Fernanda E. Pinto

Chemical profile of mango (Mangifera indica L.) using electrospray ionisation mass spectrometry (ESI-MS).

Mangifera indica L., mango fruit, is consumed as a dietary supplement with purported health benefits; it is widely used in the food industry. Herein, the chemical profile of the Ubá mango at four distinct maturation stages was evaluated during the process of growth and maturity using negative-ion mode electrospray ionisation Fourier transform ion cyclotron resonance mass spectrometry (ESI(-)FT-ICR MS) and physicochemical characterisation analysis (total titratable acidity (TA), total soluble solids (TSS), TSS/TA ratio, and total polyphenolic content). Primary (organic acids and sugars) and secondary metabolites (polyphenolic compounds) were mostly identified in the third maturation stage, thus indicating the best stage for harvesting and consuming the fruit. In addition, the potential cancer chemoprevention of the secondary metabolites (phenolic extracts obtained from mango samples) was evaluated using the induction of quinone reductase activity, concluding that fruit polyphenols have the potential for cancer chemoprevention.

Morphometry to identify subtypes of leukocytes.

INTRODUCTION Recent studies in image cytometry evaluated the replacement of specific markers by morphological parameters. The aim of this study was to develop and evaluate a method to identify subtypes of leukocytes using morphometric data of the nuclei. METHOD The analyzed images were generated with a laser scanning cytometer. Two free programs were used for image analysis and statistical evaluation: Cellprofiler and Tanagra respectively. A sample of leukocytes with 200 sets of images (DAPI, CD45 and CD14) was analyzed. Using feature selection, the 20 best parameters were chosen to conduct cross-validation. RESULTS The morphometric data identified the subpopulations of the analyzed leukocytes with a sensitivity and specificity of 0.95 per sample. CONCLUSION The present study is the first that identifies subpopulations of leukocytes by nuclear morphology.

Replacement of specific markers for apoptosis and necrosis by nuclear morphology for affordable cytometry.

OBJECTIVE The objective of the present study was to employ high throughput image analysis to detect necrosis and apoptosis. Specific markers were replaced by morphological parameters of cells and nuclei. METHOD Fresh blood was taken from a healthy female and given a treatment to induce cell necrosis and apoptosis. Afterward, the samples were stained with AnnexinV-FITC, DRAQ5 and DAPI. Slides were made and analyzed using the cytometer iCys. Pictures were scanned. The analyzed sample consisted of 73 sets of images of DAPI, DRAQ5 and AnnexinV-FITC, respectively. For image analysis and subsequent statistical processing, the CellProfiler and CellProfilerAnalyst were used. Each sample was analyzed twice. The first analysis was conducted using the markers (DAPI, DRAQ5 and Annexin) for an unequivocal identification and subsequent count of necrotic, apoptotic and live cells (gold standard). Thereafter, a second analysis was performed for the nuclear morphology and texture (morphometric analysis). After the machine learning process was completed, the software calculated the quantity of cells in each of the three groups. A comparison between the result of the gold standard and the morphometric analysis was performed using linear regression and a Bland-Altman test. RESULTS The linear regression between the two compared analyses was r(2)=0.57 for apoptosis, r(2)=0.84 for necrosis and r(2)=0.79 for living cells. CONCLUSION It may be concluded that it is possible to replace specific markers against morphology without losing the reproducible high-throughput character of a cytometric analysis.

Chemical profile of pineapple cv. Vitória in different maturation stages using electrospray ionization mass spectrometry

BACKGROUND Pineapple is the fruit of Ananas comosus var. comosus plant, being cultivated in tropical areas and has high energy content and nutritional value. Herein, 30 samples of pineapple cv. Vitória were analyzed as a function of the maturation stage (0-5) and their physico-chemical parameters monitored. In addition, negative-ion mode electrospray ionization mass spectrometry [ESI(-)FT-ICR MS] was used to identify and semi-quantify primary and secondary metabolites present in the crude and phenolic extracts of pineapple, respectively. RESULTS Physico-chemical tests show an increase in the total soluble solids (TSS) values and in the TSS/total titratable acidity ratio as a function of the maturity stage, where a maximum value was observed in stage 3 (¾ of the fruit is yellow, which corresponds to the color of the fruit peel). ESI(-)FT-ICR MS analysis for crude extracts showed the presence mainly of sugars as primary metabolites present in deprotonated molecule form ([M - H]- and [2 M - H]- ions) whereas, for phenolic fractions, 11 compounds were detected, being the most abundant in the third stage of maturation. This behavior was confirmed by quantitative analysis of total polyphenols. CONCLUSION ESI-FT-ICR MS was efficient in identifying primary (carbohydrates and organic acids) and secondary metabolites (13 phenolic compounds) presents in the crude and phenolic extract of the samples, respectively.

Efficacy of guaco mouthwashes (Mikania glomerata and Mikania laevigata) on the disinfection of toothbrushes

Guaco Mikania glomerata Spreng. and M. laevigata Sch. Bip. ex Baker, Asteraceae, has antimicrobial activity and may be helpful in reducing the incidence of oral diseases. This double-blinded randomized clinical trial aimed to evaluate the efficacy of guaco mouthwashes on the disinfection of toothbrushes used by preschool children, tested positive for mutans streptococci (MS), as well as the quantification of its coumarin contents by high performance liquid chromatography. Ethanol extracts were obtained by percolation. The mouthwashes were prepared with 2.5% g/mL M. glomerata and M. laevigata ethanol extracts, standardized for their coumarin content (% mg/mg). Antimicrobial effect of the mouthwashes and extracts were assessed in vitro against Streptococcus mutans (ATCC 25175TM), using 2.4 to 500 µg/mL to calculate the minimum inhibitory concentration (MIC). For the in vivo study, 24 patients were randomly assigned to a 4-stage changeover system with a one-week interval between each stage. All solutions were used in all stages by a different group of children. After brushing without toothpaste, toothbrushes (n=96) were sprayed with water and solutions of M. glomerata (2.5%), M. laevigata (2.5%) and chlorhexidine (0.12%). Microbiological analysis was carried out after 4 h and 30 days, respectively. MIC values were 400, 125 and 14 µg/mL, respectively, for both crude ethanol extracts, mouthwashes of M. glomerata and M. laevigata. Statistical analysis showed that all solutions decreased contamination of toothbrushes by mutans streptococci (chlorhexidine 50.7±17.7%; M. glomerata 37.3±23.7% and M. laevigata 28.7±25.1% of inhibition). Treatment with chlorhexidine and M. glomerata were statistically similar (p>0.05). M. glomerata mouthwash could be useful in herbal strategy programs against mutans streptococci and the marker coumarin may be not related to the activity observed.

Study of the Corrosive Behavior of the AISI 1020 Steel in Acid Crude Oil by Microscopic Techniques (LM, AFM and SEM/EDX) and Raman Spectroscopy

Microscopic techniques were combined to study the influence of corrosion rate on the morphologic behavior of AISI 1020 steel specimens submitted to thermal degradation of a typical acid crude oil (total acid number (TAN) = 2.1390 mg KOH g and total sulfur (S) = 0.7778 wt.%). The techniques used were light microscopy (LM), scanning electron microscopy/energy dispersive spectroscopy (SEM/EDX), atomic force microscopy (AFM) as well as Raman spectroscopy. Assays were performed in six different degradation time (t = 6, 12, 24, 36, 48 and 72 h) at 320 °C. After the exposure of the specimens to petroleum, a reduction above 37% in the TAN after t = 72 h was observed, with a maximum corrosion rate during the first periods of degradation (t = 6 and 12 h). Correlating the TAN and corrosion rate data with the microscopic data, the images of LM, AFM, and SEM/EDX showed that after 6 h of exposure to petroleum, a passivation film was formed on the surface of the steel. This film consisted of two layers, an external one, formed of FeS, and an internal one, composed of iron oxides and oxyhydroxides. However, after 48 h of thermal degradation, this morphology was altered to a single layer of FeS coating the steel surface.

Study of the Naphthenic Corrosion of AISI 316 and AISI 1020 Steels by Light, Scanning Electron and Atomic Force Microscopies (LM, SEM and AFM)

Corrosion processes were evaluated for AISI 316 and AISI 1020 steels by scanning electron microscopy (SEM), atomic force microscopy (AFM) and light microscopy. Coupons were immersed in four crude oil samples with different total acid numbers (TANs) for 48 days at room temperature. The steels were also exposed to three blends (B1-B3), produced by quaternary mixtures of the oils, with lower TANs than their respective original oils. SEM micrographs showed pitting-type corrosion in most cases. AFM imaging showed drastic changes in the peak-to-peak values, topographic profiles and phase images of the AISI 1020 coupons exposed to all oils and blend B3 as compared to the unexposed steel. Defects were produced on the surface of almost all the samples exposed to naphthenic corrosion. Exposure to the oil blends reduced the extent of the naphthenic corrosion on the coupons, especially blends B1 and B2. The oil mixtures (blends) reduced corrosion.

Hexane partition from Annona crassiflora Mart. promotes cytotoxity and apoptosis on human cervical cancer cell lines

SummaryCervical cancer is the third most commonly diagnosed tumor type and the fourth cause of cancer-related death in females. Therapeutic options for cervical cancer patients remain very limited. Annona crassiflora Mart. is used in traditional medicine as antimicrobial and antineoplastic agent. However, little is known about its antitumoral properties. In this study the antineoplastic effect of crude extract and derived partitions from A. crassiflora Mart in cervical cancer cell lines was evaluated. The crude extract significantly alters cell viability of cervical cancer cell lines as well as proliferation and migration, and induces cell death in SiHa cells. Yet, the combination of the crude extract with cisplatin leads to antagonistic effect. Importantly, the hexane partition derived from the crude extract presented cytotoxic effect both in vitro and in vivo, and initiates cell responses, such as DNA damage (H2AX activity), apoptosis via intrinsic pathway (cleavage of caspase-9, caspase-3, poly (ADP-ribose) polymerase (PARP) and mitochondrial membrane depolarization) and decreased p21 expression by ubiquitin proteasome pathway. Concluding, this work shows that hexane partition triggers several biological responses such as DNA damage and apoptosis, by intrinsic pathways, and was also able to promote a direct decrease in tumor perimeter in vivo providing a basis for further investigation on its antineoplastic activity on cervical cancer.

Bauhinia variegata candida Fraction Induces Tumor Cell Death by Activation of Caspase-3, RIP, and TNF-R1 and Inhibits Cell Migration and Invasion In Vitro

Metastasis remains the most common cause of death in cancer patients. Inhibition of metalloproteinases (MMPs) is an interesting approach to cancer therapy because of their role in the degradation of extracellular matrix (ECM), cell-cell, and cell-ECM interactions, modulating key events in cell migration and invasion. Herein, we show the cytotoxic and antimetastatic effects of the third fraction (FR3) from Bauhinia variegata candida (Bvc) stem on human cervical tumor cells (HeLa) and human peripheral blood mononuclear cells (PBMCs). FR3 inhibited MMP-2 and MMP-9 activity, indicated by zymogram. This fraction was cytotoxic to HeLa cells and noncytotoxic to PBMCs and decreased HeLa cell migration and invasion. FR3 is believed to stimulate extrinsic apoptosis together with necroptosis, assessed by western blotting. FR3 inhibited MMP-2 activity in the HeLa supernatant, differently from the control. The atomic mass spectrometry (ESI-MS) characterization suggested the presence of glucopyranosides, D-pinitol, fatty acids, and phenolic acid. These findings provide insight suggesting that FR3 contains components with potential tumor-selective cytotoxic action in addition to the action on the migration of tumor cells, which may be due to inhibition of MMPs.

Haematological and serum biochemical reference values for captive blue-fronted amazon parrot

The parrot species Amazona aestiva is categorised as least concern by the Red List of Threatened Species and is one of the most popular parrot species to be traded illegally. Such illegal trade and degradation of its habitat is a threat to the species, with a decrease in the species’ population already reported. This study aimed to determine haematology and biochemistry reference values for A. aestiva to assist veterinarian and conservation programmes in the care of this species. Sampling was conducted in April 2014; a total of 32 birds were manually restrained, and their blood samples were collected and divided into two aliquots. Haematological parameters evaluated included red blood cell (RBC) count, white blood cell (WBC) count, differential leukocyte count, haematocrit, thrombocyte count, haemoglobin concentration and RBC indexes. The biochemical parameters measured were alkaline phosphatase, alanine aminotransferase, gamma glutamyltransferase, aspartate aminotransferase, uric acid, cholesterol, triglyceride, calcium, phosphorus, creatinine, albumin, total protein and urea. Comparison between the male and female groups showed no significant difference in haematological parameters, and amongst the biochemical parameters, only calcium level was significantly different. No basophils or eosinophils were detected. Reference values of all measured haematological and biochemical parameters were recorded for the species. Although the results across studies may vary, it is important that all obtained information is revealed for future use. The data gathered in the present study add to the existing knowledge about A. aestiva and will be useful in the conservation and management programmes and for veterinarians.