A voltammetric peptide biosensor for Cu2+ metal ion quantification in coffee seeds.

A prion-derived copper(II)-binding peptide was assembled onto a gold electrode for the building of a voltammetric biosensor for measuring the Cu2+ metal ion in biological samples. The chosen sequence was H-CVNITKQHTVTTTT-NH2, with an appended cysteine residue for binding to the gold surface as a self-assembled monolayer and a histidine residue as the anchorage point for copper(II) complexation. The biosensor showed a linear range of 10-7 to 10-6 M with an 8.0 × 10-8 M detection limit and a 1.0 × 10-7 M quantification limit, with good precision, trueness, and absence of matrix effect. The quantification of Cu2+ was performed in the presence of other transition metal ions, such as Zn2+, Cd2+, Fe2+, or Ni2+, which indicates the excellent selectivity of the biosensor. When the modified electrode was applied for measuring copper(II) in calcined coffee seeds, a difference in copper amount was observed between two Coffea arabica cultivars that were submitted to a treatment with a copper-based antifungal, showing the applicability of the biosensor in the agricultural field.

ABCB1 and ERCC1 gene polymorphisms are associated with nephro- and hepatotoxicity to carboplatin/paclitaxel-based chemotherapy in patients with gynecologic cancers.

BACKGROUND: Paclitaxel/carboplatin combination is the standard chemotherapeutic protocol for gynecologic cancers, but severe toxicities may compromise treatment. There is great inter-individual variability regarding the incidence and severity of toxicities, which may be due to single-nucleotide polymorphisms (SNPs) affecting drug disposition or cellular sensitivity. Here we investigate the impact of selected SNPs in ERCC1, ABCB1, CYP2C8, and CYP3A5 genes on the incidence of severe toxicities, including nephro- and hepatotoxicity. METHODS: A cohort of 507 gynecological cancer patients receiving paclitaxel/carboplatin was recruited at the Brazilian National Cancer Institute (INCA-Brazil). Clinical data were obtained during routine consultations or from electronic medical records. Toxicities were graded according to the Common Terminology Criteria for Adverse Events (CTCAE 5.0). Genotyping was performed using real-time PCR. RESULTS: ABCB1 c.1236C>T was associated with moderate-to-severe (grades 2-4) nephrotoxicity (ORadjusted 2.40; 95% CI 1.39-4.15), even after adjustment for age (≥ 65) and diabetes. The risk association between ABCB1 c.1236C>T and moderate-to-severe nephrotoxicity following paclitaxel/carboplatin chemotherapy was also present among non-diabetic patients (ORadjusted 2.16; 95% CI 1.22-3.82). ERCC1 c.118C>T was the only individual variable associated with an increased risk for moderate-to-severe (grades 2-4) hepatotoxicity (OR 3.71; 95% CI 1.08-12.77), severe nausea (OR 4.18; 95% CI 1.59-10.95), and severe myalgia (OR 1.95; 95% CI 1.12-3.40). CONCLUSIONS: ABCB1 c.1236C>T and ERCC1 c.118C>T might serve as potential biomarkers for the risk of moderate-to-severe toxicities to carboplatin/paclitaxel chemotherapy of gynecological cancers.

Acute Hemodynamic Responses to Repetitions to Failure Using Different Resistance Exercises and Protocols in Normotensive Men: A crossover study.

The present crossover design study investigated acute hemodynamic responses to two sets of leg press (LP) and bench press (BeP) at 10 and 20 repetition maximum (RM) in ten normotensive young men. At the end of each set, an increase in systolic blood pressure (SBP), heart rate (HR), and rate pressure product (RPP) was observed (p < .01), with no differences between intensities, but SBP was greater during the LP exercise (p < .01). Lower resting values of diastolic blood pressure (DBP) were observed in the post-BeP exercise period (p < .05), suggesting that DBP post-exercise hypotension may be more evident after upper-limb exercise.

Impact of gene polymorphisms on the systemic toxicity to paclitaxel/carboplatin chemotherapy for treatment of gynecologic cancers.

PURPOSE: Gynecologic malignancies are often detected in advanced stages, requiring chemotherapy with taxane/platinum combinations, which may cause severe toxicities, such as neutropenia and peripheral neuropathy. Gene polymorphisms are suspected as possible causes for the interindividual variability on chemotherapy toxicities. OBJECTIVE: To evaluate the role of ABCB1 1236C>T, 3435C>T; CYP2C8*3; CYP3A5*3C variants on paclitaxel/carboplatin toxicities. METHODS: A cohort of 503 gynecologic cancer patients treated with paclitaxel/carboplatin at the Brazilian National Cancer Institute (INCA-Brazil) was recruited (2013-2017). Polymorphisms were genotyped by real-time PCR, and toxicities were evaluated by patients' interviews at each chemotherapy cycle and by data collection from electronic records. The association of clinical features and genotypes with severe toxicities was estimated using Pearson's Chi square tests and multiple regression analyses, with calculation of adjusted odds ratios (ORadjusted), and respective 95% confidence intervals (95% CI). RESULTS: CYP2C8*3 was significantly associated with increased risks of severe (grades 3-4) neutropenia (ORadjusted 2.11; 95% CI 1.24-3.6; dominant model) and severe thrombocytopenia (ORadjusted 4.93; 95% CI 1.69-14.35; recessive model), whereas ABCB1 variant genotypes (ORadjusted 2.13; 95% CI 1.32-3.42), in association with CYP2C8*3 wild type (GG) (ORadjusted 1.93; 95% CI 1.17-3.19), were predictive of severe fatigue. CONCLUSIONS: The present study suggests that CYP2C8*3 is a potential predictor of hematological toxicities related to paclitaxel/carboplatin treatment. Since hematological toxicities, especially neutropenia, may lead to dose delay or treatment interruption, such prognostic evaluation may contribute to clinical management of selected patients with paclitaxel-based chemotherapy.

Combined use of ELISA and Western blot with recombinant N protein is a powerful tool for the immunodiagnosis of avian infectious bronchitis.

BACKGROUND: The avian infectious bronchitis virus (IBV) remains a significant source of loss in the poultry industry and early diagnosis is required to prevent the disease from spreading. This study examined the combined use of an ELISA and Western blot (WB) to detect antibodies against the nucleocapsid protein (N) of IBV. The coding sequence for N was amplified by RT-PCR and expressed in Escherichia coli. A soluble recombinant N protein (rN) of approximately 50 kDa was obtained. A total of 389 sera were tested against the rN in ELISA and the results were compared with those of the commercial IDEXX IBV Ab test. ELISA-rN achieved a 90.34% sensitivity and 90.16% specificity. WB confirmed all false negative sera in ELISA-rN or IDEXX test as truly positive. The current study indicate that the combined use of rN in ELISA and WB is a powerful tool for the immunodiagnosis of avian infectious bronchitis. METHODS: Constructed recombinant pAE/n expression vectors were used to transform E. coli BL21(DE3) Star competent cells (Invitrogen). The rN of infectious bronchitis virus was purified by affinity chromatography using HisTrap HP 1 mL columns pre-packed with pre-charged Ni Sepharose in the ÄKTAprime Automated Liquid Chromatography system (GE Healthcare). A total of 389 serum samples from chickens were used to develop and evaluate the ELISA-rN test. To standardize the indirect ELISA development, serum dilutions (1:100, 1:200 and 1:400) and different concentrations of purified rN antigen (50, 100 and 200 ng/well) were tested. Positive and negative sera for IBV were used as controls. The results were compared with those obtained from a commercial kit. Serum samples scored as negative with the commercial kit but as positive with the ELISA-rN were further analysed by Western blot analyses using the rN protein as an antigen. The results of the ELISA-rN were compared to the commercial kit results using receiver-operating characteristics curves, area under the curve, and confidence intervals with the software GraphPad Prism version 6.0 for Windows (GraphPad Software, USA). RESULTS: The expected cDNA fragment of approximately 1240 bp was successfully amplified by PCR using primers designed to select for the coding region of the N protein. The rN was expressed as a soluble protein to avoid the refolding steps and, after purification a yield of 10 mg/L of rN was obtained. The SDS-PAGE results demonstrated the presence of two distinct bands that had a molecular mass of approximately 45 and 50 KDa. Out of 244 sera that scored positive in the commercial ELISA IDEXX IBV Ab Test, 220 were also positive in the ELISA-rN, yielding an ELISA-rN test sensitivity of 90.16%. Out of 145 sera that scored negative in the IDEXX IBV Ab Test, 131 also scored negative in the ELISA-rN, indicating a specificity of 90.34%. Sera that tested negative in the ELISA-rN and positive in the commercial test also reacted with the rN protein in Western blot. CONCLUSIONS: The association between the ELISA and Western blot techniques developed in this study with a subunit of IBV (rN) were able to detect antibodies that the commercial ELISA did not detect suggesting that the ELISA-rN has greater sensitivity.

Cardamonin, a schistosomicidal chalcone from Piper aduncum L. (Piperaceae) that inhibits Schistosoma mansoni ATP diphosphohydrolase.

BACKGROUND: Schistosomiasis is one of the world's major public health problems, and praziquantel (PZQ) is the only available drug to treat this neglected disease with an urgent demand for new drugs. Recent studies indicated that extracts from Piper aduncum L. (Piperaceae) are active against adult worms of Schistosoma mansoni, the major etiological agent of human schistosomiasis. PURPOSE: We investigated the in vitro schistosomicidal activity of cardamonin, a chalcone isolated from the crude extract of P. aduncum. Also, this present work describes, for the first time, the S. mansoni ATP diphosphohydrolase inhibitory activity of cardamonin, as well as, its molecular docking with S. mansoni ATPDase1, in order to investigate its mode of inhibition. METHODS: In vitro schistosomicidal assays and confocal laser scanning microscopy were used to evaluate the effects of cardamonin on adult schistosomes. Cell viability was measured by MTT assay, and the S. mansoni ATPase activity was determined spectrophotometrically. Identification of the cardamonin binding site and its interactions on S. mansoni ATPDase1 were made by molecular docking experiments. RESULTS: A bioguided fractionation of the crude extract of P. aduncum was carried out, leading to identification of cardamonin as the active compound, along with pinocembrin and uvangoletin. Cardamonin (25, 50, and 100 µM) caused 100% mortality, tegumental alterations, and reduction of oviposition and motor activity of all adult worms of S. mansoni, without affecting mammalian cells. Confocal laser scanning microscopy showed tegumental morphological alterations and changes on the numbers of tubercles of S. mansoni worms in a dose-dependent manner. Cardamonin also inhibited S. mansoni ATP diphosphohydrolase (IC50 of 23.54 µM). Molecular docking studies revealed that cardamonin interacts with the Nucleotide-Binding of SmATPDase 1. The nature of SmATPDase 1-cardamonin interactions is mainly hydrophobic and hydrogen bonding. CONCLUSION: This report provides evidence for the in vitro schistosomicidal activity of cardamonin and demonstrated, for the first time, that this chalcone is highly effective in inhibiting S. mansoni ATP diphosphohydrolase, opening the route to further studies of chalcones as prototypes for new S. mansoni ATP diphosphohydrolase inhibitors.

Antiviral activity of a Bacillus sp. P34 peptide against pathogenic viruses of domestic animals.

P34 is an antimicrobial peptide produced by a Bacillus sp. strain isolated from the intestinal contents of a fish in the Brazilian Amazon basin with reported antibacterial activity. The aim of this work was to evaluate the peptide P34 for its in vitro antiviral properties against canine adenovirus type 2 (CAV-2), canine coronavirus (CCoV), canine distemper virus (CDV), canine parvovirus type 2 (CPV-2), equine arteritis virus (EAV), equine influenza virus (EIV), feline calicivirus (FCV) and feline herpesvirus type 1 (FHV-1). The results showed that the peptide P34 exhibited antiviral activity against EAV and FHV-1. The peptide P34 inhibited the replication of EAV by 99.9% and FHV-1 by 94.4%. Virucidal activity was detected only against EAV. When P34 and EAV were incubated for 6 h at 37 °C the viral titer reduced from 10(4.5) TCID50 to 10(2.75) TCID50, showing a percent of inhibition of 98.6%. In conclusion, our results demonstrated that P34 inhibited EAV and FHV-1 replication in infected cell cultures and it showed virucidal activity against EAV. Since there is documented resistance to the current drugs used against herpesviruses and there is no treatment for equine viral arteritis, it is advisable to search for new antiviral compounds to overcome these infections.

Anthelmintic activity of crude extract and essential oil of Tanacetum vulgare (Asteraceae) against adult worms of Schistosoma mansoni.

Schistosomiasis, a parasitic disease caused by trematode flatworms of the genus Schistosoma, affects more than 200 million people worldwide, and its control is dependent on a single drug, praziquantel. Tanacetum vulgare (Asteraceae) is used in folk medicine as a vermifuge. This study aimed to investigate the in vitro schistosomicidal activity of the crude extract (TV) and the essential oil (TV-EO) from the aerial parts of T. vulgare. TV-EO was obtained by hydrodistillation and analyzed by GC/MS, which allowed the identification of ß-thujone (84.13%) as the major constituent. TV and TV-EO, at 200 µg/mL, decreased motor activity and caused 100% mortality of all adult worms. At 100 and 50 µg/mL, only TV caused death of all adult worms, while TV-EO was inactive. TV (200 µg/mL) was also able to reduce viability and decrease production of developed eggs. Confocal laser scanning microscopy showed morphological alterations in the tegument of the S. mansoni surface after incubation with TV (50 and 100 µg/mL). Quantitative analysis on the schistosomes tegument showed that TV caused changes in the numbers of tubercles of S. mansoni male worms in a dose-dependent manner. The findings suggest that T. vulgare is a potential source of schistosomicidal compounds.

Peroxidase-linked assay for detection of antibodies against bovine leukosis virus.

A peroxidase linked assay (PLA) was designed to screen bovine sera for the presence of specific antibodies against bovine leukosis virus (BLV). Out of 201 samples of bovine sera analyzed, 52.2% were considered positive by PLA, 26.4% by AGID, and 38.9% by ELISA. Western blotting analyses excluded 27 samples found to be positive by PLA. PLA showed 100% of sensitivity when compared with AGID and ELISA. Specificity was 64.8% and 78%, respectively (kappa coefficients were 0.70 and 0.83). These findings indicate that PLA can be used as an alternative method for the diagnosis of BLV infection in cattle.

Toxic metals (Pb and Cd) and their respective antagonists (Ca and Zn) in infant formulas and milk marketed in Brasilia, Brazil.

In non-ideal scenarios involving partial or non-breastfeeding, cow's milk-based dairy products are mainstream in infant feeding. Therefore, it is important to study the concentrations of potentially neurotoxic contaminants (Pb and Cd) and their respective counteracting elements (Ca and Zn) in infant dairy products. Fifty-five brands of infant formulas and milk sold in Brasilia, Brazil were analyzed. The dairy products came from areas in the central-west (26%), southeast (29%) and south of Brazil (36%) extending as far as Argentina (7%) and the Netherlands (2%). For toxic Pb and Cd, median concentrations in powdered samples were 0.109 mg/kg and 0.033 mg/kg, respectively; in fluid samples median Pb concentration was 0.084 mg/kg, but median Cd concentration was below the limit of detection and overall values were below reference safety levels. However, 62% of these samples presented higher Pb concentration values than those established by FAO/WHO. Although the inverse correlation between Cd and Zn (Spearman r = -0.116; P = 0.590) was not statistically significant, the positive correlation between Ca and Pb was (Spearman r = 0.619; P < 0.0001). Additionally, there was a significant correlation between Pb and Cd. Furthermore, the study also revealed that provision of the essential trace element Zn in infant formulas can provide adequate amounts of the recommended daily requirements. Infant formulas and milk sold for consumption by infants and children can be an efficient tool to monitor neurotoxic metal risk exposure among young children.

The binding of zinc(II) to Mung Bean Nuclease. A voltammetric study.

Binding of zinc to Mung Bean Nuclease was investigated by anodic stripping voltammetry and cyclic voltammetry. These methods rely on the direct monitoring of the oxidation current of zinc in the absence and presence of Mung Bean Nuclease. Titration curves of Zn(2+) with the enzyme were obtained in concentrations ranging from 1.08x10(-9) to 1.07x10(-8) M and 1.16x10(-8) to 1.04x10(-7) M. The acquired data were used to calculate the dissociation constant and the stoichiometry of the complex. The binding sites of zinc in the Mung Bean Nuclease molecule were investigated using cyclic voltammetry. Two types of binding sites for zinc were identified and were attributed to a mononuclear exposed zinc-binding site with catalytic function and to an inaccessible binuclear zinc-binding site with structural functions.

Ca(II) -gamma-thionin complex: interaction studies by differential pulse voltammetry and MALDI-TOF/MS.

In this work we investigated the formation of a complex between gamma-thionin SI alpha1 and Ca(2+) using differential pulse voltammetry and MALDI TOF/MS. A stoichiometry rate of one calcium ion per one SI alpha1 molecule was obtained. K(d) values of 1.31 x 10(-9) mol L(-1), 2.63 x 10(-8) mol L(-1) and 2.71 x 10(-8) mol L(-1) were determined for the Ca(2+)- SI alpha1 complex by three different methodologies. These findings contribute to a better understanding of the SI alpha1 inhibition mechanism towards insect and mammalian alpha-amylases.