Polyhydroxybutyrate production by an extremely halotolerant Halomonas elongata strain isolated from the hypersaline meromictic Fara Fund Lake (Transylvanian Basin, Romania).

AIM: This study aimed at unprecedented physical and chemical evaluation of the 'green plastics' polyhydroxyalkanoates (PHAs), in an extremely halotolerant Halomonas elongata strain 2FF under high-salt concentration. METHODS AND RESULTS: The investigated bacterial strain was isolated from the surface water of the hypersaline Fara Fund Lake. The 16S rRNA gene sequence phylogeny and phenotypic analysis indicated that the isolate belonged to H. elongata. PHA inclusions were observed by Sudan Black B, Nile Red staining, and transmission electron microscopy during growth at high salinity (10%, w/v, NaCl) on 1% (w/v) d-glucose. The produced polymer was quantitatively and qualitatively assessed using crotonic acid assay, elemental analysis, Fourier transform infrared and Raman spectroscopies. Additionally, X-ray powder diffraction, 1 H-NMR spectroscopy, and differential scanning calorimetry were applied. The investigations showed that the intracellular polymer was polyhydroxybutyrate (PHB) of which the strain produced up to 40 wt% of total cell dry weight after 48 h. The analysis of phaC gene from the isolated H. elongata strain indicated that the encoded PHA synthase belongs to Class I PHA synthase family. CONCLUSIONS: Overall, our investigations pointed out that the halotolerant H. elongata strain 2FF was capable to produce significant amounts of PHB from d-glucose, and PHAs from various carbon substrates at high-salt concentrations. SIGNIFICANCE AND IMPACT OF THE STUDY: The tested strain showed the ability for significant production of natural, biodegradable polymers under nutrient limitation and hypersaline conditions suggesting its potentiality for further metabolic and molecular investigations towards enhanced biopolymer production. Additionally, this study reports on the unprecedented use of Raman and XPRD techniques to investigate PHAs of an extremely halotolerant bacterium, thus expanding the repertoire of physical methods to study green plastics derived from extremophilic microorganisms.

EFFECT OF EXPERIMENTAL DYSGLYCEMIA ON UNDER-CARBOXYLATED OSTEOCALCIN PRODUCTION IN HUMAN PRIMARY OSTEOBLAST-LIKE CELL CULTURES.

CONTEXT: The undercarboxylated form of osteocalcin (ucOC) and osteoprotegerin (OPG) are bone-derived molecules involved in the endocrine crosstalk governing the bone, the adipose tissue and the pancreas. In addition, glucocorticoids are major determinants of both insulin resistance and osteoporosis. OBJECTIVE: We aimed to investigate the response of ucOC and OPG to dysglycemia and/or dexamethasone (DXM) in primary human osteoblastic cell (HOC) cultures. DESIGN AND METHODS: Third-passage sub-confluent primary HOC cultures were treated with glucose: 2.8 mmol/L, 5.6 mmol/L, 11.1 mmol/L and 28 mmol/L, respectively. Alternatively, HOC cultures were subjected to DXM 1 µmol/L. In more complex experiments, HOC cultures were pre-treated with glucose (5.6 mmol/L) with/without insulin (1 pmol/L) followed by DXM (1 µmol/L). 24-hours post-treatment, culture medium ucOC and OPG were measured by ELISA. RESULTS: ucOC production differed significantly (p<0.05) between cell groups, decreasing in a dose-dependent manner as glucose concentration in the medium increased. Insulin prevented this effect. OPG levels appeared not to be significantly influenced by the hyperglycemic culture medium and were not related to ucOC concentration (p>0.05). Addition of DXM resulted in significantly lower ucOC concentrations compared to vehicle-treated cells (p<0.05). However, the effect of insulin co-treatment on ucOC was not counteracted by DXM (p<0.05). CONCLUSIONS: An obvious alteration of OC production/metabolism was observed as glucose levels changed in the bone microenvironment, to potentially be involved in diabetes-related osteopenia. DXM suppressed ucOC levels however not in insulin-rich environment.

RELATIONSHIP OF OXIDATIVE STRESS TO URINARY ANGIOTENSIN CONVERTING ENZYME 2 IN TYPE 2 DIABETES MELLITUS PATIENTS.

CONTEXT: Angiotensin converting enzyme 2 (ACE2) is highly expressed in the kidney and cleaves angiotensin II to Angiotensin (1-7), annihilating the deleterious effects of angiotensin II which is known to be a strong activator of oxidative stress. OBJECTIVE: We aimed to evaluate the relationship of oxidative stress to urinary ACE2 (uACE2) in type 2 diabetes mellitus (T2DM) patients. DESIGN: We included consecutive normo or microalbuminuric T2DM patients in an observational transversal study. Routine laboratory investigations, plasma malondialdehyde (MDA, fluorimetric thiobarbituric method) as a marker of prooxidant capacity and superoxide dismutase (SOD, cytochrome reduction method) and catalase (CAT) activity (in erythrocyte lysate by the modification of absorbance method) as two measures of serum antioxidant capacity and uACE2 (ELISA method) were assessed. RESULTS: MDA showed a negative correlation with SOD (r=-0.44, p=0.001), CAT (r=-0.37, p=0.006), uACE2 (r=-0.33, p=0.016) and a positive correlation with glycated haemoglobin (HbA1c) (r=0.49, p<0.001) and associated cardiovascular disease (r=0.42, p=0.001). CAT as also positively correlated to uACE2 (r=0.29, p=0.037). SOD was also negatively correlated with glycemia (r=-0.71, p<0.001) and HbA1c (r=-0.53, p<0.001). Patients with lower MDA (when divided according to median value of 3.88 nmol/mL) had higher uACE2 57.15(40.3-71.2) pg/mL compared to 38.5(31.8-45.95) pg/mL in patients with higher MDA (p<0.001). In multivariate logistic regression uACE2 was the only predictor for MDA above or below its median (OR=0.94, 95%CI[0.90-0.98], p=0.002). CONCLUSION: Increased prooxidant serum capacity is associated with lower uACE2 levels in T2DM patients.

Adsorption mechanisms of L-Glutathione on Au and controlled nano-patterning through Dip Pen Nanolithography.

Dip Pen Nanolithography technique has been employed for patterning L-Glutathione tripeptide (l-y-glutamyl-l-cysteinyl-glycine) nanostructures at specific locations on metallic Au(111) substrate. The formed supramolecular architectures were designed through straight lines and dots serving as precursors for building blocks assemblies in nano-bio-electronics applications or as template structures for functionalized particles in the form of host-guest networks. Tween 20 polyoxyethylene surfactant concentrations ranging from 0.005 to 0.1% (v/v) into initial l-Glutathione tripeptide (2 mg mL(-1)) ink solutions were sequentially tested for the improvement of the ink delivery process and to assure an optimum uniformity and homogeneity over the patterned space. A strong relationship was found between the coated atomic force microscope (AFM) cantilever within the highly effective Tween 20 activator adjuvant and the molecular diffusion along concentration gradients. An increase in the driving force for ink transport from the AFM tip has been demonstrated within the highest 0.1% (v/v) TW 20 surfactant concentration, favoring the patterning of GSH molecules routinely with sub-100 nm resolution. Self-assembled monolayers of GSH were also fabricated and characterized in the light of X-ray photoemission spectroscopy (XPS) and ellipsometric optical measurements. Adsorption from water of l-Glutathione to the gold substrate is proven to be made by the thiol group of cysteine. Theoretical DFT approaches were applied for quantum chemical studies dedicated to electronic processes underneath molecular GSH/Au(111) systems.

Adiponectin predicts cardiovascular events in diabetes dialysis patients.

OBJECTIVES: Adiponectin is an insulin-sensitizing, anti-inflammatory adipokine with anti-atherogenic actions in the general population. In dialysis patients it is unclear whether adiponectin conserves its protective value or is, on the contrary, associated to worse prognosis. We assessed the predictive value of adiponectin for atherosclerosis related cardiovascular events in type 2 diabetic dialysis patients. DESIGN AND METHODS: Prevalent diabetic dialysis patients from three dialysis units (n=77) were enrolled in a 3years' prospective observational study. Serum adiponectin, clinical and laboratory parameters were determined at baseline; new occurrence of atherosclerosis related events (coronary events, atherosclerosis obliterans, and stroke) was recorded. RESULTS: Baseline adiponectin was 17.25(9.53-31.97) µg/mL and significantly correlated to HDL cholesterol (r=0.29, p=0.01), triglycerides (r=-0.40, p=0.0004), ferritin (r=-0.29, p=0.02), transferrin (r=-0.28, p=0.02), and uric acid (r=-0.24, p=0.04). In multivariate analysis association to triglycerides (p=0.001), HDL cholesterol (p=0.01) and ferritin (p=0.04) remained significant. 36 new fatal and non-fatal new cardiovascular events occurred, 29 patient died. Cox proportional regression analysis showed that adiponectin below or above a ROC-derived cut-off of 27.33µg/mL significantly influenced event-free survival: hazard ratio (HR) 2.48, 95% confidence interval (CI) (1.09-5.66), p=0.031 along with fasting glucose HR 1.01, 95%CI(1.00-1.02), p=0.01 and history of cardiovascular events at inclusion HR 3.16, 95%CI(1.36-7.32), p=0.007. In multivariate analysis baseline adiponectin HR 5.02, 95%CI(0.98-25.06), p=0.05 and glycemia HR 1.01, 95%CI(1.00-1.02), p=0.01 influenced event-free survival. Adiponectin also predicted cardiovascular events in patients without cardiovascular disease at inclusion but was not associated to overall mortality. CONCLUSIONS: In diabetes dialysis patients low adiponectin favors occurrence of atherosclerosis related cardiovascular events.

Low plasma adiponectin levels predict increased urinary albumin/creatinine ratio in type 2 diabetes patients.

BACKGROUND: Experimental studies have shown that adiponectin has antiproteinuric and nephroprotective effects. The purpose of the study was to assess the value of plasma adiponectin as a predictor of proteinuria in type 2 diabetes (T2D) patients. METHODS: In this one-year prospective follow-up study, we included T2D patients with positive visual test for microalbuminuria (Micral) and negative visual test for proteinuria. Exclusion criteria were: glomerular filtration ratio (GFR) < 30 ml/min, acute infection/inflammation, uncontrolled hypertension, and atherosclerotic complications. The main outcome measure was the change in urinary albumin/creatinine ratio (UACR) after 1 year follow-up (Δ UACR). RESULTS: Fifty-six patients (66% males) completed the study. Their initial mean UACR was 81.58 ± 26.42 mg/g and mean GFR was 81.15 ± 3.96 ml/min. At baseline, simple regression disclosed significant correlations between UACR and plasma adiponectin (r = 0.54, P = 0.00002) and GFR (r = -0.28, P = 0.03); in multiple regression analysis, plasma adiponectin remained the only predictor of UACR (P = 0.00007). Baseline plasma adiponectin was significantly correlated to body mass index (r = -0.28, P = 0.04), waist circumference (r = -0.27, P = 0.05), HDL cholesterol (r = 0.35, P = 0.01), and LDL cholesterol (r = 0.27, P = 0.04). Baseline plasma adiponectin significantly correlated in simple (r = -0.38, P = 0.004) and multiple regression (P = 0.04) to Δ UACR. When patients were divided according to Δ UACR in nonprogressors (Δ UACR < 0) and progressors (Δ UACR > 0), logistic regression showed that baseline GFR (OR = 1.04, CI95%: 1.00-1.09, P = 0.04) and plasma adiponectin (OR = 1.16, CI95%: 1.02-1.32, P = 0.02) were the only factors that predicted whether the patient would be a progressor or not. CONCLUSION: In T2D patients, lower plasma adiponectin levels seem to be predictive of increased UACR.

Risk factors of renal failure progression two years prior to dialysis.

AIM: The respective contribution of sex, type of nephropathy, degree of proteinuria, blood pressure, protein and sodium daily intakes, blood lipid profile, protidemia, hemoglobinemia, acidosis and CaPO4 product on the rate of renal failure progression is debated. PATIENTS AND METHODS: The link between these parameters and the decrease of creatinine clearance, deltaCcr (according to Cockroft) was assessed in uni- and multivariate analysis in a population of 49 patients (26 women; age 60+/-15 years, weight 79+/-15 kg) selected out of 173 presently treated hemodialysis patients on the basis of availability of a quarterly follow-up for 2 years before starting dialysis. The patients were advised a moderate protein and salt restriction which could be retrospectively assessed (on urinary excretion of urea and sodium) at, respectively, 0.82 g/kg/day and 6.5 g/day. RESULTS: The 2-year deltaCcr was 14+/-14 ml/min. It was not different in men and women. This decrease in Ccr was neither significantly different in gomerular disease (17+/-8, n = 14), diabetic nephropathy (12+/-6, n = 7), nephroangiosclerosis (15+/-8, n = 5), interstitial nephritis (12+/-10, n = 14), and PKD (11 +/-12, n = 9). Patients with antihypertensive drugs (n = 42) had a faster progression than those without drugs (n = 7): deltaCcr = 15+/-14 vs 7+/-7 ml/min (p < 0.05) in spite of comparable blood pressure but with higher proteinuria. Linear regression of deltaCcr with the initial and 2-year averaged values of the quantitative parameters showed a significant positive link for both values with cholesterol, hemoglobine and proteinuria and a negative one with protidemia. A positive link was observed with the initial value of bicarbonate and the 2-year mean of diastolic and mean blood pressures. No link at all was observed with urea and Na excretion, CaPO4 product and triglycerides. Multiple regression disclosed a significant link only for protidemia (negative with both initial and 2-year averaged value), diastolic BP (only for the 2-year averaged value and hemoglobinemia (for the initial value). When the patients were classified according to a threshold value of their protidemia, DBP, hemoglobinemia, and cholesterolemia those with the combination of 2 risk factors of progression (protidemia > or = 66 g/l, DBP > or = 90 mmHg, hemoglobinemia > 11 g/dl, proteinuria > or = 3 g/d, CT > 5 mmol/l) had a significantly greater decrease of Ccr than those with the 3 other combinations at the exception of the association of low protidemia with DBP. CONCLUSION: Diastolic hypertension and low protidemia are the 2 most important factors predicting progression of renal failure. A predictive synergy was furthermore pointed out between low protidemia or diastolic hypertension with proteinuria and cholesterol. On the contrary anemia attenuates progression linked to low protidemia, diastolic hypertension, proteinuria and high cholesterol.

[Factors in the progression of renal insufficiency during the 2 years preceding the use of dialysis]. / Facteurs de progression de l'insuffisance rénale dans les deux ans précédant la mise en dialyse.

UNLABELLED: The respective contribution of sex, type of nephropathy, degree of proteinuria, blood pressure, protein and sodium daily intake, lipid profile, protidemia, hemoglobinemia, acidosis and CaPO4 product on the rate of renal failure progression is debated. The link between these parameters and the decrease of creatinine clearance, delta Ccr (according to Cockroft) was assessed in uni and multivariate analysis in a population of 49 patients (26 men, 23 women; age 60 +/- 15 years, weight 73 +/- 15 kg) selected out of 173 presently treated hemodialysis patients on the basis of availability of a quarterly follow-up for two years before starting dialysis. The patients were advised a moderate protein and salt restriction which could be retrospectively assessed (on urinary excretion of urea and sodium) at respectively 0.82 g/kg/day and 6.5 g/day. The two years delta Ccr was 14 +/- 14 ml/min. It was not different in men and women (specially when expressed in % of initial value). This decrease in Ccr was neither significantly different in glomerular disease (17 +/- 8, n = 14), diabetic nephropathy (12 +/- 6, n = 7), nephroangiosclerosis (15 +/- 8, n = 5), interstitial nephritis (12 +/- 10, n = 14), and PKD (11 +/- 12, n = 9). Patients with antihypertensive drugs (n = 42) had a faster progression than those without drugs (n = 7): delta Ccr = 15 +/- 14 vs 7 +/- 7 ml/min (p < 0.05) in spite of comparable blood pressure but with higher proteinuria. Linear regression of delta Ccr with the initial and two year averaged values of the quantitative parameters showed a significant positive link for both values with cholesterol, hemoglobin and proteinuria and a negative one with protidemia. A positive link was observed with the initial value of bicarbonate and the two year mean of diastolic and mean blood pressures. No link at all was observed with urea and Na excretion, CaPO4 product and triglycerides. Multiple regression disclosed a significant link only for protidemia (negative with both initial and two years averaged value), diastolic BP (only for the two year averaged value and hemoglobinemia (for the initial value). When the patients were classified according to a threshold value of their protidemia, DBP, hemoglobinemia, and cholesterolemia those with the combination of two risk factors of progression (pro-tidemia < 66 g/l, DBP > or = 90 mmHg, hemoglobinemia > 11 g/dl, proteinuria > 3g/d, CT > 5 mmol/l) had a significantly greater decrease of Ccr than those with the three other combinations at the exception of the association of low protidemia with DBP. CONCLUSION: 1. diastolic hypertension and low protidemia are the two most important factors predicting progression of renal failure; 2. a predictive synergy was furthermore pointed out between on one hand low protidemia and diastolic hypertension and on the other hand proteinuria and cholesterol; 3. on the contrary, anemia attenuates progression linked to low protidemia, diastolic hypertension, proteinuria and high cholesterol.