Development of a new hazelnut sandwich ELISA based on detection of Cor a 9, a major hazelnut allergen

The emerging health problem of food-induced allergic reactions, including life-threatening anaphylaxis, presents an important challenge to the food. In the framework of the current ALLERRISK project, analytical approaches for allergen detection are evaluated and developed. Cor a 9 is a major hazelnut protein with nutrient reservoir function, and that also known as a major food allergen. Presence of Cor a 9 indicates contamination of a product with hazelnut and, consequently, potential risk of allergenicity. Crude extract of hazelnut was obtained from milled mixture of hazelnuts from 10 commercially available brands. Then the extract was concentrated and applied to gel filtration and Con A affinity chromatography columns for Cor a 9 isolation and purification. The final product was characterized by analytical gel permeation chromatography, SDS PAGE and MALDI mass spectrometry. The purified Cor a 9 was used as an antigen for the production of polyclonal antibodies in chickens according to the protocol developed in our laboratory. A part of the most active antibodies was further purified by gel permeation chromatography and characterized by SDS PAGE before further use. The prepared antibodies were applied for development of a sandwich ELISA using polyclonal antibody-enzyme adducts as a tracer. Optimization of reagent concentrations led us to achieve detection limit ~10 ng/ml. Influence of ionic strength, pH and buffer composition on analytical parameters of the developed ELISA were thoroughly studied. The developed ELISA can be successfully applied for quantitative detection of Cor a 9 in large pH (pH = 5 – 10) and ionic strength (0.05 – 1.5 M salts solution) ranges. Cross-reactivity with series of nuts, ovalbumin, whey, and wheat proteins was also investigated (Figure 2) and showed that the developed assay was very specific. Moreover, presence of several other proteins (peanut, cashew, ovalbumin, whey and wheat) at high concentrations (1 mg/ml) in the presence of Cor a 9 did not influence the calibration curve of Cor a 9. Detection of hazelnut protein in cookies with known amount of hazelnut inside initial dough showed good recovery (35-40%) and absence of significant signal in blank samples (i.e. without spiking of hazelnut)

Anxiety in patients after cesarean section

Introduction: Depression is a frequent consequence of cesarean section. In the same time, even if some women don’t suffer from post-cesarean depression, the majority of them present anxiety signs, especially in the immediate postoperative period. Aims: to estimate the level of anxiety in patients with cesarean section. Evaluation of anxiety level in patients before cesarean section. Evaluation of anxiety level in patients with cesarean section in the confinement and late postoperative periods. Methods: The study included 100 patients, who were submitted to cesarean section. The first part of the study was performed during the early postoperative period (confinement period) and the second part (late postoperative period) - 6 months later after the cesarean section. Estimation of state and trait anxiety was performed by the means of Spielberger-Hanin test. Results: Trait anxiety was light in 76,0%±4,27 patients, moderate - in 23,0%±4,20 cases and severe - in 1,0%±0,99 cases. Confinement period: The majority of patients presented a moderate (58,0%±4,93) and a severe state of anxiety (14,0%±3,46) during the first days after cesarean section. It results that the level of state anxiety in the majority of cases depends directly upon postoperative stress and not upon by the level of trait anxiety. Late postoperative period: State anxiety during the late postoperative period was: moderate in 36,0%±4,80 cases and severe in 4,0%±1,95 cases. Compared with state anxiety during the confinement period, state anxiety during the late postoperative period has significantly decreased. Conclusion: 23% of the patients had a moderate trait of anxiety and 1% had severe trait anxiety before cesarean section. a) During the confinement period, state anxiety is dominated by the moderate form (58%), depending directly upon postoperative stress. b) During the late postoperative period, the level of state anxiety is decreased, predominant being light state anxiety (60%)

The non-specific effects of estrogens

Conducător ştiinţific: doctor în medicină, conferenţiar universitar, Nicolae Demişcan Catedra Fiziologia Omului şi Biofizică, USMF “Nicolae Testemiţanu”The effects of estrogens upon the cardiovascular system are: prevention of atherosclerosis, improvement of the lipid profile and vasodilation. The effects of estrogens upon the digestive system are: reduction of the intestinal peristalsis and increase of absorption. Estrogens also have neurotrophic and neuroprotective effects, along with the prevention of brain ischemia, they have an antidiabetic action, increase the retention of water and sodium, protect the skeletal system and increase blood coagulation. Estrogens also maintain the normal structure of skin in women by promoting keratinocytes and fibroblasts proliferation, and by stimulating its vascularization. La nivelul sistemului cardiovascular, estrogenii au efect antiaterosclerotic, de ameliorare a profilului lipidic şi de vasodilatare. La nivel digestiv, estrogenii micşorează peristaltismul intestinal, favorizînd absorbţia. Estrogenii mai posedă efect neurotrofic, neuroprotector şi antiischemic cerebral, au acţiune antidiabetică, favorizează retenţia de apă şi sodiu, au efect protector asupra ţesutului osos, majorează coagulabilitatea sîngelui. La nivelul pielii, estrogenii menţin structura ei normală la femei prin promovarea proliferării keratinocitelor, fibroblastelor, prin stimularea vascularizării pielii

Pathogenetic aspects of chronic complications in diabetes mellitus

Catedra Fiziopatologie şi Fiziopatologie clinicăChronic hyperglycemia in Diabetes Mellitus (DM) represents the basis of the production of chronic lesions, specific for this pathology. Its negative effects are realized by the means of forcing some abnormal metabolic pathways, among which the most important ones are: 1. Enzymatic and non-enzymatic protein glycosylation; 2. Excessive activation of the polyol pathway; 3. Stimulation of free oxygen radicals' production and the decrease of the antioxidative capacity. Enzymatic and non-enzymatic protein glycosylation leads to the production of AGE (advanced glycosylation end-products); these ones, in their turn, form the atherosclerotic plate by the means of macrophages' stimulation. Other secondary modifications of protein glycosylation are: altered structure and function of proteins which compose the walls of the blood vessels and the myelin sheath; stimulation of endothelins' production; increase of glycosylated proteins' susceptibility to oxidative stress; thickening of glomerular basement membrane and of the capillary walls, modifications that lead to diabetic micro- and macroangiopathy; modifications of collagen's properties; modifications of crystaline's properties until its opacification (a phenomenon which causes diabetic retinopathy). Glycation also causes dysmetabolic cardiomyopathy, diabetic renal disease and diabetic neuropathy. Along with an excessive production of sorbitol and fructose, resulted from polyol pathway's activation, hyperglycemia leads to cellular hyperosmolarity, which favours nervous cells' affecting, in its turn, together with the decrease of nervous conduction speed and with the production of some segmentary demielinisations. These changes bring about diabetic neuropathy, including cardiovascular neuropathy too. Thus, a great number of chronic diabetic complications (such as: cataract development, diabetic neuropathy, vascular alterations which lead to atherosclerosis, retinopathy and so on) can be caused by the polyol pathway. Hyperglycemic conditions activate the process of glucose autooxidation, which causes the production of free oxygen radicals. Thus, oxidative stress can lead to macroangiopathy (by the means of the increased synthesis of TX A2 and of trombi's forming), as well as to diabetic renal disease. DM is also responsible for some changes in the blood properties; erythrocytes become more rigid because of the modifications of cellular membranes, related to protein glycosylation, including hemoglobin glycosylation too. Changes of blood platelets' functions, along with the increase of their agregability, as an answer to the changes to which vascular collagen has been submitted to, and an increased synthesis of TX A2 are also being produced. Vasodilation mediated by endothelial nitric oxide is lowered too. Chronic hyperglycemia in Diabetes Mellitus (DM) represents the basis of the production of chronic lesions, specific for this pathology. Its negative effects are realized by the means of forcing some abnormal metabolic pathways, among which the most important ones are: 1. Enzymatic and non-enzymatic protein glycosylation; 2. Excessive activation of the polyol pathway; 3. Stimulation of free oxygen radicals' production and the decrease of the antioxidative capacity. Enzymatic and non-enzymatic protein glycosylation leads to the production of AGE (advanced glycosylation end-products); these ones, in their turn, form the atherosclerotic plate by the means of macrophages' stimulation. Other secondary modifications of protein glycosylation are: altered structure and function of proteins which compose the walls of the blood vessels and the myelin sheath; stimulation of endothelins' production; increase of glycosylated proteins' susceptibility to oxidative stress; thickening of glomerular basement membrane and of the capillary walls, modifications that lead to diabetic micro- and macroangiopathy; modifications of collagen's properties; modifications of crystaline's properties until its opacification (a phenomenon which causes diabetic retinopathy). Glycation also causes dysmetabolic cardiomyopathy, diabetic renal disease and diabetic neuropathy. Along with an excessive production of sorbitol and fructose, resulted from polyol pathway's activation, hyperglycemia leads to cellular hyperosmolarity, which favours nervous cells' affecting, in its turn, together with the decrease of nervous conduction speed and with the production of some segmentary demielinisations. These changes bring about diabetic neuropathy, including cardiovascular neuropathy too. Thus, a great number of chronic diabetic complications (such as: cataract development, diabetic neuropathy, vascular alterations which lead to atherosclerosis, retinopathy and so on) can be caused by the polyol pathway. Hyperglycemic conditions activate the process of glucose autooxidation, which causes the production of free oxygen radicals. Thus, oxidative stress can lead to macroangiopathy (by the means of the increased synthesis of TX A2 and of trombi's forming), as well as to diabetic renal disease. DM is also responsible for some changes in the blood properties; erythrocytes become more rigid because of the modifications of cellular membranes, related to protein glycosylation, including hemoglobin glycosylation too. Changes of blood platelets' functions, along with the increase of their agregability, as an answer to the changes to which vascular collagen has been submitted to, and an increased synthesis of TX A2 are also being produced. Vasodilation mediated by endothelial nitric oxide is lowered too

Whey proteins interact with lipids during autoxidation primarly via the production of reactive carbonyl species

Nowadays, it is well recognized that polyunsaturated fatty acids (PUFAs) can provide extensive nutritional and health benefits. Thus, ω-3 PUFAs have been considered to contribute to the prevention of several diseases. Recognition of the potential benefits of ω-3 fatty acids has stimulated increased interest towards the fortification of foods with oils rich in these particular fatty acids. However, enrichment of food products with such unsaturated fatty acids should be carefully evaluated since they are highly susceptible to oxidation. Moreover, exposure of proteins to peroxidizing lipids or their secondary breakdown products may induce severe changes in proteins, including polymerization, insolubilization and formation of lipid – protein complexes. Several amino acids, but mainly mainly cysteine, methionine, histidine, tyrosine and lysine are affected by the secondary lipid oxidation products, therefore leading to reductions of their availability. The objective of this study was to characterize changes induced in whey protein isolate (a mixture of α-lactalbumin and β-lactoglobulin) through oxidation with oils with different unsaturation degrees and different initial oxidation status. The incubation of whey proteins with oils caused an increase in protein bound carbonyl content through the interaction of lipid oxidation products with the amino acids. Changes in the amino acid composition were therefore also observed mostly upon incubation with fish and highly oxidized soybean oil. Interaction of proteins with lipid oxidation products was taking place via interaction of the reactive carbonyl species formed thus leading to protein aggregates formation. Protein aggregation was therefore one of the most prominent consequence of the interaction of whey proteins with oxidizing lipids

Stability of whey protein derived peptides upon severe protein glycation

Cow’s milk and dairy products are major nutrients in the human diet, especially during infancy. Though at one time whey proteins were considered as by-product of the cheese making process, nowadays, due to their wide ranging nutritional, biological, and functional properties, whey proteins are often used in food technology as low-cost protein ingredients. However, whey-protein fractions, such as β-lactoglobulin (β-LG) and α-lactalbumin (α-LA) represent the major allergens in cow’s milk. Therefore, the use of whey proteins in food might pose a serious threat to the milk allergic consumers. It is well reported that food processing may modify the allergenicity and detectability of proteins. This can be due to hydrolysis or chemical reactions with other food components (carbohydrates, fatty acids etc), leading to modification or destruction of the allergen’s structure. Therefore, the objective of this study was in the first stage to investigate the influence of glycation on the molecular changes induced in whey proteins. This was done with a special focus on the modifications induced on the lysine residues, free amino groups, the formation of protein bound carbonyls, formation of fluorescent compounds and brown polymers and on the protein aggregation. Matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) was used to get a better insight into the molecular changes that took place on the protein level. Unexpectedly, this study led to the identification of protein segments in the epitope region that remained unmodified during the experiments that mimic typical food processing conditions. The 57Val – Lys76 and 31Val – Arg56 from β–LG, remained unchanged disregarding the severe heating treatment in the presence of glucose and bulk proteins and they could be identified by either direct MALDI-TOF MS and MS/MS or after a more tedious separation using reversed phase chromatography. It is proposed that these peptide segments can be used as analytical targets for the development of more robust methods for the assessment of the presence of whey proteins in processed foodstuffs. Moreover, MALDI-TOF MS and MS/MS holds potential to be used as a screening tool for the identification of such stable peptides

The risk factors for medical and psychoemotional disturbances after cesarean section

Catedra Obstetrică și Ginecologie, USMF „Nicolae Testemițanu”The aim of the present study was the analysis and the evaluation of the risk factors for the development of medical and psychoemotional disturbances during the early and late postoperatory periods in 100 patients after cesarean section. The level of influence of certain risk factors over medical and psychoemotional aspects was determined separately for each period with the help of Pearson correlation tables. Scopul studiului dat a fost analiza și evaluarea factorilor de risc pentru apariția disfuncțiilor medicale și psihoemoționale în perioada postoperatorie precoce și cea la distanță la 78 100 de paciente după operația cezariană. Cu ajutorul tabelelor de corelație Pearson a fost determinat nivelul de influență a anumitor factori de risc asupra aspectelor medicale și psihoemoționale, separat pentru fiecare perioadă

Operaţia cezariană: Aspecte medicale şi psihoemoţionale

Prin intermediul studiului dat s-au evaluat aspectele medicale (funcţia de lactaţie, funcţia menstruală, funcţia sexuală) şi psihoemoţionale (anxietatea reactivă şi de personalitate,depresia) la 100 de paciente după operaţia cezariană,cercetarea efectuându-se atât în perioada de lăuzie(postoperatorie precoce), cât şi în perioada postoperatorie la distanţă