Transcriptional and Cellular Diversity of the Human Heart.

BACKGROUND: The human heart requires a complex ensemble of specialized cell types to perform its essential function. A greater knowledge of the intricate cellular milieu of the heart is critical to increase our understanding of cardiac homeostasis and pathology. As recent advances in low-input RNA sequencing have allowed definitions of cellular transcriptomes at single-cell resolution at scale, we have applied these approaches to assess the cellular and transcriptional diversity of the nonfailing human heart. METHODS: Microfluidic encapsulation and barcoding was used to perform single nuclear RNA sequencing with samples from 7 human donors, selected for their absence of overt cardiac disease. Individual nuclear transcriptomes were then clustered based on transcriptional profiles of highly variable genes. These clusters were used as the basis for between-chamber and between-sex differential gene expression analyses and intersection with genetic and pharmacologic data. RESULTS: We sequenced the transcriptomes of 287 269 single cardiac nuclei, revealing 9 major cell types and 20 subclusters of cell types within the human heart. Cellular subclasses include 2 distinct groups of resident macrophages, 4 endothelial subtypes, and 2 fibroblast subsets. Comparisons of cellular transcriptomes by cardiac chamber or sex reveal diversity not only in cardiomyocyte transcriptional programs but also in subtypes involved in extracellular matrix remodeling and vascularization. Using genetic association data, we identified strong enrichment for the role of cell subtypes in cardiac traits and diseases. Intersection of our data set with genes on cardiac clinical testing panels and the druggable genome reveals striking patterns of cellular specificity. CONCLUSIONS: Using large-scale single nuclei RNA sequencing, we defined the transcriptional and cellular diversity in the normal human heart. Our identification of discrete cell subtypes and differentially expressed genes within the heart will ultimately facilitate the development of new therapeutics for cardiovascular diseases.

Gout and the risk of advanced chronic kidney disease in the UK health system: a national cohort study.

OBJECTIVE: Evaluate the association between gout and risk of advanced chronic kidney disease (CKD). DESIGN: Retrospective matched cohort study. SETTING: UK Clinical Practice Research Datalink. PARTICIPANTS: The analysis included data for 68 897 patients with gout and 554 964 matched patients without gout. Patients were aged ≥18 years, registered at UK practices, had ≥12 months of clinical data and had data linked with Hospital Episode Statistics. Patients were excluded for history of advanced CKD, juvenile gout, cancer, HIV, tumour lysis syndrome, Lesch-Nyhan syndrome or familial Mediterranean fever. PRIMARY AND SECONDARY OUTCOME MEASURES: Advanced CKD was defined as first occurrence of: (1) dialysis, kidney transplant, diagnosis of end-stage kidney disease (ESKD) or stage 5 CKD (diagnostic codes in Read system or International Classification of Diseases, Tenth Revision); (2) estimated glomerular filtration rate (eGFR) <10 mL/min/1.73 m²; (3) doubling of serum creatinine from baseline and (4) death associated with CKD. RESULTS: Advanced CKD incidence was higher for patients with gout (8.54 per 1000 patient-years; 95% CI 8.26 to 8.83) versus without gout (4.08; 95% CI 4.00 to 4.16). Gout was associated with higher advanced CKD risk in both unadjusted analysis (HR, 2.00; 95% CI 1.92 to 2.07) and after adjustment (HR, 1.29; 95% CI 1.23 to 1.35). Association was strongest for ESKD (HR, 2.13; 95% CI 1.73 to 2.61) and was present for eGFR <10 mL/min/1.73 m² (HR, 1.45; 95% CI 1.30 to 1.61) and serum creatinine doubling (HR, 1.13; 95% CI 1.08 to 1.19) but not CKD-associated death (HR, 1.14; 95% CI 0.99 to 1.31). Association of gout with advanced CKD was replicated in propensity-score matched analysis (HR, 1.23; 95% CI 1.17 to 1.29) and analysis limited to patients with incident gout (HR, 1.28; 95% CI 1.22 to 1.35). CONCLUSIONS: Gout is associated with elevated risk of CKD progression. Future studies should investigate whether controlling gout is protective and reduces CKD risk.

A Multifaceted Role for Myd88-Dependent Signaling in Progression of Murine Mammary Carcinoma.

Previous data obtained in our laboratory suggested that there may be constitutive signaling through the myeloid differentiation primary response gene 88 (Myd88)-dependent signaling cascade in murine mammary carcinoma. Here, we extended these findings by showing that, in the absence of an added Toll-like receptor (TLR) agonist, the myddosome complex was preformed in 4T1 tumor cells, and that Myd88 influenced cytoplasmic extracellular signal-regulated kinase (Erk)1/Erk2 levels, nuclear levels of nuclear factor-kappaB (NFκB) and signal transducer and activator of transcription 5 (STAT5), tumor-derived chemokine (C-C motif) ligand 2 (CCL2) expression, and in vitro and in vivo tumor growth. In addition, RNA-sequencing revealed that Myd88-dependent signaling enhanced the expression of genes that could contribute to breast cancer progression and genes previously associated with poor outcome for patients with breast cancer, in addition to suppressing the expression of genes capable of inhibiting breast cancer progression. Yet, Myd88-dependent signaling in tumor cells also suppressed expression of genes that could contribute to tumor progression. Collectively, these data revealed a multifaceted role for Myd88-dependent signaling in murine mammary carcinoma.

Sexual behaviours, HIV testing, and the proportion of men at risk of transmitting and acquiring HIV in London, UK, 2000-13: a serial cross-sectional study.

BACKGROUND: HIV incidence in men who have sex with men (MSM) in the UK has remained unchanged over the past decade despite increases in HIV testing and antiretroviral therapy (ART) coverage. In this study, we examine trends in sexual behaviours and HIV testing in MSM and explore the risk of transmitting and acquiring HIV. METHODS: In this serial cross-sectional study, we obtained data from ten cross-sectional surveys done between 2000 and 2013, consisting of anonymous self-administered questionnaires and oral HIV antibody testing in MSM recruited in gay social venues in London, UK. Data were collected between October and January for all survey years up to 2008 and between February and August thereafter. All men older than 16 years were eligible to take part and fieldworkers attempted to approach all MSM in each venue and recorded refusal rates. Data were collected on demographic and sexual behavioural characteristics. We analysed trends over time using linear, logistic, and quantile regression. FINDINGS: Of 13 861 questionnaires collected between 2000 and 2013, we excluded 1985 (124 had completed the survey previously or were heterosexual reporting no anal intercourse in the past year, and 1861 did not provide samples for antibody testing). Of the 11 876 eligible MSM recruited, 1512 (13%) were HIV positive, with no significant trend in HIV positivity over time. 35% (531 of 1505) of HIV-positive MSM had undiagnosed infection, which decreased non-linearly over time from 34% (45 of 131) to 24% (25 of 106; p=0·01), while recent HIV testing (ie, in the past year) increased from 26% (263 of 997) to 60% (467 of 777; p<0·0001). The increase in recent testing in undiagnosed men (from 29% to 67%, p<0·0001) and HIV-negative men (from 26% to 62%, p<0·0001) suggests that undiagnosed infection might increasingly be recently acquired infection. The proportion of MSM reporting unprotected anal intercourse (UAI) in the past year increased from 43% (513 of 1187) to 53% (394 of 749; p<0·0001) and serosorting (exclusively) increased from 18% (207 of 1132) to 28% (177 of 6369; p<0·0001). 268 (2%) of 11 570 participants had undiagnosed HIV and reported UAI in the past year were at risk of transmitting HIV. Additionally 259 (2%) had diagnosed infection and reported UAI and non-exclusive serosorting in the past year. Although we did not collect data on antiretroviral therapy or viral load, surveillance data suggests that a small proportion of men with diagnosed infection will have detectable viral load and hence might also be at risk of transmitting HIV. 2633 (25%) of 10 364 participants were at high risk of acquiring HIV (defined as HIV-negative MSM either reporting one or more casual UAI partners in the past year or not exclusively serosorting). The proportions of MSM at risk of transmission or acquisition changed little over time (p=0·96 for MSM potentially at risk of transmission and p=0·275 for MSM at high risk of acquiring HIV). Undiagnosed men reporting UAI and diagnosed men not exclusively serosorting had consistently higher partner numbers than did other MSM over the period (median ranged from one to three across surveys in undiagnosed men reporting UAI, two to ten in diagnosed men not exclusively serosorting, and none to two in other men). INTERPRETATION: An increasing proportion of undiagnosed HIV infections in MSM in London might have been recently acquired, which is when people are likely to be most infectious. High UAI partner numbers of MSM at risk of transmitting HIV and the absence of a significant decrease in the proportion of men at high risk of acquiring the infection might explain the sustained HIV incidence. Implementation of combination prevention interventions comprising both behavioural and biological interventions to reduce community-wide risk is crucial to move towards eradication of HIV. FUNDING: Public Health England.

The signs, symptoms and help-seeking experiences of neutropenic sepsis patients before they reach hospital: a qualitative study.

PURPOSE: Neutropenic sepsis (NS) is a medical emergency in which urgent treatment with antibiotics is known to improve outcomes, yet there are minimal data about what happens to patients with NS before they reach hospital. We aimed to examine the pre-hospital experiences of patients with NS, identifying its early presenting features and exploring the factors potentially delaying patients' arrival at hospital. METHODS: We conducted in-depth, qualitative interviews with 22 cancer patients admitted to hospital for treatment of NS and 10 patient carers. The setting was a tertiary referral centre in Southern England. RESULTS: Thirty seven percent of patients took over 12 h to present to hospital after symptom onset. The mean delay in presentation was 11 h (range 0-68 h). Thematic analysis of the interviews, using grounded theory, revealed wide-ranging, potentially modifiable factors delaying patients' presentation to hospital. For example, information provided to patients about NS from different sources was inconsistent, with 'mixed messages' about urgency triggering delays. All patients self-monitored their temperature and understood the implication of a fever but few appreciated the potential significance of feeling unwell in the absence of fever. Attempts to obtain treatment were sometimes thwarted by nonspecialists' failure to recognise possible neutropenia in a patient with apparently mild signs, and several patients with NS were discharged without treatment. Some patients denied their symptoms to themselves and others to avoid hospital admission; palliative patients seemed particularly prone to these attitudes, while their carers were keen to seek medical attention. CONCLUSIONS: This investigation of patients' and carers' experiences of NS identifies numerous strategies for improving patient education, support and pre-hospital management, all of which may reduce pre-hospital delays and consequently decrease morbidity and mortality from NS.

Metabolic alterations during the growth of tumour spheroids.

Solid tumours undergo considerable alterations in their metabolism of nutrients in order to generate sufficient energy and biomass for sustained growth and proliferation. During growth, the tumour microenvironment exerts a number of influences (e.g. hypoxia and acidity) that affect cellular biology and the flux or utilisation of fuels including glucose. The tumour spheroid model was used to characterise the utilisation of glucose and describe alterations to the activity and expression of key glycolytic enzymes during the tissue growth curve. Glucose was avidly consumed and associated with the production of lactate and an acidified medium, confirming the reliance on glycolytic pathways and a diminution of oxidative phosphorylation. The expression levels and activities of hexokinase, phosphofructokinase-1, pyruvate kinase and lactate dehydrogenase in the glycolytic pathway were measured to assess glycolytic capacity. Similar measurements were made for glucose-6-phosphate dehydrogenase, the entry point and regulatory step of the pentose-phosphate pathway (PPP) and for cytosolic malate dehydrogenase, a key link to TCA cycle intermediates. The parameters for these key enzymes were shown to undergo considerable variation during the growth curve of tumour spheroids. In addition, they revealed that the dynamic alterations were influenced by both transcriptional and posttranslational mechanisms.