•Antimicrobial peptides were generated from the hydrolysis of wheat gluten proteins.•The hydrolysates exhibited activity against the all six tested fungi.•The hydrolysates extended the shelf life of ...bread in three days.•28 peptides were sequenced and then the best four peptides were synthesized.•The peptides were able to induce damage to the fungal plasma membrane.
Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme: substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60–90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.
Calotropis procera cysteine peptidases (CpCPs) have been used for reducing cow’s milk allergenicity or as rennet in cheesemaking. Due to their residual presence in food products, the present study ...evaluated, for the first time, different protocols for their immobilization on different supports. Although the yield of immobilization on sulfopropyl-agarose (99%, at pH 7.0) was better than when using DEAE- and MANAE-agarose (40% and 15%, respectively), the derivatives had low recovered activity (4%). On the other hand, MANAE-agarose at pH 10.0 (200 mM buffer) exhibited the highest recovered enzymatic activity (~23%). Regarding the covalent immobilization, the peptidases immobilized on glyoxyl-agarose (glyoxyl-CpCPs) showed broader pH stability (pH 3.0–10.0), 60-fold more stable at 60 °C, and retained 70% of their initial activities after five reaction cycles, even though the immobilization has induced some structural changes analyzed by Fourier-Transform Infrared (FTIR) spectroscopy as well as altered some of enzyme kinetic parameters (Vmax, Km, Kcat, and catalytic efficiency). In addition, this biocatalyst (glyoxyl-CpCPs) hydrolyzed the major cow’s milk allergens (whey proteins) to a greater extent (65%) than the soluble enzymes (8%) and a commercial hypoallergenic formula (50%).
Display omitted
•Calotropis procera cysteine peptidases (CpCPs) were immobilized on different supports.•Glyoxyl-agarose was the best among all ionic and covalent supports.•The immobilization induced structural changes and altered the enzyme kinetics.•But the biocatalyst had better pH stability and was 60-fold more stable at 60 °C.•It reduced the milk protein antigenicity to a level lower than of a commercial formula.
Germin-like proteins (GLPs) have been identified in several plant tissues. However, only one work describes GLP in latex fluids. Therefore, the goal of this study was to investigate GLPs in latex and ...get new insights concerning the structural and functional aspects of these proteins. Two complete sequences with high identity (>50%) with other GLPs, termed CpGLP1 and CpGLP2, were obtained and consecutively presented 216 and 206 amino acid residues, corresponding to molecular masses of 22.7 and 21.7kDa, pI 6.8 and 6.5. The three-dimensional models revealed overall folding similar to those reported for other plant GLPs. Both deduced sequences were grouped into the GER 2 subfamily. Molecular docking studies indicated a putative binding site consisting of three highly conserved histidines and a glutamate residue, which interacted with oxalate. This interaction was later supported by enzymatic assays. Superoxide dismutase (common activity in GLPs) was not detected for CpGLP1 and CpGLP2 by zymogram. The two proteins were detected in the latex, but not in non-germinated or germinated seeds and calli. These results give additional support that germin-like proteins are broadly distributed in plants and they are tissue-specific. This particularity deserves further studies to better understand their functions in latex.
The germin-like protein (GLP) purified from Thevetia peruviana, Peruvianin-I, is the only one described as having proteolytic activity. Therefore, the goal of this study was to investigate the ...structural features responsible for its enzymatic activity. Although the amino acid sequence of Peruvianin-I showed high identity with other GLPs, it exhibited punctual mutations, which were responsible for the absence of oxalate oxidase activity. The phylogenetic analysis showed that Peruvianin-I does not belong to any classification of GLP subfamilies. Moreover, Peruvianin-I contains a catalytic triad found in all plant cysteine peptidases. Molecular docking simulations confirmed the role of the catalytic triad in its proteolytic activity. Synchrotron radiation circular dichroism assays confirmed that Peruvianin-I was stable at pH ranging from 5.0 to 8.0 and that it presented significant structural changes only above 60 °C. The addition of iodoacetamide caused changes in its native conformation, but only a slight effect was observed after adding a reducing agent. This study reports an unusual protein with germin-like structure, lacking typical oxalate oxidase activity. Instead, the proteolytic activity observed suggests that the protein is a cysteine peptidase. These structural peculiarities make Peruvianin‑I an interesting model for further understanding of the action of laticifer fluids in plant defense.
Display omitted
•A low-cost new conductive ink based-graphite powder and commercial glue is proposed.•A new flexible electrochemical sensor was proposed using Ecoflex™ as substrate.•The proposed ...sensor was applied in the dopamine detection in synthetic sweat samples.•The proposed sensor was applied in the paracetamol detection in synthetic sweat and drug samples.
Flexible, fully printed electrochemical sensors can be employed for non-invasive clinical analyses, which require biocompatibility and wearability. Herein, we report on a flexible electrochemical sensor printed on an Ecoflex™ substrate with a conductive ink made with craft glue, graphite, acetone, ethyl acetate, and glycerin. Under optimized conditions for the electrochemical sensor, square wave voltammetry was used to detect paracetamol in the range from 7.0 to 100 μmol L−1 and dopamine from 3.0 to 100 μmol L−1, with detection limits of 1.4 μmol L−1 and 78 nmol L−1, respectively. The sensor was suitable for rapid detection of paracetamol and dopamine in artificial sweat with recovery between 90 and 110%, being unresponsive to interference from other molecules. The electrochemical sensors can be screen printed at low cost and with mass production and adapted for microfluidic and wearable (bio)devices.
Dogs are considered sentinels in areas of Trypanosoma cruzi transmission risk to humans. ELISA is generally the method of choice for diagnosing T. cruzi exposure in dogs, but its performance ...substantially depends on the antigenic matrix employed. In previous studies, our group has developed four chimeric antigens (IBMP-8.1, 8.2, 8.3, and 8.4) and evaluated their potential for diagnosing T. cruzi exposure in humans. For human sera, these chimeric antigens presented superior diagnostic performances as compared to commercial tests available in Brazil, Spain, and Argentina. Therefore, in this study we have evaluated the potential of these antigenic proteins for detection of anti-T. cruzi IgG antibodies in dog sera.
The IBMP-ELISA assays were optimized by checkerboard titration. Subsequently, the diagnostic potential was validated through analysis of ROC curves and the performance of the tests was determined using double entry tables. Cross-reactivity was also evaluated for babesiosis, ehrlichiosis, dirofilariosis, anaplasmosis, and visceral leishmaniasis. Best performance was shown by IBMP-8.3 and IBMP-8.4, although all four antigens demonstrated a high diagnostic performance with 46 positive and 149 negative samples tested. IBMP-8.3 demonstrated 100% sensitivity, followed by IBMP-8.4 (96.7-100%), IBMP-8.2 (73.3-87.5%), and IBMP-8.1 (50-100%). The highest specificities were achieved with IBMP-8.2 (100%) and IBMP-8.4 (100%), followed by IBMP-8.3 (96.7-97.5%) and IBMP 8.1 (89.1-100%).
The use of chimeric antigenic matrices in immunoassays for anti-T. cruzi IgG antibody detection in sera of infected dogs was shown to be a promising tool for veterinary diagnosis and epidemiological studies. The chimeric antigens used in this work allowed also to overcome the common hurdles related to serodiagnosis of T. cruzi infection, especially regarding variation of efficiency parameters according to different strains and cross-reactivity with other infectious diseases.
The bacterial community present in the female lower genital tract plays an important role in maternal and neonatal health. Imbalances in this microbiota have been associated with negative ...reproductive outcomes, such as spontaneous preterm birth (sPTB), but the mechanisms underlying the association between a disturbed microbiota and sPTB remain poorly understood. An intrauterine infection ascending from the vagina is thought to be an important contributor to the onset of preterm labour. Our objective was to characterize the vaginal microbiota of pregnant women who had sPTB (n = 46) and compare to those of pregnant women who delivered at term (n = 170). Vaginal swabs were collected from women at 11-16 weeks of gestational age. Microbiota profiles were created by PCR amplification and pyrosequencing of the cpn60 universal target region.
Profiles clustered into seven community state types: I (Lactobacillus crispatus dominated), II (Lactobacillus gasseri dominated), III (Lactobacillus iners dominated), IVA (Gardnerella vaginalis subgroup B or mix of species), IVC (G. vaginalis subgroup A dominated), IVD (G. vaginalis subgroup C dominated) and V (Lactobacillus jensenii dominated). The microbiota of women who experienced preterm birth (< 37 weeks gestation) had higher richness and diversity and higher Mollicutes prevalence when compared to those of women who delivered at term. The two groups did not cluster according to CST, likely because CST assignment is driven in most cases by the dominance of one particular species, overwhelming the contributions of more rare taxa. In conclusion, we did not identify a specific microbial community structure that predicts sPTB, but differences in microbiota richness, diversity and Mollicutes prevalence were observed between groups.
Although a causal relationship remains to be determined, our results confirm previous reports of an association between Mollicutes and sPTB and further suggest that a more diverse microbiome may be important in the pathogenesis of some cases.
The vaginal microbiome plays an important role in maternal and neonatal health. Imbalances in this microbiota (dysbiosis) during pregnancy are associated with negative reproductive outcomes, such as ...pregnancy loss and preterm birth, but the underlying mechanisms remain poorly understood. Consequently a comprehensive understanding of the baseline microbiome in healthy pregnancy is needed. We characterized the vaginal microbiomes of healthy pregnant women at 11-16 weeks of gestational age (n = 182) and compared them to those of non-pregnant women (n = 310). Profiles were created by pyrosequencing of the cpn60 universal target region. Microbiome profiles of pregnant women clustered into six Community State Types: I, II, III, IVC, IVD and V. Overall microbiome profiles could not be distinguished based on pregnancy status. However, the vaginal microbiomes of women with healthy ongoing pregnancies had lower richness and diversity, lower prevalence of Mycoplasma and Ureaplasma and higher bacterial load when compared to non-pregnant women. Lactobacillus abundance was also greater in the microbiomes of pregnant women with Lactobacillus-dominated CSTs in comparison with non-pregnant women. This study provides further information regarding characteristics of the vaginal microbiome of low-risk pregnant women, providing a baseline for forthcoming studies investigating the diagnostic potential of the microbiome for prediction of adverse pregnancy outcomes.
Dose optimisation has been revisited in the literature due to the frequent use of cone beam computed tomography (CBCT). Although the reduction of the field-of-view (FOV) size has shown to be an ...effective strategy, this indirectly increases the negative effect from the exomass. The aim of this study was to evaluate the diagnostic accuracy of an optimised CBCT protocol in the detection of simulated vertical root fracture (VRF) in the presence of metal in the exomass and/or inside the FOV. Twenty teeth were endodontically instrumented and VRF was induced in half of them. All teeth were individually placed in a human mandible covered with a soft tissue equivalent material, metallic materials were placed at different dispositions in the exomass and/or endomass, and CBCT scans were obtained at two dose protocols: standard and optimised. Five radiologists evaluated the images and indicated the presence of VRF using a 5-point scale. Area under the ROC curve (AUC), sensitivity, and specificity were calculated and compared using ANOVA (α = 0.05). Overall, AUC, sensitivity, and specificity did not differ significantly (p > 0.05) between the dose protocols. In conclusion, optimised dose protocols should be considered in the detection of simulated VRF irrespective of the occurrence of artefacts from metallic materials in the exomass and/or inside the FOV.
•Decentralised sewage monitoring was used to assess COVID-19 prevalence.•Relative prevalence index (RPI) was proposed to assess severity of pandemic in different regions.•Decay of SARS-CoV-2 RNA in ...sewage was estimated based on modelling.•Monitored areas had different peak length, week of peak occurrence, number of peaks.•The most vulnerable regions in the city were the hardest hit by the pandemic.
Brazil has become one of the epicentres of the COVID-19 pandemic, with cases heavily concentrated in large cities. Testing data is extremely limited and unreliable, which restricts health authorities’ ability to deal with the pandemic. Given the stark demographic, social and economic heterogeneities within Brazilian cities, it is important to identify hotspots so that the limited resources available can have the greatest impact. This study shows that decentralised monitoring of SARS-CoV-2 RNA in sewage can be used to assess the distribution of COVID-19 prevalence in the city. The methodology developed in this study allowed the identification of hotspots by comprehensively monitoring sewers distributed through Belo Horizonte, Brazil's third largest city. Our results show that the most vulnerable neighbourhoods in the city were the hardest hit by the pandemic, indicating that, for many Brazilians, the situation is much worse than reported by official figures.
Display omitted
PDF
