The visualization of large organized chromatin domains enriched in the H3K9me2 mark within a single chromosome in a single cell

Despite considerable efforts, our understanding of the organization of higher order chromatin conformations in single cells and how these relate to chromatin marks remains poor. We have earlier invented the Chromatin In Situ Proximity (ChrISP) technique to determine proximities between chromatin fibers within a single chromosome. Here we used ChrISP to identify chromosome 11-specific hubs that are enriched in the H3K9me2 mark and that project toward the nuclear membrane in finger-like structures. Conversely, chromosome 11-specfic chromatin hubs, visualized by the presence of either H3K9me1 or H3K9me3 marks, are chromosome-wide and largely absent at the nuclear periphery. As the nuclear periphery-specific chromatin hubs were lost in the induced reduction of H3K9me2 levels, they likely represent Large Organization Chromatin in Lysine Methylation (LOCK) domains, previously identified by ChIP-seq analysis. Strikingly, the downregulation of the H3K9me2/3 marks also led to the chromosome-wide compaction of chromosome 11, suggesting a pleiotropic function of these features not recognized before. The ChrISP-mediated visualization of dynamic chromatin states in single cells thus provides an analysis of chromatin structures with a resolution far exceeding that of any other light microscopic technique.     

Suppression of immune cell function in vitro by recombinant human transforming growth factor-beta

The influence of recombinant human transforming growth factor-beta (rHuTGF-beta) on B-cell function and antigen-specific T-cell responses in vitro was investigated. The addition of 0.1 ng/ml of rHuTGF-beta to cultures of peripheral blood mononuclear cells (PBMC) stimulated with tetanus toxoid resulted in a 50% inhibition of the PBMC proliferative response as determined by [3H]thymidine incorporation. Further, rHuTGF-beta at 0.37 ng/ml caused a greater than 50% reduction in the number of immunoglobulin G-secreting cells among PBMC induced by pokeweed mitogen. rHuTGF-beta also inhibited the generation of allospecific cytotoxic T lymphocytes (CTL) in the mixed-lymphocyte reaction but had no effect on the cytolytic function of CTL generated in the absence of exogenous HuTGF-beta. The results indicate additional immunoregulatory activities for HuTGF-beta and suggest that this factor may play an important role in the regulation of the antigen-dependent immune response.     

Thermoregulatory responses of diabetic rats

1. Thermal responses and skin microcirculation were measured in streptozotocin-induced diabetic (SD) rats during acute and chronic exposure to ambient (Ta) temperatures ranging from about 5 to 35 degrees C. 2. At 28 degrees C, SD rats had higher rate of oxygen consumptions (VO2), tail skin blood flow (SKBF), but lower rectal temperatures (Tre) than saline-injected controls. 3. Chronic exposure of the SD rats to 35 and 5 degrees C caused a sharp rise and decline in Tre, respectively. 4. At 35 degrees C, hyperthermia in the SD rats was associated with greater increase in VO2 than controls, but changes in SKBF were similar in both groups. 5. At 5 degrees C, VO2 changed similarly in both the SD and control rats, but vasoconstriction was greater in the controls. 6. The data suggest that hypothermia in SD rats may be associated with impairment of vasoconstriction and hyperthermia may be related to an increase VO2 not accompanied by greater vasodilation.     

The regulation of RhoA at focal adhesions by StarD13 is important for astrocytoma cell motility

Malignant astrocytomas are highly invasive into adjacent and distant regions of the normal brain. Rho GTPases are small monomeric G proteins that play important roles in cytoskeleton rearrangement, cell motility, and tumor invasion. In the present study, we show that the knock down of StarD13, a GTPase activating protein (GAP) for RhoA and Cdc42, inhibits astrocytoma cell migration through modulating focal adhesion dynamics and cell adhesion. This effect is mediated by the resulting constitutive activation of RhoA and the subsequent indirect inhibition of Rac. Using Total Internal Reflection Fluorescence (TIRF)-based Förster Resonance Energy Transfer (FRET), we show that RhoA activity localizes with focal adhesions at the basal surface of astrocytoma cells. Moreover, the knock down of StarD13 inhibits the cycling of RhoA activation at the rear edge of cells, which makes them defective in retracting their tail. This study highlights the importance of the regulation of RhoA activity in focal adhesions of astrocytoma cells and establishes StarD13 as a GAP playing a major role in this process.     

Tyrosine Phosphorylation of Wiskott-Aldrich Syndrome Protein (WASP) by Hck Regulates Macrophage Function

We have shown previously that tyrosine phosphorylation of Wiskott-Aldrich syndrome protein (WASP) is important for diverse macrophage functions including phagocytosis, chemotaxis, podosome dynamics, and matrix degradation. However, the specific tyrosine kinase mediating WASP phosphorylation is still unclear. Here, we provide evidence that Hck, which is predominantly expressed in leukocytes, can tyrosine phosphorylate WASP and regulates WASP-mediated macrophage functions. We demonstrate that tyrosine phosphorylation of WASP in response to stimulation with CX3CL1 or via Fcγ receptor ligation were severely reduced in Hck^−/− bone marrow-derived macrophages (BMMs) or in RAW/LR5 macrophages in which Hck expression was silenced using RNA-mediated interference (Hck shRNA). Consistent with reduced WASP tyrosine phosphorylation, phagocytosis, chemotaxis, and matrix degradation are reduced in Hck^−/− BMMs or Hck shRNA cells. In particular, WASP phosphorylation was primarily mediated by the p61 isoform of Hck. Our studies also show that Hck and WASP are required for passage through a dense three-dimensional matrix and transendothelial migration, suggesting that tyrosine phosphorylation of WASP by Hck may play a role in tissue infiltration of macrophages. Consistent with a role for this pathway in invasion, WASP^−/− BMMs do not invade into tumor spheroids with the same efficiency as WT BMMs and cells expressing phospho-deficient WASP have reduced ability to promote carcinoma cell invasion. Altogether, our results indicate that tyrosine phosphorylation of WASP by Hck is required for proper macrophage functions.     

BMI modifies associations of IL-6 genotypes with insulin resistance: the Framingham Study

Objective: The ?174 interleukin (IL)‐6 gene polymorphism has been proposed as a risk factor for type 2 diabetes, but data are conflicting. Because white fat is a major source of IL‐6 in resting individuals, we tested the hypothesis that BMI modifies the association among the IL‐6 genotype, insulin resistance (IR) (measured using the homeostasis model), and risk of diabetes. Research Methods and Procedures: Outcomes were assessed in a community‐based cohort study of 1525 adults (mean age, 55.6 years; 753 men), who participated in the Framingham Offspring Study during the 1991 to 1995 examinations. Results: We found a significant interaction between IL‐6 genotype and BMI on levels of IR in men (p < 0.0001), with obese homozygotes for the minor C allele being most resistant. The IL‐6‐BMI interaction was not significant (p = 0.46) in women. Among men with the CC genotype, increasing BMI was associated with increased prevalence of diabetes [odds ratio (OR) per unit increase in BMI, 1.30; 95% confidence interval (CI), 1.11 to 1.50] but not among those with the GG (OR, 1.10; 95% CI, 0.98 to 1.22) or GC genotype (OR, 1.05; 95% CI, 0.97 to 1.14). Discussion: The ?174 IL‐6 promoter polymorphism modifies the association of obesity with IR and diabetes risk in men. Weight loss regimens targeted at reducing the risk of diabetes may be of particular benefit for men with a ?174 IL‐6 CC genotype.     

Heritability of cardiovascular and personality traits in 6,148 Sardinians

In family studies, phenotypic similarities between relatives yield information on the overall contribution of genes to trait variation. Large samples are important for these family studies, especially when comparing heritability between subgroups such as young and old, or males and females. We recruited a cohort of 6,148 participants, aged 14–102 y, from four clustered towns in Sardinia. The cohort includes 34,469 relative pairs. To extract genetic information, we implemented software for variance components heritability analysis, designed to handle large pedigrees, analyze multiple traits simultaneously, and model heterogeneity. Here, we report heritability analyses for 98 quantitative traits, focusing on facets of personality and cardiovascular function. We also summarize results of bivariate analyses for all pairs of traits and of heterogeneity analyses for each trait. We found a significant genetic component for every trait. On average, genetic effects explained 40% of the variance for 38 blood tests, 51% for five anthropometric measures, 25% for 20 measures of cardiovascular function, and 19% for 35 personality traits. Four traits showed significant evidence for an X-linked component. Bivariate analyses suggested overlapping genetic determinants for many traits, including multiple personality facets and several traits related to the metabolic syndrome; but we found no evidence for shared genetic determinants that might underlie the reported association of some personality traits and cardiovascular risk factors. Models allowing for heterogeneity suggested that, in this cohort, the genetic variance was typically larger in females and in younger individuals, but interesting exceptions were observed. For example, narrow heritability of blood pressure was approximately 26% in individuals more than 42 y old, but only approximately 8% in younger individuals. Despite the heterogeneity in effect sizes, the same loci appear to contribute to variance in young and old, and in males and females. In summary, we find significant evidence for heritability of many medically important traits, including cardiovascular function and personality. Evidence for heterogeneity by age and sex suggests that models allowing for these differences will be important in mapping quantitative traits.     

Melanopsin regulates visual processing in the mouse retina

The discovery of melanopsin-dependent inner retinal photoreceptors in mammals has precipitated a fundamental reassessment of such non-image forming (NIF) light responses as circadian photoentrainment and the pupil light reflex. By contrast, it remains unclear whether these new photoreceptors also play a role in classical image-forming vision. The retinal ganglion cells that subserve inner retinal photoreception (ipRGCs) project overwhelmingly to brain areas involved in NIF responses, indicating that, in terms of central signaling, their predominant function is non-image forming. However, ipRGCs also exhibit intraretinal communication via gap junction coupling, which could allow them to modulate classical visual pathways within this tissue. Here, we explore this second possibility by using melanopsin knockout (Opn4-/-) mice to examine the role of inner retinal photoreceptors in diurnal regulation of retinal function. By using electroretinography in wild-type mice, we describe diurnal rhythms in both the amplitude and speed of the retinal cone pathway that are a function of both prior light exposure and circadian phase. Unexpectedly, loss of the melanopsin gene abolishes circadian control of these parameters, causing significant attenuation of the diurnal variation in cone vision. Our results demonstrate for the first time a melanopsin-dependent regulation of visual processing within the retina, revealing an important function for inner retinal photoreceptors in optimizing classical visual pathways according to time of day.