Trichoderma longibrachiatum and thermothelomyces thermophilus co-culture: improvement the saccharification profile of different sugarcane bagasse varieties.

OBJECTIVES: The aim of the present work was to perform the co-culture between Trichoderma longibrachiatum LMBC 172, a mesophilic fungus, with Thermothelomyces thermophilus LMBC 162, a thermophilic fungus, by submerged fermentation in a bioreactor. RESULTS: There was an increase in protein production, reaching the value of 35.60 ± 3.76 µg/ml at 72 h. An increase in the amount of proteins of 27.5% in relation to the isolated cultivation of T. longibrachiatum and 19.7% in comparison when T. thermophilus was isolated and cultivated. After that, the saccharification profile of three varieties of sugarcane (sugarcane in natura, culms of sugarcane SP80-3280, and culms of Energy cane) submitted in two pretreatments (autohydrolysis and chemical) was performed. The (e) chemical pretreatment was the better in generating of fermentable sugars from sugarcane bagasse and culms of Energy cane, while with the autohydrolysis pretreatment was obtained the better values to culms of SP80-3280 sugarcane. The sugars found were glucose, xylose, arabinose, and cellobiose. CONCLUSION: These results suggest that the co-culture between these microorganisms has the potential to produce an enzymatic cocktail with high performance in the hydrolysis of materials from the sugar-alcohol industry.

Use of carbohydrate-directed enzymes for the potential exploitation of sugarcane bagasse to obtain value-added biotechnological products.

Microorganisms, such as fungi and bacteria, are crucial players in the production of enzymatic cocktails for biomass hydrolysis or the bioconversion of plant biomass into products with industrial relevance. The biotechnology industry can exploit lignocellulosic biomass for the production of high-value chemicals. The generation of biotechnological products from lignocellulosic feedstock presents several bottlenecks, including low efficiency of enzymatic hydrolysis, high cost of enzymes, and limitations on microbe metabolic performance. Genetic engineering offers a route for developing improved microbial strains for biotechnological applications in high-value product biosynthesis. Sugarcane bagasse, for example, is an agro-industrial waste that is abundantly produced in sugar and first-generation processing plants. Here, we review the potential conversion of its feedstock into relevant industrial products via microbial production and discuss the advances that have been made in improving strains for biotechnological applications.

Enzymatic Pretreatment with Laccases from Lentinus sajor-caju Induces Structural Modification in Lignin and Enhances the Digestibility of Tropical Forage Grass (Panicum maximum) Grown under Future Climate Conditions.

Since laccase acts specifically in lignin, the major contributor to biomass recalcitrance, this biocatalyst represents an important alternative to the pretreatment of lignocellulosic biomass. Therefore, this study investigates the laccase pretreatment and climate change effects on the hydrolytic performance of Panicum maximum. Through a Trop-T-FACE system, P. maximum grew under current (Control (C)) and future climate conditions: elevated temperature (2 °C more than the ambient canopy temperature) combined with elevated atmospheric CO2 concentration(600 µmol mol-1), name as eT+eC. Pretreatment using a laccase-rich crude extract from Lentinus sajor caju was optimized through statistical strategies, resulting in an increase in the sugar yield of P. maximum biomass (up to 57%) comparing to non-treated biomass and enabling hydrolysis at higher solid loading, achieving up to 26 g L-1. These increments are related to lignin removal (up to 46%) and lignin hydrophilization catalyzed by laccase. Results from SEM, CLSM, FTIR, and GC-MS supported the laccase-catalyzed lignin removal. Moreover, laccase mitigates climate effects, and no significant differences in hydrolytic potential were found between C and eT+eC groups. This study shows that crude laccase pretreatment is a potential and sustainable method for biorefinery solutions and helped establish P. maximum as a promising energy crop.

Structural model and functional properties of an exo-polygalacturonase from Neosartorya glabra.

A purified exo-polygalacturonase of Neosartorya glabra (EplNg) was successfully characterized. EplNg native presented 68.2 kDa, with 32% carbohydrate content. The deglycosylated form showed 46.3 kDa and isoelectric point of 5.4. The identity of EplNg was confirmed as an exo-polygalacturonase class I (EC 3.2.1.67) using mass spectrometry and Western-Blotting. Capillary electrophoresis indicated that only galacturonic acid was released by the action of EplNg on sodium polypectate, confirming an exoenzyme character. The structural model confers that EplNg has a core formed by twisted parallel ß-sheets structure. Among twelve putative cysteines, ten were predicted to form disulfide bridges. The catalytic triad predicted is composed of Asp223, Asp245, and Asp246 aligned along with a distance in 4-5 Å, suggesting that EplNg probably does not perform the standard inverting catalytic mechanism described for the GH28 family. EplNg was active from 30 to 90 °C, with maximum activity at 65 °C, pH 5.0. The Km and Vmax determined using sodium polypectate were 6.9 mg·mL-1 and Vmax 690 µmol·min-1.mg-1, respectively. EplNg was active and stable over a wide range of pH values and temperatures, confirming the interesting properties EplNg and provide a basis for the development of the enzyme in different biotechnological processes.

Prospection of Fungal Lignocellulolytic Enzymes Produced from Jatoba (Hymenaea courbaril) and Tamarind (Tamarindus indica) Seeds: Scaling for Bioreactor and Saccharification Profile of Sugarcane Bagasse.

The lignocellulosic biomass comprises three main components: cellulose, hemicellulose, and lignin. Degradation and conversion of these three components are attractive to biotechnology. This study aimed to prospect fungal lignocellulolytic enzymes with potential industrial applications, produced through a temporal analysis using Hymenaea courbaril and Tamarindus indica seeds as carbon sources. α-L-arabinofuranosidase, acetyl xylan esterase, endo-1,5-α-L-arabinanase, ß-D-galactosidase, ß-D-glucosidase, ß-glucanase, ß-D-xylosidase, cellobiohydrolase, endoglucanase, lichenase, mannanase, polygalacturonase, endo-1,4-ß-xylanase, and xyloglucanase activities were determined. The enzymes were produced for eight filamentous fungi: Aspergillus fumigatus, Trametes hirsuta, Lasiodiplodia sp., two strains of Trichoderma longibrachiatum, Neocosmospora perseae, Fusarium sp. and Thermothelomyces thermophilus. The best producers concerning enzymatic activity were T. thermophilus and T. longibrachiatum. The optimal conditions for enzyme production were the media supplemented with tamarind seeds, under agitation, for 72 h. This analysis was essential to demonstrate that cultivation conditions, static and under agitation, exert strong influences on the production of several enzymes produced by different fungi. The kind of sugarcane, pretreatment used, microorganisms, and carbon sources proved limiting sugar profile factors.

Molecular Characterization of Xyloglucanase cel74a from Trichoderma reesei.

BACKGROUND: The filamentous fungus Trichoderma reesei is used on an industrial scale to produce enzymes of biotechnological interest. This fungus has a complex cellulolytic system involved in the degradation of lignocellulosic biomass. However, several aspects related to the regulation of the expression of holocellulolytic genes and the production of cellulases by this fungus are still understood. METHODS: Here, we constructed a null mutant strain for the xyloglucanase cel74a gene and performed the characterization of the Δcel74a strain to evaluate the genetic regulation of the holocellulases during sugarcane bagasse (SCB) cultivation. RESULTS: Our results demonstrate that the deletion of xyloglucanase cel74a may impact the regulation of holocellulase expression during SCB cultivation. The expression of cellulases cel7a, cel7b, and cel6a was reduced in Δcel74a strain, while the hemicellulases xyn1 and xyn2 were increased in the presence of SCB. The cel74a mutation also affected the xyloglucan hydrolysis patterns. In addition, CEL74A activity was modulated in the presence of calcium, suggesting that this ion may be required for efficient degradation of xyloglucan. CONCLUSIONS: CEL74A affects the regulation of holocellulolytic genes and the efficient degradation of SCB in T. reesei. This data makes a significant contribution to our understanding of the carbon utilization of fungal strains as a whole.

Aspergillus Fumigatus ZnfA, a Novel Zinc Finger Transcription Factor Involved in Calcium Metabolism and Caspofungin Tolerance.

Invasive pulmonary aspergillosis is a life-threatening fungal infection especially in the immunocompromised patients. The low diversity of available antifungal drugs coupled with the emergence of antifungal resistance has become a worldwide clinical concern. The echinocandin Caspofungin (CSP) is recommended as a second-line therapy but resistance and tolerance mechanisms have been reported. However, how the fungal cell articulates the response to CSP is not completely understood. This work provides a detailed characterization of ZnfA, a transcription factor (TF) identified in previous screening studies that is involved in the A. fumigatus responses to calcium and CSP. This TF plays an important role in the regulation of iron homeostasis and cell wall organization in response to high CSP concentrations as revealed by Chromatin Immunoprecipitation coupled to DNA sequencing (ChIP-seq) analysis. Furthermore, ZnfA acts collaboratively with the key TF CrzA in modulating the response to calcium as well as cell wall and osmotic stresses. This study therefore describes the existence of an additional, previously unknown TF that bridges calcium signaling and the CSP cellular response and further exposes the complex connections that exist among different pathways which govern stress sensing and signaling in A. fumigatus.

Physiology Responses and Players' Stay on the Court During a Futsal Match: A Case Study With Professional Players.

Physiological responses in futsal have not been studied together with temporal information about the players' stay on the court. The aim of this study was to compare heart rate (HR) and blood lactate concentration ([La-]) responses between 1-H and 2-H considering the time of permanency of the players on the court at each substitution in a futsal match. HR was recorded during entire match and [La-] was analyzed after each substitution of seven players. %HRmean (89.61 ± 2.31 vs. 88.03 ± 4.98 %HRmax) and [La-] mean (8.46 ± 3.01 vs. 8.17 ± 2.91 mmol·L-1) did not differ between 1-H and 2-H (ES, trivial-small). Time in intensity zones of 50-100 %HRmax differed only in 60-70 %HRmax (ES, moderate). HR coefficient of variation throughout the match was low (7%) and among the four outfield players on the court (quartets, 5%). Substitutions (2 player's participation in each half), time of permanence on the court (7.15 ± 2.39 vs. 9.49 ± 3.80 min), ratio between time in- and out-ratio on the court (In:Outcourt = 1:1.30 ± 1:0.48 vs. 1:1.05 ± 1:0.55 min) also were similar between 1-H and 2-H (ES, moderate and small, respectively). Balancing the number of substitutions, and the In:Outcourt ratio of players in both halves of the match, playing lower time at 1-H, ~8 min for each participation in the match, made it possible to maintain intensity of the match in 2-H similar to the 1H. These results are a good guidance to coaches and for application in future studies.

Aspergillus fumigatus G-Protein Coupled Receptors GprM and GprJ Are Important for the Regulation of the Cell Wall Integrity Pathway, Secondary Metabolite Production, and Virulence.

G-protein coupled receptors (GPCRs) are extracellular signaling receptors that sense environmental cues. Fungi sense their environment primarily through GPCR-mediated signaling pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. Aspergillus fumigatus is an important human pathogen that causes aspergillosis, a heterogeneous group of diseases that present a wide range of clinical manifestations. Here, we investigate in detail the role of the GPCRs GprM and GprJ in growth and gene expression. GprM and GprJ are important for melanin production and the regulation of the cell wall integrity (CWI) pathway. Overexpression of gprM and gprJ causes a 20 and 50% reduction in growth rate compared to the wild-type (WT) strain and increases sensitivity to cell wall-damaging agents. Phosphorylation of the CWI protein kinase MpkA is increased in the ΔgprM and ΔgprJ strains and decreased in the overexpression mutants compared to the WT strain. Furthermore, differences in cell wall polysaccharide concentrations and organization were observed in these strains. Transcriptome sequencing suggests that GprM and GprJ negatively regulate genes encoding secondary metabolites (SMs). Mass spectrometry analysis confirmed that the production of fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, and fumitremorgin is reduced in the ΔgprM and ΔgprJ strains, at least partially through the activation of MpkA. Overexpression of grpM also resulted in the regulation of many transcription factors, with AsgA predicted to function downstream of GprM and MpkA signaling. Finally, we show that the ΔgprM and ΔgprJ mutants are reduced in virulence in the Galleria mellonella insect model of invasive aspergillosis.IMPORTANCEA. fumigatus is the main etiological agent of invasive pulmonary aspergillosis, a life-threatening fungal disease that occurs in severely immunocompromised humans. Withstanding the host environment is essential for A. fumigatus virulence, and sensing of extracellular cues occurs primarily through G-protein coupled receptors (GPCRs) that activate signal transduction pathways, which, in turn, regulate fungal development, metabolism, virulence, and mycotoxin biosynthesis. The A. fumigatus genome encodes 15 putative classical GPCRs, with only three having been functionally characterized to date. In this work, we show that the two GPCRs GprM and GprJ regulate the phosphorylation of the mitogen-activated protein kinase MpkA and thus control the regulation of the cell wall integrity pathway. GprM and GprJ are also involved in the regulation of the production of the secondary metabolites fumagillin, pyripyropene, fumigaclavine C, fumiquinazoline, melanin, and fumitremorgin, and this regulation partially occurs through the activation of MpkA. Furthermore, GprM and GprJ are important for virulence in the insect model Galleria mellonella This work therefore functionally characterizes two GPCRs and shows how they regulate several intracellular pathways that have been shown to be crucial for A. fumigatus virulence.

The Biomolecular Spectrum Drives Microbial Biology and Functions in Agri-Food-Environments.

Microbial biomolecules have huge commercial and industrial potential. In nature, biological interactions are mostly associated with biochemical and biological diversity, especially with the discovery of associated biomolecules from microbes. Within cellular or subcellular systems, biomolecules signify the actual statuses of the microorganisms. Understanding the biological prospecting of the diverse microbial community and their complexities and communications with the environment forms a vital basis for active, innovative biotechnological breakthroughs. Biochemical diversity rather than the specific chemicals that has the utmost biological importance. The identification and quantification of the comprehensive biochemical diversity of the microbial molecules, which generally consequences in a diversity of biological functions, has significant biotechnological potential. Beneficial microbes and their biomolecules of interest can assist as potential constituents for the wide-range of natural product-based preparations and formulations currently being developed on an industrial scale. The understanding of the production methods and functions of these biomolecules will contribute to valorisation of agriculture, food bioprocessing and biopharma, and prevent human diseases related to the environment.

Enhance of Cellulase Production and Biomass Degradation by Transformation of the Trichoderma reesei RUT-C30∆ zface1 Strain

Abstract The second-generation bioethanol employs lignocellulosic materials degraded by microbial cellulases in their production. The fungus Trichoderma reesei is one of the main microorganisms producing cellulases, and its genetic modification can lead to the optimization in obtaining hydrolytic enzymes. This work carried out the deletion of the sequence that encodes the zinc finger motif of the transcription factor ACE1 (cellulase expression repressor I) of the fungus T. reesei RUT-C30. The transformation of the RUT-C30 lineage was confirmed by amplification of the 989 bp fragment relative to the selection marker, and by the absence of the zinc finger region amplification in mutants, named T. reesei RUT-C30Δzface1. The production of cellulases by mutants was compared to RUT-C30 and measured with substrates carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel®) and Whatman filter paper (PF). The results demonstrated that RUT-C30Δzface1 has cellulolytic activity increased 3.2-fold in Avicel and 2.1-fold in CMC and PF. The mutants presented 1.4-fold higher sugar released in the hydrolysis of the biomass assays. These results suggest that the partial deletion of ace1 gene is an important strategy in achieving bioethanol production on an industrial scale at a competitive price in the fuel market.

Aspergillus fumigatus calcium-responsive transcription factors regulate cell wall architecture promoting stress tolerance, virulence and caspofungin resistance.

Aspergillus fumigatus causes invasive aspergillosis, the most common life-threatening fungal disease of immuno-compromised humans. The treatment of disseminated infections with antifungal drugs, including echinocandin cell wall biosynthesis inhibitors, is increasingly challenging due to the rise of drug-resistant pathogens. The fungal calcium responsive calcineurin-CrzA pathway influences cell morphology, cell wall composition, virulence, and echinocandin resistance. A screen of 395 A. fumigatus transcription factor mutants identified nine transcription factors important to calcium stress tolerance, including CrzA and ZipD. Here, comparative transcriptomics revealed CrzA and ZipD regulated the expression of shared and unique gene networks, suggesting they participate in both converged and distinct stress response mechanisms. CrzA and ZipD additively promoted calcium stress tolerance. However, ZipD also regulated cell wall organization, osmotic stress tolerance and echinocandin resistance. The absence of ZipD in A. fumigatus caused a significant virulence reduction in immunodeficient and immunocompetent mice. The ΔzipD mutant displayed altered cell wall organization and composition, while being more susceptible to macrophage killing and eliciting an increased pro-inflammatory cytokine response. A higher number of neutrophils, macrophages and activated macrophages were found in ΔzipD infected mice lungs. Collectively, this shows that ZipD-mediated regulation of the fungal cell wall contributes to the evasion of pro-inflammatory responses and tolerance of echinocandin antifungals, and in turn promoting virulence and complicating treatment options.

Endo-ß-1,3-glucanase (GH16 Family) from Trichoderma harzianum Participates in Cell Wall Biogenesis but Is Not Essential for Antagonism Against Plant Pathogens.

Trichoderma species are known for their ability to produce lytic enzymes, such as exoglucanases, endoglucanases, chitinases, and proteases, which play important roles in cell wall degradation of phytopathogens. ß-glucanases play crucial roles in the morphogenetic-morphological process during the development and differentiation processes in Trichoderma species, which have ß-glucans as the primary components of their cell walls. Despite the importance of glucanases in the mycoparasitism of Trichoderma spp., only a few functional analysis studies have been conducted on glucanases. In the present study, we used a functional genomics approach to investigate the functional role of the gluc31 gene, which encodes an endo-ß-1,3-glucanase belonging to the GH16 family in Trichoderma harzianum ALL42. We demonstrated that the absence of the gluc31 gene did not affect the in vivo mycoparasitism ability of mutant T. harzianum ALL42; however, gluc31 evidently influenced cell wall organization. Polymer measurements and fluorescence microscopy analyses indicated that the lack of the gluc31 gene induced a compensatory response by increasing the production of chitin and glucan polymers on the cell walls of the mutant hyphae. The mutant strain became more resistant to the fungicide benomyl compared to the parental strain. Furthermore, qRT-PCR analysis showed that the absence of gluc31 in T. harzianum resulted in the differential expression of other glycosyl hydrolases belonging to the GH16 family, because of functional redundancy among the glucanases.

The pH Signaling Transcription Factor PAC-3 Regulates Metabolic and Developmental Processes in Pathogenic Fungi.

The zinc finger transcription factor PAC-3/RIM101/PacC has a defined role in the secretion of enzymes and proteins in response to ambient pH, and also contributes to the virulence of species. Herein we evaluated the role of PAC-3 in the regulation of Neurospora crassa genes, in a model that examined the plant-fungi interactions. N. crassa is a model fungal species capable of exhibiting dynamic responses to its environment by employing endophytic or phytopathogenic behavior according to a given circumstance. Since plant growth and productivity are highly affected by pH and phosphorus (P) acquisition, we sought to verify the impact that induction of a Δpac-3 mutation would have under limited and sufficient Pi availability, while ensuring that the targeted physiological adjustments mimicked ambient pH and nutritional conditions required for efficient fungal growth and development. Our results suggest direct regulatory functions for PAC-3 in cell wall biosynthesis, homeostasis, oxidation-reduction processes, hydrolase activity, transmembrane transport, and modulation of genes associated with fungal virulence. Pi-dependent modulation was observed mainly in genes encoding for transporter proteins or related to cell wall development, thereby advancing the current understanding regarding colonization and adaptation processes in response to challenging environments. We have also provided comprehensive evidence that suggests a role for PAC-3 as a global regulator in plant pathogenic fungi, thus presenting results that have the potential to be applied to various types of microbes, with diverse survival mechanisms.

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.

Extracellular vesicles carry cellulases in the industrial fungus Trichoderma reesei.

BACKGROUND: Trichoderma reesei is the most important industrial producer of lignocellulolytic enzymes. These enzymes play an important role in biomass degradation leading to novel applications of this fungus in the biotechnology industry, specifically biofuel production. The secretory pathway of fungi is responsible for transporting proteins addressed to different cellular locations involving some cellular endomembrane systems. Although protein secretion is an extremely efficient process in T. reesei, the mechanisms underlying protein secretion have remained largely uncharacterized in this organism. RESULTS: Here, we report for the first time the isolation and characterization of T. reesei extracellular vesicles (EVs). Using proteomic analysis under cellulose culture condition, we have confidently identified 188 vesicular proteins belonging to different functional categories. Also, we characterized EVs production using transmission electron microscopy in combination with light scattering analysis. Biochemical assays revealed that T. reesei extracellular vesicles have an enrichment of filter paper (FPase) and ß-glucosidase activities in purified vesicles from 24, 72 and 96, and 72 and 96 h, respectively. Furthermore, our results showed that there is a slight enrichment of small RNAs inside the vesicles after 96 h and 120 h, and presence of hsp proteins inside the vesicles purified from T. reesei grown in the presence of cellulose. CONCLUSIONS: This work points to important insights into a better understanding of the cellular mechanisms underlying the regulation of cellulolytic enzyme secretion in this fungus.

New Genomic Approaches to Enhance Biomass Degradation by the Industrial Fungus Trichoderma reesei.

The filamentous fungi Trichoderma reesei is one of the most well-studied cellulolytic microorganisms. It is the most important fungus for the industrial production of enzymes to biomass deconstruction being widely used in the biotechnology industry, mainly in the production of biofuels. Here, we performed an analytic review of the holocellulolytic system presented by T. reesei as well as the transcriptional and signaling mechanisms involved with holocellulase expression in this fungus. We also discuss new perspectives about control of secretion and cellulase expression based on RNA-seq and functional characterization data of T. reesei growth in different carbon sources, which comprise glucose, cellulose, sophorose, and sugarcane bagasse.

The Duality of the MAPK Signaling Pathway in the Control of Metabolic Processes and Cellulase Production in Trichoderma reesei.

In this study, through global transcriptional analysis by RNA-Sequencing, we identified the main changes in gene expression that occurred in two functional mutants of the MAPK genes tmk1 and tmk2 in Trichoderma reesei during sugarcane bagasse degradation. We found that the proteins encoded by these genes regulated independent processes, sometimes in a cross-talk manner, to modulate gene expression in T. reesei. In the Δtmk2 strain, growth in sugarcane bagasse modulated the expression of genes involved in carbohydrate metabolism, cell growth and development, and G-protein-coupled receptor-mediated cell signaling. On the other hand, deletion of tmk1 led to decreased expression of the major genes for cellulases and xylanases. Furthermore, TMK1 found to be involved in the regulation of the expression of major facilitator superfamily transporters. Our results revealed that the MAPK signaling pathway in T. reesei regulates many important processes that allow the fungus to recognize, transport, and metabolize different carbon sources during plant cell wall degradation.

Influência do estatuto posicional sobre o comportamento tático de jogadores de futebol / Influence of the positional role on the tactical actions of soccer players / Influencia del rango posicional sobre el comportamiento táctico de los jugadores de fútbol

Este estudo comparou o comportamento tático de jogadores de futebol da categoria Sub-15, de acordo com o estatuto posicional. Vinte e cinco jogadores de dois clubes portugueses foram avaliados através do Sistema de Avaliação Tática no Futebol. Utilizou-se estatística descritiva e o teste Qui-quadrado (χ²), com o nível de significância de p<0,05. Os resultados apresentaram diferença significativa nas variáveis: Unidade ofensiva, Contenção e Concentra- ção (Defensores); Mobilidade (Meio-campistas); Ações táticas ofensivas no meio de campo defensivo (Defensores); Cometer faltas, ceder lateral ou escanteio (Meio-campistas e Atacantes); Continuar sem a posse de bola (Defensores). Conclui-se que o comportamento tático dos jogadores apresentou níveis de especialização nas suas posições.

This study compared the tactical behavior of U-15 soccer players according to the positional role. Twenty-five players from two Portuguese clubs were evaluated through the System of Tactical Assessment in Soccer (FUT-SAT). Descriptive statistics and chi-square test (χ²) were used, with significance level of p<0.05. Results displayed significant statistical differences for the following variables: Offensive Unit, Delay and Concentration (Defenders); Depth Mobility (Midfielders); Offensive tactical actions in defensive midfield (Defenders); Commit a foul, give away a corner or throw-in (Midfielders and Forwards); Ball possession of the opponent (Defenders). We conclude that the tactical behavior of the players presented levels of specialization in their positions.

Este estudio comparó el comportamiento táctico de los jugadores de fútbol de la categoría Sub-15 en relación al rango posicional. Evaluamos a 25 jugadores de dos clubes Portugueses por medio del Sistema de Evaluación de la Táctica en Fútbol (FUT-SAT). Usamos la estadística descriptiva y el test de Chi-cuadrado (χ²), con elnivel de significancia de p<0,05. Los resultados presentaron diferencia significativa en las variables: Unidad ofensiva, Contención y Concentración (Defensas); Movilidad (Mediocampistas); Acciones tácticas ofensivas en el medio de campo defensivo (Defensas); Cometer faltas, ceder un saque de banda o córner (Mediocampistas y Atacantes); continuar sin la posesión del balón (Defensas). Se concluyó que el comportamiento táctico de los jugadores presentó niveles de especialización en sus posiciones.

The Aspergillus fumigatus CrzA Transcription Factor Activates Chitin Synthase Gene Expression during the Caspofungin Paradoxical Effect.

Aspergillus fumigatus is an opportunistic fungal pathogen that causes invasive aspergillosis (IA), a life-threatening disease in immunocompromised humans. The echinocandin caspofungin, adopted as a second-line therapy in combating IA, is a ß-1,3-glucan synthase inhibitor, which, when used in high concentrations, reverts the anticipated A. fumigatus growth inhibition, a phenomenon called the "caspofungin paradoxical effect" (CPE). The CPE has been widely associated with increased chitin content in the cell wall due to a compensatory upregulation of chitin synthase-encoding genes. Here, we demonstrate that the CPE is dependent on the cell wall integrity (CWI) mitogen-activated protein kinase MpkAMPK1 and its associated transcription factor (TF) RlmARLM1, which regulate chitin synthase gene expression in response to different concentrations of caspofungin. Furthermore, the calcium- and calcineurin-dependent TF CrzA binds to and regulates the expression of specific chitin synthase genes during the CPE. These results suggest that the regulation of cell wall biosynthetic genes occurs by several cellular signaling pathways. In addition, CrzA is also involved in cell wall organization in the absence of caspofungin. Differences in the CPE were also observed between two A. fumigatus clinical isolates, which led to the identification of a novel basic leucine zipper TF, termed ZipD. This TF functions in the calcium-calcineurin pathway and is involved in the regulation of cell wall biosynthesis genes. This study therefore unraveled additional mechanisms and novel factors governing the CPE response, which ultimately could aid in developing more effective antifungal therapies.IMPORTANCE Systemic Aspergillus fumigatus infections are often accompanied by high mortality rates. The fungal cell wall is important for infection as it has immunomodulatory and immunoevasive properties. Paradoxical growth of A. fumigatus in the presence of high concentrations of the cell wall-disturbing agent caspofungin has been observed for more than a decade, although the mechanistic nature of this phenomenon remains largely uncharacterized. Here, we show that the CWI pathway components MpkA and RlmA as well as the calcium/calcineurin-responsive transcription factor CrzA regulate the expression of cell wall biosynthetic genes during the caspofungin paradoxical effect (CPE). Furthermore, an additional, novel calcium/calcineurin-responsive transcription factor was identified to play a role in cell wall biosynthesis gene expression during the CPE. This work paints a crucial role for calcium metabolism in the CPE and provides further insight into the complex regulation of cell wall biosynthesis, which could ultimately lead to the development of more efficient antifungal therapies.