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In 2015, glyphosate was classified as "Group 2A - probably carcinogenic to humans" by the International Agency for Research on Cancer (IARC). Therefore, public concerns about the environmental and health risks of this substance have rapidly increased. Considering its toxicokinetic characteristics, urinary levels of glyphosate could be a powerful tool for human biomonitoring. Nevertheless, the physicochemical properties of this molecule and the complexity of the matrix make this purpose particularly challenging. In order to solve this problem, the presented study describes a simple LC-MS/MS method for the quantification of glyphosate in human urine after pre-column derivatization with FMOC-Cl. Method development was focused on the optimization of the derivatization reaction in human urine, adjusting critical variables such as pH of borate buffer, FMOC-Cl concentration and derivatization time. Besides, chromatographic separation and spectrometric parameters were also established. The analytical method was fully validated according international guidelines for selectivity, carry over, linearity, accuracy, precision, lower limit of quantitation, matrix effect and stability under different conditions. All performance parameters were within the acceptance criteria. In addition, the method was successfully applied to 52 urine samples obtained from exposed subjects from northern Argentina, laying the foundation for future epidemiological studies.

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AIMS: To evaluate the ability of the haloarchaeon Haloferax volcanii to produce Ag and Au nanoparticles (NPs) and to characterize the obtained material in order to find relevant properties for future potential applications. METHODS AND RESULTS: Nanoparticles were produced by incubating H. volcanii cells with the corresponding metal salt. In the presence of precursor salts, cultures evidenced a colour change associated to the formation of metallic nanostructures with plasmonic bands located in the visible range of the spectrum. X-ray fluorescence analysis confirmed the presence of Ag and Au in the NPs which were spherical, with average sizes of 25 nmol l-1 (Ag) and 10 nmol l-1 (Au), as determined by electronic microscopy. Fourier transformed infrared spectroscopy indicated that both types of NPs showed a stable protein capping. Ag NPs evidenced antibacterial activity and Au NPs improved the specificity of polymerase chain reaction reactions. Au and Ag NPs were able to reduce 4-nitrophenol when incubated with NaBH4 . CONCLUSIONS: Haloferax volcanii is able to synthesize metallic NPs with interesting properties for technological applications. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data demonstrate the ability of H. volcanii to synthesize metal NPs and constitutes a solid starting point to deepen the study and explore novel applications.

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INTRODUCTION: Oral direct thrombin and Xa inhibitors are worldwide distributed for prevention and treatment of thrombosis. It is important to recognize their effects on lupus anticoagulant (LA) testing. The aim of the study is to describe the rate of false-positive results of LA tests on plasmas of patients with previous negative LA tests results that receive dabigatran etexilate (DAB) 110 mg/twice a day, rivaroxaban (RIV) 10 mg/day or 15 mg/twice a day, or enoxaparin 40 mg/day. METHODS: Blood was taken between 1.5 and 4 h post administration. Tests evaluated are as follows: prothrombin time, APTT, dilute Russell viper venom time (DRVVT) screen, APTT, and DRVVT mixing studies, index of circulating anticoagulant (ICA) with normal plasma, screen/confirm normalized ratio (NR) for DRVVT and silica clotting time (SCT). RESULTS: Plasmas from patients taking DAB (n = 22) presented 100% prolonged APTT and DRVVT with ICA above the cutoff point and 81.8% positive screen/confirm NR, 100% prolonged SCT screen, but 4.5% positive confirmatory NR. All patients receiving RIV at 15 mg/twice a day (n = 4) presented positive DRVVT screen, mixing, and confirmatory tests, 75% and 100% prolonged APTT and SCT screen, with negative screen/confirm NR. Those taking RIV 10 mg/day (n = 22) showed 81.8% prolonged DRVVT screen, 82.3% and 76.5% of them with positive mixing and confirmatory studies. Patients receiving enoxaparin also presented high prevalence of APTT and DRVVT false-positive results. CONCLUSION: Dabigatran etexilate, RIV, and enoxaparin affect tests for LA not only in screening and mixing, but also in confirmatory studies. We considered that LA testing should not to be performed when patients are taken these drugs, particularly if blood is collected at peak, in order to avoid false-positive results.

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AIMS: The effect of various nitrogen sources and nutritional starvation was examined on the production of an extracellular protease secreted by the haloalkaliphilic archaeon Natrialba magadii. METHODS AND RESULTS: Cell growth and proteolytic activity were measured in cells grown with different nitrogen sources. Proteolytic activity was produced in complex and easily metabolized nitrogen sources such as yeast extract, casein and casamino acids; meanwhile, ammonium repressed enzyme production. The time course and amount of protease accumulated showed an inverse correlation with growth rate and nutrient concentration. Starvation did not induce extracellular protease production. CONCLUSION: The accumulation of Nab. magadii extracellular protease is stimulated by nutrient limitation and slow growth rate indicating that it is probably induced in response to a deficit in the energetic status of the cells. Nutritional starvation did not induce protease accumulation suggesting that de novo synthesis of this protease and/or factor/s necessary for its activation are required. This enzyme may be regulated by nitrogen catabolite repression and it does not require protein substrates for induction. SIGNIFICANCE AND IMPACT OF THE STUDY: These results contribute to the basic knowledge on protease regulation in haloalkaliphilic archaea and will help to optimize the production of this extremozyme for biotechnological applications such as protease-catalysed peptide synthesis.

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A serine protease secreted by the haloalkaliphilic archaeon Natrialba magadii at the end of the exponential growth phase was isolated. This enzyme was purified 83 fold with a total yield of 25% by ethanol precipitation, affinity chromatography, and gel filtration. The native molecular mass of the enzyme determined by gel filtration was 45 kDa. Na. magadii extracellular protease was dependent on high salt concentrations for activity and stability, and it had an optimum temperature of 60 degrees C in the presence of 1.5M NaCl. The enzyme was stable and had a broad pH profile (6-12) with an optimum pH of 8-10 for azocasein hydrolysis. The protease was strongly inhibited by diisopropyl fluorophosphate (DFP), phenylmethyl sulfonylfluoride (PMSF), and chymostatin, indicating that it is a serine protease. It was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and activated by thiol-containing reducing agents such as dithiotreitol (DTT) and 2-mercaptoethanol. This protease degraded casein and gelatin and showed substrate specificity for synthetic peptides containing Phe, Tyr, and Leu at the carboxyl terminus, showing that it has chymotrypsin-like activity. Na. magadii protease presented no cross-reactivity with polyclonal antibodies raised against the extracellular protease of Natronococcus occultus, suggesting that although these proteases share several biochemical traits, they might be antigenically unrelated.

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Non-modulators are a subset of essential hypertensive individuals in whom renal hemodynamic and adrenal aldosterone responses to angiotensin II fail to modulate appropriately during high dietary salt intake. The main aim of this study was to investigate the familial aggregation of non-modulation and several erythrocyte Na+ transport systems in normotensive and hypertensive individuals as well as offspring of hypertensive parents. An additional aim was to evaluate the effect of treatment with enalapril on erythrocyte Na+ transport. We studied 15 normotensive subjects (6 males, 27+/-6 years), 14 untreated modulating essential hypertensive subjects (7 males, 38+/-7 years), 12 untreated non-modulating essential hypertensive subjects (7 males, 38+/-6 years), 14 modulating offspring of hypertensive parents (8 males, 25+/-6 years), and 14 non-modulating offspring of hypertensive parents (8 males, 26+/-4 years). Blood pressure was recorded with an oscillometric device and renal plasma flow and glomerular filtration rate by clearances of para-aminohippurate and inulin, respectively. Non-modulating subjects were identified as individuals who failed to increase effective renal plasma flow by 30% and decrease filtration fraction by at least 30% 10 days after changing from a low (20 mmol/d) to a high (250 mmol/d) sodium intake. Erythrocyte Na+ transport was characterized by measurements of the Na+-K+ pump, Na+-Li+ countertransport, Na+-K+-Cl- cotransport, passive Na+ permeability, and Na+ content. After the initial studies, hypertensive individuals were treated with enalapril (20 mg/d P.O.) for 6 months, after which erythrocyte Na+ transport measurements were again made. The main findings were that Na+-Li+ countertransport is increased in non-modulating hypertensive subjects and non-modulating offspring of hypertensive parents, that the increase in blood pressure in response to high salt intake is greater in non-modulating than modulating hypertensive subjects, and that enalapril decreases Na+-Li+ countertransport activity to normal in non-modulating hypertensive subjects. These findings provide support for a possible genetic role in the development of salt sensitivity and suggest that Na+-Li+ countertransport and non-modulation are related phenotypes.

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BACKGROUND: Non-modulating hypertensives are a subset of sodium-sensitive hypertensives characterized by a failure to modulate renal, vascular and adrenal glomerulosa responsivenesses to angiotensin II appropriately. OBJECTIVE: To investigate the plasma renin activity (PRA) and urinary kallikrein-like activity (Ku) under different sodium conditions in essential hypertensive patients and in the modulating and non-modulating subsets of hypertensives. Additionally, in these groups of patients, the effects on blood pressure of a sustained Na+ restriction were evaluated. METHODS: Fifteen normotensives (10 men, aged 29 +/- 5 years) and 54 untreated hypertensives (30 men, aged 34 +/- 7 years) were each administered subsequently three different diets containing 240, 140 and 50 mmol/day Na+, each diet for 10 days. At the end of each period, the PRA, Ku, 24 h urinary volume and urinary Na+ excretion were measured. Afterwards, the essential hypertensives were classified as 29 modulating essential hypertensives (MHT, 20 men, aged 32 +/- 7 years) and 25 non-modulating essential hypertensives (NMHT, 10 men, aged 36 +/- 8 years). Non-modulating ones were identified as individuals who failed to increase their effective renal plasma flow and to decrease their filtration fraction by at least 30% from baseline values, 10 days after changing from a low (10 mmol/day) to a high (260 mmol/day) Na+ intake. Blood pressure was measured with a Dinamap 8100 Critikon device. Both PRA and Ku were measured during normal Na+ intake by standard methods. Patients were administered a low-Na+ diet (10-50 mmol/day) for 12 months. RESULTS: In essential hypertensives, Ku was lower under the three Na+ diets than it was in normotensives (P < 0.01) whereas the PRA was higher in hypertensives only during the low Na+ intake (P < 0.01). The non-modulating patients showed significantly higher PRA levels (4.0 +/- 0.8 ng ml h, P < 0.05) than did modulating ones (2.6 +/- 1.0 ng ml h) or normotensives (2.3 +/- 1.0 ng ml h). Conversely, non-modulating hypertensives had lower Ku (4.1 +/- 1.0 IU/24 h, P < 0.025) than did modulating ones (6.2 +/- 1.0 IU/24 h) or normotensives (7.8 +/- 2.0 IU/24 h). Blood pressure was significantly reduced during low Na+ intake only in normotensives (month 6: 143 +/- 4/94 +/- 2 mmHg; month 12: 139 +/- 5/89 +/- 3 mmHg) compared with baseline values (169 +/- 4/102 +/- 6 mmHg, P < 0.025). CONCLUSIONS: It was shown that, in non-modulating hypertensives, in addition to an increased PRA, a reduced kallikrein-like activity coexists and seems to be associated with the impaired Na+ handling. Moreover, in these untreated patients the Na+ restriction was able to exert an antihypertensive effect even for long periods.

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We studied total exchangeable sodium, ion transport activity at maximal rate, and erythrocyte Na+ content in response to angiotensin converting enzyme inhibition in mild-to-moderate essential hypertensive patients with normal renal function. Twenty-five patients (mean age 56 years, range 40-62 years) who had abnormal red blood cell Na(+)-K(+)-Cl- cotransport or red blood cell Li(+)-Na+ countertransport were treated with either enalapril (20 mg daily) or hydrochlorothiazide (50 mg daily) during a 30-day period. During the period of enalapril treatment, Na(+)-K+ pump and Na(+)-K(+)-Cl- cotransport increased significantly from 4,282 +/- 255 to 5,236 +/- 325 mumol/l red blood cell/hr (p less than 0.01) and 166 +/- 21 to 220 +/- 24 mumol/l red blood cell/hr (p less than 0.05), respectively. Mean intracellular Na+ content in erythrocytes decreased from 11.4 +/- 0.40 to 10.0 +/- 0.33 mmol/l (p less than 0.01) and exchangeable Na+ from 39.8 +/- 0.6 mmol/kg to 35.6 +/- 0.6 mmol/kg (p less than 0.001). Sodium reduction correlated with the recovery of Na(+)-K(+)-Cl- cotransport activity (r = -0.65, p less than 0.01). During treatment, systolic and diastolic blood pressures were reduced significantly (p less than 0.01). In 12 patients treated with hydrochlorothiazide, Na(+)-K(+)-Cl- cotransport, Na(+)-K+ pump, Na(+)-Li+ countertransport, and Na+ permeability did not change significantly while Na+ content decreased from 11.7 +/- 0.3 to 10.3 +/- 0.2 mmol/l (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

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Creatinine clearance and microalbuminuria were measured before and after an oral protein load in 17 children with a history of haemolytic uraemic syndrome, 11 with a single kidney, and 15 controls, all of them normotensive and without evidence of renal damage, to look for indirect evidence of glomerular hyperfiltration. While creatinine clearance increased significantly after the protein load in controls, it did not change in patients with either haemolytic uraemic syndrome or a single kidney. Basal microalbuminuria was significantly higher in those with haemolytic uraemic syndrome when compared with those with a single kidney and controls. It increased significantly in all groups after a water load; this increase was significantly higher in haemolytic uraemic syndrome. After the protein load microalbuminuria returned to baseline. In conclusion, children with a history of haemolytic uraemic syndrome have an abnormal renal functional reserve like children with a single kidney. Only patients with haemolytic uraemic syndrome exhibited an increased microalbuminuria, however, suggesting that it may be the expression of a pathophysiological mechanism involved in haemolytic uraemic syndrome and not in single kidney, that could account for their different prognosis.

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We measured the effective renal plasma flow, the glomerular filtration rate and the fractional excretion of sodium under low and high sodium intakes in the normotensive offspring of hypertensives and in essential hypertensive patients. During the high sodium intake, 57% of the offspring and 42% of the hypertensive patients showed an increase in effective renal plasma flow and fractional excretion of sodium. In the remaining hypertensives, enalapril improved the renal haemodynamic and sodium-handling responses. Enalapril also reduced the blood pressure in all hypertensives, whether they showed a normal or an abnormal response to a high sodium intake. In the hypertensive patients with an abnormal response to a high sodium intake, the intra-erythrocyte sodium content was higher than in the normal responders. Thus, abnormal renal haemodynamic and sodium-handling responses to a high sodium intake were observed in 50% of the hypertensive patients and the offspring. The high intra-erythrocyte sodium content observed in the hypertensive patients suggests that extrarenal alterations in sodium handling were present.

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Several abnormalities in Na metabolism have been implicated in the pathogenesis of essential hypertension. In addition, recent work by several investigators has showed that some Na transport systems in red cell membranes may be altered in those patients. In order to confirm such abnormalities we evaluated simultaneously four different and clearly defined Na transport systems in red cell membranes: the ouabain sensitive Na pump (P) and the Na-K cotransport (Co) in nystatin loaded cells, the maximal rate of Na-Li countertransport (CTT) in lithium loaded cells and the rate constant of Na passive permeability (pp) in 58 normotensive control subjects with no family history of hypertension (N), 19 normotensive subjects with family history of hypertension (NH) and 34 essential hypertensive patients (HE). The mean (mean +/- SEM microns/lc/h) value of the P and pp was found to be comparable in the three groups. Co was found significantly decreased in both HE (241 +/- 28) and in NH (227 +/- 42) when compared to the control group (290 +/- 10). Although NH also showed CTT values (377 +/- 87) higher than controls, the difference did not reach statistical significance. Our results indicate that approximately 90.9% of both HE and NH presented abnormalities in one or more of the various Na transport systems studied. Normotensive patients with a positive family history and alterations in some of the Na transport systems in red cell membranes may prove an interesting experimental model to assess the importance of such alterations for the development of hypertension.

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Several abnormalities in Na metabolism have been implicated in the pathogenesis of essential hypertension. In addition, recent work by several investigators has showed that some Na transport systems in red cell membranes may be altered in those patients. In order to confirm such abnormalities we evaluated simultaneously four different and clearly defined Na transport systems in red cell membranes: the ouabain sensitive Na pump (P) and the Na-K cotransport (Co) in nystatin loaded cells, the maximal rate of Na-Li countertransport (CTT) in lithium loaded cells and the rate constant of Na passive permeability (pp) in 58 normotensive control subjects with no family history of hypertension (N), 19 normotensive subjects with family history of hypertension (NH) and 34 essential hypertensive patients (HE). The mean (mean +/- SEM microns/lc/h) value of the P and pp was found to be comparable in the three groups. Co was found significantly decreased in both HE (241 +/- 28) and in NH (227 +/- 42) when compared to the control group (290 +/- 10). Although NH also showed CTT values (377 +/- 87) higher than controls, the difference did not reach statistical significance. Our results indicate that approximately 90.9

of both HE and NH presented abnormalities in one or more of the various Na transport systems studied. Normotensive patients with a positive family history and alterations in some of the Na transport systems in red cell membranes may prove an interesting experimental model to assess the importance of such alterations for the development of hypertension.

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Red blood cell (RBC) sodium transport systems were studied by cation flux methodology, measuring both the ouabain-sensitive Na-K pump and the ouabain-insensitive Na-K cotransport (CoT), as well as the Na-lithium (Li) countertransport (CTT), in eight patients on chronic hemodialysis and a control group of eight normal individuals. Intracellular sodium content and passive Na permeability were also determined. The effect of L-carnitine on RBC sodium transport in the uremic group was evaluated by supplementation with oral (1 g/day) and i.v. (1 g post-hemodialysis) L-carnitine for 60 days. Mean Na efflux through the ouabain-sensitive Na-K pump was 30.7% lower in uremic patients than in controls (3.49 +/- 1.52 vs. 5.04 +/- 0.72 mmol/liter RBCxhr; P less than 0.025). Intracellular Na content was higher in uremic patients (11.57 +/- 3.38 vs. 8.86 +/- 0.88 mEq/liter RBC; P less than 0.05), but no differences were found in Na-K CoT, Na-Li CTT or passive Na permeability. L-carnitine treatment increased the ouabain-sensitive Na efflux in uremic patients (4.76 +/- 1.6 vs. 3.49 +/- 1.52 mmol/liter RBCxhr; P less than 0.05), with no change in CoT, CTT, intracellular Na content or passive Na permeability. We conclude that L-carnitine deficiency may play a major role in uremic Na-K pump disfunction.

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