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Endothelial cells (ECs) are constantly submitted in vivo to hemodynamical forces derived from the blood circulation, including shear stress (SS). ECs are able to detect SS and consequently adapt their phenotype, thus affecting many endothelial functions. If a plethora of shear stress-regulated molecular networks have been described in peripheral ECs, less is known about the molecular responses of microvascular brain ECs which constitute the blood–brain barrier (BBB). In this work, we investigated the response of human cerebral microvascular ECs to laminar physiological shear stress using the well characterized hCMEC/D3 cell line. Interestingly, we showed that hCMEC/D3 cells responded to shear stress by aligning perpendicularly to the flow direction, contrary to peripheral endothelial cells which aligned in the flow direction. Whole proteomic profiles were compared between hCMEC/D3 cells cultured either in static condition or under 5 or 10 dyn.cm −2 SS for 3 days. 3592 proteins were identified and expression levels were significantly affected for 3% of them upon both SS conditions. Pathway analyses were performed which revealed that most proteins overexpressed by SS refer to the antioxidant defense, probably mediated by activation of the NRF2 transcriptional factor. Regarding down-regulated proteins, most of them participate to the pro-inflammatory response, cell motility and proliferation. These findings confirm the induction of EC quiescence by laminar physiological SS and reveal a strong protective effect of SS on hCMEC/D3 cells, suggesting a similar effect on the BBB. Our results also showed that SS did not significantly increase expression levels nor did it affect the localization of junctional proteins and did not afect either the functional activity of several ABC transporters (P-glycoprotein and MRPs). This work provides new insights on the response of microvascular brain ECs to SS and on the importance of SS for optimizing in vitro BBB models.

(1) A 24 h urinary free cortisol (UFF) is one of the first-line exams recommended for the diagnosis of Cushing’s syndrome. In a hospital hormonology department, this activity can exceed several hundred dosages per week. The UFF is generally determined via an immunoassay with an automate using a chemiluminescence or electrochemiluminescence detection system. To increase the cortisol concentration in the analyzed sample, the automated analysis is preceded by urine extraction, which does not prevent there from being some interferences due to other steroids with close structures. (2) This paper describes the development of on-line solid phase extraction coupled to liquid chromatography and mass spectrometry for the analysis of urinary free cortisol. The on-line extraction was based on the TurboflowTM chromatography coupled to the analytical column by two valves, easily available for the laboratories. (3) The choice of the Accucore Polar Premium® analytical column made it possible to avoid analytical interferences with exogenous or endogenous molecules having the same SRM transition (363 → 121) as cortisol. (4) The method was fully validated in the range of clinically relevant concentrations from the lower limit of quantification (LLOQ) to 411.75 nmol·L−1.

In the MITICA (Mother‐to‐Infant TransmIssion of microbiota in Central‐Africa) study, 48 mothers and their 50 infants were followed from delivery to 6 months between December 2017 and June 2019 in Bangui (Central‐African Republic). Blood tests and stool analyses were performed in mothers at delivery, and their offspring at birth, 11 weeks and 25 weeks. Stool cultures were performed in specific growth media for Salmonella, Shigella, E. coli, Campylobacter, Enerobacter, Vibrio cholerae, Citrobacter and Klebsiella, as well as rotavirus, yeasts and parasitological exams. The median vitamin C levels in mothers at delivery were 15.3 μmol/L (inter‐quartile‐range [IQR] 6.2–27.8 μmol/L). In infants, the median vitamin C levels at birth were 35.2 μmol/L (IQR 16.5–63.9 μmol/L). At 11 and 25 weeks, the median vitamin C levels were 41.5 μmol/L (IQR 18.7–71.6 μmol/L) and 18.2 μmol/L (IQR 2.3–46.6 μmol/L), respectively. Hypovitaminosis C was defined as seric vitamin C levels <28 μmol/L and vitamin C deficiency was defined as vitamin C levels <11 μmol/L according to the WHO definition. In mothers, the prevalence of hypovitaminosis‐C and vitamin C deficiency at delivery was 34/45 (75.6%) and 19/45 (42.2%), respectively. In infants, the prevalence of hypovitaminosis‐C and vitamin C deficiency at 6 months was 18/33 (54.6%) and 11/33 (33.3%), respectively. Vitamin C levels in mothers and infants were correlated at birth (Spearman's rho = 0.5; P value = 0.002), and infants had significantly higher levels of vitamin C (median = 35.2 μmol/L; IQR 16.5–63.9 μmol/L), compared to mothers (median = 15.3 μmol/L; IQR 6.2–27.8 μmol/L; P value <0.001). The offspring of vitamin C‐deficient mothers had significantly lower vitamin C levels at delivery (median = 18.7 μmol/L; IQR 13.3–30.7 μmol/L), compared to the offspring of non‐deficient mothers (median = 62.2 μmol/L; IQR 34.6–89.2 μmol/L; P value <0.001). Infants with hypovitaminosis‐C were at significantly higher risk of having a positive stool culture during the first 6 months of life (adjusted OR = 5.3, 95% CI 1.1; 26.1; P value = 0.038).

Steatosis can sensitise the liver to various challenges and favour the development of non-alcoholic fatty liver disease (NAFLD). In this context, fructose feeding promotes endotoxin translocation from the gut, contributing to disease progression via an inflammatory process. Citrulline is protective against fructose-induced NAFLD; we hypothesised that this property might be related to its anti-inflammatory and antioxidative action against endotoxin-induced hepatic injuries. This hypothesis was evaluated in a model of perfused liver isolated from NAFLD rats. Male Sprague–Dawley rats (n 30) were fed either a standard rodent chow or a 60 % fructose diet alone, or supplemented with citrulline (1 g/kg per d) for 4 weeks. After an evaluation of their metabolic status, fasted rats received an intraperitoneal injection of lipopolysaccharide (LPS) (2·5 mg/kg). After 1 h, the livers were isolated and perfused for 1 h to study liver function and metabolism, inflammation and oxidative status. In vivo, citrulline significantly decreased dyslipidaemia induced by a high-fructose diet and insulin resistance. In the isolated perfused rat livers, endotoxaemia resulted in higher cytolysis (alanine aminotransferase release) and higher inflammation (Toll-like receptor 4) in livers of fructose-fed rats, and it was prevented by citrulline supplementation. Oxidative stress and antioxidative defences were similar in all three groups. Amino acid exchanges and metabolism (ammonia and urea release) were only slightly different between the three groups. In this context of mild steatosis, our results suggest that fructose-induced NAFLD leads to an increased hepatic sensitivity to LPS-induced inflammation. Citrulline-induced restriction of the inflammatory process may thus contribute to the prevention of NAFLD.

AimsAltered pancreatic exocrine function can be observed in patients with type 1 or type 2 diabetes. In the present study, we evaluated the potential nutritional consequences of this dysfunction.MethodsSerum concentrations of nutritional markers, including albumin, cholesterol, triacylglycerol, vitamins A, D, and E, were assessed in a cohort of 468 patients (137 with type 1 diabetes and 331 with type 2 diabetes), after exclusion of the patients with a CRP > 10 mg/l. These patients were compared with 47 patients with diseases of the exocrine pancreas and diabetes (type 3c diabetes or pancreatogenic diabetes). Fecal elastase-1 and chymotrypsin concentrations were measured and patients with type 1 and type 2 diabetes were divided into three groups according to whether zero (group NN), one (group LN), or both (group LL) concentrations were decreased.ResultsSeveral markers differed significantly between the groups of patients, including BMI, albumin, phosphorus, and fat-soluble vitamins. Patients with pancreatogenic diabetes had markedly more profound alterations than patients with type 1 or type 2 diabetes and altered exocrine function. However, patients with type 1 or type 2 diabetes and decreased concentrations of both elastase-1 and chymotrypsin had lower albumin, phosphorus, and vitamin A than patients with normal pancreatic exocrine function.ConclusionsModest nutritional alterations were found in patients with type 1 or type 2 diabetes and altered exocrine function. Patients with type 1 or type 2 diabetes and altered exocrine function may thus deserve to be screened for nutritional deficiencies.

(1) A 24 h urinary free cortisol (UFF) is one of the first-line exams recommended for the diagnosis of Cushing’s syndrome. In a hospital hormonology department, this activity can exceed several hundred dosages per week. The UFF is generally determined via an immunoassay with an automate using a chemiluminescence or electrochemiluminescence detection system. To increase the cortisol concentration in the analyzed sample, the automated analysis is preceded by urine extraction, which does not prevent there from being some interferences due to other steroids with close structures. (2) This paper describes the development of on-line solid phase extraction coupled to liquid chromatography and mass spectrometry for the analysis of urinary free cortisol. The on-line extraction was based on the Turboflow[sup.TM] chromatography coupled to the analytical column by two valves, easily available for the laboratories. (3) The choice of the Accucore Polar Premium[sup.®] analytical column made it possible to avoid analytical interferences with exogenous or endogenous molecules having the same SRM transition (363 → 121) as cortisol. (4) The method was fully validated in the range of clinically relevant concentrations from the lower limit of quantification (LLOQ) to 411.75 nmol·L[sup.−1] .

In critically ill patients, acute injury alters gut function, causing greater risk for sepsis and malnutrition. Peptide-enriched diets may promote nitrogen absorption, whereas ω3-enriched diets reduce alterations in gut barrier function. The aim of this study was to assess the effectiveness of a peptide- and ω3-enriched diet on the metabolic response to injury and the gut barrier function in a model of prolonged catabolism in the rat. Given the intestinal trophic effect of glutamine, we tested for a synergistic effect of glutamine. We randomized 40 male Sprague-Dawley rats (250 g) into four groups to enterally receive a standard high-protein diet (S), or a peptide- and ω3-enriched diet either alone (IMN) or supplemented with glutamine and alanine supplied as dipeptide (DIP) or as free amino acids (AAs) for 4 d. Metabolic response to injury was induced by turpentine injections on days 1 and 3. At sacrifice, nutritional and inflammatory biomarkers and intestinal and liver function were assessed. Weight gain (+45–62%) and nitrogen balance (+33–56%) were significantly higher in all groups than in the S group. In jejunal mucosa, total glutathione was significantly higher (+20–30%) and myeloperoxidase activity significantly lower in all groups compared with the S group. Hepatic triacylglycerol content was significantly lower in the AA (0.30 ± 0.04 μM/g) and DIP (0.43 ± 0.08 μM/g) groups than in the S group (0.71 ± 0.08 μM/g). In this model of prolonged catabolism, compared with a standard diet, a peptide- and ω3-enriched diet improved metabolic response to injury, with better nitrogen balance and weight recovery, and decreased intestinal myeloperoxidase activity. Only marginal additional effects of glutamine supplementation were observed with decreased hepatic fat content. •Peptides have better absorption than proteins and ω3 fatty acids exert protective effects on the gut.•Peptide- and ω3-enriched enteral nutrition may be beneficial in situations of aggression.•In a model of prolonged catabolism, this enteral diet improved the metabolic response to injury.•Addition of glutamine had little or no additive effects.

N-carbamoyl putrescine (NCP), the decarboxylation derivative of citrulline, metabolically related to polyamines, may exert biological effects in mammals. The aim of this study was (i) to evaluate the nutritional properties of NCP in healthy rats and (ii) to determine the effect of NCP administration on muscle metabolism in malnourished old rats. The nutritional properties of NCP were first evaluated in 20 8-week-old male rats randomized to receive for two weeks a standard diet either alone (C group) or supplemented with NCP, 5 or 50 mg/kg/d. In a second study, 29 malnourished 18-month-old male rats were studied either before or after a 4-day refeeding with a standard diet either alone (REN group) or supplemented with NCP, 1 or 10 mg/kg/d. NCP had no effect on weight gain and body composition in either of the two studies. In healthy rats, muscle protein content was significantly increased in the soleus with NCP 5 mg/kg/d. A decrease in plasma glutamine and kidney spermine was observed at the 50 mg/kg/d dose; otherwise, no significant changes in plasma chemistry and tissue polyamines were observed. In malnutrition-induced sarcopenic old rats, refeeding with NCP 10 mg/kg/d was associated with higher tibialis weight and a trend for increased protein content in extensor digitorum longus (EDL). While the muscle protein synthesis rate was similar between groups, ribosomal protein S6 kinase was increased in tibialis and higher in the EDL in NCP-treated rats. The muscle RING-finger protein-1 expression was decreased in tibialis and urinary 3-methyl-histidine to creatinine ratio slightly lower with the supply of NCP. However, this initial period of refeeding was also associated with elevated fasted plasma triglycerides and glucose, significant in NCP groups, suggesting glucose intolerance and possibly insulin resistance. NCP was well-tolerated in healthy young-adults and in malnourished old rats. In healthy adults, NCP at 5 mg/kg/d induced a significant increase in protein content in the soleus, a type I fiber-rich muscle. In malnourished old rats, NCP supply during refeeding, may help to preserve lean mass by limiting protein breakdown; however, these effects may be limited in our model by a possible immediate refeeding-associated glucose intolerance.

Ornithine α-ketoglutarate (OKG) displays anabolic properties at the hepatic level, but the mechanisms involved remain unclear. This study investigated in vivo the ability of OKG to modulate hepatic gene expression of three liver-secreted proteins: albumin, transthyretin, and retinol binding protein. One hundred eighty rats were fed for 5 d with a balanced regimen enriched with OKG (5 g·kg-1 ·d-1 ) or an isonitrogenous mixture (alanine, glycine, and serine). Hepatic mRNA levels and plasma concentrations of the three proteins studied were determined at the end of the nutrition period and after 1, 2, and 3 d of food deprivation. Results were compared by analysis of variance and Bonferroni-Dunn tests. At the end of the nutrition period, hepatic mRNA levels and plasma concentrations of the three proteins were not modified by OKG supplementation. However, OKG largely increased mRNA levels of albumin, transthyretin, and retinol binding protein on the first day of starvation compared with control animals (+68%, +64% and +51%, respectively; P < 0.01 versus control). OKG precociously increased albuminemia (on day 2) but had no effect on plasma concentrations of transthyretin and retinol binding protein. Neither regulation of polyamine hepatic concentration nor alteration in hepatic amino acid content seemed to be implicated in these actions. This study is the first to demonstrate that OKG regulates in vivo liver gene expression during acute malnutrition by modulating hepatic mRNA levels.

Ornithine α-ketoglutarate (OKG) displays anabolic properties at the hepatic level, but the mechanisms involved remain unclear. This study investigated in vivo the ability of OKG to modulate hepatic gene expression of three liver-secreted proteins: albumin, transthyretin, and retinol binding protein. One hundred eighty rats were fed for 5 d with a balanced regimen enriched with OKG (5 g · kg −1 · d −1) or an isonitrogenous mixture (alanine, glycine, and serine). Hepatic mRNA levels and plasma concentrations of the three proteins studied were determined at the end of the nutrition period and after 1, 2, and 3 d of food deprivation. Results were compared by analysis of variance and Bonferroni-Dunn tests. At the end of the nutrition period, hepatic mRNA levels and plasma concentrations of the three proteins were not modified by OKG supplementation. However, OKG largely increased mRNA levels of albumin, transthyretin, and retinol binding protein on the first day of starvation compared with control animals (+68%, +64% and +51%, respectively; P < 0.01 versus control). OKG precociously increased albuminemia (on day 2) but had no effect on plasma concentrations of transthyretin and retinol binding protein. Neither regulation of polyamine hepatic concentration nor alteration in hepatic amino acid content seemed to be implicated in these actions. This study is the first to demonstrate that OKG regulates in vivo liver gene expression during acute malnutrition by modulating hepatic mRNA levels.