the engage and explain phases of the interview. Supervision also appeared to provide some safeguards for suspects who did not have a legal advisor. Because the performance of PEACE-trained officers did not differ from those who had not received this training, further improvement in training is needed, especially regarding a number of communication skills. It is argued that such improvement would be facilitated by a more consistent approach to interviewer training and to the effective supervision of interviews. Copyright © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

This article investigates the importance of place-of-origin communities to an understanding of the impact of migrant remittances on local development, employing the concept of social capital. Two aspects of social capital (each present prior to migration) are shown to be of significance–family relationships and community organisation. Drawing on recent research in two peasant communities in Oaxaca, Mexico, social capital is used to examine the relevance of non-migrants in shaping the outcomes of migration in communities of origin. Evaluation of migrant remittances and home-town associations demonstrates the developmental significance of non-migrants and communities of origin. [ABSTRACT FROM AUTHOR]

Aims: Clinical assessment of dehydration in children is often inaccurate. We aimed to determine if a scoring system based on standardised clinical signs would reduce the variability between doctors' assessment of dehydration. Methods: A clinical scoring system was developed using seven physiological variables based on previously published research. Estimated percentage dehydration and severity scores were recorded for 100 children presenting to a Paediatric Emergency Department with symptoms of gastroenteritis and dehydration by three doctors of different seniority (resident medical officer, registrar and consultant). Agreement was measured using intra-class correlation coefficient (ICC) for percentage ratings and total clinical scores and kappa for individual characteristics. Results: Estimated percentage dehydration ranged from 0–9%, mean 2.96%, across the three groups. Total clinical scores from 0–10, mean 2.20. There was moderate agreement amongst clinicians for the percentage dehydration (ICC 0.40). The level of agreement on the clinical scoring system was identical (ICC 0.40). Consultants gave statistically lower scores than the other two groups (Consultant (Con) vs. Resident P = 0.001, Con vs. Registrar P = 0.013). There was a marked difference in agreement across characteristics comprising the scoring system, from kappa 0.02 for capillary refill time to 0.42 for neurological status. Conclusion: The clinical scoring system used did not reduce the variability of assessment of dehydration compared to doctors' conventional methods. In order to reduce variability improving education may be more important than production of a scoring system as experience appears to be a key determinant in the assessment of a potentially dehydrated child. [ABSTRACT FROM AUTHOR]

The involvement of gangs, guns and ganja (marijuana) in Jamaica has, since independence in 1962, largely been confined to the capital, Kingston, and more specifically to the downtown, impoverished sections of the city known locally as the ghetto. This paper examines the characteristics of the ghetto; the context that it provides for political patronage among Kingston's most marginalised citizens; the evolution of certain downtown constituencies into garrison communities; and the separation between politics and drug violence that has marked the last twenty years, as Colombian cocaine has displaced locally-produced ganja as the key drug to be consumed and traded. [ABSTRACT FROM AUTHOR]

Since the early 1980s, the introduction of International Monetary Fund-directed structural adjustment packages to stabilize the Jamaican economy has reduced the scope of the government, cut back its capacity to intervene in the housing market, opened the economy to foreign goods (but limited capital), and re-produced the colonial version of a non-dynamic, labour-surplus urban economy in Kingston. This paper traces the impact of structural adjustment on unemployment and class formation in Kingston, and the relationship of these issues to housing problems. Rented, poor-quality housing, underpinned by low socio-economic status and historically high rates of unemployment, has created an overt spatial concentration of poverty, located in West and East Kingston. Nevertheless, overall unemployment is currently lower than at independence in 1962, and virtually all housing indicators have recorded improvements over the same time period. These improvements have been due to a deceleration in the growth of Kingston's population since the mid-1960s; government commitment, despite structural adjustment, to improve the quality of collective consumption; and the determination of Kingston's citizens to build better homes for themselves, often aided by loans from local building societies and remittances from family members resident overseas. However, at least a quarter of Kingston's population remains both unemployed and concentrated into areas of poor quality housing. These circumstances in Kingston are compared with those in adjacent Latin American cites under structural adjustment. [ABSTRACT FROM AUTHOR]

Coexpression analysis is a powerful, widely used methodology for the investigation of underlying patterns in gene expression data. This 'guilt-by-association' approach aims to find groups of genes with closely correlated expression profiles. Observation of consistent correlations across phenotypically diverse samples indicates that these genes have a shared function. We have recently described the application of weighted gene coexpression network analysis (WGCNA) to a 295 sample production CHO cell line microarray dataset and elucidated groups of genes related to growth rate and cell-specific productivity (Qp). In this study, we present the CHO gene coexpression database (CGCDB), a web-based system, designed specifically for researchers in the CHO community to provide user-friendly access to these gene-gene coexpression patterns. In addition to correlation between genes, the direct correlations between probesets and either growth rate or Qp are provided. Results are presented to the user via an interactive network diagram and in a downloadable tabular format. It is hoped that this resource will allow researchers to prioritize cell line engineering and/or biomarker candidates to enhance CHO-based cell culture for the production of biotherapeutics. Availability: . Biotechnol. Bioeng. 2012; 109:1368-1370. © 2011 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The article provides information concerning on the African American community in Liverpool, England. The African American community in the city has its origin in the mid-nineteenth century, when local ship owners have started to hire West African seamen, many of whom settled in the city. This ethnography examines the uses of African Americans in the context of racism, nationalism, and localism in the city.