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This paper describes the implementation of curricula for Liberia's first-ever psychiatry training programme in 2019 and the actions of the only two Liberian psychiatrists in the country at the time in developing and executing a first-year postgraduate psychiatry training programme (i.e. residency) with support from international collaborators. It explores cultural differences in training models among collaborators and strategies to synergise them best. It highlights the assessment of trainees’ (residents’) basic knowledge on entry into the programme and how it guided immediate and short-term priority teaching objectives, including integrated training in neuroscience and neurology. The paper describes the strengths and challenges of this approach as well as opportunities for continued growth.

Background: Malignancy is a long-term complication of lung transplantation (LTx); however, contemporary Australian data and detailed evaluation of nonreportable cancers are lacking. Methods: Retrospective review of LTx recipients’ medical records and registry data linkage were performed to identify histologically proven malignancies. Baseline clinico-demographic variables were collected, and cancer incidence was compared with reported data for the general Australian population. Results: There were 1,715 LTx in 1,631 patients between 1989 and 2021, with a follow-up of 9,696 person-years. Eight hundred and ninety-three (54.8%) patients were male, and the median age at first LTx was 54.7 years. There were 886 deaths with a median overall survival of 7.5 years (95% confidence intervals (CI) 6.8-8.3 years). One thousand seven hundred and seventy-four separate invasive cancer events occurred across 407 patients, of which, 1,588 (89.5%) were nonmelanoma skin cancers (NMSCs). This translated to a 9-fold increased incidence of NMSCs and a 4-fold increased incidence of other cancers compared with the general population. Cancer mortality reached parity with chronic lung allograft dysfunction 10 years postfirst transplant and was independently associated with age (hazard ratios (HR) per year increase in age 1.02 [95% CI 1.01-1.03], p = 0.001), Epstein-Barr virus primary mismatch (HR 3.24 [95% CI 1.68-6.25], vs nonmismatch, p = 0.002), and cancer count (HR per cancer event 1.19 [95% CI 1.13-1.24], p

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal disease caused by iduronate-2-sulfatase (IDS) deficiency, leading to accumulation of glycosaminoglycans (GAGs) and the emergence of progressive disease. Enzyme replacement therapy is the only currently approved treatment, but it leaves neurological disease unaddressed. Cerebrospinal fluid (CSF)-directed administration of AAV9.CB7.hIDS (RGX-121) is an alternative treatment strategy, but it is unknown if this approach will affect both neurologic and systemic manifestations. We compared the effectiveness of intrathecal (i.t.) and intravenous (i.v.) routes of administration (ROAs) at a range of vector doses in a mouse model of MPS II. While lower doses were completely ineffective, a total dose of 1 × 109 gc resulted in appreciable IDS activity levels in plasma but not tissues. Total doses of 1 × 1010 and 1 × 1011 gc by either ROA resulted in supraphysiological plasma IDS activity, substantial IDS activity levels and GAG reduction in nearly all tissues, and normalized zygomatic arch diameter. In the brain, a dose of 1 × 1011 gc i.t. achieved the highest IDS activity levels and the greatest reduction in GAG content, and it prevented neurocognitive deficiency. We conclude that a dose of 1 × 1010 gc normalized metabolic and skeletal outcomes, while neurologic improvement required a dose of 1 × 1011 gc, thereby suggesting the prospect of a similar direct benefit in humans.

DNA methylation is an epigenetic factor that is modifiable and can change over a lifespan. While many studies have identified methylation sites (CpGs) related to aging, the relationship of these to gene function and age-related disease phenotypes remains unclear. This research explores this question by testing for the conjoint association of age-related CpGs with gene expression and the relation of these to body fat phenotypes. The study included blood-based gene transcripts and intragenic CpG methylation data from Illumina 450 K arrays in 74 healthy adults from the Norfolk Island population. First, a series of regression analyses were performed to detect associations between gene transcript level and intragenic CpGs and their conjoint relationship with age. Second, we explored how these age-related expression CpGs (eCpGs) correlated with obesity-related phenotypes, including body fat percentage, body mass index, and waist-to-hip ratio. We identified 35 age-related eCpGs associated with age. Of these, ten eCpGs were associated with at least one body fat phenotype. Collagen Type XI Alpha 2 Chain (COL11A2), Complement C1s (C1s), and four and a half LIM domains 2 (FHL2) genes were among the most significant genes with multiple eCpGs associated with both age and multiple body fat phenotypes. The COL11A2 gene contributes to the correct assembly of the extracellular matrix in maintaining the healthy structural arrangement of various components, with the C1s gene part of complement systems functioning in inflammation. Moreover, FHL2 expression was upregulated under hypermethylation in both blood and adipose tissue with aging. These results suggest new targets for future studies and require further validation to confirm the specific function of these genes on body fat regulation.

Abstract Previous studies have documented natural infections of SARS-CoV-2 in various domestic and wild animals. More recently, studies have been published noting the susceptibility of members of the Cervidae family, and infections in both wild and captive cervid populations. In this study, we investigated the presence of SARS-CoV-2 in mammalian wildlife within the state of Vermont. 739 nasal or throat samples were collected from wildlife throughout the state during the 2021 and 2022 harvest season. Data was collected from red and gray foxes (Vulpes vulples and Urocyon cineroargentus, respectively), fishers (Martes pennati), river otters (Lutra canadensis), coyotes (Canis lantrans), bobcats (Lynx rufus rufus), black bears (Ursus americanus), and white-tailed deer (Odocoileus virginianus). Samples were tested for the presence of SARS-CoV-2 via quantitative RT-qPCR using the CDC N1/N2 primer set and/or the WHO-E gene primer set. Surprisingly, we initially detected a number of N1 and/or N2 positive samples with high cycle threshold values, though after conducting environmental swabbing of the laboratory and verifying with a second independent primer set (WHO-E) and PCR without reverse transcriptase, we showed that these were false positives due to plasmid contamination from a construct expressing the N gene in the general laboratory environment. Our final results indicate that no sampled wildlife were positive for SARS-CoV-2 RNA, and highlight the importance of physically separate locations for the processing of samples for surveillance and experiments that require the use of plasmid DNA containing the target RNA sequence. These negative findings are surprising, given that most published North America studies have found SARS-CoV-2 within their deer populations. The absence of SARS-CoV-2 RNA in populations sampled here may provide insights in to the various environmental and anthropogenic factors that reduce spillover and spread in North American’s wildlife populations.

The ability to differentiate species is not adequate for modern forage breeding programs. The measurement of persistence is currently a bottleneck in the breeding system that limits the throughput of cultivars to the marketplace and prevents it from being selected as a trait. The use of hyperspectral data obtained through remote sensing offers the potential to reduce guesswork by identifying the distribution of pasture species, but only if such data alone can distinguish the subtle differences within species, i.e., cultivars. The implementation of this technology faces many challenges due to the spectral and temporal variability of species. To understand the spectral variability between and within species groups, differentiation using hyperspectral data from monoculture plots of turf species was utilized. Spectral data were collected over a year using an ASD FieldSpec® and canopy pasture probe (CAPP). The plots consisted of monocultures of various species, and cultivars (a total of 10 plots). Linear discriminant analysis (LDA) was conducted on the full spectrum and reduced band data. This technique successfully differentiated the species with high accuracy (>98%). We demonstrate the potential of hyperspectral data and analysis techniques to accurately separate differences down to cultivar level. We also show that diurnal variation is measurable in the spectra but does not preclude differentiation.

Abstract Background Immune checkpoint inhibitors (ICI) are associated with immune-mediated adverse effects, potentially involving any organ. ICI has also been associated with an increased risk of cardiovascular disease in cancer populations. Objective To characterize the incidence and risk of major atherosclerotic cardiovascular events associated with ICI use in a high-risk and advanced melanoma population. Methods We conducted a retrospective cohort study of patients with high-risk or advanced melanoma (AJCC stage II, III or IV) presenting to an academic tertiary hospital between 2015–2020. The main outcome was major atherosclerotic cardiovascular events (MACE) including acute myocardial infarction, ischemic stroke, acute limb ischemia and coronary revascularization. Results The study cohort consisted of 646 patients, including 289 who had been treated with ICI. The incidence of MACE was higher in the ICI treated group (3.6 vs. 0.9 events per 100-person years). After adjusting for age, sex, smoking history and prior BRAF and/or MEK inhibitor use, ICI treatment was associated with an increased risk of MACE (HRadj 2.8, 95% CI 1.1–6.9, p = 0.03). Elevated risk was especially pronounced in patients with a past history of MACE (HR 14.4, 95% CI 1.9–112.3, p = 0.01). Conclusion Patients with high-risk or advanced melanoma are at an increased risk of atherosclerotic cardiovascular events following ICI treatment, particularly those with a history of cardiovascular disease.

The regulation of the distinct intrinsic phenotypic states in melanoma remain poorly characterised. Here, multi-omics analysis for a panel of 68 early passage melanoma cell lines reveals that cancer cell intrinsic transcriptomic programs are associated with distinct immune features.

Hunter syndrome is a rare x-linked recessive genetic disorder that affects lysosomal metabolism due to deficiency of iduronate-2-sulfatase (IDS), with subsequent accumulation of glycosaminoglycans heparan and dermatan sulfates (GAG). Enzyme replacement therapy is the only FDA-approved remedy and is an expensive life-time treatment that alleviates some symptoms of the disease without neurocognitive benefit. We previously reported successful treatment in a mouse model of mucopolysaccharidosis type II (MPS II) using adeno-associated viral vector serotype 9 encoding human IDS (AAV9.hIDS) via intracerebroventricular injection. As a less invasive and more straightforward procedure, here we report intravenously administered AAV9.hIDS in a mouse model of MPS II. In animals administered 1.5 × 1012 vg of AAV9.hIDS at 2 months of age, we observed supraphysiological levels of IDS enzyme activity in the circulation (up to 9100-fold higher than wild-type), in the tested peripheral organs (up to 560-fold higher than wild-type), but only 4% to 50% of wild type levels in the CNS. GAG levels were normalized on both sides of the blood-brain-barrier (BBB) in most of tissues tested. Despite low levels of the IDS observed in the CNS, this treatment prevented neurocognitive decline as shown by testing in the Barnes maze and by fear conditioning. This study demonstrates that a single dose of IV-administered AAV9.hIDS may be an effective and non-invasive procedure to treat MPS II that benefits both sides of the BBB, with implications for potential use of IV-administered AAV9 for other neuronopathic lysosomal diseases.

Abstract Introduction Aerosol pollutants are known to raise the risk of development of non-communicable respiratory diseases (NCRDs) such as asthma, chronic bronchitis, chronic obstructive pulmonary disease, and allergic rhinitis. Sub-Saharan Africa’s rapid pace of urbanization, economic expansion, and population growth raise concerns of increasing incidence of NCRDs. This research characterizes the state of research on pollution and NCRDs in the 46 countries of Sub-Saharan Africa (SSA). This research systematically reviewed the literature on studies of asthma; chronic bronchitis; allergic rhinitis; and air pollutants such as particulate matter, ozone, NOx, and sulfuric oxide. Methods We searched three major databases (PubMed, Web of Science, and Scopus) using the key words “asthma”, “chronic bronchitis”, “allergic rhinitis”, and “COPD” with “carbon monoxide (CO)”, “sulfuric oxide (SO)”, “ozone (O3)”, “nitrogen dioxide (NO2)”, and “particulate matter (PM)”, restricting the search to the 46 countries that comprise SSA. Only papers published in scholarly journals with a defined health outcome in individuals and which tested associations with explicitly measured or modelled air exposures were considered for inclusion. All candidate papers were entered into a database for review. Results We found a total of 362 unique research papers in the initial search of the three databases. Among these, 14 met the inclusion criteria. These papers comprised studies from just five countries. Nine papers were from South Africa; two from Malawi; and one each from Ghana, Namibia, and Nigeria. Most studies were cross-sectional. Exposures to ambient air pollutants were measured using spectrometry and chromatography. Some studies created composite measures of air pollution using a range of data layers. NCRD outcomes were measured by self-reported health status and measures of lung function (spirometry). Populations of interest were primarily schoolchildren, though a few studies focused on secondary school students and adults. Conclusions The paucity of research on NCRDs and ambient air pollutant exposures is pronounced within the African continent. While capacity to measure air quality in SSA is high, studies targeting NCRDs should work to draw attention to questions of outdoor air pollution and health. As the climate changes and SSA economies expand and countries urbanize, these questions will become increasingly important.

Abstract Introduction Snakebites are a major cause of permanent injury and death among poor, rural populations in developing countries, including those in East Africa. This research characterizes snakebite incidence, risk factors, and subsequent health-seeking behaviors in two regions of Kenya using a mixed methods approach. Methods As a part of regular activities of a health demographic surveillance system, household-level survey on snakebite incidence was conducted in two areas of Kenya: Kwale along the Kenyan Coast and Mbita on Lake Victoria. If someone in the home was reported to have been bitten in the 5 years previous to the visit, a survey instrument was administered. The survey gathered contextual information on the bite, treatment-seeking behavior and clinical manifestations. To obtain deeper, contextual information, respondents were also asked to narrate the bite incident, subsequent behavior and outcomes. Results 8775 and 9206 households were surveyed in Kwale and Mbita, respectively. Out of these, 453 (5.17%) and 92 (1.00%) households reported that at least one person had been bitten by a snake in the past 5 years. Deaths from snakebites were rare (4.04%), but patterns of treatment seeking varied. Treatment at formal care facilities were sought for 50.8% and at traditional healers for 53.3%. 18.4% sought treatment from both sources. Victims who delayed receiving treatment from a formal facility were more likely to have consulted a traditional healer (OR 8.8995% CI [3.83, 20.64]). Delays in treatment seeking were associated with significantly increased odds of having a severe outcome, including death, paralysis or loss of consciousness (OR 3.47 95% CI [1.56; 7.70]). Conclusion Snakebite incidence and outcomes vary by region in Kenya, and treatment-seeking behaviors are complex. Work needs to be done to better characterize the spatial distribution of snakebite incidence in Kenya and efforts need to be made to ensure that victims have sufficient access to effective treatments to prevent death and serious injury.

Abstract Objectives Despite the success of immune checkpoint blockade, most metastatic melanoma patients fail to respond to therapy or experience severe toxicity. Assessment of biomarkers and immunophenotypes before or early into treatment will help to understand favourable responses and improve therapeutic outcomes. Methods We present a high‐dimensional approach for blood T‐cell profiling using three multi‐parameter cytometry panels: (1) a TruCount panel for absolute cell counts, (2) a 27‐colour spectral panel assessing T‐cell markers and (3) a 20‐colour spectral panel evaluating intracellular cytokine expression. Pre‐treatment blood mononuclear cells from patients and healthy controls were cryopreserved before staining across 11 batches. Batch effects were tracked using a single‐donor control and the suitability of normalisation was assessed. The data were analysed using manual gating and high‐dimensional strategies. Results Batch‐to‐batch variation was minimal, as demonstrated by the dimensionality reduction of batch‐control samples, and normalisation did not improve manual or high‐dimensional analysis. Application of the workflow demonstrated the capacity of the panels and showed that patients had fewer lymphocytes than controls (P = 0.0027), due to lower naive CD4+ (P = 0.015) and CD8+ (P = 0.011) T cells and follicular helper T cells (P = 0.00076). Patients showed trends for higher proportions of Ki67 and IL‐2‐expressing cells within CD4+ and CD8+ memory subsets, and increased CD57 and EOMES expression within TCRγδ+ T cells. Conclusion Our optimised high‐parameter spectral cytometry approach provided in‐depth profiling of blood T cells and found differences in patient immunophenotype at baseline. The robustness of our workflow, as demonstrated by minimal batch effects, makes this approach highly suitable for the longitudinal evaluation of immunotherapy effects.

Some crises, such as those brought on or exposed by the COVID-19 pandemic, are wicked problems—large, complex problems with no immediate answer. As such, they make rich centerpieces for learning with respect to public deliberation and issue-based dialogue. This essay reflects on an experimental, transdisciplinary health and science communication course entitled Comprehending COVID-19. The course represents a collaborative effort among 14 faculty representing 10 different academic departments to create a resource for teaching students how to deliberate the pandemic, despite its attending, oversaturated, fake-news-infused, infodemic. We offer transdisciplinary deliberation as a pedagogical framework to expand communication repertoires in ways useful for sifting through the messiness of an infodemic while also developing key deliberation skills for productively engaging participatory decision-making with concern to wicked problems.

The rampant misinformation amid the COVID-19 pandemic demonstrates an obvious need for persuasion. This article draws on the fields of digital rhetoric and rhetoric of science, technology, and medicine to explore the persuasive threats and opportunities machine communicators pose to public health. As a specific case, Alexa and the machine’s performative similarities to the Oracle at Delphi are tracked alongside the voice-based assistant’s further resonances with the discourses of expert systems to develop an account of the machine’s rhetorical energies. From here, machine communicators are discussed as optimal deliverers of inoculations against misinformation in light of the fact that their performances are attended by rhetorical energies that can enliven persuasions against misinformation.

Abstract Chronic kidney disease (CKD) is a persistent impairment of kidney function. Genome-wide association studies (GWAS) have revealed multiple genetic loci associated with CKD susceptibility but the complete genetic basis is not yet clear. Since CKD shares risk factors with cardiovascular diseases and diabetes, there may be pleiotropic loci at play but may go undetected when using single phenotype GWAS. Here, we used multi-phenotype GWAS in the Norfolk Island isolate (n = 380) to identify new loci associated with CKD. We performed a principal components analysis on different combinations of 29 quantitative traits to extract principal components (PCs) representative of multiple correlated phenotypes. GWAS of a PC derived from glomerular filtration rate, serum creatinine, and serum urea identified a suggestive peak (pmin = 1.67 × 10–7) that mapped to KCNIP4. Inclusion of other secondary CKD measurements with these three kidney function traits identified the KCNIP4 locus with GWAS significance (pmin = 1.59 × 10–9). Finally, we identified a group of two SNPs with increased minor allele frequencies as potential functional variants. With the use of genetic isolate and the PCA-based multi-phenotype GWAS approach, we have revealed a potential pleotropic effect locus for CKD. Further studies are required to assess functional relevance of this locus.

Immunotherapies now dominate the treatment landscape for melanoma, but why they only work in a subset of patients remains unclear. Here, the authors perform an immunogenomic analysis on 67 intratumor sub-regions of a PD-1 inhibitor resistant melanoma, and 2 additional metastases from a single patient, mapping the spatial relationships between genomic and immune heterogeneity at high resolution.

Identification of clinical relevant biomarkers to predict response to immune checkpoint blockade (ICB) in melanoma remains challenging. Here, the authors show that stroma remodelling and reduced cell division are associated with durable response to anti-PD1 in a mouse model of melanoma and in ICB-treated patients.

Abstract Background Allele-specific methylation (ASM) occurs when DNA methylation patterns exhibit asymmetry among alleles. ASM occurs at imprinted loci, but its presence elsewhere across the human genome is indicative of wider importance in terms of gene regulation and disease risk. Here, we studied ASM by focusing on blood-based DNA collected from 24 subjects comprising a 3-generation pedigree from the Norfolk Island genetic isolate. We applied a genome-wide bisulphite sequencing approach with a genotype-independent ASM calling method to map ASM across the genome. Regions of ASM were then tested for enrichment at gene regulatory regions using Genomic Association Test (GAT) tool. Results In total, we identified 1.12 M CpGs of which 147,170 (13%) exhibited ASM (P ≤ 0.05). When including contiguous ASM signal spanning ≥ 2 CpGs, this condensed to 12,761 ASM regions (AMRs). These AMRs tagged 79% of known imprinting regions and most (98.1%) co-localised with known single nucleotide variants. Notably, miRNA and lncRNA showed a 3.3- and 1.8-fold enrichment of AMRs, respectively (P

Targeted and immunotherapy regimens have revolutionized the treatment of advanced melanoma patients. Despite this, only a subset of patients respond durably. Recently, combination strategies of BRAF/MEK inhibitors with immune checkpoint inhibitor monotherapy (α-CTLA-4 or α-PD-1) have increased the rate of durable responses. Based on evidence from our group and others, these therapies appear synergistic, but at the cost of significant toxicity. We know from other treatment paradigms (e.g. hematologic malignancies) that combination strategies with multi-drug regimens (>4 drugs) are associated with more durable disease control. To better understand the mechanism of these improved outcomes, and to identify and prioritize new strategies for testing, we studied several multi-drug regimens combining BRAF/MEK targeted therapy and immunotherapy combinations in a Braf-mutant murine melanoma model (BrafV600E/Pten−/−). Short-term treatment with α-PD-1 and α-CTLA-4 monotherapies were relatively ineffective, while treatment with α-OX40 demonstrated some efficacy [17% of mice with no evidence of disease, (NED), at 60-days]. Outcomes were improved in the combined α-OX40/α-PD-1 group (42% NED). Short-term treatment with quadruplet therapy of immunotherapy doublets in combination with targeted therapy [dabrafenib and trametinib (DT)] was associated with excellent tumor control, with 100% of mice having NED after combined DT/α-CTLA-4/α-PD-1 or DT/α-OX40/α-PD-1. Notably, tumors from mice in these groups demonstrated a high proportion of effector memory T cells, and immunologic memory was maintained with tumor re-challenge. Together, these data provide important evidence regarding the potential utility of multi-drug therapy in treating advanced melanoma and suggest these models can be used to guide and prioritize combinatorial treatment strategies.

Background Limited information exists regarding procedural success and clinical outcomes in patients with previous coronary artery bypass grafting (CABG) undergoing percutaneous coronary intervention (PCI). We sought to compare outcomes in patients undergoing PCI with or without CABG. Methods and Results This was an observational cohort study of 123 780 consecutive PCI procedures from the Pan‐London (UK) PCI registry from 2005 to 2015. The primary end point was all‐cause mortality at a median follow‐up of 3.0 years (interquartile range, 1.2–4.6 years). A total of 12 641(10.2%) patients had a history of previous CABG, of whom 29.3% (n=3703) underwent PCI to native vessels and 70.7% (n=8938) to bypass grafts. There were significant differences in the demographic, clinical, and procedural characteristics of these groups. The risk of mortality during follow‐up was significantly higher in patients with prior CABG (23.2%; P=0.0005) compared with patients with no prior CABG (12.1%) and was seen for patients who underwent either native vessel (20.1%) or bypass graft PCI (24.2%; P

Sample-based media producers compose their art for machines. They implement aural effects and editing techniques to their source media, which very directly affect the sound of their music, but the aesthetics of which are not aimed at human ears; they are aimed at convincing copyright bots their media is not worth “flagging.” We offer machinic enculturation as a term descriptive of the phenomenon of adapting one’s media practices to machinic audiences. Then, using the basic operations of rhetoric (addition, omission, transposition, and transmutation) and a stylistic framework (antithesis, apostrophe, and reasoning by question and answer), we demonstrate a born-digital aesthetic, moored in the rhetorical prowess of some producers as they compose their music for copyright bots.

Epigenetics plays a fundamental role in cellular development and differentiation; epigenetic mechanisms, such as DNA methylation, are involved in gene regulation and the exquisite nuance of expression changes seen in the journey from pluripotency to final differentiation. Thus, DNA methylation as a marker of cell identify has the potential to reveal new insights into cell biology. We mined publicly available DNA methylation data with a machine-learning approach to identify differentially methylated loci between different white blood cell types. We then interrogated the DNA methylation and mRNA expression of candidate loci in CD4+, CD8+, CD14+, CD19+ and CD56+ fractions from 12 additional, independent healthy individuals (6 male, 6 female). 'Classic' immune cell markers such as CD8 and CD19 showed expected methylation/expression associations fitting with established dogma that hypermethylation is associated with the repression of gene expression. We also observed large differential methylation at loci which are not established immune cell markers; some of these loci showed inverse correlations between methylation and mRNA expression (such as PARK2, DCP2). Furthermore, we validated these observations further in publicly available DNA methylation and RNA sequencing datasets. Our results highlight the value of mining publicly available data, the utility of DNA methylation as a discriminatory marker and the potential value of DNA methylation to provide additional insights into cell biology and developmental processes.

Prey bacteria have evolved different strategies to counteract predation but the genetic basis remains unclear. Here, Duncan et al. identify key genes involved in Vibrio cholerae sensitivity to Bdellovibrio bacteriovorus predation, providing new insights into prey resistance mechanisms.

Abstract Background In Alzheimer’s disease, there are striking changes in CSF composition that relate to altered choroid plexus (CP) function. Studying CP tissue gene expression at the blood–cerebrospinal fluid barrier could provide further insight into the epithelial and stromal responses to neurodegenerative disease states. Methods Transcriptome-wide Affymetrix microarrays were used to determine disease-related changes in gene expression in human CP. RNA from post-mortem samples of the entire lateral ventricular choroid plexus was extracted from 6 healthy controls (Ctrl), 7 patients with advanced (Braak and Braak stage III–VI) Alzheimer’s disease (AD), 4 with frontotemporal dementia (FTD) and 3 with Huntington’s disease (HuD). Statistics and agglomerative clustering were accomplished with MathWorks, MatLab; and gene set annotations by comparing input sets to GeneGo (http://www.genego.com) and Ingenuity (http://www.ingenuity.com) pathway sets. Bonferroni-corrected hypergeometric p-values of

Abstract Background Although many genetic variants have been associated with multiple sclerosis (MS) risk, they do not explain all the disease risk and there remains uncertainty as to how these variants contribute to disease. DNA methylation is an epigenetic mechanism that can influence gene expression and has the potential to mediate the effects of environmental factors on MS. In a previous study, we found a differentially methylation region (DMR) at MHC HLA-DRB1 that was associated within relapsing-remitting MS (RRMS) patients in CD4+ T cells. This study aimed to confirm this earlier finding in an independent RRMS cohort of treatment-naïve female patients. Methods Total genomic DNA was extracted from CD4+ T cells of 28 female RRMS and 22 age-matched healthy controls subjects. DNA was bisulfite-converted and hybridised to Illumina 450K arrays. Beta values for all CpGs were analysed using the DMPFinder function in the MINFI program, and a follow-up prioritisation process was applied to identify the most robust MS-associated DMRs. Results This study confirmed our previous findings of a hypomethylated DMR at HLA-DRB1 and a hypermethylated DMR at HLA-DRB5 in this RRMS patient cohort. In addition, we identified a large independent DMR at MHC, whereby 11 CpGs in RNF39 were hypermethylated in MS cases compared to controls (max. ∆beta = 0.19, P = 2.1 × 10−4). We did not find evidence that SNP genotype was influencing the DMR in this cohort. A smaller MHC DMR was also identified at HCG4B, and two non-MHC DMRs at PM20D1 on chr1 and ERICH1 on chr8 were also identified. Conclusions The findings from this study confirm our previous results of a DMR at HLA-DRB1 and also suggest hypermethylation in an independent MHC locus, RNF39, is associated with MS. Taken together, our results highlight the importance of epigenetic factors at the MHC locus in MS independent of treatment, age and sex. Prospective studies are now required to discern whether methylation at MHC is involved in influencing risk of disease onset or whether the disease itself has altered the methylation profile.

Abstract Approaches to prolong responses to BRAF targeting drugs in melanoma patients are challenged by phenotype heterogeneity. Melanomas of a “MITF‐high” phenotype usually respond well to BRAF inhibitor therapy, but these melanomas also contain subpopulations of the de novo resistance “AXL‐high” phenotype. > 50% of melanomas progress with enriched “AXL‐high” populations, and because AXL is linked to de‐differentiation and invasiveness avoiding an “AXL‐high relapse” is desirable. We discovered that phenotype heterogeneity is supported during the response phase of BRAF inhibitor therapy due to MITF‐induced expression of endothelin 1 (EDN1). EDN1 expression is enhanced in tumours of patients on treatment and confers drug resistance through ERK re‐activation in a paracrine manner. Most importantly, EDN1 not only supports MITF‐high populations through the endothelin receptor B (EDNRB), but also AXL‐high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL‐high‐expressing cells and sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL‐high cells.

Abstract BRAF inhibitors (BRAFi) are standard of care for the treatment of BRAF V600 mutation-driven metastatic melanoma, but can lead to paradoxical activation of the mitogen-activated protein kinase (MAPK) signalling pathway. This can result in the promotion of precancerous lesions and secondary neoplasms, mainly (but not exclusively) associated with pre-existing mutations in RAS genes. We previously reported a patient with synchronous BRAF-mutated metastatic melanoma and BRAF wt /KRAS G12D-metastatic colorectal cancer (CRC), whose CRC relapsed and progressed when treated with the BRAF inhibitor dabrafenib (GSK2118436). We used tissue from the resected CRC metastasis to derive a cell line, LM-COL-1, which directly and reliably mimicked the clinical scenario including paradoxical activation of the MAPK signalling pathway resulting in increased cell proliferation upon dabrafenib treatment. Novel BRAF inhibitors (PLX8394 and PLX7904), dubbed as “paradox breakers”, were developed to inhibit V600 mutated oncogenic BRAF without causing paradoxical MAPK pathway activation. In this study we used our LM-COL-1 model alongside multiple other CRC cell lines with varying mutational backgrounds to demonstrate and confirm that the paradox breaker PLX8394 retains on-target inhibition of mutated BRAF V600 without paradoxically promoting MAPK signalling.

Abstract Background Epigenetic mechanisms provide an interface between environmental factors and the genome and are known to play a role in complex diseases such as obesity. These mechanisms, including DNA methylation, influence the regulation of development, differentiation and the establishment of cellular identity. Here we employ two approaches to identify differential methylation between two white adipose tissue depots in obese individuals before and after gastric bypass and significant weight loss. We analyse genome-wide DNA methylation data using (a) traditional paired t tests to identify significantly differentially methylated loci (Bonferroni-adjusted P ≤ 1 × 10−7) and (b) novel combinatorial algorithms to identify loci that differentiate between tissue types. Results Significant differential methylation was observed for 3239 and 7722 CpG sites, including 784 and 1129 extended regions, between adipose tissue types before and after significant weight loss, respectively. The vast majority of these extended differentially methylated regions (702) were consistent across both time points and enriched for genes with a role in transcriptional regulation and/or development (e.g. homeobox genes). Other differentially methylated loci were only observed at one time point and thus potentially highlight genes important to adipose tissue dysfunction observed in obesity. Strong correlations (r > 0.75, P ≤ 0.001) were observed between changes in DNA methylation (subcutaneous adipose vs omentum) and changes in clinical trait, in particular for CpG sites within PITX2 and fasting glucose and four CpG sites within ISL2 and HDL. A single CpG site (cg00838040, ATP2C2) gave strong tissue separation, with validation in independent subcutaneous (n = 681) and omental (n = 33) adipose samples. Conclusions This is the first study to report a genome-wide DNA methylome comparison of subcutaneous abdominal and omental adipose before and after weight loss. The combinatorial approach we utilised is a powerful tool for the identification of methylation loci that strongly differentiate between these tissues. This study provides a solid basis for future research focused on the development of adipose tissue and its potential dysfunction in obesity, as well as the role DNA methylation plays in these processes.

ABSTRACT Bdellovibrio bacteriovorus is a bacterial predator capable of killing and replicating inside most Gram-negative bacteria, including antibiotic-resistant pathogens. Despite growing interest in this organism as a potential therapeutic, many of its genes remain uncharacterized. Here, we perform a high-throughput genetic screen with B. bacteriovorus using transposon sequencing (Tn-seq) to explore the genetic requirements of predation. Two hundred one genes were deemed essential for growth in the absence of prey, whereas over 100 genes were found to be specifically required for predative growth on the human pathogens Vibrio cholerae and Escherichia coli in both planktonic and biofilm states. To further this work, we created an ordered-knockout library in B. bacteriovorus and developed new high-throughput techniques to characterize the mutants by their stage of deficiency in the predator life cycle. Using microscopy and flow cytometry, we confirmed 10 mutants defective in prey attachment and eight mutants defective in prey rounding. The majority of these genes are hypothetical and previously uncharacterized. Finally, we propose new nomenclature to group B. bacteriovorus mutants into classes based on their stage of predation defect. These results contribute to our basic understanding of bacterial predation and may be useful for harnessing B. bacteriovorus to kill harmful pathogens in the clinical setting. IMPORTANCE Bdellovibrio bacteriovorus is a predatory bacterium that can kill a wide range of Gram-negative bacteria, including many human pathogens. Given the global rise of antibiotic resistance and dearth of new antibiotics discovered in the past 30 years, this predator has potential as an alternative to traditional antibiotics. For many years, B. bacteriovorus research was hampered by a lack of genetic tools, and the genetic mechanisms of predation have only recently begun to be established. Here, we comprehensively identify and characterize predator genes required for killing bacterial prey, as well as genes that interfere in this process, which may allow us to design better therapeutic predators. Based on our study, we and other researchers may ultimately be able to genetically engineer strains that have improved killing rates, target specific species of prey, or preferentially target prey in the planktonic or biofilm state.

Cancer research has seen unprecedented advances over the past several years, with tremendous insights gained into mechanisms of response and resistance to cancer therapy. Central to this has been our understanding of crosstalk between the tumor and the microenvironment, with the recognition that complex interactions exist between tumor cells, stromal cells, overall host immunity, and the environment surrounding the host. This is perhaps best exemplified in cancer immunotherapy, where numerous studies across cancer types have illuminated our understanding of the genomic and immune factors that shape responses to therapy. In addition to their individual contributions, it is now clear that there is a complex interplay between genomic/epigenomic alterations and tumor immune responses that impact cellular plasticity and therapeutic responses. In addition to this, it is also now apparent that significant heterogeneity exists within tumors–both at the level of genomic mutations as well as tumor immune responses–thus contributing to heterogeneous clinical responses. Beyond the tumor microenvironment, overall host immunity plays a major role in mediating clinical responses. The gut microbiome plays a central role, with recent evidence revealing that the gut microbiome influences the overall immune set-point, through diverse effects on local and systemic inflammatory processes. Indeed, quantifiable differences in the gut microbiome have been associated with disease and treatment outcomes in patients and pre-clinical models, though precise mechanisms of microbiome-immune interactions are yet to be elucidated. Complexities are discussed herein, with a discussion of each of these variables as they relate to treatment response.

BackgroundWe investigated the molecular etiology of a young male proband with confirmed immunodeficiency of unknown cause, presenting with recurrent bacterial and Varicella zoster viral infections in childhood and persistent lymphopenia into early adulthood.AimTo identify causative functional genetic variants related to an undiagnosed primary immunodeficiency.MethodWhole genome microarray copy number variant (CNV) analysis was performed on the proband followed by whole exome sequencing (WES) and trio analysis of the proband and family members. A >4 kbp deletion identified by repeated CNV analysis of exome sequencing data along with three damaging missense single nucleotide variants were validated by Sanger sequencing in all family members. Confirmation of the causative role of the candidate gene was performed by qPCR and Western Blot analyses on the proband, family members and a healthy control.ResultsCNV identified our previously reported interleukin 25 amplification in the proband; however, the variant was not validated to be a candidate gene for immunodeficiency. WES trio analysis, data filtering and in silico prediction identified a novel, damaging (SIFT: 0; Polyphen 1; Grantham score: 101) and disease-causing (MutationTaster) single base mutation in the X chromosome (c.511C > T p.Arg171Trp) MSN gene not identified in the UCSC Genome Browser database. The mutation was validated by Sanger sequencing, confirming the proband was hemizygous X-linked recessive (–/T) at this locus and inherited the affected T allele from his non-symptomatic carrier mother (C/T), with other family members (father, sister) confirmed to be wild type (C/C). Western Blot analysis demonstrated an absence of moesin protein in lymphocytes derived from the proband, compared with normal expression in lymphocytes derived from the healthy control, father and mother. qPCR identified significantly lower MSN mRNA transcript expression in the proband compared to an age- and sex-matched healthy control subject in whole blood (p = 0.02), and lymphocytes (p = 0.01). These results confirmed moesin deficiency in the proband, directly causative of his immunodeficient phenotype.ConclusionThese findings confirm X-linked moesin-associated immunodeficiency in a proband previously undiagnosed up to 24 years of age. This study also highlights the utility of WES for the diagnosis of rare or novel forms of primary immunodeficiency disease.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of advanced melanoma. The first ICI to demonstrate clinical benefit, ipilimumab, targets cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4); however, the long-term overall survival is just 22%. More than 40 years ago intralesional (IL) bacillus Calmette–Guérin (BCG), a living attenuated strain of Mycobacterium bovis, was found to induce tumor regression by stimulating cell-mediated immunity following a localized and self-limiting infection. We evaluated these two immune stimulants in combination with melanoma with the aim of developing a more effective immunotherapy and to assess toxicity. In this phase I study, patients with histologically confirmed stage III/IV metastatic melanoma received IL BCG injection followed by up to four cycles of intravenous ipilimumab (anti-CTLA-4) (ClinicalTrials.gov number NCT01838200). The trial was discontinued following treatment of the first five patients as the two patients receiving the escalation dose of BCG developed high-grade immune-related adverse events (irAEs) typical of ipilimumab monotherapy. These irAEs were characterized in both patients by profound increases in the repertoire of autoantibodies directed against both self- and cancer antigens. Interestingly, the induced autoantibodies were detected at time points that preceded the development of symptomatic toxicity. There was no overlap in the antigen specificity between patients and no evidence of clinical responses. Efforts to increase response rates through the use of novel immunotherapeutic combinations may be associated with higher rates of irAEs, thus the imperative to identify biomarkers of toxicity remains strong. While the small patient numbers in this trial do not allow for any conclusive evidence of predictive biomarkers, the observed changes warrant further examination of autoantibody repertoires in larger patient cohorts at risk of developing irAEs during their course of treatment. In summary, dose escalation of IL BCG followed by ipilimumab therapy was not well tolerated in advanced melanoma patients and showed no evidence of clinical benefit. Measuring autoantibody responses may provide early means for identifying patients at risk from developing severe irAEs during cancer immunotherapy.

Epilepsy is a neurological disorder characterized by an increased predisposition for seizures. Although this definition suggests that it is a single disorder, epilepsy encompasses a group of disorders with diverse aetiologies and outcomes. A genetic basis for epilepsy syndromes has been postulated for several decades, with several mutations in specific genes identified that have increased our understanding of the genetic influence on epilepsies. With 70-80% of epilepsy cases identified to have a genetic cause, there are now hundreds of genes identified to be associated with epilepsy syndromes which can be analyzed using next generation sequencing (NGS) techniques such as targeted gene panels, whole exome sequencing (WES) and whole genome sequencing (WGS). For effective use of these methodologies, diagnostic laboratories and clinicians require information on the relevant workflows including analysis and sequencing depth to understand the specific clinical application and diagnostic capabilities of these gene sequencing techniques. As epilepsy is a complex disorder, the differences associated with each technique influence the ability to form a diagnosis along with an accurate detection of the genetic etiology of the disorder. In addition, for diagnostic testing, an important parameter is the cost-effectiveness and the specific diagnostic outcome of each technique. Here, we review these commonly used NGS techniques to determine their suitability for application to epilepsy genetic diagnostic testing.

Epigenome-wide association studies seek to identify DNA methylation sites associated with clinical outcomes. Difference in observed methylation between specific cell-subtypes is often of interest; however, available samples often comprise a mixture of cells. To date, cell-subtype estimates have been obtained from mixed-cell DNA data using linear regression models, but the accuracy of such estimates has not been critically assessed. We evaluated linear regression performance for cell-subtype specific methylation estimation using a 450K methylation array dataset of both mixed-cell and cell-subtype sorted samples from six healthy males. CpGs associated with each cell-subtype were first identified using t-tests between groups of cell-subtype sorted samples. Subsequent reduced panels of reliably accurate CpGs were identified from mixed-cell samples using an accuracy heuristic (D). Performance was assessed by comparing cell-subtype specific estimates from mixed-cells with corresponding cell-sorted mean using the mean absolute error (MAE) and the Coefficient of Determination (R2). At the cell-subtype level, methylation levels at 3272 CpGs could be estimated to within a MAE of 5% of the expected value. The cell-subtypes with the highest accuracy were CD56+ NK (R2 = 0.56) and CD8+T (R2 = 0.48), where 23% of sites were accurately estimated. Hierarchical clustering and pathways enrichment analysis confirmed the biological relevance of the panels. Our results suggest that linear regression for cell-subtype specific methylation estimation is accurate only for some cell-subtypes at a small fraction of cell-associated sites but may be applicable to EWASs of disease traits with a blood-based pathology. Although sample size was a limitation in this study, we suggest that alternative statistical methods will provide the greatest performance improvements.

Studies comparing coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI) have largely been performed in the bare-metal stent (BMS) and first-generation drug eluting stent (F-DES) era. Second-generation DES (S-DES) have shown improved outcomes when compared to F-DES, but data comparing CABG with PCI using S-DES is limited. We compared mortality following CABG versus PCI for patients with multivessel disease and analyzed different stent types.A total of 6,682 patients underwent multivessel revascularization at Harefield Hospital, UK. We stratified CABG patients into single arterial graft (SAG) or multiple arterial grafts (MAG); and PCI patients into BMS, F-DES or S-DES groups. We analyzed all-cause mortality at 5 years.4,388 patients had CABG (n[SAG] = 3,358; n[MAG] = 1,030) and 2,294 patients had PCI (n[BMS] = 416; n[F-DES] = 752; n[S-DES] = 1,126). PCI had higher 5-year mortality with BMS (HR = 2.27, 95% CI:1.70-3.05, p

Direct visualization of HIV-1 replication would improve our understanding of the viral life cycle. We adapted established technology and reagents to develop an imaging approach, ViewHIV, which allows evaluation of early HIV-1 replication intermediates, from reverse transcription to integration. These methods permit the simultaneous evaluation of both the capsid protein (CA) and viral DNA genome (vDNA) components of HIV-1 in both the cytosol and nuclei of single cells. ViewHIV is relatively rapid, uses readily available reagents in combination with standard confocal microscopy, and can be done with virtually any HIV-1 strain and permissive cell lines or primary cells. Using ViewHIV, we find that CA enters the nucleus and associates with vDNA in both transformed and primary cells. We also find that CA’s interaction with the host polyadenylation factor, CPSF6, enhances nuclear entry and potentiates HIV-1’s depth of nuclear invasion, potentially aiding the virus’s integration into gene-dense regions.

Human rhinovirus (HRV) causes upper respiratory infections and asthma exacerbations. We screened multiple orthologous RNAi reagents and identified host proteins that modulate HRV replication. Here, we show that RNASEK, a transmembrane protein, was needed for the replication of HRV, influenza A virus, and dengue virus. RNASEK localizes to the cell surface and endosomal pathway and closely associates with the vacuolar ATPase (V-ATPase) proton pump. RNASEK is required for endocytosis, and its depletion produces enlarged clathrin-coated pits (CCPs) at the cell surface. These enlarged CCPs contain endocytic cargo and are bound by the scissioning GTPase, DNM2. Loss of RNASEK alters the localization of multiple V-ATPase subunits and lowers the levels of the ATP6AP1 subunit. Together, our results show that RNASEK closely associates with the V-ATPase and is required for its function; its loss prevents the early events of endocytosis and the replication of multiple pathogenic viruses.

Herpes simplex virus type 1 (HSV-1) is a significant human pathogen that infects a large portion of the human population. Cells deploy a variety of defenses to limit the extent to which the virus can replicate. One such factor is the promyelocytic leukemia (PML) protein, the nucleating and organizing factor of nuclear domain 10 (ND10). PML responds to a number of stimuli and is implicated in intrinsic and innate cellular antiviral defenses against HSV-1. While the role of PML in a number of cellular pathways is controlled by post-translational modifications, the effects of phosphorylation on its antiviral activity toward HSV-1 have been largely unexplored. Consequently, we mapped phosphorylation sites on PML, mutated these and other known phosphorylation sites on PML isoform I (PML-I), and examined their effects on a number of PML’s activities. Our results show that phosphorylation at most sites on PML-I is dispensable for the formation of ND10s and colocalization between PML-I and the HSV-1 regulatory protein, ICP0, which antagonizes PML-I function. However, inhibiting phosphorylation at sites near the SUMO-interaction motif (SIM) of PML-I impairs its ability to respond to HSV-1 infection. Overall, our data suggest that PML phosphorylation regulates its antiviral activity against HSV-1.

ABSTRACT The type III secretion system (T3SS) is a bacterial virulence factor expressed by dozens of Gram-negative pathogens but largely absent from commensals. The T3SS is an attractive target for antimicrobial agents that may disarm pathogenic bacteria while leaving commensal populations intact. We previously identified piericidin A1 as an inhibitor of the Ysc T3SS in Yersinia pseudotuberculosis. Piericidins were first discovered as inhibitors of complex I of the electron transport chain in mitochondria and some bacteria. However, we found that piericidin A1 did not alter Yersinia membrane potential or inhibit flagellar motility powered by the proton motive force, indicating that the piericidin mode of action against Yersinia type III secretion is independent of complex I. Instead, piericidin A1 reduced the number of T3SS needle complexes visible by fluorescence microscopy at the bacterial surface, preventing T3SS translocator and effector protein secretion. Furthermore, piericidin A1 decreased the abundance of higher-order YscF needle subunit complexes, suggesting that piericidin A1 blocks YscF needle assembly. While expression of T3SS components in Yersinia are positively regulated by active type III secretion, the block in secretion by piericidin A1 was not accompanied by a decrease in T3SS gene expression, indicating that piericidin A1 may target a T3SS regulatory circuit. However, piericidin A1 still inhibited effector protein secretion in the absence of the T3SS regulator YopK, YopD, or YopN. Surprisingly, while piericidin A1 also inhibited the Y. enterocolitica Ysc T3SS, it did not inhibit the SPI-1 family Ysa T3SS in Y. enterocolitica or the Ysc family T3SS in Pseudomonas aeruginosa. Together, these data indicate that piericidin A1 specifically inhibits Yersinia Ysc T3SS needle assembly. IMPORTANCE The bacterial type III secretion system (T3SS) is widely used by both human and animal pathogens to cause disease yet remains incompletely understood. Deciphering how some natural products, such as the microbial metabolite piericidin, inhibit type III secretion can provide important insight into how the T3SS functions or is regulated. Taking this approach, we investigated the ability of piericidin to block T3SS function in several human pathogens. Surprisingly, piericidin selectively inhibited the Ysc family T3SS in enteropathogenic Yersinia but did not affect the function of a different T3SS within the same species. Furthermore, piericidin specifically blocked the formation of T3SS needles on the bacterial surface without altering the localization of several other T3SS components or regulation of T3SS gene expression. These data show that piericidin targets a mechanism important for needle assembly that is unique to the Yersinia Ysc T3SS.

Infection of human cells with Yersinia pseudotuberculosis expressing a functional type III secretion system (T3SS) leads to activation of host NF-κB. We show that the Yersinia T3SS activates distinct NF-κB pathways dependent upon bacterial subcellular localization. We found that wildtype Yersinia able to remain extracellular triggered NF-κB activation independently of the non-canonical NF-κB kinase NIK in HEK293T cells. In contrast, Yersinia lacking the actin-targeting effectors YopEHO, which become internalized into host cells, induce a NIK-dependent response and nuclear entry of the non-canonical NF-κB subunit p52. Blocking actin polymerization and uptake of effector mutant bacteria using cytochalasin D shifted the host NF-κB response from NIK-independent to primarily NIK-dependent. We observed similar results using Pseudomonas aeruginosa, which expresses a related T3SS and the actin-targeting effector ExoT. As the NF-κB response of HEK293T cells to effectorless Yersinia has been used both as a screening tool for chemical inhibitors of the T3SS and for bacterial forward genetic screens, a better understanding of this response is important for tool optimization and interpretation.

Awareness of the role that nutraceuticals play in the treatment and prevention of disease has led to an explosion of research in this exciting arena that seems to overflow into the food, cosmetic, nutraceutical, and pharmaceutical industries. Nutrients, supplements and herbal compounds have shown promise as either alternatives to modern medicine or complementary tools in the treatment and prevention of disease. This review provides a brief outlay of the advantages and challenges of nutraceutical delivery via dermatological, oral and ophthalmic routes. Emphasis is directed towards nutraceutical formulation strategies adopted to overcome physicochemical challenges and instability of natural bioactives in order to improve their delivery and bioavailability to the body. This paper highlights how novel techniques have achieved products with greater commercial viability and efficacy than their conventional counterparts. Importance of multicomponent products where individual bioactives potency is not subdued by each other has been marked. Ultimately it is the adoption and merging of the different formulation technologies and prudent scientific validation that will dictate the future success of nutraceuticals. This is especially pertinent in a market where an informed consumer demands an innovative all-in-one product that does not compromise in its results.

Botulism is a serious foodborne neuroparalytic disease, caused by botulinum neurotoxin (BoNT), produced by the anaerobic bacterium Clostridium botulinum. Seven toxin serotypes (A–H) have been described. The majority of human cases of botulism are caused by serotypes A and B followed by E and F. We report here a group of serotype B specific monoclonal antibodies (mAbs) capable of binding toxin under physiological conditions. Thus, they serve as capture antibodies for a sandwich (capture) ELISA. The antibodies were generated using recombinant peptide fragments corresponding to the receptor-binding domain of the toxin heavy chain as immunogen. Their binding properties suggest that they bind a complex epitope with dissociation constants (KD’s) for individual antibodies ranging from 10 to 48 × 10−11 M. Assay performance for all possible combinations of capture-detector antibody pairs was evaluated and the antibody pair resulting in the lowest level of detection (L.O.D.), ~20 pg/mL was determined. Toxin was detected in spiked dairy samples with good recoveries at concentrations as low as 0.5 pg/mL and in ground beef samples at levels as low as 2 ng/g. Thus, the sandwich ELISA described here uses mAb for both the capture and detector antibodies (binding different epitopes on the toxin molecule) and readily detects toxin in those food samples tested.

In this article, a remix produced by the artist Pogo and a mashup made by the artist Kutiman are examined as positioned within an aesthetic public sphere. Using the taxonomy afforded by the quadpartitia ratio, these mashups and remixes are pulled apart to reveal insights into how traces of authorship can be detected by juxtaposing original and mashed-up or remixed images. Ultimately, this article will show that locating traces of authorship in mashups and remixes not only adds depth to aesthetic appreciation but also illustrates the aesthetic dependence of mashups and remixes upon the media images that circulate within a given aesthetic public sphere.

BackgroundST‐segment elevation myocardial infarction is increasingly common in octogenarians, and optimal management in this cohort is uncertain. This study aimed to describe the outcomes of octogenarians with ST‐segment elevation myocardial infarction treated by primary percutaneous coronary intervention. Methods and ResultsWe analyzed 10 249 consecutive patients with ST‐segment elevation myocardial infarction treated with primary percutaneous coronary intervention between 2005 and 2011 at 8 tertiary cardiac centers across London, United Kingdom. The primary end point was all‐cause mortality at a median follow‐up of 3 years. In total, 1051 patients (10.3%) were octogenarians, with an average age of 84.2 years, and the proportion increased over the study period (P=0.04). In‐hospital mortality (7.7% vs 2.4%, P

Multiphenotype genome-wide association studies (GWAS) may reveal pleiotropic genes, which would remain undetected using single phenotype analyses. Analysis of large pedigrees offers the added advantage of more accurately assessing trait heritability, which can help prioritise genetically influenced phenotypes for GWAS analysis. In this study we performed a principal component analysis (PCA), heritability (h2) estimation and pedigree-based GWAS of 37 cardiovascular disease -related phenotypes in 330 related individuals forming a large pedigree from the Norfolk Island genetic isolate. PCA revealed 13 components explaining >75% of the total variance. Nine components yielded statistically significant h2 values ranging from 0.22 to 0.54 (P

Some crises, such as those brought on or exposed by the COVID-19 pandemic, are wicked problems--large, complex problems with no immediate answer. As such, they make rich centerpieces for learning with respect to public deliberation and issue-based dialogue. This essay reflects on an experimental, transdisciplinary health and science communication course entitled Comprehending COVID-19." The course represents a collaborative effort among 14 faculty representing 10 different academic departments to create a resource for teaching students how to deliberate the pandemic, despite its attending, oversaturated, fake-news-infused, infodemic. We offer transdisciplinary deliberation as a pedagogical framework to expand communication repertoires in ways useful for sifting through the messiness of an infodemic while also developing key deliberation skills for productively engaging participatory decision-making with concern to wicked problems.

Primary cells are often used to study viral replication and host-virus interactions as their antiviral pathways have not been altered or inactivated; however, their use is restricted by their short lifespan. Conventional methods to extend the life of primary cultures typically utilize viral oncogenes. Many of these oncogenes, however, perturb or inactivate cellular antiviral pathways, including the interferon (IFN) response. It has been previously shown that expression of the telomerase reverse transcriptase (TERT) gene extends the life of certain cell types. The effect that TERT expression has on the innate antiviral response to RNA- and DNA-containing viruses has not been examined. In the current study, we introduced the human TERT (hTERT) gene into a primary human embryonic lung (HEL-299) cell strain, which is known to respond to the type I IFN, IFN-β. We show that the resulting HEL-TERT cell line is capable of replicating beyond 100 population doublings without exhibiting signs of senescence. Treatment with IFN-β resulted in the upregulation of four model IFN stimulated genes (ISGs) in HEL-299 and HEL-TERT cells. Both cell lines supported the replication of herpes simplex virus type 1 (HSV-1) and vesicular stomatitis virus (VSV) and impaired the replication of both viruses upon IFN-β pretreatment. Introduction of the viral oncoprotein, simian virus 40 (SV40) large T-antigen, which is frequently used to immortalize cells, largely negated this effect. Taken together, our data indicate that expression of hTERT does not alter type 1 IFN signaling and/or the growth of two viruses, making this cell line a useful reagent for studying viral replication and virus-cell interactions.