Towards alpha- or beta-D-C-glycosyl compounds by tin-catalyzed addition of glycosyl radicals to acrylonitrile and vinylphosphonate,and flexible reduction of tetra-O-acetyl-alpha-D-glucopyranosyl bromide with cyanoborohydride

Photo-induced radical addition of acetylated alpha-D-glucopyranosyl bromide (1). to acrylonitrile or diethyl vinylphosphonate, in the presence of catalytic amounts of tri-n-butyltin chloride and sodium (or tetra-n-butylammonium) cyanoborohydride in excess, allowed efficient preparations of alpha-configurated nonononitrile and 2-(alpha-D-glucopyranosyl)-ethylphosphonate (79, 70% yields, respectively). These conditions led to 2-(alpha-D-manno-, and galactopyranosyl)-ethylphosphonates in 68 and 76% yields. Similarly, radical addition of acetylated 1-bromo-beta-D-glucopyranosyl chloride (2). to acrylonitrile or diethyl vinylphosphonate afforded mainly intermediate chlorides which, upon radical reduction with excess tri-n-butyltin hydride, afforded the corresponding beta anomers (40 and 38%, respectively) by sequential C-C and C-H bond formation. Stereocontrol relies on the alpha-stereoselective quenching of D-glycopyranos-1-yl radicals. We found also that UV light irradiation of 1 with excess NaBH(3)CN in tert-butanol afforded either 1,3,4,6-tetra-O-acetyl-2-deoxy-alpha-D-arabino-hexopyranose (65% after crystallization) or, when 10% mol thiophenol was added, 2,3,4,6-tetra-O-acetyl-1,5-anhydro-D-glucitol (79%). These are simple, tin-free, and easily controlled conditions, which compare well with known preparations of these reduced sugars.     

Rosiglitazone attenuates hypoxia-induced pulmonary arterial remodeling

Thiazolidinediones (TZDs) are insulin-sensitizing agents that also decrease systemic blood pressure, attenuate the formation of atherosclerotic lesions, and block remodeling of injured arterial walls. Recently, TZDs were shown to prevent pulmonary arterial (PA) remodeling in rats treated with monocrotaline. Presently we report studies testing the ability of the TZD rosiglitazone (ROSI) to attenuate pathological arterial remodeling in the lung and prevent the development of pulmonary hypertension (PH) in rats subjected to chronic hypoxia. PA remodeling was reduced in ROSI-treated animals exposed to hypoxia compared with animals exposed to hypoxia alone. ROSI treatment blocked muscularization of distal pulmonary arterioles and reversed remodeling and neomuscularization in lungs of animals previously exposed to chronic hypoxia. Decreased PA remodeling in ROSI-treated animals was associated with decreased smooth muscle cell proliferation, decreased collagen and elastin deposition, and increased matrix metalloproteinase-2 activity in the PA wall. Cells expressing the c-Kit cell surface marker were observed in the PA adventitia of untreated animals exposed to hypoxia but not in ROSI-treated hypoxic rats. Right ventricular hypertrophy and cardiomyocyte hypertrophy were also blunted in ROSI-treated hypoxic animals. Interestingly, mean PA pressures were elevated equally in the untreated and ROSI-treated groups, indicating that ROSI had no effect on the development of PH. However, mean PA pressure was normalized acutely in both groups of hypoxia-exposed animals by Fasudil, an agent that inhibits RhoA/Rho kinase-mediated vasoconstriction. We conclude that ROSI can attenuate and reverse PA remodeling and neomuscularization associated with hypoxic PH. However, this agent fails to block the development of PH, apparently because of its inability to repress sustained Rho kinase-mediated arterial vasoconstriction.     

Increased serine protease activity and cathelicidin promotes skin inflammation in rosacea

Acne rosacea is an inflammatory skin disease that affects 3% of the US population over 30 years of age and is characterized by erythema, papulopustules and telangiectasia. The etiology of this disorder is unknown, although symptoms are exacerbated by factors that trigger innate immune responses, such as the release of cathelicidin antimicrobial peptides. Here we show that individuals with rosacea express abnormally high levels of cathelicidin in their facial skin and that the proteolytically processed forms of cathelicidin peptides found in rosacea are different from those present in normal individuals. These cathelicidin peptides are a result of a post-translational processing abnormality associated with an increase in stratum corneum tryptic enzyme (SCTE) in the epidermis. In mice, injection of the cathelicidin peptides found in rosacea, addition of SCTE, and increasing protease activity by targeted deletion of the serine protease inhibitor gene Spink5 each increases inflammation in mouse skin. The role of cathelicidin in enabling SCTE-mediated inflammation is verified in mice with a targeted deletion of Camp, the gene encoding cathelicidin. These findings confirm the role of cathelicidin in skin inflammatory responses and suggest an explanation for the pathogenesis of rosacea by demonstrating that an exacerbated innate immune response can reproduce elements of this disease.     

Label-free Quantitative Urinary Proteomics Identifies the Arginase Pathway as a New Player in Congenital Obstructive Nephropathy

Obstructive nephropathy is a frequently encountered situation in newborns. In previous studies, the urinary peptidome has been analyzed for the identification of clinically useful biomarkers of obstructive nephropathy. However, the urinary proteome has not been explored yet and should allow additional insight into the pathophysiology of the disease. We have analyzed the urinary proteome of newborns (n = 5/group) with obstructive nephropathy using label free quantitative nanoLC-MS/MS allowing the identification and quantification of 970 urinary proteins. We next focused on proteins exclusively regulated in severe obstructive nephropathy and identified Arginase 1 as a potential candidate molecule involved in the development of obstructive nephropathy, located at the crossroad of pro- and antifibrotic pathways. The reduced urinary abundance of Arginase 1 in obstructive nephropathy was verified in independent clinical samples using both Western blot and MRM analysis. These data were confirmed in situ in kidneys obtained from a mouse obstructive nephropathy model. In addition, we also observed increased expression of Arginase 2 and increased total arginase activity in obstructed mouse kidneys. mRNA expression analysis of the related arginase pathways indicated that the pro-fibrotic arginase-related pathway is activated during obstructive nephropathy. Taken together we have identified a new actor in the development of obstructive nephropathy in newborns using quantitative urinary proteomics and shown its involvement in an in vivo model of disease. The present study demonstrates the relevance of such a quantitative urinary proteomics approach with clinical samples for a better understanding of the pathophysiology and for the discovery of potential therapeutic targets.Congenital obstructive nephropathy is the main cause of end stage renal disease (ESRD) in children (1). The most frequently found cause of congenital obstructive nephropathy is ureteropelvic junction (UPJ)¹ obstruction with an estimated incidence of 1 in 1000–1500 births. Milder forms of UPJ obstruction often progress to the spontaneous resolution of the pathology over time. This has led to a watchful waiting approach with surgical intervention only if renal deterioration is detected (2). Although this medical surveillance prevents unnecessary surgery, it mostly relies on invasive follow-up. Consequently with the aim to reduce this invasive follow-up, several groups have initiated research to identify noninvasive urinary biomarkers of UPJ obstruction using both targeted and nontargeted (e.g. proteome analysis based) strategies. Targeted strategies including urinary cytokine expression analyses failed to clearly determine the need for surgery in UPJ obstruction (3, 4). On the other hand, untargeted strategies have been more successful and by using urinary proteomics, biomarkers for renal and non-renal diseases have been identified (5–9). Using urinary peptidome analysis, we identified and validated a urinary peptide panel that predicted the clinical outcome of newborns with UPJ obstruction with 97% accuracy several months in advance (3, 10). An independent small-scale study confirmed the efficiency of this biomarker panel (7). These studies indicate the potential of urinary proteomics to predict the clinical fate of patients with UPJ obstruction. Although these endogenous urinary peptide biomarkers are of great potential clinical value, sequencing of these biomarkers mainly identified collagen fragments that are less informative on the pathophysiology of the disease. In contrast, studies of the high molecular weight urinary proteome (i.e. proteins) might be more informative on the pathophysiology of disease. Different approaches have been used in the past to characterize the urinary proteome, either by 2D-gel electrophoresis coupled to mass spectrometry (11, 12) or reverse phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) analysis (13–16). In-depth proteome analysis using extensive fractionation of the sample and high resolution, fast sequencing mass spectrometers have reported the identification of >2000 proteins in normal human urine (13, 15, 16). Here, we applied quantitative high-resolution label free LC-MS/MS analysis for the identification of urinary proteins associated to UPJ obstruction in newborns. Among a number of proteins uniquely associated with severe UPJ obstruction, we identified Arginase 1, not previously recognized in UPJ obstruction. Using an independent larger cohort, we further verified reduced urinary abundance of Arginase 1 using both Western blot and multiple reaction monitoring (MRM). Using the mouse model of obstructive nephropathy, we observed that the expression of arginases is modulated in situ in obstructed kidneys. Further gene expression analysis of the arginase pathway allowed us to hypothesize for a role of arginases in the development of fibrotic lesions in obstructive nephropathy.     

CRISPR typing and subtyping for improved laboratory surveillance of Salmonella infections

Laboratory surveillance systems for salmonellosis should ideally be based on the rapid serotyping and subtyping of isolates. However, current typing methods are limited in both speed and precision. Using 783 strains and isolates belonging to 130 serotypes, we show here that a new family of DNA repeats named CRISPR (clustered regularly interspaced short palindromic repeats) is highly polymorphic in Salmonella. We found that CRISPR polymorphism was strongly correlated with both serotype and multilocus sequence type. Furthermore, spacer microevolution discriminated between subtypes within prevalent serotypes, making it possible to carry out typing and subtyping in a single step. We developed a high-throughput subtyping assay for the most prevalent serotype, Typhimurium. An open web-accessible database was set up, providing a serotype/spacer dictionary and an international tool for strain tracking based on this innovative, powerful typing and subtyping tool.     

White Matter Reorganization and Functional Response after Focal Cerebral Ischemia in the Rat

After stroke, the brain has shown to be able to achieve spontaneous functional recovery despite severe cerebral damage. This phenomenon is poorly understood. To address this issue, focal transient ischemia was induced by 60 min middle cerebral artery occlusion in Wistar rats. The evolution of stroke was followed using two magnetic resonance imaging modalities: diffusion spectrum imaging (acquired before, one and four weeks after stroke) and functional magnetic resonance imaging (acquired before and five weeks after stroke). To confirm the imaging observations, immunohistochemical staining for myelin, astrocytes and macrophages/microglia was added. At four weeks after stroke, a focal alteration of the diffusion anisotropy was observed between the ipsilesional ventricle and the lesion area. Using tractography this perturbation was identified as reorganization of the ipsilesional internal capsule. Functional imaging at five weeks after ischemia demonstrated activation of the primary sensorimotor cortex in both hemispheres in all rats except one animal lacking a functional response in the ipsilesional cortex. Furthermore, fiber tracking showed a transhemispheric fiber connection through the corpus callosum, which-in the rat without functional recovery-was lost. Our study shows the influence of the internal capsule reorganization, combined with inter-hemispheric connections though the corpus callosum, on the functional activation of the brain from stroke. In conclusion, tractography opens a new door to non-invasively investigate the structural correlates of lack of functional recovery after stroke.     

Assessment of the genetic diversity of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans as a basis to identify putative pathogenicity genes and a type III secretion system of the SPI-1 family by multiple suppression subtractive hybridizations

Fluorescent amplified fragment length polymorphism revealed that strains of Xanthomonas axonopodis pv. phaseoli and Xanthomonas fuscans subsp. fuscans are genetically distinct and can be grouped into four genetic lineages. Four suppression subtractive hybridizations were then performed to isolate DNA fragments present in these bean pathogens and absent from closely related xanthomonads. Virulence gene candidates were identified such as homologs of hemagglutinins, TonB-dependent receptors, zinc-dependent metalloproteases, type III effectors, and type IV secretion system components. Unexpectedly, homologs of the type III secretion apparatus components (SPI-1 family), usually reported in animal pathogens and insect symbionts, were also detected.     

Venus flytrap biomechanics: Forces in the Dionaea muscipula trap

Biomechanics of morphing structures in the Venus flytrap has attracted the attention of scientists during the last 140 years. The trap closes in a tenth of a second if a prey touches a trigger hair twice. The driving force of the closing process is most likely due to the elastic curvature energy stored and locked in the leaves, which is caused by a pressure differential between the upper and lower layers of the leaf. The trap strikes, holds and compresses the prey. We have developed new methods for measuring all these forces involved in the hunting cycle. We made precise calibration of the piezoelectric sensor and performed direct measurements of the average impact force of the trap closing using a high speed video camera for the determination of time constants. The new equation for the average impact force was derived. The impact average force between rims of two lobes in the Venus flytrap was found equal to 149 mN and the corresponding pressure between the rims was about 41 kPa. Direct measurements of the constriction force in the trap of Dionaea muscipula was performed during gelatin digestion. This force increases in the process of digestion from zero to 450 mN with maximal constriction pressure created by the lobes reaching to 9 kPa. The insects and different small prey have little chance to escape after the snap of the trap. The prey would need to overpower the “escaping” force which is very strong and can reach up to 4 N.     

Platelet-derived growth factor BB induces nuclear export and proteasomal degradation of CREB via phosphatidylinositol 3-kinase/Akt signaling in pulmonary artery smooth muscle cells

Cyclic AMP response element binding protein (CREB) content is diminished in smooth muscle cells (SMCs) in remodeled pulmonary arteries from animals with pulmonary hypertension and in the SMC layers of atherogenic systemic arteries and cardiomyocytes from hypertensive individuals. Loss of CREB can be induced in cultured SMCs by chronic exposure to hypoxia or platelet-derived growth factor BB (PDGF-BB). Here we investigated the signaling pathways and mechanisms by which PDGF elicits depletion of SMC CREB. Chronic PDGF treatment increased CREB ubiquitination in SMCs, while treatment of SMCs with the proteasome inhibitor lactacystin prevented decreases in CREB content. The nuclear export inhibitor leptomycin B also prevented depletion of SMC CREB alone or in combination with lactacystin. Subsequent studies showed that PDGF activated extracellular signal-regulated kinase, Jun N-terminal protein kinase, and phosphatidylinositol 3 (PI3)-kinase pathways in SMCs. Inhibition of these pathways blocked SMC proliferation in response to PDGF, but only inhibition of PI3-kinase or its effector, Akt, blocked PDGF-induced CREB loss. Finally, chimeric proteins containing enhanced cyan fluorescent protein linked to wild-type CREB or CREB molecules with mutations in several recognized phosphorylation sites were introduced into SMCs. PDGF treatment reduced the levels of each of these chimeric proteins except for one containing mutations in adjacent serine residues (serines 103 and 107), suggesting that CREB loss was dependent on CREB phosphorylation at these sites. We conclude that PDGF stimulates nuclear export and proteasomal degradation of CREB in SMCs via PI3-kinase/Akt signaling. These results indicate that in addition to direct phosphorylation, proteolysis and intracellular localization are key mechanisms regulating CREB content and activity in SMCs.     

Multilocus sequence analysis and type III effector repertoire mining provide new insights into the evolutionary history and virulence of Xanthomonas oryzae

Multilocus sequence analysis (MLSA) and type III effector (T3E) repertoire mining were performed to gain new insights into the genetic relatedness of Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), two major bacterial pathogens of rice. Based on a collection of 45 African and Asian strains, we first sequenced and analysed three housekeeping genes by MLSA, Bayesian clustering and a median-joining network approach. Second, we investigated the distribution of 32 T3E genes, which are known to be major virulence factors of plant pathogenic bacteria, in all selected strains, by polymerase chain reaction and dot-blot hybridization methods. The diversity observed within housekeeping genes, as well as within T3E repertoires, clearly showed that both pathogens belong to closely related, but distinct, phylogenetic groups. Interestingly, these evolutionary groups are differentiated according to the geographical origin of the strains, suggesting that populations of Xoo and Xoc might be endemic in Africa and Asia, and thus have evolved separately. We further revealed that T3E gene repertoires of both pathogens comprise core and variable gene suites that probably have distinct roles in pathogenicity and different evolutionary histories. In this study, we carried out a functional analysis of xopO, a differential T3E gene between Xoo and Xoc, to determine the involvement of this gene in tissue specificity. Altogether, our data contribute to a better understanding of the evolutionary history of Xoo and Xoc in Africa and Asia, and provide clues for functional studies aiming to understand the virulence, host and tissue specificity of both rice pathogens.