Synergistic effect of sildenafil combined with controlled hypothermia to alleviate microglial activation after neonatal hypoxia-ischemia in rats.

BACKGROUND AND PURPOSE: The only validated treatment to prevent brain damage associated with hypoxia-ischemia (HI) encephalopathy of the newborn is controlled hypothermia with limited benefits. Additional putative neuroprotective drug candidates include sildenafil citrate, a phosphodiesterase-type 5 inhibitor. The main objective of this preclinical study is to assess its ability to reduce HI-induced neuroinflammation, in particular through its potential effect on microglial activation. METHODS: HI was induced in P10 Sprague-Dawley rats by unilateral carotid permanent artery occlusion and hypoxia (HI) and treated by either hypothermia (HT) alone, Sildenafil (Sild) alone or combined treatment (SildHT). Lesion size and glial activation were analyzed by immunohistochemistry, qRT-PCR, and proteomic analyses performed at P13. RESULTS: None of the treatments was associated with a significant early reduction in lesion size 72h after HI, despite significant changes in tissue loss distribution. Significant reductions in both Iba1 + (within the ipsilateral hemisphere) and GFAP + cells (within the ipsilateral hippocampus) were observed in SildHT group, but not in the other treatment groups. In microglia-sorted cells, pro-inflammatory markers, i.e. Il1b, Il6, Nos2, and CD86 were significantly downregulated in SildHT treatment group only. These changes were restricted to the ipsilateral hemisphere, were not evidenced in sorted astrocytes, and were not sex dependent. Proteomic analyses in sorted microglia refined the pro-inflammatory effect of HI and confirmed a biologically relevant impact of SildHT on specific molecular pathways including genes related to neutrophilic functions. CONCLUSIONS: Our findings suggest that Sildenafil combined with controlled hypothermia produces maximum effect in mitigating microglial activation induced by HI through complex proteomic regulation. The reduction of neuroinflammation induced by Sildenafil may represent an interesting therapeutic strategy for neonatal neuroprotection.

Site-Selective Unnatural Amino Acid Incorporation at Single or Multiple Positions to Control Sugar-Protein Connectivity in Glycoconjugate Vaccine Candidates.

In cellulo site-specific unnatural amino acid incorporation based on amber stop codon reassignment is a powerful tool to modify proteins at defined positions. This technique is herein applied to the selective functionalization of the Pneumococcal surface adhesin A protein at three distinct positions. Nϵ -propargyloxycarbonyl-l-lysine residues were incorporated and their alkyne groups reacted using click-chemistry with a synthetic azido-functionalized tetrasaccharide representative of one repeat unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Anti-PsaA antibody response induced in mice by the trivalent glycoconjugate was determined in comparison with corresponding monovalent and randomly functionalized conjugates. Our results suggest that controlled was superior to random conjugation for preserving antigenicity. In definitive, the reported strategy offers a unique opportunity to study the impact of carbohydrate antigen-carrier protein connectivity on immunogenicity.

Molecular characterization of accripin11, a soluble shell protein with an acidic C-terminus, identified in the prismatic layer of the Mediterranean fan mussel Pinna nobilis (Bivalvia, Pteriomorphia).

We have identified a novel shell protein, accripin11, as a major soluble component of the calcitic prisms of the fan mussel Pinna nobilis. Initially retrieved from a cDNA library, its full sequence is confirmed here by transcriptomic and proteomic approaches. The sequence of the mature protein is 103 residues with a theoretical molecular weight of 11 kDa and is moderately acidic (pI 6.74) except for its C-terminus which is highly enriched in aspartic acid. The protein exhibits a peculiar cysteine pattern in its central domain. The full sequence shares similarity with six other uncharacterized molluscan shell proteins from the orders Ostreida, Pteriida and Mytilida, all of which are pteriomorphids and produce a phylogenetically restricted pattern of nacro-prismatic shell microstructures. This suggests that accripin11 is a member of a family of clade-specific shell proteins. A 3D model of accripin11 was predicted with AlphaFold2, indicating that it possesses three short alpha helices and a disordered C-terminus. Recombinant accripin11 was tested in vitro for its ability to influence the crystallization of CaCO3 , while a polyclonal antibody was able to locate accripin11 to prismatic extracts, particularly in the acetic acid-soluble matrix. The putative functions of accripin11 are further discussed in relation to shell biomineralization.

Role of Caspase-10-P13tBID axis in erythropoiesis regulation.

Red blood cell production is negatively controlled by the rate of apoptosis at the stage of CFU-E/pro-erythroblast differentiation, depending on the balance between erythropoietin (EPO) levels and activation of the Fas/FasL pathway. At this stage, activation of transient caspases through depolarization via mitochondrial outer membrane permeabilization (MOMP) is also required for terminal erythroid differentiation. Molecular mechanisms regulating the differential levels of MOMP during differentiation and apoptosis, however, remain poorly understood. Here we show a novel and essential role for the caspase-10-P13-tBID axis in erythroid terminal differentiation. Caspase-10 (but not caspase-8, which is activated during apoptosis) is activated at the early stages of erythroid terminal differentiation leading to the cleavage of P22-BID into P18-tBID, and later into P13-tBID. Erythropoietin (EPO) by inducing casein kinase I alpha (CKIα) expression, which in turn phosphorylates P18-tBID, prevents the generation of MYR-P15-tBID (leading to apoptosis) and allows the generation of P13-tBID by caspase-10. Unlike P15-tBID, P13-tBID is not myristoylated and as such, does not irreversibly anchor the mitochondrial membrane resulting in a transient MOMP. Likewise, transduction of a P13-tBID fragment induces rapid and strong erythroid terminal differentiation. Thus, EPO modulates the pattern of BID cleavage to control the level of MOMP and determines the fate of erythroblasts between apoptosis and differentiation. This pathway is impaired in 5q- myelodysplastic syndromes because of CK1α haplo-insufficiency and may contribute to erythroid differentiation arrest and high sensitivity of this disease to lenalidomide (LEN).

Whole-Genome Sequencing Highlights Conservative Genomic Strategies of a Stress-Tolerant, Long-Lived Scleractinian Coral, Porites australiensis Vaughan, 1918.

Massive corals of the genus Porites, common, keystone reef builders in the Indo-Pacific Ocean, are distinguished by their relative stress tolerance and longevity. In order to identify genetic bases of these attributes, we sequenced the complete genome of a massive coral, Porites australiensis. We developed a genome assembly and gene models of comparable quality to those of other coral genomes. Proteome analysis identified 60 Porites skeletal matrix protein genes, all of which show significant similarities to genes from other corals and even to those from a sea anemone, which has no skeleton. Nonetheless, 30% of its skeletal matrix proteins were unique to Porites and were not present in the skeletons of other corals. Comparative genomic analyses showed that genes widely conserved among other organisms are selectively expanded in Porites. Specifically, comparisons of transcriptomic responses of P. australiensis and Acropora digitifera, a stress-sensitive coral, reveal significant differences in regard to genes that respond to increased water temperature, and some of the genes expanded exclusively in Porites may account for the different thermal tolerances of these corals. Taken together, widely shared genes may have given rise to unique biological characteristics of Porites, massive skeletons and stress tolerance.

The mitochondrially-localized nucleoside diphosphate kinase D (NME4) is a novel metastasis suppressor.

BACKGROUND: Mitochondrial nucleoside diphosphate kinase (NDPK-D, NME4, NM23-H4) is a multifunctional enzyme mainly localized in the intermembrane space, bound to the inner membrane. RESULTS: We constructed loss-of-function mutants of NDPK-D, lacking either NDP kinase activity or membrane interaction and expressed mutants or wild-type protein in cancer cells. In a complementary approach, we performed depletion of NDPK-D by RNA interference. Both loss-of-function mutations and NDPK-D depletion promoted epithelial-mesenchymal transition and increased migratory and invasive potential. Immunocompromised mice developed more metastases when injected with cells expressing mutant NDPK-D as compared to wild-type. This metastatic reprogramming is a consequence of mitochondrial alterations, including fragmentation and loss of mitochondria, a metabolic switch from respiration to glycolysis, increased ROS generation, and further metabolic changes in mitochondria, all of which can trigger pro-metastatic protein expression and signaling cascades. In human cancer, NME4 expression is negatively associated with markers of epithelial-mesenchymal transition and tumor aggressiveness and a good prognosis factor for beneficial clinical outcome. CONCLUSIONS: These data demonstrate NME4 as a novel metastasis suppressor gene, the first localizing to mitochondria, pointing to a role of mitochondria in metastatic dissemination.

The 'Shellome' of the Crocus Clam Tridacna crocea Emphasizes Essential Components of Mollusk Shell Biomineralization.

Molluscan shells are among the most fascinating research objects because of their diverse morphologies and textures. The formation of these delicate biomineralized structures is a matrix-mediated process. A question that arises is what are the essential components required to build these exoskeletons. In order to understand the molecular mechanisms of molluscan shell formation, it is crucial to identify organic macromolecules in different shells from diverse taxa. In the case of bivalves, however, taxon sampling in previous shell proteomics studies are focused predominantly on representatives of the class Pteriomorphia such as pearl oysters, edible oysters and mussels. In this study, we have characterized the shell organic matrix from the crocus clam, Tridacna crocea, (Heterodonta) using various biochemical techniques, including SDS-PAGE, FT-IR, monosaccharide analysis, and enzyme-linked lectin assay (ELLA). Furthermore, we have identified a number of shell matrix proteins (SMPs) using a comprehensive proteomics approach combined to RNA-seq. The biochemical studies confirmed the presence of proteins, polysaccharides, and sulfates in the T. crocea shell organic matrix. Proteomics analysis revealed that the majority of the T. crocea SMPs are novel and dissimilar to known SMPs identified from the other bivalve species. Meanwhile, the SMP repertoire of the crocus clam also includes proteins with conserved functional domains such as chitin-binding domain, VWA domain, and protease inhibitor domain. We also identified BMSP (Blue Mussel Shell Protein, originally reported from Mytilus), which is widely distributed among molluscan shell matrix proteins. Tridacna SMPs also include low-complexity regions (LCRs) that are absent in the other molluscan genomes, indicating that these genes may have evolved in specific lineage. These results highlight the diversity of the organic molecules - in particular proteins - that are essential for molluscan shell formation.

The shell matrix and microstructure of the Ram's Horn squid: Molecular and structural characterization.

Molluscs are one of the most diversified phyla among metazoans. Most of them produce an external calcified shell, resulting from the secretory activity of a specialized epithelium of the calcifying mantle. This biomineralization process is controlled by a set of extracellular macromolecules, the organic matrix. In spite of several studies, these components are mainly known for bivalves and gastropods. In the present study, we investigated the physical and biochemical properties of the internal planispiral shell of the Ram's Horn squid Spirula spirula. Scanning Electron Microscope investigations of the shell reveal a complex microstructural organization. The saccharides constitute a quantitatively important moiety of the matrix, as shown by Fourier-transform infrared and solid-state nuclear magnetic resonance spectroscopies. NMR identified ß-chitin and additional polysaccharides for a total amount of 80% of the insoluble fraction. Proteomics was applied to both soluble and insoluble matrices and in silico searches were performed, first on heterologous metazoans models, and secondly on an unpublished transcriptome of Spirula spirula. In the first case, several peptides were identified, some of them matching with tyrosinase, chitinase 2, protease inhibitor, or immunoglobulin. In the second case, 39 hits were obtained, including transferrin, a serine protease inhibitor, matrilin, or different histones. The very few similarities with known molluscan shell matrix proteins suggest that Spirula spirula uses a unique set of shell matrix proteins for constructing its internal shell. The absence of similarity with closely related cephalopods demonstrates that there is no obvious phylogenetic signal in the cephalopod skeletal matrix.

The shell matrix of the european thorny oyster, Spondylus gaederopus: microstructural and molecular characterization.

Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterised. We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe's cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences. This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids - fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.

Identification of hemicatenane-specific binding proteins by fractionation of HeLa nuclei extracts.

DNA hemicatenanes (HCs) are four-way junctions in which one strand of a double-stranded helix is catenated with one strand of another double-stranded DNA. Frequently mentioned as DNA replication, recombination and repair intermediates, they have been proposed to participate in the spatial organization of chromosomes and in the regulation of gene expression. To explore potential roles of HCs in genome metabolism, we sought to purify proteins capable of binding specifically HCs by fractionating nuclear extracts from HeLa cells. This approach identified three RNA-binding proteins: the Tudor-staphylococcal nuclease domain 1 (SND1) protein and two proteins from the Drosophila behavior human splicing family, the paraspeckle protein component 1 and the splicing factor proline- and glutamine-rich protein. Since these proteins were partially pure after fractionation, truncated forms of these proteins were expressed in Escherichia coli and purified to near homogeneity. The specificity of their interaction with HCs was re-examined in vitro. The two truncated purified SND1 proteins exhibited specificity for HCs, opening the interesting possibility of a link between the basic transcription machinery and HC structures via SND1.

Proteome analysis of formalin-fixed paraffin-embedded colorectal adenomas reveals the heterogeneous nature of traditional serrated adenomas compared to other colorectal adenomas.

Traditional serrated adenoma (TSA) remains the least understood of all the colorectal adenomas, although these lesions have been associated with a significant cancer risk, twice that of the conventional adenoma (CAD) and of the sessile serrated adenoma (SSA/P). This study was performed to investigate the proteomic profiles of the different colorectal adenomas to better understand the pathogenesis of TSA. We performed a global quantitative proteome analysis using the label-free quantification (LFQ) method on 44 colorectal adenoma (12 TSAs, 15 CADs, and 17 SSA/Ps) and 17 normal colonic mucosa samples, archived as formalin-fixed paraffin-embedded blocks. Unsupervised consensus hierarchical clustering applied to the whole proteomic profile of the 44 colorectal adenomas identified four subtypes: C1 and C2 were well-individualized clusters composed of all the CADs (15/15) and most of the SSA/Ps (13/17), respectively. This is consistent with the fact that CADs and SSA/Ps are homogeneous and distinct colorectal adenoma entities. In contrast, TSAs were subdivided into C3 and C4 clusters, consistent with the more heterogeneous entity of TSA at the morphologic and molecular levels. Comparison of the proteome expression profile between the adenoma subtypes and normal colonic mucosa further confirmed the heterogeneous nature of TSAs, which overlapped either on CADs or SSA/Ps, whereas CADs and SSAs formed homogeneous and distinct entities. Furthermore, we identified LEFTY1 a new potential marker for TSAs that may be relevant for the pathogenesis of TSA. LEFTY1 is an inhibitor of the Nodal/TGFß pathway, which we found to be one of the most overexpressed proteins specifically in TSAs. This finding was confirmed by immunohistochemistry. Our study confirms that CADs and SSA/Ps form homogeneous and distinct colorectal adenoma entities, whereas TSAs are a heterogeneous entity and may arise from either SSA/Ps or from normal mucosa evolving through a process related to the CAD pathway. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

Activation of platelets and neutrophils in septic shock results in the formation of microvascular clots containing an intricate scaffold of fibrin with neutrophil extracellular traps (NETs) DNA. NETs contain multiple components that might impact endogenous fibrinolysis, resulting in failure to lyse clots in the microcirculation and residual systemic microthrombosis. We propose herein that the reservoir of human neutrophil elastase (HNE) on NETs may directly interfere with the fibrinolytic mechanism via a plasminogen proteolytic pathway. To investigate this mechanism, we constructed fibrin-NETs matrices by seeding and activating neutrophils onto a fibrin surface and monitored plasminogen activation or degradation. We demonstrate that the elastase activity of HNE-DNA complexes is protected from inhibition by plasma antiproteases and sustains its ability to degrade plasminogen. Using mass spectrometry proteomic analysis, we identified plasminogen fragments composed of kringle (K) domains (K1+2+3, k1+2+3+4) and the serine protease (SP) region (K5-SP). We further demonstrate that patients with septic shock with disseminated intravascular coagulation have circulating HNE-DNA complexes, HNE-derived plasminogen fragments, a low plasminogen concentration, and a reduced capacity to generate plasmin onto fibrin. In conclusion, we show that NETs bearing active HNE-DNA complexes reduce plasminogen into fragments, thus impairing fibrinolysis by decreasing the local plasminogen concentration, plasminogen binding to fibrin, and localized plasmin formation.-Barbosa da Cruz, D., Helms, J., Aquino, L. R., Stiel, L., Cougourdan, L., Broussard, C., Chafey, P., Riès-Kautt, M., Meziani, F., Toti, F., Gaussem, P., Anglés-Cano, E. DNA-bound elastase of neutrophil extracellular traps degrades plasminogen, reduces plasmin formation, and decreases fibrinolysis: proof of concept in septic shock plasma.

From genomic to LC-MS/MS evidence: Analysis of PfEMP1 in Benin malaria cases.

BACKGROUND: PfEMP1 is the major protein from parasitic origin involved in the pathophysiology of severe malaria, and PfEMP1 domain subtypes are associated with the infection outcome. In addition, PfEMP1 variability is endless and current publicly available protein repositories do not reflect the high diversity of the sequences of PfEMP1 proteins. The identification of PfEMP1 protein sequences expressed with samples remains challenging. The aim of our study is to identify the different PfEMP1 proteins variants expressed within patient samples, and therefore identify PfEMP1 proteins domains expressed by patients presenting uncomplicated malaria or severe malaria in malaria endemic setting in Cotonou, Benin. METHODS: We performed a multi-omic approach to decipher PfEMP1 expression at the patient's level in different clinical settings. Using a combination of whole genome sequencing approach and RNA sequencing, we were able to identify new PfEMP1 sequences and created a new custom protein database. This database was used for protein identification in mass spectrometry analysis. RESULTS: The differential expression analysis of RNAsequencing data shows an increased expression of the var domains transcripts DBLα1.7, DBLα1.1, DBLα2 and DBLß12 in samples from patients suffering from Cerebral Malaria compared to Uncomplicated Malaria. Our approach allowed us to attribute PfEMP1 sequences to each sample and identify new peptides associated to PfEMP1 proteins in mass spectrometry. CONCLUSION: We highlighted the diversity of the PfEMP1 sequences from field sample compared to reference sequences repositories and confirmed the validity of our approach. These findings should contribute to further vaccine development strategies based on PfEMP1 proteins.

CSRP3 mediates polyphenols-induced cardioprotection in hypertension.

Berries contain bioactive polyphenols, whose capacity to prevent cardiovascular diseases has been established recently in animal models as well in human clinical trials. However, cellular processes and molecular targets of berries polyphenols remain to be identified. The capacity of a polyphenol-enriched diet (i.e., blueberries, blackberries, raspberries, strawberry tree fruits and Portuguese crowberries berries mixture) to promote animal survival and protect cardiovascular function from salt-induced hypertension was evaluated in a chronic salt-sensitive Dahl rat model. The daily consumption of berries improved survival of Dahl/salt-sensitive rats submitted to high-salt diet and normalized their body weight, renal function and blood pressure. In addition, a prophylactic effect was observed at the level of cardiac hypertrophy and dysfunction, tissue cohesion and cardiomyocyte hypertrophy. Berries also protected the aorta from fibrosis and modulated the expression of aquaporin-1, a channel involved in endothelial water and nitric oxide permeability. Left ventricle proteomics analysis led to the identification of berries and salt metabolites targets, including cystein and glycin-rich protein 3 (CSRP3), a protein involved in myocyte cytoarchitecture. In neonatal rat ventricular cardiomyocytes, CSRP3 was validated as a target of a berries-derived polyphenol metabolite, 4-methylcatechol sulfate, at micromolar concentrations, mimicking physiological conditions of human plasma circulation. Accordingly, siRNA silencing of CSRP3 and 4-methylcatechol sulfate pretreatment reversed cardiomyocyte hypertrophy and CSRP3 overexpression induced by phenylephrine. Our systemic study clearly supports the modulation of CSRP3 by a polyphenol-rich berries diet as an efficient cardioprotective strategy in hypertension-induced heart failure.

Soluble Amyloid Precursor Protein Alpha Interacts with alpha3-Na, K-ATPAse to Induce Axonal Outgrowth but Not Neuroprotection: Evidence for Distinct Mechanisms Underlying these Properties.

Amyloid precursor protein (APP) is cleaved not only to generate the amyloid peptide (Aß), involved in neurodegenerative processes, but can also be metabolized by alpha secretase to produce and release soluble N-terminal APP (sAPPα), which has many properties including the induction of axonal elongation and neuroprotection. The mechanisms underlying the properties of sAPPα are not known. Here, we used proteomic analysis of mouse cortico-hippocampal membranes to identify the neuronal specific alpha3 (α3)-subunit of the plasma membrane enzyme Na, K-ATPase (NKA) as a new binding partner of sAPPα. We showed that sAPPα recruits very rapidly clusters of α3-NKA at neuronal surface, and its binding triggers a cascade of events promoting sAPPα-induced axonal outgrowth. The binding of sAPPα with α3-NKA was not observed for sAPPα-induced Aß1-42 oligomers neuroprotection, neither the downstream events particularly the interaction of sAPPα with APP before endocytosis, ERK signaling, and the translocation of SET from the nucleus to the plasma membrane. These data suggest that the mechanisms of the axonal growth promoting and neuroprotective properties of sAPPα appear to be specific and independent. The signals at the cell surface specific to trigger these mechanisms require further study.

PFI1785w: A highly conserved protein associated with pregnancy associated malaria.

Pregnancy-associated malaria (PAM) is one of the severe forms of Plasmodium falciparum infection. The main antigen VAR2CSA is the target of vaccine development. However, the large size of VAR2CSA protein and its high degree of variability limit to the efficiency of the vaccination. Using quantitative mass spectrometry method, we detected and quantified proteotypic peptides from 5 predicted PAM associated proteins. Our results confirmed that PFI1785w is over-expressed in PAM samples. Then, we investigated PFI1785w variability among a set of parasite samples from various endemic areas. PFI1785w appear to be more conserved than VAR2CSA. PFB0115w, another PAM associated protein, seems also associated with the pathology. Further vaccination strategies could integrate other proteins in addition to the major VAR2CSA antigen to improve immune response to vaccination.

Molecular analysis of vascular smooth muscle cells from patients with giant cell arteritis: Targeting endothelin-1 receptor to control proliferation.

OBJECTIVE: The pathophysiology of giant cell arteritis (GCA) and the mechanisms underlying vascular remodeling, are poorly understood. We aimed to compare vascular smooth muscle cells (VSMCs) from patients with GCA and controls by a proteomic and gene expression profile approach and to identify the signaling pathways involved in proliferation. METHODS: VSMCs were cultured from temporal artery biopsies (TABs) from patients with biopsy-proven GCA (TAB+-GCA), biopsy-negative GCA (TAB--GCA), and diagnosis other than GCA (GCA-control). VSMCs from normal human aorta (HAoSMC) were used as controls. 2D-differential in-gel electrophoresis and Affymetrix chips were used to compare proteomes and gene expression profiles of VSMCs. Proliferation was assessed by BrdU incorporation assay. TAB+-GCA and GCA-control TABs underwent immunohistochemistry staining for endothelin-1 (ET-1) and receptors ETAR and ETBR. RESULTS: We identified 16, 30 and 2 protein spots differentially expressed between TAB+-GCA and GCA-control VSMCs, TAB+-GCA and TAB--GCA VSMCs and TAB--GCA and GCA-control VSMCs, respectively (fold change ≥1.5 and p≤0.05). Among the 153 proteins differentially expressed between TAB+-GCA and HAoSMC VSMCs, many were linked with ET-1. Genes differentially expressed between TAB+-GCA and GCA-control VSMCs were involved in proliferation. ET-1 was identified as a link between genes of interest. Proliferation was reduced for TAB+-GCA VSMCs on treatment with the endothelin antagonist macitentan and its active metabolite. Patients showing transmural expression of ET-1 in temporal artery lesions received a significantly higher glucocorticoid daily dose after 6-month follow-up. CONCLUSION: Inhibiting the proliferation with macitentan, combined with glucocorticoids, might be a promising therapeutic approach for patients with GCA.

Urtica dioica pollen allergy: Clinical, biological, and allergomics analysis.

BACKGROUND: The most emblematic members of Urticaceae at allergic risk level are wall pellitories (Parietaria), whereas nettle (Urtica) pollen is considered as poorly allergenic. No allergen from nettle pollen has yet been characterized, whereas 4 are listed for Parietaria pollen by the International Union of Immunological Societies. Clinical and biological profiles of 2 adult men who developed symptoms against nettle pollen and/or leaves were studied. OBJECTIVE: To characterize the allergic reaction and identify the potential nettle pollen sensitizing allergens. METHODS: IgE-mediated reaction to nettle pollen extract was evaluated by skin prick test, immunoassay, nasal provocation, and basophil activation test. To characterize specific nettle pollen allergens, an allergomic (IgE immunoproteomic) analysis was performed combining 1- and 2-dimensional electrophoresis, IgE immunoblots of nettle pollen extract, identification of allergens by mass spectrometry, and database queries. RESULTS: The results of biological and immunochemical analyses revealed that the allergic rhinitis was due to Urtica dioica pollen in both patients. The allergomic analysis of nettle pollen extract allowed the characterization of 4 basic protein allergens: a thaumatin-like protein (osmotin) with a relative molecular mass of 27 to 29 kDa, a pectinesterase (relative molecular mass, 40 kDa), and 2 other basic proteins with relative molecular masses of 14 to 16 kDa and 43 kDa. There is no or only very weak allergen associations between pellitory and nettle pollen. CONCLUSION: Exposure to nettle pollen can be responsible of allergic symptoms, and several allergens were characterized. Unravelling the allergens of this underestimated allergy might help to improve diagnosis and care for patients, to predict cross-reactivities and design adapted specific immunotherapy.

Proteomic analysis of vascular smooth muscle cells in physiological condition and in pulmonary arterial hypertension: Toward contractile versus synthetic phenotypes.

Vascular smooth muscle cells (VSMCs) are highly specialized cells that regulate vascular tone and participate in vessel remodeling in physiological and pathological conditions. It is unclear why certain vascular pathologies involve one type of vessel and spare others. Our objective was to compare the proteomes of normal human VSMC from aorta (human aortic smooth muscle cells, HAoSMC), umbilical artery (human umbilical artery smooth muscle cells, HUASMC), pulmonary artery (HPASMC), or pulmonary artery VSMC from patients with pulmonary arterial hypertension (PAH-SMC). Proteomes of VSMC were compared by 2D DIGE and MS. Only 19 proteins were differentially expressed between HAoSMC and HPASMC while 132 and 124 were differentially expressed between HUASMC and HAoSMC or HPASMC, respectively (fold change 1.5≤ or -1.5≥, p < 0.05). As much as 336 proteins were differentially expressed between HPASMC and PAH-SMC (fold change 1.5≤ or -1.5≥, p < 0.05). HUASMC expressed increased amount of α-smooth muscle actin compared to either HPASMC or HAoSMC (although not statistically significant). In addition, PAH-SMC expressed decreased amount of smooth muscle myosin heavy chain and proliferation rate was increased compared to HPASMC thus supporting that PAH-SMC have a more synthetic phenotype. Analysis with Ingenuity identified paxillin and (embryonic lethal, abnormal vision, drosophila) like 1 (ELAVL1) as molecules linked with a lot of proteins differentially expressed between HPASMC and PAH-SMC. There was a trend toward reduced proliferation of PAH-SMC with paxillin-si-RNA and increased proliferation with ELAVL1-siRNA. Thus, VSMCs have very diverse protein content depending on their origin and this is in link with phenotypic differentiation. Paxillin targeting may be a promising treatment of PAH. ELAVL1 also participate in the regulation of PAH-SMC proliferation.