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Cytokine Treatment of Renal Cell Carcinoma
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The importance of B7-H molecules for the T cell/tumor communication and its impact on renal cell carcinoma (RCC) progression and prognosis has been recently described. Cytokine treatment of RCC has earlier been shown to be beneficial in preclinical settings, but its clinical implementation has not proven to be as effective. This might be partially explained by the yet incomplete picture of cellular alterations in tumor cells upon cytokine treatment investigated in detail in this study.
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[
"Dysregulated bone morphogenetic proteins (BMP) may contribute to the development and progression of renal cell carcinoma (RCC). Herein, we report that BMP-6 promotes the growth of RCC by interleukin (IL)-10-mediated M2 polarization of tumor-associated macrophages (TAM). BMP-6-mediated IL-10 expression in macrophages required Smad5 and STAT3. In human RCC specimens, the three-marker signature BMP-6/IL-10/CD68 was associated with a poor prognosis. Furthermore, patients with elevated IL-10 serum levels had worse outcome after surgery. Together, our results suggest that BMP-6/macrophage/IL-10 regulates M2 polarization of TAMs in RCC.",
"Renal cell carcinoma (RCC) is considered a cytokine-responsive tumor. The clinical course of a patient may thus be influenced by the patient's capacity to produce distinct cytokines. Therefore, cytokine gene polymorphisms in RCC patients were analyzed to determine haplotype combinations with prognostic significance. A selection of 21 single nucleotide polymorphisms within the promoter regions of 13 cytokine genes were analyzed in a cross-sectional single-center study of 80 metastatic RCC patients. Univariate and multivariate analyses and the Cox forward-stepwise regression model were chosen to assess genetic risk factors. Multivariate Cox regression analysis confirmed by a bootstrap technique identified the heterozygous IL4 genotype -589T-33T/-589C-33C as an independent prognostic risk factor (risk ratio, 3.1; P < .01; 95% CI, 1.4 to 6.9; adjusted for age, sex, and nuclear grading) in metastatic RCC patients. IL4 haplotype -589T-33T and -589C-33C were found with a frequency of 0.069 and 0.925, respectively, which represents a two-fold decrease of IL4 haplotype -589T-33T (P < .01) and an increase of IL4 haplotype -589C-33C frequency (P < .05) in metastatic RCC compared with other white reference study populations. The median overall survival was decreased 3.5-fold (P < .05) in heterozygote patients carrying IL4 haplotype -589T-33T and -589C-33C (3.78 months) compared with patients homozygote for IL4 haplotype -589C-33C (13.44 months). In addition, a linkage disequilibrium between the IL4 gene and the KIF3A gene was detected. Our findings indicate that IL4 promoter variants influence prognosis in patients with metastatic RCC and suggest that genetically determined interleukin-4 (IL-4) production affects the clinical course of the disease possibly through regulation of immune surveillance.",
"Immune cytokines have been shown to play important roles in regulating immune cell functions as well as neoplastic cells. Interleukin-4 (IL4), primarily known as a B-cell growth factor, can also activate and differentiate other immune cells. This cytokine has recently been shown to have immunotherapeutic benefit in tumor-bearing hosts. The present study assessed the effect on human renal cell carcinoma cell lines of recombinant IL4 alone and in combination with recombinant gamma-interferon (IFN) or recombinant alpha-tumor necrosis factor (TNF). IL4 inhibited cell growth of all lines at 250-500 units/ml in a differential manner. Expression of IL4 receptors was demonstrated on renal cell carcinomas. Overall, IFN (500 units/ml) alone inhibited cell growth; however, TNF (500 units/ml) was not as strong an inhibitor. When IL4 was combined with IFN or TNF there was a significant augmentation of cell growth inhibition and modulation of cell morphology of the cell lines. Tumor-associated ganglioside antigens (NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer, GalNAc beta 1-4 (NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer, and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer) HLA class I, HLA-DR, and beta 2-microglobulin on the cell surface of renal cancer lines were assessed by flow cytometry and radiometric binding assay. IL4 alone or in combination with other cytokines modulated HLA class I and HLA-DR expression. IL4 and IFN consistently enhanced NeuAc alpha 2-8NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer and GalNAc beta 1-4(NeuAc alpha 2-8NeuAc alpha 2-3)Gal beta 1-4Glc beta 1-1'Cer expression on individual cell lines. The study demonstrated that IL4 alone or in combination with other cytokines can significantly inhibit growth, and modulate the expression of surface major histocompatibility and tumor-associated antigens of renal cell carcinomas.",
"Tumor-associated macrophages (TAMs) derived from peripheral blood monocytes recruited into the renal cell carcinoma (RCC) microenvironment. In response to inflammatory stimuli, macrophages undergo M1 (classical) or M2 (alternative) activation. M1 cells produce high levels of inflammatory cytokines, such as tumor necrosis factor-α, interleukin (IL)-12, IL-23 and IL-6, while M2 cells produce anti-inflammatory cytokines, such as IL-10, thus contributing to RCC-related immune dysfunction. The presence of extensive TAM infiltration in RCC microenvironment contributes to cancer progression and metastasis by stimulating angiogenesis, tumor growth, and cellular migration and invasion. Moreover, TAMs are involved in epithelial-mesenchymal transition of RCC cancer cells and in the development of tumor resistance to targeted agents. Interestingly, macrophage autophagy seems to play an important role in RCC. Based on this scenario, TAMs represent a promising and effective target for cancer therapy in RCC. Several strategies have been proposed to suppress TAM recruitment, to deplete their number, to switch M2 TAMs into antitumor M1 phenotype and to inhibit TAM-associated molecules. In this review, we summarize current data on the essential role of TAMs in RCC angiogenesis, invasion, impaired anti-tumor immune response and development of drug resistance, thus describing the emerging TAM-centered therapies for RCC patients.",
"While previous reports clearly demonstrated antiproliferative effects of IL-4 on renal cell carcinoma (RCC) in vitro, the administration of IL-4 to patients with metastatic RCC in clinical trials could not recapitulate the promising preclinical results. In the present study we wanted to examine the context of IL-4 action and to establish conditions of enhanced IL-4 efficacy. Primary and permanent human RCC cells were cultured in either serum-supplemented or chemically defined, serum-free culture medium in the presence or absence of cytokines. Cell proliferation was assessed as [(3)H]-thymidine incorporation. Cell apoptosis was measured using the fluorescent DNA intercalator 7-aminoactinomycin D and flow cytometry. In addition, culture media conditioned by RCC were subjected to cytokine antibody array and cytokine multiplex analysis. Our results indicate that the previously reported antiproliferative effects of IL-4 are serum-dependent. Under serum-free conditions, IL-4 failed to exhibit growth-inhibitory effects or was even growth-stimulatory. In a chemically defined, serum-free medium (AIM-V), however, IL-4 inhibited the TNF-alpha induced proliferation of RCC. IL-4 and TNF-alpha synergistically induced apoptosis of RCC as well as a complex cytokine response by RCC, which included the synergistic upregulation of RANTES and MCP-1. IL-4 alone has little effect on the spontaneous proliferation of RCC but can prevent the enhancement of proliferation induced by growth promoters like FBS and TNF-alpha. The concomitant growth inhibitory, apoptosis-inducing, and cytokine-enhancing effects of IL-4 in combination with TNF-alpha on RCC support the view that Th2 cytokines may be required for productive immune responses against RCC.",
"We investigated the biologic meaning of tumor-associated macrophages (TAM) in renal cell carcinoma (RCC). The study group comprised of 83 patients with RCC. TAM was isolated by plastic adherence following enzymatic digestion of surgically removed tumor tissues. In some of the patients, monocytes were also isolated from peripheral blood mononuclear cells by plastic adherence. When TAM and monocytes were compared in the same patients, TAM produced interleukin 6 (IL-6), tumor necrosis factor alpha (TNFalpha) and interleukin 1beta (IL-1beta) without lipopolysaccharide (LPS) stimulation, while monocytes hardly produced IL-6, TNFalpha and IL-1beta without LPS stimulation. However, with LPS stimulation, monocytes produced more IL-6, TNFalpha and IL-1beta than TAM. In stage T1 RCC patients, there was a significant positive correlation between TNFalpha production of TAM and tumor size. In order to investigate the effects of TAM on cancer cells, TAM was co-cultured with A498, K562 and in some cases, with short-term established RCC lines for 96 h. As a result, TAM largely enhanced cell proliferation. These results suggested that TAM may play an important role in certain steps of tumor progression.",
"Bindarit (or 2-[(1-benzyl-indazol-3-yl)methoxy]-2-methyl propionic acid) reduces heat induced denaturation of bovine and rat serum albumin in vitro (EC50 = 8.5 and 65 micrograms/ml, respectively) and inhibits heat induced serum albumin denaturation after in vivo (12.5-25-50 mg/kg po) administration in rats. To assess the relationship between protein denaturation and the development of chronic inflammatory diseases, the drug (0.5 or 0.12% medicated diet) was studied in comparison with indomethacin (1 mg/kg po daily) in rats injected with complete Freund's adjuvant. Bindarit appeared different from aspirin-like drugs, antiinflammatory steroids and immunosuppressants because it does not reduce primary inflammation of arthritic rats and was shown to be completely inactive on cyclo and lipooxygenase activity in vitro and on immune reactions of mice in vivo. Nevertheless, the drug strongly reduced the development of the secondary phase of adjuvant induced arthritis. The most significant effect of bindarit in this phase was a strong inhibition of serum albumin denaturation in arthritic rats. Assessment of both electrophoretic and quantitative changes suggests that the reduction of albumin during inflammation is due, at least in part, to a denaturation of native albumin, which loses its electrophoretic mobility. The involvement of protein denaturation in the production of new antigenic determinants, their pathogenic relevance in the development of adjuvant arthritis and the possibility that protein stabilization by bindarit could be the mechanism of action of the drug are discussed."
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Extracellular vesicles in the pathogenesis of idiopathic pulmonary fibrosis
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Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and irreversible interstitial lung disease caused by aberrant deposition of extracellular matrix in the lungs with significant morbidity and mortality. The therapeutic choices for IPF remain limited. Extracellular vesicles (EVs), as messengers for intercellular communication, are cell-secreted lipid bilayer nanoscale particles found in body fluids, and regulate the epithelial phenotype and profibrotic signaling pathways by transporting bioactive cargo to recipients in the pathogenesis of IPF. Furthermore, an increasing number of studies suggests that EVs derived from stem cells can be employed as a cell-free therapeutic approach for IPF, given their intrinsic tissue-homing capabilities and regeneration characteristics. This review highlights new sights of EVs in the pathogenesis of IPF, their potential as diagnostic and prognostic biomarkers, and prospects as novel drug delivery systems and next-generation therapeutics against IPF. Notably, bringing engineering strategies to EVs holds great promise for enhancing the therapeutic effect of anti-pulmonary fibrosis and promoting clinical transformation.
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[
"The pathogenesis of idiopathic pulmonary fibrosis (IPF) involves a complex interplay of cell types and signaling pathways. Recurrent alveolar epithelial cell (AEC) injury may occur in the context of predisposing factors (e.g., genetic, environmental, epigenetic, immunologic, and gerontologic), leading to metabolic dysfunction, senescence, aberrant epithelial cell activation, and dysregulated epithelial repair. The dysregulated epithelial cell interacts with mesenchymal, immune, and endothelial cells via multiple signaling mechanisms to trigger fibroblast and myofibroblast activation. Recent single-cell RNA sequencing studies of IPF lungs support the epithelial injury model. These studies have uncovered a novel type of AEC with characteristics of an aberrant basal cell, which may disrupt normal epithelial repair and propagate a profibrotic phenotype. Here, we review the pathogenesis of IPF in the context of novel bioinformatics tools as strategies to discover pathways of disease, cell-specific mechanisms, and cell-cell interactions that propagate the profibrotic niche.",
"The heterogeneity of small extracellular vesicles and presence of non-vesicular extracellular matter have led to debate about contents and functional properties of exosomes. Here, we employ high-resolution density gradient fractionation and direct immunoaffinity capture to precisely characterize the RNA, DNA, and protein constituents of exosomes and other non-vesicle material. Extracellular RNA, RNA-binding proteins, and other cellular proteins are differentially expressed in exosomes and non-vesicle compartments. Argonaute 1-4, glycolytic enzymes, and cytoskeletal proteins were not detected in exosomes. We identify annexin A1 as a specific marker for microvesicles that are shed directly from the plasma membrane. We further show that small extracellular vesicles are not vehicles of active DNA release. Instead, we propose a new model for active secretion of extracellular DNA through an autophagy- and multivesicular-endosome-dependent but exosome-independent mechanism. This study demonstrates the need for a reassessment of exosome composition and offers a framework for a clearer understanding of extracellular vesicle heterogeneity.",
"Pulmonary fibrosis (PF), the end point of interstitial lung diseases, is characterized by myofibroblast over differentiation and excessive extracellular matrix accumulation, leading to progressive organ dysfunction and usually a terminal outcome. Studies have shown that umbilical cord-derived mesenchymal stromal cells (uMSCs) could alleviate PF; however, the underlying mechanism remains to be elucidated. The therapeutic effects of uMSC-derived extracellular vesicles (uMSC-EVs) on PF were evaluated using bleomycin (BLM)-induced mouse models. Then, the role and mechanism of uMSC-EVs in inhibiting myofibroblast differentiation were investigated in vivo and in vitro. Treatment with uMSC-EVs alleviated the PF and enhanced the proliferation of alveolar epithelial cells in BLM-induced mice, thus improved the life quality, including the survival rate, body weight, fibrosis degree, and myofibroblast over differentiation of lung tissue. Moreover, these effects of uMSC-EVs on PF are likely achieved by inhibiting the transforming growth factor-β (TGF-β) signaling pathway, evidenced by decreased expression levels of TGF-β2 and TGF-βR2. Using mimics of uMSC-EV-specific miRNAs, we found that miR-21 and miR-23, which are highly enriched in uMSC-EVs, played a critical role in inhibiting TGF-β2 and TGF-βR2, respectively. The effects of uMSCs on PF alleviation are likely achieved via EVs, which reveals a new role of uMSC-EV-derived miRNAs, opening a novel strategy for PF treatment in the clinical setting.",
"Exosomes are small biological membrane vesicles that measure 30 to 100 nm in diameter. They are involved in a wide array of biological activities, such as cell-cell communication, signal transduction, transport of genetic materials, and modulation of immune response. Evidence indicates that they can be used as not only therapeutic agents targeted against disease but also diagnostic biomarkers for pathologic conditions. In this review, we endeavor to present exosomes as immunologic agents that can be used as pioneering cancer vaccines to prime the immune system and explicate their therapeutic and diagnostic capabilities. An extensive literature search for studies that involved the use of exosomes as immunotheranostic nanoparticles was conducted using PubMed, ISI Web of Knowledge, and Google Scholar. Clinical trials that involved exosomes were also compiled by searching the clinicaltrials.gov database. In its therapeutic facet of application, exosomes can be used as vehicles for drug or gene delivery. These biological vesicles have been found to have excellent host biodistribution and biocompatibility, issues often presented with gene delivery vehicles. Diagnostically, exosomes may prove to be useful biomarkers that are able to surpass current setbacks of modern diagnostic testing, which include invasive methods. Finally, current evidence has implied that the use of exosomes could form the basis for the development of future cell-free cancer vaccines. Exosomes have numerous functions, and their double-edged features make the scope of their clinical applications, as both a diagnostic and therapeutic tool, immense.",
"There is growing interest in stem cell-derived exosomes for their therapeutic and regenerative benefits given their manufacturing and regulatory advantages over cell-based therapies. As existing fibrosis impedes the viability and efficacy of stem cell/exosome-based strategies for treating chronic diseases, here we tested the effects of the anti-fibrotic drug, serelaxin, on the therapeutic efficacy of human amnion epithelial cell (AEC)-derived exosomes in experimental lung disease. Female Balb/c mice were subjected to either the 9.5-week model of ovalbumin and naphthalene (OVA/NA)-induced chronic allergic airway disease (AAD) or 3-week model of bleomycin (BLM)-induced pulmonary fibrosis; then administered increasing concentrations of AEC-exosomes (5 μg or 25μg), with or without serelaxin (0.5mg/kg/day) for 7-days. 1x106 AECs co-administered with serelaxin over the corresponding time-period were included for comparison in both models, as was pirfenidone-treatment of the BLM model. Control groups received saline/corn oil or saline, respectively. Both experimental models presented with significant tissue inflammation, remodelling, fibrosis and airway/lung dysfunction at the time-points studied. While AEC-exosome (5 μg or 25μg)-administration alone demonstrated some benefits in each model, serelaxin was required for AEC-exosomes (25μg) to rapidly normalise chronic AAD-induced airway fibrosis and airway reactivity, and BLM-induced lung inflammation, epithelial damage and subepithelial/basement membrane fibrosis. Combining serelaxin with AEC-exosomes (25μg) also demonstrated broader protection compared to co-administration of serelaxin with 1x106 AECs or pirfenidone. Serelaxin enhanced the therapeutic efficacy of AEC-exosomes in treating basement membrane-induced fibrosis and related airway dysfunction.",
"T cells secrete bioactive exosomes (EXO), but the potential immunoregulatory effect of T-cell EXO is largely unknown. In this study, we generated activated ovalbumin (OVA)-specific CD4(+) T cells in vitro via coculture of OVA-pulsed dendritic cells (DC(OVA)) with naive CD4(+) T cells derived from OVA-specific T-cell receptor (TCR) transgenic OTII mice. CD4(+) T-cell EXO were then purified from the CD4(+) T-cell culture supernatants by differential ultracentrifugation. CD4(+) T-cell EXO exhibited the 'saucer' shape that is characteristic of EXO with a diameter between 50 and 100 nm, as assessed by electron microscopy, and contained the EXO-associated proteins LAMP-1, TCR and lymphocyte function associated antigen-1 (LFA-1), as determined by western blot. Flow cytometric analysis showed that CD4(+) T-cell EXO expressed CD4(+) T-cell markers (CD4, TCR, LFA-1, CD25 and Fas ligand), but to a lesser extent than CD4(+) T cells. We demonstrated that DC(OVA) took up CD4(+) T-cell EXO via peptide/major histocompatibility complex (pMHC) II/TCR and CD54/LFA-1 interactions. OVA-specific CD4(+) T-cell EXO from OTII mice, but not ConA-stimulated polyclonal CD4(+) T-cell EXO from wild-type C57BL/6 mice inhibited DC(OVA)-stimulated in vitro CD4(+) T-cell proliferation and in vivo CD8(+) cytotoxic T lymphocyte (CTL) responses and antitumor immunity against OVA-expressing B16 melanoma BL6-10(OVA) cells. In addition, EXO derived from a T-cell hybridoma cell line, MF72.2D9, expressing an OVA-specific CD4(+) TCR, had a similar inhibitory effect as OTII CD4(+) T-cell EXO on CTL-mediated antitumor immunity. Taken together, our data indicate that antigen-specific T-cell EXO may serve as a new type of immunosuppressive reagent for use in transplant rejection and treatment of autoimmune diseases.",
"Idiopathic Pulmonary Fibrosis (IPF) is a type of chronic interstitial pneumonia, often fatal, with elusive causes and a bleak prognosis. Its treatment options are limited and largely ineffective. Early detection and precise diagnosis are pivotal in managing the disease effectively and enhancing patient survival rates. Recently, the quest for trustworthy biomarkers for IPF has gained momentum. Notably, emerging studies indicate that circular RNAs (circRNAs) found in exosomes may hold significant potential as valuable diagnostic markers. In this study, we initially explored the expression profile of circRNAs in exosomes sourced from the blood of IPF patients and healthy volunteers, employing a human circRNA microarray. We then utilized RT-qPCR to corroborate the dysregulated circRNAs identified by the microarray during the training phase. Next, the circRNAs that displayed a significant increase during the training phase were selected for further validation in a larger cohort encompassing 113 IPF patients and 76 healthy volunteers. Ultimately, the expression level and function of hsa_circ_0044226 were substantiated through a series of in vivo and in vitro experiments. Utilizing a human circRNA microarray, we identified 11 dysregulated circRNAs in the exosomes derived from the blood of IPF patients and control volunteers. Subsequent RT-qPCR analysis revealed significant increases in three circRNAs (hsa_circ_0044226, hsa_circ_0004099, hsa_circ_0008898) within the IPF patients. Notably, hsa_circ_0044226 was markedly elevated in patients experiencing acute exacerbation of IPF (AE-IPF) compared to those with stable IPF (S-IPF). Additionally, an upregulation of hsa_circ_0044226 was observed in the blood exosomes derived from a bleomycin-induced IPF mouse model. The expression levels of hsa_circ_0044226, hsa_circ_0004099, and hsa_circ_0008898 in plasma exosomes introduce a new paradigm of biomarkers for the diagnosis and progression of IPF.",
"Endothelial-to-mesenchymal transition (EndMT), the process by which endothelial cells lose their characteristics and acquire mesenchymal phenotypes, participates in the pathogenic mechanism of idiopathic pulmonary fibrosis. Recently, exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-Exos) has been introduced as a promising treatment in organ fibrosis. This study aimed to explore the effects as well as the molecular mechanism for hucMSC-Exo in pulmonary fibrosis. The intravenous administration of hucMSC-Exos alleviated bleomycin-induced pulmonary fibrosis in vivo. Moreover, hucMSC-Exos elevated miR-218 expression and restored endothelial properties weakened by TGF-β in endothelial cells. Knockdown of miR-218 partially abrogated the inhibition effect of hucMSC-Exos on EndMT. Our mechanistic study further demonstrated that MeCP2 was the direct target of miR-218. Overexpressing MeCP2 aggravated EndMT and caused increased CpG islands methylation at BMP2 promoter, which lead to BMP2 post-transcriptional gene silence. Transfection of miR-218 mimic increased BMP2 expression as well, which was downregulated by overexpression of MeCP2. Taken together, these findings indicate exosomal miR-218 derived from hucMSCs may possess anti-fibrotic properties and inhibit EndMT through MeCP2/BMP2 pathway, providing a new avenue of preventive application in pulmonary fibrosis.",
"There is a growing interest in the cell-cell communication roles in cancer mediated by secreted vesicles termed exosomes. In this study, we examined whether exosomes produced by cancer cells could transmit information to normal stromal fibroblasts and trigger a cellular response. We found that some cancer-derived exosomes could trigger elevated α-smooth muscle actin expression and other changes consistent with the process of fibroblast differentiation into myofibroblasts. We show that TGF-β is expressed at the exosome surface in association with the transmembrane proteoglycan betaglycan. Although existing in a latent state, this complex was fully functional in eliciting SMAD-dependent signaling. Inhibiting either signaling or betaglycan expression attenuated differentiation. While the kinetics and overall magnitude of the response were similar to that achieved with soluble TGF-β, we identified important qualitative differences unique to the exosomal route of TGF-β delivery, as exemplified by a significant elevation in fibroblast FGF2 production. This hitherto unknown trigger for instigating cellular differentiation in a distinctive manner has major implications for mechanisms underlying cancer-recruited stroma, fibrotic diseases, and wound-healing responses.",
"Cells continuously secrete a large number of microvesicles, macromolecular complexes, and small molecules into the extracellular space. Of the secreted microvesicles, the nanoparticles called exosomes are currently undergoing intense scrutiny. These are small vesicles (30-120 nm) containing nucleic acid and protein, perceived to be carriers of this cargo between diverse locations in the body. They are distinguished in their genesis by being budded into endosomes to form multivesicular bodies (MVBs) in the cytoplasm. The exosomes are released to extracellular fluids by fusion of these multivesicular bodies with the cell surface, resulting in secretion in bursts. Exosomes are secreted by all types of cells in culture, and also found in abundance in body fluids including blood, saliva, urine, and breast milk. In this review, we summarize strategies for exosome isolation, our understanding to date of exosome composition, functions, and pathways, and discuss their potential for diagnostic and therapeutic applications. Currently, the control of exosome formation, the makeup of the \"cargo\", biological pathways and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication--exosomes are thought to function as the messengers, delivering various effectors or signaling macromolecules between supposedly very specific cells. Both seasoned and newer investigators of nanovesicles have presented various viewpoints on what exosomes are, with some differences but a large common area. It would be useful to develop a codified definition of exosomes in both descriptive and practical terms. We hope this in turns leads to a consistent set of practices for their isolation, characterization and manipulation."
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what is a ptspl gene
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SPLs, a family of transcription factors specific to plants, play vital roles in plant growth and development through regulation of various physiological and biochemical processes. Although Populus trichocarpa is a model forest tree, the PtSPL gene family has not been systematically studied.
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"Throughout development the Arabidopsis shoot apical meristem successively undergoes several major phase transitions such as the juvenile-to-adult and floral transitions until, finally, it will produce flowers instead of leaves and shoots. Members of the Arabidopsis SBP-box gene family of transcription factors have been implicated in promoting the floral transition in dependence of miR156 and, accordingly, transgenics constitutively over-expressing this microRNA are delayed in flowering. To elaborate their roles in Arabidopsis shoot development, we analysed two of the 11 miR156 regulated Arabidopsis SBP-box genes, i.e. the likely paralogous genes SPL9 and SPL15. Single and double mutant phenotype analysis showed these genes to act redundantly in controlling the juvenile-to-adult phase transition. In addition, their loss-of-function results in a shortened plastochron during vegetative growth, altered inflorescence architecture and enhanced branching. In these aspects, the double mutant partly phenocopies constitutive MIR156b over-expressing transgenic plants and thus a major contribution to the phenotype of these transgenics as a result of the repression of SPL9 and SPL15 is strongly suggested.",
"miRNAs are a class of versatile small RNAs that control gene expression post-transcriptionally, governing many facets of plant cell functions. They interact with their target mRNA at a site of sequence complementarity and modulate their expression levels. Here, we provide evidence, based on transient assays and stable transgenic lines, that the 3' UTR of the Arabidopsis SBP box gene SPL3 contains a functional miRNA-responsive element (MRE) that is complementary to miR156 and miRNA157. Seedlings of transgenic lines constitutively over-expressing an SPL3 transgene either carrying an unaltered or a disrupted MRE accumulate considerable levels of SPL3 transcripts. However, while the unaltered MRE UTR does not allow the expression of detectable levels of SPL3 protein, the altered MRE does. Translational inhibition thus provides an important mechanism for miRNA-mediated post-transcriptional repression of SPL3. As a consequence of precocious translation of the constitutively expressed SPL3 transgene, due to the absence of a functional MRE, plants exhibit very early flowering in addition to frequent morphological changes.",
"MicroRNAs (miRNAs), a group of small non-coding RNAs, have recently become the subject of intense study. They are a class of post-transcriptional negative regulators playing vital roles in plant development and growth. However, little is known about their regulatory roles in the responses of trees to the stressful environments incurred over their long-term growth. Here, we report the cloning of small RNAs from abiotic stressed tissues of Populus trichocarpa (Ptc) and the identification of 68 putative miRNA sequences that can be classified into 27 families based on sequence homology. Among them, nine families are novel, increasing the number of the known Ptc-miRNA families from 33 to 42. A total of 346 targets was predicted for the cloned Ptc-miRNAs using penalty scores of </=2.5 for mismatched patterns in the miRNA:mRNA duplexes as the criterion. Six of the selected targets were validated experimentally. The expression of a majority of the novel miRNAs was altered in response to cold, heat, salt, dehydration, and mechanical stresses. Microarray analysis of known Ptc-miRNAs identified 19 additional cold stress-responsive Ptc-miRNAs from 14 miRNA gene families. Interestingly, we found that individual miRNAs of a family responded differentially to stress, which suggests that the members of a family may have different functions. These results reveal possible roles for miRNAs in the regulatory networks associated with the long-term growth of tree species and provide useful information for developing trees with a greater level of stress resistance.",
"Gibberellins (GAs) are important plant growth regulators, regulating many plant developmental processes, including seed germination, root and stem elongation, rosette expansion, floral induction and anther development. The diverse effects of GAs on plant development make it critical to maintain an appropriate endogenous GA level and a fine-tuned GA signalling. Some global regulators in GA signalling have been identified but little is known about genes specifically involved in local implementation of GA signalling. Here we report that the Arabidopsis thaliana SBP-box gene SQUAMOSA-PROMOTER-BINDING-PROTEIN-LIKE8 (SPL8) acts as a local regulator in a subset of GA-dependent developmental processes. Previous knowledge holds that SPL8 is involved in reproductive development as deduced from its loss-of-function phenotype (Unte et al. (2003) Plant Cell 15:1009-1019). We now determined that constitutive overexpression of SPL8 affects fertility due to non-dehiscent anthers, likely resulting from a constitutive GA response, suggesting a positive role of SPL8 in GA-mediated anther development. On the other hand, SPL8 gain- and loss-of-function mutants showed opposite responses to GA and its biosynthetic inhibitor paclobutrazol (PAC) with respect to seed germination and root elongation during the seedling stage. Genes involved in GA biosynthesis and signalling are transcriptionally affected by altered SPL8 expression. Our study uncovered a tissue-dependent regulatory role for SPL8 in the response to GA signalling in plant development.",
"The growth of plant organs is influenced by a stream of the phytohormone auxin that flows from the shoot apex to the tip of the root. However, until now it has not been known how auxin regulates the cell proliferation and enlargement that characterizes organ growth. Here we show that auxin controls the growth of roots by modulating cellular responses to the phytohormone gibberellin (GA). GA promotes the growth of plants by opposing the effects of nuclear DELLA protein growth repressors, one of which is Arabidopsis RGA (for repressor of gal-3). GA opposes the action of several DELLA proteins by destabilizing them, reducing both the concentration of detectable DELLA proteins and their growth-restraining effects. We also show that auxin is necessary for GA-mediated control of root growth, and that attenuation of auxin transport or signalling delays the GA-induced disappearance of RGA from root cell nuclei. Our observations indicate that the shoot apex exerts long-distance control on the growth of plant organs through the effect of auxin on GA-mediated DELLA protein destabilization.",
"Expression of miR398 is induced in response to copper deficiency and is involved in the degradation of mRNAs encoding copper/zinc superoxide dismutase in Arabidopsis thaliana. We found that SPL7 (for SQUAMOSA promoter binding protein-like7) is essential for this response of miR398. SPL7 is homologous to Copper response regulator1, the transcription factor that is required for switching between plastocyanin and cytochrome c(6) in response to copper deficiency in Chlamydomonas reinhardtii. SPL7 bound directly to GTAC motifs in the miR398 promoter in vitro, and these motifs were essential and sufficient for the response to copper deficiency in vivo. SPL7 is also required for the expression of multiple microRNAs, miR397, miR408, and miR857, involved in copper homeostasis and of genes encoding several copper transporters and a copper chaperone, indicating its central role in response to copper deficiency. Consistent with this idea, the growth of spl7 plants was severely impaired under low-copper conditions.",
"The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data."
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Downregulation of MutS protein homolog 2 by histone deacetylase 6
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MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSβ) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained.
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"An astounding number of important regulatory and structural proteins are subject to modification by the attachment of ubiquitin or ubiquitin-like proteins. This modification acts as a targeting signal, delivering the modified protein to different locations in the cell and modifying its activity, macromolecular interactions, or half-life. Deubiquitination, or the removal of this modification, is being recognized as an important regulatory strategy. This reaction is catalyzed by processing proteases known as deubiquitinating enzymes (DUBs). More than 60 DUBs are already known, although little is known about their biological roles. This review concentrates on recent findings and new insights into this fascinating class of enzymes.",
"The ubiquitin-proteasome pathway has a well-defined beginning and end. Target proteins are initially recognized by upstream components and tagged with polyubiquitin chains. The 26S proteasome then degrades these polyubiquitinated proteins. Until recently, it was not known what, if any, steps occurred between the initial polyubiquitination of target proteins and their final degradation. Several new papers investigating the function of the Cdc48-Ufd1-Npl4 complex indicate that there is indeed a middle to the ubiquitin-proteasome pathway. The Cdc48-Ufd1-Npl4 complex functions in the recognition of several polyubiquitin-tagged proteins and facilitates their presentation to the 26S proteasome for processive degradation or even more specific processing. The elucidation of Cdc48, Ufd1 and Npl4 action not only provides long-sought functions for these specific proteins, but illuminates a poorly understood part of the ubiquitin-proteasome pathway.",
"The properties of the nucleosomes of a salt-soluble, transcriptionally active gene-enriched fraction of chicken erythrocyte chromatin were evaluated by hydroxyapatite dissociation chromatography. We have demonstrated previously that the salt-soluble, transcriptionally active gene-enriched polynucleosomes are enriched in dynamically acetylated and ubiquitinated histones, and in an atypical U-shaped nucleosome that possessed about 20% less protein than a typical nucleosome. Further, newly synthesized histones H2A and H2B exchange preferentially with the nucleosomal histones H2A and H2B of this salt-soluble chromatin fraction. Analysis of the histones eluting from the hydroxyapatite-bound chromatin demonstrated that hyperacetylated and ubiquitinated (u), including multi-ubiquitinated, H2A-H2B.1 dimers dissociated at lower concentrations of NaCl than unmodified dimers or dimers with histone variants H2A.Z and/or H2B.2. Cross-linking studies revealed that at least 50% of uH2B.1 was paired with uH2A. uH2A-uH2B.1 dimers dissociated at lower NaCl concentrations than H2A-uH2B.1 dimers. Hyperacetylated histone (H3-H4)2 tetramers also eluted at lower concentrations of NaCl than unmodified tetramers. Our results support the idea that acetylation and ubiquitination of histones H2A and H2B.1 increase the lability of H2A-H2B.1 dimers in transcriptionally active nucleosomes. In contrast, our observations suggest that histone variants H2A.Z and H2B.2. stabilize the association of the H2A-H2B dimer in nucleosomes. The elevated lability of the H2A-H2B dimer may facilitate processes such as the exchange of these dimers with newly synthesized histones, the elongation process of transcription and transcription factor binding.",
"Histone deacetylases (HDACs) are thought to function as critical mediators of transcriptional repression. However, the physiological targets and posttranslational modifications of the class II HDACs are largely unknown. Here we show that the C terminus of HDAC 6 is both necessary and sufficient for specific association with polyubiquitin. This region contains a putative zinc finger but lacks significant similarity to other known ubiquitin binding domains. Thus, we have designated this region as a PAZ domain, for Polyubiquitin Associated Zinc finger. Although the PAZ domain possesses homology with the zinc finger of deubiquitinating enzymes, it is dispensable for the deubiquitinating activity we find associated with HDAC6 following immunopurification. We also show that both HDAC 5 and HDAC 6 are ubiquitinated in vitro and in vivo. However, both of these proteins are stable in vivo and do not appear to be targeted for rapid degradation by the proteasome. Thus, HDAC6 is linked to the ubiquitin system via ubiquitin conjugation, polyubiquitin binding, and copurification with deubiquitinating enzymes.",
"Histone deacetylase 2 (HDAC2) is a member of a large family of enzymes that alter gene expression by catalyzing the removal of acetyl groups from core histones. Originally isolated as a transcriptional co-repressor, HDAC2 possesses extensive amino acid sequence homology to HDAC1 (the founding member and most extensively studied HDAC enzyme). Because of this high degree of sequence similarity between HDAC1 and HDAC2, coupled with the fact that the two always co-exist in the same complexes, it is difficult to assess whether different properties exist between these two proteins. We report here that HDAC2 is a phosphoprotein similar to HDAC1. In addition, like HDAC1, the phospho-acceptor sites in HDAC2 are located in the C-terminal portion of the protein. However, unlike HDAC1, which can be phosphorylated by protein kinase CK2, cAMP-dependent protein kinase, and protein kinase G, HDAC2 is phosphorylated uniquely by protein kinase CK2 in vitro. Studies using unfractionated cell extracts with CK2 inhibitors suggest that protein kinase CK2 is the major source of HDAC2 kinase. Finally, and perhaps most interesting, HDAC2 phosphorylation promotes enzymatic activity, selectively regulates complex formation, but has no effect on transcriptional repression. Together, our data indicate that like many HDACs, HDAC2 is regulated by post-translational modification, particularly phosphorylation. Furthermore, we demonstrate for the first time that there are similarities and differences in the regulation of HDAC1 and HDAC2 by phosphorylation.",
"The relationship between transcription and ubiquitination of the histones was investigated. Previous studies have shown that ubiquitinated (u) histone H2B and, to a lesser extent, mono- and polyubiquitinated histone H2A are enriched in transcriptionally active gene-enriched chromatin fractions. Here, we show that treatment of T-47D-5 human breast cancer cells with actinomycin D (10 micrograms/mL) or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, inhibitors of heterogeneous nuclear RNA synthesis, selectively reduced the level of uH2B, but not uH2A, uH2A.Z, or polyubiquitinated H2A, in chromatin. Treatment of the cells with low levels (0.04 micrograms/mL) of actinomycin D slightly reduced the level of uH2B, suggesting that inhibition of ribosomal RNA synthesis does not have a profound effect on the level of uH2B in chromatin. The level of the ubiquitinated histones was not affected by treating the cells with inhibitors of DNA synthesis (sodium butyrate or aphidicolin), but heat-shock treatment resulted in the loss of all the monoubiquitinated histone species. These results demonstrate that maintenance of the levels of uH2B in chromatin is dependent upon ongoing transcription, particularly the synthesis of hnRNA. Thus, histone H2B would be ubiquitinated when the nucleosome was opened during transcription. Ubiquitination of histone H2B may impede nucleosome refolding, facilitating subsequent rounds of transcription.",
"We have a new approach, two-dimensional hybridization mapping of nucleosomes, to compare the structures of mononucleosomes from different regions of the Drosophila melanogaster genome. Approximately one in two nucleosomes of the transcribed copia and heat-shock 70 (hsp 70) genes in nonshocked cultured cells contains ubiquitin-H2A (uH2A) semihistone, a covalent conjugate of histone H2A and a small protein, ubiquitin. In striking contrast, less than one in 25 nucleosomes of tandemly repeated, nontranscribed 1.688 satellite DNA contains uH2A, suggesting that most of the nucleosomal uH2A is located in transcribed genes. Approximately 25% of all nucleosomes are ubiquitinated in nonsynchronized cultured Drosophila cells. The hsp 70 genes in nonshocked cells occur in nucleosomes, are greatly enriched in uH2A and are not digested preferentially by staphylococcal nuclease. In contrast, the same genes in chromatin from heat-shocked cells are highly sensitive to staphylococcal nuclease and no longer possess nucleosomal organization recognizable with this probe. Histone ubiquitination in transcribed nucleosomes may prevent formation of higher order chromosomal structures by modifying nucleosome-nucleosome interactions. The observed loss of nucleosomal organization in very actively transcribed genes, such as the hsp 70 genes in shocked cells, may be related to the recent finding that ubiquitin conjugates are substrates for the cytoplasmic ATP-dependent proteolytic system. We have also found that 1.688 satellite mononucleotomes contain a specific approximately 50,000 dalton nonhistone protein, D1, in addition to being extremely under-ubiquitinated. D1 may be involved in formation of the highly compact structure of satellite heterochromatin."
] |
Intracranial Pressure Changes in Children and Adolescents
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The specific aim of this review is to report the features of intracranial pressure changes [spontaneous intracranial hypotension (SIH) and idiopathic intracranial hypertension (IIH)] in children and adolescents, with emphasis on the presentation, diagnosis, and treatment modalities. Headache associated with intracranial pressure changes are relatively rare and less known in children and adolescents. SIH is a specific syndrome involving reduced intracranial pressure with orthostatic headache, frequently encountered connective tissue disorders, and a good prognosis with medical management, initial epidural blood patching, and sometimes further interventions may be required. IIH is an uncommon condition in children and different from the disease in adults, not only with respect to clinical features (likely to present with strabismus and stiff neck rather than headache or pulsatile tinnitus) but also different in outcome. Consequently, specific ICP changes of pediatric ages required specific attention both of exact diagnosis and entire management.
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[
"Idiopathic intracranial hypertension (IIH) is defined as increased intracranial pressure in the absence of intracranial mass or obstructive hydrocephalus. Over 80% of patients are overweight women. IIH is usually encountered in the neurology and ophthalmology practise as headaches, visual disturbance and papilloedema are the characteristic features of this syndrome. Patients with IIH also experience tinnitus, hearing loss, balance disturbance, cerebrospinal fluid (CSF) otorrhoea or rhinorrhoea and in some cases these otorhinological symptoms can be presenting features of this syndrome. IIH is also associated with obstructive sleep apnoea. Otolaryngologists should be familiar with this important condition as it can manifest a variety of symptoms that are more frequently seen in their clinics. Sometimes otolaryngologists may be involved in the surgical management of this condition, such as repair of CSF rhinorrhoea or otorrhoea or endoscopic optic nerve decompression. The aim of this review article is to familiarise the otolaryngologists with the important features of this unusual syndrome which may remain unrecognised in the otolaryngology practice.",
"Twelve patients are described who were found to have asymptomatic intracranial hypertension monitored over an extended (6 months to 6 years) period. There were three groups: 5 patients with treated hydrocephalus with an apparently functioning shunt, 4 patients with untreated hydrocephalus, clinically and radiologically nonprogressive, and 3 patients with pseudotumor cerebri, 2 treated and 1 untreated. Although the magnitude of the intracranial pressure changes varied, all patients had abnormal baseline pressures together with repeated A and B waves. In no case was there any clinical manifestation of raised intracranial pressure and in all cases ventricular size remained constant over the period of evaluation. The clinical and pathophysiological implications of these findings are discussed.",
"Pseudotumor cerebri, a disorder of intracranial pressure regulation, is characterized by elevated cerebrospinal fluid pressures. The symptoms and signs are those of elevated intracranial pressure alone. Extensive endocrine studies in patients with the primary disease have generally been normal. The major threat to the patient with pseudotumor cerebri is loss of vision; severe deficits occur in 4% to 12% of patients. The causative mechanism is not known. There are no controlled studies of treatment and interpretation of uncontrolled studies is limited by the high rate of spontaneous remission. A short course of steroid therapy is recommended for the patient with significant symptoms that have not remitted after 7 to 10 days of observation and serial lumbar punctures. Shunting is considered in refractory patients or when loss of vision is threatened.",
"IIH is a disorder associated with increased intracranial pressure with no clinical, laboratory, or radiologic evidence of an intracranial space-occupying lesion. The aim of this study was to establish ONSD standards of healthy pediatric subjects and compare the normal measurements with those of patients with IIH. One hundred fifteen MR imaging studies of children 4 months to 17 years of age were blinded and reviewed by a pediatric neuroradiologist. A total of 230 optic nerves were measured. Eighty-six MR imaging examinations were performed in apparently healthy subjects. This control group included subjects who underwent MR imaging for various reasons, and their MR imaging findings were interpreted as normal. Twenty-nine MR imaging examinations were performed in patients with documented IIH. The ONSD was measured 1 cm anterior to the optic foramina on an axial T2 sequence. For statistical analysis, both patients and controls were stratified into 4 age groups (I, 0-3 years; II, 3-6 years; III, 6-12 years; IV, 12-18 years). The mean ONSD of the control group in all age groups (I, 3.1 mm; II, 3.41 mm; III, 3.55 mm; IV, 3.56 mm) was significantly smaller than the mean ONSD of patients (I, 4.35 mm; II, 4.37 mm; III, 4.25 mm; IV, 4.69 mm). A positive correlation between age and ONSD (r = 0.414, P < .01) was found in the control group. According to our study, in pediatric patients with IIH, the ONSD is significantly larger than that in healthy controls regardless of age group and sex. This measurement might prove to be an auxiliary tool in the diagnosis of increased intracranial pressure in pediatric patients.",
"Facial nerve palsies are uncommon in infants. We report on 10-week-old monozygotic twins, diagnosed with cystic fibrosis by newborn screening, who developed facial palsy and increased intracranial pressure. Cranial imaging and cerebrospinal fluid analysis produced normal results. Levels of serum vitamin A were below normal range. Low levels of vitamin A are associated with facial nerve paralysis, and are at least partly implicated in the development of increased intracranial pressure in infants with cystic fibrosis."
] |
Secondary Bile Acids and Colon Cancer
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Bile acids were first proposed as carcinogens in 1939. Since then, accumulated evidence has linked exposure of cells of the gastrointestinal tract to repeated high physiologic levels of bile acids as an important risk factor for gastrointestinal cancers. High exposure to bile acids may occur in a number of settings, but most importantly, is prevalent among individuals who have a high dietary fat intake. A rapid effect on cells of high bile acid exposure is the generation of reactive oxygen species and reactive nitrogen species, disruption of the cell membrane and mitochondria, induction of DNA damage, mutation and apoptosis, and development of reduced apoptosis capability upon chronic exposure. Here, we review the substantial evidence of the mechanism of secondary bile acids and their role in colon cancer.
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[
"A secondary bile acid, namely, deoxycholic acid (DCA), has been known to promote colon tumors; on the other hand, it also induces apoptosis in several human colon cancer cell lines. A hydrophobic primary bile acid, namely, chenodeoxycholic acid (CDCA), exhibits a similar property of apoptosis induction; DCA and CDCA also trigger some specific intracellular signal pathways in the human colon cancer cell line HCT116. In this article, we report that hydrophobic bile acids induce different cellular responses depending on their concentration, that is, a sublethal concentration of hydrophobic bile acids can suppress the apoptosis induced by a higher concentration of DCA. Pretreatment with DCA or CDCA at a concentration of < or = 200 microM for 8 h suppressed the apoptosis induced by 500 microM DCA in HCT116 cells. Under this condition, the association of caspase-9 and Apaf-1 and subsequent activation of caspase-9 were inhibited, but the release of cytochrome c from the mitochondria was not. At 200 microM, DCA and CDCA induced the phosphorylation of Akt and ERK1/2, although these phosphorylations do not appear to be indispensable for the cytoprotection. It is interpreted that prolonged exposure to sublethal concentrations of hydrophobic bile acids induces resistance to apoptosis, leading to promotion of colorectal tumorigenesis.",
"The structure of the bile acid molecule is described and correlated with physiochemical properties of bile acids such as solubility, ionization, and micelle formation. Recent measurements of the critical micellar concentration (CMC) of a large number of bile acids indicate that the CMC is influenced by both side chain and nuclear structure. Bile acids with hydroxy substituents on both sides of the steroid nucleus are non-amphipathic and do not form micelles, and decreasing the length of the side chain causes an exponential increase in the CMC. Bile acid ionization, measured by titration in alcohol-water mixtures, is shown to be uninfluenced by nuclear substituents; the pKa of all unconjugated bile acids is about 5. Interactions of bile acid solutions with Ca2+ are discussed; recent work indicates that cholyl conjugates bind Ca2+ as monomers in solution. Model systems relevant to biological processes are classified, as are some of the physicochemical parameters of these systems. Biological processes involving bile acids are tabulated, and corresponding model systems are assigned to each. Some biological processes such as bile acid transport show marked species differences, suggesting that physicochemical parameters are insufficient to explain biological differences. It is recommended that the physical chemist study a variety of bile acids, that the biologist study a variety of species, and that both collaborate to attempt to factor out the extent to which physicochemical properties of bile acids can explain their biological properties.",
"The mechanism and regulation of the degradation of cholesterol into bile acids has attracted increased interest, in particular after the recent discovery that nuclear receptors (farnesoid X receptor and liver X receptor) are involved in the regulation of bile acid synthesis. Recently, it has also been shown that the biosynthesis of bile acids is not exclusively restricted to the liver, and that degradation may start by a hydroxylation of cholesterol in the brain or in other extrahepatic organs. During the past 2 years the genes coding for three of the six enzymes catalysing the first steps in bile acid biosynthesis have been cloned and characterized. These genes and their gene products will be described here.",
"The relation between meat consumption and colorectal cancer risk remains controversial. In this report, we quantitatively reviewed the prospective observational studies that have analyzed the relation between meat consumption and colorectal cancer. We conducted electronic searches of MEDLINE, EMBASE, and CANCERLIT databases through to the end of June 1999 and manual searches of references from retrieved articles. We used both fixed and random-effects meta-analytical techniques to estimate the overall association and to investigate possible sources of heterogeneity among studies. Thirteen studies were eligible for inclusion in the meta-analysis. Pooled results indicate that a daily increase of 100 g of all meat or red meat is associated with a significant 12-17% increased risk of colorectal cancer. The marginally significant between-study heterogeneity for all meat and red meat was explained by a number of study-level covariates. A significant 49% increased risk was found for a daily increase of 25 g of processed meat. The individual study estimates for processed meat showed no detectable heterogeneity. On the basis of this quantitative review of prospective studies, the overall association between meat consumption and risk of colorectal cancer appears to be positive, with marginal heterogeneity between studies. The finding for processed meat and data from experimental studies suggests that it may also be an important predictor of colorectal cancer risk. However, because only a few of the studies reviewed here attempted to examine the independent effect of meat intake on colorectal cancer risk, the possibility that the overall association may be confounded or modified by other factors cannot be excluded."
] |
Gene Duplication and Retention in Angiosperm Genome Evolution
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Recurrent gene duplication and retention played an important role in angiosperm genome evolution. It has been hypothesized that these processes contribute significantly to plant adaptation but so far this hypothesis has not been tested at the genome scale.
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[
"Changes in ploidy occurred early in the diversification of some animal and plant lineages and represent an ongoing phenomenon in others. While the prevalence of polyploid lineages indicates that this phenomenon is a common and successful evolutionary transition, whether polyploidization itself has a significant effect on patterns and rates of diversification remains an open question. Here we review evidence for the creative role of polyploidy in evolution. We present new estimates for the incidence of polyploidy in ferns and flowering plants based on a simple model describing transitions between odd and even base chromosome numbers. These new estimates indicate that ploidy changes may represent from 2 to 4% of speciation events in flowering plants and 7% in ferns. Speciation via polyploidy is likely to be one of the more predominant modes of sympatric speciation in plants, owing to its potentially broad-scale effects on gene regulation and developmental processes, effects that can produce immediate shifts in morphology, breeding system, and ecological tolerances. Theoretical models support the potential for increased adaptability in polyploid lineages. The evidence suggests that polyploidization can produce shifts in genetic systems and phenotypes that have the potential to result in increased evolutionary diversification, yet conclusive evidence that polyploidy has changed rates and patterns of diversification remains elusive.",
"The role that natural selection plays in governing the locations and early evolution of copy-number mutations remains largely unexplored. We used high-density full-genome tiling arrays to create a fine-scale genomic map of copy-number polymorphisms (CNPs) in Drosophila melanogaster. We inferred a total of 2658 independent CNPs, 56% of which overlap genes. These include CNPs that are likely to be under positive selection, most notably high-frequency duplications encompassing toxin-response genes. The locations and frequencies of CNPs are strongly shaped by purifying selection, with deletions under stronger purifying selection than duplications. Among duplications, those overlapping exons or introns, as well as those falling on the X chromosome, seem to be subject to stronger purifying selection.",
"Many Saccharomyces cerevisiae duplicate genes that were derived from an ancient whole-genome duplication (WGD) unexpectedly show a small synonymous divergence (K(S)), a higher sequence similarity to each other than to orthologues in Saccharomyces bayanus, or slow evolution compared with the orthologue in Kluyveromyces waltii, a non-WGD species. This decelerated evolution was attributed to gene conversion between duplicates. Using approximately 300 WGD gene pairs in four species and their orthologues in non-WGD species, we show that codon-usage bias and protein-sequence conservation are two important causes for decelerated evolution of duplicate genes, whereas gene conversion is effective only in the presence of strong codon-usage bias or protein-sequence conservation. Furthermore, we find that change in mutation pattern or in tDNA copy number changed codon-usage bias and increased the K(S) distance between K. waltii and S. cerevisiae. Intriguingly, some proteins showed fast evolution before the radiation of WGD species but little or no sequence divergence between orthologues and paralogues thereafter, indicating that functional conservation after the radiation may also be responsible for decelerated evolution in duplicates.",
"Various population genetic models of duplicated genes are introduced. The problems covered in this review include the fixation process of a duplicated copy, copy number polymorphism, the fates of duplicated genes and single nucleotide polymorphism in duplicated genes. Because of increasing evidence for concerted evolution by gene conversion, this review introduces recently developed gene conversion models. In the first half, models assuming independent evolution of duplicated genes are introduced, and then the effect of gene conversion is considered in the second half.",
"In Arabidopsis, tandemly arrayed genes (TAGs) comprise >10% of the genes in the genome. These duplicated genes represent a rich template for genetic innovation, but little is known of the evolutionary forces governing their generation and maintenance. Here we compare the organization and evolution of TAGs between Arabidopsis and rice, two plant genomes that diverged ~150 million years ago. TAGs from the two genomes are similar in a number of respects, including the proportion of genes that are tandemly arrayed, the number of genes within an array, the number of tandem arrays, and the dearth of TAGs relative to single copy genes in centromeric regions. Analysis of recombination rates along rice chromosomes confirms a positive correlation between the occurrence of TAGs and recombination rate, as found in Arabidopsis. TAGs are also biased functionally relative to duplicated, nontandemly arrayed genes. In both genomes, TAGs are enriched for genes that encode membrane proteins and function in \"abiotic and biotic stress\" but underrepresented for genes involved in transcription and DNA or RNA binding functions. We speculate that these observations reflect an evolutionary trend in which successful tandem duplication involves genes either at the end of biochemical pathways or in flexible steps in a pathway, for which fluctuation in copy number is unlikely to affect downstream genes. Despite differences in the age distribution of tandem arrays, the striking similarities between rice and Arabidopsis indicate similar mechanisms of TAG generation and maintenance.",
"We conducted a detailed analysis of duplicate genes in three complete genomes: yeast, Drosophila, and Caenorhabditis elegans. For two proteins belonging to the same family we used the criteria: (1) their similarity is > or =I (I = 30% if L > or = 150 a.a. and I = 0.01n + 4.8L(-0.32(1 + exp(-L/1000))) if L < 150 a.a., where n = 6 and L is the length of the alignable region), and (2) the length of the alignable region between the two sequences is > or = 80% of the longer protein. We found it very important to delete isoforms (caused by alternative splicing), same genes with different names, and proteins derived from repetitive elements. We estimated that there were 530, 674, and 1,219 protein families in yeast, Drosophila, and C. elegans, respectively, so, as expected, yeast has the smallest number of duplicate genes. However, for the duplicate pairs with the number of substitutions per synonymous site (K(S)) < 0.01, Drosophila has only seven pairs, whereas yeast has 58 pairs and nematode has 153 pairs. After considering the possible effects of codon usage bias and gene conversion, these numbers became 6, 55, and 147, respectively. Thus, Drosophila appears to have much fewer young duplicate genes than do yeast and nematode. The larger numbers of duplicate pairs with K(S) < 0.01 in yeast and C. elegans were probably largely caused by block duplications. At any rate, it is clear that the genome of Drosophila melanogaster has undergone few gene duplications in the recent past and has much fewer gene families than C. elegans.",
"Due to the selection pressure imposed by highly variable environmental conditions, stress sensing and regulatory response mechanisms in plants are expected to evolve rapidly. One potential source of innovation in plant stress response mechanisms is gene duplication. In this study, we examined the evolution of stress-regulated gene expression among duplicated genes in the model plant Arabidopsis thaliana. Key to this analysis was reconstructing the putative ancestral stress regulation pattern. By comparing the expression patterns of duplicated genes with the patterns of their ancestors, duplicated genes likely lost and gained stress responses at a rapid rate initially, but the rate is close to zero when the synonymous substitution rate (a proxy for time) is > approximately 0.8. When considering duplicated gene pairs, we found that partitioning of putative ancestral stress responses occurred more frequently compared to cases of parallel retention and loss. Furthermore, the pattern of stress response partitioning was extremely asymmetric. An analysis of putative cis-acting DNA regulatory elements in the promoters of the duplicated stress-regulated genes indicated that the asymmetric partitioning of ancestral stress responses are likely due, at least in part, to differential loss of DNA regulatory elements; the duplicated genes losing most of their stress responses were those that had lost more of the putative cis-acting elements. Finally, duplicate genes that lost most or all of the ancestral responses are more likely to have gained responses to other stresses. Therefore, the retention of duplicates that inherit few or no functions seems to be coupled to neofunctionalization. Taken together, our findings provide new insight into the patterns of evolutionary changes in gene stress responses after duplication and lay the foundation for testing the adaptive significance of stress regulatory changes under highly variable biotic and abiotic environments.",
"Until around 1990, most multigene families were thought to be subject to concerted evolution, in which all member genes of a family evolve as a unit in concert. However, phylogenetic analysis of MHC and other immune system genes showed a quite different evolutionary pattern, and a new model called birth-and-death evolution was proposed. In this model, new genes are created by gene duplication and some duplicate genes stay in the genome for a long time, whereas others are inactivated or deleted from the genome. Later investigations have shown that most non-rRNA genes including highly conserved histone or ubiquitin genes are subject to this type of evolution. However, the controversy over the two models is still continuing because the distinction between the two models becomes difficult when sequence differences are small. Unlike concerted evolution, the model of birth-and-death evolution can give some insights into the origins of new genetic systems or new phenotypic characters.",
"Gene duplications have a major role in the evolution of new biological functions. Theoretical studies often assume that a duplication per se is selectively neutral and that, following a duplication, one of the gene copies is freed from purifying (stabilizing) selection, which creates the potential for evolution of a new function. In search of systematic evidence of accelerated evolution after duplication, we used data from 26 bacterial, six archaeal, and seven eukaryotic genomes to compare the mode and strength of selection acting on recently duplicated genes (paralogs) and on similarly diverged, unduplicated orthologous genes in different species. We find that the ratio of nonsynonymous to synonymous substitutions (Kn/Ks) in most paralogous pairs is <<1 and that paralogs typically evolve at similar rates, without significant asymmetry, indicating that both paralogs produced by a duplication are subject to purifying selection. This selection is, however, substantially weaker than the purifying selection affecting unduplicated orthologs that have diverged to the same extent as the analyzed paralogs. Most of the recently duplicated genes appear to be involved in various forms of environmental response; in particular, many of them encode membrane and secreted proteins. The results of this analysis indicate that recently duplicated paralogs evolve faster than orthologs with the same level of divergence and similar functions, but apparently do not experience a phase of neutral evolution. We hypothesize that gene duplications that persist in an evolving lineage are beneficial from the time of their origin, due primarily to a protein dosage effect in response to variable environmental conditions; duplications are likely to give rise to new functions at a later phase of their evolution once a higher level of divergence is reached.",
"Ubiquitin-ligases or E3s are components of the ubiquitin proteasome system (UPS) that coordinate the transfer of ubiquitin to the target protein. A major class of ubiquitin-ligases consists of RING-finger domain proteins that include the substrate recognition sequences in the same polypeptide; these are known as single-subunit RING finger E3s. We are studying a particular family of RING finger E3s, named ATL, that contain a transmembrane domain and the RING-H2 finger domain; none of the member of the family contains any other previously described domain. Although the study of a few members in A. thaliana and O. sativa has been reported, the role of this family in the life cycle of a plant is still vague. To provide tools to advance on the functional analysis of this family we have undertaken a phylogenetic analysis of ATLs in twenty-four plant genomes. ATLs were found in all the 24 plant species analyzed, in numbers ranging from 20-28 in two basal species to 162 in soybean. Analysis of ATLs arrayed in tandem indicates that sets of genes are expanding in a species-specific manner. To get insights into the domain architecture of ATLs we generated 75 pHMM LOGOs from 1815 ATLs, and unraveled potential protein-protein interaction regions by means of yeast two-hybrid assays. Several ATLs were found to interact with DSK2a/ubiquilin through a region at the amino-terminal end, suggesting that this is a widespread interaction that may assist in the mode of action of ATLs; the region was traced to a distinct sequence LOGO. Our analysis provides significant observations on the evolution and expansion of the ATL family in addition to information on the domain structure of this class of ubiquitin-ligases that may be involved in plant adaptation to environmental stress."
] |
Piwi-related Argonaute proteins in Caenorhabditis elegans
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In metazoans, Piwi-related Argonaute proteins play important roles in maintaining germline integrity and fertility and have been linked to a class of germline-enriched small RNAs termed piRNAs. Caenorhabditis elegans encodes two Piwi family proteins called PRG-1 and PRG-2, and PRG-1 interacts with the C. elegans piRNAs (21U-RNAs). Previous studies found that mutation of prg-1 causes a marked reduction in the expression of 21U-RNAs, temperature-sensitive defects in fertility and other phenotypic defects.
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[
"The regulation of higher-order chromosome structure is central to cell division and sexual reproduction. Heterochromatin assembly at the centromeres facilitates both kinetochore formation and sister chromatid cohesion, and the formation of specialized chromatin structures at telomeres serves to maintain the length of telomeric repeats, to suppress recombination, and to aid in formation of a bouquet-like structure that facilitates homologous chromosome pairing during meiosis. In fission yeast, genes encoding the Argonaute, Dicer, and RNA-dependent RNA polymerase factors involved in RNA interference (RNAi) are required for heterochromatin formation at the centromeres and mating type region. In this study, we examine the effects of deletions of the fission yeast RNAi machinery on chromosome dynamics during mitosis and meiosis. We find that the RNAi machinery is required for the accurate segregation of chromosomes. Defects in mitotic chromosome segregation are correlated with loss of cohesin at centromeres. Although the telomeres of RNAi mutants maintain silencing, length, and localization of the heterochromatin protein Swi6, we discovered defects in the proper clustering of telomeres in interphase mitotic cells. Furthermore, a small proportion of RNAi mutant cells display aberrant telomere clustering during meiotic prophase. This study demonstrates that the fission yeast RNAi machinery is required for the proper regulation of chromosome architecture during mitosis and meiosis.",
"RNA interference (RNAi) is an ancient, highly conserved mechanism in which small RNA molecules (siRNAs) guide the sequence-specific silencing of gene expression . Several silencing machinery protein components have been identified, including helicases, RNase-related proteins, double- and single-stranded RNA binding proteins, and RNA-dependent RNA polymerase-related proteins . Work on these factors has led to the revelation that RNAi mechanisms intersect with cellular pathways required for development and fertility . Despite rapid progress in understanding key steps in the RNAi pathway, it is clear that many factors required for both RNAi and related developmental mechanisms have not yet been identified. Here, we report the characterization of the C. elegans gene rde-3. Genetic analysis of presumptive null alleles indicates that rde-3 is required for siRNA accumulation and for efficient RNAi in all tissues, and it is essential for fertility and viability at high temperatures. RDE-3 contains conserved domains found in the polymerase beta nucleotidyltransferase superfamily, which includes conventional poly(A) polymerases, 2'-5' oligoadenylate synthetase (OAS), and yeast Trf4p . These findings implicate a new enzymatic modality in RNAi and suggest possible models for the role of RDE-3 in the RNAi mechanism.",
"In plants and mammals, small RNAs indirectly mediate epigenetic inheritance by specifying cytosine methylation. We found that small RNAs themselves serve as vectors for epigenetic information. Crosses between Drosophila strains that differ in the presence of a particular transposon can produce sterile progeny, a phenomenon called hybrid dysgenesis. This phenotype manifests itself only if the transposon is paternally inherited, suggesting maternal transmission of a factor that maintains fertility. In both P- and I-element-mediated hybrid dysgenesis models, daughters show a markedly different content of Piwi-interacting RNAs (piRNAs) targeting each element, depending on their parents of origin. Such differences persist from fertilization through adulthood. This indicates that maternally deposited piRNAs are important for mounting an effective silencing response and that a lack of maternal piRNA inheritance underlies hybrid dysgenesis.",
"In the fission yeast Schizosaccharomyces pombe, the RNA interference (RNAi) machinery is required to generate small interfering RNAs (siRNAs) that mediate heterochromatic gene silencing. Efficient silencing also requires the TRAMP complex, which contains the noncanonical Cid14 poly(A) polymerase and targets aberrant RNAs for degradation. Here we use high-throughput sequencing to analyze Argonaute-associated small RNAs (sRNAs) in both the presence and absence of Cid14. Most sRNAs in fission yeast start with a 5' uracil, and we argue these are loaded most efficiently into Argonaute. In wild-type cells most sRNAs match to repeated regions of the genome, whereas in cid14Delta cells the sRNA profile changes to include major new classes of sRNAs originating from ribosomal RNAs and a tRNA. Thus, Cid14 prevents certain abundant RNAs from becoming substrates for the RNAi machinery, thereby freeing the RNAi machinery to act on its proper targets.",
"Small non-coding RNAs, including microRNAs (miRNAs), serve an important role in controlling gene expression during development and disease. However, little detailed information exists concerning the relative expression patterns of small RNAs during development of animals such as Caenorhabditis elegans. We performed a deep analysis of small RNA expression in C. elegans using recent advances in sequencing technology, and found that a significant number of known miRNAs showed major changes in expression during development and between males and hermaphrodites. Additionally, we identified 66 novel miRNA candidates, about 35% of which showed transcripts from their 'star sequence', suggesting that they are bona fide miRNAs. Also, hundreds of novel Piwi-interacting RNAs (piRNAs)/21U-RNAs with dynamic expression during development, together with many longer transcripts encompassing 21U-RNA sequences, were detected in our libraries. Our analysis reveals extensive regulation of non-coding small RNAs during development of hermaphrodites and between different genders of C. elegans, and suggests that these RNAs, including novel miRNA candidates, are involved in developmental processes. These findings should lead to a better understanding of the biological roles of small RNAs in C. elegans development.",
"Endogenous small RNAs (endo-siRNAs) interact with Argonaute (AGO) proteins to mediate sequence-specific regulation of diverse biological processes. Here, we combine deep-sequencing and genetic approaches to explore the biogenesis and function of endo-siRNAs in C. elegans. We describe conditional alleles of the Dicer-related helicase, drh-3, that abrogate both RNA interference and the biogenesis of endo-siRNAs, called 22G-RNAs. DRH-3 is a core component of RNA-dependent RNA polymerase (RdRP) complexes essential for several distinct 22G-RNA systems. We show that, in the germline, one system is dependent on worm-specific AGOs, including WAGO-1, which localizes to germline nuage structures called P granules. WAGO-1 silences certain genes, transposons, pseudogenes, and cryptic loci. Finally, we demonstrate that components of the nonsense-mediated decay pathway function in at least one WAGO-mediated surveillance pathway. These findings broaden our understanding of the biogenesis and diversity of 22G-RNAs and suggest additional regulatory functions for small RNAs."
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Does insomnia therapy improve sleep and pain in chronic low back pain?
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Insomnia, which is very common in patients with chronic low back pain (LBP), has long been viewed as a pain symptom that did not merit specific treatment. Recent data suggest that adding insomnia therapy to pain-targeted treatment should improve outcome; however, this has not been empirically tested in LBP or in any pain condition treated with a standardized pain medication regimen. We sought to test the hypothesis that adding insomnia therapy to pain-targeted treatment might improve sleep and pain in LBP.
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[
"To identify symptoms that change in prevalence and severity during midlife and evaluate their relationships to menopausal status, hormonal levels, and other factors. In a longitudinal, population-based study of 438 Australian-born women observed for 7 years with an 89% retention rate, 172 advanced from premenopause to perimenopause or postmenopause. Annual measures included a 33-item symptom check list; psychosocial, lifestyle, and health-related factors; menstrual status; hormone usage; and blood levels of follicle-stimulating hormone and estradiol (E2). Increasing from early to late perimenopause were the number of women who reported five or more symptoms (+14%), hot flushes (+27%), night sweats (+17%) and vaginal dryness (+17%) (all P <.05). Breast soreness-tenderness decreased with the menopausal transition (-21%). Trouble sleeping increased by +6%. The major change in prevalence was from early to late perimenopause, except for insomnia, which showed a gradual increase. Those variables most related to onset of hot flushes were number of symptoms at early perimenopause (P <.05), having an unskilled or no occupation (P <.05), more than 10 pack-years of smoking (P <.01), and decreased E2 (P <.01). The onset of night sweats increased with the change in E2 (P <.05). The onset of vaginal dryness decreased with more years of education (P <.05). Trouble sleeping was predicted by prior lower well-being (P <.01), belief at baseline that women with many interests hardly notice menopause (P <.01), and hot flushes (P <.01). Although middle-aged women are highly symptomatic, the symptoms that appear to be specifically related to hormonal changes of menopausal transition are vasomotor symptoms, vaginal dryness, and breast tenderness. Insomnia reflected bothersome hot flushes and psychosocial factors.",
"Insomnia is the most prevalent sleep disorder in the general population, and is commonly encountered in medical practices. Insomnia is defined as the subjective perception of difficulty with sleep initiation, duration, consolidation, or quality that occurs despite adequate opportunity for sleep, and that results in some form of daytime impairment.1 Insomnia may present with a variety of specific complaints and etiologies, making the evaluation and management of chronic insomnia demanding on a clinician's time. The purpose of this clinical guideline is to provide clinicians with a practical framework for the assessment and disease management of chronic adult insomnia, using existing evidence-based insomnia practice parameters where available, and consensus-based recommendations to bridge areas where such parameters do not exist. Unless otherwise stated, \"insomnia\" refers to chronic insomnia, which is present for at least a month, as opposed to acute or transient insomnia, which may last days to weeks.",
"Sixty participants with insomnia secondary to chronic pain were assigned randomly to either a cognitive-behavioral therapy (CBT) or a self-monitoring/waiting-list control condition. The therapy consisted of a multicomponent 7-week group intervention aimed at promoting good sleep habits, teaching relaxation skills, and changing negative thoughts about sleep. Treated participants were significantly more improved than control participants on self-report measures of sleep onset latency, wake time after sleep onset, sleep efficiency, and sleep quality, and they showed less motor activity in ambulatory recordings of nocturnal movement. At a 3-month follow-up assessment, treated participants showed good maintenance of most therapeutic gains. These results provide the 1st evidence from a randomized controlled trial that CBT is an effective treatment for insomnia that is secondary to chronically painful medical conditions.",
"To evaluate 6 months' eszopiclone treatment upon patient-reported sleep, fatigue and sleepiness, insomnia severity, quality of life, and work limitations. Randomized, double blind, controlled clinical trial. 54 research sites in the U.S. 830 primary insomnia patients who reported mean nightly total sleep time (TST) < or = 6.5 hours/night and/or mean nightly sleep latency (SL) >30 min. Eszopiclone 3 mg or matching placebo. Patient-reported sleep measures, Insomnia Severity Index, Medical Outcomes Study Short-Form Health Survey (SF-36), Work Limitations Questionnaire, and other assessments measured during baseline, treatment Months 1-6, and 2 weeks following discontinuation of treatment. Patient-reported sleep and daytime function were improved more with eszopiclone than with placebo at all months (P <0.001). Eszopiclone reduced Insomnia Severity Index scores to below clinically meaningful levels for 50% of patients (vs 19% with placebo; P <0.05) at Month 6. SF-36 domains of Physical Functioning, Vitality, and Social Functioning were improved with eszopiclone vs placebo for the Month 1-6 average (P < 0.05). Similarly, improvements were observed for all domains of the Work Limitations Questionnaire with eszopiclone vs placebo for the Month 1-6 average (P <0.05). This is the first placebo-controlled investigation to demonstrate that long-term nightly pharmacologic treatment of primary insomnia with any hypnotic enhanced quality of life, reduced work limitations, and reduced global insomnia severity, in addition to improving patient-reported sleep variables.",
"A multicenter, double-blind, parallel-group study was designed to assess the efficacy and safety of zolpidem extended-release coadministered with escitalopram in patients with insomnia and comorbid generalized anxiety disorder. Patients (N = 383) received open-label escitalopram 10 mg/d and were randomized to either adjunct zolpidem extended-release 12.5 mg or placebo. The primary efficacy measure was change from baseline to week 8 in subjective total sleep time. Secondary efficacy measures included subjective sleep onset latency, number of awakenings, wake time after sleep onset, sleep quality, the Hamilton Rating Scale for Anxiety, the Beck Anxiety Inventory, the Sleep Impact Scale, the Massachusetts General Hospital Cognitive and Physical Functioning Questionnaire, and the Sheehan Disability Scale. The last-observation-carried-forward method was used to impute missing values for most efficacy measures. Safety was monitored at each visit. At week 8 and all time points, there was a significant improvement in the zolpidem extended-release/escitalopram group compared with placebo/escitalopram for total sleep time (P < 0.0001). Most of the secondary efficacy measures also significantly favored zolpidem at most visits (P < 0.0001). The most common treatment-emergent adverse events in both groups were nausea, dizziness, headache, fatigue, and dry mouth. Concurrent zolpidem extended-release/escitalopram, compared with placebo/escitalopram, significantly improved insomnia and sleep-related next-day symptoms, but not anxiety symptoms, in patients with comorbid insomnia and generalized anxiety disorder.",
"The purpose of this post hoc analysis was to compare the treatment effect size of eszopiclone 3 mg for insomnia in patients with a diagnosis of primary insomnia and in several of the psychiatric and medical conditions that are most commonly comorbid with insomnia. Data were analyzed from 5 large, multicenter, randomized, double-blind, placebo-controlled studies of adult outpatients of at least 1 month duration published between 2006 and 2009. Diary-derived indices of sleep and daytime functioning and the Insomnia Severity Index were compared for patients with primary insomnia (DSM-IV-TR criteria, n = 828) and for those with insomnia comorbid with major depressive disorder (MDD, DSM-IV-TR criteria, n = 545), generalized anxiety disorder (GAD, DSM-IV-TR criteria, n = 595), perimenopause/postmenopause (Stages of Reproductive Aging Workshop criteria, n = 410), and rheumatoid arthritis (American College of Rheumatology criteria, n = 153). Cohen d effect sizes were calculated for each individual study as the between-treatment difference score divided by the pooled standard deviation. Effect sizes ranged from 0.40 to 0.69 (small-medium) as early as week 1 and were maintained at 0.26-0.63 at week 4 for sleep latency, wake time after sleep onset, and total sleep time. Sleep latency and total sleep time effect sizes increased from week 1 to week 4 in the primary insomnia group. At week 4, effect sizes on all 3 parameters and the Insomnia Severity Index tended to be highest for the primary insomnia patients and tended to be lowest for patients with comorbid GAD and MDD. The effect sizes for daytime functioning were small for all insomnia patient groups. Eszopiclone 3 mg is an effective treatment for insomnia across 5 clinically diverse patient populations; however, magnitude of effect is mediated by underlying comorbidity and their treatments, with largest measures of effect seen in primary insomnia and lowest in MDD and GAD. These consistent results, and the fact that clinical trials were conducted in patients being treated as appropriate for their comorbid clinical conditions, support the results' real-world generalizability and utility to clinical practice.",
"Acute and chronic administration of the selective serotonin reuptake inhibitors (SSRIs) have been widely reported to disrupt sleep in laboratory studies. This study examines the naturalistic, longitudinal effects of paroxetine and fluvoxamine on sleep quality in the home setting. Fourteen healthy volunteers free of medical and neuropsychiatric symptoms entered a 31-day protocol: 7 days of drug-free baseline (days 1-7), 19 days of drug treatment (steady state during days 18-26), and 5 days of acute withdrawal (days 27-31). On day 8, the subjects were randomly assigned to receive either 100 mg/day of fluvoxamine or 20 mg/day of paroxetine (half receiving each drug) in divided morning and evening oral doses. Investigators remained blinded to drug assignment until all sleep data had been analyzed. Sleep was monitored using the Nightcap ambulatory sleep monitor. Four standard and 3 novel measures were computed and compared using multivariate analysis of variance, analysis of variance, and Bonferroni-corrected comparison of means. Sleep disruption was most clearly demonstrated using the novel measures eyelid quiescence index, rhythmicity, and eyelid movements per minute in non-rapid eye movement sleep, but was also apparent as determined by standard measures of sleep efficiency, number of awakenings, and sleep onset latency. Paroxetine disrupted sleep more than fluvoxamine, and paroxetine-induced sleep disruption persisted into the withdrawal phase. Rapid eye movement sleep was suppressed during treatment (especially for fluvoxamine) and rebounded during withdrawal (especially for paroxetine). We confirm laboratory polysomnographic findings of SSRI-induced sleep quality changes and demonstrate the Nightcap's efficacy as an inexpensive longitudinal monitor for objective sleep changes induced by psychotropic medication."
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Laryngeal framework surgery for unilateral vocal fold paralysis.
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Laryngeal framework surgery is the current gold standard treatment for unilateral vocal fold paralysis. It provides a permanent solution to glottic insufficiency caused by injury to the recurrent laryngeal nerve. Various modifications to the original Isshiki type I laryngoplasty procedure have been described to improve voice and swallowing outcomes. The success of this procedure is highly dependent on the experience of the surgeon as it epitomizes the intersection of art and science in the field. The following article reviews the evidence, controversies, and complications related to laryngoplasty for unilateral vocal fold paralysis. It also provides a detailed analysis of how and when arytenoid-positioning procedures should be considered, and summarizes the literature on postoperative outcomes.
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"There continues to be controversy about the added role of arytenoid adduction (AA) in the rehabilitation of unilateral vocal cord paralysis (UVCP). Some authors feel that the added morbidity of AA is not warranted in UVCP rehabilitation. Objective analysis of acoustic and aerodynamic measures were performed before and after surgery to try to resolve this controversy. This is an institutional review board-approved retrospective study of 85 patients with UVCP undergoing surgical rehabilitation by injection laryngoplasty (n = 45), medialization laryngoplasty (n = 14), or medialization laryngoplasty with arytenoid adduction (n = 26). Acoustic and aerodynamic parameters were studied before and after surgery. The medialization laryngoplasty (ML) group (medialization alone, injection laryngoplasty ML/IL) data were compared to the AA-ML group using a paired t test for the individual measures and analysis of variance (ANOVA) for the multivariate analysis of acoustic and aerodynamic measures. In all treatment arms there were statistically significant improvements in all acoustic and aerodynamic measures after intervention (P < .05). For ML and IL, the mean difference between preoperative from postoperative jitter was 1.504%, shimmer 3.265%, noise to harmonic ratio (NHR) 0.036, mean phonation time 4.523 seconds, transglottic flow 0.130 L/s, and subglottic pressure 0.616 cm H2O. For AA-ML the mean difference between preoperative and postoperative jitter was 2.431%, shimmer 6.068%, NHR 0.082, mean phonation time 6.74 seconds, flow 0.181 L/s, and subglottic pressure 0.611 cm H2. Preoperatively, the average phonatory function of the AA-ML group was worse than the ML group. Comparison between the two treatment arms, individual acoustic and aerodynamic measures, were not different (paired t test, P < .05). However, mulitvariate analysis (ANOVA, P < .05) of acoustic and aerodynamic measures, showed a statistically significant difference between the two groups. The degree of change was significantly better in the AA-ML group. AA-ML and IL/ML improve phonatory function, but not to normal. We were unable to demonstrate a statistical difference between groups using a single measure, but using mutlivariate analysis, there is a statistical significance between the groups. AA-ML patients had worse preoperative function and had better postoperative function. When clinically indicated, AA-ML procedure does appear to correct the physiology of the incompetent larynx better than ML alone.",
"In a series of 12 newborn kittens, horseradish peroxidase (HRP) was used to trace retrograde axoplasmic flow in the motor neurons to laryngeal muscles. The animals were sacrificed 24 hours after injection of HRP into a specific laryngeal muscle, and the brain stems were stained for peroxidase. This clear-cut colorimetric method permitted the localization of the motor neurons in two nuclei of the ipsilateral brain stem. These are the nucleus ambiguus and the retrofacial nucleus. The primary source of laryngeal motor supply is the nucleus ambiguus. All the laryngeal muscles were represented here in two divisions. Adductor neurons were located in the dorsal division and were more loosely arranged in the lateral reticular formation. The abductor neurons of the posterior cricoarytenoid muscle formed the compact ventral division of the nucleus ambiguus and were fewer in number than adductor neurons by a factor of four to one. Since the expiratory and inspiratory centers are also located dorsally and ventrally in the brain stem reticular formation, the motor cells of the nucleus ambiguus are conveniently arranged to receive their afferent input. This arrangement is probably the result of phylogenetic development of abductor laryngeal function and pulmonary function in lower forms. A second source of laryngeal innervation is the retrofacial nucleus. This small nucleus is situated rostral to the nucleus ambiguus and is made up of small and medium-sized neurons of at least two types. Only the cricothyroid (CT) and posterior cricoarytenoid (PCA) muscles were shown to have significant innervation from this nucleus. The CT neurons were located peripherally while the PCA cells occupied the central portion of the nucleus. The functional significance of this nucleus is unknown, but it is suggested that it may have something to do with the various types of muscle units that have been demonstrated physiologically in the CT and PCA muscles.",
"Laryngoplastic phonosurgery is a new term used to describe all forms of laryngeal framework surgery to improve the voice. One of the most important new surgical techniques is medialization laryngoplasty (ML) for the correction of unilateral vocal cord paralysis. This article focuses on ML techniques, results, complications and indications; the artenoid adduction procedure (AA) is described as a complementary technique. In addition, some of the more experimental laryngoplastic procedures are reviewed.",
"Surgical management of unilateral vocal cord paralysis has evolved over the last three decades. The recent use of type I thyroplasty has resulted in improvements in voice, swallowing, and respiration. The study was performed to evaluate our experience in 28 patients undergoing arytenoid adduction as part of their surgical rehabilitation of unilateral vocal cord paralysis. Patients undergoing arytenoid adduction with or without silastic medialization for unilateral vocal cord paralysis were entered into a prospective data base. Evaluation included symptomatic improvement in hoarseness, aspiration, dysphagia, dyspnea, and the radiographic documentation of pneumonia. Objective evaluation included mean phonatory air flow and acoustic analysis. Complications associated with surgery were recorded. A satisfactory result was obtained in 27 of 28 (96%) patients. By symptom, improvement in hoarseness was evident in 96%, dyspnea 80%, dysphagia 94%, and aspiration 84%. Improvements in phonatory flow rate (p < .001), estimated mean laryngeal airway resistance (p < .001), and maximally prolonged phonation (p < .01) were identified. Complications occurred in 18% and consisted of local wound sepsis (n = 1), hematoma (n = 1), seroma (n = 1), and transient airway edema (n = 2). There were no episodes of airway obstruction requiring tracheostomy or implant extrusion. Arytenoid adduction as part of type I thyroplasty is a safe and effective procedure. Subjective analysis confirms marked improvement in laryngeal function in the form of speech, swallowing, and respiration. Objective analysis confirms improvement in voice parameters. Future directions will focus on determination of those patients best served by arytenoid adduction.",
"To describe the method of inserting a wedge-shaped adjustable balloon implant (wABI) via a minithyrotomy for medialization thyroplasty and evaluate its effect on a range of phonatory parameters using the excised larynx bench apparatus. Repeated measures with each larynx serving as its own control. A prototype wABI was deployed in six excised canine larynges of various sizes through a minithyrotomy and then filled with saline. Mucosal wave, aerodynamic, and acoustic parameters were measured for three conditions: normal, vocal fold paralysis, and paralysis with the wABI. Phonation threshold pressure (P < .001), flow (P < .001), and power (P = .002) were significantly lower for wABI compared to paralysis trials; values did not differ significantly from normal trials. Percent jitter (P = .002) and percent shimmer (P = .007) were also significantly decreased compared to the paralysis condition, and values were not significantly different compared to normal. The mucosal wave was preserved after insertion of the wABI. Effective vocal fold medialization with preservation of the mucosal wave was observed with the wABI in this preliminary excised larynx experiment. The wABI offers the potential for a minimally invasive insertion in addition to postoperative adjustability. Further studies in living animals and humans are warranted to evaluate clinical utility. NA.",
"Medialization thyroplasty (MT) and arytenoid adduction (AA) are effective treatments for medializing the paralyzed vocal cord, but indications and benefits of each procedure are controversial. We performed a formal evidence-based review to answer this question: In patients with unilateral vocal cord paralysis, does AA combined with MT significantly improve subjective and objective voice outcomes compared with MT alone?Study design We performed a MEDLINE literature search using specific search terms to identify pertinent articles, which were reviewed and graded according to the evidence quality. We identified 219 potentially pertinent articles; detailed review yielded 10 studies for further analysis. Only 3 studies directly compared MT with AA plus MT: overall, there was no clear benefit in subjective or objective outcomes for AA plus MT. Very limited grade C evidence indicates no clear benefit, or lack of benefit, in subjective or objective outcomes if AA is added to MT, compared with MT alone."
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In vitro antifungal activity of dirutheniumpentadithiocarbamate complexes C1-C9
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The in vitro antifungal activity of nine dirutheniumpentadithiocarbamate complexes C1-C9 was investigated and assessed for its activity against four different fungal species with clinical interest and related to invasive fungal infections (IFIs), such as Candida spp. [C. albicans (two clinical isolates), C. glabrata, C. krusei, C. parapsolisis, C. tropicalis, C.dubliniensis (six clinical isolates)], Paracoccidioides brasiliensis (seven clinical isolates), Cryptococcus neoformans and Sporothrix schenckii. All synthesized complexes C1-C9 and also the free ligands L1-L9 were submitted to in vitro tests against those fungi and the results are very promising, since some of the obtained MIC (minimal inhibitory concentration) values were very low (from 10-6 mol mL-1 to 10-8 mol mL-1) against all investigated clinically relevant fungal pathogens, except for C. glabrata, that the MIC values are close to the ones obtained for fluconazole, the standard antifungal agent tested. Preliminary structure-activity relations (SAR) might be suggested and a strong influence from steric and lipophilic parameters in the antifungal activity can be noticed. Cytotoxicity assays (IC50) showed that the complexes are not as toxic (IC50 values are much higher-30 to 200 fold-than MIC values). These ruthenium complexes are very promising lead compounds for novel antifungal drug development, especially in IFIs, one of most harmful emerging infection diseases (EIDs).
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[
"The reaction of ruthenium(III) complexes, [RuX(3)(EPh(3))(3)] (E=As, X=Cl or Br; E=P, X=Cl) and [RuBr(3)(PPh(3))(2)(CH(3)OH)] with bidendate Schiff base ligands derived by condensing salicylaldehyde with methylamine (Hsalmet), cyclohexylamine (Hsalchx), 2-aminopyridine (Hsalampy) have been carried out. The complexes were characterized by analytical and spectral studies (IR, electronic and EPR) and are formulated as [RuX(EPh(3))(LL')(2)] (where LL'=monobasic bidentate Schiff base ligand; E=P or As, X=Cl or Br). An octahedral geometry has been tentatively proposed for the new complexes. Dioxygen affinity of some of the Ru(III) Schiff base complexes was studied by cyclic voltammetry. The representative Schiff bases and their complexes were tested in vitro to evaluate their activity against fungi, namely, Aspergillus flavus (A. flavus) and Fusarium species.",
"Invasive aspergillosis is an increasingly frequent opportunistic infection in immunocompromised patients. Only two agents, amphotericin B and itraconazole, are licensed for therapy. Itraconazole acts through inhibition of a P-450 enzyme undertaking sterol 14alpha demethylation. In vitro resistance in Aspergillus fumigatus to itraconazole correlated with in vivo outcome has not been previously described. For three isolates (AF72, AF90, and AF91) of A. fumigatus from two patients with invasive aspergillosis itraconazole MICs were elevated. A neutropenic murine model was used to establish the validity of the MICs. The isolates were typed by random amplification of polymorphic DNA. Analysis of sterols, inhibition of cell-free sterol biosynthesis from [14C] mevalonate, quantitation of P-450 content, and [3H]itraconazole concentration in mycelial pellets were used to determine the mechanisms of resistance. The MICs for the three resistant isolates were >16 microg/ml. In vitro resistance was confirmed in vivo for all three isolates. Molecular typing showed the isolates from the two patients to be genetically distinct. Compared to the susceptible isolate from patient 1, AF72 had a reduced ergosterol content, greater quantities of sterol intermediates, a similar susceptibility to itraconazole in cell-free ergosterol biosynthesis, and a reduced intracellular [3H]itraconazole concentration. In contrast, AF91 and AF92 had slightly higher ergosterol and lower intermediate sterol concentrations, fivefold increased resistance in cell-free systems to the effect of itraconazole on sterol 14alpha demethylation, and intracellular [3H] itraconazole concentrations found in susceptible isolates. Resistance to itraconazole in A. fumigatus is detectable in vitro and is present in wild-type isolates, and at least two mechanisms of resistance are responsible.",
"The in vitro activity of itraconazole was determined against 7,299 isolates of Candida spp., 1,615 isolates of Cryptococcus neoformans, and 445 isolates of Aspergillus spp. obtained from over 200 medical centers worldwide. Itraconazole was active against all Candida spp. (96% of MICs were < or =1 microg/ml) with the exception of C. glabrata (77% of MICs were < or =1 microg/ml). Itraconazole inhibited 94% of C. krusei and 84% of other fluconazole-resistant Candida species, exclusive of C. glabrata, at a MIC of < or =1 microg/ml. Itraconazole was not active against fluconazole-resistant isolates of C. glabrata. Only modest activity was seen against C. neoformans (80% of MICs were < or =1 microg/ml); however, itraconazole showed excellent activity against Aspergillus spp. (94% of MICs were < or =1 microg/ml). These results provide an update on the antifungal activity of itraconazole against major opportunistic fungal pathogens. In light of the new intravenous formulation of itraconazole these data suggest that this agent remains a viable systemically active antifungal agent.",
"Allogeneic hematopoietic stem-cell transplant recipients often receive fluconazole or an amphotericin B preparation for antifungal prophylaxis. Because of concerns about fungal resistance with fluconazole and toxicity with amphotericin B, alternative prophylactic regimens have become necessary. To compare the efficacy and safety of intravenous and oral itraconazole with the efficacy and safety of intravenous and oral fluconazole for long-term prophylaxis of fungal infections. Open-label, multicenter, randomized trial. Five transplantation centers in the United States. 140 patients undergoing allogeneic hematopoietic stem-cell transplantation. Itraconazole (200 mg intravenously every 12 hours for 2 days followed by 200 mg intravenously every 24 hours or a 200-mg oral solution every 12 hours) or fluconazole (400 mg intravenously or orally every 24 hours) from day 1 until day 100 after transplantation. Proven invasive or superficial fungal infection, drug-related side effects, mortality from fungal infection, and overall mortality. Proven invasive fungal infections occurred in 6 of 71 itraconazole recipients (9%) and in 17 of 67 fluconazole recipients (25%) during the first 180 days after transplantation (difference, -16 percentage points [95% CI, -29.2 to -4.7 percentage points]; P = 0.01). Superficial fungal infections occurred in 3 of 71 itraconazole recipients (4%) and in 2 of 67 fluconazole recipients (3%). In a multivariable analysis using factors known to affect the risk for invasive fungal infection after hematopoietic stem-cell transplantation, prophylaxis with itraconazole was still associated with fewer invasive fungal infections (odds ratio, 0.300 [CI, 0.111 to 0.814]; P = 0.02) caused by either yeasts or molds. More fungal pathogens were found to be resistant to fluconazole than to itraconazole. Except for more frequent gastrointestinal side effects (nausea, vomiting, diarrhea, or abdominal pain) in patients given itraconazole (24% vs. 9%; difference, 15 percentage points [CI, 2.9 to 27.0 percentage points]; P = 0.02), both itraconazole and fluconazole were well tolerated. The overall mortality rate was similar in each group (32 of 71 patients in the itraconazole group [45%] vs. 28 of 67 patients in the fluconazole group [42%]; difference, 3 percentage points [CI, -13.2 to 19.8 percentage points]; P > 0.2), but fewer deaths were related to fungal infection in patients given itraconazole (6 of 71 [9%]) than in patients given fluconazole (12 of 67 [18%]) (difference, 9 percentage points [CI, -20.6 to 1.8 percentage points]; P = 0.13). Itraconazole is more effective than fluconazole for long-term prophylaxis of invasive fungal infections after allogeneic hematopoietic stem-cell transplantation. Except for gastrointestinal side effects, itraconazole is well tolerated.",
"The reaction of ammonium pyrrolidinedithiocarbamate, [NH4{S2CN(CH2)4}], with SnCl2, [Sn(C6H5)2Cl2], [Sn(C6H5)3Cl], [Sn(C4H9)2Cl2] and [Sn(C6H11)3Cl] produced in good yield the compounds [Sn{S2CN(CH2)4}2Cl2] (1), [Sn{S2CN(CH2)4}2Ph2] (2), [Sn{S2CN(CH2)4}Ph3] (3), [Sn{S2CN(CH2)4}2 n-Bu2] (4) and [Sn{S2CN(CH2)4}Cy3] (5). The complexes were characterised by infrared, multinuclear NMR (1H, 13C{1H} and 119Sn{1H}) and 119Sn Mössbauer spectroscopies. In addition, the crystal structure of 4 was determined by X-ray crystallography. The in vitro antifungal activity of the tin(IV) complexes as well of the ligand was performed on human pathogenic fungi, Candida albicans, in concentrations of 0.025; 0.050; 0.100; 0.200; 0.400; 0.800; 1.600 and 3.200 mM. The microorganism presented resistance to the dithiocarbamate ligand and all tin(IV) complexes tested were actives. The highest activity was found for compounds 1 and 4.",
"We examined the in vitro activity of caspofungin, posaconazole, voriconazole, ravuconazole, itraconazole, and amphotericin B against 448 recent clinical mold isolates. The endpoint for reading caspofungin was the minimum effective concentration (MEC). Among the triazoles, posaconazole was most active, inhibiting 95% of isolates at <or=1 microg/ml, followed by ravuconazole (91%), voriconazole (90%), and itraconazole (79%). Caspofungin and amphotericin B inhibited 93% and 89% of isolates at <or=1 microg/ml, respectively, with caspofungin demonstrating an MEC 90 of 0.12 microg/ml. All three new triazoles and caspofungin inhibited >95% of Aspergillus spp. at <or=1 microg/ml compared to 83% for itraconazole and 91% for amphotericin B. Amphotericin B inhibited only 38% of Aspergillus terreus isolates at <or=1 microg/ml, whereas the three new triazoles and caspofungin inhibited all A. terreus at <or=0.5 microg/ml. The new triazoles and caspofungin have excellent in vitro activity against a very large collection of recent clinical isolates of Aspergillus spp., and some in vitro activity against selected other filamentous fungi.",
"The isolates in a collection of 170 Aspergillus fumigatus isolates recovered from 114 patients and 21 different medical centers in The Netherlands over a period of 53 years were tested for the presence of resistance to itraconazole and voriconazole according to the guidelines of NCCLS document M38-P and by the E-test. Three isolates were highly resistant to itraconazole, and voriconazole MICs were low for all isolates.",
"Antifungal agents can be classified by their site of action in fungal cells, which can have important implications for both efficacy and tolerability. Currently available agents include the polyenes, nucleoside analogs, and the azoles. With the exception of 5-fluorocytosine, all agents act by interfering with the structural or functional integrity of the fungal plasma membrane. However, the non-selective nature of this therapeutic target results in concomitant cross-inhibition (or toxicity) in mammalian cells. New compounds that interfere with the fungal cell wall--a target not present in mammalian cells--therefore constitute an important focus of current clinical research. Caspofungin, the first representative of a new class of antifungals that inhibit beta-(1,3)-D-glucan synthesis, exerts potent activity against Candida and Aspergillus spp. and appears to be generally well tolerated. This paper reviews the data on caspofungin.",
"The pharmacokinetics of intravenous itraconazole (ITC) was studied in dialysis patients. Dialysis had no effect on the half-life and clearance of ITC or OH-ITC. However, dialysis allowed the clearance of hydroxypropyl-beta-cyclodextrin (HP-beta-CD). The area under the concentration-time curve from time zero to infinity (AUC(0- infinity)) for HP-beta-CD administered before dialysis was lower than the AUC(0- infinity) when it was administered after dialysis (P < 0.01). Administration of ITC intravenously just prior to hemodialysis appears to produce adequate systemic exposures of ITC and OH-ITC while allowing dialysis clearance of HP-beta-CD. Studies of multiple administrations are warranted.",
"A tetrazolium salt has been used to develop a quantitative colorimetric assay for mammalian cell survival and proliferation. The assay detects living, but not dead cells and the signal generated is dependent on the degree of activation of the cells. This method can therefore be used to measure cytotoxicity, proliferation or activation. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) and show a high degree of precision. No washing steps are used in the assay. The main advantages of the colorimetric assay are its rapidity and precision, and the lack of any radioisotope. We have used the assay to measure proliferative lymphokines, mitogen stimulations and complement-mediated lysis."
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G protein-coupled receptor kinase-interactor 2 is an essential terminator of Toll-like receptor-induced NF-B signaling
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G protein-coupled receptor kinase-interactor 2 (GIT2) regulates thymocyte positive selection, neutrophil-direction sensing, and cell motility during immune responses by regulating the activity of the small GTPases ADP ribosylation factors (Arfs) and Ras-related C3 botulinum toxin substrate 1 (Rac1). Here, we show that Git2-deficient mice were more susceptible to dextran sodium sulfate (DSS)-induced colitis, Escherichia coli, or endotoxin-shock challenge, and a dramatic increase in proinflammatory cytokines was observed in Git2 knockout mice and macrophages. GIT2 is a previously unidentified negative regulator of Toll-like receptor (TLR)-induced NF-κB signaling. The ubiquitination of TNF receptor associated factor 6 (TRAF6) is critical for the activation of NF-κB. GIT2 terminates TLR-induced NF-κB and MAPK signaling by recruiting the deubiquitinating enzyme Cylindromatosis to inhibit the ubiquitination of TRAF6. Finally, we show that the susceptibility of Git2-deficient mice to DSS-induced colitis depends on TLR signaling. Thus, we show that GIT2 is an essential terminator of TLR signaling and that loss of GIT2 leads to uncontrolled inflammation and severe organ damage.
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[
"Members of the Toll-like receptor (TLR) family are key regulators of both innate and adaptive immune responses. The function of TLRs in various human diseases has been investigated by comparison of the incidence of disease among people having different polymorphisms in genes that participate in TLR signaling. These studies have shown that TLR function affects several diseases, including sepsis, immunodeficiencies, atherosclerosis and asthma. As this body of data grows, it will provide new insights into disease pathogenesis as well as valuable information on the merits of various therapeutic options.",
"Toll-like receptors (TLR) are innate immune receptors involved in recognition of the intestinal microflora; they are expressed by numerous cell types in the intestine, including epithelial cells, myeloid cells, and lymphocytes. Little is known about the relative contributions of TLR signaling in distinct cellular compartments to intestinal homeostasis. We aimed to define the roles of TLR signals in distinct cell types in the induction and regulation of chronic intestinal inflammation. We assessed the roles of the shared TLR signaling adaptor protein, MyD88, in several complementary mouse models of inflammatory bowel disease, mediated by either innate or adaptive immune activation. MyD88-deficient mice and bone marrow chimeras were used to disrupt TLR signals selectively in distinct cellular compartments in the intestine. MyD88-dependent activation of myeloid cells was required for the development of chronic intestinal inflammation. By contrast, although epithelial cell MyD88 signals were required for host survival, they were insufficient to induce intestinal inflammation in the absence of an MyD88-competent myeloid compartment. MyD88 expression by T cells was not required for their pathogenic and regulatory functions in the intestine. Cellular compartmentalization of MyD88 signals in the intestine allow the maintenance of host defense and prevent deleterious inflammatory responses.",
"Targeted disruption of the c-src proto-oncogene in mice has shown that src expression is required for normal bone resorption, since the src-deficient mutants develop osteopetrosis. To evaluate the mechanisms by which src-deficiency affects osteoclast function, we treated src-deficient mice with the stimulants of bone resorption, IL-1, parathyroid hormone, and parathyroid hormone-related protein, and analyzed the effects by quantitative bone histomorphometry and electron microscopy. Increased numbers of multinucleated cells with the morphological characteristics of osteoclasts appeared on bone surfaces, but these cells did not form ruffled borders or normal resorption lacunae. To confirm these in vivo findings, we cultured src-mutant bone marrow cells on dentine slices in the presence of 1,25 dihydroxyvitamin D3. Increased numbers of multinucleated cells were formed, but unlike normal murine bone marrow cells, they did not form resorption pits. These results indicate that osteoclasts appear in the absence of pp60c-src, but that pp60c-src expression is required for mature osteoclasts to form ruffled borders and resorb bone.",
"Thymocytes are highly motile cells that migrate under the influence of chemokines in distinct thymic compartments as they mature. The motility of thymocytes is tightly regulated; however, the molecular mechanisms that control thymocyte motility are not well understood. Here we report that G protein-coupled receptor kinase-interactor 2 (GIT2) was required for efficient positive selection. Notably, Git2(-/-) double-positive thymocytes showed greater activation of the small GTPase Rac, actin polymerization and migration toward the chemokines CXCL12 (SDF-1) and CCL25 in vitro. By two-photon laser-scanning microscopy, we found that the scanning activity of Git2(-/-) thymocytes was compromised in the thymic cortex, which suggests GIT2 has a key role in regulating the chemokine-mediated motility of double-positive thymocytes.",
"Bone resorption and remodeling is an intricately controlled, physiological process that requires the function of osteoclasts. The processes governing both the differentiation and activation of osteoclasts involve signals induced by osteoprotegerin ligand (OPGL), a member of tumor necrosis factor (TNF) superfamily, and its cognate receptor RANK. The molecular mechanisms of the intracellular signal transduction remain to be elucidated. Here we report that mice deficient in TNF receptor-associated factor 6 (TRAF6) are osteopetrotic with defects in bone remodeling and tooth eruption due to impaired osteoclast function. Using in vitro assays, we demonstrate that TRAF6 is crucial not only in IL-1 and CD40 signaling but also, surprisingly, in LPS signaling. Furthermore, like TRAF2 and TRAF3, TRAF6 is essential for perinatal and postnatal survival. These findings establish unexpectedly diverse and critical roles for TRAF6 in perinatal and postnatal survival, bone metabolism, LPS, and cytokine signaling.",
"The pleiotropic biological activities of interleukin-1 (IL-1) are mediated by its type I receptor (IL-1RI). When the ligand binds, IL-1RI initiates a signaling cascade that results in the activation of the transcription regulator nuclear factor kappa B (NF-kappa B). A protein kinase designated IRAK (IL-1 receptor-associated kinase) was purified, and its complementary DNA was molecularly cloned. When human embryonic kidney cells (cell line 293) over-expressing IL-1RI or HeLa cells were exposed to IL-1, IRAK rapidly associated with the IL-1RI complex and was phosphorylated. The primary amino acid sequence of IRAK shares similarity with that of Pelle, a protein kinase that is essential for the activation of a NF-kappa B homolog in Drosophila.",
"Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered."
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Genetic characterization of a highly pathogenic avian influenza virus subtype H5N1 isolated from a moribund domestic duck in central Vietnam
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This study reports the genetic characterization of a highly pathogenic avian influenza virus subtype H5N1 isolated from a moribund domestic duck in central Vietnam during 2012. In the moribund duck's flock, within 6 days after vaccination with a commercial H5N1 vaccine (Re-5) to 59-day-old birds, 120 out of 2,000 ducks died. Genetic analysis revealed a substantial number of mutations in the HA gene of the isolate in comparison with the vaccine strains, Re-1 and Re-5. Similar mutations were also found in selected Vietnamese H5N1 strains isolated since 2009. Mutations in the HA gene involved positions at antigenic sites associated with antibody binding and also neutralizing epitopes, with some of the mutations resulting in the modification of N-linked glycosylation of the HA. Those mutations may be related to the escape of virus from antibody binding and the infection of poultry, interpretations which may be confirmed through a reverse genetics approach. The virus also carried an amino acid substitution in the M2, which conferred a reduced susceptibility to amantadine, but no neuraminidase inhibitor resistance markers were found in the viral NA gene. Additional information including vaccination history in the farm and the surrounding area is needed to fully understand the background of this outbreak. Such understanding and expanded monitoring of the H5N1 influenza viruses circulating in Vietnam is an urgent need to provide updated information to improve effective vaccine strain selection and vaccination protocols, aiding disease control, and biosecurity to prevent H5N1 infection in both poultry and humans.
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[
"Scientists, educators and the general public often need to know times of divergence between species. But they rarely can locate that information because it is buried in the scientific literature, usually in a format that is inaccessible to text search engines. We have developed a public knowledgebase that enables data-driven access to the collection of peer-reviewed publications in molecular evolution and phylogenetics that have reported estimates of time of divergence between species. Users can query the TimeTree resource by providing two names of organisms (common or scientific) that can correspond to species or groups of species. The current TimeTree web resource (TimeTree2) contains timetrees reported from molecular clock analyses in 910 published studies and 17 341 species that span the diversity of life. TimeTree2 interprets complex and hierarchical data from these studies for each user query, which can be launched using an iPhone application, in addition to the website. Published time estimates are now readily accessible to the scientific community, K-12 and college educators, and the general public, without requiring knowledge of evolutionary nomenclature. TimeTree2 is accessible from the URL http://www.timetree.org, with an iPhone app available from iTunes (http://itunes.apple.com/us/app/timetree/id372842500?mt=8) and a YouTube tutorial (http://www.youtube.com/watch?v=CxmshZQciwo).",
"We announce the release of an advanced version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which currently contains facilities for building sequence alignments, inferring phylogenetic histories, and conducting molecular evolutionary analysis. In version 6.0, MEGA now enables the inference of timetrees, as it implements the RelTime method for estimating divergence times for all branching points in a phylogeny. A new Timetree Wizard in MEGA6 facilitates this timetree inference by providing a graphical user interface (GUI) to specify the phylogeny and calibration constraints step-by-step. This version also contains enhanced algorithms to search for the optimal trees under evolutionary criteria and implements a more advanced memory management that can double the size of sequence data sets to which MEGA can be applied. Both GUI and command-line versions of MEGA6 can be downloaded from www.megasoftware.net free of charge."
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Training for people with schizophrenia
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The purpose of this article was to conduct a review of the types of training offered to people with schizophrenia in order to help them develop strategies to cope with or compensate for neurocognitive or sociocognitive deficits.
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[
"The aim of the present study is to test Brenner's model of cognitive functioning in schizophrenia. It is assumed that elementary cognitive disorders (attention and encoding) and complex cognitive disorders (recall, concept formation) reinforce each other. Cognitive disorders are supposed to cause detrimental effects on functional outcome. We used cognitive rehabilitation as a strategy to induce cognitive changes in 27 patients assigned to treatment groups following the cognitive modules of the Integrated Psychological Treatment (IPT). Ten schizophrenic patients without cognitive impairments worked as a control group. With only one minor conceptual change (replacing concept formation with executive function, a more comprehensive construct), we found that our data fitted with Brenner's model. A relationship has been found between neuropsychological improvements and higher levels of autonomy and social functioning. These findings have important implications not only for cognitive assessment but also for selecting targets in cognitive rehabilitation.",
"Cognitive remediation training has been shown to improve both cognitive and social cognitive deficits in people with schizophrenia, but the mechanisms that support this behavioral improvement are largely unknown. One hypothesis is that intensive behavioral training in cognition and/or social cognition restores the underlying neural mechanisms that support targeted skills. However, there is little research on the neural effects of cognitive remediation training. This study investigated whether a 50 h (10-week) remediation intervention which included both cognitive and social cognitive training would influence neural function in regions that support social cognition. Twenty-two stable, outpatient schizophrenia participants were randomized to a treatment condition consisting of auditory-based cognitive training (AT) [Brain Fitness Program/auditory module ~60 min/day] plus social cognition training (SCT) which was focused on emotion recognition [~5-15 min per day] or a placebo condition of non-specific computer games (CG) for an equal amount of time. Pre and post intervention assessments included an fMRI task of positive and negative facial emotion recognition, and standard behavioral assessments of cognition, emotion processing, and functional outcome. There were no significant intervention-related improvements in general cognition or functional outcome. fMRI results showed the predicted group-by-time interaction. Specifically, in comparison to CG, AT+SCT participants had a greater pre-to-post intervention increase in postcentral gyrus activity during emotion recognition of both positive and negative emotions. Furthermore, among all participants, the increase in postcentral gyrus activity predicted behavioral improvement on a standardized test of emotion processing (MSCEIT: Perceiving Emotions). Results indicate that combined cognition and social cognition training impacts neural mechanisms that support social cognition skills.",
"The objective was to investigate the impact of a verbal memory training task on psychiatric symptoms and cognition in schizophrenia. As part of a larger, 6-month cognitive remediation program, 57 patients with schizophrenia were randomly assigned to receive performance-based, hierarchical training on a verbal memory task based on a dichotic listening (DL) with distracter paradigm. These patients were compared with 68 patients who had been randomly assigned to a control condition. Training on the DL task was not associated with changes in general psychopathology or auditory hallucinations (AH) specifically. Training was associated with improvements in verbal memory, but not attention. The current investigation adds to the growing literature on the effectiveness of cognitive remediation training and indicates that training on the DL task enhances verbal episodic memory. The results do not support the use of DL training as a method for reducing AH.",
"The purpose of this study was to evaluate whether social cognition enhancement training (SCET) is effective in improving social context appraisal and perspective-taking abilities of patients with schizophrenia. A total of 34 patients were randomly assigned to receive SCET plus standard psychiatric rehabilitation training or standard psychiatric rehabilitation training only. SCET was conducted twice weekly on a group basis for 6 months. Some social cognitive abilities were improved rapidly after 2-month treatment whereas some improved only after 6-month treatment. These results show promise for the impact of SCET on the social cognitive functioning of individuals with chronic schizophrenia, and further investigation is merited.",
"The cognitive deficits associated with schizophrenia commonly include impairment in attention, which may contribute to difficulties with learning, memory, and executive function. This study evaluated the effectiveness of computer-aided training of attentional skills in schizophrenia. Two groups of schizophrenic subjects (9 men and 1 woman) were matched for age, estimated premorbid IQ, and positive and negative symptom scores. Both groups were assessed using a battery of attentional tests. Subjects then received either six 1-hr. computer-aided cognitive rehabilitation sessions (experimental condition) or six sessions of graphics-based computer games (control condition). Both groups were reassessed with attentional measures. There was significant improvement on only one test, a letter-cancellation task. This improvement was evident in both groups suggesting that this was a practise effect. Apart from the letter-cancellation test, subjects undertaking the computer-aided rehabilitation treatment did not show significant improvement on any attentional tasks.",
"Many individuals diagnosed with schizophrenia have significant neurocognitive deficits, especially in the areas of attention, memory, and executive function. These deficits may exacerbate patients' psychiatric symptoms. Cognitive remediation has shown efficacy in improving neurocognitive functioning and may lead to amelioration of psychiatric symptoms in persons with schizophrenia. Forty-two schizophrenic inpatients were randomly assigned to either a cognitive remediation group (n=21) or a control group (n=21) and were assessed using a neuropsychological battery and symptom scales for depression, anxiety, and positive and negative symptoms of schizophrenia at baseline and after two months of participation in a cognitive remediation program. The intervention group showed significant improvements in neuropsychological functioning, depression and negative symptoms of schizophrenia after the intervention compared to the control group. Results of the study provide support for cognitive remediation as an efficacious intervention to improve neurocognitive functioning and decrease psychiatric symptoms of schizophrenia.",
"Cognitive impairment has always been regarded as an important characteristic of schizophrenia. Many domains of cognition are disrupted with varying degrees of deficit: attention, executive functions, verbal and visuospatial working memory, learning, and memory. However, it is only recently that cognitive dysfunction has been recognized as a primary and enduring core deficit in schizophrenia (rather than the previous focus on positive and negative symptoms). This article discusses cognitive impairment and the therapeutic effects of newer antipsychotic agents on cognitive functioning in patients with schizophrenia. Cognitive dysfunction occurs before the first psychotic episode and persists throughout the course of the illness. It involves every aspect of cognitive functioning and has an important impact on long-term social and occupational outcomes. Improvement of cognitive functioning by antipsychotic treatment can be due indirectly to the improvement of therapeutic profiles of the newer antipsychotic agents (eg, higher efficacy on positive and negative symptoms, fewer side effects, less anticholinergic effects) or directly to effects on cerebral functioning (eg, by restoring dopamine prefrontal activity). However, further research is needed regarding the therapeutic effects of the newer antipsychotic drugs on cognitive functioning and their impact on psychosocial outcome. Although newer medications may improve cognitive functioning, they do not normalize neurocognitive deficits in schizophrenia. In addition, various nonpharmacologic, psychological interventions have been used in the rehabilitation of patients with cognitive deficits."
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Adiponectin and metabolic and cardiovascular disease
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Under disease conditions including obesity (insulin resistance) and diabetes, dysregulation of adipokines such as tumor necrosis factor (TNF)-α, leptin, resistin, and adiponectin contribute to the development of metabolic and cardiovascular disease. Unlike other adipokines, adiponectin has been shown to be a therapeutic target for metabolic syndrome and cardiovascular disease. Circulating levels of adiponectin are markedly reduced in obese, diabetic, hypertensive, and coronary artery disease patients as well as experimental animal models of insulin resistance and diabetes. Recently, the small molecule adiponectin receptors (AdipoRs) agonist was discovered and suggested that the agonist is a novel therapeutic target for the treatment of type 2 diabetes linked to obesity in an experimental mouse model. This review will focus on signaling pathways involved in adiponectin and its receptors and the role of adiponectin in metabolic and cardiovascular disease including insulin resistance, cardiomyopathy, and cardiovascular dysfunction.
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[
"Age-related macular degeneration (AMD) is a leading cause of central blindness in the elderly population. The wet type of AMD is characterized by extensive growth of new vessels. One of the effective strategies to treat wet AMD is to limit the choroidal neovascularization (CNV). We studied the effects of adiponectin peptide I (APNpI) on new vessel growth in laser-induced rat model of wet AMD and on rat choroidal endothelial cell (CEC) culture. CNV size and vessel density were investigated by microscopy. Immunohistochemical staining (IHC) for von Willebrand Factor (vWF), APN, APN receptors 1 (AdipoR1), 2 (AdipoR2), VEGF, VEGF receptor 2 (VEGF-R2), proliferating cell nuclear antigen (PCNA) was performed in CNV area. The mRNA expression of VEGF and VEGF-R2 in RPE-choroid was investigated by RT-PCR and real-time PCR. APNpI inhibited area of CNV by 4 fold, number of vWF positive vessels by 99% and area of subretinal tissue by 40%. The expression of VEGF and VEGF-R2 at mRNA and protein levels decreased after APNpI treatment in vivo. Proliferative index (PCNA) was 5 folds less in laser spots of APNpI treated rats compared to controls. In conclusion, APNpI inhibited formation of new vessels in rat model of CNV by decreasing VEGF, VEGF-R2 expression and cell proliferation. Thus, APNpI may have potential therapeutic use for AMD treatment since it significantly inhibited CNV.",
"The objective of the present study was to investigate the effect of alcohol and nicotine consumption on the pathogenesis of choroidal neovascularization (CNV) in rats after laser-photocoagulation. Confocal microscopic analysis demonstrated an increase in CNV complex size in rats fed with alcohol (2.3-fold), nicotine (1.9-fold), and the combination of alcohol and nicotine (2.7-fold) compared with the control groups. Immunohistochemical analysis revealed that alcohol and nicotine consumption increased MAC deposition and VEGF expression in laser spots. Expression of CD59 by RT-PCR and Western blot was drastically reduced in the animals that were fed with alcohol, nicotine and alcohol and nicotine compared to those fed with water alone and this was associated with exacerbation of CNV.",
"The unique vascular supply of the retina, the ability to visualize the vasculature in vivo, and the ability to selectively express genes in the retina make the retina an ideal model system to study molecular mechanisms of angiogenesis. In addition, this area of investigation has great clinical significance, because retinal and choroidal neovascularization are the most common causes of severe visual loss in developed countries and new treatments are needed. As a result, interest in ocular neovascularization is rapidly growing and there has been considerable recent progress. Use of genetically engineered mice in recently developed murine models provides a means to investigate the role of individual gene products in neovascularization in two distinct vascular beds, the retinal vasculature and the choroidal vasculature. It appears that angiogenesis in different vascular beds has common themes, but also has tissue-specific aspects. This review summarizes recent progress in the field of ocular neovascularization and the prospects that it provides for the development of new treatments.",
"Adiponectin is an adipocyte-specific adipocytokine with anti-atherogenic and anti-diabetic properties. Here, we investigated whether adiponectin regulates angiogenic processes in vitro and in vivo. Adiponectin stimulated the differentiation of human umbilical vein endothelium cells (HUVECs) into capillary-like structures in vitro and functioned as a chemoattractant in migration assays. Adiponectin promoted the phosphorylation of AMP-activated protein kinase (AMPK), protein kinase Akt/protein kinase B, and endothelial nitric oxide synthesis (eNOS) in HUVECs. Transduction with either dominant-negative AMPK or dominant-negative Akt abolished adiponectin-induced eNOS phosphorylation as well as adiponectin-stimulated HUVEC migration and differentiation. Dominant-negative AMPK also inhibited adiponectin-induced Akt phosphorylation, suggesting that AMPK is upstream of Akt. Dominant-negative Akt or the phosphatidylinositol 3-kinase inhibitor LY294002 blocked adiponectin-stimulated Akt and eNOS phosphorylation, migration, and differentiation without altering AMPK phosphorylation. Finally, adiponectin stimulated blood vessel growth in vivo in mouse Matrigel plug implantation and rabbit corneal models of angiogenesis. These data indicate that adiponectin can function to stimulate the new blood vessel growth by promoting cross-talk between AMP-activated protein kinase and Akt signaling within endothelial cells.",
"Adiponectin (ADPN), which is a secretory protein of adipose tissue, attenuates endothelial inflammatory responses in vitro. Among human subjects, plasma ADPN concentrations are reduced among patients with atherosclerotic complications but are substantially increased among patients with advanced renal failure. The clinical and biochemical correlates of plasma ADPN levels were investigated and the predictive power of ADPN levels with respect to survival rates and cardiovascular events was prospectively tested in a cohort of 227 hemodialysis patients, who were monitored for 31 +/- 13 mo. Plasma ADPN levels were 2.5 times higher (P < 0.0001) among dialysis patients (15.0 +/- 7.7 microg/ml) than among healthy subjects (6.3 +/- 2.0 microg/ml), were independent of age, and were higher (P = 0.03) among women (15.2 +/- 7.9 microg/ml) than among men (14.0 +/- 7.4 microg/ml). For both genders, plasma ADPN levels were inversely related to body mass index values, plasma leptin levels, insulin levels, serum triglyceride levels, and homeostatic model assessment index values. Furthermore, plasma ADPN levels were directly related to HDL cholesterol levels and inversely related to von Willebrand factor levels. Plasma ADPN levels were lower (P < 0.05) among patients who experienced new cardiovascular events (13.7 +/- 7.3 microg/ml) than among event-free patients (15.8 +/- 7.8 microg/ml). There was a 3% risk reduction for each 1 microg/ml increase in plasma ADPN levels, and the relative risk of adverse cardiovascular events was 1.56 times (95% confidence interval, 1.12 to 1.99 times) higher among patients in the first ADPN tertile, compared with those in the third tertile. Plasma ADPN levels are an inverse predictor of cardiovascular outcomes among patients with end-stage renal disease. Furthermore, ADPN is related to several metabolic risk factors in a manner consistent with the hypothesis that this protein acts as a protective factor for the cardiovascular system.",
"Metabolic syndrome is thought to result from obesity and obesity-linked insulin resistance. Obesity in adulthood is characterized by adipocyte hypertrophy. Adipose tissue participates in the regulation of energy homeostasis as an important endocrine organ that secretes a number of biologically active \"adipokines.\"Heterozygous peroxisome proliferator-activated receptor-gamma knockout mice were protected from high-fat diet induced obesity, adipocyte hypertrophy, and insulin resistance. Systematic gene profiling analysis of these mice revealed that adiponectin/Acrp30 was overexpressed. Functional analyses including generation of adiponectin transgenic or knockout mice have revealed that adiponectin serves as an insulin-sensitizing adipokine. In fact, obesity-linked down-regulation of adiponectin was a mechanism whereby obesity could cause insulin resistance and diabetes. Recently, we have cloned adiponectin receptors in the skeletal muscle (AdipoR1) and liver (AdipoR2), which appear to comprise a novel cell-surface receptor family. We showed that AdipoR1 and AdipoR2 serve as receptors for globular and full-length adiponectin and mediate increased AMP-activated protein kinase, peroxisome proliferator-activated receptor-alpha ligand activities, and glucose uptake and fatty-acid oxidation by adiponectin. Obesity decreased expression levels of AdipoR1/R2, thereby reducing adiponectin sensitivity, which finally leads to insulin resistance, the so-called \"vicious cycle.\" Most recently, we showed that osmotin, which is a ligand for the yeast homolog of AdipoR (PHO36), activated AMPK via AdipoR in C2C12 myocytes. This may facilitate efficient development of adiponectin receptor agonists. Adiponectin receptor agonists and adiponectin sensitizers should serve as versatile treatment strategies for obesity-linked diseases such as diabetes and metabolic syndrome.",
"To examine the extent to which the defect in insulin action in subjects with non-insulin-dependent diabetes mellitus (NIDDM) can be accounted for by impairment of muscle glycogen synthesis, we performed combined hyperglycemic-hyperinsulinemic clamp studies with [13C]glucose in five subjects with NIDDM and in six age- and weight-matched healthy subjects. The rate of incorporation of intravenously infused [1-13C]glucose into muscle glycogen was measured directly in the gastrocnemius muscle by means of a nuclear magnetic resonance (NMR) spectrometer with a 15.5-minute time resolution and a 13C surface coil. The steady-state plasma concentrations of insulin (approximately 400 pmol per liter) and glucose (approximately 10 mmol per liter) were similar in both study groups. The mean (+/- SE) rate of glycogen synthesis, as determined by 13C NMR, was 78 +/- 28 and 183 +/- 39 mumol-glucosyl units per kilogram of muscle tissue (wet weight) per minute in the diabetic and normal subjects, respectively (P less than 0.05). The mean glucose uptake was markedly reduced in the diabetic (30 +/- 4 mumol per kilogram per minute) as compared with the normal subjects (51 +/- 3 mumol per kilogram per minute; P less than 0.005). The mean rate of nonoxidative glucose metabolism was 22 +/- 4 mumol per kilogram per minute in the diabetic subjects and 42 +/- 4 mumol per kilogram per minute in the normal subjects (P less than 0.005). When these rates are extrapolated to apply to the whole body, the synthesis of muscle glycogen would account for most of the total-body glucose uptake and all of the nonoxidative glucose metabolism in both normal and diabetic subjects. We conclude that muscle glycogen synthesis is the principal pathway of glucose disposal in both normal and diabetic subjects and that defects in muscle glycogen synthesis have a dominant role in the insulin resistance that occurs in persons with NIDDM.",
"Endothelial dysfunction is a crucial feature in the evolution of atherosclerosis. Adiponectin is an adipocyte-specific plasma protein with antiatherogenic and antidiabetic properties. In the present study, we investigated the relation between adiponectin and endothelium-dependent vasodilation. We analyzed endothelial function in 202 hypertensive patients, including those who were not taking any medication. Forearm blood flow was measured by strain-gauge plethysmography. Plasma adiponectin level was highly correlated with the vasodilator response to reactive hyperemia in the total (r=0.257, P<0.001) and no-medication (r=0.296, P=0.026) groups but not with nitroglycerin-induced hyperemia, indicating that adiponectin affected endothelium-dependent vasodilation. Multiple regression analysis of data from all hypertensive patients revealed that plasma adiponectin level was independently correlated with the vasodilator response to reactive hyperemia. Vascular reactivity was also analyzed in aortic rings from adiponectin-knockout (KO) and wild-type (WT) mice. Adiponectin-KO mice showed obesity, hyperglycemia, and hypertension compared with WT mice after 4 weeks on an atherogenic diet. Endothelium-dependent vasodilation in response to acetylcholine was significantly reduced in adiponectin-KO mice compared with WT mice, although no significant difference was observed in endothelium-independent vasodilation in response to sodium nitroprusside. Our observations suggest that hypoadiponectinemia is associated with impaired endothelium-dependent vasorelaxation and that the measurement of plasma adiponectin level might be helpful as a marker of endothelial dysfunction.",
"Previous studies revealed that carboxyl-terminal fragment containing the globular domain of adiponectin exists in human plasma. Although it is proposed that the globular fragment is generated by proteolytic cleavage, the place and responsible enzyme of the cleavage are still unclear. In this study, we evaluated the activity to cleave adiponectin in culture medium of several cell lines in vitro. Adiponectin cleavage into several carboxyl-terminal fragments containing the globular domain was observed in the medium of phorbol 12-myristate 13-acetate-stimulated monocytic cell lines THP-1 and U937. The molecular masses of the major products were 25, 20, and 18 kDa. The cleavage was thought to be mediated by leukocyte elastase (also known as neutrophil elastase) based on the following observations. First, the cleavage was inhibited by serine-protease inhibitors [phenylmethylsulfonylfluoride, Pefabloc SC (Roche Diagnostics, Basel, Switzerland) and aprotinin] and by the leukocyte elastase-specific peptide inhibitor MeOSuc-AAPV-CMK. Second, no activity was detected after THP-1 cells had fully differentiated into macrophages. Third, purified leukocyte elastase cleaved adiponectin with the same cleavage pattern as THP-1 cells. Finally, leukocyte elastase secreted by activated neutrophils cleaved adiponectin into the globular fragments. Amino-terminal sequence analysis revealed that cleavage sites of adiponectin by purified leukocyte elastase were between 38Thr and 39Cys, 40Ala and 41Gly, 44Ala and 45Gly, 91Ala and 92Glu, and 110Ala and 111Ala (the numbering of the positions of the amino acids starts at the signal sequence), suggesting that the cleavage occurs in the collagenous domain. These data indicate that the cleavage of adiponectin by leukocyte elastase secreted from activated monocytes and/or neutrophils could be a candidate for the mechanism of the generation of the globular fragment of adiponectin."
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Polycomb repressive complex 2 binds to RNA but inhibits its enzymatic activity until it associates with JARID2
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Polycomb repressive complex 2 (PRC2) is a histone methyltransferase that is localized to thousands of mammalian genes. Though important to human disease and as a drug target, how PRC2 is recruited remains unclear. One model invokes cis-regulatory RNA. Herein, we biochemically and functionally probe PRC2's recognition of RNA using the X-inactivation model. We observe surprisingly high discriminatory capabilities. While SUZ12 and JARID2 subunits can bind RNA, EZH2 has highest affinity and is somewhat promiscuous. EED regulates the affinity of EZH2 for RNA, lending greater specificity to PRC2-RNA interactions. Intriguingly, while RNA is crucial for targeting, RNA inhibits EZH2's catalytic activity. JARID2 weakens PRC2's binding to RNA and relieves catalytic inhibition. We propose that RNA guides PRC2 to its target but inhibits its enzymatic activity until PRC2 associates with JARID2 on chromatin. Our study provides a molecular view of regulatory interactions between RNA and PRC2 at the chromatin interface.
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[
"Histone methylation is an important epigenetic phenomenon that participates in a diverse array of cellular processes and has been found to be associated with cancer. Recent identification of several histone demethylases has proved that histone methylation is a reversible process. Through a candidate approach, we have biochemically identified JMJD3 as an H3K27 demethylase. Transfection of JMJD3 into HeLa cells caused a specific reduction of trimethyl H3K27, but had no effect on di- and monomethyl H3K27, or histone lysine methylations on H3K4 and H3K9. The enzymatic activity requires the JmjC domain and the conserved histidine that has been suggested to be important for a cofactor binding. In vitro biochemical experiments demonstrated that JMJD3 directly catalyzes the demethylation. In addition, we found that JMJD3 is upregulated in prostate cancer, and its expression is higher in metastatic prostate cancer. Thus, we identified JMJD3 as a demethylase capable of removing the trimethyl group from histone H3 lysine 27 and upregulated in prostate cancer.",
"The enzymes catalyzing lysine and arginine methylation of histones are essential for maintaining transcriptional programs and determining cell fate and identity. Until recently, histone methylation was regarded irreversible. However, within the last few years, several families of histone demethylases erasing methyl marks associated with gene repression or activation have been identified, underscoring the plasticity and dynamic nature of histone methylation. Recent discoveries have revealed that histone demethylases take part in large multiprotein complexes synergizing with histone deacetylases, histone methyltransferases, and nuclear receptors to control developmental and transcriptional programs. Here we review the emerging biochemical and biological functions of the histone demethylases and discuss their potential involvement in human diseases, including cancer.",
"Posttranslational modifications of histone N-terminal tails impact chromatin structure and gene transcription. While the extent of histone acetylation is determined by both acetyltransferases and deacetylases, it has been unclear whether histone methylation is also regulated by enzymes with opposing activities. Here, we provide evidence that LSD1 (KIAA0601), a nuclear homolog of amine oxidases, functions as a histone demethylase and transcriptional corepressor. LSD1 specifically demethylates histone H3 lysine 4, which is linked to active transcription. Lysine demethylation occurs via an oxidation reaction that generates formaldehyde. Importantly, RNAi inhibition of LSD1 causes an increase in H3 lysine 4 methylation and concomitant derepression of target genes, suggesting that LSD1 represses transcription via histone demethylation. The results thus identify a histone demethylase conserved from S. pombe to human and reveal dynamic regulation of histone methylation by both histone methylases and demethylases.",
"An important development in understanding the influence of chromatin on gene regulation has been the finding that DNA methylation and histone post-translational modifications lead to the recruitment of protein complexes that regulate transcription. Early interpretations of this phenomenon involved gene regulation reflecting predictive activating or repressing types of modification. However, further exploration reveals that transcription occurs against a backdrop of mixtures of complex modifications, which probably have several roles. Although such modifications were initially thought to be a simple code, a more likely model is of a sophisticated, nuanced chromatin 'language' in which different combinations of basic building blocks yield dynamic functional outcomes."
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are inotropes used in heart failure?
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Inotropes are widely used in hospitalized systolic heart failure (HF) patients, especially those with low systolic blood pressure (SBP) or cardiac index. In addition, inotropes are considered to be harmful in nonischemic HF.
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"Pulmonary artery catheters (PACs) have been used to guide therapy in multiple settings, but recent studies have raised concerns that PACs may lead to increased mortality in hospitalized patients. To determine whether PAC use is safe and improves clinical outcomes in patients hospitalized with severe symptomatic and recurrent heart failure. The Evaluation Study of Congestive Heart Failure and Pulmonary Artery Catheterization Effectiveness (ESCAPE) was a randomized controlled trial of 433 patients at 26 sites conducted from January 18, 2000, to November 17, 2003. Patients were assigned to receive therapy guided by clinical assessment and a PAC or clinical assessment alone. The target in both groups was resolution of clinical congestion, with additional PAC targets of a pulmonary capillary wedge pressure of 15 mm Hg and a right atrial pressure of 8 mm Hg. Medications were not specified, but inotrope use was explicitly discouraged. The primary end point was days alive out of the hospital during the first 6 months, with secondary end points of exercise, quality of life, biochemical, and echocardiographic changes. Severity of illness was reflected by the following values: average left ventricular ejection fraction, 19%; systolic blood pressure, 106 mm Hg; sodium level, 137 mEq/L; urea nitrogen, 35 mg/dL (12.40 mmol/L); and creatinine, 1.5 mg/dL (132.6 micromol/L). Therapy in both groups led to substantial reduction in symptoms, jugular venous pressure, and edema. Use of the PAC did not significantly affect the primary end point of days alive and out of the hospital during the first 6 months (133 days vs 135 days; hazard ratio [HR], 1.00 [95% confidence interval {CI}, 0.82-1.21]; P = .99), mortality (43 patients [10%] vs 38 patients [9%]; odds ratio [OR], 1.26 [95% CI, 0.78-2.03]; P = .35), or the number of days hospitalized (8.7 vs 8.3; HR, 1.04 [95% CI, 0.86-1.27]; P = .67). In-hospital adverse events were more common among patients in the PAC group (47 [21.9%] vs 25 [11.5%]; P = .04). There were no deaths related to PAC use, and no difference for in-hospital plus 30-day mortality (10 [4.7%] vs 11 [5.0%]; OR, 0.97 [95% CI, 0.38-2.22]; P = .97). Exercise and quality of life end points improved in both groups with a trend toward greater improvement with the PAC, which reached significance for the time trade-off at all time points after randomization. Therapy to reduce volume overload during hospitalization for heart failure led to marked improvement in signs and symptoms of elevated filling pressures with or without the PAC. Addition of the PAC to careful clinical assessment increased anticipated adverse events, but did not affect overall mortality and hospitalization. Future trials should test noninvasive assessments with specific treatment strategies that could be used to better tailor therapy for both survival time and survival quality as valued by patients.",
"Heart failure (HF) is a syndrome characterized by high mortality, frequent hospitalization, reduced quality of life, and a complex therapeutic regimen. Knowledge about HF is accumulating so rapidly that individual clinicians may be unable to readily and adequately synthesize new information into effective strategies of care for patients with this syndrome. Trial data, though valuable, often do not give direction for individual patient management. These characteristics make HF an ideal candidate for practice guidelines. The 2010 Heart Failure Society of America comprehensive practice guideline addresses the full range of evaluation, care, and management of patients with HF.",
"This study was designed to determine whether nesiritide, administered for acute decompensated congestive heart failure (CHF), affects healthcare costs by hospital length of stay (LOS), readmissions and short-term mortality, compared to dobutamine. Dobutamine is a commonly used inotropic treatment for CHF. Although dobutamine may have favorable hemodynamic and symptomatic effects, its use may be associated with side effects such as tachycardia, cardiac arrhythmias and myocardial ischemia. Nesiritide (B-type natriuretic peptide) is a new intravenous (IV) drug that produces hemodynamic and symptomatic improvement through balanced vasodilatory effects, neurohormonal suppression and enhanced natriuresis and diuresis. From an open-label randomized study of nesiritide versus standard care (SC) in patients with CHF requiring hospitalization, we compared short-term outcome data from patients given nesiritide (0.015 or 0.03 microg/kg per min) with a subgroup of SC patients given dobutamine. A total of 261 patients are included in this analysis. Compared to dobutamine, both nesiritide doses were administered for a shorter total duration (p < 0.001), and the total duration of all IV vasoactive therapy (including study drug) was also shorter (p less-than-or-equal 0.012). Although there was no difference in LOS, there was a trend toward decreased readmissions in the two nesiritide groups (8% and 11%, respectively, vs. 20% in the dobutamine group). Six-month mortality was lower in the nesiritide groups. Treatment of decompensated CHF with nesiritide may lead to lower healthcare costs and reduced mortality compared to treatment with dobutamine.",
"The appropriate role of intravenous inodilator therapy (inotropic agents with vasodilator properties) in the management of acute heart failure syndromes (AHFS) has long been a subject of controversy, mainly because of the lack of prospective, placebo-controlled trials and a lack of alternative therapies. The use of intravenous inodilator infusions, however, remains common, but highly variable. As new options emerge for the treatment of AHFS, the available information should be reviewed to determine which approaches are supported by evidence, which are used empirically without evidence, and which should be considered inappropriate. For these purposes, we reviewed data available from randomized controlled trials on short-term, intermittent, and long-term use of intravenous inodilator agents (dobutamine, dopamine, and milrinone) in AHFS. Randomized controlled trials failed to show benefits with current medications and suggested that acute, intermittent, or continuous use of inodilator infusions may increase morbidity and mortality in patients with AHFS. Their use should be restricted to patients who are hypotensive as a result of low cardiac output despite a high left ventricular filling pressure.",
"Clinical trials for acute heart failure syndromes (AHFS) have traditionally enrolled patients well after emergency department (ED) presentation. We hypothesized a large proportion of patients would undergo changes in clinical profiles during the first 24 h of hospitalization, and these changes would be associated with adverse events. We evaluated a prospective cohort of patients with clinical data available at ED presentation and 12-24 h after ED treatment for AHFS. Patients were categorized into distinct clinical profiles at these time points based on (1) systolic blood pressure: a-hypertensive (>160 mmHg); b-normotensive (100-159 mmHg); or c-hypotensive (<100 mmHg); (2) moderate-to-severe renal dysfunction (GFR ≤ 60 ml/min/1.73 m(2)); and (3) presence of troponin positivity. A composite outcome of 30-day cardiovascular events was determined by phone follow-up. In the 370 patients still hospitalized with data available at the 12-24 h time point, 196 (53.0%) had changed their clinical profiles, with 117 (59.7%) improving and 79 (40.3%) worsening. The composite 30-day event rate was 16.9%. Patients whose clinical profile started and stayed abnormal had a significantly greater proportion of events than those who started and stayed normal (26.1% vs. 11.3%; P = 0.03). Patients with abnormal clinical profiles at presentation that remain abnormal throughout the first 12-24 h of hospitalization are at increased risk of 30-day adverse events. Future clinical trials may need to consider targeting these patients, as they may be the most likely to benefit from experimental therapy.",
"Because acute decompensated heart failure causes substantial morbidity and mortality, there is a need for agents that at least improve hemodynamics and relieve symptoms without adversely affecting survival. To assess the effect of a short-term intravenous infusion of levosimendan or dobutamine on long-term survival. The Survival of Patients With Acute Heart Failure in Need of Intravenous Inotropic Support (SURVIVE) study was a randomized, double-blind trial comparing the efficacy and safety of intravenous levosimendan or dobutamine in 1327 patients hospitalized with acute decompensated heart failure who required inotropic support. The trial was conducted at 75 centers in 9 countries and patients were randomized between March 2003 and December 2004. Intravenous levosimendan (n = 664) or intravenous dobutamine (n = 663). All-cause mortality at 180 days. All-cause mortality at 180 days occurred in 173 (26%) patients in the levosimendan group and 185 (28%) patients in the dobutamine group (hazard ratio, 0.91; 95% confidence interval, 0.74-1.13; P = .40). The levosimendan group had greater decreases in B-type natriuretic peptide level at 24 hours that persisted through 5 days compared with the dobutamine group (P<.001 for all time points). There were no statistical differences between treatment groups for the other secondary end points (all-cause mortality at 31 days, number of days alive and out of the hospital, patient global assessment, patient assessment of dyspnea at 24 hours, and cardiovascular mortality at 180 days). There was a higher incidence of cardiac failure in the dobutamine group. There were higher incidences of atrial fibrillation, hypokalemia, and headache in the levosimendan group. Despite an initial reduction in plasma B-type natriuretic peptide level in patients in the levosimendan group compared with patients in the dobutamine group, levosimendan did not significantly reduce all-cause mortality at 180 days or affect any secondary clinical outcomes. clinicaltrials.gov Identifier: NCT00348504.",
"Although patients hospitalized with heart failure have relatively low in-hospital mortality, the post-discharge rehospitalization and mortality rates remain high despite advances in treatment. Most patients admitted for heart failure have normal or high blood pressure, but 15-25 % have low systolic blood pressure with or without signs and/or symptoms of hypoperfusion. All pharmacological agents known to improve the prognosis of patients with heart failure also reduce blood pressure, and this limits their use in patients with heart failure and low blood pressure (HF-LBP). However, patients with HF-LBP have much higher in-hospital and post-discharge mortality. In these patients, a conceptually important therapeutic target is to improve cardiac output in order to alleviate signs of hypoperfusion. Accordingly, the majority of these patients will require an inotrope as cardiac dysfunction is the cause of their low cardiac output. However, the short-term use of currently available inotropes has been associated with further decreases in blood pressure and increases in heart rate, myocardial oxygen consumption and arrhythmias. Agents that improve cardiac contractility without this undesirable effects should be developed. To the best of our knowledge, the epidemiology, pathophysiology and therapy of patients with HF-LBP have not been addressed thoroughly. In June 2010, a workshop that included scientists and clinicians was held in Rome, Italy. The objectives of this meeting were to (1) develop a working definition for HF-LBP, (2) describe its clinical characteristics and pathophysiology, (3) review current therapies and their limitations, (4) discuss novel agents in development and (5) create a framework for the design and conduct of future clinical trials.",
"Acute heart failure syndromes (AHFS) are associated with the rapid onset of heart failure (HF) signs and symptoms. Hospitalizations for AHFS continue to rise and are associated with significant mortality and morbidity. Several pharmacological agents are currently approved for the treatment of AHFS, but their use is associated with an increase in short-term mortality. There is a need for new agents that can be given in the acute setting with increased efficacy and safety. Istaroxime is a unique agent with both inotropic and lusitropic properties which is currently being studied for the treatment of AHFS. Istaroxime inhibits the sodium-potassium adenosine triphosphatase (ATPase) and stimulates the sarcoplasmic reticulum calcium ATPase isoform 2 (SERCA-2) thereby improving contractility and diastolic relaxation. Early data from human studies reveal that istaroxime decreases pulmonary capillary wedge pressure (PCWP) and possibly improves diastolic function without causing a significant change in heart rate (HR), blood pressure, ischemic or arrhythmic events. Most commonly reported side effects were related to gastrointestinal intolerance and were dose related. In conclusion, istaroxime is a novel agent being investigated for the treatment of AHFS whose mechanism of action and cellular targets make it a promising therapy. Further studies with longer infusion times in patients with hypotension are required to confirm its efficacy and safety.",
"The primary objective of the new ESC-HF Pilot Survey was to describe the clinical epidemiology of outpatients and inpatients with heart failure (HF) and the diagnostic/therapeutic processes applied across 12 participating European countries. This pilot study was specifically aimed at validating the structure, performance, and quality of the data set, for continuing the survey into a permanent registry. The ESC-HF Pilot study is a prospective, multicentre, observational survey conducted in 136 cardiology centres from 12 European countries selected to represent the different health systems and care attitudes across Europe. All outpatients with HF and patients admitted for acute HF were included during the enrolment period (1 day per week for 8 consecutive months). From October 2009 to May 2010, 5118 patients were included in this pilot survey, of which 1892 (37%) were admitted for acute HF and 3226 (63%) for chronic HF. Ischaemic aetiology was reported in about half of the patients. In patients admitted for acute HF, the most frequent clinical profile was decompensated HF (75% of cases), whereas pulmonary oedema and cardiogenic shock were reported, respectively, in 13.3 and 2.3% of the cases. The total in-hospital mortality rate was 3.8% and was cardiovascular in 90.1% of the cases. Lowest and highest mortality rates were observed in hypertensive HF and in cardiogenic shock, respectively. More than 80% of patients with chronic HF were treated with renin-angiotensin-aldosterone system blockers and β-adrenergic blockers. However, target doses of such drugs were reached in one-third to one-fourth of the patients only. The ESC-HF Pilot Survey is an example of the possibility of utilizing an observational methodology to get insights into the current clinical practice in Europe, whose picture will be completed by the 1-year follow-up. Moreover, this study offered the opportunity to refine the organizational structure of a long-term, extended European network.",
"Acute heart failure represents an increasingly common cause of hospitalization, and may require the use of inotropic drugs in patients with low cardiac output and evidence of organ hypoperfusion. However, currently available therapies may have deleterious effects and increase mortality. An ideal inotropic drug should restore effective tissue perfusion by enhancing myocardial contractility without causing adverse effects. Such a drug is not available yet. New agents with different biological targets are under clinical development. In particular, istaroxime seems to dissociate the inotropic effect exerted by digitalis (inhibition of the membrane sodium/potassium adenosine triphosphatase) from the arrhythmic effect and to ameliorate diastolic dysfunction (via sarcoendoplasmic reticulum calcium adenosine triphosphatase activation). Additionally, the myosin activator omecamtiv mecarbil appears to have promising characteristics, while genetic therapy has been explored in animal studies only. Further investigations are needed to confirm and expand the effectiveness and safety of these agents in patients with acute heart failure and low cardiac output."
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Inherent dynamics of head domain in determining ATP-recognition of P2X4 receptors
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P2X receptors are ATP-gated ion channels involved in many physiological functions, and determination of ATP-recognition (AR) of P2X receptors will promote the development of new therapeutic agents for pain, inflammation, bladder dysfunction and osteoporosis. Recent crystal structures of the zebrafish P2X4 (zfP2X4) receptor reveal a large ATP-binding pocket (ABP) located at the subunit interface of zfP2X4 receptors, which is occupied by a conspicuous cluster of basic residues to recognize triphosphate moiety of ATP. Using the engineered affinity labeling and molecular modeling, at least three sites (S1, S2 and S3) within ABP have been identified that are able to recognize the adenine ring of ATP, implying the existence of at least three distinct AR modes in ABP. The open crystal structure of zfP2X4 confirms one of three AR modes (named AR1), in which the adenine ring of ATP is buried into site S1 while the triphosphate moiety interacts with clustered basic residues. Why architecture of ABP favors AR1 not the other two AR modes still remains unexplored. Here, we examine the potential role of inherent dynamics of head domain, a domain involved in ABP formation, in AR determinant of P2X4 receptors. In silico docking and binding free energy calculation revealed comparable characters of three distinct AR modes. Inherent dynamics of head domain, especially the downward motion favors the preference of ABP for AR1 rather than AR2 and AR3. Along with the downward motion of head domain, the closing movement of loop139-146 and loop169-183, and structural rearrangements of K70, K72, R298 and R143 enabled ABP to discriminate AR1 from other AR modes. Our observations suggest the essential role of head domain dynamics in determining AR of P2X4 receptors, allowing evaluation of new strategies aimed at developing specific blockers/allosteric modulators by preventing the dynamics of head domain associated with both AR and channel activation of P2X4 receptors.
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"Inflammation is the most fundamental body reaction to noxious stimuli. No vascularized tissue, organ or apparatus is free from this response. Several mediators of inflammation, originating from outside (exogenous) or inside (endogenous) the body, are known. Among the endogenous factors, extracellular nucleotides and nucleosides are attracting interest for their ubiquity and striking ability to modulate diverse immune responses. Until recently, it was doubted that the central nervous system (CNS), reportedly an 'immunoprivileged organ', could be the site of immune reactions. Nowadays, it is acknowledged that inflammation and immunity have a key role in a vast range of CNS diseases. Likewise, it is clear that purinergic signalling profoundly affects neuroinflammation. Here, we provide a brief update of the state of the art in this expanding field.",
"Unlike other methods for docking ligands to the rigid 3D structure of a known protein receptor, Glide approximates a complete systematic search of the conformational, orientational, and positional space of the docked ligand. In this search, an initial rough positioning and scoring phase that dramatically narrows the search space is followed by torsionally flexible energy optimization on an OPLS-AA nonbonded potential grid for a few hundred surviving candidate poses. The very best candidates are further refined via a Monte Carlo sampling of pose conformation; in some cases, this is crucial to obtaining an accurate docked pose. Selection of the best docked pose uses a model energy function that combines empirical and force-field-based terms. Docking accuracy is assessed by redocking ligands from 282 cocrystallized PDB complexes starting from conformationally optimized ligand geometries that bear no memory of the correctly docked pose. Errors in geometry for the top-ranked pose are less than 1 A in nearly half of the cases and are greater than 2 A in only about one-third of them. Comparisons to published data on rms deviations show that Glide is nearly twice as accurate as GOLD and more than twice as accurate as FlexX for ligands having up to 20 rotatable bonds. Glide is also found to be more accurate than the recently described Surflex method.",
"Normal mode analysis (NMA) is a powerful tool for predicting the possible movements of a given macromolecule. It has been shown recently that half of the known protein movements can be modelled by using at most two low-frequency normal modes. Applications of NMA cover wide areas of structural biology, such as the study of protein conformational changes upon ligand binding, membrane channel opening and closure, potential movements of the ribosome, and viral capsid maturation. Another, newly emerging field of NMA is related to protein structure determination by X-ray crystallography, where normal mode perturbed models are used as templates for diffraction data phasing through molecular replacement (MR). Here we present ElNémo, a web interface to the Elastic Network Model that provides a fast and simple tool to compute, visualize and analyse low-frequency normal modes of large macro-molecules and to generate a large number of different starting models for use in MR. Due to the 'rotation-translation-block' (RTB) approximation implemented in ElNémo, there is virtually no upper limit to the size of the proteins that can be treated. Upon input of a protein structure in Protein Data Bank (PDB) format, ElNémo computes its 100 lowest-frequency modes and produces a comprehensive set of descriptive parameters and visualizations, such as the degree of collectivity of movement, residue mean square displacements, distance fluctuation maps, and the correlation between observed and normal-mode-derived atomic displacement parameters (B-factors). Any number of normal mode perturbed models for MR can be generated for download. If two conformations of the same (or a homologous) protein are available, ElNémo identifies the normal modes that contribute most to the corresponding protein movement. The web server can be freely accessed at http://igs-server.cnrs-mrs.fr/elnemo/index.html.",
"VMD is a molecular graphics program designed for the display and analysis of molecular assemblies, in particular biopolymers such as proteins and nucleic acids. VMD can simultaneously display any number of structures using a wide variety of rendering styles and coloring methods. Molecules are displayed as one or more \"representations,\" in which each representation embodies a particular rendering method and coloring scheme for a selected subset of atoms. The atoms displayed in each representation are chosen using an extensive atom selection syntax, which includes Boolean operators and regular expressions. VMD provides a complete graphical user interface for program control, as well as a text interface using the Tcl embeddable parser to allow for complex scripts with variable substitution, control loops, and function calls. Full session logging is supported, which produces a VMD command script for later playback. High-resolution raster images of displayed molecules may be produced by generating input scripts for use by a number of photorealistic image-rendering applications. VMD has also been expressly designed with the ability to animate molecular dynamics (MD) simulation trajectories, imported either from files or from a direct connection to a running MD simulation. VMD is the visualization component of MDScope, a set of tools for interactive problem solving in structural biology, which also includes the parallel MD program NAMD, and the MDCOMM software used to connect the visualization and simulation programs. VMD is written in C++, using an object-oriented design; the program, including source code and extensive documentation, is freely available via anonymous ftp and through the World Wide Web.",
"P2X receptors are trimeric ATP-activated ion channels permeable to Na+, K+ and Ca2+. The seven P2X receptor subtypes are implicated in physiological processes that include modulation of synaptic transmission, contraction of smooth muscle, secretion of chemical transmitters and regulation of immune responses. Despite the importance of P2X receptors in cellular physiology, the three-dimensional composition of the ATP-binding site, the structural mechanism of ATP-dependent ion channel gating and the architecture of the open ion channel pore are unknown. Here we report the crystal structure of the zebrafish P2X4 receptor in complex with ATP and a new structure of the apo receptor. The agonist-bound structure reveals a previously unseen ATP-binding motif and an open ion channel pore. ATP binding induces cleft closure of the nucleotide-binding pocket, flexing of the lower body β-sheet and a radial expansion of the extracellular vestibule. The structural widening of the extracellular vestibule is directly coupled to the opening of the ion channel pore by way of an iris-like expansion of the transmembrane helices. The structural delineation of the ATP-binding site and the ion channel pore, together with the conformational changes associated with ion channel gating, will stimulate development of new pharmacological agents.",
"Until now, the only well documented, fast excitatory neurotransmitter in the brain has been glutamate. Although there is evidence for adenosine 5'-triphosphate (ATP) acting as a transmitter in the peripheral nervous system, suggestions for such a role in the central nervous system have so far not been supported by any direct evidence. Here we report the recording of evoked and miniature synaptic currents in the rat medial habenula. The fast rise time of the currents showed that they were mediated by a ligand-activated ion channel rather than a second messenger system, thus limiting the known transmitter candidates. Evidence was found for the presence on the cells of glutamate, gamma-aminobutyric acid, acetylcholine and ATP receptors, but not for 5-hydroxytryptamine (5HT3) or glycine receptors. The evoked currents were unaffected by blockers of glutamate, gamma-aminobutyric acid or acetylcholine receptors but were blocked by the ATP receptor-blocker, suramin and the desensitizing ATP receptor-agonist alpha,beta-methylene-ATP. Our evidence identifies for the first time synaptic currents in the brain, mediated directly by ATP receptors."
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Separation of superficial and cerebral absorption changes in functional near-infrared spectroscopy
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In functional near-infrared spectroscopy (fNIRS) superficial hemodynamics can mask optical signals related to brain activity. We present a method to separate superficial and cerebral absorption changes based on the analysis of changes in moments of time-of-flight distributions and a two-layered model. The related sensitivity factors were calculated from individual optical properties. The method was validated on a two-layer liquid phantom. Absorption changes in the lower layer were retrieved with an accuracy better than 20%. The method was successfully applied to in vivo data and compared to the reconstruction of homogeneous absorption changes.
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"In conventional functional near-infrared spectroscopy (fNIRS), systemic physiological fluctuations evoked by a body's motion and psychophysiological changes often contaminate fNIRS signals. We propose a novel method for separating functional and systemic signals based on their hemodynamic differences. Considering their physiological origins, we assumed a negative and positive linear relationship between oxy- and deoxyhemoglobin changes of functional and systemic signals, respectively. Their coefficients are determined by an empirical procedure. The proposed method was compared to conventional and multi-distance NIRS. The results were as follows: (1) Nonfunctional tasks evoked substantial oxyhemoglobin changes, and comparatively smaller deoxyhemoglobin changes, in the same direction by conventional NIRS. The systemic components estimated by the proposed method were similar to the above finding. The estimated functional components were very small. (2) During finger-tapping tasks, laterality in the functional component was more distinctive using our proposed method than that by conventional fNIRS. The systemic component indicated task-evoked changes, regardless of the finger used to perform the task. (3) For all tasks, the functional components were highly coincident with signals estimated by multi-distance NIRS. These results strongly suggest that the functional component obtained by the proposed method originates in the cerebral cortical layer. We believe that the proposed method could improve the reliability of fNIRS measurements without any modification in commercially available instruments.",
"Recent studies have shown that time-resolved optical measurements of the head can estimate changes in the absorption coefficient with depth discrimination. Thus, changes in tissue oxygenation, which are specific to intracranial tissues, can be assessed using this advanced technique, and this method allows us to avoid the influence of changes to extracerebral tissue oxygenation on the measured signals. We report the results of time-resolved optical imaging that was carried out during carotid endarterectomy. This surgery remains the \"gold standard\" treatment for carotid stenosis, and intraoperative brain oxygenation monitoring may improve the safety of this procedure. A time-resolved optical imager was utilized within the operating theater. This instrument allows for the simultaneous acquisition of 32 distributions of the time-of-flight of photons at two wavelengths on both hemispheres. Analysis of the statistical moments of the measured distributions of the time-of-flight of photons was applied for estimating changes in the absorption coefficient as a function of depth. Time courses of changes in oxy- and deoxyhemoglobin of the extra- and intracerebral compartments during cross-clamping of the carotid arteries were obtained. A decrease in the oxyhemoglobin concentration and an increase in the deoxyhemoglobin concentrations were observed in a large area of the head. Large changes were observed in the hemisphere ipsilateral to the site of clamped carotid arteries. Smaller amplitude changes were noted at the contralateral site. We also found that changes in the hemoglobin signals, as estimated from intracerebral tissue, are very sensitive to clamping of the internal carotid artery, whereas its sensitivity to clamping of the external carotid artery is limited. We concluded that intraoperative multichannel measurements allow for imaging of brain tissue hemodynamics. However, when monitoring the brain during carotid surgery, a single-channel measurement may be sufficient.",
"Near-infrared spectroscopy (NIRS) of the human brain is aiming at the non-invasive determination of concentration changes of oxy- and deoxyhemoglobin in the cortex. However, it usually relies on the assumption of spatially homogeneous absorption changes. To overcome this limitation we performed instrumental and methodological developments of time-resolved NIRS with the aim to achieve depth resolution. We present our recently developed time-domain near-infrared brain imager based on picosecond diode lasers and time-correlated single photon counting (TCSPC) which can be used at the bedside. To achieve depth localization of absorption changes we analysed statistical moments (integral, mean time of flight and variance) of measured time-of-flight distributions of diffusely reflected photons. In particular, variance has a selective sensitivity to deep absorptions changes and provides a suitable representation of cerebral signals. The separation of cerebral and extracerebral changes of hemoglobin concentrations is demonstrated for a motor stimulation experiment.",
"Functional near infrared spectroscopy (fNIRS) is a portable monitor of cerebral hemodynamics with wide clinical potential. However, in fNIRS, the vascular signal from the brain is often obscured by vascular signals present in the scalp and skull. In this paper, we evaluate two methods for improving in vivo data from adult human subjects through the use of high-density diffuse optical tomography (DOT). First, we test whether we can extend superficial regression methods (which utilize the multiple source-detector pair separations) from sparse optode arrays to application with DOT imaging arrays. In order to accomplish this goal, we modify the method to remove physiological artifacts from deeper sampling channels using an average of shallow measurements. Second, DOT provides three-dimensional image reconstructions and should explicitly separate different tissue layers. We test whether DOT's depth-sectioning can completely remove superficial physiological artifacts. Herein, we assess improvements in signal quality and reproducibility due to these methods using a well-characterized visual paradigm and our high-density DOT system. Both approaches remove noise from the data, resulting in cleaner imaging and more consistent hemodynamic responses. Additionally, the two methods act synergistically, with greater improvements when the approaches are used together.",
"During performance of attention-demanding cognitive tasks, certain regions of the brain routinely increase activity, whereas others routinely decrease activity. In this study, we investigate the extent to which this task-related dichotomy is represented intrinsically in the resting human brain through examination of spontaneous fluctuations in the functional MRI blood oxygen level-dependent signal. We identify two diametrically opposed, widely distributed brain networks on the basis of both spontaneous correlations within each network and anticorrelations between networks. One network consists of regions routinely exhibiting task-related activations and the other of regions routinely exhibiting task-related deactivations. This intrinsic organization, featuring the presence of anticorrelated networks in the absence of overt task performance, provides a critical context in which to understand brain function. We suggest that both task-driven neuronal responses and behavior are reflections of this dynamic, ongoing, functional organization of the brain.",
"Optical techniques enable portable, non-invasive functional neuroimaging. However, low lateral resolution and poor discrimination between brain hemodynamics and systemic contaminants have hampered the translation of near infrared spectroscopy from research instrument to widespread neuroscience tool. In this paper, we demonstrate that improvements in spatial resolution and signal-to-noise, afforded by recently developed high-density diffuse optical tomography approaches, now permit detailed phase-encoded mapping of the visual cortex's retinotopic organization. Due to its highly organized structure, the visual cortex has long served as a benchmark for judging neuroimaging techniques, including the original development of functional magnetic resonance imaging (fMRI) and positron emission tomography. Using phase-encoded visual stimuli that create traveling waves of cortical activations, we are able to discriminate the representations of multiple visual angles and eccentricities within an individual hemisphere, reproducing classic fMRI results. High contrast-to-noise and repeatable imaging allow the detection of inter-subject differences. These results represent a significant advancement in the level of detail that can be obtained from non-invasive optical imaging of functional brain responses. In addition, these phase-encoded paradigms and the maps they generate form a standardized model with which to judge new developments in optical algorithms and systems, such as new image reconstruction techniques and registration with anatomic imaging. With these advances in techniques and validation paradigms, optical neuroimaging can be extended into studies of higher-order brain function and of clinical utility with greater performance and confidence.",
"The time-resolved Beer-Lambert law proposed for oxygen monitoring using pulsed light was extended to the non-time-resolved case in a scattered medium such as living tissues with continuous illumination. The time-resolved Beer-Lambert law was valid for the phantom model and living tissues in the visible and near-infrared regions. The absolute concentration and oxygen saturation of haemoglobin in rat brain and thigh muscle could be determined. The temporal profile of rat brain was reproduced by Monte Carlo simulation. When the temporal profiles of rat brain under different oxygenation states were integrated with time, the absorbance difference was linearly related to changes in the absorption coefficient. When the simulated profiles were integrated, there was a linear relationship within the absorption coefficient which was predicted for fractional inspiratory oxygen concentration from 10 to 100% and, in the case beyond the range of the absorption coefficient, the deviation from linearity was slight. We concluded that an optical pathlength which is independent of changes in the absorption coefficient is a good approximation for near-infrared oxygen monitoring."
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Domain-level ortholog clustering
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Identification of ortholog groups is a crucial step in comparative analysis of multiple genomes. Although several computational methods have been developed to create ortholog groups, most of those methods do not evaluate orthology at the sub-gene level. In our method for domain-level ortholog clustering, DomClust, proteins are split into domains on the basis of alignment boundaries identified by all-against-all pairwise comparison, but it often fails to determine appropriate boundaries.
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[
"The concept of homology drives speculation on a gene's function in any given species when its biological roles in other species are characterized. With reference to a specific species radiation homologous relations define orthologs, i.e. descendants from a single gene of the ancestor. The large-scale delineation of gene genealogies is a challenging task, and the numerous approaches to the problem reflect the importance of the concept of orthology as a cornerstone for comparative studies. Here, we present the updated OrthoDB catalog of eukaryotic orthologs delineated at each radiation of the species phylogeny in an explicitly hierarchical manner of over 100 species of vertebrates, arthropods and fungi (including the metazoa level). New database features include functional annotations, and quantification of evolutionary divergence and relations among orthologous groups. The interface features extended phyletic profile querying and enhanced text-based searches. The ever-increasing sampling of sequenced eukaryotic genomes brings a clearer account of the majority of gene genealogies that will facilitate informed hypotheses of gene function in newly sequenced genomes. Furthermore, uniform analysis across lineages as different as vertebrates, arthropods and fungi with divergence levels varying from several to hundreds of millions of years will provide essential data for uncovering and quantifying long-term trends of gene evolution. OrthoDB is freely accessible from http://cegg.unige.ch/orthodb.",
"The identification of orthologous genes forms the basis for most comparative genomics studies. Existing approaches either lack functional annotation of the identified orthologous groups, hampering the interpretation of subsequent results, or are manually annotated and thus lag behind the rapid sequencing of new genomes. Here we present the eggNOG database ('evolutionary genealogy of genes: Non-supervised Orthologous Groups'), which contains orthologous groups constructed from Smith-Waterman alignments through identification of reciprocal best matches and triangular linkage clustering. Applying this procedure to 312 bacterial, 26 archaeal and 35 eukaryotic genomes yielded 43 582 course-grained orthologous groups of which 9724 are extended versions of those from the original COG/KOG database. We also constructed more fine-grained groups for selected subsets of organisms, such as the 19 914 mammalian orthologous groups. We automatically annotated our non-supervised orthologous groups with functional descriptions, which were derived by identifying common denominators for the genes based on their individual textual descriptions, annotated functional categories, and predicted protein domains. The orthologous groups in eggNOG contain 1 241 751 genes and provide at least a broad functional description for 77% of them. Users can query the resource for individual genes via a web interface or download the complete set of orthologous groups at http://eggnog.embl.de.",
"Orthologous relationships form the basis of most comparative genomic and metagenomic studies and are essential for proper phylogenetic and functional analyses. The third version of the eggNOG database (http://eggnog.embl.de) contains non-supervised orthologous groups constructed from 1133 organisms, doubling the number of genes with orthology assignment compared to eggNOG v2. The new release is the result of a number of improvements and expansions: (i) the underlying homology searches are now based on the SIMAP database; (ii) the orthologous groups have been extended to 41 levels of selected taxonomic ranges enabling much more fine-grained orthology assignments; and (iii) the newly designed web page is considerably faster with more functionality. In total, eggNOG v3 contains 721,801 orthologous groups, encompassing a total of 4,396,591 genes. Additionally, we updated 4873 and 4850 original COGs and KOGs, respectively, to include all 1133 organisms. At the universal level, covering all three domains of life, 101,208 orthologous groups are available, while the others are applicable at 40 more limited taxonomic ranges. Each group is amended by multiple sequence alignments and maximum-likelihood trees and broad functional descriptions are provided for 450,904 orthologous groups (62.5%).",
"Pfam is a comprehensive collection of protein domains and families, represented as multiple sequence alignments and as profile hidden Markov models. The current release of Pfam (22.0) contains 9318 protein families. Pfam is now based not only on the UniProtKB sequence database, but also on NCBI GenPept and on sequences from selected metagenomics projects. Pfam is available on the web from the consortium members using a new, consistent and improved website design in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/), as well as from mirror sites in France (http://pfam.jouy.inra.fr/) and South Korea (http://pfam.ccbb.re.kr/)."
] |
Combined Fluorescent Molecular Tomography-MRI System for Heart and Aorta Molecular Imaging
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Combining Fluorescent Molecular Tomography (FMT) with anatomical imaging, e.g. MRI facilitates interpreting functional information. Furthermore, using a heterogeneous model for light propagation has been shown in simulations to be superior to homogeneous modeling to quantify fluorescence. Here, we present a combined FMT-MRI system and apply it to heart and aorta molecular imaging, a challenging area due to strong tissue heterogeneity and the presence of air-voids due to lungs. First investigating performance in a phantom and mouse corpse, the MRI-enabled heterogeneous models resulted in an improved quantification of fluorescence reconstructions. The system was then used in mice for in vivo atherosclerosis molecular imaging. Results show that, when using the heterogeneous model, reconstructions were in agreement with the ex vivo measurements. Therefore, the proposed system might serve as a powerful imaging tool for atherosclerosis in mice.
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[
"We report a parallel Monte Carlo algorithm accelerated by graphics processing units (GPU) for modeling time-resolved photon migration in arbitrary 3D turbid media. By taking advantage of the massively parallel threads and low-memory latency, this algorithm allows many photons to be simulated simultaneously in a GPU. To further improve the computational efficiency, we explored two parallel random number generators (RNG), including a floating-point-only RNG based on a chaotic lattice. An efficient scheme for boundary reflection was implemented, along with the functions for time-resolved imaging. For a homogeneous semi-infinite medium, good agreement was observed between the simulation output and the analytical solution from the diffusion theory. The code was implemented with CUDA programming language, and benchmarked under various parameters, such as thread number, selection of RNG and memory access pattern. With a low-cost graphics card, this algorithm has demonstrated an acceleration ratio above 300 when using 1792 parallel threads over conventional CPU computation. The acceleration ratio drops to 75 when using atomic operations. These results render the GPU-based Monte Carlo simulation a practical solution for data analysis in a wide range of diffuse optical imaging applications, such as human brain or small-animal imaging.",
"Fluorescence tomography (FT) is a promising molecular imaging technique that can spatially resolve both fluorophore concentration and lifetime parameters. However, recovered fluorophore parameters highly depend on the size and depth of the object due to the ill-posedness of the FT inverse problem. Structural a priori information from another high spatial resolution imaging modality has been demonstrated to significantly improve FT reconstruction accuracy. In this study, we have constructed a combined magnetic resonance imaging (MRI) and FT system for small animal imaging. A photo-multiplier tube is used as the detector to acquire frequency domain FT measurements. This is the first MR-compatible time-resolved FT system that can reconstruct both fluorescence concentration and lifetime maps simultaneously. The performance of the hybrid system is evaluated with phantom studies. Two different fluorophores, indocyanine green and 3-3' diethylthiatricarbocyanine iodide, which have similar excitation and emission spectra but different lifetimes, are utilized. The fluorescence concentration and lifetime maps are both reconstructed with and without the structural a priori information obtained from MRI for comparison. We show that the hybrid system can accurately recover both fluorescence intensity and lifetime within 10% error for two 4.2 mm-diameter cylindrical objects embedded in a 38 mm-diameter cylindrical phantom when MRI structural a priori information is utilized.",
"Local molecular and physiological processes can be imaged in vivo through perturbations in the fluorescence lifetime (FLT) of optical imaging agents. In addition to providing functional information, FLT methods can quantify specific molecular events and multiplex diagnostic and prognostic information. We have developed a fluorescence lifetime diffuse optical tomography (DOT) system for in vivo preclinical imaging. Data is captured using a time-resolved intensified charge coupled device (ICCD) system to measure fluorescence excitation and emission in the time domain. Data is then converted to the frequency domain, and we simultaneously reconstruct images of yield and lifetime using an extension to the normalized Born approach. By using differential phase measurements, we demonstrate DOT imaging of short lifetimes (from 350 ps) with high precision (+/-5 ps). Furthermore, this system retains the efficiency, speed, and flexibility of transmission geometry DOT. We demonstrate feasibility of FLT-DOT through a progressive series of experiments. Lifetime range and repeatability are first measured in phantoms. Imaging of subcutaneous implants then verifies the FLT-DOT approach in vivo in the presence of inhomogeneous optical properties. Use in a common research scenario is ultimately demonstrated by imaging accumulation of a targeted near-infrared (NIR) fluorescent-labeled peptide probe (cypate-RGD) in a mouse with a subcutaneous tumor."
] |
Gender differences in topographical anatomy of the levator ani and rhabdosphincter
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Detailed knowledge of the anatomy of the rhabdosphincter and adjacent tissues is mandatory during urologic surgery to ensure reliable oncologic and functional outcomes. To characterize the levator ani (LA) function for the urethral sphincter, we described connective tissue morphology between the LA and urethral rhabdosphincter. The interface tissue between the LA and rhabdosphincter area in males contained abundant irregularly arrayed elastic fibers and smooth muscles. The male rhabdosphincter was positioned alongside the LA to divide the elevation force and not in-series along the axis of LA contraction. The male perineal membrane was thin but solid and extends along the inferior margin or bottom of the rhabdosphincter area. In contrast, the female rhabdosphincter, including the compressor urethrae and urethrovaginal sphincter muscles, was embedded in the elastic fiber mesh that is continuous with the thick, multilaminar perineal membrane. The inferomedial edge of the female LA was attached to the upper surface of the perineal membrane and not directly attached to the rhabdosphincter. We presented new diagrams showing the gender differences in topographical anatomy of the LA and rhabdosphincter.
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[
"Which parts of the male urethra correspond to the female urethra? To resolve this question, we need to understand fetal topographical changes in the urethra, its external sphincter and vagina. The vagina joins the mid-course of the primitive urethra and, later \"descends\" to the vaginal vestibulum. We examined histological sections of 14 female and 4 male mid-term fetuses. The inferior end of the vagina was consistently embedded in the posterior wall of the urethra at 9-12 weeks. The supero-inferior level of the vaginal merging was lower in larger fetuses. Thus, the sequential variation in levels appeared to reflect the process of vaginal descent. However, in spite of penetration of the vaginal end into the posterior urethral wall, we found no sign of destruction of the urethral wall after vaginal descent in the low-merging types. Before vaginal descent, the female external sphincter extended posterolaterally around the urethra. The vaginal descent is classically regarded as a relative topographical change, but it is likely to be a result of elongation of the proximal urethra in the superior side of the vaginal merging. Conversely, the distal urethra is likely to be incorporated into the vaginal vestibulum by 15 weeks. During these processes, most of the female external sphincter seems to be expelled from the original anterior position into the vestibular wall as the urethrovaginal sphincter. The adult female urethra seems to correspond to the male prostatic urethra superior to the prostatic colliculus.",
"To investigate why the development of a completely circular striated sphincter is so rare, we examined histological sections of 11 female and 11 male mid-term human fetuses. In male fetuses, the striated muscle initially extended in the frontal, rather than in the horizontal plane. However, a knee-like portion was absent in the female fetal urethra because, on the inferior side of the vaginal end, a wide groove for the future vestibule opened inferiorly. Accordingly, it was difficult for the developing striated muscle to surround the groove, even though there was not a great difference in width or thickness between the female vestibule and the male urethra. The development of a completely circular striated sphincter seems to be impossible in females because of interruption of the frontal plane by the groove-like vestibule. However, we cannot rule out the possibility that before descent of the vagina, the urethral striated muscle extends posteriorly.",
"The aims of this study were, using fixed and fresh cadavers, to clarify how closely the pelvic splanchnic nerve, levator ani nerve (LAN), and sacrospinous ligament (SSL) are located and to examine how to avoid nerve injury during the posterior tension-free vaginal mesh procedure (TVMP), in which mesh is applied through the SSL. Macroscopic dissection of fixed cadavers demonstrated that the LAN crossed the inside of the SSL at a point 0-18 mm medial to the ischial spine and entered the muscle at a point 12-26 mm inferior to the ischial spine and 40-55 mm anterolateral to the coccyx. Dissection after TVMP of fresh cadavers revealed that the point of penetration of mesh through the SSL was close to the LAN. To preserve the LAN, penetration of the SSL should be within 5 mm of the lower margin at a point 20-25 mm medial to the ischial spine.",
"The connective tissue located between the uterine cervix and sacrospinous ligament (the uterospinous connective tissue; USCT) has recently been noted as the level 1 supportive tissue instead of the classical uterosacral ligament. We examined whether or not the USCT changes its histological architecture by vaginal delivery in correlation with the levels 2 and 3 supportive tissues. In the pelvic floors of 17 female cadavers (9 nuliparous and 8 multiparous), we compared histological architectures among the USCT, arcus tendineus fasciae pelvis (ATFP) and perineal membrane (PM). The USCT was evident as a string-like tissue structure in multiparous women or a thick mesh in nuliparous women. It consistently contained fewer elastic and smooth muscle fibers than other levels. In contrast, the ATFP usually contained abundant elastic fibers and smooth muscle. Likewise, the PM also displayed a constant morphology. Although all three sites were likely to be injured by delivery, the USCT seemed to be more severely damaged and/or more difficult to be recovered than the ATFP and PM.",
"When performing nerve-sparing abdominoperitoneal resection or intersphincteric resection of lower rectal cancer, difficulty is sometimes encountered during dissection, separation and treatment in the area anterior to the anorectum passing through the levator hiatus between the bilateral levator ani slings owing to missing the surgical plane or venous bleeding. The rectourethralis muscle, which is a mass of smooth muscle, occupies the levator hiatus. The present histological study using nine male cadaveric specimens demonstrated that: (i) the external anal sphincter is likely to be tightly connected to the rectourethralis muscle; (ii) the rectal muscularis propria communicates with the rectourethralis muscle; (iii) the anorectal veins take a tortuous course across the rectourethralis muscle; (iv) Denonvilliers' fascia ends at the rectourethralis muscle; and (v) the rectourethralis muscle provides posterior attachment for the rhabdosphincter. Moreover, the cavernous nerve has been reported to penetrate the rectourethralis muscle. Therefore, careful treatment of the muscle seems to be necessary to avoid male sexual dysfunction. Owing to muscle fiber communications between the rectal muscularis propria and the rectourethralis muscle, and the fact that Denonvilliers' fascia terminates in the rectourethralis muscle, the surgical plane would tend to deeply penetrate the muscle mass. However, mass ligation of the anterior tissues for control of venous bleeding should be avoided. When the tumor is non-anterior, an abdominal surgical plane behind Denonvilliers' fascia is recommended to avoid excess invasion into the rectourethralis muscle.",
"Myofibrils are linked to the cell membrane at myotendinous junctions located at the ends of muscle fibers, and at costameres, sites positioned periodically along lateral surfaces of muscle cells. Both of these sites are enriched in proteins that link active components of myofibrils to the cell membrane. Costameres are also enriched in desmin intermediate filaments that link passive components of myofibrils to the lateral surfaces of muscle cells. In this study, the possibility that desmin is also found between the terminal Z-disk of myofibrils and the myotendinous junction membrane is examined by immunocytochemistry and by KI-extraction procedures. Data presented show that desmin is located in the filamentous core of cellular processes at myotendinous junctions at sites 30 nm or more from the membrane. This core lies deep to subsarcolemmal material previously shown to contain talin, vinculin, and dystrophin. The distance from desmin to the membrane suggests desmin does not interact directly with membrane proteins at the junction. Immunoblots and indirect immunofluorescence of junctional regions of muscle compared to nonjunctional regions show no apparent enrichment of desmin at junctional sites, although vinculin, another costameric and junctional component, is significantly enriched at junctional regions. These findings show that passive elements of myofibrils may be continuous from myotendinous junctions of muscle origin to insertion via desmin filaments located between terminal Z-disks and the junctional membrane. This can provide a system in parallel to that involving thin filaments, vinculin, and talin for linking myofibrils to the cell membrane at myotendinous junctions.",
"This study was undertaken to identify with some precision the location of various paraurethral structures along the length of the urethra. The locations of four areas were identified. The intramural urethra (the area where this structure traverses the bladder wall) extends from the 0 to 15% of total urethral length. The midurethra extends from about 20 to 60% of total urethral length and is the region where one finds the striated urethral sphincter, posterior pubourethral ligaments, and vaginolevator attachments. The urogenital diaphragm with its compressor urethrae and urethrovaginal sphincter is found from 54 to 76% of urethral length. The distal urethra courses from 79 to 100% where the urethra passes between the vestibular bulbs and bulbocavernosus muscles. Some functional considerations of these findings are discussed.",
"Smooth muscle cells undergo substantial increases in length, passively stretching during increases in intraluminal pressure in vessels and hollow organs. Active contractile responses to counteract increased transmural pressure were first described almost a century ago (Bayliss, 1902) and several mechanisms have been advanced to explain this phenomenon. We report here that elongation of smooth muscle cells results in ryanodine receptor-mediated Ca(2+) release in individual myocytes. Mechanical elongation of isolated, single urinary bladder myocytes to approximately 120% of slack length (DeltaL = 20) evoked Ca(2+) release from intracellular stores in the form of single Ca(2+) sparks and propagated Ca(2+) waves. Ca(2+) release was not due to calcium-induced calcium release, as release was observed in Ca(2+)-free extracellular solution and when free Ca(2+) ions in the cytosol were strongly buffered to prevent increases in [Ca(2+)](i). Stretch-induced calcium release (SICR) was not affected by inhibition of InsP(3)R-mediated Ca(2+) release, but was completely blocked by ryanodine. Release occurred in the absence of previously reported stretch-activated currents; however, SICR evoked calcium-activated chloride currents in the form of transient inward currents, suggesting a regulatory mechanism for the generation of spontaneous currents in smooth muscle. SICR was also observed in individual myocytes during stretch of intact urinary bladder smooth muscle segments. Thus, longitudinal stretch of smooth muscle cells induces Ca(2+) release through gating of RYR. SICR may be an important component of the physiological response to increases in luminal pressure in smooth muscle tissues."
] |
Fracture classification and characterization of the radial head with 3D computed tomography and 3D physical modeling.
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To tests the hypothesis that classification and characterization of fractures of the radial head is more accurate with 3D than 2D computed tomography images and radiographs, using a prospective study design with intraoperative inspection as the reference standard. Treating surgeons and first assistants completed a questionnaire assigning a fracture type according to the Broberg and Morrey modification of Mason's classification, evaluating selected fracture characteristics, and electing preferred management based upon radiographs and 2D images alone; then adding 3D-CT; then 3D printed physical models; and finally intra-operative visualization. The addition of the 3D CT and physical models improved the sensitivity for fracture line separating the entire head from the neck, comminution of the radial neck, fracture involving the articular surface, articular fracture gap greater than 2 mm, impacted fracture fragments, greater than 3 articular fragments, and articular fragments judged too small to repair. There were no significant differences in diagnostic performance with the addition of 3D models. The addition of 3D CT and models improved the reliability of Broberg and Morrey classification. We conclude that 3DCT and 3D physical modeling provide more accurate fracture classification and characterization of fracture of the radial head with less proposed variability in treatment. We did not demonstrate a clear advantage for modeling over 3DCT reconstructions.
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[
"Fractures of the proximal radius need to be classified in an appropriate and reproducible manner. The aim of this study was to assess the reliability of the three most widely used classification systems. Elbow radiographs images of patients with proximal radius fractures were classified according to Mason, Morrey, and Arbeitsgemeinschaft für osteosynthesefragen/Association for the Study of Internal Fixation (AO/ASIF) classifications by four observers with different experience with this subject to assess their intra- and inter-observer agreement. Each observer analyzed the images on three different occasions on a computer with numerical sequence randomly altered. We found that intra-observer agreement of Mason and Morrey classifications were satisfactory (kappa = 0.582 and 0.554, respectively), while the AO/ASIF classification had poor intra-observer agreement (kappa = 0.483). Inter-observer agreement was higher in the Mason (kappa = 0.429-0.560) and Morrey (kappa = 0.319-0.487) classifications than in the AO/ASIF classification (kappa = 0.250-0.478), which showed poor reliability. Inter- and intra-observer agreement of the Mason and Morey classifications showed overall satisfactory reliability when compared to the AO/ASIF system. The Mason classification is the most reliable system.",
"Existing classifications of fractures of the head of the humerus are inadequate in terms of interobserver reliability and the predictability of the clinical outcome. From a combined study of 73 fracture specimens in museums and 84 CT-three-dimensional reconstructions in patients, we have devised a classification which appears to be more useful clinically. Common patterns of fracture and a plausible mechanism of injury were observed. In 3-D most proximal humeral fractures can be organised into five basic types. These correspond in some degree to the Codman/Neer classification, but differ significantly in regard to the more complex patterns of fracture. We observed a logical progression from simple to complex fractures. An interobserver reliability study was carried out which indicated the improved usefulness of this new 3-D concept in providing a common language among clinicians for classifying these injuries. When surgery is indicated, the 3-D concept is also invaluable in guiding the restitution of anatomy through either open or percutaneous means.",
"This study tests the hypothesis that 3-dimensional computed tomography (CT) reconstructions improve interobserver agreement on classification and treatment of coronoid fractures compared with 2-dimensional CT. A total of 29 orthopedic surgeons evaluated 10 coronoid fractures on 2 occasions (first with radiographs and 2-dimensional CT and then with radiographs and 3-dimensional CT), separated by a minimum of 2 weeks. Surgeons classified fractures according to the classifications of Regan and Morrey and of O'Driscoll et al., identified specific characteristics, recommended the most appropriate treatment approach, and made treatment recommendations. The kappa multirater measure (kappa) was calculated to estimate agreement between observers. Regardless of the imaging modality used, there was fair to moderate agreement for most of the observations. Three-dimensional CT improved interobserver agreement in Regan and Morrey's classsication (kappa(3-dimensional) = 0.51 vs kappa(2-dimensional) = 0.40; p < .001) and O'Driscoll et al.'s classifications (kappa(3-dimensional) = 0.48 vs kappa(2-dimensional) = 0.42; p = .009). There were trends toward better reliability for 3-dimensional reconstruction in recognition of coronoid tip fractures (kappa(3-dimensional) = 0.19, kappa(2-dimensional) = 0.03; p = .268), comminution (kappa(3-dimensional) = 0.41 vs kappa(2-dimensional) = 0.29; p = .133), and impacted fragments (kappa(3-dimensional) = 0.39 vs kappa(2-dimensional) = 0.27; p = .094), and in surgeons' opinions on the need for something other than screws or plate for surgical fixation (kappa(3-dimensional) = 0.31 vs kappa(2-dimensional) = 0.15; p = .138). Interobserver agreement on treatment approach was better with 2-dimensional CT (kappa(3-dimensional) = 0.27, kappa(2-dimensional) = 0.32; p = .015). Three-dimensional CT reconstructions improve interobserver agreement with respect to fracture classification compared with 2-dimensional CT. Diagnostic III.",
"The purpose of this study was to assess the interobserver reliability and intraobserver reproducibility of the AO/ASIF and Rüedi and Allgöwer classifications for fractures of the distal tibia, and to determine the benefit of a computed tomography (CT) scan and experience on observer agreement for several fracture characteristics, including classification. The radiographs of forty-three fractures of the distal tibia, fourteen of which had CT scans, were assessed by groups of experienced and less-experienced observers. Each case was classified according to the AO/ASIF and Rüedi and Allgöwer systems. Several other fracture characteristics also were assessed. The kappa coefficient of agreement was calculated and used to compare the interobserver reliability and intraobserver reproducibility of the classification systems and to determine the benefit of experience and CT scans. The intraclass correlation coefficient was used to assess noncategoric data. Interobserver and intraobserver agreements were good when classifying fractures into AO/ASIF types and significantly better than that for the Rüedi and Allgöwer system. However, agreement was poor when classifying the fractures into AO/ASIF groups. For most assessments, the experienced group tended to have higher levels of interobserver agreement, but not intraobserver agreement. Viewing the CT scans improved agreement on the percentage of articular surface involved, but it did not improve interobserver reliability or intraobserver reproducibility for either of the classification systems. The AO/ASIF classification for fractures of the distal tibia has good observer agreement at the type level, but poor agreement at the group level. Experience tends to improve interobserver agreement, but not intraobserver agreement. Viewing CT scans does not improve agreement on classification, but it tends to improve agreement on articular surface involvement.",
"The Broberg and Morrey modification of the Mason classification of radial head fractures has substantial interobserver variation. This study used a large web-based collaborative of experienced orthopaedic surgeons to test the hypothesis that three-dimensional reconstructions of computed tomography (CT) scans improve the interobserver reliability of the classification of radial head fractures according to the Broberg and Morrey modification of the Mason classification. Eighty-five orthopaedic surgeons evaluated twelve radial head fractures. They were randomly assigned to review either radiographs and two-dimensional CT scans or radiographs and three-dimensional CT images to determine the fracture classification, fracture characteristics, and treatment recommendations. The kappa multirater measure (κ) was calculated to estimate agreement between observers. Three-dimensional CT had moderate agreement and two-dimensional CT had fair agreement among observers for the Broberg and Morrey modification of the Mason classification, a difference that was significant. Observers assessed seven fracture characteristics, including fracture line, comminution, articular surface involvement, articular step or gap of ≥2 mm, central impaction, recognition of more than three fracture fragments, and fracture fragments too small to repair. There was a significant difference in kappa values between three-dimensional CT and two-dimensional CT for fracture fragments too small to repair, recognition of three fracture fragments, and central impaction. The difference between the other four fracture characteristics was not significant. Among treatment recommendations, there was fair agreement for both three-dimensional CT and two-dimensional CT. Although three-dimensional CT led to some small but significant decreases in interobserver variation, there is still considerable disagreement regarding classification and characterization of radial head fractures. Three-dimensional CT may be insufficient to optimize interobserver agreement."
] |
BAC-FISH and optical mapping determine the arrangement of the tomato pseudomolecules
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The order and orientation (arrangement) of all 91 sequenced scaffolds in the 12 pseudomolecules of the recently published tomato (Solanum lycopersicum, 2n = 2x = 24) genome sequence were positioned based on marker order in a high-density linkage map. Here, we report the arrangement of these scaffolds determined by two independent physical methods, bacterial artificial chromosome-fluorescence in situ hybridization (BAC-FISH) and optical mapping. By localizing BACs at the ends of scaffolds to spreads of tomato synaptonemal complexes (pachytene chromosomes), we showed that 45 scaffolds, representing one-third of the tomato genome, were arranged differently than predicted by the linkage map. These scaffolds occur mostly in pericentric heterochromatin where 77% of the tomato genome is located and where linkage mapping is less accurate due to reduced crossing over. Although useful for only part of the genome, optical mapping results were in complete agreement with scaffold arrangement by FISH but often disagreed with scaffold arrangement based on the linkage map. The scaffold arrangement based on FISH and optical mapping changes the positions of hundreds of markers in the linkage map, especially in heterochromatin. These results suggest that similar errors exist in pseudomolecules from other large genomes that have been assembled using only linkage maps to predict scaffold arrangement, and these errors can be corrected using FISH and/or optical mapping. Of note, BAC-FISH also permits estimates of the sizes of gaps between scaffolds, and unanchored BACs are often visualized by FISH in gaps between scaffolds and thus represent starting points for filling these gaps.
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[
"We report the ∼2.66-Gb genome sequence of a female Yunnan black goat. The sequence was obtained by combining short-read sequencing data and optical mapping data from a high-throughput whole-genome mapping instrument. The whole-genome mapping data facilitated the assembly of super-scaffolds >5× longer by the N50 metric than scaffolds augmented by fosmid end sequencing (scaffold N50 = 3.06 Mb, super-scaffold N50 = 16.3 Mb). Super-scaffolds are anchored on chromosomes based on conserved synteny with cattle, and the assembly is well supported by two radiation hybrid maps of chromosome 1. We annotate 22,175 protein-coding genes, most of which were recovered in the RNA-seq data of ten tissues. Comparative transcriptomic analysis of the primary and secondary follicles of a cashmere goat reveal 51 genes that are differentially expressed between the two types of hair follicles. This study, whose results will facilitate goat genomics, shows that whole-genome mapping technology can be used for the de novo assembly of large genomes.",
"Pfam is a widely used database of protein families, currently containing more than 13,000 manually curated protein families as of release 26.0. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/). Here, we report on changes that have occurred since our 2010 NAR paper (release 24.0). Over the last 2 years, we have generated 1840 new families and increased coverage of the UniProt Knowledgebase (UniProtKB) to nearly 80%. Notably, we have taken the step of opening up the annotation of our families to the Wikipedia community, by linking Pfam families to relevant Wikipedia pages and encouraging the Pfam and Wikipedia communities to improve and expand those pages. We continue to improve the Pfam website and add new visualizations, such as the 'sunburst' representation of taxonomic distribution of families. In this work we additionally address two topics that will be of particular interest to the Pfam community. First, we explain the definition and use of family-specific, manually curated gathering thresholds. Second, we discuss some of the features of domains of unknown function (also known as DUFs), which constitute a rapidly growing class of families within Pfam.",
"Fluorescence in situ hybridization (FISH) is an effective and accurate cytogenetic tool for mapping single copy and repetitive DNA sequences on chromosomes. Attempts to increase the detection sensitivity of very small chromosomal targets, and to improve the spatial resolution of signals derived from flanking sequences, have led to the development of a variety of novel techniques: it is now possible to perform in situ hybridizations on interphase nuclei, meiotic pachytene chromosomes and isolated chromatin (DNA fibres). The recent application of these techniques has indicated that a spatial resolution of 1 kb between adjacent targets and a sensitivity of targets smaller than 1 kb is now feasible. Here, we describe the benefits of these novel chromosome analysis techniques and discuss their relevance for the study of plant genomes.",
"Genome sequencing projects have been initiated for a wide range of eukaryotes. A few projects have reached completion, but most exist as draft assemblies. As one of the main reasons to sequence a genome is to obtain its catalog of genes, an important question is how complete or completable the catalog is in unfinished genomes. To answer this question, we have identified a set of core eukaryotic genes (CEGs), that are extremely highly conserved and which we believe are present in low copy numbers in higher eukaryotes. From an analysis of a phylogenetically diverse set of eukaryotic genome assemblies, we found that the proportion of CEGs mapped in draft genomes provides a useful metric for describing the gene space, and complements the commonly used N50 length and x-fold coverage values."
] |
A novel NKX2-5 mutation associated with sudden cardiac death from ventricular arrhythmia.
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To date, several disease-related mutations in NKX2-5, a cardiac-specific homeobox gene, have been documented in patients with a variety of congenital heart diseases (CHDs). The most commonly reported phenotypes are secundum atrial septal defect (ASD) and atrioventricular conduction disease (AVCD). Reports of sudden cardiac death (SCD) have been attributed to progressive conduction disease preventable with pacemaker therapy. A retrospective chart review of individuals from three generations of a family with a novel NKX2-5 mutation associated with CHD, ventricular arrhythmias, and SCD despite pacemaker therapy was conducted. The review documented NKX2-5 Gln181His missense mutation in 11 phenotypically affected members of a single family with a strong family history of SCD, CHD, and AVCD. Before genotyping, four family members died suddenly, two despite pacemaker therapy. The ages at SCD were respectively 23, 29, 44, and 45 years. Observed phenotypic characteristics of genotype-positive patients included ASD, ventricular septal defect, aortic coarctation, tricuspid atresia, supraventricular tachycardia, progressive AVCD, and ventricular tachycardia documented on implantable cardiac defibrillator (ICD) recording. The age at presentation ranged from 5 months to 44 years, and AVCD was seen as early as infancy. Four phenotypically unaffected family members tested negative for the mutation. The findings of this review strongly suggest a new association of this NKX2-5 mutation with SCD from ventricular arrhythmia. This observation has significant implications for the choice of therapy for affected individuals, specifically the use of ICDs, and broadens the observed phenotypic spectrum of NKX2-5 mutations.
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"NKX2-5 is a homeodomain-containing transcription factor important in cardiac development. Familial mutations in the NKX2-5 gene are associated with cardiac abnormalities, but mutations are rare in sporadic cases. We studied the pathology and molecular genetics of NKX2-5 in diseased heart tissues of 68 patients with complex congenital heart disease (CHD), particularly atrial (ASD), ventricular (VSD), and atrioventricular septal defects (AVSD). We also studied DNA extracted from 16 normal hearts, as well as lymphocytic DNA from 50 healthy volunteers, 7 families, and 4 unrelated individuals with CHD. Direct sequencing revealed 53 NKX2-5 mutations in the diseased heart tissues, including nonsynonymous substitutions in the homeodomain of NKX2-5. We found common mutations among unrelated patients, but certain mutations were specific to VSDs and AVSDs. Many patients had multiple NKX2-5 mutations, up to 14 nonsynonymous mutations per patient in VSDs. Importantly, these nonsynonymous mutations were mainly absent in normal heart tissues of the same CHD patients, thus indicating somatic origin and mosaicism of mutations. Further, observed mutations were completely absent in normal hearts and lymphocytic DNA of healthy individuals. Our findings provide new insights for somatic NKX2-5 mutations to be of importance in congenital heart disease.",
"Heterozygous mutations in the human transcription factor gene NKX2.5 are associated with either isolated or combined congenital heart disease (CHD), primarily secundum atrial septal defect-II (ASD-II), ventricular septal defect (VSD) or tetralogy of Fallot (TOF). Thus, NKX2.5 has an important role at different stages of cardiac development. The frequency of NKX2.5 mutations in a broader phenotypic spectrum of CHD is not completely determined. Here, we report the identification of two novel mutations in the NKX2.5 gene in a screening of 121 patients with a broad spectrum of CHDs. However, mutations were only associated with familial ASD-II and in both, patients also showed atrioventricular (AV) block. We found one missense mutation (R190L) in two siblings with ASD-II and a frame-shift mutation (A255fsX38) at the C-terminus in a mother and daughter. In addition, a single patient with hypoplastic left heart syndrome (HLHS) had the reported sequence variant R25C. Importantly, sporadic cases of CHD that share phenotypic aspects of NKX2.5 mutation carriers were negative for genetic analysis. Thus, even important for cardiac development, germline mutations in NKX2.5 are rare in patients with sporadic CHD and genetic and/or pathophysiologic heterogeneity is likely for sporadic forms of CHD.",
"The homeodomain family of transcription factors plays a fundamental role in a diverse set of functions that include body plan specification, pattern formation and cell fate determination during metazoan development. Members of this family are characterized by a helix-turn-helix DNA-binding motif known as the homeodomain. Homeodomain proteins regulate various cellular processes by specifically binding to the transcriptional control region of a target gene. These proteins have been conserved across a diverse range of species, from yeast to human. A number of inherited human disorders are caused by mutations in homeodomain-containing proteins. In this study, we present an evolutionary classification of 129 human homeodomain proteins. Phylogenetic analysis of these proteins, whose sequences were aligned based on the three-dimensional structure of the homeodomain, was performed using a distance matrix approach. The homeodomain proteins segregate into six distinct classes, and this classification is consistent with the known functional and structural characteristics of these proteins. An ancestral sequence signature that accurately describes the unique sequence characteristics of each of these classes has been derived. The phylogenetic analysis, coupled with the chromosomal localization of these genes, provides powerful clues as to how each of these classes arose from the ancestral homeodomain.",
"The purpose of this study was to estimate the frequency of NKX2.5 mutations in specific cardiovascular anomalies and investigate genotype-phenotype correlations in individuals with NKX2.5 mutations. Recent reports have implicated mutations in the transcription factor NKX2.5 as a cause of various congenital heart defects (CHD). We tested genomic deoxyribonucleic acid from 608 prospectively recruited patients with conotruncal anomalies (n = 370), left-sided lesions (n = 160), secundum atrial septal defect (ASD) (n = 71), and Ebstein's malformation (n = 7) for NKX2.5 mutations. Twelve distinct mutations in the NKX2.5 coding region were identified in 18 of 608 patients (3%), including 9 of 201 (4%) with tetralogy of Fallot, 3 of 71 (4%) with a secundum ASD, one each with truncus arteriosus, double-outlet right ventricle, L-transposition of the great arteries, interrupted aortic arch, hypoplastic left heart syndrome, and aortic coarctation, but in no patients with D-transposition of the great arteries (n = 86) or valvar aortic stenosis (n = 21). Eleven of the mutations were amino acid-altering missense nucleotide substitutions or deletions, and one was predicted to cause premature termination of translation. None of the mutations were in the homeodomain. Sixteen of the 18 individuals with NKX2.5 mutations in this study had no family history of congenital cardiovascular anomalies, and one had first-degree atrioventricular (AV) block. NKX2.5 mutations occur in a small percentage of patients with various CHD. Most of the mutations identified in this study were missense, outside the homeodomain, and not associated with AV block. These findings suggest that NKX2.5 mutations in non-homeodomain regions may be important in the development of human structural cardiac defects.",
"Mutations in the gene encoding the homeobox transcription factor NKX2-5 were found to cause nonsyndromic, human congenital heart disease. A dominant disease locus associated with cardiac malformations and atrioventricular conduction abnormalities was mapped to chromosome 5q35, where NKX2-5, a Drosophila tinman homolog, is located. Three different NKX2-5 mutations were identified. Two are predicted to impair binding of NKX2-5 to target DNA, resulting in haploinsufficiency, and a third potentially augments target-DNA binding. These data indicate that NKX2-5 is important for regulation of septation during cardiac morphogenesis and for maturation and maintenance of atrioventricular node function throughout life."
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Fumarilacetoacetate hydrolase(+/-) pigs: a large animal model of hereditary tyrosinemia type I.
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Hereditary tyrosinemia type I (HT1) is caused by deficiency in fumarylacetoacetate hydrolase (FAH), an enzyme that catalyzes the last step of tyrosine metabolism. The most severe form of the disease presents acutely during infancy, and is characterized by severe liver involvement, most commonly resulting in death if untreated. Generation of FAH(+/-) pigs was previously accomplished by adeno-associated virus-mediated gene knockout in fibroblasts and somatic cell nuclear transfer. Subsequently, these animals were outbred and crossed to produce the first FAH(-/-) pigs. FAH-deficiency produced a lethal defect in utero that was corrected by administration of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexanedione (NTBC) throughout pregnancy. Animals on NTBC were phenotypically normal at birth; however, the animals were euthanized approximately four weeks after withdrawal of NTBC due to clinical decline and physical examination findings of severe liver injury and encephalopathy consistent with acute liver failure. Biochemical and histological analyses, characterized by diffuse and severe hepatocellular damage, confirmed the diagnosis of severe liver injury. FAH(-/-) pigs provide the first genetically engineered large animal model of a metabolic liver disorder. Future applications of FAH(-/-) pigs include discovery research as a large animal model of HT1 and spontaneous acute liver failure, and preclinical testing of the efficacy of liver cell therapies, including transplantation of hepatocytes, liver stem cells, and pluripotent stem cell-derived hepatocytes.
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"Isolation of hepatic progenitor cells is a promising approach for cell replacement therapy of chronic liver disease. The winged helix transcription factor Foxl1 is a marker for progenitor cells and their descendants in the mouse liver in vivo. Here, we purify progenitor cells from Foxl1-Cre; RosaYFP mice and evaluate their proliferative and differentiation potential in vitro. Treatment of Foxl1-Cre; RosaYFP mice with a 3,5-diethoxycarbonyl-1,4-dihydrocollidine diet led to an increase of the percentage of YFP-labeled Foxl1(+) cells. Clonogenic assays demonstrated that up to 3.6% of Foxl1(+) cells had proliferative potential. Foxl1(+) cells differentiated into cholangiocytes and hepatocytes in vitro, depending on the culture condition employed. Microarray analyses indicated that Foxl1(+) cells express stem cell markers such as Prom1 as well as differentiation markers such as Ck19 and Hnf4a. Thus, the Foxl1-Cre; RosaYFP model allows for easy isolation of adult hepatic progenitor cells that can be expanded and differentiated in culture.",
"The liver and exocrine pancreas share a common structure, with functioning units (hepatic plates and pancreatic acini) connected to the ductal tree. Here we show that Sox9 is expressed throughout the biliary and pancreatic ductal epithelia, which are connected to the intestinal stem-cell zone. Cre-based lineage tracing showed that adult intestinal cells, hepatocytes and pancreatic acinar cells are supplied physiologically from Sox9-expressing progenitors. Combination of lineage analysis and hepatic injury experiments showed involvement of Sox9-positive precursors in liver regeneration. Embryonic pancreatic Sox9-expressing cells differentiate into all types of mature cells, but their capacity for endocrine differentiation diminishes shortly after birth, when endocrine cells detach from the epithelial lining of the ducts and form the islets of Langerhans. We observed a developmental switch in the hepatic progenitor cell type from Sox9-negative to Sox9-positive progenitors as the biliary tree develops. These results suggest interdependence between the structure and homeostasis of endodermal organs, with Sox9 expression being linked to progenitor status.",
"The management of tyrosinaemia type 1 (HT1, fumarylacetoacetase deficiency) has been revolutionised by the introduction of nitisinone but dietary treatment remains essential and the management is not easy. In this review detailed recommendations for the management are made based on expert opinion, published case reports and investigational studies as the evidence base is limited and there are no prospective controlled studies.The added value of this paper is that it summarises in detail current clinical knowledge about HT1 and makes recommendations for the management.",
"Maturation of induced pluripotent stem cells (hiPSCs) to hepatocyte-like cells (HLCs) has been proposed to address the shortage of human hepatocytes for therapeutic applications. The purpose of this study was to evaluate hiPSCs, HLCs and hepatocytes, all of human origin, in terms of performance metrics of relevance to cell therapies. hiPSCs were differentiated to HLCs in vitro using an established four-stage approach. We observed that hiPSCs had low oxygen consumption and possessed small, immature mitochondria located around the nucleus. With maturation to HLCs, mitochondria showed characteristic changes in morphology, ultrastructure, and gene expression. These changes in mitochondria included elongated morphology, swollen cristae, dense matrices, cytoplasmic migration, increased expression of mitochondrial DNA transcription and replication-related genes, and increased oxygen consumption. Following differentiation, HLCs expressed characteristic hepatocyte proteins including albumin and hepatocyte nuclear factor 4-alpha, and intrinsic functions including cytochrome P450 metabolism. But HLCs also expressed high levels of alpha fetoprotein, suggesting a persistent immature phenotype or inability to turn off early stage genes. Furthermore, the levels of albumin production, urea production, cytochrome P450 activity, and mitochondrial function of HLCs were significantly lower than primary human hepatocytes. - hiPSCs offer an unlimited source of human HLCs. However, reduced functionality of HLCs compared to primary human hepatocytes limits their usefulness in clinical practice. Novel techniques are needed to complete differentiation of hiPSCs to mature hepatocytes.",
"The Wnt target gene Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5) marks actively dividing stem cells in Wnt-driven, self-renewing tissues such as small intestine and colon, stomach and hair follicles. A three-dimensional culture system allows long-term clonal expansion of single Lgr5(+) stem cells into transplantable organoids (budding cysts) that retain many characteristics of the original epithelial architecture. A crucial component of the culture medium is the Wnt agonist RSPO1, the recently discovered ligand of LGR5. Here we show that Lgr5-lacZ is not expressed in healthy adult liver, however, small Lgr5-LacZ(+) cells appear near bile ducts upon damage, coinciding with robust activation of Wnt signalling. As shown by mouse lineage tracing using a new Lgr5-IRES-creERT2 knock-in allele, damage-induced Lgr5(+) cells generate hepatocytes and bile ducts in vivo. Single Lgr5(+) cells from damaged mouse liver can be clonally expanded as organoids in Rspo1-based culture medium over several months. Such clonal organoids can be induced to differentiate in vitro and to generate functional hepatocytes upon transplantation into Fah(-/-) mice. These findings indicate that previous observations concerning Lgr5(+) stem cells in actively self-renewing tissues can also be extended to damage-induced stem cells in a tissue with a low rate of spontaneous proliferation.",
"Several reliable and reproducible methods are available to induce oval cells in rat liver. Effective methods often involve inhibiting proliferation in hepatocytes using an alkylating agent, then subjecting the rat to partial hepatectomy (PH). The surgery is difficult to perform reproducibly in mice. Approaches that do not include partial hepatectomy, such as administration of D-galactosamine, are ineffective in mice. We found that a choline-deficient, ethionine-supplemented (CDE) diet, which is very effective in rats, leads to high morbidity and mortality when administered to mice. This article outlines an alternative protocol by which a CDE diet can be administered to mice. This diet is shown to be highly effective for oval cell induction, without causing high mortality. It takes less time and is at least as effective as other commonly used protocols for inducing oval cells in mice."
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Multiple locus sequencing typing of enterotoxigenic Escherichia coli clinical isolates from children in northern Colombia.
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Enterotoxigenic Escherichia coli (ETEC) are major causes of childhood diarrhea in low and middle income countries including Colombia, South America. To understand the diversity of ETEC strains in the region, clinical isolates obtained from northern Colombia children were evaluated for multiple locus sequencing typing, serotyping, classical and nonclassical virulence genes, and antibiotic susceptibility. Among 40 ETEC clinical isolates evaluated, 21 (52.5%) were positive for LT gene, 13 (32.5%) for ST gene, and 6 (15%) for both ST and LT. The most prevalent colonization surface antigens (CS) were CS21 and CFA/I identified in 21 (50%) and 13 (32.5%) isolates, respectively. The eatA, irp2, and fyuA were the most common nonclassical virulence genes present in more than 60% of the isolates. Ampicillin resistance (80% of the strains) was the most frequent phenotype among ETEC strains followed by trimethoprim-sulfamethoxazole resistance (52.5%). Based on multiple locus sequencing typing (MLST), we recognize that 6 clonal groups of ETEC clinical isolates circulate in Colombia. ETEC clinical isolates from children in northern Colombia are highly diverse, yet some isolates circulating in the community belong to well-defined clonal groups that share a unique set of virulence factors, serotypes, and MLST sequence types.
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"An enterotoxin-producing strain of Escherichia coli isolated from a case of cholera-like diarrhea (E. coli strain H-10407) was found to possess a surface-associated colonization factor. Colonization was manifested as the ability of small inocula (10(5) bacteria) to attain large (10(9)) populations in the infant rabbit intestine with a concomitant diarrheal response. A laboratory-passed derivative of E. coli H-10407, designated H-10407-P, failed to exhibit an increase in population in the infant rabbit and also failed to induce diarrhea. Cell-free culture supernatant fluids of E. coli H-10407 and H-10407-P produced equivalent enterotoxic responses in infant and in adult rabbits. Specific anti-colonization factor antiserum was produced by adsorbing hyperimmune anti-H-10407 serum with both heat-killed and living cells E. coli H-10407-P. This specific adsorbed serum protected infant rabbits from challenge with living E. coli H-10407 although the serum did not possess bactericidal activity. The anti-colonization factor serum did not agglutinate a strain of E. coli K-12 possessing the K88 colonization factor peculiar to E. coli enterotoxigenic for swine. By electron microscopy it was demonstrated that E. coli H-10407, but not H10407-, possessed pilus-like surface structures which agglutinated with the specific adsorbed (anti-colonization factor) antiserum. E. coli H-10407 possessed three species of plasmid deoxyribonucleic acid, measuring 60 X 10(6), 42 X 10(6), and 3.7 X 10(6) daltons, respectively. E. coli H-10407-P possessed only the 42 X 10(6)- and the 3.7 X 10(6)-dalton plasmid species. Spontaneous loss of the specific H-10407 surface-associated antigen was accompanied by loss of the 60 X 10(6)-dalton species of plasmid deoxyribonucleic acid and loss of colonizing ability. Thus, it is concluded that the E. coli colonization factor described here is a virulence factor which may play an important and possibly essential role in naturally occurring E. coli enterotoxic diarrhea in man.",
"Enterotoxigenic Escherichia coli (ETEC) causes substantial diarrheal morbidity and mortality in young children in countries with limited resources. We determined the phenotypic profiles of 915 ETEC diarrheal isolates derived from Egyptian children under 3 years of age who participated in a 3-year population-based study. For each strain, we ascertained enterotoxin and colonization factor (CF) expression, the O:H serotype, and antimicrobial susceptibility. Sixty-one percent of the strains expressed heat-stable enterotoxin (ST) only, 26% expressed heat-labile enterotoxin (LT) alone, and 12% expressed both toxins. The most common CF phenotypes were colonization factor antigen I (CFA/I) (10%), coli surface antigen 6 (CS6) (9%), CS14 (6%), and CS1 plus CS3 (4%). Fifty-nine percent of the strains did not express any of the 12 CFs included in our test panel. Resistance of ETEC strains to ampicillin (63%), trimethoprim-sulfamethoxazole (52%), and tetracycline (43%) was common, while resistance to quinolone antibiotics was rarely detected. As for the distribution of observed serotypes, there was an unusually wide diversity of O antigens and H types represented among the 915 ETEC strains. The most commonly recognized composite ETEC phenotypes were ST CS14 O78:H18 (4%), ST (or LTST) CFA/I O128:H12 (3%), ST CS1+CS3 O6:H16 (2%), and ST CFA/I O153:H45 (1.5%). Temporal plots of diarrheal episodes associated with ETEC strains bearing common composite phenotypes were consistent with discrete community outbreaks either within a single or over successive warm seasons. These data suggest that a proportion of the disease that is endemic to young children in rural Egypt represents the confluence of small epidemics by clonally related ETEC strains that are transiently introduced or that persist in a community reservoir.",
"Escherichia coli is a diverse bacterial species which is widely distributed in the environment but also exists as a commensal and pathogen of different host species. Human intestinal pathogenic E. coli causes over 160 million cases of diarrhea and an estimated 1 million deaths per year. The majority of deaths are attributable to one pathovar of E. coli, namely, enterotoxigenic E. coli. The pathogenesis of enterotoxigenic E. coli is dependent on the production of a colonization factor to promote adhesion to the intestinal epithelium and the elaboration of heat-labile or heat-stable toxins which induce a secretory diarrhea. Despite the high morbidity and mortality associated with enterotoxigenic E. coli infection, little is known of the genetic background of this global pathogen. Here we demonstrate by multilocus sequence typing that enterotoxigenic E. coli isolates are present in all phylogenetic lineages of E. coli, indicating that acquisition of the toxin genes may be sufficient to generate an enterotoxigenic E. coli strain. In addition, screening of diarrheal isolates for the presence of additional genes previously associated with the virulence of enterotoxigenic E. coli revealed that they were not abundant. These observations have significant implications for disease epidemiology and for the design of effective vaccines.",
"The enterotoxigenic Escherichia coli (ETEC) strains are major causes of morbidity and mortality due to diarrheal illness in developing countries. At present, there is no broadly protective vaccine for this diverse group of pathogens. The EtpA protein, identified in ETEC H10407 in a recent search for candidate immunogens, is a large glycosylated exoprotein secreted via two-partner secretion (TPS). Similar to structurally related molecules, EtpA functions in vitro as an adhesin. The studies reported here use a recently developed murine model of ETEC intestinal colonization to examine the immunogenicity and protective efficacy of EtpA. We report that mice repeatedly exposed to ETEC are protected from subsequent colonization and that they mount immune responses to both EtpA and its presumed two-partner secretion transporter (EtpB) during the course of experimental infection. Furthermore, isogenic etpA deletion mutants were impaired in the colonization of mice, and intranasal immunization of mice with recombinant EtpA conferred protection against ETEC H10407 in this model. Together, these data suggest that EtpA is required for optimal colonization of the intestine, findings paralleling those of previous in vitro studies demonstrating its role in adherence. EtpA and other TPS proteins may be viable targets for ETEC vaccine development.",
"Enterotoxigenic Escherichia coli (ETEC) strains remain a formidable cause of diarrheal disease. To identify novel surface proteins of ETEC, we performed TnphoA mutagenesis of prototype ETEC strain H10407 and discovered a secreted protein not previously recognized in ETEC. DNA sequencing of the interrupted locus in mutant TnphoA.977 revealed a candidate 4,095-bp open reading frame without significant homology to commensal E. coli K-12 genomic DNA. Translation of this sequence revealed that it encoded a predicted peptide of 147.7 kDa that bears significant homology to members of the autotransporter family of bacterial virulence factors, particularly the serine protease autotransporters of the Enterobacteriaceae proteins. The gene identified in H10407, eatA (ETEC autotransporter A), encodes a potential serine protease motif (GDSGSP) in the secreted amino-terminal domain, and the predicted peptide shows more than 80% homology with SepA, a virulence protein secreted by Shigella flexneri. DNA hybridization and PCR demonstrated that eatA resides on the 92-kDa pCS1 virulence plasmid of H10407 and that it is present in multiple clinical ETEC strains. Immunoblots with antisera directed against a recombinant EatA passenger protein fragment identified a 110-kDa protein in supernatants purified from H10407 but not from the TnphoA.977 mutant or H10407-P, which lacks pCS1. EatA possesses serine protease activity that is abolished by mutations within a serine protease catalytic triad formed by residues H(134), D(162), and S(267). Finally, interruption of the eatA gene retarded fluid accumulation in the rabbit ileal loop model, suggesting that this autotransporter contributes to the virulence of ETEC.",
"In a follow-up study, enterotoxigenic Escherichia coli (ETEC) infections in 145 children from two communities located in northeastern Argentina were monitored for 2 years. The occurrence of diarrhea was monitored by weekly household visits. Of 730 fecal specimens collected, 137 (19%) corresponded to diarrheal episodes. ETEC was isolated from a significantly higher proportion of symptomatic (18.3%) than asymptomatic (13.3%) children (P = 0.04541). Individuals of up to 24 months of age were found to have a higher risk of developing ETEC diarrhea than older children (odds ratio [OR], 3.872; P = 0.00021). When the toxin profiles were considered, only heat stable enterotoxin (ST)-producing ETEC was directly associated with diarrhea (P = 0.00035). Fifty-five percent of the ETEC isolated from symptomatic children and 19% of the ETEC isolated from asymptomatic children expressed one of the colonization factors (CFs) investigated, i.e., CF antigen I (CFA/I), CFA/II, CFA/III, and CFA/IV; coli surface antigens CS7 and CS17; and putative CFs PCFO159, PCFO166, and PCFO20, indicating a clear association between diarrhea and ETEC strains that carry these factors (P = 0.0000034). The most frequently identified CFs were CFA/IV (16%), CFA/I (10%), and CS17 (9%). CFs were mostly associated with ETEC strains that produce ST and both heat-labile enterotoxin and ST. Logistic regression analysis, applied to remove confounding effects, revealed that the expression of CFs was associated with illness independently of the toxin type (OR, 4.81; P = 0.0003). When each CF was considered separately, CS17 was the only factor independently associated with illness (OR, 16.6; P = 0.0151). Most CFs (the exception was CFA/IV) fell within a limited array of serotypes, while the CF-negative isolates belonged to many different O:H types. These results demonstrate that some CFs are risk factors for the development of ETEC diarrhea.",
"The right sampling of homologous sequences for phylogenetic or molecular evolution analyses is a crucial step, the quality of which can have a significant impact on the final interpretation of the study. There is no single way for constructing datasets suitable for phylogenetic analysis, because this task intimately depends on the scientific question we want to address, Moreover, database mining softwares such as BLAST which are routinely used for searching homologous sequences are not specifically optimized for this task. To fill this gap, we designed BLAST-Explorer, an original and friendly web-based application that combines a BLAST search with a suite of tools that allows interactive, phylogenetic-oriented exploration of the BLAST results and flexible selection of homologous sequences among the BLAST hits. Once the selection of the BLAST hits is done using BLAST-Explorer, the corresponding sequence can be imported locally for external analysis or passed to the phylogenetic tree reconstruction pipelines available on the Phylogeny.fr platform. BLAST-Explorer provides a simple, intuitive and interactive graphical representation of the BLAST results and allows selection and retrieving of the BLAST hit sequences based a wide range of criterions. Although BLAST-Explorer primarily aims at helping the construction of sequence datasets for further phylogenetic study, it can also be used as a standard BLAST server with enriched output. BLAST-Explorer is available at http://www.phylogeny.fr."
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A tool for assessing trend accuracy of continuous glucose monitoring data
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Continuous glucose monitoring (CGM) devices are being increasingly used to monitor glycemia in people with diabetes. One advantage with CGM is the ability to monitor the trend of sensor glucose (SG) over time. However, there are few metrics available for assessing the trend accuracy of CGM devices. The aim of this study was to develop an easy to interpret tool for assessing trend accuracy of CGM data. SG data from CGM were compared to hourly blood glucose (BG) measurements and trend accuracy was quantified using the dot product. Trend accuracy results are displayed on the Trend Compass, which depicts trend accuracy as a function of BG. A trend performance table and Trend Index (TI) metric are also proposed. The Trend Compass was tested using simulated CGM data with varying levels of error and variability, as well as real clinical CGM data. The results show that the Trend Compass is an effective tool for differentiating good trend accuracy from poor trend accuracy, independent of glycemic variability. Furthermore, the real clinical data show that the Trend Compass assesses trend accuracy independent of point bias error. Finally, the importance of assessing trend accuracy as a function of BG level is highlighted in a case example of low and falling BG data, with corresponding rising SG data. This study developed a simple to use tool for quantifying trend accuracy. The resulting trend accuracy is easily interpreted on the Trend Compass plot, and if required, performance table and TI metric.
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"Hypoglycemia has been identified as a primary barrier to optimal management of diabetes. This observation, in conjunction with the introduction of continuous glucose monitoring (CGM) devices, has set the stage for achieving tight glycemic control with systems that adjust the insulin pump settings based on measured glucose concentrations. Because system safety and system reliability are key considerations, there is a need for algorithms that reduce the risk of hypoglycemia in closed-loop, open-loop, and advisory-mode systems. More specifically, the algorithm presented here is formulated as a component of the independent safety system module proposed in the modular control-to-range architecture. We developed two algorithms for attenuating insulin pump injections, which we refer to as Brakes and Power Brakes: Brakes is a pump attenuation function computed using CGM information only, while Power Brakes is an attenuation function in which a metabolic state observer with insulin input is used in addition to CGM information to inform the level of pump attenuation. These algorithms modulate the insulin pump delivery so that the insulin injection rate is dramatically reduced when the risk of hypoglycemia is high. Additionally, we combined these algorithms with an alert system that indicates a level of hypoglycemic risk to the user. We demonstrated the effectiveness of Brakes and Power Brakes in reducing the incidence of hypoglycemia in two simulated scenarios: an elevated basal rate scenario and a scenario in which a bolus is delivered for a meal that is skipped. For these scenarios, the incidence of hypoglycemia using Power Brakes was reduced by 88 and 94%, respectively, where we defined hypoglycemia based on the American Diabetes Association guidelines for defining and reporting as 70 mg/dl. In the elevated basal rate scenario, no rebounds above 180 mg/dl (the desired upper limit of the control-to-range protocol) following hypoglycemia were shown to occur. We demonstrated the way the hypoglycemia alert system can trigger the intake of carbohydrates to reduce the incidence of hypoglycemia by 98%. This article offers, for the first time, a method for smoothly reducing insulin delivery rate to prevent hypoglycemia in individuals with type 1 diabetes mellitus based on a mathematically formal assessment of hypoglycemic risk. In silico, we demonstrate the way this method can prevent hypoglycemia while avoiding hyperglycemia rebounds that exceed 180 mg/dl. In conjunction with the pump attenuation algorithms, this article also proposes a system for alerting an individual of their hypoglycemic risk that contains three \"levels\" of alerts in the form of a traffic light. This alert system can be used in an advisory mode setting to alert the user to take action when hypoglycemia is imminent (\"red\" light) or in a closed-loop setting where initiation of rescue action begins when the red light alert is triggered.",
"Integrated closed-loop control (CLC), combining continuous glucose monitoring (CGM) with insulin pump (continuous subcutaneous insulin infusion [CSII]), known as artificial pancreas, can help optimize glycemic control in diabetes. We present a fundamental modular concept for CLC design, illustrated by clinical studies involving 11 adolescents and 27 adults at the Universities of Virginia, Padova, and Montpellier. We tested two modular CLC constructs: standard control to range (sCTR), designed to augment pump plus CGM by preventing extreme glucose excursions; and enhanced control to range (eCTR), designed to truly optimize control within near normoglycemia of 3.9-10 mmol/L. The CLC system was fully integrated using automated data transfer CGM→algorithm→CSII. All studies used randomized crossover design comparing CSII versus CLC during identical 22-h hospitalizations including meals, overnight rest, and 30-min exercise. sCTR increased significantly the time in near normoglycemia from 61 to 74%, simultaneously reducing hypoglycemia 2.7-fold. eCTR improved mean blood glucose from 7.73 to 6.68 mmol/L without increasing hypoglycemia, achieved 97% in near normoglycemia and 77% in tight glycemic control, and reduced variability overnight. In conclusion, sCTR and eCTR represent sequential steps toward automated CLC, preventing extremes (sCTR) and further optimizing control (eCTR). This approach inspires compelling new concepts: modular assembly, sequential deployment, testing, and clinical acceptance of custom-built CLC systems tailored to individual patient needs.",
"Continuous glucose monitors (CGMs) generate data streams that are both complex and voluminous. The analyses of these data require an understanding of the physical, biochemical, and mathematical properties involved in this technology. This article describes several methods that are pertinent to the analysis of CGM data, taking into account the specifics of the continuous monitoring data streams. These methods include: (1) evaluating the numerical and clinical accuracy of CGM. We distinguish two types of accuracy metrics-numerical and clinical-each having two subtypes measuring point and trend accuracy. The addition of trend accuracy, e.g., the ability of CGM to reflect the rate and direction of blood glucose (BG) change, is unique to CGM as these new devices are capable of capturing BG not only episodically, but also as a process in time. (2) Statistical approaches for interpreting CGM data. The importance of recognizing that the basic unit for most analyses is the glucose trace of an individual, i.e., a time-stamped series of glycemic data for each person, is stressed. We discuss the use of risk assessment, as well as graphical representation of the data of a person via glucose and risk traces and Poincaré plots, and at a group level via Control Variability-Grid Analysis. In summary, a review of methods specific to the analysis of CGM data series is presented, together with some new techniques. These methods should facilitate the extraction of information from, and the interpretation of, complex and voluminous CGM time series.",
"The control of diabetes is an interdisciplinary endeavor, which includes a significant biomedical engineering component, with traditions of success beginning in the early 1960s. It began with modeling of the insulin-glucose system, and progressed to large-scale in silico experiments, and automated closed-loop control (artificial pancreas). Here, we follow these engineering efforts through the last, almost 50 years. We begin with the now classic minimal modeling approach and discuss a number of subsequent models, which have recently resulted in the first in silico simulation model accepted as substitute to animal trials in the quest for optimal diabetes control. We then review metabolic monitoring, with a particular emphasis on the new continuous glucose sensors, on the analyses of their time-series signals, and on the opportunities that they present for automation of diabetes control. Finally, we review control strategies that have been successfully employed in vivo or in silico, presenting a promise for the development of a future artificial pancreas and, in particular, discuss a modular architecture for building closed-loop control systems, including insulin delivery and patient safety supervision layers. We conclude with a brief discussion of the unique interactions between human physiology, behavioral events, engineering modeling and control relevant to diabetes."
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Infection-induced systemic inflammatory response syndrome in a patient with rheumatoid arthritis treated by tocilizumab
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A 65-year-old woman with rheumatoid arthritis treated by tocilizumab (TCZ) presented with tongue squamous cell carcinoma. While surgery was performed without any complications the aspiration pneumonia rapidly worsened by postoperative day 2 and severe pulmonary suppuration in the right lung field with infection-induced systemic inflammatory response syndrome (SIRS) was diagnosed. Antibiotic and respirator treatment improved her condition. The anti-inflammatory effect of TCZ may mask the symptoms and signs of severe infection with SIRS.
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"Background. Degenerative changes caused by rheumatoid arthritis (RA) typically occur in the hands and feet, but are not rare in the hip. The purpose of our research was to assess outcome in RA patients undergoing total hip arthroplasty. Material and methods. From 1992 to 2000 we performed total arthroplasty in 166 RA patients (121 women and 45 men) with degenerative changes in the hip joint. The average age of these patients was 57.4 years (range 35 to 74). Outcome was evaluated clinically in terms of pain relief, locomotion, and range of motion in the hip, evaluated according to the Harris hip function scale. Radiological analysis was also performed. Results. The greatest problem during surgery proved to be the lack of strong bone edges on the acetabulum (78%). The average results from the Harris scale went up 46 points, to 83.2+/-11.3. During the final radiological analysis there were no visible gaps or gaps only in Zone I of the acetabulum in 80% of the joints. We had to perform revision surgery in 3 hips (1.7%) due to infection, and in 8 hips (4.5%) due to aseptic loosening of the acetabulum. Conclusions. 2/3 of the hips affected by rheumatic changes show destruction of the acetabulum, which hinders proper emplacement of the acetabulum. Loosening of the hip endoprosthesis was found radiologically in 13% of the hips, which was accompanied by clinical symptoms in half the cases. The conditions for stable insertion of the shaft were better than for the acetabulum.",
"Despite great advancement in the understanding of the pathophysiology and in the development of novel therapeutic approaches, mortality of sepsis still remains unacceptably high. Adequate laboratory diagnostics represents a major requirement for the improvement of this situation. For a better understanding of the immunological dysregulation in this disease, several markers are now available for routine diagnostics in the clinical laboratory. They include the cytokines interleukin (IL) -6, IL-8, procalcitonin and the LPS-binding protein (LBP). These novel markers will be compared to the conventional procedure of diagnosing inflammatory and infectious disease, such as measurements of C-reactive protein (CRP) as a major acute phase protein and differential blood counting. Important questions addressed in this review are the usefulness of these markers for early diagnosis, their role as prognostic markers and in the risk assessment of patients. Furthermore, we will discuss whether these parameters are to differentiate between systemic inflammatory response syndrome (SIRS) and sepsis at its different degrees. In the case of an infectious nature of the disease, it is important to differentiate between viral or bacterial origin and to monitor the responsiveness of antibiotic therapies. The literature was analysed with focus on the evidence for diagnostic and analytical performance. For this purpose international definition and staging criteria were used in context of criteria for assay performance including sensitivity, specificity, negative and positive predictive values, ROC analysis and other analytical criteria.",
"This study was undertaken to investigate whether sequential measurement of blood interleukin (IL)-6 levels using chemiluminescent enzyme immunoassay (CLEIA) would be useful for the management of patients with systemic inflammatory response syndrome (SIRS)/sepsis. Forty consecutive patients with SIRS/sepsis admitted to ICU were involved in the study. Blood IL-6 level was measured everyday throughout their ICU stay at the clinical laboratory by CLEIA method. The platelet count and the sequential organ failure assessment (SOFA) score were measured consecutively. The blood IL-6 levels were elevated in SIRS/sepsis patients and were extremely high in patients with septic shock. There was no significant difference in the blood IL-6 level on admission between survivors (n=27) and non-survivors (n=13). However, the mean blood IL-6 level during ICU stay was significantly higher in the non-survivors (p<0.05). There were significant correlation between the peak IL-6 blood level and the lowest platelet count, and between the peak IL-6 blood level and the maximum SOFA score, respectively. The platelet count became lowest 2.0+/-2.0 days later on average, and the SOFA score became maximal 2.5+/-1.4 days later on average following the day when IL-6 reached its peak value. Sequential measurement of blood IL-6 levels by CLEIA is useful in evaluating the severity and in predicting the outcome of the patients with SIRS/sepsis.",
"We evaluated the effects of binary combinations of four cytokines on production of the positive acute phase proteins alpha-1 antichymotrypsin, haptoglobin and fibrinogen, and the negative acute phase proteins albumin and alpha-fetoprotein (AFP) in two human hepatoma cell lines. The effects of the cytokine combinations on the five proteins varied; each protein exhibited a unique and specific pattern of response to the cytokine combinations. In Hep G2 cells, antichymotrypsin was induced by all four cytokines, IL-6, IL-1, TNF-alpha, and transforming growth factor beta 1 alone, and their effects in binary combinations could be attributed to additive or minimally synergistic interactions. Fibrinogen was induced only by IL-6 and this induction was inhibited by IL-1 alpha, TNF-alpha or transforming growth factor beta 1. Haptoglobin was also induced only by IL-6, but TNF-alpha was the only cytokine that inhibited this induction at all concentrations of IL-6. Each of the four cytokines alone down regulated production of AFP and albumin. However, binary combinations of the four cytokines were simply additive, for the most part, in inhibiting AFP production, whereas the inhibitory effects of combinations of cytokines on albumin production differed significantly from simple additive effects. These observations, taken together with studies of effects of cytokine combinations on other acute phase proteins, indicate that the various acute phase proteins respond differently to different combinations of cytokines and that the potential exists for highly specific regulation of synthesis of individual plasma proteins by cytokine interactions. These findings imply that the acute phase response in vivo represents the integrated sum of multiple, separately regulated changes in gene expression.",
"To understand the acute phase responses to surgical intervention in patients with rheumatoid arthritis (RA) treated with the anti-interleukin (IL)6 receptor antibody, tocilizumab. In a retrospective 1:1 pair-matched case-control study, 22 tocilizumab-treated RA cases and 22 cases treated with conventional disease-modifying antirheumatic drugs (DMARDs) and matched for type of surgery, age and sex were evaluated for body temperature every day, and blood C-reactive protein (CRP) levels and white blood cell (WBC), neutrophil and lymphocyte counts on days -1, 1, 3 and weeks 1 and 2 after joint surgery. Safety issues were also monitored. No complications of infection or delay of wound healing occurred in either patient group. Tocilizumab partially, but significantly, suppressed the increase in body temperature on postoperative days 1 and 2, compared with DMARDs (average (SD) maximum increase in temperature was 0.45 (0.1) degrees C in the tocilizumab group and 0.78 (0.1) degrees C in the DMARD group; p<0.01). Tocilizumab completely suppressed the increase in CRP after surgery, whereas all cases treated with DMARDs showed a significant increase of CRP at postoperative day 1 (5.5 (0.6) mg/dl; p<0.001). WBC, neutrophil and lymphocyte counts showed no remarkable change after surgery, and there was no significant difference in any cell counts between the patient groups. Within this small number of cases, safe operations on patients were performed during tocilizumab treatment. Tocilizumab suppressed fever and increase of CRP after surgery, whereas there was no influence on the transition in number of leukocytes. This characteristic postoperative response should be considered during tocilizumab treatment.",
"Postoperative serum interleukin-6 (SIL-6) and C-reactive protein (SCRP) levels were examined in 71 patients who underwent various types of abdominal surgery. Similar time-dependent changes in SIL-6 and SCRP levels were observed in 12 patients despite differences in surgical procedures and liver function among the patients. SIL-6 started to increase within 3 hours after the beginning of the operation and reached a peak after 24 hours. SCRP started to increase after 12 hours and was maximum at 48 to 72 hours. The increase in SIL-6 at 24 hours (delta IL-6) showed a close correlation with that of SCRP at 48 hours (delta CRP) in 53 patients without liver cirrhosis. In 18 patients with liver cirrhosis, delta CRP relative to delta IL-6 was less than that in patients without cirrhosis and was poorly correlated with the latter. delta IL-6 was correlated with the length of time of the operation and blood loss in both groups, but delta CRP showed no significant correlation with these factors in either group. These findings indicate that the increase in IL-6 triggered by a surgical procedure may function as a hepatocyte-stimulating factor and that monitoring of SIL-6 may be more helpful than monitoring of SCRP for estimation of inflammatory status and early detection of an acute-phase response.",
"Fever is defined as a regulated rise in body temperature. The regulation of this phenomenon is accomplished by the actions of two types of endogenous cytokines, some functioning as pyrogens and others as antipyretics. Previous data obtained with the use of traditional pharmacological techniques, such as the injection of neutralizing antibodies, implicate interleukin (IL)-1 and IL-6 as endogenous pyrogens or inducers of fever. In almost all instances in which the endogenous actions of IL-1 or IL-6 are antagonized, fevers are attenuated. Other cytokines, such as tumor necrosis factor-alpha (TNF-alpha) and IL-10, are thought to act as endogenous antipyretics or inhibitors of fever. In several studies, the inhibition of TNF action has enhanced fever. Recently, mice genetically engineered to lack cytokines or their receptors in all tissues of the body have been used to examine the regulation of IL-1, IL-6, TNF, and IL-10 on fever. Data obtained with these mice shed new light on our understanding of cytokine interactions in fever and, in some instances, contradict data obtained with pharmacological methods. This review summarizes the responses of cytokine and cytokine receptor knockout mice to fevers induced by lipopolysaccharide, turpentine, and sepsis."
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A multi-layer multi-column model of lesioned and malformed cortex
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The paper introduces a multi-layer multi-column model of the cortex that uses four different neuron types and short-term plasticity dynamics. It was designed with details of neuronal connectivity available in the literature and meets these conditions: (1) biologically accurate laminar and columnar flows of activity, (2) normal function of low-threshold spiking and fast spiking neurons, and (3) ability to generate different stages of epileptiform activity. With these characteristics the model allows for modeling lesioned or malformed cortex, i.e. examine properties of developmentally malformed cortex in which the balance between inhibitory neuron subtypes is disturbed.
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"Whole-cell patch-clamp recordings followed by histochemical staining and single-cell RT-PCR were obtained from 180 Martinotti interneurones located in layers II to VI of the somatosensory cortex of Wistar rats (P13-P16) in order to examine their anatomical, electrophysiological and molecular properties. Martinotti cells (MCs) mostly displayed ovoid-shaped somata, bitufted dendritic morphologies, and axons with characteristic spiny boutons projecting to layer I and spreading horizontally across neighbouring columns more than 1 mm. Electron microscopic examination of MC boutons revealed that all synapses were symmetrical and most synapses (71%) were formed onto dendritic shafts. MCs were found to contact tuft, apical and basal dendrites in multiple neocortical layers: layer II/III MCs targeted mostly layer I and to a lesser degree layer II/III; layer IV MCs targeted mostly layer IV and to a lesser degree layer I; layer V and VI MCs targeted mostly layer IV and layer I and to a lesser degree the layer in which their somata was located. MCs typically displayed spike train accommodation (90%; n = 127) in response to depolarizing somatic current injections, but some displayed non-accommodating (8%) and a few displayed irregular spiking responses (2%). Some accommodating and irregular spiking MCs also responded initially with bursts (17%). Accommodating responses were found in all layers, non-accommodating mostly in upper layers and bursting mostly in layer V. Single-cell multiplex RT-PCR performed on 63 MCs located throughout layers II-VI, revealed that all MCs were somatostatin (SOM) positive, and negative for parvalbumin (PV) as well as vasoactive intestinal peptide (VIP). Calbindin (CB), calretinin (CR), neuropeptide Y (NPY) and cholecystokinin (CCK) were co- expressed with SOM in some MCs. Some layer-specific trends seem to exist. Finally, 24 accommodating MCs were examined for the expression of 26 ion channel genes. The ion channels with the highest expression in these MCs were (from highest to lowest); Cabeta1, Kv3.3, HCN4, Cabeta4, Kv3.2, Kv3.1, Kv2.1, HCN3, Caalpha1G, Kv3.4, Kv4.2, Kv1.1 and HCN2. In summary, this study provides the first detailed analysis of the anatomical, electrophysiological and molecular properties of Martinotti cells located in different neocortical layers. It is proposed that MCs are crucial interneurones for feedback inhibition in and between neocortical layers and columns.",
"Properties of local synaptic connections in neocortex, studied with dual intracellular recordings in vitro and correlated with cell and synaptic morphology are summarized. The different durations and sensitivities to somatic membrane potential of pyramid-pyramid excitatory postsynaptic potentials (EPSPs) apparently reflect the positions of the synapses on the postsynaptic dendrites. Their time-, frequency- and voltage-dependent properties enable supra-linear summation of several low-frequency inputs arising in the same dendritic region, even if only loosely coincident, but they depress during repetitive firing in any one input. Pyramidal input to classical fast spiking and low threshold spiking interneurones are strikingly different. Here low presynaptic firing rates results in many transmission failures. EPSPs are brief and inputs must be near coincident for summation. However, these synapses display pronounced. frequency-dependent, incrementing facilitation at higher presynaptic frequencies. Once initiated by a brief high-frequency burst, this facilitation is maintained at lower frequencies. GABAA receptor-mediated inhibitory postsynaptic potentials (IPSPs) arising proximally are of very different durations depending on the type of interneurone activated and can prevent and subsequently synchronize firing in their many postsynaptic partners with very different delays (eg. 10-100 ms). Low threshold spiking interneurones, in contrast, generate brief IPSPs only in more distal dendritic regions and have little effect on somatic excitability acting to shunt input distally.",
"1. The cellular mechanisms of synchronous synaptic activity were studied in isolated slices of rat SmI neocortex in which gamma-aminobutyric acid (GABA)-mediated inhibition was slightly suppressed. Intracellular measurements were made from single neurons, and extracellular recordings monitored the timing and intensity of population events. 2. Neurons in cortical layers II-VI were classified by the attributes of their single action potentials and repetitive firing patterns during injection of intracellular current pulses. Regular-spiking (RS) cells occurred in all layers and had relatively long-duration spikes and strong frequency adaptation. Intrinsically bursting (IB) cells occurred only in layers IV and V and generated bursts of greater than or equal to 3 spikes; some IB cells of lower-layer V produced repetitive bursts during long depolarizing pulses. Fast-spiking (FS) cells had brief spikes and little or no adaptation and fired at high frequencies. 3. When GABAA-mediated inhibition was slightly reduced with low doses of bicuculline methiodide (BMI, 0.8-1.0 microM), synchronous events were evoked by stimulating layer VI with single shocks. Synchronous events were characterized by prominent, often all-or-none extracellular field potentials that propagated horizontally for variable distances up to several millimeters. Large field potentials were invariably correlated with excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) in single neurons. Both PSPs and field potentials often had long (up to 250 ms) and variable latencies, and sometimes two or more events were generated by single stimuli. In all cases the PSPs and field potentials were synchronous. Both field potentials and single cells sometimes generated short epochs (3-7 peaks) of rhythmic events at 20-50 Hz. 4. The physiological class of single neurons was correlated with the relative dominance of excitation and inhibition during each synchronous event. In phase with each synchronous event, most RS cells were very strongly inhibited with only small amounts of concurrent excitation. By contrast, IB cells were strongly and consistently excited, with relatively little inhibition. FS cells were also phasically excited. 5. Anatomic studies have identified RS and IB cells as pyramidal cells and FS cells as GABAergic nonpyramidal cells. This implies that, during the synchronous events of the present study, the majority of pyramidal cells were dominated by IPSPs. Synchronous excitation of FS cells, the presumed inhibitory interneurons, is consistent with this. Only a subset of the pyramidal neurons, almost all of them IB cells of the middle layers, displayed strong, synchronous excitation and clusters of action potentials.(ABSTRACT TRUNCATED AT 400 WORDS)",
"Cortical neurons receive synaptic inputs from thousands of afferents that fire action potentials at rates ranging from less than 1 hertz to more than 200 hertz. Both the number of afferents and their large dynamic range can mask changes in the spatial and temporal pattern of synaptic activity, limiting the ability of a cortical neuron to respond to its inputs. Modeling work based on experimental measurements indicates that short-term depression of intracortical synapses provides a dynamic gain-control mechanism that allows equal percentage rate changes on rapidly and slowly firing afferents to produce equal postsynaptic responses. Unlike inhibitory and adaptive mechanisms that reduce responsiveness to all inputs, synaptic depression is input-specific, leading to a dramatic increase in the sensitivity of a neuron to subtle changes in the firing patterns of its afferents.",
"Many studies of cortical interneurones use immature rodent tissue, while many recordings in vivo are made in adult cats. To determine the extent to which interneuronal circuitry studied with one approach can transfer to another, we compared layer 4 interneurones and their local connections across two age groups and two species and with similar connections in layers 3 and 5, using two common recording techniques: dual whole cell recordings at 20 degrees C and dual sharp electrode recordings at 35 degrees C. In each group, a range of morphological and electrophysiological characteristics was observed. In all groups, however, positive correlations were found between the width of the action potential and rise times and widths at half-amplitude of EPSPs and IPSPs and the EPSP paired pulse ratio. Multipolar interneurones with narrow spikes generated the fastest IPSPs in pyramidal cells and received the briefest, most strongly depressing EPSPs, while bitufted interneurones with broader spikes and adapting and burst firing patterns activated the broadest IPSPs and received the slowest, most strongly facilitating/augmenting EPSPs. Correlations were similar in all groups, with no significant differences between adult rat and cat, or between layers, but events were four times slower in juveniles at 20 degrees C. Comparisons with previous studies indicate that this is due in part to age, but in large part to temperature. Studies in adults were extended with detailed analysis of synaptic dynamics, which appeared to decay more rapidly than at juvenile connections. EPSPs exhibited the complexity in time course of facilitation, augmentation and depression previously described in other adult neocortical connections. That is, the time course of recovery from facilitation or depression rarely followed a simple smooth exponential decay. Facilitation and depression were not always maximal at the shortest interspike intervals, and recovery was often interrupted by peaks and troughs in mean EPSP amplitude with a periodicity around 80 Hz.",
"The prefrontal cortex is specially adapted to generate persistent activity that outlasts stimuli and is resistant to distractors, presumed to be the basis of working memory. The pyramidal network that supports this activity is unknown. Multineuron patch-clamp recordings in the ferret medial prefrontal cortex showed a heterogeneity of synapses interconnecting distinct subnetworks of different pyramidal cells. One subnetwork was similar to the pyramidal network commonly found in primary sensory areas, consisting of accommodating pyramidal cells interconnected with depressing synapses. The other subnetwork contained complex pyramidal cells with dual apical dendrites displaying nonaccommodating discharge patterns; these cells were hyper-reciprocally connected with facilitating synapses displaying pronounced synaptic augmentation and post-tetanic potentiation. These cellular, synaptic and network properties could amplify recurrent interactions between pyramidal neurons and support persistent activity in the prefrontal cortex.",
"Using dual intracellular recordings in slices of adult rat and cat neocortex, the frequency-filtering characteristics of 'depressing' synapses made by pyramidal axons at interspike intervals between 5 and 50 ms were studied. At 'depressing' connections from excitatory cells to some inhibitory interneurons (n = 6), recovery from short interspike interval depression was near exponential. Extrapolation of exponentials fitted to this recovery demonstrated a residual 10-20% depression at intervals >50 ms. This slowly decaying component was larger for later excitatory postsynaptic potentials (EPSPs) in trains which were typically more strongly depressed. At >80% of connections between spiny excitatory cells and at pyramid to parvalbumin-immunopositive interneuron connections, however, recovery exhibited a more complex time course. A narrow 'notch' (half-width 5 ms), peaking at intervals of 13-25 ms during which the EPSP was depressed further, interrupted recovery from short interval depression. This 'notch' was equally apparent for all EPSPs in brief trains and was mediated presynaptically."
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Short-, mid- and long-term mortality by copeptin in unselected admitted patients
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Rapid risk stratification is a core task in emergency medicine. Identifying patients at high and low risk shortly after admission could help clinical decision-making regarding treatment, level of observation, allocation of resources and post discharge follow-up. The purpose of the present study was to determine short-, mid- and long-term mortality by plasma measurement of copeptin in unselected admitted patients.
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"The antidiuretic hormone (ADH) vasopressin is a simple peptide hormone with a number of complex, essential physiological actions. It is becoming clear that this hormone is developing an important therapeutic role in a number of different conditions. These include vasodilatory shock due to sepsis or cardiac surgery, cardiac arrest, and prolonged/excessive bleeding caused by, for example, variceal haemorrhage. This article reviews the physiology of ADH relevant to these actions and scrutinises the evidence for its therapeutic applications.",
"This study aimed to independently derive an intracerebral hemorrhage grading scale (ICH-GS) for prediction of 3 outcome measures. We evaluated 378 patients with primary ICH at hospital arrival and during the next 30 days. Independent predictors were identified by multivariate models of in-hospital and 30-day mortality. Points were allotted to each predictor based on its prognostic performance. ICH-GS was also evaluated to predict good 30-day functional status and ICH-GS was compared with the ICH score as the reference scoring system. Independent predictors were age, Glasgow Coma Scale, ICH location, ICH volume, and intraventricular extension, all components of the ICH score. Nevertheless, different cutoffs and scoring improved substantially the prognostic power of the predictors. Compared with the ICH score, ICH-GS explained more variance in the 3 outcome measures, had higher sensitivity in predicting in-hospital and 30-day mortality, and performed equally well in predicting good functional outcome at 30 days follow up. The derived ICH-GS is a simple yet robust scale in predicting in-hospital and 30-day mortality, as well as good 30-day functional status, with equivalent performance.",
"Circulating biomarkers can offer insight into subclinical cardiovascular stress and thus have the potential to aid in risk stratification and tailoring of therapy. We measured plasma levels of 4 cardiovascular biomarkers, midregional pro-atrial natriuretic peptide (MR-proANP), midregional pro-adrenomedullin (MR-proADM), C-terminal pro-endothelin-1 (CT-proET-1), and copeptin, in 3717 patients with stable coronary artery disease and preserved left ventricular ejection fraction who were randomized to trandolapril or placebo as part of the Prevention of Events With Angiotensin Converting Enzyme (PEACE) trial. After adjustment for clinical cardiovascular risk predictors and left ventricular ejection fraction, elevated levels of MR-proANP, MR-proADM, and CT-proET-1 were independently associated with the risk of cardiovascular death or heart failure (hazard ratios per 1-SD increase in log-transformed biomarker levels of 1.97, 1.48, and 1.47, respectively; P≤0.002 for each biomarker). These 3 biomarkers also significantly improved metrics of discrimination when added to a clinical model. Trandolapril significantly reduced the risk of cardiovascular death or heart failure in patients who had elevated levels of ≥2 biomarkers (hazard ratio, 0.53; 95% confidence interval, 0.36-0.80), whereas there was no benefit in patients with elevated levels of 0 or 1 biomarker (hazard ratio, 1.09; 95% confidence interval, 0.74-1.59; P(interaction)=0.012). In patients with stable coronary artery disease and preserved left ventricular ejection fraction, our results suggest that elevated levels of novel biomarkers of cardiovascular stress may help identify patients who are at higher risk of cardiovascular death and heart failure and may be useful to select patients who derive significant benefit from angiotensin-converting enzyme inhibitor therapy.",
"High plasma copeptin level has been associated with one-month mortality after traumatic brain injury. However, not much is known regarding its relation with long-term outcome. Thus, we investigated the ability of copeptin to predict 1-year outcome in patients with traumatic brain injury. One hundred and six healthy controls and 106 patients with acute severe traumatic brain injury were included. Plasma samples were obtained on admission. Its concentration was measured by enzyme-linked immunosorbent assay. Forty-eight patients (45.3%) suffered from unfavorable outcome (Glasgow Outcome Scale score of 1-3) and 31 patients (29.2%) died in 1 year after traumatic brain injury. Upon admission, plasma copeptin level in patients was substantially higher than that in healthy controls. A forward stepwise logistic regression selected plasma copeptin level as an independent predictor for 1-year unfavorable outcome and mortality of patients. A receiver operating characteristic curve analysis showed plasma copeptin level predicted 1-year unfavorable outcome and mortality obviously. The predictive value of the copeptin concentration was thus similar to that of Glasgow Coma Scale score for the prediction of unfavorable outcome and mortality after 1 year. In a combined logistic-regression model, copeptin improved the area under curve of Glasgow Coma Scale score for the prediction of unfavorable outcome and mortality after 1 year, but the differences were not significant. Thus, copeptin level is a useful, complementary tool to predict functional outcome and mortality 1 year after traumatic brain injury.",
"Spontaneous intracerebral hemorrhage (ICH) accounts for a high mortality and morbidity. Early prediction of outcome is crucial for optimized care and treatment decision. Copeptin, the C-terminal part of provasopressin, has emerged as a new prognostic marker in a variety of diseases, but its prognostic value in ICH is unknown. In 40 consecutive patients who were admitted to the hospital within 72 hours after a spontaneous ICH, the plasma copeptin level was measured with a sandwich immunoassay upon admission. The prognostic value of copeptin to predict 30 day mortality and functional outcome after 90 days was assessed. A favorable outcome was defined as a Barthel score above 85 and a score below 3 on the Modified Rankin Scale. Copeptin correlated positively with hematoma volume (r = 0.32, p < 0.05) and negatively with the Glasgow Coma Scale (GCS) on admission (r = -0.35, p < 0.05). Copeptin levels were higher in patients who died within 30 days than in 30-day survivors (179.0 pmol/l (IQR 33.7- 566.0) vs. 12.9 pmol/l (IQR 5.2 - 42.8), p = 0.003). Copeptin levels were also higher in patients with an unfavorable functional outcome at 90 days compared to patients with a favorable outcome (32.4 pmol/l (IQR 9.5-97.8) vs. 11.9 pmol/l (IQR 3.2-19.8), p = 0.04). For the prediction of death, receiver-operating-characteristics analysis revealed an area under the curve (AUC) for copeptin of 0.88 (95%CI 0.75-1.00). The predictive value of the copeptin concentration was thus similar to that of GCS (AUC 0.82 (95%CI 0.59-1.00) p = 0.53), of the ICH Score (AUC 0.89, (95%CI 0.76-1.00), p = 0.94) and the ICH Grading Scale (AUC 0.86 (95%CI 0.69-1.00), p = 0.81). Copeptin is a new prognostic marker in patients with an ICH. If this finding can be confirmed in larger studies, copeptin might be an additional valuable tool for risk stratification and decision-making in the acute phase of ICH.",
"The early diagnosis of acute myocardial infarction (AMI) can be particularly challenging in patients with known coronary artery disease (CAD) due to pre-existing ECG changes and chronic increases in cardiac troponin (cTn) levels. Of 1170 consecutive patients presenting with symptoms suggestive of AMI, 433 (37%) with pre-existing CAD were analysed in a prospective multicentre study and the diagnostic and prognostic impact of copeptin in combination with either fourth generation cardiac troponin T (cTnT) or high-sensitivity cTnT (hs-cTnT) was evaluated. AMI was the final diagnosis in 78 patients with pre-existing CAD (18%). Copeptin was significantly higher in patients with AMI than in those without (26 pmol/l (IQR 9-71) vs 7 pmol/l (IQR 4-16), p<0.001). The diagnostic accuracy for AMI as quantified by the area under the receiver operating characteristic curve (AUC) was significantly higher for the combination of copeptin and cTnT than for cTnT alone (0.94 vs 0.86, p<0.001). The combination of copeptin and hs-cTnT (0.94) was trending to superiority compared with hs-cTnT alone (0.92, p=0.11). The combination of copeptin and the cTn assays was able to improve the negative predictive value up to 99.5% to rule out AMI. Copeptin was a strong and independent predictor of 1-year mortality (HR 4.18-4.63). Irrespective of cTn levels, patients with low levels of copeptin had an excellent prognosis compared with patients with raised levels of both copeptin and cTn (360-day mortality 2.8-3.6% vs 23.1-33.8%, p<0.001). In patients with pre-existing CAD, copeptin significantly improves the diagnostic accuracy if used in addition to cTnT, but only trended to superiority compared with hs-cTnT alone. Copeptin provides independent prognostic information, largely by overcoming the challenging interpretation of mild increases in hs-cTnT. ClinicalTrials Gov number NCT00470587.",
"The effects of the non-peptide vasopressin V(2) receptor antagonist 5-dimethylamino-1-[4-(2-methylbenzoylamino)benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride (OPC-31260) on the cerebral oedema induced by general cerebral hypoxia were studied in rats. The general cerebral hypoxia was produced by bilateral common carotid ligation in Sprague-Dawley rats of the CFY strain. By 6 h after the ligation, half of the rats had died, but the survival rate was significantly higher following OPC-31260 administration. Electron microscopic examinations revealed typical ischaemic changes after the carotid ligation. The carotid ligation increased the brain contents of water and Na(+) and enhanced the plasma vasopressin level. The increased brain water and Na(+) accumulation was prevented by OPC-31260 administration, but the plasma vasopressin level was further enhanced by OPC-31260. These results demonstrate the important role of vasopressin in the development of the disturbances in brain water and electrolyte balance in response to general cerebral hypoxia. The carotid ligation-induced cerebral oedema was significantly reduced following oral OPC-31260 administration. The protective mechanism exerted by OPC-31260 stems from its influence on the renal vasopressin V(2) receptors. These observations might suggest an effective approach to the treatment of global hypoxia-induced cerebral oedema in humans."
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CAPER/TBX3 repressor complex regulates CDKN2A-p16INK transcription and mRNA stability
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Cellular senescence is a crucial tumor suppressor mechanism. We discovered a CAPERα/TBX3 repressor complex required to prevent senescence in primary cells and mouse embryos. Critical, previously unknown roles for CAPERα in controlling cell proliferation are manifest in an obligatory interaction with TBX3 to regulate chromatin structure and repress transcription of CDKN2A-p16INK and the RB pathway. The IncRNA UCA1 is a direct target of CAPERα/TBX3 repression whose overexpression is sufficient to induce senescence. In proliferating cells, we found that hnRNPA1 binds and destabilizes CDKN2A-p16INK mRNA whereas during senescence, UCA1 sequesters hnRNPA1 and thus stabilizes CDKN2A-p16INK. Thus CAPERα/TBX3 and UCA1 constitute a coordinated, reinforcing mechanism to regulate both CDKN2A-p16INK transcription and mRNA stability. Dissociation of the CAPERα/TBX3 co-repressor during oncogenic stress activates UCA1, revealing a novel mechanism for oncogene-induced senescence. Our elucidation of CAPERα and UCA1 functions in vivo provides new insights into senescence induction, and the oncogenic and developmental properties of TBX3.
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[
"Senescence induction could be used as an effective treatment for hepatocellular carcinoma (HCC). However, major senescence inducers (p53 and p16(Ink4a)) are frequently inactivated in these cancers. We tested whether transforming growth factor-beta (TGF-beta) could serve as a potential senescence inducer in HCC. First, we screened for HCC cell lines with intact TGF-beta signaling that leads to small mothers against decapentaplegic (Smad)-targeted gene activation. Five cell lines met this condition, and all of them displayed a strong senescence response to TGF-beta1 (1-5 ng/mL) treatment. Upon treatment, c-myc was down-regulated, p21(Cip1) and p15(Ink4b) were up-regulated, and cells were arrested at G(1). The expression of p16(Ink4a) was not induced, and the senescence response was independent of p53 status. A short exposure of less than 1 minute was sufficient for a robust senescence response. Forced expression of p21(Cip1) and p15(Ink4b) recapitulated TGF-beta1 effects. Senescence response was associated with reduced nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) induction and intracellular reactive oxygen species (ROS) accumulation. The treatment of cells with the ROS scavenger N-acetyl-L-cysteine, or silencing of the NOX4 gene, rescued p21(Cip1) and p15(Ink4b) accumulation as well as the growth arrest in response to TGF-beta. Human HCC tumors raised in immunodeficient mice also displayed TGF-beta1-induced senescence. More importantly, peritumoral injection of TGF-beta1 (2 ng) at 4-day intervals reduced tumor growth by more than 75%. In contrast, the deletion of TGF-beta receptor 2 abolished in vitro senescence response and greatly accelerated in vivo tumor growth. TGF-beta induces p53-independent and p16(Ink4a)-independent, but Nox4-dependent, p21(Cip1)-dependent, p15(Ink4b)-dependent, and ROS-dependent senescence arrest in well-differentiated HCC cells. Moreover, TGF-beta-induced senescence in vivo is associated with a strong antitumor response against HCC.",
"Nuclear hormone receptors are ligand-dependent transcription factors that regulate genes critical to such biological processes as development, reproduction, and homeostasis. Interestingly, these receptors can function as molecular switches, alternating between states of transcriptional repression and activation, depending on the absence or presence of cognate hormone, respectively. In the absence of hormone, several nuclear receptors actively repress transcription of target genes via interactions with the nuclear receptor corepressors SMRT and NCoR. Upon binding of hormone, these corepressors dissociate away from the DNA-bound receptor, which subsequently recruits a nuclear receptor coactivator (NCoA) complex. Prominent among these coactivators is the SRC (steroid receptor coactivator) family, which consists of SRC-1, TIF2/GRIP1, and RAC3/ACTR/pCIP/AIB-1. These cofactors interact with nuclear receptors in a ligand-dependent manner and enhance transcriptional activation by the receptor via histone acetylation/methylation and recruitment of additional cofactors such as CBP/p300. This review focuses on the mechanism of action of SRC coactivators in terms of interactions with receptors and activation of transcription. Specifically, the roles of the highly conserved LXXLL motifs in mediating SRC function will be detailed. Additionally, potential diversity among SRC family members, as well as several recently cloned SRC-associated cofactors, will be discussed.",
"The main regulator of the human tumor suppresser gene p21(waf1/cip1) is the transcription factor p53, but more recently it has been suggested to be a primary anti-proliferative target for the nuclear receptor VDR in the presence of its ligand 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). To identify VDR responding regions, we analyzed 20 overlapping regions covering the first 7.1 kb of the p21(waf1/cip1) promoter in MCF-7 human breast cancer cells using chromatin immuno-precipitation assays (ChIP) with antibodies against p53 and VDR. We confirmed two known p53 binding regions at approximate positions -1400 and -2300 and identified a novel site at position -4500. In addition, we found three VDR-associated promoter regions at positions -2300, -4500 and -6900, i.e. two regions showed binding for both p53 and VDR. In silico screening and in vitro binding assays using recombinant and in vitro translated proteins identified five p53 binding sites within the three p53-positive promoter regions and also five 1alpha,25(OH)2D3 response elements within the three VDR-positive regions. Reporter gene assays confirmed the expected responsiveness of the respective promoter regions to the p53 inducer 5-fluorouracil and 1alpha,25(OH)2D3. Moreover, re-ChIP assays confirmed the functionality of the three 1alpha,25(OH)2D3-reponsive promoter regions by monitoring simultaneous occupancy of VDR with the co-activator proteins CBP, SRC-1 and TRAP220. Taken together, we demonstrated that the human p21((waf1/cip1)) gene is a primary 1alpha,25(OH)2D3-responding gene with at least three VDR binding promoter regions, in two of which also p53 co-localizes.",
"The T-box transcription factor TBX3 is necessary for early embryonic development and for the normal development of the mammary gland. Homozygous mutations, in mice, are embryonic lethal while heterozygous mutations result in perturbed mammary gland development. In humans, mutations that result in the haploinsufficiency of TBX3 causes Ulnar Mammary Syndrome (UMS) characterized by mammary gland hypoplasia as well as other congenital defects. In addition to its role in mammary gland development, various studies have also supported a role for Tbx3 in breast cancer development. TBX3 is over-expressed in various breast cancer cell lines as well as cancer tissue and has been found to contribute to breast cancer cell migration. Previous studies have suggested that TBX3 contributes to cancer development by its ability to bypass senescence by repressing the expression of p14(ARF)-tumor suppressor. Although many studies have shown that a dysregulation of TBX3 expression may contribute to cancer progression, no direct evidence shows TBX3 causes breast cancer. In this study, we created doxycycline inducible double transgenic mice (MMTV-rtTA;tet-myc-TBX3-IRES-Luciferase) to test whether TBX3 over-expression can induce tumor formation within the mammary gland. Although over-expression of TBX3, alone, did not induce tumor formation it did promote accelerated mammary gland development by increasing mammary epithelial cell proliferation. We also show that TBX3 directly binds to and represses NFκBIB, an inhibitor of the NF-κB pathway known to play a role in regulating cell proliferation. Lastly, we also show that the over-expression of TBX3 is associated with an increase in mammary stem-like cells. Overall, our data suggests that over-expression of TBX3 may contribute to breast cancer development by promoting accelerated mammary gland development through the inhibition of the NF-κB pathway and stimulation of both mammary epithelial cell and stem-like cell proliferation.",
"To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.",
"A complex network of regulatory pathways links transcription to cell growth and proliferation. Here we show that cellular quiescence alters chromatin structure by promoting trimethylation of histone H4 at lysine 20 (H4K20me3). In contrast to pericentric or telomeric regions, recruitment of the H4K20 methyltransferase Suv4-20h2 to rRNA genes and IAP elements requires neither trimethylation of H3K9 nor interaction with HP1 proteins but depends on long noncoding RNAs (lncRNAs) that interact with Suv4-20h2. Growth factor deprivation and terminal differentiation lead to upregulation of these lncRNAs, increase in H4K20me3, and chromatin compaction. The results uncover a lncRNA-mediated mechanism that guides Suv4-20h2 to specific genomic loci to establish a more compact chromatin structure in growth-arrested cells.",
"The vitamin D3 24-hydroxylase gene (CYP24) is one of the most strongly induced genes known. Despite this, its induction by the hormone 1alpha,25-dihydroxyvitamin D3 (1alpha,25OH2D3) has been characterized only partially. Therefore, we monitored the spatio-temporal, 1alpha,25OH2D3-dependent chromatin acetylation status of the human CYP24 promoter by performing chromatin immunoprecipitation (ChIP) assays with antibodies against acetylated histone 4. This was achieved by performing PCR on 25 contiguous genomic regions spanning the first 7.7 kb of the promoter. ChIP assays using antibodies against the 1alpha,25OH2D3 receptor (VDR) revealed that, in addition to the proximal promoter, three novel regions further upstream associated with VDR. Combined in silico/in vitro screening identified in three of the four promoter regions sequences resembling known VDREs and reporter gene assays confirmed the inducibility of these regions by 1alpha,25OH2D3)=. In contrast, the fourth VDR-associated promoter region did not contain any recognizable classical VDRE that could account for the presence of the protein on this region. However, re-ChIP assays monitored on all four promoter regions simultaneous association of VDR with retinoid X receptor, coactivator, mediator and RNA polymerase II proteins. These proteins showed a promoter region-specific association pattern demonstrating the complex choreography of the CYP24 gene promoter activation over 300 minutes. Thus, this study reveals new information concerning the regulation of the CYP24 gene by 1alpha,25OH2D3, and is a demonstration of the simultaneous participation of multiple, structurally diverse response elements in promoter activation in a living cell."
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Synthesis of acridine and tetrahydroacridine VATG-032 as potent autophagy inhibitors.
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Autophagy is a dynamic cell survival mechanism by which a double-membrane vesicle, or autophagosome, sequesters portions of the cytosol for delivery to the lysosome for recycling. This process can be inhibited using the antimalarial agent chloroquine (CQ), which impairs lysosomal function and prevents autophagosome turnover. Despite its activity, CQ is a relatively inadequate inhibitor that requires high concentrations to disrupt autophagy, highlighting the need for improved small molecules. To address this, we screened a panel of antimalarial agents for autophagy inhibition and chemically synthesized a novel series of acridine and tetrahydroacridine derivatives. Structure-activity relationship studies of the acridine ring led to the discovery of VATG-027 as a potent autophagy inhibitor with a high cytotoxicity profile. In contrast, the tetrahydroacridine VATG-032 showed remarkably little cytotoxicity while still maintaining autophagy inhibition activity, suggesting that both compounds act as autophagy inhibitors with differential effects on cell viability. Further, knockdown of autophagy-related genes showed no effect on cell viability, demonstrating that the ability to inhibit autophagy is separate from the compound cytotoxicity profiles. Next, we determined that both inhibitors function through lysosomal deacidification mechanisms and ultimately disrupt autophagosome turnover. To evaluate the genetic context in which these lysosomotropic inhibitors may be effective, they were tested in patient-derived melanoma cell lines driven by oncogenic BRAF (v-raf murine sarcoma viral oncogene homolog B). We discovered that both inhibitors sensitized melanoma cells to the BRAF V600E inhibitor vemurafenib. Overall, these autophagy inhibitors provide a means to effectively block autophagy and have the potential to sensitize mutant BRAF melanomas to first-line therapies.
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[
"Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading unnecessary or dysfunctional cellular organelles and proteins in all living cells. Autophagy is particularly active during metabolic stress. In the cancer cell it fulfils a dual role, having tumor-promoting and tumor-suppressing properties. Functional autophagy prevents necrosis and inflammation, which can lead to genetic instability. On the other hand, autophagy might be important for tumor progression by providing energy through its recycling mechanism during unfavorable metabolic circumstances. A central checkpoint that negatively regulates autophagy is mTOR, and anticancer drugs inhibiting the PI3K/Akt/mTOR axis putatively stimulate autophagy. However, whether autophagy contributes to the antitumor effect of these drugs or to drug resistance is largely unknown. The antimalarial drugs chloroquine and hydroxychloroquine inhibit autophagy, leading to increased cytotoxicity in combination with several anticancer drugs in preclinical models. The therapeutic clinical roles of autophagy induction and inhibition remain to be defined. To improve our understanding of autophagy in human cancers new methods for measuring autophagy in clinical samples need to be developed. This Review delineates the possible role of autophagy as a novel target for anticancer therapy.",
"Chloroquine (CQ), the worldwide used anti-malarial drug, has recently being focused as a potential anti-cancer agent as well as a chemosensitizer when used in combination with anti-cancer drugs. It has been shown to inhibit cell growth and/or to induce cell death in various types of cancer. 5-Fluorouracil (5-FU) is the chemotherapeutic agent of first choice in colorectal cancer, but in most cases, resistance to 5-FU develops through various mechanisms. Here, we focused on the combination of CQ as a mechanism to potentiate the inhibitory effect of 5-FU on human colon cancer cells. HT-29 cells were treated with CQ and/or 5-FU, and their proliferative ability, apoptosis and autophagy induction effects, and the affection of the cell cycle were evaluated. The proliferative ability of HT-29 was analyzed by the MTS assay. Apoptosis was quantified by flow-cytometry after double-staining of the cells with AnnexinV/PI. The cell cycle was evaluated by flow-cytometry after staining of cells with PI. Autophagy was quantified by flow-cytometry and Western blot analysis. Finally, to evaluate the fate of the cells treated with CQ and/or 5-FU, the colony formation assay was performed. 5-FU inhibited the proliferative activity of HT-29 cells, which was mostly dependent on the arrest of the cells to the G0/G1-phase but also partially on apoptosis induction, and the effect was potentiated by CQ pre-treatment. The potentiation of the inhibitory effect of 5-FU by CQ was dependent on the increase of p21Cip1 and p27Kip1 and the decrease of CDK2. Since CQ is reported to inhibit autophagy, the catabolic process necessary for cell survival under conditions of cell starvation or stress, which is induced by cancer cells as a protective mechanism against chemotherapeutic agents, we also analyzed the induction of autophagy in HT-29. HT-29 induced autophagy in response to 5-FU, and CQ inhibited this induction, a possible mechanism of the potentiation of the anti-cancer effect of 5-FU. Our findings suggest that the combination therapy with CQ should be a novel therapeutic modality to improve efficacy of 5-FU-based chemotherapy, possibly by inhibiting autophagy-dependent resistance to chemotherapy.",
"In the present study, we examined the relationship between Beclin-1 expression and HIF-1alpha expression in esophageal squamous cell carcinoma(ESCC). There was a loss of Beclin-1 protein expression in 33% of ESCCs. Beclin-1 expression significantly correlated with depth of invasion, lymph node metastasis and clinical stage. Among the 54 patients, The survival rate of the Beclin-1-positive group was better than that of the Beclin-1-negative group. Twenty-five of the 54 (46%) tumor specimens showed high levels of HIF-1alpha immunoreactivity. Beclin-1 expression was associated with HIF-1alpha expression. The survival rate of patients with Beclin-1-positive and HIF-1alpha-low tumors was significantly higher than that of the other groups. These results suggest that Beclin-1 and HIF-1alpha expression are important determinants of survival in ESCCs.",
"A prominent feature of late-onset neurodegenerative diseases is accumulation of misfolded protein in vulnerable neurons. When levels of misfolded protein overwhelm degradative pathways, the result is cellular toxicity and neurodegeneration. Cellular mechanisms for degrading misfolded protein include the ubiquitin-proteasome system (UPS), the main non-lysosomal degradative pathway for ubiquitinated proteins, and autophagy, a lysosome-mediated degradative pathway. The UPS and autophagy have long been viewed as complementary degradation systems with no point of intersection. This view has been challenged by two observations suggesting an apparent interaction: impairment of the UPS induces autophagy in vitro, and conditional knockout of autophagy in the mouse brain leads to neurodegeneration with ubiquitin-positive pathology. It is not known whether autophagy is strictly a parallel degradation system, or whether it is a compensatory degradation system when the UPS is impaired; furthermore, if there is a compensatory interaction between these systems, the molecular link is not known. Here we show that autophagy acts as a compensatory degradation system when the UPS is impaired in Drosophila melanogaster, and that histone deacetylase 6 (HDAC6), a microtubule-associated deacetylase that interacts with polyubiquitinated proteins, is an essential mechanistic link in this compensatory interaction. We found that compensatory autophagy was induced in response to mutations affecting the proteasome and in response to UPS impairment in a fly model of the neurodegenerative disease spinobulbar muscular atrophy. Autophagy compensated for impaired UPS function in an HDAC6-dependent manner. Furthermore, expression of HDAC6 was sufficient to rescue degeneration associated with UPS dysfunction in vivo in an autophagy-dependent manner. This study suggests that impairment of autophagy (for example, associated with ageing or genetic variation) might predispose to neurodegeneration. Morover, these findings suggest that it may be possible to intervene in neurodegeneration by augmenting HDAC6 to enhance autophagy.",
"Previous study reported that resveratrol has anti-tumor activity. In this study, we investigated the involvement of autophagy in the resveratrol-induced apoptotic death of human U251 glioma cells. The growth inhibition of U251 cells induced by resveratrol was assessed with methyl thiazolyl tetrazolium (MTT). The activation of autophagy and proapoptotic effect were characterized by monodansylcadaverine labeling and Hoechst stain, respectively. Mitochondrialtransmembrane potential (DeltaPsim) was measured as a function of drug treatment using 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). The role of autophagy and apoptosis in the resveratrol-induced death of U251 cells was assessed using autophagic and caspase inhibitors. Immunofluorescence, flow cytometry, and Western blot analysis were used to study the apoptotic and autophagic mechanisms. Methyl thiazolyl tetrazolium (MTT) assays indicated that resveratrol decreased the viability of U251 cells in a dose- and time-dependent manner. Flow cytometry analysis indicated that resveratrol increased cell population at sub-G1 phase, an index of apoptosis. Furthermore, resveratrol-induced cell death was associated with a collapse of the mitochondrial membrane potential. The pan-caspase inhibitor Z-VAD-fmk suppressed resveratrol-induced U251 cell death. Resveratrol stimulated autophagy was evidenced by punctuate monodansylcadaverine(MDC) staining and microtubule-associated protein light chain 3 (LC3) immunoreactivty. Resveratrol also increased protein levels of beclin 1 and membrane form LC3 (LC3-II). Autophagy inhibitors 3-methylademine (3-MA) and bafilomycin A1 sensitized the cytotoxicity of resveratrol. Together, these findings indicate that resveratrol induces autophagy in human U251 glioma cells and autophagy suppressed resveratrol-induced apoptosis. This study thus suggests that autophagy inhibitors can increase the cytotoxicity of resveratrol to glioma cells.",
"Chloroquine (CQ), N'-(7-chloroquinolin-4-yl)-N,N-diethyl-pentane-1,4-diamine, is widely used as an effective and safe anti-malarial and anti-rheumatoid agent. CQ was discovered 1934 as \"Resochin\" by Andersag and co-workers at the Bayer laboratories. Ironically, CQ was initially ignored for a decade because it was considered too toxic to use in humans. CQ was \"re-discovered\" during World War II in the United States in the course of anti-malarial drug development. The US government-sponsored clinical trials during this period showed unequivocally that CQ has a significant therapeutic value as an anti-malarial drug. Consequently, CQ was introduced into clinical practice in 1947 for the prophylaxis treatment of malaria (Plasmodium vivax, ovale and malariae). CQ still remains the drug of choice for malaria chemotherapy because it is highly effective and well tolerated by humans. In addition, CQ is widely used as an anti-inflammatory agent for the treatment of rheumatoid arthritis, lupus erythematosus and amoebic hepatitis. More recently, CQ has been studied for its potential as an enhancing agent in cancer therapies. Accumulating lines of evidence now suggest that CQ can effectively sensitize cell-killing effects by ionizing radiation and chemotherapeutic agents in a cancer-specific manner. The lysosomotrophic property of CQ appears to be important for the increase in efficacy and specificity. Although more studies are needed, CQ may be one of the most effective and safe sensitizers for cancer therapies. Taken together, it appears that the efficacy of conventional cancer therapies can be dramatically enhanced if used in combination with CQ and its analogs.",
"Autophagy is a cellular degradation pathway for the clearance of damaged or superfluous proteins and organelles. The recycling of these intracellular constituents also serves as an alternative energy source during periods of metabolic stress to maintain homeostasis and viability. In tumour cells with defects in apoptosis, autophagy allows prolonged survival. Paradoxically, autophagy defects are associated with increased tumorigenesis, but the mechanism behind this has not been determined. Recent evidence suggests that autophagy provides a protective function to limit tumour necrosis and inflammation, and to mitigate genome damage in tumour cells in response to metabolic stress.",
"This phase II study evaluated the efficacy and safety of sorafenib plus dacarbazine in patients with advanced melanoma. This randomized, double-blind, placebo-controlled, multicenter study enrolled chemotherapy-naive patients with stage III (unresectable) or IV melanoma. A total of 101 patients received placebo plus dacarbazine (n = 50) or sorafenib plus dacarbazine (n = 51). On day 1 of a 21-day cycle, patients received intravenous dacarbazine 1,000 mg/m(2) for a maximum of 16 cycles. Oral sorafenib 400 mg or placebo was administered twice a day continuously. The primary end point was progression-free survival (PFS) by independent assessment. Secondary and tertiary end points included time to progression (TTP), response rate, and overall survival (OS). Median PFS in the sorafenib plus dacarbazine arm was 21.1 weeks versus 11.7 weeks in the placebo plus dacarbazine arm (hazard ratio [HR], 0.665; P = .068). There were statistically significant improvements in PFS rates at 6 and 9 months, and in TTP (median, 21.1 v 11.7 weeks; HR, 0.619) in favor of the sorafenib plus dacarbazine arm. No difference in OS was observed (median, 51.3 v 45.6 weeks in the placebo plus dacarbazine and sorafenib plus dacarbazine arms, respectively; HR, 1.022). The regimen was well tolerated and had a manageable toxicity profile. Sorafenib plus dacarbazine was well tolerated in patients with advanced melanoma and yielded an encouraging improvement in PFS. Based on these findings, additional studies with the combination are warranted in this patient population.",
"Recent studies have suggested that autophagy plays a pivotal role in regulation of cancer development and progression. High expression of the autophagy-related Beclin 1 protein predicted favorable patient outcome in several tumors. Here, a randomized controlled trial (RCT)-derived 128 nasopharyngeal carcinoma (NPC) patients were subjected to analysis of Beclin 1 expression and survival probability. In this RCT, 61 patients treated with induction chemotherapy plus concurrent chemoradiotherapy were used as a training set to generate a Beclin 1 cutoff score for patient outcome by receiver operating characteristic (ROC) curve analysis. For validation, the ROC-derived cutoff point was subjected to analysis of the association of Beclin 1 expression with patient outcome and clinical characteristics in testing set. The testing set comprised of 67 patients received induction chemotherapy plus radiotherapy. In the testing set and overall patients, our univariate and multivariate analysis showed that higher Beclin 1 expression, defined by the training set ROC analysis-generated cutoff score, predicted poorer overall survival, progression-free survival and distant metastasis-free survival. However, we failed to detect a correlation between Beclin 1 and local failure-free survival. Moreover, a positive relationship between Beclin 1 and HI F-1alpha expression was found. Importantly, among patients with elevated HI F-1alpha expression, a subset with lower Beclin 1 expression displayed a significant overall survival advantage than those with higher expression (p = 0.036). Contrary to previous studies, our results demonstrated that high autophagic Beclin 1 expression was an inferior prognostic marker for NPC. HI F-1alpha-associated Beclin 1 high expression might facilitate NPC cells surviving from chemoradiotherapy, suggesting a novel therapeutic molecular target for NPC.",
"This phase III, randomized, double-blind, placebo-controlled study was conducted to evaluate the efficacy and safety of sorafenib with carboplatin and paclitaxel (CP) in patients with advanced melanoma who had progressed on a dacarbazine- or temozolomide-containing regimen. A total of 270 patients were randomly assigned to receive intravenous paclitaxel 225 mg/m2 plus intravenous carboplatin at area under curve 6 (AUC 6) on day 1 of a 21-day cycle followed by either placebo (n = 135) or oral sorafenib 400 mg (n = 135) twice daily on days 2 to 19. The primary efficacy end point was progression-free survival (PFS); secondary and tertiary end points included overall survival and incidence of best response, respectively. The median PFS was 17.9 weeks for the placebo plus CP arm and 17.4 weeks for the sorafenib plus CP arm (hazard ratio, 0.91; 99% CI, 0.63 to 1.31; two-sided log-rank test P = .49). Response rate was 11% with placebo versus 12% with sorafenib. Dermatologic events, grade 3 thrombocytopenia, diarrhea, and fatigue were more common in patients treated with sorafenib plus CP versus placebo plus CP. In this study, the addition of sorafenib to CP did not improve any of the end points over placebo plus CP and cannot be recommended in the second-line setting for patients with advanced melanoma. Both regimens had clinically acceptable toxicity profiles with no unexpected adverse events. A trial of similar design for the first-line treatment of patients with advanced melanoma (intergroup trial E2603) is currently ongoing."
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Comparison of qPCR and droplet digital PCR for human cytomegalovirus detection in patient plasma samples
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Human cytomegalovirus (CMV) has historically been the major infectious cause of morbidity and mortality among patients receiving hematopoietic cell or organ transplant. Standard care in a transplant setting involves frequent monitoring of CMV viral load over weeks to months to determine when antiviral treatment may be required. Quantitative PCR (qPCR) is the standard molecular diagnostic method for monitoring. Recently, digital PCR (dPCR) has shown promise in viral diagnostics, although current dPCR systems have lower throughput than qPCR systems. Here, we compare qPCR and droplet digital PCR (ddPCR) for CMV detection in patient plasma samples. Droplet digital PCR exhibits increased precision over qPCR at viral loads of ≥4 log10 with equivalent sensitivity. However, retrospective analysis of longitudinal samples from transplant patients with CMV viral loads near therapeutic thresholds did not provide evidence that the improved precision of ddPCR would be of clinical benefit. Given the throughput advantages of current qPCR systems, a widespread switch to dPCR for CMV monitoring would appear premature.
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[
"Plasma human immunodeficiency virus type 1 (HIV-1) RNA suppression <50 copies/mL is regarded as the optimal outcome of highly active antiretroviral therapy (HAART). Current viral load (VL) assays show increased sensitivity, but the significance of RNA detection <50 copies/mL is unclear. This study investigated the virologic outcomes of 1247 patients with VL <50 copies/mL at an arbitrary time point during HAART (= T0), according to whether the actual, unreported (T0)VL was 40-49 copies/mL, RNA detected <40 copies/mL (RNA(+)), or RNA not detected (RNA(-)), as measured by the Abbott Real Time assay. Predictors of rebound >50 and >400 copies/mL over 12 months following T0 were analyzed with Cox proportional hazards models incorporating the (T0)VL and demographic and clinical data. Rebound rates >50 copies/mL were 34.2% for (T0)VL 40-49 copies/mL, 11.3% for RNA(+), and 4.0% for RNA(-); rebound rates >400 copies/mL were 13.0%, 3.8%, and 1.2%, respectively. The adjusted hazard ratios for rebound >50 copies/mL were 4.67 (95% confidence interval, 2.91-7.47; P < .0001) and 1.97 (1.25-3.11; P < .0001) with (T0)VL 40-49 copies/mL and RNA(+), respectively, relative to RNA(-), and 6.91 (2.90-16.47; P < .0001) and 2.88 (1.24-6.69; P < .0001), respectively, for rebound >400 copies/mL. The association was independent of adherence levels. In treated patients monitored by RealTime, a VL of 40-49 copies/mL and, to a lesser extent, RNA detection <40 copies/mL predict rebound >50 and >400 copies/mL independently of other recognized determinants. The goal of HAART may need to be revised to a lower cutoff than 50 copies/mL.",
"Viralload (VL) assessment of cytomegalovirus (CMV) by real-time PCR is an important tool for diagnosing and monitoring CMV viremia in patients with compromised immune systems. We report results from a sample exchange organized by members of the Association for Molecular Pathology that compared PCR results from 23 laboratories; 22 such laboratories used a laboratory-developed real-time PCR assay and one laboratory used a competitive PCR assay. The samples sent to each laboratory were comprised of a dilution panel of CMV virion-derived reference materials that ranged from 0 to 500,000 copies/ml. Accuracy, linearity, and intralaboratory precision were established for the different laboratory-developed assays. Overall, PCR results were linear for each laboratory (R(2) > 0.97 in all but two). While 13 laboratories showed no significant quantitative assay bias, 10 laboratories reported VLs that were significantly different compared with expected values (bias range, -0.82 to 1.4 logs). The intralaboratory precision [mean coefficient of variance of 2% to 5% (log-scale)] suggested that changes in VLs of less than 3- to fivefold may not be significantly different. There was no significant association between laboratory-specific technical variables (PCR platform, calibrator, extraction method) and assay linearity or accuracy. These data suggested that, within each laboratory, relative VL values were linear, but additional method standardization and a CMV DNA reference standard are needed to allow laboratories to achieve comparable numeric results.",
"Unlike quantitative polymerase chain reaction (qPCR), digital PCR (dPCR) achieves sensitive and accurate absolute quantitation of a DNA sample without the need for a standard curve. A single PCR reaction is divided into many separate reactions that each have a positive or negative signal. By applying Poisson statistics, the number of DNA molecules in the original sample is directly calculated from the number of positive and negative reactions. The recent availability of multiple commercial dPCR platforms has led to increased interest in clinical diagnostic applications, such as low viral load detection and low abundance mutant detection, where dPCR could be superior to traditional qPCR. Here we review current literature that demonstrates dPCR's potential utility in viral diagnostics, particularly through absolute quantification of target DNA sequences and rare mutant allele detection.",
"Previous studies have shown that detection of cytomegalovirus (CMV) DNA in plasma is less sensitive than the antigenemia assay for CMV surveillance in blood. In 1,983 blood samples, plasma PCR assays with three different primer sets (UL125 alone, UL126 alone, and UL55/UL123-exon 4) were compared to the pp65 antigenemia assay and blood cultures. Plasma PCR detected CMV more frequently in blood specimens than either the antigenemia assay or cultures, but of the three PCR assays, the double-primer assay (UL55/UL123-exon 4) performed best with regard to sensitivity, specificity, and predictive values compared to antigenemia: 122 of 151 antigenemia-positive samples were detected (sensitivity, 80.1%), and there were 122 samples that were PCR positive-antigenemia negative (specificity, 93%). Samples with discrepant results had a low viral load (median, 0.5 cells per slide; 1,150 copies per ml) and were often obtained from patients receiving antiviral therapy. CMV could be detected by other methods in 15 of 29 antigenemia positive-PCR negative samples compared to 121 of 122 PCR positive-antigenemia negative samples (P < 0.001). On a per-subject basis, 21 of 25 patients (antigenemia positive-PCR negative) and all 57 (PCR positive-antigenemia negative) could be confirmed at different time points during follow-up. The higher sensitivity of the double-primer assay resulted in earlier detection compared to antigenemia in a time-to-event analysis of 42 CMV-seropositive stem cell transplant recipients, and two of three patients with CMV disease who were antigenemia negative were detected by plasma PCR prior to the onset of disease. Interassay variability was low, and the dynamic range was >5 log(10). Automated DNA extraction resulted in high reproducibility, accurate CMV quantitation (R = 0.87, P < 0.001), improved sensitivity, and increased speed of sample processing. Thus, primer optimization and improved DNA extraction techniques resulted in a plasma-based PCR assay that is significantly more sensitive than pp65 antigenemia and blood cultures for detection of CMV in blood specimens."
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Sociality in the Swiss Alps: A dual impact of altitude and development time
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Eusociality is taxonomically rare, yet associated with great ecological success. Surprisingly, studies of environmental conditions favouring eusociality are often contradictory. Harsh conditions associated with increasing altitude and latitude seem to favour increased sociality in bumblebees and ants, but the reverse pattern is found in halictid bees and polistine wasps. Here, we compare the life histories and distributions of populations of 176 species of Hymenoptera from the Swiss Alps. We show that differences in altitudinal distributions and development times among social forms can explain these contrasting patterns: highly social taxa develop more quickly than intermediate social taxa, and are thus able to complete the reproductive cycle in shorter seasons at higher elevations. This dual impact of altitude and development time on sociality illustrates that ecological constraints can elicit dynamic shifts in behaviour, and helps explain the complex distribution of sociality across ecological gradients.
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[
"Bumble bees are important pollinators of crops and other plants. However, many aspects of their basic biology remain relatively unexplored. For example, one important and unusual natural history feature in bumble bees is the massive size variation seen between workers of the same nest. This size polymorphism may be an adaptation for division of labor, colony economics, or be nonadaptive. It was also suggested that perhaps this variation allows for niche specialization in workers foraging at different temperatures: larger bees might be better suited to forage at cooler temperatures and smaller bees might be better suited to forage at warmer temperatures. This we tested here using a large, enclosed growth chamber, where we were able to regulate the ambient temperature. We found no significant effect of ambient or nest temperature on the average size of bees flying to and foraging from a suspended feeder. Instead, bees of all sizes successfully flew and foraged between 16°C and 36°C. Thus, large bees foraged even at very hot temperatures, which we thought might cause overheating. Size variation therefore could not be explained in terms of niche specialization for foragers at different temperatures.",
"Bergmann's rule states that, within species of mammals, individuals tend to be larger in cooler environments. However, the validity of the rule has been debated. We examined the relationship between size and latitude as well as size and temperature within various species of mammals. We also tested the idea that smaller mammals follow Bergmann's rule more strongly than larger mammals, as expected if heat conservation is the cause of the rule. When all studies were included, the percentage of species showing a positive correlation between size and latitude was significantly >50% (78 of 110 species). Similarly, the percentage of species showing a negative correlation between size and temperature was significantly >50% (48 of 64). Analyses using only significant studies or only studies that sampled extensively also support Bergmann's rule. The size-latitude and size-temperature trends were consistent within all orders and most families for which data are available. We did not find support for the hypothesis that smaller mammals conform more strongly to Bergmann's rule than larger mammals. Thus, we found broad support for Bergmann's rule as a general trend for mammals; however, our analyses do not support heat conservation as the explanation.",
"Paper nests of social wasps are intriguing constructions for both, biologists and engineers. We demonstrate that moisture and latent heat significantly influence the thermal performance of the nest construction. Two colonies of the hornet Vespa crabro were investigated in order to clarify the relation of the temperature and the moisture regime inside the nest. Next to fairly stable nest temperatures the hornets maintain a high relative humidity inside the nest. We found that in consequence a partial vapor-pressure gradient between nest and ambient drives a constant vapor flux through the envelope. The vapor flux is limited by the diffusion resistance of the envelope. The driving force of vapor flux is heat, which is consumed through evaporation inside the nest. The colony has to compensate this loss with metabolic heat production in order to maintain a stable nest temperature. However, humidity fluctuations inside the nest induce circadian adsorption and desorption cycles, which stabilize the nest temperature and thus contribute significantly to temperature homeostasis. Our study demonstrates that both mechanisms influence nest thermoregulation and need to be considered to understand the thermodynamic behavior of nests of wasps and social insects in general.",
"Some of the most sophisticated of all animal-built structures are the mounds of African termites of the subfamily Macrotermitinae, the fungus-growing termites. They have long been studied as fascinating textbook examples of thermoregulation or ventilation of animal buildings. However, little research has been designed to provide critical tests of these paradigms, derived from a very small number of original papers. Here I review results from recent studies on Macrotermes bellicosus that considered the interdependence of ambient temperature, thermoregulation, ventilation and mound architecture, and that question some of the fundamental paradigms of termite mounds. M. bellicosus achieves thermal homeostasis within the mound, but ambient temperature has an influence too. In colonies in comparably cool habitats, mound architecture is adapted to reduce the loss of metabolically produced heat to the environment. While this has no negative consequences in small colonies, it produces a trade-off with gas exchange in large colonies, resulting in suboptimally low nest temperatures and increased CO(2) concentrations. Along with the alteration in mound architecture, the gas exchange/ventilation mechanism also changes. While mounds in the thermally appropriate savannah have a very efficient circular ventilation during the day, the ventilation in the cooler forest is a less efficient upward movement of air, with gas exchange restricted by reduced surface exchange area. These results, together with other recent findings, question entrenched ideas such as the thermosiphon-ventilation mechanism or the assumption that mounds function to dissipate internally produced heat. Models trying to explain the proximate mechanisms of mound building, or building elements, are discussed.",
"Knowledge about the phylogeny and ecology of communities along environmental gradients helps to disentangle the role of competition-driven processes and environmental filtering for community assembly. In this study, we evaluated patterns in species richness, phylogenetic structure and life-history traits of bee communities along altitudinal gradients in the Alps, Germany. We found a linear decline in species richness and abundance but increasing phylogenetic clustering in communities with increasing altitude. The proportion of social- and ground-nesting species, as well as mean body size and altitudinal range of bee communities, increased with increasing altitude, whereas the mean geographical distribution decreased. Our results suggest that community assembly at high altitudes is dominated by environmental filtering effects, whereas the relative importance of competition increases at low altitudes. We conclude that inherent phylogenetic and ecological species attributes at high altitudes pose a threat for less competitive alpine specialists with ongoing climate change.",
"Many of the females of the first summer brood of the sweat bee Halictus rubicundus mate, soon vanish from the nesting site, undergo diapause, and return unworn the following spring to found their own nests without ever functioning as workers. Roughly half a season's foundresses may originate in this way. This finding contradicts previous assumptions regarding timing of reproductive production in sweat bees and offers a remarkable example of intraspecific social diversity; some females in the population behave as solitary bees while others are social. Such behavior may be widespread in bees and wasps, for it is almost undetectable with ordinary field techniques. These findings require the reevaluation of previous studies of sweat bee life cycles and of theories and models of the evolution of primitive insect societies.",
"Bumble bees (Bombus) are a cold-adapted, largely alpine group that can elucidate patterns of Holarctic historical biogeography, particularly in comparison to the alpine plants with which they likely coevolved. A recently published molecular phylogeny of bumble bees provides uniquely comprehensive species sampling for exploring historical patterns of distribution and diversification. Using this phylogeny and detailed data on extant distributions, I reconstruct the historical distribution of bumble bees in a temporal framework, estimating divergence times using fossil data and molecular rates derived from the literature. The nearly comprehensive phylogeny allows assessment of the tempo of diversification within the bumble bees using lineage-through-time plots and diversification statistics, which have been performed with special consideration to confidence intervals. These analyses reveal movements of Bombus concordant with geographic and climatic events of the late Cenozoic. The initial diversification of extant bumble bee lineages was estimated at around 25 to 40 Ma, near the Eocene-Oligocene boundary 34 Ma, a period of dramatic global cooling. Dispersal-vicariance analysis (DIVA) predicted an Old World Bombus ancestor, with early diversification events largely restricted to the eastern Old World. The numerous intercontinental dispersal events occurred mostly in the direction of Old World to New World and North America to South America. Early movements from the Palearctic into the Nearctic most likely took place after 20 Ma and may have coincided with a period of Miocene cooling that gave rise to taiga habitat across Beringia. Subsequent dispersal between these regions is estimated to have occurred among boreal and tundra-adapted species mostly in the last 5 million years. Radiations are estimated in both Nearctic and Neotropical regions at approximately 6 to 8 Ma and after 3.5 Ma, concordant with the opening of land corridors between the continents."
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Obstructive sleep apnea syndrome and CPAP therapy in morbidly obese patients
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In patients undergoing surgical interventions under general anesthesia, obstructive sleep apnea syndrome (OSA) can cause serious perioperative cardiovascular or respiratory complications leading to fatal consequences, even sudden death. In this study we test the hypothesis that morbidly obese patients diagnosed by a polysomnography test and using continuous positive airway pressure (CPAP) therapy have fewer and less severe perioperative complications and a shorter hospital stay than patients who have a medical history that meets at least three STOP-Bang criteria and are not using CPAP therapy.
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"Obstructive sleep apnea (OSA) is associated with increased BP and other cardiometabolic risk factors. The aim of the present study was to determine whether arterial stiffness and central BP (two important cardiovascular risk factors) would change, independent of peripheral BP following either the initiation of or withdrawal from nasal continuous positive airway pressure (CPAP) treatment in subjects with OSA. Arterial stiffness and peripheral and central BP were measured at baseline, and then either at 2 months after starting CPAP therapy (intervention group, n=20) or 7 nights after withdrawal from CPAP therapy (withdrawal group, n=20) using pulse wave analysis. In the intervention group, there were reductions in arterial stiffness (ie, the aortic augmentation index fell by 2.5%) and central systolic BP (fell by 4.2 mm Hg) without a concomitant reduction in peripheral BP. The change in arterial stiffness was associated with CPAP compliance (r=-0.47). In contrast, in the withdrawal group there were no overall changes in arterial stiffness or BP. However, there was an early morning increase in diastolic BP and heart rate relative to late evening. These results suggest that clinically important changes in arterial stiffness and central BP may occur following effective CPAP treatment of OSA without parallel changes in peripheral BP.",
"Obstructive sleep apnoea (OSA) is often undiagnosed before elective surgery and may predispose patients to perioperative complications. A literature search of PubMed-Medline, Web of Science, Scopus, EMBASE, Cochrane Database of Systematic Reviews, and Cochrane Central Register of Controlled Trials up to November 2010 was conducted. Our search was restricted to cohort or case-control studies in adults diagnosed with OSA by screening questionnaire, oximetry, or polysomnography. Studies without controls, involving upper airway surgery, and with OSA diagnosed by ICD-9 codes alone were excluded. The primary postoperative outcomes were desaturation, acute respiratory failure (ARF), reintubation, myocardial infarction/ischaemia, arrhythmias, cardiac arrest, intensive care unit (ICU) transfer, and length of stay. Thirteen studies were included in the final analysis (n=3942). OSA was associated with significantly higher odds of any postoperative cardiac events [45/1195 (3.76%) vs 24/1420 (1.69%); odds ratio (OR) 2.07; 95% confidence interval (CI) 1.23-3.50, P=0.007] and ARF [33/1680 (1.96%) vs 24/3421 (0.70%); OR 2.43, 95% CI 1.34-4.39, P=0.003]. Effects were not heterogeneous for these outcomes (I(2)=0-15%, P>0.3). OSA was also significantly associated with higher odds of desaturation [189/1764 (10.71%) vs 105/1881 (5.58%); OR 2.27, 95% CI 1.20-4.26, P=0.01] and ICU transfer [105/2062 (5.09%) vs 58/3681 (1.57%), respectively; OR 2.81, 95% CI 1.46-5.43, P=0.002]. Both outcomes showed a significant degree of heterogeneity of the effect among studies (I(2)=57-68%, P<0.02). Subgroup analyses had similar conclusions as main analyses. The incidence of postoperative desaturation, respiratory failure, postoperative cardiac events, and ICU transfers was higher in patients with OSA.",
"The mechanisms by which obesity and obstructive sleep apnoea (OSA) may contribute to endothelial dysfunction are unclear. We sought to follow up a sample of obese subjects undergoing either bariatric surgery or continuous positive airway pressure (CPAP) therapy to treat OSA. We hypothesised improved vascular function with both therapeutic approaches, consistent with a reversible OSA effect on the circulation. Twenty-seven obese (BMI ≥30 kg/m(2)) subjects with OSA underwent either bariatric surgery without CPAP (n = 12, median BMI 43.7 kg/m(2) IQR 9.4) or CPAP (n = 15, median BMI 33.8 kg/m(2) IQR 6.6). Polysomnography and vascular testing (flow-mediated dilation of the brachial artery measured with high-resolution ultrasound, endothelium-dependent change in skin blood flow measured with laser Doppler flowmetry, and arterial stiffness measured with applanation tonometry) took place at baseline and after 6 months. Both groups showed significant improvements in the apnoea-hypopnea index and overnight oxygen saturation. Endothelium-dependent microvascular reactivity was 45.6% (IQR 37.5) at baseline in the CPAP group, which increased to 69.1% (IQR 62.3) post-treatment (P < 0.05). No significant changes were observed in the surgery group, despite significant weight loss (post-surgery BMI 32.7 kg/m2 IQR 8.6 (P < 0.01); no change in BMI was observed in the CPAP group. There were no significant changes in brachial artery flow-mediated dilation in either group. This pilot study demonstrates that 6 months of CPAP may be sufficient to improve endothelium-dependent microvascular reactivity, while substantial surgically induced weight loss did not result in improvements. Further research should be directed towards comparative effectiveness trials using these novel surrogate outcomes, as well as hard cardiovascular outcomes."
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Particle conformation regulates antibody cross-GII.4 blockade.
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GII.4 noroviruses (NoVs) are the primary cause of epidemic viral acute gastroenteritis. One primary obstacle to successful NoV vaccination is the extensive degree of antigenic diversity among strains. The major capsid protein of GII.4 strains is evolving rapidly, resulting in the emergence of new strains with altered blockade epitopes. In addition to characterizing these evolving blockade epitopes, we have identified monoclonal antibodies (MAbs) that recognize a blockade epitope conserved across time-ordered GII.4 strains. Uniquely, the blockade potencies of MAbs that recognize the conserved GII.4 blockade epitope were temperature sensitive, suggesting that particle conformation may regulate functional access to conserved blockade non-surface-exposed epitopes. To map conformation-regulating motifs, we used bioinformatics tools to predict conserved motifs within the protruding domain of the capsid and designed mutant VLPs to test the impacts of substitutions in these motifs on antibody cross-GII.4 blockade. Charge substitutions at residues 310, 316, 484, and 493 impacted the blockade potential of cross-GII.4 blockade MAbs with minimal impact on the blockade of MAbs targeting other, separately evolving blockade epitopes. Specifically, residue 310 modulated antibody blockade temperature sensitivity in the tested strains. These data suggest access to the conserved GII.4 blockade antibody epitope is regulated by particle conformation, temperature, and amino acid residues positioned outside the antibody binding site. The regulating motif is under limited selective pressure by the host immune response and may provide a robust target for broadly reactive NoV therapeutics and protective vaccines.
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"Noroviruses (NoVs) are the main agents of gastroenteritis in humans and the primary pathogens of shellfish-related outbreaks. Some NoV strains bind to shellfish tissues by using carbohydrate structures similar to their human ligands, leading to the hypothesis that such ligands may influence bioaccumulation. This study compares the bioaccumulation efficiencies and tissue distributions in oysters (Crassostrea gigas) of three strains from the two principal human norovirus genogroups. Clear differences between strains were observed. The GI.1 strain was the most efficiently concentrated strain. Bioaccumulation specifically occurred in digestive tissues in a dose-dependent manner, and its efficiency paralleled ligand expression, which was highest during the cold months. In comparison, the GII.4 strain was very poorly bioaccumulated and was recovered in almost all tissues without seasonal influence. The GII.3 strain presented an intermediate behavior, without seasonal effect and with less bioaccumulation efficiency than that of the GI.1 strain during the cold months. In addition, the GII.3 strain was transiently concentrated in gills and mantle before being almost specifically accumulated in digestive tissues. Carbohydrate ligand specificities of the strains at least partly explain the strain-dependent bioaccumulation characteristics. In particular, binding to the digestive-tube-specific ligand should contribute to bioaccumulation, whereas we hypothesize that binding to the sialic acid-containing ligand present in all tissues would contribute to retain virus particles in the gills or mantle and lead to rapid destruction.",
"CaliciNet, the outbreak surveillance network for noroviruses in the United States, was launched in March 2009. As of January 2011, twenty state and local health laboratories had been certified to submit norovirus sequences and epidemiologic outbreak data to CaliciNet. During the network's first year, 552 outbreaks were submitted to CaliciNet, of which 78 (14%) were associated with foodborne transmission. A total of 395 (72%) outbreaks were typed as GII.4, of which 298 (75%) belonged to a new variant, GII.4 New Orleans, which first emerged in October 2009. Analysis of the complete capsid and P2 region sequences confirmed that GII.4 New Orleans is distinct from previous GII.4 variants, including GII.4 Minerva (2006b).",
"Noroviruses are the principal cause of epidemic gastroenteritis worldwide with GII.4 strains accounting for 80% of infections. The major capsid protein of GII.4 strains is evolving rapidly, resulting in new epidemic strains with altered antigenic potentials. To test if antigenic drift may contribute to GII.4 persistence, human memory B cells were immortalized and the resulting human monoclonal antibodies (mAbs) characterized for reactivity to a panel of time-ordered GII.4 virus-like particles (VLPs). Reflecting the complex exposure history of the volunteer, human anti-GII.4 mAbs grouped into three VLP reactivity patterns; ancestral (1987-1997), contemporary (2004-2009), and broad (1987-2009). NVB 114 reacted exclusively to the earliest GII.4 VLPs by EIA and blockade. NVB 97 specifically bound and blocked only contemporary GII.4 VLPs, while NBV 111 and 43.9 exclusively reacted with and blocked variants of the GII.4.2006 Minerva strain. Three mAbs had broad GII.4 reactivity. Two, NVB 37.10 and 61.3, also detected other genogroup II VLPs by EIA but did not block any VLP interactions with carbohydrate ligands. NVB 71.4 cross-neutralized the panel of time-ordered GII.4 VLPs, as measured by VLP-carbohydrate blockade assays. Using mutant VLPs designed to alter predicted antigenic epitopes, two evolving, GII.4-specific, blockade epitopes were mapped. Amino acids 294-298 and 368-372 were required for binding NVB 114, 111 and 43.9 mAbs. Amino acids 393-395 were essential for binding NVB 97, supporting earlier correlations between antibody blockade escape and carbohydrate binding variation. These data inform VLP vaccine design, provide a strategy for expanding the cross-blockade potential of chimeric VLP vaccines, and identify an antibody with broadly neutralizing therapeutic potential for the treatment of human disease. Moreover, these data support the hypothesis that GII.4 norovirus evolution is heavily influenced by antigenic variation of neutralizing epitopes and consequently, antibody-driven receptor switching; thus, protective herd immunity is a driving force in norovirus molecular evolution.",
"Over the last fifteen years there have been five pandemics of norovirus (NoV) associated gastroenteritis, and the period of stasis between each pandemic has been progressively shortening. NoV is classified into five genogroups, which can be further classified into 25 or more different human NoV genotypes; however, only one, genogroup II genotype 4 (GII.4), is associated with pandemics. Hence, GII.4 viruses have both a higher frequency in the host population and greater epidemiological fitness. The aim of this study was to investigate if the accuracy and rate of replication are contributing to the increased epidemiological fitness of the GII.4 strains. The replication and mutation rates were determined using in vitro RNA dependent RNA polymerase (RdRp) assays, and rates of evolution were determined by bioinformatics. GII.4 strains were compared to the second most reported genotype, recombinant GII.b/GII.3, the rarely detected GII.3 and GII.7 and as a control, hepatitis C virus (HCV). The predominant GII.4 strains had a higher mutation rate and rate of evolution compared to the less frequently detected GII.b, GII.3 and GII.7 strains. Furthermore, the GII.4 lineage had on average a 1.7-fold higher rate of evolution within the capsid sequence and a greater number of non-synonymous changes compared to other NoVs, supporting the theory that it is undergoing antigenic drift at a faster rate. Interestingly, the non-synonymous mutations for all three NoV genotypes were localised to common structural residues in the capsid, indicating that these sites are likely to be under immune selection. This study supports the hypothesis that the ability of the virus to generate genetic diversity is vital for viral fitness.",
"GII.4 noroviruses are a significant source of acute gastroenteritis worldwide, causing the majority of human norovirus outbreaks. Evolution of the GII.4 major capsid protein occurs rapidly, resulting in the emergence of new strains that produce successive waves of pandemic disease. A new pandemic isolate, GII.4 2012 Sydney, largely replaced previously circulating strains in late 2012. We compare the antigenic properties of GII.4 2012 Sydney with previously circulating strains. To determine whether GII.4-2012 Sydney is antigenically different from recently circulating strains GII.4-2006 Minerva and GII.4-2009 New Orleans in previously identified blockade epitopes, we compared reactivity and blockade profiles of GII.4-2006, GII.4-2009, and GII.4-2012 virus-like particles in surrogate neutralization/blockade assays using monoclonal antibodies and human polyclonal sera. Using monoclonal antibodies that map to known blockade epitopes in GII.4-2006 and GII.4-2009 and human outbreak polyclonal sera, we demonstrate either complete loss or significantly reduced reactivity and blockade of GII.4.2012 compared to GII.4-2006 and GII.4-2009. GII.4-2012 Sydney is antigenically different from GII.4-2006 Minerva and GII.4-2009 New Orleans in at least 2 key blockade epitopes. Viral evolution in key potential neutralization epitopes likely allowed GII.4-2012 to escape from human herd immunity and emerge as the new predominant strain.",
"Noroviruses account for 96% of viral gastroenteritis cases worldwide, with GII.4 strains responsible >80% of norovirus outbreaks. Histo-blood group antigens (HBGAs) are norovirus binding ligands, and antigenic and preferential HBGA binding profiles vary over time as new GII.4 strains emerge. The capsid P2 subdomain facilitates HBGA binding, contains neutralizing antibody epitopes, and likely evolves in response to herd immunity. To identify amino acids regulating HBGA binding and antigenic differences over time, we created chimeric virus-like particles (VLPs) between the GII.4-1987 and GII.4-2006 strains by exchanging amino acids in putative epitopes and characterized their antigenic and HBGA binding profiles using anti-GII.4-1987 and -2006 mouse monoclonal antibodies (MAbs) and polyclonal sera, 1988 outbreak human sera, and synthetic HBGAs. The exchange of amino acids 393 to 395 between GII.4-1987 and GII.4-2006 resulted in altered synthetic HBGA binding compared to parental strains. Introduction of GII.4-1987 residues 294, 297 to 298, 368, and 372 (epitope A) into GII.4-2006 resulted in reactivity with three anti-GII.4-1987 MAbs and reduced reactivity with four anti-GII.4-2006 MAbs. The three anti-GII.4-1987 MAbs also blocked chimeric VLP-HBGA interaction, while an anti-GII.4-2006 blocking antibody did not, indicating that epitope A amino acids comprise a potential neutralizing epitope for GII.4-1987 and GII.4-2006. We also tested GII.4-1987-immunized mouse polyclonal sera and 1988 outbreak human sera for the ability to block chimeric VLP-HBGA interaction and found that epitope A amino acids contribute significantly to the GII.4-1987 blockade response. Our data provide insights that help explain the emergence of new GII.4 epidemic strains over time, may aid development of norovirus therapeutics, and may help predict the emergence of future epidemic strains.",
"Small round-structured viruses (SRSV) are a group of RNA viruses that can cause gastroenteritis in persons of all ages. To determine the incidence of SRSV-associated gastroenteritis in The Netherlands and to study the genetic variability of outbreak strains, all outbreaks that were reported to the epidemiologists of the regional health services in 1996 were investigated using a standardized protocol. In 60 (87%) of the 69 reported outbreaks, SRSV could be detected, showing the etiologic significance of SRSV in outbreaks of gastroenteritis in The Netherlands. Of these outbreaks, 84% occurred in semiclosed communities, such as nursing homes (59%) and hospital wards (25%). Sequence analysis of the outbreak strains revealed that the majority of the strains from January to November 1996 formed a tight cluster within genogroup II SRSV. In November 1996, a shift toward genogroup I SRSV occurred, suggesting a change to a new predominant strain."
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Longitudinal changes in B cell subsets during coronavirus CNS infection
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Various infections in the central nervous system (CNS) trigger B cell accumulation; however, the relative dynamics between viral replication and alterations in distinct B cell subsets are largely unknown. Using a glia-tropic coronavirus infection, which is initiated in the brain but rapidly spreads to and predominantly persists in the spinal cord, this study characterizes longitudinal changes in B cell subsets at both infected anatomical sites. The phase of T cell-dependent, antibody-independent control of infectious virus was associated with a similar recruitment of naive/early-activated IgD(+) IgM(+) B cells into both the brain and spinal cord. This population was progressively replaced by CD138(-) IgD(-) IgM(+) B cells, isotype-switched CD138(-) IgD(-) IgM(-) memory B cells (B(mem)), and CD138(+) antibody-secreting cells (ASC). A more rapid transition to B(mem) and ASC in spinal cord than in brain was associated with higher levels of persisting viral RNA and transcripts encoding factors promoting B cell migration, differentiation, and survival. The results demonstrate that naive/early-activated B cells are recruited early during coronavirus CNS infection but are subsequently replaced by more differentiated B cells. Furthermore, viral persistence, even at low levels, is a driving force for accumulation of isotype-switched B(mem) and ASC.
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"Multiple sclerosis (MS) is an autoimmune disease of the CNS characterized by disruption of the blood-brain barrier (BBB). This breach in CNS immune privilege allows undeterred trafficking of myelin-specific lymphocytes into the CNS where they induce demyelination. Although the mechanism of BBB compromise is not known, the chemokine CXCL12 has been implicated as a molecular component of the BBB whose pattern of expression is specifically altered during MS and which correlates with disease severity. The inflammatory cytokine IL-1beta has recently been shown to contribute not only to BBB permeability but also to the development of IL-17-driven autoimmune responses. Using experimental autoimmune encephalomyelitis, the rodent model of MS, we demonstrate that IL-1beta mediates pathologic relocation of CXCL12 during the induction phase of the disease, before the development of BBB disruption. We also show that CD4, CD8, and, surprisingly gammadelta T cells are all sources of IL-1beta. In addition, gammadelta T cells are also targets of this cytokine, contributing to IL-1beta-mediated production of IL-17. Finally, we show that the level of CNS IL-1R determines the clinical severity of experimental autoimmune encephalomyelitis. These data suggest that T cell-derived IL-1beta contributes to loss of immune privilege during CNS autoimmunity via pathologic alteration in the expression of CXCL12 at the BBB.",
"B cell entry to lymph nodes and Peyer's patches depends on chemokine receptor signaling, but the principal chemokine involved has not been defined. Here we show that the homing of CXCR4-/- B cells is suppressed in CCL19 (ELC)- and CCL21 (SLC)-deficient paucity of lymph node T cells mice, but not in wild-type mice. We also find that CXCR4 can contribute to T cell homing. Using intravital microscopy, we find that B cell adhesion to high endothelial venules (HEVs) is disrupted when CCR7 and CXCR4 are predesensitized. In Peyer's patches, B cell entry is dependent on CXCR5 in addition to CCR7/CXCR4. CXCL12 (SDF1) is displayed broadly on HEVs, whereas CXCL13 (BLC) is found selectively on Peyer's patch follicular HEVs. These findings establish the principal chemokine and chemokine receptor requirements for B cell entry to lymph nodes and Peyer's patches.",
"The migration of B and T lymphocytes in the mouse has been studied by using 1) short-term in vivo homing studies, and 2) an in vitro assay of lymphocyte binding to specialized lymphoid organ venules (post-capillary, high endothelial venules (HEV)) in frozen sections of lymph nodes and Peyer's patches. The homing characteristics of B and T cell populations are largely independent of their organ of origin. B cells from any source distribute preferentially to Peyer's patches, whereas T cells home preferentially to peripheral lymph nodes. This organ specificity of migration appears to be determined at the site of lymphocyte exit from the blood by selective recognition of organ-specific determinants on the endothelial cells of HEV. In addition, the in vivo tendency of B cells to migrate preferentially to the spleen, and of T cells to localize better in lymph nodes is confirmed. The results indicate that, in a hypothetical situation in which an equal number of B and T lymphocytes localized in peripheral lymph nodes (or bound in vitro to peripheral node HEV), there would be about 2.5 B cells for every T cell in the mesenteric node, four to six B cells per T cell in Peyer's patches, and seven to nine B cells per T cell in the spleen. Comparison of these homing preferences with the distribution of B and T lymphocyte populations in situ suggests that selective lymphocyte migration may help determine the proportions of functionally distinct lymphocyte classes in particular lymphoid organs or sites of chronic inflammation, and thus may serve to influence the character of local immune responses.",
"An antigenic variant of the neurotropic murine coronavirus JHMV, designated 2.2-V-1, causes marked demyelination in the relative absence of encephalitis. It is thus useful for the study of the pathogenesis of demyelinating lesions. To better understand the sequential events leading to demyelination, we have examined murine brain and spinal cord tissue at daily intervals after intracerebral inoculation, evaluating them for the distribution of viral antigen, leukocyte infiltration, and demyelination. Immunohistochemical staining indicated that virus established primary infection in the ependymal cells in both brain and spinal cord before spreading into nearby structures and along white matter tracts by cell-to-cell contact. Spread from brain to spinal cord appeared to occur via cerebrospinal fluid. Viral replication was focally cytocidal for ependymal cells, and essentially noncytocidal for other neural cells including glia. In brain, viral antigen and inflammation reached a peak at day 5 postinfection, and rapidly subsided by day 10 postinfection. In spinal cord, viral antigen was less abundant than in brain and was maximal between days 7 and 9 postinfection. The inflammatory response and demyelination, however, were more severe persisting from day 7 through day 19. In the spinal cord, demyelinating lesions developed initially in areas closer to the central canal and were detected most prominently in the anterior funiculi. This finding suggests that the permissiveness of the ependymal cell is crucial to viral entry and that sequential infection of glial cells leads to the characteristic distribution of demyelination.",
"As activated B cells differentiate into plasma cells they complete a final series of migration steps that take them to locations where they can efficiently carry out their effector function, secreting immunoglobulin (Ig) M or IgG into circulation or releasing dimeric IgA adjacent to the epithelium for transcytosis. Recent experiments have established a key role for chemokines in directing antibody secreting cell (ASC) movement within the secondary lymphoid organs where they are generated, as well as in guiding the cells to the bone marrow or mucosal surfaces. This review discusses the chemokines involved in directing ASC movements, particularly focusing on the role of CXCR4 and CXCL12/SDF1. The function of CCR9 and CCR10 in IgA ASC homing and contributions made by integrins and lectins are also discussed.",
"Protection from influenza A virus (IAV) challenge requires switched, high affinity Abs derived from long-lived memory B cells and plasma cells. These B cell subsets are generated in germinal centers (GCs), hallmark structures of T helper cell-driven B cell immunity. A full understanding of the GC reaction after respiratory IAV infection is lacking, as is the characterization of T follicular helper (T(FH)) cells that support GCs. Here, GC B cell and T(FH) cell responses were studied in mice following pulmonary challenge with IAV. Marked GC reactions were induced in draining lymph nodes (dLNs), lung, spleen and nasal-associated lymphoid tissue (NALT), although the magnitude and kinetics of the response was site-specific. Examination of switching within GCs demonstrated IgG2(+) cells to compose the largest fraction in dLNs, lung and spleen. IgA(+) GC B cells were infrequent in these sites, but composed a significant subset of the switched GC population in NALT. Further experiments demonstrated splenectomized mice to withstand a lethal recall challenge, suggesting the spleen to be unnecessary for long-term protection in spite of strong GC responses in this organ. Final studies showed that T(FH) cell numbers were highest in dLNs and spleen, and peaked in all sites prior to the height of the GC reaction. T(FH) cells purified from dLNs generated IL-21 and IFNγ upon activation, although CD4(+)CXCR5(-) T effector cells produced higher levels of all cytokines. Collectively, these findings reveal respiratory IAV infection to induce strong T helper cell-driven B cell responses in various organs, with each site displaying unique attributes.",
"Adaptive immunity depends on T-cell exit from the thymus and T and B cells travelling between secondary lymphoid organs to survey for antigens. After activation in lymphoid organs, T cells must again return to circulation to reach sites of infection; however, the mechanisms regulating lymphoid organ exit are unknown. An immunosuppressant drug, FTY720, inhibits lymphocyte emigration from lymphoid organs, and phosphorylated FTY720 binds and activates four of the five known sphingosine-1-phosphate (S1P) receptors. However, the role of S1P receptors in normal immune cell trafficking is unclear. Here we show that in mice whose haematopoietic cells lack a single S1P receptor (S1P1; also known as Edg1) there are no T cells in the periphery because mature T cells are unable to exit the thymus. Although B cells are present in peripheral lymphoid organs, they are severely deficient in blood and lymph. Adoptive cell transfer experiments establish an intrinsic requirement for S1P1 in T and B cells for lymphoid organ egress. Furthermore, S1P1-dependent chemotactic responsiveness is strongly upregulated in T-cell development before exit from the thymus, whereas S1P1 is downregulated during peripheral lymphocyte activation, and this is associated with retention in lymphoid organs. We find that FTY720 treatment downregulates S1P1, creating a temporary pharmacological S1P1-null state in lymphocytes, providing an explanation for the mechanism of FTY720-induced lymphocyte sequestration. These findings establish that S1P1 is essential for lymphocyte recirculation and that it regulates egress from both thymus and peripheral lymphoid organs."
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TetR-like transcriptional regulator protein from Pseudomonas fluorescens isolated from Tanzanian tilapia ponds
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Pseudomonas fluorescens isolates from Tanzanian tilapia ponds were found to possess a gene encoding a TetR-like transcriptional regulator protein. Phylogenetic analysis revealed close similarity to five previously reported GeneBank sequences which cluster separately from the other 70 members of this family. It is assumed that this TetR-like protein belongs to a new family of TetR-like proteins that has no direct link to the class 1 integron.
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[
"Transcriptional regulation plays a critical role in the life cycle of Mycobacterium smegmatis and its related species, M. tuberculosis, the causative microbe for tuberculosis. However, the key transcriptional factors involved in broad regulation of diverse genes remain to be characterized in mycobacteria. In the present study, a TetR-like family transcriptional factor, Ms6564, was characterized in M. smegmatis as a master regulator. A conserved 19 bp-palindromic motif was identified for Ms6564 binding using DNaseI footprinting and EMSA. A total of 339 potential target genes for Ms6564 were further characterized by searching the M. smegmatis genome based on the sequence motif. Notably, Ms6564 bound with the promoters of 37 cell cycle and DNA damage/repair genes and regulated positively their expressions. The Ms6564-overexpressed recombinant strain yielded 5-fold lower mutation rates and mutation frequencies, whereas deletion of Ms6564 resulted in ∼5-fold higher mutation rates for the mutant strain compared with the wild-type strain. These findings suggested that Ms6564 may function as a global regulator and might be a sensor necessary for activation of DNA damage/repair genes.",
"WebLogo generates sequence logos, graphical representations of the patterns within a multiple sequence alignment. Sequence logos provide a richer and more precise description of sequence similarity than consensus sequences and can rapidly reveal significant features of the alignment otherwise difficult to perceive. Each logo consists of stacks of letters, one stack for each position in the sequence. The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of symbols within the stack reflects the relative frequency of the corresponding amino or nucleic acid at that position. WebLogo has been enhanced recently with additional features and options, to provide a convenient and highly configurable sequence logo generator. A command line interface and the complete, open WebLogo source code are available for local installation and customization.",
"The genome sequence of Mycobacterium tuberculosis was analysed by searching for homologues of genes known to be involved in the reversal or repair of DNA damage in Escherichia coli and related organisms. Genes necessary to perform nucleotide excision repair (NER), base excision repair (BER), recombination, and SOS repair and mutagenesis were identified. In particular, all of the genes known to be directly involved in the repair of oxidative and alkylative damage are present in M. tuberculosis. In contrast, we failed to identify homologues of genes involved in mismatch repair. This finding has potentially significant implications with respect to genome stability, strain variability at repeat loci and the emergence of chromosomally encoded drug resistance mutations.",
"Letters of the English alphabet have heretofore been used to name tetracycline resistance determinants. Since all 26 letters have now been used, a nomenclature employing numerals is recommended for future determinants, and one laboratory has offered to coordinate the assignment of numerals."
] |
Expression levels of rice VQ motif-containing genes in response to pathogen infection vary among pathogens
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Expression levels of rice VQ motif-containing genes in response to pathogen infection vary among pathogens, and some of the genes are co-expressed with defense-response WRKY genes. Recent studies have revealed that some VQ (FxxxVQxLTG) motif-containing proteins in plants partner with WRKY transcription factors to participate in their functions. Accumulating information suggests that WRKY proteins play important roles in the response of rice plants to pathogen infection. However, the functions of rice VQ motif-containing proteins are unknown. To explore whether VQ motif-containing proteins are involved in defense against pathogens in rice, we performed a comprehensive expression analysis of the genes for these proteins. The rice VQ motif-containing family consists of 40 genes, all of which encode proteins harboring a 21-amino acid VQ-containing motif, which in turn contains the known VQ motif. On the basis of their phylogenetic relationships and tissue-specific and developmental stage-specific expression characteristics, we transcriptionally analyzed 13 representative genes in rice responses to three pathogens: Xanthomonas oryzae pv. oryzae, which causes bacterial blight disease; X. oryzae pv. oryzicola, which causes bacterial streak disease; and Magnaporthe oryzae, which causes fungal blast disease. The expression of some of the genes changed markedly in response to infection by at least one of the pathogen species, and some of the genes also showed markedly different expression in resistant and susceptible reactions. In addition, some defense-responsive VQ motif-containing genes were co-expressed with defense-response WRKY genes. These results provide a new perspective on the putative roles of rice VQ motif-containing proteins and their putative WRKY partners in rice-pathogen interactions.
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[
"A pathway database (DB) is a DB that describes biochemical pathways, reactions, and enzymes. The EcoCyc pathway DB (see http://ecocyc.org) describes the metabolic, transport, and genetic-regulatory networks of Escherichia coli. EcoCyc is an example of a computational symbolic theory, which is a DB that structures a scientific theory within a formal ontology so that it is available for computational analysis. It is argued that by encoding scientific theories in symbolic form, we open new realms of analysis and understanding for theories that would otherwise be too large and complex for scientists to reason with effectively.",
"Large-scale two-hybrid projects were used in an approach to examine protein-protein interactions. Despite the various limitations of this approach, these projects revealed a wealth of novel interactions, and the protein interactome may be much larger than expected.",
"Cytoscape is an open source software project for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework. Although applicable to any system of molecular components and interactions, Cytoscape is most powerful when used in conjunction with large databases of protein-protein, protein-DNA, and genetic interactions that are increasingly available for humans and model organisms. Cytoscape's software Core provides basic functionality to layout and query the network; to visually integrate the network with expression profiles, phenotypes, and other molecular states; and to link the network to databases of functional annotations. The Core is extensible through a straightforward plug-in architecture, allowing rapid development of additional computational analyses and features. Several case studies of Cytoscape plug-ins are surveyed, including a search for interaction pathways correlating with changes in gene expression, a study of protein complexes involved in cellular recovery to DNA damage, inference of a combined physical/functional interaction network for Halobacterium, and an interface to detailed stochastic/kinetic gene regulatory models.",
"Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is a devastating disease of rice worldwide. The qualitative or pathogen race-specific resistance to this pathogen conferred by major disease resistance (MR) genes has been widely used in rice improvement. Accumulating genetic and molecular data have revealed that the molecular mechanisms of rice qualitative resistance to Xoo are largely different from those of qualitative resistance in other plant-pathogen pathosystems. In this review, we focus on the unique features of rice qualitative resistance to Xoo based on MR genes that have been identified and characterized. The distinctiveness of the rice-Xoo interaction provides a unique pathosystem to elucidate the diverse molecular mechanisms in plant qualitative resistance.",
"WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Proteins containing a short VQ (FxxxVQxLTG) motif have been recently shown to interact with WRKY transcription factors, implying that AtVQ proteins are important in the plant defense responses in Arabidopsis, either as positive or negative cofactors of WRKY transcription factors. Thirty-nine Oryza sativa genes containing the VQ motif (OsVQs) were identified and the genome structures of OsVQ proteins were characterized through genome-wide analysis in rice. Also, phylogenetic tree analysis was performed with the VQ domain of Arabidopsis and rice. The expression patterns of these OsVQ genes in plants under several stress treatments were assessed, specifically, following infection with the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), treatment with abscisic acid (ABA), or exposure to drought. The cellular localization of a few OsVQ proteins was examined using rice protoplast system. Based on our results, we suggest that OsVQ proteins function as important co-regulators during the plant defense response to biotic and abiotic stresses."
] |
Chemically modified peptides spanning the membrane proximal external region of gp41 suppress antiepitope responses.
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Broadly neutralizing monoclonal antibodies (bNAbs) 2F5 and 4E10 bind to the membrane proximal external region (MPER) of gp41 and also cross-react with phospholipids. In this study, we investigated if chemical modifications on the MPER adjacent to 2F5 and 4E10 epitopes using mimetics of inflammation-associated posttranslational modifications to induce 2F5- and 4E10-like bNAbs can break tolerance. We synthesized a series of chemically modified peptides spanning the MPER. The serine, threonine, and tyrosine residues in the peptides were modified with sulfate, phosphate, or nitrate moieties and presented in liposomes for rabbit immunizations. All immunizations resulted in high antisera titers directed toward both the modified and unmodified immunogens. Tyrosine modification was observed to significantly suppress antiepitope responses. Sera with strong anti-gp140 titers were purified by affinity chromatography toward the MPER peptide and found to possess a higher affinity toward the MPER than did the bNAbs 2F5 and 4E10. Modest neutralization was observed in the H9 neutralization assay, but neutralization was not observed in the TZM-bl cell or peripheral blood mononuclear cell (PBMC) neutralization assay platforms. Although neutralizing antibodies were not induced by this approach, we conclude that chemical modifications can increase the immune responses to poorly immunogenic antigens, suggesting that chemical modification in an appropriate immunization protocol should be explored further as an HIV-1 vaccine strategy.
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[
"The identification and epitope mapping of broadly neutralizing anti-human immunodeficiency virus type 1 (HIV-1) antibodies (Abs) is important for vaccine design, but, despite much effort, very few such Abs have been forthcoming. Only one broadly neutralizing anti-gp41 monoclonal Ab (MAb), 2F5, has been described. Here we report on two MAbs that recognize a region immediately C-terminal of the 2F5 epitope. Both MAbs were generated from HIV-1-seropositive donors, one (Z13) from an antibody phage display library, and one (4E10) as a hybridoma. Both MAbs recognize a predominantly linear and relatively conserved epitope, compete with each other for binding to synthetic peptide derived from gp41, and bind to HIV-1(MN) virions. By flow cytometry, these MAbs appear to bind relatively weakly to infected cells and this binding is not perturbed by pretreatment of the infected cells with soluble CD4. Despite the apparent linear nature of the epitopes of Z13 and 4E10, denaturation of recombinant envelope protein reduces the binding of these MAbs, suggesting some conformational requirements for full epitope expression. Most significantly, Z13 and 4E10 are able to neutralize selected primary isolates from diverse subtypes of HIV-1 (e.g., subtypes B, C, and E). The results suggest that a rather extensive region of gp41 close to the transmembrane domain is accessible to neutralizing Abs and could form a useful target for vaccine design.",
"The membrane-proximal region of the ectodomain of the gp41 envelope glycoprotein of human immunodeficiency virus type 1 (HIV-1) is the target of three of the five broadly neutralizing anti-HIV-1 antibodies thus far isolated. We have determined crystal structures of the antigen-binding fragment for one of these antibodies, 2F5, in complex with 7-mer, 11-mer, and 17-mer peptides of the gp41 membrane-proximal region, at 2.0-, 2.1-, and 2.2-A resolutions, respectively. The structures reveal an extended gp41 conformation, which stretches over 30 A in length. Contacts are made with five complementarity-determining regions of the antibody as well as with nonpolymorphic regions. Only one exclusive charged face of the gp41 epitope is bound by 2F5, while the nonbound face, which is hydrophobic, may be hidden due to occlusion by other portions of the ectodomain. The structures reveal that the 2F5 antibody is uniquely built to bind to an epitope that is proximal to a membrane surface and in a manner mostly unaffected by large-scale steric hindrance. Biochemical studies with proteoliposomes confirm the importance of lipid membrane and hydrophobic context in the binding of 2F5 as well as in the binding of 4E10, another broadly neutralizing antibody that recognizes the membrane-proximal region of gp41. Based on these structural and biochemical results, immunization strategies for eliciting 2F5- and 4E10-like broadly neutralizing anti-HIV-1 antibodies are proposed.",
"Infection by HIV-1 involves the fusion of viral and cellular membranes with subsequent transfer of viral genetic material into the cell. The HIV-1 envelope glycoprotein that mediates fusion consists of the surface subunit gp120 and the transmembrane subunit gp41. gp120 directs virion attachment to the cell-surface receptors, and gp41 then promotes viral-cell membrane fusion. A soluble, alpha-helical, trimeric complex within gp41 composed of N-terminal and C-terminal extraviral segments has been proposed to represent the core of the fusion-active conformation of the HIV-1 envelope. A thermostable subdomain denoted N34(L6)C28 can be formed by the N-34 and C-28 peptides connected by a flexible linker in place of the disulfide-bonded loop region. Three-dimensional structure of N34(L6)C28 reveals that three molecules fold into a six-stranded helical bundle. Three N-terminal helices within the bundle form a central, parallel, trimeric coiled coil, whereas three C-terminal helices pack in the reverse direction into three hydrophobic grooves on the surface of the N-terminal trimer. This thermostable subdomain displays the salient features of the core structure of the isolated gp41 subunit and thus provides a possible target for therapeutics designed selectively to block HIV-1 entry.",
"Mutations were introduced into the ectodomain of the human immunodeficiency virus type 1 (HIV-1) transmembrane envelope glycoprotein, gp41, within a region immediately adjacent to the membrane-spanning domain. This region, which is predicted to form an alpha-helix, contains highly conserved hydrophobic residues and is unusually rich in tryptophan residues. In addition, this domain overlaps the epitope of a neutralizing monoclonal antibody, 2F5, as well as the sequence corresponding to a peptide, DP-178, shown to potently neutralize virus. Site-directed mutagenesis was used to create deletions, substitutions, and insertions centered around a stretch of 17 hydrophobic and uncharged amino acids (residues 666 to 682 of the HXB2 strain of HIV-1) in order to determine the role of this region in the maturation and function of the envelope glycoprotein. Deletion of the entire stretch of 17 amino acids abrogated the ability of the envelope glycoprotein to mediate both cell-cell fusion and virus entry without affecting the normal maturation, transport, or CD4-binding ability of the protein. This phenotype was also demonstrated by substituting alanine residues for three of the five tryptophan residues within this sequence. Smaller deletions, as well as multiple amino acid substitutions, were also found to inhibit but not block cell-cell fusion. These results demonstrate the crucial role of a tryptophan-rich motif in gp41 during a post-CD4-binding step of glycoprotein-mediated fusion. The basis for the invariant nature of the tryptophans, however, appears to be at the level of glycoprotein incorporation into virions. Even the substitution of phenylalanine for a single tryptophan residue was sufficient to reduce Env incorporation and drop the efficiency of virus entry approximately 10-fold, despite the fact that the same mutation had no significant effect on syncytium formation.",
"HIV-1 (human immunodeficiency virus type 1) uses its trimeric gp160 envelope (Env) protein consisting of non-covalently associated gp120 and gp41 subunits to mediate entry into human T lymphocytes. A facile virus fusion mechanism compensates for the sparse Env copy number observed on viral particles and includes a 22-amino-acid, lentivirus-specific adaptation at the gp41 base (amino acid residues 662-683), termed the membrane proximal external region (MPER). We show by NMR and EPR that the MPER consists of a structurally conserved pair of viral lipid-immersed helices separated by a hinge with tandem joints that can be locked by capping residues between helices. This design fosters efficient HIV-1 fusion via interconverting structures while, at the same time, affording immune escape. Disruption of both joints by double alanine mutations at Env positions 671 and 674 (AA) results in attenuation of Env-mediated cell-cell fusion and hemifusion, as well as viral infectivity mediated by both CD4-dependent and CD4-independent viruses. The potential mechanism of disruption was revealed by structural analysis of MPER conformational changes induced by AA mutation. A deeper acyl chain-buried MPER middle section and the elimination of cross-hinge rigid-body motion almost certainly impede requisite structural rearrangements during the fusion process, explaining the absence of MPER AA variants among all known naturally occurring HIV-1 viral sequences. Furthermore, those broadly neutralization antibodies directed against the HIV-1 MPER exploit the tandem joint architecture involving helix capping, thereby disrupting hinge function.",
"Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.",
"The new program DYANA (DYnamics Algorithm for Nmr Applications) for efficient calculation of three-dimensional protein and nucleic acid structures from distance constraints and torsion angle constraints collected by nuclear magnetic resonance (NMR) experiments performs simulated annealing by molecular dynamics in torsion angle space and uses a fast recursive algorithm to integrate the equations of motions. Torsion angle dynamics can be more efficient than molecular dynamics in Cartesian coordinate space because of the reduced number of degrees of freedom and the concomitant absence of high-frequency bond and angle vibrations, which allows for the use of longer time-steps and/or higher temperatures in the structure calculation. It also represents a significant advance over the variable target function method in torsion angle space with the REDAC strategy used by the predecessor program DIANA. DYANA computation times per accepted conformer in the \"bundle\" used to represent the NMR structure compare favorably with those of other presently available structure calculation algorithms, and are of the order of 160 seconds for a protein of 165 amino acid residues when using a DEC Alpha 8400 5/300 computer. Test calculations starting from conformers with random torsion angle values further showed that DYANA is capable of efficient calculation of high-quality protein structures with up to 400 amino acid residues, and of nucleic acid structures."
] |
Optimal storage and shipment conditions for dried blood spots for HIV drug resistance genotyping in resource-limited settings
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Dried blood spots (DBS) are an alternative specimen type for HIV drug resistance genotyping in resource-limited settings. Data relating to the impact of DBS storage and shipment conditions on genotyping efficiency under field conditions are limited. We compared the genotyping efficiencies and resistance profiles of DBS stored and shipped at different temperatures to those of plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood and a fifth card from finger-prick blood were prepared from 103 HIV patients with a median viral load (VL) of 57,062 copies/ml (range, 1,081 to 2,964,191). DBS were stored at ambient temperature for 2 or 4 weeks or frozen at -80 °C and shipped from Uganda to the United States at ambient temperature or frozen on dry ice for genotyping using a broadly sensitive in-house method. Plasma (97.1%) and DBS (98.1%) stored and shipped frozen had similar genotyping efficiencies. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiencies. Genotyping efficiency was reduced for DBS stored at ambient temperature for 4 weeks (89.3%, P = 0.03) or prepared from finger-prick blood and stored at ambient temperature for 2 weeks (77.7%, P < 0.001) compared to DBS prepared from venous blood and handled similarly. Resistance profiles were similar between plasma and DBS specimens. This report delineates the optimal DBS collection, storage, and shipping conditions and opens a new avenue for cost-saving ambient-temperature DBS specimen shipments for HIV drug resistance (HIVDR) surveillances in resource-limited settings.
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[
"In low resource settings the inability to diagnose HIV in infants early presents a major obstacle to providing care for HIV-infected children. Dried blood spot samples offer a solution to the scarcity of skills available for venesecting young infants but pose challenges to laboratories because their processing requirements are distinct from that of the liquid blood samples widely used for viral detection assays. Different methods of excising 287 paired HIV-positive and HIV-negative dried blood spot samples (n=574) for testing by the Roche Amplicor HIV-1 DNA assay version 1.5 were assessed. A manual punch in conjunction with three different cleaning protocols (n=372) and an automated punch (BSD 1000 GenePunch) using a single cleaning protocol (n=202) was assessed for the risk of cross contamination between samples. A single false positive result obtained using the automated punch may have been attributable to contamination during the excision step of the assay. Excision of dried blood spot samples is associated with a very low risk of cross contamination regardless of the instrument or cleaning intervention used. The process of excising dried blood spot samples should not compromise the results of HIV viral detection assays performed on dried blood spots in routine laboratories which is encouraging for increasing access to an accurate diagnosis of HIV in infants.",
"As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.",
"HIV-1 viral load (VL) testing is not widely available in resource-limited settings. The use of finger prick dried blood spot (FP-DBS) samples could remove barriers related to sample collection and transport. Measurement of VL using DBS from EDTA venous blood (VB-DBS) in place of plasma has previously been validated using the NucliSENS Easy-Q HIV-1 v2.0 assay, but information on the accuracy of FP-DBS samples for measuring VL is limited. This prospective study, conducted at Thyolo District Hospital in southern Malawi, compared VL levels measured on FP-DBS samples and plasma using the NucliSENS Easy-Q HIV-1 v2.0 assay. Comparability was assessed by means of agreement and correlation (131 patients with VLs of ≥100 copies/ml), sensitivity, and specificity (612 patients on antiretroviral treatment [ART]). Samples of EDTA venous blood and FP-DBS from 1,009 HIV-infected individuals were collected and prepared in the laboratory. Bland-Altman analysis found good agreement between plasma and FP-DBS VL levels, with a mean difference of -0.35 log10, and 95% limits of agreement from -1.26 to 0.55 log10. FP-DBS had a sensitivity of 88.7% (95% confidence interval [CI], 81.1 to 94.4%) and a specificity of 97.8% (95% CI, 96.1 to 98.9%) using a 1,000-copies/ml cut point and a sensitivity of 83.0% (95% CI, 73.4 to 90.1%) and a specificity of 100% (95% CI, 99.3 to 100%) using a 5,000-copies/ml cut point. This study shows that FP-DBS is an acceptable alternative to plasma for measuring VL using the NucliSENS Easy-Q HIV-1 v2.0. We are conducting a second study to assess the proficiency of health workers at preparing FP-DBS in primary health care clinics.",
"Exposure to antiretroviral drugs in resource-constrained settings is likely to result in the emergence of drug-resistant HIV-1 variants, limiting treatment options. Genotypic drug resistance testing assists clinical management and outcomes assessment, but a sensitive and reproducible genotypic assay feasible for resource-constrained settings is needed. A sensitive, reproducible genotyping assay to detect HIV-1 drug resistance mutations in reverse transcriptase and protease was developed and validated using blood stored as dried blood spots or as frozen RNA extracts from Zambian children infected with subtype C. HIV-1 genotypes derived from samples stored as dried blood spots were compared to those derived from paired liquid plasma samples in American young adults infected with HIV-1 subtype B. The method reproducibly amplified patient-specific sequences and detected drug resistance mutations from all of the dried blood spots or excess frozen RNA extracts with detectable viremia over a broad range of viral loads (193-3 million HIV-1 RNA copies/mL) in both HIV-1 subtypes B and C infection. This method captured the genetic variation typical of HIV-1 infection, including mutations at usual sites of drug resistance, polymorphisms, and mixtures. This sensitive and reproducible genotypic assay is feasible for detection of antiretroviral resistance in resource-constrained settings.",
"Study 903 was a 144-week, randomized, double-blind, active-controlled study of tenofovir disoproxil fumarate (TDF) therapy in treatment-naive HIV-1-infected patients. Patients received either TDF (n = 299) or stavudine (d4T) (n = 301) with lamivudine (3TC) and efavirenz (EFV). Resistance analyses were performed at baseline and at virological failure to determine the effects of baseline resistance and the patterns of resistance at virological failure. Plasma HIV-1 from patients at baseline and at virological failure (>400 HIV-1 RNA copies/mL at week 144 or early discontinuation) was analysed phenotypically and by population sequencing. Sixteen per cent of patients were classified as having virological failure (47 on TDF and 49 on d4T; P = 0.91). Patients with non-B HIV-1 subtypes or baseline nucleoside reverse transcriptase inhibitor (NRTI)-associated mutations responded similarly to the overall population. Resistance to EFV (K103N and others) or 3TC (M184V) developed most frequently (8.3% and 5.8%, respectively) and similarly in the two arms. In the d4T arm, a variety of NRTI mutations developed: K65R (n = 2), L74V (n = 2), V75M (n = 1), and T69A + Y115H (n = 1). K65R developed in eight TDF patients (2.7%); in seven of these eight patients, within 48 weeks. All eight patients began new regimens with a protease inhibitor (PI) and NRTIs, including two patients who remained on TDF; five of the eight patients achieved HIV RNA <50 copies/mL in second-line therapy with the remaining patients having no follow-up or being nonadherent. Treatment of HIV-1 with TDF, 3TC and EFV was highly effective, with <3% of patients developing resistance to TDF over 144 weeks."
] |
Evolution of the A1 family of eukaryotic aspartic proteinases
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The A1 family of eukaryotic aspartic proteinases (APs) forms one of the 16 AP families. Although one of the best characterized families, the recent increase in genome sequence data has revealed many fungal AP homologs with novel sequence characteristics. This study was performed to explore the fungal AP sequence space and to obtain an in-depth understanding of fungal AP evolution. Using a comprehensive phylogeny of approximately 700 AP sequences from the complete proteomes of 87 fungi and 20 nonfungal eukaryotes, 11 major clades of APs were defined of which clade I largely corresponds to the A1A subfamily of pepsin-archetype APs. Clade II largely corresponds to the A1B subfamily of nepenthesin-archetype APs. Remarkably, the nine other clades contain only fungal APs, thus indicating that fungal APs have undergone a large sequence diversification. The topology of the tree indicates that fungal APs have been subject to both "birth and death" evolution and "functional redundancy and diversification." This is substantiated by coclustering of certain functional sequence characteristics. A meta-analysis toward the identification of Cluster Determining Positions (CDPs) was performed in order to investigate the structural and biochemical basis for diversification. Seven CDPs contribute to the secondary structure of the enzyme. Three other CDPs are found in the vicinity of the substrate binding cleft. Tree topology, the large sequence variation among fungal APs, and the apparent functional diversification suggest that an amendment to update the current A1 AP classification based on a comprehensive phylogenetic clustering might contribute to refinement of the classification in the MEROPS peptidase database.
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[
"Candida albicans is the most common fungal pathogen in humans, causing both debilitating mucosal infections and potentially life-threatening systemic infections. Until recently, C. albicans was thought to be strictly asexual, existing only as an obligate diploid. A cryptic mating cycle has since been uncovered in which diploid a and alpha cells undergo efficient cell and nuclear fusion, resulting in tetraploid a/alpha mating products. Whereas mating between a and alpha cells has been established (heterothallism), we report here two pathways for same-sex mating (homothallism) in C. albicans. First, unisexual populations of a cells were found to undergo autocrine pheromone signalling and same-sex mating in the absence of the Bar1 protease. In both C. albicans and Saccharomyces cerevisiae, Bar1 is produced by a cells and inactivates mating pheromone alpha, typically secreted by alpha cells. C. albicans Deltabar1 a cells were shown to secrete both a and alpha mating pheromones; alpha-pheromone activated self-mating in these cells in a process dependent on Ste2, the receptor for alpha-pheromone. In addition, pheromone production by alpha cells was found to promote same-sex mating between wild-type a cells. These results establish that homothallic mating can occur in C. albicans, revealing the potential for genetic exchange even within unisexual populations of the organism. Furthermore, Bar1 protease has an unexpected but pivotal role in determining whether sexual reproduction can potentially be homothallic or is exclusively heterothallic. These findings also have implications for the mode of sexual reproduction in related species that propagate unisexually, and indicate a role for specialized sexual cycles in the survival and adaptation of pathogenic fungi.",
"Botrytis cinerea causes gray mold rot on over 200 plant species worldwide, resulting in great economic loss every year. Cooperation of proteins secreted by B. cinerea plays an important role in its successful infection to host plants. The ambient pH, as one of the most important environmental parameters, can regulate expression of secreted proteins in various fungal pathogens. In the present study, we mainly investigated the effect of ambient pH on secretome of B. cinerea strain B05.10 with a comparative proteomic method based on 2-DE. Distinct differences in secretome of B. cinerea were found between pH 4 and 6 treatments, and 47 differential spots, corresponding to 21 unique proteins, were identified using MALDI-TOF/TOF. At pH 4, more proteins related to proteolysis were induced, whereas most of up-accumulated proteins were cell wall degrading enzymes at pH 6. Analysis of gene expression using quantitative real-time PCR suggests that production of most of these proteins was regulated at the level of transcription. These findings indicate that B. cinerea can adjust protein profile of secretome responding to different ambient pH values and provide evidence to deeply understand the complicated infecting mechanisms of B. cinerea on a wide range of plant hosts.",
"Rhizopus oryzae is the primary cause of mucormycosis, an emerging, life-threatening infection characterized by rapid angioinvasive growth with an overall mortality rate that exceeds 50%. As a representative of the paraphyletic basal group of the fungal kingdom called \"zygomycetes,\" R. oryzae is also used as a model to study fungal evolution. Here we report the genome sequence of R. oryzae strain 99-880, isolated from a fatal case of mucormycosis. The highly repetitive 45.3 Mb genome assembly contains abundant transposable elements (TEs), comprising approximately 20% of the genome. We predicted 13,895 protein-coding genes not overlapping TEs, many of which are paralogous gene pairs. The order and genomic arrangement of the duplicated gene pairs and their common phylogenetic origin provide evidence for an ancestral whole-genome duplication (WGD) event. The WGD resulted in the duplication of nearly all subunits of the protein complexes associated with respiratory electron transport chains, the V-ATPase, and the ubiquitin-proteasome systems. The WGD, together with recent gene duplications, resulted in the expansion of multiple gene families related to cell growth and signal transduction, as well as secreted aspartic protease and subtilase protein families, which are known fungal virulence factors. The duplication of the ergosterol biosynthetic pathway, especially the major azole target, lanosterol 14alpha-demethylase (ERG11), could contribute to the variable responses of R. oryzae to different azole drugs, including voriconazole and posaconazole. Expanded families of cell-wall synthesis enzymes, essential for fungal cell integrity but absent in mammalian hosts, reveal potential targets for novel and R. oryzae-specific diagnostic and therapeutic treatments.",
"It is now well established that mating can occur between diploid a and alpha cells of Candida albicans. There is, however, controversy over when, and with what efficiency, nuclear fusion follows cell fusion to create stable tetraploid a/alpha cells. In this study, we have analysed the mating process between C. albicans strains using both cytological and genetic approaches. Using strains derived from SC5314, we used a number of techniques, including time-lapse microscopy, to demonstrate that efficient nuclear fusion occurs in the zygote before formation of the first daughter cell. Consistent with these observations, zygotes micromanipulated from mating mixes gave rise to mononuclear tetraploid cells, even when no selection for successful mating was applied to them. Mating between different clinical isolates of C. albicans revealed that while all isolates could undergo nuclear fusion, the efficiency of nuclear fusion varied in different crosses. We also show that nuclear fusion in C. albicans requires the Kar3 microtubule motor protein. Deletion of the CaKAR3 gene from both mating partners had little or no effect on zygote formation but reduced the formation of stable tetraploids more than 600-fold, as determined by quantitative mating assays. These findings demonstrate that nuclear fusion is an active process that can occur in C. albicans at high frequency to produce stable, mononucleate mating products.",
"Many species of human pathogenic fungi secrete proteases in vitro or during the infection process. Secreted endoproteases belong to the aspartic proteases of the pepsin family, serine proteases of the subtilisin family, and metalloproteases of two different families. To these proteases has to be added the non-pepsin-type aspartic protease from Aspergillus niger and a unique chymotrypsin-like protease from Coccidioides immitis. Pathogenic fungi also secrete aminopeptidases, carboxypeptidases and dipeptidyl-peptidases. The function of fungal secreted proteases and their importance in infections vary. It is evident that secreted proteases are important for the virulence of dermatophytes since these fungi grow exclusively in the stratum corneum, nails or hair, which constitutes their sole nitrogen and carbon sources. The aspartic proteases secreted by Candida albicans are involved in the adherence process and penetration of tissues, and in interactions with the immune system of the infected host. For Aspergillus fumigatus, the role of proteolytic activity has not yet been proved. Although the secreted proteases have been intensively investigated as potential virulence factors, knowledge on protease substrate specificities is rather poor and few studies have focused on the research of inhibitors. Knowledge of substrate specificities will increase our understanding about the action of each protease secreted by pathogenic fungi and will help to determine their contribution to virulence.",
"Many posttranslational modifications (N-myristoylation or glycosylphosphatidylinositol (GPI) lipid anchoring) and localization signals (the peroxisomal targeting signal PTS1) are encoded in short, partly compositionally biased regions at the N- or C-terminus of the protein sequence. These sequence signals are not well defined in terms of amino acid type preferences but they have significant interpositional correlations. Although the number of verified protein examples is small, the quantification of several physical conditions necessary for productive protein binding with the enzyme complexes executing the respective transformations can lead to predictors that recognize the signals from the amino acid sequence of queries alone. Taxon-specific prediction functions are required due to the divergent evolution of the active complexes. The big-Pi tool for the prediction of the C-terminal signal for GPI lipid anchor attachment is available for metazoan, protozoan and plant sequences. The myristoyl transferase (NMT) predictor recognizes glycine N-myristoylation sites (at the N-terminus and for fragments after processing) of higher eukaryotes (including their viruses) and fungi. The PTS1 signal predictor finds proteins with a C-terminus appropriate for peroxisomal import (for metazoa and fungi). Guidelines for application of the three WWW-based predictors (http://mendel.imp.univie.ac.at/) and for the interpretation of their output are described."
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Forkhead Box A2 regulates glucose homeostasis
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Forkhead Box A2 (FOXA2) exerts an influence on glucose homeostasis via activity in the liver. In addition, a key genome-wide association study (GWAS) recently demonstrated that genetic variation, namely rs6048205, at the FOXA2 locus is robustly associated with fasting glucose levels. Our hypothesis was that this DNA-binding protein regulates the expression of a set of molecular pathways critical to endocrine traits.
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"The human genome encodes the blueprint of life, but the function of the vast majority of its nearly three billion bases is unknown. The Encyclopedia of DNA Elements (ENCODE) project has systematically mapped regions of transcription, transcription factor association, chromatin structure and histone modification. These data enabled us to assign biochemical functions for 80% of the genome, in particular outside of the well-studied protein-coding regions. Many discovered candidate regulatory elements are physically associated with one another and with expressed genes, providing new insights into the mechanisms of gene regulation. The newly identified elements also show a statistical correspondence to sequence variants linked to human disease, and can thereby guide interpretation of this variation. Overall, the project provides new insights into the organization and regulation of our genes and genome, and is an expansive resource of functional annotations for biomedical research.",
"We performed a genome-wide association study (GWAS) of systemic lupus erythematosus (SLE) in a Chinese Han population by genotyping 1,047 cases and 1,205 controls using Illumina Human610-Quad BeadChips and replicating 78 SNPs in two additional cohorts (3,152 cases and 7,050 controls). We identified nine new susceptibility loci (ETS1, IKZF1, RASGRP3, SLC15A4, TNIP1, 7q11.23, 10q11.22, 11q23.3 and 16p11.2; 1.77 x 10(-25) < or = P(combined) < or = 2.77 x 10(-8)) and confirmed seven previously reported loci (BLK, IRF5, STAT4, TNFAIP3, TNFSF4, 6q21 and 22q11.21; 5.17 x 10(-42) < or = P(combined) < or = 5.18 x 10(-12)). Comparison with previous GWAS findings highlighted the genetic heterogeneity of SLE susceptibility between Chinese Han and European populations. This study not only advances our understanding of the genetic basis of SLE but also highlights the value of performing GWAS in diverse ancestral populations.",
"The identification and characterization of mammalian core promoters and transcription start sites is a prerequisite to understanding how RNA polymerase II transcription is controlled. New experimental technologies have enabled genome-wide discovery and characterization of core promoters, revealing that most mammalian genes do not conform to the simple model in which a TATA box directs transcription from a single defined nucleotide position. In fact, most genes have multiple promoters, within which there are multiple start sites, and alternative promoter usage generates diversity and complexity in the mammalian transcriptome and proteome. Promoters can be described by their start site usage distribution, which is coupled to the occurrence of cis-regulatory elements, gene function and evolutionary constraints. A comprehensive survey of mammalian promoters is a major step towards describing and understanding transcriptional control networks.",
"The transcription factor hepatocyte nuclear factor 3 (HNF-3) is involved in the coordinate expression of several liver genes. HNF-3 DNA binding activity is composed of three different liver proteins which recognize the same DNA site. The HNF-3 proteins (designated alpha, beta, and gamma) possess homology in the DNA binding domain and in several additional regions. To understand the cell-type-specific expression of HNF-3 beta, we have defined the regulatory sequences that elicit hepatoma-specific expression. Promoter activity requires -134 bp of HNF-3 beta proximal sequences and binds four nuclear proteins, including two ubiquitous factors. One of these promoter sites interacts with a novel cell-specific factor, LF-H3 beta, whose binding activity correlates with the HNF-3 beta tissue expression pattern. Furthermore, there is a binding site for the HNF-3 protein within its own promoter, suggesting that an autoactivation mechanism is involved in the establishment of HNF-3 beta expression. We propose that both the LF-H3 beta and HNF-3 sites play an important role in the cell-type-specific expression of the HNF-3 beta transcription factor.",
"Hepatocyte Nuclear Factor-1 alpha (HNF-1 alpha) and HNF-1 beta are homeodomain-containing transcription factors which interact with the GTTAATNATTAAC motif essential to the function of more than 15 promoters selectively expressed in the liver. These homeoproteins can form homo- and heterodimers in solution and share identical DNA-binding domains but have different transcriptional activation properties. During retinoic acid (RA) induced differentiation of F9 embryonal carcinoma (EC) cells, which stimulates aspects of pre-implantation embryogenesis, both HNF-1 beta mRNA and immunoreactive DNA-binding activity are strongly induced approximately 24 h post RA-treatment. In contrast, HNF-1 alpha mRNA increases approximately 4-fold after 5 days, concomitant with elevation of HNF-1 alpha DNA-binding activity and expression of the HNF-1 target gene alpha-fetoprotein. These results indicate that HNF-1 alpha and -1 beta expression can be controlled by regulatory hierarchies downstream of primary RA-response genes, and suggest that independent regulatory mechanisms for these factors can confer distinct and interactive developmental functions.",
"Initially thought to play a restricted role in calcium homeostasis, the pleiotropic actions of vitamin D in biology and their clinical significance are only now becoming apparent. However, the mode of action of vitamin D, through its cognate nuclear vitamin D receptor (VDR), and its contribution to diverse disorders, remain poorly understood. We determined VDR binding throughout the human genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After calcitriol stimulation, we identified 2776 genomic positions occupied by the VDR and 229 genes with significant changes in expression in response to vitamin D. VDR binding sites were significantly enriched near autoimmune and cancer associated genes identified from genome-wide association (GWA) studies. Notable genes with VDR binding included IRF8, associated with MS, and PTPN2 associated with Crohn's disease and T1D. Furthermore, a number of single nucleotide polymorphism associations from GWA were located directly within VDR binding intervals, for example, rs13385731 associated with SLE and rs947474 associated with T1D. We also observed significant enrichment of VDR intervals within regions of positive selection among individuals of Asian and European descent. ChIP-seq determination of transcription factor binding, in combination with GWA data, provides a powerful approach to further understanding the molecular bases of complex diseases.",
"Recently, common noncoding variants in the TCF7L2 gene were strongly associated with increased risk of type 2 diabetes in samples from Iceland, Denmark, and the U.S. We genotyped 13 single nucleotide polymorphisms (SNPs) across TCF7L2 in 8,310 individuals in family-based and case-control designs from Scandinavia, Poland, and the U.S. We convincingly confirmed the previous association of TCF7L2 SNPs with the risk of type 2 diabetes (rs7903146T odds ratio 1.40 [95% CI 1.30-1.50], P = 6.74 x 10(-20)). In nondiabetic individuals, the risk genotypes were associated with a substantial reduction in the insulinogenic index derived from an oral glucose tolerance test (risk allele homozygotes have half the insulin response to glucose of noncarriers, P = 0.003) but not with increased insulin resistance. These results suggest that TCF7L2 variants may act through insulin secretion to increase the risk of type 2 diabetes.",
"Transcription factors (TFs) direct gene expression by binding to DNA regulatory regions. To explore the evolution of gene regulation, we used chromatin immunoprecipitation with high-throughput sequencing (ChIP-seq) to determine experimentally the genome-wide occupancy of two TFs, CCAAT/enhancer-binding protein alpha and hepatocyte nuclear factor 4 alpha, in the livers of five vertebrates. Although each TF displays highly conserved DNA binding preferences, most binding is species-specific, and aligned binding events present in all five species are rare. Regions near genes with expression levels that are dependent on a TF are often bound by the TF in multiple species yet show no enhanced DNA sequence constraint. Binding divergence between species can be largely explained by sequence changes to the bound motifs. Among the binding events lost in one lineage, only half are recovered by another binding event within 10 kilobases. Our results reveal large interspecies differences in transcriptional regulation and provide insight into regulatory evolution.",
"Transcription factor 7-like 2 (TCF7L2) has been strongly implicated in type 2 diabetes and cancer. Our goal was to identify the DNA sequences bound by this transcription factor in vivo. We applied chromatin immunoprecipitation and sequencing to globally identify and map human DNA sequences bound by TCF7L2 in the colorectal carcinoma cell line, HCT116, where it is abundantly expressed. We identified 1,095 discrete binding sites across the genome, of which a subset were within 5 kb of 548 annotated NCBI Reference Sequence (RefSeq) genes. Despite using a cancer cell line, the most significant functions represented using pathway analysis software were related to diabetes, genetic disorders and coronary artery disease. As one of the enriched categories was related to genetic disorders, we queried our results against all published genome-wide association studies (GWAS) and found a highly significant over-representation of reported loci from among the genes bound by TCF7L2 within 5 kb (p = 7.50 × 10⁻¹⁵). This observation was primarily driven by excess loci revealed from GWAS of metabolic and cardiovascular traits; however, there was no or only minor enrichment of GWAS-derived loci for cancer, and inflammatory or neurological diseases. Of the specific traits, the most enriched loci were for type 2 diabetes and height. When defining the distance from genes at 50 kb or 500 kb, this enrichment pattern persisted, with some additional evidence for enrichment of cancer-related loci. A highly significant proportion of genes bound by TCF7L2 are known disease-associated loci. These findings suggest that TCF7L2 is a central node in the regulation of human diabetes and other disease-associated genes.",
"Transcription of erythroid-expressed genes and normal erythroid development in vivo are dependent on a regulatory protein (GATA-1) that recognizes a consensus GATA motif. GATA-1 expression is itself restricted to erythroid progenitors and to two related hematopoietic lineages, megakaryocytes and mast cells. During cellular maturation the levels of GATA-1 RNA and protein increase progressively. In an effort to delineate mechanisms by which this pivotal transcription factor is itself regulated we have characterized the mouse GATA-1 gene and cis-elements within its promoter. We find that the isolated promoter retains cell specificity exhibited by the intact gene. Full promoter activity requires the presence of proximal CACCC box sequences and an upstream, double GATA motif that binds a single GATA-1 molecule in an asymmetric fashion. Using in vivo footprinting of mouse erythroleukemic cells we detect protein binding in vivo to both cis-elements. On the basis of these findings we propose that a positive feedback loop mediated through GATA-1 serves two complementary functions: maintenance of the differentiated state by locking the promoter into an \"on\" state, and programming the progressive increase in protein content throughout cellular maturation."
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Activation of hypoxia-inducible factor-1 in intestinal epithelial cells does not enhance colon cancer
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The hypoxic response is mediated by two transcription factors, hypoxia-inducible factor (HIF)-1α and HIF-2α. These highly homologous transcription factors are induced in hypoxic foci and regulate cell metabolism, angiogenesis, cell proliferation, and cell survival. HIF-1α and HIF-2α are activated early in cancer progression and are important in several aspects of tumor biology. HIF-1α and HIF-2α have overlapping and distinct functions. In the intestine, activation of HIF-2α increases inflammation and colon carcinogenesis in mouse models. Interestingly, in ischemic and inflammatory diseases of the intestine, activation of HIF-1α is beneficial and can reduce intestinal inflammation. HIF-1α is a critical transcription factor regulating epithelial barrier function following inflammation. The beneficial value of pharmacological agents that chronically activate HIF-1α is decreased due to the tumorigenic potential of HIFs. The present study tested the hypothesis that chronic activation of HIF-1α may enhance colon tumorigenesis. Two models of colon cancer were assessed, a sporadic and a colitis-associated colon cancer model. Activation of HIF-1α in intestinal epithelial cells does not increase carcinogenesis or progression of colon cancer. Together, the data provide proof of principle that pharmacological activation of HIF-1α could be a safe therapeutic strategy for inflammatory bowel disease.
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"Hypoxia-inducible factor (HIF), a key modulator of the transcriptional response to hypoxia, is increased in colon cancer. However, the role of HIF in colon carcinogenesis in vivo remains unclear. In this study, we found that intestinal epithelium-specific disruption of the von Hippel-Lindau tumor suppressor protein (VHL) resulted in constitutive HIF signaling, and increased HIF expression augmented colon tumorigenesis in the Apc(min/+) intestinal tumor model. Intestine-specific disruption of Vhl increased colon tumor multiplicity and progression from adenomas to carcinomas. These effects were ameliorated in mice with double disruption of Vhl and HIF-2α. Activation of HIF signaling resulted in increased cell survival in normal colon tissue; however, tumor apoptosis was not affected. Interestingly, a robust activation of cyclin D1 was observed in tumors of Apc(min/+) mice in which HIF-2α was activated in the intestine. Consistent with this result, bromodeoxyuridine incorporation indicated that cellular proliferation was increased in colon tumors following HIF activation. Further analysis showed that dysregulation of the intestinal iron absorption transporter divalent metal transporter-1 (DMT-1) was a critical event in HIF-2α-mediated colon carcinogenesis. These data provide a mechanistic basis for the widely reported link between iron accumulation and colon cancer risk. Together, our findings show that a chronic increase in HIF-2α in the colon initiates protumorigenic signaling, which may have important implications in developing preventive and therapeutic strategies for colon cancer.",
"Sites of inflammation are characterized by significant changes in metabolic activity. Shifts in energy supply and demand can result in diminished delivery and/or availability of oxygen, leading to inflammation-associated tissue hypoxia and metabolic acidosis. These shifts in tissue metabolism, as indicated by previous studies, are frequently associated with vasculitis and profound recruitment of inflammatory cell types, particularly myeloid cells such as neutrophils (PMN) and monocytes. Here, we review recent work addressing the influence of hypoxia on development of inflammatory lesions, with particular emphasis on molecular pathways regulated by hypoxia-inducible factor (HIF).",
"Hypoxic inflammation (decreased oxygen tension at sites of inflammation) is a feature of inflammatory bowel disease (IBD). The hypoxia response is mediated by the transcription factors hypoxia-inducible factor (HIF) 1α and endothelial PAS domain protein 1 (EPAS1 or HIF2α), which are induced in intestinal tissues of patients with IBD. HIF1α limits intestinal barrier dysfunction, but the role of EPAS1 has not been assessed under conditions of hypoxic inflammation or in models of IBD. Acute colitis was induced by administration of Citrobacter rodentium or dextran sulfate sodium (DSS) to transgenic hypoxia reporter mice (oxygen-dependent degradation-luciferase), mice with conditional overexpression of Epas1 (Epas1(LSL/LSL)), mice with intestinal epithelium-specific deletion of Epas1 (Epas1(ΔIE) ), or wild-type littermates (controls). Colon tissues from these mice and from patients with ulcerative colitis or Crohn's disease were assessed by histologic and immunoblot analyses, immunohistochemistry, and quantitative polymerase chain reaction. Levels of hypoxia and EPAS1 were increased in colon tissues of mice after induction of colitis and patients with ulcerative colitis or Crohn's disease compared with controls. Epas1(ΔIE) mice had attenuated colonic inflammation and were protected from DSS-induced colitis. Intestine-specific overexpression of EPAS1, but not HIF-1α, led to spontaneous colitis, increased susceptibility to induction of colitis by C rodentium or DSS, and reduced survival times compared with controls. Disruption of intestinal epithelial EPAS1 attenuated the inflammatory response after administration of DSS or C rodentium, and intestine-specific overexpression of EPAS1 increased this response. We found EPAS1 to be a positive regulator of tumor necrosis factor-α production by the intestinal epithelium. Blocking tumor necrosis factor-α completely reduced hypoxia-induced intestinal inflammation. EPAS1 is a transcription factor that activates mediators of inflammation, such as tumor necrosis factor-α, in the intestinal epithelium and promotes development of colitis in mice.",
"Epithelial cells of the kidney represent a primary target for hypoxic injury in ischemic acute renal failure (ARF); however, the underlying transcriptional mechanism(s) remain undefined. In this study, human proximal tubular epithelial cells (HK-2) exposed to hypoxia in vitro demonstrated a non-lethal but dysfunctional phenotype, closely reflective of the epithelial pathobiology of ARF. HK-2 cells exposed to hypoxia demonstrated increased paracellular permeability, decreased proliferation, loss of tight junctional integrity, and significant actin disassembly in the absence of cell death. Microarray analysis of transcriptomic changes underlying this response identified a distinct cohort of 48 genes with a closely shared hypoxia-dependent expression profile. Within this hypoxia-sensitive cluster were genes identified previously as hypoxia-inducible factor-1 (HIF-1)-dependent (e.g. vascular endothelial growth factor and adrenomedullin) as well as genes not previously known to be hypoxia-responsive (e.g. stanniocalcin 2). In hypoxia, HIF-1 bound to evolutionarily conserved hypoxia-response elements (HRE) in the promoters of these genes as well as to the HRE consensus motif. A further subset of these genes, not associated with transcriptional regulation by HIF-1, was also present, suggesting alternative HIF-1-independent pathways. Overexpression of HIF-1 alpha in normoxia induced the expression of a significant number of the hypoxia-dependent genes; however, it did not induce the pathophysiologic epithelial response. In summary, hypoxia-elicited alterations in renal proximal tubular epithelial cells in vitro closely resemble the epithelial pathophysiology of ARF. Our data indicate that although this event may rely heavily on HIF-1-dependent gene transcription, it is likely that separate hypoxia-dependent transcriptional regulators also play a role.",
"Mucosal epithelial cells are uniquely equipped to maintain barrier function even under adverse conditions. Previous studies have implicated hypoxia in mucosal tissue damage resulting from both acute and chronic inflammation. Given the importance of the transcriptional regulator hypoxia-inducible factor-1 (HIF-1) for adaptive hypoxia responses, we hypothesized that HIF-1 may serve as a barrier-protective element during mucosal inflammation. Initial studies of hapten-based murine colitis revealed extensive mucosal hypoxia and concomitant HIF-1 activation during colitis. To study this in more detail, we generated 2 mouse lines with intestinal epithelium-targeted expression of either mutant Hif1a (inability to form HIF-1) or mutant von Hippel-Lindau gene (Vhlh; constitutively active HIF-1). Studies of colitis in these mice revealed that decreased HIF-1 expression correlated with more severe clinical symptoms (mortality, weight loss, colon length), while increased HIF levels were protective in these parameters. Furthermore, colons with constitutive activation of HIF displayed increased expression levels of HIF-1-regulated barrier-protective genes (multidrug resistance gene-1, intestinal trefoil factor, CD73), resulting in attenuated loss of barrier during colitis in vivo. Taken together, these studies provide insight into tissue microenvironmental changes during model inflammatory bowel disease and identify HIF-1 as a critical factor for barrier protection during mucosal insult.",
"Mucosal organs such as the intestine are supported by a rich and complex underlying vasculature. For this reason, the intestine, and particularly barrier-protective epithelial cells, are susceptible to damage related to diminished blood flow and concomitant tissue hypoxia. We sought to identify compensatory mechanisms that protect epithelial barrier during episodes of intestinal hypoxia. Initial studies examining T84 colonic epithelial cells revealed that barrier function is uniquely resistant to changes elicited by hypoxia. A search for intestinal-specific, barrier-protective factors revealed that the human intestinal trefoil factor (ITF) gene promoter bears a previously unappreciated binding site for hypoxia-inducible factor (HIF)-1. Hypoxia resulted in parallel induction of ITF mRNA and protein. Electrophoretic mobility shift assay analysis using ITF-specific, HIF-1 consensus motifs resulted in a hypoxia-inducible DNA binding activity, and loading cells with antisense oligonucleotides directed against the alpha chain of HIF-1 resulted in a loss of ITF hypoxia inducibility. Moreover, addition of anti-ITF antibody resulted in a loss of barrier function in epithelial cells exposed to hypoxia, and the addition of recombinant human ITF to vascular endothelial cells partially protected endothelial cells from hypoxia-elicited barrier disruption. Extensions of these studies in vivo revealed prominent hypoxia-elicited increases in intestinal permeability in ITF null mice. HIF-1-dependent induction of ITF may provide an adaptive link for maintenance of barrier function during hypoxia.",
"Inflammatory bowel diseases are associated with increased expression of zinc-dependent Matrix Metalloproteinase 9 (MMP-9). A stark dysregulation of intestinal mucosal homeostasis has been observed in patients with chronic inflammatory bowel diseases. We therefore sought to determine the contribution of MMP-9 to the pathogenesis of Citrobacter rodentium-induced colitis and its effects on gut microbiome homeostasis. Wild-type and MMP-9-/- mice aged 5-6 weeks were challenged with C. rodentium by orogastric gavage and sacrificed either 10 or 30 days post-infection. Disease severity was assessed by histological analysis of colonic epithelial hyperplasia and by using an in vivo intestinal permeability assay. Changes in the inflammatory responses were measured by using qPCR, and the composition of the fecal microbiome evaluated with both qPCR and terminal restriction fragment length polymorphism. Activation and localization of MMP-9 to the apical surface of the colonic epithelium in response to C. rodentium infection was demonstrated by both zymography and immunocytochemistry. The pro-inflammatory response to infection, including colonic epithelial cell hyperplasia and barrier dysfunction, was similar, irrespective of genotype. Nonmetric multidimensional scaling of terminal restriction fragments revealed a different fecal microbiome composition and C. rodentium colonization pattern between genotypes, with MMP-9-/- having elevated levels of protective segmented filamentous bacteria and interleukin-17, and lower levels of C. rodentium. MMP-9-/- but not wild-type mice were also protected from reductions in fecal microbial diversity in response to the bacterial enteric infection. These results demonstrate that MMP-9 expression in the colon causes alterations in the fecal microbiome and has an impact on the pathogenesis of bacterial-induced colitis in mice."
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Quantitative characterization of the dark noise of fluorescent protein promoters in Bacillus subtilis
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Fluorescent protein promoter reporters are important tools that are widely used for diverse purposes in microbiology, systems biology and synthetic biology and considerable engineering efforts are still geared at improving the sensitivity of the reporter systems. Here we focus on dark noise, i.e. the signal that is generated by the empty vector control. We quantitatively characterize the dark noise of a few common bacterial reporter systems by single cell microscopy. All benchmarked reporter systems generated significant amounts of dark noise that exceed the cellular autofluorescence to different extents. We then reengineered a multicolor set of fluorescent ectopic integration vectors for Bacillus subtilis by introducing a terminator immediately upstream of the promoter insertion site, resulting in an up to 2.7-fold reduction of noise levels. The sensitivity and dynamic range of the new high-performance pXFP_Star reporter system is only limited by cellular autofluorescence. Moreover, based on studies of the rapE promoter of B. subtilis we show that the new pXFP_Star reporter system reliably reports on the weak activity of the rapE promoter whereas the original reporter system fails because of transcriptional interference. Since the pXFP_Star reporter system properly isolates the promoter from spurious transcripts, it is a particularly suitable tool for quantitative characterization of weak promoters in B. subtilis.
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"Quantification of promoter activity or protein expression in gene regulatory networks is generally achieved via measurement of fluorescent protein (FP) intensity, which is related to the true FP concentration by an unknown scaling factor, thereby limiting analysis and interpretation. Here, using approaches originally developed for eukaryotic cells, we show that two-photon (2p) fluorescence fluctuation microscopy, specifically scanning number and brightness (sN&B) analysis, can be applied to determine the absolute concentrations of diffusing FPs in live bacterial cells. First, we demonstrate the validity of the approach, despite the small size of the bacteria, using the central pixels and spatial averaging. We established the lower detection limit at or below 75 nM (~3 molecules of FP/vol(ex)) and the upper detection limit at approximately 10 μM, which can be extended using intensity measurements. We found that the uncertainty inherent in our measurements (<5%) was smaller than the high cell-cell variations observed for stochastic leakage from FP fusions of the lac promoter in the repressed state or the 10 to 25% variation observed on induction. This demonstrates that a reliable and absolute measure of transcriptional noise can be made using our approach, which should make it particularly appropriate for the investigation of stochasticity in gene expression networks.",
"Bacteria form architecturally complex communities known as biofilms in which cells are held together by an extracellular matrix. Biofilms harbor multiple cell types, and it has been proposed that within biofilms individual cells follow different developmental pathways, resulting in heterogeneous populations. Here we demonstrate cellular differentiation within biofilms of the spore-forming bacterium Bacillus subtilis, and present evidence that formation of the biofilm governs differentiation. We show that motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm, and that the localization and percentage of each cell type is dynamic throughout development of the community. Importantly, mutants that do not produce extracellular matrix form unstructured biofilms that are deficient in sporulation. We propose that sporulation is a culminating feature of biofilm formation, and that spore formation is coupled to the formation of an architecturally complex community of cells.",
"Bacteria adapt to environmental stimuli by adjusting their transcriptomes in a complex manner, the full potential of which has yet to be established for any individual bacterial species. Here, we report the transcriptomes of Bacillus subtilis exposed to a wide range of environmental and nutritional conditions that the organism might encounter in nature. We comprehensively mapped transcription units (TUs) and grouped 2935 promoters into regulons controlled by various RNA polymerase sigma factors, accounting for ~66% of the observed variance in transcriptional activity. This global classification of promoters and detailed description of TUs revealed that a large proportion of the detected antisense RNAs arose from potentially spurious transcription initiation by alternative sigma factors and from imperfect control of transcription termination.",
"Spore formation by the bacterium Bacillus subtilis has long been studied as a model for cellular differentiation, but predominantly as a single cell. When analyzed within the context of highly structured, surface-associated communities (biofilms), spore formation was discovered to have heretofore unsuspected spatial organization. Initially, motile cells differentiated into aligned chains of attached cells that eventually produced aerial structures, or fruiting bodies, that served as preferential sites for sporulation. Fruiting body formation depended on regulatory genes required early in sporulation and on genes evidently needed for exopolysaccharide and surfactin production. The formation of aerial structures was robust in natural isolates but not in laboratory strains, an indication that multicellularity has been lost during domestication of B. subtilis. Other microbial differentiation processes long thought to involve only single cells could display the spatial organization characteristic of multicellular organisms when studied with recent natural isolates.",
"E. coli is widely used for systems biology research; there exists a need, however, for tools that can be used to accurately and comprehensively measure expression dynamics in individual living cells. To address this we present a library of transcriptional fusions of gfp to each of about 2,000 different promoters in E. coli K12, covering the great majority of the promoters in the organism. Each promoter fusion is expressed from a low-copy plasmid. We demonstrate that this library can be used to obtain highly accurate dynamic measurements of promoter activity on a genomic scale, in a glucose-lactose diauxic shift experiment. The library allowed detection of about 80 previously uncharacterized transcription units in E. coli, including putative internal promoters within previously known operons, such as the lac operon. This library can serve as a tool for accurate, high-resolution analysis of transcription networks in living E. coli cells.",
"Early during endospore formation in the bacterium Bacillus subtilis, two distinct cell types are formed. The initiation of this developmental pathway requires several physiological conditions (e.g., nutrient deprivation) and is controlled by the Spo0A transcription factor. We have found that in a culture of sporulating cells, there are two subpopulations, one that has initiated the developmental program and activated the expression of early developmental genes and one in which early developmental gene expression remains uninduced. We measured the expression of developmental (spo) genes in single cells of B. subtilis by using spo-lacZ fusions. Cells containing a spo-lacZ fusion were stained with a dye that fluoresces upon hydrolysis by beta-galactosidase, and the fluorescence in individual cells was measured with a flow cytometer. For Spo+ cells, we found that the proportion of the population expressing early developmental genes correlates well with the fraction of the population that eventually produces spores. In addition, mutations that cause a decrease in the amount of activated (phosphorylated) Spo0A transcription factor cause a decrease in the size of the subpopulation expressing early developmental genes that are directly activated by Spo0A approximately P. Again, the size of the subpopulation correlates well with the fraction of cells that produce spores. These results indicate that a threshold level of activated Spo0A (Spo0A approximately P) or of a component of the phosphorylation pathway must accumulate to induce sporulation gene expression and that most of the cells that are able to induce the expression of early genes that are directly activated by Spo0A approximately P go on to produce mature spores.(ABSTRACT TRUNCATED AT 250 WORDS)",
"Induction of prophage lambda inhibits the expression of the gal operon from its cognate promoters. The effect is observed only in cis, and is due to frequent transcription of the gal promoter region by RNA polymerase molecules initiating upstream at the prophage PL promoter. The frequency of transcription initiation at PL is some 30 times greater than that at the gal promoter, Pg1. PL is one of the strongest procaryotic promoters. This \"promoter occlusion\" is essentially complete when the distance between gal and PL is small (less than or equal to 10 kb); and when PL is fully active (that is, in the absence of the cl or cro repressors). We discuss the possibility that promoter occlusion at two lambda promoters, Pint and PR', might play a role in the sequential expression of viral functions."
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Dissociation of language and multiple-demand systems during cognitive processing: a synergistic combination of functional MRI methods
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What is the relationship between language and other high-level cognitive functions? Neuroimaging studies have begun to illuminate this question, revealing that some brain regions are quite selectively engaged during language processing, whereas other "multiple-demand" (MD) regions are broadly engaged by diverse cognitive tasks. Nonetheless, the functional dissociation between the language and MD systems remains controversial. Here, we tackle this question with a synergistic combination of functional MRI methods: we first define candidate language-specific and MD regions in each subject individually (using functional localizers) and then measure blood oxygen level-dependent signal fluctuations in these regions during two naturalistic conditions ("rest" and story-comprehension). In both conditions, signal fluctuations strongly correlate among language regions as well as among MD regions, but correlations across systems are weak or negative. Moreover, data-driven clustering analyses based on these inter-region correlations consistently recover two clusters corresponding to the language and MD systems. Thus although each system forms an internally integrated whole, the two systems dissociate sharply from each other. This independent recruitment of the language and MD systems during cognitive processing is consistent with the hypothesis that these two systems support distinct cognitive functions.
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[
"A fundamental assumption in neuroscience is that brain structure determines function. Accordingly, functionally distinct regions of cortex should be structurally distinct in their connections to other areas. We tested this hypothesis in relation to face selectivity in the fusiform gyrus. By using only structural connectivity, as measured through diffusion-weighted imaging, we were able to predict functional activation to faces in the fusiform gyrus. These predictions outperformed two control models and a standard group-average benchmark. The structure-function relationship discovered from the initial participants was highly robust in predicting activation in a second group of participants, despite differences in acquisition parameters and stimuli. This approach can thus reliably estimate activation in participants who cannot perform functional imaging tasks and is an alternative to group-activation maps. Additionally, we identified cortical regions whose connectivity was highly influential in predicting face selectivity within the fusiform, suggesting a possible mechanistic architecture underlying face processing in humans.",
"Previous neuroimaging research has identified a number of brain regions sensitive to different aspects of linguistic processing, but precise functional characterization of these regions has proven challenging. We hypothesize that clearer functional specificity may emerge if candidate language-sensitive regions are identified functionally within each subject individually, a method that has revealed striking functional specificity in visual cortex but that has rarely been applied to neuroimaging studies of language. This method enables pooling of data from corresponding functional regions across subjects rather than from corresponding locations in stereotaxic space (which may differ functionally because of the anatomical variability across subjects). However, it is far from obvious a priori that this method will work as it requires that multiple stringent conditions be met. Specifically, candidate language-sensitive brain regions must be identifiable functionally within individual subjects in a short scan, must be replicable within subjects and have clear correspondence across subjects, and must manifest key signatures of language processing (e.g., a higher response to sentences than nonword strings, whether visual or auditory). We show here that this method does indeed work: we identify 13 candidate language-sensitive regions that meet these criteria, each present in >or=80% of subjects individually. The selectivity of these regions is stronger using our method than when standard group analyses are conducted on the same data, suggesting that the future application of this method may reveal clearer functional specificity than has been evident in prior neuroimaging research on language.",
"Aerobic glycolysis is defined as glucose utilization in excess of that used for oxidative phosphorylation despite sufficient oxygen to completely metabolize glucose to carbon dioxide and water. Aerobic glycolysis is present in the normal human brain at rest and increases locally during increased neuronal activity; yet its many biological functions have received scant attention because of a prevailing energy-centric focus on the role of glucose as substrate for oxidative phosphorylation. As an initial step in redressing this neglect, we measured the regional distribution of aerobic glycolysis with positron emission tomography in 33 neurologically normal young adults at rest. We show that the distribution of aerobic glycolysis in the brain is differentially present in previously well-described functional areas. In particular, aerobic glycolysis is significantly elevated in medial and lateral parietal and prefrontal cortices. In contrast, the cerebellum and medial temporal lobes have levels of aerobic glycolysis significantly below the brain mean. The levels of aerobic glycolysis are not strictly related to the levels of brain energy metabolism. For example, sensory cortices exhibit high metabolic rates for glucose and oxygen consumption but low rates of aerobic glycolysis. These striking regional variations in aerobic glycolysis in the normal human brain provide an opportunity to explore how brain systems differentially use the diverse cell biology of glucose in support of their functional specializations in health and disease.",
"The brain mechanisms mediating visuospatial attention were investigated by recording event-related potentials (ERPs) during a line-orientation discrimination task. Nonpredictive peripheral cues were used to direct participant's attention involuntarily to a spatial location. The earliest attentional modulation was observed in the P1 component (peak latency about 130 ms), with the valid trials eliciting larger P1 than invalid trials. Moreover, the attentional modulations on both the amplitude and latency of the P1 and N1 components had a different pattern as compared to previous studies with voluntary attention tasks. In contrast, the earliest visual ERP component, C1 (peak latency about 80 ms), was not modulated by attention. Low-resolution brain electromagnetic tomography (LORETA) showed that the earliest attentional modulation occurred in extrastriate cortex (middle occipital gyrus, BA 19) but not in the primary visual cortex. Later attention-related reactivations in the primary visual cortex were found at about 110 ms after stimulus onset. The results suggest that involuntary as well as voluntary attention modulates visual processing at the level of extrastriate cortex; however, at least some different processes are involved by involuntary attention compared to voluntary attention. In addition, the possible feedback from higher visual cortex to the primary visual cortex is faster and occurs earlier in involuntary relative to voluntary attention task.",
"In speaking and comprehending language, word information is retrieved from memory and combined into larger units (unification). Unification operations take place in parallel at the semantic, syntactic and phonological levels of processing. This article proposes a new framework that connects psycholinguistic models to a neurobiological account of language. According to this proposal the left inferior frontal gyrus (LIFG) plays an important role in unification. Research in other domains of cognition indicates that left prefrontal cortex has the necessary neurobiological characteristics for its involvement in the unification for language. I offer here a psycholinguistic perspective on the nature of language unification and the role of LIFG.",
"Magnetic resonance imaging enables the noninvasive mapping of both anatomical white matter connectivity and dynamic patterns of neural activity in the human brain. We examine the relationship between the structural properties of white matter streamlines (structural connectivity) and the functional properties of correlations in neural activity (functional connectivity) within 84 healthy human subjects both at rest and during the performance of attention- and memory-demanding tasks. We show that structural properties, including the length, number, and spatial location of white matter streamlines, are indicative of and can be inferred from the strength of resting-state and task-based functional correlations between brain regions. These results, which are both representative of the entire set of subjects and consistently observed within individual subjects, uncover robust links between structural and functional connectivity in the human brain.",
"During rest, the cerebral cortex displays rich, coordinated patterns of spontaneous activity. The mechanism that shapes these patterns is largely unknown. Here we demonstrate that a Hebbian-like, sustained process plays a role in focusing these coherent patterns. Human subjects used an fMRI-based neurofeedback (NF) paradigm to intensely activate the dorsal anterior cingulate cortex for a single epoch (30 min). Resting-state correlations between all of the cortical voxels' BOLD time courses (functional connectivity) were mapped before, immediately after, and one day after the NF session. We found that the single epoch of cortical activation induced a lasting restructuring of the functional connections according to a Hebbian-like rule. Therefore, the change (increase and decrease) in functional connectivity strength of cortical voxels during rest reflected the level of their prior coactivation during the NF epoch. Interestingly, the effect was significantly enhanced 1 d after the NF activation epoch. The effect was evident in each subject individually, indicating its potential as a diagnostic window into the personal history of prior brain activations of both healthy and abnormal individuals."
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Near-infrared confocal resonance Raman spectroscopy of the red-shifted channelrhodopsin from Chlamydomonas augustae
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Channelrhodopsins (ChRs), which form a distinct branch of the microbial rhodopsin family, control phototaxis in green algae. Because ChRs can be expressed and function in neuronal membranes as light-gated cation channels, they have rapidly become an important optogenetic tool in neurobiology. While channelrhodopsin-2 from the unicellular alga Chlamydomonas reinhardtii (CrChR2) is the most commonly used and extensively studied optogenetic ChR, little is known about the properties of the diverse group of other ChRs. In this study, near-infrared confocal resonance Raman spectroscopy along with hydrogen-deuterium exchange and site-directed mutagenesis were used to study the structure of red-shifted ChR1 from Chlamydomonas augustae (CaChR1). These measurements reveal that (i) CaChR1 has an all-trans-retinal structure similar to those of the light-driven proton pump bacteriorhodopsin (BR) and sensory rhodopsin II but different from that of the mixed retinal composition of CrChR2, (ii) lowering the pH from 7 to 2 or substituting neutral residues for Glu169 or Asp299 does not significantly shift the ethylenic stretch frequency more than 1-2 cm(-1) in contrast to BR in which a downshift of 7-9 cm(-1) occurs reflecting neutralization of the Asp85 counterion, and (iii) the CaChR1 protonated Schiff base (SB) has stronger hydrogen bonding than BR. A model is proposed to explain these results whereby at pH 7 the predominant counterion to the SB is Asp299 (the homologue to Asp212 in BR) while Glu169 (the homologue to Asp85 in BR) exists in a neutral state. We observe an unusual constancy of the resonance Raman spectra over the broad range from pH 9 to 2 and discuss its implications. These results are in accord with recent visible absorption and current measurements of CaChR1 [Sineshchekov, O. A., et al. (2013) Intramolecular proton transfer in channelrhodopsins. Biophys. J. 104, 807-817; Li, H., et al. (2014) Role of a helix B lysine residue in the photoactive site in channelrhodopsins. Biophys. J. 106, 1607-1617].
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[
"Proteorhodopsins are an extensive family of photoactive membrane proteins found in proteobacteria distributed throughout the world's oceans which are often classified as green- or blue-absorbing (GPR and BPR, respectively) on the basis of their visible absorption maxima. GPR and BPR have significantly different properties including photocycle lifetimes and wavelength dependence on pH. Previous studies revealed that these different properties are correlated with a single residue, Leu105 in GPR and Gln105 in BPR, although the molecular basis for the different properties of GPR and BPR has not yet been elucidated. We have studied the unexcited states of GPR and BPR using resonance Raman spectroscopy which enhances almost exclusively chromophore vibrations. We find that both spectra are remarkably similar, indicating that the retinylidene structure of GPR and BPR are almost identical. However, the frequency of a band assigned to the retinal C13-methyl-rock vibration is shifted from 1006 cm (-1) in GPR to 1012 cm (-1) in BPR. A similar shift is observed in the GPR mutant L105Q indicating Leu and Gln residues interact differently with the retinal C13-methyl group. The environment of the Schiff base of GPR and BPR differ as indicated by differences in the H/D induced down-shift of the Schiff base vibration. Residues located in transmembrane helices (D-G) do not contribute to the observed differences in the protein-chromophore interaction between BPR and GPR based on the Raman spectra of chimeras. These results support a model whereby the substitution of the hydrophilic Gln105 in BPR with the smaller hydrophobic Leu105 in GPR directly alters the environment of both the retinal C13 group and the Schiff base.",
"Proteorhodopsins are a recently discovered class of microbial rhodopsins, ubiquitous in marine bacteria. Over 4000 variants have thus far been discovered, distributed throughout the oceans of the world. Most variants fall into one of two major groups, green- or blue-absorbing proteorhodopsin (GPR and BPR, respectively), on the basis of both the visible absorption maxima (530 versus 490 nm) and photocycle kinetics ( approximately 20 versus approximately 200 ms). For a well-studied pair, these differences appear to be largely determined by the identity of a single residue at position 105 (leucine/GPR and glutamine/BPR). We find using a combination of visible and infrared spectroscopy that a second difference is the protonation state of a glutamic acid residue located at position 142 on the extracellular side of helix D. In BPR, Glu142 (the GPR numbering system is used) is deprotonated and can act as an alternate proton acceptor, thus explaining the earlier observations that neutralization of the Schiff base counterion, Asp97, does not block the formation of the M intermediate. In contrast, Glu142 in GPR is protonated and cannot act in this state as an alternate proton acceptor for the Schiff base. On the basis of these findings, a mechanism is proposed for proton pumping in BPR. Because the pKa of Glu142 is near the pH of its native marine environment, changes in pH may act to modulate its function in the cell.",
"Channelrhodopsin-2 (ChR2) is a light-gated cation channel and a member of the family of retinylidene photoreceptors. Since the demonstration of light-induced depolarization of ChR2-expressing animal cell membranes, it was increasingly exploited for light triggering of action potentials. ChR2 conducts cations upon light absorption that embodies retinal isomerization as the primary reaction and a structurally unknown opening mechanism. It is evident from spectroscopic data that protonation reactions at the Schiff base are part of the photocycle, comparable to other microbial-type rhodopsins. However, the connection between the processes at the chromophore site and the channel's pore remained enigmatic. Here, we use slow mutants of ChR2 that we generated by disturbing a postulated hydrogen bond when mutating C128 in the transmembrane (TM) helix 3 and D156 in TM helix 4. The lifetime of the mutants' open state is increased more than 100 times. We investigated the spectral properties of the slow mutants. Whereas the deprotonation of the Schiff base (yielding P390) occurs on the same time scale as that of the wild type, reprotonation to P520 is retarded in the slow mutants and their photocycle is split, leading to the presence of two photointermediates, P390 and P520, in the open state. The photoreactions of P390 and P520 lead to a quenching of the current in electrophysiological measurements. We conclude that the putative hydrogen bond between C128 and D156 is an important structural determinant of the channel's closing reaction. Furthermore, we show that the D156A mutant is even more suitable for light control of excitable cells than C128A.",
"Factors affecting the C = N stretching frequency of protonated retinal Schiff base (RSBH+) were studied with a series of synthetic chromophores and measured under different conditions. Interaction of RSBH+ with nonconjugated positive charges in the vicinity of the ring moiety or a planar polyene conformation (in contrast to the twisted retinal conformation in solution) shifted the absorption maxima but did not affect the C = N stretching frequency. The latter, however, was affected by environmental perturbations in the vicinity of the Schiff base linkage. Diminished ion pairing (i.e., of the positively charged nitrogen to its anion) achieved either by substituting a more bulky counteranion or by designing models with a homoconjugation effect lowered the C = N stretch energy. Decreasing solvation of the positively charged nitrogen leads to a similar trend. These effects in the vicinity of the Schiff base linkage also perturb the deuterium isotope effect observed upon deuteriation of the Schiff base. The results are interpreted by considering the mixing of the C = N stretching and C = N-H bending vibration. The C = N mode is shifted due to electrostatic interaction with nonconjugated positive charges in the vicinity of the Schiff base linkage, an interaction that does not influence the isotope effect. Weak hydrogen bonding between the Schiff base linkage in bacteriorhodopsin (bR) and its counteranion or, alternatively, poor solvation of the positively charged Schiff base nitrogen can account for the C = N stretching frequency of 1640 cm-1 and the deuterium isotope effect of 17 cm-1 observed in this pigment.(ABSTRACT TRUNCATED AT 250 WORDS)",
"We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by approximately 100-fold relative to wild-type bacteriorhodopsin. Time-resolved visible absorption spectroscopy and resonance Raman experiments, respectively, show the presence of long-lived O-like and N-like intermediates in the photocycles of the above mutants. We infer the existence of an equilibrium between the N and O intermediates in the photocycles of these mutants. The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or O intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon. Since the 13-cis----all-trans reisomerization of retinal is completed during the decay of the N and O intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle. This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal.",
"Temporally precise, noninvasive control of activity in well-defined neuronal populations is a long-sought goal of systems neuroscience. We adapted for this purpose the naturally occurring algal protein Channelrhodopsin-2, a rapidly gated light-sensitive cation channel, by using lentiviral gene delivery in combination with high-speed optical switching to photostimulate mammalian neurons. We demonstrate reliable, millisecond-timescale control of neuronal spiking, as well as control of excitatory and inhibitory synaptic transmission. This technology allows the use of light to alter neural processing at the level of single spikes and synaptic events, yielding a widely applicable tool for neuroscientists and biomedical engineers.",
"Proteorhodopsins (PRs), bacterial photoactive proton pumps, were originally detected in the uncultured marine gamma-proteobacterial SAR86 group. PRs are now known to occur in both the gamma and alpha marine proteobacterial lineages. Recent environmental shotgun sequence analysis in the Sargasso Sea has added yet more diversity, and a potentially broader taxonomic distribution, to the PR family. Much remains to be learned, however, about within-taxon PR variability and the broader organismal distribution of different PR types. We report here genomic analyses of large genome fragments from different subgroups of the SAR86 lineage, recovered from naturally occurring bacterioplankton populations in coastal Red Sea and open ocean Pacific waters. Sequence comparisons were performed on large bacterial artificial chromosomes (BACs) bearing both rRNA and PR genes, derived from different SAR86 subgroups. Our analyses indicated the presence of different PR sequence types within the same SAR86 rRNA subgroup. The data suggested that the distribution of particular PR types does not necessarily parallel the phylogenetic relationship inferred from highly conserved genes such as rRNA. Further analyses of the genomic regions flanking PR also revealed a potential pathway for the biosynthesis of retinal, the PR chromophore that is required to generate the functionally active photoprotein. Finally, comparison of our results with recently reported Sargasso Sea environmental shotgun sequence assemblies demonstrated the utility of BAC clones for interpreting environmental shotgun sequence data, much of which is represented in short contigs that have an overall low depth of coverage."
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Cannabinoid receptor 2 and the immune system
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It has been well appreciated that the endocannabinoid system can regulate immune responses via the cannabinoid receptor 2 (CB2), which is primarily expressed by cells of the hematopoietic system. The endocannabinoid system is composed of receptors, ligands and enzymes controlling the synthesis and degradation of endocannabinoids. Along with endocannabinoids, both plant-derived and synthetic cannabinoids have been shown to bind to and signal through CB2 via G proteins leading to both inhibitory and stimulatory signals depending on the biological process. Because no cannabinoid ligand has been identified that only binds to CB2, the generation of mice deficient in CB2 has greatly expanded our knowledge of how CB2 contributes to immune cell development and function in health and disease. In regards to humans, genetic studies have associated CB2 with a variety of human diseases. Here, we review the endocannabinoid system with an emphasis on CB2 and its role in the immune system.
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"Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS) in a complex and dose-dependent manner. Although CNS depression and analgesia are well documented effects of the cannabinoids, the mechanisms responsible for these and other cannabinoid-induced effects are not so far known. The hydrophobic nature of these substances has suggested that cannabinoids resemble anaesthetic agents in their action, that is, they nonspecifically disrupt cellular membranes. Recent evidence, however, has supported a mechanism involving a G protein-coupled receptor found in brain and neural cell lines, and which inhibits adenylate cyclase activity in a dose-dependent, stereoselective and pertussis toxin-sensitive manner. Also, the receptor is more responsive to psychoactive cannabinoids than to non-psychoactive cannabinoids. Here we report the cloning and expression of a complementary DNA that encodes a G protein-coupled receptor with all of these properties. Its messenger RNA is found in cell lines and regions of the brain that have cannabinoid receptors. These findings suggest that this protein is involved in cannabinoid-induced CNS effects (including alterations in mood and cognition) experienced by users of marijuana.",
"The endocannabinoid system represents a novel therapeutic target for autoimmune and chronic inflammatory diseases. IL-12 and IL-23 are functionally related heterodimeric cytokines that play a crucial role in the pathogenesis of multiple sclerosis (MS). In the present study we investigated the effects of the endocannabinoid anandamide (AEA) on the inducible expression of the biologically active cytokines IL-12p70 and IL-23, and their forming subunits, in activated microglial cells. We also studied the signalling pathways involved in the regulation of IL-12p70/IL-23 expression and addressed the possible interactions of AEA with these pathways. Here, we show that AEA was capable to inhibit the production of biologically active IL-12p70 and IL-23, and their subunits, by activated human and murine microglial cultures. Treatment of activated microglial cells with inhibitors of several mitogen-activated protein kinase (MAPK) reveals that AEA acts through the ERK1/2 and JNK pathways to down-regulate IL-12p70 and IL-23. These effects were partially mediated by CB2 receptor activation. Together, our results provide the first demonstration of a role of AEA in inhibiting IL-12p70/IL-23 axis in human and murine microglial cells via the CB2 receptor and suggest that the pharmacological manipulation of the endocannabinoid system is a potential tool for treating brain inflammatory and autoimmune diseases, like MS.",
"Many of the pharmacological effects of Delta(9)-tetrahydrocannabinol are mediated through CB(1) and CB(2) cannabinoid receptors. However, with the discovery of endogenous cannabinoids, some discrepancies have arisen. Furthermore, unlike the CB(1) receptor, the sequences of the mouse and human CB(2) receptor are divergent, raising the possibility of species specificity. The gene for the rat CB(2) receptor was cloned, expressed, and its properties compared with those of mouse and human CB(2) receptors. Sequence analysis of the coding region of the rat CB(2) genomic clone indicates 90% nucleic acid identity (93% amino acid identity) between rat and mouse and 81% nucleic acid identity (81% amino acid identity) between rat and human. The rat CB(2) receptor was stably expressed in human embryonic kidney-293 cells to examine its pharmacology. The rat CB(2) showed low affinity for anandamide, an endogenous ligand shown to act at the CB(1) receptor. In contrast, high-affinity binding for SR144528 (CB(2)-selective antagonist) as well as several cannabinoid receptor agonists was observed. Coupling to adenylate cyclase was observed. Aspects of the pharmacology of palmitoylethanolamide were also examined. It bound to CB(1) and CB(2) receptors with low affinity and stimulated GTPgammaS binding in the cerebellum and CB(2)-expressing cell lines with low potency. The data in this study suggest that the discrepancies in affinities between rat and human may represent species differences. The rat CB(2) receptor genomic clone will be a useful tool for studying the function and regulation of CB(2) in rats.",
"The cannabinoid system is known to be important in neuronal regulation, but is also capable of modulating immune function. Although the CNS resident microglial cells have been shown to express the CB2 subtype of cannabinoid receptor during non-immune-mediated pathological conditions, little is known about the expression of the cannabinoid system during immune-mediated CNS pathology. To examine this question, we measured CB2 receptor mRNA expression in the CNS of mice with experimental autoimmune encephalomyelitis (EAE) and, by real-time PCR, found a 100-fold increase in CB2 receptor mRNA expression during EAE onset. We next determined whether microglial cells specifically express the CB2 receptor during EAE, and found that activated microglial cells expressed 10-fold more CB2 receptor than microglia in the resting state. To determine the signals required for the up-regulation of the CB2 receptor, we cultured microglial cells with combinations of gamma-interferon (IFN-gamma) and granulocyte) macrophage-colony stimulating factor (GM-CSF), which both promote microglial cell activation and are expressed in the CNS during EAE, and found that they synergized, resulting in an eight to 10-fold increase in the CB2 receptor. We found no difference in the amount of the CB2 receptor ligand, 2-arachidonylglycerol (2-AG), in the spinal cord during EAE. These data demonstrate that microglial cell activation is accompanied by CB2 receptor up-regulation, suggesting that this receptor plays an important role in microglial cell function in the CNS during autoimmune-induced inflammation.",
"2-Arachidonoylglycerol (2-AG), an endogenous cannabinoid receptor ligand, was shown to induce rapid phosphorylation of p42/44 mitogen-activated protein kinase (MAP kinase) in HL-60 cells. We confirmed that the enzyme activity of p42/44 MAP kinase in HL-60 cells was augmented markedly when the cells were stimulated with 2-AG. The addition of SR144528, a cannabinoid CB2 receptor-specific antagonist, to the cells prior to the addition of 2-AG abolished the response induced by 2-AG, indicating that the CB2 receptor is involved in the response. G protein G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the cells nullified the response induced by 2-AG. CP55940 and anandamide also induced the activation of p42/44 MAP kinase, although the activation by anandamide was less pronounced than that by 2-AG or CP55940. These results suggest that 2-AG may play an important physiological role in this type of cell through the activation of the p42/44 MAP kinase cascade.",
"Two cannabinoid receptors, designated neuronal (or CB1) and peripheral (or CB2), have recently been cloned. Activation of CB1 receptors leads to inhibition of adenylate cyclase and N-type voltage-dependent Ca2+ channels. Here we show, using a CB2 transfected Chinese hamster ovary cell line, that this receptor binds a variety of tricyclic cannabinoid ligands as well as the endogenous ligand anandamide. Activation of the CB2 receptor by various tricyclic cannabinoids inhibits adenylate cyclase activity and this inhibition is pertussis toxin sensitive indicating that this receptor is coupled to the Gi/G(o) GTP-binding proteins. Interestingly, contrary to results with CB1, anandamide did not inhibit the CB2 coupled adenylate cyclase activity and delta 9-tetrahydrocannabinol had only marginal effects. These results characterize the CB2 receptor as a functional and distinctive member of the cannabinoid receptor family.",
"In spite of the rapid advances in our understanding of vanilloid-receptor pharmacology in the PNS, the function of vanilloid receptors in the brain has remained elusive. Recently, the endocannabinoid anandamide has been proposed to function as an endogenous agonist at the vanilloid receptor VR1. This is an exciting hypothesis because the localization of VR1 overlaps with that of anandamide and its preferred cannabinoid receptor CB(1) in various brain areas. The interaction of anandamide and/or related lipid metabolites with these two completely separate receptor systems in the brain clearly places VR1 in a much broader role than pain perception. At a practical level, the overlapping ligand recognition properties of VR1 and CB(1) might be exploited by medicinal chemistry. For example, arvanil, a 'chimeric' ligand that combines structural features of capsaicin and anandamide, promises to be an interesting lead for new drugs that interact at both vanilloid and cannabinoid receptors."
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First-line persistence and healthcare costs of HIV-1 in patients in the United States
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Recent treatment patterns and cost data associated with HIV in the United States are limited. This study assessed first-line persistence and healthcare costs of HIV-1 in patients by treatment line and CD4 cell count.
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"For antiretroviral therapy, the 95% adherence \"threshold\" is based on nucloside-exposed patients who are receiving partially suppressive, unboosted protease inhibitor regimens. Using unannounced pill counts and electronic medication monitoring, viral suppression is common with a 54%-100% mean adherence level to nonnucleoside reverse-transcriptase-inhibitor regimens. Although perfect adherence is an important goal, viral suppression is possible with moderate adherence to potent regimens.",
"To compare the prevalence of resistance by adherence level in patients treated with non-nucleoside reverse transcriptase inhibitors (NNRTI) or protease inhibitors (PI). Also to examine the mechanism of differential class-specific adherence-resistance relationships, focusing on the patient-derived capacity of wild-type and drug-resistant recombinant variants to replicate in vitro in the presence of variable drug levels. Participants received unannounced pill count measures to assess adherence, viral load monitoring, and genotypic resistance testing. The replicative capacity of drug-susceptible and drug-resistant recombinants was determined using a single-cycle recombinant phenotypic susceptibility assay. Drug exposure was estimated using population-averaged pharmacological measurements adjusted by participant-specific levels of adherence. In the NNRTI-treated group, 69% had resistance at 0-48% adherence compared to 13% at 95-100% (P = 0.01). PI resistance was less common than NNRTI resistance at 0-48% adherence (69% versus 23%; P = 0.01). In multivariate analysis, the odds for PI resistance increased (P = 0.03) while the odds for NNRTI resistance decreased (P = 0.04) with improving adherence. Individuals with drug-resistant variants were more likely to have levels of drug exposure where the resistant variant was more fit than the drug-susceptible variant in vitro, while those with drug-susceptible virus were more likely to have levels of drug exposure where the drug-susceptible virus was more fit than the drug-resistant variant (P = 0.005). NNRTI resistance was more common than PI resistance at low levels of adherence. Class-specific adherence-resistance relationships are associated with the relative replicative capacity of drug-resistant versus wild-type variants to replicate in the presence of clinically relevant drug levels.",
"We hypothesized that the percent adherence to antiretroviral therapy necessary to maintain HIV suppression would decrease with longer duration of viral suppression. Eligible participants were identified from the REACH cohort of marginally housed HIV infected adults in San Francisco. Adherence to antiretroviral therapy was measured through pill counts obtained at unannounced visits by research staff to each participant's usual place of residence. Marginal structural models and targeted maximum likelihood estimation methodologies were used to determine the effect of adherence to antiretroviral therapy on the probability of virologic failure during early and late viral suppression. A total of 221 subjects were studied (median age 44.1 years; median CD4+ T cell nadir 206 cells/mm(3)). Most subjects were taking the following types of antiretroviral regimens: non-nucleoside reverse transcriptase inhibitor based (37%), ritonavir boosted protease inhibitor based (28%), or unboosted protease inhibitor based (25%). Comparing the probability of failure just after achieving suppression vs. after 12 consecutive months of suppression, there was a statistically significant decrease in the probability of virologic failure for each range of adherence proportions we considered, as long as adherence was greater than 50%. The estimated risk difference, comparing the probability of virologic failure after 1 month vs. after 12 months of continuous viral suppression was 0.47 (95% CI 0.23-0.63) at 50-74% adherence, 0.29 (CI 0.03-0.50) at 75-89% adherence, and 0.36 (CI 0.23-0.48) at 90-100% adherence. The risk of virologic failure for adherence greater than 50% declines with longer duration of continuous suppression. While high adherence is required to maximize the probability of durable viral suppression, the range of adherence capable of sustaining viral suppression is wider after prolonged periods of viral suppression.",
"To evaluate the effects of HIV infection on T cell turnover, we examined levels of DNA synthesis in lymph node and peripheral blood mononuclear cell subsets by using ex vivo labeling with BrdUrd. Compared with healthy controls (n = 67), HIV-infected patients (n = 57) had significant increases in the number and fraction of dividing CD4(+) and CD8(+) T cells. Higher percentages of dividing CD4(+) and CD8(+) T cells were noted in patients with the higher viral burdens. No direct correlation was noted between rates of T cell turnover and CD4(+) T cell counts. Marked reductions in CD4(+) and CD8(+) T cell proliferation were seen in 11/11 patients 1-12 weeks after initiation of highly active antiretroviral therapy (HAART). These reductions persisted for the length of the study (16-72 weeks). Decreases in naive T cell proliferation correlated with increases in the levels of T cell receptor rearrangement excision circles. Division of CD4(+) and CD8(+) T cells increased dramatically in association with rapid increases in HIV-1 viral loads in 9/9 patients 5 weeks after termination of HAART and declined to pre-HAART-termination levels 8 weeks after reinitiation of therapy. These data are consistent with the hypothesis that HIV-1 infection induces a viral burden-related, global activation of the immune system, leading to increases in lymphocyte proliferation."
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Genetic predictors of antidepressant response.
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Major depressive disorder is the most common psychiatric disorder, worldwide, yet response and remission rates are still unsatisfactory. The identification of genetic predictors of antidepressant (AD) response could provide a promising opportunity to improve current AD efficacy through the personalization of treatment. The major steps and findings along this path are reviewed together with their clinical implications and limitations.
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[
"Disturbances in serotonergic neurotransmission system have been implicated in the etiology of mood disorders. As the importance of genetic factors is well established, genes encoding for proteins of the serotonergic pathway are important candidates to unravel the underlying genetic contribution. We examined two polymorphisms in the serotonin-2A-receptor gene (5-HT2A; T102C and His452Tyr) and the insertion/deletion polymorphism in the promoter region of the serotonin transporter (5-HTTLPR) in a sample of 173 patients with major depression and 121 healthy controls. No statistical significant differences between patients and controls were found for any of the three investigated polymorphisms, neither in the distribution of the genotypes nor in allele frequencies. However, concerning the 5-HTTLPR polymorphism, the frequency of S/S (short allele) homozygotes was higher (23.1%) than in the control group (14.0%), but this failed to reach significance. Moreover we observed a different treatment response in patients with one or two C-alleles of the T102C polymorphism, with a significantly higher decrease in HAMD-17 (ANOVA: d.f. = 1, F = 5,288, P = 0.023) after 4 weeks of antidepressant treatment. Overall our results suggest that the investigated 5-HT2A and 5-HTTLPR polymorphisms are not major susceptibility factors in the etiology of major depression. However, subtypes might be identified at least on a basis of differential treatment response.",
"Polymorphisms in the serotonin transporter gene (5-HTT) may influence antidepressant response to selective serotonin reuptake inhibitors (SSRIs). The norepinephrine transporter (NET) is the analogous target for norepinephrine reuptake inhibitors (NRIs). To determine whether antidepressant responses to SSRIs or NRIs are associated with genetic polymorphisms of the corresponding monoamine transporters. A 6-week naturalistic treatment study with blinded outcome evaluation of 241 Korean inpatients and outpatients with major depression at an academic psychiatry service. Patients were recruited to the study from March 1998 through February 2003. Treatment with an SSRI (fluoxetine or sertraline; n = 136) or an NRI (nortriptyline; n = 105) antidepressant. Adherence was assessed by measuring plasma concentration at 4 weeks. Patients were genotyped for s/l polymorphisms in 5-HTT promoter region (5-HTTLPR), 5-HTT intron 2 s/l variation, and NET G1287A variation of exon 9. An SSRI and NRI response (defined as > or =50% decrease in Hamilton Rating Scale for Depression score at 6 weeks). NRI response was associated with the NET G1287A polymorphism (odds ratio [OR], 7.54; 95% confidence interval [CI], 2.53-22.49; P<.001). An SSRI response was associated with the 5-HTT intron 2 s/l variation (OR, 20.11; 95% CI, 4.27-94.74; P<.001). The 5-HTTLPR was also associated with an SSRI response (OR, 3.34; 95% CI, 1.41-7.91; P = .006). In contrast to studies in white patients, the favorable allele for SSRI response was S 5-HTTLPR. The S 5-HTTLPR was associated also with NRI response (OR, 3.73; 95% CI, 1.32-10.53; P = .01). The NET polymorphism was not associated with an SSRI response. The NET G1287A GG genotype (56% of the population) was associated with better response to the NRI (83.3% [35/42]) than to SSRI (58.7% [44/75]) (OR, 3.52; 95% CI, 1.39-8.95; P = .006). Some genotype combinations were associated with high rates of antidepressant response and others with low rates of response. Monoamine transporter gene polymorphisms were associated with response to antidepressants with homologous monoamine transporter targets. Combinations of polymorphisms were informative for response and nonresponse. Confirmation of these preliminary findings would permit refined pharmacogenetic selection of antidepressant treatment.",
"The efficacy of antidepressant drug treatment in depression is unsatisfactory; 1 in 3 patients does not fully recover even after several treatment trials. Genetic factors and clinical characteristics contribute to the failure of a favorable treatment outcome. To identify genetic and clinical determinants of antidepressant drug treatment outcome in depression. Genomewide pharmacogenetic association study with 2 independent replication samples. We performed a genomewide association study in patients from the Munich Antidepressant Response Signature (MARS) project and in pooled DNA from an independent German replication sample. A set of 328 single-nucleotide polymorphisms highly related to outcome in both genomewide association studies was genotyped in a sample of the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) study. A total of 339 inpatients with a depressive episode (MARS sample), a further 361 inpatients with depression (German replication sample), and 832 outpatients with major depression (STAR*D sample). We generated a multilocus genetic variable that described the individual number of alleles of the selected single nucleotide polymorphisms associated with beneficial treatment outcome in the MARS sample (\"response\" alleles) to evaluate additive genetic effects on antidepressant drug treatment outcome. Multilocus analysis revealed a significant contribution of a binary variable that categorized patients as carriers of a high vs low number of response alleles in the prediction of antidepressant drug treatment outcome in both samples (MARS and STAR*D). In addition, we observed that patients with a comorbid anxiety disorder combined with a low number of response alleles showed the least favorable outcome. These results demonstrate the importance of multiple genetic factors combined with clinical features in the prediction of antidepressant drug treatment outcome, which underscores the multifactorial nature of this trait.",
"Several investigations have suggested that alterations in serotonin 6 (5-HT6) receptors might be associated with the pathophysiology of major depressive disorder (MDD), and that 5-HT6 receptors might be a therapeutic target for serotonin selective reuptake inhibitor (SSRI) in MDD. To evaluate the association between HTR6 and the efficacy of SSRI treatment in Japanese MDD patients, we conducted a case-control study in a Japanese population sample. We selected five tagging SNPs (rs6693503, rs1805054, rs4912138, rs3790757 and rs9659997), and performed an association analysis of HTR6 and the efficacy of SSRI treatment in 260 MDD patients. We did not detect an association between tagging SNPs in HTR6 and the therapeutic response to SSRI in MDD in allele/genotype or haplotype analysis. HTR6 may not play an important role in the pathophysiology of SSRI response in the Japanese population. Because our sample was relatively small, statistical errors were possible in the results of our association analyses. To overcome these limitations, a replication study using a larger sample may be required for conclusive results.",
"The serotonin transporter (5-HTT) is the site of primary action for the selective serotonin reuptake inhibitors (SSRIs). Previous Western reports have demonstrated that the lallele of the 5-HTT gene-linked polymorphic-region (5-HTTLPR) polymorphism is associated with better SSRI antidepressive effects than the s allele, however, another study of a Korean population has produced a contrasting finding. The present study tested the hypothesis that the 5-HTTLPR genetic polymorphism is associated with SSRI antidepressant response by evaluating total and cluster depressive symptoms for 121 Chinese patients diagnosed with major depression. Analysis of the results reveals that patients with the l/l genotype had a significantly better response to SSRI (fluoxetine) when compared with s allele carriers, as evaluated on the basis of total (P = 0.013), core (P = 0.011), and psychic-anxiety (P = 0.005) and somatic-anxiety (P = 0.002) Hamilton Depression Rating Scale-score percentage change. Our findings confirm reports that the l allele is associated with better SSRI response.",
"With a multitude of antidepressants available, predictors of response to different classes of antidepressants are of considerable interest. The purpose of the present study was to determine whether norepinephrine transporter gene (NET) and serotonin transporter gene (5-HTT) polymorphisms are associated with the antidepressant response to milnacipran, a dual serotonin/norepinephrine reuptake inhibitor. Ninety-six Japanese patients with major depressive disorder were treated with milnacipran, 50-100 mg/day, for 6 weeks. Severity of depression was assessed with the Montgomery-Asberg Depression Rating Scale. Assessments were carried out at baseline and at 1, 2, 4, and 6 weeks of treatment. The method of polymerase chain reaction was used to determine allelic variants. Eighty patients completed the study. The presence of the T allele of the NET T-182C polymorphism was associated with a superior antidepressant response, whereas the A/A genotype of the NET G1287A polymorphism was associated with a slower onset of therapeutic response. In contrast, no influence of 5-HTT polymorphisms on the antidepressant response to milnacipran was detected. The results suggest that NET but not 5-HTT polymorphisms in part determine the antidepressant response to milnacipran.",
"Serotonin systems appear to play a key role in the pathogenesis of major depression and the therapeutic mechanisms of antidepressants. The firing rate of dorsal raphe serotonergic neurons is controlled by somatodendritic 5-hydroxytryptamine 1A (HTR1A) autoreceptors, and desensitization of these receptors is implicated in the antidepressant mechanism of selective serotonin reuptake inhibitors. We tested whether a functional polymorphism (C-1019G) in the promoter region of the HTR1A gene and serotonin-related genetic variants are related to fluoxetine antidepressant effect. We genotyped the HTR1A C-1019G polymorphism as well as polymorphisms in the serotonin transporter gene-linked polymorphic region (SERTPR), variable-number tandem-repeat polymorphisms in intron 2 (STin2) of the serotonin transporter gene, serotonin 2A receptor (T102C), tryptophan hydroxylase (A218C), and G-protein beta3 subunit (C825T) in 224 Chinese patients from southern Taiwan with major depression, who accepted 4-week fluoxetine treatment and therapeutic evaluation. Our results demonstrated that the HTR1A -1019C/C carriers (P=0.009) and SERTPR l/l carriers (P<0.001) showed a better response to fluoxetine, while other polymorphisms were not associated with fluoxetine therapeutic response. The major limitation of this study is the lack of a placebo control. Future prospective study with placebo control may help to predict and individualize antidepressant treatment."
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Glucosamine Modification of Lipopolysaccharide Increases Resistance to CAMPs
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Bordetella pertussis, the causative agent of whooping cough, has many strategies for evading the human immune system. Lipopolysaccharide (LPS) is an important Gram-negative bacterial surface structure that activates the immune system via Toll-like receptor 4 and enables susceptibility to cationic antimicrobial peptides (CAMPs). We show modification of the lipid A region of LPS with glucosamine increased resistance to numerous CAMPs, including LL-37. Furthermore, we demonstrate that this glucosamine modification increased resistance to outer membrane perturbation.
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[
"Paneth cells are specialized small intestine epithelial cells that contain lysozyme, possess phagocytic properties, and secrete cytoplasmic granules into the intestinal crypt lumen after the entry of bacteria. Recent studies by Ouellette and associates (A. J. Ouellette, R. M. Greco, M. James, D. Frederick, J. Naftilan, and J. T. Fallon, J. Cell Biol. 108:1687-1695, 1989) indicated that murine Paneth cells produce prodefensin mRNA, but the properties of its peptide product were not reported. We purified two closely related defensins, cryptdin 1 and cryptdin 2, from a subcellular fraction of murine small intestine cells that was enriched in Paneth cells. Both peptides contained 35 amino acid residues, including the characteristic defensin \"signature\" of six invariantly conserved cysteines. Cryptdins 1 and 2 were approximately 90 to 95% homologous to each other and to the carboxy-terminal domain of the 93-amino-acid defensin precursor, cryptdin A, described by Ouellette and associates (Ouellette et al., J. Cell Biol. 108:1687-1695, 1989). Both cryptdins exerted bactericidal activity against Listeria monocytogenes EGD and Escherichia coli ML-35p in vitro. Their potency exceeded that of human neutrophil defensin HNP-1 but was considerably lower than that of NP-1, a defensin produced by rabbit neutrophils and alveolar macrophages. Both cryptdins killed mouse-avirulent Salmonella typhimurium 7953S (phoP) much more effectively than its phoP+, mouse-virulent, isogenic counterpart, S. typhimurium 14028S. Our data indicate that mouse intestinal prodefensins are processed into 35-amino-acid mature defensins (cryptdins) with broad-spectrum antimicrobial properties. The production of defensins and lysozyme by Paneth cells may enable them to protect the small intestine from bacterial overgrowth by autochthonous flora and from invasion by potential pathogens that cause infection via the peroral route, such as L. monocytogenes and Salmonella species.",
"We examined the susceptibilities of Bordetella pertussis strains to several antimicrobial peptides by determining the concentration required to inhibit or kill 50% of the bacterial population. The peptides are ranked in decreasing potency as follows: cecropin B > cecropin A >> melittin > cecropin P1 > (ala8,13,18)-magainin II amide > mastoparan = defensin HNP1 > protamine > or = magainin II = magainin I. By using a radial diffusion assay to compare susceptibilities between strains, wild-type B. pertussis BP338 was more resistant than the avirulent bvg mutant strain BP347 and the brk mutant strain BPM2041 to killing by cecropin P1. In contrast, compared with the wild type, the avirulent BP347 strain was highly resistant to killing by protamine and defensin HNP1."
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New technologies for the diagnosis and treatment of postmenopausal osteoporosis.
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This review describes new technologies for the diagnosis and treatment, including fracture risk prediction, of postmenopausal osteoporosis. Four promising technologies and their potential for clinical translation and basic science studies are discussed. These include reference point indentation (RPI), Fourier transform infrared spectroscopy (FTIR), Raman spectroscopy, and magnetic resonance imaging (MRI). While each modality exploits different physical principles, the commonality is that none of them require use of ionizing radiation. To provide context for the new developments, brief summaries are provided for the current state of biomarker assays, fracture risk assessment (FRAX), and other fracture risk prediction algorithms and quantitative ultrasound (QUS) measurements.
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"Small increases in bone mass are commonly seen with existing treatments for osteoporosis, which reduce bone remodelling and primarily prevent bone loss. Since these drugs reduce but do not eliminate risk of fractures, an anabolic agent that would increase bone mass and potentially cure the underlying skeletal problem is needed. We did a 3-year randomised controlled trial to find out the effects of 1-34 human parathyroid hormone (hPTH [1-34], 400 U/25 micrograms daily subcutaneously) in postmenopausal women with osteoporosis taking hormone-replacement therapy (n = 17). The controls were women taking hormone-replacement therapy only (n = 17). The primary outcome was bone-mineral density of the lumbar vertebrae, with bone-mineral density at other sites and vertebral fractures as secondary endpoints. Patients taking hormone-replacement therapy and PTH (1-34) had continuous increase in vertebral bone-mineral density during the 3 years, whereas there was no significant change in the control group. The total increase in vertebral bone-mineral density was 13.0% (p < 0.001); 2.7% at the hip (p = 0.05); and 8.0% in total-body bone mineral (p = 0.002). No loss of bone mass was found at any skeletal site. Increased bone mass was associated with a reduction in the rate of vertebral fractures, which was significant when fractures were taken as a 15% reduction in vertebral height (p = 0.04). During the first 6 months of treatment, serum osteocalcin concentration, which reflects bone formation, increased by more than 55%, whereas excretion of crosslinked n-telopeptide, which reflects bone resorption, increased by only 20%, which suggests some uncoupling of bone formation and resorption. By 6 months, there were similar increases in both markers, which gradually returned towards baseline as the study progressed. Vertebral bone-mineral density increased most during the first year of PTH treatment. We found that PTH has a pronouned anabolic effect on the central skeleton in patients on hormone-replacement therapy. PTH also increases total-body bone mineral, with no detrimental effects at any skeletal site. The increased vertebral mass was associated with a reduced rate of vertebral fracture, despite increased bone turnover. Bone-mass changes may be consistent with a reduction in all osteoporotic fractures. If confirmed in larger studies, these data have important implications for the treatment of postmenopausal osteoporosis.",
"A number of recent studies have suggested that non-invasive measures of bone turnover are associated with bone loss at the forearm in postmenopausal women. Whether bone turnover markers are predictive of bone loss from the clinically important sites of lumbar spine and femoral neck remain unclear, and was the aim of this 4-year prospective study. One hundred and forty-one normal, postmenopausal women (mean age 52.0 +/- 3.3 years, mean menopause duration 20.4 +/- 5.7 months) were recruited for the study in 1988. Fasting early morning samples of blood and urine were collected at the baseline visit and stored at -20 degrees C prior to analysis. Serum was assayed for osteocalcin, oestradiol, oestrone, oestrone sulphate, testosterone, sex hormone binding globulin, dehydroepiandrosterone sulphate and total alkaline phosphatase. Urine was assayed for calcium, hydroxyproline, oestrone glucuronide and the collagen cross-links pyridinoline and deoxypyridinoline using high-performance liquid chromatography. Bone density was measured at the lumbar spine and femoral neck using dual photon absorptiometry at time 0, 12, 24 and 48 months. The mean annual percentage change in bone density (SE) was -1.41% (0.18) at the lumbar spine and -0.86% (0.22) at the femoral neck. There was no evidence of bimodality or a fast loser subgroup as the rates of change were normally distributed. Both simple and multiple stepwise regression analyses revealed no significant correlation between the rates of change in bone density with any biochemical marker, either individually or in combination, despite the study having sufficient power (80%) to detect a correlation of 0.5 between any biochemical marker levels and bone loss. We conclude that single measurements of these markers of bone turnover and endogenous sex hormones appear unlikely to be clinically useful in predicting early postmenopausal bone loss from either the spine or the hip.",
"We measured urinary N-telopeptide of type I collagen (U-NTX) to monitor response to bisphosphonates for osteoporosis. Decrease in U-NTX was associated with increase in spine bone density. A lesser response in U-NTX was more likely in those with secondary osteoporosis or with poor compliance. U-NTX may be a useful early indicator of treatment non-compliance or secondary osteoporosis. This study aims to determine the utility of the bone resorption marker, U-NTX, in the clinical setting, to monitor the response to bisphosphonate therapy (alendronate and risedronate) for osteoporosis. A retrospective evaluation of data collected as part of the bone turnover marker monitoring service in the Metabolic Bone Centre, Sheffield, UK. Treatment compliance, underlying causes of osteoporosis, change in U-NTX/creatinine (Cr) at 4 months and change in spine and hip bone mineral density (BMD) by dual-energy X-ray absorptiometry were recorded. Treatment response was defined as either a change in U-NTX/Cr greater than a pre-defined least significant change (LSC) of 54 % or to within the lower half of a pre-defined pre-menopausal reference interval (≤ 30 nM BCE/mmol Cr). A greater decrease in U-NTX/Cr at 4 months was associated with a greater increase in spine BMD at 18 months (r = -0.33; P < 0.0001, Pearson's correlation). The mean U-NTX/Cr at 4 months was higher in patients with secondary osteoporosis compared with those with primary osteoporosis (P < 0.01, ANOVA). A lesser response in U-NTX/Cr increased the likelihood of secondary osteoporosis or poor treatment compliance (P = 0.04, Fisher's exact test). A lack of response in U-NTX/Cr to within the lower half of the reference interval was a better indicator of secondary osteoporosis and treatment non-compliance than a change in U-NTX/Cr greater than LSC. Treatment monitoring using U-NTX/Cr has a place in clinical practice for the early identification of non-compliance or presence of secondary osteoporosis.",
"Bisphosphonates are widely used in the treatment of osteoporosis. It has been suggested that bisphosphonate treatment may be associated with atypical femoral fractures (AFFs), severely suppressed bone turnover rate, and decreased mineralization. We studied bone properties using bone quantitative histomorphometry and Fourier transform infrared spectroscopic imaging (FTIRI) on patients with AFFs. Further, the incidence of AFFs was estimated. Patient records of Kuopio University Hospital, Finland from January 2007 to June 2009 were reviewed to identify all patients who had sustained and had been operated for AFF (n = 8). The incidence of AFFs among patients on bisphosphonates was 0.61 fractures/1,000 patients per year, compared to 0.0067/1,000 per year among untreated patients. The patients that underwent bone biopsy (n = 4) were postmenopausal women (aged 55.5-81.1 years) who had been treated with bisphosphonates for over 4 years. Histomorphometry revealed low trabecular bone volume. Bone formation and resorption parameters tended to be low. Trabecular bone single labels were detected in one patient in the region of interest. In the extended label search, trabecular bone double labels were found in two patients. Based on FTIRI results, higher phosphate-to-amide I ratio and collagen maturity were found compared to normal samples. The heterogeneity of phosphate-to-amide I ratio was low. Overall incidence of atypical femoral fractures is low. The poor fracture resistance in some patients on long-term bisphosphonate-therapy could be explained by low bone formation, and changes in bone composition, i.e., higher degree of mineralization, increased collagen maturity, and decreased heterogeneity of the degree of mineralization.",
"Reasonable application of laboratory parameters in prevention, diagnosis, treatment and therapy monitoring of osteoporosis. Physicians from different specialist disciplines (general medicine, geriatrics, gynaecology, urology, internal medicine-especially endocrinology and metabolism, nephrology, laboratory medicine, rheumatology, nuclear medicine, orthopaedics, paediatrics, rehabilitation and physical medicine, radiology, social medicine, transplantation medicine, accident surgery), moreover social insurances, hospitals and self-help groups. Evaluation of aetiology of bone disorders, widening of the therapeutic spectrum for diseases of bone and knowledge on biochemical markers of bone turnover. Improvements in judging the success of therapy and in monitoring the compliance of patients. Research perspectives. Scientific literature and guidelines, consensus meetings. RÉSUMÉ: Basic and specialized laboratory investigations are important in differentiation between primary and secondary osteoporosis for an adequate therapy. Biochemical markers of bone turnover are an additional aid in evaluation of individual fracture risk. These markers identify responders to bone therapy faster than surveillance of bone mineral density, which helps to improve patient's compliance too. Characteristics, preanalytic precautions and applications are presented for selected markers of bone resorption and formation and for parameters regulating bone metabolism.",
"We report the predictive value for hip fracture of a new marker of bone resorption, serum C-telopeptide of type I collagen (CTX), measured on a new automated analyzer, Elecsys. Baseline urinary and serum samples from 212 patients who subsequently had a hip fracture and from 642 controls were analyzed in a nested case control study within the EPIDOS prospective cohort. Each fracture patient was matched with three control patients of the same age who did not fracture. Mean follow-up was 3.3 years (maximum 4.9 years). We measured urinary CTX, urinary free deoxypyridinoline, and serum CTX. Urinary markers were assessed to know whether the magnitude of prediction of hip fracture by this serum marker was similar compared with that given by urinary markers. In the whole group, serum CTX was not predictive of hip fracture risk. When the analysis was restricted to samples taken in the early afternoon (between 1 and 2 P.M.), representing 115 fracture cases and 293 controls, serum CTX was significantly predictive with a relative hazard of 1.86 (95% confidence interval 1.01-3.76) for values above the premenopausal range (mean + 2 SD). For comparison, in the whole group, the relative hazard for fracture of women having a T-score >/= 2 for urinary CTX and free deoxypyridinoline was 1.67 (1.19-2.32), and 2. 07 (1.49-2.9), respectively. Serum CTX from morning samples did not predict hip fractures probably because it was not controlled for time and fasting/nonfasting state. We conclude that serum CTX sampled under controlled conditions significantly predicts the subsequent risk of hip fracture in ambulatory elderly women, with the same magnitude as urinary markers of resorption.",
"The aim was to analyse data on the use of biochemical bone turnover markers (BTM) in postmenopausal osteoporosis. We carried out a comparative analysis of the most important papers concerning BTM in postmenopausal osteoporosis that have been published recently. The BTM levels are influenced by several factors. They are moderately correlated with BMD and subsequent bone loss. Increased levels of bone resorption markers are associated with a higher risk of fracture. Changes in the BTM during the anti-osteoporotic treatment (including combination therapy) reflect the mechanisms of action of the drugs and help to establish their effective doses. Changes in the BTM during the anti-resorptive treatment are correlated with their anti-fracture efficacy. Biological samples should be obtained in a standardised way. BTM cannot be used for prediction of the accelerated bone loss at the level of the individual. BTM help to detect postmenopausal women who are at high risk of fracture; however, adequate practical guidelines are lacking. BTM measurements taken during the anti-resorptive therapy help to identify non-compliers. They may improve adherence to the anti-resorptive therapy and the fall in the BTM levels that exceeds the predefined threshold improves patients' persistence with the treatment. There are no guidelines concerning the use of BTM in monitoring anti-osteoporotic therapy in postmenopausal women.",
"The development of new biochemical markers has made it possible to assess the effects of therapeutic agents on bone turnover more rapidly and precisely. In this early phase II study, we analyzed the effects of short-term, high-dose treatment with risedronate, a potent pyridinyl bisphosphonate, on markers of bone resorption and formation. Resorption markers included urinary free deoxypyridinoline (D-Pyr) crosslinks, N-terminal telopeptide (NTx) and C-terminal telopeptide (CTx) type I collagen crosslinks. Bone formation markers included osteocalcin (OC), bone-specific alkaline phosphatase (BSAP) and the C-terminal peptide of type I procollagen (PICP). All three resorption markers showed rapid, significant (p<0.05) decreases from baseline following daily administration of 30 mg risedronate for 2 weeks. The mean decreases at 2 weeks were 28% for D-Pyr, 61% for NTx and 73% for CTx, respectively. Over the next 10 weeks after treatment, D-Pyr approached baseline while NTx and CTx remained well below baseline values. The markers of bone formation showed little change during therapy but decreased significantly at 4-10 weeks after therapy - an expected outcome of bisphosphonate therapy. Moreover, there was a significant correlation between the early effects on bone resorption markers and the delayed effects on formation markers. This study demonstrates that the approved dose of risedonate (30 mg/day) for Paget's disease is effective at decreasing bone turnover after 2 weeks of treatment, as observed by the sensitive response of bone turnover markers.",
"Bone mineral density (BMD) is used to follow gain or loss of bone mass but cannot detect changes within a short period of time. Biochemical markers of bone turnover may be of value for prediction of individual bone loss. We studied the relation between common inexpensive markers of bone turnover (serum alkaline phosphatase (ALP), osteocalcin (OC), urinary hydroxyproline (OHPr), and calcium (Ca)), BMD, age, and menopause in a combined cross-sectional and longitudinal design comprising 429 pre- and postmenopausal randomly selected women aged 21-79 years (mean 50 years). A follow-up was initiated after 5 years (including 192 of these women), which focused on changes in bone mass and the ability of these four common markers of bone turnover (sampled at baseline) to predict future bone loss. A marked increase was observed for all markers at the beginning of menopause. During the postmenopausal period ALP and Ca decreased to near premenopausal levels, while OC and OHPr remained high even 15 years after menopause. We also found inverse correlations at baseline between the bone markers and BMD, independent of the selected marker or skeletal site, r=-0.14 to -0.46, P<0.05. The correlations between ALP, OC, OHPr, and subsequent bone loss over 5 years, was significant for arm, r=-0.23 to -0.36, P<0.01. Baseline levels of all bone markers correlated significantly at group level with the 5-year follow-up of BMD for all sites. The ability of markers to predict individual bone loss was estimated by a multivariate regression model, which included baseline BMD, age, and body mass index as independent variables. ROC analysis showed a validity of approximately 76% for the forearm model, but was lower for the hip (55%) and lumbar spine (65%). These data show that the common inexpensive biochemical markers of bone turnover ALP, OC, OHPr, and Ca were related to the current bone mass and, moreover, provides information about future bone loss at the individual level. Future investigations should include an evaluation of the clinical relevance of markers of bone turnover in relation to fracture risk.",
"The present study was undertaken to determine whether the addition of an androgen to estrogen therapy in postmenopausal women would alter the skeletal response as determined by measurements of markers of bone formation and resorption. Postmenopausal women were treated for 9 weeks with either a combination of 1.25 mg esterified estrogen and 2.5 mg methyltestosterone (E+A) or 1.25 mg conjugated equine estrogen (CEE). Both groups showed a similar decrease in urinary excretion of the bone resorption markers, deoxypyridinoline, pyridinoline, and hydroxyproline. Patients treated with CEE showed decreases in the serum markers of bone formation, bone-specific alkaline phosphatase, osteocalcin, and C-terminal procollagen peptide. In contrast, subjects treated with E+A showed increases in these markers of bone formation. CEE increased, and E+A decreased serum levels of sex hormone-binding globulin as well as triglycerides and high density lipoprotein levels. Only CEE significantly reduced low density lipoproteins. Both regimens were effective in reducing postmenopausal somatic symptoms, but only E+A had a significant effect on psychological symptoms. We conclude that short term administration of androgen with estrogen may reverse the inhibitory effects of estrogen on bone formation. Long term studies are needed to determine the relative benefits and risks of the combination of estrogen and androgen and whether this results in greater increases in bone mass and strength."
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Effect of Hemin on T Cell Polarization in Chronically Transfused Sickle Cell Disease
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Patients with sickle cell disease (SCD) often require transfusions to treat and prevent worsening anemia and other SCD complications. However, transfusions can trigger alloimmunization against transfused RBCs with serious clinical sequelae. Risk factors for alloimmunization in SCD remain poorly understood. We recently reported altered regulatory T cell (Treg) and Th responses with higher circulating Th1 (IFN-γ(+)) cytokines in chronically transfused SCD patients with alloantibodies as compared with those without alloantibodies. Because monocytes play a critical role in polarization of T cell subsets and participate in clearance of transfused RBCs, we tested the hypothesis that in response to the RBC breakdown product hemin, monocyte control of T cell polarization will differ between alloimmunized and non-alloimmunized SCD patients. Exogenous hemin induced Treg polarization in purified T cell/monocyte cocultures from healthy volunteers through the monocyte anti-inflammatory heme-degrading enzyme heme oxygenase-1. Importantly, hemin primarily through its effect on CD16+ monocytes induced an anti-inflammatory (higher Treg/lower Th1) polarization state in the non-alloimmunized SCD group, whereas it had little effect in the alloimmunized group. Non-alloimmunized SCD CD16+ monocytes expressed higher basal levels of heme oxygenase-1. Furthermore, IL-12, which contributed to a proinflammatory polarization state (low Treg/high Th1) in SCD, was dampened in hemin-treated stimulated monocytes from non-alloimmunized SCD patients, but not in the alloimmunized group. These data suggest that unlike alloimmunized patients, non-alloimmunized SCD CD16+ monocytes in response to transfused RBC breakdown products promote an anti-inflammatory state that is less conducive to alloimmunization.
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"Th17 cells are a recently defined subset of proinflammatory T cells that contribute to pathogen clearance and tissue inflammation by means of the production of their signature cytokine IL-17A (henceforth termed IL-17). Although the in vitro requirements for human Th17 development are reasonably well established, it is less clear what their in vivo requirements are. Here, we show that the production of IL-17 by human Th17 cells critically depends on both the activation status and the anatomical location of accessory cells. In vivo activated CD14+ monocytes were derived from the inflamed joints of patients with active rheumatoid arthritis (RA). These cells were found to spontaneously and specifically promote Th17, but not Th1 or Th2 responses, compared with resting CD14+ monocytes from the blood. Surprisingly, unlike Th17 stimulation by monocytes that were in vitro activated with lipopolysaccharide, intracellular IL-17 expression was induced by in vivo activated monocytes in a TNF-alpha- and IL-1beta-independent fashion. No role for IL-6 or IL-23 production by either in vitro or in vivo activated monocytes was found. Instead, in vivo activated monocytes promoted Th17 responses in a cell-contact dependent manner. We propose that, in humans, newly recruited memory CD4(+) T cells can be induced to produce IL-17 in nonlymphoid inflamed tissue after cell-cell interactions with activated monocytes. Our data also suggest that different pathways may be utilized for the generation of Th17 responses in situ depending on the site or route of accessory cell activation.",
"Red blood cell alloimmunization is a major complication of transfusion therapy. Host immune markers that can predict antibody responders remain poorly described. As regulatory T cells (Tregs) play a role in alloimmunization in mouse models, we analyzed the Treg compartment of a cohort of chronically transfused patients with sickle cell disease (SCD, n = 22) and β-thalassemia major (n = 8) with and without alloantibodies. We found reduced Treg activity in alloantibody responders compared with nonresponders as seen in mice. Higher circulating anti-inflammatory IL-10 levels and lower IFN-γ levels were detected in non-alloimmunized SCD patients. Stimulated sorted CD4+ cells from half of the alloimmunized patients had increased frequency of IL-4 expression compared with nonresponders, indicating a skewed T helper (Th) 2 humoral immune response in a subgroup of antibody responders. All patients had increased Th17 responses, suggesting an underlying inflammatory state. Although small, our study indicates an altered immunoregulatory state in alloantibody responders which may help future identification of potential molecular risk factors for alloimmunization.",
"Red blood cell (RBC) alloimmunization is common in transfused patients. Most studies report on the rate of alloimmunization in chronically transfused patients, which can be as high as 60 percent. Less is known on the incidence of clinically relevant antibodies in accidentally transfused patients. Because the probability of repeat transfusion increases with longer life expectancy, it was wondered to which extend non-chronically transfused alloimmunized patients are prone to form additional antibodies after repeat transfusion events. A 20-year retrospective multicenter study was performed analyzing additional alloantibody formation, against the RH, KEL, FY, JK, and MNS blood group systems. After additional transfusions, 21.4 percent of 653 patients produced additional antibodies, resulting in 157 new antibody specificities. At the end of the study 33.4 percent of patients had multiple antibodies. Eighty of 140 patients (57%) who formed additional antibodies did so after one transfusion episode of a median of 2 units of RBCs. Based on the antigen profile of 316 patients, 83 percent of antibodies could have been prevented by extended matching for the C, E, c, K, Fy(a), and Jk(a) antigens. Considering the current available donors in our region, 1 to 10 percent of potential donors would be available for 39 percent of patients and greater than 10 percent of potential donors for 61 percent of patients. It has been shown that nonhematooncologic alloimmunized patients are high antibody responders, with a more than 20 times increased risk to form antibodies compared to first-time alloimmunization risk. If extended matching for C, c, E, K, Fy(a), and Jk(a) antigens in the future is considered, this group should be taken into account.",
"Circulating blood monocytes supply peripheral tissues with macrophage and dendritic cell (DC) precursors and, in the setting of infection, also contribute directly to immune defense against microbial pathogens. In humans and mice, monocytes are divided into two major subsets that either specifically traffic into inflamed tissues or, in the absence of overt inflammation, constitutively maintain tissue macrophage/DC populations. Inflammatory monocytes respond rapidly to microbial stimuli by secreting cytokines and antimicrobial factors, express the CCR2 chemokine receptor, and traffic to sites of microbial infection in response to monocyte chemoattractant protein (MCP)-1 (CCL2) secretion. In murine models, CCR2-mediated monocyte recruitment is essential for defense against Listeria monocytogenes, Mycobacterium tuberculosis, Toxoplasma gondii, and Cryptococcus neoformans infection, implicating inflammatory monocytes in defense against bacterial, protozoal, and fungal pathogens. Recent studies indicate that inflammatory monocyte recruitment to sites of infection is complex, involving CCR2-mediated emigration of monocytes from the bone marrow into the bloodstream, followed by trafficking into infected tissues. The in vivo mechanisms that promote chemokine secretion, monocyte differentiation and trafficking, and finally monocyte-mediated microbial killing remain active and important areas of investigation.",
"Infection with the human immunodeficiency virus HIV-1 is associated with the expansion of a CD14lowCD16high monocyte subset in peripheral blood. This subset, which represents a minor subpopulation of monocytes in healthy individuals, increases during HIV infection and, in patients with AIDS, may represent up to 40% of the total circulating monocyte cell population. The CD14lowCD16high circulating monocytes co-express MAX.1, p150,95 and HLA-DR which are typical of tissue macrophage markers. These cells also express higher levels of intracellular interleukin (IL)-1 alpha and tumor necrosis factor (TNF)-alpha than the CD14highCD16low monocyte population from the same patients. The CD14lowCD16high cells also express low levels of CD35, CD11a and CD4 in common with normal monocytes. When cultured in vitro, monocytes from HIV-seropositive individuals differentiated within a few hours into an elongated fibroblastoid shape characteristic of migratory cells. Our results suggest that the expansion of the CD14lowCD16high monocyte subset, which produce high amount sof TNF-alpha and IL-1 alpha, may participate in the immune dysfunction observed during HIV infection.",
"Transfusion therapy is a life-sustaining treatment for patients with sickle cell disease (SCD), but can cause serious complications including alloimmunization. We previously reported diminished regulatory T cells (Tregs) and skewed Th2 responses in alloimmunized SCD patients. We hypothesized that the B cell regulatory (Breg) compartment, which controls Treg and Th differentiation, may also be compromised in allosensitized SCD patients. Phenotypically, we did not find differences in the frequency or numbers of CD24(hi) CD38(hi) and CD24(hi) CD27(+) B cell subsets, both previously identified as human Bregs, between alloimmunized and non-alloimmunized SCD patients on regular transfusions. However, at the functional level, CD19+ B cells from alloimmunized SCD patients expressed lower levels of IL-10 following stimulation as compared with non-alloimmunized patients (P < 0.05), and had reduced ability in inhibiting autologous CD14+ monocyte TNF-α expression (P < 0.05). These findings suggest that Bregs from alloimmunized and non-alloimmunized SCD patients differ in their ability to produce IL-10 and dampen monocyte activation, all consistent with an altered immunoregulatory state in alloimmunized SCD patients.",
"Transgenic Leishmania parasites that encode the murine chemokine monocyte chemoattractant protein 1 (MCP-1) were generated. These parasites transcribed MCP-1 mRNA and secreted MCP-1 protein. Infection of BALB/c, C57BL/6, or MCP-1 knockout (KO) mice with these parasites resulted in minimal lesion development with fewer parasites in the infected foot, lymph node, and spleen compared to wild-type-infected mice. In contrast, transgenic parasites caused substantial lesions with relatively high numbers of parasites in CC chemokine receptor 2 (CCR2) KO mice, indicating that the parasites are viable and healthy and that the lack of lesion development is CCR2 dependent. Prior infection of mice with transgenic parasites offered no protection to subsequent wild-type L. major challenge, suggesting that the transgenic parasites are controlled by an early innate immune response. Consistent with innate immunity, flow cytometry of cells from the ears of mice infected with transgenic parasites revealed an increase in the number of CCR2-positive macrophages by day 7 postinfection. The enumeration of transgenic parasites in ear lesions demonstrated a significant reduction in parasite numbers, which coincided with the increased CCR2-positive macrophage migration. CCR2-positive macrophages isolated from ears of mice infected with transgenic parasites contained virtually no parasites. In vitro studies revealed that optimal parasite killing required the recruitment of CCR2-positive macrophages, followed by stimulation with a combination of both MCP-1 and gamma interferon (IFN-gamma). This work suggests that the parasite-derived MCP-1 can recruit a restrictive population of CCR2-positive macrophages into lesions that can be optimally stimulated by MCP-1 and IFN-gamma to efficiently kill Leishmania parasites."
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Physiological characterization of the cholinergic vestibular efferent neurons in the brainstem
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The functional role of efferent innervation of the vestibular end-organs in the inner ear remains elusive. This study provides the first physiological characterization of the cholinergic vestibular efferent (VE) neurons in the brainstem by utilizing a transgenic mouse model, expressing eGFP under a choline-acetyltransferase (ChAT)-locus spanning promoter in combination with targeted patch clamp recordings. The intrinsic electrical properties of the eGFP-positive VE neurons were compared to the properties of the lateral olivocochlear (LOC) brainstem neurons, which gives rise to efferent innervation of the cochlea. Both VE and the LOC neurons were marked by their negative resting membrane potential <-75 mV and their passive responses in the hyperpolarizing range. In contrast, the response properties of VE and LOC neurons differed significantly in the depolarizing range. When injected with positive currents, VE neurons fired action potentials faithfully to the onset of depolarization followed by sparse firing with long inter-spike intervals. This response gave rise to a low response gain. The LOC neurons, conversely, responded with a characteristic delayed tonic firing upon depolarizing stimuli, giving rise to higher response gain than the VE neurons. Depolarization triggered large TEA insensitive outward currents with fast inactivation kinetics, indicating A-type potassium currents, in both the inner ear-projecting neuronal types. Immunohistochemistry confirmed expression of Kv4.3 and 4.2 ion channel subunits in both the VE and LOC neurons. The difference in spiking responses to depolarization is related to a two-fold impact of these transient outward currents on somatic integration in the LOC neurons compared to in VE neurons. It is speculated that the physiological properties of the VE neurons might be compatible with a wide-spread control over motion and gravity sensation in the inner ear, providing likewise feed-back amplification of abrupt and strong phasic signals from the semi-circular canals and of tonic signals from the gravito-sensitive macular organs.
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"Electrical properties of cochlear efferent (olivocochlear) neurons were investigated with the use of the whole cell patch recording technique in slice preparations of the neonatal rat (postnatal days 5-11). Lateral and medial olivocochlear (LOC and MOC, respectively) neurons were retrogradely labeled with a fluorescent tracer injected into the cochlea. Stained neurons were identified under a fluorescence microscope, and they were subjected to whole cell recording. LOC and MOC neurons showed different electrophysiological properties. Both showed spike trains of tonic pattern in response to injection of depolarizing current pulses at the resting membrane potential (-60 to -70 mV). However, when the membrane was slightly hyperpolarized (-72 to -76 mV), LOC neurons showed spike trains with a long first interspike interval (ISI), whereas MOC neurons showed spike trains with a long latency to the first spike. Extracellular application of 4-aminopyridine (4-AP; 0.5-2 mM) shortened these ISIs and latencies. In voltage-clamp experiments, two transient outward currents with different (fast and slow) decay kinetics were observed in LOC neurons. The fast outward current (I(A-LOC)) was inactivated by the preceding depolarization, and decayed with a time constant (tau) of 86 ms (at 0 mV). The preceding potential, which reduced the current size to the half-maximum (V1/2), was -72 mV. The slow current (I(KD)) decayed with a tau of 853 ms (at 0 mV). I(A-LOC) was sensitive to 4-AP (2 mM), and was less sensitive to tetraethylammonium chloride (TEA; 20 mM). I(KD) was partially blocked by TEA (20 mM), but was insensitive to 4-AP (2 mM). The recovery from inactivation of I(A-LOC) was time dependent with a time constant (tau(rec)) of 32 ms at -90 mV. MOC neurons also showed a transient outward current that consisted of a single transient component (I(A-MOC)) with a steady outward current. I(A-MOC) was inactivated by the preceding depolarization. Decay tau of I(A-MOC) was 33 ms (at 0 mV), and V1/2 was -75 mV. I(A-MOC) was sensitive to 4-AP (0.5-1 mM). The time-dependent recovery from inactivation of I(A-MOC) was faster than that of I(A-LOC), and tau(rec) was 15 ms at -90 mV. The different kinetics of transient outward currents between LOC and MOC neurons seems to be responsible for the difference in firing properties of these two neurons.",
"Several distinct mechanisms have evolved in the auditory periphery to extract frequency information from a sound. In the mammalian cochlea, a travelling wave on the basilar membrane enhanced by a physiologically vulnerable neuromechanical interaction performs the primary frequency separation. In lizards, tuning is likely to depend on structures in the papilla other than the basilar membrane, and tuning in the auditory nerve is correlated with the length of the stereocilia. In turtles and possibly some bird species, an electrical resonance in the receptor cells is responsible for frequency selectivity. In addition to those organs obviously specialized to detect acoustic stimuli, afferents of the vestibular system can exhibit tuning to low-frequency airborne sounds, despite the absence of mechanical frequency separation by accessory structures. I report here that in the frog saccule, a vestibular organ apparently constructed for the detection of vibratory accelerations, frequency tuning may arise from an electrical resonance intrinsic to the hair cells. The mechanism is similar to that found in turtle and ensures that a stimulus with frequency corresponding to the membrane resonant frequency will produce the largest signal in the cell. This type of tuning may thus be quite widespread. Oscillatory mechanisms have been reported in sensory cells of other modalities in several lower vertebrates, and may even contribute to their sensitivity, although such mechanisms do imply that the signal-to-noise ratio is degraded near threshold.",
"The influence of the efferent vestibular system (EVS) upon the background discharge and response dynamics of horizontal semicircular canal afferents was examined in the toadfish. In one set of experiments the EVS was activated using a behavioral paradigm; in the second, electrical shocks were applied to the efferent vestibular nucleus in the brain stem. The afferent's background discharge and responses to rotation were recorded before and during efferent stimulation. Both EVS activation paradigms gave qualitatively similar results: a facilitation of the afferent's rate, while the animal was at rest or in motion, and a reduction in response sensitivity. Afferents were not affected uniformly: low-gain, velocity-sensitive afferents were weakly influenced, while high-gain and acceleration afferents having low rates were the most excited. The afferents' phase of response was unmodified by electrical EVS stimulation. In many afferents a prominent form of response nonlinearity is discharge silencing over large portions of the stimulus cycle. Efferent-evoked rate increase was often sufficient to produce a full-cycle bidirectional response. Caloric facilitation of afferent rate confirmed that the EVS-induced sensitivity decrease was rate independent. These results show a dual action of the efferent system: (1) facilitating the afferent's rate and (2) reducing its sensitivity to adequate stimulation that may be correlated with the dual EVS synaptic innervation of the labyrinth, namely postsynaptic efferent-afferent synapses and presynaptic efferent-hair cell synapses.",
"In the preceding article (McCue and Guinan, 1994) we described a class of vestibular primary afferent fibers in the cat that responds vigorously to sounds at moderately high sound levels. Like their cochlear homologs, vestibular afferents and their associated hair cells receive efferent projections from brainstem neurons. In this report, we explore efferent influences on the background activity and tone-burst responses of the acoustically responsive vestibular afferents. Shock-burst stimulation of efferents excited acoustically responsive vestibular afferents; no inhibition was seen. A fast excitatory component built up within 100-200 msec of shock-burst onset and decayed with a similar time course at the end of each shock burst. During repeated 400 msec shock bursts at 1.5 sec intervals, a slow excitatory component grew over 20-40 sec and then decayed, even though the shock bursts continued. Efferent stimulation excited acoustically responsive vestibular afferents without appreciably changing an afferent's sound threshold or its average sound-evoked response. This evidence supports the hypothesis that excitation is due to efferent synapses on afferent fibers rather than on hair cells. Efferent stimulation enhanced the within-cycle modulation of afferent discharges evoked by a tone; that is, it increased the \"AC gain.\" No appreciable change was noted in the degree of phase locking to low-frequency tones as measured by the synchronization index. Little or no improvement in the bidirectionality (linearity) of transduction was seen. Vestibular afferent responses to tones normally had one peak per cycle; however, during efferent stimulation, two peaks per cycle were sometimes seen. We hypothesize that this is caused by two driving components acting at different sound phases with the components differentially affected by efferent activity. We discuss the relationship of our findings to efferent influences on acoustic responses in cochlear afferent fibers. The acoustically responsive vestibular afferents provide a mammalian model for studying purely excitatory efferent effects in a hair cell system.",
"Hair cells in the auditory, vestibular, and lateral-line systems respond to mechanical stimulation and transmit information to afferent nerve fibers. The sensitivity of mechanoelectrical transduction is modulated by the efferent pathway, whose activity usually reduces the responsiveness of hair cells. The basis of this effect remains unknown. We employed immunocytological, electrophysiological, and micromechanical approaches to characterize the anatomy of efferent innervation and the effect of efferent activity on the electrical and mechanical properties of hair cells in the bullfrog's sacculus. We found that efferent fibers form extensive synaptic terminals on all macular and extramacular hair cells. Macular hair cells expressing the Ca(2+)-buffering protein calretinin contain half as many synaptic ribbons and are innervated by twice as many efferent terminals as calretinin-negative hair cells. Efferent activity elicits inhibitory postsynaptic potentials in hair cells and thus inhibits their electrical resonance. In hair cells that exhibit spiking activity, efferent stimulation suppresses the generation of action potentials. Finally, efferent activity triggers a displacement of the hair bundle's resting position. The hair cells of the bullfrog's sacculus receive a rich efferent innervation with the heaviest projection to calretinin-containing cells. Stimulation of efferent axons desensitizes the hair cells and suppresses their spiking activity. Although efferent activation influences mechanoelectrical transduction, the mechanical effects on hair bundles are inconsistent.",
"Anterograde labeling techniques were used to examine peripheral innervation patterns of vestibular efferent neurons in the crista ampullares of the gerbil. Vestibular efferent neurons were labeled by extracellular injections of biocytin or biotinylated dextran amine into the contralateral or ipsilateral dorsal subgroup of efferent cell bodies (group e) located dorsolateral to the facial nerve genu. Anterogradely labeled efferent terminal field varicosities consist mainly of boutons en passant with fewer of the terminal type. The bouton swellings are located predominately in apposition to the basolateral borders of the afferent calyces and type II hair cells, but several boutons were identified close to the hair cell apical border on both types. Three-dimensional reconstruction and morphological analysis of the terminal fields from these cells located in the sensory neuroepithelium of the anterior, horizontal, and posterior cristae were performed. We show that efferent neurons densely innervate each end organ in widespread terminal fields. Subepithelial bifurcations of parent axons were minimal, with extensive collateralization occurring after the axons penetrated the basement membrane of the neuroepithelium. Axonal branching ranged between the 6th and 27th orders and terminal field collecting area far exceeds that of the peripheral terminals of primary afferent neurons. The terminal fields of the efferent neurons display three morphologically heterogeneous types: central, peripheral, and planum. All cell types possess terminal fields displaying a high degree of anisotropy with orientations typically parallel to or within +/-45 degrees of the longitudinal axis if the crista. Terminal fields of the central and planum zones predominately project medially toward the transverse axis from the more laterally located penetration of the basement membrane by the parent axon. Peripheral zone terminal fields extend predominately toward the planum semilunatum. The innervation areas of efferent terminal fields display a trend from smallest to largest for the central, peripheral, and planum types, respectively. Neurons that innervate the central zone of the crista do not extend into the peripheral or planum regions. Conversely, those neurons with terminal fields in the peripheral or planum regions do not innervate the central zone of the sensory neuroepithelium. The central zone of the crista is innervated preferentially by efferent neurons with cell bodies located in the ipsilateral group e. The peripheral and planum zones of the crista are innervated preferentially by efferent neurons with cell bodies located in the contralateral group e. A model incorporating our anatomic observations is presented describing an ipsilateral closed-loop feedback between ipsilateral efferent neurons and the periphery and an open-loop feed-forward innervation from contralateral efferent neurons. A possible role for the vestibular efferent neurons in the modulation of semicircular canal afferent response dynamics is proposed.",
"The cells of origin of the olivocochlear bundles in the guinea pig were labeled by retrograde axonal transport with either the fluorescent dye 'Fast Blue' or horseradish peroxidase (HRP). Labeled cells were found bilaterally in the superior olivary complex, as described previously. Moreover, an extremely large population of labeled cells occurred within the cell mass of the lateral superior olivary nucleus (LSO) ipsilaterally to the injection site. Preliminary electron microscopical observations confirmed that the labeled LSO cells were distinctly smaller than the unlabeled LSO cells. The labeled cells contained a large nucleus that exhibited extensive invaginations."
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In vitro and in vivo characterization of murine gammaherpesvirus 68 microRNAs.
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Gammaherpesviruses, including Epstein-Barr virus (EBV), Kaposi sarcoma-associated herpesvirus (KSHV, or HHV-8), and murine gammaherpesvirus 68 (MHV68, γHV68, or MuHV-4), are B cell-tropic pathogens that each encode at least 12 microRNAs (miRNAs). It is predicted that these regulatory RNAs facilitate infection by suppressing host target genes involved in a wide range of key cellular pathways. However, the precise contribution that gammaherpesvirus miRNAs make to viral life cycle and pathogenesis in vivo is unknown. MHV68 infection of mice provides a highly useful system to dissect the function of specific viral elements in the context of both asymptomatic infection and disease. Here, we report (i) analysis of in vitro and in vivo MHV68 miRNA expression, (ii) generation of an MHV68 miRNA mutant with reduced expression of all 14 pre-miRNA stem-loops, and (iii) comprehensive phenotypic characterization of the miRNA mutant virus in vivo. The profile of MHV68 miRNAs detected in infected cell lines varied with cell type and did not fully recapitulate the profile from cells latently infected in vivo. The miRNA mutant virus, MHV68.Zt6, underwent normal lytic replication in vitro and in vivo, demonstrating that the MHV68 miRNAs are dispensable for acute replication. During chronic infection, MHV68.Zt6 was attenuated for latency establishment, including a specific defect in memory B cells. Finally, MHV68.Zt6 displayed a striking attenuation in the development of lethal pneumonia in mice deficient in IFN-γ. These data indicate that the MHV68 miRNAs may facilitate virus-driven maturation of infected B cells and implicate the miRNAs as a critical determinant of gammaherpesvirus-associated disease.
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"Virus infections cause a much more profound perturbation of the lymphoid tissue than can be accounted for by the exigencies of the antigen-specific response. The extent of this 'immunological dissonance' is seen most dramatically in mice infected with a persistent gamma-herpesvirus, MHV-68. A profile of massive, continuing proliferation of both T and B cells in the lymph nodes and spleen leads to a dramatic increase in the prevalence of a CD62Llow CD8+ T cell subset in the blood, a pattern first detected two to three weeks after intranasal exposure to the inducing virus. This syndrome, which seems identical to human infectious mononucleosis (IM), persists for a further month or more. Part of the IM-like phase of MHV-68 infection reflects the selective expansion of Vbeta4+ CD8+ T cells, with the Vbeta4 effect being apparent for several different MHC class I H-2 types but not in mice that are deficient in MHC class II glycoprotein expression. Depleting CD4(+) T helper cells in MHV-68-infected mice leads to the decreased proliferation of the CD8+ T cells in the spleen and fewer CD62Llow CD8+ T lymphocytes than would be expected in peripheral blood, but fails to diminish the prominence of the V4beta+ CD8+ population. The results so far of this unique experimental mouse model of IM suggest that both cytokine-mediated effects and a viral superantigen are operating to promote the dramatic expansion and persistence of activated CD8+ T cells in the vascular compartment.",
"This work describes analyses of the function of the murid herpesvirus 4 strain 68 (MHV-68) M2 gene. A frameshift mutation was made in the M2 open reading frame that caused premature termination of translation of M2 after amino acid residue 90. The M2 mutant showed no defect in productive replication in vitro or in lungs after infection of mice. Likewise, the characteristic transient increase in spleen cell number, Vbeta4 T-cell-receptor-positive CD8(+) T-cell mononucleosis, and establishment of latency were unaffected. However, the M2 mutant virus was defective in its ability to cause the transient sharp rise in latently infected cells normally seen in the spleen after infection of mice. We also demonstrate that expression of M2 is restricted to B cells in the spleen and that M2 encodes a 30-kDa protein localizing predominantly in the cytoplasm and plasma membrane of B cells.",
"Seventeen miRNAs encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) have been identified and their functions have begun to be characterized. Among these miRNAs, we report here that miR-K12-7 directly targets the replication and transcription activator (RTA) encoded by open reading frame 50. We found that miR-K12-7 targeted the RTA 3' untranslated region (RTA3'UTR) in a seed sequence-dependent manner. miR-K12-7-5p derived from miR-K12-7 mediates the inhibition of RTA expression, and the mutation of the seed match site totally abrogated the inhibitory effect of miR-K12-7 on RTA3'UTR. The inhibition of RTA expression by miR-K12-7 was further confirmed in the latently KSHV-infected 293/Bac36 cell line through transient transfection of miR-K12-7 expression plasmid or specific inhibitor of miR-K12-7-5p, respectively. The transient transfection of miR-K12-7 into 293/Bac36 cells reduced RTA expression and the expression of the downstream early genes regulated by RTA, and also the production of progeny virus was significantly reduced after treatment with chemical inducers. Our study revealed that another miRNA, miR-K12-7-5p, targets the viral immediate early gene RTA and that this miRNA contributes to the maintenance of viral latency.",
"Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring herpesvirus of wild rodents and is genetically related to human herpesvirus 8 and Epstein-Barr virus. The ability of MHV-68 to establish acute and persistent infection within laboratory mice offers a unique opportunity to investigate immunological and virological aspects of gammaherpesvirus pathogenesis.",
"Infection of mice with murine gammaherpesvirus 68 (gamma HV68; also referred to as MHV68) provides a tractable small-animal model with which to address the requirements for the establishment and maintenance of gammaherpesvirus infection in vivo. The M2 gene of gamma HV68 is a latency-associated gene that encodes a protein lacking discernible homology to any known viral or cellular proteins. M2 gene transcripts have been detected in latently infected splenocytes (S. M. Husain, E. J. Usherwood, H. Dyson, C. Coleclough, M. A. Coppola, D. L. Woodland, M. A. Blackman, J. P. Stewart, and J. T. Sample, Proc. Natl. Acad. Sci. USA 96:7508-7513, 1999; H. W. Virgin IV, R. M. Presti, X. Y. Li, C. Liu, and S. H. Speck, J. Virol. 73:2321-2332, 1999) and peritoneal exudate cells (H. W. Virgin IV, R. M. Presti, X. Y. Li, C. Liu, and S. H. Speck, J. Virol. 73:2321-2332, 1999), as well as in a latently gamma HV68-infected B-lymphoma cell line (S. M. Husain, E. J. Usherwood, H. Dyson, C. Coleclough, M. A. Coppola, D. L. Woodland, M. A. Blackman, J. P. Stewart, and J. T. Sample, Proc. Natl. Acad. Sci. USA 96:7508-7513, 1999). Here we describe the generation of gamma HV68 mutants with disruptions in the M2 gene. Mutation of the M2 gene did not affect the ability of the virus to replicate in tissue culture, nor did it affect gamma HV68 virulence in B6.Rag1 deficient mice. However, we found that M2 was differentially required for acute replication in vivo. While mutation of M2 did not affect acute phase of virus replication in the lungs of mice following intranasal inoculation, acute-phase virus replication in the spleen was decreased compared to that of the wild-type and marker rescue viruses following intraperitoneal inoculation. Upon intranasal inoculation, M2 mutant viruses exhibited a significant decrease in the establishment of latency in the spleen on day 16 postinfection, as measured by the frequency of viral genome-positive cells. In addition, M2 mutant viral genome-positive cells reactivated from latency inefficiently compared to wild-type and marker rescue viruses. By day 42 after intranasal inoculation, the frequencies of M2 mutant and wild-type viral genome-positive cells were nearly equivalent and little reactivation was detected from either population. In sharp contrast to the results obtained following intranasal inoculation, after intraperitoneal inoculation, no significant defect was observed in the establishment or reactivation from latency with the M2 mutant viruses. These results indicate that the requirements for the establishment of latency are affected by the route of infection.",
"The fundamental question of whether a primed immune system is capable of preventing latent gammaherpesvirus infection remains unanswered. Recent studies showing that vaccination can reduce acute replication and short-term latency but cannot alter long-term latency further call into question the possibility of achieving sterilizing immunity against gammaherpesviruses. Using the murine gammaherpesvirus 68 (gammaHV68) system, we demonstrate that it is possible to effectively vaccinate against long-term latency. By immunizing mice with a gammaHV68 mutant virus that is deficient in its ability to reactivate from latency, we reduced latent infection of wild-type challenge virus to a level below the limit of detection. Establishment of latency was inhibited by vaccination regardless of whether mice were challenged intraperitoneally or intranasally. Passive transfer of antibody from vaccinated mice could partially reconstitute the effect, demonstrating that antibody is an important component of vaccination. These results demonstrate the potential of a memory immune response against gammaherpesviruses to alter long-term latency and suggest that limiting long-term latent infection in a clinically relevant situation is an attainable goal.",
"MicroRNAs are small noncoding RNAs that posttranscriptionally regulate gene expression. Kaposi sarcoma (KS)-associated herpesvirus (KSHV) encodes 12 distinct microRNA genes, all of which are located within the latency-associated region that is highly expressed in all KSHV-associated malignancies. We amplified, cloned, and sequenced a 2.8-kbp-long region containing a cluster of 10 microRNAs plus a 646-bp fragment of K12/T0.7 containing the remaining 2 microRNAs from 5 primary effusion lymphoma-derived cell lines and from 17 patient samples. The patients included 2 with classic KS, 12 with AIDS-KS (8 from the United States, 1 from Europe, 3 from Africa, and 4 from Central/South America), and 2 with multicentric Castleman disease (MCD). Additionally, we analyzed the K1, open reading frame 75, and K15 genes to determine KSHV subtypes, and we performed a phylogenetic analysis. Phylogenetic analysis of the 2.8-kbp microRNA region revealed 2 distinct clusters of sequences: a major (A/C) and a variant (B/Q) cluster. The variant cluster included sequences from 3 patients of African origin and both patients with MCD. Some microRNAs were highly conserved, whereas others had changes that could affect processing and, therefore, biological activity. These data demonstrate that KSHV microRNA genes are under tight selection in vivo and suggest that they contribute to the biological activity and possibly the pathogenesis of KSHV-associated malignancies."
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-emitting isotopes in the treatment of B cell non-Hodgkin's lymphoma
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Rituximab, the CD20-directed antibody, has become a standard component of treatment regimens for patients with B cell non-Hodgkin's lymphoma (NHL). The use of rituximab has resulted in greatly improved response and survival rates with less toxicity relative to standard chemotherapeutic regimes. However, relapse and recurrence is common, particularly in indolent varieties which remain incurable, requiring alternate therapeutic options. The subsequent coupling of β-emitting isotopes such as (131)I and (90)Y to anti-CD20 monoclonal antibodies (mAbs), including rituximab, has been steadily growing over the last decade and demonstrates even greater therapeutic efficacy with more durable responses. (177)Lutetium-labelled rituximab offers a number of convenient advantages over (131)I and (90)Y anti-CD20 mAbs for treatment of NHL, and a number of alpha-emitting isotopes lie at the frontier of consolidation therapy for residual, micrometastatic disease.
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"Many patients with non-Hodgkin's lymphomas are not cured by current therapies, and new approaches to treatment are needed. As part of an ongoing phase 1 study, we examined the effect of radioimmunotherapy with 131I-labeled B-cell-specific anti-CD20 monoclonal antibody in 10 patients with CD20-positive B-cell lymphomas in whom primary chemotherapy had failed. Anti-B1 (anti-CD20) mouse monoclonal antibody trace-labeled with 131I (15 mg containing 5 mCi) was given intravenously at approximately one-week intervals: first, without pretreatment with unlabeled anti-B1 antibody, to all 10 patients; then, with pretreatment with 135 mg of unlabeled antibody, to 8 patients; and then, with pretreatment with 685 mg, to 2 patients. Serial quantitative gamma-camera images and measures of whole-body radioactivity were obtained after each tracer dose. All known disease sites larger than 2 cm could be imaged. The effect of a pretreatment dose of unlabeled anti-B1 antibody on targeting of the tumor with the radiolabeled antibody was variable. The pretreatment dose of unlabeled antibody that produced the highest ratio of the tumor dose to the whole-body dose in tracer studies was then used to deliver higher doses of radioactivity for radioimmunotherapy in nine patients. Three patients received doses designed to deliver 25 cGy to the whole body (two patients treated twice, six to eight weeks apart), four patients received 35 cGy (one patient treated twice), and two patients received 45 cGy (one patient treated twice); each dose contained 34 to 66 mCi of activity. Six of the nine treated patients had tumor responses, including patients with bulky or chemotherapy-resistant disease: four patients had complete remissions, and two had partial responses. Three patients had objective responses to tracer infusions before they received radioimmunotherapeutic doses. Of the four patients with complete remissions, one remained in remission for eight months and the other three continue to have no disease progression (for 11, 9, and 8 months). There was mild or no myelosuppression. Radioimmunotherapy with [131I]anti-B1 antibody is a promising new treatment for lymphoma.",
"The radiobiology of radioimmunotherapy is an important determinant of both the toxicity and the efficacy associated with the treatment of B-cell non-Hodgkin's lymphoma with radiolabeled anti-CD20 monoclonal antibodies. The properties of the target, CD20, and the mechanisms of action of both the monoclonal antibodies and the associated exponentially decreasing low-dose-rate radiotherapy are described. The radiation dose and dose-rate effects are discussed and related to both the tumor responses and normal organ toxicity. Finally, the use of either unlabeled or radiolabeled anti-CD20 monoclonal antibodies as a component of combined modality therapy (including the sequential or concurrent use of sensitizers) and future directions of the field are discussed.",
"Radioimmunotherapy (RIT) is a new treatment modality for B-cell non-Hodgkin's lymphoma (NHL). Recent clinical trials have clearly established its efficacy in NHL patients refractory to standard chemotherapy or immunotherapy with the widely used unconjugated rituximab monoclonal antibody (mAb). The Food and Drug Administration has approved (90)Y-ibritumomab tiuxetan anti-B-cell NHL mAb as the first commercially available radiolabeled antibody for cancer therapy. This comes only a few years after the introduction of rituximab into clinical practice as the first unconjugated antibody for cancer treatment, underscoring the success of both immunotherapy and RIT in the treatment of NHL. With the approval of (90)Y-ibritumomab tiuxetan, and based on the results of numerous clinical trials with radiolabeled anti-B-cell NHL mAbs, RIT promises to become integral to nuclear medicine practice. In this article, the basic concepts of RIT are reviewed with important milestones in its development for B-cell NHL treatment and particular emphasis on phase II and III clinical trials establishing its efficacy in clearly defined patient populations. Finally, the prospects for the expected widespread clinical use of RIT in the management of B-cell NHL, alone or in combination with other more established therapies, are discussed. This article provides both investigative and clinical nuclear medicine physicians with a better understanding of RIT capabilities and limitations in B-cell NHL and their role as consultants in the care of NHL patients.",
"Radioimmunotherapy (RIT) with yttrium-90 ((90)Y)-labeled anti-CD20 antibody ((90)Y ibritumomab tiuxetan; Zevalin, IDEC Pharmaceuticals Corporation, San Diego, CA) has a high rate of tumor response in patients with relapsed or refractory, low-grade, follicular, or transformed B-cell non-Hodgkin's lymphoma (NHL). This study presents the safety data from 349 patients in five studies of outpatient treatment with (90)Y ibritumomab tiuxetan. Patients received rituximab 250 mg/m(2) on days 1 and 8, and either 0.4 mCi/kg (15 MBq/kg) or 0.3 mCi/kg (11 MBq/kg) of (90)Y ibritumomab tiuxetan on day 8 (maximum dose, 32 mCi). Patients were observed for up to 4 years after therapy or until progressive disease. Infusion-related toxicities were typically grade 1 or 2 and were associated with rituximab. No significant organ toxicity was noted. Toxicity was primarily hematologic, with nadir counts occurring at 7 to 9 weeks and lasting approximately 1 to 4 weeks depending on the method of calculation. After the 0.4-mCi/kg dose, grade 4 neutropenia, thrombocytopenia, and anemia occurred in 30%, 10%, and 3% of patients, respectively, and after the 0.3-mCi/kg dose, these grade 4 toxicities occurred in 35%, 14%, and 8% of patients, respectively. The risk of hematologic toxicity increased with degree of baseline bone marrow involvement with NHL. Seven percent of patients were hospitalized with infection (3% with neutropenia) and 2% had grade 3 or 4 bleeding events. Myelodysplasia or acute myelogenous leukemia was reported in five patients (1%) 8 to 34 months after treatment. Single-dose (90)Y ibritumomab tiuxetan RIT has an acceptable safety profile in relapsed NHL patients with less than 25% lymphoma marrow involvement, adequate marrow reserve, platelets greater than 100,000 cells/ micro L, and neutrophils greater than 1,500 cells/ micro L.",
"(131)I- and (90)Y-labelled anti-CD20 antibodies have been shown to be effective in the treatment of low-grade, B-cell non-Hodgkin's lymphoma (NHL). However, the most appropriate radionuclide in terms of high efficiency and low toxicity has not yet been established. In this study we evaluated an immunoconjugate formed by the anti-CD20 antibody rituximab and the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). DOTA-rituximab was prepared as a kit formulation and can be labelled in a short time (<20 min) with either (177)Lu or (90)Y. Immunoconjugates with different numbers of DOTA molecules per rituximab were prepared using p-SCN-Bz-DOTA. In vitro immunoreactivity and stability were tested and preliminary dosimetric results were acquired in two patients. The immunological binding properties of DOTA-rituximab to the CD20 antigen were found to be retained after conjugation with up to four chelators. The labelled product was stable against a 10(5) times excess of diethylenetriaminepentaacetic acid (DTPA, 37 degrees C, 7 days). Two patients with relapsed NHL were treated with 740 MBq/m(2) body surface (177)Lu-DOTA-rituximab. Scintigraphic images showed specific uptake at tumour sites and acceptable dosimetric results. The mean whole-body dose was found to be 314 mGy. The administration of (177)Lu-DOTA-rituximab was tolerated well. Our results show that DOTA-rituximab (4:1) can be labelled with (177)Lu with sufficient stability while the immunoconjugate retains its immunoreactivity. (177)Lu-DOTA-rituximab is an interesting, well-tolerated radiolabelled antibody with clinical activity in a low dose range, and provides an approach to the efficient treatment with few side effects for patients with relapsed NHL.",
"Unlike beta particle-emitting isotopes, alpha emitters can selectively kill individual cancer cells with a single atomic decay. HuM195, a humanized anti-CD33 monoclonal antibody, specifically targets myeloid leukemia cells and has activity against minimal disease. When labeled with the beta-emitters (131)I and (90)Y, HuM195 can eliminate large leukemic burdens in patients, but it produces prolonged myelosuppression requiring hematopoietic stem cell transplantation at high doses. To enhance the potency of native HuM195 yet avoid the nonspecific cytotoxicity of beta-emitting constructs, the alpha-emitting isotope (213)Bi was conjugated to HuM195. Eighteen patients with relapsed and refractory acute myelogenous leukemia or chronic myelomonocytic leukemia were treated with 10.36 to 37.0 MBq/kg (213)Bi-HuM195. No significant extramedullary toxicity was seen. All 17 evaluable patients developed myelosuppression, with a median time to recovery of 22 days. Nearly all the (213)Bi-HuM195 rapidly localized to and was retained in areas of leukemic involvement, including the bone marrow, liver, and spleen. Absorbed dose ratios between these sites and the whole body were 1000-fold greater than those seen with beta-emitting constructs in this antigen system and patient population. Fourteen (93%) of 15 evaluable patients had reductions in circulating blasts, and 14 (78%) of 18 patients had reductions in the percentage of bone marrow blasts. This study demonstrates the safety, feasibility, and antileukemic effects of (213)Bi-HuM195, and it is the first proof-of-concept for systemic targeted alpha particle immunotherapy in humans.",
"Rituximab is routinely used for the treatment of neoplasia, although the mechanism of action remains uncertain. In the current study, CD20-induced apoptosis was investigated with a panel of anti-CD20 monoclonal antibodies (mAb) in a wide range of cell lines. A hierarchy of mAb activity was apparent, with the B1 mAb generally the most potent. Apoptosis through CD20 was dependent on the nature of mAb binding and correlated with the extent of homotypic cell adhesion induced. However, using anti-CD20 mAb, which vary in the extent to which they redistribute wild-type and mutant CD20 molecules to membrane rafts, we showed that CD20-induced apoptosis was independent of translocation to TX-100 insoluble rafts. Using crmA-transfected cells and caspase inhibitors, we showed that phosphatidylserine translocation and mitochondrial permeability transition evoked during CD20-induced apoptosis appeared caspase independent. Furthermore, in cytoplasts which lack mitochondria and in Bcl(2)-transfected cells, phosphatidylserine was still translocated to the cell surface after CD20 stimulation. Together, these data imply that CD20 can evoke apoptosis without the involvement of mitochondria and caspases and irrespective of redistribution into TX-100 insoluble membrane rafts."
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Mitochondrial dysfunction and oxidative stress in unrelated pathologies
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Beyond the disorders recognized as mitochondrial diseases, abnormalities in function and/or ultrastructure of mitochondria have been reported in several unrelated pathologies. These encompass ageing, malformations, and a number of genetic or acquired diseases, as diabetes and cardiologic, haematologic, organ-specific (e.g., eye or liver), neurologic and psychiatric, autoimmune, and dermatologic disorders. The mechanistic grounds for mitochondrial dysfunction (MDF) along with the occurrence of oxidative stress (OS) have been investigated within the pathogenesis of individual disorders or in groups of interrelated disorders. We attempt to review broad-ranging pathologies that involve mitochondrial-specific deficiencies or rely on cytosol-derived prooxidant states or on autoimmune-induced mitochondrial damage. The established knowledge in these subjects warrants studies aimed at elucidating several open questions that are highlighted in the present review. The relevance of OS and MDF in different pathologies may establish the grounds for chemoprevention trials aimed at compensating OS/MDF by means of antioxidants and mitochondrial nutrients.
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"Mitochondrial diseases involve the respiratory chain, which is under the dual control of nuclear and mitochondrial DNA (mtDNA). The complexity of mitochondrial genetics provides one explanation for the clinical heterogeneity of mitochondrial diseases, but our understanding of disease pathogenesis remains limited. Classification of Mendelian mitochondrial encephalomyopathies has been laborious, but whole-exome sequencing studies have revealed unexpected molecular aetiologies for both typical and atypical mitochondrial disease phenotypes. Mendelian mitochondrial defects can affect five components of mitochondrial biology: subunits of respiratory chain complexes (direct hits); mitochondrial assembly proteins; mtDNA translation; phospholipid composition of the inner mitochondrial membrane; or mitochondrial dynamics. A sixth category-defects of mtDNA maintenance-combines features of Mendelian and mitochondrial genetics. Genetic defects in mitochondrial dynamics are especially important in neurology as they cause optic atrophy, hereditary spastic paraplegia, and Charcot-Marie-Tooth disease. Therapy is inadequate and mostly palliative, but promising new avenues are being identified. Here, we review current knowledge on the genetics and pathogenesis of the six categories of mitochondrial disorders outlined above, focusing on their salient clinical manifestations and highlighting novel clinical entities. An outline of diagnostic clues for the various forms of mitochondrial disease, as well as potential therapeutic strategies, is also discussed.",
"Oxidative stress plays an important role in multiple sclerosis (MS). The present study was designed to evaluate the modifications of plasma proteins by estimation markers of oxidative/nitrosative stress: carbonyl groups and 3-nitrotyrosines (3-NT) levels in relapsing-remitting (RR) (n=10) and secondary progressive (SP) (n=10) clinical course of multiple sclerosis. Moreover, we estimated the level of uric acid (UA) in plasma of MS patients. Compared to controls (n=10), the levels of carbonyl groups in plasma proteins were elevated (P<0.0001) as well in RRMS as in SPMS. The highest concentration of 3-NT was observed in plasma proteins obtained from SPMS patients (P<0.0005). The level of uric acid in plasma was significantly lower in RRMS (P<0.0001) than SPMS. This is the first report which presented differences between SPMS and RRMS patients in 3-NT and protein carbonyl groups in plasma proteins.",
"In spite of relatively large amount of evidence that oxidative stress is implicated in the pathogenesis of systemic sclerosis, there is no study analyzing antioxidants profile of the saliva of these patients. The aim of this study was to compare salivary antioxidants in subjects with systemic sclerosis and the healthy controls. The unstimulated and stimulated salivary flow and the specific activity of peroxidase, superoxide dismutase 1, the total amount of uric acid, and total antioxidant status were determined in two subgroups of systemic sclerosis women and healthy controls. A significant increase in the specific activity of peroxidase, a significant decrease in the total amount of uric acid and total antioxidants status in unstimulated saliva as well as a significant increase in all antioxidants examined in stimulated saliva of group with normal salivary flow rate as compared to the healthy controls were observed. Our results showed a significant decrease in the specific activity of peroxidase in unstimulated and a significant decrease in all antioxidants examined in stimulated saliva of the group with hyposalivation as compared to the group with normal salivary flow rate. Our results prove that impairment of the salivary glands in the course of systemic sclerosis may be attributed to free radicals, and it is correlated with disease duration.",
"Large scale clinical trials have demonstrated that an intensive antihyperglycemic treatment in diabetes mellitus (DM) in individuals reduces the incidence of micro- and macrovascular complications, e.g. nephropathy, retinopathy, DM-accelerated atherosclerosis, myocardial infarction, or limb amputations. Here, we investigated the effect of short- and long-term insulin administration on mitochondrial function in peripheral tissues of streptozotocin (STZ)-induced hyperglycemic rats. In addition, the in vitro effect of methylglyoxal (MG), advanced glycation end products (AGEs) and human diabetic plasma on mitochondrial activity was investigated in skeletal muscle and liver mitochondria and in rat skin primary fibroblasts. Hyperglycemic STZ rats showed tissue-specific patterns of energy deficiency, evidenced by reduced activities of complexes I, II and/or IV after 30 days of hyperglycemia in heart, skeletal muscle and liver; moreover, cardiac tissue was found to be the most sensitive to the diabetic condition, since energy metabolism was impaired after 10 days of the hyperglycemia. Insulin-induced tight glycemic control was effective in protecting against the hyperglycemia-induced inhibition of mitochondrial enzyme activities. Furthermore, the long-term hormone replacement (30 days) also increased these activities in kidney from STZ-treated animals, where the hyperglycemic state did not modify the electron transport activity. Results from in vitro experiments indicate that mitochondrial impairment could result from oxidative stress-induced accumulation of MG and/or AGEs. Further investigations demonstrated that human plasma AGE accumulation elicits reduced mitochondrial function in skin fibroblast. These data suggest that persistent hyperglycemia results in tissue-specific patterns of energy deficiency and that early and continuous insulin therapy is necessary to maintain proper mitochondrial metabolism.",
"Oncocytic tumors are a distinctive class of proliferative lesions composed of cells with a striking degree of mitochondrial hyperplasia that are particularly frequent in the thyroid gland. To understand whether specific mitochondrial DNA (mtDNA) mutations are associated with the accumulation of mitochondria, we sequenced the entire mtDNA in 50 oncocytic lesions (45 thyroid tumors of epithelial cell derivation and 5 mitochondrion-rich breast tumors) and 52 control cases (21 nononcocytic thyroid tumors, 15 breast carcinomas, and 16 gliomas) by using recently developed technology that allows specific and reliable amplification of the whole mtDNA with quick mutation scanning. Thirteen oncocytic lesions (26%) presented disruptive mutations (nonsense or frameshift), whereas only two samples (3.8%) presented such mutations in the nononcocytic control group. In one case with multiple thyroid nodules analyzed separately, a disruptive mutation was found in the only nodule with oncocytic features. In one of the five mitochondrion-rich breast tumors, a disruptive mutation was identified. All disruptive mutations were found in complex I subunit genes, and the association between these mutations and the oncocytic phenotype was statistically significant (P=0.001). To study the pathogenicity of these mitochondrial mutations, primary cultures from oncocytic tumors and corresponding normal tissues were established. Electron microscopy and biochemical and molecular analyses showed that primary cultures derived from tumors bearing disruptive mutations failed to maintain the mutations and the oncocytic phenotype. We conclude that disruptive mutations in complex I subunits are markers of thyroid oncocytic tumors.",
"Pearson syndrome is a multiorgan mitochondrial cytopathy that results from defective oxidative phosphorylation owing to mitochondrial DNA deletions. Prognosis is severe and death occurs in infancy or early childhood. This article describes 2 cases with a severe neonatal onset of the disease. A review of the literature reveals the atypical presentation of the disease in the neonatal period, which is often overlooked and underdiagnosed.",
"Numerous evidence and proofs suggest that the oxidative stress contributes to the pathogenesis of schizophrenia (SZ) and bipolar disorder (BD). The aim of this study is to determine the glutathione levels and the antioxidant enzyme activities in blood samples of patients suffering from SZ and patients with bipolar disorder in comparison with the healthy controlled subjects. It was a case-controlled study carried on upon three groups: forty-six SZ patients (41 men and 5 women, mean age=33.2±7years), thirty BD patients (25 men and 5 women, mean age=31.3±8years) and forty healthy controls (33 men and 7 women, mean age=32.3±7years). The glutathione levels are the total glutathione (GSHt), the reduced glutathione (GSHr), and the oxidized glutathione (GSSG) and the antioxidant enzyme activities that are the superoxide dismutase (SOD), the glutathione peroxidase (GPx), and the catalase (CAT) are determined by the spectrophotometer. We noticed that the GSHt and the GSHr levels significantly decreased in both SZ and BD patients in comparison with the healthy control subjects. As for SOD and CAT activities they remained lower for the patients with SZ when compared both with the controls or the BD patients. We noticed as well that the CAT activity was significantly lower in the BD group than that in the control group, whereas, GPx activity showed no significant change in each group. Hence, this report of the decreased plasma levels of GSHt and GSHr, and the impaired antioxidant enzyme activities in SZ and BD patients aims at highlighting the GSH deficit that seems to be contributing to these disorders, and showing that it may be an important indirect biomarker of the oxidative stress for the SZ and BD.",
"Cancer cells utilize complex mechanisms to remodel their bioenergetic properties. We exploited the intrinsic genomic stability of xeroderma pigmentosum C (XPC) to understand the inter-relationships between genomic instability, reactive oxygen species (ROS) generation, and metabolic alterations during neoplastic transformation. We showed that knockdown of XPC (XPC(KD)) in normal human keratinocytes results in metabolism remodeling through NADPH oxidase-1 (NOX-1) activation, which in turn leads to increased ROS levels. While enforcing antioxidant defenses by overexpressing catalase, CuZnSOD, or MnSOD could not block the metabolism remodeling, impaired NOX-1 activation abrogates both alteration in ROS levels and modifications of energy metabolism. As NOX-1 activation is observed in human squamous cell carcinomas (SCCs), the blockade of NOX-1 could be a target for the prevention and the treatment of skin cancers.",
"Oxidative stress is an imbalance between the production of reactive oxygen species (ROS) and physiological antioxidant defences. It occurs frequently in conditions characterized by immune activation and inflammation. Plasma levels of oxidized end products have never been evaluated in primary Sjøgren's syndrome (pSS). The aim of this study was to investigate the level of oxidative stress in primary Sjøgren's syndrome. As a secondary outcome, the association between oxidative stress and fatigue was explored. A cross-sectional study of 26 pSS patients was carried out. Oxidative stress was assessed using two markers of protein oxidation, protein carbonyl (PC) and advanced oxidation protein products (AOPP). Reference values for the oxidative stress markers were obtained from 15 healthy subjects. AOPP and PC levels were increased in the pSS patients compared to the healthy subjects. This is a novel finding. There were no associations between oxidative stress measures and fatigue in the patients. Patients with pSS have increased levels of oxidative stress compared to healthy subjects.",
"Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes (T2D) are growing public health problems, and are strongly associated. The link between the two conditions remains poorly understood. Hepatic interleukin-6 (IL-6), a major proinflammatory cytokine, expression is increased in animal models of NAFLD, while in mice, selective sustained upregulation of IL-6 in the liver results in systemic insulin resistance. The extent and clinical significance of hepatic IL-6 expression in human NAFLD, as well as potential mechanisms by which steatosis may increase IL-6 production in the liver, have not been examined. To ascertain the occurrence and significance of IL-6 expression in the liver in human NAFLD. Plasma was obtained at time of liver biopsy from 50 consecutive patients with suspected NAFLD. Histology was assessed blindly. Hepatic IL-6 expression was assessed by immunohistochemistry, while plasma IL-6 levels were determined by an enzyme-linked immunosorbent assay. IL-6 expression was markedly increased in the livers of patients with nonalcoholic steatohepatitis (NASH) as compared to patients with simple steatosis (P < 0.005) or normal biopsies (P < 0.010), confirming the presence of hepatic IL-6 expression in human NASH. A positive correlation was observed between hepatocyte IL-6 expression and degree of inflammation and stage of fibrosis. Furthermore, liver IL-6 expression positively correlated with plasma IL-6 levels and degree of systemic insulin resistance. Culture of liver cells with saturated, but not mono- or polyunsaturated, FFA resulted in a significant increase in IL-6 messenger RNA (mRNA) and protein expression. Collectively, these data suggest that increased hepatic IL-6 production may play an important role in NASH development, as well as in systemic insulin resistance and diabetes."
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Genome-wide expression profiling of the adult female Culicoides sonorensis midge unigene
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Unlike other important vectors such as mosquitoes and sandflies, genetic and genomic tools for Culicoides biting midges are lacking, despite the fact that they vector a large number of arboviruses and other pathogens impacting humans and domestic animals world-wide. In North America, female Culicoides sonorensis midges are important vectors of bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV), orbiviruses that cause significant disease in livestock and wildlife. Libraries of tissue-specific transcripts expressed in response to feeding and oral orbivirus challenge in C. sonorensis have previously been reported, but extensive genome-wide expression profiling in the midge has not. Here, we successfully used deep sequencing technologies to construct the first adult female C. sonorensis reference transcriptome, and utilized genome-wide expression profiling to elucidate the genetic response to blood and sucrose feeding over time. The adult female midge unigene consists of 19,041 genes, of which less than 7% are differentially expressed during the course of a sucrose meal, while up to 52% of the genes respond significantly in blood-fed midges, indicating hematophagy induces complex physiological processes. Many genes that were differentially expressed during blood feeding were associated with digestion (e.g. proteases, lipases), hematophagy (e.g., salivary proteins), and vitellogenesis, revealing many major metabolic and biological factors underlying these critical processes. Additionally, key genes in the vitellogenesis pathway were identified, which provides the first glimpse into the molecular basis of anautogeny for C. sonorensis. This is the first extensive transcriptome for this genus, which will serve as a framework for future expression studies, RNAi, and provide a rich dataset contributing to the ultimate goal of informing a reference genome assembly and annotation. Moreover, this study will serve as a foundation for subsequent studies of genome-wide expression analyses during early orbivirus infection and dissecting the molecular mechanisms behind vector competence in midges.
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"The fat body is the main organ of intermediary metabolism in insects and the principal source of hemolymph proteins. As part of our ongoing efforts to understand mosquito fat body physiology and to identify novel targets for insect control, we have conducted a transcriptome analysis of the fat body of Aedes aegypti before and in response to blood feeding. We created two fat body non-normalized EST libraries, one from mosquito fat bodies non-blood fed (NBF) and another from mosquitoes 24 hrs post-blood meal (PBM). 454 pyrosequencing of the non-normalized libraries resulted in 204,578 useable reads from the NBF sample and 323,474 useable reads from the PBM sample. Alignment of reads to the existing reference Ae. aegypti transcript libraries for analysis of differential expression between NBF and PBM samples revealed 116,912 and 115,051 matches, respectively. De novo assembly of the reads from the NBF sample resulted in 15,456 contigs, and assembly of the reads from the PBM sample resulted in 15,010 contigs. Collectively, 123 novel transcripts were identified within these contigs. Prominently expressed transcripts in the NBF fat body library were represented by transcripts encoding ribosomal proteins. Thirty-five point four percent of all reads in the PBM library were represented by transcripts that encode yolk proteins. The most highly expressed were transcripts encoding members of the cathepsin b, vitellogenin, vitellogenic carboxypeptidase, and vitelline membrane protein families. The two fat body transcriptomes were considerably different from each other in terms of transcript expression in terms of abundances of transcripts and genes expressed. They reflect the physiological shift of the pre-feeding fat body from a resting state to vitellogenic gene expression after feeding.",
"In insects, the functional molecules responsible for the taste system are still obscure. The gene for a 28.5 kDa protein purified from taste sensilla of the blowfly Phormia regina belongs to a gene family that includes takeout of Drosophila melanogaster. Molecular phylogenetic analysis revealed that the Phormia Takeout-like protein is most similar to the protein encoded by a member of the Drosophila takeout gene family, CG14661, whose expression and function have not been identified yet. Western blot analyses revealed that Phormia Takeout-like protein was exclusively expressed in antennae and labellum of the adult blowfly in both sexes. Immunohistochemical experiments demonstrated that Takeout-like protein was localized around the lamella structure of the auxiliary cells and in the sensillar lymph of the labellar taste sensillum. In antennae, Takeout-like protein was distributed at the base of the olfactory sensilla as well. No significant differences in Takeout-like protein expression were found between the sexes. Our results suggest that Phormia Takeout-like protein is involved in some early events concerned with chemoreception in both the taste and olfactory systems.",
"In anautogenous mosquitoes, a blood meal is required for activation of genes encoding yolk protein precursors (YPP). Vitellogenin (Vg), the major YPP gene, is transcribed at a very high level following blood meal activation. It is expressed exclusively in the female fat body, the tissue producing most of mosquito hemolymph and immune proteins. In this paper, we analyzed the upstream region of the Aedes aegypti Vg gene in order to identify regulatory elements responsible for its unique expression pattern. To achieve this goal, we analyzed the gene using transgenic Drosophila and Aedes as well as DNA-binding assays. These analyses revealed three regulatory regions in the 2.1 kb upstream portion of the Vg gene. The proximal region containing binding sites to EcR/USP, GATA, C/EBP and HNF3/fkh is required for the correct tissue- and stage-specific expression at a low level. The median region carrying sites for early ecdysone response factors E74 and E75 is responsible for hormonal enhancement of Vg expression. Finally, the distal GATA-rich region is necessary for extremely high expression levels characteristic of the Vg gene. The present work elucidates the molecular basis of blood meal-dependent expression of this mosquito gene, laying the foundation for mosquito-specific expression cassettes with predictable stage and tissue specificity.",
"Blood feeding tightly regulates the reproductive cycle in anautogenous mosquitoes. Vitellogenesis (the synthesis of yolk protein precursors) is a key event in the mosquito reproductive cycle and is activated in response to a blood meal. Before blood feeding, Aedes aegypti is in a state of reproductive arrest during which the yolk protein precursor genes (YPPs) are repressed. The regulatory region of the major YPP gene vitellogenin (Vg) has multiple GATA-binding sites required for the high expression level of this gene. However, a GATA factor (AaGATAr) likely acts as a repressor, preventing activation of this gene before a blood meal. Here we report in vivo data confirming the role of AaGATAr as a repressor of the Vg gene at the state of previtellogenic arrest. Using an RNA interference (RNAi)-mediated technique in conjunction with the Sindbis viral expression system, we show that knockdown of the AaGATAr gene results in an increased basal level of expression of the Vg gene and an elevated response to the steroid hormone 20-hydroxyecdysone in mosquitoes in a state of arrest. These experiments have revealed a component in the molecular mechanism by which anautogeny is maintained in A. aegypti.",
"Mosquitoes generate an enormous burden on human health worldwide. Disease-transmitting species use a reproductive strategy, termed anautogeny, that requires a blood meal to initiate egg maturation. Whereas this strategy is important for driving disease transmission, the molecular mechanisms underlying this phenomenon are still poorly understood. The production of yolk protein precursors (YPPs), a central event in egg maturation, is called vitellogenesis. YPPs are synthesized in the fat body, the insect analogue of the vertebrate liver. Mosquito vitellogenesis is regulated by the steroid hormone 20 hydroxyecdysone (20E). However, 20E alone is not capable of activating vitellogenesis in vivo. Here, we report that amino acid signaling through the nutrient-sensitive target of rapamycin (TOR) pathway is essential for the activation of YPP gene expression. An increase in extracellular amino acid levels, similar to the increase observed after a blood meal, is critical for 20E stimulation of YPP gene expression. Treatment with the TOR kinase inhibitor rapamycin significantly inhibits YPP expression. We used RNA interference to knockdown the expression of two key proteins of the TOR signaling pathway, TOR, and tuberous sclerosis complex 2. Knockdown of TOR inhibited amino acid stimulation while knockdown of tuberous sclerosis complex 2, a negative regulator of TOR signaling, resulted in enhanced YPP expression. Thus, amino acid-based TOR signaling regulates the activation of egg development after a blood meal, an adaptation to the unique life style of mosquitoes.",
"The fat body of vitellogenic mosquitoes was found to synthesize and secrete another protein in addition to vitellogenin, that accumulated in developing oocytes. In the tissues, this protein has Mr = 53000 on SDS-PAGE under reducing or non-reducing conditions. This protein is glycosylated as shown by [3H]mannose incorporation and experiments with tunicamycin. Polyclonal antibodies were produced using the ovarian 53-kDa peptide. Immunoblot analysis demonstrated the immunological identity of 53 kDa peptides from the fat body and the ovary. Furthermore, the 53-kDa protein (53KP) is synthesized and secreted exclusively by the vitellogenic fat body. Radioimmunoassay showed that 53KP is produced by the female fat body as early as 4 h and reaches its peak near 24 h after the initiation of vitellogenesis. Synthesis then drops to low levels by 36 h and declines to background levels by 48 h. In vitro experiments conducted on fat bodies of previtellogenic females demonstrated that the synthesis and secretion of 53KP can be stimulated by a physiological dose of 20-hydroxyecdysone (10-6 M). Immunocytochemical studies of the ovary demonstrate that 53KP is present in channels between follicle cells, in the perioocytic space and in yolk granules of the developing oocytes. This suggests that 53KP is accumulated in the oocytes by a pathway similar to that of vitellogenin.",
"The analysis of the free amino acids in the hemolymph of female Culex pipiens pallens L. indicated that asparagine, glutamine, glycine, leucine, serine, threonine, tyrosine, and valine noticeably increased in concentration during a blood meal digestion at 22 degrees C. The concentrations started to rise at about 4 h, reaching maximal level at 12-24 h, and then gradually returned to the prefeeding level by the fifth day after the blood meal. Before the sharp increase, the concentrations of asparagine, glutamine, glycine, serine, threonine, and tyrosine declined slightly during the first 1-2 h, probably because of hemolymph dilution by water absorbed from the blood meal. Histidine and lysine also showed a minor decrease soon after blood feeding, followed by a moderate but noticeable increase, delaying maximal concentrations until 48 h after the blood meal. Proline and alanine both exhibited a high hemolymph content and changed greatly, but with a large variation between the two samples analyzed. The changing pattern of individual hemolymph amino acids depended to a large extent upon the content of each amino acid in the blood meal given to the mosquitoes. Other free amino acids detected in the hemolymph were at very small concentrations and except for phenylalanine did not show any changes after a blood meal. The total free amino acid concentration was 50 nmol (or 6 micrograms) per microliters hemolymph before a blood meal, and increased to a maximal concentration of 88 nmol (or 11 micrograms) per microliters hemolymph at 18 h after the blood meal."
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Cytokines, Pyroptosis, and Tumor Microenvironment
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In the realm of cancer research, the tumor microenvironment (TME) plays a crucial role in tumor initiation and progression, shaped by complex interactions between cancer cells and surrounding non-cancerous cells. Cytokines, as essential immunomodulatory agents, are secreted by various cellular constituents within the TME, including immune cells, cancer-associated fibroblasts, and cancer cells themselves. These cytokines facilitate intricate communication networks that significantly influence tumor initiation, progression, metastasis, and immune suppression. Pyroptosis contributes to TME remodeling by promoting the release of pro-inflammatory cytokines and sustaining chronic inflammation, impacting processes such as immune escape and angiogenesis. However, challenges remain due to the complex interplay among cytokines, pyroptosis, and the TME, along with the dual effects of pyroptosis on cancer progression and therapy-related complications like cytokine release syndrome. Unraveling these complexities could facilitate strategies that balance inflammatory responses while minimizing tissue damage during therapy. This review delves into the complex crosstalk between cytokines, pyroptosis, and the TME, elucidating their contribution to tumor progression and metastasis. By synthesizing emerging therapeutic targets and innovative technologies concerning TME, this review aims to provide novel insights that could enhance treatment outcomes for cancer patients.
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"Programmed cell death (PCD) refers to the way in which cells die depending on specific genes encoding signals or activities. Apoptosis, autophagy, and pyroptosis are all mechanisms of PCD. Among these mechanisms, pyroptosis is mediated by the gasdermin family, accompanied by inflammatory and immune responses. The relationship between pyroptosis and cancer is complex, and the effects of pyroptosis on cancer vary in different tissues and genetic backgrounds. On one hand, pyroptosis can inhibit the occurrence and development of tumors; on the other hand, as a type of proinflammatory death, pyroptosis can form a suitable microenvironment for tumor cell growth and thus promote tumor growth. In addition, the induction of tumor pyroptosis is also considered a potential cancer treatment strategy. Studies have shown that DFNA5 (nonsyndromic hearing impairment protein 5)/GSDME (Gasdermin-E) mRNA methylation results in lower expression levels of DFNA5/GSDME in most tumor cells than in normal cells, making it difficult to activate the pyroptosis in most tumor cells. During the treatment of malignant tumors, appropriate chemotherapeutic drugs can be selected according to the expression levels of DFNA5/GSDME, which can be upregulated in tumor cells, thereby increasing the sensitivity to chemotherapeutic drugs and reducing drug resistance. Therefore, induced pyroptosis may play a predominant role in the treatment of cancer. Here, we review the latest research on the anti- and protumor effects of pyroptosis and its potential applications in cancer treatment.",
"Pyroptosis, an inflammatory programmed cell death, distinguishes itself from apoptosis and necroptosis and has drawn increasing attention. Recent studies have revealed a correlation between the expression levels of many pyroptosis-related genes and both tumorigenesis and progression. Despite advancements in cancer treatments such as surgery, radiotherapy, chemotherapy, and immunotherapy, the persistent hallmark of cancer enables malignant cells to elude cell death and develop resistance to therapy. Recent findings indicate that pyroptosis can overcome apoptosis resistance amplify treatment-induced tumor cell death. Moreover, pyroptosis triggers antitumor immunity by releasing pro-inflammatory cytokines, augmenting macrophage phagocytosis, and activating cytotoxic T cells and natural killer cells. Additionally, it transforms \"cold\" tumors into \"hot\" tumors, thereby enhancing the antitumor effects of various treatments. Consequently, pyroptosis is intricately linked to tumor development and holds promise as an effective strategy for boosting therapeutic efficacy. As the principal executive protein of pyroptosis, the gasdermin family plays a pivotal role in influencing pyroptosis-associated outcomes in tumors and can serve as a regulatory target. This review provides a comprehensive summary of the relationship between pyroptosis and gasdermin family members, discusses their roles in tumor progression and the tumor immune microenvironment, and analyses the underlying therapeutic strategies for tumor treatment based on pyroptotic cell death.",
"Pyroptosis, an inflammatory form of lytic cell death, is a type of cell death mediated by the gasdermin (GSDM) protein family. Upon recognizing exogenous or endogenous signals, cells undergo inflammasome assembly, GSDM cleavage, the release of proinflammatory cytokines and other cellular contents, eventually leading to inflammatory cell death. In this review, we discuss the roles of the GSDM family for anti-cancer functions and various antitumor drugs that could activate the pyroptosis pathways.",
"Gasdermins, a family of five pore-forming proteins (GSDMA-GSDME) in humans expressed predominantly in the skin, mucosa and immune sentinel cells, are key executioners of inflammatory cell death (pyroptosis), which recruits immune cells to infection sites and promotes protective immunity1,2. Pore formation is triggered by gasdermin cleavage1,2. Although the proteases that activate GSDMB, C, D and E have been identified, how GSDMA-the dominant gasdermin in the skin-is activated, remains unknown. Streptococcus pyogenes, also known as group A Streptococcus (GAS), is a major skin pathogen that causes substantial morbidity and mortality worldwide3. Here we show that the GAS cysteine protease SpeB virulence factor triggers keratinocyte pyroptosis by cleaving GSDMA after Gln246, unleashing an active N-terminal fragment that triggers pyroptosis. Gsdma1 genetic deficiency blunts mouse immune responses to GAS, resulting in uncontrolled bacterial dissemination and death. GSDMA acts as both a sensor and substrate of GAS SpeB and as an effector to trigger pyroptosis, adding a simple one-molecule mechanism for host recognition and control of virulence of a dangerous microbial pathogen.",
"Pyroptosis is rapidly emerging as a mechanism of anti-microbial host defense, and of extracellular release of the inflammasome-dependent cytokines interleukin (IL)-1β and IL-18, which contributes to autoinflammatory pathology. Caspases 1, 4, 5 and 11 trigger this regulated form of necrosis by cleaving the pyroptosis effector gasdermin D (GSDMD), causing its pore-forming amino-terminal domain to oligomerize and perforate the plasma membrane. However, the subcellular events that precede pyroptotic cell lysis are ill defined. In this study, we triggered primary macrophages to undergo pyroptosis from three inflammasome types and recorded their dynamics and morphology using high-resolution live-cell spinning disk confocal laser microscopy. Based on quantitative analysis of single-cell subcellular events, we propose a model of pyroptotic cell disintegration that is initiated by opening of GSDMD-dependent ion channels or pores that are more restrictive than recently proposed GSDMD pores, followed by osmotic cell swelling, commitment of mitochondria and other membrane-bound organelles prior to sudden rupture of the plasma membrane and full permeability to intracellular proteins. This study provides a dynamic framework for understanding cellular changes that occur during pyroptosis, and charts a chronological sequence of GSDMD-mediated subcellular events that define pyroptotic cell death at the single-cell level.",
"Inflammatory caspases (caspase-1, -4, -5 and -11) are critical for innate defences. Caspase-1 is activated by ligands of various canonical inflammasomes, and caspase-4, -5 and -11 directly recognize bacterial lipopolysaccharide, both of which trigger pyroptosis. Despite the crucial role in immunity and endotoxic shock, the mechanism for pyroptosis induction by inflammatory caspases is unknown. Here we identify gasdermin D (Gsdmd) by genome-wide clustered regularly interspaced palindromic repeat (CRISPR)-Cas9 nuclease screens of caspase-11- and caspase-1-mediated pyroptosis in mouse bone marrow macrophages. GSDMD-deficient cells resisted the induction of pyroptosis by cytosolic lipopolysaccharide and known canonical inflammasome ligands. Interleukin-1β release was also diminished in Gsdmd(-/-) cells, despite intact processing by caspase-1. Caspase-1 and caspase-4/5/11 specifically cleaved the linker between the amino-terminal gasdermin-N and carboxy-terminal gasdermin-C domains in GSDMD, which was required and sufficient for pyroptosis. The cleavage released the intramolecular inhibition on the gasdermin-N domain that showed intrinsic pyroptosis-inducing activity. Other gasdermin family members were not cleaved by inflammatory caspases but shared the autoinhibition; gain-of-function mutations in Gsdma3 that cause alopecia and skin defects disrupted the autoinhibition, allowing its gasdermin-N domain to trigger pyroptosis. These findings offer insight into inflammasome-mediated immunity/diseases and also change our understanding of pyroptosis and programmed necrosis.",
"IL-1β requires processing by caspase-1 to generate the active, pro-inflammatory cytokine. Acute IL-1β secretion from inflammasome-activated macrophages requires caspase-1-dependent GSDMD cleavage, which also induces pyroptosis. Mechanisms of IL-1β secretion by pyroptotic and non-pyroptotic cells, and the precise functions of caspase-1 and GSDMD therein, are unresolved. Here, we show that, while efficient early secretion of endogenous IL-1β from primary non-pyroptotic myeloid cells in vitro requires GSDMD, later IL-1β release in vitro and in vivo proceeds independently of GSDMD. IL-1β maturation is sufficient for slow, caspase-1/GSDMD-independent secretion of ectopic IL-1β from resting, non-pyroptotic macrophages, but the speed of IL-1β release is boosted by inflammasome activation, via caspase-1 and GSDMD. IL-1β cleavage induces IL-1β enrichment at PIP2-enriched plasma membrane ruffles, and this is a prerequisite for IL-1β secretion and is mediated by a polybasic motif within the cytokine. We thus reveal a mechanism in which maturation-induced IL-1β trafficking facilitates its unconventional secretion."
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Identification of a canine ortholog of human melanoma differentiation associated gene-7/interleukin-24.
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Human melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24) displays potent growth suppressing and cell killing activity against a wide variety of human and rodent cancer cells. In this study, we identified a canine ortholog of the human mda-7/IL-24 gene located within a cluster of IL-10 family members on chromosome 7. The full-length mRNA sequence of canine mda-7 was determined, which encodes a 186-amino acid protein that has 66% similarity to human MDA-7/IL-24. Canine MDA-7 is constitutively expressed in cultured normal canine epidermal keratinocytes (NCEKs), and its expression levels are increased after lipopolysaccharide stimulation. In cultured NCEKs, the canine mda-7 pre-mRNA is differentially spliced, via exon skipping and alternate 5'-splice donor sites, to yield five splice variants (canine mda-7sv1, canine mda-7sv2, canine mda-7sv3, canine mda-7sv4 and canine mda-7sv5) that encode four protein isoforms of the canine MDA-7 protein. These protein isoforms have a conserved N-terminus (signal peptide sequence) and are dissimilar in amino acid sequences at their C-terminus. Canine MDA-7 is not expressed in primary canine tumor samples, and most tumor derived cancer cell lines tested, like its human counterpart. Unlike human MDA-7/IL-24, canine mda-7 mRNA is not expressed in unstimulated or lipopolysaccharide (LPS), concanavalin A (ConA) or phytohemagglutinin (PHA) stimulated canine peripheral blood mononuclear cells (PBMCs). Furthermore, in-silico analysis revealed that canonical canine MDA-7 has a potential 28 amino acid signal peptide sequence that can target it for active secretion. This data suggests that canine mda-7 is indeed an ortholog of human mda-7/IL-24, its protein product has high amino acid similarity to human MDA-7/IL-24 protein and it may possess similar biological properties to human MDA-7/IL-24, but its expression pattern is more restricted than its human ortholog.
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"Subtraction hybridization identified melanoma differentiation-associated gene-7 (mda-7) as a gene induced during terminal differentiation in human melanoma cells. On the basis of structure, chromosomal localization and cytokine-like properties, mda-7 is classified as IL-24. Administration of mda-7/IL-24 by means of a replication-incompetent adenovirus (Ad.mda-7) induces apoptosis selectively in diverse human cancer cells without inducing harmful effects in normal fibroblast or epithelial cells. The present studies investigated the mechanism underlying this differential apoptotic effect. Infection of melanoma cells, but not normal immortal melanocytes, with Ad.mda-7 induced a time- and dose-dependent increase in expression, mRNA and protein, of a family of growth arrest and DNA damage (GADD)-inducible genes, which correlated with induction of apoptosis. Among the members of the GADD family of genes, GADD153, GADD45 alpha, and GADD34 displayed marked, and GADD45 gamma showed minimal induction. Treatment of melanoma cells with SB203580, a selective inhibitor of the p38 mitogen-activated protein kinase (MAPK) pathway, effectively inhibited Ad.mda-7-induced apoptosis. Additional support for an involvement of the p38 MAPK pathway in Ad.mda-7-mediated apoptosis was documented by using an adenovirus expressing a dominant negative mutant of p38 MAPK. Infection with Ad.mda-7 increased the phosphorylation of p38 MAPK and heat shock protein 27 in melanoma cells but not in normal immortal melanocytes. In addition, SB203580 effectively inhibited Ad.mda-7-mediated induction of the GADD family of genes in a time- and dose-dependent manner, and it effectively blocked Ad.mda-7-mediated down-regulation of the antiapoptotic protein BCL-2. Inhibition of GADD genes by an antisense approach either alone or in combination also effectively blocked Ad.mda-7-induced apoptosis in melanoma cells. These results support the hypothesis that Ad.mda-7 mediates induction of the GADD family of genes by means of the p38 MAPK pathway, thereby resulting in the selective induction of apoptosis in human melanoma cells.",
"Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a cancer-specific, growth-suppressing and apoptosis-inducing gene with broad-spectrum antitumor activity. However, when administered by means of a replication-incompetent adenovirus, Ad.mda-7, several colorectal carcinoma cell lines are resistant to its antiproliferative and antisurvival effects. We have presently endeavored to determine if K-ras mutations, present in approximately 40-50% of colorectal cancers and which may mediate resistance to chemotherapy and radiotherapy, represent a predisposing genetic factor mitigating reduced sensitivity to Ad.mda-7. To suppress ras expression, three structurally different replication-incompetent adenoviral vectors were engineered that express (1) an intracellular, neutralizing single-chain antibody (scAb) to p21 ras (Ad.K-ras scAb), (2) an antisense (AS) K-ras gene (Ad.K-ras AS) or (3) both mda-7/IL-24 and a K-ras AS gene in a single bipartite virus (Ad.m7.KAS). Simultaneous inhibition of K-ras and expression of mda-7/IL-24 enhanced killing of colorectal carcinoma cells with mutated K-ras, but not with wild-type K-ras. The extent of killing depended on the degree of K-ras downregulation, with Ad.K-ras AS being generally more efficient than Ad.K-ras scAb in combination with Ad.mda-7. These findings support an effective dual-combinatorial approach for the therapy of colorectal cancers that employs a unique cancer-specific suppressor gene (mda-7/IL-24) with targeted inhibition of oncogene (ras) expression.",
"The human mda-7/IL-24 gene product is normally expressed in melanocytes and certain lymphocyte populations. Loss of expression, a distinctive feature of many tumor suppressor genes, has been documented at RNA and protein levels in association with melanoma progression both in vitro as well as in human tumor-derived material. The MDA-7/IL-24 protein undergoes post-translational processing, including removal of an amino-terminal 48-residue signal peptide and extensive glycosylation prior to its secretion by producing cells. Its inherent cytokine properties have been documented in multiple reports, which have identified and characterized its cognate receptors and activation of the JAK/STAT signaling pathway following ligand/receptor docking. A notable and incompletely understood property of MDA-7/IL-24 is its ability to induce apoptosis in transformed cells, while having marginal growth suppressive effects on normal primary or immortalized cell lines. MDA-7/IL-24 has been delivered to cells, tumor xenografts and patients in clinical trials via a nonreplicating adenovirus (Ad.mda-7). Studies utilizing eukaryotically expressed and purified MDA-7/IL-24 protein from several sources have recapitulated some of the molecule's reported properties including receptor binding and JAK/STAT activation. Here, we report the properties and characteristics of a bacterially expressed and purified GST-MDA-7 fusion protein. These studies reveal that GST-MDA-7 retains its cancer-selective apoptosis-inducing properties, thereby providing a new reagent that will assist in defining the mechanism of action of this novel cytokine. In addition, retention of tumor-specific activity of GST-MDA-7 suggests that this protein may also have therapeutic applications.",
"Melanoma differentiation associated gene-7(mda-7) encodes IL-24, a cytokine that can selectively trigger apoptosis in transformed cells. Recombinant mda-7 adenovirus (Ad.mda-7) effectively kills glioma cells, offering a novel gene therapy strategy to address deadly brain tumors. In this study, we defined the proximal mechanisms by which Ad-mda-7 kills glioma cells. Key factors implicated included activation of the endoplasmic reticulum stress kinase protein kinase R-like endoplasmic reticulum kinase (PERK), Ca(++) elevation, ceramide generation and reactive oxygen species (ROS) production. PERK inhibition blocked ceramide or dihydroceramide generation, which were critical for Ca(++) induction and subsequent ROS formation. Activation of autophagy and cell death relied upon ROS formation, the inhibition of which ablated Ad.mda-7-killing activity. In contrast, inhibiting TRX induced by Ad.MDA-7 enhanced tumor cytotoxicity and improved animal survival in an orthotopic tumor model. Our findings indicate that mda-7/IL-24 induces an endoplasmic reticulum stress response that triggers production of ceramide, Ca(2+), and ROS, which in turn promote glioma cell autophagy and cell death.",
"Human interleukin-24 (IL-24) is unique among the IL-10 superfamily as there is considerable evidence that it possesses multiple anti-cancer properties, including direct tumor cell cytotoxicity, helper T cell (TH1) immune stimulation, and anti-angiogenic activities. The primary sequence of human IL-24 differs from homologous cytokines, because it possesses three consensus N-linked glycosylation sites and the potential for a single disulfide bond. To address the significance of these modifications in human IL-24, we analyzed the relationship between post-translational modifications and the cytokine activity of the human IL-24 protein. In contrast to related interleukins, we identified a relationship between net glycosylation, protein solubility, and cytokine activity. In addition, abrogation of the two cysteine residues by mutagenesis dramatically altered the ability of IL-24 to secrete from host cells and resulted in the concomitant loss of IL-24 activity. We conclude that, unlike other IL-10 family members, human IL-24 must be glycosylated to maintain solubility and bioavailability. Further, a single, unique disulfide bond is required for secretion and activity. These structure-function relationships show that, although IL-24 is a member of the IL-19 subfamily of IL-10-like cytokines by sequence similarity, its surface properties and its distinctive disulfide arrangement make it unique. These observations could explain the novel biological activities measured of this cytokine. Understanding the structural basis of IL-24 activity will be important in the interpretation of the function of this cytokine and in the development of scale-up strategies for biophysical and clinical applications.",
"Despite recent advances in treatment strategies, the overall 5-year survival rate for patients with common epithelial cancers is poor largely because of the difficulty in treating metastatic cancers. Therefore, therapeutic agents are urgently needed that can effectively inhibit both primary epithelial tumors and their metastases. One such agent that has shown promise in preclinical studies is the tumor suppressor/cytokine, melanoma differentiation associated gene-7 also known as interleukin-24 (mda-7/IL-24). Preclinical studies from our and other laboratories have shown that overexpression of MDA-7/IL-24 causes a strong tumor- suppressive effect in many human cancer cells but spares normal cells. This gene therapy also enhances the tumor-suppressive activity of radiotherapy and chemotherapy. Secreted MDA-7 protein that is glycosylated also has been shown to have potent antiangiogenic activity both in vitro and in vivo. Studies examining the immune properties of mda-7 have shown that MDA-7/IL-24 unlike the related IL-10, functions as a Th1 cytokine. Recently, an MDA-7 protein-mediated \"bystander effect\" on tumor cells has been documented. Building on these findings we successfully completed a Phase I clinical trial of adenovirus-based mda-7 cancer therapy that confirmed the safety of this gene therapy. Phase II trials evaluating the efficacy of mda-7-based gene therapy are warranted. The outcome of such ongoing mda-7-based gene therapy trials will allow us to better understand this therapy's clinical utility.",
"Terminal prostate cancer is refractory to conventional anticancer treatments because of frequent overexpression of antiapoptotic proteins Bcl-2 and/or Bcl-x(L). Adenovirus-mediated delivery of melanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24), a secreted cytokine having cancer-selective apoptosis-inducing properties, profoundly inhibits prostate cancer cell growth. However, forced overexpression of Bcl-2 or Bcl-x(L) renders prostate cancer cells resistant to Ad.mda-7. We constructed a conditionally replication-competent adenovirus in which expression of the adenoviral E1A gene, necessary for replication, is driven by the cancer-specific promoter of progression elevated gene-3 (PEG-3) and which simultaneously expresses mda-7/IL-24 in the E3 region of the adenovirus (Ad.PEG-E1A-mda-7), a cancer terminator virus (CTV). This CTV generates large quantities of MDA-7/IL-24 as a function of adenovirus replication uniquely in cancer cells. Infection of Ad.PEG-E1A-mda-7 (CTV) in normal prostate epithelial cells and parental and Bcl-2- or Bcl-x(L)-overexpressing prostate cancer cells confirmed cancer cell-selective adenoviral replication, mda-7/IL-24 expression, growth inhibition, and apoptosis induction. Injecting Ad.PEG-E1A-mda-7 (CTV) into xenografts derived from DU-145-Bcl-x(L) cells in athymic nude mice completely eradicated not only primary tumors but also distant tumors (established in the opposite flank), thereby implementing a cure. These provocative findings advocate potential therapeutic applications of this novel virus for advanced prostate cancer patients with metastatic disease."
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Antinociceptive effect of systemic naloxone in a mouse model of neuropathic pain
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Opioid antagonists, such as naloxone and naltrexone, exhibit agonistic properties at the mutated μ receptor, MOR-S196ACSTA. In our previous study, systemic naloxone (10 mg·kg(-1) , s.c.) elicited antinociceptive effect without the induction of tolerance, dependence or rewarding effect in mice 2 weeks after intrathecal administration of double-stranded adeno-associated virus-MOR-S196ACSTA-eGFP. Here, we have investigated if this antinociceptive paradigm would be effective in a mouse model of neuropathic pain.
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"Recent developments in the study of the structure and function of opioid receptors raise significant challenges for the definition of individual receptor types and the development of a nomenclature that precisely describes isoforms that may subserve different functions in vivo. Presentations at the 2013 meeting of the International Narcotics Research Conference in Cairns, Australia, considered some of the new discoveries that are now unravelling the complexities of opioid receptor signalling. Variable processing of opioid receptor messenger RNAs may lead to the presence of several isoforms of the μ receptor. Each opioid receptor type can function either as a monomer or as part of a homo- or heterodimer or higher multimer. Additionally, recent evidence points to the existence of agonist bias in the signal transduction pathways activated through μ receptors, and to the presence of regulatory allosteric sites on the receptors. This brief review summarizes the recent discoveries that raise challenges for receptor definition and the characterization of signal transduction pathways activated by specific receptor forms. This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2.",
"Cholecystokinin (CCK) may act as an endogenous anti-opioid and blockade of CCK receptors can enhance the potency and efficacy of morphine. This effect is blocked by opioid delta (delta) receptor antagonists, suggesting a tonic inhibitory action of CCK to diminish the release and/or availability of endogenous enkephalins. The present studies have further evaluated this possibility by studying the antiallodynic actions of a CCKB antagonist (L365,260) alone, or in the presence of thiorphan (a neutral endopeptidase inhibitor) in a model of peripheral neuropathy. Animals subjected to nerve injury, but not sham controls, exhibited long lasting, stable mechanical allodynia. Intrathecal (i.t.) administration of L365,260 or thiorphan alone did not alter allodynia. However, co-administration of these compounds produced a significant antiallodynic action which was antagonized by receptor selective doses of naltrindole, an opioid delta receptor antagonist. In addition, antisera to [Leu5]enkephalin, but not to [Met5]enkephalin, also blocked the antiallodynic action of thiorphan plus L365,260. These data suggest that blockade of CCKB receptors may enhance the actions or availability of endogenous [Leu5]enkephalin or a like substance which can elicit a significant antiallodynic action via opioid delta receptors when its degradation is by inhibited by thiorphan. The data suggest that delta opioids are involved in regulation of some aspects of nerve-injury induced pain.",
"Relief of pain is rewarding. Using a model of experimental postsurgical pain we show that blockade of afferent input from the injury with local anesthetic elicits conditioned place preference, activates ventral tegmental dopaminergic cells, and increases dopamine release in the nucleus accumbens. Importantly, place preference is associated with increased activity in midbrain dopaminergic neurons and blocked by dopamine antagonists injected into the nucleus accumbens. The data directly support the hypothesis that relief of pain produces negative reinforcement through activation of the mesolimbic reward-valuation circuitry.",
"Many clinical case reports have suggested that sustained opioid exposure can elicit unexpected, paradoxical pain. Here, we explore the possibility that (1) opioid-induced pain results from tonic activation of descending pain facilitation arising in the rostral ventromedial medulla (RVM) and (2) the presence of such pain manifests behaviorally as antinociceptive tolerance. Rats implanted subcutaneously with pellets or osmotic minipumps delivering morphine displayed time-related tactile allodynia and thermal hyperalgesia (i. e., opioid-induced \"pain\"); placebo pellets or saline minipumps did not change thresholds. Opioid-induced pain was observed while morphine delivery continued and while the rats were not in withdrawal. RVM lidocaine, or bilateral lesions of the dorsolateral funiculus (DLF), did not change response thresholds in placebo-pelleted rats but blocked opioid-induced pain. The intrathecal morphine antinociceptive dose-response curve (DRC) in morphine-pelleted rats was displaced to the right of that in placebo-pelleted rats, indicating antinociceptive \"tolerance.\" RVM lidocaine or bilateral DLF lesion did not alter the intrathecal morphine DRC in placebo-pelleted rats but blocked the rightward displacement seen in morphine-pelleted animals. The subcutaneous morphine antinociceptive DRC in morphine-pelleted rats was displaced to the right of that in placebo-pelleted rats; this right shift was blocked by RVM lidocaine. The data show that (1) opioids elicit pain through tonic activation of bulbospinal facilitation from the RVM, (2) increased pain decreases spinal opioid antinociceptive potency, and (3) blockade of pain restores antinociceptive potency, revealing no change in antinociceptive signal transduction. These studies offer a mechanism for paradoxical opioid-induced pain and allow the development of approaches by which the loss of analgesic activity of opioids might be inhibited.",
"Orofacial pain has been well-characterized clinically, but evaluation of orofacial pain in animals has not kept pace. The objective of this study was to describe behavioral responses to facial thermal stimulation and inflammation with/without an analgesic using a novel operant paradigm. Animals were trained to voluntarily place their face against a stimulus thermode (37.7-57.2 degrees C) providing access to positive reinforcement. These contingencies present a conflict between positive reward and tolerance for nociceptive stimulation. Inflammation was induced and morphine was provided as an analgesic in a subset of animals. Six outcome measures were determined: reward intake, reward licking contacts, stimulus facial contacts, facial contact duration, ratio of reward/stimulus contacts, and ratio of facial contact duration/event. Animals displayed aversive behaviors to the higher temperatures, denoted by a significant decrease in reward intake, total facial contact duration, and reward licking events. The number of facial contacts increased with increasing temperature, replacing long drinking bouts with more frequent short drinks, as reflected by a low ratio of facial contact duration/event. The number of reward licking/facial contact events was significantly decreased as the thermal stimulus intensity increased, providing another pain index derived from this operant method. These outcomes were significantly affected in the direction of increased nociception following inflammation, and these indices of hyperalgesia were reversed with morphine administration. These data reflect an orofacial pain behavior profile that was based on an animal's responses in an operant escape paradigm. This technique allows evaluation of nociceptive processing and modulation throughout the neuraxis.",
"Although injury-induced afferent discharge declines significantly over time, experimental neuropathic pain persists unchanged for long periods. These observations suggest that processes that initiate experimental neuropathic pain may differ from those that maintain such pain. Here, the role of descending facilitation arising from developing plasticity in the rostral ventromedial medulla (RVM) in the initiation and maintenance of experimental neuropathic pain was explored. Tactile and thermal hypersensitivity were induced in rats by spinal nerve ligation (SNL). RVM lidocaine blocked SNL-induced tactile and thermal hypersensitivity on post-SNL days 6-12 but not on post-SNL day 3. Lesion of RVM cells expressing mu-opioid receptors with dermorphin-saporin did not prevent the onset of SNL-induced tactile and thermal hypersensitivity, but these signs reversed to baseline levels beginning on post-SNL day 4. Similarly, lesions of the dorsolateral funiculus (DLF) did not prevent the onset of SNL-induced tactile and thermal hypersensitivity, but these signs reversed to baseline levels beginning on post-SNL day 4. Lesions of the DLF also blocked the SNL-induced increase in spinal dynorphin content, which has been suggested to promote neuropathic pain. These data distinguish mechanisms that initiate the neuropathic state as independent of descending supraspinal influences and additional mechanism(s) that require supraspinal facilitation to maintain such pain. In addition, the data indicate that these time-dependent descending influences can underlie some of the SNL-induced plasticity at the spinal level. Such time-dependent descending influences driving associated spinal changes, such as the upregulation of dynorphin, are key elements in the maintenance, but not initiation, of neuropathic states.",
"Osteoarthritis (OA) is a chronic pain condition characterized by pain during joint use as well as pain at rest (i.e., ongoing pain). Although injection of monosodium iodoacetate (MIA) into the intra-articular space of the rodent knee is a well established model of OA pain that is characterized by changes in weight bearing and hypersensitivity to tactile and thermal stimuli, it is not known if this procedure elicits ongoing pain. Further, the time-course and possible underlying mechanisms of these components of pain remain poorly understood. In these studies, we demonstrated the presence of ongoing pain in addition to changes in weight bearing and evoked hypersensitivity. Twenty-eight days following MIA injection, spinal clonidine blocked changes in weight bearing and thermal hypersensitivity and produced place preference indicating that MIA induces ongoing and evoked pain. These findings demonstrate the presence of ongoing pain in this model that is present at a late-time point after MIA allowing for mechanistic investigation.",
"The human experience of pain is multidimensional and comprises sensory, affective, and cognitive dimensions. Preclinical assessment of pain has been largely focused on the sensory features that contribute to nociception. The affective (aversive) qualities of pain are clinically significant but have received relatively less mechanistic investigation in preclinical models. Recently, operant behaviors such as conditioned place preference, avoidance, escape from noxious stimulus, and analgesic drug self-administration have been used in rodents to evaluate affective aspects of pain. An important advance of such operant behaviors is that these approaches may allow the detection and mechanistic investigation of spontaneous neuropathic or ongoing inflammatory/nociceptive (i.e., nonevoked) pain that is otherwise difficult to assess in nonverbal animals. Operant measures may allow the identification of mechanisms that contribute differentially to reflexive hypersensitivity or to pain affect and may inform the decision to progress novel mechanisms to clinical trials for pain therapy. Additionally, operant behaviors may allow investigation of the poorly understood mechanisms and neural circuits underlying motivational aspects of pain and the reward of pain relief.",
"In this study, we developed a rat model of incisional pain. A 1-cm longitudinal incision was made through skin, fascia and muscle of the plantar aspect of the hindpaw in halothane-anesthetized rats. Withdrawal responses were measured using von Frey filaments at different areas around the wound before surgery and for the next 6 days. A cumulative pain score based on the weight bearing behavior of the animals was also utilized. The results of tests for withdrawal responses and scores based on weight bearing suggest that a surgical incision of the rat foot causes a reliable and quantifiable mechanical hyperalgesia lasting for several days after surgery. An incision that only included skin and fascia but not muscle in the foot caused less severe hyperalgesia during the initial postoperative period. Distinct areas around the wound had different withdrawal thresholds during the study period. Even remote sites as much as 10 mm from the wound showed persistent mechanical hyperalgesia. Selective denervations of the rat hindpaw prior to foot incision revealed both the sural and tibial nerves were responsible for transmitting input from the incision that produces hyperalgesia. This model should allow us to understand mechanisms of sensitization caused by surgery and investigate new therapies for postoperative pain in humans.",
"The capsaicin receptor TRPV1 is an emerging target for the treatment of pain with a unique expression profile in peripheral nociceptors and the ability to show polymodal activation, TRPV1 is an important integrator of responses to inflammatory mediators. Sensitization of TRPV1 during chronic pain is believed to contribute to the transduction of noxious signaling for normally innocuous stimuli and consequently the search for novel TRPV1 therapeutics is intense. The current understanding of the physiological role the receptor, as well as the potential therapeutic utility and emerging liabilities of TRPV1 modulators are discussed."
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Alosetron: a novel 5-HT3 receptor antagonist for Irritable Bowel Syndrome
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Irritable bowel syndrome (IBS) is the most common functional gastrointestinal disorder and affects up to 12% to 15% of adults in the United States, with a higher prevalence among women and those younger than 50 years. IBS adversely impacts quality of life and medical expenditures, with significant costs arising from healthcare visits and reduced workplace productivity. Recent studies have shown that the adverse effects of IBS are so significant that many patients are willing to accept risks of adverse events from effective treatment to gain symptom relief. Alosetron is a 5-HT3 receptor antagonist approved by the US Food and Drug Administration (FDA) for women with severe diarrhea-predominant IBS that has not responded to traditional therapies. Alosetron yields overall improvements in IBS symptoms in 51% of patients vs 36% treated with placebo, with efficacy continuing undiminished over the course of a 48-week randomized, controlled trial. In real-world clinical practice, patients receiving alosetron had significant improvements in multiple IBS-related clinical parameters, including the new FDA IBS-diarrhea composite endpoint, lower gastrointestinal symptoms, fecal incontinence, and quality of life. Ischemic colitis and complications of constipation have been rare in occurrence. After nearly a decade of alosetron use under the risk management plan, adjudication of ischemic colitis and complications of constipation cases indicate that their incidence rates have remained low and stable.
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"To review the evidence supporting the biopsychosocial model in understanding patients with gastrointestinal disorders (GI). Essay of personal experience and review of related literature through a MEDLINE search. Through clinical examples of three common gastrointestinal disorders, a case is made to refocus our understanding from a biomedical or disease-based model of illness to a biopsychosocial model. With the latter model, the psychosocial and biological predeterminants are seen to interact in the clinical expression of illness and disease. With gastroesophageal reflux disease, the evidence shows that stress can lead to amplification of heartburn symptoms that is independent of the degree of reflux. Functional gastrointestinal pain is \"an illness without disease,\" where structural or physiological disturbance of the GI system does not exist. Rather, the symptoms are understood in terms of visceral hypersensitivity as modulated by central nervous system activity. With the Crohn's disease example, the clinical expression of the disorder is not explained by the degree of disease activity. Rather, the symptoms and impaired quality of life relate to preexisting psychosocial determinants. The observed association of stress with disease activation in Crohn's disease is explained by stress-related alterations in psychoimmunological function via the hypothalamic-pituitary-adrenal axis. Gastrointestinal disorders, as a model for other medical conditions, exemplify the important role of an integrated, biopsychosocial model of illness.",
"Irritable bowel syndrome (IBS) is a common gastrointestinal motility disorder that typically affects persons of working age and is costly to employers. The financial burden attributable to the direct (use of healthcare resources) and indirect (missed days from work [absenteeism] and loss of productivity while at work [presenteeism]) costs of IBS is similar to that of other common long-term medical disorders, such as asthma, migraine, hypertension, and congestive heart failure. The symptoms of IBS are significantly bothersome and place a substantial burden on the personal and working lives of patients. As with other long-term medical conditions that have a significant impact on productivity, directed efforts by employers can address IBS in the workplace and thereby potentially decrease its impact. In this article, the symptoms of IBS and its impact on patients and on society as a whole are discussed; options are outlined by which employers can help reduce the total costs of IBS, including lost productivity (both absenteeism and presenteeism), in the workplace.",
"Psychotherapy and antidepressants are effective in patients with severe irritable bowel syndrome (IBS), but the cost-effectiveness of either treatment in routine practice has not been established. Patients with severe IBS were randomly allocated to receive 8 sessions of individual psychotherapy, 20 mg daily of the specific serotonin reuptake inhibitor (SSRI) antidepressant, paroxetine, or routine care by a gastroenterologist and general practitioner. Primary outcome measures of abdominal pain, health-related quality of life, and health care costs were determined after 3 months of treatment and 1 year later. A total of 257 subjects (81% response rate) from 7 hospitals were recruited; 59 of 85 patients (69%) randomized to psychotherapy and 43 of 86 (50%) of the paroxetine group completed the full course of treatment. Both psychotherapy and paroxetine were superior to treatment as usual in improving the physical aspects of health-related quality of life (SF-36 physical component score improvement, 5.2 [SEM, 1.26], 5.8 [SEM, 1.0], and -0.3 [SEM, 1.17]; P < 0.001), but there was no difference in the psychological component. During the follow-up year, psychotherapy but not paroxetine was associated with a significant reduction in health care costs compared with treatment as usual (psychotherapy, $976 [SD, $984]; paroxetine, $1252 [SD, $1616]; and treatment as usual, $1663 [SD, $3177]). For patients with severe IBS, both psychotherapy and paroxetine improve health-related quality of life at no additional cost.",
"This study assessed the impact of recruitment on irritable bowel syndrome clinical trials, by determining whether irritable bowel syndrome patients recruited from advertisement or a specialty clinic differ in clinical and physiologic measures. We prospectively surveyed 657 irritable bowel syndrome patients who either: (i) were referred from a functional bowel disease clinic (52%); or (ii) responded to advertisement for clinical trials (48%), using questionnaires about bowel and psychological symptoms, and quality of life. In a subset of 42 irritable bowel syndrome patients (29 advertisement and 15 clinic patients), rectal discomfort thresholds were measured before and after repetitive sigmoid stimulation. While the advertisement population more commonly consulted primary care physicians, the clinic population more commonly consulted gastroenterologists. The clinic population reported more prevalent and severe abdominal pain, and higher psychological symptom scores, while the advertisement population had greater quality of life. In the visceral perception studies, both subgroups were hypersensitive to rectal distension. Compared to the clinic population, the advertisement population had less severe abdominal pain and psychological symptoms, better quality of life but similar visceral perception. The differences in clinical self-reports may have consequences for enrolment of these different patient populations into clinical trials.",
"There have been few Asian studies regarding anxiety and depression associated with irritable bowel syndrome (IBS). The aim of this study was to evaluate the frequency and importance of anxiety and depression in Korean patients with IBS. A total of 124 IBS patients and 91 healthy subjects were enrolled consecutively. All participants were asked to complete self-administered questionnaires: one addressing symptom severity, the Short Form 36, and the Hospital Anxiety and Depression Scale (HADS). The patients were also asked to complete the IBS-specifi c quality of life (IBS-QOL) questionnaire. Anxiety and depression were observed in 38.6% and 38.6% of IBS patients, respectively, and in 24.2% and 16.5% of healthy subjects, respectively (p<0.05 for both). The mean HADS scores for anxiety and depression in IBS patients were 6.8±4.5 and 7.1±4.4, respectively. Both anxiety and depression were associated with self-reported symptom severity (p<0.012 and p<0.001, respectively). As determined by multivariate analysis, symptom severity was the most important factor in the prediction of anxiety and depression. Self-reported symptom severity and depression were clearly and independently associated with the overall IBS-QOL score. Anxiety and depression were frequently observed in Korean IBS patients and were related to the severity of their symptoms and the impairment of the patient's QOL. Our data suggest that assessing anxiety and depression is important when evaluating IBS patients.",
"In November 2000, alosetron HCl (Lotronex), a treatment for irritable bowel syndrome (IBS), was removed from the U.S. market in part because of the occurrence of colon ischemia in treated patients. Since the relation between colon ischemia and IBS is poorly understood, we evaluated the incidence of colon ischemia among people with and without IBS. Using medical claims data from a large health care organization in the United States, we identified 87,449 people with an IBS diagnosis between January 1995 and December 1999. We calculated age- and sex-specific incidence rates in the general population and in IBS patients. There were 740 cases of colon ischemia during 8.5 million person-years of observation in 5.4 million persons. The crude incidence rate was 42.8 cases per 100,000 person-years for IBS patients. By comparison, the incidence rate was 7.2 per 100,000 person-years in the general population. After adjustment for age, sex, and calendar year, the incidence of colon ischemia in people with IBS was 3.4 times higher than in persons without (95% CI 2.6-4.5). Rates of colon ischemia among patients carrying a diagnosis of IBS are substantially higher than in the general population. Colon ischemia, though unusual in IBS patients, may nonetheless constitute a distinct part of the IBS natural history. Alternatively, it may be a consequence of therapy, or a manifestation of other bowel pathology that is sometimes confused with IBS.",
"Patients' responses to quality-of-life questionnaires are dependent on the cultural milieu. The aims of this study were to translate the Irritable Bowel Syndrome Quality of Life questionnaire, which was developed in the West, into Korean and to validate the translated questionnaire in patients with irritable bowel syndrome. Translation of the original questionnaire was performed according to accepted linguistic validation guidelines. Korean patients had no difficulty understanding the questions. Data from the translated questionnaire were well correlated with results from the SF-36 questionnaire and reflected the stress status of the patients as measured on the Perceived Stress Scale. Some questionnaire items seemed, on first analysis, to be problematic in this subset of patients, but these results may relate, in part, to cultural differences between Korea and the West. We conclude that the translated questionnaire is reliable and offers an accurate measure of quality of life for Korean patients with irritable bowel syndrome."
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Chemosensitization strategies for cisplatin resistance
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The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.
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"The regulation of glutathione and various glutathione-dependent enzymes has been studied in two ovarian adenocarcinoma cell lines derived from a patient before (PE01) and after (PE04) the onset of drug resistance to cis-platinum, chlorambucil and 5-fluorouracil. Reduced glutathione levels were higher in the drug resistant cells (PE04). This could possibly be attributed to a much higher (6.5-fold) gamma-glutamyl-transpeptidase activity. In addition, glutathione-S-transferase (GST) and glutathione peroxidase were 2.9- and 2.3-fold higher in this cell line. Analysis of the GST subunit composition showed both cell lines contained high levels of the acidic GST and lower concentrations of a basic isozyme. The difference in GST activity between PE01 and PE04 did not appear to be related to the levels of these GST subunits. GSH, glutathione peroxidase and gamma-glutamylcysteinyl synthetase were all found to be regulated during the cell cycle, higher levels being detected in logarithmic versus confluent cultures of PE01 and PE04 and MCF7. This did affect some of the differences between PE01 and PE04 and therefore may be a contributing factor to the differential sensitivity of these cells to cytotoxic compounds. The above data provide the first evidence that tumour cells obtained from a patient before and after the onset of drug resistance have significant differences in glutathione-dependent enzyme content.",
"Loss of expression of the hMLH1 and hPMS2 subunits of the MutL alpha-mismatch repair complex is a frequent event (9/10) in independent cisplatin resistant derivatives of a human ovarian carcinoma cell line. However, only hMLH1 mRNA is decreased in these MutL alpha-deficient lines. No alterations in the levels of the hMSH2 and hMSH6 (GTBP) subunits of the MutS alpha-complex are observed. An increase in the proportion of ovarian tumours negative for the hMLH1 subunit is observed in samples taken at second look laparotomy after chemotherapy (36%: 4/11), compared to untreated tumours (10%: 4/39). No significant difference is observed for hMSH2, hMSH6 or hPMS2. Furthermore, cisplatin and doxorubicin-resistant ovarian lines deficient in hMLH1 expression are cross-resistant to 6-thioguanine and the methylating agent N-methyl-N-nitrosourea (MNU). Depletion of O6-alkylguanine-DNA-alkyltransferase (ATase) activity confers only limited increased sensitivity to MNU. Thus the mismatch repair deficient lines retain DNA damage tolerance even after ATase depletion. The hMLH1 deficient lines also lose ability to engage G1 and G2 cell cycle arrest after cisplatin damage. Together these data suggest that loss of hMLH1 expression may be a high frequency event following exposure of ovarian tumour cells to cisplatin and may be critically involved in the development of drug resistance. Thus, the hMLH1 status of these cells appears to be highly correlated with the ability to engage cell death and cell cycle arrest after DNA damage induced by cisplatin.",
"Mitochondrial outer-membrane permeabilization by pro-apoptotic Bcl-2 family members plays a crucial role in apoptosis induction. However, whether this directly causes the release of the different mitochondrial apoptogenic factors simultaneously is currently unknown. Here we report that in cells or with isolated mitochondria, pro-apoptotic Bcl-2 proteins cause the release of cytochrome c, Smac/Diablo and HtrA2/Omi but not endonuclease G (EndoG) and apoptosis-inducing factor (AIF). In cells treated with Bax/Bak-dependent pro-apoptotic drugs, neither the caspase inhibitor zVAD-fmk nor loss of Apaf-1 affected the efflux of cytochrome c, Smac/Diablo and HtrA2/Omi, but both prevented the release of EndoG and AIF. Our findings identify the mitochondrial response to pro-apoptotic stimuli as a selective process leading to a hierarchical ordering of the effectors involved in cell death induction. Moreover, as in Caenorhabditis elegans, EndoG and AIF act downstream of caspase activation. Thus EndoG and AIF seem to define a 'caspase-dependent' mitochondria-initiated apoptotic DNA degradation pathway that is conserved between mammals and nematodes.",
"We have previously obtained, by exposure to near continuous increasing concentrations of cisplatin, a panel of human ovarian cancer cell lines that exhibit a wide range of primary resistance to the drug (9- to > 400-fold). These cells had strikingly increased (4- to 50-fold) levels of glutathione (GSH) as compared with the drug-sensitive cells of origin (A. K. Godwin et al., Proc. Natl. Acad. Sci. USA, 89: 3070-3074, 1992). Utilizing this panel of resistant cell lines, we evaluated cross-resistance to classical alkylating agents, natural product drugs, and irradiation. We observed that cross-resistance to carboplatin paralleled that of cisplatin, culminating in approximately 250-fold resistance. Similarly, melphalan cross-resistance continued to increase to > 400-fold and again paralleled the primary cisplatin resistance. Cell lines with low to very high levels of resistance to cisplatin are 8- to 850-fold resistant to the epipodophyllotoxin derivative etoposide. Cross-resistance is also observed for other natural product drugs, including Adriamycin (approximately 80-fold), mitoxantrone (approximately 440-fold), and taxol (approximately 40-fold). Cross-resistance to irradiation is, however, modest (< 2-fold). The cells with the greatest primary resistance to cisplatin most commonly had the highest cross-resistance to the other drugs examined. The cross-resistance to the natural product category drugs was found not to be mediated by the products of either the multidrug resistance 1 (MDR1) or multidrug resistance-associated protein (MRP) genes based on lack of coordinate increased expression or amplification of these genes as assessed by Northern and Southern blot analyses. Furthermore, verapamil failed to markedly increase drug sensitivity. Although there was no indication that these natural product drug efflux pumps were operative, we observed decreased doxorubicin accumulation in these cell lines cross-resistant to natural products. In addition, alternations in DNA topoisomerase II mRNA levels, which have been observed in a variety of human tumor cell lines selected in vitro for resistance to etoposide or teniposide, were not detected. Only intracellular levels of GSH correlated with cross-resistance to these diverse anticancer agents and partial loss of resistance was associated with a marked decrease in glutathione levels. In the absence of alternative mechanisms, we speculate that the very broad clinically relevant cross-resistance seen in this model system may, at least in part, be the direct result of GSH-mediated drug inactivation or may be due to a combination of GSH conjugation to drug and conjugate efflux mediated by the putative ATP-dependent glutathione S-conjugate export pump.",
"Apoptosis is a genetically regulated biological process that plays a major role in chemotherapy-induced tumor cell killing. It may be triggered by two major intracellular signaling cascades, the mitochondrial pathway and the death receptor pathway, both leading to caspase activation and cleavage of specific cellular substrates. The p53 gene is involved in the regulation of apoptosis. Caspase activation following wild-type p53 induction is associated with the release of the apoptogenic factors cytochrome c and Smac/DIABLO from the mitochondria, that is in turn controlled by the pro-apoptotic and anti-apoptotic Bcl-2 family proteins. In ovarian cancer p53 status is a strong predictor of response to platinum-based chemotherapy. Patients whose tumors have p53 mutations experience a lower chance of achieving a complete response following platinum-based regimens when compared to patients without p53 mutations. Conversely, experimental and clinical data seem to show that paclitaxel enhances apoptosis through a p53-independent pathway, that probably involves the Bax gene. Whereas patients with wild-type p53 tumors have a good chance to respond to platinum, patients with mutant p53 tumors may have a clinical benefit from the addition of paclitaxel to platinum-based chemotherapy. Therefore determining p53 status can be useful in predicting therapeutic response to specific drugs. Moreover the understanding of cellular mechanisms regulating apoptosis might offer a strong rationale for the combination of chemotherapy with other biological treatments.",
"The MUC1 transforming protein is overexpressed by most human carcinomas. The present studies demonstrate that the MUC1 C-terminal subunit (MUC1 C-ter) localizes to mitochondria in HCT116/MUC1 colon carcinoma cells and that heregulin stimulates mitochondrial targeting of MUC1 C-ter. We also show that MUC1 attenuates cisplatin-induced (1) release of mitochondrial apoptogenic factors, (2) activation of caspase-3, and (3) induction of apoptosis. Moreover, knockdown of MUC1 expression in A549 lung and ZR-75-1 breast carcinoma cells by MUC1siRNA was associated with increased sensitivity to genotoxic drugs in vitro and in vivo. These findings indicate that MUC1 attenuates the apoptotic response to DNA damage and that this oncoprotein confers resistance to genotoxic anticancer agents.",
"Improved understanding of the molecular mechanisms by which small-molecule inhibitors of histone deacetylases (HDAC) induce programs, such as cellular differentiation and apoptosis, would undoubtedly assist their clinical development as anticancer agents. As modulators of gene transcript levels, HDAC inhibitors (HDACi) typically affect only 5% to 10% of actively transcribed genes with approximately as many mRNA transcripts being up-regulated as down-regulated. Using microRNA (miRNA) array analysis, we report rapid alteration of miRNA levels in response to the potent hydroxamic acid HDACi LAQ824 in the breast cancer cell line SKBr3. Within 5 hours of exposure to a proapoptotic dose of LAQ824, significant changes were measured in 40% of the >60 different miRNA species expressed in SKBr3 cells with 22 miRNA species down-regulated and 5 miRNAs up-regulated. To explore a potential functional link between HDACi induced mRNA up-regulation and miRNA down-regulation, antisense experiments were done against miR-27a and miR-27b, both abundantly expressed and down-regulated in SKBr3 cells by LAQ824. Correlating a set of genes previously determined by cDNA array analysis to be rapidly up-regulated by LAQ824 in SKBr3 with a database of potential 3' untranslated region miRNA binding elements, two genes containing putative miR-27 anchor elements were identified as transcriptionally up-regulated following miR-27 antisense transfection, ZBTB10/RINZF, a Sp1 repressor, and RYBP/DEDAF, an apoptotic facilitator. These findings emphasize the importance of post-transcriptional mRNA regulation by HDACi in addition to their established effects on promoter-driven gene expression."
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Caloric restriction modulates extracellular matrix and metastatic potential in triple-negative breast cancer
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Caloric restriction (CR) has been shown to cause tumor regression in models of triple-negative breast cancer (TNBC), and the regression is augmented when coupled with ionizing radiation (IR). In this study, we sought to determine if the molecular interaction between CR and IR could be mediated by microRNA (miR). miR arrays revealed 3 miRs in the miR-17~92 cluster as most significantly down regulated when CR is combined with IR. In vivo, CR and IR down regulated miR-17/20 in 2 TNBC models. To elucidate the mechanism by which this cluster regulates the response to CR, cDNA arrays were performed and the top 5 statistically significant gene ontology terms with high fold changes were all associated with extracellular matrix (ECM) and metastases. In silico analysis revealed 4 potential targets of the miR-17~92 cluster related to ECM: collagen 4 alpha 3, laminin alpha 3, and metallopeptidase inhibitors 2 and 3, which were confirmed by luciferase assays. The overexpression or silencing of miR-17/20a demonstrated that those miRs directly affected the ECM proteins. Furthermore, we found that CR-mediated inhibition of miR-17/20a can regulate the expression of ECM proteins. Functionally, we demonstrate that CR decreases the metastatic potential of cells which further demonstrates the importance of the ECM. In conclusion, CR can be used as a potential treatment for cancer because it may alter many molecular targets concurrently and decrease metastatic potential for TNBC.
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"As normal cells progress toward malignancy, they must switch to an angiogenic phenotype to attract the nourishing vasculature that they depend on for their growth. In cultured fibroblasts from Li-Fraumeni patients, this switch was found to coincide with loss of the wild-type allele of the p53 tumor suppressor gene and to be the result of reduced expression of thrombospondin-1 (TSP-1), a potent inhibitor of angiogenesis. Transfection assays revealed that p53 can stimulate the endogenous TSP-1 gene and positively regulate TSP-1 promoter sequences. These data indicate that, in fibroblasts, wild-type p53 inhibits angiogenesis through regulation of TSP-1 synthesis.",
"Although little is known as yet about the processes that coordinate cell-signalling pathways, matrix proteins are probably major players in this type of global control. The CCN (cyr61, ctgf, nov) proteins are an important family of matricellular regulatory factors involved in internal and external cell signalling. This family participates in angiogenesis, chondrogenesis, and osteogenesis, and they are probably involved in the control of cell proliferation and differentiation. Runping Gao and David Brigstock (Hepatol Res 2003; 27: 214-20) recently showed that CCN2 (CTGF, connective tissue growth factor) is a cell-adhesion factor for hepatic stellate cells. On exposure to transforming growth factor beta, hepatic stellate cells produce distinct CCN2 isoforms. Gao and Brigstock assign to CCN2 module 3 the capacity to mediate binding to low-density-lipoprotein receptor-related protein (LRP), which was previously reported to interact with CCN2 and to be involved in various types of signalling. They also establish that CCN2 binding to LRP is heparin dependent and that module 4 of CCN2 promotes LRP-independent adhesion of hepatic stellate cells. The differential binding of CCN2 isoforms to LRP highlights the importance of functional interactions between individual modules, and reinforces the concept that different module combinations might confer agonistic or antagonistic activities. WHERE NEXT? It is essential to understand how the distinct configuration of the various CCN isoform affects their biological activities and bioavailability, and to explore the mechanisms and the regulatory processes involved in the production of truncated CCN isoforms. A better understanding of the structural basis for their multifunctionality is a prerequisite to wider use of CCN proteins in molecular medicine.",
"Human adenocarcinomas commonly harbor mutations in the KRAS and MYC proto-oncogenes and the TP53 tumor suppressor gene. All three genetic lesions are potentially pro-angiogenic, as they sustain production of vascular endothelial growth factor (VEGF). Yet Kras-transformed mouse colonocytes lacking p53 formed indolent, poorly vascularized tumors, whereas additional transduction with a Myc-encoding retrovirus promoted vigorous vascularization and growth. In addition, VEGF levels were unaffected by Myc, but enhanced neovascularization correlated with downregulation of anti-angiogenic thrombospondin-1 (Tsp1) and related proteins, such as connective tissue growth factor (CTGF). Both Tsp1 and CTGF are predicted targets for repression by the miR-17-92 microRNA cluster, which was upregulated in colonocytes coexpressing K-Ras and c-Myc. Indeed, miR-17-92 knockdown with antisense 2'-O-methyl oligoribonucleotides partly restored Tsp1 and CTGF expression; in addition, transduction of Ras-only cells with a miR-17-92-encoding retrovirus reduced Tsp1 and CTGF levels. Notably, miR-17-92-transduced cells formed larger, better-perfused tumors. These findings establish a role for microRNAs in non-cell-autonomous Myc-induced tumor phenotypes.",
"Triple-negative breast cancer (TNBC) accounts for 15% to 20% of breast cancers. It is a heterogeneous disease, not only on the molecular level, but also on the pathologic and clinical levels. TNBC is associated with a significantly higher probability of relapse and poorer overall survival in the first few years after diagnosis when compared with other breast cancer subtypes. This is observed despite its usual high sensitivity to chemotherapy. In the advanced setting, responses observed with chemotherapy lack durability. Early-stage clinical studies suggested impressive potential when a poly (ADP-ribose) polymerase (PARP) inhibitor is given for the treatment of advanced TNBC with BRCA gene dysfunction. The molecular complexity of TNBC has led to proposed subclassifications, which will be of great value for the development of targeted therapies. In this review, we discuss the biology of TNBC at the pathologic and the molecular levels. We also elaborate on the role of systemic therapies and the results of the first phase III clinical trial evaluating the addition of iniparib, a novel investigational anticancer agent that does not possess characteristics typical of the PARP inhibitor class, in combination with chemotherapy in advanced TNBC.",
"MicroRNAs (miRNAs) are an abundant class of small non-protein-coding RNAs that function as negative gene regulators. They regulate diverse biological processes, and bioinformatic data indicates that each miRNA can control hundreds of gene targets, underscoring the potential influence of miRNAs on almost every genetic pathway. Recent evidence has shown that miRNA mutations or mis-expression correlate with various human cancers and indicates that miRNAs can function as tumour suppressors and oncogenes. miRNAs have been shown to repress the expression of important cancer-related genes and might prove useful in the diagnosis and treatment of cancer."
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Sex-related differences in migraine epidemiology, symptoms and natural history
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This paper reviews sex-related differences in migraine epidemiology, symptoms, natural history and co-morbid disorders. Migraine is more than twice as common in females as in males, and women experience more frequent, longer lasting and more painful attacks, have more disability and a risk of transition from episodic to chronic migraine greater than men, but the mechanisms behind these differences are still poorly understood. The role of sex hormones, genes, and the differences in brain function and structure are discussed. Finally, we evaluate the many gender-related questions about treatment of migraine in women. In future research data should be analyzed separately for men and women to ensure that differences between the sexes could be identified.
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"Previous studies have suggested that migraine is a risk factor for brain lesions, but methodological issues hampered drawing definite conclusions. Therefore, we initiated the magnetic resonance imaging (MRI) ‘CAMERA’ (Cerebral Abnormalities in Migraine, an Epidemiological Risk Analysis) study. We summarize our previously published results. A total of 295 migraineurs and 140 controls were randomly selected from a previously diagnosed population-based sample (n = 6039), who underwent an interview, physical examination and a brain MRI scan. Migraineurs, notably those with aura, had higher prevalence of subclinical infarcts in the posterior circulation [odds ratio (OR) 13.7; 95% confidence interval (CI) 1.7, 112]. Female migraineurs were at independent increased risk of white matter lesions (WMLs; OR 2.1; 95% CI 1.0, 4.1), and migraineurs had a higher prevalence of brainstem hyperintense lesions (4.4% vs. 0.7%, P = 0.04). We observed a higher lifetime prevalence of (frequent) syncope and orthostatic insufficiency in migraineurs; future research needs to clarify whether autonomic nervous system dysfunction could explain (part of) the increased risk of WMLs in female migraineurs. Finally, in migraineurs aged < 50 years, compared with controls, we found evidence of increased iron concentrations in putamen (P = 0.02), globus pallidus (P = 0.03) and red nucleus (P = 0.03). Higher risks in those with higher attack frequency or longer disease duration were found consistent with a causal relationship between migraine and lesions. This summary of our population-based data illustrates that migraine is associated with a significantly increased risk of brain lesions. Longitudinal studies are needed to assess whether these lesions are progressive and have relevant (long-term) functional correlates.",
"Migraine is a chronic-recurrent disorder that progresses in some individuals. Transformed migraine is the result of this progression. Since migraine does not progress in most patients, identifying the risk factors for progression has emerged as a very important public health priority. If risk factors can be identified, that might provide a foundation for more aggressive preventive intervention. Risk factors for progression may be divided into non-remediable (gender, age, race) and remediable categories. In this paper, we focus on several already identified remediable risk factors, including frequency of migraine attacks, obesity, acute medication overuse, caffeine overuse, stressful life events, depression, and sleep disorders. We present the evidence for each risk factor and discuss possible interventions to address them.",
"Migraine is common, with an estimated lifetime prevalence of 7-17%. Population-based studies have reported an association between various psychiatric conditions and migraine. This is a population-based study exploring the association between migraine and psychiatric disorders in a large cohort and assessing various health-related outcomes. (1) Determine the prevalence of various psychiatric conditions in association with migraine; (2) describe the patterns of association of these comorbidities with a variety of health-related outcomes. Data from the 2002 Canadian Community Health Survey were used. This is a national health survey which included administration of the World Mental Health Composite International Diagnostic Interview to a sample of 36,984 subjects. Health-related outcomes included 2-week disability, restriction of activities, quality-of-life, and mental health care utilization. The prevalence of physician-diagnosed migraine (n=36,984) was 15.2% for females and 6.1% for males. Migraine was most common in those between ages 25 and 44 years and in those of lower income. Migraine was associated with major depressive disorder, bipolar disorder, panic disorder, and social phobia, all occurring more than twice as often in those with migraines compared with those without. Migraine was not associated with drug, alcohol, or substance dependence. The higher prevalence of psychiatric disorders in migraineurs was not related to sociodemographic variables. Psychiatric disorders were less common in those over 65 years, in those who were in a relationship, and in those of higher income whether migraine was present or not. Health-related outcomes were worst in those with both migraines and a psychiatric disorder and intermediate in those with either condition alone. Migraine is associated with major depressive disorder, bipolar disorder, panic disorder, and social phobia. Migraine in association with various mental health disorders results in poorer health-related outcomes compared with migraine or a psychiatric condition alone. Understanding the psychiatric correlates of migraine is important in order to adequately manage this patient population and to guide public health policies regarding health services utilization and health-care costs.",
"Ergotamine exposure during pregnancy has been suggested to cause birth defects which have a vascular disruptive aetiology. The present case provides additional support for the possible adverse fetal effects of exposure to ergotamine, caffeine, and propranolol during the first four months of pregnancy. At birth the infant showed evidence of early arrested cerebral maturation and paraplegia. The nature of these defects suggests a primary vascular disruptive aetiology. We hypothesise that ergotamine, acting either alone or in synergy with propranolol and caffeine, produced fetal vasoconstriction resulting in tissue ischaemia and subsequent malformation. This case raises the possibility that fetal malformation may result from concomitant use of multiple vasoconstrictive agents during pregnancy.",
"The high prevalence of migraine in women during their reproductive years means that new drug treatments for migraine, such as the serotonin 5-HT(1B/1D) receptor agonists (the 'triptans'), are likely to be widely used by women of childbearing potential. Scrutiny of these agents in an effort to detect any signal of teratogenicity is thus important. A systematic review of the medical literature was conducted to identify information regarding the safety of sumatriptan during pregnancy. This agent was chosen to be investigated because it has been available for the longest and is the most widely used of the triptan class. Information was obtained regarding the impact of migraine on pregnancy outcome, and data on sumatriptan use in pregnancy were obtained from animal studies, preclinical drug trials, postmarketing surveillance efforts, prospective pregnancy registries, national birth registries and teratogen information services. Synthesis of information from these sources is sufficient to rule out a large increase in birth defects from sumatriptan use during pregnancy and is reassuring for cases where inadvertent exposure to sumatriptan during pregnancy has occurred. However, current information is not sufficient to rule out small increases in the risk for birth defects. For this reason, caution should be exercised in making a positive recommendation for the use of sumatriptan during pregnancy."
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P2X7 Receptor Inhibits Th17-, Th1/17- and Th1-Mediated Alloimmunity to Collagen Type V
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IL17-dependent autoimmunity to collagen type V (Col V) has been associated with lung transplant obliterative bronchiolitis. Unlike the T helper 1 (Th1)-dependent immune responses to Tetanus Toxoid (TT), the Th17 response to Col V in lung transplant patients and its Th1/17 variant observed in coronary artery disease patients requires IL-1β, tumor necrosis factor α and CD14(+) cells. Given the involvement of the P2X7 receptor (P2X7R) in monocyte IL-1β responses, we investigated its role in Th17-, Th1/17- and Th1-mediated proinflammatory responses. Transfer of antigen-pulsed peripheral blood mononucleated cells (PBMCs) from Col V-reactive patients into SCID mouse footpads along with P2X7R antagonists revealed a selective inhibition of Col V-, but not TT-specific swelling responses. P2X7R inhibitors blocked IL-1β induction from monocytes, including both Col V-α1 peptide-induced (T-dependent), as well as native Col V-induced (T-independent) responses. Significantly higher P2X7R expression was found on CXCR3(neg) CCR4(+)/6(+) CD4(+) [Th17] versus CXCR3(+)CCR4/6(neg) CD4(+) [Th1] subsets in PBMCs, suggesting that the paradigm of selective dependence on P2X7R might extend beyond Col V autoimmunity. Indeed, P2X7R inhibitors suppressed not only anti-Col V, but also Th1/17-mediated alloimmunity, in a heart transplant patient without affecting anti-viral Epstein-Barr virus responses. These results suggest that agents targeting the P2X7R might effectively treat Th17-related transplant pathologies, while maintaining Th1-immunity to infection.
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"Human cutaneous dendritic cells (DCs) have the ability to prime and bias Th17 lymphocytes. However, the factors that stimulate cutaneous DCs to induce Th17 responses are not well known. Alarmins, such as ATP, likely play a pivotal role in the induction and maintenance of cutaneous immune responses by stimulating DC maturation, chemotaxis, and secretion of IL-1β and IL-6, Th17-biasing cytokines. In this study, using a well-established human skin model, we have demonstrated that signaling purinergic receptors, predominantly the P2X7 receptor (P2X7R), via an ATP analog initiate innate proinflammatory inflammation, DC17 differentiation, and the subsequent induction of Th17-biased immunity. Moreover, our results suggest a potential role for P2X7R signaling in the initiation of psoriasis pathogenesis, a Th17-dependent autoimmune disease. In support of this, we observed the increased presence of P2X7R in nonlesional and lesional psoriatic skin compared with normal healthy tissues. Interestingly, there was also a P2X7R variant that was highly expressed in lesional psoriatic skin compared with nonlesional psoriatic and normal healthy skin. Furthermore, we demonstrated that psoriatic responses could be initiated via P2X7R signaling in nonlesional skin following treatment with a P2X7R agonist. Mechanistic studies revealed a P2X7R-dependent mir-21 angiogenesis pathway that leads to the expression of vascular endothelial growth factor and IL-6 and that may be involved in the development of psoriatic lesions. In conclusion, we have established that purinergic signaling in the skin induces innate inflammation, leading to the differentiation of human Th17 responses, which have implications in the pathogenesis and potential treatment of psoriasis.",
"Danger signals released upon cell damage can cause excessive immune-mediated tissue destruction such as that found in acute graft-versus-host disease (GVHD), allograft rejection and systemic inflammatory response syndrome. Given that ATP is found in small concentrations in the extracellular space under physiological conditions, and its receptor P2X(7)R is expressed on several immune cell types, ATP could function as a danger signal when released from dying cells. We observed increased ATP concentrations in the peritoneal fluid after total body irradiation, and during the development of GVHD in mice and in humans. Stimulation of antigen-presenting cells (APCs) with ATP led to increased expression of CD80 and CD86 in vitro and in vivo and actuated a cascade of proinflammatory events, including signal transducer and activator of transcription-1 (STAT1) phosphorylation, interferon-γ (IFN-γ) production and donor T cell expansion, whereas regulatory T cell numbers were reduced. P2X(7)R expression increased when GVHD evolved, rendering APCs more responsive to the detrimental effects of ATP, thereby providing positive feedback signals. ATP neutralization, early P2X(7)R blockade or genetic deficiency of P2X(7)R during GVHD development improved survival without immune paralysis. These data have major implications for transplantation medicine, as pharmacological interference with danger signals that act via P2X(7)R could lead to the development of tolerance without the need for intensive immunosuppression.",
"Delayed-type hypersensitivity response (DTH) is a rapid in vivo manifestation of T cell-dependent immune response to a foreign antigen (Ag) that the host immune system has experienced in the recent past. DTH reactions are often divided into a sensitization phase, referring to the initial antigen experience, and a challenge phase, which usually follows several days after sensitization. The lack of a delayed-type hypersensitivity response to a recall Ag demonstrated by skin testing is often regarded as an evidence of anergy. The traditional DTH assay has been effectively used in diagnosing many microbial infections. Despite sharing similar immune features such as lymphocyte infiltration, edema, and tissue necrosis, the direct DTH is not a feasible diagnostic technique in transplant patients because of the possibility of direct injection resulting in sensitization to donor antigens and graft loss. To avoid this problem, the human-to-mouse \"trans-vivo\" DTH assay was developed (1,2). This test is essentially a transfer DTH assay, in which human peripheral blood mononuclear cells (PBMCs) and specific antigens were injected subcutaneously into the pinnae or footpad of a naïve mouse and DTH-like swelling is measured after 18-24 hr (3). The antigen presentation by human antigen presenting cells such as macrophages or DCs to T cells in highly vascular mouse tissue triggers the inflammatory cascade and attracts mouse immune cells resulting in swelling responses. The response is antigen-specific and requires prior antigen sensitization. A positive donor-reactive DTH response in the Tv-DTH assay reflects that the transplant patient has developed a pro-inflammatory immune disposition toward graft alloantigens. The most important feature of this assay is that it can also be used to detect regulatory T cells, which cause bystander suppression. Bystander suppression of a DTH recall response in the presence of donor antigen is characteristic of transplant recipients with accepted allografts (2,4-14). The monitoring of transplant recipients for alloreactivity and regulation by Tv-DTH may identify a subset of patients who could benefit from reduction of immunosuppression without elevated risk of rejection or deteriorating renal function. A promising area is the application of the Tv-DTH assay in monitoring of autoimmunity(15,16) and also in tumor immunology (17).",
"We studied late graft rejection in a patient who had received a kidney transplant 9-10 years earlier from his mother and who had been off all immunosuppressive drugs for 7 years at the time of graft rejection onset. The mother differed for one HLA-A (A3) and one HLA-B (B62) antigen but had only a subtype mismatch at the HLA-DR beta 1 locus (donor: DR beta 1*1104; recipient: DR beta 1*1102). A gradual rise in serum creatinine from 1.8 to 2.0 mg/dl at year 9 prompted a biopsy, which was negative for rejection (focal infiltrates but no tubulitis). Ten months later the patient's creatinine had risen to > 3.4 mg/dl, and a second biopsy revealed extensive tubulitis, cellular rejection, and glomerular sclerosis. Sonicates of donor leukocytes triggered no delayed-type hypersensitivity (DTH) response above background (PBMC only) in the patient's peripheral blood leukocytes obtained prior to year 9. A gradual recovery of antidonor DTH response between year 9 and 10 closely paralleled the change from tolerant to rejection status. Antidonor antibody was also undetectable in serum prior to year 9, but a donor-reactive antibody did develop at year 10.2 shortly after the peak of DTH response. The serum level of soluble donor HLA class I B62 antigen rose > 10-fold over prerejection level at the time of the biopsy-proven rejection, suggesting a possible trigger for both the cellular and humoral immune response. Nonetheless, we found no evidence for the development of humoral or cellular immunity to maternal HLA class I. Instead, DTH analysis of memory T cells of the patient obtained after rejection showed that a single maternal HLA DR beta 1*1104 allopeptide, differing by two amino acids in sequence from the peptide of the recipient (DR beta 1*1102), stimulated a strong memory DTH response. Similarly, we found an anti-HLA class II donor-specific antibody in serum that appeared to be crossreactive with DR beta 1*1104 and DR beta 1*1101 but not with the recipient DR beta 1*1102 antigen. The data support the idea of a profound unresponsive state at both the cellular (DTH) and humoral level toward maternal HLA class I antigens that was not reversed even during late cellular rejection, despite the release of high levels of soluble HLA class I. Furthermore, the data suggest that DTH recovery was a close correlate of the onset of rejection and this \"indirect\" alloresponse, like the anti-donor alloantibody response that followed, was directed not to noninherited maternal HLA-A,B antigens but to the maternal HLA DR beta 1*1104 subtype.",
"We hypothesized that donor/recipient sharing of the human leukocyte antigen (HLA) involved in allopeptide presentation to the T regulatory cell increases the incidence of immune regulation, thus contributing to long-term graft survival. Peripheral blood mononuclear cells (PBMC) were obtained from 40 living related donor (LRD) and 31 cadaver renal transplant recipients. The trans vivo delayed type hypersensitivity (DTH) assay was used to assign patients to regulator, nonregulator, and sensitized categories. In a large cohort (n=1934 patients), primary graft survival and rejection episodes were analyzed using a log rank test for comparison with the DTH results. The highest incidence of regulated anti-donor DTH was observed in the LRD HLA-identical group (6/6; 100%) followed by the LRD HLA 1 haplotype matched group (18/27; 67%). Within the cadaver population, two DR-matched recipients had a higher frequency of regulated anti-donor DTH (6/11; 55%) than 1 & 0 DR-matched recipients (3/18; 17%). In a multivariate model, matching for HLA-DR alone, or for DR plus DQ was significantly (p=0.045, p=0.041) correlated with DTH regulation. The better HLA-matched groups showed the highest incidence of DTH regulation and, in a larger retrospective analysis, displayed better graft survival and freedom from acute rejection (p<0.0001). HLA matching, and HLA-DR matching in particular, correlates with the incidence of immune regulation after kidney transplantation.",
"α1(V) is an extensively modified collagen chain important in disease. Comprehensive mapping of α1(V) post-translational modifications reveals unexpectedly large numbers of X-position hydroxyprolines in Gly-X-Y amino acid triplets. The unexpected abundance of X-position hydroxyprolines suggests a mechanism for differential modification of collagen properties. Positions, numbers, and occupancy of modified sites can provide insights into α1(V) biological properties. Aberrant expression of the type V collagen α1(V) chain can underlie the connective tissue disorder classic Ehlers-Danlos syndrome, and autoimmune responses against the α1(V) chain are linked to lung transplant rejection and atherosclerosis. The α1(V) collagenous COL1 domain is thought to contain greater numbers of post-translational modifications (PTMs) than do similar domains of other fibrillar collagen chains, PTMs consisting of hydroxylated prolines and lysines, the latter of which can be glycosylated. These types of PTMs can contribute to epitopes that underlie immune responses against collagens, and the high level of PTMs may contribute to the unique biological properties of the α1(V) chain. Here we use high resolution mass spectrometry to map such PTMs in bovine placental α1(V) and human recombinant pro-α1(V) procollagen chains. Findings include the locations of those PTMs that vary and those PTMs that are invariant between these α1(V) chains from widely divergent sources. Notably, an unexpectedly large number of hydroxyproline residues were mapped to the X-positions of Gly-X-Y triplets, contrary to expectations based on previous amino acid analyses of hydrolyzed α1(V) chains from various tissues. We attribute this difference to the ability of tandem mass spectrometry coupled to nanoflow chromatographic separations to detect lower-level PTM combinations with superior sensitivity and specificity. The data are consistent with the presence of a relatively large number of 3-hydroxyproline sites with less than 100% occupancy, suggesting a previously unknown mechanism for the differential modification of α1(V) chain and type V collagen properties.",
"There are clinical situations in which it may be advantageous to monitor delayed type hypersensitivity (DTH) responses, an index of cell-mediated immunity, without exposing patients directly to the challenge antigens. For example, transplant patients may be at risk for becoming sensitized to donor antigens if injected with donor antigen during traditional skin tests. We describe an alternative method for human DTH testing, which involves the transfer of human peripheral blood mononuclear cells plus antigen into the pinnae or footpads of naive mice. This induces a measurable DTH-like swelling response, which we refer to as the \"trans vivo DTH response.\" As proof of principle, we provide data obtained during trans vivo DTH studies with tetanus toxoid, cytomegalovirus (CMV) and alloantigens. In general, human T cells must be co-localized with antigen and human macrophages to produce swelling responses, and such responses are antigen-specific and require prior antigen sensitization. Not only does this assay offer a simple, reliable clinical monitoring device, but it also provides a model with which to study the in vivo mechanisms of human DTH responses."
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Relationship between hippocampal subfield volumes and chronic pain in nondemented older adults
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Although previous studies have demonstrated that the hippocampus plays a role in pain processing, the role of hippocampal subfields is uncertain. The goal of this study was to examine the relationship between hippocampal subfield volumes and chronic pain in nondemented older adults. The study sample included 86 community-residing adults age 70 or older who were free of dementia and recruited from the Einstein Aging Study. Chronic pain was defined as pain over the last 3 months, that was moderate or severe (minimum rating of 4 out of 10) most, or all of the time. Hippocampal subfield volumes were estimated using FreeSurfer software. We modeled the association between chronic pain and hippocampal and subfield volume using linear regression. The sample had a mean age of 80 and was 58% female. Chronic pain, present in 55% of the sample, was associated with smaller right and total hippocampal volumes, particularly in women, after adjusting for age, education, and intracranial volume (eTICV). In addition, in women, volume was significantly reduced in participants with chronic pain in right CA2-3 (β=-0.35, p=0.010), right CA4-DG (β=-0.35, p=0.011), left presubiculum (β=-0.29, p=0.030), and left fimbria (β=-0.30, p=0.023). In men, chronic pain was not associated with the volume of any of the hippocampal subfield volumes. Chronic pain in women is associated with a reduction in the volume of right hippocampus and also selected hippocampal subfields. Future studies should clarify the mechanisms underlying the association between regional hippocampal volumes and chronic pain, particularly in women.
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"Neurons in the basal ganglia are connected to areas of prefrontal cerebral cortex involved in higher cognitive functions, and these connections occur primarily via the thalamus. In patients with bipolar disorder, regardless of age, neuroimaging studies have consistently reported an increased number of white matter hyperintensities, indicating possible alterations in striatum-thalamus and thalamus-prefrontal cortex connections. In the current study, we acquired high-resolution magnetic resonance imaging (MRI) and diffusion tensor (DT) scans of 40 patients with bipolar spectrum (BPS) illnesses (bipolar type I = 17, bipolar type II = 7, cyclothymia = 16) and 36 sex- and age-matched control subjects. Two researchers, without knowledge of diagnosis, outlined the caudate, putamen, and thalamus on contiguous axial MRI slices. We measured the volumes of the basal ganglia, thalamus, and gray/white matter of the frontal cortex. Bipolar spectrum patients as a single group did not differ from control subjects in thalamus and the basal ganglia volumes, but the cyclothymia patients had reductions in the volumes of putamen and the thalamus compared with control subjects. The BPS patients had significantly reduced volume of the white and the gray matter of the frontal cortex. Furthermore, compared with control subjects, BPS patients as a group showed alterations in anisotropy of the internal capsule adjacent to the striatum and thalamus and the frontal white matter. Our findings indicate that BPS patients may have distinct anatomical alterations in brain structures involved in the regulation of mood and cognition, as well as alterations in these structures' connection to related brain areas.",
"Previous magnetic resonance imaging (MRI) findings have demonstrated psychopathological symptom-related smaller gray matter volumes in various cingulate gyrus subregions in schizophrenia and bipolar disorder. However, it is unclear whether these gray matter abnormalities show a subregional specificity to either disorder and whether they show postonset progression. To determine whether there are initial and progressive gray matter volume deficits in cingulate gyrus subregions in patients with first-episode schizophrenia (FESZ) and patients with first-episode affective psychosis (FEAFF, mainly manic) and their specificity to FESZ or FEAFF. A naturalistic cross-sectional study at first hospitalization for psychosis and a longitudinal follow-up approximately 1(1/2) years later. Patients were from a private psychiatric hospital. Thirty-nine patients with FESZ and 41 with FEAFF at first hospitalization for psychosis and 40 healthy control subjects (HCs) recruited from the community underwent high-spatial-resolution MRI, with follow-up scans in 17 FESZ patients, 18 FEAFF patients, and 18 HCs. Individual subjects were matched for age, sex, parental socioeconomic status, and handedness. Cingulate gyrus gray matter volumes in 3 anterior subregions (subgenual, affective, and cognitive) and 1 posterior subregion, and whether there was a paracingulate sulcus. At first hospitalization, patients with FESZ showed significantly smaller left subgenual (P = .03), left (P = .03) and right (P = .005) affective, right cognitive (P = .04), and right posterior (P = .003) cingulate gyrus gray matter subregions compared with HCs. Moreover, at the 1(1/2)-year follow-up, patients with FESZ showed progressive gray matter volume decreases in the subgenual (P = .002), affective (P < .001), cognitive (P < .001), and posterior (P = .02) cingulate subregions compared with HCs. In contrast, patients with FEAFF showed only initial (left, P < .001; right, P = .002) and progressive subgenual subregion abnormalities (P < .001). Finally, patients with FESZ showed a less asymmetric paracingulate pattern than HCs (P = .02). Patients with FEAFF and FESZ showed differences in initial gray matter volumes and in their progression. Initial and progressive changes in patients with FEAFF were confined to the subgenual cingulate, a region strongly associated with affective disorder, whereas patients with FESZ evinced widespread initial and progressively smaller volumes.",
"We present a technique for automatically assigning a neuroanatomical label to each voxel in an MRI volume based on probabilistic information automatically estimated from a manually labeled training set. In contrast to existing segmentation procedures that only label a small number of tissue classes, the current method assigns one of 37 labels to each voxel, including left and right caudate, putamen, pallidum, thalamus, lateral ventricles, hippocampus, and amygdala. The classification technique employs a registration procedure that is robust to anatomical variability, including the ventricular enlargement typically associated with neurological diseases and aging. The technique is shown to be comparable in accuracy to manual labeling, and of sufficient sensitivity to robustly detect changes in the volume of noncortical structures that presage the onset of probable Alzheimer's disease.",
"A differential role has been suggested for two important areas in the neural circuitry of stress, central nucleus of the amygdala (CeA) and bed nucleus of stria terminalis (BNST) in the extended amygdala, in regulating fear versus anxiety. Since chronic stress enhances anxiety and consolidation of aversive memories, we examined the effects of chronic immobilization stress (CIS) on neuronal morphology in the CeA and BNST of rats. In contrast to previous reports of stress-induced atrophy in the hippocampus, CIS does not cause dendritic atrophy in CeA and BNST neurons. While dendritic arborization in CeA neurons remains unaffected, it increases in BNST neurons after CIS. These results suggest a role for dendritic remodeling of BNST neurons in stress-induced facilitation of anxiety.",
"Accumulating evidence indicates that phobic and posttraumatic anxiety disorders likely result from a failure to extinguish fear memories. Extinction normally depends on a new learning that competes with the original fear memory and is driven by medial prefrontal cortex (mPFC) projections to the amygdala. Although mPFC stimulation was reported to inhibit the central medial (CEm) amygdala neurons that mediate fear responses via their brainstem and hypothalamic projections, it is unclear how this inhibition is generated. Because the mPFC has very sparse projections to CEm output neurons, the mPFC-evoked inhibition of the CEm is likely indirect. Thus, this study tested whether it resulted from a feedforward inhibition of basolateral amygdala (BLA) neurons that normally relay sensory inputs to the CEm. However, our results indicate that mPFC inputs excite rather than inhibit BLA neurons, implying that the inhibition of CEm cells is mediated by an active gating mechanism downstream of the BLA.",
"A variety of findings have implicated the portion of the anterior cingulate cortex ventral to the corpus callosum in the pathophysiology of familial depressive disorder. There are, as yet, few data to address the specificity of these abnormalities to depressive disorders or to characterize their stability over time. The authors studied 10 subjects who were judged to have had major depressive disorder with psychotic features, who underwent magnetic resonance imaging (MRI) protocols, and who participated in a longitudinal study of recent-onset psychosis. These were group-matched to 10 subjects with schizophrenia and to 10 well comparison subjects. Volumetric measures were made of the posterior and anterior portions of the subgenual prefrontal cortex for these 30 subjects. Follow-up scans done an average of 4 years after intake were available for seven subjects with major depressive disorder, nine subjects with schizophrenia, and five well comparison subjects. Volumes of the left side of the posterior subgenual prefrontal cortex differed significantly by group and were smallest for the group with psychotic major depressive disorder. Volumes of the anterior subgenual prefrontal cortex did not differ significantly by group. Patients with major depressive disorder were more likely to show increases in posterior subgenual prefrontal cortex volume on follow-up than were comparison subjects or patients with schizophrenia. These findings add to the evidence that abnormalities in the subgenual region of the anterior cingulate play a role in at least some types of mood disorder.",
"Numerous studies have reported a smaller hippocampal volume in Alzheimer's disease (AD) patients than in aging controls. However, in mild cognitive impairment (MCI), the results are inconsistent. Moreover, the left-right asymmetry of the hippocampus receives less research attention. In this article, meta-analyses are designed to determine the extent of hippocampal atrophy in MCI and AD, and to evaluate the asymmetry pattern of the hippocampal volume in control, MCI, and AD groups. From 14 studies including 365 MCI patients and 382 controls, significant atrophy is found in both the left [Effect size (ES), 0.92; 95% confidence interval (CI), 0.72-1.11] and right (ES, 0.78; 95% CI, 0.57-0.98) hippocampus, which is lower than that in AD (ES, 1.60, 95% CI, 1.37-1.84, in left; ES, 1.52, 95% CI, 1.31-1.72, in right). Comparing with aging controls, the average volume reduction weighted by sample size is 12.9% and 11.1% in left and right hippocampus in MCI, and 24.2% and 23.1% in left and right hippocampus in AD, respectively. The findings show a bilateral hippocampal volume loss in MCI and the extent of atrophy is less than that in AD. By comparing the left and right hippocampal volume, a consistent left-less-than-right asymmetry pattern is found, but with different extents in control (ES, 0.39), MCI (ES, 0.56), and AD (ES, 0.30) group.",
"Recent developments in MRI data acquisition technology are starting to yield images that show anatomical features of the hippocampal formation at an unprecedented level of detail, providing the basis for hippocampal subfield measurement. However, a fundamental bottleneck in MRI studies of the hippocampus at the subfield level is that they currently depend on manual segmentation, a laborious process that severely limits the amount of data that can be analyzed. In this article, we present a computational method for segmenting the hippocampal subfields in ultra-high resolution MRI data in a fully automated fashion. Using Bayesian inference, we use a statistical model of image formation around the hippocampal area to obtain automated segmentations. We validate the proposed technique by comparing its segmentations to corresponding manual delineations in ultra-high resolution MRI scans of 10 individuals, and show that automated volume measurements of the larger subfields correlate well with manual volume estimates. Unlike manual segmentations, our automated technique is fully reproducible, and fast enough to enable routine analysis of the hippocampal subfields in large imaging studies.",
"Pain is a complex experience consisting of sensory-discriminative, affective-motivational, and cognitive-evaluative dimensions. Now it has been gradually known that noxious information is processed by a widely-distributed, hierarchically- interconnected neural network, referred to as neuromatrix, in the brain. Thus, identifying the multiple neural networks subserving these functional aspects and harnessing this knowledge to manipulate the pain response in new and beneficial ways are challenging tasks. Albeit with elaborate research efforts on the cortical responses to painful stimuli or clinical pain, involvement of the hippocampal formation (HF) in pain is still a matter of controversy. Here, we integrate previous animal and human studies from the viewpoint of HF and pain, sequentially representing anatomical, behavioral, electrophysiological, molecular/biochemical and functional imaging evidence supporting the role of HF in pain processing. At last, we further expound on the relationship between pain and memory and present some unresolved issues.",
"Suicidal behavior has been associated with hypothalamic-pituitary-adrenal overactivity in humans, as measured by increased corticosteroid secretion. To investigate whether this overactivity is reflected at the pituitary level, we have studied the localization of pro-opiomelanocortin (POMC) mRNA, and glucocorticoid receptor (GR) mRNA, in human anterior pituitaries, and quantified these messages relative to controls. Pituitaries from 7 suicide victims and 11 cardiac deaths were sectioned into 10-microns slides, stained with thionin and processed for in situ hybridization using a riboprobe complementary to human POMC mRNA. To correct for possible postmortem cell loss, hybridization with P1B15, a cDNA complementary to rat cyclophillin mRNA, was used in adjacent sections. POMC mRNA containing cells were found to be localized in clusters and were highly associated with corticotropin-releasing hormone (CRH) receptors. In contrast, GR mRNA containing cells were distributed through the pituitary, although areas of increased density were associated with POMC mRNA cells. Quantification with a computerized image analysis system revealed a 25% increase in POMC message in suicide victims. Analysis of the corticotrophic cell clumps showed that the suicide victims had higher POMC mRNA density per cell (p = 0.04) and larger corticotrophic cell size (p = 0.04) than the cardiac death victims. No differences in GR mRNA were detected between the two groups, although GR and POMC mRNA levels were highly and significantly correlated (r = 0.8, p < 0.001). There were no differences in P1B15 message between the two groups. We conclude that in situ hybridization is a useful tool to study gene regulation in human neuroendocrine tissue and that suicide victims show evidence of chronic hypothalamic-pituitary-adrenal axis activation."
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Use of urinary miR-126 as a biomarker for diabetic nephropathy
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Background. The purpose of the present study was to assess the feasibility of using miR-126 in the urine as a biomarker for diabetic nephropathy. Methods. miRNAs were extracted from the urine samples of T2DM patients with diabetic nephropathy (DN; n = 92), T2DM without DN (n = 86), and 85 healthy volunteers using quantitative reverse transcriptase polymerase chain reaction (real-time polymerase chain reaction) analysis. Stability of urinary miR-126 and factors that affected the stability were assessed. A subgroup analysis was also carried out to compare the urinary miR-126 level in T2DM patients well controlled by the treatment versus those who were not well controlled. Results. Urinary miR-126 was stable when the urine samples were kept at room temperature for extended period of time, 4°C, -20°C, and -80°C for up to 12 hours or subjected to 10 freeze-and-thaw cycle. Urinary miR-126 was significantly higher in T2DM patients with DN (5.76 ± 0.33 versus 3.25 ± 0.45 in T2DM patients without DN). Successful treatment significantly reduced urinary miR-126 in T2DM patients with DN to 3.89 ± 0.52 (P < 0.05). Conclusion. miR-126 in the urine is stable and it could be used as a biomarker of DN and to monitor the treatment response.
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"Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ( approximately 22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.",
"A cell line has been derived from a human prostatic carcinoma xenograft, CWR22R. This represents one of very few available cell lines representative of this disease. The cell line is derived from a xenograft that was serially propagated in mice after castration-induced regression and relapse of the parental, androgen-dependent CWR22 xenograft. Flow cytometric and cytogenetic analysis showed that this cell line represents one hyper DNA-diploid stem line with two clonal, evolved cytogenetic sublines. The basic karyotype is close to that of the grandparent xenograft, CWR22, and is relatively simple with 50 chromosomes. In nude mice, the line forms tumors with morphology similar to that of the xenografts, and like the parental CWR22 and CWR22R xenografts, this cell line expresses prostate specific antigen. Growth is weakly stimulated by dihydroxytestosterone and lysates are immunoreactive with androgen receptor antibody by Western blot analysis. Growth is stimulated by epidermal growth factor but is not inhibited by transforming growth factor-beta1."
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Muscleblind-like splicing is a key regulator of erythroid terminal differentiation
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The scope and roles of regulated isoform gene expression during erythroid terminal development are poorly understood. We identified hundreds of differentiation-associated isoform changes during terminal erythropoiesis. Sequences surrounding cassette exons of skipped exon events are enriched for motifs bound by the Muscleblind-like (MBNL) family of splicing factors. Knockdown of Mbnl1 in cultured murine fetal liver erythroid progenitors resulted in a strong block in erythroid differentiation and disrupted the developmentally regulated exon skipping of Ndel1 mRNA, which is bound by MBNL1 and critical for erythroid terminal proliferation. These findings reveal an unanticipated scope of the alternative splicing program and the importance of Mbnl1 during erythroid terminal differentiation.
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[
"Myotonic dystrophy (DM1) is an autosomal dominant neuromuscular disorder associated with a (CTG)(n) expansion in the 3'-untranslated region of the DM1 protein kinase (DMPK) gene. To explain disease pathogenesis, the RNA dominance model proposes that the DM1 mutation produces a gain-of-function at the RNA level in which CUG repeats form RNA hairpins that sequester nuclear factors required for proper muscle development and maintenance. Here, we identify the triplet repeat expansion (EXP) RNA-binding proteins as candidate sequestered factors. As predicted by the RNA dominance model, binding of the EXP proteins is specific for dsCUG RNAs and proportional to the size of the triplet repeat expansion. Remarkably, the EXP proteins are homologous to the Drosophila muscleblind proteins required for terminal differentiation of muscle and photoreceptor cells. EXP expression is also activated during mammalian myoblast differentiation, but the EXP proteins accumulate in nuclear foci in DM1 cells. We propose that DM1 disease is caused by aberrant recruitment of the EXP proteins to the DMPK transcript (CUG)(n) expansion.",
"Differentiating erythroid cells execute a unique gene expression program that insures synthesis of the appropriate proteome at each stage of maturation. Standard expression microarrays provide important insight into erythroid gene expression but cannot detect qualitative changes in transcript structure, mediated by RNA processing, that alter structure and function of encoded proteins. We analyzed stage-specific changes in the late erythroid transcriptome via use of high-resolution microarrays that detect altered expression of individual exons. Ten differentiation-associated changes in erythroblast splicing patterns were identified, including the previously known activation of protein 4.1R exon 16 splicing. Six new alternative splicing switches involving enhanced inclusion of internal cassette exons were discovered, as well as 3 changes in use of alternative first exons. All of these erythroid stage-specific splicing events represent activated inclusion of authentic annotated exons, suggesting they represent an active regulatory process rather than a general loss of splicing fidelity. The observation that 3 of the regulated transcripts encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may indicate significant changes in the RNA processing machinery of late erythroblasts. Together, these results support the existence of a regulated alternative pre-mRNA splicing program that is critical for late erythroid differentiation.",
"Erythropoietin (EPO) is a glycoprotein that is mainly produced in the adult kidney, and it was initially highlighted for its action on the hematopoietic system. Moreover, EPO is also expressed in several non-hematopoietic tissues, where it plays a role in the protection from apoptosis and inflammation due to hypoxia, toxicity or injury. These protective effects are mainly known and studied in cardioprotection and neuroprotection but are also reported in retina degeneration, auditory injury and pancreatic-related diseases. The tissue protective effect of EPO is mainly mediated through the interaction with the heterodimeric receptor EPOR/βcR. Human recombinant EPO (HuREPO), which has been developed to treat anemia, is not adequate for tissue protection. The low affinity of the alternative receptor for EPO involves the injection of excessive concentration of erythropoiesis-stimulating agents (ESAs), implicating side effects due to the cross-talk with hematopoietic activity. For these reasons, EPO derivatives with less affinity for the EPO homodimeric receptor are under development. In this review, we provide an overview of the erythroid and non-erythroid functions of EPO by detailing the molecular mechanisms activated by the binding of EPO to its receptors in different tissues.",
"Partially purified human burst-forming unit-erythroid (BFU-E) cells from peripheral blood were cultured for 6 to 8 days to obtain colony-forming unit-erythroid (CFU-E) cells. When these BFU-E-derived CFU-E were further purified and recultured in liquid suspension cultures with erythropoietin (EPO), they matured and differentiated into reticulocytes in vitro. A maximum rate of hemoglobin synthesis was observed at day 10 of cumulative culture time by measuring 59Fe incorporation into heme. Withdrawal of EPO from erythroblast cultures at various times during development showed that between day 10 and day 11 (when the majority of the cells are in the polychromatic erythroblast stage), these cells became independent of EPO. The timing of the disappearance of the EPO requirement in these cells coincided with the marked decline in proliferation. Measurement of EPO receptor messenger RNA (mRNA) levels by Northern analysis showed that there is a slight decline during the day 8 to day 10 time period, followed by a rapid decline between days 10 and 14. Binding of 125I-EPO to erythroblasts also showed a steady decline of the cell surface binding during maturation and terminal differentiation. The half-life of the human EPO receptor was 90 minutes in the presence of the transcriptional inhibitor actinomycin D and the half-life measured at two different times during the 8- to 14-day culture period remained constant. These results indicate that human EPO receptor mRNA must be transcribed continuously to maintain the levels seen by Northern analysis. The human cell system described here is well suited for the study of a wide variety of biochemical events during late erythroid differentiation.",
"A large German family with autosomal dominantly inherited elliptocytosis, associated with truncated beta-spectrin missing the phosphorylated C-terminal peptide, has been described (Eber, S.W., Morris, S. A., Schroeter, W., and Gratzer, W. B. (1988) J. Clin. Invest. 81, 523-530). We have attempted to delineate the molecular defect of this abnormality at the gene level. Southern blot analyses revealed no evidence of a partial gene deletion or rearrangement. We used polymerase chain reaction (PCR) and amplified several relevant portions of the beta-spectrin gene, using genomic DNA from two different heterozygous patients. No abnormality was found in the last four exons of the beta-spectrin gene produced by PCR. Examination of the introns connecting the last four exons revealed a T to A substitution in the 5' splice site following the exon X in four of eight clones prepared from two affected individuals, but not from a normal subject of this family. To examine the effect of the T to A substitution in these patients, we made cDNA from the reticulocyte mRNA of the patient and examined its composition by PCR. Two distinct PCR fragments produced from the patient's beta-spectrin cDNA were found. One matched the predicted size for normal spectrin, while the other was 197 base pairs shorter. The mutant cDNA sequence revealed that the entire exon preceding the T to A substitution was spliced out, while the two terminal exons were preserved. The deletion of this exon resulted in a frameshift giving a different terminal amino acid (serine instead of leucine) as well as a new termination codon causing a deletion of 129 amino acids including potentially phosphorylated residues.",
"Porphobilinogen deaminase [PBGD; porphobilinogen ammonia-lyase (polymerizing), EC 4.3.1.8] is a cytosolic enzyme involved in the heme biosynthetic pathway. Two isoforms of PBGD, encoded by two mRNAs differing solely in their 5' end, are known: one is found in all cells and the other is present only in erythroid cells. We have previously shown that the human PBGD is encoded by a single gene and have now cloned and characterized this gene, which is split into 15 exons spread over 10 kilobases of DNA. We demonstrate that the two mRNAs arise from two overlapping transcription units. The first one (upstream) is active in all tissues and its promoter has some of the structural features of a housekeeping promoter; the second, located 3 kilobases downstream, is active only in erythroid cells and its promoter displays structural homologies with the beta-globin gene promoters.",
"Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them."
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Unipolar growth in the Rhizobiales Agrobacterium tumefaciens
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The synthesis of peptidoglycan (PG) in bacteria is a crucial process controlling cell shape and vitality. In contrast to bacteria such as Escherichia coli that grow by dispersed lateral insertion of PG, little is known of the processes that direct polar PG synthesis in other bacteria such as the Rhizobiales. To better understand polar growth in the Rhizobiales Agrobacterium tumefaciens, we first surveyed its genome to identify homologs of (~70) well-known PG synthesis components. Since most of the canonical cell elongation components are absent from A. tumefaciens, we made fluorescent protein fusions to other putative PG synthesis components to assay their subcellular localization patterns. The cell division scaffolds FtsZ and FtsA, PBP1a, and a Rhizobiales- and Rhodobacterales-specific l,d-transpeptidase (LDT) all associate with the elongating cell pole. All four proteins also localize to the septum during cell division. Examination of the dimensions of growing cells revealed that new cell compartments gradually increase in width as they grow in length. This increase in cell width is coincident with an expanded region of LDT-mediated PG synthesis activity, as measured directly through incorporation of exogenous d-amino acids. Thus, unipolar growth in the Rhizobiales is surprisingly dynamic and represents a significant departure from the canonical growth mechanism of E. coli and other well-studied bacilli.
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[
"Murein synthesized in ether-permeabilized cells of Escherichia coli deficient in individual penicillin-binding proteins (PBPs) and in the presence of certain beta-lactam antibiotics was analyzed by high-pressure liquid chromatography separation of the muramidase split products. PBP 1b was found to to be the major murein synthesizing activity that was poorly compensated for by PBP 1a. A PBP 2 mutant as well as mecillinam-inhibited cells showed increased activity in the formation of oligomeric muropeptides as well as UDP-muramylpeptidyl-linked muropeptides, the reaction products of transpeptidation, bypassing the lipid intermediate. In contrast, penicillin G and furazlocillin severely inhibited these reactions but stimulated normal dimer production. It is concluded that two distinct transpeptidases exist in E. coli: one, highly sensitive to penicillin G and furazlocillin, catalyzes the formation of hyper-cross-linked muropeptides, and a second one, quite resistant to these antibiotics, synthesizes muropeptide dimers.",
"Penicillin-binding protein 7 (PBP7) and its proteolytic degradation product PBP8 are shown to be soluble proteins, which can be set free from whole cells of Escherichia coli by an osmotic shock. The proteins are loosely associated with the membranes and are totally released into the supernatant in the presence of 1 M NaCl. Partial purification of PBP8 was accomplished by hydroxyapatite, heparin-Sepharose and MonoS chromatography. Murein meso-diaminopimelate-D-alanine DD-endopeptidase activity was demonstrated for both PBP7 and PBP8, which specifically hydrolyse the DD-diaminopimelate-alanine bonds in high-molecular-mass murein sacculi but fail to cleave these bonds in isolated dimeric muropeptides. The enzyme is inhibited by the 'penem' beta-lactam antibiotic CGP31608 at a concentration of 0.25 micrograms/ml by 50%. Thus besides PBP4 and the mepA gene product, a third endopeptidase exists in E. coli.",
"During cytokinesis in Escherichia coli, the peptidoglycan (PG) layer produced by the divisome must be split to promote cell separation. Septal PG splitting is mediated by the amidases: AmiA, AmiB, and AmiC. To efficiently hydrolyze PG, the amidases must be activated by LytM domain factors. EnvC specifically activates AmiA and AmiB, while NlpD specifically activates AmiC. Here, we used an exportable, superfolding variant of green fluorescent protein (GFP) to demonstrate that AmiB, like its paralog AmiC, is recruited to the division site by an N-terminal targeting domain. The results of colocalization experiments indicate that EnvC is recruited to the division site well before its cognate amidase AmiB. Moreover, we show that EnvC and AmiB have differential FtsN requirements for their localization. EnvC accumulates at division sites independently of this essential division protein, whereas AmiB localization is FtsN dependent. Interestingly, we also report that AmiB and EnvC are recruited to division sites independently of one another. The same is also true for AmiC and NlpD. However, unlike EnvC, we find that NlpD shares an FtsN-dependent localization with its cognate amidase. Importantly, when septal PG synthesis is blocked by cephalexin, both EnvC and NlpD are recruited to septal rings, whereas the amidases fail to localize. Our results thus suggest that the order in which cell separation amidases and their activators localize to the septal ring relative to other components serves as a fail-safe mechanism to ensure that septal PG synthesis precedes the expected burst of PG hydrolysis at the division site, accompanied by amidase recruitment.",
"Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI(q)-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZalpha. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.",
"MreB is thought to be a bacterial actin homolog that defines the morphology of rod-shaped bacteria. Rhodobacter sphaeroides changes shape, from a rod to coccobacillus, and undergoes extensive cytoplasmic membrane invagination when it switches from aerobic to photoheterotrophic growth. The role of MreB in defining R. sphaeroides shape was therefore investigated. Attempts at deleting or insertionally inactivating mreB were unsuccessful under all growth conditions. Immunofluorescence microscopy showed MreB localized to mid-cell in elongating cells under both aerobic and photoheterotrophic conditions. Three-dimensional reconstruction showed that MreB formed a ring at mid-cell. MreB remained at mid-cell as septation began but localized to new sites in the daughter cells before the completion of septation. MreB localized to putative septation sites in cephalexin-treated filamentous cells. Genomic single-copy mreB was replaced with gfp-mreB, and green fluorescent protein (GFP)-MreB localized in the same pattern, as seen with immunofluorescence microscopy. Some of the cells expressing GFP-MreB were abnormal, principally displaying an increase in cell width, suggesting that the fusion was not fully functional in all cells. GFP-MreB localized to swellings at mid-cell in cells treated with the penicillin-binding protein 2 inhibitor amdinocillin. These data suggest that MreB is essential in R. sphaeroides, performing a role at mid-cell in elongating cells, and in early septation, putatively in the cytoplasmic control of the peptidoglycan synthetic complexes.",
"The Escherichia coli Tat system serves to export folded proteins harbouring an N-terminal twin-arginine signal peptide across the cytoplasmic membrane. Previous work has demonstrated that strains mutated in genes encoding essential Tat pathway components are highly defective in the integrity of their cell envelope. Here, we report the isolation, by transposon mutagenesis, of tat mutant strains that have their outer membrane integrity restored. This outer membrane repair of the tat mutant arises as a result of upregulation of the amiB gene, which encodes a cell wall amidase. Overexpression of the genes encoding the two additional amidases, amiA and amiC, does not compensate for the outer membrane defect of the tatC strain. Analysis of the amiA and amiC coding sequences indicates that the proteins may be synthesized with plausible twin-arginine signal sequences, and we demonstrate that they are translocated to the periplasm by the Tat pathway. A Tat+ strain that has mislocalized AmiA and AmiC proteins because of deletion of their signal peptides displays an identical defective cell envelope phenotype. The presence of genes encoding amidases with twin-arginine signal sequences in the genomes of other Gram-negative bacteria suggests that a similar cell envelope defect may be a common feature of tat mutant strains.",
"Prokaryotes come in a wide variety of shapes, determined largely by natural selection, physical constraints, and patterns of cell growth and division. Because of their relative simplicity, bacterial cells are excellent models for how genes and proteins can directly determine morphology. Recent advances in cytological methods for bacteria have shown that distinct cytoskeletal filaments composed of actin and tubulin homologs are important for guiding growth patterns of the cell wall in bacteria, and that the glycan strands that constitute the wall are generally perpendicular to the direction of growth. This cytoskeleton-directed cell wall patterning is strikingly reminiscent of how plant cell wall growth is regulated by microtubules. In rod-shaped bacilli, helical cables of actin-like MreB protein stretch along the cell length and orchestrate elongation of the cell wall, whereas the tubulin-like FtsZ protein directs formation of the division septum and the resulting cell poles. The overlap and interplay between these two systems and the peptidoglycan-synthesizing enzymes they recruit are the major driving forces of cylindrical shapes. Round cocci, on the other hand, have lost their MreB cables and instead must grow mainly via their division septum, giving them their characteristic round or ovoid shapes. Other bacteria that lack MreB homologs or even cell walls use distinct cytoskeletal systems to maintain their distinct shapes. Here I review what is known about the mechanisms that determine the shape of prokaryotic cells.",
"As one of the final steps in the bacterial growth cycle, daughter cells must be released from one another by cutting the shared peptidoglycan wall that separates them. In Escherichia coli, this delicate operation is performed by several peptidoglycan hydrolases, consisting of multiple amidases, lytic transglycosylases, and endopeptidases. The interactions among these enzymes and the molecular mechanics of how separation occurs without lysis are unknown. We show here that deleting the endopeptidase PBP 4 from strains lacking AmiC produces long chains of unseparated cells, indicating that PBP 4 collaborates with the major peptidoglycan amidases during cell separation. Another endopeptidase, PBP 7, fulfills a secondary role. These functions may be responsible for the contributions of PBPs 4 and 7 to the generation of regular cell shape and the production of normal biofilms. In addition, we find that the E. coli peptidoglycan amidases may have different substrate preferences. When the dd-carboxypeptidase PBP 5 was deleted, thereby producing cells with higher levels of pentapeptides, mutants carrying only AmiC produced a higher percentage of cells in chains, while mutants with active AmiA or AmiB were unaffected. The results suggest that AmiC prefers to remove tetrapeptides from peptidoglycan and that AmiA and AmiB either have no preference or prefer pentapeptides. Muropeptide compositions of the mutants corroborated this latter conclusion. Unexpectedly, amidase mutants lacking PBP 5 grew in long twisted chains instead of straight filaments, indicating that overall septal morphology was also defective in these strains.",
"Four low-molecular-weight penicillin binding proteins (LMW PBPs) of Escherichia coli are closely related and have similar DD-carboxypeptidase activities (PBPs 4, 5, and 6 and DacD). However, only one, PBP 5, has a demonstrated physiological function. In its absence, certain mutants of E. coli have altered diameters and lose their uniform outer contour, resulting in morphologically aberrant cells. To determine what differentiates the activities of these LMW PBPs, we constructed fusion proteins combining portions of PBP 5 with fragments of other DD-carboxypeptidases to see which hybrids restored normal morphology to a strain lacking PBP 5. Functional complementation occurred when truncated PBP 5 was combined with the terminal membrane anchor sequences of PBP 6 or DacD. However, complementation was not restored by the putative carboxy-terminal anchor of PBP 4 or by a transmembrane region of the osmosensor protein ProW, even though these hybrids were membrane bound. Site-directed mutagenesis of the carboxy terminus of PBP 5 indicated that complementation required a generalized amphipathic membrane anchor but that no specific residues in this region seemed to be required. A functional fusion protein was produced by combining the N-terminal enzymatic domain of PBP 5 with the C-terminal beta-sheet domain of PBP 6. In contrast, the opposite hybrid of PBP 6 to PBP 5 was not functional. The results suggest that the mode of PBP 5 membrane anchoring is important, that the mechanism entails more than a simple mechanical tethering of the enzyme to the outer face of the inner membrane, and that the physiological differences among the LMW PBPs arise from structural differences in the DD-carboxypeptidase enzymatic core."
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Drosophila caspase 1 regulates mitochondrial morphology and autophagy flux
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Increasing evidence reveals that a subset of proteins participates in both the autophagy and apoptosis pathways, and this intersection is important in normal physiological contexts and in pathological settings. In this paper, we show that the Drosophila effector caspase, Drosophila caspase 1 (Dcp-1), localizes within mitochondria and regulates mitochondrial morphology and autophagic flux. Loss of Dcp-1 led to mitochondrial elongation, increased levels of the mitochondrial adenine nucleotide translocase stress-sensitive B (SesB), increased adenosine triphosphate (ATP), and a reduction in autophagic flux. Moreover, we find that SesB suppresses autophagic flux during midoogenesis, identifying a novel negative regulator of autophagy. Reduced SesB activity or depletion of ATP by oligomycin A could rescue the autophagic defect in Dcp-1 loss-of-function flies, demonstrating that Dcp-1 promotes autophagy by negatively regulating SesB and ATP levels. Furthermore, we find that pro-Dcp-1 interacts with SesB in a nonproteolytic manner to regulate its stability. These data reveal a new mitochondrial-associated molecular link between nonapoptotic caspase function and autophagy regulation in vivo.
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[
"Cysteine proteases of the ICE/CED-3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila. We report here the identification and characterization of drICE, a D. melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr. drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.",
"The core effectors of apoptosis encompass proteolytic enzymes of the caspase family, which reside as latent precursors in most nucleated metazoan cells. A majority of studies on apoptosis are based on the assumption that caspase precursors are activated by cleavage, a common mechanism for most protease zymogen activations. Although this appears to be true for the executioner caspases, recent research points to a distinct activation mechanism for the initiator caspases that trigger the apoptotic pathways. This mechanism is proximity-induced dimerization without cleavage, and its elucidation has led to the revision of concepts of feedback regulation of apoptosis.",
"Essentially, all metazoan cells can undergo apoptosis, but some cells are more sensitive than others to apoptotic stimuli. To date, it is unclear what determines the apoptotic potential of the cell. We set up an in vivo system for monitoring and comparing the activity levels of the two main effector caspases in Drosophila melanogaster, Drice and Dcp-1. Both caspases were activated by the apoptosome after irradiation. However, whereas each caspase alone could induce apoptosis, Drice was a more effective inducer of apoptosis than Dcp-1, which instead had a role in establishing the rate of cell death. These functional differences are attributed to their intrinsic properties rather than merely their tissue specificities. Significantly, the levels of the procaspases are directly proportional to their activity levels and play a key role in determining the cell's sensitivity to apoptosis. Finally, we provide evidence for the existence of a cellular execution threshold of caspase activity, which must be reached to induce apoptosis.",
"Autophagy is a process of self-cannibalization. Cells capture their own cytoplasm and organelles and consume them in lysosomes. The resulting breakdown products are inputs to cellular metabolism, through which they are used to generate energy and to build new proteins and membranes. Autophagy preserves the health of cells and tissues by replacing outdated and damaged cellular components with fresh ones. In starvation, it provides an internal source of nutrients for energy generation and, thus, survival. A powerful promoter of metabolic homeostasis at both the cellular and whole-animal level, autophagy prevents degenerative diseases. It does have a downside, however--cancer cells exploit it to survive in nutrient-poor tumors.",
"Outer membranes isolated from yeast mitochondria were capable mediating the in vitro insertion of porin. As with the outer membrane of intact mitochondria, the insertion was ATP-dependent, and the inserted porin was resistant to trypsin treatment after detergent solubilization. However, the extent of porin insertion into isolated outer membranes was much less per mg of outer membrane protein than with intact mitochondria. The greater efficiency of intact mitochondria was not due to contact site-mediated translocation as isolated contact sites were less able to insert porin than isolated outer membranes, and blockade of the contact site channel in intact mitochondria did not affect porin insertion. However, mitochondria that had been subjected to osmotic shock sufficient to rupture the outer membrane and deplete the contents of the intermembrane space (i.e. mitoplasts) lost most of their ability to insert porin. Since outer membranes are isolated from mitoplasts, the low insertion activity of mitoplasts explains the low efficiency of insertion into isolated outer membranes. These results also indicate that, unlike proteins that are imported to the inner membrane and matrix of the mitochondria, porin's assembly is severely reduced by breaching the outer membrane and depletion of the intermembrane space contents.",
"We describe a microinjection-based phiC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the phiC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.",
"Apoptosis is coordinated by members of the caspase family of aspartic acid-specific proteases. Other members of this protease family also play essential roles in inflammation where they participate in the maturation of pro-inflammatory cytokines. To date, almost 400 substrates for the apoptosis-associated caspases have been reported and there are likely to be hundreds more yet to be discovered. Thus, the fraction of the proteome that is degraded (the degradome) by caspases during the demolition phase of apoptosis appears to be quite substantial. Despite this, we still know surprisingly little concerning how caspases provoke some of the signature events in apoptosis, such as membrane phosphatidylserine externalization, cellular retraction, chromatin condensation and apoptotic body production. The inflammatory caspases appear to be much more specific proteases than those involved in apoptosis and only two confirmed substrates for these proteases have been described to date. Here, we have compiled a comprehensive list of caspase substrates and describe a searchable web resource (The Casbah; www.casbah.ie) which contains information pertaining to all currently known caspase substrates. We also discuss some of the unresolved issues relating to caspase-dependent events in apoptosis and inflammation."
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Deleterious mutations in Eighty-five Requiring 3A are associated with autism spectrum disorder
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Whole-exome sequencing studies in autism spectrum disorder (ASD) have identified de novo mutations in novel candidate genes, including the synaptic gene Eighty-five Requiring 3A (EFR3A). EFR3A is a critical component of a protein complex required for the synthesis of the phosphoinositide PtdIns4P, which has a variety of functions at the neural synapse. We hypothesized that deleterious mutations in EFR3A would be significantly associated with ASD.
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[
"Tuberous sclerosis is a single-gene disorder caused by heterozygous mutations in the TSC1 (9q34) or TSC2 (16p13.3) gene and is frequently associated with mental retardation, autism and epilepsy. Even individuals with tuberous sclerosis and a normal intelligence quotient (approximately 50%) are commonly affected with specific neuropsychological problems, including long-term and working memory deficits. Here we report that mice with a heterozygous, inactivating mutation in the Tsc2 gene (Tsc2(+/-) mice) show deficits in learning and memory. Cognitive deficits in Tsc2(+/-) mice emerged in the absence of neuropathology and seizures, demonstrating that other disease mechanisms are involved. We show that hyperactive hippocampal mammalian target of rapamycin (mTOR) signaling led to abnormal long-term potentiation in the CA1 region of the hippocampus and consequently to deficits in hippocampal-dependent learning. These deficits included impairments in two spatial learning tasks and in contextual discrimination. Notably, we show that a brief treatment with the mTOR inhibitor rapamycin in adult mice rescues not only the synaptic plasticity, but also the behavioral deficits in this animal model of tuberous sclerosis. The results presented here reveal a biological basis for some of the cognitive deficits associated with tuberous sclerosis, and they show that treatment with mTOR antagonists ameliorates cognitive dysfunction in a mouse model of this disorder.",
"Research during the past decade has seen significant progress in the understanding of the genetic architecture of autism spectrum disorders (ASDs), with gene discovery accelerating as the characterization of genomic variation has become increasingly comprehensive. At the same time, this research has highlighted ongoing challenges. Here we address the enormous impact of high-throughput sequencing (HTS) on ASD gene discovery, outline a consensus view for leveraging this technology, and describe a large multisite collaboration developed to accomplish these goals. Similar approaches could prove effective for severe neurodevelopmental disorders more broadly.",
"The rolling blackout (rbo) gene encodes an integral plasma membrane lipase required for Drosophila phototransduction. Photoreceptors are enriched for the RBO protein, and temperature-sensitive rbo mutants show reversible elimination of phototransduction within minutes, demonstrating an acute requirement for the protein. The block is activity dependent, indicating that the action of RBO is use dependent. Conditional rbo mutants show activity-dependent depletion of diacylglycerol and concomitant accumulation of phosphatidylinositol phosphate and phosphatidylinositol 4,5-bisphosphate within minutes of induction, suggesting rapid downregulation of phospholipase C (PLC) activity. The RBO requirement identifies an essential regulatory step in G-protein-coupled, PLC-dependent inositol lipid signaling mediating activation of TRP and TRPL channels during phototransduction.",
"Recent advances in the genetics of autism spectrum disorders (ASDs) are offering new valuable insights into molecular and cellular mechanisms of pathology. At the same time, the emerging data challenge long-standing diagnostic conventions and the notion of phenotypic specificity. This review addresses the particular issues that attend gene discovery in neuropsychiatric and neurodevelopmental disorders and ASDs in particular, summarizes recent findings in human genetics broadly that are driving the reevaluation of the conventional wisdom regarding the allelic architecture of common psychiatric conditions, reviews selected discoveries in ASDs and their relevance to models of pathology, highlights the conceptual and practical issues raised by the observation of a convergence of ASD genetic risks with distinct psychiatric disorders, and considers the important interplay of studies of neurobiology and genetics in clarifying and extending our understanding of social disability syndromes.",
"Autism spectrum disorder (ASD) is a complex developmental syndrome of unknown etiology. Recent studies employing exome- and genome-wide sequencing have identified nine high-confidence ASD (hcASD) genes. Working from the hypothesis that ASD-associated mutations in these biologically pleiotropic genes will disrupt intersecting developmental processes to contribute to a common phenotype, we have attempted to identify time periods, brain regions, and cell types in which these genes converge. We have constructed coexpression networks based on the hcASD \"seed\" genes, leveraging a rich expression data set encompassing multiple human brain regions across human development and into adulthood. By assessing enrichment of an independent set of probable ASD (pASD) genes, derived from the same sequencing studies, we demonstrate a key point of convergence in midfetal layer 5/6 cortical projection neurons. This approach informs when, where, and in what cell types mutations in these specific genes may be productively studied to clarify ASD pathophysiology."
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Glycerol-mediated defence response in Theobroma cacao
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Previous work has implicated glycerol-3-phosphate (G3P) as a mobile inducer of systemic immunity in plants. We tested the hypothesis that the exogenous application of glycerol as a foliar spray might enhance the disease resistance of Theobroma cacao through the modulation of endogenous G3P levels. We found that exogenous application of glycerol to cacao leaves over a period of 4 days increased the endogenous level of G3P and decreased the level of oleic acid (18:1). Reactive oxygen species (ROS) were produced (a marker of defence activation) and the expression of many pathogenesis-related genes was induced. Notably, the effects of glycerol application on G3P and 18:1 fatty acid content, and gene expression levels, in cacao leaves were dosage dependent. A 100 mm glycerol spray application was sufficient to stimulate the defence response without causing any observable damage, and resulted in a significantly decreased lesion formation by the cacao pathogen Phytophthora capsici; however, a 500 mm glycerol treatment led to chlorosis and cell death. The effects of glycerol treatment on the level of 18:1 and ROS were constrained to the locally treated leaves without affecting distal tissues. The mechanism of the glycerol-mediated defence response in cacao and its potential use as part of a sustainable farming system are discussed.
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[
"The comparison of the chromosome numbers of today's species with common reconstructed paleo-ancestors has led to intense speculation of how chromosomes have been rearranged over time in mammals. However, similar studies in plants with respect to genome evolution as well as molecular mechanisms leading to mosaic synteny blocks have been lacking due to relevant examples of evolutionary zooms from genomic sequences. Such studies require genomes of species that belong to the same family but are diverged to fall into different subfamilies. Our most important crops belong to the family of the grasses, where a number of genomes have now been sequenced. Based on detailed paleogenomics, using inference from n = 5-12 grass ancestral karyotypes (AGKs) in terms of gene content and order, we delineated sequence intervals comprising a complete set of junction break points of orthologous regions from rice, maize, sorghum, and Brachypodium genomes, representing three different subfamilies and different polyploidization events. By focusing on these sequence intervals, we could show that the chromosome number variation/reduction from the n = 12 common paleo-ancestor was driven by nonrandom centric double-strand break repair events. It appeared that the centromeric/telomeric illegitimate recombination between nonhomologous chromosomes led to nested chromosome fusions (NCFs) and synteny break points (SBPs). When intervals comprising NCFs were compared in their structure, we concluded that SBPs (1) were meiotic recombination hotspots, (2) corresponded to high sequence turnover loci through repeat invasion, and (3) might be considered as hotspots of evolutionary novelty that could act as a reservoir for producing adaptive phenotypes.",
"The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a major role in defense response in cacao. This fundamental knowledge can contribute to breeding of disease resistant cacao varieties through the application of molecular markers or the use of transgenic strategies.",
"We sequenced and assembled the draft genome of Theobroma cacao, an economically important tropical-fruit tree crop that is the source of chocolate. This assembly corresponds to 76% of the estimated genome size and contains almost all previously described genes, with 82% of these genes anchored on the 10 T. cacao chromosomes. Analysis of this sequence information highlighted specific expansion of some gene families during evolution, for example, flavonoid-related genes. It also provides a major source of candidate genes for T. cacao improvement. Based on the inferred paleohistory of the T. cacao genome, we propose an evolutionary scenario whereby the ten T. cacao chromosomes were shaped from an ancestor through eleven chromosome fusions.",
"Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed.",
"Nonhost interactions are prevalent between plants and specialized phytopathogens. Although it has great potential for providing crop plants with durable resistance, nonhost resistance is poorly understood. Here, we show that nonhost resistance is controlled, at least in part, by general resistance. Arabidopsis plants are resistant to the nonhost pathogen Pseudomonas syringae pv phaseolicola NPS3121 and completely arrest bacterial multiplication in the plant. Ten Arabidopsis mutants were isolated that were compromised in nonhost (nho) resistance to P. s. phaseolicola. Among these, nho1 is caused by a single recessive mutation that defines a novel gene. nho1 is defective in nonspecific resistance to Pseudomonas bacteria, because it also supported the growth of P. s. tabaci and P. fluorescens bacteria, both of which are nonpathogenic on Arabidopsis. In addition, the nho1 mutation also compromised resistance mediated by RPS2, RPS4, RPS5, and RPM1. Interestingly, the nho1 mutation had no effect on the growth of the virulent bacteria P. s. maculicola ES4326 and P. s. tomato DC3000, but it partially restored the in planta growth of the DC3000 hrpS(-) mutant bacteria. Thus, the virulent bacteria appear to evade or suppress NHO1-mediated resistance by means of an Hrp-dependent virulence mechanism.",
"Sorghum, an African grass related to sugar cane and maize, is grown for food, feed, fibre and fuel. We present an initial analysis of the approximately 730-megabase Sorghum bicolor (L.) Moench genome, placing approximately 98% of genes in their chromosomal context using whole-genome shotgun sequence validated by genetic, physical and syntenic information. Genetic recombination is largely confined to about one-third of the sorghum genome with gene order and density similar to those of rice. Retrotransposon accumulation in recombinationally recalcitrant heterochromatin explains the approximately 75% larger genome size of sorghum compared with rice. Although gene and repetitive DNA distributions have been preserved since palaeopolyploidization approximately 70 million years ago, most duplicated gene sets lost one member before the sorghum-rice divergence. Concerted evolution makes one duplicated chromosomal segment appear to be only a few million years old. About 24% of genes are grass-specific and 7% are sorghum-specific. Recent gene and microRNA duplications may contribute to sorghum's drought tolerance."
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Amlodipine and atorvastatin in the treatment of oxidative stress in the human body
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Oxidized low-density lipoprotein (LDL) plays central roles in the formation and progression of atherosclerotic lesions. Malondialdehyde (MDA)-modified LDL (MDA-LDL) is speculated to be generated as a result of oxidative stress in the human body. Because both amlodipine and atorvastatin have been reported to reduce oxidative stress, it is expected that both drugs would have a favorable influence to reduce oxidative stress.
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[
"Evidence to support an important role of oxidative modification in mediating the atherogenicity of LDL continues to grow. New hypotheses suggest mechanisms by which Ox-LDL or products of Ox-LDL can affect many components of the atherogenic process, including vasomotor properties and thrombosis, as well as lesion initiation and progression itself. These ideas suggest new approaches, that in combination with lowering of plasma cholesterol, could lead to the prevention of atherosclerosis and its complications.",
"In many countries, combination therapy with amlodipine and atorvastatin is indicated for the treatment of patients with hypertension and hypercholesterolemia. The aim of this study was to investigate the impact of this combination therapy on plasma adiponectin levels. Combination therapy with amlodipine and atorvastatin would increase plasma adiponectin levels. A total of 25 patients with coronary artery disease and concomitant hypertension and hypercholesterolemia were evaluated. The combination of amlodipine and atorvastatin in 8 different dosage strengths were flexibly titrated over a period of 14 weeks. Lipid profile and plasma adiponectin were measured. Brachial flow-mediated dilation (FMD) was determined by vascular ultrasound. As compared with baseline, combination therapy with amlodipine and atorvastatin significantly reduced systolic and diastolic blood pressure, total cholesterol, and low-density lipoprotein cholesterol (all P < 0.05). Furthermore, there were significant increases in adiponectin levels (mean [95% confidence interval (CI)], 12.1 [10.7-13.7] vs 8.1 [6.5-10.0] μg/mL; P < 0.001) and brachial FMD (4.4 ± 0.6% vs 5.6 ± 0.5%; P = 0.046) over 14 weeks of treatment. The change in adiponectin levels correlated significantly with the changes in diastolic blood pressure (r = -0.49; P = 0.014) and FMD (r = 0.55; P = 0.007). The results of this study indicate that along with its antihypertensive and cholesterol-lowering effects, combination therapy with amlodipine and atorvastatin appears to increase plasma adiponectin levels and improve endothelial function.",
"The effect of minimally modified LDL (MM-LDL) on the ability of large vessel endothelial cells (EC) to interact with monocytes and neutrophils was examined. These LDL preparations, obtained by storage or by mild iron oxidation, were indistinguishable from native LDL to the LDL receptor and were not recognized by the scavenger receptor. Treatment of EC with as little as 0.12 micrograms/ml MM-LDL caused a significant increase in the production of chemotactic factor for monocytes (sevenfold) and increased monocyte binding (three- to fivefold). Monocyte binding was maximal after 4 h of EC exposure to MM-LDL, persisted for 48 h, and was inhibited by cycloheximide. In contrast, neutrophil binding was not increased after 1-24 h of exposure. Activity in the MM-LDL preparations was found primarily in the polar lipid fraction. MM-LDL was toxic for EC from one rabbit but not toxic for the cells from another rabbit or any human umbilical vein EC. The resistant cells became sensitive when incubated with lipoprotein in the presence of cycloheximide, whereas the sensitive strain became resistant when preincubated with sublethal concentrations of MM-LDL. We conclude that exposure of EC to sublethal levels of MM-LDL enhances monocyte endothelial interactions and induces resistance to the toxic effects of MM-LDL.",
"In this study, we questioned whether in vivo probucol could prevent the progression of atherosclerosis in homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia. At 2 months of age, eight WHHL rabbits were divided into two groups. Group A (n = 4) was fed standard rabbit chow for 6 months. Group B (n = 4) was fed standard rabbit chow containing 1% probucol for 6 months. At the end of the experiments, average plasma concentrations of cholesterol were 704 +/- 121 mg/dl in group A and 584 +/- 61 mg/dl in group B, respectively. The percentage of surface area of total thoracic aorta with visible plaques in group A versus group B was 54.2% +/- 18.8% versus 7.0% +/- 6.3%, respectively. What was noteworthy was that the percentage of plaque in the descending thoracic aorta was almost negligible (0.2% +/- 0.2%) in group B rabbits compared to that in group A rabbits (41.1% +/- 20.2%). Low density lipoproteins (LDL) isolated from WHHL rabbits under treatment with probucol (group B) were shown to be highly resistant to oxidative modification by cupric ion and to be minimally recognized by macrophages. On the contrary, LDL from group A rabbits incubated with cupric ion showed a 7.4-fold increase in peroxides (thiobarbituric acid-reactive substances) and a 4.3-fold increase in the synthesis of cholesteryl ester in macrophages compared to those of LDL from group B rabbits. Thus, probucol could definitely prevent the progression of atherosclerosis in homozygous WHHL rabbits in vivo by limiting oxidative LDL modification and foam cell transformation of macrophages.",
"Macrophages express a specific receptor that recognizes acetylated low density lipoprotein (LDL) and certain other chemically modified forms of LDL but not native LDL. LDL oxidatively modified either by incubation with endothelial cells in Ham's F-10 medium or by incubation with 5 microM copper(II) ion in the absence of cells is recognized by this same receptor. This oxidative modification, whether cell-induced or copper-catalyzed, is accompanied by many changes in the physical and chemical properties of LDL, including an increase in density, conversion of phosphatidylcholine to lysophosphatidylcholine, generation of lipid peroxides, and degradation of apolipoprotein B-100. Which changes are essential for eliciting the recognition by the receptor is not known. In the present paper it is shown that fragments of the degraded apolipoprotein from delipidated, oxidized LDL can be almost quantitatively resolubilized using n-octyl beta-D-glucopyranoside. These 125I-labeled, solubilized apoproteins were degraded rapidly by mouse peritoneal macrophages, and that degradation was competitively inhibited by unlabeled acetyl-LDL and endothelial cell-modified LDL but not by native LDL. These results show that the acetyl-LDL receptor recognizes an epitope on the apoprotein moiety, either newly generated or exposed as a result of oxidative modification, rather than some oxidized lipid moiety. Further, the results suggest that the lipids of oxidatively modified LDL do not play an obligatory role in determining the conformation of that epitope.",
"It has been postulated that low density lipoprotein (LDL) becomes fully atherogenic only if it first undergoes oxidative modification. The oxidatively modified form, but not native LDL, is recognized by the acetyl-LDL or \"scavenger\" receptor and could, therefore, be taken up rapidly by tissue macrophages to generate the fatty-streak lesion of atherosclerosis. However, there is thus far very little direct evidence for oxidative modification in vivo. The studies reported here take advantage of the fact that probucol is an effective antioxidant transported in lipoproteins, including LDL, and blocks the oxidative modification of LDL in vitro. We now show that the rate of degradation of LDL in the macrophage-rich fatty-streak lesions of the LDL receptor-deficient rabbit treated with probucol (1% by weight in the diet) is reduced to about one-half of that in the lesions of receptor-deficient rabbits not given probucol (but matched for plasma cholesterol levels). In contrast, the rates of degradation in the nonlesioned areas of the aorta were no different in probucol-treated and control animals. Most of the LDL degradation in fatty-streak lesions takes place in macrophages, whereas in nonlesioned aorta, which contains very few macrophages, the degradation is almost exclusively in endothelial cells and smooth muscle cells. Thus, the results are compatible with the postulate that the native LDL taken up and degraded by foam cells in the developing fatty-streak lesions was in part first converted to a form recognized by the scavenger receptor (by oxidative or analogous modification). Finally, and most importantly, we show that treatment with probucol significantly reduced the rate of development of fatty-streak lesions even though plasma cholesterol levels were no lower than lovastatin-treated (control) rabbits."
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PEX7 is targeted to the peroxisome in a PEX5- and cargo-dependent pathway
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Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and transported to the organelle by shuttling receptors. Matrix proteins containing a type 1 signal are carried to the peroxisome by PEX5, whereas those harboring a type 2 signal are transported by a PEX5-PEX7 complex. The pathway followed by PEX5 during the protein transport cycle has been characterized in detail. In contrast, not much is known regarding PEX7. In this work, we show that PEX7 is targeted to the peroxisome in a PEX5- and cargo-dependent manner, where it becomes resistant to exogenously added proteases. Entry of PEX7 and its cargo into the peroxisome occurs upstream of the first cytosolic ATP-dependent step of the PEX5-mediated import pathway, i.e., before monoubiquitination of PEX5. PEX7 passing through the peroxisome becomes partially, if not completely, exposed to the peroxisome matrix milieu, suggesting that cargo release occurs at the trans side of the peroxisomal membrane. Finally, we found that export of peroxisomal PEX7 back into the cytosol requires export of PEX5 but, strikingly, the two export events are not strictly coupled, indicating that the two proteins leave the peroxisome separately.
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[
"In eukaryotes, microbodies called peroxisomes play important roles in cellular activities during the life cycle. Previous studies indicate that peroxisomal functions are important for plant infection in many phytopathogenic fungi, but detailed relationships between fungal pathogenicity and peroxisomal function still remain unclear. Here we report the importance of peroxisomal protein import through PTS2 (Peroxisomal Targeting Signal 2) in fungal development and pathogenicity of Magnaporthe oryzae. Using an Agrobacterium tumefaciens-mediated transformation library, a pathogenicity-defective mutant was isolated from M. oryzae and identified as a T-DNA insert in the PTS2 receptor gene, MoPEX7. Gene disruption of MoPEX7 abolished peroxisomal localization of a thiolase (MoTHL1) containing PTS2, supporting its role in the peroxisomal protein import machinery. ΔMopex7 showed significantly reduced mycelial growth on media containing short-chain fatty acids as a sole carbon source. ΔMopex7 produced fewer conidiophores and conidia, but conidial germination was normal. Conidia of ΔMopex7 were able to develop appressoria, but failed to cause disease in plant cells, except after wound inoculation. Appressoria formed by ΔMopex7 showed a defect in turgor generation due to a delay in lipid degradation and increased cell wall porosity during maturation. Taken together, our results suggest that the MoPEX7-mediated peroxisomal matrix protein import system is required for fungal development and pathogenicity M. oryzae.",
"Peroxisomes are organelles responsible for multiple metabolic pathways including the biosynthesis of plasmalogens and the oxidation of branched-chain as well as very-long-chain fatty acids (VLCFAs). Peroxisomal disorders (PDs) are heterogeneous groups of diseases and affect many organs with varying degrees of involvement. Even pathogenetically distinct PDs share some common symptoms. However, several PDs have uniquely characteristic clinical findings. The durations of survival in PDs are also variable. Infants with PDs are usually presented with developmental delay, visual and hearing impairment. Generalized hypotonia is present in severe cases. Epileptic seizures are also a common characteristic of patients with certain PDs. Nonetheless, the classification and evolution of epilepsy in PDs have not been elucidated in detail. Here, we review the relevant literatures and provide an overview of PDs with particular emphasis on the characteristics of seizures in infants.",
"Peroxisomes are essential organelles responsible for many metabolic reactions, such as the oxidation of very long chain and branched fatty acids, D-amino acids and polyamines, as well as the production and turnover of hydrogen peroxide. They comprise a class of organelles called microbodies, including glycosomes, glyoxysomes and Woronin bodies. Dysfunction of human peroxisomes causes severe and often fatal peroxisome biogenesis disorders (PBDs). Peroxisomal matrix protein import is mediated by receptors that shuttle between the cytosol and peroxisomal matrix using ubiquitination/deubiquitination reactions and ATP hydrolysis for receptor recycling. We focus on the machinery involved in the peroxisomal matrix protein import cycle, highlighting recent advances in peroxisomal matrix protein import, cargo release and receptor recycling/degradation.",
"Peroxin 5 (PEX5), the peroxisomal protein shuttling receptor, binds newly synthesized peroxisomal matrix proteins in the cytosol and promotes their translocation across the organelle membrane. During the translocation step, PEX5 itself becomes inserted into the peroxisomal docking/translocation machinery. PEX5 is then monoubiquitinated at a conserved cysteine residue and extracted back into the cytosol in an ATP-dependent manner. We have previously shown that the ubiquitin-PEX5 thioester conjugate (Ub-PEX5) released into the cytosol can be efficiently disrupted by physiological concentrations of glutathione, raising the possibility that a fraction of Ub-PEX5 is nonenzymatically deubiquitinated in vivo. However, data suggesting that Ub-PEX5 is also a target of a deubiquitinase were also obtained in that work. Here, we used an unbiased biochemical approach to identify this enzyme. Our results suggest that ubiquitin-specific protease 9X (USP9X) is by far the most active deubiquitinase acting on Ub-PEX5, both in female rat liver and HeLa cells. We also show that USP9X is an elongated monomeric protein with the capacity to hydrolyze thioester, isopeptide, and peptide bonds. The strategy described here will be useful in identifying deubiquitinases acting on other ubiquitin conjugates."
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Mediterranean fever mutations in Egyptian systemic-onset juvenile idiopathic arthritis
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Systemic-onset juvenile idiopathic arthritis (SoJIA) is a chronic auto-inflammatory disease of childhood, with a complex genetic trait, which is characterized by arthritis associated with systemic manifestations. Familial Mediterranean fever (FMF) is another auto-inflammatory disorder that is monogenic. There are speculations as to whether Mediterranean fever (MEFV) mutations are among the genetic determinants of SoJIA. Our aim was to explore the frequency and clinical significance of MEFV mutations in Egyptian SoJIA patients. A group of healthy children were assigned to the control group in an attempt to estimate the carrier rate of MEFV mutations in Egypt.
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[
"Familial Mediterranean Fever (FMF) is the prototype of a group of inherited inflammatory disorders. The gene (MEFV) responsible for this disease, comprises 10 exons and 781 codons. Twenty-nine mutations, most located in the last exon, have been identified so far. It is unclear whether all are true disease-causing mutations. Five founder mutations, V726A, M694V, M694I, M680I and E148Q account for 74% of FMF chromosomes from typical cases (Armenians, Arabs, Jews, and Turks). Rare mutations are preferentially found in populations not usually affected by FMF (eg Europeans not from the above ancestries). The various combinations of MEFV mutations define severe to mild genotypes. The trend is that genotypes including two mutations located within mutational 'hot-spots' (codons 680 or 694) of the gene are associated with severe phenotypes, whereas mild phenotypes are associated with some other mutations, E148Q being the mildest and least penetrant. Understanding the correlation between the FMF phenotype and genotype is further obscured by the existence of complex alleles, modifier loci, genetic heterogeneity and possible epigenetic factors. Additionally, mutations in the MEFV gene are thought to be involved in non FMF disorders. Carrier rates for FMF mutations may be as high as 1:3 in some populations, suggesting that the disease is underdiagnosed. This review update emphasises that both clinical and genetic features are to be taken into account for patient diagnosis, colchicine treatment and prognosis.",
"Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by self-limiting recurrent attacks of fever and serosal inflammation, leading to abdominal, thoracic or articular pain. To detect variable clinical presentations and genotypic distribution of different groups of FMF patients and the efficacy of colchicine therapy in treatment of these groups of FMF after one year. A cross-sectional study was conducted on 70 patients already diagnosed with FMF and following-up at the Rheumatology Clinic, Children's Hospital - Cairo University. Diagnosis of FMF was determined according to Tel Hashomer criteria for FMF. All patients were subjected to a questionnaire including detailed history with emphasis on clinical manifestations and colchicine dose to control attacks. Mutational analysis was performed for all study subjects covering 12 mutations in the MEFV gene: E148Q, P369S, F479L, M680I (G/C), M680I (G/A), I692del, M694V, M694I, K695R, V726A, A744S and R761H. Response to colchicine treatment was evaluated as complete, incomplete and unresponsive. Out of the 70 patients- 40 males and 30 females- fever was the most common presenting feature, followed by abdominal pain, and arthritis; documented in 95.7%, 94.3%, and 77.1% of cases respectively. Mutational analysis detected gene mutation on both alleles in 20 patients (homozygotes), on only 1 allele in 40 patients (heterozygotes), and on none of the alleles (uncharacterized cases). Mild to moderate disease severity score (according to Tel Hashomer key to severity score) was detected in a significant proportion of heterozygotes and the uncharacterized group than the homozygotes. All patients received colchicine therapy; 22.9% of them showed complete response, 74.3% showed incomplete response and 2.9% showed no response to therapy. The colchicine dose needed to control attacks was significantly lower in heterozygotes than the homozygotes(P=0.04). Also patients' response to colchicine therapy was significantly better in the heterozygous group(P=0.023). Fever, abdominal pain and arthritis are the most common presenting features for homozygous, Heterozygous and uncharacterized patients. E148Q, V726A, and M680I were the most common mutations detected in the heterozygous group. Homozygosity were found for M680I, M694V, and M694I mutations in 13 patients (65% of homozygotes). Heterozygotes presenting with severe phenotype should be further analyzed for less common second MEFV mutation using gene sequencing. The colchicine dose required to control the attacks was significantly lower and patients' response to colchicine therapy was significantly better in the heterozygous group than homozygous group.",
"To describe the clinical manifestations of familial Mediterranean fever (FMF) in 91 patients from 47 families and provide data from the genetic study. P:atients and methods. We conducted a retrospective chart review of 91 patients (including 83 children aged <15 yr) from 47 families through a questionnaire and a specific database. The genetic analysis included complete screening of known mutations of the MEFV gene on chromosome 16p13.3. A positive diagnosis required at least two mutations, one on each chromosome. Our panel included 52 females and 39 males, with a mean age of 7.27 yr. Of the 47 families, 31 were non-Ashkenazi Jews, 10 were Armenians and six were from other ethnic groups. Clinical features included fever (100%), peritonitis (86%), pleuritis (56%), arthritis (34%) and myalgias (27%). We observed a high rate of cutaneous manifestations (47%); erythema, oedema and recurrent oral ulcers were the most frequent. Phenotype-genotype correlations showed a significant association of M694V homozygosity with earlier age of onset (P: = 0.044), fever >39 degrees C (P: = 0. 002), pleural crisis (P: = 0.0044), splenomegaly (P: = 0.0005) and arthritis (P: = 0.001). Associations with mucocutaneous features were as follows: erysipelas-like erythema (P: = 0.012), oedema (P: = 0.61, not significant) and oral ulcers (P: = 0.45, not significant). New phenotype-genotype correlations emerged from our study: homozygosity for the M694V mutation was associated with intensity of fever, splenomegaly and with erysipelas-like erythema. Apart from erysipelas-like erythema, no significant association was found between other cutaneous features and the genotype."
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African-American and American Indian families' perceptions of infant safe sleep practices.
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To investigate, by using qualitative methods, beliefs among African-American and American Indian families about infant safe sleep practices, barriers to acceptance of prevention recommendations, and more effective messaging strategies. Seventy-three mothers and supporters participated in focus groups. Participants discussed infant sleep practices and effectiveness of safe sleep messages. Data were coded, and themes were developed and revised in an iterative manner as patterns became more apparent. Themes included reasons for and influences on sleep decisions, and concerns about safe sleep recommendations. Parental sleep decisions seemed to be driven by perceptions of what would make their infant most comfortable and safe, and what would be most convenient. Parents were aware of safe sleep recommendations but unaware of the rationale. Because they generally did not believe that their infants were at risk for a sleep-related death, day-to-day decisions seemed to focus on what was most effective in getting their infant to sleep. There appeared to be no distinctions in opinions among African-American and American Indian families. African-American and American Indian families seemed to have similar concerns about infant comfort and safety, and their perceptions about what would be most effective in achieving these goals appeared to be important influences on their sleep practices. Adherence with safe sleep recommendations may be enhanced if health care providers and educational materials discussed rationale underlying recommendations and addressed common parental concerns. It may be beneficial to target educational interventions towards fathers, as they may be untapped sources in implementing safe sleep practices.
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[
"Racial misclassification of American Indian and Alaska Native (AI/AN) individuals as non-AI/AN in cancer registries presents problems for cancer surveillance, research, and public health practice. The aim of this study was to investigate the efficiency of tribal linkages in enhancing the quality of racial information in state cancer registries. Registry Plus Link Plus 2.0 probabilistic record linkage software was used to link the Michigan state cancer registry data (1985-2004; 1,031,168 cancer cases) to the tribal membership roster (40,340 individuals) in July of 2007. A data set was created containing AI/AN cancer cases identified by the state registry, Indian Health Service (IHS) linkages, and tribal linkage. The differences between these three groups of individuals were compared by distribution of demographic, diagnostic, and county-level characteristics using multilevel analysis (conducted in 2007-2008). From 1995 to 2004, the tribal enrollment file showed linkages to 670 cancer cases (583 individuals) and the tribal linkage led to the identification of 190 AI/AN cancer cases (168 individuals) that were classified as non-AI/AN in the registry. More than 80% of tribal members were reported as non-AI/AN to the registry. Individuals identified by IHS or tribal linkages were different from those reported to be AI/AN in terms of stage at diagnosis, tumor confirmation, and characteristics of the county of diagnosis, including contract health services availability, tribal health services availability, and proportion of AI/AN residents. The data linkage between tribal and state cancer registry data sets improved racial classification validity of AI/AN Michigan cancer cases. Assessing tribal linkages is a simple, noninvasive way to improve the accuracy of state cancer data for AI/AN populations and to generate tribe-specific cancer information.",
"To investigate, by using qualitative methods, perceptions about sudden infant death syndrome (SIDS) in African-American parents and how these influence decisions. Eighty-three mothers participated in focus groups or individual interviews. Interviews probed reasons for decisions about infant sleep environment and influences affecting these decisions. Data were coded, and themes were developed and revised in an iterative manner as patterns became more apparent. Themes included lack of plausibility, randomness, and vigilance. Many mothers believed that the link between SIDS and sleep position was implausible. Because the cause of SIDS was unknown, they did not understand how certain behaviors could be defined as risk factors. This confusion was reinforced by perceived inconsistency in the recommendations. Most mothers believed that SIDS occurred randomly (\"God's will\") and that the only way to prevent it was vigilance. Many African-American mothers may not understand the connection between SIDS and sleep behaviors or believe that behavior (other than vigilance) cannot affect risk. These beliefs, if acted on, may affect rates of safe sleep practices. Efforts to explain a plausible link between SIDS and safe sleep recommendations and to improve consistency of the message may result in increased adherence to these recommendations.",
"African American infants continue to be at more than twice the risk for sudden infant death syndrome (SIDS) than Caucasian Americans. In addition, African Americans are twice as likely to place their infants prone for sleep. To investigate, using qualitative methods, factors influencing African American parents' decisions regarding infant sleep position. Eighty-three mothers participated in focus groups or individual interviews. Questions probed reasons for infant sleep position decisions and influences on decision making. All of the mothers in this study were aware of the recommendation to place infants supine for sleep. Mothers largely chose infant sleep position for either safety or comfort reasons. Many mothers who chose the prone position did so because they perceived it to be safer than supine. Sleep position decisions were influenced by parental needs (largely the need for sleep), perception of SIDS risk and biological plausibility, and trusted sources of information. Mothers sought information from multiple trusted sources before making any decision. Even if mothers had trust in their pediatrician, they were comfortable making decisions counter to their pediatrician's recommendations if they felt it to be in the best interests of their infant. African American mothers are generally aware of the Back to Sleep recommendation. However, many may not believe that the supine position is the safest position. Other mothers may use the prone position because of infant comfort or parent's need for longer sleep. Trust in the pediatrician may not be sufficient reason for parents to use the supine position.",
"This paper measures agreement between survey and administrative measures of race/ethnicity for Medicaid enrollees. Level of agreement and the demographic and health-related characteristics associated with misclassification on the administrative measure are examined. Minnesota Medicaid enrollee files matched to self-report information from a telephone/mail survey of 4,902 enrollees conducted in 2003. Measures of agreement between the two measures of race/ethnicity are computed. Using logistic regression, we also assess whether misclassification of race/ethnicity on administrative files is associated with demographic factors, health status, health care utilization, or ratings of quality of health care. Race/ethnicity fields from administrative Medicaid files were extracted and merged with self-report data. The administrative data correctly classified 94 percent of cases on race/ethnicity. Persons who self-identified as Hispanic and those whose home language was English had the greater odds (compared with persons who self-identified as white and those whose home language was not English) of being misclassified in administrative data. Persons classified as unknown/other on administrative data were more likely to self-identify as white. In this case study in Minnesota, researchers can be reasonably confident that the racial designations on Medicaid administrative data comport with how enrollees self-identify. Moreover, misclassification is not associated with common measures of health status, utilization, and ratings of quality of care. Further replication is recommended given variation in how race information is collected and coded by Medicaid agencies in different states."
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The dyslexia-associated gene KIAA0319 in rats is impaired at speech sound discrimination
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In utero RNAi of the dyslexia-associated gene Kiaa0319 in rats (KIA-) degrades cortical responses to speech sounds and increases trial-by-trial variability in onset latency. We tested the hypothesis that KIA- rats would be impaired at speech sound discrimination. KIA- rats needed twice as much training in quiet conditions to perform at control levels and remained impaired at several speech tasks. Focused training using truncated speech sounds was able to normalize speech discrimination in quiet and background noise conditions. Training also normalized trial-by-trial neural variability and temporal phase locking. Cortical activity from speech trained KIA- rats was sufficient to accurately discriminate between similar consonant sounds. These results provide the first direct evidence that assumed reduced expression of the dyslexia-associated gene KIAA0319 can cause phoneme processing impairments similar to those seen in dyslexia and that intensive behavioral therapy can eliminate these impairments.
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"Neuroanatomical and behavioral evidence indicate that the cerebellum is particularly vulnerable to the toxic effects of prenatal alcohol exposure. Recent research has shown impairments in eyeblink conditioning in rats following binge-like neonatal ethanol exposure. The neural substrates of eyeblink conditioning have been localized to the cerebellum and related brainstem mechanisms. The present study considered whether heavy prenatal alcohol exposure would result in similar impairments in eyeblink conditioning in children. A related purpose was to determine if eyeblink conditioning could discriminate between children with prenatal alcohol exposure and children diagnosed with attention deficit hyperactive disorder or developmental dyslexia. Fifty-three age-matched children [10 prenatal alcohol exposure (FAE), 16 attention deficit hyperactive disordered (ADHD), 14 children with dyslexia (DYS), 13 normal controls] were assessed on eyeblink conditioning in the delay paradigm. Children in the FAE and DYS groups failed to learn the conditioned response, producing longer latencies and poorly timed responses to the conditioning stimulus. Children with ADHD were impaired on measures of adaptively timed responses, although conditioned responses matched normal controls. The results suggest that children prenatally exposed to alcohol have deficits in cerebellar processing similar to those with dyslexia, and that these functional deficits are related to disabilities in learning.",
"Recent in vitro investigations have suggested that GABA is involved in the development of the mammalian central nervous system. To evaluate the roles of GABA in neurogenesis in vivo, we generated mice lacking both the isoforms of glutamic acid decarboxylase (GAD), GAD65 and GAD67, by mating GAD65- and GAD67-mutant mice generated by homologous recombination in this laboratory. Similar to GAD67-deficient mice, the GAD65/67-deficient mice did not survive after birth because of cleft palate. We thus analyzed these mice at the fetal and newborn stages. GABA was scarcely detectable in the GAD65/67-deficient brains, indicating that the GAD-independent GABA synthetic pathway was not active. The activity of ornithine decarboxylase, which is possibly involved in such a pathway, did not increase with the GAD deficiency. Histological and immunohistochemical studies of the GAD65/67-deficient brain did not reveal any discernible disorders of histogenesis. The discrepancy between the results of previous in vitro investigations, performed mostly on rat tissue, and those of the present analysis on mutant mice may be attributed to the different species used or to the possibility that other mediators can compensate for GABA functions in vivo.",
"We study the impact of correlated neuronal firing rate variability on the accuracy with which an encoded quantity can be extracted from a population of neurons. Contrary to widespread belief, correlations in the variabilities of neuronal firing rates do not, in general, limit the increase in coding accuracy provided by using large populations of encoding neurons. Furthermore, in some cases, but not all, correlations improve the accuracy of a population code.",
"Of the many signals in the developing nervous system, GABA (gamma-aminobutyric acid) has been shown to be one of the earliest neurotransmitters present. Unlike in the adult, where this transmitter acts synaptically to inhibit neurons, during development, GABA can depolarize progenitor cells and their progeny due to their high intracellular chloride concentration. This early form of GABA signalling may provide the main excitatory drive for the immature cortical network and play a central role in regulating cortical development. Many features of GABA signalling are conserved in different species and are recapitulated during neurogenesis in the adult brain, demonstrating the importance of this versatile molecule in driving cortical formation. Here, we present recent evidence supporting the multiple functions of GABA during embryonic development and adult neurogenesis, from regulating progenitor proliferation to influencing the migration and maturation of newborn neurons.",
"Learning to read proceeds smoothly for most children, yet others struggle to translate verbal language into its written form. Poor readers often have a host of auditory, linguistic, and attention deficits, including abnormal neural representation of speech and inconsistent performance on psychoacoustic tasks. We hypothesize that this constellation of deficits associated with reading disorders arises from the human auditory system failing to respond to sound in a consistent manner, and that this inconsistency impinges upon the ability to relate phonology and orthography during reading. In support of this hypothesis, we show that poor readers have significantly more variable auditory brainstem responses to speech than do good readers, independent of resting neurophysiological noise levels. Thus, neural variability may be an underlying biological contributor to well established behavioral and neural deficits found in poor readers.",
"Studies in sensory neuroscience reveal the critical importance of accurate sensory perception for cognitive development. There is considerable debate concerning the possible sensory correlates of phonological processing, the primary cognitive risk factor for developmental dyslexia. Across languages, children with dyslexia have a specific difficulty with the neural representation of the phonological structure of speech. The identification of a robust sensory marker of phonological difficulties would enable early identification of risk for developmental dyslexia and early targeted intervention. Here, we explore whether phonological processing difficulties are associated with difficulties in processing acoustic cues to speech rhythm. Speech rhythm is used across languages by infants to segment the speech stream into words and syllables. Early difficulties in perceiving auditory sensory cues to speech rhythm and prosody could lead developmentally to impairments in phonology. We compared matched samples of children with and without dyslexia, learning three very different spoken and written languages, English, Spanish, and Chinese. The key sensory cue measured was rate of onset of the amplitude envelope (rise time), known to be critical for the rhythmic timing of speech. Despite phonological and orthographic differences, for each language, rise time sensitivity was a significant predictor of phonological awareness, and rise time was the only consistent predictor of reading acquisition. The data support a language-universal theory of the neural basis of developmental dyslexia on the basis of rhythmic perception and syllable segmentation. They also suggest that novel remediation strategies on the basis of rhythm and music may offer benefits for phonological and linguistic development.",
"Coincident pre- and postsynaptic activity generates long-term potentiation (LTP), a possible cellular model of learning and memory. LTP has two components: (1) an increase in the excitatory postsynaptic potential (EPSP), and (2) an increase in the ability of the EPSP to generate a spike (E-S coupling of LTP). We have used pharmacological and genetic approaches to address the molecular nature of E-S coupling in CA1 pyramidal neurons. Blockade of the Ca2+-sensitive phosphatase, calcineurin, prevents induction of E-S coupling without interfering with LTP of the EPSP. Calcineurin produces its effect on E-S coupling by inducing a long-lasting depression (LTD) of the GABA(A)-mediated inhibitory postsynaptic potentials (IPSPs). This LTD of the IPSP was prevented by blockade of NMDA receptors. Thus, the tetanus that elicits NMDA-dependent LTP mediates a coordinately regulated double function. It produces LTP of the EPSP and, concomitantly, LTD of the IPSP that leads to enhancement of E-S coupling.",
"Understanding speech in background noise is challenging for every listener, including those with normal peripheral hearing. This difficulty is attributable in part to the disruptive effects of noise on neural synchrony, resulting in degraded representation of speech at cortical and subcortical levels as reflected by electrophysiological responses. These problems are especially pronounced in clinical populations such as children with learning impairments. Given the established effects of noise on evoked responses, we hypothesized that listening-in-noise problems are associated with degraded processing of timing information at the brainstem level. Participants (66 children; ages, 8-14 years; 22 females) were divided into groups based on their performance on clinical measures of speech-in-noise (SIN) perception and reading. We compared brainstem responses to speech syllables between top and bottom SIN and reading groups in the presence and absence of competing multitalker babble. In the quiet condition, neural response timing was equivalent between groups. In noise, however, the bottom groups exhibited greater neural delays relative to the top groups. Group-specific timing delays occurred exclusively in response to the noise-vulnerable formant transition, not to the more perceptually robust, steady-state portion of the stimulus. These results demonstrate that neural timing is disrupted by background noise and that greater disruptions are associated with the inability to perceive speech in challenging listening conditions.",
"The 6p21-p22 chromosomal region has been identified as a developmental dyslexia locus both in linkage and association studies, the latter generating evidence for the doublecortin domain containing 2 (DCDC2) as a candidate gene at this locus (and also for KIAA0319). Here, we report an association between DCDC2 and reading and spelling ability in 522 families of adolescent twins unselected for reading impairment. Family-based association was conducted on 21 single nucleotide polymorphisms (SNPs) in DCDC2 using quantitative measures of lexical processing (irregular-word reading), phonological decoding (non-word reading) and spelling-based measures of dyslexia derived from the Components of Reading Examination test. Significant support for association was found for rs1419228 with regular-word reading and spelling (P=0.002) as well as irregular-word reading (P=0.004), whereas rs1091047 was significantly associated (P=0.003) with irregular-word reading (a measure of lexical storage). Four additional SNPs (rs9467075, rs9467076, rs7765678 and rs6922023) were nominally associated with reading and spelling. This study provides support for DCDC2 as a risk gene for reading disorder, and suggests that this risk factor acts on normally varying reading skill in the general population.",
"In the postnatal subventricular zone (SVZ), astrocyte-like cells tightly encapsulate chains of migrating neuronal precursors, although an influence of the astrocyte-like cells on precursor migration has not yet been demonstrated. Cell migration was studied in acute sagittal brain slices to determine whether GABA signaling between astrocyte-like cells and neuronal precursors controls the speed of neuronal precursor migration in the anterior SVZ and rostral migratory stream of juvenile and adult mice. Application of GABA at 10 microm, a nondesensitizing concentration for GABA(A) receptors (GABA(A)Rs), reduced the rate (mean of approximately 50 microm/hr) of cell migration by 21% via GABA(A)R activation. Application of the GABA(A)R antagonist bicuculline enhanced the migration rate by 30%, suggesting that endogenous GABA tonically reduces the speed of cell migration via GABA(A)R activation. Using immunohistochemistry, we found that astrocyte-like cells express the high-affinity GABA transporter subtype GAT4 on processes ensheathing neuronal precursors that contain GABA. Inhibition of GABA uptake into astrocyte-like cells or enhancement of GABA release from neuronal precursors during high K(+) application further reduced the migration rate by increasing ambient GABA levels. GABA altered the migration speed by interfering with intracellular Ca(2+) signaling independently of cell depolarization, because high K(+) application did not alter the speed of cell migration in the presence of bicuculline. These data indicate that astrocyte-like cells create a microenvironment in which their uniquely positioned GABA transporters control the degree of GABA(A)R activation and the migration of neuronal precursors."
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Microbial characterization of evaporitic domes in the Central Andes, Chile
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The Central Andes in northern Chile contains a large number of closed basins whose central depression is occupied by saline lakes and salt crusts (salars). One of these basins is Salar de Llamara (850 m a.s.l.), where large domed structures of seemingly evaporitic origin forming domes can be found. In this work, we performed a detailed microbial characterization of these domes. Mineralogical studies revealed gypsum (CaSO(4)) as a major component. Microbial communities associated to these structures were analysed by 454 16S rDNA amplicon sequencing and compared between winter and summer seasons. Bacteroidetes Proteobacteria and Planctomycetes remained as the main phylogenetic groups, an increased diversity was found in winter. Comparison of the upper air-exposed part and the lower water-submerged part of the domes in both seasons showed little variation in the upper zone, showing a predominance of Chromatiales (Gammaproteobacteria), Rhodospirillales (Alphaproteobacteria), and Sphingobacteriales (Bacteroidetes). However, the submerged part showed marked differences between seasons, being dominated by Proteobacteria (Alpha and Gamma) and Verrucomicrobia in summer, but with more diverse phyla found in winter. Even though not abundant by sequence, Cyanobacteria were visually identified by scanning electron microscopy (SEM), which also revealed the presence of diatoms. Photosynthetic pigments were detected by high-performance liquid chromatography, being more diverse on the upper photosynthetic layer. Finally, the system was compared with other endoevaporite, mats microbialite and Stromatolites microbial ecosystems, showing higher similitude with evaporitic ecosystems from Atacama and Guerrero Negro. This environment is of special interest for extremophile studies because microbial life develops associated to minerals in the driest desert all over the world. Nevertheless, it is endangered by mining activity associated to copper and lithium extraction; thus, its environmental protection preservation is strongly encouraged.
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"Living marine stromatolites at Highborne Cay, Bahamas, are formed by microbial mat communities that facilitate precipitation of calcium carbonate and bind and trap small carbonate sand grains. This process results in a laminated structure similar to the layering observed in ancient stromatolites. In the modern marine system at Highborne Cay, lamination, lithification and stromatolite formation are associated with cycling between three types of microbial communities at the stromatolite surface (Types 1, 2 and 3, which range from a leathery microbial mat to microbially fused sediment). Examination of 923 universal small-subunit rRNA gene sequences from these communities reveals that taxonomic richness increases during transition from Type 1 to Type 3 communities, supporting a previous model that proposed that the three communities represent different stages of mat development. The phylogenetic composition also changes significantly between these community types and these community changes occur in concert with variation in biogeochemical rates. The dominant bacterial groups detected in the stromatolites include Alphaproteobacteria, Planctomycetes, Cyanobacteria and Bacteroidetes. In addition, the stromatolite communities were found to contain novel cyanobacteria that may be uniquely associated with modern marine stromatolites. The implications of these findings are discussed in the context of current models for stromatolite formation.",
"A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and β-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.",
"In this paper, we report the presence of sedimentary microbial ecosystems in wetlands of the Salar de Atacama. These laminated systems, which bind, trap and precipitate mineral include: microbial mats at Laguna Tebenquiche and Laguna La Brava, gypsum domes at Tebenquiche and carbonate microbialites at La Brava. Microbial diversity and key biogeochemical characteristics of both lakes (La Brava and Tebenquiche) and their various microbial ecosystems (non-lithifying mats, flat and domal microbialites) were determined. The composition and abundance of minerals ranged from trapped and bound halite in organic-rich non-lithifying mats to aragonite-dominated lithified flat microbialites and gypsum in lithified domal structures. Pyrosequencing of the V4 region of the 16s rDNA gene showed that Proteobacteria comprised a major phylum in all of the microbial ecosystems studied, with a marked lower abundance in the non-lithifying mats. A higher proportion of Bacteroidetes was present in Tebenquiche sediments compared to La Brava samples. The concentration of pigments, particularly that of Chlorophyll a, was higher in the Tebenquiche than in La Brava. Pigments typically associated with anoxygenic phototrophic bacteria were present in lower amounts. Organic-rich, non-lithifying microbial mats frequently formed snake-like, bulbous structures due to gas accumulation underneath the mat. We hypothesize that the lithified microbialites might have developed from these snake-like microbial mats following mineral precipitation in the surface layer, producing domes with endoevaporitic communities in Tebenquiche and carbonate platforms in La Brava. Whereas the potential role of microbes in carbonate platforms is well established, the contribution of endoevaporitic microbes to formation of gypsum domes needs further investigation.",
"Previous investigations of the salinity effects on the microbial community composition have largely been limited to dynamic estuaries and coastal solar salterns. In this study, the effects of salinity and mineralogy on microbial community composition was studied by using a 900-cm sediment core collected from a stable, inland hypersaline lake, Lake Chaka, on the Tibetan Plateau, north-western China. This core, spanning a time of 17,000 years, was unique in that it possessed an entire range of salinity from freshwater clays and silty sands at the bottom to gypsum and glauberite in the middle, to halite at the top. Bacterial and archaeal communities were studied along the length of this core using an integrated approach combining mineralogy and geochemistry, molecular microbiology (16S rRNA gene analysis and quantitative polymerase chain reaction), cultivation and lipid biomarker analyses. Systematic changes in microbial community composition were correlated with the salinity gradient, but not with mineralogy. Bacterial community was dominated by the Firmicutes-related environmental sequences and known species (including sulfate-reducing bacteria) in the freshwater sediments at the bottom, but by halophilic and halotolerant Betaproteobacteria and Bacteroidetes in the hypersaline sediments at the top. Succession of proteobacterial groups along the salinity gradient, typically observed in free-living bacterial communities, was not observed in the sediment-associated community. Among Archaea, the Crenarchaeota were predominant in the bottom freshwater sediments, but the halophilic Halobacteriales of the Euryarchaeota was the most important group in the hypersaline sediments. Multiple isolates were obtained along the whole length of the core, and their salinity tolerance was consistent with the geochemical conditions. Iron-reducing bacteria were isolated in the freshwater sediments, which were capable of reducing structural Fe(III) in the Fe(III)-rich clay minerals predominant in the source sediment. These data have important implications for understanding how microorganisms respond to increased salinity in stable, inland water bodies."
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Inter- and intra-regional neural synchronization and temporal variance of resting state activity in anesthesia
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Recent studies at the cellular and regional levels have pointed out the multifaceted importance of neural synchronization and temporal variance of neural activity. For example, neural synchronization and temporal variance has been shown by us to be altered in patients in the vegetative state (VS). This finding nonetheless leaves open the question of whether these abnormalities are specific to VS or rather more generally related to the absence of consciousness. The aim of our study was to investigate the changes of inter- and intra-regional neural synchronization and temporal variance of resting state activity in anesthetic-induced unconsciousness state. Applying an intra-subject design, we compared resting state activity in functional magnetic resonance imaging (fMRI) between awake versus anesthetized states in the same subjects. Replicating previous studies, we observed reduced functional connectivity within the default mode network (DMN) and thalamocortical network in the anesthetized state. Importantly, intra-regional synchronization as measured by regional homogeneity (ReHo) and temporal variance as measured by standard deviation (SD) of the BOLD signal were significantly reduced in especially the cortical midline regions, while increased in the lateral cortical areas in the anesthetized state. We further found significant frequency-dependent effects of SD in the thalamus, which showed abnormally high SD in Slow-5 (0.01-0.027 Hz) in the anesthetized state. Our results show for the first time of altered temporal variance of resting state activity in anesthesia. Combined with our findings in the vegetative state, these findings suggest a close relationship between temporal variance, neural synchronization and consciousness.
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"Neuronal dynamics unfolding within the cerebral cortex exhibit complex spatial and temporal patterns even in the absence of external input. Here we use a computational approach in an attempt to relate these features of spontaneous cortical dynamics to the underlying anatomical connectivity. Simulating nonlinear neuronal dynamics on a network that captures the large-scale interregional connections of macaque neocortex, and applying information theoretic measures to identify functional networks, we find structure-function relations at multiple temporal scales. Functional networks recovered from long windows of neural activity (minutes) largely overlap with the underlying structural network. As a result, hubs in these long-run functional networks correspond to structural hubs. In contrast, significant fluctuations in functional topology are observed across the sequence of networks recovered from consecutive shorter (seconds) time windows. The functional centrality of individual nodes varies across time as interregional couplings shift. Furthermore, the transient couplings between brain regions are coordinated in a manner that reveals the existence of two anticorrelated clusters. These clusters are linked by prefrontal and parietal regions that are hub nodes in the underlying structural network. At an even faster time scale (hundreds of milliseconds) we detect individual episodes of interregional phase-locking and find that slow variations in the statistics of these transient episodes, contingent on the underlying anatomical structure, produce the transfer entropy functional connectivity and simulated blood oxygenation level-dependent correlation patterns observed on slower time scales.",
"Resting state studies of spontaneous fluctuations in the functional MRI (fMRI) blood oxygen level dependent (BOLD) signal have shown great promise in mapping the brain's intrinsic, large-scale functional architecture. An important data preprocessing step used to enhance the quality of these observations has been removal of spontaneous BOLD fluctuations common to the whole brain (the so-called global signal). One reproducible consequence of global signal removal has been the finding that spontaneous BOLD fluctuations in the default mode network and an extended dorsal attention system are consistently anticorrelated, a relationship that these two systems exhibit during task performance. The dependence of these resting-state anticorrelations on global signal removal has raised important questions regarding the nature of the global signal, the validity of global signal removal, and the appropriate interpretation of observed anticorrelated brain networks. In this study, we investigate several properties of the global signal and find that it is, indeed, global, not residing preferentially in systems exhibiting anticorrelations. We detail the influence of global signal removal on resting state correlation maps both mathematically and empirically, showing an enhancement in detection of system-specific correlations and improvement in the correspondence between resting-state correlations and anatomy. Finally, we show that several characteristics of anticorrelated networks including their spatial distribution, cross-subject consistency, presence with modified whole brain masks, and existence before global regression are not attributable to global signal removal and therefore suggest a biological basis.",
"Schizophrenia has been associated with abnormal task-related brain activation in sensory and motor regions as well as social cognition network. Recently, two studies investigated temporal correlation between resting-state functional magnetic resonance imaging (R-fMRI) low-frequency oscillations (LFOs) in schizophrenia but reported mixed results. This may be due to the different frequency bands used in these studies. Here we utilized R-fMRI to measure the amplitude of low-frequency fluctuations (ALFF) and fractional ALFF (fALFF) in three different frequency bands (slow-5: 0.01-0.027 Hz; slow-4: 0.027-0.08 Hz; and typical band: 0.01-0.08 Hz) in 69 patients with schizophrenia and 62 healthy controls. We showed that there were significant differences in ALFF/fALFF between the two bands (slow-5 and slow-4) in regions including basal ganglia, midbrain, and ventromedial prefrontal cortex. Importantly, we also identified significant interaction between frequency bands and groups in inferior occipital gyrus, precuneus, and thalamus. The results suggest that the abnormalities of LFOs in schizophrenia is dependent on the frequency band and suggest that future studies should take the different frequency bands into account when measure intrinsic brain activity.",
"Despite evidence pointing to a ubiquitous tendency of human minds to wander, little is known about the neural operations that support this core component of human cognition. Using both thought sampling and brain imaging, the current investigation demonstrated that mind-wandering is associated with activity in a default network of cortical regions that are active when the brain is \"at rest.\" In addition, individuals' reports of the tendency of their minds to wander were correlated with activity in this network.",
"Anticorrelated relationships in spontaneous signal fluctuation have been previously observed in resting-state functional magnetic resonance imaging (fMRI). In particular, it was proposed that there exists two systems in the brain that are intrinsically organized into anticorrelated networks, the default mode network, which usually exhibits task-related deactivations, and the task-positive network, which usually exhibits task-related activations during tasks that demands external attention. However, it is currently under debate whether the anticorrelations observed in resting state fMRI were valid or were instead artificially introduced by global signal regression, a common preprocessing technique to remove physiological and other noise in resting-state fMRI signal. We examined positive and negative correlations in resting-state connectivity using two different preprocessing methods: a component base noise reduction method (CompCor, Behzadi et al., 2007), in which principal components from noise regions-of-interest were removed, and the global signal regression method. Robust anticorrelations between a default mode network seed region in the medial prefrontal cortex and regions of the task-positive network were observed under both methods. Specificity of the anticorrelations was similar between the two methods. Specificity and sensitivity for positive correlations were higher under CompCor compared to the global regression method. Our results suggest that anticorrelations observed in resting-state connectivity are not an artifact introduced by global signal regression and might have biological origins, and that the CompCor method can be used to examine valid anticorrelations during rest.",
"The traditional approach to studying brain function is to measure physiological responses to controlled sensory, motor and cognitive paradigms. However, most of the brain's energy consumption is devoted to ongoing metabolic activity not clearly associated with any particular stimulus or behaviour. Functional magnetic resonance imaging studies in humans aimed at understanding this ongoing activity have shown that spontaneous fluctuations of the blood-oxygen-level-dependent signal occur continuously in the resting state. In humans, these fluctuations are temporally coherent within widely distributed cortical systems that recapitulate the functional architecture of responses evoked by experimentally administered tasks. Here, we show that the same phenomenon is present in anaesthetized monkeys even at anaesthetic levels known to induce profound loss of consciousness. We specifically demonstrate coherent spontaneous fluctuations within three well known systems (oculomotor, somatomotor and visual) and the 'default' system, a set of brain regions thought by some to support uniquely human capabilities. Our results indicate that coherent system fluctuations probably reflect an evolutionarily conserved aspect of brain functional organization that transcends levels of consciousness."
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what is gallstone ileus
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Gallstone ileus (GI) results from the passage of a stone through a cholecystoenteric fistula, subsequently causing a bowel obstruction. The ideal treatment procedure for GI remains controversial.
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"Laparoscopic appendectomy is a safe alternative to open appendectomy to treat appendicitis. The author reports his experience in performing laparoscopic appendectomy with the use of only one trocar in pediatric patients. Between 1 January 1994 and 30 October 1995 at the Department of General and Pediatric Surgery, Division of Pediatric Surgery of the \"Federico II\" University of Naples, we performed 51 laparoscopic appendectomies. Patient age varied from 4 to 16 years with a mean age of 7 years. In the last 25 patients of our series we performed the one-trocar appendectomy, positioning only one trocar infraumbilically with the use of a 10-mm operative telescope. The appendix is identified, dissected when necessary, grasped laparoscopically with a 450-mm operative atraumatic instrument introduced through the operative channel of the laparoscope, and then exteriorized through the umbilical cannula. The appendectomy was performed using traditional method outside the abdominal cavity. We had no intra- or perioperative mortality or morbidity. The mean overall hospitalization time was 2 days (1-4 days). At a maximal follow-up of 20 months the children have no clinical problems nor any visible scar related to the laparoscopic appendectomy. In conclusion, the author considers the one-trocar appendectomy an appropriate alternative procedure to other techniques of laparoscopic appendectomy.",
"Gallstone is a common disease with a 10% prevalence in the United States and Western Europe. However, it is only symptomatic in 20-30% of patients, with biliary pain \"colic\" being the most common symptom. Complications of asymptomatic gallstone disease are generally rare, with an incidence of <1 %/yr. The most common complications of gallstone disease are acute cholecystitis, acute pancreatitis, ascending cholangitis, and gangrenous gallbladder. Less frequent complications include Mirizzi syndrome, cholecystocholedochal fistula, and gallstone ileus. Mirizzi syndrome and cholecystocholedochal fistula are two manifestations of the same process that starts with impaction of a gallstone in the gallbladder neck that results in obstruction of the bile duct, causing jaundice. The gallstone may erode into the bile duct, causing cholecystocholedochal fistula. Gallstone ileus refers to small bowel obstruction resulting from the impaction of one or more gallstones after they have migrated through a cholecystoenteric fistula. An accurate diagnosis is essential to the management and prevention of further complications. A variety of imaging and endoscopic modalities are used to make the diagnosis once the condition is suspected clinically. Treatment should be tailored to each individual patient. Management choices include ERCP, lithotripsy (endoscopic or extracorporeal), and surgery. Prognosis is frequently related to early recognition, management of any comorbid conditions, and careful selection of treatment modalities.",
"Single incision laparoscopic surgery (SILS) is a rapidly developing field that may represent the future of laparoscopic surgery. The major advantage of SILS over standard laparoscopic surgery is in cosmesis, with surgery becoming essentially scarless if the incision is hidden within the umbilicus. Only one incision is required so the risk of potential complications like port site hernias, haematomas and wound infection is reduced. The trade-off for this is a technically more challenging procedure with different underlying principles to that of traditional laparoscopic surgery. A wide variety of new equipment has been developed to support SILS and the range of procedures that are amenable to the technique is increasing. To date most of the published data relating to SILS are in the form of case series, with the first large randomised controlled trials due to be completed by the end of 2012. The existing evidence suggests that SILS is similar to standard laparoscopic surgery in terms of complication rates, completion rates and post-operative pain scores. However, the duration of SILS is longer than equivalent laparoscopic procedures. This article discusses SILS with regard to its applications in general surgery and reviews the evidence currently available."
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Vibrio vulnificus hemolysin is a -trefoil lectin.
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Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs.
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[
"The CCP4 (Collaborative Computational Project, number 4) program suite is a collection of programs and associated data and subroutine libraries which can be used for macromolecular structure determination by X-ray crystallography. The suite is designed to be flexible, allowing users a number of methods of achieving their aims and so there may be more than one program to cover each function. The programs are written mainly in standard Fortran77. They are from a wide variety of sources but are connected by standard data file formats. The package has been ported to all the major platforms under both Unix and VMS. The suite is distributed by anonymous ftp from Daresbury Laboratory and is widely used throughout the world.",
"Interactions between carbohydrates and proteins mediate intracellular traffic, cell adhesion, cell recognition and immune system function. Two recent papers describe how arrays of oligosaccharide and polysaccharide molecules can be used to investigate these interactions more fully.",
"The cholesterol-dependent cytolysins (CDCs) are characterized by an undecapeptide sequence (ECTGLAWEWWR) that is located near the C terminus and within domain 4 of these proteins. Pyolysin (PLO), the CDC of Arcanobacterium pyogenes, has a variant undecapeptide sequence (EATGLAWDPWW). Site-directed mutants were constructed in undecapeptide residues in a recombinant PLO molecule containing a hexahistidine tag (His-PLO). Mutations in each of the three undecapeptide tryptophan residues resulted in low haemolytic activity, confirming the importance of these residues in the protein. Deletion of a proline residue (P(499)), inserted in PLO, or substitution of this residue with either phenylalanine or glycine resulted in mutant proteins with undetectable or low haemolytic activities, indicating that P(499) is essential for His-PLO haemolytic activity. Substitution of the PLO undecapeptide sequence with a consensus undecapeptide resulted in a His-PLO protein with only 0.1% activity, confirming that the variant PLO undecapeptide is required for the full cytolytic activity of this toxin. The presence of the conserved undecapeptide cysteine residue either alone (His-PLO.C(492)) or in a consensus sequence resulted in His-PLO molecules which were activated in the presence of reducing compounds, confirming the importance of this residue in the thiol-activated nature of many CDC toxins. The ability of His-PLO mutant proteins to bind cholesterol mimicked haemolytic activity, with the exception of His-PLO.C(492), which, despite having reduced haemolytic activity, showed an increased ability to bind cholesterol compared to His-PLO. Despite reductions in haemolytic activity and cholesterol-binding, all mutant proteins were still able to bind to erythrocyte membranes, suggesting that other regions of PLO may recognize host-cell membranes, through receptors other than cholesterol.",
"Pathogens selectively target host cells using adhesion molecules and secreted virulence factors that may utilize protein, lipid, or carbohydrate ligands on the cell surface. The human intestinal pathogen Vibrio cholerae secretes a pore-forming toxin, V.cholerae cytolysin (VCC), which contains two domains that are structurally similar to known carbohydrate-binding proteins. These tandem domains are attached to the carboxy-terminus of the cytolytic domain and contain a β-trefoil fold and a β-prism fold. VCC has been shown to bind glycosylated proteins, and removal of the β-prism domain leads to a large decrease in lytic activity against rabbit erythrocytes. Despite these clues, the identity of the glycan receptors of VCC and the role of glycan binding in toxin activity remain unknown. To better understand this specificity, we used a combination of structural and functional approaches to characterize the carbohydrate-binding activity of the VCC toxin. We first probed the monosaccharide-binding activity of VCC and demonstrated that the toxin exhibits millimolar affinity for aldohexoses. To understand this specificity, we solved the crystal structure of the VCC β-prism domain bound to methyl-α-mannose. Next, we utilized a mammalian glycan screen to determine that the β-prism domain preferentially binds complex N-glycans with a heptasaccharide GlcNAc(4)Man(3) core (NGA2). Fluorescence anisotropy and surface plasmon resonance indicated an approximately 100-nM affinity of the β-prism domain for the heptasaccharide core. Our results suggest that carbohydrate-binding domains on the VCC toxin facilitate high-affinity targeting of mammalian cell membranes, which may contribute to the ability of VCC to lyse cells at picomolar concentrations.",
"The haemolytic action of Vibrio vulnificus haemolysin (VVH) was compared to that of streptolysin O (SLO). Both were cholesterol-binding haemolysins, but differed in the release of haemoglobin (Hb). In the first step of haemolysis, the haemolysins were temperature-independently bound to the cholesterol site on the target erythrocyte membrane. This was followed by the rapid release of K+, which is an intra-erythrocyte marker. Hb was then released, in different ways. In the case of VVH, Hb was released slowly after a relatively long lag, whereas with SLO, Hb was released as rapidly as K+. Haemolysis by VVH was inhibited by the addition of 30 mM-dextran 4 (mean Mr 4000), which is considered to be an effective colloid-osmotic protectant. The results therefore indicated that haemolysis by VVH (like that by Escherichia coli alpha-haemolysin and Staphylococcus aureus alpha-toxin) was caused by a colloid-osmotic mechanism. Both K+ and Hb release caused by VVH proceeded temperature-dependently, and the membrane fluidity of liposomes prepared with lipids extracted from sheep red blood cell membranes increased above 20 degrees C. These results suggest that the temperature-dependence of the haemolysis by VVH is due to the requirement for an increase in the membrane fluidity during the formation of a transmembrane pore.",
"We describe MUSCLE, a new computer program for creating multiple alignments of protein sequences. Elements of the algorithm include fast distance estimation using kmer counting, progressive alignment using a new profile function we call the log-expectation score, and refinement using tree-dependent restricted partitioning. The speed and accuracy of MUSCLE are compared with T-Coffee, MAFFT and CLUSTALW on four test sets of reference alignments: BAliBASE, SABmark, SMART and a new benchmark, PREFAB. MUSCLE achieves the highest, or joint highest, rank in accuracy on each of these sets. Without refinement, MUSCLE achieves average accuracy statistically indistinguishable from T-Coffee and MAFFT, and is the fastest of the tested methods for large numbers of sequences, aligning 5000 sequences of average length 350 in 7 min on a current desktop computer. The MUSCLE program, source code and PREFAB test data are freely available at http://www.drive5. com/muscle.",
"Vibrio vulnificus hemolysin (VVH) is thought to be a member of the cholesterol-dependent cytolysin (CDC) family of pore-forming toxins. To date, the structure-function relationships of CDCs produced by Gram-negative bacteria remain largely unknown. We show here that the aromatic ring of phenylalanine residue conserved in Vibrionaceae hemolysins is essential for oligomerization of VVH. We generated the VVH mutants; substituted Phe 334 for Ile (F334I), Ala (F334A), Tyr (F334Y), or Trp (F334W); and tested their binding and oligomerizing activity on Chinese hamster ovary cells. Binding in all mutants fell by approximately 50% compared with that in the wild type. Oligomerizing activities were completely eliminated in F334I and F334A mutants, whereas this ability was partially retained in F334Y and F334W mutants. These findings indicate that both hydrophobicity and an aromatic ring residue at the 334th position were needed for full binding activity and that the oligomerizing activity of this toxin was dependent on the existence of an aromatic ring residue at the 334th position. Our findings might help further understanding of the structure-and-function relationships in Vibrionaceae hemolysins.",
"Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.",
"CCP4mg is a project that aims to provide a general-purpose tool for structural biologists, providing tools for X-ray structure solution, structure comparison and analysis, and publication-quality graphics. The map-fitting tools are available as a stand-alone package, distributed as 'Coot'.",
"We describe a graphical system for automatically generating multiple 2D diagrams of ligand-protein interactions from 3D coordinates. The diagrams portray the hydrogen-bond interaction patterns and hydrophobic contacts between the ligand(s) and the main-chain or side-chain elements of the protein. The system is able to plot, in the same orientation, related sets of ligand-protein interactions. This facilitates popular research tasks, such as analyzing a series of small molecules binding to the same protein target, a single ligand binding to homologous proteins, or the completely general case where both protein and ligand change."
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Impact of specific KRAS mutation in codons 12 and 13 on clinical outcome of chemotherapy and radiotherapy in colorectal cancer patients
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Knowledge obtained via high-throughput technologies, used for tumor genome sequencing or identifying gene expression and methylation signatures, is clinically applicable thanks to molecular characterization in the context of tumor development and progression. This study was conducted to assess the impact of specific KRAS mutation in codons 12 and 13 on clinical outcome of chemotherapy and radiotherapy in colorectal cancer patients.
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"Flow cytometry was performed on available archival material from 232 patients with rectal cancer enrolled in the National Surgical Adjuvant Breast and Bowel Project protocol R-01. Tumor ploidy was not found to be significantly related to such pathologic and clinical parameters as Dukes' stage; nodal status; nuclear or histologic grades; patient's age, sex, or overall survival rate with an average study time of 79 months. A trend was evident that patients with poor histologic grade, or those with Dukes' B and C tumors that were aneuploid fared worse than those with diploid cancers. However, measurements of survival were found to be more strongly and consistently related to such conventional prognostic parameters as tumor differentiation, Dukes' stage, and nodal status. Further, numbers of nodes with metastases (ie, 1 to 4 or 5+) more significantly discriminated Dukes' C cases than estimation of tumor ploidy. Although tumor ploidy may reflect some features of rectal cancers, their natural history and prognosis are explained better by assessment of conventional parameters used for these purposes.",
"Colorectal cancers (CRC) are thought to have genetic instability in the form of either microsatellite instability (MSI) or chromosomal instability (CIN). Recently, tumours have been described without either MSI or CIN, that is, microsatellite and chromosome stable (MACS) CRCs. We investigated the (i) frequency of the MACS-CRCs and (ii) whether this genotype predicted responsiveness to neoadjuvant chemoradiotherapy. To examine the frequency of MACS-CRCs, DNA content (ploidy) was examined in 89 sporadic microsatellite-stable CRCs using flow cytometry. The tumours were also screened for mutations in KRAS/BRAF/TP53/PIK3CA by QMC-PCR. To examine the value of tumour ploidy in predicting response to chemoradiotherapy, DNA content was tested in a separate group of 62 rectal cancers treated with neoadjuvant chemoradiotherapy. Fifty-one of 89 CRCs (57%) were aneuploid and 38 (43%) were diploid. There was no significant association between mutations in TP53/KRAS/BRAF/PIK3CA and ploidy. Testing of association between mutations revealed only mutual exclusivity of KRAS/BRAF mutation (P < 0.001). Of the 62 rectal cancers treated with neoadjuvant chemoradiotherapy, 22 had responded (Mandard tumour regression grade 1/2) and 40 failed to respond (Grade 3-5). Twenty-five of 62 (40%) tumours were diploid, but there was no association between ploidy and response to therapy. We conclude that MACS-CRCs form a significant proportion of microsatellite-stable CRCs with a mutation profile overlapping that of CRCs with CIN. A diploid genotype does not, however, predict the responsiveness to radiotherapy.",
"Patients with metastatic colorectal cancer that harbors KRAS mutations in exon 2 do not benefit from anti-epidermal growth factor receptor (EGFR) therapy. Other activating RAS mutations may also be negative predictive biomarkers for anti-EGFR therapy. In this prospective-retrospective analysis, we assessed the efficacy and safety of panitumumab plus oxaliplatin, fluorouracil, and leucovorin (FOLFOX4) as compared with FOLFOX4 alone, according to RAS (KRAS or NRAS) or BRAF mutation status. A total of 639 patients who had metastatic colorectal cancer without KRAS mutations in exon 2 had results for at least one of the following: KRAS exon 3 or 4; NRAS exon 2, 3, or 4; or BRAF exon 15. The overall rate of ascertainment of RAS status was 90%. Among 512 patients without RAS mutations, progression-free survival was 10.1 months with panitumumab-FOLFOX4 versus 7.9 months with FOLFOX4 alone (hazard ratio for progression or death with combination therapy, 0.72; 95% confidence interval [CI], 0.58 to 0.90; P=0.004). Overall survival was 26.0 months in the panitumumab-FOLFOX4 group versus 20.2 months in the FOLFOX4-alone group (hazard ratio for death, 0.78; 95% CI, 0.62 to 0.99; P=0.04). A total of 108 patients (17%) with nonmutated KRAS exon 2 had other RAS mutations. These mutations were associated with inferior progression-free survival and overall survival with panitumumab-FOLFOX4 treatment, which was consistent with the findings in patients with KRAS mutations in exon 2. BRAF mutations were a negative prognostic factor. No new safety signals were identified. Additional RAS mutations predicted a lack of response in patients who received panitumumab-FOLFOX4. In patients who had metastatic colorectal cancer without RAS mutations, improvements in overall survival were observed with panitumumab-FOLFOX4 therapy. (Funded by Amgen and others; PRIME ClinicalTrials.gov number, NCT00364013.).",
"A method has been developed that allows flow cytometry to be used for measuring the cellular DNA content of paraffin-embedded human tumors. Thick (i.e., 30 micron) sections were cut from tissue blocks using a microtome and dewaxed in xylene. The sections were then rehydrated by sequentially immersing them in 100, 95, 70, and 50% ethanol before finally washing in distilled water. Single cell suspensions were then prepared by incubation in 0.5% pepsin, pH 1.5, at 37 degrees C for 30 min. The cells were counted, washed, and stained with 1 microgram/ml 4',6'-diamidino-2-phenylindole for 30 min, and DNA content was measured using an ICP 22 flow cytometer. There was a good correlation between the DNA histograms produced using this method and those obtained using unfixed tissue from the same tumor stained with ethidium bromide plus mithramycin. This method allows the retrospective study of archival material where the clinical outcome is already known, and it should, therefore, be particularly useful for determining the prognostic significance of abnormal DNA content measured by flow cytometry."
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Targeting androgen receptor signalling in breast cancer
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The androgen receptor (AR) has re-emerged as a potential therapeutic target in breast cancer. This stems from recent progress made in preclinical models, that have recognized important differences in the effect of AR expression on patient outcomes among different breast cancer subtypes. In parallel, the clinical development of new generations of AR directed therapies for prostate cancer has begun to mature. The availability of these new agents has translated into new trials to treat breast cancer. It is critical that studies of the effect of AR expression and signaling in breast cancer be context and subtype specific in order to successfully target AR signalling as a therapeutic strategy for breast cancer. We will review developments in preclinical studies, and recent clinical trials targeting AR in breast cancer.
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"Patients with hormone receptor-negative breast cancer generally do not benefit from endocrine-targeted therapies. However, a subset with androgen receptor (AR) expression is predicted to respond to antiandrogen therapies. This phase II study explored bicalutamide in AR-positive, estrogen receptor (ER), and progesterone receptor (PgR)-negative metastatic breast cancer. Tumors from patients with ER/PgR-negative advanced breast cancer were tested centrally for AR [immunohistochemistry (IHC) > 10% nuclear staining considered positive]. If either the primary or a metastatic site was positive, patients were eligible to receive the AR antagonist bicalutamide at a dose of 150 mg daily. Clinical benefit rate (CBR), the primary endpoint, was defined as the total number of patients who show a complete response (CR), partial response (PR), or stable disease (SD) > 6 months; secondary endpoints included progression-free survival (PFS) and toxicity. Correlative studies included measurement of circulating endocrine markers and IHC surrogates for basal-like breast cancer. Of 424 patients with ER/PgR-negative breast cancer, 12% tested AR-positive. The 6-month CBR was 19% [95% confidence interval (CI), 7%-39%] for bicalutamide. The median PFS was 12 weeks (95% CI, 11-22 weeks). Bicalutamide was well-tolerated with no grade 4/5 treatment-related adverse events observed. AR was expressed in 12% of patients with ER/PgR-negative breast cancer screened for this trial. The CBR of 19% observed with bicalutamide shows proof of principle for the efficacy of minimally toxic androgen blockade in a select group of patients with ER/PgR-negative, AR-positive breast cancer.",
"Endocrine therapies for breast cancer that target the estrogen receptor (ER) are ineffective in the 25%-30% of cases that are ER negative (ER-). Androgen receptor (AR) is expressed in 60%-70% of breast tumors, independent of ER status. How androgens and AR regulate breast cancer growth remains largely unknown. We find that AR is enriched in ER- breast tumors that overexpress HER2. Through analysis of the AR cistrome and androgen-regulated gene expression in ER-/HER2+ breast cancers we find that AR mediates ligand-dependent activation of Wnt and HER2 signaling pathways through direct transcriptional induction of WNT7B and HER3. Specific targeting of AR, Wnt or HER2 signaling impairs androgen-stimulated tumor cell growth suggesting potential therapeutic approaches for ER-/HER2+ breast cancers.",
"Enzalutamide (formerly called MDV3100) targets multiple steps in the androgen-receptor-signaling pathway, the major driver of prostate-cancer growth. We aimed to evaluate whether enzalutamide prolongs survival in men with castration-resistant prostate cancer after chemotherapy. In our phase 3, double-blind, placebo-controlled trial, we stratified 1199 men with castration-resistant prostate cancer after chemotherapy according to the Eastern Cooperative Oncology Group performance-status score and pain intensity. We randomly assigned them, in a 2:1 ratio, to receive oral enzalutamide at a dose of 160 mg per day (800 patients) or placebo (399 patients). The primary end point was overall survival. The study was stopped after a planned interim analysis at the time of 520 deaths. The median overall survival was 18.4 months (95% confidence interval [CI], 17.3 to not yet reached) in the enzalutamide group versus 13.6 months (95% CI, 11.3 to 15.8) in the placebo group (hazard ratio for death in the enzalutamide group, 0.63; 95% CI, 0.53 to 0.75; P<0.001). The superiority of enzalutamide over placebo was shown with respect to all secondary end points: the proportion of patients with a reduction in the prostate-specific antigen (PSA) level by 50% or more (54% vs. 2%, P<0.001), the soft-tissue response rate (29% vs. 4%, P<0.001), the quality-of-life response rate (43% vs. 18%, P<0.001), the time to PSA progression (8.3 vs. 3.0 months; hazard ratio, 0.25; P<0.001), radiographic progression-free survival (8.3 vs. 2.9 months; hazard ratio, 0.40; P<0.001), and the time to the first skeletal-related event (16.7 vs. 13.3 months; hazard ratio, 0.69; P<0.001). Rates of fatigue, diarrhea, and hot flashes were higher in the enzalutamide group. Seizures were reported in five patients (0.6%) receiving enzalutamide. Enzalutamide significantly prolonged the survival of men with metastatic castration-resistant prostate cancer after chemotherapy. (Funded by Medivation and Astellas Pharma Global Development; AFFIRM ClinicalTrials.gov number, NCT00974311.).",
"We sought to determine the prevalence of androgen receptor (AR) expression in a predominantly estrogen receptor (ER)-negative subset of breast cancers and delineate the immunohistochemical and clinical associations, including whether AR expression has prognostic significance in ER-negative tumors. We identified 69 ER-negative and 19 ER-positive breast cancer cases with concurrent immunohistochemical prognostic panels (ER, PR, HER-2/neu, Ki-67, and p53); immunohistochemical analysis was performed for AR using standard techniques. Clinical data were extracted from medical records. chi 2 tests were used to assess associations between variables. AR was found in 49% (34/69) of ER-negative and 89% (17/19) of ER-positive cases. In ER-negative tumors, AR was associated with increased age (P = .02), postmenopausal status (P < .001), tumor grade (P = .03), tumor size (P = .03), and HER-2/neu overexpression (P = .003). In ER-positive tumors, AR was associated with progesterone receptor expression (P < .03). In univariate analysis of ER-negative tumors, patients with AR-positive tumors had significantly better disease-free survival (P = .049). AR is expressed in a significant number of ER-negative cases and shows significant associations with important clinical and pathologic prognostic factors.",
"MDV3100 is an androgen-receptor antagonist that blocks androgens from binding to the androgen receptor and prevents nuclear translocation and co-activator recruitment of the ligand-receptor complex. It also induces tumour cell apoptosis, and has no agonist activity. Because growth of castration-resistant prostate cancer is dependent on continued androgen-receptor signalling, we assessed the antitumour activity and safety of MDV3100 in men with this disease. This phase 1-2 study was undertaken in five US centres in 140 patients. Patients with progressive, metastatic, castration-resistant prostate cancer were enrolled in dose-escalation cohorts of three to six patients and given an oral daily starting dose of MDV3100 30 mg. The final daily doses studied were 30 mg (n=3), 60 mg (27), 150 mg (28), 240 mg (29), 360 mg (28), 480 mg (22), and 600 mg (3). The primary objective was to identify the safety and tolerability profile of MDV3100 and to establish the maximum tolerated dose. The trial is registered with ClinicalTrials.gov, number NCT00510718. We noted antitumour effects at all doses, including decreases in serum prostate-specific antigen of 50% or more in 78 (56%) patients, responses in soft tissue in 13 (22%) of 59 patients, stabilised bone disease in 61 (56%) of 109 patients, and conversion from unfavourable to favourable circulating tumour cell counts in 25 (49%) of the 51 patients. PET imaging of 22 patients to assess androgen-receptor blockade showed decreased (18)F-fluoro-5alpha-dihydrotestosterone binding at doses from 60 mg to 480 mg per day (range 20-100%). The median time to progression was 47 weeks (95% CI 34-not reached) for radiological progression. The maximum tolerated dose for sustained treatment (>28 days) was 240 mg. The most common grade 3-4 adverse event was dose-dependent fatigue (16 [11%] patients), which generally resolved after dose reduction. We recorded encouraging antitumour activity with MDV3100 in patients with castration-resistant prostate cancer. The results of this phase 1-2 trial validate in man preclinical studies implicating sustained androgen-receptor signalling as a driver in this disease. Medivation, the Prostate Cancer Foundation, National Cancer Institute, the Howard Hughes Medical Institute, Doris Duke Charitable Foundation, and Department of Defense Prostate Cancer Clinical Trials Consortium.",
"Persistence of ligand-mediated androgen receptor signaling has been documented in castration-resistant prostate cancers (CRPCs). Abiraterone acetate (AA) is a potent and selective inhibitor of CYP17, which is required for androgen biosynthesis in the testes, adrenal glands, and prostate tissue. This trial evaluated the efficacy and safety of AA in combination with prednisone to reduce the symptoms of secondary hyperaldosteronism that can occur with AA monotherapy. Fifty-eight men with progressive metastatic CRPC who experienced treatment failure with docetaxel-based chemotherapy received AA (1,000 mg daily) with prednisone (5 mg twice daily). Twenty-seven (47%) patients had received prior ketoconazole. The primary outcome was > or = 50% prostate-specific antigen (PSA) decline, with objective response by Response Evaluation Criteria in Solid Tumors (RECIST) criteria, and changes in Eastern Cooperative Oncology Group (ECOG) performance status (PS) and circulating tumor cell (CTC) numbers. Safety was also evaluated. A > or = 50% decline in PSA was confirmed in 22 (36%) patients, including 14 (45%) of 31 ketoconazole-naïve and seven (26%) of 27 ketoconazole-pretreated patients. Partial responses were seen in four (18%) of 22 patients with RECIST-evaluable target lesions. Improved ECOG PS was seen in 28% of patients. Median time to PSA progression was 169 days (95% CI, 82 to 200 days). CTC conversions with treatment from > or = 5 to < 5 were noted in 10 (34%) of 29 patients. The majority of AA-related adverse events were grade 1 to 2, and no AA-related grade 4 events were seen. AA plus prednisone was well tolerated, with encouraging antitumor activity in heavily pretreated CRPC patients. The incidence of mineralocorticoid-related toxicities (hypertension or hypokalemia) was reduced by adding low-dose prednisone. The combination of AA plus prednisone is recommended for phase III investigations.",
"Clinical experience suggests that many women with triple-negative metastatic breast cancer (MBC) relapse quickly. This has implications for clinical practice and trial design. We evaluated the duration of first-, second-, and third-line chemotherapy as a surrogate for duration of treatment response. We performed a retrospective multicenter chart review of patients with triple-negative MBC receiving palliative chemotherapy. Primary outcome was duration of palliative chemotherapy, and secondary outcome was to identify prognostic variables. A total of 111 patients were analyzed. Median age at diagnosis was 51 years (range, 26-82 years). Fourteen percent of patients presented with MBC. Twenty-seven percent received neoadjuvant chemotherapy, and 48% received adjuvant chemotherapy. Median distant disease-free interval (DDFI) was 18 months (range, 0-172 months). At presentation of MBC, 68% had visceral and 71% had multiple sites of disease. Median survival with MBC was 13.3 months (range, 0.8-99.8 months). Median duration of first-line palliative therapy was 11.9 weeks (range, 0-73.1 weeks). Eighty-seven patients (78%) went on to receive second-line therapy with a median duration of 9 weeks (range, 0-120.9 weeks), and 55 (49%) received third-line therapy with a median duration of 4 weeks (range, 0-59 weeks). Multivariate analysis revealed that age < 50 years, ALP > 120 U/L, history of previous chemotherapy, DDFI < 12 months, and visceral presentation were all independently associated with a poor prognosis. Despite the poorer overall prognosis of patients with triple-negative disease, there remains considerable heterogeneity in individual outcomes. Many women with recurrent triple-negative disease will progress quickly on first-, second-, and third-line palliative treatment. Future clinical trials in this population must take into account their shorter time to progression when determining optimal trial design.",
"Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castration-resistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer.",
"The principal objective of this trial was to evaluate the antitumor activity of abiraterone acetate, an oral, specific, irreversible inhibitor of CYP17 in docetaxel-treated patients with castration-resistant prostate cancer (CRPC). In this multicenter, two-stage, phase II study, abiraterone acetate 1,000 mg was administered once daily continuously. The primary end point was achievement of a prostate-specific antigen (PSA) decline of > or = 50% in at least seven of 35 patients. Per an attained phase II design, more than 35 patients could be enrolled if the primary end point was met. Secondary objectives included: PSA declines of > or = 30% and > or = 90%; rate of RECIST (Response Evaluation Criteria in Solid Tumors) responses and duration on study; time to PSA progression; safety and tolerability; and circulating tumor cell (CTC) enumeration. Docetaxel-treated patients with CRPC (N = 47) were enrolled. PSA declines of > or = 30%, > or = 50% and > or = 90% were seen in 68% (32 of 47), 51% (24 of 47), and 15% (seven of 47) of patients, respectively. Partial responses (by RECIST) were reported in eight (27%) of 30 patients with measurable disease. Median time to PSA progression was 169 days (95% CI, 113 to 281 days). The median number of weeks on study was 24, and 12 (25.5%) of 47 patients remained on study > or = 48 weeks. CTCs were enumerated in 34 patients; 27 (79%) of 34 patients had at least five CTCs at baseline. Eleven (41%) of 27 patients had a decline from at least five to less than 5 CTCs, and 18 (67%) of 27 had a > or = 30% decline in CTCs after starting treatment with abiraterone acetate. Abiraterone acetate was well tolerated. Abiraterone acetate has significant antitumor activity in post-docetaxel patients with CRPC. Randomized, phase III trials of abiraterone acetate are underway to define the future role of this agent.",
"Each year, the American Cancer Society estimates the number of new cancer cases and deaths expected in the United States in the current year and compiles the most recent data regarding cancer incidence, mortality, and survival based on incidence data from the National Cancer Institute, the Centers for Disease Control and Prevention, and the North American Association of Central Cancer Registries and mortality data from the National Center for Health Statistics. Incidence and death rates are age-standardized to the 2000 US standard million population. A total of 1,529,560 new cancer cases and 569,490 deaths from cancer are projected to occur in the United States in 2010. Overall cancer incidence rates decreased in the most recent time period in both men (1.3% per year from 2000 to 2006) and women (0.5% per year from 1998 to 2006), largely due to decreases in the 3 major cancer sites in men (lung, prostate, and colon and rectum [colorectum]) and 2 major cancer sites in women (breast and colorectum). This decrease occurred in all racial/ethnic groups in both men and women with the exception of American Indian/Alaska Native women, in whom rates were stable. Among men, death rates for all races combined decreased by 21.0% between 1990 and 2006, with decreases in lung, prostate, and colorectal cancer rates accounting for nearly 80% of the total decrease. Among women, overall cancer death rates between 1991 and 2006 decreased by 12.3%, with decreases in breast and colorectal cancer rates accounting for 60% of the total decrease. The reduction in the overall cancer death rates translates to the avoidance of approximately 767,000 deaths from cancer over the 16-year period. This report also examines cancer incidence, mortality, and survival by site, sex, race/ethnicity, geographic area, and calendar year. Although progress has been made in reducing incidence and mortality rates and improving survival, cancer still accounts for more deaths than heart disease in persons younger than 85 years. Further progress can be accelerated by applying existing cancer control knowledge across all segments of the population and by supporting new discoveries in cancer prevention, early detection, and treatment."
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Plasticity of NG2 IR cells in the locus coeruleus
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The locus coeruleus (LC) nucleus modulates adaptive behavioral responses to stress and dysregulation of LC neuronal activity is implicated in stress-induced mental illnesses. The LC is composed primarily of noradrenergic neurons together with various glial populations. A neuroglia cell-type largely unexplored within the LC is the NG2 cell. NG2 cells serve primarily as oligodendrocyte precursor cells throughout the brain. However, some NG2 cells are in synaptic contact with neurons suggesting a role in information processing. The aim of this study was to neurochemically and anatomically characterize NG2 cells within the rat LC. Furthermore, since NG2 cells have been shown to proliferate in response to traumatic brain injury, we investigated whether such NG2 cells plasticity also occurs in response to emotive insults such as stress. Immunohistochemistry and confocal microscopy revealed that NG2 cells were enriched within the pontine region occupied by the LC. Close inspection revealed that a sub-population of NG2 cells were located within unique indentations of LC noradrenergic somata and were immunoreactive for the neuronal marker NeuN whilst NG2 cell processes formed close appositions with clusters immunoreactive for the inhibitory synaptic marker proteins gephyrin and the GABA-A receptor alpha3-subunit, on noradrenergic dendrites. In addition, LC NG2 cell processes were decorated with vesicular glutamate transporter 2 immunoreactive puncta. Finally, 10 days of repeated restraint stress significantly increased the density of NG2 cells within the LC. The study demonstrates that NG2 IR cells are integral components of the LC cellular network and they exhibit plasticity as a result of emotive challenges.
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"Oligodendrocytes are the myelinating cells of the central nervous system. Although the CNS possesses the ability to repair demyelinating insults, in certain cases, such as the chronic lesions found in multiple sclerosis, remyelination fails. Cycling cells capable of becoming oligodendrocytes have been identified in both the developing and the adult mammalian forebrain. Many studies have focused on differences in gene expression profiles as oligodendrocyte progenitors differentiate into myelinating oligodendrocytes by isolating cells at different developmental stages from animals at a single age. However, few have studied the differences that exist between the progenitors of the neonatal CNS and those of the adult CNS. This study examined the response of neonatal and adult O4(+) cells to platelet-derived growth factor-AA, basic fibroblast growth factor, and insulin-like growth factor-1 and revealed marked differences. Whereas adult cells readily differentiated in vitro, the majority of neonatal progenitors remained immature. Microarray analysis was used to examine differences between acutely isolated neonatal and adult progenitors further. Gene expression profiles showed that the adult O4(+) cells are more developmentally mature than neonatal cells. Neonatal cells expressed higher levels of genes involved in proliferation. Adult O4(+) cells expressed higher levels of transcripts for genes involved in cell death and survival. Therefore, O4(+) cells from the adult differ greatly from those of the neonate, and the developmental stage of the animal models utilized must be taken into consideration when applying principles from neonatal systems to the adult.",
"Neurogenesis is known to persist in the adult mammalian central nervous system (CNS). The identity of the cells that generate new neurons in the postnatal CNS has become a crucial but elusive issue. Using a transgenic mouse, we show that NG2 proteoglycan-positive progenitor cells that express the 2',3'-cyclic nucleotide 3'-phosphodiesterase gene display a multipotent phenotype in vitro and generate electrically excitable neurons, as well as astrocytes and oligodendrocytes. The fast kinetics and the high rate of multipotent fate of these NG2+ progenitors in vitro reflect an intrinsic property, rather than reprogramming. We demonstrate in the hippocampus in vivo that a sizeable fraction of postnatal NG2+ progenitor cells are proliferative precursors whose progeny appears to differentiate into GABAergic neurons capable of propagating action potentials and displaying functional synaptic inputs. These data show that at least a subpopulation of postnatal NG2-expressing cells are CNS multipotent precursors that may underlie adult hippocampal neurogenesis.",
"Remyelination occurs in demyelinated CNS regions in diseases such as multiple sclerosis. Identification of the cell type(s) responsible for this remyelination, however, has been elusive. Here, we examine one potential source of remyelinating oligodendrocytes-immature, cycling cells endogenous to adult white matter-and demonstrate that this population responds to demyelination by differentiating into myelinating oligodendrocytes. Dividing cells in subcortical white matter of adult rats were labeled by stereotactic injection of a replication-deficient lacZ-encoding retrovirus (BAG). Following a focal demyelination induced with lysolecithin, many of the BAG-labeled cells differentiated into myelinating oligodendrocytes engaging in repair of the lesion. Identification of endogenous cells capable of remyelination provides a target for the study of CNS repair processes in demyelinating diseases.",
"A detailed comparison in the developing rat central nervous system between the distribution of the NG2 proteoglycan and the alpha-receptor for platelet-derived growth factor (PDGF) shows that these two molecules are co-expressed by glial progenitor cells of the O2A lineage and can serve as reliable markers for identification of O2A cells in vivo. Our mapping experiments indicate that NG2-positive, PDGF alpha-receptor positive O2A cells are abundant throughout the developing central nervous system in both white and gray matter. The earliest cells immunoreactive for either of the two markers are found adjacent to the central canal of the embryonic day 15 (E15) spinal cord. These cells express only PDGF alpha-receptor and not NG2. By E17, process-bearing cells expressing both NG2 and PDGF alpha-receptor in a highly co-localized fashion are found throughout the central nervous system. The first postnatal week marks the peak in the number of NG2 and PDGF alpha-receptor immunoreactive cells, as well as the peak in the level of expression and the extent of co-localization of the two molecules. After the first week, the level of expression of both NG2 and PDGF alpha-receptor declines, although both molecules continue to be expressed in the adult brain. On O2A cells in the mature brain, NG2 and PDGF alpha-receptor are not as well co-localized at the subcellular level as they are on O2A cells in the younger brain. The functional consequences of co-localization and subsequent dissociation of NG2 and PDGF alpha-receptor on maturing O2A progenitors are investigated in the accompanying paper (Nishiyama et al.: J Neurosci Res 43:315-330, 1996).",
"Despite their abundance, still little is known about the rather frequent, constantly proliferating progenitors spread throughout the adult mouse brain parenchyma. The majority of these progenitors express the basic-helix-loop-helix transcription factor Olig2, and their number further increases after injury. Here, we examine the progeny of this progenitor population by genetic fate mapping using tamoxifen-inducible Cre-recombination in the Olig2 locus to turn on permanent reporter gene expression in the adult brain. Consistent with Olig2 expression in proliferating NG2(+) progenitors, most reporter(+) cells seen shortly after initiating recombination at adult stages incorporated BrdU and contained the proteoglycan NG2 in both the gray (GM) and the white matter (WM) of the cerebral cortex. However, at longer time points after induction, we observed profound differences in the identity of reporter(+) cells in the WM and GM. Whereas most of the Olig2(+) progenitors had generated mature, myelinating oligodendrocytes in the WM, hardly any reporter(+) cells showing mature oligodendrocyte characteristics were detectable even up to 6 months after recombination in the GM. In the GM, most reporter(+) cells remained NG2(+), even after injury, but stopped proliferating rather soon after recombination. Thus, our results demonstrate the continuous generation of mature, myelinating oligodendrocytes in the WM, whereas cells in the GM generated mostly postmitotic NG2(+) glia.",
"Insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF) and ciliary neurotrophic factor (CNTF) are multifunctional growth factors which are found in the CNS. Oligodendroglia are the cells that form and maintain myelin sheaths and many in vitro experiments have shown that these growth factors promote the proliferation, differentiation and survival of cells in the oligodendroglial lineage. Since myelin breakdown is often severe in multiple sclerosis (MS), the possibility of growth factor use in the treatment of MS has been considered and recently, IGF-I treatment has been shown to reduce lesion severity and promote myelin regeneration in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. This review briefly summarizes the structural characteristics of these growth factors and the actions which might help reduce oligodendrocyte-myelin sheath injury in MS and promote myelin regeneration.",
"We used cDNA arrays to investigate molecular aspects of the differentiation of an immortalized line of oligodendroglial progenitors, and of immunopan-purified primary cultures of oligodendroglial progenitors, to immature oligodendroglia. Developmental regulation of the proteolipid and 2-hydroxyacylsphingosine 1-galactosyltransferase genes was tighter in the primary than in the immortalized cells. Our data suggest that increased expression of genes encoding the following proteins are involved in oligodendroglial differentiation: Fyn, Erk, p85, G-alpha-12 guanine nucleotide binding, and transducin beta-2 signal transduction molecules; glial maturation factor; the proteasomal subunits C8 and C3; the proteasomal targeting molecule polyubiquitin; the cell cycle regulatory proteins Set, protein phosphatase 2A, and nuclear tyrosine phosphatase (PRL-1); and the high-affinity glutamate cotransporter EAAC-1."
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Agrobacterium tumefaciens VirA truncation and signal transduction
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Histidine kinases serve as critical environmental sensing modules, and despite their designation as simple two-component modules, their functional roles are remarkably diverse. In Agrobacterium tumefaciens pathogenesis, VirA serves with VirG as the initiating sensor/transcriptional activator for inter-kingdom gene transfer and transformation of higher plants. Through responses to three separate signal inputs, low pH, sugars, and phenols, A. tumefaciens commits to pathogenesis in virtually all flowering plants. However, how these three signals are integrated to regulate the response and why these signals might be diagnostic for susceptible cells across such a broad host-range remains poorly understood. Using a homology model of the VirA linker region, we provide evidence for coordinated long-range transmission of inputs perceived both outside and inside the cell through the creation of targeted VirA truncations. Further, our evidence is consistent with signal inputs weakening associations between VirA domains to position the active site histidine for phosphate transfer. This mechanism requires long-range regulation of inter-domain stability and the transmission of input signals through a common integrating domain for VirA signal transduction.
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[
"Allosteric regulation of protein function occurs when the regulatory trigger, such as the binding of a small-molecule effector or inhibitor, takes place some distance from the protein's, or protein complex's, active site. This distance can be a few A, or tens of A. Many proteins are regulated in this way and exhibit a variety of allosteric mechanisms. Here we review how analyses of experimentally determined models of protein 3D structures, using either X-ray crystallography or NMR spectroscopy, have revealed some of the mechanisms involved.",
"The virulence genes of Agrobacterium tumefaciens are induced by specific plant phenolic metabolites and sugars (G. A. Cangelosi, R. G. Ankenbauer, and E. W. Nester, Proc. Natl. Acad. Sci. USA, in press). In this report, monosaccharides, derivatives, and analogs which induce the vir regulon have been identified and the structural requirements for monosaccharide-mediated induction have been determined. Pyranose sugars with equatorial hydroxyls at C-1, C-2, and C-3 displayed strong vir gene-inducing activity; the C-4 hydroxyl could be epimeric and a wide variety of substitutions at C-5 were permissible. The acidic monosaccharide derivatives D-galacturonic acid and D-glucuronic acid were the strongest inducers among the monosaccharides tested. Eight of the 11 inducing compounds are known plant metabolites, and 7 are monomers of major plant cell wall polysaccharides. A role for monosaccharides and plant phenolic compounds as wound-specific plant metabolites which signal the ChvE/VirA/VirG regulatory system is proposed.",
"The activation of the Agrobacterium virulence system is known to be induced by certain phenolic compounds. We have tested the vir-inducing ability of fifty compounds, by using a virB-lacZ gene fusion, and analysed the relationship between structure and activity of these compounds. In this way we have identified several new vir-inducers: coniferylalcohol, 3,5-dimethoxy-4-hydroxybenzene, homovanillic acid, ferulic acid, 3-ethoxy-4-hydroxybenzaldehyde and guaiacol, all of which are compounds with strong or moderate activity and four compounds with weak vir-inducing activity. In view of the specificity of vir-inducers, our data extended observations of others and enabled us to define the specific structural features of a vir-inducer molecule. In addition we show here that induction of the octopine Ti vir-genes is (i) optimal at 29 degrees C and totally abolished at 37 degrees C, and (ii) strongly inhibited at low concentrations of sodium chloride. The implications for plant transformation are discussed.",
"VirA and VirG activate the Agrobacterium tumefaciens vir regulon in response to phenolic compounds, monosaccharides, and acidity released from plant wound sites. VirA contains an amino-terminal periplasmic domain and three cytoplasmic domains: a linker, a protein kinase, and a phosphoryl receiver. We constructed internal deletions of virA that truncate one or more domains and tested the ability of the resulting proteins to mediate environmentally responsive vir gene activation in vivo. The periplasmic domain is required for sensing of monosaccharides (in agreement with earlier results), while the linker domain is required for sensing of phenolic compounds and acidity. The phosphoryl receiver domain of VirA plays an inhibitory role in signal transduction that may be modulated by phosphorylation. The carboxy terminus of the protein was also dispensable for tumorigenesis, while the periplasmic domain was required.",
"Phenolic plant metabolites such as acetosyringone induce transcription of the virulence (vir) genes of Agrobacterium tumefaciens through the transmembrane VirA protein. We report here that certain sugars induce the vir genes synergistically with phenolic inducers by way of a distinct regulatory pathway that includes VirA and a chromosomally encoded virulence protein, ChvE. Sequence comparison showed that ChvE is a periplasmic galactose-binding protein corresponding to the GBP1 protein isolated from Agrobacterium radiobacter. Like homologous sugar-binding proteins in Escherichia coli, ChvE was required for chemotaxis toward galactose and several other sugars. These sugars strongly induced vir gene expression in wild-type cells when acetosyringone was absent or present in low concentrations. Mutations in chvE abolished vir gene induction by sugars and resulted in a limited host range for infection but did not affect vir gene induction by acetosyringone. A mutant lacking the periplasmic domain of VirA exhibited the same regulatory phenotype and limited host range as chvE mutants. These data show that the vir genes are regulated by two separate classes of plant-derived inducers by way of distinct regulatory pathways that can be separated by mutation. Induction by sugars is essential for infection of some but not all plant hosts.",
"The VirA protein of Agrobacterium tumefaciens is a transmembrane sensory kinase that phosphorylates the VirG response regulator in response to chemical signals released from plant wound sites. VirA contains both a two-component kinase module and, at its carboxyl terminus, a receiver module. We previously provided evidence that this receiver module inhibited the activity of the kinase module and that inhibition might be neutralized by phosphorylation. In this report, we provide additional evidence for this model by showing that overexpressing the receiver module in trans can restore low-level basal activity to a VirA mutant protein lacking the receiver module. We also show that ablation of the receiver module restores activity to the inactive VirA (delta324-413) mutant, which has a deletion within a region designated the linker module. This indicates that deletion of the linker module does not denature the kinase module, but rather locks the kinase into a phenotypically inactive conformation, and that this inactivity requires the receiver module. These data provide genetic evidence that the kinase and receiver modules of VirA attain their native conformations autonomously. The receiver module also restricts the variety of phenolic compounds that have stimulatory activity, since removal of this module causes otherwise nonstimulatory phenolic compounds such as 4-hydroxyacetophenone to stimulate vir gene expression.",
"The EnvZ/OmpR histidyl-aspartyl phosphorelay (HAP) system in Escherichia coli regulates the expression of ompF and ompC, the major outer membrane porin genes, in response to environmental osmolarity changes. Here, we report that dimers of EnvZc, the cytoplasmic domain of EnvZ, undergo spontaneous subunit exchange in solution. By introducing a cysteine substitution (S260C) in the dimerization domain of EnvZc, we were able to crosslink the two subunits in a dimer and trap the heterodimer formed between two different mutant EnvZc. By using a complementing system with two autophosphorylation-defective EnvZc mutants, one containing the H243V mutation at the autophosphorylation site and the other containing the G405A mutation in the ATP-binding domain, we demonstrated that an EnvZc(G405A) subunit can be phosphorylated by an EnvZc(H243V) subunit only when a heterodimer is formed. The rate of subunit exchange is concentration-dependent, with higher rates at higher concentrations of protein. The disulfide-crosslinked EnvZc(G405A) homodimer could not be phosphorylated by EnvZc(H243V), since the heterodimer formation between the two mutant proteins was blocked, indicating that autophosphorylation cannot occur by dimer-dimer interaction. By using MBP-deltaL-EnvZc(S260C) fusion protein (deltaL: the linker region, spanning residues 180-222, was deleted), it was found that in the disulfide-crosslinked MBP-deltaL-EnvZc(S260C)/deltaL-EnvZc(S260C/G405A) heterodimer, only the deltaL-EnvZc(S260C/G405A) subunit was phosphorylated but not the MBP-deltaL-EnvZc(S260C) subunit. Together, the present results provide biochemical evidence that EnvZ autophosphorylation occurs in trans and only within an EnvZ dimer."
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Prefrontal cortex 6-hydroxydopamine depletion modulates impulsive choice, novelty-preference, and preferences for cocaine in juvenile rats
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Reduced cortical dopamine levels have been observed in individuals with attention deficit hyperactivity disorder (ADHD). Global dopamine depletions by 6-hydroxydopamine (6-OHDA; with noradrenergic protection) in neonatal rats produces locomotor hyperactivity, with less known about how cortical depletion modulates risky behaviors. Here, we determined the effect of a medial prefrontal cortex (PFC) 6-OHDA depletions (30-60%) or sham microinjection at postnatal day 11 on behavior in male and female juvenile rats. Separate groups were studied for delay discounting (impulsive choice), novelty-preference, and preferences for cues and environments associated with cocaine (10, 20, and 40 mg/kg), their extinction, and reinstatement with place conditioning. Because PFC D1 receptors play a role in these behaviors, confocal microscopy was used to measure D1-immunoreactive projections to the nucleus accumbens core. Both 6-OHDA males and females increased delay discounting relative to sham controls, although only 6-OHDA females increased novelty preferences. Preferences for cocaine-associated environments, their extinction, and reinstatement with a priming dose of cocaine were reduced in 6-OHDA subjects overall. However, impulsive choice at 5s positively correlated with preferences for cocaine-associated environments in 6-OHDA subjects, but not sham controls. As possible compensation for low dopamine levels, D1-immunoreactivity on traced neurons increased in 6-OHDA females; dopamine levels did not remain low in adolescent 6-OHDA males and D1 did not change. We believe that these modest depletions restricted to the PFC demonstrate the role of dopamine, and not norepinephrine, in understanding these behaviors in other animal models where cortical dopamine is reduced during development.
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[
"Several lines of evidence support the notion that a substantial reorganization of cortical connections, involving a programmed synaptic pruning, takes place during adolescence in humans. A review of neurobiological abnormalities in schizophrenia indicates that the neurobiological parameters that undergo peripubertal regressive changes may be abnormal in this disorder. An excessive pruning of the prefrontal corticocortical, and corticosubcortical synapses, perhaps involving the excitatory glutamatergic inputs to pyramidal neurons, may underlie schizophrenia. A reciprocal failure of pruning in certain subcortical structures, such as lenticular nuclei, may also occur. Several developmental trajectories, related to early brain insults as well as genetic factors affecting postnatal neurodevelopment, could lead to the illness. These models would have heuristic value and may be consistent with several known facts of the schizophrenic illness, such as its onset in adolescence and the gender differences in its onset and natural course. The relationship between these models and other etiological models of schizophrenia are summarized and approaches to test relevant hypotheses are discussed.",
"Puberty is a central process in the complex set of changes that constitutes the transition from childhood to adolescence. Research on the role of pubertal change in this transition has been impeded by the difficulty of assessing puberty in ways acceptable to young adolescents and others involved. Addressing this problem, this paper describes and presents norms for a selfreport measure of pubertal status. The measure was used twice annually over a period of three years in a longitudinal study of 335 young adolescent boys and girls. Data on a longitudinal subsample of 253 subjects are reported. The scale shows good reliability, as indicated by coefficient alpha. In addition, several sources of data suggest that these reports are valid. The availability of such a measure is important for studies, such as those based in schools, in which more direct measures of puberty may not be possible.",
"This study investigated whether age and ADHD symptoms affected choice preferences in children and adolescents when they chose between (1) small immediate rewards and larger delayed rewards and (2) small certain rewards and larger probabilistic uncertain rewards. A temporal discounting (TD) task and a probabilistic discounting (PD) task were used to measure the degree to which the subjective value of a large reward decreased as one had to wait longer for it (TD), and as the probability of obtaining it decreased (PD). Rewards used were small amounts of money. In the TD task, the large reward (10 cents) was delayed by between 0 and 30s, and the immediate reward varied in magnitude (0-10 cents). In the PD task, receipt of the large reward (10 cents) varied in likelihood, with probabilities of 0, 0.25, 0.5, 0.75, and 1.0 used, and the certain reward varied in magnitude (0-10 cents). Age and diagnostic group did not affect the degree of PD of rewards: All participants made choices so that total gains were maximized. As predicted, young children, aged 6-11 years (n = 25) demonstrated steeper TD of rewards than adolescents, aged 12-17 years (n = 21). This effect remained significant even when choosing the immediate reward did not shorten overall task duration. This, together with the lack of interaction between TD task version and age, suggests that steeper discounting in young children is driven by reward immediacy and not by delay aversion. Contrary to our predictions, participants with ADHD (n = 22) did not demonstrate steeper TD of rewards than controls (n = 24). These results raise the possibility that strong preferences for small immediate rewards in ADHD, as found in previous research, depend on factors such as total maximum gain and the use of fixed versus varied delay durations. The decrease in TD as observed in adolescents compared to children may be related to developmental changes in the (dorsolateral) prefrontal cortex. Future research needs to investigate these possibilities.",
"Early onset schizophrenia (with onset before adulthood) represents a rarer and possibly more severe form of the disorder which has received particular attention in the last two decades. Current evidence strongly suggest continuity with adult onset schizophrenia, with phenomenological, cognitive, genetic and neuroimaging data pointing towards similar neurobiological correlates and clinical deficits but worse long term outcome. Future research in early onset cases is likely to increase further our insight into the neurodevelopmental origins of schizophrenia and the complex gene-environment interactions affecting its emergence.",
"Despite the importance of understanding the long-term outcome of children with attention-deficit/hyperactivity disorder (ADHD), the available literature is predominantly based on male samples. This study estimated the lifetime burden of comorbid psychopathology in a large sample of girls with and without ADHD followed up over five years. We conducted a blind, five-year prospective longitudinal study of girls with (n=140) and without (n=122) ADHD, aged 6-18 years at baseline, consecutively ascertained from either community pediatricians or psychiatrists at an academic medical center. At the five-year follow-up, 123 (88%) and 112 (92%) of the ADHD and control children, respectively, were re-assessed at a mean age of 16.7 years. Psychiatric disorders were assessed using blinded structured diagnostic interviews. At follow-up, females with ADHD were at significantly higher risk than controls to manifest disruptive behavior, mood and anxiety disorders, and substance dependence. The magnitude of increased risk was greatest for major depression and oppositional-defiant disorder, followed by substance dependence and anxiety disorders. These prospective follow-up findings documenting high morbidity associated with ADHD extend to females previously reported findings in male samples and underscore the importance of early recognition and intervention efforts for youth with ADHD of both genders.",
"The peak age of onset for many psychiatric disorders is adolescence, a time of remarkable physical and behavioural changes. The processes in the brain that underlie these behavioural changes have been the subject of recent investigations. What do we know about the maturation of the human brain during adolescence? Do structural changes in the cerebral cortex reflect synaptic pruning? Are increases in white-matter volume driven by myelination? Is the adolescent brain more or less sensitive to reward? Finding answers to these questions might enable us to further our understanding of mental health during adolescence.",
"Dysregulation of the prefrontal cortex (PFC) has been implicated in impulse control disorders, including attention deficit hyperactivity disorder. A growing body of evidence suggests that impulsivity is non-unitary in nature, and recent data indicate that the ventral and dorsal regions of the PFC are differentially involved in distinct aspects of impulsive behaviour, findings which may reflect differences in the monoaminergic regulation of these regions. In the current experiment, levels of dopamine, serotonin and their metabolites were measured in the medial PFC (n = 12) and orbitofrontal cortex (OFC) (n = 19) of rats using in vivo microdialysis during the delay-discounting model of impulsive choice, where impulsivity is defined as selection of small immediate over larger delayed rewards. Yoked groups were also dialysed to control for instrumental responding and reward delivery. Significant increases in 5-hydroxytryptamine efflux were observed in the mPFC, but not in the OFC, during task performance but not under yoked control conditions. In the OFC, 3,4-di-hydroxy-phenylocetic acid (DOPAC) levels increased in animals performing the task but not in yoked animals, whereas mPFC DOPAC levels increased in all subjects. These data suggest a double dissociation between serotonergic and dopaminergic modulation of impulsive decision-making within distinct areas of frontal cortex."
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Inhibitory responses to the medial superior olive from the lateral nuclei of the trapezoid body
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The medial superior olive (MSO) senses microsecond differences in the coincidence of binaural signals, a critical cue for detecting sound location along the azimuth. An important component of this circuit is provided by inhibitory neurons of the medial and lateral nuclei of the trapezoid body (MNTB and LNTB, respectively). While MNTB neurons are fairly well described, little is known about the physiology of LNTB neurons. Using whole cell recordings from gerbil brainstem slices, we found that LNTB and MNTB neurons have similar membrane time constants and input resistances and fire brief action potentials, but only LNTB neurons fire repetitively in response to current steps. We observed that LNTB neurons receive graded excitatory and inhibitory synaptic inputs, with at least some of the latter arriving from other LNTB neurons. To address the relative timing of inhibition to the MSO from the LNTB versus the MNTB, we examined inhibitory responses to auditory nerve stimulation using a slice preparation that retains the circuitry from the auditory nerve to the MSO intact. Despite the longer physical path length of excitatory inputs driving contralateral inhibition, inhibition from both pathways arrived with similar latency and jitter. An analysis of paired whole cell recordings between MSO and MNTB neurons revealed a short and reliable delay between the action potential peak in MNTB neurons and the onset of the resulting IPSP (0.55 ± 0.01 ms, n = 4, mean ± SEM). Reconstructions of biocytin-labeled neurons showed that MNTB axons ranged from 580 to 858 μm in length (n = 4). We conclude that while both LNTB and MNTB neurons provide similarly timed inhibition to MSO neurons, the reliability of inhibition from the LNTB at higher frequencies is more constrained relative to that from the MNTB due to differences in intrinsic properties, the strength of excitatory inputs, and the presence of feedforward inhibition.
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[
"In the auditory system, large somatic synapses convey strong excitation that supports temporally precise information transfer. The information transfer of such synapses has predominantly been investigated in the endbulbs of Held in the anterior ventral cochlear nucleus and the calyx of Held in the medial nucleus of the trapezoid body. These large synapses either work as relays or integrate over a small number of inputs to excite the postsynaptic neuron beyond action potential (AP) threshold. In the monaural system, another large somatic synapse targets neurons in the ventral nucleus of the lateral lemniscus (VNLL). Here, we comparatively analyze the mechanisms of synaptic information transfer in endbulbs in the VNLL and the calyx of Held in juvenile Mongolian gerbils. We find that endbulbs in the VNLL are functionally surface-scaled versions of the calyx of Held with respect to vesicle availability, release efficacy, and synaptic peak currents. This functional scaling is achieved by different calcium current kinetics that compensate for the smaller AP in VNLL endbulbs. However, the average postsynaptic current in the VNLL fails to elicit APs in its target neurons, even though equal current suffices to generate APs in neurons postsynaptic to the calyx of Held. In the VNLL, a postsynaptic A-type outward current reduces excitability and prevents AP generation upon a single presynaptic input. Instead, coincidence detection of inputs from two converging endbulbs is ideal to reliably trigger APs. Thus, even large endbulbs do not guarantee one-to-one AP transfer. Instead, information flow appears regulated by circuit requirements.",
"K+ channels shape individual action potentials and determine their pattern of firing. In auditory relays, both high- and low-voltage-activated K+ channels (HVA and LVA) are critical for preservation of auditory timing cues. We examined how these channels participate in firing in the medial nucleus of the trapezoid body. Principal cells at physiological temperature were voltage clamped using spike waveforms previously recorded in response to calyceal firing. Current components were isolated by digital subtraction of traces recorded in the channel antagonists dendrotoxin-I or tetraethylammonium. During orthodromic spikes delivered at 300 and 600 Hz, both currents activated with a slight delay, peaking just after the crest of the spike. The decay of HVA was sufficiently fast to match the time course of the spike. By contrast, with 300-Hz stimuli, LVA continued to decay after the spikes reached a stable interspike potential. Although LVA currents partially inactivate during prolonged voltage steps, their peak amplitudes remained stable or increased during trains of spikelike stimuli. At 600 Hz, LVA did not fully deactivate between the spikes and therefore generated a leak current. To determine the effect of blocking LVA channels on spiking, prerecorded postsynaptic conductances were injected, with and without dendrotoxin-I. After block of LVA channels, strong synaptic conductances produced broader spikes, greater spike jitter, and prolonged depolarized states. HVA blockade with tetraethylammonium also broadened spikes but led to less error in timing. These results reveal multiple roles for LVA channels in spike repolarization and timing during synaptic activity.",
"Information processing in the brain relies on precise timing of signal propagation. The highly conserved neuronal network for computing spatial representations of acoustic signals resolves microsecond timing of sounds processed by the two ears. As such, it provides an excellent model for understanding how precise temporal regulation of neuronal signals is achieved and maintained. The well described avian and mammalian brainstem circuit for computation of interaural time differences is composed of monaural cells in the cochlear nucleus (CN; nucleus magnocellularis in birds) projecting to binaurally innervated coincidence detection neurons in the medial superior olivary nucleus (MSO) in mammals or nucleus laminaris (NL) in birds. Individual axons from CN neurons issue a single axon that bifurcates into an ipsilateral branch and a contralateral branch that innervate segregated dendritic regions of the MSO/NL coincidence detector neurons. We measured conduction velocities of the ipsilateral and contralateral branches of these bifurcating axon collaterals in the chicken by antidromic stimulation of two sites along each branch and whole-cell recordings in the parent neurons. At the end of each experiment, the individual CN neuron and its axon collaterals were filled with dye. We show that the two collaterals of a single axon adjust the conduction velocities individually to achieve the specific conduction velocities essential for precise temporal integration of information from the two ears, as required for sound localization. More generally, these results suggest that individual axonal segments in the CNS interact locally with surrounding neural structures to determine conduction velocity.",
"The onset of responsiveness to eighth nerve stimulation was examined in n. magnocellularis and n. laminaris, (second- and third-order neurons) of the chick brainstem auditory system. Extracellular microelectrode mapping techniques were used to examine postsynaptic responses in in vitro brainstem preparations. Two specific questions were addressed. First, what is the earliest time at which postsynaptic action potentials can be evoked in n. magnocellularis and n. laminaris by eighth nerve stimulation? Second, does responsiveness to eighth nerve stimulation develop along a spatial gradient in n. magnocellularis and, if so, how does this gradient compare with other developmental events observed in the chick auditory system? Postsynaptic responses in n. magnocellularis were first recorded at 11 days of incubation. Nucleus laminaris responses to direct stimulation of n. magnocellularis were also first recorded at 11 days, although n. laminaris responses to eight nerve stimulation were not seen until 12 days of incubation. A gradient of response development within n. magnocellularis was indicated by mapping of responsive sites on days 11-13. At 11 days, responses to eighth nerve stimulation were restricted to the most anteromedial portion of n. magnocellularis. Between 11 and 13 days, cells in increasingly more posterolateral portions of n. magnocellularis became responsive. This anteromedial-to-posterolateral gradient in n. magnocellularis is correlated with the basal-to-apical gradient of morphogenesis observed in the basilar papilla and morphogenetic gradients previously observed in n. magnocellularis and n. laminaris.",
"Antisera to highly purified basic protein (BP) from rat and chicken brain were prepared and their purity and specificity demonstrated by double immunodiffusion and cross-immunoadsorption. These antisera were used for immunohistochemical localization of BP in the brains of adult and developing rat and chick. Myelin basic protein was exclusively localized to myelin or the myelin forming elements of the CNS. It was present in high concentrations in white matter and absent in areas free of myelin. Neuronal parikarya and dendrites were negative as were axons cut in cross section and at Nodes of Ranvier. The latter was best observed in cross sections of human spinal cord demonstrating also the immunoreactivity of the antibodies with human BP. The internodal distance in a fine (1.5 micrometer) rat cortical fiber was determined to be approximately 45 micrometers. Myelin basic protein was shown to extend into cranial roots, in contrast to myelin proteolipid protein which abruptly lose fluorescence as the nerves emerged from the brain. During development, BP was first observed on the fourteenth day of incubation in chick and at birth in the rat. The protein appeared in oligodendrocytes and in association with fibers near these cells. Fluorescent processes were frequently observed connecting the oligodendrocytes with the fibers. As myelination progressed, the intensity of the immunohistochemical reaction decreased in the oligodendrocytes while the brightness in fibers increase. Eventually, the oligodendrocytes became undetectable. Fibers with immature myelin exhibited a beaded or varicosed appearance with the highest concentration of immunofluorescence in the outer portion of the varicosities. The varicosities were postulated to represent dilations in the newly forming sheath between intervals of compaction along the axon undergoin myelination. These dilations might represent areas of increased cytoplasmic volume which could serve as channels for transport and/or storage sites for myelin proteins prior to incorporation into the membrane. The varicosities became less prominent with the thickening of the myelin sheath and mature myelinated fibers became smooth. The process of synthesis of BP, transport of the protein to the varicosed fibers, and maturation of the myelin sheath was seen to progress in a more or less caudal to rostral direction as myelination of the CNS takes place. In the rat, this was accomplished over approximately a 30-day period starting near the time of birth. In the chick, most of the myelination was accomplished in the three or four days immediately before hatching. At this time, innumerable oligodendrocytes were observed producing BP simultaneously in the major white fiber tracts. It is postulated that in chick some degree of oligodendrocytic cell death occurs normally during myelination.",
"It has been shown previously that impulses in axons of the descending branches of myelinated afferents in rat dorsal columns may suffer a blockade of transmission along their course in the dorsal columns. This paper tests the effect of the mechanism of primary afferent depolarization on the orthodromic movement of impulses in descending dorsal column primary afferent axons originating in the L1 dorsal root. Orthodromic impulses were recorded in the L5 and 6 dorsal columns after stimulation of the L1 dorsal root. Twenty-seven out of 82 axons (33%) suffered a temporary transmission block if primary afferent depolarization had been induced by L5 stimulation before the L1 stimulus. The tendency to block peaked at 10-15 ms and persisted for up to 30-40 ms. The number of single unit orthodromic impulses originating from the L1 root and recorded during a search of the dorsal columns 15 mm caudal to L1 increased by a factor of 3.1 after the systemic administration of bicuculline (1 mg/kg). The number of single unit orthodromic impulses originating from the L1 root and recorded in axons descending in the dorsal columns 20 mm caudal to the root increased by a factor of 8.7 after the systemic administration of picrotoxin (5 mg/kg). It is concluded that the transmission of impulses in the long range caudally running axons from dorsal roots to dorsal columns may be blocked during primary afferent depolarization and that conduction may be restored by the administration of GABA antagonists.",
"Myelin, the insulating layers of membrane wrapped around axons by oligodendrocytes, is essential for normal impulse conduction. It forms during late stages of fetal development but continues into early adult life. Myelination correlates with cognitive development and can be regulated by impulse activity through unknown molecular mechanisms. Astrocytes do not form myelin, but these nonneuronal cells can promote myelination in ways that are not understood. Here, we identify a link between myelination, astrocytes, and electrical impulse activity in axons that is mediated by the cytokine leukemia inhibitory factor (LIF). These findings show that LIF is released by astrocytes in response to ATP liberated from axons firing action potentials, and LIF promotes myelination by mature oligodendrocytes. This activity-dependent mechanism promoting myelination could regulate myelination according to functional activity or environmental experience and may offer new approaches to treating demyelinating diseases."
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Prostate cancer androgen receptor variants: etiology, characterization, and biomarker validation.
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As prostate cancer (PCa) progresses to the lethal castration resistant and metastatic form, genetic and epigenetic adaptation, clonal selection, and evolution of the tumor microenvironment contribute to the emergence of unique biological characteristics under the selective pressure of external stresses. These stresses include the therapies applied in the clinic or laboratory and the exposures of cancers to hormonal, paracrine, or autocrine stimuli in the context of the tumor micro- and macro-environment. The androgen receptor (AR) is a key gene involved in PCa etiology and oncogenesis, including disease development, progression, response to initial hormonal therapies, and subsequent resistance to hormonal therapies. Alterations in the AR signaling pathway have been observed in certain selection contexts and contribute to the resistance to agents that target hormonal regulation of the AR, including standard androgen deprivation therapy, antiandrogens such as enzalutamide, and androgen synthesis inhibition with abiraterone acetate. One such resistance mechanism is the synthesis of constitutively active AR variants lacking the canonical ligand-binding domain. This review focuses on the etiology, characterization, biological properties, and emerging data contributing to the clinical characteristics of AR variants, and suggests approaches to full-length AR and AR variant biomarker validation, assessment, and systemic targeting in the clinic.
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[
"The androgen receptor (AR) signaling pathway plays an important role during the development of the normal prostate gland, but also during the progression of prostate cancer on androgen ablation therapy. Mutations in the AR gene emerge to keep active the AR signaling pathway and to support prostate cancer cells growth and survival despite the low levels of circulating androgens. Indeed, mutations affecting the ligand binding domain (LBD) of the AR have been shown to generate so-called \"promiscuous\" receptors that present widened ligand specificity and allow the stimulation of these receptors by a larger spectrum of endogenous hormones. Another class of mutations, arising in the amino-terminal domain (NTD) of the receptor, modulate AR interactions with coregulators involved in cell proliferation regulation. Besides characteristics of these well-known types of mutations, the properties of other classes of AR mutants recently described in prostate cancer are currently under investigation. Most interestingly, in addition to their potential role in the mechanisms which allow prostate cancer cells to escape androgen ablation therapy, data suggest that certain AR mutations are present early in the natural history of the disease and may play a role in many aspects of prostate cancer progression. Surprisingly, singular truncated AR devoid of their carboxy-terminal end (CTE) region seem to exert specific paracrine effects and to induce a clonal cooperation with neighboring prostate cancer cells, which may facilitate both the invasion and metastasis processes. In this article, we review the functional properties of different classes of AR mutants and their potential impact on the natural history of prostate cancer. Hum Mutat 0, 1-14, 2008. (c) 2008 Wiley-Liss, Inc.",
"Castration-recurrent prostate cancer (CRPC) is suspected to depend on androgen receptor (AR). The AF-1 region in the amino-terminal domain (NTD) of AR contains most, if not all, of the transcriptional activity. Here we identify EPI-001, a small molecule that blocked transactivation of the NTD and was specific for inhibition of AR without attenuating transcriptional activities of related steroid receptors. EPI-001 interacted with the AF-1 region, inhibited protein-protein interactions with AR, and reduced AR interaction with androgen-response elements on target genes. Importantly, EPI-001 blocked androgen-induced proliferation and caused cytoreduction of CRPC in xenografts dependent on AR for growth and survival without causing toxicity.",
"Androgen receptor (AR) splice variants lacking the ligand binding domain (ARVs), originally isolated from prostate cancer cell lines derived from a single patient, are detected in normal and malignant human prostate tissue, with the highest levels observed in late stage, castration-resistant prostate cancer. The most studied variant (called AR-V7 or AR3) activates AR reporter genes in the absence of ligand and therefore, could play a role in castration resistance. To explore the range of potential ARVs, we screened additional human and murine prostate cancer models using conventional and next generation sequencing technologies and detected several structurally diverse AR isoforms. Some, like AR-V7/AR3, display gain of function, whereas others have dominant interfering activity. We also find that ARV expression increases acutely in response to androgen withdrawal, is suppressed by testosterone, and in some models, is coupled to full-length AR (AR-FL) mRNA production. As expected, constitutively active, ligand-independent ARVs such as AR-V7/AR3 are sufficient to confer anchorage-independent (in vitro) and castration-resistant (in vivo) growth. Surprisingly, this growth is blocked by ligand binding domain-targeted antiandrogens, such as MDV3100, or by selective siRNA silencing of AR-FL, indicating that the growth-promoting effects of ARVs are mediated through AR-FL. These data indicate that the increase in ARV expression in castrate-resistant prostate cancer is an acute response to castration rather than clonal expansion of castration or antiandrogen-resistant cells expressing gain of function ARVs, and furthermore, they provide a strategy to overcome ARV function in the clinic.",
"Biosynthesis of extragonadal androgen may contribute to the progression of castration-resistant prostate cancer. We evaluated whether abiraterone acetate, an inhibitor of androgen biosynthesis, prolongs overall survival among patients with metastatic castration-resistant prostate cancer who have received chemotherapy. We randomly assigned, in a 2:1 ratio, 1195 patients who had previously received docetaxel to receive 5 mg of prednisone twice daily with either 1000 mg of abiraterone acetate (797 patients) or placebo (398 patients). The primary end point was overall survival. The secondary end points included time to prostate-specific antigen (PSA) progression (elevation in the PSA level according to prespecified criteria), progression-free survival according to radiologic findings based on prespecified criteria, and the PSA response rate. After a median follow-up of 12.8 months, overall survival was longer in the abiraterone acetate-prednisone group than in the placebo-prednisone group (14.8 months vs. 10.9 months; hazard ratio, 0.65; 95% confidence interval, 0.54 to 0.77; P<0.001). Data were unblinded at the interim analysis, since these results exceeded the preplanned criteria for study termination. All secondary end points, including time to PSA progression (10.2 vs. 6.6 months; P<0.001), progression-free survival (5.6 months vs. 3.6 months; P<0.001), and PSA response rate (29% vs. 6%, P<0.001), favored the treatment group. Mineralocorticoid-related adverse events, including fluid retention, hypertension, and hypokalemia, were more frequently reported in the abiraterone acetate-prednisone group than in the placebo-prednisone group. The inhibition of androgen biosynthesis by abiraterone acetate prolonged overall survival among patients with metastatic castration-resistant prostate cancer who previously received chemotherapy. (Funded by Cougar Biotechnology; COU-AA-301 ClinicalTrials.gov number, NCT00638690.).",
"Progression of prostate cancer following castration is associated with increased androgen receptor (AR) expression and signaling despite AR blockade. Recent studies suggest that these activities are due to the generation of constitutively active AR splice variants, but the mechanisms by which these splice variants could mediate such effects are not fully understood. Here we have identified what we believe to be a novel human AR splice variant in which exons 5, 6, and 7 are deleted (ARv567es) and demonstrated that this variant can contribute to cancer progression in human prostate cancer xenograft models in mice following castration. We determined that, in human prostate cancer cell lines, ARv567es functioned as a constitutively active receptor, increased expression of full-length AR (ARfl), and enhanced the transcriptional activity of AR. In human xenografts, human prostate cancer cells transfected with ARv567es cDNA formed tumors that were resistant to castration. Furthermore, the ratio of ARv567es to ARfl expression within the xenografts positively correlated with resistance to castration. Importantly, we also detected ARv567es frequently in human prostate cancer metastases. In summary, these data indicate that constitutively active AR splice variants can contribute to the development of castration-resistant prostate cancers and may serve as biomarkers for patients who are likely to suffer from early recurrence and are candidates for therapies directly targeting the AR rather than ligand.",
"Constitutively active androgen receptor variants (AR-V) lacking the ligand binding domain (LBD) may promote the development of castration-resistant prostate cancer (CRPC). The expression of AR-Vs in the clinically most important metastatic site, the bone, has, however, not been well documented. Our aim was therefore to compare levels of AR-Vs in hormone-naive (HN) and CRPC bone metastases in comparison to primary PC and non-malignant prostate tissue, as well as in relation to AR protein expression, whole-genome transcription profiles and patient survival. Hormone-naïve (n = 10) and CRPC bone metastases samples (n = 30) were obtained from 40 patients at metastasis surgery. Non-malignant and malignant prostate samples were acquired from 13 prostatectomized men. Levels of full length AR (ARfl) and AR-Vs termed AR-V1, AR-V7, and AR-V567es mRNA were measured with RT-PCR and whole-genome transcription profiles with an Illumina Beadchip array. Protein levels were examined by Western blotting and immunohistochemistry. Transcripts for ARfl, AR-V1, and AR-V7 were detected in most primary tumors and metastases, and levels were significantly increased in CRPC bone metastases. The AR-V567es transcript was detected in 23% of the CRPC bone metastases only. A sub-group of CRPC bone metastases expressed LBD-truncated AR proteins at levels comparable to the ARfl. Detectable AR-V567es and/or AR-V7 mRNA in the upper quartile, seen in 1/3 of all CRPC bone metastases, was associated with a high nuclear AR immunostaining score, disturbed cell cycle regulation and short survival. Expression of AR-Vs is increased in CRPC compared to HN bone metastases and associated with a particularly poor prognosis. Further studies are needed to test if patients expressing such AR-Vs in their bone metastases benefit more from drugs acting on or down-stream of these AR-Vs than from therapies inhibiting androgen synthesis.",
"Mutations in the NH(2)-terminal regulatory domain of the beta-catenin gene lead to aberrant stabilization and accumulation of the protein and increased TCF/LEF-dependent transcription. Although these mutations are common in some cancers, they are infrequent in prostate and breast cancer. We have found that metastatic prostate cancer specimens, obtained through a rapid autopsy tissue procurement program, expressed a novel M(r) 75,000 proteolytic fragment of beta-catenin (beta-cat(75)). beta-Cat(75) was also expressed in multiple prostate and breast cancer cell lines and was closely associated with the activity of the calcium-dependent protease, calpain. In a prostate cancer cDNA microarray, m-calpain RNA levels were found to be significantly increased in metastatic disease compared with normal prostate. We showed calpain-dependent generation of beta-cat(75) in cell culture and in vitro. Molecular mapping revealed that calpain cleavage removed the NH(2)-terminal regulatory domain of the beta-catenin protein. Treatment of MCF-7 cells with ionomycin led to increased accumulation of beta-cat(75) in the nucleus and TCF-dependent transcriptional activity. Overexpression of a similar beta-catenin fragment that lacks the NH(2)-terminal 132 amino acids and has transforming potential activated TCF-dependent transcription. Given the low frequency of mutation-induced activation of beta-catenin in prostate and breast cancers, proteolytic cleavage of beta-catenin by calpain may represent a novel mechanism by which the protein is activated during tumorigenesis."
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Ionic conductances in the paraventricular thalamic nucleus
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Neurons in the midline and intralaminar thalamic nuclei are components of an interconnected brainstem, limbic and prefrontal cortex neural network that is engaged during arousal, vigilance, motivated and addictive behaviors, and stress. To better understand the cellular mechanisms underlying these functions, here we review some of the recently characterized electrophysiological and neuropharmacological properties of neurons in the paraventricular thalamic nucleus (PVT), derived from whole cell patch clamp recordings in acute rat brain slice preparations. PVT neurons display firing patterns and ionic conductances (IT and IH) that exhibit significant diurnal change. Their resting membrane potential (RMP) is maintained by various ionic conductances that include inward rectifier (Kir), hyperpolarization-activated nonselective cation (HCN) and TWIK-related acid sensitive (TASK) K(+) channels. Firing patterns are regulated by high voltage-activated (HVA) and low voltage-activated (LVA) Ca(2+) conductances. Moreover, transient receptor potential (TRP)-like nonselective cation channels together with Ca(2+)- and Na(+)-activated K(+) conductances (KCa; KNa) contribute to unique slow afterhyperpolarizing potentials (sAHPs) that are generally not detectable in lateral thalamic or reticular thalamic nucleus neurons. The excitability of PVT neurons is also modulated by activation of neurotransmitter receptors associated with afferent pathways to PVT and other thalamic midline nuclei. We report on receptor-mediated actions of GABA, glutamate, monoamines and several neuropeptides: arginine vasopressin, gastrin-releasing peptide, thyrotropin releasing hormone and the orexins (hypocretins). This review represents an initial survey of intrinsic and transmitter-sensitive ionic conductances that are deemed to be unique to this population of midline thalamic neurons, information that is fundamental to an appreciation of the role these thalamic neurons may play in normal central nervous system (CNS) physiology and in CNS disorders that involve the dorsomedial thalamus.
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[
"Marked species differences in the distribution of central tachykinin receptors are reported but uncertainty remains about the ability of available ligands to detect NK2 and NK3 receptors in human brain. We compared the distribution of NK1, NK2, and NK3 receptors in sections from rodent, primate, and human brain using the 125I-labeled ligands substance P (SP) for the NK1 receptor, neurokinin A (NKA) for the NK2 receptor, and neurokinin B (NKB) and eledoisin for NK3 receptors. Duration of exposure to autoradiographic film was from 7 days for [125I]SP up to 90 days for the other ligands. High levels of specific [125I]SP binding were seen throughout the brains of all species studied. Specific [125I]NKA binding was detected in brains from neonatal rat, and to a lesser level in adult rat, gerbil, and guinea pig; it was not detected in monkey or human brain, but was present in circular muscle of human duodenum, confirming that this ligand binds to human NK2 receptors under our experimental conditions. Specific [125I]NKB and [125I]eledoisin binding was widespread in brain sections from rats, gerbils, and guinea pigs, and very low levels were also detected in marmoset, squirrel monkey, and rhesus monkey brain after prolonged (up to 90 days) exposure. We failed to identify specific eledoisin binding in human brain, even after prolonged exposures. These findings demonstrate that the NK1 receptor is the predominant tachykinin receptor expressed in primate and human brain, but that low levels of NK3 receptor are present in nonhuman, primate brain.",
"Neural circuits for essential natural behaviors are shaped by selective pressure to coordinate reliable execution of flexible goal-directed actions. However, the structural and functional organization of survival-oriented circuits is poorly understood due to exceptionally complex neuroanatomy. This is exemplified by AGRP neurons, which are a molecularly defined population that is sufficient to rapidly coordinate voracious food seeking and consumption behaviors. Here, we use cell-type-specific techniques for neural circuit manipulation and projection-specific anatomical analysis to examine the organization of this critical homeostatic circuit that regulates feeding. We show that AGRP neuronal circuits use a segregated, parallel, and redundant output configuration. AGRP neuron axon projections that target different brain regions originate from distinct subpopulations, several of which are sufficient to independently evoke feeding. The concerted anatomical and functional analysis of AGRP neuron projection populations reveals a constellation of core forebrain nodes, which are part of an extended circuit that mediates feeding behavior.",
"Some GABA(A) receptors (GABA(A)Rs) are activated by low transmitter levels present in the extracellular space and generate an uninterrupted conductance referred to as \"tonic.\" This tonic conductance is highly sensitive to all factors regulating the amount of GABA surrounding the neurons. Only a few GABA(A)Rs with particular subunit combinations are well suited to mediate the tonic conductance. These same receptors constitute important and specific targets for various endogenous and exogenous neuroactive compounds and possible therapeutic targets.",
"The orexins (also known as hypocretins) are peptide neurotransmitters made by hypothalamic neurons that are thought to play an important role in regulating wake-sleep states. One terminal area for orexin neurons is the tuberomammillary nucleus, a histaminergic cell group that is wake-active, but the relationship of the orexinergic terminals to the tuberomammillary neurons has not been examined in detail. We studied the ultrastructure of orexin A-immunoreactive axons and terminals in the tuberomammillary nucleus using pre- and post-embedding electron microscopic protocols. We confirmed an abundant projection of orexin-immunoreactive boutons to both dorsal and ventral divisions of the tuberomammillary nucleus. These terminals made asymmetric synaptic contacts with proximal and intermediate dendrites of tuberomammillary neurons. They contained small, clear synaptic vesicles and up to 30-40 dense core vesicles were seen per terminal in a single section. Both pre- and post-embedding immunostaining revealed that orexin immunoreactivity was localized to the dense core vesicles, which were always at a distance from the synaptic specialization. We also found glutamate immunoreactivity in the small synaptic vesicles which were at the active zone of the synapses of many of the same terminals. Orexinergic afferents to the tuberomammillary neurons contain separate populations of orexinergic and glutamatergic vesicles, suggesting that the release of these neurotransmitters may be differentially regulated.",
"As we commemorate the 25th anniversary of the journal Peptides, it is timely to review the functional significance of the bombesin (BB)-like peptides and receptors in the CNS. Over two decades ago we published an article in the journal Peptides demonstrating that BB-like peptides are present in high densities in certain rat brain regions (such as the paraventricular nucleus of the hypothalamus). Subsequently, one of the mammalian forms of BB, gastrin-releasing peptide (GRP) containing cell bodies were found in the suprachiasmatic nucleus of the hypothalamus and nucleus of the solitary tract of the hindbrain. Another related peptide, namely neuromedin (NM)B, was detected in the olfactory bulb and dentate gyrus. BB and GRP bind with high affinity to BB(2) receptors, whereas NMB binds with high affinity to BB(1) receptors. The actions of BB or GRP are blocked by BB(2) receptor antagonists such as (Psi(13,14)-Leu(14))BB whereas PD168368 is a BB(1) receptor antagonist. Exogenous administration of BB into the rat brain causes hypothermia, hyperglycemia, grooming and satiety. BB-like peptides activate the sympathetic nervous system and appear to modulate stress, fear and anxiety responses. GRP and NMB modulate distinct biological processes through discrete brain regions or circuits, and globally these peptidergic systems may serve in an integrative or homeostatic function.",
"Neuropeptide S (NPS) and its receptor (NPSR) constitute a novel neuropeptide system that is involved in regulating arousal and anxiety. The NPS precursor mRNA is highly expressed in a previously undescribed group of neurons located between the locus coeruleus (LC) and Barrington's nucleus. We report here that the majority of NPS-expressing neurons in the LC area and the principal sensory trigeminal nucleus are glutamatergic neurons, whereas many NPS-positive neurons in the lateral parabrachial nucleus coexpress corticotropin-releasing factor (CRF). In addition, we describe a comprehensive map of NPSR mRNA expression in the rat brain. High levels of expression are found in areas involved in olfactory processing, including the anterior olfactory nucleus, the endopiriform nucleus, and the piriform cortex. NPSR mRNA is expressed in several regions mediating anxiety responses, including the amygdaloid complex and the paraventricular hypothalamic nucleus. NPSR mRNA is also found in multiple key regions of sleep neurocircuitries, such as the thalamus, the hypothalamus, and the preoptic region. In addition, NPSR mRNA is strongly expressed in major output and input regions of hippocampus, including the parahippocampal regions, the lateral entorhinal cortex, and the retrosplenial agranular cortex. Multiple hypothalamic nuclei, including the dorsomedial and the ventromedial hypothalamic nucleus and the posterior arcuate nucleus, express high levels of NPSR mRNA, indicating that NPS may regulate energy homeostasis. These data suggest that the NPS system may play a key role in modulating a variety of physiological functions, especially arousal, anxiety, learning and memory, and energy balance.",
"Well-characterized antisera to porcine beta-endorphin were used to localize immunoreactive sites in cryostat sections of formaldehyde-fixed rat brain by indirect immunohistochemistry. Specificity was established by absorption of immune sera with synthetic peptide fragments. Specific immunoreactivity was localized to neuronal perikarya in the basal tuberal hypothalamus, and to varicose nerve fibers which were distributed to midline nuclear areas throughout the diencephalon and anterior pons. These patterns of reactivity were unaffected by preabsorption of the immune sera with millimolar concentrations of Met5- or Leu5-enkephalin or alpha-endorphin. The beta-endorphin immunoreactive structures were morphologically separate from those cells and fibers reported to react with antisera to the enkephalins. One anti-beta-endorphin serum gave additional immunoreactivity with myelinated axons in limbic cortical zones; when absorbed with purified rat myelin basic protein, only the specific patterns of immunoreactivity remained. Thus, discrete beta-endorphin-containing neuronal circuits exist in rat brain and are anatomically distinguishable from enkephalin-containing nerve cell and fiber pathways."
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Discovery of a novel neurotensin receptor-specific nonpeptide compound
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Compounds active at neurotensin receptors (NTS1 and NTS2) exert analgesic effects on different types of nociceptive modalities, including thermal, mechanical, and chemical stimuli. The NTS2 preferring peptide JMV-431 (2) and the NTS2 selective nonpeptide compound levocabastine (6) have been shown to be effective in relieving the pain associated with peripheral neuropathies. With the aim of identifying novel nonpeptide compounds selective for NTS2, we examined analogues of SR48692 (5a) using a FLIPR calcium assay in CHO cells stably expressing rat NTS2. This led to the discovery of the NTS2 selective nonpeptide compound 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)cyclohexane carboxylic acid (NTRC-739, 7b) starting from the nonselective compound 5a.
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[
"We describe the characteristics of SR 48692, a selective, nonpeptide antagonist of the neurotensin receptor. In vitro, this compound competitively inhibits 125I-labeled neurotensin binding to the high-affinity binding site present in brain tissue from various species with IC50 values of 0.99 +/- 0.14 nM (guinea pig), 4.0 +/- 0.4 nM (rat mesencephalic cells), 7.6 +/- 0.6 nM (COS-7 cells transfected with the cloned high-affinity rat brain receptor), 13.7 +/- 0.3 nM (newborn mouse brain), 17.8 +/- 0.9 nM (newborn human brain), 8.7 +/- 0.7 nM (adult human brain), and 30.3 +/- 1.5 nM (HT-29 cells). It also displaces 125I-labeled neurotensin from the low-affinity levocabastine-sensitive binding sites but at higher concentrations (34.8 +/- 8.3 nM for adult mouse brain and 82.0 +/- 7.4 nM for adult rat brain). In guinea pig striatal slices, SR 48692 blocks K(+)-evoked release of [3H]dopamine stimulated by neurotensin with a potency (IC50 = 0.46 +/- 0.02 nM) that correlates with its binding affinity. In a cell line derived from a human colon carcinoma (HT-29), SR 48692 competitively antagonizes neurotensin-induced intracellular Ca2+ mobilization with a pA2 (-log Kapp) values of 8.13 +/- 0.03, which is consistent with results obtained in binding studies. Moreover, SR 48692 is devoid of any intrinsic agonist activity. This compound is also active in vivo, since it reverses at low dose (80 micrograms/kg) the turning behavior induced by intrastriatal injection of neurotensin in mice with similar potency whatever the route of administration (i.p. or orally) and with a long duration of action (6 hr). Thus, being a potent and selective neurotensin receptor antagonist, SR 48692 may be considered as a powerful tool for investigating the role of neurotensin in physiological and pathological processes.",
"The effect of scopolamine and atropine upon the increase in extracellular 3,4-dihydroxyphenylacetic acid induced by central injection of neurotensin was examined in the nucleus accumbens and the striatum of anaesthetized rats using in vivo differential pulse voltammetry with carbon fibre electrodes. Scopolamine (1 and 3 mg/kg, i.p.) and atropine (20 micrograms, i.c.v.) did not alter the 3,4-dihydroxyphenylacetic acid level in the nucleus accumbens or the striatum, measured for 60 min after administration. Neurotensin (10 micrograms, i.c.v.) increased the 3,4-dihydroxyphenylacetic acid peak height in both regions. Pretreatment with scopolamine (1 mg/kg) 15 min before neurotensin injection blocked the increase in extracellular 3,4-dihydroxyphenylacetic acid in the striatum but not in the nucleus accumbens whilst scopolamine (3 mg/kg) partially attenuated the effect of neurotensin in the nucleus accumbens and blocked the increase in 3,4-dihydroxyphenylacetic acid in the striatum. Atropine partially attenuated the effect produced by neurotensin in the nucleus accumbens and blocked the increase in 3,4-dihydroxyphenylacetic acid induced by the peptide in the striatum. However, the increase in extracellular 3,4-dihydroxyphenylacetic acid induced by haloperidol (1 mg/kg, s.c.) was not altered by scopolamine (1 mg/kg) or atropine. Also, the increase in dopamine metabolism in the nucleus accumbens and the striatum after centrally injected haloperidol (10 micrograms, i.c.v.) was not altered by atropine (20 micrograms, i.c.v.). Together, the results demonstrate a functional interaction between muscarinic antagonists and neurotensin on in vivo dopamine metabolism in the nucleus accumbens and the striatum but with a greater effect in the latter region.",
"The ontogenesis of neurotensin binding sites was studied in human brain of subjects deceased from Sudden Infant Death Syndrome. Monoiodo-Tyr3 neurotensin specifically recognized 2 distinct classes of binding sites in human brain homogenate. The high affinity sites were already present at birth and increased to a maximal level of 240 fmol/mg protein 1 month after birth. Thereafter, the density of these sites decreased to reach a value of 8 fmol/mg protein in 15-month-old brain, a value similar to that found in adult brain. The dissociation constant of the high-affinity sites (about 0.3 nM) did not vary from birth to adulthood. The high-affinity binding sites were sensitive to GTP which decreased their affinity for neurotensin by a factor of 3, indicating that these sites are functional receptors coupled to GTP-binding proteins. By contrast, the low-affinity sites were insensitive to GTP and could be partly blocked by the antihistaminic drug levocabastine. These sites were absent in human brain during the first post-natal year and could be detected only in brain homogenate of 15-month-old infants. The transient increase in high-affinity neurotensin binding sites after birth suggests that neurotensin could act as a regulatory peptide during brain development.",
"Neurotensin (NT) has been implicated in the central regulation of LH and PRL secretion in the rat. We investigated the importance of NT release to the neural events that trigger the preovulatory LH surge and coincident PRL surge, using as our animal model ovariectomized (OVX) rats treated with estrogen and progesterone to induce reliable and robust surges. To interfere with the action of endogenous NT in the basal forebrain, we administered a NT antiserum (NTAS) in a series of bilateral microinjections aimed at the anterior border of the medial preoptic area. One week after OVX, rats bearing cerebral guide cannulae received Silastic capsules (3 x 15 mm; sc) containing 17 beta-estradiol. Two days later, beginning at 0830 h, conscious rats were administered either NTAS or control serum bilaterally in a series of four 100-nl injections spaced at 30-min intervals. After an initial blood sample, rats received progesterone (4 mg, sc) at 1200 h; blood samples were then taken at 1-h intervals from 1400-2100 h. Blood samples were obtained from conscious, freely moving rats via a chronic atrial catheter implanted previously. Plasma levels of LH and PRL were measured by RIA, and the location of microinjection sites was verified histologically. Administration of NTAS caused a 66% reduction in the magnitude of the LH surge without altering its timing, whereas the PRL surge was unaffected. These results provide strong evidence that NT in the basal forebrain participates in the steroid-induced LH surge and suggest that NT plays a role in the preovulatory LH surge."
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GTPases of the Rho, Ras, Rab and Arf families regulate intestinal epithelial homeostasis
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The intestinal epithelium forms a stable barrier protecting underlying tissues from pathogens in the gut lumen. This is achieved by specialized integral membrane structures such as tight and adherens junctions that connect neighboring cells and provide stabilizing links to the cytoskeleton. Junctions are constantly remodeled to respond to extracellular stimuli. Assembly and disassembly of junctions is regulated by interplay of actin remodeling, endocytotic recycling of junctional proteins, and various signaling pathways. Accumulating evidence implicate small G proteins of the Ras superfamily as important signaling molecules for the regulation of epithelial junctions. They function as molecular switches circling between an inactive GDP-bound and an active GTP-bound state. Once activated, they bind different effector molecules to control cellular processes required for correct junction assembly, maintenance and remodelling. Here, we review recent advances in understanding how GTPases of the Rho, Ras, Rab and Arf families contribute to intestinal epithelial homeostasis.
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"Intestinal barrier dysfunction and translocation of endotoxins are involved in the pathogenesis of alcoholic liver disease. Exposure to ethanol and its metabolite, acetaldehyde at relatively high concentrations have been shown to disrupt intestinal epithelial tight junctions in the conventional two dimensional cell culture models. The present study investigated quantitatively and qualitatively the effects of ethanol at concentrations detected in the blood after moderate ethanol consumption, of its metabolite acetaldehyde and of the combination of both compounds on intestinal barrier function in a three-dimensional cell culture model. Caco-2 cells were grown in a basement membrane matrix (Matrigel™) to induce spheroid formation and were then exposed to the compounds at the basolateral side. Morphological differentiation of the spheroids was assessed by immunocytochemistry and transmission electron microscopy. The barrier function was assessed by the flux of FITC-labeled dextran from the basal side into the spheroids' luminal compartment using confocal microscopy. Caco-2 cells grown on Matrigel assembled into fully differentiated and polarized spheroids with a central lumen, closely resembling enterocytes in vivo and provide an excellent model to study epithelial barrier functionality. Exposure to ethanol (10-40 mM) or acetaldehyde (25-200 µM) for 3 h, dose-dependently and additively increased the paracellular permeability and induced redistribution of ZO-1 and occludin without affecting cell viability or tight junction-encoding gene expression. Furthermore, ethanol and acetaldehyde induced lysine residue and microtubules hyperacetylation. These results indicate that ethanol at concentrations found in the blood after moderate drinking and acetaldehyde, alone and in combination, can increase the intestinal epithelial permeability. The data also point to the involvement of protein hyperacetylation in ethanol- and acetaldehyde-induced loss of tight junctions integrity.",
"The role of guanine nucleotides in ras p21 function was determined by using the ability of p21 protein to induce maturation of Xenopus oocytes as a quantitative assay for biological activity. Two oncogenic mutant human N-ras p21 proteins, Asp12 and Val12, actively induced maturation, whereas normal Gly12 p21 was relatively inactive in this assay. Both mutant proteins were found to be associated with guanosine triphosphate (GTP) in vivo. In contrast, Gly12 p21 was predominantly guanosine diphosphate (GDP)-bound because of a dramatic stimulation of Gly12 p21-associated guanosine triphosphatase (GTPase) activity. A cytoplasmic protein was shown to be responsible for this increase in activity. This protein stimulated GTP hydrolysis by purified Gly12 p21 more than 200-fold in vitro, but had no effect on Asp12 or Val12 mutants. A similar factor could be detected in extracts from mammalian cells. It thus appears that, in Xenopus oocytes, this protein maintains normal p21 in a biologically inactive, GDP-bound state through its effect on GTPase activity. Furthermore, it appears that the major effect of position 12 mutations is to prevent this protein from stimulating p21 GTPase activity, thereby allowing these mutants to remain in the active GTP-bound state.",
"Maintenance of intestinal epithelial barrier functions is crucial to prevent systemic contamination by microbes that penetrate from the gut lumen. GTPases of the Rho-family such as RhoA, Rac1, and Cdc42 are known to be critically involved in the regulation of intestinal epithelial barrier functions. However, it is still unclear whether inactivation or activation of these GTPases exerts barrier protection or not. We tested the effects of Rho GTPase activities on intestinal epithelial barrier functions by using the bacterial toxins cytotoxic necrotizing factor 1 (CNF-1), toxin B, C3 transferase (C3 TF), and lethal toxin (LT) in an in vitro model of the intestinal epithelial barrier. Incubation of cell monolayers with CNF-1 for 3 h induced exclusive activation of RhoA whereas Rac1 and Cdc42 activities were unchanged. As revealed by FITC-dextran flux and measurements of transepithelial electrical resistance (TER) intestinal epithelial permeability was significantly increased under these conditions. Inhibition of Rho kinase via Y27632 blocked barrier destabilization of CNF-1 after 3 h. In contrast, after 24 h of incubation with CNF-1 only Rac1 and Cdc42 but not RhoA were activated which resulted in intestinal epithelial barrier stabilization. Toxin B to inactivate RhoA, Rac1, and Cdc42 as well as Rac1 inhibitor LT increased intestinal epithelial permeability. Similar effects were observed after inhibition of RhoA/Rho kinase signaling by C3 TF or Y27632. Taken together, these data demonstrate that both activation and inactivation of RhoA signaling increased paracellular permeability whereas activation of Rac1 and Cdc42 correlated with stabilized barrier functions.",
"The zebrafish genomic sequence database was analyzed for the presence of genes encoding members of the Rho small GTPases. The analysis shows the presence of 32 zebrafish Rho genes representing one or more homologs of the human RHOA, RND3, RHOF, RHOG, RHOH, RHOJ, RHOU, RHOV, CDC42, RAC1, RAC2, RAC3, RND1, RHOBTB1, RHOBTB2, RHOBTB3, and RHOT1 genes. By expression analysis using reverse transcriptase-PCR we show that at least 20 of the predicted zebrafish small GTPase genes are expressed in the adult stage. Interestingly, only 5 of these were found to be expressed at early embryonic stages, including rhoab, rhoad, cdc42a, cdc42c, and rac1a. We observed a strong upregulation of zebrafish rhogb expression after Mycobacterium marinum infection of adult fish. This complete annotation study provides a firm basis for the use of zebrafish as a model for analysis of Rho GTPase function in vertebrate development and the innate immune system.",
"Enteropathogenic Escherichia coli delivers a subset of effectors into host cells via a type III secretion system, and this step is required for the progression of disease. Here, we show that the type III effectors, EspG and its homolog Orf3, trigger actin stress fiber formation and the destruction of the microtubule networks beneath adherent bacteria. Both effectors were shown to possess the ability to interact with tubulins, and to stimulate microtubule destabilization in vitro. A recent study showed that microtubule-bound GEF-H1, a RhoA-specific guanine nucleotide exchange factor, was converted to its active form by microtubule destabilization, and this sequence of events resulted in RhoA stimulation. Indeed, EspG- and Orf3-induced stress fiber formation was inhibited by the expression of dominant-negative forms of GEF-H1 and RhoA, but not of Rac1 and Cdc42, and by treatment with a ROCK inhibitor. These results indicate that the impact of EspG/Orf3 on microtubule networks triggers the activation of the RhoA-ROCK signaling pathway via GEF-H1 activity. This report reveals for the first time that a pathogen can exploit the host factor GEF-H1.",
"GTPases of the Rho family are molecular switches that play important roles in converting and amplifying external signals into cellular effects. Originally demonstrated to control the dynamics of the F-actin cytoskeleton, Rho GTPases have been implicated in many basic cellular processes that influence cell proliferation, differentiation, motility, adhesion, survival, or secretion. To elucidate the evolutionary history of the Rho family, we have analyzed over 20 species covering major eukaryotic clades from unicellular organisms to mammals, including platypus and opossum, and have reconstructed the ontogeny and the chronology of emergence of the different subfamilies. Our data establish that the 20 mammalian Rho members are structured into 8 subfamilies, among which Rac is the founder of the whole family. Rho, Cdc42, RhoUV, and RhoBTB subfamilies appeared before Coelomates and RhoJQ, Cdc42 isoforms, RhoDF, and Rnd emerged in chordates. In vertebrates, gene duplications and retrotranspositions increased the size of each chordate Rho subfamily, whereas RhoH, the last subfamily, arose probably by horizontal gene transfer. Rac1b, a Rac1 isoform generated by alternative splicing, emerged in amniotes, and RhoD, only in therians. Analysis of Rho mRNA expression patterns in mouse tissues shows that recent subfamilies have tissue-specific and low-level expression that supports their implication only in narrow time windows or in differentiated metabolic functions. These findings give a comprehensive view of the evolutionary canvas of the Rho family and provide guides for future structure and evolution studies of other components of Rho signaling pathways, in particular regulators of the RhoGEF family.",
"RhoG is a low-molecular-weight GTPase highly expressed in lymphocytes that activates gene transcription and promotes cytoskeletal reorganization in vitro. To study the in vivo function of RhoG, we generated mice homozygous for a targeted disruption of the RhoG gene. Despite the absence of RhoG, the development of B and T lymphocytes was unaffected. However, there was an increase in the level of serum immunoglobulin G1 (IgG1) and IgG2b as well as a mild increase of the humoral immune response to thymus-dependent antigens. In addition, B- and T-cell proliferation in response to antigen receptor cross-linking was slightly increased. Although RhoG deficiency produces a mild phenotype, our experiments suggest that RhoG may contribute to the negative regulation of immune responses. The lack of a strong phenotype could indicate a functional redundancy of RhoG with other Rac proteins in lymphocytes.",
"Chemokines are critical regulatory factors that direct migration, proliferation and maturation of receptor expressing target cells within gut mucosa. The aim of the present study was to define the cellular mechanisms whereby engagement of the essential chemokine CXCL12 to CXCR4 regulates restitutive epithelial cell migration. Non-transformed IEC-6 cells or polarized T84 epithelial monolayers were wounded and F-actin accumulation assessed using fluorescence microscopy and flow cytometry. Immunoblot analysis, pull-down assays, fluorescence microscopy and wound healing assays defined activation of Rho, Rho-kinase (ROCK), and myosin light chain (MLC) and the role for those Rho effectors in CXCL12-regulated epithelial restitution. CXCL12 increased RhoGTP and F-actin localization to the leading edge of wounded IEC-6 and T84 monolayers. CXCL12 congruently stimulated an increase in active MLC that was inhibited by blockade of ROCK and myosin light chain kinase and regulated epithelial migration. Our data in model intestinal epithelia suggest CXCR4 and CXCL12 may function as an autocrine and paracrine mucosal signaling network regulating the competency of the epithelial barrier to withstand injury and mediate repair following damage.",
"Epithelial cells form organized sheets to protect underlying tissues and maintain the physiological environment by the assembly of tight junctions (TJs) and adherens junctions (AJs), which mainly regulate paracellular molecular passage and selective cell-cell adhesion, respectively. At the cytoplasmic surface, TJs and AJs associate with a specific actomyosin cytoskeletal structure called the perijunctional actomyosin ring (PJAR), which encircles cells in a belt-like manner. ZO family proteins play important roles in regulating TJ and PJAR organization. We recently found that ARHGEF11, a member of the RGS-RhoGEF family of proteins, associates with TJs by binding to ZO-1. ARHGEF11 mediates ZO-1-dependent junction assembly and barrier formation in mammary epithelial cells. Another recent study demonstrated that ARHGEF11-dependent apical actomyosin contraction is coupled to planar cell polarity signaling in neuroepithelial cells for the control of neural tube formation. These findings suggest that ARHGEF11 generally regulates apical junctions and junction-associated actomyosin in various epithelial tissues.",
"Tight control of cell-cell communication is essential for the generation of a normally patterned embryo. A critical mediator of key cell-cell signaling events during embryogenesis is the highly conserved Wnt family of secreted proteins. Recent biochemical and genetic analyses have greatly enriched our understanding of how Wnts signal, and the list of canonical Wnt signaling components has exploded. The data reveal that multiple extracellular, cytoplasmic, and nuclear regulators intricately modulate Wnt signaling levels. In addition, receptor-ligand specificity and feedback loops help to determine Wnt signaling outputs. Wnts are required for adult tissue maintenance, and perturbations in Wnt signaling promote both human degenerative diseases and cancer. The next few years are likely to see novel therapeutic reagents aimed at controlling Wnt signaling in order to alleviate these conditions."
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Antibiotics for multi-drug-resistant bacterial pathogens
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Bacterial infections are raising serious concern across the globe. The effectiveness of conventional antibiotics is decreasing due to global emergence of multi-drug-resistant (MDR) bacterial pathogens. This process seems to be primarily caused by an indiscriminate and inappropriate use of antibiotics in non-infected patients and in the food industry. New classes of antibiotics with different actions against MDR pathogens need to be developed urgently. In this context, this review focuses on several ways and future directions to search for the next generation of safe and effective antibiotics compounds including antimicrobial peptides, phage therapy, phytochemicals, metalloantibiotics, lipopolysaccharide, and efflux pump inhibitors to control the infections caused by MDR pathogens.
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"An antimicrobial peptide database (APD) has been established based on an extensive literature search. It contains detailed information for 525 peptides (498 antibacterial, 155 antifungal, 28 antiviral and 18 antitumor). APD provides interactive interfaces for peptide query, prediction and design. It also provides statistical data for a select group of or all the peptides in the database. Peptide information can be searched using keywords such as peptide name, ID, length, net charge, hydrophobic percentage, key residue, unique sequence motif, structure and activity. APD is a useful tool for studying the structure-function relationship of antimicrobial peptides. The database can be accessed via a web-based browser at the URL: http://aps.unmc.edu/AP/main.html.",
"Gene-encoded peptide antibiotics are ubiquitous components of host defenses in mammals, birds, amphibia, insects, and plants. Their de novo synthesis or release from storage sites can be induced rapidly, which makes them particularly important in the initial phases of resistance to microbial invasion. The endogenous antimicrobial peptides of animals are products of single genes and are synthesized as preproproteins. Multistep processing yields the mature peptide, which generally acts by inducing microbial membrane permeabilization. Several families of antimicrobial peptides have been identified that differ with respect to the presence of disulfide linkages, amino acid composition, structural conformation, and spectrum of activity. The arginine-rich three disulfide-containing beta-sheet defensins are remarkably abundant and widely distributed in animals and plants. The antibiotic peptides of higher eukaryotes merit further study for their role in natural immunity and their potential as novel therapeutic compounds.",
"The bacterial RNA polymerase (RNAP) is an essential enzyme that is responsible for making RNA from a DNA template and is targeted by several antibiotics. Rifampicin was the first of such antibiotics to be described and is one of the most efficient anti-tuberculosis drugs in use. In the past five years, structural studies of bacterial RNAP and the resolution of several complexes of drugs bound to RNAP subunits have revealed molecular details of the drug-binding sites and the mechanism of drug action. This knowledge opens avenues for the development of antibiotics. Here these drugs are reviewed, together with their mechanisms and their potential interest for therapeutic applications.",
"Inhibition of bacterial RNA polymerase (RNAP) is an established strategy for antituberculosis therapy and broad-spectrum antibacterial therapy. Crystal structures of RNAP-inhibitor complexes are available for four classes of antibiotics: rifamycins, sorangicin, streptolydigin, and myxopyronin. The structures define three different targets, and three different mechanisms, for inhibition of bacterial RNAP: (1) rifamycins and sorangicin bind near the RNAP active center and block extension of RNA products; (2) streptolydigin interacts with a target that overlaps the RNAP active center and inhibits conformational cycling of the RNAP active center; and (3) myxopyronin interacts with a target remote from the RNAP active center and functions by interfering with opening of the RNAP active-center cleft to permit entry and unwinding of DNA and/or by interfering with interactions between RNAP and the DNA template strand. The structures enable construction of homology models of pathogen RNAP-antibiotic complexes, enable in silico screening for new antibacterial agents, and enable rational design of improved antibacterial agents.",
"Antimicrobial peptides are an abundant and diverse group of molecules that are produced by many tissues and cell types in a variety of invertebrate, plant and animal species. Their amino acid composition, amphipathicity, cationic charge and size allow them to attach to and insert into membrane bilayers to form pores by 'barrel-stave', 'carpet' or 'toroidal-pore' mechanisms. Although these models are helpful for defining mechanisms of antimicrobial peptide activity, their relevance to how peptides damage and kill microorganisms still need to be clarified. Recently, there has been speculation that transmembrane pore formation is not the only mechanism of microbial killing. In fact several observations suggest that translocated peptides can alter cytoplasmic membrane septum formation, inhibit cell-wall synthesis, inhibit nucleic-acid synthesis, inhibit protein synthesis or inhibit enzymatic activity. In this review the different models of antimicrobial-peptide-induced pore formation and cell killing are presented.",
"Rifampicin is an antibiotic that inhibits the function of RNA polymerase in eubacteria. Mutations affecting the beta subunit of RNA polymerase can confer resistance to rifampicin. A large number of rifampicin-resistant (hereafter called Rifr) mutants have been isolated in Escherichia coli to probe the involvement of RNA polymerase in a variety of physiological processes. We have undertaken a comprehensive analysis of Rifr mutations to identify their structural and functional effects on RNA polymerase. Forty-two Rifr isolates with a variety of phenotypes were mapped to defined intervals within the rpoB gene using a set of deletions of the rpoB gene. The mutations were sequenced. Seventeen mutational alterations affecting 14 amino acid residues were identified. These alleles are located in three distinct clusters in the center of the rpoB gene. We discuss the implications of our results with regards to the structure of the rifampicin binding site.",
"The mechanism of rifampicin inhibition of Escherichia coli RNA polymerase was studied with a newly developed steady state assay for RNA chain initiation and by analysis of the products formed with several 5'-terminal nucleotides. The major effect of rifampicin was found to be a total block of the translocation step that would ordinarily follow formation of the first phosphodiester bond. These effects were incorporated into a steric model for rifampicin inhibition. Additional minor effects of the enzyme bound inhibitor were to increase slightly the lifetime of RNA polymerase on the lambdaPR' promoter and to increase by two the apparent Michaelis constants of the initiating triphosphates. The products formed by RNA polymerase in the presence of rifampicin belong nearly exclusively to the class pppPupN. No evidence for the accumulation of such molecules was obtained in vivo.",
"A new drug target - the 'switch region' - has been identified within bacterial RNA polymerase (RNAP), the enzyme that mediates bacterial RNA synthesis. The new target serves as the binding site for compounds that inhibit bacterial RNA synthesis and kill bacteria. Since the new target is present in most bacterial species, compounds that bind to the new target are active against a broad spectrum of bacterial species. Since the new target is different from targets of other antibacterial agents, compounds that bind to the new target are not cross-resistant with other antibacterial agents. Four antibiotics that function through the new target have been identified: myxopyronin, corallopyronin, ripostatin, and lipiarmycin. This review summarizes the switch region, switch-region inhibitors, and implications for antibacterial drug discovery.",
"Sorangicin A, a macrolide polyether antibiotic and the ansamycin antibiotic rifampicin inhibit DNA-dependent RNA polymerase to a similar extent. Resistance to sorangicin A is due to a mutation in the RNA polymerase which renders the enzyme less sensitive. Parallel investigations with rifampicin revealed partial cross-resistance, which was more marked in sorangicin A-resistant mutants than in rifampicin-resistant mutants.",
"To delineate the role of capsaicin (8-methyl-N-vanillyl-6-nonenamide) as an inhibitor of the NorA efflux pump and its impact on invasion of macrophages by Staphylococcus aureus. Capsaicin in combination with ciprofloxacin was tested for activity against S. aureus SA-1199B (NorA overproducing), SA-1199 (wild-type) and SA-K1758 (norA knockout). The role of NorA in the intracellular invasion of S. aureus and the ability of capsaicin to inhibit this invasion was established in J774 macrophage cell lines. The three-dimensional structure of NorA was predicted using an in silico approach and docking studies of capsaicin were performed. Capsaicin significantly reduced the MIC of ciprofloxacin for S. aureus SA-1199 and SA-1199B. Furthermore, capsaicin also extended the post-antibiotic effect of ciprofloxacin by 1.1 h at MIC concentration. There was a decrease in mutation prevention concentration of ciprofloxacin when combined with capsaicin. Inhibition of ethidium bromide efflux by NorA-overproducing S. aureus SA-1199B confirmed the role of capsaicin as a NorA efflux pump inhibitor (EPI). The most significant finding of this study was the ability of capsaicin to reduce the intracellular invasion of S. aureus SA-1199B (NorA overproducing) in J774 macrophage cell lines by 2 log(10). This study, for the first time, has shown that capsaicin, a novel EPI, not only inhibits the NorA efflux pump of S. aureus but also reduces the invasiveness of S. aureus, thereby reducing its virulence."
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Infectious spleen and kidney necrosis virus ORF005L disrupts mitochondrial function and causes apoptosis in fish fry-1 cells
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Infectious spleen and kidney necrosis virus (ISKNV) belongs to the genus Megalocytivirus in the family Iridoviridae. This virus is the etiological agent of a serious and pandemic disease in fish. Cells infected with ISKNV undergo apoptosis. In this study, the ISKNV ORF005L gene was characterized and functionally investigated. Bioinformatics analysis revealed that the ORF005L protein contains a region similar to the catalytic domain of CTD-like phosphatases. Real-time quantitative-PCR results showed the transcription profile of ORF005L in ISKNV-infected cells; in these cells, ORF005L was initially transcribed at 24 h post-infection. The green fluorescent protein-tagged ORF005L protein was localized in the mitochondria. Sub-mitochondrial fractions were subjected to Western blot, and the results showed that ORF005L was specifically located in the inner membrane of the mitochondria. The ORF005L in fathead minnow cells was transiently expressed, resulting in the decrease in mitochondrial transmembrane potential, which induced cell apoptosis. ORF005L was knocked down by specific dsRNA, thereby significantly reducing the apoptosis of mandarin fish fry-1 cells induced by ISKNV infection. These results indicated that the ORF005L of ISKNV could disrupt mitochondrial function and cause apoptosis. This study may provide novel insights into the pathogenesis of Megalocytivirus infection.
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"Intranasal exposure of mice to influenza virus led to cell death in bronchial/bronchiolar epithelial cells, alveolar cells and lymphoid cells. These cells displayed fragmentation of nuclei and chromatin condensation. Nick end-labelling of DNA in situ confirmed that such apoptotic cells had fragmented DNA. These results suggest that the influenza virus induces apoptosis in vivo.",
"Inhibition of mitochondrial oxidative phosphorylation progresses to uncoupling when opening of cyclosporin A-sensitive permeability transition pores increases permeability of the mitochondrial inner membrane to small solutes. Involvement of the mitochondrial permeability transition (MPT) in necrotic and apoptotic cell death is implicated by demonstrations of protection by cyclosporin A against oxidative stress, ischemia/reperfusion, tumor necrosis factor-alpha exposure, Fas ligation, calcium overload, and a variety of toxic chemicals. Confocal microscopy directly visualizes the MPT in single mitochondria within living cells from the translocation of impermeant fluorophores, such as calcein, across the inner membrane. Simultaneously, mitochondria release potential-indicating fluorophores. Subsequently, mitochondria swell, causing outer membrane rupture and release of cytochrome c and other proapoptotic proteins from the intermembrane space. In situ a sequence of decreased NAD(P)H, increased free calcium, and increased reactive oxygen species formation within mitochondria promotes the MPT and subsequent cell death. Necrotic and apoptotic cell death after the MPT depends, in part, on ATP levels. If ATP levels fall profoundly, glycine-sensitive plasma membrane permeabilization and rupture ensue. If ATP levels are partially maintained, apoptosis follows the MPT. The MPT also signals mitochondrial autophagy, a process that may be important in removing damaged mitochondria. Cellular features of necrosis, apoptosis, and autophagy frequently occur together after death signals and toxic stresses. A new term, necrapoptosis, describes such death processes that begin with a common stress or death signal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmed cellular resorption (apoptosis), depending on modifying factors such as ATP.",
"The ectopic overexpression of Bcl-2 restricts both influenza A virus-induced apoptosis and influenza A virus replication in MDCK cells, thus suggesting a role for Bcl-2 family members during infection. Here we report that influenza A virus cannot establish an apoptotic response without functional Bax, a downstream target of Bcl-2, and that both Bax and Bak are directly involved in influenza A virus replication and virus-induced cell death. Bak is substantially downregulated during influenza A virus infection in MDCK cells, and the knockout of Bak in mouse embryonic fibroblasts yields a dramatic rise in the rate of apoptotic death and a corresponding increase in levels of virus replication, suggesting that Bak suppresses both apoptosis and the replication of virus and that the virus suppresses Bak. Bax, however, is activated and translocates from the cytosol to the mitochondria; this activation is required for the efficient induction of apoptosis and virus replication. The knockout of Bax in mouse embryonic fibroblasts blocks the induction of apoptosis, restricts the infection-mediated activation of executioner caspases, and inhibits virus propagation. Bax knockout cells still die but by an alternative death pathway displaying characteristics of autophagy, similarly to our previous observation that influenza A virus infection in the presence of a pancaspase inhibitor leads to an increase in levels of autophagy. The knockout of Bax causes a retention of influenza A virus NP within the nucleus. We conclude that the cell and virus struggle to control apoptosis and autophagy, as appropriately timed apoptosis is important for the replication of influenza A virus.",
"A major component of the cellular antiviral system is the latent protein kinase PKR, which is activated by binding to either double-stranded RNA (dsRNA) or the cellular PACT protein. Activated PKR phosphorylates the translation initiation factor eIF2, thereby inhibiting viral and cellular protein synthesis and virus replication. To evade the antiviral effects of PKR, many viruses, including influenza A virus, have evolved multiple mechanisms. For influenza A virus, the non-structural (NS1A) protein plays a major role in blocking activation of PKR during virus infection. The mechanism by which the NS1A protein inhibits PKR activation in infected cells has not been established. In the present study, we first carried out a series of in vitro experiments to determine whether the NS1A protein could utilize a common mechanism to inhibit PKR activation by both PACT and dsRNA, despite their different modes of activation. We demonstrated that the direct binding of the NS1A protein to the N-terminal 230 amino acid region of PKR can serve as such a common mechanism and that this binding does not require the RNA-binding activity of the NS1A protein. The lack of requirement for NS1A RNA-binding activity for the inhibition of PKR activation in vivo was established by two approaches. First, we showed that an NS1A protein lacking RNA-binding activity, like the wild-type (wt) protein, blocked PKR activation by PACT in vivo, as well as the downstream effects of PKR activation in cells, namely, eIF2 phosphorylation and apoptosis. In addition, we demonstrated that PKR activation is inhibited in cells infected with a recombinant influenza A virus expressing NS1A mutant protein that cannot bind RNA, as is the case in cells infected with wild-type influenza A virus.",
"The influenza virus PB1-F2 is an 87-amino acid mitochondrial protein that previously has been shown to induce cell death, although the mechanism of apoptosis induction has remained unclear. In the process of characterizing its mechanism of action we found that the viral PB1-F2 protein sensitizes cells to apoptotic stimuli such as tumor necrosis factor alpha, as demonstrated by increased cleavage of caspase 3 substrates in PB1-F2-expressing cells. Moreover, treatment of purified mouse liver mitochondria with recombinant PB1-F2 protein resulted in cytochrome c release, loss of the mitochondrial membrane potential, and enhancement of tBid-induced mitochondrial permeabilization, suggesting a possible mechanism for the observed cellular sensitization to apoptosis. Using glutathione-S-transferase pulldowns with subsequent mass spectrometric analysis, we identified the mitochondrial interactors of the PB1-F2 protein and showed that the viral protein uniquely interacts with the inner mitochondrial membrane adenine nucleotide translocator 3 and the outer mitochondrial membrane voltage-dependent anion channel 1, both of which are implicated in the mitochondrial permeability transition during apoptosis. Consistent with this interaction, blockers of the permeability transition pore complex (PTPC) inhibited PB1-F2-induced mitochondrial permeabilization. Based on our findings, we propose a model whereby the proapoptotic PB1-F2 protein acts through the mitochondrial PTPC and may play a role in the down-regulation of the host immune response to infection.",
"Iridovirus is a causative agent of epizootics among cultured rock bream (Oplegnathus fasciatus) in Korea. Here, we report the complete genomic sequence of rock bream iridovirus (RBIV). The genome of RBIV was 112080 bp long and contained at least 118 putative open reading frames (ORFs), and its genome organization was similar to that of infectious spleen and kidney necrosis virus (ISKNV). Of the RBIV's 118 ORFs, 85 ORFs showed 60-99% amino acid identity to those of ISKNV. Phylogenetic analysis of major capsid protein (MCP), DNA repair protein RAD2, and DNA polymerase type-B family indicated that RBIV is closely related to red sea bream iridovirus (RSIV), Grouper sleepy disease iridovirus (GSDIV), Dwarf gourami iridovirus (DGIV), and ISKNV. The genome sequence provides useful information concerning the evolution and divergence of iridoviruses in cultured fish.",
"Influenza viruses continue to pose a severe threat worldwide, causing thousands of deaths and an enormous economic loss every year. The major problem in fighting influenza is the high genetic variability of the virus, resulting in the rapid formation of variants that escape the acquired immunity against previous virus strains, or have resistance to antiviral agents. Every virus depends on its host cell and, hence, cellular functions that are essential for viral replication might be suitable targets for antiviral therapy. As a result, intracellular signaling cascades induced by the virus, in particular mitogen-activated protein kinase pathways, have recently come into focus."
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Type 1 interferon (IFN) signature in HIV-1 infection
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With successful antiretroviral therapy, HIV-1-infected subjects can achieve undetectable peripheral viral loads and immune homeostasis. However, in a subset of individuals on therapy, peripheral monocytes have a gene expression profile characteristic of a type 1 interferon α (IFN) response. This type 1 IFN response correlates with a number of pathogenic conditions including neural cell injury and in combination with HCV infection, cognitive impairment. Lessons from the non-human primate models of pathogenic and nonpathogenic SIV suggest that returning the initial IFN spike in acute SIV infection to normal allows the immune system to control infection and return to homeostasis. An IFN "alarm" signature, defined as monocyte activation with overexpression of the type1 IFN genes IFI27 and CD169, would be useful for identifying a subset of subjects with HIV-1 infection that could progress to a number of pathologies associated with immune activation including cognitive dysfunction. This strategy is being actively pursued for autoimmune diseases that are characterized by an IFN signature. Therapies to block the IFN signature are under investigation as a means to reset the immune system and in a subset of HIV-1-infected subjects may be an adjuvant to standard antiviral therapy to return cognitive function.
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"In human immunodeficiency virus (HIV)-1 infection, highly increased T-cell turnover was proposed to cause exhaustion of lymphocyte production and consequently development of AIDS. Here, we investigated cell proliferation, as measured by expression of the Ki-67 nuclear antigen, in peripheral blood CD4(+) and CD8(+) lymphocyte subpopulations before and during highly active antiretroviral therapy (HAART). In untreated HIV-1 infection, both the percentage and number of Ki-67(+) CD4(+) and CD8(+) lymphocytes were significantly increased, compared with values obtained from healthy individuals. A more than 10-fold increase in the percentage of dividing naive CD4(+) T cells in the blood was found when the number of these cells were below 100 per microL. HAART induced an immediate decline in Ki-67 antigen expression, despite often very low CD4(+) T-cell numbers, arguing against increased proliferation being a homeostatic response. After approximately 24 weeks of HAART treatment, a transient increase in the number of proliferating cells was seen, but only in the CD4(+) CD27(+) memory pool. In the CD8(+) T-cell compartment, the number of dividing cells was elevated 20- to 25-fold. This increase was most notable in the CD27(+) CD 45RO(+) and CD27(-) CD45RO(+) memory CD8(+) T-cell pool, corresponding with the degree of expansion of these subsets. Reduction of plasma HIV-RNA load by HAART was accompanied by a decrease in numbers and percentages of dividing cells in all CD8(+) T-cell subsets. Taken together, our results indicate that peripheral T-cell proliferation is a consequence of generalized immune activation. (Blood. 2000;95:249-255)",
"Infection with the human immunodeficiency virus HIV-1 is associated with the expansion of a CD14lowCD16high monocyte subset in peripheral blood. This subset, which represents a minor subpopulation of monocytes in healthy individuals, increases during HIV infection and, in patients with AIDS, may represent up to 40% of the total circulating monocyte cell population. The CD14lowCD16high circulating monocytes co-express MAX.1, p150,95 and HLA-DR which are typical of tissue macrophage markers. These cells also express higher levels of intracellular interleukin (IL)-1 alpha and tumor necrosis factor (TNF)-alpha than the CD14highCD16low monocyte population from the same patients. The CD14lowCD16high cells also express low levels of CD35, CD11a and CD4 in common with normal monocytes. When cultured in vitro, monocytes from HIV-seropositive individuals differentiated within a few hours into an elongated fibroblastoid shape characteristic of migratory cells. Our results suggest that the expansion of the CD14lowCD16high monocyte subset, which produce high amount sof TNF-alpha and IL-1 alpha, may participate in the immune dysfunction observed during HIV infection.",
"To characterise activation of the type I interferon (IFN) pathway in patients with systemic lupus erythematosus (SLE), dermatomyositis (DM), polymyositis (PM), rheumatoid arthritis (RA) and systemic scleroderma (SSc) and to evaluate the potential to develop a molecular diagnostic tool from the peripheral blood that reflects this activation in disease-affected tissues. Overexpressed transcripts were identified in the whole blood (WB) of 262 patients with SLE, 44 with DM, 33 with PM, 28 with SSc and 89 with RA and compared with 24 healthy subjects using Affymetrix microarrays. A five gene type I IFN signature was assessed in these subjects to identify subpopulations showing both activation and concordance of the type I IFN pathway in the peripheral blood and disease-affected tissues of each disease and to correlate activation of this pathway in the WB with clinical measurements. A common set of 36 type I IFN inducible transcripts were identified among the most overexpressed in the WB of all subjects. Significant activation of the type I IFN pathway in subgroups of each of the five diseases studied was observed. Baseline disease activity measurements correlated with a type I IFN gene signature in the WB of subjects with SLE, PM and SSc, as did various serum autoantibody levels in subjects with SLE and DM. This signature was also well correlated between disease-affected tissue and WB in subjects with SLE, DM, PM and SSc. The results indicate that the type I IFN pathway is activated in patient subsets of five rheumatic diseases and suggest that these subsets may benefit from anti-IFN therapy.",
"IL-27 is a novel anti-HIV cytokine that inhibits HIV-1 replication in both CD4 T cells and monocyte-derived macrophages (MDM) as IFN-alpha does. To elucidate the mechanism of the antiviral activity, we compared the activity and the gene expression profile of IL-27-treated cells with that of IFN-alpha-treated cells. CD4 T cells and monocytes were isolated from peripheral blood mononuclear cells of healthy donors. CD4 T cells were stimulated with phytohemagglutinin, and MDM were induced from monocytes using macrophage-colony stimulating factor. HIV-1 replication was monitored by p24 antigen capture assay. The gene expression profiles were analysed using DNA microarray analysis. The increase in the expression of IFN-inducible genes (IFIG) was confirmed by the Quantigene plex assay. Both cytokines preferentially inhibited HIV-1 replication in MDM compared with CD4 T cells. Quantitative real time polymerase chain reaction, enzyme-linked immunosorbent assay and neutralization assay using anti-IFN indicated that IFN-alpha, IFN-beta and IFN-gamma had no significant impact on IL-27-mediated HIV inhibition. DNA microarray analysis illustrated that IFN-alpha induced 33 and 18 IFIG in MDM and CD4 T cells, respectively. IL-27 induced 28 IFIG in MDM and five IFIG in CD4 T cells. The quantitative assay confirmed that IL-27 activated genes of RNA-dependent kinase, oligoadenylate synthetase, myxovirus protein, and apolipoprotein B messenger RNA-editing enzyme-catalytic polypeptide-like 3G. IL-27 differentially regulates the gene expression between CD4 T cells and MDM. IL-27 significantly induces antiviral genes in MDM as does IFN-alpha, suggesting that IL-27 inhibits HIV replication in MDM via mechanism(s) similar to that of IFN-alpha.",
"Immune activation is a strong predictor of disease progression in HIV infection. Combinatorial plasma biomarker signatures that represent surrogate markers of immune activation in both viremic and aviremic HIV patients on combination antiretroviral therapy (cART) have not been defined. Here, we identify a plasma inflammatory biomarker signature that distinguishes between both viremic and aviremic HIV patients on cART and healthy controls and examine relationships of this signature to markers of disease progression. Multiplex profiling and ELISA were used to detect 15 cytokines/chemokines, soluble IL-2R (sIL-2R), and soluble CD14 (sCD14) in plasma from 57 HIV patients with CD4 nadir <300 cells/µl and 29 healthy controls. Supervised and unsupervised analyses were used to identify biomarkers explaining variance between groups defined by HIV status or drug abuse. Relationships between biomarkers and disease markers were examined by Spearman correlation. The majority (91%) of HIV subjects were on cART, with 38% having undetectable viral loads (VL). Hierarchical clustering identified a biomarker cluster in plasma consisting of two interferon-stimulated gene products (CXCL9 and CXCL10), T cell activation marker (sIL-2R), and monocyte activation marker (sCD14) that distinguished both viremic and aviremic HIV patients on cART from controls (p<0.0001) and were top-ranked in variables important in projection plots. IL-12 and CCL4 were also elevated in viremic and aviremic patients compared to controls (p<0.05). IL-12 correlated with IFNα, IFNγ, CXCL9, and sIL-2R (p<0.05). CXCL10 correlated positively with plasma VL and percentage of CD16+ monocytes, and inversely with CD4 count (p = 0.001, <0.0001, and 0.04, respectively). A plasma inflammatory biomarker signature consisting of CXCL9, CXCL10, sIL-2R, and sCD14 may be useful as a surrogate marker to monitor immune activation in both viremic and aviremic HIV patients on cART during disease progression and therapeutic responses.",
"Acute SIV infection is characterized by explosive infection of memory CD4 T cells in peripheral and mucosal tissues. Interestingly, relatively few memory CD4 T cells are infected until as late as days 7-8 after challenge. However, by day 10 postinfection, most of the memory CD4 T cells are infected and carry viral DNA. The rapidity with which infection expands within 2-3 days to encompass virtually the entire memory CD4 T cell compartment suggests significant alterations in the susceptibility of memory CD4 T cells to infection during this period. The mechanism(s) underlying this increased permissiveness to infection is not known. In this study, we show that IL-15 secretion significantly correlates with the up-regulated expression of CD4 on memory CD4 T cells that is associated with increased permissiveness to SIV infection. Activation and proliferation of memory CD8, but not memory CD4 T cells, preceded the amplification of viral infection. Although memory CD4 T cells did not express normal activation markers, they displayed a significant up-regulation in the density of CD4 but not CCR5 expression between days 7 and 10 postinfection that correlated with increased plasma IL-15 levels and infection in these cells. Culture of purified CD4 T cells with IL-15 and/or SIV was associated with a significant increase in the expression of CD4 and infection of these sorted cells. Our results demonstrate that IL-15 contributes to the increased susceptibility of memory CD4 T cells to SIV during the early phase of acute SIV infection.",
"B cell depletion therapy is efficacious in rheumatoid arthritis (RA) patients failing on tumor necrosis factor (TNF) blocking agents. However, approximately 40% to 50% of rituximab (RTX) treated RA patients have a poor response. We investigated whether baseline gene expression levels can discriminate between clinical non-responders and responders to RTX. In 14 consecutive RA patients starting on RTX (test cohort), gene expression profiling on whole peripheral blood RNA was performed by Illumina® HumanHT beadchip microarrays. Supervised cluster analysis was used to identify genes expressed differentially at baseline between responders and non-responders based on both a difference in 28 joints disease activity score (ΔDAS28 < 1.2) and European League against Rheumatism (EULAR) response criteria after six months RTX. Genes of interest were measured by quantitative real-time PCR and tested for their predictive value using receiver operating characteristics (ROC) curves in an independent validation cohort (n = 26). Genome-wide microarray analysis revealed a marked variation in the peripheral blood cells between RA patients before the start of RTX treatment. Here, we demonstrated that only a cluster consisting of interferon (IFN) type I network genes, represented by a set of IFN type I response genes (IRGs), that is, LY6E, HERC5, IFI44L, ISG15, MxA, MxB, EPSTI1 and RSAD2, was associated with ΔDAS28 and EULAR response outcome (P = 0.0074 and P = 0.0599, respectively). Based on the eight IRGs an IFN-score was calculated that reached an area under the curve (AUC) of 0.82 to separate non-responders from responders in an independent validation cohort of 26 patients using Receiver Operator Characteristics (ROC) curves analysis according to ΔDAS28 < 1.2 criteria. Advanced classifier analysis yielded a three IRG-set that reached an AUC of 87%. Comparable findings applied to EULAR non-response criteria. This study demonstrates clinical utility for the use of baseline IRG expression levels as a predictive biomarker for non-response to RTX in RA."
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NMR structural studies of the melanocortin-2 and -4 receptors in an amphipol environment
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While amphipols have been proven useful for refolding of seven transmembrane helical (7-TM) proteins including G-protein-coupled receptors (GPCRs) and it could be shown that an amphipol environment is in principle suitable for NMR structural studies of the embedded protein, high-resolution NMR insights into amphipol refolded and isotopically labeled GPCRs are still very limited. Here we report on the recent progress toward NMR structural studies of the melanocortin-2 and -4 receptors, two class A GPCRs which so far have not been reported to be incorporated into an amphipol environment. Making use of the established 7-TM protein bacteriorhodopsin (BR) we initially tested and optimized amphipol refolding conditions. Most promising conditions were transferred to the refolding of the two melanocortin receptors. Analytical-scale refolding experiments on the melanocortin-2 receptor show very similar behavior to the results obtained on BR. Using cell-free protein expression we could generate sufficient amounts of isotopically labeled bacteriorhodopsin as well as melanocortin-2 and -4 receptors for an initial NMR analysis. Upscaling of the amphipol refolding protocol to protein amounts needed for NMR structural studies was, however, not straightforward and impeded detailed NMR insights for the two GPCRs. While well-resolved and dispersed NMR spectra could only be obtained for bacteriorhodopsin, a comparison of NMR data recorded on the melanocortin-4 receptor in SDS and in an amphipol environment indicates that amphipol refolding induces larger structural modifications in the receptor.
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"The lipid/protein stoichiometries of a naturally crystalline biological membrane, the purple membrane (PM) of Halobacterium salinarum, have been obtained by a combination of (31)P- and (1)H-NMR analyses of the lipid extract. In total, 10 lipid molecules per retinal were found to be present in the PM lipid extract: 2-3 molecules of phosphatidylglycerophosphate methyl ester (PGP-Me), 3 of glycolipid sulfate, 1 of phosphatidylglycerol, 1 of archaeal glycocardiolipin (GlyC), 2 of squalene plus minor amounts of phosphatidylglycerosulfate (PGS) and bisphosphatidylglycerol (archaeal cardiolipin) (BPG) and a negligible amount of vitamin MK8. The novel data of the present study are necessary to identify the lipids in the electron density map, and to shed light on the structural relationships of the lipid and protein components of the PM.",
"Although nearly half of today's major pharmaceutical drugs target human integral membrane proteins (hIMPs), only 30 hIMP structures are currently available in the Protein Data Bank, largely owing to inefficiencies in protein production. Here we describe a strategy for the rapid structure determination of hIMPs, using solution NMR spectroscopy with systematically labeled proteins produced via cell-free expression. We report new backbone structures of six hIMPs, solved in only 18 months from 15 initial targets. Application of our protocols to an additional 135 hIMPs with molecular weight <30 kDa yielded 38 hIMPs suitable for structural characterization by solution NMR spectroscopy without additional optimization.",
"Amphipols are short amphipathic polymers designed to stabilize membrane proteins in aqueous solutions in the absence of detergent. Bacteriorhodopsin (BR), a light-driven proton pump, has been denatured, either by direct solubilization of the purple membrane in sodium dodecylsulfate (SDS) solution or by a procedure that involves delipidation with organic solvent followed by transfer to SDS, and renatured in amphipol A8-35. The effect of different renaturation procedures and of the presence or absence of lipids and the cofactor retinal have been investigated. The resulting samples have been characterized by absorbance spectroscopy, size-exclusion chromatography, thermostability measurements, and determination of photocycle kinetics. Transfer to A8-35 can be achieved by SDS precipitation, dilution, or dialysis, the first route resulting in the highest yield of refolding. Functional BR can be refolded whether in the presence or absence of lipids, higher yields being achieved in their presence. Retinal is not required for the protein to refold, but it stabilizes the refolded form and, thereby, improves folding yields. Lipids are not required for BR to perform its complete photocycle, but their presence speeds up the return to the ground state. Taken together, these data indicate that a membrane or membrane-mimetic environment is not required for correct decoding of the chemical information contained in the sequence of BR; functional folding is possible even in the highly foreign environment of lipid-free amphipols. BR interactions with lipids, however, contribute to an effective photocycle.",
"The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems."
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Torsades de pointes in patients receiving voriconazole
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Voriconazole can prolong the QT interval contributing to life-threatening cardiac arrhythmia. Torsades de pointes is an uncommon but serious complication of voriconazole use which may be under-recognised. We present torsades de pointes in two patients with underlying haematological malignancy being treated for invasive fungal infection with voriconazole. Patients receiving voriconazole should be screened and monitored for evidence of QT prolongation, and if prolongation detected, consideration given to alternative treatments or more intensive cardiac monitoring.
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"To review the chemistry, pharmacology, and safety of fluoroquinolones. A MEDLINE search (1966-July 2007) was conducted using the key words fluoroquinolones or quinolones with safety, adverse effects, hypoglycemia, hyperglycemia, dysglycemia, QTc prolongation, torsades, seizures, phototoxicity, tendon rupture, Clostridium difficile, and pseudomembranous colitis for articles published in the English language. Medicinal chemistry, in vitro, animal, and human trials were reviewed for information on the chemistry, pharmacology, and safety of each fluoroquinolone. Clinical trials were reviewed and included to compare the safety of systemic fluoroquinolones on the market. Literature on the pathology of serious adverse effects was also reviewed. Gatifloxacin has been shown to increase the risk of hospitalization for dysglycemia in patients with and without diabetes. Hyperglycemia may occur with any fluoroquinolone, especially if not properly dose adjusted. Hypoglycemia may occur with any fluoroquinolone and has a higher frequency in patients receiving concomitant oral hypoglycemic drugs or insulin. Use of any fluoroquinolone should be avoided in patients with risk factors for QTc interval prolongation or tendinopathy. All fluoroquinolones should be used with caution in patients with a history of seizure disorders and may cause phototoxicity or C. difficile-associated diarrhea (CDAD). Clinicians should be aware of possible alterations in blood glucose, QTc interval prolongation, seizures, phototoxicity, tendinopathy, or CDAD with the use of any fluoroquinolone, especially in patients with other risk factors for these conditions. Clinicians should closely monitor for these adverse effects and appropriately adjust doses to minimize these risks. To provide safe treatment for patients needing antibiotic therapy, an assessment of the risk-benefit ratio may be warranted in the decision to use a fluoroquinolone.",
"Tricyclic antidepressants remain a common cause of fatal drug poisoning as a result of their cardiovascular toxicity manifested by ECG abnormalities, arrhythmias and hypotension. Dosulepin and amitriptyline appear to be particularly toxic in overdose. The principal mechanism of toxicity is cardiac sodium channel blockade, which increases the duration of the cardiac action potential and refractory period and delays atrioventricular conduction. Electrocardiographic changes include prolongation of the PR, QRS and QT intervals, nonspecific ST segment and T wave changes, atrioventricular block, right axis deviation of the terminal 40 ms vector of the QRS complex in the frontal plane (T 40 ms axis) and the Brugada pattern (downsloping ST segment elevation in leads V1-V3 in association with right bundle branch block). Maximal changes in the QRS duration and the T 40 ms axis are usually present within 12 hours of ingestion but may take up to a week to resolve. Sinus tachycardia is the most common arrhythmia due to anticholinergic activity and inhibition of norepinephrine uptake by tricyclic antidepressants but bradyarrhythmias (due to atrioventricular block) and tachyarrhythmias (supraventricular and ventricular) may occur. Torsade de pointes occurs uncommonly. Hypotension results from a combination of reduced myocardial contractility and reduced systemic vascular resistance due to alpha-adrenergic blockade. Life-threatening arrhythmias and death due to tricyclic antidepressant poisoning usually occurs within 24 hours of ingestion. Rapid deterioration is common. Level of consciousness at presentation is the most sensitive clinical predictor of serious complications. Although a QRS duration >100 ms and a rightward T 40 ms axis appear to be better predictors of cardiovascular toxicity than the plasma tricyclic drug concentration, they have at best moderate sensitivity and specificity for predicting complications.",
"A 15-year-old patient with acute lymphoblastic leukemia and Fusarium infection was treated with voriconazole. She developed asymptomatic bradycardia, QT interval prolongation, and nonsustained, polymorphic ventricular tachycardia, which recurred upon rechallenge with the drug. Voriconazole levels and metabolism were within expected normal values. This non-concentration-dependent, voriconazole-associated ventricular tachycardia mandates cardiac rhythm monitoring during voriconazole treatment.",
"Fluoroquinolones are widely used and well tolerated antibacterial agents. However, prolongation of the QT interval is an adverse effect associated with the use of fluoroquinolones. According to the available case reports and clinical studies, moxifloxacin carries the greatest risk of QT prolongation from all available quinolones in clinical practice and it should be used with caution in patients with predisposing factors for Torsades de pointes (Tdp). Although gemifloxacin, levofloxacin and ofloxacin are associated with a lower risk of QT prolongation compared with moxifloxacin, they should also be used with caution in patients with risk factors for QT prolongation. Ciprofloxacin appears to be associated with the lowest risk for QT prolongation and the lowest rate of Tdp. The overall risk of Tdp is small with the use of fluoroquinolones. Clinicians can minimise that risk by avoiding prescriptions of multiple medications associated with QT interval prolongation, especially in high-risk patients.",
"Severe and occasionally fatal arrhythmias, commonly presenting as Torsade de Pointes [TdP] have been reported with Class III-antiarrhythmics, but also with non-antiarrhythmic drugs. Most cases result from an action on K(+) channels encoded by the HERG gene responsible for the IKr repolarizing current, leading to a long QT and repolarization abnormalities. The hydrophobic central cavity of the HERG-K+ channels, allows a large number of structurally unrelated drugs to bind and cause direct channel inhibition. Some examples are dofetilide, quinidine, sotalol, erythromycin, grepafloxacin, cisapride, dolasetron, thioridazine, haloperidol, droperidol and pimozide. Other drugs achieve channel inhibition indirectly by impairing channel traffic from the endoplasmic reticulum to the cell membrane, decreasing channel membrane density (pentamidine, geldalamicin, arsenic trioxide, digoxin, and probucol). Whereas, ketoconazole, fluoxetine and norfluoxetine induce both direct channel inhibition and impaired channel trafficking. Congenital long QT syndrome, subclinical ion-channel mutations, subjects and relatives of subjects with previous history of drug-induced long QT or TdP, dual drug effects on cardiac repolarization [long QT plus increased QT dispersion], increased transmural dispersion of repolarization and T wave abnormalities, use of high doses, metabolism inhibitors and/or combinations of QT prolonging drugs, hypokalemia, structural cardiac disease, sympathomimetics, bradycardia, women and older age, have been shown to increase the risk for developing drug-induced TdP. Because most of these reactions are preventable, careful evaluation of risk factors and increased knowledge of drugs use associated with repolarization abnormalities is strongly recommended. Future genetic testing and development of practical and simple provocation tests are in route to prevent iatrogenic TdP.",
"QTc interval-prolonging drugs have been linked to cardiac arrhythmias, cardiac arrest and sudden death. In this study we aimed to quantify the risk of cardiac arrest associated with the use of non-antiarrhythmic QTc-prolonging drugs in an academic hospital setting. We performed a case-control study in which patients, for whom intervention of the advanced life support resuscitation team was requested for cardiac arrest between 1995 and 2003 in the Academic Medical Centre, Amsterdam, were compared with controls regarding current use of non-antiarrhythmic QTc-prolonging drugs. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using unconditional logistic regression, adjusting for potential confounding factors. A statistically significant increased risk of cardiac arrest (OR 2.1, 95% CI 1.2, 3.5) was observed in patients who received QTc-prolonging drugs (42/140). The risk was more pronounced in patients receiving doses > 1 defined daily dose (OR 2.5, 95% CI 1.1, 5.9), patients taking > 1 QTc-prolonging drug simultaneously (OR 4.8, 95% CI 1.6, 14) and patients taking pharmacokinetic interacting drugs concomitantly (OR 4.0, 95% CI 1.2, 13). Use of non-antiarrhythmic QTc-prolonging drugs in hospitalized patients with several underlying disease is associated with an increased risk of cardiac arrest. The effect is dose related and pharmacokinetic drug-drug interactions increase the risk substantially. Physicians caring for inpatients should be made aware of the fact that these non-antiarrhythmic drugs may be hazardous, so that potential risks can be weighed against treatment benefits and additional cardiac surveillance can be requested, if necessary.",
"The human ether-a-go-go-related gene (hERG) encodes a channel that conducts the rapidly activating delayed rectifier K(+) current (I(Kr)), which is important for cardiac repolarization. Mutations in hERG reduce I(Kr) and cause congenital long QT syndrome (LQTS). More frequently, common medications can reduce I(Kr) and cause LQTS as a side effect. Protein trafficking abnormalities are responsible for most hERG mutation-related LQTS and are recently recognized as a mechanism for drug-induced LQTS. Whereas hERG trafficking has been studied in recombinant expression systems, there has been no reported study on cardiac I(Kr) trafficking at the protein level. In the present study, we identified that I(Kr) is present in cultured neonatal rat ventricular myocytes and can be robustly recorded using Cs(+) as the charge carrier. We further discovered that 4,4'-(isopropylidenedithio)-bis-(2,6-di-t-butylphenol) (probucol), a cholesterol-lowering drug that induces LQTS, disrupted I(Kr) trafficking and prolonged the cardiac action potential duration. Probucol did not directly block I(Kr). Probucol also disrupted hERG trafficking and did not block hERG channels expressed in human embryonic kidney 293 cells. We conclude that probucol induces LQTS by disrupting ether-a-go-go-related gene trafficking, and that primary culture of neonatal rat cardiomyocytes represents a useful system for studying native I(Kr) trafficking."
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Tracheal intubation in patients with drug overdose due to self harm
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Tracheal intubation may be performed in patients with drug overdose due to self-harm; however, the details of the causative drug are unknown. The purpose of this study was to clarify the relationship between drugs or its blood levels of patients with drug overdose and the need for tracheal intubation based on the actual measurement results.
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"The aims of present study are to clarify the clinical features of patients with benzodiazepine use disorder (BZsUD), and to examine the characteristics of psychiatric treatments which may cause BZsUD. We conducted a medical chart and interview survey to 87 outpatients with benzodiazepine use disorder, who had consecutively visited the four hospitals, specialized in addiction problems, located in metropolitan area, during a month of December, 2011. Consequently, 88.5% of the patients with BZsUD reported to obtain BZs to be abused from general psychiatric clinics, and 83.9% contracted BZsUD in process of general psychiatric treatment. Among the patients who contracted BZsUD in process of psychiatric treatment, 43.8% were speculated to have the other substance-related disorders such as methamphetamine or alcohol-related disorder at start of the psychiatric treatment. Further, approximately 70% of them reported that surplus BZs were prescribed without consideration of storing drugs, and over 40% also reported that BZs were prescribed without a medical examination. The findings of the present study may let us to propose the following four measures to be taken for prevention of BZsUD: First, latent substance-related disorders should not be overlooked, second, short-acting and high-potency BZs should not be prescribed if possible, third, storing and abusing BZs should always be considered, and finally, BZs should be prescribed with a medical examination.",
"Little is known about psychotropic medication prescriptions prior to drug overdose. We aimed to examine the possible associations between the risk of overdose and exposures to various psychotropic medications. We conducted a matched case-control study of 3 groups of patients aged 12-74 years, using a large-scale health insurance claims database in Japan (population 1.2 million). A total of 351 cases with drug poisoning were compared with two control groups without overdose: 1755 patients with any treatment (general controls) and those with depression (high-risk controls). Current, past, and nonusers were patients most recently exposed to psychotropic medications ≤ 90 days, 91-180 days, and ≥ 180 days before the index date. Current sedative-hypnotic use was associated with an increased odds of overdose relative to general control nonusers (odds ratio [OR], 21.5; 95 % confidence interval [CI], 9.7-47.8) and high-risk control nonusers (OR, 2.6; 95 % CI, 1.9-3.5). In the comparison of cases and high-risk controls, the ORs for overdose were higher among excessive dosage users than among usual dosage users (OR, 4.3; 95 % CI, 3.0-6.1), among barbiturate users than among benzodiazepine/Z-drug only users (OR, 4.5; 95 % CI, 2.3-8.7), and among multiple provider episodes than among single provider episodes (OR, 4.4; 95 % CI, 1.7-11.0). Psychiatrists prescribed more than 77 % of potentially questionable prescriptions. These results highlight the need for psychiatrists to monitor prescribed medications and balance the benefits and risks of pharmacological treatments.",
"Japan Coma Scale (JCS) is a grading system used to evaluate disturbed consciousness in prehospital care settings. We aimed to identify the association between the JCS levels at the scene with in-hospital mortality, as well as the discrimination ability for the outcomes. A retrospective cohort study based on the nationwide trauma database in Japan. Multicentre cohort study using data from the Japan Trauma Data Bank, which is a nationwide, prospective, observational trauma registry derived from 235 hospitals. Adult trauma victims transferred directly from the scene of injury to the hospital from January 2004 to December 2017 were eligible for inclusion. Primary outcome was the association between the JCS levels at the scene with in-hospital mortality. We conducted a multivariate logistic regression analysis to calculate the adjusted ORs of JCS levels with 95% CIs for in-hospital mortality. We also calculated the c-statistics for in-hospital mortality. 164 723 patients were included in the analysis. In a multivariate logistic regression analysis, the corresponding adjusted ORs of JCS levels 2 and 3 referred to level 1 for in-hospital mortality were 4.1 (95% CI 3.8 to 4.4) and 26.0 (95% CI 24.8 to 27.2). The c-statistics of the JCS level for in-hospital mortality was 0.845 (95% CI 0.842 to 0.849). Data from large multicentre prospective registry revealed strong associations of the JCS level at the scene of injury with in-hospital mortality as well as the good discriminatory performance for this outcome.",
"Cigarette smoking is a significant risk factor for higher incidences of numerous adverse health consequences. Related health disorders are also found in non-smokers exposed to secondhand smoke (SHS). To investigate the influence of cigarette smoking and exposure to SHS on the composition of human skin gas, a trace biogas emanating from human skin, dermal emissions of volatile compounds were semi-quantitatively measured for volunteers who smoke a cigarette and those exposed to SHS. This was performed using a passive flux sampler (PFS) coupled with gas chromatography mass spectrometry (GCMS). Numerous chemicals were detected, including acetaldehyde, toluene, 3-methyl furan (3-MF), 2,5-dimethyl furan (2,5-DMF), 3-ethenyl pyridine (3-EP), and nicotine, in the samples collected from the smokers after a smoking event, and a remarkable increase in the amount of chemicals collected was observed just after smoking. These chemicals were also found in the samples collected from volunteers exposed to SHS. Assessment of current smoking status is important for managing the negative effects of active and passive smoking, and for the development of public health policy. The tobacco specific chemicals such as 3-MF, 2,5-DMF, 3-EP, and nicotine, emanating from human skin surfaces, represent a potential non-invasive biomarker for monitoring current smoking status of active and passive smokers after establishing a more quantitative procedure."
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Oxytocin and Vasopressin in the Development of the Social Brain
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The related neuropeptides oxytocin and vasopressin are involved in species-typical behavior, including social recognition behavior, maternal behavior, social bonding, communication, and aggression. A wealth of evidence from animal models demonstrates significant modulation of adult social behavior by both of these neuropeptides and their receptors. Over the last decade, there has been a flood of studies in humans also implicating a role for these neuropeptides in human social behavior. Despite popular assumptions that oxytocin is a molecule of social bonding in the infant brain, less mechanistic research emphasis has been placed on the potential role of these neuropeptides in the developmental emergence of the neural substrates of behavior. This review summarizes what is known and assumed about the developmental influence of these neuropeptides and outlines the important unanswered questions and testable hypotheses. There is tremendous translational need to understand the functions of these neuropeptides in mammalian experience-dependent development of the social brain. The activity of oxytocin and vasopressin during development should inform our understanding of individual, sex, and species differences in social behavior later in life.
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"The lasting effects of a 9-day neonatal exposure to vasopressin and oxytocin were examined in the rat to discover if peptide administration results in organizational effects. When tested in young adulthood, brain growth, not body growth, appeared to be impaired. Basal and challenge tests of urine production, carried out to see the development of the hormonal antidiuretic function of vasopressin, revealed no lasting changes, and therefore did not confirm earlier findings of an induced mild polyurea. Behavioral testing of learning by making use of a one-trail step-through paradigm with a 24-h retention trial--a test that is sensitive to vasopressin--did not show impairments. Open field tests, however, showed enhanced emotionality in the vasopressin-treated females, as well as an initially increased ambulation in the males, and increased grooming in both sexes, the latter also having been reported to be induced by vasopressin administration in the septal areas. Oxytocin treatment did not produce lasting changes. Our conclusion, therefore, is that peripherally circulating vasopressin can affect the organizational development of the rat brain. It remains to be established whether this is an effect obtained through changes in the general peripheral physiology or a reflection of plasticity phenomena at the level of central vasopressin neurotransmission.",
"Defining how arginine vasopressin (AVP) acts centrally to regulate homeostasis and behavior is problematic, as AVP is made in multiple nuclei in the hypothalamus (i.e., paraventricular [PVN], supraoptic [SON], and suprachiasmatic [SCN]) and extended amygdala (i.e., bed nucleus of the stria terminalis [BNST] and medial amygdala [MeA]), and these groups of neurons have extensive projections throughout the brain. To understand the function of AVP, it is essential to know the site of origin of various projections. In mice, we used gonadectomy to eliminate gonadal steroid hormone-dependent expression of AVP in the BNST and MeA and electrolytic lesions to eliminate the SCN, effectively eliminating those AVP-immunoreactive projections; we also quantified AVP-immunoreactive fiber density in gonadectomized and sham-operated male and female mice to examine sex differences in AVP innervation. Our results suggest that the BNST/MeA AVP system innervates regions containing major modulatory neurotransmitters (e.g., serotonin and dopamine) and thus may be involved in regulating behavioral state. Furthermore, this system may be biased toward the regulation of male behavior, given the numerous regions in which males have a denser AVP-immunoreactive innervation than females. AVP from the SCN is found in regions important for the regulation of hormone output and behavior. Innervation from the PVN and SON is found in brain regions that likely work in concert with the well-known peripheral AVP actions of controlling homeostasis and stress response; female-biased sex differences in this system may be related to the heightened stress response observed in females.",
"Anomalies in hormonal and neurotransmitter status during early stages of brain development, can lead to lifespan alterations in the functioning of central systems. The neuropeptide vasopressin is nowadays recognized as a putative neurotransmitter, after years of study on its neurosecretory hormonal aspect in water metabolism. Since vasopressin is moreover present early in the brain, and has various mitogenic, metabolic and physiological actions, one might expect vasopressin to be of importance for normal brain development as well. Indeed, the absence of brain vasopressin in the Brattleboro mutant rat coincides with impaired brain development, and some physiological and behavioral defects of these rats are not adjusted by treatment with vasopressin. Regionally the cerebellum seems to be the most affected brain area, both morphologically and biochemically. Only when vasopressin supplementation was done prenatally, this disturbed growth could be restored, which suggests an early role for vasopressin in neurogenesis. Enhanced levels of vasopressin during the perinatal period on the other hand, have been shown to affect permanently the 'setting' of peripheral vasopressin functions in cardiovascular and renal regulatory systems. It is not excluded as yet that after such treatments central organization of vasopressin systems is not impaired as well.",
"Previous studies have revealed that the neuropeptide hormone oxytocin (OT) has developmental effects on subsequent social behavior and on mechanisms underlying social behavior such as OT neurons and estrogen receptor alpha. This suggests that OT might also have developmental effects on neural responses to social stimuli. This was tested in socially monogamous prairie voles (Microtus ochrogaster) by manipulating OT on the first day of life and then assessing the response to a heterosexual pairing in adulthood. The response to cohabitation was assessed by quantifying neural activation in regions of the brain associated with sociosexual behavior and anxiety using c-Fos immunoreactivity. Additionally, immunocytochemistry was used to label OT and vasopressin neurons and plasma was assayed for both neuropeptides. Treatment effects were evident in females, but not in males. Blockade of OT receptors with an OT antagonist on the first day of life resulted in neural activation of the central amygdala in response to a pairing with a novel male in adulthood. The central amygdala does not normally express c-Fos after a heterosexual pairing in reproductively naïve prairie voles. Treatment effects also were observed in vasopressin immunoreactivity in the SON with OT-treated females showing a decrease.",
"Oxytocin has numerous prosocial and antipsychotic-like effects in animals. Prosocial effects of acute intranasal oxytocin administration have also been reported in human subjects. We conducted a randomized, placebo-controlled trial testing the effects of twice daily intranasal oxytocin treatment for 14 days on psychotic symptoms and social cognition in patients with schizophrenia. PANSS scores declined significantly and several social cognition measures improved significantly or nearly significantly in oxytocin (N=11) but not placebo (N=9) recipients. Our results suggest that, in addition to reducing classic psychotic symptoms, oxytocin may diminish certain social cognition deficits that are not improved by current antipsychotic medications.",
"Studies concerning the development of the magnocellular system are scarce and discordant in literature. We carried out an immunohistochemical study on supraotic and paraventricular hypothalamic nuclei using antivasopressin and antioxytocin antibodies in developing rats between the 15th day of intrauterine life and the 6th day of postnatal life. In addition, we performed RT-PCR experiments to establish the stage at which these hormones appear and neurosecretory activity commences. The results showed that supraoptic and paraventricular nuclei appear, respectively, on the 16th and the 18th day of intrauterine life and both immediately synthetize vasopressin neurohormone. By contrast, synthesis of oxytocin takes place from the 2nd day after birth. Probably, these nuclei synthetize oxytocin in conjunction with the decline of placental maternal oxytocin.",
"Oxytocin (OXT) has been hypothesized to play a role in aetiology of autism based on a demonstrated involvement in the regulation of social behaviours. It is postulated that OXT reduces activation of the amygdala, inhibiting social anxiety, indicating a neural mechanism for the effects of OXT in social cognition. Genetic variation at the oxytocin receptor gene (OXTR) has been reported to be associated with autism. We examined 18 SNPs at the OXTR gene for association in three independent autism samples from Ireland, Portugal and the United Kingdom. We investigated cis-acting genetic effects on OXTR expression in lymphocytes and amygdala region of the brain using an allelic expression imbalance (AEI) assay and by investigating the correlation between RNA levels and genotype in the amygdala region. No marker survived multiple correction for association with autism in any sample or in a combined sample (n=436). Results from the AEI assay performed in the lymphoblast cell lines highlighted two SNPs associated with relative allelic abundance in OXTR (rs237897 and rs237895). Two SNPs were found to be effecting cis-acting variation through AEI in the amygdala. One was weakly correlated with total gene expression (rs13316193) and the other was highlighted in the lymphoblast cell lines (rs237895). Data presented here does not support the role of common genetic variation in OXTR in the aetiology of autism spectrum disorders in Caucasian samples."
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