Structural characterization of DNA amplicons by ATR-FTIR spectroscopy as a guide for screening metainflammatory disorders in blood plasma.

Attenuated total reflectance (ATR) Fourier transform infrared (FTIR) spectroscopy is a promising rapid, reagent-free, and low-cost technique considered for clinical translation. It allows to characterize biofluids proteome, lipidome, and metabolome at once. Metainflammatory disorders share a constellation of chronic systemic inflammation, oxidative stress, aberrant adipogenesis, and hypoxia, that significantly increased cardiovascular and cancer risk. As a result, these patients have elevated concentration of cfDNA in the bloodstream. Considering this, DNA amplicons were analyzed by ATR-FTIR at 3 concentrations with 1:100 dilution: (IU/mL): 718, 7.18, and 0.0718. The generated IR spectrum was used as a guide for variable selection. The main peaks in the biofingerprint (1800-900 cm-1) give important information about the base, base-sugar, phosphate, and sugar-phosphate transitions of DNA. To validate our method of selecting variables in blood plasma, 38 control subjects and 12 with metabolic syndrome were used. Using the wavenumbers of the peaks in the biofingerprint of the DNA amplicons, was generated a discriminant analysis model with Mahalanobis distance in blood plasma, and 100 % discrimination accuracy was obtained. In addition, the interval 1475-1188 cm-1 showed the greatest sensitivity to variation in the concentration of DNA amplicons, so curve fitting with Gaussian funcion was performed, obtaining adjusted-R2 of 0.993. PCA with Mahalanobis distance in the interval 1475-1188 cm-1 obtained an accuracy of 96 % and PLS-DA modeling in the interval 1475-1088 cm-1 obtained AUC = 0.991 with sensitivity of 95 % and specificity of 100 %. Therefore, ATR-FTIR spectroscopy with variable selection guided by DNA IR peaks is a promising and efficient method to be applied in metainflammatory disorders.

The moderating role of macronutrient intake in relation to body composition and genotoxicity: A study with gym users.

In a cross-sectional study of gymnasium users (both sexes, ages = 41.9 ± 14.8 years), we examined the moderating role of macronutrient intake in relation to body composition and genotoxicity. A questionnaire was administered to evaluate characteristics of the participants. To assess macronutrient consumption, we used 24-h food recalls on three non-consecutive days. Body composition (body fat percentage and muscle mass) was evaluated with a bioimpedance scale. Genotoxicity was assessed with the buccal micronucleus cytome assay. Multiple linear regression models were applied, adjusting for age; sex; tobacco and alcohol consumption; and (with regard to exercise habits) frequency, training time, intensity, and types. Micronucleus frequency was directly associated with body fat and inversely associated with muscle mass. Our study shows that carbohydrate and fat intakes affect body fat percentage and micronucleus frequency in gymnasium users.

Discrimination of dyslipidemia types with ATR-FTIR spectroscopy and chemometrics associated with multivariate analysis of the lipid profile, anthropometric, and pro-inflammatory biomarkers.

BACKGROUND: Obesity, dyslipidemia, and low-grade inflammatory state form a triad of self-sustaining metabolic dysfunction. Attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy is a simple, rapid, and non-destructive technique that generates spectral fingerprints of biomolecules that can be correlated with metabolic changes. We verified the efficiency of ATR-FTIR spectroscopy in blood plasma (n = 74) to discriminate the types of dyslipidemias and suggest metabolic inflammatory changes. METHODS: Principal Component Analysis (PCA) was performed on the biochemical and anthropometric data to verify whether the dyslipidemia types share a similar biochemical profile plausible of discrimination in chemometric modeling. To discriminate the types of dyslipidemias based on spectral data, Orthogonal Partial Least-Squares Discriminant Analysis (OPLS-DA) was used and validated with leave-one-out cross-validation. RESULTS: Although no significant difference was obtained between the types of dyslipidemia and normal subjects by CRP, leptin, and cfDNA, there was a significant difference between normal subjects vs combined hyperlipidemia (CH) + hypercholesterolemia (HCL) + hypertriglyceridemia (HTG) (p < 0.05) by the 1245 cm-1 peak [νas(PO2-)] (possible indication of chronic inflammation by increased cfDNA). The area under the curve of the region between 1770 and 1720 cm-1 was significantly increased for CH in relation to other dyslipidemias and normal subjects. Furthermore, there were significant differences for the main representative peaks of lipids, proteins, carbohydrates, and nucleic acids between the types of dyslipidemias and between the types of dyslipidemias and normal subjects. The OPLS-DA model achieved 100 % accuracy with 1 latent variable and Standard Error of Cross-Validation (SECV) < 0.004 for all types of dyslipidemia  and the control group. CONCLUSIONS: Our results suggest that ATR-FTIR spectroscopy associated with chemometric modeling is a plausible applicant for screening the types of dyslipidemias. However, more extensive studies should be conducted to verify the real applicability in clinical analysis laboratories or medical clinics.

Detection of metabolic syndrome with ATR-FTIR spectroscopy and chemometrics in blood plasma.

Metabolic Syndrome (MetS) is a constellation of 3 or more risk factor (abdominal obesity, high triglycerides, low HDL-c, high blood pressure, and elevated blood glucose) for atherosclerotic cardiovascular disease. Considering these systemic metabolic changes in the biochemical pathways of all biomolecules, Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) spectroscopy is a rapid, low-cost, and reagent-free alternative technique capable of identifying spectral biomarkers that differentiate subjects with MetS from control. In this study, plasma samples from 74 subjects (14 MetS, 60 control) were analyzed on the ATR-FTIR spectrophotometer. The objective was to differentiate subjects with MetS from control with supervised chemometrics modeling (Orthogonal Partial Least Squares-Discriminant Analysis, OPLS-DA). Additionally, the inflammatory status of subjects with MetS and control (supervised by C-reactive protein - CRP, leptin, and cell-free DNA - cfDNA) was verified. The OPLS-DA model achieved 100% sensitivity and specificity in cross-validation. For 1 latent variable (93.4% of variance), RMSECV < 0.002, PRESS CV < 0.0001, and R2 > 0.9999 was obtained. Significant spectrochemical differences (p < 0.05) were found between MetS and control subjects in the following biomolecular regions (cm-1): 1717-1703 [ν(CO) and δ(NH)], 1166-1137 [ν(C-OH) + ν(CO) and ν(CC) + Î´(OH) + ν(CO)], 1113-1040 [ν(PO2-) and ν(C-OH)], and 1027-1008 [ν(CO) and v(CH2OH)]. In the OPLS-DA model loadings, amide I [1720-1600 cm-1, ν(CO)] and amide II [1570-1480 cm-1, δ(NH) + ν(CH)] had significantly greater weight than all other regions. There was a significant difference in inflammatory status between MetS patient and control (p < 0.05 for CRP and leptin, and p < 0.01 for cfDNA).

Iron supplementation does not aggravate impaired glucose tolerance and sugar overload-induced genotoxicity in rats.

High sugar intake is a major risk factor for metabolic disorders. Genotoxicity is an important factor in diabetes onset, and iron (Fe) may be an aggravating element. However, this relationship is still poorly established. Thus, this study evaluated whether Fe supplementation could aggravate obesity, impaired glucose tolerance, and sugar overload-induced genotoxicity in rats. A total of 24 rats were treated with different diets: standard diet (SD, n = 8), invert sugar overload (320 g/L, HSD, n = 8), or Fe plus invert sugar overload (2.56 mg/L of Fe2+, Fe-HSD, n = 8) for four months. After treatment, the Fe-HSD group showed no excessive weight gain or impaired glucose tolerance. DNA damage in blood, as assessed by comet assay, gradually increased in HSD during treatment (p < 0.001), whereas Fe-HSD showed a nonlinear increase in DNA damage. Moreover, Fe-HSD presented 0.6-fold more DNA damage compared with SD (p = 0.0055) in the 1st month of treatment. At months 2 and 3, results show a ≥ 1.4-fold increase in HSD and Fe-HSD DNA damage, respectively, compared with SD (p < 0.01). At the end of the experiment, only HSD DNA damage differed from SD (1.5-fold more, p = 0.0196). Fe supplementation did not aggravate the invert sugar-induced DNA damage (p > 0.05). In the pancreas, results showed no differences in DNA damage. Mutagenicity, evaluated by micronucleus testing, was not observed regardless of treatment (p = 0.428). Fe supplementation, in the evaluated concentration, did not aggravate weight gain, impaired glucose tolerance, and sugar overload-induced genotoxicity in rats.

Rapid and low-cost liquid biopsy with ATR-FTIR spectroscopy to discriminate the molecular subtypes of breast cancer.

Breast cancer (BC) is the most prevalent cancer worldwide. The prognosis and survival of these patients are directly related to the diagnostic stage. Even so, the gold standard screening method (mammography) has a long waiting period, high rates of false positives, anxiety for patients, and consequently delays the diagnosis by core needle biopsy (invasive method). Alternatively, the Attenuated Total Reflection Fourier Transform Infrared (ATR-FTIR) spectroscopy is a noninvasive, low-cost, rapid, and reagent-free technique that generates the spectral metabolomic profile of biomolecules. This makes it possible to assess systemic repercussions, such as the BC carcinogenesis process. Blood plasma samples (n = 56 BC and n = 18 controls) were analyzed in the spectrophotometer in the ATR-FTIR mode. For the exploratory analysis of the data, interval Principal Component Analysis (iPCA) was used, and for predictive chemometric modeling, the Orthogonal Partial Least Squares Discriminant Analysis (OPLS-DA) algorithm with validation by leave-one-out cross-validation. iPCA in the region of 1118-1052 cm-1 (predominantly DNA/RNA bands) showed significant clustering of molecular subtypes and control. The OPLS-DA model achieved 100% accuracy with only 1 latent variable and Root Mean Square Error of Cross-Validation (RMSECV) < 0.005 for all molecular subtypes and control. The wavenumbers (cm-1) with the highest iPCA peaks (loadings: 1117, 1089, 1081, 1075, 1057, and 1052) were used as input to MANOVA (Wilks' Lambda, p < 0.001 between molecular subtypes and control). The rapid and low-cost detection of BC molecular subtypes by ATR-FTIR spectroscopy would plausibly allow initial screening and clinical management, improving prognosis, reducing mortality and costs for the health system.

Higher IgG level correlated with vitamin D receptor in the hippocampus of a pristane-induced lupus model.

INTRODUCTION/OBJECTIVES: Patients with systemic lupus erythematosus (SLE) may have neurological complications, characterizing neuropsychiatric lupus (NPSLE). Studies have investigated alternative therapies such as vitamin D, which has an effect on the immune system and brain, to control manifestations of SLE. Experimental lupus models may be a good alternative to best study the immunological mechanisms underlying the development of NPSLE, and the animal model of pristane-induced lupus (PIL) may mimic SLE symptoms in humans. Our objective was to evaluate central nervous system involvement and vitamin D supplementation in a PIL model. METHOD: Female BALB/c mice were divided into controls (CO; n = 7), PIL (n = 9), and PIL supplemented with vitamin D (VD; n = 7). The hippocampus area was measured and immunoassays were performed for detecting vitamin D receptor (VDR) and IgG. RESULTS: The PIL group had a higher hippocampal IgG infiltrate when compared to the CO group. Vitamin D showed potential for reducing IgG infiltration. The hippocampus area was similar in all groups. No differences in VDR expression were observed between groups. A positive correlation was observed between the expression of VDR and IgG in the hippocampus. CONCLUSION: Our data suggest that increased IgG infiltration into the hippocampus indicated an inflammatory process that may have stimulated VDR expression. Key Points • IgG infiltrate is higher in PIL animals than controls • VDR increases along with IgG infiltrate • Hippocampal VDR expression does not increase with vitamin D supplementation.

Falciform ligament tubular graft for mesenteric-portal vein reconstruction during pancreaticoduodenectomy.

BACKGROUND: Portal vein resection and reconstruction in locally advanced pancreatic cancer represents a potentially curative treatment in selected patients without increasing surgical mortality. However, vascular reconstruction after segmental venous resection is challenging. The parietal peritoneum has emerged as a venous substitute but few reports include its use as a tubular graft. We report a retrospective series of portal vein reconstruction using a falciform ligament tubular graft during pancreaticoduodenectomy. MATERIAL AND METHODS: Technical aspects and short-term morbidity and mortality after pancreaticoduodenectomy with falciform ligament tubular graft interposition were analyzed. RESULTS: Among 21 patients who used parietal peritoneum for venous substitution between 2015 and 2019, eight underwent pancreaticoduodenectomy with venous resection and reconstruction using interposition of falciform ligament tubular graft. The mean duration of surgery and clamping time were 350 and 27 min, respectively. No perioperative blood transfusion was required. All the grafts were patent the day after surgery. No complication related to venous obstruction was detected during the hospital stay. Two patients had postoperative pancreatic fistula. No further intervention was needed. The 90-day mortality was null. CONCLUSIONS: The use of interposition of falciform ligament tubular graft for portal venous reconstruction during pancreaticoduodenectomy seems to be a reliable, inexpensive, and safe procedure.

Liver and kidney function markers among gym users: the role of dietary supplement usage.

Dietary supplements have been increasingly used by gym users and are often consumed without the guidance of a health professional. Moreover, the indiscriminate supplements use can have adverse health effects, such as changes in liver and kidney function. The aim of this study was to verify the association between dietary supplements intake with alterations in the liver and kidney function among gym users. A cross-sectional study was conducted with 594 gym users (mean age 37 (sd 14) years, 55·2 % women) from a city in southern Brazil. A questionnaire was used to evaluate the use of dietary supplements. The markers of the liver (alanine aminotransferase, aspartate aminotransferase (AST), alkaline phosphatase, γ-glutamyltransferase) and renal (creatinine and urea) function were also evaluated on a subsample of the study population. Data were analysed by binary logistic regression, adjusted for sex, age and education. The prevalence of dietary supplement intake was 36·0 %. Individuals who intake dietary supplements showed a higher prevalence to present slight alterations in the AST enzyme and in the urea after adjustments for potential confounders. In conclusion, the use of dietary supplement was associated with slight alterations in AST enzyme and in the urea among gym users. These findings show the importance of using supplements correctly, especially with guidance from professionals trained to avoid possible risks to health.

Complete and partial replacement of the inferior vena cava with autologous peritoneum in cancer surgery.

Resection of the inferior vena cava may be required in the courses of oncological surgeries for the tumors originating from or invading it. Management of the remaining defect depends on the extension of the resection. Partial or complete replacement of the inferior vena cava, with a patch or interposition graft, may be required. Standard techniques for the reconstruction with a prosthetic material or the autologous veins can be associated with the prosthetic graft infection, high cost, long-standing anticoagulation, technical difficulties, and/or need for extra incisions. The use of the autologous peritoneum represents an easy and inexpensive alternative for the partial and complete inferior vena cava reconstructions.

Potential for bifenthrin removal using microalgae from a natural source.

The accumulation of environmental pesticides can cause problems in aquatic ecosystems and adverse effects in humans. These compounds can be found in water due to runoff from agricultural, industrial and domestic applications. In Southern Brazil, tobacco cultivation is one of the most important economic agricultural activities. The bifenthrin pesticide, classified as having moderate toxicity (class II), is commonly used as an insecticide in this culture. In this context, the present research aimed to study the performance of microalgae-induced bioremediation processes. Microalgae were isolated from a natural water source in the city of Santa Cruz do Sul, RS, Brazil, which is an artificial reservoir used for public water supply. For this purpose, biodegradation, biosorption, influence of pH, percentage of inoculum and photoperiod were evaluated in batch experiments for 20 cultivation days. After the phycoremediation process, the bifenthrin pesticide (m/z = 181) was quantified by gas chromatography with mass spectrometry (GC-MS). The results indicated that microalgae isolated from the water of the lake were able to contribute to the removal of approximately 99% of bifenthrin through biodegradation and biosorption processes. Photodegradation was identified (>77%) and the best condition for the phycoremediation was 20% inoculum with a photoperiod of 18:6 h.

Toxicity evaluation of hospital laundry wastewaters treated by microbial fuel cells and constructed wetlands.

Hospital laundries generate high wastewater volumes with the presence of several contaminants. Nevertheless, few studies have investigated the toxicity of these effluents and looked for treatment alternatives that might reduce this eventual toxicity. So, the present study assessed the performance of an integrated system combining a microbial fuel cell (MBFC) and a constructed wetland (CW) to reduce toxic effects of wastewaters generated at a hospital laundry. After collection, raw effluents remained 7 days at the first unit (MBFC) of integrated system. Afterward, they were transferred to the second unit (CW) unit where remained more 7 days totaling a hydraulic detention time of 14 days. The toxicity evaluation involved three different organisms: Daphnia magna (acute ecotoxicity), Lactuca sativa (phytotoxicity) and Allium cepa (phytotoxicity, cytotoxicity, mutagenicity, and genotoxicity). Got results revealed an extremely acute ecotoxicity against D. magna, high phytotoxic effects in the L. sativa and A. cepa assays, and genotoxicity in the A. cepa assay for the untreated effluents. Furthermore, no significant incidence of micronuclei was observed in the raw wastewaters. Regarding the treatment, after the first stage, it was possible to verify that MBFC reduced the toxicity of the wastewaters only in some tested assays (endpoints) while after the CW (second stage) the effluents presented a complete absence of toxicity of the investigated bioassays. Therefore, the use of the integrated system combining two environmentally friendly technologies can be considered promising, since both MBFC and CW presented a complimentary effect with excellent results regarding the reduction of the overall toxicity of hospital laundry wastewaters.

Biomonitoring of urban wastewaters treated by an integrated system combining microalgae and constructed wetlands.

The objectives of the present study were to apply different, toxicological assays for monitoring the toxicity of treated and untreated urban effluents produced at a university campus. The research was conducted at the wastewater treatment plant of the University of Santa Cruz do Sul, (UNISC), from october 2018 to april 2019. An integrated system with, anaerobic reactor (AR), microalgae (MA) and constructed wetlands (CWs) was, proposed for detoxification of the wastewaters produced at the university campus with a hydraulic detention time of 17 days. Daphnia, magna (ecotoxicity) and Allium cepa (phytotoxicity, cytotoxicity, and, genotoxicity) were used as tools to monitor the efficiency of the integrated system. Obtained results showed that the integrated system (MA, + CWs) presented good COD and BOD5 reductions, besides removal rates of, almost 98% for N-NH3, being much more efficient than the UNISC wastewater, treatment plant (UWTTP). The results of ecotoxicity presented the raw wastewaters (RW) as slightly toxic and an absence of ecotoxicity in all the treatments steps. Regarding phytotoxicity, the results showed no significant differences between the treatments. The cytogenetic assays indicated a significant increase in mitotic index (MI) (p < 0.001) after treatment by CWs compared to the final treatment UWTTP while the results, regarding binucleated cells (BNC) did not present significant differences, among the treatments. Micronucleus (MN) indexes were significantly different between the UWWPT and the integrated system (p < 0.01). In relation to chromosome aberrations (CA) the results indicate a significant difference between the CWs and UWWTP treatments (p < 0.01) and, RW and CWs (p < 0.001), confirming the detoxifying potential of the integrated system when compared to UWWPT. Thus, the results of the present research highlight the relevance in the proposition of the integrated system as an alternative of cleaner technology to the detriment of conventional technologies applied in wastewater treatment.

Análise do potencial microbiano de uma biopilha na biorremediação de solos contaminados por hidrocarbonetos de petróleo / Analysis of microbial potential of a biopile in soil bioremediation contaminated by petroleum hydrocarbons

RESUMO A presente pesquisa avaliou o potencial microbiano de uma biopilha na biorremediação de solos contaminados por hidrocarbonetos, montada em uma base de distribuição de combustíveis na região metropolitana de Porto Alegre, Rio Grande do Sul. Desta biopilha, foram avaliadas as concentrações dos hidrocarbonetos totais de petróleo (HTP) e de compostos benzeno, tolueno, etilbenzeno e xilenos (BTEX), em nove pontos, durante quatro etapas de operação e em três profundidades por ponto. De cada coleta, também foram reservadas amostras de solo para realização das análises microbiológicas. A partir dessas amostras, buscou-se identificar fungos e leveduras pela análise de suas estruturas reprodutivas em lâminas de microcultivo, e bactérias pela técnica da Reação em Cadeira da Polimerase (PCR) e sequenciamento do gene que codifica o RNAr 16S. Ainda, avaliou-se a capacidade dos microrganismos isolados em degradar óleo diesel comercial, utilizando o indicador redox 2,6-diclorofenol indofenol (DCPIP). Como resultado deste estudo, observaram-se elevados percentuais de redução nas concentrações de HTP e BTEX na biopilha, como 92 e 100%, respectivamente. Das amostras de solo da biopilha, foram isoladas 101 cepas de microrganismos, das quais foram identificadas 19 cepas de fungos filamentosos, 34 de bactérias e 1 de levedura. Os resultados evidenciaram a capacidade de alguns gêneros de fungos, como Aspergillus, Trichoderma, Penicillium, Cladosporium e Verticillium, e bactérias, como Bacillus spp. e Streptomyces sp., em degradar hidrocarbonetos constituintes do óleo diesel comercial.

ABSTRACT This research evaluated the microbial potential of a biopile in bioremediation of soils contaminated by hydrocarbons, mounted on a fuel distribution base in the metropolitan region of Porto Alegre, Rio Grande do Sul. Of this biopile were evaluated concentrations of total petroleum hydrocarbons (TPH) and benzene compounds, toluene, ethylbenzene and xylenes (BTEX) at nine points, for four stages of operation and a point in three depths. Of each collection were also reserved sampled soil to perform the microbiological testing. From the samples, we sought to identify fungi and yeasts by analyzing their reproductive structures microcultivation blades, and bacteria by the technique of Polymerase Reaction Chair (PCR) and gene sequencing encoding the 16S rRNA. Still, we evaluated the ability of microorganisms to degrade commercial diesel oil, using the redox indicator 2,6-dichlorophenol indophenol (DCPIP). As result of this study, there was a high percentage reduction in the concentration of TPH and BTEX in biopilha as 92 and 100%, respectively. Of soil samples were isolated from biopilha 101 strains of microorganisms, of which 19 were identified strains of filamentous fungi and 34 bacterial yeast. The results showed the ability of some genera of fungi such as Aspergillus, Trichoderma, Penicillium, Cladosporium and Verticillium and bacteria such as Bacillus spp. and Streptomyces sp. to degrade hydrocarbons constituents of commercial diesel oil.

Performance of an integrated system combining microalgae and vertical flow constructed wetlands for urban wastewater treatment.

The present study investigated the performance of an integrated system, combining the sequential use of microalgae (MA) and vertical flow constructed wetland (VFCW) for the treatment of wastewaters produced at a university campus. Ecotoxicity and phytotoxicity assays were performed using respectively Daphnia magna and Lactuca sativa, whereas the genotoxicity of the wastewaters was assessed by using D. magna and Allium cepa. The results revealed that the major environmental impacts of the studied wastewaters are associated with the high eutrophication potential, due to high N-NH3 (68.8 ± 25.7 mg L-1), total P (7.71 ± 2.5 mg L-1), and BOD5 (526.4 ± 177 mg L-1) values, pathogenic load, and genototoxicity (p < 0.0001). The results also showed that the integrated system (MA + VFCW) was not able to satisfactory reduce the total p values (only 4%). Nevertheless, the MA + VFCW system achieved very promising results for the nitrogen removal, with emphasis on N-NH3 removal (100%) and the highest BOD5 removal (57%). Neither the raw wastewaters nor the treated wastewaters were phytotoxic. The integrated system completely eliminated the ecotoxicity (100%) and genotoxicity (n.s.) of the raw wastewater and showed decontamination potential. Thus, the integrated system emerges as an innovative environmental technology and, with minor adjustments, might be efficiently used in large scale and eventually replace conventional wastewater treatment systems.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity.

INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

Coagulase-negative staphylococci in Southern Brazil: looking toward its high diversity

Abstract: INTRODUCTION: Coagulase-negative staphylococci (CoNS) are the most prevalent pathogens in nosocomial infections and may serve as a reservoir of mobile genetic elements such as the staphylococcal cassette chromosome mec (SCCmec) encoding methicillin resistance. Molecular characterization of SCCmec types combined with advanced molecular typing techniques may provide essential information for understanding the evolution and epidemiology of CoNS infections. We therefore aimed to investigate the SCCmec distribution, multidrug-resistance (MDR), and biofilm formation in CoNS blood culture isolates from a hospital in Southern Brazil. METHODS: We analyzed 136 CoNS blood culture isolates obtained during 2002-2004 from patients admitted to a tertiary care hospital in Brazil. SCCmec types I to V were determined using multiplex PCR. The clonal relationship of Staphylococcus epidermidis was determined using pulsed field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). Molecular epidemiological data were interpreted along with data on biofilm formation, presence of the icaD gene, and MDR. RESULTS: The most prevalent species were S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus hominis harboring mainly SCCmec types II, III, and V. Overall, the presence of multiple SCCmec was associated with non-MDR, except for S. epidermidis. S. epidermidis isolates showed a high prevalence of icaD, but had low phenotypic biofilm formation. PFGE and MLST revealed high genetic diversity in the S. epidermidis population. CONCLUSIONS: Our results suggest a major shift in SCCmec types within a short period and reveal a different behavior of S. epidermidis with regard to the association between the presence of multiple SCCmec types and MDR profile.

DNA damage and cytotoxicity in pathology laboratory technicians exposed to organic solvents.

The aim of this study was to evaluate potential DNA damage and cytotoxicity in pathology laboratory technicians exposed to organic solvents, mainly xylene. Peripheral blood and buccal cells samples were collected from 18 technicians occupationally exposed to organic solvents and 11 non-exposed individuals. The technicians were sampled at two moments: Monday and Friday. DNA damage and cytotoxicity were evaluated using the Comet Assay and the Buccal Micronucleus Cytome assay. Fifteen subjects (83.5%) of the exposed group to solvents complained about some symptom probably related to contact with vapours of organic solvents. DNA damage in the exposed group to solvents was nearly 2-fold higher on Friday than on Monday, and in both moments the individuals of this group showed higher levels of DNA damage in relation to controls. No statistical difference was detected in buccal cell micronucleus frequency between the laboratory technicians and the control group. However, in the analysis performed on Friday, technicians presented higher frequency (about 3-fold) of karyolytic and apoptotic-like cells (karyorrhectic and pyknotic) in relation to control group. Considering the damage frequency and the working time, a positive correlation was found in the exposed group to solvents (r=0.468; p=0.05). The results suggest that pathology laboratory workers inappropriately exposed to organic solvents have increased levels of DNA damage.

Ischemic versus pharmacologic hepatic preconditioning.

BACKGROUND: Hepatic ischemia-reperfusion injury has a significant impact on liver resection and transplantation. Many strategies have been developed to reduce the effects of ischemia-reperfusion injury, including pharmacologic and ischemic preconditioning; however, studies comparing these two methods are lacking. MATERIAL AND METHODS: An experimental study was performed in a swine model. Eighteen swine were randomly assigned to three different groups: an ischemic preconditioning (IschPC) group, a pharmacologic preconditioning (PharmPC) group, and a control group. All animals underwent a 40-min liver ischemia, followed by 40 min of reperfusion. The IschPC group received a short period of ischemia (10 min) and a short period of reperfusion (15 min) before prolonged ischemia. The PharmPC group received inhaled sevoflurane for 30 min before prolonged ischemia. The control group did not receive any intervention before prolonged ischemia. Blood samples and liver tissue were obtained after ischemic and reperfusion periods. Injury was evaluated by measure of DNA damage (using COMET assay) and serum biochemical markers (transaminases, alkaline phosphatase, amylase, bilirubin, and C-reactive protein [CRP]). RESULTS: No significant difference was found in serum biochemical markers, except for the C-reactive protein level that was lower in the PharmPC group than in the control group soon after hepatic ischemia. Soon after prolonged ischemia, DNA damage index, both in blood samples and in liver tissue samples, was similar among the groups. However, an increase in DNA damage after reperfusion was higher in the control group than in the PharmPC group (P < 0.05). The increase in DNA damage in the IschPC group was half of that observed in the control, but this difference was not statistically significant. CONCLUSIONS: Our results suggest an early protective effect of PharmPC (lower levels of C-reactive protein soon after ischemia). The protective effect observed after reperfusion was higher with PharmPC than with ischemic preconditioning. The simultaneous use of both methods could potentiate protection for ischemia-reperfusion.

Evaluation of four different DNA extraction methods in coagulase-negative staphylococci clinical isolates.

Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS.