Infrared spectroscopy combined with chemometrics in transflectance mode: An alternative approach in the photodiagnosis of COVID-19 using saliva.

The Coronavirus Disease 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has required the search for sensitive, rapid, specific, and lower-cost diagnostic methods to meet the high demand. The gold standard method of laboratory diagnosis is real-time reverse transcription polymerase chain reaction (RT-PCR). However, this method is costly and results can take time. In the literature, several studies have already described the potential of Fourier transform infrared spectroscopy (FTIR) as a tool in the biomedical field, including the diagnosis of viral infections, while being fast and inexpensive. In view of this, the objective of this study was to develop an FTIR model for the diagnosis of COVID-19. For this analysis, all private clients who had performed a face-to-face collection at the Univates Clinical Analysis Laboratory (LAC Univates) within a period of six months were invited to participate. Data from clients who agreed to participate in the study were collected, as well as nasopharyngeal secretions and a saliva sample. For the development of models, the RT-PCR result of nasopharyngeal secretions was used as a reference method. Absorptions with high discrimination (p < 0.001) between GI (28 patients, RT-PCR test positive to SARS-CoV-2 virus) and GII (173 patients who did not have the virus detected in the test) were most relevant at 3512 cm-1, 3385 cm-1 and 1321 cm-1 after 2nd derivative data transformation. To carry out the diagnostic modeling, chemometrics via FTIR and Discriminant Analysis of Orthogonal Partial Least Squares (OPLS-DA) by salivary transflectance mode with one latent variable and one orthogonal signal correction component were used. The model generated predictions with 100 % sensitivity, specificity and accuracy. With the proposed model, in a single application of an individual's saliva in the FTIR equipment, results related to the detection of SARS-CoV-2 can be obtained in a few minutes of spectral evaluation.

Rapid Classification of Serum from Patients with Paracoccidioidomycosis Using Infrared Spectroscopy, Univariate Statistics, and Linear Discriminant Analysis (LDA).

Paracoccidioidomycosis (PCM) is a systemic mycosis that is diagnosed by visualizing the fungus in clinical samples or by other methods, like serological techniques. However, all PCM diagnostic methods have limitations. The aim of this study was to develop a diagnostic tool for PCM based on Fourier transform infrared (FTIR) spectroscopy. A total of 224 serum samples were included: 132 from PCM patients and 92 constituting the control group (50 from healthy blood donors and 42 from patients with other systemic mycoses). Samples were analyzed by attenuated total reflection (ATR) and a t-test was performed to find differences in the spectra of the two groups. The wavenumbers that had p < 0.05 had their diagnostic potential evaluated using receiver operating characteristic (ROC) curves. The spectral region with the lowest p value was used for variable selection through principal component analysis (PCA). The selected variables were used in a linear discriminant analysis (LDA). In univariate analysis, the ROC curves with the best performance were obtained in the region 1551-1095 cm-1. The wavenumber that had the highest AUC value was 1264 cm-1, achieving a sensitivity of 97.73%, specificity of 76.01%, and accuracy of 94.22%. The total separation of groups was obtained in the PCA performed with a spectral range of 1551-1095 cm-1. LDA performed with the eight wavenumbers with the greatest weight from the group discrimination in the PCA obtained 100% accuracy. The methodology proposed here is simple, fast, and highly accurate, proving its potential to be applied in the diagnosis of PCM. The proposed method is more accurate than the currently known diagnostic methods, which is particularly relevant for a neglected tropical mycosis such as paracoccidioidomycosis.

Prediction of melanin content of Fonsecaea pedrosoi using Fourier transform infrared spectroscopy (FTIR) and chemometrics.

Fungal melanin contributes to the survival and virulence of pathogenic fungi, such as Fonsecaea pedrosoi, which is responsible for causing chromoblastomycosis. The objective of this study was to employ Fourier transform infrared spectroscopy (FTIR) to predict the melanin content of F. pedrosoi. The melanin content, in percentage, was previously determined using gravimetry for twenty-six clinical isolates. Quintuplicate spectra of each isolate were obtained using attenuated total reflection (ATR) within the range of 4000 to 650 cm-1. To predict the melanin content, modeling was performed using partial least squares regression (PLS) in the region 1800 - 750 cm-1. Two models were tested: PLS and successive projections algorithms for interval selection in partial least squares (iSPA-PLS). The best modeling results were achieved using iSPA-PLS with one factor. The calibration set exhibited a determination coefficient (R2) of 0.9745 and a root mean square error of cross-validation (RMSECV) of 0.0977. In the prediction set, the R2 value was 0.9711, and the root mean square error of prediction (RMSEP) was 0.0999. Modeling with FTIR and multivariate calibration provides a valuable means of predicting fungal melanin content, which is simpler and more robust, thereby contributing to the advancement of this field of study.

Immobilization of Horseradish Peroxidase on Ca Alginate-Starch Hybrid Support: Biocatalytic Properties and Application in Biodegradation of Phenol Red Dye.

In this study, horseradish peroxidase was extracted, purified, and immobilized on a calcium alginate-starch hybrid support by covalent bonding and entrapment. The immobilized HRP was used for the biodegradation of phenol red dye. A 3.74-fold purification was observed after precipitation with ammonium sulfate and dialysis. An immobilization yield of 88.33%, efficiency of 56.89%, and activity recovery of 50.26% were found. The optimum pH and temperature values for immobilized and free HRP were 5.0 and 50 °C and 6.5 and 60 °C, respectively. The immobilized HRP showed better thermal stability than its free form, resulting in a considerable increase in half-life time (t1/2) and deactivation energy (Ed). The immobilized HRP maintained 93.71% of its initial activity after 45 days of storage at 4 °C. Regarding the biodegradation of phenol red, immobilized HRP resulted in 63.57% degradation after 90 min. After 10 cycles of reuse, the immobilized HRP was able to maintain 43.06% of its initial biodegradative capacity and 42.36% of its enzymatic activity. At the end of 15 application cycles, a biodegradation rate of 8.34% was observed. In conclusion, the results demonstrate that the immobilized HRP is a promising option for use as an industrial biocatalyst in various biotechnological applications.

Immobilization of commercial horseradish peroxidase in calcium alginate-starch hybrid support and its application in the biodegradation of phenol red dye.

In this study, horseradish peroxidase (HRP) was immobilized for the first time on Ca alginate-starch hybrid beads and employed for the biodegradation of phenol red dye. The optimal protein loading was 50 mg/g of support. Immobilized HRP demonstrated improved thermal stability and maximum catalytic activity at 50 °C and pH 6.0, with an increase in half-life (t1/2) and enzymatic deactivation energy (Ed) compared to free HRP. After 30 days of storage at 4 °C, immobilized HRP retained 109% of its initial activity. Compared to free HRP, the immobilized enzyme exhibited higher potential for phenol red dye degradation, as evidenced by the removal of 55.87% of initial phenol red after 90 min, which was 11.5 times greater than free HRP. In sequential batch reactions, the immobilized HRP demonstrated good potential efficiency for the biodegradation of phenol red dye. The immobilized HRP was used for a total of 15 cycles, degrading 18.99% after 10 cycles and 11.69% after 15 cycles, with a residual enzymatic activity of 19.40% and 12.34%, respectively. Overall, the results suggest that HRP immobilized on Ca alginate-starch hybrid supports shows promise as a biocatalyst for industrial and biotechnological applications, particularly for the biodegradation of recalcitrant compounds such as phenol red dye.

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.

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.

Using infrared spectroscopy of serum and chemometrics for diagnosis of paracoccidioidomycosis.

Paracoccidioidomycosis (PCM) is a systemic granulomatous mycosis endemic to Latin America, whose etiologic agents are fungi of the genus Paracoccidioides. PCM is usually diagnosed by microscopic observation of the fungus in biological samples, combined or not with other techniques such as serological methods. However, all currently used diagnostic methods have limitations. The objective of this study was to develop a method based on Fourier transform infrared spectroscopy (FTIR) and chemometric analysis for PCM diagnosis. We included 224 serum samples: 132 PCM sera, 24 aspergillosis sera, 10 cryptococcosis sera, 8 histoplasmosis sera, and 50 sera from healthy blood donors. Samples were analyzed by attenuated total reflection (ATR), and chemometric analyses including exploratory analysis through principal component analysis (PCA) and a classification method (PCM and non-PCM) through orthogonal partial least squares discriminant analysis (OPLS-DA). The spectra were similar, with the main bands up to approximately 1652 cm-1 and 1543 cm-1 (amide I and amide II bands). This same region was mainly responsible for the partial separation of the samples in PCA. The OPLS-DA model correctly classified all serum samples with only one latent variable, with a determination coefficient (R²) higher than 0.999 for both the calibration set and prediction set. Sensitivity and specificity were 100% for both sets, showing better performance than the reference diagnostic methods. Therefore, the use of FTIR/ATR together with OPLS-DA modeling proved to be a promising method for PCM diagnosis.

Saliva may predict quality of life in psoriasis as measured by Fourier transform infrared spectroscopy (FTIR) and chemometrics.

BACKGROUND: Psoriasis is a chronic skin disease, with several comorbidities, such as psoriatic arthritis, inflammatory bowel disease, metabolic syndrome, and impaired quality of life and work activity. The Dermatology Life Quality Index (DLQI) is the most commonly used quality of life index in psoriatic patients, as it is a marker of severe disease. This study evaluated the association between salivary Fourier transform Infrared Spectroscopy (FTIR) metabolic fingerprints and severity of psoriasis as measured by DLQI, using chemometric algorithms. MATERIALS AND METHODS: Saliva was collected from 56 (27 with DLQI ≤ 10 [GI]; 29 with DLQI > 10 [GII]) psoriatic patients diagnosed and assessed by DLQI for disease severity by a dermatologist and analyzed by the transflectance technique in mid-infrared. Hierarchic cluster analysis (HCA), principal component analysis (PCA), orthogonal partial least squares discriminant analysis (OPLS-DA) and orthogonal partial least squares (OPLS) algorithms were used to associate salivary FTIR spectra with the respective DLQI scores. RESULTS: Second derivative (2D) discriminated GI and GII at 2522 cm-1 (p < 0.0001). HCA and PCA partially discriminated GI from GII at 4000-450 cm-1 (p = 0.042 and 0.00821, respectively). Data processing with 1st derivative (1D), 3 latent variables (LV) and 1 orthogonal signal correction (OSC) component at 2550-1801 cm-1 generated an FTIR/OPLS-DA model with 100% accuracy to classify the severity of psoriasis, and an FTIR/OPLS model to quantify DLQI (range 0-28) with high performance: root mean square error of prediction (RMSEP) < 0.01 and coefficient of determination (R2) > 0.9999. CONCLUSIONS: Salivary FTIR combined with chemometric algorithms such as OPLS-DA and OPLS can be used as a clinical tool to classify or predict the severity of psoriasis according to DLQI in patients with confirmed psoriasis.

Rapid classification of chromoblastomycosis agents genera by infrared spectroscopy and chemometrics supervised by sequencing of rDNA regions.

Chromoblastomycosis (CBM) is a skin and subcutaneous infection caused by species of seven fungal genera. Identification of CBM species is performed by DNA sequencing of one or more genes, which becomes a time-consuming work. Fourier Transform Infrared Spectroscopy (FTIR) has been used for the identification of other microorganisms, however, only one CBM genus was evaluated by FTIR analysis to date. Therefore, the study is aimed to differentiate the CBM agents for identification at genera level using FTIR supervised by Internal Transcribed Spacer (ITS) rDNA region. Seventy-seven isolates of the main five CBM genera were prepared for Attenuated Total Reflection FTIR (ATR-FTIR) with a new methodology using slices of dry fungus in glass fixing-modeling proposed in this study. The algorithm Hierarchical Cluster Analysis (HCA) was used to analyze the differences and similarities between species through the spectra. Orthogonal Partial Least Square Discriminant Analysis (OPLS-DA) allowed to correctly classify all samples of five CBM genera. The ATR-FTIR/OPLS-DA models highlighted important contributions of regions attributed to NH and OH stretching, amide I of proteins, polysaccharides bands and fingerprint region for the complete differentiation of the genera investigated. Thus, FTIR can be a fast and inexpensive alternative for identification of CBM agents.

Prediction of itraconazole minimum inhibitory concentration for Fonsecaea pedrosoi using Fourier Transform Infrared Spectroscopy (FTIR) and chemometrics.

Fonsecaea pedrosoi is one of the main agents of chromoblastomycosis, a chronic subcutaneous mycosis. Itraconazole (ITC) is the most used antifungal in its treatment, however, in vitro antifungal susceptibility tests are important to define the best therapy. These tests are standardized by the Clinical and Laboratory Standards Institute (CLSI), but these protocols have limitations such as the high complexity, cost and time to conduct. An alternative to in vitro susceptibility test, which overcomes these limitations, is FTIR. This study determined the minimum inhibitory concentration (MIC) of itraconazole for F. pedrosoi, using FTIR and chemometrics. The susceptibility to ITC of 36 strains of F. pedrosoi was determined according to CLSI and with the addition of tricyclazole (TCZ), to inhibit 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis. Strains were grown in Sabouraud agar and prepared for Attenuated Total Reflection (ATR)/FTIR. Partial least squares (PLS) regression was performed using leave-one-out cross-validation (by steps of quintuplicates), then tested on an external validation set. A coefficient of determination (R²) higher than 0.99 was obtained for both the MIC-ITC and MIC-ITC+TCZ ATR/PLS models, confirming a high correlation of the reference values with the ones predicted using the FTIR spectra. This is the first study to propose the use of FTIR and chemometric analyses according to the M38-A2 CLSI protocol to predict ITC MICs of F. pedrosoi. Considering the limitations of the conventional methods to test in vitro susceptibility, this is a promising methodology to be used for other microorganisms and drugs.

Melanin: Quantification and protection against oxidative stress in chromoblastomycosis agents.

Chromoblastomycosis (CBM) is a chronic cutaneous and subcutaneous infection caused by melanized fungal species. We quantified the extractable melanin of 77 strains of CBM agents distributed within five genera. Moreover, resistance to oxidative stress was evaluated in strains exposed or not to the melanin inhibitor tricyclazole. The median percentage of melanin mass extracted from dry fungal mass varied from 0.69 (Rhinocladiella similis) to 3.81 (Phialophora americana). Inhibition of melanin synthesis decreased survival rates to hydrogen peroxide. Together, these data highlight the importance of melanin in CBM agents.

Differentiation of Candida albicans, Candida glabrata, and Candida krusei by FT-IR and chemometrics by CHROMagar™ Candida.

Pathogenic Candida species are detected in clinical infections. CHROMagar™ is a phenotypical method used to identify Candida species, although it has limitations, which indicates the need for more sensitive and specific techniques. Infrared Spectroscopy (FT-IR) is an analytical vibrational technique used to identify patterns of metabolic fingerprint of biological matrixes, particularly whole microbial cell systems as Candida sp. in association of classificatory chemometrics algorithms. On the other hand, Soft Independent Modeling by Class Analogy (SIMCA) is one of the typical algorithms still little employed in microbiological classification. This study demonstrates the applicability of the FT-IR-technique by specular reflectance associated with SIMCA to discriminate Candida species isolated from vaginal discharges and grown on CHROMagar™. The differences in spectra of C. albicans, C. glabrata and C. krusei were suitable for use in the discrimination of these species, which was observed by PCA. Then, a SIMCA model was constructed with standard samples of three species and using the spectral region of 1792-1561cm-1. All samples (n=48) were properly classified based on the chromogenic method using CHROMagar™ Candida. In total, 93.4% (n=45) of the samples were correctly and unambiguously classified (Class I). Two samples of C. albicans were classified correctly, though these could have been C. glabrata (Class II). Also, one C. glabrata sample could have been classified as C. krusei (Class II). Concerning these three samples, one triplicate of each was included in Class II and two in Class I. Therefore, FT-IR associated with SIMCA can be used to identify samples of C. albicans, C. glabrata, and C. krusei grown in CHROMagar™ Candida aiming to improve clinical applications of this technique.

Avaliação da qualidade do ar de um centro cirúrgico de um hospital do sul do Brazil / Evaluation of the quality of air in a surgical center of a hospital in the south of Brazil / Evaluación de la calidad del aire de un centro quirúrgico de un hospital del sur de Brasil

Objetivo Avaliar a qualidade do ar das salas do centro cirúrgico de um hospital no Sul do Brasil. Métodos Foi realizada uma avaliação dos parâmetros de confortabilidade (temperatura, luminosidade, concentração de dióxido de carbono e umidade relativa do ar), análise microbiológica e cromatográfica do material particulado presente nos filtros dos aparelhos de ar condicionados. Resultados Em relação aos aspectos de confortabilidade, três salas cirúrgicas não apresentaram temperatura de acordo com a legislação vigente, mas encontram-se dentro do padrão ideal nos demais fatores ambientais. Na análise do material particulado, obteve-se um maior número de bioaerossóis fúngicos de colônias viscosas do que as filamentosas. Conclusões Constatou-se que os principais aspectos da qualidade do ar no hospital são atendidos, no entanto há a necessidade de modificação de aspectos construtivos nas salas de cirurgia para que se reduza a possibilidade de contaminações por via atmosférica e possa reduzir a concentração de dióxido de carbono no ambiente.(AU)

Objective The aim of this study was to evaluate the quality of air in surgical centre rooms of a hospital in the South of Brazil. Methods An evaluation of the parameters of comfortability (temperature, luminosity, concentration of carbon dioxide and relative humidity), microbiological analysis and chromatographic of the particulate material present in the filters of air conditioners was carried out. Results Regarding the aspects of comfortability, three surgical rooms did not present temperature in accordance to the current legislation, but were found within the ideal patterns in other environmental factors. In the analysis of particulate material, a larger number of fungal bio-aerosols of viscous colonies was obtained than filamentous. Conclusion It was found that the main aspects related to the quality of air in the hospital being studied are being accomplished; however there is a need of modifying building aspects in the surgical rooms in order to reduce the possibility of contamination by air and decrease the concentration of carbon dioxide in the environment.(AU)

Objetivo Evaluar la calidad del aire de las salas del centro quirúrgico de un hospital en el "Vale do Rio Pardo", en el sur de Brasil. Métodos Fue realizada una evaluación de los parámetros de confortabilidad (temperatura, luminosidad, concentración de dióxido de carbono y humedad relativa del aire), análisis microbiológico y cromatográfico del material particulado presente en los filtros de los aparatos de aire acondicionados. Resultados En relación a los aspectos de confortabilidad, tres salas quirúrgicas no presentaron temperatura de acuerdo con la legislación vigente, pero se encuentran dentro del padrón ideal en los demás factores ambientales. En el análisis del material particulado, se obtuvo un número más grande de bioaerosoles fúngicos de colonias viscosas de que las filamentosas. Conclusión Se constató que los principales aspectos relacionados a la calidad del aire en el hospital en estudio, son atendidos, sin embargo, ahí la necesidad de cambio de aspectos constructivos en las salas de cirugía para que se reduzca la posibilidad de contaminaciones por vía atmosférica y pueda reducir la concentración de dióxido de carbono en el ambiente.(AU)

[Evaluation of the quality of air in a surgical center of a hospital in the south of Brazil]. / Avaliação da qualidade do ar de um centro cirúrgico de um hospital do sul do Brazil.

Objective The aim of this study was to evaluate the quality of air in surgical centre rooms of a hospital in the South of Brazil. Methods An evaluation of the parameters of comfortability (temperature, luminosity, concentration of carbon dioxide and relative humidity), microbiological analysis and chromatographic of the particulate material present in the filters of air conditioners was carried out. Results Regarding the aspects of comfortability, three surgical rooms did not present temperature in accordance to the current legislation, but were found within the ideal patterns in other environmental factors. In the analysis of particulate material, a larger number of fungal bio-aerosols of viscous colonies was obtained than filamentous. Conclusion It was found that the main aspects related to the quality of air in the hospital being studied are being accomplished; however there is a need of modifying building aspects in the surgical rooms in order to reduce the possibility of contamination by air and decrease the concentration of carbon dioxide in the environment.

The influence of active and passive smoking on the cardiorespiratory fitness of adults.

BACKGROUND: The aim of this study was to analyze the influence of active and passive smoking on cardiorespiratory responses in asymptomatic adults during a sub-maximal-exertion incremental test. METHODS: The participants (n = 43) were divided into three different groups: active smokers (n = 14; aged 36.5 ± 8 years), passive smokers (n = 14; aged 34.6 ± 11.9 years) and non-smokers (n = 15; aged 30 ± 8.1 years). They all answered the Test for Nicotine Dependence and underwent anthropometric evaluation, spirometry and ergospirometry according to the Bruce Treadmill Protocol. RESULTS: VO2max differed statistically between active and non-smokers groups (p < 0.001) and between non-smokers and passive group (p=0.022). However, there was no difference between the passive and active smokers groups (p=0.053). Negative and significant correlations occurred between VO2max and age (r = - 0.401, p = 0.044), percentage of body fat (r = - 0.429, p = 0.011), and waist circumference (WC) (r = - 0.382, p = 0.025). CONCLUSION: VO2max was significantly higher in non-smokers compared to active smokers and passive smokers. However, the VO2max of passive smokers did not differ from active smokers.

Cultivation of Desmodesmus subspicatus in a tubular photobioreactor for bioremediation and microalgae oil production.

The microalgae Desmodesmus subspicatus (Chlorophyta) was cultivated in a tubular photobioreactor using effluent from the wastewater treatment plant of the University of Santa Cruz do Sul, Brazil to demonstrate the reactor's operation. The algae's ability to remove nutrients from wastewater and the oleaginous potential of the algae's biomass were also evaluated. Total phosphorus and ammonia nitrogen were measured. The photobioreactor consisted of a system of three acrylic tubes, a reservoir, connections and a CO2 supply. The gas supply was semicontinuous with CO2 added from a cylinder. The culture's growth was estimated from cell numbers counted on a daily basis. Lipid content in the biomass was analysed using gas chromatography. A maximum cell density of 9.11 x 10(6) cellsmL-1 and a dry weight of 234.00 mg L-1 were obtained during cultivation without CO2, and these values rose to 42.48 x 10(6) cells mL-1 and 1277.44 mg L-1, respectively, when CO2 was added to the cultivation. Differences in the quality of the effluent and the presence of CO2 did not result in different lipid profiles. The presence ofpalmitic acid and oleic acid was notable. The average extracted oil content was 18% and 12% for cultivation with and without the input of CO2, respectively.

Application of 6-nitrocoumarin as a substrate for the fluorescent detection of nitroreductase activity in Sporothrix schenckii.

INTRODUCTION: Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. METHODS: Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. RESULTS: All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. CONCLUSION: The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection.

APPLICATION OF 6-NITROCOUMARIN AS A SUBSTRATE FOR THE FLUORESCENT DETECTION OF NITROREDUCTASE ACTIVITY IN Sporothrix schenckii / Aplicação de 6-nitrocumarina como substrato fluorescente para detecção de atividade nitroredutásica em Sporothrix schenckii

Introduction Sporothrix schenckii is a thermal dimorphic pathogenic fungus causing a subcutaneous mycosis, sporotrichosis. Nitrocoumarin represents a fluorogenic substrate class where the microbial nitroreductase activity produces several derivatives, already used in several other enzyme assays. The objective of this study was the analysis of 6-nitrocoumarin (6-NC) as a substrate to study the nitroreductase activity in Sporothrix schenckii. Methods Thirty-five samples of S. schenckii were cultivated for seven, 14 and 21 days at 35 °C in a microculture containing 6-nitrocoumarin or 6-aminocoumarin (6-AC) dissolved in dimethyl sulfoxide or dimethyl sulfoxide as a negative control, for posterior examination under an epifluorescence microscope. The organic layer of the seven, 14 and 21-day cultures was analyzed by means of direct illumination with 365 nm UV light and by means of elution on G silica gel plate with hexane:ethyl acetate 1:4 unveiled with UV light. Results All of the strains showed the presence of 6-AC (yellow fluorescence) and 6-hydroxylaminocoumarin (blue fluorescence) in thin layer chromatography, which explains the green fluorescence observed in the fungus structure. Conclusion The nitroreductase activity is widely distributed in the S. schenckii complex and 6-NC is a fluorogenic substrate of easy access and applicability for the nitroreductase activity detection. .

Introdução Sporothrix schenckii é um fungo dimórfico térmico, agente etiológico de micose subcutânea, a esporotricose. Nitrocumarina representa classe de substratos fluorogênicos em que a atividade nitroredutásica microbiana produz vários derivados, já utilizados em vários outros ensaios enzimáticos. O objetivo deste estudo foi analisar 6-nitrocumarina (6-NC) como substrato para estudo da atividade nitroredutásica em Sporothrix schenckii. Métodos Trinta e cinco isolados de S. schenckii foram cultivados por sete, 14 e 21 dias a 35 °C em um microcultivo contendo 6-nitrocumarina ou 6-aminocumarina (6-AC) solubilizada em dimetilsulfóxido ou dimetilsulfóxido como controle negativo, para posterior análise em microscópio de epifluorescência. A fase orgânica da cultura de sete, 14 e 21 dias foi analisada por meio de iluminação direta com luz UV de 365 nm e por eluição em placas de sílica gel G com hexano:acetato de etila 1:4 e revelada com luz UV. Resultados Todos os isolados mostraram a presença de 6-AC (fluorescência amarela) e 6-hidroxilaminocumarina (fluorescência azul) em cromatografia em camada delgada, que explica a fluorescência verde observada na estrutura dos fungos. Conclusão A atividade nitroredutásica é amplamente distribuída no complexo S. schenckii e 6-NC é um substrato fluorogênico de fácil obtenção e aplicabilidade para detecção da atividade nitroredutásica. .

A fast and cost-effective methodology for Fonsecaea pedrosoi ATCC46428 staining using ESIPT fluorescent dyes.

The microscopic morphology of Fonsecaea pedrosoi ATCC46428 was observed using two benzazole derivatives, 2-(2'-hydroxyphenyl)benzoxazole and 2-(5'-amino-2'-hydroxyphenyl)benzoxazole, which emit intense fluorescence by a proton transfer mechanism in the electronically excited state (ESIPT). The cell surface could be successfully stained with fluorescent dye solutions of 10 microM-10 mM using two different fast and cost-effective procedures. At these concentrations, any structure or dye crystallization could be observed. Concerning the external microstructural details, only the amino derivative allowed the differentiation between hyphae and conidia. These dyes presented some advantages comparing to commercial dyes, since the stained cells showed high chemical, thermal and photochemical stability during the experiments and also after several months of storage at room temperature and normal light exposition. Procedure 1 presented the advantage to be used when heating can change the chemical or biochemical cell composition. On the other hand Procedure 2 showed to be useful as a routine methodology for cells staining. The results allowed to propose a simple and highly sensitive assay to study the F. pedrosoi micromorphology by epifluorescence microscopy. This methodology can probably be extended for other fungi of clinical interest.