Shear-induced endothelial mechanotransduction: the interplay between reactive oxygen species (ROS) and nitric oxide (NO) and the pathophysiological implications

Hemodynamic shear stress, the blood flow-generated frictional force acting on the vascular endothelial cells, is essential for endothelial homeostasis under normal physiological conditions. Mechanosensors on endothelial cells detect shear stress and transduce it into biochemical signals to trigger vascular adaptive responses. Among the various shear-induced signaling molecules, reactive oxygen species (ROS) and nitric oxide (NO) have been implicated in vascular homeostasis and diseases. In this review, we explore the molecular, cellular, and vascular processes arising from shear-induced signaling (mechanotransduction) with emphasis on the roles of ROS and NO, and also discuss the mechanisms that may lead to excessive vascular remodeling and thus drive pathobiologic processes responsible for atherosclerosis. Current evidence suggests that NADPH oxidase is one of main cellular sources of ROS generation in endothelial cells under flow condition. Flow patterns and magnitude of shear determine the amount of ROS produced by endothelial cells, usually an irregular flow pattern (disturbed or oscillatory) producing higher levels of ROS than a regular flow pattern (steady or pulsatile). ROS production is closely linked to NO generation and elevated levels of ROS lead to low NO bioavailability, as is often observed in endothelial cells exposed to irregular flow. The low NO bioavailability is partly caused by the reaction of ROS with NO to form peroxynitrite, a key molecule which may initiate many pro-atherogenic events. This differential production of ROS and RNS (reactive nitrogen species) under various flow patterns and conditions modulates endothelial gene expression and thus results in differential vascular responses. Moreover, ROS/RNS are able to promote specific post-translational modifications in regulatory proteins (including S-glutathionylation, S-nitrosylation and tyrosine nitration), which constitute chemical signals that are relevant in cardiovascular pathophysiology. Overall, the dynamic interplay between local hemodynamic milieu and the resulting oxidative and S-nitrosative modification of regulatory proteins is important for ensuing vascular homeostasis. Based on available evidence, it is proposed that a regular flow pattern produces lower levels of ROS and higher NO bioavailability, creating an anti-atherogenic environment. On the other hand, an irregular flow pattern results in higher levels of ROS and yet lower NO bioavailability, thus triggering pro-atherogenic effects.     

Birds are islands for parasites

Understanding the mechanisms driving the extraordinary diversification of parasites is a major challenge in evolutionary biology. Co-speciation, one proposed mechanism that could contribute to this diversity is hypothesized to result from allopatric co-divergence of host–parasite populations. We found that island populations of the Galápagos hawk (Buteo galapagoensis) and a parasitic feather louse species (Degeeriella regalis) exhibit patterns of co-divergence across variable temporal and spatial scales. Hawks and lice showed nearly identical population genetic structure across the Galápagos Islands. Hawk population genetic structure is explained by isolation by distance among islands. Louse population structure is best explained by hawk population structure, rather than isolation by distance per se, suggesting that lice tightly track the recent population histories of their hosts. Among hawk individuals, louse populations were also highly structured, suggesting that hosts serve as islands for parasites from an evolutionary perspective. Altogether, we found that host and parasite populations may have responded in the same manner to geographical isolation across spatial scales. Allopatric co-divergence is likely one important mechanism driving the diversification of parasites.     

alpha-N-acetylgalactosamine-capping of chondroitin sulfate core region oligosaccharides primed on xylosides

We previously reported that cultured mammalian cells incubated with 4- methylumbelliferyl (MU) or p -nitrophenyl (pNP) beta-xyloside synthesize an alpha-GalNAc-terminated pentasaccharide resembling the glycosaminoglycan-core protein linkage region. Here we show that human melanoma M21 cells and human neuroblastoma cells incubated with Xylbeta- MU/pNP also make an alpha-GalNAc-terminated heptasaccharide containing one chondroitin disaccharide repeat. High performance liquid chromatography and matrix-assisted laser desorption ionization mass spectrometry analysis of intact or glycosidase-digested xyloside showed the structure as: GalNAcalphaGlcAbeta1,3GalNAcbeta1,4GlcAbeta1,3Galbe ta1,3Galbeta1, 4Xylbeta-MU/pNP. The alpha-GalNAc-terminated xylosides can account for approximately 10% of the total Xylbeta-MU/pNP products ( approximately 1.5 nmol/h/mg). These results show that GalNAcalphaGlcAbeta-modification is relatively abundant, but not unique to the GAG-linkage tetrasaccharide. alpha-GalNAc addition to the GlcA residue does not appear to be an extension of general phase II detoxification of xenobiotics that involve glucuronidation, since M21 cells incubated with MU synthesize only 0.3 pmol GlcAbeta-MU/h/mg protein, and undetectable amount of GalNAcalphaGlcAbeta-MU (<40 fmol/h/mg). Further, subcellular fractionation shows that the alpha- N- acetylgalactosaminyltransferase activity colocalizes in the Golgi with other glycosyl transferases and not in the ER, where xenobiotic detoxification glucuronosyltransferases are found. Although GalNAcalphaGlcAbeta-terminal modification has not been detected on naturally occurring GAG chains, the substantial amount of alpha-GalNAc transferase activity suggests that the alpha-GalNAc transferase could utilize other GlcA-containing glycoconjugates as acceptors.      

Animal-Driven Nutrient Supply Declines Relative to Ecosystem Nutrient Demand Along a Pond Hydroperiod Gradient

Ecosystems - In many lentic ecosystems, hydroperiod, or the duration of inundation, controls animal community composition and biomass. Although hydroperiod-imposed differences in wetland animal...     

High-Affinity Accumulation of a Maytansinoid in Cells via Weak Tubulin Interaction

The microtubule-targeting maytansinoids accumulate in cells and induce mitotic arrest at 250- to 1000-fold lower concentrations than those required for their association with tubulin or microtubules. To identify the mechanisms of this intracellular accumulation and exceptional cytotoxicity of maytansinoids we studied interaction of a highly cytotoxic maytansinoid, S-methyl DM1 and several other maytansinoids with cells. S-methyl DM1 accumulated inside the cells with a markedly higher apparent affinity than to tubulin or microtubules. The apparent affinities of maytansinoids correlated with their cytotoxicities. The number of intracellular binding sites for S-methyl DM1 in MCF7 cells was comparable to the number of tubulin molecules per cell (~ 4–6 × 10⁷ copies). Efflux of ³ [H]-S-methyl DM1 from cells was enhanced in the presence of an excess of non-labeled S-methyl DM1, indicating that re-binding of ³ [H]-S-methyl DM1 to intracellular binding sites contributed to its intracellular retention. Liposomes loaded with non-polymerized tubulin recapitulated the apparent high-affinity association of S-methyl DM1 to cells. We propose a model for the intracellular accumulation of maytansinoids in which molecules of the compounds diffuse into a cell and associate with tubulin. Affinities of maytansinoids for individual tubulin molecules are weak, but the high intracellular concentration of tubulin favors, after dissociation of a compound-tubulin complex, their re-binding to a tubulin molecule, or to a tip of a microtubule in the same cell, over their efflux. As a result, a significant fraction of microtubule tips is occupied with a maytansinoid when added to cells at sub-nanomolar concentrations, inducing mitotic arrest and cell death.     

Serum Glycoprotein Biomarker Discovery and Qualification Pipeline Reveals Novel Diagnostic Biomarker Candidates for Esophageal Adenocarcinoma

We report an integrated pipeline for efficient serum glycoprotein biomarker candidate discovery and qualification that may be used to facilitate cancer diagnosis and management. The discovery phase used semi-automated lectin magnetic bead array (LeMBA)-coupled tandem mass spectrometry with a dedicated data-housing and analysis pipeline; GlycoSelector (http://glycoselector.di.uq.edu.au). The qualification phase used lectin magnetic bead array-multiple reaction monitoring-mass spectrometry incorporating an interactive web-interface, Shiny mixOmics (http://mixomics-projects.di.uq.edu.au/Shiny), for univariate and multivariate statistical analysis. Relative quantitation was performed by referencing to a spiked-in glycoprotein, chicken ovalbumin. We applied this workflow to identify diagnostic biomarkers for esophageal adenocarcinoma (EAC), a life threatening malignancy with poor prognosis in the advanced setting. EAC develops from metaplastic condition Barrett''s esophagus (BE). Currently diagnosis and monitoring of at-risk patients is through endoscopy and biopsy, which is expensive and requires hospital admission. Hence there is a clinical need for a noninvasive diagnostic biomarker of EAC. In total 89 patient samples from healthy controls, and patients with BE or EAC were screened in discovery and qualification stages. Of the 246 glycoforms measured in the qualification stage, 40 glycoforms (as measured by lectin affinity) qualified as candidate serum markers. The top candidate for distinguishing healthy from BE patients'' group was Narcissus pseudonarcissus lectin (NPL)-reactive Apolipoprotein B-100 (p value = 0.0231; AUROC = 0.71); BE versus EAC, Aleuria aurantia lectin (AAL)-reactive complement component C9 (p value = 0.0001; AUROC = 0.85); healthy versus EAC, Erythroagglutinin Phaseolus vulgaris (EPHA)-reactive gelsolin (p value = 0.0014; AUROC = 0.80). A panel of 8 glycoforms showed an improved AUROC of 0.94 to discriminate EAC from BE. Two biomarker candidates were independently verified by lectin magnetic bead array-immunoblotting, confirming the validity of the relative quantitation approach. Thus, we have identified candidate biomarkers, which, following large-scale clinical evaluation, can be developed into diagnostic blood tests. A key feature of the pipeline is the potential for rapid translation of the candidate biomarkers to lectin-immunoassays.Biomarkers play a central role in health care by enabling accurate diagnosis and prognosis; hence there is extensive research on the identification and development of novel biomarkers. However, despite numerous biomarker publications over the years (1), only a handful of new cancer biomarkers have successfully completed the journey from discovery, qualification, to verification and validation (2–4). One possible way to overcome this challenge is to develop an integrated biomarker pipeline that facilitates the smooth and successful transition from discovery to validation (5–10). The first and foremost consideration in an integrated pipeline is the sample source. In general, most of the proteomics based workflows use tissues or proximal fluids during the discovery phase, with the goal of extending the findings to plasma. Although this approach avoid the high complexity serum/plasma proteome and the associated requisite multi-dimensional sample separation in discovery stages, it often leads to failure when the candidates are not detected in plasma because of the limited sensitivity of the available analytical methods, or the absence of candidates in the plasma (11). To overcome this pitfall, we have developed an integrated glycoprotein biomarker pipeline, which can simply and rapidly isolate glycosylated proteins from serum to enable high throughput analysis of differentially glycosylated proteins in discovery and qualification stages.The workflow utilizes naturally occurring glycan binding proteins, lectins, in a semi-automated high throughput workflow called lectin magnetic bead array-tandem mass spectrometry (LeMBA-MS/MS)¹ (12, 13). Although lectins have been well-utilized in glycobiology and biomarker discovery (14–17), the LeMBA-MS/MS workflow demonstrates several unique features. First, serum glycoproteins are isolated in a single-step using 20 individual lectin-coated magnetic beads in microplate format. Second, we have optimized the concentrations of salts and detergents for sample denaturation to avoid co-isolation of protein complexes without adversely affecting lectin pull-down efficiency. Third, a liquid handler is used for sample processing to facilitate high-throughput screening and increase reproducibility. In addition, we have optimized on-bead trypsin digestion and incorporated lectin-exclusion lists during nano-LC-MS/MS to identify nonglycosylated peptides from the isolated glycoproteins. With these innovations, LeMBA-MS/MS demonstrates nanomolar sensitivity and linearity, and applicability across species (12). Compared with existing single, serial or multi-lectin affinity chromatography (18, 19), LeMBA-MS/MS offers the capability to simultaneously screen 20 lectins in a semi-automated, high throughput manner. On the other hand, because LeMBA-MS/MS identifies the nonglycosylated peptides, it cannot be used for glycan site assignment and glycan structure elucidation (20–23). However, the main advantage of LeMBA, we believe, is as a part of an integrated translational biomarker pipeline leading to lectin immunoassays. The lack of glycan structure details is not critical for clinical translation, as exemplified by the alpha-fetoprotein-L3 (AFP-L3) test, which measures the Lens culinaris agglutinin (LCA) binding fraction of serum alpha-fetoprotein (24, 25), and has been approved by the U.S. Food and Drug Administration for detection of hepatocellular carcinoma.In this study, we report the extension of the glycoprotein biomarker pipeline to the qualification phase with LeMBA-MRM-MS, and introduce statistical analysis pipelines GlycoSelector (http://glycoselector.di.uq.edu.au/) and Shiny mixOmics (http://mixomics-projects.di.uq.edu.au/Shiny) for the discovery and qualification phases, respectively. The utility of this integrated serum glycoprotein biomarker pipeline is demonstrated using esophageal adenocarcinoma (EAC) with unmet clinical need for an in vitro diagnostic test. EAC is a lethal malignancy of the lower esophagus with very poor 5-year survival rate of less than 25% (26). EAC is becoming increasingly common and its incidence is associated with the prevalent precursor metaplastic condition Barrett''s esophagus (BE), but with a low annual conversion rate of up to 1% (27). A common set of risk factors are described for BE and EAC, include gastroesophageal reflux disease (GERD), obesity, male gender, and smoking (28, 29). The current endoscopy-biopsy based diagnosis is invasive and costly, leading to an ineffective surveillance program. A blood test employing serum biomarkers that can distinguish patients with EAC from those with either BE or healthy tissue would, potentially, change the paradigm for the way in which BE and EAC are managed in the population (30). Serum glycan profiling studies have shown differential expression of glycan structures between healthy, BE, early dysplastic and EAC patients (31–35). However, diagnostic serum glycoproteins showing differential glycosylation hence differential lectin binding remain to be discovered, making it a suitable disease model for this study.     

应用ARIMA模型预测宝安区某街道其它感染性腹泻发病率的探讨

目的：探讨应用ARIMA模型预测宝安区某街道其它感染性腹泻发病率的可行性。方法：应用SPSS13．0软件对2005年～2009年宝安区某街道其它感染性腹泻逐月发病率进行ARIMA模型建模拟合,用所得到的模型对2010年各月发病率进行预测,并评价其预测效果。结果：宝安区某街道其它感染性腹泻发病率每年11月为发病高峰,ARIMA（0,1,1）（0,1,0）12模型是其拟合的最佳模型,其预测结果和实际值绝对误差的绝对值最大为930．47,最小为1．96,平均值214．83,平均相对误差百分比39．04％。结论：模型虽然起到一定的预测效果,但预测精度仍存在误差,可通过积累新的周期数据对ARIMA模型进行修正和重新拟合,也可尝试新的预测方法或其他模型,才能加强和保证预测的精度。     

应用ARIMA模型预测宝安区某街道其它感染性腹泻发病率的探讨

目的:探讨应用ARIMA模型预测宝安区某街道其它感染性腹泻发病率的可行性。方法:应用SPSSl3.0软件对2005年～2009年宝安区某街道其它感染性腹泻逐月发病率进行ARIMA模型建模拟合,用所得到的模型对2010年各月发病率进行预测,并评价其预测效果。结果:宝安区桌街道其它感染性腹泻发病率每年11月为发病高峰,ARIMA(0,1,1)(0,1,0)12模型是其拟合的最佳模型,其预测结果和实际值绝对误差的绝对值最大为930.47,最小为1.96,平均值214.83,平均相对误差百分比39.04%。结论:模型虽然起到一定的预测效果,但预测精度仍存在误差,可通过积累新的周期数据对ARIMA模型进行修正和重新拟合,也可尝试新的预测方法或其他模型,才能加强和保证预测的精度。     

Rapid changes in phospho-MAP/tau epitopes during neuronal stress: cofilin-actin rods primarily recruit microtubule binding domain epitopes

Abnormal mitochondrial function is a widely reported contributor to neurodegenerative disease including Alzheimer's disease (AD), however, a mechanistic link between mitochondrial dysfunction and the initiation of neuropathology remains elusive. In AD, one of the earliest hallmark pathologies is neuropil threads comprising accumulated hyperphosphorylated microtubule-associated protein (MAP) tau in neurites. Rod-like aggregates of actin and its associated protein cofilin (AC rods) also occur in AD. Using a series of antibodies--AT270, AT8, AT100, S214, AT180, 12E8, S396, S404 and S422--raised against different phosphoepitopes on tau, we characterize the pattern of expression and re-distribution in neurites of these phosphoepitope labels during mitochondrial inhibition. Employing chick primary neuron cultures, we demonstrate that epitopes recognized by the monoclonal antibody 12E8, are the only species rapidly recruited into AC rods. These results were recapitulated with the actin depolymerizing drug Latrunculin B, which induces AC rods and a concomitant increase in the 12E8 signal measured on Western blot. This suggests that AC rods may be one way in which MAP redistribution and phosphorylation is influenced in neurons during mitochondrial stress and potentially in the early pathogenesis of AD.