Diagnosis and management of tuberculosis infection in inclusion health populations in London.

BACKGROUND: Tuberculosis in the UK is more prevalent in people with social risk factors- e.g. previous incarceration, homelessness - and in migrants from TB endemic countries. The management of TB infection is part of TB elimination strategies, but is challenging to provide to socially excluded groups and the evidence base for effective interventions is small. METHODS: We evaluated a TB infection screening and treatment programme provided by a peer-led service (Find&Treat) working in inclusion health settings (e.g. homeless hostels) in London. IGRA (interferon-gamma release assay) testing and TB infection treatment were offered to eligible adults using a community-based model. The primary outcome was successful progression through the cascade of care. We also evaluated socio-demographic characteristics associated with a positive IGRA. RESULTS: 42/312 (13.5%) participants had a positive IGRA and no one had evidence of active TB. 35/42 completed a medical evaluation; 22 started treatment, and 17 completed treatment. Having a positive IGRA was associated with previous incarceration and being born outside of the UK. DISCUSSION: Provision of TB infection diagnosis and management to this socially excluded population has several challenges including maintaining people in care and drug-drug interactions. Peer-support workers provided this service safely and effectively with appropriate support. Further work to generate data to inform risks and benefits of treatment for TB infection in this group is needed to facilitate joint decision making.

Detection of chromosomal alteration after infusion of gene-edited allogeneic CAR T cells.

A chromosome 14 inversion was found in a patient who developed bone marrow aplasia following treatment with allogeneic chimeric antigen receptor (CAR) Tcells containing gene edits made with transcription activator-like effector nucleases (TALEN). TALEN editing sites were not involved at either breakpoint. Recombination signal sequences (RSSs) were found suggesting recombination-activating gene (RAG)-mediated activity. The inversion represented a dominant clone detected in the context of decreasing absolute CAR Tcell and overall lymphocyte counts. The inversion was not associated with clinical consequences and wasnot detected in the drug product administered to this patient or in any drug product used in this or other trials using the same manufacturing processes. Neither was the inversion detected in this patient at earlier time points or in any other patient enrolled in this or other trials treated with this or other product lots. This case illustrates that spontaneous, possibly RAG-mediated, recombination events unrelated to gene editing can occur in adoptive cell therapy studies, emphasizes the need for ruling out off-target gene editing sites, and illustrates that other processes, such as spontaneous V(D)J recombination, can lead to chromosomal alterations in infused cells independent of gene editing.

Fabrication and characterization of machined multi-core fiber tweezers for single cell manipulation: erratum.

We have found an error in our work reported in [Opt. Express26(3), 3557 (2018)], which we correct in this erratum. We used incorrect data for the experimentally measured values of power of the fibre trap and power of the conventional optical tweezers (OT) used to 'break' the fibre trap. Using the correct data, Ffibre and Q (force and quality) of the multicore fibre tweezer are re-calculated. In this erratum, we communicate the correct values of Ffibre and Q and publish a revised Fig. 7 that contains results based on the correct data. Based on the revised result, two statements, in the abstract and conclusions, are also revised. The fabrication method, technique and general conclusion remain unaffected.

Fabrication and characterization of machined multi-core fiber tweezers for single cell manipulation.

Optical tweezing is a non-invasive technique that can enable a variety of single cell experiments; however, it tends to be based on a high numerical aperture (NA) microscope objective to both deliver the tweezing laser light and image the sample. This introduces restrictions in system flexibility when both trapping and imaging. Here, we demonstrate a novel, high NA tweezing system based on micro-machined multicore optical fibers. Using the machined, multicore fiber tweezer, cells are optically manipulated under a variety of microscopes, without requiring a high NA objective lens. The maximum NA of the fiber-based tweezer demonstrated is 1.039. A stable trap with a maximum total power 30 mW has been characterized to exert a maximum optical force of 26.4 pN, on a trapped, 7 µm diameter yeast cell. Single cells are held 15-35 µm from the fiber end and can be manipulated in the x, y and z directions throughout the sample. In this way, single cells are controllably trapped under a Raman microscope to categorize the yeast cells as live or dead, demonstrating trapping by the machined multicore fiber-based tweezer decoupled from the imaging or excitation objective lens.

Tablet and phone applications--A reflection on the experience of development.

Tablet devices are now ubiquitous. Medical illustrators have the skills to produce a wide range of media content. These devices offer the potential of using their creative abilities in new and exciting ways. There is much to explore. The primary difficulty lies in understanding the necessary computer technical skills to realise a vision.

Abundance, distribution, and dynamics of Anopheles species (Diptera: Culicidae) in Iowa, United States.

Malaria was once endemic in the United States prior to its elimination in 1951. However, due to consistent introductions of travel-associated malaria cases and the presence of several native Anopheles species (Diptera: Culicidae) that are competent vectors of malaria, the potential for local (autochthonous) malaria transmission remains a persistent threat in the United States. While several intermittent cases of local malaria transmission have occurred in the United States in the decades since elimination, the emergence of autochthonous transmission in 4 states in 2023 demonstrates the continued risk for future outbreaks. Moreover, these recent examples also highlight significant gaps in current mosquito surveillance efforts that have predominantly focused on threats of arboviral disease, such that our understanding of Anopheles distributions relies only on historical records and offers limited insight into the ecological factors that influence their abundance. Herein, we summarize mosquito surveillance data collected over the last 20 years (2004-2023) across 59 Iowa counties to provide essential information into the spatial distribution, temporal abundance, and trap preferences of Anopheles species in the state. Further analyses of the 2 most abundant species, Anopheles punctipennis Say and Anopheles quadrimaculatus Say, reveal the additional influence of precipitation and forested habitats in defining An. punctipennis abundance. Together, we believe these results provide an increased understanding of previously neglected Anopheles species that have the potential for autochthonous malaria transmission in Iowa and that can be extended to other regions of the United States to enhance preparedness for future malaria outbreaks.

Hepatitis B virus (HBV) screening, linkage and retention-in-care in inclusion health populations: Evaluation of an outreach screening programme in London.

OBJECTIVES: We evaluated a hepatitis B virus (HBV) screening programme, delivered by a specialist pan-London multidisciplinary outreach team, to understand population characteristics and care cascade among people who experience extreme social exclusion (Inclusion Health (IH) groups). METHODS: Point-of-care HBV screening was performed in temporary accommodation for people experiencing homelessness (PEH) and people seeking asylum (initial accommodation centres, IACs) via a mobile unit staffed by peers with lived experience, nurses, and doctors. We analysed demographics and HBV characteristics of adults screened between May 2020 and January 2022. We ascertained linkage-to-care (LTC), retention-in-care (RIC) and loss-to-follow-up (LTFU). People LTFU were contacted by peers to re-engage in care. RESULTS: 2473 people were screened: 809 in IACs, 1664 in other temporary accommodation. Overall hepatitis B surface antigen (HBsAg) prevalence was 1.7% (43/2473), highest in IACs (3.5%, 28/809). LTC within 3 months was 56% (24/43) and RIC, 87% (26/30). LTC was higher when referred to a local IH-specialist hepatitis service, compared to other services (77%, 17/22 vs 33%, 7/21; p = 0.006). LTFU was 30% (13/43), reduced to 21% (9/43) after intervention by peers. CONCLUSION: Our findings support outreach screening among IH populations and peer-supported linkage to IH-specialist hepatitis services. We recommend increased HBV testing and HBV-specific IH specialist services.

Integrated miRNA, mRNA and protein expression analysis reveals the role of post-transcriptional regulation in controlling CHO cell growth rate.

BACKGROUND: To study the role of microRNA (miRNA) in the regulation of Chinese hamster ovary (CHO) cell growth, qPCR, microarray and quantitative LC-MS/MS analysis were utilised for simultaneous expression profiling of miRNA, mRNA and protein. The sample set under investigation consisted of clones with variable cellular growth rates derived from the same population. In addition to providing a systems level perspective on cell growth, the integration of multiple profiling datasets can facilitate the identification of non-seed miRNA targets, complement computational prediction tools and reduce false positive and false negative rates. RESULTS: 51 miRNAs were associated with increased growth rate (35 miRNAs upregulated and 16 miRNAs downregulated). Gene ontology (GO) analysis of genes (n=432) and proteins (n=285) found to be differentially expressed (DE) identified biological processes driving proliferation including mRNA processing and translation. To investigate the influence of miRNA on these processes we combined the proteomic and transcriptomic data into two groups. The first set contained candidates where evidence of translational repression was observed (n=158). The second group was a mixture of proteins and mRNAs where evidence of translational repression was less clear (n=515). The TargetScan algorithm was utilised to predict potential targets within these two groups for anti-correlated DE miRNAs. CONCLUSIONS: The evidence presented in this study indicates that biological processes such as mRNA processing and protein synthesis are correlated with growth rate in CHO cells. Through the integration of expression data from multiple levels of the biological system a number of proteins central to these processes including several hnRNPs and components of the ribosome were found to be post-transcriptionally regulated. We utilised the expression data in conjunction with in-silico tools to identify potential miRNA-mediated regulation of mRNA/proteins involved in CHO cell growth rate. These data have allowed us to prioritise candidates for cell engineering and/or biomarkers relevant to industrial cell culture. We also expect the knowledge gained from this study to be applicable to other fields investigating the role of miRNAs in mammalian cell growth.

CGCDB: a web-based resource for the investigation of gene coexpression in CHO cell culture.

UNLABELLED: 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: www.cgcdb.org.

Sustained productivity in recombinant Chinese hamster ovary (CHO) cell lines: proteome analysis of the molecular basis for a process-related phenotype.

BACKGROUND: The ability of mammalian cell lines to sustain cell specific productivity (Qp) over the full duration of bioprocess culture is a highly desirable phenotype, but the molecular basis for sustainable productivity has not been previously investigated in detail. In order to identify proteins that may be associated with a sustained productivity phenotype, we have conducted a proteomic profiling analysis of two matched pairs of monoclonal antibody-producing Chinese hamster ovary (CHO) cell lines that differ in their ability to sustain productivity over a 10 day fed-batch culture. RESULTS: Proteomic profiling of inherent differences between the two sets of comparators using 2D-DIGE (Difference Gel Electrophoresis) and LC-MS/MS resulted in the identification of 89 distinct differentially expressed proteins. Overlap comparisons between the two sets of cell line pairs identified 12 proteins (AKRIB8, ANXA1, ANXA4, EIF3I, G6PD, HSPA8, HSP90B1, HSPD1, NUDC, PGAM1, RUVBL1 and CNN3) that were differentially expressed in the same direction. CONCLUSION: These proteins may have an important role in sustaining high productivity of recombinant protein over the duration of a fed-batch bioprocess culture. It is possible that many of these proteins could be useful for future approaches to successfully manipulate or engineer CHO cells in order to sustain productivity of recombinant protein.

Do 18-month-olds really attribute mental states to others? A critical test.

In the research reported here, we investigated whether 18-month-olds would use their own past experience of visual access to attribute perception and consequent beliefs to other people. Infants in this study wore either opaque blindfolds (opaque condition) or trick blindfolds that looked opaque but were actually transparent (trick condition). Then both groups of infants observed an actor wearing one of the same blindfolds that they themselves had experienced, while a puppet removed an object from its location. Anticipatory eye movements revealed that infants who had experienced opaque blindfolds expected the actor to behave in accordance with a false belief about the object's location, but that infants who had experienced trick blindfolds did not exhibit that expectation. Our results suggest that 18-month-olds used self-experience with the blindfolds to assess the actor's visual access and to update her belief state accordingly. These data constitute compelling evidence that 18-month-olds infer perceptual access and appreciate its causal role in altering the epistemic states of other people.

Development and characterization of a Chinese hamster ovary cell-specific oligonucleotide microarray.

The Chinese hamster ovary (CHO) cell line is one of the most widely used mammalian cell lines for biopharmaceutical production. We have developed and characterized a gene expression microarray (WyeHamster2a) specific for CHO cells that has enabled the study of ~3,500 sequences. Analysis of multiple sets of replicate scans showed that data derived from the WyeHamster2a array is highly reproducible confirming it as a robust tool for profiling. Twelve gene sequences were selected for follow-up RT-qPCR to confirm the accuracy and precision of the microarray results. In all but the most subtle gene expression differences, the microarray proved to be a reliable measure of differential gene expression. Finally, we were able to quantify the difference between using a bona fide CHO-specific microarray for profiling CHO cells versus an alternate, commercially available, rodent microarray such as a mouse or rat-specific format.

Mosquito Guttersnipe: A New Sampling Tool for Roof Gutters, Tree Holes, and Other Elevated Mosquito Habitats.

Mosquitoes pose health risks to human populations by serving as vectors of diseases. Mosquito control organizations are responsible for inspecting and controlling vector populations to reduce the risk of infection of these diseases. Current sampling methods are effective for numerous types of mosquito habitat, but not conducive for sampling small overhead habitat such as roof gutters or tree holes. We have developed and tested a tool called the Mosquito GutterSnipe to sample these overhead habitats. Volumetric and larval capacity testing of the tool prototype demonstrated comparable sampling integrity to standard mosquito dipping methods. The GutterSnipe can be employed as a reliable way to sample previously overlooked mosquito habitat. Its current model is cost effective and easy to produce for mosquito control organizations and easy to use for inspectors.

Practising critical resilience as an advanced peer support worker in London: A qualitative evaluation of a peer-led hepatitis C intervention amongst people experiencing homelessness who inject drugs.

BACKGROUND: Peer support has been used as a mechanism to facilitate active engagement with healthcare amongst underserved populations. The HepCare project upskilled experienced peer support workers (PSWs) to become equal members of a service provider team, taking on advanced clinical roles normally carried out by medical or nursing specialists. METHOD: A participatory case study approach was taken to the study following the methodological guidance of Merriam (1998). The subject of the case in our study is the advanced peer support workers (APSWs) functioning in the HepCare project as service providers. The object of the case is an exploration of their transition to service provider in the HCV screening and treatment support service. Five peer led in-depth interviews with APSWs were supplemented by a survey of health professionals, interviews with service users, documentary evidence in the form of job descriptions, observational notes and a blog from the field. Thematic analysis of the data was conducted, refined and finalised in a workshop with the research team and APSW participants. RESULTS: Three themes were generated from the data that explore the peer support worker's transition to APSW in the programme: Transition to Integration, Retaining 'Peerness', and Practising Critical Resilience. The advocacy and support enacted by the APSWs through the HepCare project, offer purpose and meaning alongside integration into a new social group. This is buffered by the supportive context of the programme and facilitates a motivating sense of worth. CONCLUSION: The programme offers policy guidance for the structured career development of APSWs and a platform for enactment of critical resilience as they transition to their advanced role, in the healthcare provider team.

Microarray and proteomics expression profiling identifies several candidates, including the valosin-containing protein (VCP), involved in regulating high cellular growth rate in production CHO cell lines.

A high rate of cell growth (micro) leading to rapid accumulation of viable biomass is a desirable phenotype during scale up operations and the early stages of production cultures. In order to identify genes and proteins that contribute to higher growth rates in Chinese hamster ovary (CHO) cells, a combined approach using microarray and proteomic expression profiling analysis was carried out on two matched pairs of CHO production cell lines that displayed either fast or slow growth rates. Statistical analysis of the microarray and proteomic data separately resulted in the identification of 118 gene transcripts and 58 proteins that were differentially expressed between the fast- and slow-growing cells. Overlap comparison of both datasets identified a priority list of 21 candidates associated with a high growth rate phenotype in CHO. Functional analysis (by siRNA) of five of these candidates identified the valosin-containing protein (VCP) as having a substantial impact on CHO cell growth and viability. Knockdown of HSPB1 and ENO1 also had an effect on cell growth (negative and positive, respectively). Further functional validation in CHO using both gene knockdown (siRNA) and overexpression (cDNA) confirmed that altered VCP expression impacted CHO cell proliferation, indicating that VCP and other genes and proteins identified here may play an important role in the regulation of CHO cell growth during log phase culture and are potential candidates for CHO cell line engineering strategies.

The oxytocin-prostaglandins pathways in the horse (Equus caballus) placenta during pregnancy, physiological parturition, and parturition with fetal membrane retention.

Despite their importance in mammalian reproduction, substances in the oxytocin-prostaglandins pathways have not been investigated in the horse placenta during most of pregnancy and parturition. Therefore, we quantified placental content of oxytocin (OXT), oxytocin receptor (OXTR), and prostaglandin E2 and F2 alpha during days 90-240 of pregnancy (PREG), physiological parturition (PHYS), and parturition with fetal membrane retention (FMR) in heavy draft horses (PREG = 13, PHYS = 11, FMR = 10). We also quantified OXTR and prostaglandin endoperoxide synthase-2 (PTGS2) mRNA expression and determined the immunolocalization of OXT, OXTR, and PTGS2. For relative quantification of OXT and OXTR, we used western blotting with densitometry. To quantify the prostaglandins, we used enzyme immunoassays. For relative quantification of OXTR and PTGS2, we used RT-qPCR. For immunolocalization of OXT, OXTR, and PTGS2, we used immunohistochemistry. We found that OXT was present in cells of the allantochorion and endometrium in all groups. PTGS2 expression in the allantochorion was 14.7-fold lower in FMR than in PHYS (p = 0.007). These results suggest that OXT is synthesized in the horse placenta. As PTGS2 synthesis is induced by inflammation, they also suggest that FMR in heavy draft horses may be associated with dysregulation of inflammatory processes.

Proteomic profiling of CHO cells with enhanced rhBMP-2 productivity following co-expression of PACEsol.

Chinese hamster ovary (CHO) cells are widely used for the production of recombinant protein biopharmaceuticals. The purpose of this study was to investigate differences in the proteome of CHO DUKX cells expressing recombinant human bone morphogenetic protein-2 (rhBMP-2) (G5 cells) compared to cells also expressing soluble exogenous paired basic amino acid cleaving enzyme soluble paired basic amino acid cleaving enzyme (PACEsol) (3C9 cells), which has been previously found to improve the post-translational processing of the mature rhBMP-2 dimer. PACEsol co-expression was also associated with a significant increase (almost four-fold) in cellular productivity of rhBMP-2 protein. Differential proteomic expression profiling using 2-D DIGE and MALDI-TOF MS was performed to compare 3C9 and G5 cells, and revealed a list of 60 proteins that showed differential expression (up/downregulated), with a variety of different cellular functions. A substantial number of these altered proteins were found to have chaperone activity, involved with protein folding, assembly and secretion, as well as a number of proteins involved in protein translation. These results support the use of proteomic profiling as a valuable tool towards understanding the biology of bioprocess cultures.

Transcriptional profiling of gene expression changes in a PACE-transfected CHO DUKX cell line secreting high levels of rhBMP-2.

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry for the production of recombinant human proteins including complex polypeptides such as recombinant human bone morphogenic protein 2 (rhBMP-2). Large-scale manufacture of rhBMP-2 has associated production difficulties resulting from incomplete processing of the recombinant human protein due to insufficient endogenous levels of the paired basic amino acid cleaving enzyme (PACE) in CHO. In order to resolve this issue, CHO DUKX cells expressing rhBMP-2 were transfected with the soluble version of human PACE (PACEsol) resulting in improved amino-terminal homogeneity and a fourfold increase in rhBMP-2 productivity. In this article, we present a microarray expression profile analysis comparing the parental lineage to the higher producing subclone co-expressing PACEsol using a proprietary CHO-specific microarray. Using this technology we observed 1,076 significantly different genes in the high-productivity cells co-expressing PACEsol. Following further analysis of the differentially expressed genes, the Unfolded Protein Response (UPR) component of the endoplasmic reticulum stress response pathway was identified as a key candidate for effecting increased productivity in this cell system. Several additional ER- and Golgi-localised proteins were identified which may also contribute to this effect. The results presented here support the use of large-scale microarray expression profiling as a viable and valuable route towards understanding the behaviour of bioprocess cultures in vitro.

The metabolic syndrome--what is it and how should it be managed?

Following a hypertension symposium in Portland, ME, in October 2005, a roundtable was convened to discuss the metabolic syndrome and its significance. Dr. Marvin Moser of the Yale University School of Medicine, New Haven, CT, moderated the discussion. Participating in the discussion were Dr. Bonita Falkner of the Jefferson Medical College of Thomas Jefferson University, Philadelphia, PA; Dr. Michael A. Weber of SUNY Downstate College of Medicine, New York, NY; and Dr. Leonard Mark Keilson of the University of Vermont College of Medicine.