A poised chromatin platform for TGF-β access to master regulators

Specific chromatin marks keep master regulators of differentiation silent yet poised for activation by extracellular signals. We report that nodal TGF-β signals use the poised histone mark H3K9me3 to trigger differentiation of mammalian embryonic stem cells. Nodal receptors induce the formation of companion Smad4-Smad2/3 and TRIM33-Smad2/3 complexes. The PHD-Bromo cassette of TRIM33 facilitates binding of TRIM33-Smad2/3 to H3K9me3 and H3K18ac on the promoters of mesendoderm regulators Gsc and Mixl1. The crystal structure of this cassette, bound to histone H3 peptides, illustrates that PHD recognizes K9me3, and Bromo binds an adjacent K18ac. The interaction between TRIM33-Smad2/3 and H3K9me3 displaces the chromatin-compacting factor HP1γ, making nodal response elements accessible to Smad4-Smad2/3 for Pol II recruitment. In turn, Smad4 increases K18 acetylation to augment TRIM33-Smad2/3 binding. Thus, nodal effectors use the H3K9me3 mark as a platform to switch master regulators of stem cell differentiation from the poised to the active state.     

A Virulence and Antimicrobial Resistance DNA Microarray Detects a High Frequency of Virulence Genes in Escherichia coli Isolates from Great Lakes Recreational Waters

Escherichia coli is generally described as a commensal species with occasional pathogenic strains. Due to technological limitations, there is currently little information concerning the prevalence of pathogenic E. coli strains in the environment. For the first time, using a DNA microarray capable of detecting all currently described virulence genes and commonly found antimicrobial resistance genes, a survey of environmental E. coli isolates from recreational waters was carried out. A high proportion (29%) of 308 isolates from a beach site in the Great Lakes carried a pathotype set of virulence-related genes, and 14% carried antimicrobial resistance genes, findings consistent with a potential risk for public health. The results also showed that another 8% of the isolates had unusual virulence gene combinations that would be missed by conventional screening. This new application of a DNA microarray to environmental waters will likely have an important impact on public health, epidemiology, and microbial ecology in the future.     

Targeted deletion of integrin-linked kinase reveals a role in T-cell chemotaxis and survival

Integrin-linked kinase (ILK) is a serine/threonine kinase that is important in cell-matrix interactions and cell signaling. To examine the role of ILK in leukocyte trafficking and survival, we generated T cell-specific ILK knockouts by breeding ILK(flox/flox) mice to transgenic mice expressing Cre recombinase under control of the Lck proximal promoter. Thymic T cells from Lck-Cre(+)/ILK(flox/flox) mice had a marked reduction (>95%) in ILK protein levels. Thymic cellularity was comparable in 3- to 4-week-old mice, but a threefold diminution of thymic T cells became evident by 6 to 8 weeks of age in the T cell-specific ILK knockout mice due to increased cell death of double-positive (DP) T cells. Analysis of peripheral T cells by quantitative PCR and by breeding Lck-Cre(+)/ILK(flox/flox) mice to a YFP-transgenic reporter strain demonstrated an approximate 20-fold enrichment of ILK-competent cells, suggesting these cells have a competitive advantage in trafficking to and/or survival in peripheral lymphatic organs. We explored mechanisms related to altered cell trafficking and survival that might explain the decreases in thymic cellularity and enrichment for ILK-competent cells in the spleen and lymph nodes. We observed a >50% reduction in chemotaxis of ILK-deficient T cells to the chemokines CXCL12 (stromal cell-derived factor [SDF]-1alpha) and CCL19 (macrophage inflammatory protein [MIP]-3beta), as well as enhanced apoptosis of ILK-deficient cells upon stress. Signaling studies in ILK-deficient T cells demonstrated diminished phosphorylation of Akt on the activating phosphorylation site, Ser 473, and a concordant decrease in Akt kinase activity following stimulation with the chemokine SDF-1. Rac1 activation was also markedly diminished in ILK-deficient T cells following chemokine stimulation. These data extend the role of ILK to immune-cell trafficking and survival via modulation of Akt- and Rac-dependent substrates, and have implications for cell recruitment in both homeostatic and pathological processes.     

Trabeculectomy with mitomycin C in glaucoma associated with uveitis

The patients with uveitic glaucoma are at high risk for failure following drainage surgery because of young age of these patients, preoperative long-term control of inflammation and postoperative complications. Twenty-two trabeculectomies performed in 22 patients with uveitic glaucoma were retrospectively evaluated to analyze the effect of intraoperative application of mitomycin C (MMC). Success rates, postoperative levels of intraocular pressure (IOP) and postoperative complications were studied. After a mean follow-up of 10.6 months (range, 5-28 months), 15 patients (68.2%) achieved IOP of 21mmHg or less without antiglaucoma medications. There were statistically significant reduction in IOP postoperatively during the period studied (p < 0.001). Early postoperative complications included chorioidal detachment (9.1%), shallow anterior chamber (9.1%), hyphema (13.6%), macular edema (4.5%) and raised IOP (27.3%). Late postoperative complications included exacerbation of uveitis (4.5%), macular edema (4.5%), cataract (22.7%) and raised IOP (31.8%). The eyes with raised IOP needed additional antiglaucoma medication. The results of this retrospective and uncontrolled study suggest that intraoperative application of MMC may be a good option for enhancement of short-term trabeculectomy success rates in patients with uveitic glaucoma.     

Full-thickeness anterior blepharotomy and transpalpebral fat decompression in Graves' orbitopathy

A chief morbidity of Graves eye disease is eyelid retraction and exophthalmus. Transpalpebral orbital fat removal accomplished with full thickness anterior blepharotomy was performed in 4 patients (5 orbits). Preoperative and postoperative ocular exposure symptoms, visual acuity, upper eyelid retraction and proptosis were evaluated. In all 5 operated orbits preoperative symptoms resolved; good results were achieved from the functional and cosmetic point of view. Full-thickness anterior blepharotomy combined with fat decompression is a safe and effective surgery for patients with upper eyelid retraction and exophthalmus due to enlarged orbital fat compartment.     

Evidence of involvement of leptin and IL-6 peptides in the action of interferon-beta in secondary progressive multiple sclerosis

Leptin is a peptide hormone which acts on cells of immune system by influencing the production of cytokines. Serum leptin levels and cytokine production by peripheral blood mononuclear cells (PBMC) were measured in 18 secondary progressive multiple sclerosis (SPMS) patients under IFN-beta-1b treatment. There were no overall effects on leptin, interleukin-6 (IL-6), IL-10 and IL-12 p40 after 2, 6 and 12 months of treatment. However, leptin and IL-6 decreased after 6 and 12 months of treatment in 12 patients who did not show progression of disability. Thus, our pilot data show that the beneficial effect of IFN-beta on some SPMS patients might be associated with the reduced levels of leptin and reduced IL-6 production by PBMC.     

Ultrastructure of the contact surfaces of Passalurus ambiguus (Rudolphi, 1819) (Nematoda)

The ultrastructure of the contact surfaces (integument and intestinal wall) of the nematode Passalurus ambiguus has been studied. The integument is composed according to the scheme common for all nematodes and includes a cuticle, hypodermis and a muscular layer. The specificity is with regard to the epicuticle, the different number of the cuticular sublayers in the anterior, central and the posterior parts of the worm body and the absence of a basal cuticular membrane. The intestinal wall consists of epithelial cells with microvilli. The ultrastructural characteristics of both contact surfaces indicate their main functions--absorption, secretion, transport, protection, movement, etc.     

CD40 ligation prevents onset of tolerogenic properties in human dendritic cells treated with CTLA-4-Ig

The synthetic immunomodulator cytotoxic T lymphocyte antigen 4-Ig (CTLA-4-Ig) initiates effects in human monocyte-derived dendritic cells (DC) that rely on immunosuppressive tryptophan catabolism. However, it is unable to induce suppressive properties in DC matured by CD40 engagement. Thus, CD40-driven events may physiologically set human DC free from restraint by regulatory cells expressing surface CTLA-4.     

The NK1 receptor localizes to the plasma membrane microdomains, and its activation is dependent on lipid raft integrity

The spatial targeting of receptors to discrete domains within the plasma membrane allows their preferential coupling to specific effectors, which is essential for rapid and accurate discrimination of signals. Efficiency of signaling is further increased by protein and lipid segregation within the plasma membrane. We have previously demonstrated the importance of raft-mediated signaling in the regulation of smooth and skeletal muscle cell contraction. Since G protein-coupled receptors (GPCRs) are key components in the regulation of smooth muscle contraction-relaxation cycles, it is important to determine whether GPCR signaling is mediated by lipid rafts and raft-associated molecules. Neurokinin 1 receptor (NK1R) is expressed in central and peripheral nervous system as well as in endothelial and smooth muscle cells and involved in mediation of pain, inflammation, exocrine secretion, and smooth muscle contraction. The NK1 receptor was transiently expressed in HEK293 and HepG2 cell lines and its localization in membrane microdomains investigated using biochemical methods and immunofluorescent labeling. We show that the NK1 receptor, similar to the earlier described beta(2)-adrenergic receptor and G proteins, localizes to lipid rafts and caveolae. Protein kinase C (PKC) is one of the downstream effectors of the NK1 activation. Its active form translocates from the cytoplasm to the plasma membrane. Upon stimulation of the NK1 receptor with Substance P, the activated PKC relocated to lipid rafts. Using cholesterol extraction and replenishment assays we show that activation of NK1 receptor is dependent on the microarchitecture of the plasma membrane: NK1R-mediated signaling was abolished after cholesterol depletion of the receptor-expressing cells with methyl-beta-cyclodextrin. Our results demonstrate that reorganization of the plasma membrane has an effect on the activation of the raft-associated NK1R and the down-stream events such as recruitment of protein kinases.     

Multiscale modeling of the cardiovascular system: application to the study of pulmonary and coronary perfusions in the univentricular circulation

The objective of this study is to compare the coronary and pulmonary blood flow dynamics resulting from two configurations of systemic-to-pulmonary artery shunts currently utilized during the Norwood procedure: the central (CS) and modified Blalock Taussig (MBTS) shunts. A lumped parameter model of the neonatal cardiovascular circulation and detailed 3-D models of the shunt based on the finite volume method were constructed. Shunt sizes of 3, 3.5 and 4 mm were considered. A multiscale approach was adopted to prescribe appropriate and realistic boundary conditions for the 3-D models of the Norwood circulation. Results showed that the average shunt flow rate is higher for the CS option than for the MBTS and that pulmonary flow increases with shunt size for both options. Cardiac output is higher for the CS option for all shunt sizes. Flow distribution between the left and the right pulmonary arteries is not completely balanced, although for the CS option the discrepancy is low (50-51% of the pulmonary flow to the right lung) while for the MBTS it is more pronounced with larger shunt sizes (51-54% to the left lung). The CS option favors perfusion to the right lung while the MBTS favors the left. In the CS option, a smaller percentage of aortic flow is distributed to the coronary circulation, while that percentage rises for the MBTS. These findings may have important implications for coronary blood flow and ventricular function.