Immunoproteomics of peptidases from Lactobacillus helveticus

  Format du poster : 21 x 29,7Immunoproteomics of peptidases from Lactobacillus helveticus. 10. International Conference on Agri-Food Antibodie

Immunoproteomics of peptidases from Lactobacillus helveticus

Milk proteins beyond their high nutritional quality are a source of peptides with biological activity. These bioactive peptides would exert different activities in vivo, affecting the cardiovascular, endocrine, immune, digestive or nervous systems. They can be released during food processing by enzymatic hydrolysis through the action of numerous proteolytic enzymes, naturally occurring in milk or provided by exogenous supply or microbial ecosystems. However, the nature and the origin of the bacterial enzymes and among them peptidases involved in the production of such bioactive peptides are not well established. Therefore, methods of detection have to be developed to monitor these peptidases directly in cheeses during ripening. Our aim was to produce and characterise monoclonal antibodies reacting against peptidases of Lactobacillus helveticus, a lactic acid bacterium highly proteolytic, used in fermented milk products as producers of bioactive peptides. We used a strategy of pre-enrichment in intracellular peptidases to be able to produce various antibodies against peptidases from the same pool of enzymes. Two fractions enriched in peptidases, PepLHA and PepLHB were obtained from the cell free extract of Lactobacillus helveticus LH1 (FTL122, UR342 collection) and were used to immunise two sets of mice. Fusion PepLHA produced 53 hybrid clones and fusion PepLHB produced 202 hybrid clones both giving a positive reaction against fraction A and/or B. These clones were classified into 8 and 11 different families for PepLHA and PepLHB respectively. The antibodies corresponding to each family were characterised according to two methodologies: affinity columns for PepLHA, and western blot on 2D electrophoresis gels for PepLHB, and analyses by nano LC coupled on line with ESI-QTOF MS/MS. We have then produced monoclonal antibodies that react against interesting peptidases, i.e. PepN, PepC, PepX and PepQ that will be used in ELISA test

Immunoproteomics of peptidases from Lactobacillus helveticus

  Format du poster : 21 x 29,7Immunoproteomics of peptidases from Lactobacillus helveticus. 10. International Conference on Agri-Food Antibodie

Immunoproteomics of peptidases from Lactobacillus helveticus

Milk proteins beyond their high nutritional quality are a source of peptides with biological activity. These bioactive peptides would exert different activities in vivo, affecting the cardiovascular, endocrine, immune, digestive or nervous systems. They can be released during food processing by enzymatic hydrolysis through the action of numerous proteolytic enzymes, naturally occurring in milk or provided by exogenous supply or microbial ecosystems. However, the nature and the origin of the bacterial enzymes and among them peptidases involved in the production of such bioactive peptides are not well established. Therefore, methods of detection have to be developed to monitor these peptidases directly in cheeses during ripening. Our aim was to produce and characterise monoclonal antibodies reacting against peptidases of Lactobacillus helveticus, a lactic acid bacterium highly proteolytic, used in fermented milk products as producers of bioactive peptides. We used a strategy of pre-enrichment in intracellular peptidases to be able to produce various antibodies against peptidases from the same pool of enzymes. Two fractions enriched in peptidases, PepLHA and PepLHB were obtained from the cell free extract of Lactobacillus helveticus LH1 (FTL122, UR342 collection) and were used to immunise two sets of mice. Fusion PepLHA produced 53 hybrid clones and fusion PepLHB produced 202 hybrid clones both giving a positive reaction against fraction A and/or B. These clones were classified into 8 and 11 different families for PepLHA and PepLHB respectively. The antibodies corresponding to each family were characterised according to two methodologies: affinity columns for PepLHA, and western blot on 2D electrophoresis gels for PepLHB, and analyses by nano LC coupled on line with ESI-QTOF MS/MS. We have then produced monoclonal antibodies that react against interesting peptidases, i.e. PepN, PepC, PepX and PepQ that will be used in ELISA test

Fate of Sb(V) and Sb(III) species along a gradient of pH and oxygen concentration in the Carnoulès mine waters (Southern France)

International audienceThe speciation and behaviour of antimony were investigated in surface waters downstream from the abandoned Pb–Zn Carnoulès mine (Gard, France). These waters exhibit a permanent gradient of oxygen concentration and pH, ranging from acid suboxic in Reigous Creek at the outlet of sulfide tailings impoundment, to near neutral oxygenated at downstream sites along the rivers Amous and Gardon. The concentration of total dissolved (<0.22 μm) antimony, acquired through a seven-year monitoring, decreased from 7.7–409.9 μg L−1 at the source of Reigous Creek to 0.22–0.45 μg L−1 in the Gardon River, showing natural Sb attenuation. Speciation analysis carried out during three surveys indicated that Sb(III) represented up to 70% of the total dissolved Sb concentration at the source of Reigous Creek, while Sb(V) represented less than 50%. Field characterization showed that Sb(III) and Sb(V) species were attenuated through dilution and were also removed from the dissolved phase during downstream transport. Speciation analysis in suspended particulate matter extracts gave a distribution of particulate Sb into 70 to 100% of Sb(III) and less than 30% of Sb(V). The removal of Sb(III) and Sb(V) species from the dissolved phase was concordant with the oversaturation of Reigous Creek water relative to Sb(III)- and Sb(V)-oxides and Sb(III)– and Sb(V)–Fe oxides. Sb(III) was more efficiently removed than Sb(V) or As(III) and it was no more detectable in the dissolved phase at downstream sites in the rivers Amous and Gardon. Conversely, the concentration of Sb(V) in the rivers Amous and Gardon still denoted contamination arising from the Carnoulès mine. The range of log Kd values, from 2.4 L kg−1 to 4.9 L kg−1, indicated that Sb was mainly transported in the dissolved phase downstream the Reigous Creek input. Altogether, these results give a better understanding of the fate of Sb downstream from sulfide-rich mining wastes

Myocardial expression of a dominant-negative form of Daxx decreases infarct size and attenuates apoptosis in an in vivo mouse model of ischemia/reperfusion injury.

BACKGROUND: Apoptosis has been described extensively in acute myocardial infarction and chronic heart failure. Because Daxx (death-associated protein) appears to be essential for stress-induced cell death and acts as an antisurvival molecule, we tested the hypothesis that Daxx is involved in myocardial ischemia/reperfusion-induced cell death in vivo. METHODS AND RESULTS: Transgenic mice overexpressing a dominant-negative form of Daxx (Daxx-DN) under the control of the beta-actin promoter and control wild-type mice underwent an ischemia/reperfusion protocol: 40 minutes of left coronary artery occlusion and 60 minutes of reperfusion. Area at risk and infarct size were measured after dual staining by triphenyltetrazolium chloride and phthalocyanine blue dye. Apoptosis was measured in the ischemic versus the nonischemic part of the left ventricle by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling staining, enzyme-linked immunosorbent assay, and Western blotting of caspase-3, caspase-8, and poly(ADP-ribose) polymerase. The mitogen-activated protein kinase status was investigated by Western blot analysis. Comparison between groups was assessed by ANOVA or Student t test (statistical significance: P<0.05). Left ventricle tissues from transgenic mice expressed Daxx-DN at the protein level. Area at risk/left ventricle values were comparable among groups. Infarct size/area at risk was 45% reduced in Daxx-DN versus wild-type mice (P<0.001). This cardioprotection was maintained for a 4-hour reperfusion. Ischemia/reperfusion-induced apoptosis was significantly decreased and ERK1/2 prosurvival pathway was activated in ischemic Daxx-DN hearts. CONCLUSIONS: Our study clearly indicates that Daxx participates in myocardial ischemia/reperfusion proapoptotic signaling in vivo

Characterisation of a high-risk profile for maternal thrombotic and severe haemorrhagic complications in pregnant women with antiphospholipid syndrome in France (GR2): a multicentre, prospective, observational study

International audienceBackgroundProspective data about the risks of thrombotic and severe haemorrhagic complications during pregnancy and post partum are unavailable for women with antiphospholipid syndrome. We aimed to assess thrombotic and haemorrhagic events in a prospective cohort of pregnant women with antiphospholipid syndrome.MethodsThis multicentre, prospective, observational study was done at 76 centres in France. To be eligible for this study, women had to have diagnosis of antiphospholipid syndrome; have conceived before April 17, 2020; have an ongoing pregnancy that had reached 12 weeks of gestation; and be included in the study before 18 weeks of gestation. Exclusion criteria were active systemic lupus erythematosus nephropathy, or a multifetal pregnancy. Severe haemorrhage was defined as the need for red blood cell transfusion or maternal intensive care unit admission because of bleeding or invasive procedures, defined as interventional radiology or surgery, to control bleeding. The GR2 study is registered with ClinicalTrials.gov, NCT02450396.FindingsBetween May 26, 2014, and April 17, 2020, 168 pregnancies in 27 centres met the inclusion criteria for the study. 89 (53%) of 168 women had a history of thrombosis. The median term at inclusion was 8 weeks gestation. 16 (10%) of 168 women (95%CI 5–15) had a thrombotic (six [4%] women; 95% CI 1–8) or severe haemorrhagic event (12 [7%] women; 95% CI 4–12). There were no deaths during the study. The main risk factors for thrombotic events were lupus anticoagulant positivity at inclusion (six [100%] of six women with thrombosis vs 78 [51%] of 152 of those with no thrombosis; p=0·030) and placental insufficiency (four [67%] of six women vs 28 [17%] of 162 women; p=0·013). The main risk factors for severe haemorrhagic events were pre-existing maternal hypertension (four [33%] of 12 women vs 11 [7%] of 156 women; p=0·014), lupus anticoagulant positivity at inclusion (12 [100%] of 12 women vs 72 [49%] of 146 women; p<0·0001) and during antiphospholipid history (12 [100%] of 12 women vs 104 [67%] of 156 women; p=0·019), triple antiphospholipid antibody positivity (eight [67%] of 12 women vs 36 [24%] of 147 women; p=0·0040), placental insufficiency (five [42%] of 12 women vs 27 [17%] of 156 women; p=0·038), and preterm delivery at 34 weeks or earlier (five [45%] of 11 women vs 12 [8%] of 145 women; p=0·0030).InterpretationDespite treatment adhering to international recommendations, a proportion of women with antiphospholipid syndrome developed a thrombotic or severe haemorrhagic complication related to pregnancy, most frequently in the post-partum period. Lupus anticoagulant and placental insufficiency were risk factors for these life-threatening complications. These complications are difficult to prevent, but knowledge of the antenatal characteristics associated with them should increase awareness and help physicians manage these high-risk pregnancies