Repetitive DNAs and chromosome evolution in Megaleporinus obtusidens and M. reinhardti (Characiformes: Anostomidae).

The high dynamism of repetitive DNAs is a major driver of chromosome evolution. In particular, the accumulation of repetitive DNA sequences has been reported as part of the differentiation of sex-specific chromosomes. In turn, the fish species of the genus Megaleporinus are a monophyletic clade in which the presence of differentiated ZZ/ZW sex chromosomes represents a synapomorphic condition, thus serving as a suitable model to evaluate the dynamic evolution of repetitive DNA classes. Therefore, transposable elements (TEs) and in tandem repeats were isolated and located on chromosomes of Megaleporinus obtusidens and M. reinhardti to infer their role in chromosome differentiation with emphasis on sex chromosome systems. Despite the conserved karyotype features of both species, the location of repetitive sequences - Rex 1, Rex 3, (TTAGGG)n, (GATA)n, (GA)n, (CA)n, and (A)n - varied both intra and interspecifically, being mainly accumulated in Z and W chromosomes. The physical mapping of repetitive sequences confirmed the remarkable dynamics of repetitive DNA classes on sex chromosomes that might have promoted chromosome diversification and reproductive isolation in Megaleporinus species.

Development and validation of LC-MS/MS methods for the simultaneous quantification of sofosbuvir and its major metabolite (GS-331007) in blood plasma and cerebrospinal and seminal fluid: Application to a pilot clinical trial with a focus on Zika.

Zika still poses a threat to global health owing to its association with serious neurological conditions and the absence of a vaccine and treatment. Sofosbuvir, an anti-hepatitis C drug, has shown anti-Zika effects in animal and cell models. Thus, this study aimed to develop and validate novel LC-MS/MS methods for the quantification of sofosbuvir and its major metabolite (GS-331007) in human plasma and cerebrospinal (CSF) and seminal fluid (SF), and apply the methods to a pilot clinical trial. The samples were prepared by liquid-liquid extraction and separated using isocratic mode on Gemini C18 columns. Analytical detection was performed using a triple quadrupole mass spectrometer equipped with an electrospray ionization source. The validated ranges for sofosbuvir were 0.5-2,000 ng/mL (plasma) and 0.5-100 ng/mL (CSF and SF), while for the metabolite they were 2.0-2,000 ng/mL (plasma), 5.0-200 ng/mL (CSF) and 10-1,500 ng/mL (SF). The intra-day and inter-day accuracies (90.8-113.8%) and precisions (1.4-14.8%) were within the acceptance range. The developed methods fulfilled all validation parameters concerning selectivity, matrix effect, carryover, linearity, dilution integrity, precision, accuracy and stability, confirming the suitability of the method for the analysis of clinical samples.

Cesarean Delivery and Risk of Excess Weight Among Brazilian Preschool Children.

OBJECTIVE: To study the relationship between cesarean Delivery (CD) and overweight in preschool children, considering the presence of birth aspects and demographic characteristics that have been associated with the risk of excess weight. METHODS: This retrospective cross-sectional analysis used representative data of children from 2 to 6 years of age at municipal daycare centers in Taubaté, São Paulo, Brazil. The sample included 752 preschoolers. Weight and height were collected in daycare centers, and the nutritional status classification was performed using z scores of Body Mass Index (zBMI). Data analysis was performed in the bivariate and multivariate manner, the latter with the inclusion of another possible risk factor. Adjustments were made for daycare centers time, sex, birth length and weight and maternal schooling. RESULTS: The mean age of preschool children was 4.7 years, and 53.8% were boys. The prevalence of overweight children was 21.7%. The median zBMI of the children born by CD was higher than that of vaginal Delivery (p = 0.0194). The prevalence of overweight showed association with CD (p = 0.0196; PR = 1.397 with 95% CI of 1065-1831). Logistic binary multivariate regression analysis confirmed the association, even in the presence of other possible risk factors (CR: 1.580, 95% CI: 1.072-2.330). CONCLUSION FOR PRACTICE: Birth by CD is one of the factors that are associated with the early development of overweight in preschoolers.

DNA transposon invasion and microsatellite accumulation guide W chromosome differentiation in a Neotropical fish genome.

Sex chromosome differentiation is subject to independent evolutionary processes among different lineages. The accumulation of repetitive DNAs and consequent crossing-over restriction guide the origin of the heteromorphic sex chromosome region. Several Neotropical fish species have emerged as interesting models for understanding evolution and genome diversity, although knowledge of their genomes is scarce. Here, we investigate the content of repetitive DNAs between males and females of Apareiodon sp. based on large-scale genomic data focusing on W sex chromosome differentiation. In Apareiodon, females are the heterogametic sex (ZW) and males are the homogametic sex (ZZ). The genome size estimate for Apareiodon was 1.2 Gb (with ~ 42× and ~ 47× coverage for males and females, respectively). In Apareiodon sp., approximately 36% of the genome was composed of repetitive DNAs and transposable elements (TEs) were the most abundant class. Read coverage analysis revealed different amounts of repetitive DNAs in males and females. The female-enriched clusters were located on the W sex chromosome and were mostly composed of microsatellite expansions and DNA transposons. Landscape analysis of TE contents demonstrated two major waves of invasions of TEs in the Apareiodon genome. Estimation of TE insertion times correlated with in situ locations permitted the inference that helitron, Tc1-mariner, and CMC EnSpm DNA transposons accumulated repeated copies during W chromosome differentiation between 20 and 12 million years ago. DNA transposons and microsatellite expansions appeared to be major players in W chromosome differentiation and to guide modifications in the genome content of the heteromorphic sex chromosomes.

Secure and Sustainable Sourcing of Plant Tissues for the Exhaustive Exploration of Their Chemodiversity.

The main challenge of plant chemical diversity exploration is how to develop tools to study exhaustively plant tissues. Their sustainable sourcing is a limitation as bioguided strategies and dereplication need quite large amounts of plant material. We examine if alternative solutions could overcome these difficulties by obtaining a secure, sustainable, and scalable source of tissues able to biosynthesize an array of metabolites. As this approach would be as independent of the botanical origin as possible, we chose eight plant species from different families. We applied a four steps culture establishment procedure, monitoring targeted compounds through mass spectrometry-based analytical methods. We also characterized the capacities of leaf explants in culture to produce diverse secondary metabolites. In vitro cultures were successfully established for six species with leaf explants still producing a diversity of compounds after the culture establishment procedure. Furthermore, explants from leaves of axenic plantlets were also analyzed. The detection of marker compounds was confirmed after six days in culture for all tested species. Our results show that the first stage of this approach aiming at easing exploration of plant chemodiversity was completed, and leaf tissues could offer an interesting alternative providing a constant source of natural compounds.

Tri-Ponderal Mass Index: A Screening Tool for Risk of Central Fat Accumulation in Brazilian Preschool Children.

Background and Objectives: To verify the use of the tri-ponderalmass index (TMI) as a screening tool for risk of central fat accumulation in preschool children. Materials and Methods: An observational, analytical study was carried out on samples from children 2 to 5 years of age. The body mass index (BMI) and the tri-ponderalmass index (TMI: Weight/height3) were calculated. The waist circumference-to-height ratio (WHtR) was used to classify central fat accumulation risk. Preschoolers whose WHtRwas in the upper tertile of the sample were classified as at risk for central fat accumulation. A comparison of the two indicators (BMI and TMI) was made from the area under the receiver operator characteristics (ROC) curve (AUC) in the discrimination of the WHtR. Results: The sample used for analysis was 919 preschoolers. The mean age of the children was 3.9 years (SD = 0.7). The difference in AUC was 5% higher for TMI (p < 0.0001). In the individual analysis of the ROC curve of the TMI, favoring a higher sensitivity, the cutoff point of 14.0 kg/m3 showed a sensitivity of 99.3% (95% CI: 97.6-99.9). Conclusion: Considering WHtR as a marker of possible future metabolic risk among preschool children, TMI proved to be a useful tool, superior to BMI, in screening for risk of central fat accumulation in preschool children.

Insight into the α-Amylase Inhibitory Activity of Plant Lipid Transfer Proteins.

We previously characterized the inhibitory activity of human salivary α-amylase (HSA) and Callosobruchus maculatus intestinal α-amylases by the plant lipid transfer protein from Vigna unguiculata ( Vu-LTP). Herein, we further study this inhibitory activity. First by an analysis of protein α-amylase inhibitors complexed with α-amylase, we find that positively charged amino acids of inhibitors interact with the active site of α-amylases and we know that Vu-LTP is rich in positively charged amino acid residues. For this reason, we model Vu-LTP, and based on its three-dimensional structure, we choose five peptides to be synthesized. Herein, we report that two peptides of Vu-LTP are responsible for HSA inhibition. A comparison of primary and tertiary structures of LTPs with and without inhibitory activity against α-amylase, superimposed with the sequence of Vu-LTP mapped for HSA inhibition, reinforces our suggestion that positively charged amino acids in loops are responsible for the inhibition. To prove our observation, one modified peptide is synthesized in which Arg39 is replaced by Gln. This modified peptide loses the HSA inhibitory property presented by the unmodified peptide. Therefore, we describe a new biological active for Vu-LTP, i.e. the α-amylase inhibitory activity that is not a fortuitous biological activity and probably has evolved to perform a biological function which is still unknown. A good candidate should be defense against insects. The results of this study also expand the possible biotechnological applications of LTPs.

Fermentation strategy for second generation ethanol production from sugarcane bagasse hydrolyzate by Spathaspora passalidarum and Scheffersomyces stipitis.

Alcoholic fermentation of released sugars in pretreatment and enzymatic hydrolysis of biomass is a central feature for second generation ethanol (E2G) production. Saccharomyces cerevisiae used industrially in the production of first generation ethanol (E1G) convert sucrose, fructose, and glucose into ethanol. However, these yeasts have no ability to ferment pentose (xylose). Therefore, the present work has focused on E2G production by Scheffersomyces stipitis and Spathaspora passalidarum. The fermentation strategy with high pitch, cell recycle, fed-batch mode, and temperature decrease for each batch were performed in a hydrolyzate obtained from a pretreatment at 130°C with NaOH solution (1.5% w/v) added with 0.15% (w/w) of anthraquinone (AQ) and followed by enzymatic hydrolysis. The process strategy has increased volumetric productivity from 0.35 to 0.38 g · L-1 · h-1 (first to third batch) for S. stipitis and from 0.38 to 0.81 g · L-1 · h-1 for S. passalidarum (first to fourth batch). Mass balance for the process proposed in this work showed the production of 177.33 kg ethanol/ton of sugar cane bagasse for S. passalidarum compared to 124.13 kg ethanol/ton of sugar cane bagasse for S. stipitis fermentation. The strategy proposed in this work can be considered as a promising strategy in the production of second generation ethanol. Biotechnol. Bioeng. 2017;114: 2211-2221. © 2017 Wiley Periodicals, Inc.

Breastfeeding, comnlementarv food introduction and overweight in preschool children.

Growing phenomenon, which involves high morbidity and consequently high costs for health systems, obesity has been found also among the pediatric population and is currently considered a public health problem. The aim of this study was to verify if in children in the early preschool age we can see the prevalence of overweight and if introducing complementary feeding as well as the type of food introduced, are associated with this condition in this age group. It is an observational analytic study with children born in 2011-2012 that attended public schools in Taubat6 -SP during 2014. In addition to the weight and height of children, information about the history of feeding and birth were collectedusing a standardized questionnaire.The nutritional status was defined as having overweight children with z-scores for body mass index (zIMC) > 1.We conducted bivariate analysis and then linear regression analysis of multiple variables.The prevalence of overweight was elevated (27.5%). Only birth weight showed significant correlation with respect to zIMC (r = 0.22, p < 0.0001). The multivariable analysis showed no relationship with the various foods, but showed birth weightas a high risk factor, the male and the total duration of breastfeeding as protective factors. As a result, we conclude that the early introduction of new foods is not a risk factor for the development of overweight at the beginning of pre-school age.

Vicilin-like peptides from Capsicum baccatum L. seeds are α-amylase inhibitors and exhibit antifungal activity against important yeasts in medical mycology.

The objective of this study was to isolate antimicrobial peptides from Capsicum baccatum seeds and evaluate their antimicrobial activity and inhibitory effects against α-amylase. Initially, proteins from the flour of C. baccatum seeds were extracted in sodium phosphate buffer, pH 5.4, and precipitated with ammonium sulfate at 90% saturation. The D1 and D2 fractions were subjected to antifungal tests against the yeasts Saccharomyces cerevisiae, Candida albicans, Candida tropicalis, and Kluyveromyces marxiannus, and tested against α-amylases from Callosobruchus maculates and human saliva. The D2 fraction presented higher antimicrobial activity and was subjected to further purification and seven new different fractions (H1-H7) were obtained. Peptides in the H4 fraction were sequenced and the N-terminal sequences revealed homology with previously reported storage vicilins from seeds. The H4 fraction exhibited strong antifungal activity and also promoted morphological changes in yeast, including pseudohyphae formation. All fractions, including H4, inhibited mammalian α-amylase activity but only the H4 fraction was able to inhibit C. maculatus α-amylase activity. These results suggest that the fractions isolated from the seeds of C. baccatum can act directly in plant defenses against pathogens and insects.

Functional expression and activity of the recombinant antifungal defensin PvD1r from Phaseolus vulgaris L. (common bean) seeds.

BACKGROUND: Defensins are basic, cysteine-rich antimicrobial peptides that are important components of plant defense against pathogens. Previously, we isolated a defensin, PvD1, from Phaseolus vulgaris L. (common bean) seeds. RESULTS: The aim of this study was to overexpress PvD1 in a prokaryotic system, verify the biologic function of recombinant PvD1 (PvD1r) by comparing the antimicrobial activity of PvD1r to that of the natural defensin, PvD1, and use a mutant Candida albicans strain that lacks the gene for sphingolipid biosynthesis to unravel the target site of the PvD1r in C. albicans cells. The cDNA encoding PvD1, which was previously obtained, was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform bacterial cells (Rosetta Gami 2 (DE3) pLysS) leading to recombinant protein expression. After expression had been induced, PvD1r was purified, cleaved with enterokinase and repurified by chromatographic steps. N-terminal amino acid sequencing showed that the overall process of the recombinant production of PvD1r, including cleavage with the enterokinase, was successful. Additionally, modeling revealed that PvD1r had a structure that was similar to the defensin isolated from plants. Purified PvD1 and PvD1r possessed inhibitory activity against the growth of the wild-type pathogenic yeast strain C. albicans. Both defensins, however, did not present inhibitory activity against the mutant strain of C. albicans. Antifungal assays with the wild-type C. albicans strains showed morphological changes upon observation by light microscopy following growth assays. PvD1r was coupled to FITC, and the subsequent treatment of wild type C. albicans with DAPI revealed that the labeled peptide was intracellularly localized. In the mutant strain, no intracellular labeling was detected. CONCLUSION: Our results indicate that PvD1r retains full biological activity after recombinant production, enterokinase cleavage and purification. Additionally, our results from the antimicrobial assay, the microscopic analysis and the PvD1r-FITC labeling assays corroborate each other and lead us to suggest that the target of PvD1 in C. albicans cells is the sphingolipid glucosylceramide.

Neural respiratory drive during maximal voluntary ventilation in individuals with hypertension: A case-control study.

Neural respiratory drive (NRD) is measured using a non-invasive recording of respiratory electromyographic signal. The parasternal intercostal muscle can assess the imbalance between the load and capacity of respiratory muscles and presents a similar pattern to diaphragmatic activity. We aimed to analyze the neural respiratory drive in seventeen individuals with hypertension during quite breathing and maximal voluntary ventilation (MVV) (103.9 ± 5.89 vs. 122.6 ± 5 l/min) in comparison with seventeen healthy subjects (46.5 ± 2.5 vs. 46.4 ± 2.4 years), respectively. The study protocol was composed of quite breathing during five minutes, maximum inspiratory pressure followed by maximal ventilatory ventilation (MVV) was recorded once for 15 seconds. Anthropometric measurements were collected, weight, height, waist, hip, and calf circumferences, waist-to-hip ratio (WHR), waist-to-height ratio (WHtR), BMI, and conicity index (CI). Differences between groups were analyzed using the unpaired t-test or Mann-Whitney test to determine the difference between groups and moments. A significance level of 5% (p<0,05) was adopted for all statistical analyses. The group of individuals with hypertension presented higher values when compared to the healthy group for neural respiratory drive (EMGpara% 17.9±1.3 vs. 13.1±0.8, p = 0.0006) and neural respiratory drive index (NRDi (AU) 320±25 vs. 205.7±15,p = 0.0004) during quiet breathing and maximal ventilatory ventilation (EMGpara% 29.3±2.7 vs. 18.3±0.8, p = 0.000, NRDi (AU) 3140±259.4 vs. 1886±73.1,p<0.0001), respectively. In conclusion, individuals with hypertension presented higher NRD during quiet breathing and maximal ventilatory ventilation when compared to healthy individuals.

Activity of recombinant and natural defensins from Vigna unguiculata seeds against Leishmania amazonensis.

Antimicrobial peptides (AMPs), which are differentiated from other antibiotic peptides, such as gramicidins and polymyxins, because they are synthesized by large enzymatic complex and bear modified amino acids including d-amino acids, are short polymers of l-amino acids synthesized by ribosomes upon which all living organisms rely to defend themselves from invaders or competitor microorganisms. AMPs have received a great deal of attention from the scientific community as potential new drugs for neglected diseases such as Leishmaniasis. In plants, they include several families of compounds, including the plant defensins. The aim of the present study was to improve the expression of recombinant defensin from Vigna unguiculata seeds (Vu-Defr) and to test its activity against Leishmania amazonensis promatigotes. Recombinant expression was performed in LB and TB media and under different conditions. The purification of Vu-Defr was achieved by immobilized metal ion affinity and reversed-phase chromatography. The purified Vu-Defr was analyzed by circular dichroism (CD), and its biological activity was tested against L. amazonenis promastigotes. To demonstrate that the recombinant production of Vu-Defr did not interfere with its fold and biological activity, the results of all experiments were compared with the results from the natural defensin (Vu-Def). The CD spectra of both peptides presented good superimposition indicating that both peptides present very similar secondary structure and that the Vu-Defr was correctly folded. L. amazonensis treated with Vu-Defr led to the elimination of 54.3% and 46.9% of the parasites at 24 and 48h of incubation time, respectively. Vu-Def eliminated 50% and 54.8% of the parasites at 24 and 48 h, respectively. Both were used at a concentration of 100 µg/mL. These results suggested the potential for plant defensins to be used as new antiparasitic substances.

Evaluation of mobile applications focused on the care of patients with anxiety disorders: A systematic review in app stores in Brazil.

OBJECTIVE: To identify and evaluate the quality of mobile apps available in Brazil focused on the care of patients with anxiety disorders. METHODS: A comprehensive search was conducted until October 2021 on Play Store (Android) and Apple Store (iOS) in Brazil, using the terms "anxiety," "phobia," "panic attack," and "social phobia." Two independent authors identified the apps and performed data extraction and quality assessment using the Mobile App Rating Scale (MARS). Pearson's correlation was used to analyze the relationship between user star rating and the quality defined by the MARS instrument. RESULTS: A total of 3,278 potential apps were identified, of which 71 fully met the eligibility criteria. Most apps were made available on the Play Store (91.74%), in English (69.01%), and updated in the last two years (90.14%). Approximately half of the apps (50.70%) did not inform the developer's country and most of them did not report the user star rating (70.42%). The target population was indicated as free by most apps (85.92%), with generalized anxiety disorder being the most addressed disorder (74.65%), followed by panic disorder (33.80%). The three main purposes of the apps were education (83.10%), self-assessment (38.03%), and meditation/breathing (32.39%). Only 31 apps (43.66%) had acceptable quality (above 3.0) and the average total MARS quality score of 2.93 (2.20 to 3.90), with the functionality section receiving the highest score (3.90) and the lowest scoring sections being engagement (2.16). The apps that were rated stars by users (29.58%) showed a negative Pearson correlation (ρ = -0.100), evidencing a difference in the user's evaluation and that performed using the MARS instrument. CONCLUSION: Gaps in the quality of apps focused on the care of patients with anxiety disorders were evidenced since most were classified as having low quality through the MARS instrument. Thus, users are recommended to use these apps with caution.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel α-amylase inhibitory properties.

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1) . Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein's biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1) , showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of α-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian α-amylase activity in vitro.

Nutritional status of pre-school children from low income families.

BACKGROUND: We evaluated growth and nutritional status of preschool children between 2 and 6 years old from low income families from 14 daycare centers. METHODS: Cross-sectional study with 1544 children from daycare centers of Santo Andre, Brazil. Body weight (W), height (H) and body mass index (BMI) were classified according to the 2000 National Center for Health Statistics (CDC/NCHS). Cutoff points for nutritional disorders: -2 z scores and 2.5 and 10 percentiles for malnutrition risk, 85 to 95 percentile for overweight and above BMI 95 percentile for obesity. Stepwise Forward Regression method was used including age, gender, birth weight, breastfeeding duration, age of mother at birth and period of time they attended the daycare center. RESULTS: Children presented mean z scores of H, W and BMI above the median of the CDC/NCHS reference. Girls were taller and heavier than boys, while we observed similar BMI between both genders. The z scores tended to rise with age. A Pearson Coefficient of Correlation of 0.89 for W, 0.93 for H and 0.95 for BMI was documented indicating positive association of age with weight, height and BMI. The frequency of children below -2 z scores was lower than expected: 1.5% for W, 1.75% for H and 0% for BMI, which suggests that there were no malnourished children. The other extremity of the distribution evidenced prevalence of overweight and obesity of 16.8% and 10.8%, respectively. CONCLUSION: Low income preschool children are in an advanced stage of nutritional transition with a high prevalence of overweight.

Evaluation of mid-upper arm circumference in pre-school children: comparison between NCHS/CDC-2000 and WHO-2006 references.

We aimed to evaluate the classification of arm circumference (AC) in pre-school children by using National Center for Health Statistics (NCHS/CDC-2000) and World Health Organization (WHO-2006) references. We evaluated 205 children: weight, height and AC were assessed and the body mass index (BMI) was calculated. The BMI values were classified into Z-scores by the WHO referential. The AC was classified into Z-cores by two references, comparing the whole-sample value and among groups (tercis) of BMI Z-score. The correlation was also evaluated between differences of AC with BMI Z-score. The WHO referential classified the AC in Z-scores greater than the NCHS/CDC, which is more specific and less sensitive than the NCHS/CDC for lean children and at the same time more sensitive and less specific for children with overweight. In conclusion, a significant difference in the AC classification occurs according to the referential used.

Origin of the Giant Enhanced Raman Scattering by Sulfur Chains Encapsulated inside Single-Wall Carbon Nanotubes.

In this work, we explain the origin and the mechanism responsible for the strong enhancement of the Raman signal of sulfur chains encapsulated by single-wall carbon nanotubes by running resonance Raman measurements in a wide range of excitation energies for two nanotube samples with different diameter distributions. The Raman signal associated with the vibrational modes of the sulfur chain is observed when it is confined by small-diameter metallic nanotubes. Moreover, a strong enhancement of the Raman signal is observed for excitation energies corresponding to the formation of excited nanotube-chain-hybrid electronic states. Our hypothesis was further tested by high pressure Raman measurements and confirmed by density functional theory calculations of the electronic density of states of hybrid systems formed by sulfur chains encapsulated by different types of single-wall carbon nanotubes.

Purification of a defensin isolated from Vigna unguiculata seeds, its functional expression in Escherichia coli, and assessment of its insect alpha-amylase inhibitory activity.

Plant defensins make up a family of cationic antimicrobial peptides with a characteristic three-dimensional folding pattern stabilized by four disulfide bridges. The aim of this work was the purification and functional expression of a defensin from cowpea seeds and the assessment of its alpha-amylase inhibitory activity. The cDNA encoding the cowpea defensin was cloned into the pET-32 EK/LIC vector, and the resulting construct was used to transform Escherichia coli cells. The recombinant peptide was purified via affinity chromatography on a Ni Sepharose column and by reverse-phase chromatography on a C2/C18 column using HPLC. N-terminal amino acid sequencing revealed that the recombinant peptide had a similar sequence to that of the defensin isolated from seeds. The natural and recombinant defensins were submitted to the alpha-amylase inhibition assay. The cowpea seed defensin was found to inhibit alpha-amylases from the weevils Callosobruchus maculatus and Zabrotes subfasciatus. alpha-Amylase inhibition assays also showed that the recombinant defensin inhibited alpha-amylase from the weevil C. maculatus. The cowpea seed defensin and its recombinant form were unable to inhibit mammalian alpha-amylases. The three-dimensional structure of the recombinant defensin was modeled, and the resulting structure was found to be similar to those of other plant defensins.

Designing a cocktail containing redox enzymes to improve hemicellulosic hydrolysate fermentability by microorganisms.

Bioproducts production using monomeric sugars derived from lignocellulosic biomass presents several challenges, such as to require a physicochemical pretreatment to improve its conversion yields. Hydrothermal lignocellulose pretreatment has several advantages and results in solid and liquid streams. The former is called hemicellulosic hydrolysate (HH), which contains inhibitory phenolic compounds and sugar degradation products that hinder microbial fermentation products from pentose sugars. Here, we developed and applied a novel enzyme process to detoxify HH. Initially, the design of experiments with different redox activities enzymes was carried out. The enzyme mixture containing the peroxidase (from Armoracia rusticana) together with superoxide dismutase (from Coptotermes gestroi) are the most effective to detoxify HH derived from sugarcane bagasse. Butanol fermentation by the bacteria Clostridium saccharoperbutylacetonicum and ethanol production by the yeast Scheffersomyces stipitis increased by 24.0× and 2.4×, respectively, relative to the untreated hemicellulosic hydrolysates. Detoxified HH was analyzed by chromatographic and spectrometric methods elucidating the mechanisms of phenolic compound modifications by enzymatic treatment. The enzyme mixture degraded and reduced the hydroxyphenyl- and feruloyl-derived units and polymerized the lignin fragments. This strategy uses biocatalysts under environmentally friendly conditions and could be applied in the fuel, food, and chemical industries.