Evaluation of cross-platform and interlaboratory concordance via consensus modelling of genomic measurements.

MOTIVATION: A synoptic view of the human genome benefits chiefly from the application of nucleic acid sequencing and microarray technologies. These platforms allow interrogation of patterns such as gene expression and DNA methylation at the vast majority of canonical loci, allowing granular insights and opportunities for validation of original findings. However, problems arise when validating against a "gold standard" measurement, since this immediately biases all subsequent measurements towards that particular technology or protocol. Since all genomic measurements are estimates, in the absence of a "gold standard" we instead empirically assess the measurement precision and sensitivity of a large suite of genomic technologies via a consensus modelling method called the row-linear model. This method is an application of the American Society for Testing and Materials Standard E691 for assessing interlaboratory precision and sources of variability across multiple testing sites. Both cross-platform and cross-locus comparisons can be made across all common loci, allowing identification of technology- and locus-specific tendencies. RESULTS: We assess technologies including the Infinium MethylationEPIC BeadChip, whole genome bisulfite sequencing (WGBS), two different RNA-Seq protocols (PolyA+ and Ribo-Zero) and five different gene expression array platforms. Each technology thus is characterised herein, relative to the consensus. We showcase a number of applications of the row-linear model, including correlation with known interfering traits. We demonstrate a clear effect of cross-hybridisation on the sensitivity of Infinium methylation arrays. Additionally, we perform a true interlaboratory test on a set of samples interrogated on the same platform across twenty-one separate testing laboratories. AVAILABILITY AND IMPLEMENTATION: A full implementation of the row-linear model, plus extra functions for visualisation, are found in the R package consensus at https://github.com/timpeters82/consensus. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Three-dimensional disorganization of the cancer genome occurs coincident with long-range genetic and epigenetic alterations.

A three-dimensional chromatin state underpins the structural and functional basis of the genome by bringing regulatory elements and genes into close spatial proximity to ensure proper, cell-type-specific gene expression profiles. Here, we performed Hi-C chromosome conformation capture sequencing to investigate how three-dimensional chromatin organization is disrupted in the context of copy-number variation, long-range epigenetic remodeling, and atypical gene expression programs in prostate cancer. We find that cancer cells retain the ability to segment their genomes into megabase-sized topologically associated domains (TADs); however, these domains are generally smaller due to establishment of additional domain boundaries. Interestingly, a large proportion of the new cancer-specific domain boundaries occur at regions that display copy-number variation. Notably, a common deletion on 17p13.1 in prostate cancer spanning the TP53 tumor suppressor locus results in bifurcation of a single TAD into two distinct smaller TADs. Change in domain structure is also accompanied by novel cancer-specific chromatin interactions within the TADs that are enriched at regulatory elements such as enhancers, promoters, and insulators, and associated with alterations in gene expression. We also show that differential chromatin interactions across regulatory regions occur within long-range epigenetically activated or silenced regions of concordant gene activation or repression in prostate cancer. Finally, we present a novel visualization tool that enables integrated exploration of Hi-C interaction data, the transcriptome, and epigenome. This study provides new insights into the relationship between long-range epigenetic and genomic dysregulation and changes in higher-order chromatin interactions in cancer.

Ageing-Related Changes to H3K4me3, H3K27ac, and H3K27me3 in Purified Mouse Neurons.

Neurons are central to lifelong learning and memory, but ageing disrupts their morphology and function, leading to cognitive decline. Although epigenetic mechanisms are known to play crucial roles in learning and memory, neuron-specific genome-wide epigenetic maps into old age remain scarce, often being limited to whole-brain homogenates and confounded by glial cells. Here, we mapped H3K4me3, H3K27ac, and H3K27me3 in mouse neurons across their lifespan. This revealed stable H3K4me3 and global losses of H3K27ac and H3K27me3 into old age. We observed patterns of synaptic function gene deactivation, regulated through the loss of the active mark H3K27ac, but not H3K4me3. Alongside this, embryonic development loci lost repressive H3K27me3 in old age. This suggests a loss of a highly refined neuronal cellular identity linked to global chromatin reconfiguration. Collectively, these findings indicate a key role for epigenetic regulation in neurons that is inextricably linked with ageing.

The role of chromatin remodeler SMARCA4/BRG1 in brain cancers: a potential therapeutic target.

The chromatin remodeler SMARCA4/BRG1 is a key epigenetic regulator with diverse roles in coordinating the molecular programs that underlie brain tumour development. BRG1 function in brain cancer is largely specific to the tumour type and varies further between tumour subtypes, highlighting its complexity. Altered SMARCA4 expression has been linked to medulloblastoma, low-grade gliomas such as oligodendroglioma, high-grade gliomas such as glioblastoma and atypical/teratoid rhabdoid tumours. SMARCA4 mutations in brain cancer predominantly occur in the crucial catalytic ATPase domain, which is associated with tumour suppressor activity. However, SMARCA4 is opposingly seen to promote tumourigenesis in the absence of mutation and through overexpression in other brain tumours. This review explores the multifaceted interaction between SMARCA4 and various brain cancer types, highlighting its roles in tumour pathogenesis, the pathways it regulates, and the advances that have been made in understanding the functional relevance of mutations. We discuss developments made in targeting SMARCA4 and the potential to translate these to adjuvant therapies able to enhance current methods of brain cancer treatment.

The impact of denial on health-related quality of life in patients with HIV.

BACKGROUND/PURPOSE: The purpose of this study was to examine the influence of denial coping on quality of life (QOL) over time among individuals living with HIV, as denial has been understudied as a coping strategy within the literature on HIV/AIDS. METHODS: In a sample of 65 adult men and women, we used multilevel linear modeling to test trajectories of change in physical and mental health-related QOL across baseline, 3, 6, and 12 months, including denial as a predictor and gender as a moderator. RESULTS: The use of denial coping was associated with lower physical and mental health-related QOL at baseline. Denial coping predicted an increase in QOL over time, though QOL remained low in those who practiced denial coping. Men's baseline mental health-related QOL was more negatively affected by denial coping than women's. Women tended to increase in QOL more slowly over time compared to men. CONCLUSION: Reliance on denial as a coping strategy is associated with poorer physical and mental health-related QOL in an HIV-positive population, though participants who engaged in denial also displayed more rapid improvement in their QOL over time. Men and women displayed different rates of improvement in QOL, indicating a need for gender-based treatment approaches. Future research should examine the complex role of denial on change in QOL.

BRG1 knockdown inhibits proliferation through multiple cellular pathways in prostate cancer.

BACKGROUND: BRG1 (encoded by SMARCA4) is a catalytic component of the SWI/SNF chromatin remodelling complex, with key roles in modulating DNA accessibility. Dysregulation of BRG1 is observed, but functionally uncharacterised, in a wide range of malignancies. We have probed the functions of BRG1 on a background of prostate cancer to investigate how BRG1 controls gene expression programmes and cancer cell behaviour. RESULTS: Our investigation of SMARCA4 revealed that BRG1 is over-expressed in the majority of the 486 tumours from The Cancer Genome Atlas prostate cohort, as well as in a complementary panel of 21 prostate cell lines. Next, we utilised a temporal model of BRG1 depletion to investigate the molecular effects on global transcription programmes. Depleting BRG1 had no impact on alternative splicing and conferred only modest effect on global expression. However, of the transcriptional changes that occurred, most manifested as down-regulated expression. Deeper examination found the common thread linking down-regulated genes was involvement in proliferation, including several known to increase prostate cancer proliferation (KLK2, PCAT1 and VAV3). Interestingly, the promoters of genes driving proliferation were bound by BRG1 as well as the transcription factors, AR and FOXA1. We also noted that BRG1 depletion repressed genes involved in cell cycle progression and DNA replication, but intriguingly, these pathways operated independently of AR and FOXA1. In agreement with transcriptional changes, depleting BRG1 conferred G1 arrest. CONCLUSIONS: Our data have revealed that BRG1 promotes cell cycle progression and DNA replication, consistent with the increased cell proliferation associated with oncogenesis.

Epigenetic reprogramming at estrogen-receptor binding sites alters 3D chromatin landscape in endocrine-resistant breast cancer.

Endocrine therapy resistance frequently develops in estrogen receptor positive (ER+) breast cancer, but the underlying molecular mechanisms are largely unknown. Here, we show that 3-dimensional (3D) chromatin interactions both within and between topologically associating domains (TADs) frequently change in ER+ endocrine-resistant breast cancer cells and that the differential interactions are enriched for resistance-associated genetic variants at CTCF-bound anchors. Ectopic chromatin interactions are preferentially enriched at active enhancers and promoters and ER binding sites, and are associated with altered expression of ER-regulated genes, consistent with dynamic remodelling of ER pathways accompanying the development of endocrine resistance. We observe that loss of 3D chromatin interactions often occurs coincidently with hypermethylation and loss of ER binding. Alterations in active A and inactive B chromosomal compartments are also associated with decreased ER binding and atypical interactions and gene expression. Together, our results suggest that 3D epigenome remodelling is a key mechanism underlying endocrine resistance in ER+ breast cancer.

Integrated epigenomic analysis stratifies chromatin remodellers into distinct functional groups.

BACKGROUND: ATP-dependent chromatin remodelling complexes are responsible for establishing and maintaining the positions of nucleosomes. Chromatin remodellers are targeted to chromatin by transcription factors and non-coding RNA to remodel the chromatin into functional states. However, the influence of chromatin remodelling on shaping the functional epigenome is not well understood. Moreover, chromatin remodellers have not been extensively explored as a collective group across two-dimensional and three-dimensional epigenomic layers. RESULTS: Here, we have integrated the genome-wide binding profiles of eight chromatin remodellers together with DNA methylation, nucleosome positioning, histone modification and Hi-C chromosomal contacts to reveal that chromatin remodellers can be stratified into two functional groups. Group 1 (BRG1, SNF2H, CHD3 and CHD4) has a clear preference for binding at 'actively marked' chromatin and Group 2 (BRM, INO80, SNF2L and CHD1) for 'repressively marked' chromatin. We find that histone modifications and chromatin architectural features, but not DNA methylation, stratify the remodellers into these functional groups. CONCLUSIONS: Our findings suggest that chromatin remodelling events are synchronous and that chromatin remodellers themselves should be considered simultaneously and not as individual entities in isolation or necessarily by structural similarity, as they are traditionally classified. Their coordinated function should be considered by preference for chromatin features in order to gain a more accurate and comprehensive picture of chromatin regulation.

Neurocognitive Profile of a Woman with Susac's Syndrome: Further Evidence of Cognitive Variability.

OBJECTIVE: Susac's syndrome is a rare autoimmune disease characterized by encephalopathy, retinal artery occlusions, hearing loss, and lesions to the corpus callosum. To date, only four papers (five cases) have described the neuropsychological effects of the syndrome. Extant case reports of Susac's syndrome have documented varying levels of cognitive impairment; some studies have identified diffuse cerebral dysfunction, while others describe more focal impairments in attention, memory, and executive functioning. METHOD: The goal of this case study was to provide comprehensive neurocognitive data obtained from another case of confirmed Susac's syndrome. As such, we present the case of a 42-year-old woman with a two-year history of Susac's syndrome. At the time of the neuropsychological evaluation, the patient described ongoing difficulties with memory, word-finding problems, problems with math, and poor problem-solving. Physical/sensory changes included hearing loss, tinnitus, and migraines. RESULTS: Neuropsychological test results revealed the patient to be a woman of estimated average to high average premorbid intellect who exhibited a number of focal inefficiencies in the context of a generally intact profile. Particular cognitive weaknesses emerged on select tasks involving visuoconstruction, encoding of a wordlist, and bilateral speeded finger oscillations. She also committed three failures to maintain set on a task of cognitive flexibility. There was no evidence of memory deficits. CONCLUSIONS: Our findings provide further evidence of cognitive interindividual variability in a confirmed case of Susac's syndrome.

How T cells use LFA-1 to attach and migrate.

The T lymphocyte (or T cell) has classically been perceived to be a passive circular cell attaching to other cells or fibrils of the extracellular matrix when its integrins become activated. We now understand that the modus operandi of the T cell is migration. These cells are proving to be impressively fast migrators, clocking up basal speeds of approximately 10-15 microm/min which makes them amongst the fastest movers recorded to date. Therefore, migration is the business of the T cell and in this review we will discuss how its motility is regulated and what functions this activity makes possible.

Quality of life measures for nursing home residents.

BACKGROUND: Quality of life (QOL) is a goal for nursing home residents, but measures are needed to tap this phenomenon. METHODS: In-person QOL interviews were attempted for 1988 residents, stratified by cognitive functioning, from 40 nursing homes in five states. Likert-type response options were used with reversion to dichotomous responses when necessary; z-score transformations were used to combine the formats. Tests of internal consistency and confirmatory factor analysis were performed; cluster analysis was used to shorten the scales. Correlations between domain scores were examined, and tests of convergent validity performed. Analyses were repeated for subgroups based on cognitive functioning levels. RESULTS: Long QOL scales were constructed for 1316 of the 1988 residents, including many with substantial cognitive impairment. Confirmatory factor analysis confirmed 10 QOL domains. Cronbach alphas ranged from.76 to.52. The majority (93%) of the 45 possible interscale correlations among domains were below.l4 and the rest were between.4 and.5. QOL scales were correlated with, but distinct from, residents' emotions ratings and overall satisfaction, and each was correlated with a corresponding summary rating for the domain. CONCLUSIONS: QOL can be feasibly measured from resident self-report for much of the nursing home population, including cognitively impaired residents. Additional research is suggested on the measures, but the approach has promise for regulation, continuous quality improvement, and public information.

Using resident reports of quality of life to distinguish among nursing homes.

PURPOSE: We used measures created to assess the quality of life (QOL) of nursing home residents to distinguish among nursing facilities. DESIGN AND METHODS: We statistically adjusted scores for 10 QOL domains derived from standardized interviews with nursing home residents for age, gender, activities of daily living functioning, cognitive functioning, and length of stay, and then we aggregated them to the facility level. We compared the patterns across a sample of 40 facilities. We correlated facility characteristics with QOL scores. RESULTS: The pattern of QOL scores for each of the 10 domains was generally consistent within a given facility. Although resident characteristics played a major role in explaining variance, there were significant effects of facilities as well. Some modest relationships were found between facility characteristics such as ownership, percentage of private rooms, and rural-urban location and facility QOL scores. No effect of facility size was detected. IMPLICATIONS: This article shows that it is possible to differentiate among facilities on the basis of resident self-reported QOL. On the basis of our analysis, we find that a sample of 28 residents per facility is sufficient to generate a reliable QOL score for each of the domains studied.

Calpain 2 controls turnover of LFA-1 adhesions on migrating T lymphocytes.

The immune cells named T lymphocytes circulate around the body fulfilling their role in immunosurveillance by monitoring the tissues for injury or infection. To migrate from the blood into the tissues, they make use of the integrin LFA-1 which is exclusively expressed by immune cells. These highly motile cells attach and migrate on substrates expressing the LFA-1 ligand ICAM-1. The molecular events signaling LFA-1 activation and adhesion are now reasonably well identified, but the process of detaching LFA-1 adhesions is less understood. The cysteine protease calpain is involved in turnover of integrin-mediated adhesions in less motile cell types. In this study we have explored the involvement of calpain in turnover of LFA-1-mediated adhesions of T lymphocytes. Using live cell imaging and immunohistochemistry, we demonstrate that turnover of adhesions depends on the Ca2+-dependent enzyme, calpain 2. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition results in inefficient disassembly of LFA-1 adhesions causing T lymphocyte elongation and shedding of LFA-1 clusters behind the migrating T lymphocytes. We show that calpain 2 is distributed throughout the T lymphocyte, but is most active at the trailing edge as detected by expression of its fluorescent substrate CMAC,t-BOC-Leu-Met. Extracellular Ca2+ entry is essential for the activity of calpain 2 that is constantly maintained as the T lymphocytes migrate. Use of T cells from a patient with mutation in ORAI1 revealed that the major calcium-release-activated-calcium channel is not the ion channel delivering the Ca2+. We propose a model whereby Ca2+ influx, potentially through stretch activated channels, is sufficient to activate calpain 2 at the trailing edge of a migrating T cell and this activity is essential for the turnover of LFA-1 adhesions.

T-cell integrins: more than just sticking points.

T cells use integrins in essentially all of their functions. They use integrins to migrate in and out of lymph nodes and, following infection, to migrate into other tissues. At the beginning of an immune response, integrins also participate in the immunological synapse formed between T cells and antigen-presenting cells. Because the ligands for integrins are widely expressed, integrin activity on T cells must be tightly controlled. Integrins become active following signalling through other membrane receptors, which cause both affinity alteration and an increase in integrin clustering. Lipid raft localization may increase integrin activity. Signalling pathways involving ADAP, Vav-1 and SKAP-55, as well as Rap1 and RAPL, cause clustering of leukocyte function-associated antigen-1 (LFA-1; integrin alphaLbeta2). T-cell integrins can also signal, and the pathways dedicated to the migratory activity of T cells have been the most investigated so far. Active LFA-1 causes T-cell attachment and lamellipodial movement induced by myosin light chain kinase at the leading edge, whereas RhoA and ROCK cause T-cell detachment at the trailing edge. Another important signalling pathway acts through CasL/Crk, which might regulate the activity of the GTPases Rac and Rap1 that have important roles in T-cell migration.

Interferon gamma suppresses glucocorticoid augmentation of macrophage clearance of apoptotic cells.

One of beneficial effects of glucocorticoids (GC) in inflammation may be the augmentation of macrophages' capacity for phagocytosis of apoptotic cells, a process that has a central role in resolution of inflammation. Here we define the phenotype of GC-treated monocyte-derived macrophages, comparing to IFN-gamma-treated and IL-4-treated monocyte-derived macrophages and combinatorial treatment. Our data indicate that the cytokine microenvironment at an inflammatory site will critically determine monocyte functional capacity following treatment with GC. In particular, whilst GC exert dominant regulatory effects over IFN-gamma in terms of cell surface receptor repertoire and morphology, the acquisition of a macrophage capacity for clearance of apoptotic cells is prevented by combined treatment. In terms of mechanism, GC augmentation of phagocytosis was reversed even when monocytes were pre-incubated with GC for the first 24 h of culture, a period that is critical for induction of a highly phagocytic macrophage phenotype. These findings have important implications for the effectiveness of GC in promoting acquisition of a pro-phagocytic macrophage phenotype in inflammatory diseases associated with high levels of IFN-gamma