Co-ordinated expression of the VEGF system components in granulosa cells to develop a proangiogenic autocrine milieu during ovarian follicle development.

In the present study, we investigated the temporal relationship between angiogenic and antiangiogenic vascular endothelial growth factor isoforms (VEGFxxxa and VEGFxxxb, respectively), the receptors VEGFR1 and VEGFR2, their soluble forms, and the kinases and the splicing factors regulating the synthesis of VEGF isoforms in healthy and atretic antral follicles. The results show a higher (p < 0.05) messenger RNA (mRNA) expression of VEGF120a, VEGF164a, and VEGF120b in healthy than in atretic follicles, but the mRNA expression of VEGF164b was not detected. The mRNA of serine-arginine protein kinase 1 ( SRPK1) was higher ( p < 0.05) in large healthy follicles than in large atretic follicles. In contrast, atretic follicles had higher mRNA expression of a soluble form of the receptor 2 of VEGF ( sVEGFR2) than healthy follicles ( p < 0.05). Additionally, we observed a positive relationship ( p < 0.05) between SRPK1 and serine-arginine-rich splicing factor 1 ( SRSF1) with the angiogenic isoforms VEGF120a and VEGF164a and between CDC-like kinases-1 ( CLK1) and SRSF6 with the antiangiogenic VEGF120b isoform. Principal components analysis (PCA) resulted in two PC explaining 71% of the variation, which was formed by the VEGF isoforms, the kinases and the splicing factor (PC1) and by the VEGF receptors (PC2). When PC analysis was carried out within follicular health status, there were no differences for PC1 between follicular status, whereas PC2 differed between healthy and atretic follicles. In conclusion, the higher mRNA expression for VEGF120a and VEGF164a, the low expression of sVEGFR2, and absent expression of mRNA for VEGF164b provide evidence of a proangiogenic autocrine milieu to support granulosa cells during follicle development.

Proteomic analysis of the signaling pathway mediated by the heterotrimeric Gα protein Pga1 of Penicillium chrysogenum.

BACKGROUND: The heterotrimeric Gα protein Pga1-mediated signaling pathway regulates the entire developmental program in Penicillium chrysogenum, from spore germination to the formation of conidia. In addition it participates in the regulation of penicillin biosynthesis. We aimed to advance the understanding of this key signaling pathway using a proteomics approach, a powerful tool to identify effectors participating in signal transduction pathways. RESULTS: Penicillium chrysogenum mutants with different levels of activity of the Pga1-mediated signaling pathway were used to perform comparative proteomic analyses by 2D-DIGE and LC-MS/MS. Thirty proteins were identified which showed differences in abundance dependent on Pga1 activity level. By modifying the intracellular levels of cAMP we could establish cAMP-dependent and cAMP-independent pathways in Pga1-mediated signaling. Pga1 was shown to regulate abundance of enzymes in primary metabolic pathways involved in ATP, NADPH and cysteine biosynthesis, compounds that are needed for high levels of penicillin production. An in vivo phosphorylated protein containing a pleckstrin homology domain was identified; this protein is a candidate for signal transduction activity. Proteins with possible roles in purine metabolism, protein folding, stress response and morphogenesis were also identified whose abundance was regulated by Pga1 signaling. CONCLUSIONS: Thirty proteins whose abundance was regulated by the Pga1-mediated signaling pathway were identified. These proteins are involved in primary metabolism, stress response, development and signal transduction. A model describing the pathways through which Pga1 signaling regulates different cellular processes is proposed.

Direct involvement of the CreA transcription factor in penicillin biosynthesis and expression of the pcbAB gene in Penicillium chrysogenum.

The transcription factor CreA is the main regulator responsible for carbon repression in filamentous fungi. CreA is a wide domain regulator that binds to regulatory elements in the promoters of target genes to repress their transcription. Penicillin biosynthesis and the expression of penicillin biosynthetic genes are subject to carbon repression. However, evidence of the participation of CreA in this regulation is still lacking, and previous studies on the promoter of the pcbC gene of Aspergillus nidulans indicated the lack of involvement of CreA in its regulation. Here we present clear evidence of the participation of CreA in carbon repression of penicillin biosynthesis and expression of the pcbAB gene, encoding the first enzyme of the pathway, in Penicillium chrysogenum. Mutations in cis of some of the putative CreA binding sites present in the pcbAB gene promoter fused to a reporter gene caused an important increase in the measured enzyme activity in glucose-containing medium, whereas activity in the medium with lactose was not affected. An RNAi strategy was used to attenuate the expression of the creA gene. Transformants expressing a small interfering RNA for creA showed higher penicillin production, and this increase was more evident when glucose was used as carbon source. These results confirm that CreA plays an important role in the regulation of penicillin biosynthesis in P. chrysogenum and opens the possibility of its utilization to improve the industrial production of this antibiotic.

Silencing of Amylomyces rouxii aspartic II protease by siRNA to increase tyrosinase activity.

Amylomyces rouxii is a zygomycete that produces extracellular protease and tyrosinase. The tyrosinase activity is negatively regulated by the proteases and, which attempts to purify the tyrosinase (tyr) enzyme that has been hampered by the presence of a protease that co-purified with it. In this work we identified genes encoding aspartic protease II (aspII) and VI of A. rouxii. Using an RNAi strategy based on the generation of a siRNA by transcription from two opposite-orientated promoters, the expression of these two proteases was silenced, showing that this molecular tool is suitable for gene silencing in Amylomyces. The transformant strains showed a significant attenuation of the transcripts (determined by RT-qPCR), with respective inhibition of the protease activity. In the case of aspII, inhibition was in the range of 43-90 % in different transformants, which correlated well with up to a five-fold increase in tyr activity with respect to the wild type and control strains. In contrast, silencing of aspVI caused a 43-65 % decrease in protease activity but had no significant effect on the tyr activity. The results show that aspII has a negative effect on tyr activity, and that the silencing of this protease is important to obtain strains with high levels of tyr activity.

Penicillium chrysogenum, a Vintage Model with a Cutting-Edge Profile in Biotechnology.

The discovery of penicillin entailed a decisive breakthrough in medicine. No other medical advance has ever had the same impact in the clinical practise. The fungus Penicillium chrysogenum (reclassified as P. rubens) has been used for industrial production of penicillin ever since the forties of the past century; industrial biotechnology developed hand in hand with it, and currently P. chrysogenum is a thoroughly studied model for secondary metabolite production and regulation. In addition to its role as penicillin producer, recent synthetic biology advances have put P. chrysogenum on the path to become a cell factory for the production of metabolites with biotechnological interest. In this review, we tell the history of P. chrysogenum, from the discovery of penicillin and the first isolation of strains with high production capacity to the most recent research advances with the fungus. We will describe how classical strain improvement programs achieved the goal of increasing production and how the development of different molecular tools allowed further improvements. The discovery of the penicillin gene cluster, the origin of the penicillin genes, the regulation of penicillin production, and a compilation of other P. chrysogenum secondary metabolites will also be covered and updated in this work.

Prospective predictive performance comparison between clinical gestalt and validated COVID-19 mortality scores.

Most COVID-19 mortality scores were developed at the beginning of the pandemic and clinicians now have more experience and evidence-based interventions. Therefore, we hypothesized that the predictive performance of COVID-19 mortality scores is now lower than originally reported. We aimed to prospectively evaluate the current predictive accuracy of six COVID-19 scores and compared it with the accuracy of clinical gestalt predictions. 200 patients with COVID-19 were enrolled in a tertiary hospital in Mexico City between September and December 2020. The area under the curve (AUC) of the LOW-HARM, qSOFA, MSL-COVID-19, NUTRI-CoV, and NEWS2 scores and the AUC of clinical gestalt predictions of death (as a percentage) were determined. In total, 166 patients (106 men and 60 women aged 56±9 years) with confirmed COVID-19 were included in the analysis. The AUC of all scores was significantly lower than originally reported: LOW-HARM 0.76 (95% CI 0.69 to 0.84) vs 0.96 (95% CI 0.94 to 0.98), qSOFA 0.61 (95% CI 0.53 to 0.69) vs 0.74 (95% CI 0.65 to 0.81), MSL-COVID-19 0.64 (95% CI 0.55 to 0.73) vs 0.72 (95% CI 0.69 to 0.75), NUTRI-CoV 0.60 (95% CI 0.51 to 0.69) vs 0.79 (95% CI 0.76 to 0.82), NEWS2 0.65 (95% CI 0.56 to 0.75) vs 0.84 (95% CI 0.79 to 0.90), and neutrophil to lymphocyte ratio 0.65 (95% CI 0.57 to 0.73) vs 0.74 (95% CI 0.62 to 0.85). Clinical gestalt predictions were non-inferior to mortality scores, with an AUC of 0.68 (95% CI 0.59 to 0.77). Adjusting scores with locally derived likelihood ratios did not improve their performance; however, some scores outperformed clinical gestalt predictions when clinicians' confidence of prediction was <80%. Despite its subjective nature, clinical gestalt has relevant advantages in predicting COVID-19 clinical outcomes. The need and performance of most COVID-19 mortality scores need to be evaluated regularly.

Outcomes of patients with severe and critical COVID-19 treated with dexamethasone: a prospective cohort study.

Dexamethasone implementation for COVID-19 management represented a milestone but data regarding its impact and safety have not been consistently reproduced. We aimed to evaluate in-hospital mortality before and after the implementation of corticosteroid treatment (CS-T) for severe and critical COVID-19. We conducted a cohort study that included patients admitted with severe and critical COVID-19. The primary outcome was death during hospitalization. Secondary outcomes included the length of stay (LOS), need for invasive mechanical ventilation (IMV), time to IMV initiation, IMV duration, and development of hospital-acquired infections (HAIs). Bivariate, multivariate, and propensity-score matching analysis were performed. Among 1540 patients, 688 (45%) received CS-T. Death was less frequent in the CS-T group (18 vs 31%, p < .01). Among patients on IMV, death was also less frequent in the CS-T group (25 vs 55%, p < .01). The median time to IMV was longer in the CS-T group (5 vs 3 days, p < .01). HAIs occurred more frequently in the CS-T group (20 vs 10%, p < .01). LOS, IMV, and IMV duration were similar between groups. Multivariate analysis revealed an independent association between CS-T and lower mortality (aOR 0.26, 95% CI 0.19-0.36, p < .001). Propensity-score matching analysis revealed that CS-T was independently associated with lower mortality (aOR 0.33, 95% CI 0.22-0.50, p < .01). Treatment with corticosteroids was associated with reduced in-hospital mortality among patients with severe and critical COVID-19, including those on IMV.

Outcomes in Temporary ICUs Versus Conventional ICUs: An Observational Cohort of Mechanically Ventilated Patients With COVID-19-Induced Acute Respiratory Distress Syndrome.

Throughout the COVID-19 pandemic, thousands of temporary ICUs have been established worldwide. The outcomes and management of mechanically ventilated patients in these areas remain unknown. OBJECTIVES: To investigate mortality and management of mechanically ventilated patients in temporary ICUs. DESIGN SETTING AND PARTICIPANTS: Observational cohort study in a single-institution academic center. We included all adult patients with severe COVID-19 hospitalized in temporary and conventional ICUs for invasive mechanical ventilation due to acute respiratory distress syndrome from March 23, 2020, to April 5, 2021. MAIN OUTCOMES AND MEASURES: To determine if management in temporary ICUs increased 30-day in-hospital mortality compared with conventional ICUs. Ventilator-free days, ICU-free days (both at 28 d), hospital length of stay, and ICU readmission were also assessed. RESULTS: We included 776 patients (326 conventional and 450 temporary ICUs). Thirty-day in-hospital unadjusted mortality (28.8% conventional vs 36.0% temporary, log-rank test p = 0.023) was higher in temporary ICUs. After controlling for potential confounders, hospitalization in temporary ICUs was an independent risk factor associated with mortality (hazard ratio, 1.4; CI, 1.06-1.83; p = 0.016).There were no differences in ICU-free days at 28 days (6; IQR, 0-16 vs 2; IQR, 0-15; p = 0.5) or ventilator-free days at 28 days (8; IQR, 0-16 vs 5; IQR, 0-15; p = 0.6). We observed higher reintubation (18% vs 12%; p = 0.029) and readmission (5% vs 1.6%; p = 0.004) rates in conventional ICUs despite higher use of postextubation noninvasive mechanical ventilation (13% vs 8%; p = 0.025). Use of lung-protective ventilation (87% vs 85%; p = 0.5), prone positioning (76% vs 79%; p = 0.4), neuromuscular blockade (96% vs 98%; p = 0.4), and COVID-19 pharmacologic treatment was similar. CONCLUSIONS AND RELEVANCE: We observed a higher 30-day in-hospital mortality in temporary ICUs. Although both areas had high adherence to evidence-based management, hospitalization in temporary ICUs was an independent risk factor associated with mortality.

Heterotrimeric Gα protein Pga1 from Penicillium chrysogenum triggers germination in response to carbon sources and affects negatively resistance to different stress conditions.

Heterotrimeric Gα protein Pga1 of Penicillium chrysogenum controls vegetative growth, conidiation and secondary metabolite production. In this work we studied the role of Pga1 in spore germination and resistance to different stress conditions. Strains G203R-T (expressing the dominant inactivating pga1(G203R) allele) and Δpga1 (deleted pga1) showed a delayed and asynchronic germination pattern, and a decrease in the percentage of germination, which occurred in only 70-80% of the total conidia. In contrast, in strains expressing the dominant activating pga1(G42R) allele, germination occurred at earlier times and in 100% of conidia. In addition, strains with the pga1(G42R) allele were able to bypass the carbon source (glucose or sucrose) requirement for germination in about 64% of conidia. Thus Pga1 plays an important, but not essential, role in germination, mediating carbon source sensing. Regulation of germination by Pga1 is probably mediated by cAMP, as intracellular levels of this secondary messenger undergo a peak before the onset of germination only in strains with an active Pga1. Pga1 activity is also a determinant factor in the resistance to different stress conditions. Absence or inactivation of Pga1 allow growth on SDS-containing minimal medium, increase resistance of conidia to thermal and oxidative stress, and increase resistance of vegetative mycelium to thermal and osmotic stress. In contrast, constitutive activation of Pga1 causes a decrease in the resistance of conidia to thermal stress and of vegetative mycelium to thermal and osmotic stress. Together with our previously reported results, we show in this work that Pga1 plays a central role in the regulation of the whole growth-developmental program of this biotechnologically important fungus.

High-Efficiency Electroporation for Genetic Improvement of Fungal Strains.

Electroporation is a method for the introduction of molecules (usually nucleic acids) into a cell, consisting of submitting the cells to high-voltage and short electric pulses in the presence of the exogenous DNA/molecule. It is a versatile method, adaptable to different types of cells, from bacteria to cultured cells to higher eukaryotes, and thus has applications in many diverse fields, such as environmental biology, biotechnology, genetic engineering, and medicine. Electroporation has some advantages over other genetic transformation strategies, including the simplicity of the method, a wide range of adjustable parameters (possibility of optimization), high reproducibility and avoidance of the use of chemicals toxic to cells. Here we describe an optimized electroporation procedure for the industrially important fungus Acremonium chrysogenum, using germinated conidia and fragmented young mycelium. In both cases, the transformation efficiency was higher compared to the conventional polyethylene glycol (PEG)-mediated transformation of protoplasts.

Hypoxia up-regulates VEGF ligand and downregulates VEGF soluble receptor mRNA expression in bovine granulosa cells in vitro.

Oxygen concentration (02) in antral ovarian follicles is below that found in most tissues, which is important for adequate granulosa cell function. The VEGF system is linked to angiogenesis and responds to changing 02 by stimulating neovascularization when levels are low. However, in the avascular granulosa cell layer of the follicle, VEGF action is directed to stimulating cell viability and steroidogenesis. The aim of this study was to examine the effect of 02 concentration on granulosa cell expression of the VEGF-system components. Bovine granulosa cells were isolated from medium-sized follicles (4-7 mm in diameter), placed in McCoy 5a medium supplemented with 10 ng/mL of insulin, 1 ng/mL of IGF-I, and 1 ng/mL of FSH, and cultured in four well plates (500 thousand cells per well), on three separate occasions. Culture plates were placed in gas-impermeable jars with a gas mixture containing either 2%, or 5% of O2, or under atmospheric air condition inside an incubator (20% of 02). Media was replaced at 48 h of culture and cells from the plate in each oxygen concentration were pooled for RNA extraction after 96 h. The number of mRNA copies for the VEGF-system components - including ligands (VEGF120, VEGF120b, VEGF165 and VEGF165b), enzymes (cyclin-dependent like kinases-1, CLK1 and serine-arginine protein kinase 1, SRPK1), splicing factors (serine-arginine-rich splicing factors, SRSF1 and SRSF6), and the membrane-bound (VEGFR1, VEGFR2) and soluble forms of the receptors (sVEGFR1 and sVEGFR2) were quantified by qPCR. Granulosa cells cultured with low 02 (2%) had a higher expression of VEGF ligands (P < 0.05) when compared to cells cultured at 20% 02. VEGF164b mRNA was absent in granulosa cells from all culture conditions. The 2 and 5% 02 levels, which coincide with physiological concentrations, in the ovarian follicle, induced higher SRSF6 expression than atmospheric 02 concentrations (20%, P < 0.05). In contrast, mRNA copies for SRPK1, CLK1, SRSF1, VEGFR1 or VEGFR2 did not differ between 02 culture conditions. (P > 0.05). Nonetheless, mRNA copies for the soluble receptors, sVEGFR1 and sVEGFR2, linearly increased (P < 0.05) with 02 concentration. These results suggest that when cultured under hypoxic conditions, granulosa cells may develop an autocrine milieu that favors VEGF's biological effects on their survival and function.

A method for the extraction of high quality fungal RNA suitable for RNA-seq.

Transcriptomic analysis is an OMICs technology that is becoming indispensable to understand and get a complete picture of cell functioning and adaptation to the environmental cues the cell is continuously receiving. Among the techniques available to perform transcriptomics, RNA-seq is becoming the method of choice. The quality of the RNA used for the generation of cDNA libraries and subsequent sequencing is crucial for the success of the process. Good RNA-seq performance is often limited by problems such as low RNA yield and/or integrity, RNA stability, and contamination with DNA, salts or chemicals. RNA isolation from fungi usually faces these problems and is particularly sensitive to degradation due to the high RNase activity content present in many species. Here we describe the development of a robust, highly reproducible and simple RNA purification method for filamentous fungi, which combines various strategies to get fully DNA-free RNA samples of high purity and integrity without the need to use a DNase I digestion step. The obtained RNA samples complied with all required standards to be used for RNA-seq and showed an excellent performance when subjected to Illumina-HiSeq 2500.

Effect of a heterotrimeric G protein alpha subunit on conidia germination, stress response, and roquefortine C production in Penicillium roqueforti.

Heterotrimeric G protein signaling regulates many processes in fungi, such as development, pathogenicity, and secondary metabolite biosynthesis. For example, the Galpha subunit Pga1 from Penicillium chrysogenum regulates conidiation and secondary metabolite production in this fungus. The dominant activating allele, pga1G42R, encoding a constitutively active Pga1 Galpha subunit, was introduced in Penicillium roqueforti by transformation, resulting in a phenotype characterized by low sporulation and slow growth. In this work, the effect of the constitutively active Pga1G42R Galpha subunit on conidial germination, stress tolerance, and roquefortine C production of P. roqueforti was studied. Pga1G42R triggered germination in the absence of a carbon source, in addition to negatively regulating thermal and osmotic stress tolerance. The presence of the Pga1G42R Galpha subunit also had an important effect on roquefortine C biosynthesis, increasing production and maintaining high levels of the mycotoxin throughout a culture period of 30 days. Together, the results suggest that G protein-mediated signaling participates in the regulation of these three processes in P. roqueforti.

Electroporation of germinated conidia and young mycelium as an efficient transformation system for Acremonium chrysogenum.

Three different transformation strategies were tested and compared in an attempt to facilitate and improve the genetic transformation of Acremonium chrysogenum, the exclusive producer of the pharmaceutically relevant ß-lactam antibiotic cephalosporin C. We investigated the use of high-voltage electric pulse to transform germinated conidia and young mycelium and compared these procedures with traditional PEG-mediated protoplast transformation, using phleomycin resistance as selection marker in all cases. The effect of the field strength and capacitance on transformation frequency and cell viability was evaluated. The electroporation of germinated conidia and young mycelium was found to be appropriate for transforming A. chrysogenum with higher transformation efficiencies than those obtained with the conventional protoplast-based transformation procedures. The developed electroporation strategy is fast, simple to perform, and highly reproducible and avoids the use of chemicals toxic to cells. Electroporation of young mycelium represents an alternative method for transformation of fungal strains with reduced or no sporulation, as often occurs in laboratory-developed strains in the search for high-yielding mutants for industrial bioprocesses.

Genetic Transformation of the Filamentous Fungus Pseudogymnoascus verrucosus of Antarctic Origin.

Cold-adapted fungi isolated from Antarctica, in particular those belonging to the genus Pseudogymnoascus, are producers of secondary metabolites with interesting bioactive properties as well as enzymes with potential biotechnological applications. However, at genetic level, the study of these fungi has been hindered by the lack of suitable genetic tools such as transformation systems. In fungi, the availability of transformation systems is a key to address the functional analysis of genes related with the production of a particular metabolite or enzyme. To the best of our knowledge, the transformation of Pseudogymnoascus strains of Antarctic origin has not been achieved yet. In this work, we describe for the first time the successful transformation of a Pseudogymnoascus verrucosus strain of Antarctic origin, using two methodologies: the polyethylene glycol (PEG)-mediated transformation, and the electroporation of germinated conidia. We achieved transformation efficiencies of 15.87 ± 5.16 transformants per µg of DNA and 2.67 ± 1.15 transformants per µg of DNA for PEG-mediated transformation and electroporation of germinated conidia, respectively. These results indicate that PEG-mediated transformation is a very efficient method for the transformation of this Antarctic fungus. The genetic transformation of Pseudogymnoascus verrucosus described in this work represents the first example of transformation of a filamentous fungus of Antarctic origin.

Heterotrimeric Galpha protein Pga1 of Penicillium chrysogenum controls conidiation mainly by a cAMP-independent mechanism.

Fungal heterotrimeric G proteins regulate different processes related to development, such as colony growth and asexual sporulation, the main mechanism of propagation in filamentous fungi. To gain insight into the mechanisms controlling growth and differentiation in the industrial penicillin producer Penicillioum chrysogenum, we investigated the role of the heterotrimeric Galpha subunit Pga1 in conidiogenesis. A pga1 deleted strain (Deltapga1) and transformants with constitutively activated (pga1G42R) and inactivated (pga1G203R) Pga1 alpha subunits were obtained. They showed phenotypes that clearly implicate Pga1 as an important negative regulator of conidiogenesis. Pga1 positively affected the level of intracellular cAMP, which acts as secondary messenger of Pga1-mediated signalling. Although cAMP has some inhibitory effect on conidiation, the regulation of asexual development by Pga1 is exerted mainly via cAMP-independent pathways. The regulation of conidiation by Pga1 is mediated by repression of the brlA and wetA genes. The Deltapga1 strain and transformants with the constitutively inactive Pga1G203R subunit developed a sporulation microcycle in submerged cultures triggered by the expression of brlA and wetA genes, which are deregulated in the absence of active Pga1. Our results indicate that although basic mechanisms for regulating conidiation are similar in most filamentous fungi, there are differences in the degree of involvement of specific pathways, such as the cAMP-mediated pathway, in the regulation of this process.

The unprocessed preprotein form IATC103S of the isopenicillin N acyltransferase is transported inside peroxisomes and regulates its self-processing.

Previous studies in Penicillium chrysogenum and Aspergillus nidulans suggested that self-processing of the isopenicillin N acyltransferase (IAT) is an important differential factor in these fungi. Expression of a mutant penDE(C103S) gene in P. chrysogenum gave rise to an unprocessed inactive variant of IAT (IAT(C103S)) located inside peroxisomes, which indicates that transport of the proIAT inside these organelles is not dependent on the processing state of the protein. Co-expression of the penDE(C103S) and wild-type penDE genes in P. chrysogenum (Wis54-DE(C103S) strain) led to a decrease in benzylpenicillin levels. Changes in the wild-type IAT processing profile (beta subunit formation) were observed in the Wis54-DE(C103S) strain, suggesting a regulatory role of the unprocessed IAT(C103S) in the processing of the wild-type IAT. This was confirmed in Escherichia coli, where a delay in the processing of IAT in presence of the unprocessable IAT(C103S) was observed. Our results indicate that IAT is post-translationally regulated by its preprotein, which interferes with the self-processing.

NeVOmics: An Enrichment Tool for Gene Ontology and Functional Network Analysis and Visualization of Data from OMICs Technologies.

The increasing number of OMICs studies demands bioinformatic tools that aid in the analysis of large sets of genes or proteins to understand their roles in the cell and establish functional networks and pathways. In the last decade, over-representation or enrichment tools have played a successful role in the functional analysis of large gene/protein lists, which is evidenced by thousands of publications citing these tools. However, in most cases the results of these analyses are long lists of biological terms associated to proteins that are difficult to digest and interpret. Here we present NeVOmics, Network-based Visualization for Omics, a functional enrichment analysis tool that identifies statistically over-represented biological terms within a given gene/protein set. This tool provides a hypergeometric distribution test to calculate significantly enriched biological terms, and facilitates analysis on cluster distribution and relationship of proteins to processes and pathways. NeVOmics is adapted to use updated information from the two main annotation databases: Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG). NeVOmics compares favorably to other Gene Ontology and enrichment tools regarding coverage in the identification of biological terms. NeVOmics can also build different network-based graphical representations from the enrichment results, which makes it an integrative tool that greatly facilitates interpretation of results obtained by OMICs approaches. NeVOmics is freely accessible at https://github.com/bioinfproject/bioinfo/.

The pga1 gene of Penicillium chrysogenum NRRL 1951 encodes a heterotrimeric G protein alpha subunit that controls growth and development.

The pga1 gene of Penicillium chrysogenum NRRL 1951 has been cloned and shown to participate in the developmental program of this fungus. It encodes a protein showing a high degree of identity to group I alpha subunits of fungal heterotrimeric G proteins, presenting in its sequence all the distinctive characteristics of this group. Northern analysis revealed that pga1 is highly expressed in a constitutive manner in submerged cultures, while its expression changes during development on solid media cultures; it is higher during vegetative growth and decreases significantly at the time of conidiogenesis. Attenuation of pga1 gene expression by antisense RNA, and mutations of pga1 resulting in a constitutively activated (pga1G42R allele) or constitutively inactivated (pga1G203R allele) Pga1 alpha subunit were used to study the function of Pga1 in P. chrysogenum. The phenotype of transformants expressing the antisense construction and the mutant alleles showed substantial morphological differences in colony diameter and conidiation, indicating that Pga1 controls apical extension and negatively regulates conidiogenesis on solid medium, but has no effect on submerged cultures. Pga1 is also functional in Penicillium roqueforti, controlling the same processes.

[Role of G-protein alpha sub-units in the morphogenic processes of filamentous Ascomycota fungi]. / Papel de las subunidades alfa de proteínas G en los procesos morfogénicos de hongos filamentosos de la división Ascomycota.

The phylum Ascomycota comprises about 75% of all the fungal species described, and includes species of medical, phytosanitary, agricultural, and biotechnological importance. The ability to spread, explore, and colonise new substrates is a feature of critical importance for this group of organisms. In this regard, basic processes such as conidial germination, the extension of hyphae and sporulation, make up the backbone of development in most filamentous fungi. These processes require specialised morphogenic machinery, coordinated and regulated by mechanisms that are still being elucidated. In recent years, substantial progress has been made in understanding the role of the signalling pathway mediated by heterotrimericG proteins in basic biological processes of many filamentous fungi. This review focuses on the role of the alpha subunits of heterotrimericG proteins in the morphogenic processes of filamentous Ascomycota.