Fidel Allende

Identificación bacteriana basada en el espectro de masas de proteínas: Una nueva mirada a la microbiología del siglo XXI

Bacterial identification is important for the proper treatment of infected patients hospitalized with serious infections especially in critical care units. Identification by conventional methods used in microbiology laboratories takes at least 16 hours since a culture is positive. The introduction of mass spectrometry, specifically MALDI-TOF MS (matrix-assisted laser desorption/ ionization time-of-flight mass spectrometer) in the microbiology laboratory could mean a radical change in the identification accuracy, turn around time (6 minutes per bacteria) and cost (about 5 times cheaper than conventional identification). Since its introduction in clinical microbiology laboratories in 2008, many reports about its usefulness in identifying microorganisms from colonies, as well as directly from positive blood cultures and urine samples have been published. This review describes MALDI-TOF MS methodology, its identification performance for bacteria (aerobic and anaerobic), mycobacterium and yeasts, its future applications in microbiology and its main disadvantages.

Bile acids synthesis decreases after laparoscopic sleeve gastrectomy

BACKGROUND Bariatric surgery is the most effective treatment alternative in morbid obesity. The mechanisms contributing to these benefits remain poorly understood. Bile acids (BAs) are mediators of different regulatory functions in glucose and cholesterol homeostasis and energy expenditure. Recent evidence suggests that BAs are critically important for the beneficial effects of sleeve gastrectomy (SG). OBJECTIVES The aim of this study was to evaluate the effect of SG on BA synthesis. SETTING University Hospital. Santiago, Chile. METHODS Obese patients were evaluated before and after SG (1, 3, 6, and 12 months). BA synthesis was evaluated through the serum marker, 7 α-hydroxy-4-cholesten-3-one (C4). Primary and secondary BA and C4 were determined by high performance liquid chromatography coupled with tandem mass spectrometry detection (HPLC-MS/MS). RESULTS From June 2013 to January 2014, 19 patients (age 37.6±7.8 years; BMI 35.8±3.5 kg/m(2); 79% female) were included in this study. Mean weight loss at 1, 3, 6, and 12 months was 11.3, 17.5, 23.6, and 25.4 kg, respectively, equivalent to 11.8, 18.6, 24.8, and 26.9 of total body water percentage (%TBW) (P<.0001), respectively and 43.2, 68.2, 91, and 98.8 of percentage of excess weight loss (%EWL), respectively (P<.001). Serum C4 levels at baseline, 1, 3, 6, and 12 months were 23.4±21.1, 4.9±8.2, 8.7±12.1, 13.8±12.9, and 18.8±16.8 ng/mL (P<.0001), respectively. Fibroblast growth factor 19 (FGF19) levels at baseline, 1, 3, 6, and 12 months were 71±33.3, 130.5±66.2, 117.8±57.2, 134.6±91.7, and 124.3±85.9 pg/mL (P = .019), respectively. CONCLUSION Serum levels of C4 decrease after SG, indicating a reduction in the synthesis of BA. FGF19 may play a role in decreasing BA synthesis. Further studies are necessary to characterize the effect of bariatric surgery on BA homeostasis.

The Expression of RAC1 and Mineralocorticoid Pathway- Dependent Genes are Associated With Different Responses to Salt Intake

BACKGROUND Rac1 upregulation has been implicated in salt-sensitive hypertension as a modulator of mineralocorticoid receptor (MR) activity. Rac1 could affect the expression of oxidative stress markers, such as hemoxigenase-1 (HO-1) or nuclear factor-B (NF-κB), and the expression of neutrophil gelatinase-associated lipocalin (NGAL), a cytokine upregulated upon MR activation. AIM We evaluated RAC1 expression in relation of high salt intake and association with MR, NGAL, HO-1, and NF-κB expression, mineralo- and glucocorticoids levels, and inflammatory parameters. SUBJECTS AND METHODS We studied 147 adult subjects. A food survey identified the dietary sodium (Na) intake. RAC1 expression was considered high or low according to the value found in normotensive subjects with low salt intake. We determined the gene expression of RAC1, MR, NGAL, HO-1, NF-κB, and 18S, isolated from peripheral leukocytes. We measured aldosterone, cortisol, sodium, potassium excretion, metalloproteinase (MMP9 y MMP2), and C-reactive protein. RESULTS We identified 126 subjects with high Na-intake, 18 subjects had high, and 108 low-RAC1 expression. The subjects with high-RAC1 expression showed a significant increase in MR (P = 0.0002), NGAL (P < 0.0001) HO-1 (P = 0.0004), and NF-κB (P < 0.0001) gene expression. We demonstrated an association between RAC1 expression and MR (R sp 0.64; P < 0.0001), NGAL (R sp 0.48; P < 0.0001), HO-1 (R sp 0.53; P < 0.0001), and NF-κB (R sp0.52; P < 0.0001). We did not identify any association between RAC1 and clinical or biochemical variables. CONCLUSIONS RAC1 expression was associated with an increase in MR, NGAL, NF-κB, and HO-1 expression, suggesting that RAC1 could be a mediator of cardiovascular damage induced by sodium, and may also useful to identify subjects with different responses to salt intake.

Polymorphisms in the RAC1 Gene Are Associated With Hypertension Risk Factors in a Chilean Pediatric Population

BACKGROUND The GTPase Rac1 has been implicated in hypertension as a modulator of mineralocorticoid receptor activity. Our aim is to investigate the frequency of polymorphisms rs10951982 (intron 1, G>A) and rs836478 (intron 3, T>C) in the RAC1 gene and perform association studies with clinical and biochemical parameters in a Chilean pediatric cohort. METHODS Two hundred two normotensive (NT) subjects (aged 4-16 years) were divided into 2 groups: NT subjects with hypertensive parents (NH; n = 103) and NT subjects with NT parents (NN; n = 99). We measured markers of inflammation (high-sensitivity C-reactive protein, interleukin 6 (IL-6), interleukin 8, and tumor necrosis factor α), endothelial damage (Plasminogen activator inhibitor-1 metalloproteinase-9, and metalloproteinase-2), and oxidative stress (malondialdehyde). Data were expressed as median and interquartile range (IQR). RESULTS We found differences in polymorphism rs836478 (intron 3, C>T) in both genotypic (χ(2) = 15.2, 2 df; P = 0.0005) and allelic (X(2)=5.5, 1 df; P = 0.01) frequencies in NH vs. NN subjects. NH subjects with a TT genotype showed increase MMP9 expression (median = 2.3, IQR - 1.6-3.2; vs. median = 1.6, IQR = 1.6-2.3 AU; P = 0.01) and lower IL-6 expression (median = 8.8, IQR = 7.0-11.8; vs. median = 12.1, IQR = 8.2-14.7 pg/ml; P = 0.02) compared with subjects with TC/CC genotype. No difference in the allelic frequency distribution was seen in the polymorphism rs10951982 (NH vs. NN: χ(2)=0.2, 1 df; P = 0.6). For this SNP, NN subjects with GA/AA genotype showed decreased diastolic BP indexes compared with subjects with native GG genotype (median = 1.08, IQR = 1.0-1.2; vs. median = 0.99, IQR = 0.94-1.1; P = 0.02). CONCLUSIONS We report the frequency of polymorphisms rs836478 and rs10951982 of the RAC1 gene in a Spanish-Amerindian cohort. The polymorphism rs836478 was associated with an increased expression in markers of inflammation and endothelial damage (MMP9 and IL-6) in pediatric subjects with a hypertensive genetic background.

Cortisol/cortisone ratio and matrix metalloproteinase-9 activity are associated with pediatric primary hypertension

Objective: To identify novel biomarkers associated with pediatric primary hypertension. Methods: We recruited 350 participants (4–16 years). Anthropometric parameters and aldosterone, plasma renin activity, cortisol, cortisone, Homeostasis Model Assessment Insulin Resistance (HOMA-IR), high-sensitivity C-reactive protein, adiponectin, IL-6, plasminogen activator inhibitor type 1 levels and matrix metalloproteinase-9 and matrix metalloproteinase-2 (MMP-9 and MMP-2) activities were measured. Genomic DNA was isolated. Patients with altered glucose metabolism, severe obesity [BMI-SD score (BMI-SDS) > 2.5], renovascular disease, primary aldosteronism and apparent mineralocorticoid excess syndrome were excluded. Results: In selected participants (n = 320), SBP was positively correlated with BMI-SDS (r = 0.382, P < 0.001), HOMA-IR (r = 0.211, P < 0.001), MMP-9 activity (r = 0.215, P < 0.001) and the cortisol/cortisone ratio (r = 0.231, P < 0.001). DBP showed similar correlations with these variables. No correlation was observed with aldosterone or plasma renin activity. Participants were categorized as hypertensive (n = 59) or nonhypertensive (n = 261). In the univariate analysis, hypertensive patients had higher BMI-SDS (P < 0.001), HOMA-IR (P < 0.001), high-sensitivity C-reactive protein (P < 0.001), MMP-9 activity (P < 0.001), plasminogen activator inhibitor type 1 (P < 0.001) and cortisol/cortisone ratio (P < 0.001) than nonhypertensive patients. Multiple regression analysis showed that the variables that remained associated with hypertension were higher BMI-SDS [odds ratio (OR) = 3.74; 95% confidence interval (CI) = 1.84–7.58], a higher cortisol/cortisone ratio (OR = 3.92; 95% CI = 1.98–7.71) and increased MMP-9 activity (OR = 4.23; 95% CI = 2.15–8.32). Conclusion: We report that MMP-9 activity and the cortisol/cortisone ratio were higher in pediatric primary hypertensive patients, and these associations were independent of the effect of obesity. The potential role of these novel biomarkers in predicting hypertension risk and blood pressure regulation warrants further investigation.

Identification of novel 11β-HSD1 inhibitors by combined ligand- and structure-based virtual screening

11 beta-hydroxysteroid dehydrogenase type 1 (11β-HSD1) converts cortisone to cortisol in a NADPH dependent manner. Overexpression of 11β-HSD1 in key metabolic tissues is related to the development of type 2 diabetes, obesity, hypertension and metabolic syndrome. Using crystal structures of human 11β-HSD1 in complex with inhibitors as source of structural information, a combined ligand and structure-based virtual screening approach was implemented to identify novel 11β-HSD1 inhibitors. A selected group of compounds was identified in silico and further evaluated in cell-based assays for cytotoxicity and 11β-HSD1 mediated cortisol production inhibitory capacity. The expression of 11β-HSD1 and 11β-HSD2 in human LS14 adipocytes was assessed during differentiation. Biological evaluation of 39 compounds in adipocytes and steroids quantification by HPLC-MS/MS identify 4 compounds that exhibit 11β-HSD1 mediated cortisol production inhibitory activity with potencies in the micromolar range. Two compounds showed to be selective for the 11β-HSD1 reductase activity and over 11β-HSD2 isoform, and thus represent novel leads for the development of more active derivatives with higher efficacies targeting intracellular cortisol levels in type 2 diabetes and metabolic syndrome.

Serum Cortisol and Cortisone as Potential Biomarkers of Partial 11β-Hydroxysteroid Dehydrogenase Type 2 Deficiency

BACKGROUND Pathogenic variations in HSD11B2 gene triggers the apparent mineralocorticoid excess syndrome (AME). There is scarce information regarding the phenotypes of subjects carrying heterozygous pathogenic variants in HSD11B2 gene. We investigated if serum cortisol/cortisone (F/E) ratio and cortisone are useful for identifying partial 11βHSD2 deficiency in those heterozygous subjects. METHODS We studied two patients diagnosed with AME and their families carrying either D223N or R213C mutation. We also evaluated 32 healthy control subjects (13 children and 19 adults) to obtain normal references ranges for all measured variables. Case 1: A boy carrying D223N mutation in HSD11B2 gene and Case 2: A girl carrying R213C mutation. We assessed serum F/E ratio and cortisone by HPLC-MS/MS, aldosterone, plasma-renin-activity(PRA), electrolytes, and HSD11B2 genetic analyses. RESULTS The normal values (median [interquartile range]) in children for serum F/E and cortisone (µg/dl) were 2.56 [2.21-3.69] and 2.54 [2.35-2.88], and in adults were 4.42 [3.70-4.90] and 2.23 [1.92-2.57], respectively. Case 1 showed a very high serum F/E 28.8 and low cortisone 0.46 µg/dl. His mother and sister were normotensives and heterozygous for D223N mutation with high F/E (13.2 and 6.0, respectively) and low cortisone (2.0 and 2.2, respectively). Case 2 showed a very high serum F/E 175 and suppressed cortisone 0.11 µg/dl. Her parents and sister were heterozygous for the R213C mutation with normal phenotype, but high F/E and low cortisone. Heterozygous subjects showed normal aldosterone, PRA, but lower fractional excretion of sodium and urinary Na/K ratio than controls. CONCLUSION Serum F/E ratio and cortisone allow to identify partial 11βHSD2 deficiencies, as occurs in heterozygous subjects, who would be susceptible to develop arterial hypertension.

Rocuronium pharmacokinetics and pharmacodynamics in the adductor pollicis and masseter muscles

The aim of this study was to characterize the dose–effect relationship of rocuronium at the adductor pollicis and masseter muscles.

ω-3 Long-Chain Polyunsaturated Fatty Acids and Fatty Acid Desaturase Activity Ratios as Eventual Endophenotypes for ADHD

Objective: Epidemiological studies suggest that long-chain polyunsaturated fatty acids (LC-PUFAs) may be suitable as endophenotypes for ADHD. To be appropriated vulnerability traits, endophenotypes should be altered in unaffected relatives of index cases. Serum profiles of LC-PUFAs in unaffected relatives of ADHD patients remain understudied. The main objective of this study was to compare serum LC-PUFAs in ADHD patients, unaffected relatives of index cases, and general-population unaffected participants. Method: LC-PUFA profiles of 72 participants (27 ADHD patients, 27 unaffected relatives, and 18 general-population participants) were obtained by gas chromatography–mass spectrometry (GC-MS). Groups were compared by parametrical statistics. Results: Unaffected females from the general population presented lower Docosapentaenoic acid (DPA; p = .0012) and a-linolenic acid (ALA; p = .0091) levels compared with ADHD females and unaffected relatives. In addition, docosahexaenoic acid (DHA)/ALA and DHA/DPA ratios, addressing desaturase activity, were significantly lower in ADHD patients and unaffected relatives of ADHD patients in the female-subgroup (p = .022 and .04, respectively). Conclusion: DHA/ALA, DHA/DPA, serum DPA, and serum ALA may be suitable as endophenotypes for ADHD women.

Sodium Intake Is associated With Endothelial Damage Biomarkers and Metabolic Dysregulation

BACKGROUND Mounting evidence has associated high sodium (HS) intake with hypertension, cardiovascular disease, and stroke. We investigated whether HS intake modulates the parameters of endothelial damage, inflammation, and oxidative stress. METHODS We used a cross-sectional study design including 223 Chilean subjects (6.9-65.0 years old). We measured aldosterone, renin activity, cortisol, cortisone, adiponectin, leptin, hsCRP, interleukin 6 (IL-6), tumor necrosis factor-α (TNF-α), plasminogen activator inhibitor type 1 (PAI-1), metalloproteinase (MMP)-9 and MMP-2 activity, and malondialdehyde. Sodium and creatinine were measured in 24-hour urine samples. The subjects were divided by sodium intake, high sodium (HS): ≥150 mEq/day, n = 118, and adequate sodium (AS): <150 mEq/day, n = 105. RESULTS We observed a positive correlation between urinary sodium excretion and blood pressure (r = 0.1669, P = 0.0124 for systolic and r = 0.2416, P = 0.0003 for diastolic), glycemia (r = 0.2660, P < 0.0001), and triglycerides (r = 0.1604, P = 0.0175) and a highly significant correlation between sodium excretion and PAI-1 (r = 0.2701, P < 0.0001). An inverse correlation was observed between urinary sodium and HDL-cholesterol (r = -0.2093, P = 0.0018) and adiponectin (r = -0.2679, P < 0.0001). In a linear regression model, urinary sodium excretion remained significantly associated with PAI-1 values even after adjusting for age, gender, and BMI. The HS group had higher blood pressure, glycemia, HOMA-IR, atherogenic index of plasma, and PAI-1 values than the group with AS intake. CONCLUSIONS HS intake is associated with endothelial damage (high PAI-1) and metabolic dysregulation. On the other hand, inflammation and oxidative stress parameters are not modified by sodium intake.