Artigo

Enzymatic blends produced by fungal monocultures and consortia cultured in solid-state fermentation (SSF), using sugarcane bagasse (SB) and wheat bran as substrates (1:1, w/w), were evaluated for saccharification of sugarcane bagasse pretreated by autohydrolysis (hydrothermal pretreatment—HP) and alkaline delignification (HP-Soda). The highest glucose releases were obtained after saccharifications of SB pretreated by HP using enzyme cocktails produced by Aspergillus niger and by the consortium among A. fumigatus, Ganoderma lucidum and Trametes versicolor, with 10.8 and 9.8 g L⁻¹, respectively. For SB pretreated by HP-Soda, the hydrolysate 10 (extract from A. niger, G. lucidum and Pleurotus ostreatus consortium) achieved maximal glucose concentration (11.92 g L⁻¹). After alcoholic fermentation of the hydrolysates, the greatest ethanol yield in relation to the maximum theoretical yield (60.8%) was obtained in the fermentation of hydrolysate 1 (A. niger) obtained from SB pretreated by HP-Soda. These results demonstrated that on-site produced enzyme cocktails can be applied for saccharification of pretreated sugarcane bagasse and also contribute to cost reduction of bioconversion processes.

The Brazilian Guzerá population originated from a few founders introduced from India. These animals adapted well to the harsh environments in Brazil, were selected for beef, milk, or dual-purpose (beef and milk), and were extensively used to produce crossbred animals. Here, the impact of these historical events with regard to the population structure and genetic diversity in a Guzerá meta-population was evaluated. DNA samples of 744 animals (one dairy, nine dual-purpose, and five beef herds) were genotyped for 21 microsatellite loci. Ho, He, PIC, F_is, F_it, and F_st estimates were obtained considering either farms or lineages as subpopulations. Mean Ho (0.73) and PIC (0.75) suggest that genetic diversity was efficiently conserved. F_it, F_is and F_st values (95% CI) pointed to a low fixation index, and large genetic diversity: F_it (Farms = 0.021-0.100; lineages = 0.021-0.100), F_is (Farms = -0.007-0.076; lineages = -0.014-0.070), and F_st (Farms = 0.0237-0.032; lineages = 0.029-0.038). The dual-purpose herds/selection lines are the most uniform subpopulation, while the beef one preserved larger amounts of genetic diversity among herds. In addition, the dairy herd showed to be genetically distant from other herds. Taken together, these results suggest that this Guzerá meta-population has high genetic diversity, a low degree of population subdivision, and a low inbreeding level.

Whole genome sequencing of bovine breeds has allowed identification of genetic variants in milk protein genes. However, functional repercussion of such variants at a molecular level has seldom been investigated. Here, the results of a multistep Bioinformatic analysis for functional characterization of recently identified genetic variants in Brazilian Gyr and Guzerat breeds is described, including predicted effects on the following: (i) evolutionary conserved nucleotide positions/regions; (ii) protein function, stability, and interactions; (iii) splicing, branching, and miRNA binding sites; (iv) promoters and transcription factor binding sites; and (v) collocation with QTL. Seventy-one genetic variants were identified in the caseins (CSN1S1, CSN2, CSN1S2, and CSN3), LALBA, LGB, and LTF genes. Eleven potentially regulatory variants and two missense mutations were identified. LALBA Ile60Val was predicted to affect protein stability and flexibility, by reducing the number the disulfide bonds established. LTF Thr546Asn is predicted to generate steric clashes, which could mildly affect iron coordination. In addition, LALBA Ile60Val and LTF Thr546Asn affect exonic splicing enhancers and silencers. Consequently, both mutations have the potential of affecting immune response at individual level, not only in the mammary gland. Although laborious, this multistep procedure for classifying variants allowed the identification of potentially functional variants for milk protein genes.

Purpose

The aim of this study was to investigate the association of rs6598964 (A > G), a molecular marker located in the LIN28A gene, with the performance of Brazilian soccer players using the VO₂max predicted by performance in the Yo-Yo test as the phenotype.

Methods

The study sample comprised 227 male players on a team in the first division of Brazilian soccer distributed in the following categories: U15 (n = 67, VO2max = 52.75 ± 4.74 ml/kg/min), U17 (n = 43, VO2max = 54.37 ± 5.47 ml/kg/min), U20 (n = 79, VO2max = 54.97 ± 5.13 ml/kg/min), and Professional (n = 38, VO2max = 55.84 ± 4.37 ml/kg/min). Genotype models (codominance, A-recessive, A-dominant and overdominance models) were tested using Kruskal–Wallis and Mann–Whitney tests with Bonferroni correction for multiple comparisons tests.

Results

Significantly higher predicted VO2max was observed in individuals with the A/A genotype (VO2max = 62.12 ± 3.97 mL/kg/min) compared to both the A/G (53.44 ± 8.88 mL/kg/min) and G/G (52.44 ± 6.11 mL/kg/min) genotypes (p < 0.001). Model comparisons suggested the differences in predicted VO2max were best explained by the A-recessive model.

Conclusion

This study is the first to associate the LIN28A polymorphism with endurance performance in soccer players. However, further studies are needed to confirm the associations described here and to investigate how LIN28A interacts with other genes related to athletic performance.

Cacao plantations from South America have been afflicted with the severe fungal disease known as Witches’ Broom Disease (WBD), caused by the basidiomycete Moniliophthora perniciosa. Yeasts are increasingly recognized as good fungal biocides, although their application is still mostly restricted to the postharvest control of plant and fruit decay. Their possible utilization in the field, in a preharvest phase, is nevertheless promising, particularly if the strains are locally adapted and evolved and if they belong to species considered safe for man and the environment. In this work, a group of yeast strains originating from sugarcane-based fermentative processes in Brazil, the cacao-producing country where the disease is most severe, were tested for their ability to antagonize M. perniciosa in vitro. Wickerhamomyces anomalus LBCM1105 and Saccharomyces cerevisiae strains LBCM1112 from spontaneous fermentations used to produce cachaça, and PE2 widely used in Brazil in the industrial production of bioethanol, efficiently antagonized six strains of M. perniciosa, originating from several South American countries. The two fastest growing fungal strains, both originating from Brazil, were further used to assess the mechanisms underlying the yeasts’ antagonism. Yeasts were able to inhibit fungal growth and kill the fungus at three different temperatures, under starvation, at different culture stages, or using an inoculum from old yeast cultures. Moreover, SEM analysis revealed that W. anomalus and S. cerevisiae PE2 cluster and adhere to the hyphae, push their surface, and fuse to them, ultimately draining the cells. This behavior concurs with that classified as necrotrophic parasitism/mycoparasitism. In particular, W. anomalus within the adhered clusters appear to be ligated to each other through roundish groups of fimbriae-like structures filled with bundles of microtubule-sized formations, which appear to close after cells detach, leaving a scar. SEM also revealed the formation of tube-like structures apparently connecting yeast to hypha. This evidence suggests W. anomalus cells form a network of yeast cells connecting with each other and with hyphae, supporting a possible cooperative collective killing and feeding strategy. The present results provide an initial step toward the formulation of a new eco-friendly and effective alternative for controlling cacao WBD using live yeast biocides.

The aim of this study was to evaluate the distribution of ACE-I/D polymorphisms on Brazilian football players performance in aerobic capacity, strength and speed tests.
METHODS: The participants in this study were 212 Brazilian first division male football players genotyped in DD, ID or II. Genotyping of DNA from leucocytes was performed using polymerase chain reaction and restriction fragment length polymorphism methods. We evaluated speed using a 30-meter sprint test with speed measured at 10 meters (V10), 20 meters (V20), and 30 meters (V30); muscular strength using counter-movement-jump and squat jump tests; and aerobic endurance using the Yo-Yo endurance test. The athletes were ranked in ascending order according to their performance in each test and divided into quartiles: first quartile (0-25%, weak), second (25-50%, normal), third (50-75%, good), and fourth (75-100%, excellent); these were clustered according to genotype frequency.
RESULTS: We identified significant differences in the V20 test values and in the aerobic capacity test. Higher frequencies of the ACE-DD genotype were observed in the excellent performance group in the V20. In the aerobic capacity test, higher frequencies of the ACE-II genotype were observed in excellent and good performance groups.
CONCLUSIONS: Players with higher performance in anaerobic and aerobic tests are ACE-DD and ACE-II genotypes, respectively.

Wistar Audiogenic Rat is an epilepsy model whose animals are predisposed to develop seizures induced by acoustic stimulation. This model was developed by selective reproduction and presents a consistent genetic profile due to the several generations of inbreeding. In this study, we performed an analysis of WAR RNA-Seq data, aiming identified at genetic variants that may be involved in the epileptic phenotype. Seventeen thousand eighty-five predicted variants were identified as unique to the WAR model, of which 15,915 variants are SNPs and 1,170 INDELs. We filter the predicted variants by pre-established criteria and selected five for validation by Sanger sequencing. The genetic variant c.14198T>C in the Vlgr1 gene was confirmed in the WAR model. Vlgr1 encodes an adhesion receptor that is involved in the myelination process, in the development of stereocilia of the inner ear, and was already associated with the audiogenic seizures presented by the mice Frings. The transcriptional quantification of Vlgr1 revealed the downregulation this gene in the corpus quadrigeminum of WAR, and the protein modeling predicted that the mutated residue alters the structure of a domain of the VLGR1 receptor. We believe that Vlgr1 gene may be related to the predisposition of WAR to seizures and suggest the mutation Vlgr1/Q4695R as putative causal variant, and the first molecular marker of the WAR strain.

Wickerhamomyces anomalus LBCM1105 is a yeast isolated from cachaça distillery fermentation vats, notable for exceptional glycerol consumption ability. We report its draft genome with 20.5x in-depth coverage and around 90% extension and completeness. It harbors the sequences of proteins involved in glycerol transport and metabolism.

O consumo de alimentos com alta densidade calórica e baixo teor de fibras tem levado ao acometimento de diversas doenças crônicas não transmissíveis na população brasileira. A reformulação de formulações bem aceitas pela população com ingredientes e técnicas de preparo mais saudáveis podem contribuir para a melhora do estado de saúde do brasileiro. O objetivo do presente estudo foi elaborar e avaliar a composição centesimal e sensorial de formulações de bolinhos de chuva assados acrescidos de diferentes quantidades de fibras. Foram elaboradas 10 formulações de bolinhos de chuva assados acrescidos em diferentes proporções de farinha de trigo integral e farelo de aveia. Além disso, foram realizadas análises do teor de proteínas, cinzas, lipídios, umidade, carboidrato, fibras, potássio, sódio, além do valor calórico total e análise sensorial com avaliação da aparência, sabor, consistência, cor, ideal de consistência e doçura, e intenção de compra. Houve aumento do conteúdo proteico, lipídico e de fibras nas formulações com adição de farelo de aveia e farinha de trigo integral. Sete formulações podem ser consideradas como fonte de fibras (mínimo de 3g de fibras a cada 100g de produto). A amostra com 50,5% de farelo de aveia foi a única que apresentou um índice de aceitação menor que 70%. Conclui-se que a adição de ingredientes fontes de fibras proporciona um produto final mais saudável e nutritivo, havendo um aumento de fibras presentes no produto final e preservando, no geral, as características sensoriais adequadas e boa intenção de compra. 

O seguimento de cervejas artesanais tem se desenvolvido bastante nos últimos anos. Os consumidores, cada vez mais habituados ao consumo desse tipo de cerveja mantém uma demanda constante por novidades no mercado. Mais que preços acessíveis, buscam atributos sensoriais novos e atrativos. Esse trabalho foi desenvolvido com o objetivo de produzir e avaliar cervejas artesanais estilo Pale Ale adicionadas de caldo de cana e água de coco. As cervejas foram formuladas com malte Pilsen Agrária, Fermento S04 Fermentis, lúpulo Citra Barth-Hass, água de coco 4 ºBrix e caldo de cana 21 ºBrix. Foram elaboradas três formulações contendo 2%, 5% e 10% de água de coco; três formulações contendo 2%, 5% e 10% de caldo de cana; uma formulação contendo uma combinação de 10% de água de coco e 10% de caldo de cana e uma formulação controle.  As análise físico-químicas realizadas em triplicata, após 30 dias, foram as de teor alcoólico, pH, acidez total, acidez volátil, estrato real, extrato primitivo e cor. As concentrações empregadas de água de coco e caldo de cana não determinaram diferenças significativas entre as formulações elaboradas.

Fish stocking programs have been implemented to mitigate the blockage of original riverbeds by the construction of hydropower dams, which affects the natural migration of fish populations. However, this method raises concerns regarding the genetic rescue of the original populations of migratory fish species. We investigated the spatial distribution of genetic properties, such as genetic diversity, population structure, and gene flow (migration), of the Neotropical migratory fish Prochilodus costatus in the Três Marias dam in the São Francisco River basin, Brazil, and examined the possible effects of fish stocking programs on P. costatus populations in this region. In total, 1,017 specimens were sampled from 12 natural sites and a fish stocking program, and genotyped for high-throughput sequencing at 8 microsatellite loci. The populations presented low genetic variability, with evidence of inbreeding and the presence of only four genetic pools; three pools were observed throughout the study region, and the fourth was exclusive to one area in the Paraopeba River. Additionally, we identified high unidirectional gene flow between regions, and a preferred migratory route between the Pará River and the upper portion of the São Francisco River. The fish stocking program succeeded in transposing the genetic pools from downstream to upstream of the Três Marias dam, but, regrettably, promoted genetic homogenization in the upper São Francisco River basin. Moreover, the data show the fragility of this species at the genetic level. This monitoring strategy could be a model for the development of conservation and management measures for migratory fish populations that are consumed by humans.

Second generation biorefineries demand efficient lignocellulosic hydrolysate fermenting strains and recent advances in strain isolation and engineering have progressed the bottleneck in developing production hosts from generation of strains into testing these under relevant conditions. In this paper, we introduce a methodology for high-throughput analysis of yeast strains directly in lignocellulosic hydrolysates. The Biolector platform was used to assess aerobic and anaerobic growth of 12 Saccharomyces cerevisiae strains and their ΔPdr12 mutants in wheat straw hydrolysate. The strains evaluated included lab, industrial and wild type strains and the screening could capture significant differences in growth and ethanol production among the strains. The methodology was also demonstrated with corn stover hydrolysate and the results were in line with shake flask cultures. Our study demonstrates that growth in lignocellulosic hydrolysates could be rapidly monitored using 1 ml cultures and that measuring growth and product formation under relevant conditions are crucial for evaluating strain performance.

The purpose of this study was to evaluate indicators of muscle damage and hormonal responses after soccer matches and its relation to alpha-actinin-3 (ACTN3) gene expression (XX vs. RR/RX), considering that the R allele produces alpha-actinin-3 and provides greater muscle strength and power. Thirty players (10 XX and 20 RR/RX) younger than 16 years were evaluated in this study. Blood samples were collected immediately before, after, 2, and 4 hours after the games to assess muscle damage (creatine kinase [CK] and alpha-actin) and hormonal responses (interleukin-6 [IL-6], cortisol, and testosterone). Postgame CK was higher as compared to the pregame values in both groups and it was also higher in the RR/RX (p < 0.05) than in the XX. The concentrations of alpha-actin and IL-6 were similar for both groups and did not change over time. Testosterone was increased after the game only in the RR/RX group (p < 0.05). Cortisol concentrations in group RR/RX were higher immediately after the game than before the game, and 2 and 4 hours after the game the concentration decreased (p < 0.05). The RR and RX individuals presented higher markers of muscle microtrauma and hormonal stress, probably because they performed more speed and power actions during the game, which is a self-regulated activity. From the different responses presented by RR/RX and XX genotypes, we conclude that the genotypic profile should be taken into account when planning training workloads and recovery of athletes.

The fractionation of sugarcane bagasse (SB) by hydrothermal pretreatment (HP, autohydrolysis) followed by alkaline extraction (AE) and advanced oxidative pretreatment (AOP) for production of second-generation ethanol and biogas was investigated. The AOP of SB was optimized using a Doehlert design, varying the applied H₂O₂ load, liquid-to-solid ratio (LSR), and time. The responses evaluated were yield (Y), residual cellulose (RC), delignification (DE), and enzymatic conversion (EC). The AE of SB pretreated by HP led to 61.8% DE (using 0.2 mol L⁻¹ NaOH). This high lignin removal enabled substantial savings of H₂O₂ in the AOP. The optimized AOP conditions led to 78% Y, 82.2% RC, 42.7% DE, and 88.9% EC (overall glucose yield of 60.9%). Fermentation of the enzymatic hydrolysate with Saccharomyces cerevisiae yielded 190.8 L_ethanol ton_SB⁻¹. Biogas production by anaerobic digestion of residual liquid streams of the pretreatment steps yielded 27.46 NL_CH4 kg_SB⁻¹. An energy balance was estimated for the SB fractionation.

This work was performed to verify the potential of yeast strains isolated from cachaça distilleries for two specific biotechnological applications: beer and bioethanol production. In the beer production, the strains were tested for characteristics required in brewery practices, such as: capacity to ferment maltose and maltotriose, ability to grow at lowest temperatures, low H₂S production, and flocculation profile. Among the strains tested, two of them showed appropriate characteristics to produce two different beer styles: lager and ale. Moreover, both strains were tested for cachaça production and the results confirmed the capacity of these strains to improve the quality of cachaça. In the bioethanol production, the fermentation process was performed similarly to that used by bioethanol industries: recycling of yeast biomass in the fermentative process with sulfuric acid washings (pH 2.0). The production of ethanol, glycerol, organic acids, dry cell weight, carbohydrate consumption, and cellular viability were analyzed. One strain presented fermentative parameters similar to PE2, industrial/commercial strain, with equivalent ethanol yields and cellular viability during all fermentative cycles. This work demonstrates that cachaça distilleries seem to be an interesting environment to select new yeast strains to be used in biotechnology applications as beer and bioethanol production.

The aim was to investigate a possible role of the ACTN3 R577X polymorphism in a Brazilian football player’s career progression. 2 questions were formulated: 1. Does ACTN3 polymorphism affect the probability of an individual being a professional football player? 2. Does this polymorphism affect the progression of the athlete throughout his career? The study included 353 players from first division Brazilian football clubs in the following categories: under-14 (U-14), U-15, U-17, U-20, and professional (PRO). The control group (CON) was composed of 100 healthy non-athletes. The chi-squared test was used to assess differences between the allele and genotype frequencies. Comparing football categories, the XX genotype was less frequent among professional players than in the U-20 (p<0.05) or the U-15 category (p<0.05). The RX genotype also presented more frequently in the PRO category than the U-14 category (p<0.05). Moreover, a trend towards a higher frequency of the RX genotype and a lower frequency of the XX genotype was observed in the professional category compared to U-20. These results suggest that the genotype in the ACTN3 polymorphism affects the probability of a football player progressing throughout his career and becoming professional, meaning that playing football selects against the ACTN3 XX genotype.

Genetic diversity and population studies are essential for conservation and wildlife management programs. However, monitoring requires the analysis of multiple loci from many samples. These processes can be laborious and expensive. The choice of microsatellites and PCR calibration for genotyping are particularly daunting. Here we optimized a low-cost genotyping method using multiple microsatellite loci for simultaneous genotyping of up to 384 samples using next-generation sequencing (NGS). We designed primers with adapters to the combinatorial barcoding amplicon library and sequenced samples by MiSeq. Next, we adapted a bioinformatics pipeline for genotyping microsatellites based on read-length and sequence content. Using primer pairs for eight microsatellite loci from the fish Prochilodus costatus, we amplified, sequenced, and analyzed the DNA of 96, 288, or 384 individuals for allele detection. The most cost-effective methodology was a pseudo-multiplex reaction using a low-throughput kit of 1 M reads (Nano) for 384 DNA samples. We observed an average of 325 reads per individual per locus when genotyping eight loci. Assuming a minimum requirement of 10 reads per loci, two to four times more loci could be tested in each run, depending on the quality of the PCR reaction of each locus. In conclusion, we present a novel method for microsatellite genotyping using Illumina combinatorial barcoding that dispenses exhaustive PCR calibrations, since non-specific amplicons can be eliminated by bioinformatics analyses. This methodology rapidly provides genotyping data and is therefore a promising development for large-scale conservation-genetics studies.

Cryptic relatedness is a confounding factor in genetic diversity and genetic association studies. Development of strategies to reduce cryptic relatedness in a sample is a crucial step for downstream genetic analyses. This study uses a node selection algorithm, based on network degrees of centrality, to evaluate its applicability and impact on evaluation of genetic diversity and population stratification. 1,036 Guzerá (Bos indicus) females were genotyped using Illumina Bovine SNP50 v2 BeadChip. Four strategies were compared. The first and second strategies consist on a iterative exclusion of most related individuals based on PLINK kinship coefficient (φij) and VanRaden's φij, respectively. The third and fourth strategies were based on a node selection algorithm. The fourth strategy, Network G matrix, preserved the larger number of individuals with a better diversity and representation from the initial sample. Determining the most probable number of populations was directly affected by the kinship metric. Network G matrix was the better strategy for reducing relatedness due to producing a larger sample, with more distant individuals, a more similar distribution when compared with the full data set in the MDS plots and keeping a better representation of the population structure. Resampling strategies using VanRaden's φij as a relationship metric was better to infer the relationships among individuals. Moreover, the resampling strategies directly impact the genomic inflation values in genomewide association studies. The use of the node selection algorithm also implies better selection of the most central individuals to be removed, providing a more representative sample.

The pattern of glucose repression in most Kluyveromyces marxianus strains does not correlate with fermentative behaviour; however, glucose repression and fermentative metabolism appear to be linked to the kinetics of sugar uptake. In this work, we show that lactose transport in K. marxianus CCT 7735 by lactose-grown cells is mediated by a low-affinity H⁺-sugar symporter. This system is glucose repressed and able to transport galactose with low affinity. We also observed the activity of a distinct lactose transporter in response to raffinose. Regarding glucose uptake, specificities of at least three low-affinity systems rely on the carbon source available in a given growth medium. Interestingly, it was observed only one high-affinity system is able to transport both glucose and galactose. We also showed that K. marxianus CCT 7735 regulates the expression of sugar transport systems in response to glucose availability.