Chromatin profiling reveals heterogeneity in clinical isolates of the human pathogen Aspergillus fumigatus.

Invasive Pulmonary Aspergillosis, which is caused by the filamentous fungus Aspergillus fumigatus, is a life-threatening infection for immunosuppressed patients. Chromatin structure regulation is important for genome stability maintenance and has the potential to drive genome rearrangements and affect virulence and pathogenesis of pathogens. Here, we performed the first A. fumigatus global chromatin profiling of two histone modifications, H3K4me3 and H3K9me3, focusing on the two most investigated A. fumigatus clinical isolates, Af293 and CEA17. In eukaryotes, H3K4me3 is associated with active transcription, while H3K9me3 often marks silent genes, DNA repeats, and transposons. We found that H3K4me3 deposition is similar between the two isolates, while H3K9me3 is more variable and does not always represent transcriptional silencing. Our work uncovered striking differences in the number, locations, and expression of transposable elements between Af293 and CEA17, and the differences are correlated with H3K9me3 modifications and higher genomic variations among strains of Af293 background. Moreover, we further showed that the Af293 strains from different laboratories actually differ in their genome contents and found a frequently lost region in chromosome VIII. For one such Af293 variant, we identified the chromosomal changes and demonstrated their impacts on its secondary metabolites production, growth and virulence. Overall, our findings not only emphasize the influence of genome heterogeneity on A. fumigatus fitness, but also caution about unnoticed chromosomal variations among common laboratory strains.

Impacts of intake of trichothecenes (Fusarium sporotrichioides) for dairy calves: Effects on animal growth, oxidative and inflammatory response.

The objective of the present study was to evaluate the impacts of trichothecenes (Fusarium sporotrichioides) for dairy calves on animal growth, oxidative and inflammatory responses in the presence or absence of essential oils. Twelve calves weaned at 70 days of age were divided into 2 groups: T-C (control) and T-EO (essential oils - oregano, thyme, basil and rosemary) in the period of 40 days consuming ration contaminated by trichothecenes (500 ppb). The animals in the T-EO group received a mixture of EOs via feed at a dosage of 0.75 mL per/kg of feed. Blood collections were performed on days 1, 20 and 40 for hematological and biochemical analyses; the fecal score was performed every 2 days on a scale of 1-5 and clinical examinations were performed 3 times during the experiment period. The animals were weighed at the beginning and at the end of the experiment; euthanasia of two calves per group for macroscopic and microscopic evaluation of several tissues (spleen, liver, duodenum, jejunum, ilium, cecum and colon) was performed at the end of the experiment. The calves in the T-EO group had a tendency (P = 0.07) of higher body weight when compared to the T-C. Treatment effect and treatment vs day interaction was detected for leukocytes and granulocytes variables, demonstrating a higher count of these cells in the T-EO group on both days (20 and 40), and the same behavior occurred for the distribution amplitude of erythrocytes (RDW). The enzymes alanine transferase (ALT), aspartate transferase (AST) and gamma glutamyl-transferase (GGT) showed higher serum activity in the T-C group (days 20 and 40). The levels of thiobarbituric acid reactive substances (TBARS) were lower in the serum of animals in the T-EO group. For calves in the T-EO group, glutathione S-transferase activity was higher in serum. Haptoglobulin and C-reactive protein levels were lower on days 20 and 40 in T-EO animals when compared to the T-C group. In the macroscopic and microscopic evaluations, which were collected at the end of the experiment after slaughtering the animals, liver and intestine did not show changes for the animals in the T-EO group, unlike the animals in the T-C group, which had moderately firm diffuse consistency of the liver and edema in the mesentery, as well as oxidative stress in tissues (liver, duodenum, jejunum, ileum, cecum and colon). The results concluded that the consumption of a mixture of EOs (essential oils - oregano, thyme, basil and rosemary) minimized the negative effects caused by trichothecenes in dairy calves, thus being an alternative to improving the immunological and antioxidant condition, as well as a possible adsorbent alternative.

A biofloc system avoids the adverse effects of diets with suboptimal protein levels on zootechnical performance, intestinal histomorphometry, and protein metabolism of Nile tilapia juvenile fed Spirulina biomass (Arthrospira platensis) as an alternative protein source.

This study aimed to evaluate the effect of the biofloc technology (BFT) system and the replacement of fish meal with Spirulina biomass on productive performance, intestinal histomorphometry, plasma biochemistry, and oxidative stress of Nile tilapia juveniles (Oreochromis niloticus) fed suboptimal levels of protein. Two factors were evaluated: production systems (clear water × BFT) and replacement of fish meal with Spirulina (0, 33, 66 e 100%). The design was in a 2 × 4 randomized factorial scheme with four replications, and the fish were evaluated for 48 days. Four isoproteic (28% crude protein) diets were formulated with gross energy values close to 4300 kcal kg-1. Nile tilapia juveniles (0.23 ± 0.01 g) were distributed in 16 circular tanks (70 L) at seven fish/tank. The diets were formulated with protein levels approximately 20% below that required for the species and life stage. No interaction was observed between the factors evaluated (production systems × Spirulina inclusion). Rearing the fish in the BFT system avoided the adverse effects of diets with suboptimal protein levels on performance, intestinal histomorphometry, and protein metabolism. Lower values lower lipid peroxidation and higher antioxidant capacity were observed in fish reared in the BFT system, showing evidence of improvements in antioxidant responses and lower levels of physiological oxidative stress. Spirulina completely replaced fish meal in the diets of Nile tilapia juveniles without adverse effects on intestinal morphometry, protein metabolism, and antioxidant response. Replacing 66% of fish meal with Spirulina improved the productive performance, regardless of the rearing system.

Grape seed oil in the diet of primiparous Jersey cows before and after parturition: effects on performance, health, rumen environment, and milk quality.

The objective of the study was to determine whether adding grape seed oil (GSO) to the diet of primiparous Jersey breeds during the transition period would improve animal health by measuring effects on the rumen environment, serum biochemistry, oxidative response, and the composition and quality of milk. We used 14 Jersey heifers, weighing an average of 430 kg and 240 days of gestation. The animals were divided into two groups and offered a basal diet, including GSO in the concentrate for the GSO group (dose of 25 mL per animal day) and the same dose of soybean oil (SO) for the control group. The animals were allocated and maintained in a compost barn system, receiving an anionic diet (pre-partum) and a diet for postpartum lactating animals. Dry matter intake (DMI), milk production, serum biochemistry, serum and milk oxidative stability, ruminal fluid and milk fatty acid profile, milk qualitative aspects, and ruminal parameters such as pH, bacterial activity, and protozoan count were evaluated. The addition of GSO had a positive effect on the health of the cows, especially on the oxidative stability of the cows, by increasing total thiols (P = 0.03), higher plasma ferric reducing capacity (FRAP) (P = 0.01), and total antioxidant capacity (TAC) (P = 0.01). In the oxidative stability of the milk produced by the treated animals, there was also an increase in TAC (P = 0.05) and FRAP (P = 0.03). Discreet changes were observed in the ruminal environment with a decreasing trend in pH (P = 0.04) but an increase in bacterial activity (P = 0.05) and protozoa counts (P = 0.07) in cows that consumed the additive. GSO consumption affected the fatty acid profile in milk, increasing saturated fatty acids (SFA) (P = 0.05) and reducing unsaturated fatty acids (UFA) (P = 0.03). The oil did not affect milk production or efficiency in the postpartum period. Based on this information, it is concluded that the addition of GSO positively affects the cow's antioxidant system.

A toolset of constitutive promoters for metabolic engineering of Rhodosporidium toruloides.

BACKGROUND: Rhodosporidium toruloides is a promising host for the production of bioproducts from lignocellulosic biomass. A key prerequisite for efficient pathway engineering is the availability of robust genetic tools and resources. However, there is a lack of characterized promoters to drive expression of heterologous genes for strain engineering in R. toruloides. RESULTS: This data describes a set of native R. toruloides promoters, characterized over time in four different media commonly used for cultivation of this yeast. The promoter sequences were selected using transcriptional analysis and several of them were found to drive expression bidirectionally. Promoter expression strength was determined by measurement of EGFP and mRuby2 reporters by flow cytometry. A total of 20 constitutive promoters (12 monodirectional and 8 bidirectional) were found, and are expected to be of potential value for genetic engineering of R. toruloides. CONCLUSIONS: A set of robust and constitutive promoters to facilitate genetic engineering of R. toruloides is presented here, ranging from a promoter previously used for this purpose (P7, glyceraldehyde 3-phosphate dehydrogenase, GAPDH) to stronger monodirectional (e.g., P15, mitochondrial adenine nucleotide translocator, ANT) and bidirectional (e.g., P9 and P9R, histones H3 and H4, respectively) promoters. We also identified promoters that may be useful for specific applications such as late-stage expression (e.g., P3, voltage-dependent anion channel protein 2, VDAC2). This set of characterized promoters significantly expands the range of engineering tools available for this yeast and can be applied in future metabolic engineering studies.

Synthetic and minimalist vectors for Agrobacterium tumefaciens-mediated transformation of fungi.

We present a collection of minimalist binary vectors for transformation through ATMT applicable to several fungi species. pLUO plasmid binary vectors consist of a reporter module containing fluorescent proteins, mCherry or eGFP, flanked by a multiple cloning site and a transcription terminator site. They also present a synthetic gene allowing resistance to Hygromicin B flanked by alternate promoters, one for yeast and another for filamentous fungi. Left and right borders were added for Agrobacterium tumefaciens recognition, and a minimal broad-host range RK2 replication origin. Transformation was validated in the pathogenic fungus Paracoccidioides lutzii. Hence, we developed an efficient and reliable molecular tool for fungal transformation: minimalist, synthetic, modular, and available in four different versions, and these can still be readily modified using a few primers and few cloning steps.

Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the ß-actin gene (actb), ß-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR.

Positive selection in cytochrome P450 genes is associated with gonad phenotype and mating strategy in social bees.

The honey bee, Apis mellifera differs from all other social bees in its gonad phenotype and mating strategy. Honey bee queens and drones have tremendously enlarged gonads, and virgin queens mate with several males. In contrast, in all the other bees, the male and female gonads are small, and the females mate with only one or very few males, thus, suggesting an evolutionary and developmental link between gonad phenotype and mating strategy. RNA-seq comparisons of A. mellifera larval gonads revealed 870 genes as differentially expressed in queens versus workers and drones. Based on Gene Ontology enrichment we selected 45 genes for comparing the expression levels of their orthologs in the larval gonads of the bumble bee Bombus terrestris and the stingless bee, Melipona quadrifasciata, which revealed 24 genes as differentially represented. An evolutionary analysis of their orthologs in 13 solitary and social bee genomes revealed four genes with evidence of positive selection. Two of these encode cytochrome P450 proteins, and their gene trees indicated a lineage-specific evolution in the genus Apis, indicating that cytochrome P450 genes may be involved in the evolutionary association of polyandry and the exaggerated gonad phenotype in social bees.

Mining novel cis-regulatory elements from the emergent host Rhodosporidium toruloides using transcriptomic data.

The demand for robust microbial cell factories that produce valuable biomaterials while resisting stresses imposed by current bioprocesses is rapidly growing. Rhodosporidium toruloides is an emerging host that presents desirable features for bioproduction, since it can grow in a wide range of substrates and tolerate a variety of toxic compounds. To explore R. toruloides suitability for application as a cell factory in biorefineries, we sought to understand the transcriptional responses of this yeast when growing under experimental settings that simulated those used in biofuels-related industries. Thus, we performed RNA sequencing of the oleaginous, carotenogenic yeast in different contexts. The first ones were stress-related: two conditions of high temperature (37 and 42°C) and two ethanol concentrations (2 and 4%), while the other used the inexpensive and abundant sugarcane juice as substrate. Differential expression and functional analysis were implemented using transcriptomic data to select differentially expressed genes and enriched pathways from each set-up. A reproducible bioinformatics workflow was developed for mining new regulatory elements. We then predicted, for the first time in this yeast, binding motifs for several transcription factors, including HAC1, ARG80, RPN4, ADR1, and DAL81. Most putative transcription factors uncovered here were involved in stress responses and found in the yeast genome. Our method for motif discovery provides a new realm of possibilities in studying gene regulatory networks, not only for the emerging host R. toruloides, but for other organisms of biotechnological importance.

Recent advances in plasmid-based tools for establishing novel microbial chassis.

A key challenge for domesticating alternative cultivable microorganisms with biotechnological potential lies in the development of innovative technologies. Within this framework, a myriad of genetic tools has flourished, allowing the design and manipulation of complex synthetic circuits and genomes to become the general rule in many laboratories rather than the exception. More recently, with the development of novel technologies such as DNA automated synthesis/sequencing and powerful computational tools, molecular biology has entered the synthetic biology era. In the beginning, most of these technologies were established in traditional microbial models (known as chassis in the synthetic biology framework) such as Escherichia coli and Saccharomyces cerevisiae, enabling fast advances in the field and the validation of fundamental proofs of concept. However, it soon became clear that these organisms, although extremely useful for prototyping many genetic tools, were not ideal for a wide range of biotechnological tasks due to intrinsic limitations in their molecular/physiological properties. Over the last decade, researchers have been facing the great challenge of shifting from these model systems to non-conventional chassis with endogenous capacities for dealing with specific tasks. The key to address these issues includes the generation of narrow and broad host plasmid-based molecular tools and the development of novel methods for engineering genomes through homologous recombination systems, CRISPR/Cas9 and other alternative methods. Here, we address the most recent advances in plasmid-based tools for the construction of novel cell factories, including a guide for helping with "build-your-own" microbial host.

The art of vector engineering: towards the construction of next-generation genetic tools.

When recombinant DNA technology was developed more than 40 years ago, no one could have imagined the impact it would have on both society and the scientific community. In the field of genetic engineering, the most important tool developed was the plasmid vector. This technology has been continuously expanding and undergoing adaptations. Here, we provide a detailed view following the evolution of vectors built throughout the years destined to study microorganisms and their peculiarities, including those whose genomes can only be revealed through metagenomics. We remark how synthetic biology became a turning point in designing these genetic tools to create meaningful innovations. We have placed special focus on the tools for engineering bacteria and fungi (both yeast and filamentous fungi) and those available to construct metagenomic libraries. Based on this overview, future goals would include the development of modular vectors bearing standardized parts and orthogonally designed circuits, a task not fully addressed thus far. Finally, we present some challenges that should be overcome to enable the next generation of vector design and ways to address it.

A Novel Cys2His2 Zinc Finger Homolog of AZF1 Modulates Holocellulase Expression in Trichoderma reesei.

Filamentous fungi are remarkable producers of enzymes dedicated to the degradation of sugar polymers found in the plant cell wall. Here, we integrated transcriptomic data to identify novel transcription factors (TFs) related to the control of gene expression of lignocellulosic hydrolases in Trichoderma reesei and Aspergillus nidulans Using various sets of differentially expressed genes, we identified some putative cis-regulatory elements that were related to known binding sites for Saccharomyces cerevisiae TFs. Comparative genomics allowed the identification of six transcriptional factors in filamentous fungi that have corresponding S. cerevisiae homologs. Additionally, a knockout strain of T. reesei lacking one of these TFs (S. cerevisiae AZF1 homolog) displayed strong reductions in the levels of expression of several cellulase-encoding genes in response to both Avicel and sugarcane bagasse, revealing a new player in the complex regulatory network operating in filamentous fungi during plant biomass degradation. Finally, RNA sequencing (RNA-seq) analysis showed the scope of the AZF1 homologue in regulating a number of processes in T. reesei, and chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) provided evidence for the direct interaction of this TF in the promoter regions of cel7a, cel45a, and swo Therefore, we identified here a novel TF which plays a positive effect in the expression of cellulase-encoding genes in T. reesei IMPORTANCE In this work, we used a systems biology approach to map new regulatory interactions in Trichoderma reesei controlling the expression of genes encoding cellulase and hemicellulase. By integrating transcriptomics related to complex biomass degradation, we were able to identify a novel transcriptional regulator which is able to activate the expression of these genes in response to two different cellulose sources. In vivo experimental validation confirmed the role of this new regulator in several other processes related to carbon source utilization and nutrient transport. Therefore, this work revealed novel forms of regulatory interaction in this model system for plant biomass deconstruction and also represented a new approach that could be easy applied to other organisms.

Systems and Synthetic Biology Approaches to Engineer Fungi for Fine Chemical Production.

Since the advent of systems and synthetic biology, many studies have sought to harness microbes as cell factories through genetic and metabolic engineering approaches. Yeast and filamentous fungi have been successfully harnessed to produce fine and high value-added chemical products. In this review, we present some of the most promising advances from recent years in the use of fungi for this purpose, focusing on the manipulation of fungal strains using systems and synthetic biology tools to improve metabolic flow and the flow of secondary metabolites by pathway redesign. We also review the roles of bioinformatics analysis and predictions in synthetic circuits, highlighting in silico systemic approaches to improve the efficiency of synthetic modules.

Caracterización de pacientes con pie diabético del Hospital General Docente Dr Agostinho Neto, Guantánamo / Characterization of patients with diabetic foot of the Hospital General Docente Dr Agostinho Neto, Guantánamo

Introducción: el pie diabético es un problema de salud, sin embargo, en el Servicio de Angiología y Cirugía Vascular del Hospital General Docente Dr Agostinho Neto se desconocen las características de los pacientes con esta afección. Objetivo: para profundizar en las posibles causas se realizó esta investigación con el objetivo de caracterizar a los pacientes con diagnóstico de pie diabético atendidos en este servicio durante el 2016. Método: se realizó un estudio de tipo observacional, descriptivo, y transversal en el total de pacientes atendidos con esta enfermedad (n=205). Resultados: el pie diabético fue más frecuente en mujeres, entre los 61 y 70 años de edad, con diabetes tipo 2 y más de 16 años de evolución de la enfermedad. Fue más común la forma clínica neuroinfecciosa y fue elevado el porcentaje de pacientes amputados, sobre todo las amputaciones menores. Fallecieron dos pacientes. Conclusiones: se concluyó que dicho padecimiento fue más frecuente en mujeres con edad entre 61 y 70 años, la forma clínica neuroinfecciosa la más común y que se realizaran amputaciones menores. La letalidad en los pacientes con pie diabético fue baja(AU)