Age differences in patterns of cannabis use among an online US sample of adults who consume cannabis frequently.

Background: Cannabis use is increasing among middle-aged and older US adults, populations that are particularly vulnerable to the adverse effects of cannabis. Risks for adverse effects differ by cannabis use patterns, which have become increasingly heterogeneous. Nevertheless, little is known about age differences in such patterns.Objective: To investigate age differences in cannabis use patterns, comparing younger (age 18-49), middle-aged (age 50-64), and older adults (age ≥65).Methods: A total of 4,151 US adults with past 7-day cannabis consumption completed an online survey (35.1% male; 60.1% female; 4.8% identified as "other"). Regression models examined age differences in cannabis use patterns.Results: Compared to younger adults, middle-aged and older adults were more likely to consume cannabis during evening hours (50-64: adjusted odds ratio [aOR] = 2.98, 95% CI 2.24-3.96; ≥65: aOR = 4.23, 95 CI 2.82-6.35); by only one method (50-64: aOR = 1.67, 95% CI 1.34-2.09; ≥65: aOR = 3.38, 95 CI 2.24-5.09); primarily by smoking as the only method (50-64: aOR = 1.52, 95% CI 1.29-1.78; ≥65: aOR = 2.12, 95 CI 1.64-2.74); but less likely to consume concentrated cannabis products (concentrates) with extremely high %THC (50-64: aOR = 0.71, 95% CI 0.54-0.93; ≥65: aOR = 0.30, 95 CI 0.16-0.55). Age differences in cannabis use patterns were also observed between middle-aged and older adults.Conclusion: Findings suggest that middle-aged and older adults may engage in less risky cannabis use patterns compared to younger groups (e.g. lower likelihood of consuming highly potent concentrates). However, findings also underscore the importance of recognizing risks unique to these older demographics, such as smoking-related health events. Consequently, prevention strategies targeting such use patterns are needed.

Activated carbon amendment of sand in the base of a permeable pavement reduces total nitrogen and nitrate leaching.

Urban runoff from impermeable surfaces contains various pollutants. Stormwater samples were collected for one year from car parks on the campus of Newcastle University, located in northeast England, to monitor seasonal variation in stormwater properties and leachate quality following stormwater percolation through pilot-scale, outdoor permeable pavements. The pilot study compared an innovative 'pollution munching' permeable pavement with 2 % activated carbon (AC) amendment in the sand base with a conventional, un-amended sand base permeable pavement. Faecal coliforms were detected in stormwater at an average value of 3.75 ± 0.79 log10 CFUs per 100 mL. The permeable pavements without and with AC had mean log removal values of 0.81 ± 0.35 and 0.70 ± 0.35 for these faecal bacteria. The absence of genetic markers for human host associated Bacteroides (HF183) in eleven out of twelve stormwater samples showed that the faecal bacteria were mainly from animal sources. 16S rRNA gene sequencing results confirmed the presence of nitrifying bacteria from the genera Nitrosomonas, Nitrobacter, Nitrosococcus, Nitrospira, and Nitrosospira in stormwater. Nitrification and nitrate leaching was more notable for the conventional permeable pavement and may pose a groundwater pollution risk. Two percent AC amendment of the sand base reduced nitrate and total nitrogen leaching significantly compared with the conventional permeable pavement, by 57 ± 15 % and 40 ± 20 %, respectively. The AC amendment also resulted in significantly reduced Cu and DOC leaching, and lesser accumulation of PAHs by passive samplers embedded in the permeable pavement base. Hydraulic tests showed that the AC amended base layer still met the design specifications for permeable pavements, making it a promising proposition for pollution reduction in Sustainable Drainage Systems (SuDS).

Reconstructing microvascular network skeletons from 3D images: What is the ground truth?

Structural changes to microvascular networks are increasingly highlighted as markers of pathogenesis in a wide range of disease, e.g. Alzheimer's disease, vascular dementia and tumour growth. This has motivated the development of dedicated 3D imaging techniques, alongside the creation of computational modelling frameworks capable of using 3D reconstructed networks to simulate functional behaviours such as blood flow or transport processes. Extraction of 3D networks from imaging data broadly consists of two image processing steps: segmentation followed by skeletonisation. Much research effort has been devoted to segmentation field, and there are standard and widely-applied methodologies for creating and assessing gold standards or ground truths produced by manual annotation or automated algorithms. The Skeletonisation field, however, lacks widely applied, simple to compute metrics for the validation or optimisation of the numerous algorithms that exist to extract skeletons from binary images. This is particularly problematic as 3D imaging datasets increase in size and visual inspection becomes an insufficient validation approach. In this work, we first demonstrate the extent of the problem by applying 4 widely-used skeletonisation algorithms to 3 different imaging datasets. In doing so we show significant variability between reconstructed skeletons of the same segmented imaging dataset. Moreover, we show that such a structural variability propagates to simulated metrics such as blood flow. To mitigate this variability we introduce a new, fast and easy to compute super metric that compares the volume, connectivity, medialness, bifurcation point identification and homology of the reconstructed skeletons to the original segmented data. We then show that such a metric can be used to select the best performing skeletonisation algorithm for a given dataset, as well as to optimise its parameters. Finally, we demonstrate that the super metric can also be used to quickly identify how a particular skeletonisation algorithm could be improved, becoming a powerful tool in understanding the complex implication of small structural changes in a network.

Extending Gender- and Sex-Based Analyses in Cannabis Research: Findings from an Online Sample of Gender Diverse Young Adult Consumers.

Background: Gender and sex can influence cannabis behaviors and consequences (Cannabis Use Disorder [CUD]). Research typically examines sex and gender independently. Gender analyses often exclude transgender and gender diverse (TGD) populations. The objectives of this study were to (a) replicate less frequent cannabis use among TGD young adults compared to cisgender counterparts (b) compare severity of CUD, and (c) examine the role of sex on cannabis outcomes. Method: Online survey participants between 18 and 34 (N=1213) from the United States who reported past-week cannabis consumption provided information on cannabis practices and CUD from February to April 2022. Bivariate analyses explored gender differences across frequency (daily frequency across routes of administration [ROAs]; daily use of 2+ ROAs, use throughout the day) and CUD. Adjusted regression models provided model-estimated marginal probabilities and means to examine differences across four gender-by-sex categories (cisgender men: n=385; cisgender women: n=681; male-at-birth TGD: n=26; female-at-birth TGD: n=121). Benjamini-Hochberg adjustments (10% false discovery rate) were applied. Results: Among past-week consumers, female-at-birth TGD participants demonstrated lower probability of daily flower smoking compared to cisgender men (0.54 vs. 0.67). Cisgender men reported greater probability of daily concentrate vaping (0.55) compared to cisgender women (0.45) and female-at-birth TGD participants (0.27); they were also more likely to report daily use of 2+ ROAs (cisgender men: 0.51 vs. cisgender women: 0.39 and female at-birth TGD: 0.27). TGD participants reported greater CUD severity compared to cisgender counterparts, t(1096)=-3.69, p=0.002. Model-estimated means found lower severity among cisgender women compared to cisgender men and female-at-birth TGD participants. Stratified regression models support positive associations between daily cannabis use and CUD in both TGD in cisgender groups. Among cisgender participants, greater severity was predicted by male sex, younger age, and younger age of onset. Conclusions: The present study replicates and extends a prior finding that among past-week cannabis consumers, TGD young adults report less frequent use than cisgender counterparts. Despite this, TGD participants demonstrated greater severity of CUD. While analyses were limited by the small sample of male-at-birth TGD participants, the article highlights the importance of expanding sex- and gender-focused analyses. Future work is expanding efforts to target hard-to-reach consumers.

Environmental DNA clarifies impacts of combined sewer overflows on the bacteriology of an urban river and resulting risks to public health.

There is no reference of microbiological water quality in the European Union's Water Framework Directive, adapted into English law, and consequently microbial water quality is not routinely monitored in English rivers, except for two recently designated bathing water sites. To address this knowledge gap, we developed an innovative monitoring approach for quantitative assessment of combined sewer overflow (CSO) impacts on the bacteriology of receiving rivers. Our approach combines conventional and environmental DNA (eDNA) based methods to generate multiple lines of evidence for assessing risks to public health. We demonstrated this approach by investigating spatiotemporal variation in the bacteriology of the Ouseburn in northeast England for different weather conditions in the summer and early autumn of the year 2021 across eight sampling locations that comprised rural, urban, and recreational land use settings. We characterized pollution source attributes by collecting sewage from treatment works and CSO discharge at the peak of a storm event. CSO discharge was characterized by log10 values per 100 mL (average ± stdev) of 5.12 ± 0.03 and 4.90 ± 0.03 for faecal coliforms and faecal streptococci, and 6.00 ± 0.11 and 7.78 ± 0.04 for rodA and HF183 genetic markers, for E. coli and human host associated Bacteroides, respectively, indicating about 5 % sewage content. SourceTracker analysis of sequencing data attributed 72-77 % of bacteria in the downstream section of the river during a storm event to CSO discharge sources, versus only 4-6 % to rural upstream sources. Data from sixteen summer sampling events in a public park exceeded various guideline values for recreational water quality. Quantitative microbial risk assessment (QMRA) predicted a median and 95th percentile risk of 0.03 and 0.39, respectively, of contracting a bacterial gastrointestinal disease when wading and splashing around in the Ouseburn. We show clearly why microbial water quality should be monitored where rivers flow through public parks, irrespective of their bathing water designation.

Micro to macro scale analysis of the intact human renal arterial tree with Synchrotron Tomography.

Background: The kidney vasculature is exquisitely structured to orchestrate renal function. Structural profiling of the vasculature in intact rodent kidneys, has provided insights into renal haemodynamics and oxygenation, but has never been extended to the human kidney beyond a few vascular generations. We hypothesised that synchrotron-based imaging of a human kidney would enable assessment of vasculature across the whole organ. Methods: An intact kidney from a 63-year-old male was scanned using hierarchical phase-contrast tomography (HiP-CT), followed by semi-automated vessel segmentation and quantitative analysis. These data were compared to published micro-CT data of whole rat kidney. Results: The intact human kidney vascular network was imaged with HiP-CT at 25 µm voxels, representing a 20-fold increase in resolution compared to clinical CT scanners. Our comparative quantitative analysis revealed the number of vessel generations, vascular asymmetry and a structural organisation optimised for minimal resistance to flow, are conserved between species, whereas the normalised radii are not. We further demonstrate regional heterogeneity in vessel geometry between renal cortex, medulla, and hilum, showing how the distance between vessels provides a structural basis for renal oxygenation and hypoxia. Conclusions: Through the application of HiP-CT, we have provided the first quantification of the human renal arterial network, with a resolution comparable to that of light microscopy yet at a scale several orders of magnitude larger than that of a renal punch biopsy. Our findings bridge anatomical scales, profiling blood vessels across the intact human kidney, with implications for renal physiology, biophysical modelling, and tissue engineering.

The fatal trajectory of pulmonary COVID-19 is driven by lobular ischemia and fibrotic remodelling.

BACKGROUND: COVID-19 is characterized by a heterogeneous clinical presentation, ranging from mild symptoms to severe courses of disease. 9-20% of hospitalized patients with severe lung disease die from COVID-19 and a substantial number of survivors develop long-COVID. Our objective was to provide comprehensive insights into the pathophysiology of severe COVID-19 and to identify liquid biomarkers for disease severity and therapy response. METHODS: We studied a total of 85 lungs (n = 31 COVID autopsy samples; n = 7 influenza A autopsy samples; n = 18 interstitial lung disease explants; n = 24 healthy controls) using the highest resolution Synchrotron radiation-based hierarchical phase-contrast tomography, scanning electron microscopy of microvascular corrosion casts, immunohistochemistry, matrix-assisted laser desorption ionization mass spectrometry imaging, and analysis of mRNA expression and biological pathways. Plasma samples from all disease groups were used for liquid biomarker determination using ELISA. The anatomic/molecular data were analyzed as a function of patients' hospitalization time. FINDINGS: The observed patchy/mosaic appearance of COVID-19 in conventional lung imaging resulted from microvascular occlusion and secondary lobular ischemia. The length of hospitalization was associated with increased intussusceptive angiogenesis. This was associated with enhanced angiogenic, and fibrotic gene expression demonstrated by molecular profiling and metabolomic analysis. Increased plasma fibrosis markers correlated with their pulmonary tissue transcript levels and predicted disease severity. Plasma analysis confirmed distinct fibrosis biomarkers (TSP2, GDF15, IGFBP7, Pro-C3) that predicted the fatal trajectory in COVID-19. INTERPRETATION: Pulmonary severe COVID-19 is a consequence of secondary lobular microischemia and fibrotic remodelling, resulting in a distinctive form of fibrotic interstitial lung disease that contributes to long-COVID. FUNDING: This project was made possible by a number of funders. The full list can be found within the Declaration of interests / Acknowledgements section at the end of the manuscript.

Delta-8 THC use in US adults: Sociodemographic characteristics and correlates.

BACKGROUND: Use of novel cannabis products whose primary active compound is Delta-8 tetrahydrocannabinol (Delta-8 THC), an isomer of Delta-9 THC has recently surged. While Delta-8 THC has psychoactive effects and is potentially harmful, little is known about its use. We examined sociodemographic characteristics, motivations, and consumption patterns of Delta-8 THC in US adult cannabis users. METHODS: Cannabis-using adult online survey participants (N = 4,348) provided information on Delta-8 use, and other characteristics. We assessed frequencies of sociodemographics, patterns, and correlates of Delta-8 THC use. Regression models estimated adjusted odds ratios (aOR) and 95% confidence intervals (CI) of associations between past 30-day Delta-8 THC use, sociodemographic and other characteristics. FINDINGS: Among past 30-day cannabis users, 16.7% reported Delta-8 THC use. The most common consumption method was vaping concentrated formulations of Delta-8 THC (41.2%). Primary motivations for use were its legal status and perceived therapeutic benefits. Males were more likely than females to report Delta-8 THC use (aOR = 1.4, 95% CI 1.2, 1.7). Respondents residing in states with restrictions on sales of Delta-8 THC products had lower odds of Delta-8 THC use (aOR = 0.7, 95% CI 0.57, 0.86). CONCLUSIONS: Findings provide initial insight into the current state of Delta-8 THC use in the US. Given the limited knowledge on use of Delta-8 THC, and considering emergence of reports indicating its harmful effects, there is urgent need for nationally representative data to investigate correlates of Delta-8 THC use (e.g., effectiveness of state-specific restrictions on its products). Such information can guide public-health policy around Delta-8 THC use.

Validity of the DSM-5 tobacco use disorder diagnostics in adults with problematic substance use.

BACKGROUND: DSM-5 tobacco use disorder (TUD) nosology differs from DSM-IV nicotine dependence (ND) by including craving and DSM-IV abuse criteria, a lower threshold (≥ 2 criteria), and severity levels (mild; moderate; severe). We assessed concurrent and prospective validity of the DSM-5 TUD diagnosis and severity and compared validity with DSM-IV ND diagnosis. METHODS: The sample included U.S. adults with current problematic substance use and past year cigarette smoking (N = 396). Baseline assessment collected information on DSM-IV ND and DSM-5 TUD criteria, smoking-related variables, and psychopathology. Over the following 90 days, electronic daily assessments queried smoking and cigarette craving. Variables expected to be related to TUD were validators: cigarette consumption, cigarette craving scale, Fagerström Test for Nicotine Dependence, and psychiatric disorders. Regression models estimated the association of each validator with DSM-5 TUD and severity levels, and differential association between DSM-5 TUD and DSM-IV ND diagnoses. RESULTS: DSM-5 TUD and DSM-IV ND were associated with most baseline validators (p-values < 0.05), with significantly stronger associations with DSM-5 TUD for number of days smoked (p = 0.023) and cigarette craving scale (p = 0.007). Baseline DSM-5 TUD and DSM-IV ND predicted smoking and craving on any given day during follow-up, with stronger associations for DSM-5 TUD (association difference [95% CI%]: any smoking, 0.53 [0.27, 0.77]; number of cigarettes smoked, 1.36 [0.89, 1.78]; craving scale, 0.19 [0.09, 0.28]). Validators were associated with TUD severity in a dose-dependent manner. CONCLUSION: DSM-5 TUD diagnostic measures as operationalized here demonstrated concurrent and prospective validity. Inclusion of new criteria, particularly craving, improved validity and clinical relevance.

Bcar1/p130Cas is essential for ventricular development and neural crest cell remodelling of the cardiac outflow tract.

AIMS: The adapter protein p130Cas, encoded by the Bcar1 gene, is a key regulator of cell movement, adhesion, and cell cycle control in diverse cell types. Bcar1 constitutive knockout mice are embryonic lethal by embryonic days (E) 11.5-12.5, but the role of Bcar1 in embryonic development remains unclear. Here, we investigated the role of Bcar1 specifically in cardiovascular development and defined the cellular and molecular mechanisms disrupted following targeted Bcar1 deletions. METHODS AND RESULTS: We crossed Bcar1 floxed mice with Cre transgenic lines allowing for cell-specific knockout either in smooth muscle and early cardiac tissues (SM22-Cre), mature smooth muscle cells (smMHC-Cre), endothelial cells (Tie2-Cre), second heart field cells (Mef2c-Cre), or neural crest cells (NCC) (Pax3-Cre) and characterized these conditional knock outs using a combination of histological and molecular biology techniques. Conditional knockout of Bcar1 in SM22-expressing smooth muscle cells and cardiac tissues (Bcar1SM22KO) was embryonically lethal from E14.5-15.5 due to severe cardiovascular defects, including abnormal ventricular development and failure of outflow tract (OFT) septation leading to a single outflow vessel reminiscent of persistent truncus arteriosus. SM22-restricted loss of Bcar1 was associated with failure of OFT cushion cells to undergo differentiation to septal mesenchymal cells positive for SMC-specific α-actin, and disrupted expression of proteins and transcription factors involved in epithelial-to-mesenchymal transformation (EMT). Furthermore, knockout of Bcar1 specifically in NCC (Bcar1PAX3KO) recapitulated part of the OFT septation and aortic sac defects seen in the Bcar1SM22KO mutants, indicating a cell-specific requirement for Bcar1 in NCC essential for OFT septation. In contrast, conditional knockouts of Bcar1 in differentiated smooth muscle, endothelial cells, and second heart field cells survived to term and were phenotypically normal at birth and postnatally. CONCLUSION: Our work reveals a cell-specific requirement for Bcar1 in NCC, early myogenic and cardiac cells, essential for OFT septation, myocardialization and EMT/cell cycle regulation and differentiation to myogenic lineages.

Trends over time in adult cannabis use: A review of recent findings.

In the United States, policies regarding the medical and nonmedical use of cannabis are changing rapidly. In 2021, a total of 34 US states have legalized cannabis for adult medical use, and 15 of these states have legalized adult non-medical use. These changing policies have raised questions about increasing prevalences of cannabis use, changing perceptions regarding frequent use, and potentially related outcomes such as comorbid psychiatric illness or driving under the influence of cannabis. Research regarding the correlates of any and frequent cannabis use is also developing quickly. This article reviews recent empirical studies concerning (1) adult trends in cannabis use, (2) state cannabis laws and related outcomes, and (3) emerging evidence regarding how the global coronavirus 19 pandemic may impact cannabis use patterns. We summarize recent findings and conclude with suggestions to address unanticipated effects of rapidly changing cannabis laws and policies.

The mevalonate precursor enzyme HMGCS1 is a novel marker and key mediator of cancer stem cell enrichment in luminal and basal models of breast cancer.

The definitive characterization of common cancer stem cell (CSCs) subpopulations in breast cancer subtypes with distinct genotypic and phenotypic features remains an ongoing challenge. In this study, we have used a non-biased genome wide screening approach to identify transcriptional networks that may be specific to the CSC subpopulations in both luminal and basal breast cancer subtypes. In depth studies of three CSC-enriched breast cancer cell lines representing various subtypes of breast cancer revealed a striking hyperactivation of the mevalonate metabolic pathway in comparison to control cells. The upregulation of metabolic networks is a key feature of tumour cells securing growth and proliferative capabilities and dysregulated mevalonate metabolism has been associated with tumour malignancy and cellular transformation in breast cancer. Furthermore, accumulating evidence suggests that Simvastatin therapy, a mevalonate pathway inhibitor, could affect breast cancer progression and reduce breast cancer recurrence. When detailing the mevalonate pathway in breast cancer using a single-cell qPCR, we identified the mevalonate precursor enzyme, HMGCS1, as a specific marker of CSC-enriched subpopulations within both luminal and basal tumour subtypes. Down-regulation of HMGCS1 also decreased the CSC fraction and function in various model systems, suggesting that HMGCS1 is essential for CSC-activities in breast cancer in general. These data was supported by strong associations between HMGCS1 expression and aggressive features, such as high tumour grade, p53 mutations as well as ER-negativity in lymph node positive breast cancer. Importantly, loss of HMGCS1 also had a much more pronounced effect on CSC-activities compared to treatment with standard doses of Simvastatin. Taken together, this study highlights HMGCS1 as a potential gatekeeper for dysregulated mevalonate metabolism important for CSC-features in both luminal and basal breast cancer subtypes. Pharmacological inhibition of HMGCS1 could therefore be a superior novel treatment approach for breast cancer patients via additional CSC blocking functions.

Measuring diffusion exchange across the cell membrane with DEXSY (Diffusion Exchange Spectroscopy).

INTRODUCTION: To combine numerical simulations, in vitro and in vivo experiments to evaluate the feasibility of measuring diffusion exchange across the cell membrane with diffusion exchange spectroscopy (DEXSY). METHODS: DEXSY acquisitions were simulated over a range of permeabilities in nerve tissue and yeast substrates. In vitro measurements were performed in a yeast substrate and in vivo measurements in mouse tumor xenograft models, all at 9.4 T. RESULTS: Diffusion exchange was observed in simulations over a physiologically relevant range of cell permeability values. In vitro and in vivo measures also provided evidence of diffusion exchange, which was quantified with the Diffusion Exchange Index (DEI). CONCLUSIONS: Our findings provide preliminary evidence that DEXSY can be used to make in vivo measurements of diffusion exchange and cell membrane permeability.

Spatiotemporal dynamics and heterogeneity of renal lymphatics in mammalian development and cystic kidney disease.

Heterogeneity of lymphatic vessels during embryogenesis is critical for organ-specific lymphatic function. Little is known about lymphatics in the developing kidney, despite their established roles in pathology of the mature organ. We performed three-dimensional imaging to characterize lymphatic vessel formation in the mammalian embryonic kidney at single-cell resolution. In mouse, we visually and quantitatively assessed the development of kidney lymphatic vessels, remodeling from a ring-like anastomosis under the nascent renal pelvis; a site of VEGF-C expression, to form a patent vascular plexus. We identified a heterogenous population of lymphatic endothelial cell clusters in mouse and human embryonic kidneys. Exogenous VEGF-C expanded the lymphatic population in explanted mouse embryonic kidneys. Finally, we characterized complex kidney lymphatic abnormalities in a genetic mouse model of polycystic kidney disease. Our study provides novel insights into the development of kidney lymphatic vasculature; a system which likely has fundamental roles in renal development, physiology and disease.

Cancer invasion regulates vascular complexity in a three-dimensional biomimetic model.

INTRODUCTION: There is a growing appreciation for including a complex, vascularised stroma in three-dimensional (3D) tumour models to recapitulate the native tumour microenvironment in situ. METHODS: Using a compartmentalised, biomimetic, 3D cancer model, comprising a central cancer mass surrounded by a vascularised stroma, we have tested the invasive capability of colorectal cancer cells. RESULTS: We show histological analysis of dense collagen I/laminin scaffolds, forming necrotic cores with cellular debris. Furthermore, cancer cells within this 3D matrix form spheroids, which is corroborated with high EpCAM expression. We validate the invasive growth of cancer cells into the stroma through quantitative image analysis and upregulation of known invasive gene markers, including metastasis associated in colon cancer 1, matrix metalloproteinase 7 and heparinase. Tumouroids containing highly invasive HCT116 cancer masses form less complex and less branched vascular networks, recapitulating 'leaky' vasculature associated with highly metastatic cancers. Angiogenic factors regulating this were vascular endothelial growth factor A and hepatocyte growth factor active protein. Where vascular networks were formed with less invasive cancer masses (HT29), higher expression of vascular endothelial cadherin active protein resulted in more complex and branched networks. To eliminate the cell-cell interaction between the cancer mass and stroma, we developed a three-compartment model containing an acellular ring to test the chemoattractant pull from the cancer mass. This resulted in migration of endothelial networks through the acellular ring accompanied by alignment of vascular networks at the cancer/stroma boundary. DISCUSSION: This work interrogates to the gene and protein level how cancer cells influence the development of a complex stroma, which shows to be directly influenced by the invasive capability of the cancer.

Cellular organization and molecular differentiation model of breast cancer-associated fibroblasts.

BACKGROUND: The role of cancer-associated fibroblasts (CAFs) during tumour progression is obscured by the inherently complex, heterotypic nature of fibroblast cells and behaviours in various subtypes of malignancies. Therefore, we sought to identify distinct fibroblast subpopulations at the single-cell level. METHODS: Using single-cell quantitative PCR as a powerful tool to study heterogeneity and rare cell events, in a high-throughput manner a panel of gene targets are run simultaneously on transcripts isolated from single cells obtained by fluorescence-activated cell sort. Assessment of cells with stem-like characteristics was attained by anchorage-independent, anoikis-resistant culture. RESULTS: Single-cell analysis of fibroblasts and their tumour-activated counterparts demonstrated molecularly distinct cell types defined by differential expression of characteristic mesenchymal and fibroblast activation markers. Identified subpopulations presented overlapping gene expression patterns indicating transitional molecular states during fibroblast differentiation. Using single-cell resolution data we generated a molecular differentiation model which enabled the classification of patient-derived fibroblasts, validating our modelling approach. Remarkably, a subset of fibroblasts displayed expression of pluripotency markers, which was enriched for in non-adherent conditions. Yet the ability to form single-cell derived spheres was generally reduced in CAFs and upon fibroblast activation through TGFß1 ligand and cancer cell-secreted factors. Hence, our data imply the existence of putative stem/progenitor cells as a physiological feature of undifferentiated fibroblasts. CONCLUSIONS: Within this comprehensive study we have identified distinct and intersecting molecular profiles defining fibroblast activation states and propose that underlying cellular heterogeneity, fibroblasts are hierarchically organized. Understanding the molecular make-up of cellular organization and differentiation routes will facilitate the discovery of more specific markers for stromal subtypes and targets for anti-stromal therapies.

Global gene repression by the steroid receptor coactivator SRC-1 promotes oncogenesis.

Transcriptional control is the major determinant of cell fate. The steroid receptor coactivator (SRC)-1 enhances the activity of the estrogen receptor in breast cancer cells, where it confers cell survival benefits. Here, we report that a global analysis of SRC-1 target genes suggested that SRC-1 also mediates transcriptional repression in breast cancer cells. Combined SRC-1 and HOXC11 ChIPseq analysis identified the differentiation marker, CD24, and the apoptotic protein, PAWR, as direct SRC-1/HOXC11 suppression targets. Reduced expression of both CD24 and PAWR was associated with disease progression in patients with breast cancer, and their expression was suppressed in metastatic tissues. Investigations in endocrine-resistant breast cancer cell lines and SRC-1(-/-)/PyMT mice confirmed a role for SRC-1 and HOXC11 in downregulation of CD24 and PAWR. Through bioinformatic analysis and liquid chromatography/mass spectrometry, we identified AP1 proteins and Jumonji domain containing 2C (JMD2C/KDM4C), respectively, as members of the SRC-1 interactome responsible for transcriptional repression. Our findings deepen the understanding of how SRC-1 controls transcription in breast cancers.

The function of steroid receptor coactivator-1 in normal tissues and cancer.

In 1995, the steroid receptor coactivator-1 (SRC-1) was identified as the first authentic steroid receptor coactivator. Since then, the SRC proteins have remained at the epicenter of coregulator biology, molecular endocrinology and endocrine-related cancer. Cumulative works on SRC-1 have shown that it is primarily a nuclear receptor coregulator and functions to construct highly specific enzymatic protein complexes which can execute efficient and successful transcriptional activation of designated target genes. The versatile nature of SRC-1 enables it to respond to steroid dependent and steroid independent stimulation, allowing it to bind across many families of transcription factors to orchestrate and regulate complex physiological reactions. This review highlights the multiple functions of SRC-1 in the development and maintenance of normal tissue functions as well as its major role in mediating hormone receptor responsiveness. Insights from genetically manipulated mouse models and clinical data suggest SRC-1 is significantly overexpressed in many cancers, in particular, cancers of the reproductive tissues. SRC-1 has been associated with cellular proliferation and tumor growth but its major tumorigenic contributions are promotion and execution of breast cancer metastasis and mediation of resistance to endocrine therapies. The ability of SRC-1 to coordinate multiple signaling pathways makes it an important player in tumor cells' escape of targeted therapy.

Malignancies associated with tumour necrosis factor inhibitors in registries and prospective observational studies: a systematic review and meta-analysis.

OBJECTIVES: This project was undertaken to assess the risk of malignancy in patients with rheumatoid arthritis treated with tumour necrosis factor inhibitors (TNFi) in clinical practice, as recorded in prospective, observational studies. METHODS: The authors undertook comprehensive searches of MEDLINE, EMBASE, the Cochrane Database of Systematic Reviews and American College of Rheumatology, European League against Rheumatism and British Society for Rheumatology conference abstracts according to a prespecified protocol. RESULTS: The searches identified 2039 full-text papers and 1979 conference abstracts, of which 21 full texts and eight abstracts met the inclusion criteria. The pooled estimate for the risk of all-site malignancy from seven studies was 0.95 (95% CI 0.85 to 1.05). Two studies reported there was no evidence that longer exposure to TNFi agents increased the risk of malignancy. In patients with previous malignancies there was a higher risk of a new/recurring malignancy. This risk was not increased further by exposure to TNFi, although CI were wide. Results from four studies showed that patients treated with TNFi have a significantly increased risk of developing a non-melanoma skin cancer (1.45, 95% CI 1.15 to 1.76). In addition, patients are at an increased risk of developing melanoma, as the pooled estimate from two studies was 1.79 (95% CI 0.92 to 2.67). The pooled estimate for the risk of lymphoma was 1.11 (95% CI 0.70 to 1.51). CONCLUSIONS: This systematic review and meta-analysis shows that TNFi treatments do not increase the risk of malignancy, particularly lymphoma. However, they do appear to increase the risk of skin cancer, including melanoma.

Role of xanthine oxidase in dexamethasone-induced hypertension in rats.

1. Glucocorticoid-induced hypertension (GC-HT) in the rat is associated with nitric oxide-redox imbalance. 2. We studied the role of xanthine oxidase (XO), which is implicated in the production of reactive oxygen species, in dexamethasone-induced hypertension (dex-HT). 3. Thirty male Sprague-Dawley rats were divided randomly into four treatment groups: saline, dexamethasone (dex), allopurinol plus saline, and allopurinol plus dex. 4. Systolic blood pressures (SBP) and bodyweights were recorded each alternate day. Thymus weight was used as a marker of glucocorticoid activity, and serum urate to assess XO inhibition. 5. Dex increased SBP (110 +/- 2-126 +/- 3 mmHg; P < 0.001) and decreased thymus (P < 0.001) and bodyweights (P" < 0.01). Allopurinol decreased serum urate from 76 +/- 5 to 30 +/- 3 micromol/L (P < 0.001) in saline and from 84 +/- 13 to 28 +/- 2 micromol/L in dex-treated (P < 0.01) groups. 6. Allopurinol did not prevent dex-HT. This, together with our previous findings that allopurinol failed to prevent adrenocorticotrophic hormone induced hypertension, suggests that XO activity is not a major determinant of GC-HT in the rat.