Essential-Oils-Loaded Biopolymeric Nanoparticles as Strategies for Microbial and Biofilm Control: A Current Status.

The emergence of bacterial strains displaying resistance to the currently available antibiotics is a critical global concern. These resilient bacteria can form biofilms that play a pivotal role in the failure of bacterial infection treatments as antibiotics struggle to penetrate all biofilm regions. Consequently, eradicating bacteria residing within biofilms becomes considerably more challenging than their planktonic counterparts, leading to persistent and chronic infections. Among various approaches explored, essential oils loaded in nanoparticles based on biopolymers have emerged, promising strategies that enhance bioavailability and biological activities, minimize side effects, and control release through regulated pharmacokinetics. Different available reviews analyze nanosystems and essential oils; however, usually, their main goal is the analysis of their antimicrobial properties, and progress in biofilm combat is rarely discussed, or it is not the primary objective. This review aims to provide a global vision of biofilm conformation and describes mechanisms of action attributed to each EO. Furthermore, we present a comprehensive overview of the latest developments in biopolymeric nanoparticles research, especially in chitosan- and zein-based nanosystems, targeting multidrug-resistant bacteria in both their sessile and biofilm forms, which will help to design precise strategies for combating biofilms.

Expression of Cytochrome P450 Enzymes in Pediatric Non-Rhabdomyosarcoma Soft Tissue Sarcomas: Possible Role in Carcinogenesis and Treatment Response.

The 5-year relative survival rate estimate of treated patients with non-rhabdomyosarcoma soft tissue sarcomas (NRSTS) is ∼50% since they generally present with tumor progression, relapse, metastasis, and/or chemoresistance. The expression of cytochrome P450 (CYP) enzymes in malignancies can affect the pharmacology of drugs commonly used in chemotherapy or confer susceptibility to development of chemical carcinogenesis; in addition, their specific tumor expression can be used as a therapeutic target. Using qPCR and Western blot assays, the expression of CYP1B1, CYP2E1, CYP3A4, and CYP3A5 were analyzed in a cohort of tumor tissue paired with non-malignant adjacent tissue of patients with NRSTS. The mRNA and protein expression of CYP1B1, CYP2E1, and CYP3A4 were significantly increased in tumor tissue. We propose that the expression of these isoforms is related to carcinogenesis and chemoresistance frequently observed in these neoplasms.

Repetitive DNA profile of the amphibian mitogenome.

BACKGROUND: Repetitive DNA elements such as direct and inverted repeat sequences are present in every genome, playing numerous biological roles. In amphibians, the functions and effects of the repeat sequences have not been extensively explored. We consider that the data of mitochondrial genomes in the NCBI database are a valuable alternative to generate a better understanding of the molecular dynamic of the repeat sequences in the amphibians. RESULTS: This work presents the development of a strategy to identify and quantify the total amount of repeat sequences with lengths from 5 to 30 base pairs in the amphibian mitogenomes. The results show differences in the abundance of repeat sequences among amphibians and bias to specific genomic regions that are not easily associated with the classical amphibian ancestry. CONCLUSIONS: Derived from these analyses, we show that great variability of the repeat sequences exists among amphibians, demonstrating that the mitogenomes of these organisms are dynamic.

On the use of n-octyl gallate and salicylhydroxamic acid to study the alternative oxidase role.

The alternative oxidase (AOX) catalyzes the transfer of electrons from ubiquinol to oxygen without the translocation of protons across the inner mitochondrial membrane. This enzyme has been proposed to participate in the regulation of cell growth, sporulation, yeast-mycelium transition, resistance to reactive oxygen species, infection, and production of secondary metabolites. Two approaches have been used to evaluate AOX function: incubation of cells for long periods of time with AOX inhibitors or deletion of AOX gene. However, AOX inhibitors might have different targets. To test non-specific effects of n-octyl gallate (nOg) and salicylhydroxamic acid (SHAM) on fungal physiology we measured the growth and respiratory capacity of two fungal strains lacking (Ustilago maydis-Δaox and Saccharomyces cerevisiae) and three species containing the AOX gene (U. maydis WT, Debaryomyces hansenii, and Aspergillus nidulans). For U. maydis, a strong inhibition of growth and respiratory capacity by SHAM was observed, regardless of the presence of AOX. Similarly, A. nidulans mycelial growth was inhibited by low concentrations of nOg independently of AOX expression. In contrast, these inhibitors had no effect or had a minor effect on S. cerevisiae and D. hansenii growth. These results show that nOg and SHAM have AOX independent effects which vary in different microorganisms, indicating that studies based on long-term incubation of cells with these inhibitors should be considered as inconclusive.

High fever after sublingual administration of misoprostol for treatment of post-partum haemorrhage: a hospital-based, prospective observational study in Argentina.

OBJECTIVE: To characterise the occurrence of fever (≥38.0°C) after treatment for post-partum haemorrhage (PPH) with sublingual misoprostol 800 mcg in Latin America, where elevated rates of misoprostol's thermoregulatory effects and recipients' increased susceptibility to high fever have been documented. METHODS: A prospective observational study in hospitals in Argentina enrolled consenting women with atonic PPH after vaginal delivery, eligible to receive misoprostol. Corporal temperature was assessed at 30, 60, 90 and 120 min post-treatment; other effects were recorded. The incidence of high fever ≥ 40.0°C (primary outcome) was compared to the rate observed previously in Ecuador. Logistic regressions were performed to identify clinical and population-based predictors of misoprostol-induced fever. RESULTS: Transient shivering and fever were experienced by 75.5% (37/49) of treated participants and described as acceptable by three-quarters of women interviewed (35/47). The high fever rate was 12.2% (6/49), [95% Confidence Interval (CI) 4.6, 24.8], compared to Ecuador's rate following misoprostol treatment (35.6% (58/163) [95% CI 28.3, 43.5], P = 0.002). Significant predictors of misoprostol-induced fever (model dependent) were as follows: pre-delivery haemoglobin < 11.0g/dl, rapid placental expulsion, and higher age of the woman. No serious outcomes were reported prior to discharge. CONCLUSIONS: Misoprostol to treat PPH in Argentina resulted in a significantly lower rate of high fever than in Ecuador, although both are notably higher than rates seen elsewhere. A greater understanding of misoprostol's side effects and factors involved in their occurrence, including genetics, will help alleviate concerns. The onset of shivering may be the simplest way to know if fever can also be expected.

OBJECTIF: Caractériser la survenue de fièvre (≥ 38,0°C) après traitement d'une hémorragie post-partum (HPP) avec du misoprostol sublingual à 800 mcg en Amérique latine, où des taux élevés d'effets thermorégulateurs du misoprostol et une sensibilité accrue des receveurs à une forte fièvre ont été documentés. MÉTHODES: Une étude observationnelle prospective dans des hôpitaux en Argentine a recruté des femmes consentantes atteintes d'HPP atonique après un accouchement vaginal éligibles pour recevoir du misoprostol. La température corporelle a été évaluée 30, 60, 90 et 120 minutes après le traitement; d'autres effets ont été enregistrés. L'incidence d'une fièvre élevée ≥40,0°C (critère principal) a été comparée au taux observé précédemment en Equateur. Des régressions logistiques ont été effectuées pour identifier les prédicteurs cliniques et ceux basés sur la population de la fièvre induite par le misoprostol . RÉSULTATS: Des frissons transitoires et de la fièvre ont été ressentis par 75% (37/49) des participantes traitées et décrits comme acceptables par les trois quarts des femmes interrogées (35/47). Le taux de fièvre élevé était de 12% (6/49), [intervalle de confiance (IC) à 95%: 4,6, 24,8] contre 35,6% en Equateur après traitement au misoprostol (58/163) [IC95%: 28,3, 43,5], p = 0,002). Les prédicteurs significatifs de la fièvre induite par le misoprostol (selon le modèle) étaient: hémoglobine avant l'accouchement <11,0 g/dL, expulsion placentaire rapide et âge plus élevé de la femme. Aucun résultat sévère n'a été signalé avant le sortie d'hôpital. CONCLUSIONS: Le misoprostol pour traiter l'HPP en Argentine a entraîné un taux de fièvre élevée significativement plus bas qu'en Equateur, bien que les taux dans les deux pays soient notablement plus élevés que les taux observés ailleurs. Une meilleure compréhension des effets secondaires du misoprostol et des facteurs impliqués dans leur apparition, y compris la génétique, aidera à atténuer les inquiétudes. L'apparition de frissons peut être le moyen le plus simple de savoir si l'on peut également s'attendre à de la fièvre.

Interaction of Thalassia testudinum Metabolites with Cytochrome P450 Enzymes and Its Effects on Benzo(a)pyrene-Induced Mutagenicity.

The aim of the present work was to evaluate the effects of Thalassia testudinum hydroethanolic extract, its polyphenolic fraction and thalassiolin B on the activity of phase I metabolizing enzymes as well as their antimutagenic effects. Spectrofluorometric techniques were used to evaluate the effect of tested products on rat and human CYP1A and CYP2B activity. The antimutagenic effect of tested products was evaluated in benzo[a]pyrene (BP)-induced mutagenicity assay by an Ames test. Finally, the antimutagenic effect of Thalassia testudinum (100 mg/kg) was assessed in BP-induced mutagenesis in mice. The tested products significantly (p < 0.05) inhibit rat CYP1A1 activity, acting as mixed-type inhibitors of rat CYP1A1 (Ki = 54.16 ± 9.09 µg/mL, 5.96 ± 1.55 µg/mL and 3.05 ± 0.89 µg/mL, respectively). Inhibition of human CYP1A1 was also observed (Ki = 197.1 ± 63.40 µg/mL and 203.10 ± 17.29 µg/mL for the polyphenolic fraction and for thalassiolin B, respectively). In addition, the evaluated products significantly inhibit (p < 0.05) BP-induced mutagenicity in vitro. Furthermore, oral doses of Thalassia testudinum (100 mg/kg) significantly reduced (p < 0.05) the BP-induced micronuclei and oxidative damage, together with an increase of reduced glutathione, in mice. In summary, Thalassia testudinum metabolites exhibit antigenotoxic activity mediated, at least, by the inhibition of CYP1A1-mediated BP biotransformation, arresting the oxidative and mutagenic damage. Thus, the metabolites of T. testudinum may represent a potential source of chemopreventive compounds for the adjuvant therapy of cancer.

Cytochrome P450 in the central nervous system as a therapeutic target in neurodegenerative diseases.

Cytochromes P450 (CYPs) constitute a family of enzymes that can be found in the endoplasmic reticulum (ER), mitochondria or the cell surface of the cells. CYPs are characterized by carrying out the oxidation of organic compounds and they are mainly recognized as mediators of the biotransformation of xenobiotics to polar hydrophilic metabolites that can be eliminated from the organism. However, these enzymes play a key role in many other physiological processes, being involved in diverse indispensable metabolic pathways since they metabolize many endogenous substrates. Various CYP isoforms are expressed in the brain, and it is believed that this could be in part due to the particular function of brain CYPs. In the brain, CYPs are involved in the cholesterol turnover, the biosynthesis of dopamine, serotonin, morphine, hormones, and protective lipid mediators (epoxyeicosatrienoic acids), in addition to their already recognized role in xenobiotics detoxification and psychotropic drug metabolism. Increasing evidence suggests that this group of enzymes is fundamental for the normal functioning and maintenance of brain homeostasis. This review is focused on highlighting the importance of CYP-mediated endogenous metabolism in the central nervous system (CNS) and its relationship with recent findings regarding CYP involvement in neurodegenerative diseases. Some therapeutic approaches focused on CYP regulation are also discussed.

Time-caloric restriction inhibits the neoplastic transformation of cirrhotic liver in rats treated with diethylnitrosamine.

Hepatocellular cancer is the most common type of primary liver cancer. Cirrhosis is the main risk factor that generates this malady. It has been proven that caloric restriction protocols and restricted feeding schedules are protective in experimental carcinogenic models. We tested the influence of a time-caloric restriction protocol (2 h of food access during the daytime for 18 weeks) in an experimental model of cirrhosis-hepatocarcinoma produced by weekly administration of diethylnitrosamine. Our results indicate that time-caloric restriction reduced hepatomegaly and prevented the increase in blood leukocytes promoted by diethylnitrosamine. Strikingly, time-caloric restriction preserved functional and histological characteristics of the liver in fibrotic areas compared to the cirrhotic areas of the Ad Libitum-fed group. Tumoural masses in the restricted group were well differentiated; consider a neoplastic or early stage of HCC. However, time-caloric restriction enhanced collagen deposits. With regard to the cancerous process, food restriction prevented systemic inflammation and an increase in carcinoembryonic antigen, and it favoured the occurrence of diffuse multinodular tumours. Histologically, it prevented hepatocyte inflammation response, the regenerative process, and neoplastic transformation. Time-caloric restriction stimulated circadian synchronization in fibrotic and cancerous liver sections, and it increased BMAL1 clock protein levels. We conclude that time-caloric restriction prevents fibrosis from progressing into cirrhosis, thus avoiding chronic inflammation and regenerative processes. It also prevents, probably through circadian entrainment and caloric restriction, the neoplastic transformation of tumoural lesions induced by diethylnitrosamine.

The SrkA Kinase Is Part of the SakA Mitogen-Activated Protein Kinase Interactome and Regulates Stress Responses and Development in Aspergillus nidulans.

Fungi and many other eukaryotes use specialized mitogen-activated protein kinases (MAPK) of the Hog1/p38 family to transduce environmental stress signals. In Aspergillus nidulans, the MAPK SakA and the transcription factor AtfA are components of a central multiple stress-signaling pathway that also regulates development. Here we characterize SrkA, a putative MAPK-activated protein kinase, as a novel component of this pathway. ΔsrkA and ΔsakA mutants share a derepressed sexual development phenotype. However, ΔsrkA mutants are not sensitive to oxidative stress, and in fact, srkA inactivation partially suppresses the sensitivity of ΔsakA mutant conidia to H2O2, tert-butyl-hydroperoxide (t-BOOH), and menadione. In the absence of stress, SrkA shows physical interaction with nonphosphorylated SakA in the cytosol. We show that H2O2 induces a drastic change in mitochondrial morphology consistent with a fission process and the relocalization of SrkA to nuclei and mitochondria, depending on the presence of SakA. SakA-SrkA nuclear interaction is also observed during normal asexual development in dormant spores. Using SakA and SrkA S-tag pulldown and purification studies coupled to mass spectrometry, we found that SakA interacts with SrkA, the stress MAPK MpkC, the PPT1-type phosphatase AN6892, and other proteins involved in cell cycle regulation, DNA damage response, mRNA stability and protein synthesis, mitochondrial function, and other stress-related responses. We propose that oxidative stress induces DNA damage and mitochondrial fission and that SakA and SrkA mediate cell cycle arrest and regulate mitochondrial function during stress. Our results provide new insights into the mechanisms by which SakA and SrkA regulate the remodelling of cell physiology during oxidative stress and development.

FlbD, a Myb transcription factor of Aspergillus nidulans, is uniquely involved in both asexual and sexual differentiation.

In the fungus Aspergillus nidulans, inactivation of the flbA to -E, fluG, fluF, and tmpA genes results in similar phenotypes, characterized by a delay in conidiophore and asexual spore production. flbB to -D encode transcription factors needed for proper expression of the brlA gene, which is essential for asexual development. However, recent evidence indicates that FlbB and FlbE also have nontranscriptional functions. Here we show that fluF1 is an allele of flbD which results in an R47P substitution. Amino acids C46 and R47 are highly conserved in FlbD and many other Myb proteins, and C46 has been proposed to mediate redox regulation. Comparison of ΔflbD and flbD(R47P) mutants uncovered a new and specific role for flbD during sexual development. While flbD(R47P) mutants retain partial function during conidiation, both ΔflbD and flbD(R47P) mutants are unable to develop the peridium, a specialized external tissue that differentiates during fruiting body formation and ends up surrounding the sexual spores. This function, unique among other fluffy genes, does not affect the viability of the naked ascospores produced by mutant strains. Notably, ascospore development in these mutants is still dependent on the NADPH oxidase NoxA. We generated R47K, C46D, C46S, and C46A mutant alleles and evaluated their effects on asexual and sexual development. Conidiation defects were most severe in ΔflbD mutants and stronger in R47P, C46D, and C46S strains than in R47K strains. In contrast, mutants carrying the flbD(C46A) allele exhibited conidiation defects in liquid culture only under nitrogen starvation conditions. The R47K, R47P, C46D, and C46S mutants failed to develop any peridial tissue, while the flbD(C46A) strain showed normal peridium development and increased cleistothecium formation. Our results show that FlbD regulates both asexual and sexual differentiation, suggesting that both processes require FlbD DNA binding activity and that FlbD is involved in the response to nitrogen starvation.

The role of peroxiredoxins PrxA and PrxB in the antioxidant response, carbon utilization and development in Aspergillus nidulans.

In addition to their role in the breakdown of H2O2, some peroxiredoxins (Prxs) have chaperone and H2O2 sensing functions. Acting as an H2O2 sensor, Prx Gpx3 transfers the oxidant signal to the transcription factor Yap1, involved in the antioxidant response in Saccharomyces cerevisiae. We have shown that Aspergillus nidulans Yap1 ortholog NapA is necessary for the antioxidant response, the utilization of arabinose, fructose and ethanol, and for proper development. To address the Prx roles in these processes, we generated and characterized mutants lacking peroxiredoxins PrxA, PrxB, PrxC, or TpxC. Our results show that the elimination of peroxiredoxins PrxC or TpxC does not produce any distinguishable phenotype. In contrast, the elimination of atypical 2-cysteine peroxiredoxins PrxA and PrxB produce different mutant phenotypes. ΔprxA, ΔnapA and ΔprxA ΔnapA mutants are equally sensitive to H2O2 and menadione, while PrxB is dispensable for this. However, the sensitivity of ΔprxA and ΔprxA ΔnapA mutants is increased by the lack of PrxB. Moreover, PrxB is required for arabinose and ethanol utilization and fruiting body cell wall pigmentation. PrxA expression is partially independent of NapA, and the replacement of peroxidatic cysteine 61 by serine (C61S) is enough to cause oxidative stress sensitivity and prevent NapA nuclear accumulation in response to H2O2, indicating its critical role in H2O2 sensing. Our results show that despite their high similarity, PrxA and PrxB play differential roles in Aspergillus nidulans antioxidant response, carbon utilization and development.

Filamentous actin destabilization by H2O2 favors DnmA aggregation, with crucial roles of cysteines 450 and 776 in mitochondrial and peroxisomal division in Aspergillus nidulans.

IMPORTANCE: Mitochondria constitute major sources of H2O2 and other reactive oxygen species in eukaryotic cells. The division of these organelles is crucial for multiple processes in cell biology and relies on highly regulated mechano-GTPases that are oligomerization dependent and belong to the dynamin-related protein family, like A. nidulans DnmA. Our previous work demonstrated that H2O2 induces mitochondrial constriction, division, and remodeling of the outer membrane. Here, we show that H2O2 also induces a DnmA aggregation consistent with higher-order oligomerization and its recruitment to mitochondria. The study of this response uncovered that H2O2 induces the depolymerization and reorganization of actin as well as the critical role that cysteines 450 and 776 play in DnmA function. Our results provide new insights into the mechanisms of reactive oxygen species cell signaling and how they can regulate the dynamics of the actin cytoskeleton and the division of mitochondria and peroxisomes.

A Fatal Case of Systemic Calciphylaxis in the Gastrointestinal Tract: A Case Report and Literature Review.

Calciphylaxis is an infrequent yet lethal disease often associated with end-stage kidney disease (ESKD). The most common sites include proximal and distal extremities and the trunk, with few reported in the penis and very few as gastrointestinal (GI) disease. We report a case of systemic calciphylaxis in a middle-aged male, presenting with a colostomy leak and parastomal abscess. Workup revealed severe calcification of the intestinal arteries and ischemic colon necrosis. The patient underwent colectomy, antibiotic therapy, regular hemodialysis (HD), and sodium thiosulphate infusion with clinical stability. Histopathology of the colon revealed ischemic necrosis and pericolonic vessel calcification suggestive of calciphylaxis. It is an important differential to be considered in patients with risk factors presenting with symptoms of gastrointestinal hemorrhage and necrosis with perforation.

Capillary Refill Time and Serum Lactate as Predictors of Mortality and Postoperative Extracorporeal Membrane Oxygenation Requirement in Congenital Heart Surgery.

Multiple tissue perfusion markers are described to guide therapy in critically ill pediatric patients undergoing congenital heart surgery. Given the advantages of capillary refill time, our goal is to determine its predictive capacity for mortality and postoperative extracorporeal oxygenation requirements in congenital heart surgery and compare it to serum lactate. We conducted a prospective cohort observational study in a single high-complexity university hospital. Serum lactate and capillary refill time were measured at five predetermined time points: preoperative, immediate postoperative, 6, 12, and 24 h after the surgery. Prolonged immediate postoperative, 6 h, and 12 h capillary refill time measurements turned out to be independent risk factors for both outcomes. The capillary refill time area under the curve ranged between 0.70 and 0.80, while the serum lactate resulted between 0.79 and 0.92 for both outcomes. Both tissue perfusion markers resulted in mortality and extracorporeal oxygenation requirement predictors. Given the advantages of capillary refill time over serum lactate, a monitoring strategy including these two perfusion markers should be considered for congenital heart surgeries.

Metabolic-associated Fatty Liver Disease Regulation through Nutri Epigenetic Methylation.

Metabolically associated fatty liver disease, formerly called nonalcoholic fatty liver disease, is the most common liver disease globally, representing the third cause of liver transplantation. Metabolically associated fatty liver disease is defined as having more than 5% lipid droplets in hepatocytes without other concomitant liver diseases. Various stimuli such as the secretion of inflammatory cytokines, mitochondrial and endoplasmic reticulum dysfunction due to oxidative stress, alteration of the intestine-liver axis, bacterial dysbiosis, as well as genetic and epigenetic factors can modify the progression of metabolically associated fatty liver disease to fibrosis, cirrhosis, and may reach hepatocellular carcinoma. Epigenetics is responsible for a highly sophisticated regulatory system that controls many cellular processes in response to multiple environmental factors as an adaptive mechanism unrelated to alterations in the primary deoxyribonucleic acid sequence, including gene expression, microRNAs, DNA methylation, modifications in histones, and DNA-protein interactions. Several studies have shown that epigenetic changes are associated with various diseases, including metabolically associated fatty liver disease. Nutri epigenomics is the interaction between nutrition and components at the transcriptional or post-transcriptional level. Methylation processes involve micronutrients that regulate epigenetic states in a physiological and pathological context. Micronutrients such as methionine, folate, and choline are the main components of one-carbon metabolism, functioning as methyl group donors, and their deficiency predisposes to various pathologies such as metabolically associated fatty liver disease. Understanding of epigenetic modifiers leads us to develop new therapeutic therapies for patients with metabolically associated fatty liver disease.

Inhibition of the CYP Enzymatic System Responsible of Heterocyclic Amines Bioactivation by an Asclepias subulata Extract.

Asclepias subulata plant extract has previously demonstrated antiproliferative activity and antimutagenicity against heterocyclic aromatic amines (HAAs) commonly found in cooked meat. The objective of this work was to evaluate the in vitro ability of an ethanolic extract from the medicinal plant Asclepias subulata extract (ASE), non-heated and heated (180 °C), to inhibit the activity of CYP1A1 and CYP1A2, which are largely responsible for HAAs bioactivation. Ethoxyresorufin and methoxyresorufin O-dealkylation assays were performed in rat liver microsomes exposed to ASE (0.002-960 µg/mL). ASE exerted an inhibitory effect in a dose-dependent manner. The half inhibitory concentration (IC50) for unheated ASE was 353.6 µg/mL and 75.9 µg/mL for heated ASE in EROD assay. An IC40 value of 288.4 ± 5.8 µg/mL was calculated for non-heated ASE in MROD assay. However, after heat treatment, the IC50 value was 232.1 ± 7.4 µg/mL. Molecular docking of corotoxigenin-3-O-glucopyranoside, one of the main components of ASE, with CYP1A1/2 structure, was performed. Results show that the interaction of corotoxigenin-3-O-glucopyranoside with CYP1A1/2s' α-helices, which are related with the active site and the heme cofactor, may explain the plant extract's inhibitory properties. Results showed that ASE inhibits CYP1A enzymatic subfamily and may potentially act as a chemopreventive agent by inhibiting bioactivation of promutagenic dietary HAAs.

The IV International Symposium on Fungal Stress and the XIII International Fungal Biology Conference.

For the first time, the International Symposium on Fungal Stress was joined by the XIII International Fungal Biology Conference. The International Symposium on Fungal Stress (ISFUS), always held in Brazil, is now in its fourth edition, as an event of recognized quality in the international community of mycological research. The event held in São José dos Campos, SP, Brazil, in September 2022, featured 33 renowned speakers from 12 countries, including: Austria, Brazil, France, Germany, Ghana, Hungary, México, Pakistan, Spain, Slovenia, USA, and UK. In addition to the scientific contribution of the event in bringing together national and international researchers and their work in a strategic area, it helps maintain and strengthen international cooperation for scientific development in Brazil.

Aspergillus nidulans transcription factor AtfA interacts with the MAPK SakA to regulate general stress responses, development and spore functions.

Fungi utilize a phosphorelay system coupled to a MAP kinase module for sensing and processing environmental signals. In Aspergillus nidulans, response regulator SskA transmits osmotic and oxidative stress signals to the stress MAPK (SAPK) SakA. Using a genetic approach together with GFP tagging and molecular bifluorescence we show that SakA and ATF/CREB transcription factor AtfA define a general stress-signalling pathway that plays differential roles in oxidative stress responses during growth and development. AtfA is permanently localized in the nucleus, while SakA accumulates in the nucleus in response to oxidative or osmotic stress signals or during normal spore development, where it physically interacts with AtfA. AtfA is required for expression of several genes, the conidial accumulation of SakA and the viability of conidia. Furthermore, SakA is active (phosphorylated) in asexual spores, remaining phosphorylated in dormant conidia and becoming dephosphorylated during germination. SakA phosphorylation in spores depends on certain (SskA) but not other (SrrA and NikA) components of the phosphorelay system. Constitutive phosphorylation of SakA induced by the fungicide fludioxonil prevents both, germ tube formation and nuclear division. Similarly, Neurospora crassa SakA orthologue OS-2 is phosphorylated in intact conidia and gets dephosphorylated during germination. We propose that SakA-AtfA interaction regulates gene expression during stress and conidiophore development and that SAPK phosphorylation is a conserved mechanism to regulate transitions between non-growing (spore) and growing (mycelia) states.

Sfp-type 4'-phosphopantetheinyl transferase is indispensable for fungal pathogenicity.

In filamentous fungi, Sfp-type 4'-phosphopantetheinyl transferases (PPTases) activate enzymes involved in primary (alpha-aminoadipate reductase [AAR]) and secondary (polyketide synthases and nonribosomal peptide synthetases) metabolism. We cloned the PPTase gene PPT1 of the maize anthracnose fungus Colletotrichum graminicola and generated PPTase-deficient mutants (Deltappt1). Deltappt1 strains were auxotrophic for Lys, unable to synthesize siderophores, hypersensitive to reactive oxygen species, and unable to synthesize polyketides (PKs). A differential analysis of secondary metabolites produced by wild-type and Deltappt1 strains led to the identification of six novel PKs. Infection-related morphogenesis was affected in Deltappt1 strains. Rarely formed appressoria of Deltappt1 strains were nonmelanized and ruptured on intact plant. The hyphae of Deltappt1 strains colonized wounded maize (Zea mays) leaves but failed to generate necrotic anthracnose disease symptoms and were defective in asexual sporulation. To analyze the pleiotropic pathogenicity phenotype, we generated AAR-deficient mutants (Deltaaar1) and employed a melanin-deficient mutant (M1.502). Results indicated that PPT1 activates enzymes required at defined stages of infection. Melanization is required for cell wall rigidity and appressorium function, and Lys supplied by the AAR1 pathway is essential for necrotrophic development. As PPTase-deficient mutants of Magnaporthe oryzea were also nonpathogenic, we conclude that PPTases represent a novel fungal pathogenicity factor.

H2O2 Induces Calcium and ERMES Complex-Dependent Mitochondrial Constriction and Division as Well as Mitochondrial Outer Membrane Remodeling in Aspergillus nidulans.

The dynamin-like protein DnmA and its receptor FisA are essential for H2O2-induced mitochondrial division in Aspergillus nidulans. Here, we show that in the absence of DnmA or FisA, mitochondria show few spontaneous transient constrictions, the frequency of which is extensively increased by H2O2 or the carbonyl cyanide m-chlorophenyl hydrazone (CCCP). While H2O2-induced constrictions are transient, CCCP induces a drastic and irreversible alteration of mitochondrial filaments. H2O2 induces a gradual mitochondrial depolarization, while CCCP-induced depolarization is abrupt. The calcium chelator BAPTA-AM prevents the formation of mitochondrial constrictions induced by either H2O2 or CCCP. H2O2 also induces major rearrangements of the mitochondrial outer membrane, which remain after constrictions dissipate, as well as changes in endoplasmic reticulum (ER) and nuclear morphology. Similar mitochondrial constriction, ER and nuclear morphology changes are detected during the early stages of asexual development. ER and ER-Mitochondria encounter structure (ERMES) complex-composed of proteins Mdm10, Mmm1, Mdm43 and Mdm12-are important for mitochondrial division in Saccharomyces cerevisiae. As the Mdm10 ortholog MdmB was found to be essential in A. nidulans, we evaluated its functions in ΔmdmB terminal mutants and ΔmdmB heterokaryons. ΔmdmB conidia produce a short germ tube that fails to grow further, in which inherited mitochondria become gigantic and round shaped, lacking clear contacts with the ER. In slow-growing ΔmdmB heterokaryotic mycelia, multiple hyphae contain very long mitochondria with high ROS levels, as occur in ΔdnmA and ΔfisA mutants. In this hyphae, H2O2 fails to induce mitochondrial constrictions but not outer mitochondrial membrane reshaping, indicating that these are two separate effects of H2O2. Our results indicate that H2O2 induces a generalized mitochondrial constriction response, prior to actual division, involving gradual depolarization; they also indicate that Ca2+ and the ERMES complex are critical for both mitochondrial constriction and division. This supports a view of mitochondrial dynamics as the result of a cascade of signaling events that can be initiated in vivo by H2O2.