Correction to: Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells.

The article "Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells" was originally published Online First without open access. After publication in volume 101, issue 22, page 8237-8248, the author decided to opt for Open Choice and to make the article an open access publication.

Industrial antifoam agents impair ethanol fermentation and induce stress responses in yeast cells.

The Brazilian sugarcane industry constitutes one of the biggest and most efficient ethanol production processes in the world. Brazilian ethanol production utilizes a unique process, which includes cell recycling, acid wash, and non-aseptic conditions. Process characteristics, such as extensive CO2 generation, poor quality of raw materials, and frequent contaminations, all lead to excessive foam formation during fermentations, which is treated with antifoam agents (AFA). In this study, we have investigated the impact of industrial AFA treatments on the physiology and transcriptome of the industrial ethanol strain Saccharomyces cerevisiae CAT-1. The investigated AFA included industrially used AFA acquired from Brazilian ethanol plants and commercially available AFA commonly used in the fermentation literature. In batch fermentations, it was shown that industrial AFA compromised growth rates and glucose uptake rates, while commercial AFA had no effect in concentrations relevant for defoaming purposes. Industrial AFA were further tested in laboratory scale simulations of the Brazilian ethanol production process and proved to decrease cell viability compared to the control, and the effects were intensified with increasing AFA concentrations and exposure time. Transcriptome analysis showed that AFA treatments induced additional stress responses in yeast cells compared to the control, shown by an up-regulation of stress-specific genes and a down-regulation of lipid biosynthesis, especially ergosterol. By documenting the detrimental effects associated with chemical AFA, we highlight the importance of developing innocuous systems for foam control in industrial fermentation processes.

Structural studies of the protein endostatin in fusion with BAX BH3 death domain, a hybrid that presents enhanced antitumoral activity.

Endostatin (ES) is an antiangiogenic protein that exhibits antitumor activity in animal models. However, the activity observed in animals was not observed in human clinical trials. ES-BAX is a fusion protein composed of two functional domains: ES, which presents specificity and is internalized by activated endothelial cells and the proapoptotic BH3 domain of the protein BAX, a peptide inductor of cellular death when internalized. We have previously shown (Chura-Chambi et al., Cell Death Dis, 5, e1371, 2014) that ES-BAX presents improved antitumor activity in relation to wild-type ES. Secondary and tertiary structures of ES-BAX are similar to ES, as indicated by homology-modeling studies and molecular dynamics simulations. Tryptophan intrinsic fluorescence and circular dichroism spectroscopy corroborate these data. 15 N HSQC NMR indicates that ES-BAX is structured, but some ES residues have suffered chemical shift perturbations, suggesting that the BH3 peptide interacts with some parts of the ES protein. ES and ES-BAX present similar stability to thermal denaturation. The production of stable hybrid proteins can be a new approach to the development of therapeutic agents presenting specificity for tumoral endothelium and improved antitumor effect.

Strain dynamics of contaminating bacteria modulate the yield of ethanol biorefineries.

Bioethanol is a sustainable energy alternative and can contribute to global greenhouse-gas emission reductions by over 60%. Its industrial production faces various bottlenecks, including sub-optimal efficiency resulting from bacteria. Broad-spectrum removal of these contaminants results in negligible gains, suggesting that the process is shaped by ecological interactions within the microbial community. Here, we survey the microbiome across all process steps at two biorefineries, over three timepoints in a production season. Leveraging shotgun metagenomics and cultivation-based approaches, we identify beneficial bacteria and find improved outcome when yeast-to-bacteria ratios increase during fermentation. We provide a microbial gene catalogue which reveals bacteria-specific pathways associated with performance. We also show that Limosilactobacillus fermentum overgrowth lowers production, with one strain reducing yield by ~5% in laboratory fermentations, potentially due to its metabolite profile. Temperature is found to be a major driver for strain-level dynamics. Improved microbial management strategies could unlock environmental and economic gains in this US $ 60 billion industry enabling its wider adoption.

Physiology of Saccharomyces cerevisiae during growth on industrial sugar cane molasses can be reproduced in a tailor-made defined synthetic medium.

Fully defined laboratory media have the advantage of allowing for reproducibility and comparability of results among different laboratories, as well as being suitable for the investigation of how different individual components affect microbial or process performance. We developed a fully defined medium that mimics sugarcane molasses, a frequently used medium in different industrial processes where yeast is cultivated. The medium, named 2SMol, builds upon a previously published semi-defined formulation and is conveniently prepared from some stock solutions: C-source, organic N, inorganic N, organic acids, trace elements, vitamins, Mg + K, and Ca. We validated the 2SMol recipe in a scaled-down sugarcane biorefinery model, comparing the physiology of Saccharomyces cerevisiae in different actual molasses-based media. We demonstrate the flexibility of the medium by investigating the effect of nitrogen availability on the ethanol yield during fermentation. Here we present in detail the development of a fully defined synthetic molasses medium and the physiology of yeast strains in this medium compared to industrial molasses. This tailor-made medium was able to satisfactorily reproduce the physiology of S. cerevisiae in industrial molasses. Thus, we hope the 2SMol formulation will be valuable to researchers both in academia and industry to obtain new insights and developments in industrial yeast biotechnology.

Complex yeast-bacteria interactions affect the yield of industrial ethanol fermentation.

Sugarcane ethanol fermentation represents a simple microbial community dominated by S. cerevisiae and co-occurring bacteria with a clearly defined functionality. In this study, we dissect the microbial interactions in sugarcane ethanol fermentation by combinatorically reconstituting every possible combination of species, comprising approximately 80% of the biodiversity in terms of relative abundance. Functional landscape analysis shows that higher-order interactions counterbalance the negative effect of pairwise interactions on ethanol yield. In addition, we find that Lactobacillus amylovorus improves the yeast growth rate and ethanol yield by cross-feeding acetaldehyde, as shown by flux balance analysis and laboratory experiments. Our results suggest that Lactobacillus amylovorus could be considered a beneficial bacterium with the potential to improve sugarcane ethanol fermentation yields by almost 3%. These data highlight the biotechnological importance of comprehensively studying microbial communities and could be extended to other microbial systems with relevance to human health and the environment.

Metabolic modeling predicts specific gut bacteria as key determinants for Candida albicans colonization levels.

Candida albicans is a leading cause of life-threatening hospital-acquired infections and can lead to Candidemia with sepsis-like symptoms and high mortality rates. We reconstructed a genome-scale C. albicans metabolic model to investigate bacterial-fungal metabolic interactions in the gut as determinants of fungal abundance. We optimized the predictive capacity of our model using wild type and mutant C. albicans growth data and used it for in silico metabolic interaction predictions. Our analysis of more than 900 paired fungal-bacterial metabolic models predicted key gut bacterial species modulating C. albicans colonization levels. Among the studied microbes, Alistipes putredinis was predicted to negatively affect C. albicans levels. We confirmed these findings by metagenomic sequencing of stool samples from 24 human subjects and by fungal growth experiments in bacterial spent media. Furthermore, our pairwise simulations guided us to specific metabolites with promoting or inhibitory effect to the fungus when exposed in defined media under carbon and nitrogen limitation. Our study demonstrates that in silico metabolic prediction can lead to the identification of gut microbiome features that can significantly affect potentially harmful levels of C. albicans.

Antibiotics create a shift from mutualism to competition in human gut communities with a longer-lasting impact on fungi than bacteria.

BACKGROUND: Antibiotic treatment has a well-established detrimental effect on the gut bacterial composition, but effects on the fungal community are less clear. Bacteria in the lumen of the gastrointestinal tract may limit fungal colonization and invasion. Antibiotic drugs targeting bacteria are therefore seen as an important risk factor for fungal infections and induced allergies. However, antibiotic effects on gut bacterial-fungal interactions, including disruption and resilience of fungal community compositions, were not investigated in humans. We analysed stool samples collected from 14 healthy human participants over 3 months following a 6-day antibiotic administration. We integrated data from shotgun metagenomics, metatranscriptomics, metabolomics, and fungal ITS2 sequencing. RESULTS: While the bacterial community recovered mostly over 3 months post treatment, the fungal community was shifted from mutualism at baseline to competition. Half of the bacterial-fungal interactions present before drug intervention had disappeared 3 months later. During treatment, fungal abundances were associated with the expression of bacterial genes with functions for cell growth and repair. By extending the metagenomic species approach, we revealed bacterial strains inhibiting the opportunistic fungal pathogen Candida albicans. We demonstrated in vitro how C. albicans pathogenicity and host cell damage might be controlled naturally in the human gut by bacterial metabolites such as propionate or 5-dodecenoate. CONCLUSIONS: We demonstrated that antibacterial drugs have long-term influence on the human gut mycobiome. While bacterial communities recovered mostly 30-days post antibacterial treatment, the fungal community was shifted from mutualism towards competition. Video abstract.

Effects of gamma-decanolactone on seizures induced by PTZ-kindling in mice.

Gamma-decanolactone is a monoterpene compound, and its psychopharmacological evaluation in mice revealed that it has a dose-dependent effect on the central nervous system, with hypnotic, anticonvulsant, and hypothermic activity. The aim of the present study was to investigate the effect of gamma-decanolactone on pentylenetetrazole (PTZ)-kindling in mice. Phenobarbital, an antiepileptic drug, was also tested for the purpose of comparison. After the behavioral procedures had been undertaken, the animals were killed and brain tissue was sampled to evaluate DNA damage in the brain using comet assay. The data reported here suggest that the administration of phenobarbital (10 mg/kg) and gamma-decanolactone at 0.3 g/kg, but not at 0.1 g/kg, impairs both the severity and the progression of seizures in the PTZ-kindling model. DNA damage to brain tissue decreased in gamma-decanolactone-treated kindling animals (similar to phenobarbital) as compared to nontreated animals. The results suggest that gamma-decanolactone has dose-dependent anticonvulsant properties, and may also have antiepileptogenic and neuroprotective effects in the PTZ-kindling model.

A synthetic medium to simulate sugarcane molasses.

BACKGROUND: Developing novel microbial cell factories requires careful testing of candidates under industrially relevant conditions. However, this frequently occurs late during the strain development process. The availability of laboratory media that simulate industrial-like conditions might improve cell factory development, as they allow for strain construction and testing in the laboratory under more relevant conditions. While sugarcane molasses is one of the most important substrates for the production of biofuels and other bioprocess-based commodities, there are no defined media that faithfully simulate it. In this study, we tested the performance of a new synthetic medium simulating sugarcane molasses. RESULTS: Laboratory scale simulations of the Brazilian ethanol production process, using both sugarcane molasses and our synthetic molasses (SM), demonstrated good reproducibility of the fermentation performance, using yeast strains, PE-2 and Ethanol Red™. After 4 cycles of fermentation, the final ethanol yield (gp gs-1) values for the SM ranged from 0.43 ± 0.01 to 0.44 ± 0.01 and from 0.40 ± 0.01 to 0.46 ± 0.01 for the molasses-based fermentations. The other fermentation parameters (i.e., biomass production, yeast viability, and glycerol and acetic acid yield) were also within similar value ranges for all the fermentations. Sequential pairwise competition experiments, comparing industrial and laboratory yeast strains, demonstrated the impact of the media on strain fitness. After two sequential cocultivations, the relative abundance of the laboratory yeast strain was 5-fold lower in the SM compared to the yeast extract-peptone-dextrose medium, highlighting the importance of the media composition on strain fitness. CONCLUSIONS: Simulating industrial conditions at laboratory scale is a key part of the efficient development of novel microbial cell factories. In this study, we have developed a synthetic medium that simulated industrial sugarcane molasses media. We found good agreement between the synthetic medium and the industrial media in terms of the physiological parameters of the industrial-like fermentations.

[Prevalence of anti-Leishmania spp antibodies in dogs from Garanhuns, in the middle scrub zone (Agreste) of Pernambuco]. / Prevalência de anticorpos antileishmania spp em cães de Garanhuns, Agreste de Pernambuco.

INTRODUCTION: Considering the unknown situation regarding canine visceral leishmaniasis in Garanhuns, this study had the aim of investigating occurrences of anti-Leishmania spp antibodies in domesticated and partially domesticated dogs, and the possible risk factors involved. METHODS: In the first phase of the study, 256 blood samples were collected from dogs and subjected to the indirect fluorescent antibody test (IFAT) reaction at a dilution of 1:40. Additionally, 23 IFAT-positive samples were tested using an immunochromatographic dipstick test. In the second phase, new blood samples were collected from 18 dogs that were IFAT-positive in the first phase. These animals were retested using IFAT (1:40 and 1:80) and, additionally, by means of the polymerase chain reaction to investigate the Leishmania infantum DNA. Furthermore, 16 of these samples were retested using the immunochromatographic dipstick test. RESULTS: In the first phase of the study, 16% of the samples were IFAT-positive (1:40) and only three (13%) were positive in the immunochromatographic dipstick test. In the second phase, 12 samples were IFAT-positive at the dilution of 1:40, and seven were also positive at 1:80. None of the samples were positive in the polymerase chain reaction testing or in the immunochromatographic dipstick test. Clinical signs suggestive of visceral leishmaniasis were observed in 4.9% of the IFAT-positive dogs. There were no statistical differences in relation to age, sex or clinical status of the dogs, but there was a difference in relation to place of origin. CONCLUSIONS: The domesticated and partially domesticated dogs living in Garanhuns present anti-Leishmania spp antibodies, but are mostly asymptomatic.

Prevalência de anticorpos antileishmania spp em cães de Garanhuns, Agreste de Pernambuco / Prevalence of anti-Leishmania spp antibodies in dogs from Garanhuns, in the middle scrub zone (Agreste) of Pernambuco

INTRODUÇÃO: Desconhecida a realidade da leishmaniose visceral canina em Garanhuns, objetivou-se investigar a ocorrência de anticorpos antileishmania spp em cães domiciliados e semidomiciliados e os possíveis fatores de risco envolvidos. MÉTODOS: Em uma primeira etapa foram coletadas 256 amostras de sangue de cães que foram submetidas à reação de imunofluorescência indireta (RIFI) na diluição 1:40. Adicionalmente, 23 amostras positivas na RIFI foram testadas com um teste rápido imunocromatográfico. Em uma segunda etapa, novas amostras de sangue de 18 cães positivos na RIFI na primeira fase do estudo foram coletadas, retestadas pela RIFI (1:40 e 1:80) e, adicionalmente, pela reação em cadeia da polimerase para pesquisa de DNA de Leishmania infantum. Ademais, 16 dessas amostras foram retestadas pelo teste rápido imunocromatográfico. RESULTADOS: Na primeira etapa, 16 por cento das amostras foram positivas na RIFI (1:40) e apenas três (13 por cento) foram positivas no teste rápido imunocromatográfico. Na segunda etapa, 12 amostras foram positivas na RIFI na diluição 1:40 e sete também na diluição 1:80. Nenhuma amostra foi positiva na reação em cadeia da polimerase e no teste rápido imunocromatográfico. Sinais clínicos de leishmaniose visceral ocorreram em 4,9 por cento dos cães positivos. Não houve diferença estatística entre idade, sexo e status clínico dos cães, porém entre seus locais de origem. CONCLUSÕES: Os cães domiciliados e semidomiciliados de Garanhuns apresentam anticorpos antileishmania spp, sendo, em sua grande maioria, assintomáticos.

INTRODUCTION: Considering the unknown situation regarding canine visceral leishmaniasis in Garanhuns, this study had the aim of investigating occurrences of anti-Leishmania spp antibodies in domesticated and partially domesticated dogs, and the possible risk factors involved. METHODS: In the first phase of the study, 256 blood samples were collected from dogs and subjected to the indirect fluorescent antibody test (IFAT) reaction at a dilution of 1:40. Additionally, 23 IFAT-positive samples were tested using an immunochromatographic dipstick test. In the second phase, new blood samples were collected from 18 dogs that were IFAT-positive in the first phase. These animals were retested using IFAT (1:40 and 1:80) and, additionally, by means of the polymerase chain reaction to investigate the Leishmania infantum DNA. Furthermore, 16 of these samples were retested using the immunochromatographic dipstick test. RESULTS: In the first phase of the study, 16 percent of the samples were IFAT-positive (1:40) and only three (13 percent) were positive in the immunochromatographic dipstick test. In the second phase, 12 samples were IFAT-positive at the dilution of 1:40, and seven were also positive at 1:80. None of the samples were positive in the polymerase chain reaction testing or in the immunochromatographic dipstick test. Clinical signs suggestive of visceral leishmaniasis were observed in 4.9 percent of the IFAT-positive dogs. There were no statistical differences in relation to age, sex or clinical status of the dogs, but there was a difference in relation to place of origin. CONCLUSIONS: The domesticated and partially domesticated dogs living in Garanhuns present anti-Leishmania spp antibodies, but are mostly asymptomatic.