Bacillus genus industrial applications and innovation: First steps towards a circular bioeconomy.

In recent decades, environmental concerns have directed several policies, investments, and production processes. The search for sustainable and eco-friendly strategies is constantly increasing to reduce petrochemical product utilization, fossil fuel pollution, waste generation, and other major ecological impacts. The concepts of circular economy, bioeconomy, and biorefinery are increasingly being applied to solve or reduce those problems, directing us towards a greener future. Within the biotechnology field, the Bacillus genus of bacteria presents extremely versatile microorganisms capable of producing a great variety of products with little to no dependency on petrochemicals. They are able to grow in different agro-industrial wastes and extreme conditions, resulting in healthy and environmentally friendly products, such as foods, feeds, probiotics, plant growth promoters, biocides, enzymes, and bioactive compounds. The objective of this review was to compile the variety of products that can be produced with Bacillus cells, using the concepts of biorefinery and circular economy as the scope to search for greener alternatives to each production method and providing market and bioeconomy ideas of global production. Although the genus is extensively used in industry, little information is available on its large-scale production, and there is little current data regarding bioeconomy and circular economy parameters for the bacteria. Therefore, as this work gathers several products' economic, production, and environmentally friendly use information, it can be addressed as one of the first steps towards those sustainable strategies. Additionally, an extensive patent search was conducted, focusing on products that contain or are produced by the Bacillus genus, providing an indication of global technology development and direction of the bacteria products. The Bacillus global market represented at least $18 billion in 2020, taking into account only the products addressed in this article, and at least 650 patent documents submitted per year since 2017, indicating this market's extreme importance. The data we provide in this article can be used as a base for further studies in bioeconomy and circular economy and show the genus is a promising candidate for a greener and more sustainable future.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil.

BACKGROUND: Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES: In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS: A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS: We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS: To the best of our knowledge, this is the first detection of LRV2 in the New World.

The Potential of Xylooligosaccharides as Prebiotics and Their Sustainable Production from Agro-Industrial by-Products.

In recent years, concerns about a good-quality diet have increased. Food supplements such as prebiotics have great nutritional and health benefits. Within the diverse range of prebiotics, xylooligosaccharides (XOs) show high potential, presenting exceptional properties for the prevention of systemic disorders. XOs can be found in different natural sources; however, their production is limited. Lignocellulosic biomasses present a high potential as a source of raw material for the production of XOs, making the agro-industrial by-products the perfect candidates for production on an industrial scale. However, these biomasses require the application of physicochemical pretreatments to obtain XOs. Different pretreatment methodologies are discussed in terms of increasing the production of XOs and limiting the coproduction of toxic compounds. The advance in new technologies for XOs production could decrease their real cost (USD 25-50/kg) on an industrial scale and would increase the volume of market transactions in the prebiotic sector (USD 4.5 billion). In this sense, new patents and innovations are being strategically developed to expand the use of XOs as daily prebiotics.

Fine resolution analysis of bacterial communities associated with Neochloris oleoabundans culture and insights into terpenes as contamination control agents.

Biological contamination is one of the main bottlenecks in microalgae production, reducing quality and productivity and sometimes leading to the complete loss of the cultures. Selecting terpenes can be a pathway toward eco-friendly contamination control in microalgae cultures. This work evaluated the presence of bacterial contaminants in N. oleoabundans cultures through HTS and 16 S analysis and their susceptibility to six natural terpenes (α-pinene, ß-pinene, limonene, trans-cinnamaldehyde, linalool, and eugenol). The principal phyla identified were Proteobacteria, Bacteroidetes, and Actinobacteria, and based on these data, 89 bacterial isolates of seven genera were obtained (36 Aureimonas sp., 27 Microbacterium sp., 5 Pseudomonas sp., 9 Bacillus sp., 14 Shinella sp., 1 Brevundimonas sp., and 1 Exiguobacterium sp.) at 25ºC in the presence of light. It was possible to observe that Beta-pinene 50 mg L- 1 only inhibited Bacillus sp. In contrast, Alpha-pinene, Linalool, and Trans-cinnamaldehyde, at a concentration of 6.25 mg L- 1 efficiently inhibited most isolates. The inhibition percentages found were 79-99%.

First report of putative Leishmania RNA virus 2 (LRV2) in Leishmania infantum strains from canine and human visceral leishmaniasis cases in the southeast of Brazil

BACKGROUND Leishmania RNA virus 1 (LRV1) is commonly found in South American Leishmania parasites belonging to the subgenus Viannia, whereas Leishmania RNA virus 2 (LRV2) was previously thought to be restricted to the Old-World pathogens of the subgenus Leishmania. OBJECTIVES In this study, we investigated the presence of LRV2 in strains of Leishmania (L.) infantum, the causative agent of visceral leishmaniasis (VL), originating from different hosts, clinical forms, and geographical regions. METHODS A total of seventy-one isolates were screened for LRV2 using semi-nested reverse transcription-polymerase chain reaction (RT-PCR) targeting the RNA-dependent RNA polymerase (RdRp) gene. FINDINGS We detected LRV2 in two L. infantum isolates (CUR268 and HP-EMO) from canine and human cases, respectively. MAIN CONCLUSIONS To the best of our knowledge, this is the first detection of LRV2 in the New World.

Enhancing the Recovery of Bioactive Compounds of Soybean Fermented with Rhizopus oligosporus Using Supercritical CO2: Antioxidant, Anti-Inflammatory, and Oxidative Proprieties of the Resulting Extract.

The aim of the present study was to evaluate the use of supercritical CO2 combined with cosolvent for the recovery of bioactive compounds of soybean fermented with Rhizopus oligosporus NRRL 2710. Soxhlet extractions using seven different organic solvents (n-hexane, petroleum ether, ethyl acetate, acetone, ethanol, methanol, and water) were initially performed for comparative purposes. The extracts obtained were characterized by physicochemical, antioxidant, total phenolic, and oxidative proprieties. For the Soxhlet extractions, the highest and lowest yields obtained were 45.24% and 15.56%, using methanol and hexane, respectively. The extraction using supercritical CO2 combined with ethanol as a static modifier (scCO2 + EtOH) presented, at a high pressure (25 MPa) and temperature (80 °C), a phenolic compound content of 1391.9 µg GAE g-1 and scavenging of 0.17 g, reaching a 42.87% yield. The extracts obtained by sCO2 + EtOH were characterized by high contents of essential fatty acids (linoleic acid and oleic acid) and bioactive compounds (gallic acid, trans-cinnamic acid, daidzein, and genistein). These extracts also showed a great potential for inhibiting hyaluronidase enzymes (i.e., anti-inflammatory activity). Thermogravimetric analyses of the samples showed similar profiles, with oil degradation values in the range from 145 to 540 °C, indicating progressive oil decomposition with a mass loss ranging from 93 to 98.7%. In summary, this study demonstrated the flexibility of scCO2 + EtOH as a green technology that can be used to obtain high-value-added products from fermented soybean.

A review on enzyme-producing lactobacilli associated with the human digestive process: From metabolism to application.

Complex carbohydrates, proteins, and other food components require a longer digestion process to be absorbed by the lining of the alimentary canal. In addition to the enzymes of the gastrointestinal tract, gut microbiota, comprising a large range of bacteria and fungi, has complementary action on the production of digestive enzymes. Within this universe of "hidden soldiers", lactobacilli are extensively studied because of their ability to produce lactase, proteases, peptidases, fructanases, amylases, bile salt hydrolases, phytases, and esterases. The administration of living lactobacilli cells has been shown to increase nutrient digestibility. However, it is still little known how these microbial-derived enzymes act in the human body. Enzyme secretion may be affected by variations in temperature, pH, and other extreme conditions faced by the bacterial cells in the human body. Besides, lactobacilli administration cannot itself be considered the only factor interfering with enzyme secretion, human diet (microbial substrate) being determinant in their metabolism. This review highlights the potential of lactobacilli to release functional enzymes associated with the digestive process and how this complex metabolism can be explored to contribute to the human diet. Enzymatic activity of lactobacilli is exerted in a strain-dependent manner, i.e., within the same lactobacilli species, there are different enzyme contents, leading to a large variety of enzymatic activities. Thus, we report current methods to select the most promising lactobacilli strains as sources of bioactive enzymes. Finally, a patent landscape and commercial products are described to provide the state of art of the transfer of knowledge from the scientific sphere to the industrial application.

Environmental Detection of SARS-CoV-2 Virus RNA in Health Facilities in Brazil and a Systematic Review on Contamination Sources.

SARS-CoV-2 environmental monitoring can track the rate of viral contamination and can be used to establish preventive measures. This study aimed to detect by RT-PCR the presence of SARS-CoV-2 from inert surface samples in public health settings with a literature review about surface contamination and its burden on spread virus. Samples were collected from health settings in Curitiba, Brazil, between July and December 2020. A literature review was conducted using PRISMA. A total of 711 environmental surface samples were collected from outpatient areas, dental units, doctors' offices, COVID-19 evaluation areas, and hospital units, of which 35 (4.9%) were positive for SARS-CoV-2 RNA. The frequency of environmental contamination was higher in primary care units than in hospital settings. The virus was detected on doctors' personal items. Remarkably, the previously disinfected dental chair samples tested positive. These findings agree with those of other studies in which SARS-CoV-2 was found on inanimate surfaces. Detection of SARS-CoV-2 RNA on surfaces in public health settings, including those not meant to treat COVID-19, indicates widespread environmental contamination. Therefore, the intensification of disinfection measures for external hospital areas may be important for controlling community COVID-19 dissemination.

Correlation of maternal concentrations of plasma testosterone with fetal sex in horses / Correlação das concentrações plasmáticas de testosterona materna com o sexo fetal em equinos

ABSTRACT: The objectives of this study were to evaluate the correlation of fetal sex and plasma testosterone concentrations between the 5th and 8th months of pregnancy in mares and to verify the applicability of this test to predict fetal sex. Blood samples were collected from 21 mares at 30-day intervals of between 150 and 240 days of pregnancy. Plasma testosterone was determined by radioimmunoassay and the sex of the foals confirmed at birth. The levels of maternal testosterone were higher in mares carrying female fetuses at months 5 and 8 (P < 0.05). Limit values were determined by analyzing the receiver operating characteristic (ROC) estimates: 35.5 pg/mL and 40 pg/mL for the 5th and 8th month, respectively. For the mares with plasma testosterone values equal to or above the threshold, gestation of female foals was predicted, and for those with plasma testosterone below the threshold values pregnancy of male foals was predicted. In the 5th month, the predictive values for male and female fetuses were 70% and 88.9%, respectively; the detection rates were 87.5% and 72.7%, and the total accuracy of the examination was 78.9%. In the 8th month, the predictive values for male and female fetuses were 80% and 90%, respectively; the detection rates were 88.9% and 81.8%, and the total accuracy of the examination was 85%. It was concluded that there was a correlation between fetal sex and plasma testosterone concentrations in pregnant mares. Prediction of fetal sex based on plasma concentrations of maternal testosterone can be performed in months 5 and 8 with 78.9% and 85% accuracy, respectively.

RESUMO: Os objetivos do estudo foram avaliar a correlação do sexo fetal com as concentrações plasmáticas de testosterona entre o 5° e o 8º mês de gestação na égua e verificar a aplicabilidade deste exame para a predição do sexo fetal. Amostras de sangue foram coletadas de 21 éguas, com intervalos de 30 dias, entre 150 e 240 dias de gestação. A testosterona plasmática foi determinada por radioimunoensaio e o sexo dos potros foi confirmado ao nascimento. Os valores de testosterona materna foram superiores nas éguas gestando fetos fêmeas aos cinco e oito meses (P< 0.05). Através da análise da curva ROC (receiver operating characteristic) foram determinados valores limites de 35,5 pg/mL e 40 pg/mL para o 5º e o 8° mês, respectivamente. Éguas com testosterona plasmática igual ou acima dos valores limites foram preditas como gestando fêmeas e éguas com testosterona plasmática abaixo dos valores limites foram preditas como gestando machos. Aos cinco meses, os valores preditivos para fetos machos e fêmeas foram 70% e 88,9%, respectivamente; as taxas de detecção foram 87,5% e 72,7% e a acurácia total do exame foi de 78,9%. Aos oito meses, os valores preditivos para fetos machos e fêmeas foram 80% e 90%, respectivamente; as taxas de detecção foram 88,9% e 81,8% e a acurácia total do exame foi de 85%. Conclui-se que houve correlação entre o sexo fetal e as concentrações de testosterona plasmática em éguas prenhes. A predição do sexo fetal baseada nas concentrações plasmáticas de testosterona materna pode ser realizada aos cinco e oito meses de gestação com 78,9% e 85% de acurácia, respectivamente.

Guinea Pigs Naturally Infected by Leishmania enriettii: Clinical Analyses, Parasite Isolation and Identification

Abstract Leishmania enriettii has only been found in Curitiba metropolitan region, southern Brazil were it was first observed in Cavia porcellus from the vivarium of Instituto de Biologia e Pesquisas Tecnológicas (IBPT - today named TECPAR) by Medina, 1944. Despite more than a half century from its discovery and several research articles on this species, the natural clinical signs in guinea pigs and the parasite genetic variability is still unclear. The aims of this study were to describe the clinical features, investigate the potential wild reservoirs and, in addition, we intended to understand the polymorphism trait of the species. We analyzed 26 naturally infected guinea pigs from eight Paraná state cities. All animals showed lesions compatible with leishmaniosis, such as skin nodules or ulcers on body extremities. Direct examination of the lesion samples obtained by fine-needle aspiration or punch biopsy was conducted followed by isolation and identification of parasite DNA by random amplification of polymorphic DNA (RAPD)-PCR. Through the direct exam, a large number of intracellular amastigote forms were observed in the lesions. Different strains of the parasite, isolated from the 26 animals, were grouped in 5 clusters of approximately 65% similarity. We looked for L. enriettii in other potential reservoir hosts but the parasite was not observed. These results confirm that distinct strains of L. enriettii circulate in guinea pigs from Paraná state, more specifically in the Atlantic forest region, where we believe it serves as the center for dispersion of the species.

Biohydrogen production in cassava processing wastewater using microbial consortia: Process optimization and kinetic analysis of the microbial community.

Biohydrogen production was evaluated using cassava processing wastewater (CPW) and two microbial consortia (Vir and Gal) from different Brazilian environments. The biohydrogen production was optimized using a Box-Behnken design (T, pH, C/N, and % v/v inoculum). Maximum yields were obtained with hydrolyzed substrate: 4.12 and 3.80 mol H2 / for Vir and Gal, respectively. Similarly, the kinetic parameters µ, k, and q were higher with hydrolyzed CPW in both consortia. The molecular analysis of the consortia through Illumina high-throughput sequencing showed the presence of bacteria from the families Porphyromonadaceae, Clostridiaceae, Ruminococcaceae, and Enterococcaceae. The relative abundance of microbial families varies as fermentation progresses. In both consortia, Clostridiaceae reached the maximum relative abundance in the media between 16 and 24 h, interval in which approximately 90% of the biohydrogen is generated.

A comparative study of extraction techniques for maximum recovery of bioactive compounds from Ganoderma lucidum spores / Estudio comparativo de técnicas de extracción para la máxima recuperación de compuestos bioactivos a partir esporas de Ganoderma lucidum

SUMMARY This study aimed at evaluating effective methods for breaking the hard and insoluble spores of Ganoderma lucidum to recover functional biomolecules. Rupture techniques were evaluated such as manual maceration (RM), maceration with spheres of various materials (BR), and microwave exposure plus maceration with steel/ chrome spheres (MBR1). Spore rupture was evaluated using UV-Vis spectroscopy, which showed vibrations of 2955, 1642, 1240, 1080 and 1746 cm-1 corresponding to changes in spore walls. The MBR1 extract contained the largest amounts of carbohydrates (19.80 mg.g-1 spores) and polyphenols (2.21 mg.g-1 spores), whereas the BR extract had higher antioxidant activity (57.22%Inb DPPH). The MBR1 and BR extracts contained 62.2 and 73.5% glucose, respectively. Both methods also involved significant extraction of carbohydrates and proteins. The best way to extract biomolecules from spore walls is to perform a microwave heat treatment and break the walls with steel/chrome spheres; this produces large quantities of carbohydrates with antioxidant properties.

RESUMEN El objetivo de este estudio fue evaluar varios métodos de ruptura de las esporas de Ganoderma lucidum y extraer sus propiedades bioactivas. Para este propósito se evaluaron diferentes técnicas de rompimiento como: la maceración manual (RM), la maceración con esferas de diversos materiales (BR) y la exposición a microondas junto la maceración de las esporas con esferas de acero/cromo (MBR1). La ruptura de las esporas fue evaluada por espectroscopia UV-Vis, la cual mostró que las vibraciones 2955, 1642, 1240, 1080 y 1746 cm-1 correspondieron a cambios estructurales en las paredes de las esporas. El extracto MBR1 presento el mayor contenido de carbohidratos (19,80 mg.g-1) y polifenoles (2,21 mg.g-1), mientras que el extracto BR tuvo una mayor actividad antioxidante (57,22% Inb DPPH). Los extractos MBR1 y BR también presentaron en el análisis de monosacáridos un 62,2 y 73,5% de contenido glucosa. Como conclusión la mejor metodología para extraer biomoléculas de las paredes de las esporas de G. lucidum fueron el tratamiento térmico con microondas y la ruptura de las paredes con esferas de acero/cromo, porque este proceso permitió la extracción de una mayor cantidad de carbohidratos con posibles propiedades antioxidantes.

The Antihypertensive, Antimicrobial and Anticancer Peptides from Arthrospira with Therapeutic Potential: A Mini Review.

The interest in biological peptides from Arthrospira sp. (syn Spirulina) is increasing due to its Generally Recognised as Safe "GRAS" status, the high concentration of proteins and the history of its use as a supplement and nutraceutical agent. Arthrospira peptides can be generated by the controlled hydrolysis of proteins, using proteases, followed by fractionation. The peptides obtained have a range of therapeutic effects. Amongst these bioactive peptides, three classes are of major importance: the antihypertensive (AHP), antimicrobial (AMP) and anticancer (ACP) peptides. AHPs have the ability to work as inhibitors of angiotensin-converting enzyme (ACE), and help to control several diseases such as hypertension, obesity, and cardiovascular issues, AMPs play a crucial role in the immune response, inhibiting the development of pathogens such as bacteria, fungi, viruses and others, while ACPs can aid in tumour control by the induction of apoptosis or necrosis, or the inhibition of angiogenesis. Thus, bioactive peptides are of great significance to the pharmaceutical industry. However, they can show secondary effects. This paper reviews the inhibition mechanism of antimicrobial, hypertensive and anticancer peptides from Arthrospira sp., and the possible structures of the peptides according to the type of activity and its intensity. In addition, this paper describes the purification methods of absorption mechanisms, and reviews databases for designing peptides.

Microalgal biomass pretreatment for integrated processing into biofuels, food, and feed.

Microalgae are sources of nutritional products and biofuels. However, their economical processing is challenging, because of (i) the inherently low concentration of biomass in algal cultures, below 0.5%, (ii) the high-water content in the harvested biomass, above 70%; and (iii) the variable intracellular content and composition. Cell wall structure and strength vary enormously among microalgae, from naked Dunaliella cells to robust Haematococcus cysts. High-value products justify using fast and energy-intensive processes, ranging from 0.23 kWh/kg dry biomass in high-pressure homogenization, to 6 kWh/kg dry biomass in sonication. However, in biofuels production, the energy input must be minimized, requiring slower, thermal or chemical pretreatments. Whichever the primary fraction of interest, the spent biomass can be processed into valuable by-products. This review discusses microalgal cell structure and composition, how it affects pretreatment, focusing on technologies tested for large scale or promising for industrial processes, and how these can be integrated into algal biorefineries.

L-lysine production improvement: a review of the state of the art and patent landscape focusing on strain development and fermentation technologies.

L-lysine is an essential amino acid used in various industrial sectors but mainly in food and animal feed. Intense research has been directed toward increasing its productivity. This literature review presents the state of the art and patent landscape of the industrial production of L-lysine, with a focus on the strain development and fermentation technologies, through geographic, social, and chronological analysis, using the text mining technique. The geographic analysis showed a greater tendency for countries with industrial plants with large production capacity to submit patents or publish articles, while the social analysis reflected the close relationship between educational units and companies. The technologies of each document were divided into optimization of fermentation parameters, conventional mutation, and genetic engineering. Corynebacterium glutamicum and Escherichia coli present the most attractive industrial phenotypes, and their cultivation occurs mainly in fed-batch processes with control parameters carefully selected to enhance metabolism. These strains are generally modified by conventional approaches (e.g., mutagenesis and selection of auxotrophic and/or regulatory mutants) or by genetic engineering technologies. The combination of both these approaches enables genomic breeding and the construction of strains with industrial potential, capable of accumulating more than 120 g/L of L-lysine. From the analysis of these approaches, we developed a descriptive flow of substrate uptake, amino acid metabolism, and mechanisms of excretion of a lysine-producing model cell. It is expected that the various mechanisms of L-lysine production, here shown and described, will become a guide that aids in increasing amino acid productivity without interfering with the strain stability.

Microalgal biorefineries: Integrated use of liquid and gaseous effluents from bioethanol industry for efficient biomass production.

A new method for CO2 recovery was proposed for cultivation of different microalgae. First, a chemical fixation, where CO2 was injected in alkalinized vinasse to form (bi)carbonate salts, was performed. In addition, biological fixation with CO2-enriched air injection was also accomplished for evaluation of the most promising results. Two bioreactor systems, a stirred-tank reactor and a bubble column reactor, were employed. A higher carbon transfer rate (43.35 g.L-1.h-1) was achieved in the bubble column reactor using NaOH-alkalinized vinasse, along with reductions of the chemical oxygen demand (COD), biological oxygen demand (BOD) and turbidity (TD). This allowed the cultivation of microalgae and cyanobacteria at vinasse concentrations between 70 and 100%, reaching a biomass production of 2.25 g.L-1 in 15 days of culture. The viability of chemical CO2 fixation together with the use of 100% treated vinasse from a bioethanol production unit for microalgae cultivation has been demonstrated in a successfully integrated biorefinery approach.

Prevention methods of foodborne Chagas disease: Disinfection, heat treatment and quality control by RT-PCR.

The most important mode of transmission causing outbreaks of Chagas disease in the Amazon region is the oral route due to the ingestion of contaminated food. Herein, prevention methods for foodborne diseases caused by Trypanosoma cruzi, namely, sanitization, thermal treatment were investigated and the use of reverse transcription PCR (RT-PCR) amplification for the mRNA-based detection of viable T. cruzi in açai, was developed. Three T. cruzi strains (T. cruzi I, T. cruzi III and Y) were used in the present study. The Amazonian strains T. cruzi I (425) and T. cruzi III (370) showed higher resistance to sodium hypochlorite treatment and heat treatment than the reference strain Y. The blanching of fruits (70 ±â€¯1 °C for 10 s) and pasteurization of juice (82.5 °C for 1 min) efficiently eliminated T. cruzi in food matrices. Additionally, a method that uses RT-PCR amplification of mRNA was developed for the detection of viable T. cruzi in açai, which could play a role in examining food samples, ensuring consumer health, and reducing this foodborne disease.

Biological evaluation of mimetic peptides as active molecules for a new and simple skin test in an animal model.

A skin test is a widely used tool in diagnostic evaluations to investigate cutaneous leishmaniases (CL). The actual antigen (Montenegro skin test [MST] antigen) presents some difficulties that pertain to its manufacturing and validation. To contribute to overcoming this problem, we propose the application of new-generation molecules that are based on skin antigen tests. These antigens were obtained through biotechnology pathways by manufacturing synthetic mimetic peptides. Three peptides, which were selected by phage display, were tested as skin test antigens in an animal model (Cavia porcellus) that was immunized with Leishmania amazonensis or Leishmania braziliensis. The peptide antigens, individually (PA1, PA2, PA3) or in a mix (PAMix), promoted induration reactions at 48 and 72 h after the test was performed. The indurations varied from 0.5 to 0.7 cm. In the animals immunized with L. amazonensis, the PA3 antigen showed better results than the standard MST antigen. In animals immunized with L. braziliensis, two peptide antigens (PA2 and PAMix) promoted induration reactions for a longer period of time than the standard MST antigen. These results validate our hypothesis that peptides could be used as antigens in skin tests and may replace the current antigen for CL diagnosis.

High-Throughput rRNA Gene Sequencing Reveals Highand Complex Bacterial Diversity Associated withBrazilian Coffee Bean Fermentation.

Coffee bean fermentation is a spontaneous, on-farm process involving the action of different microbial groups, including bacteria and fungi. In this study, high-throughput sequencing approach was employed to study the diversity and dynamics of bacteria associated with Brazilian coffee bean fermentation. The total DNA from fermenting coffee samples was extracted at different time points, and the 16S rRNA gene with segments around the V4 variable region was sequenced by Illumina high-throughput platform. Using this approach, the presence of over eighty bacterial genera was determined, many of which have been detected for the first time during coffee bean fermentation, including Fructobacillus, Pseudonocardia, Pedobacter, Sphingomonas and Hymenobacter. The presence of Fructobacillus suggests an influence of these bacteria on fructose metabolism during coffee fermentation. Temporal analysis showed a strong dominance of lactic acid bacteria with over 97% of read sequences at the end of fermentation, mainly represented by the Leuconostoc and Lactococcus. Metabolism of lactic acid bacteria was associated with the high formation of lactic acid during fermentation, as determined by HPLC analysis. The results reported in this study confirm the underestimation of bacterial diversity associated with coffee fermentation. New microbial groups reported in this study may be explored as functional starter cultures for on-farm coffee processing.

Effects of Cordyceps sinensis on macrophage function in high-fat diet fed rats and its anti-proliferative effects on IMR-32 human neuroblastoma cells.

Macrophages have been considered an elusive yet emerging therapeutic target in tumor development since they are an important component in tumor microenvironment. The purpose of the present study was to evaluate the effect of C. sinensis on macrophage function (a component of tumor microenvironment which can alter the virulence of cancer) in high-fat diet fed rats. IMR-32 human neuroblastoma cell cytotoxicity was also investigated. The following parameters were observed to evaluate macrophage function: superoxide anion, hydrogen peroxide, nitric oxide, lysosomal volume and phagocytic capacity. High fat diet (HFD) plus C. sinensis supplementation promoted a decreased superoxide anion and hydrogen peroxide levels as well as lysosomal volume and phagocytic capacity. Nitric oxide was increased in the same group. In summary, C. sinensis offered an important anti-tumoral perspective from the standpoint of the tumor microenvironment and in vitro IMR-32 cytotoxicity.