Fungal Alcohol Dehydrogenases: Physiological Function, Molecular Properties, Regulation of Their Production, and Biotechnological Potential.

Fungal alcohol dehydrogenases (ADHs) participate in growth under aerobic or anaerobic conditions, morphogenetic processes, and pathogenesis of diverse fungal genera. These processes are associated with metabolic operation routes related to alcohol, aldehyde, and acid production. The number of ADH enzymes, their metabolic roles, and their functions vary within fungal species. The most studied ADHs are associated with ethanol metabolism, either as fermentative enzymes involved in the production of this alcohol or as oxidative enzymes necessary for the use of ethanol as a carbon source; other enzymes participate in survival under microaerobic conditions. The fast generation of data using genome sequencing provides an excellent opportunity to determine a correlation between the number of ADHs and fungal lifestyle. Therefore, this review aims to summarize the latest knowledge about the importance of ADH enzymes in the physiology and metabolism of fungal cells, as well as their structure, regulation, evolutionary relationships, and biotechnological potential.

Hyphal Growth and Conidia Germination Are Induced by Phytohormones in the Root Colonizing and Plant Growth Promoting Fungus Metarhizium guizhouense.

Beneficial associations are very important for plants and soil-dwelling microorganisms in different ecological niches, where communication by chemical signals is relevant. Among the chemical signals, the release of phytohormones by plants is important to establish beneficial associations with fungi, and a recently described association is that of the entomopathogenic ascomycete fungus Metarhizium with plants. Here, we evaluated the effect of four different phytohormones, synthetic strigolactone (GR24), sorgolactone (SorL), 3-indolacetic acid (IAA) and gibberellic acid (GA3), on the fungus Metarhizium guizhouense strain HA11-2, where the germination rate and hyphal elongation were determined at three different times. All phytohormones had a positive effect on germination, with GA3 showing the greatest effect, and for hyphal length, on average, the group treated with synthetic strigolactone GR24 showed greater average hyphal length at 10 h of induction. This work expands the knowledge of the effect of phytohormones on the fungus M. guizhouense, as possible chemical signals for the rapid establishment of the fungus-plant association.

Características y desenlaces de los pacientes ventilados por neumonía por SARS-CoV-2 en un hospital chileno / Characteristics and outcomes of patients ventilated for SARS-CoV-2 pneumonia in a Chilean hospital / Características e resultados de pacientes ventilados por pneumonia por SARS-CoV-2 em um hospital chileno

Resumen: Introducción: la neumonía por coronavirus es emergente. Existen pocos datos del manejo ventilatorio. Presenta diferentes fenotipos pulmonares con difícil programación de la ventilación mecánica (VM). Se analizan estos pacientes en un hospital general. Material y métodos: se incluyen pacientes con neumonía por SARS-CoV-2 que ingresan a la unidad de cuidados intensivos (UCI) ventilados entre marzo y junio de 2020. Analizamos demografía, gravedad, programación ventilatoria, gases arteriales, mecánica pulmonar y desenlaces. Se describen los pacientes que recibieron posición prona (PP). Los pacientes se categorizaron por mediana de la compliance estática (Cst) y rangos ≤ 20, > 20 a ≤ 30 y > 30 en el día uno. Resultados: 118 pacientes, edad promedio 56.4 ± 1.3, 76.4% varones. El APACHE II y SOFA de ingreso: 13.6 ± 0.5 y 8.3 ± 0.2. Requirieron PP 47.5%. Los días de VM, UCI y hospital fueron 13.5 ± 0.9; 16.8 ± 0.9 y 23.8 ± 1.5. La mortalidad hospitalaria de pacientes PP y supinos fue de 32.1 y 11.3%, p = 0.005. Mortalidad global de 21.2%. Pacientes con Cst ≤ 20 mL/cmH2O presentaron una mortalidad de 44.4%. Conclusión: un porcentaje importante de los pacientes requiere PP precoz para superar la hipoxemia y aunque la mayoría responden, no asegura un buen desenlace hospitalario. Los pacientes con Cst ≤ 20 ml/cmH2O muestran mayor mortalidad.

Abstract: Introduction: coronavirus infection is an emerging pathology, there are few data regarding ventilatory management. Different pulmonary phenotypes make the MV process difficult. This encouraged us to analyze our COVID-19 patients with MV. Material and methods: all patients with SARS-CoV-2 pneumonia who were admitted ventilated to our unit through March to June 2020 were included. Demographics, severity scores, ventilatory settings, arterial gases, lung mechanics, and outcomes are analyzed. The patients who received prone position (PP) are described. Patients were categorized according to the median static compliance (Cst) and if it was ≤ 20, > 20 a ≤ 30 y > 30 in day 1. Results: 118 patients, the mean age was 56.4 ± 1.3, 76.4% males. APACHE II and SOFA on admission: 13.6 ± 0.5 and 8.3 ± 0.2. 47.5% of the patients required PP. The MV, ICU and hospital stay were 13.5 ± 0.9; 16.8 ± 0.9 and 23.8 ± 1.5 days. The in-hospital mortality of PP and supine patients was 32.1 and 11.3%, p = 0.005. Overall mortality 21.2%. Mortality of patients with Cst ≤ 20 mL/cmH2O was 44.4%. Conclusion: a significant percentage require early PP to overcome hypoxemia and although most respond, it does not ensure a good hospital outcome. The patients with compliance ≤ 20ml/ cmH2O have higher mortality.

Resumo: Introdução: a pneumonia por coronavírus é emergente. Existem poucos dados sobre o manejo ventilatório. Apresenta diferentes fenótipos pulmonares com difícil programação da VM. Esses pacientes são analisados em um hospital geral. Material e métodos: incluíram-se pacientes com pneumonia por SARS-CoV-2 internados na UTI ventilados entre março e junho de 2020. Analisamos dados demográficos, gravidade, programação ventilatória, gasometria arterial, mecânica pulmonar e desfechos. São descritos os pacientes que receberam posição prona (PP). Os pacientes foram categorizados pela média da compliance estática (Cst) e intervalos ≤ 20, >20 a ≤ 30 e >30 em 1 dia. Resultados: 118 pacientes, idade média 56.4 ± 1.3, 76.4% do sexo masculino. O APACHE II e SOFA de admissão: 13.6 ± 0.5 e 8.3 ± 0.2. Necessitaram PP 47.5%. Os dias de VM, UTI e hospital foram 13.5 ± 0.9; 16.8 ± 0.9 e 23.8 ± 1.5. A mortalidade hospitalar dos pacientes em PP e supino foi de 32.1 e 11.3%, p = 0.005. Mortalidade geral 21.2%. Pacientes com Cst ≤ 20 mL/cmH2O apresentaram mortalidade de 44.4%. Conclusão: muitos pacientes requerem PP precoce para superar a hipoxemia. A média da compliance não discrimina a mortalidade. Aqueles com Cst ≤ 20 apresentam maior mortalidade.

Peculiarities of nitronate monooxygenases and perspectives for in vivo and in vitro applications.

Nitroalkanes such as nitromethane, nitroethane, 1-nitropropane (1NP), and 2-nitropropane (2NP), derived from anthropogenic activities, are hazardous environmental pollutants due to their toxicity and carcinogenic activity. In nature, 3-nitropropionate (3NPA) and its derivatives are produced as a defense mechanism by many groups of organisms, including bacteria, fungi, insects, and plants. 3NPA is highly toxic as its conjugate base, propionate-3-nitronate (P3N), is a potent inhibitor of mitochondrial succinate dehydrogenase, essential to the tricarboxylic acid cycle, and can inhibit isocitrate lyase, a critical enzyme of the glyoxylate cycle. In response to these toxic compounds, several organisms on the phylogenetic scale express genes that code for enzymes involved in the catabolism of nitroalkanes: nitroalkane oxidases (NAOs) and nitronate monooxygenases (NMOs) (previously classified as nitropropane dioxygenases, NPDs). Two types of NMOs have been identified: class I and class II, which differ in structure, catalytic efficiency, and preferred substrates. This review focuses on the biochemical properties, structure, classification, and physiological functions of NMOs, and offers perspectives for their in vivo and in vitro applications. KEY POINTS: • Nitronate monooxygenases (NMOs) are key enzymes in nitroalkane catabolism. • NMO enzymes are involved in defense mechanisms in different organisms. • NMO applications include organic synthesis, biocatalysts, and bioremediation.

Members of the nitronate monooxygenase gene family from Metarhizium brunneum are induced during the process of infection to Plutella xylostella.

Metarhizium species are the most abundant fungi that can be isolated from soil, with a well-known biopesticide capacity. Metarhizium recognizes their hosts when the conidium interacts with insects, where the fungi are in contact with the hydrocarbons of the outermost lipid layer cuticle. These cuticular hydrocarbons comprise a mixture of n-alkanes, n-alkenes, and methyl-branched chains. Metarhizium can degrade insect hydrocarbons and use these hydrocarbons for energy production and the biosynthesis of cellular components. The metabolism of nitroalkanes involves nitronate monooxygenase activity. In this work, we isolated a family of six genes with potential nitronate monooxygenase activity from Metarhizium brunneum. The six genes were expressed in Escherichia coli, and the nitronate monooxygenase activity was verified in the recombinant proteins. Additionally, when the conidia of M. brunneum were grown in medium with nitroalkanes, virulence against Plutella xylostella increased. Furthermore, we analyzed the expression of the six Npd genes during the infection to this insect, which showed differential expression of the six Npd genes during infection.

Virulence Factors of the Entomopathogenic Genus Metarhizium.

The fungal genus Metarhizium has been used as an entomopathogen worldwide for approximately 140 years, and its mechanism of infection and its virulence factors have been studied. The present review is a compilation of virulence factors described in the literature to date and their participation in specific stages of the infection process.

Cx43-Gap Junctions Accumulate at the Cytotoxic Immunological Synapse Enabling Cytotoxic T Lymphocyte Melanoma Cell Killing.

Upon tumor antigen recognition, cytotoxic T lymphocytes (CTLs) and target cells form specialized supramolecular structures, called cytotoxic immunological synapses, which are required for polarized delivery of cytotoxic granules. In previous reports, we described the accumulation of connexin 43 (Cx43)-formed gap junctions (GJs) at natural killer (NK) cell-tumor cell cytotoxic immunological synapse. In this report, we demonstrate the functional role of Cx43-GJs at the cytotoxic immunological synapse established between CTLs and melanoma cells during cytotoxicity. Using confocal microscopy, we evaluated Cx43 polarization to the contact site between CTLs isolated from pMEL-1 mice and B16F10 melanoma cells. We knocked down Cx43 expression in B16F10 cells and evaluated its role in the formation of functional GJs and the cytotoxic activity of CTLs, by calcein transfer and granzyme B activity assays, respectively. We found that Cx43 localizes at CTL/B16F10 intercellular contact sites via an antigen-dependent process. We also found that pMEL-1 CTLs but not wild-type naïve CD8+ T cells established functional GJs with B16F10 cells. Interestingly, we observed that Cx43-GJs were required for an efficient granzyme B activity in target B16F10 cells. Using an HLA-A2-restricted/MART-1-specific CD8+ T-cell clone, we confirmed these observations in human cells. Our results suggest that Cx43-channels are relevant components of cytotoxic immunological synapses and potentiate CTL-mediated tumor cell killing.

MCPH1 is essential for cellular adaptation to the G2-phase decatenation checkpoint.

Cellular checkpoints controlling entry into mitosis monitor the integrity of the DNA and delay mitosis onset until the alteration is fully repaired. However, this canonical response can weaken, leading to a spontaneous bypass of the checkpoint, a process referred to as checkpoint adaptation. Here, we have investigated the contribution of microcephalin 1 (MCPH1), mutated in primary microcephaly, to the decatenation checkpoint, a less-understood G2 pathway that delays entry into mitosis until chromosomes are properly disentangled. Our results demonstrate that, although MCPH1 function is dispensable for activation and maintenance of the decatenation checkpoint, it is required for the adaptive response that bypasses the topoisomerase II inhibition----mediated G2 arrest. MCPH1, however, does not confer adaptation to the G2 arrest triggered by the ataxia telangiectasia mutated- and ataxia telangiectasia and rad3 related-based DNA damage checkpoint. In addition to revealing a new role for MCPH1 in cell cycle control, our study provides new insights into the genetic requirements that allow cellular adaptation to G2 checkpoints, a process that remains poorly understood.-Arroyo, M., Kuriyama, R., Guerrero, I., Keifenheim, D., Cañuelo, A., Calahorra, J., Sánchez, A., Clarke, D. J., Marchal, J. A. MCPH1 is essential for cellular adaptation to the G2-phase decatenation checkpoint.

"Sleepers" and "Creepers": A Theoretical Study of Colony Polymorphisms in the Fungus Metarhizium Related to Insect Pathogenicity and Plant Rhizosphere Colonization.

Different strains of Metarhizium exhibit a range of polymorphisms in colony phenotypes. These phenotypes range from highly conidiating colonies to colonies that produce relatively more mycelia and few conidia. These different phenotypes are exhibited in infected insects in the soil. In this paper, we provide a theoretical consideration of colony polymorphisms and suggest that these phenotypes represent a range of strategies in the soil that Metarhizium exhibits. We call these different strategies "sleepers" and "creepers". The "sleeper" phenotype produces relatively greater amounts of conidia. We use the term "sleeper" to identify this phenotype since this strategy is to remain in the soil as conidia in a relatively metabolically inactive state until a host insect or plant encounter these conidia. The "creeper" phenotype is predominantly a mycelial phenotype. In this strategy, hyphae move through the soil until a host insect or plant is encountered. We theoretically model the costs and benefits of these phenotypic polymorphisms and suggest how evolution could possibly select for these different strategies.

An Endocannabinoid Uptake Inhibitor from Black Pepper Exerts Pronounced Anti-Inflammatory Effects in Mice.

Guineensine is a dietary N-isobutylamide widely present in black and long pepper (Piper nigrum and Piper longum) previously shown to inhibit cellular endocannabinoid uptake. Given the role of endocannabinoids in inflammation and pain reduction, here we evaluated guineensine in mouse models of acute and inflammatory pain and endotoxemia. Significant dose-dependent anti-inflammatory effects (95.6 ± 3.1% inhibition of inflammatory pain at 2.5 mg/kg ip and 50.0 ± 15.9% inhibition of edema formation at 5 mg/kg ip) and acute analgesia (66.1 ± 28.1% inhibition at 5.0 mg/kg ip) were observed. Moreover, guineensine inhibited proinflammatory cytokine production in endotoxemia. Intriguingly, guineensine and LPS independently induced catalepsy, but in combination this effect was abolished. Both hypothermia and analgesia were blocked by the CB1 receptor inverse agonist rimonabant, but the pronounced hypolocomotion was CB1 receptor-independent. A subsequent screen of 45 CNS-related receptors, ion channels, and transporters revealed apparent interactions of guineensine with the dopamine transporter DAT, 5HT2A, and sigma receptors, uncovering its prospective polypharmacology. The described potent pharmacological effects of guineensine might relate to the reported anti-inflammatory effects of pepper.

Agrobacterium-Mediated Co-transformation of Multiple Genes in Metarhizium robertsii.

Fungi of the Metarhizium genus are a very versatile model for understanding pathogenicity in insects and their symbiotic relationship with plants. To establish a co-transformation system for the transformation of multiple M. robertsii genes using Agrobacterium tumefaciens, we evaluated whether the antibiotic nourseothricin has the same marker selection efficiency as phosphinothricin using separate vectors. Subsequently, in the two vectors containing the nourseothricin and phosphinothricin resistance cassettes were inserted eGFP and mCherry expression cassettes, respectively. These new vectors were then introduced independently into A. tumefaciens and used to transform M. robertsii either in independent events or in one single co-transformation event using an equimolar mixture of A. tumefaciens cultures. The number of transformants obtained by co-transformation was similar to that obtained by the individual transformation events. This method provides an additional strategy for the simultaneous insertion of multiple genes into M. robertsii.

Soporte vital extracorpóreo en adultos con insuficiencia respiratoria aguda refractaria / Extra-corporeal life support for adults with refractory severe respiratory failure / Suporte vital extracorpóreo em adultos com insuficiência respiratória aguda refratária

Resumen: Introducción: El soporte vital extracorpóreo (ECLS - por sus siglas en inglés extra-corporeal life support) se aplica exitosamente en neonatos con insuficiencia respiratoria aguda (IRA). Las dificultades técnicas, los costos y los resultados desalentadores confinaron a esta técnica como última estrategia en adultos. Nuestro objetivo es reportar nuestra experiencia con ECLS en adultos. Material y métodos: Se analizaron pacientes adultos tratados con ECLS entre febrero de 2002 y enero de 2012. Se recolectaron variables demográficas y cardiopulmonares. Los datos son expresados como mediana (rango). Se analizaron las diferencias entre los pacientes afectados con IRA aislada y con síndrome cardiopulmonar por Hantavirus (SCPH) con test U de Mann Whitney y se consideró estadísticamente significativo un valor de p < 0.05. Resultados: Se aplicó ECLS a15 pacientes. Cinco venoarteriales (VA), siete venovenoso (V-V) y tres asistencias pulmonares extracorpóreas sin bomba (P-ELA). Se describen variables demográficas, cardiopulmonares, diagnóstico, tiempo en ECLS y los desenlaces. Los días en ventilación mecánica (VM), la estadía en UCI y hospital fueron: 17 (4-49), 38 (4-93) y 46 (4-102) días respectivamente. Siete de los 15 pacientes fallecieron (47%). Dos pacientes murieron por shock séptico por bacilos Gram negativos después de un ECLS exitoso. La única diferencia estadísticamente significativa entre los pacientes con IRA aislada (ECLS-VV venovenosa o P-ELA) y los pacientes con SCPH (ECLS-VA venoarterial) fue el lactato al ingreso (p < 0.05). Conclusión: ECLS es una estrategia útil como rescate de adultos con IRA refractaria a estrategias de VM avanzada. El progreso en los equipos y el uso de algoritmos en la toma de decisiones han contribuido a reducir la morbimortalidad.

Abstract: Introduction: Extra-corporeal life support (ECLS) is an established technique for neonates with acute respiratory failure (ARF). Technical difficulties, expense and discouraging outcomes explains its confinement to a last resource tool for adults with ARF. Our objective is report the experience with adult ECLS in two hospitals. Material and methods: All consecutive adult patients treated with different ECLS techniques from 2002 to 2012 were analyzed. Demographic and cardiopulmonary variables were collected. Data are expressed as median (range). The differences between patients affected with ARF isolated and patients with hantavirus cardiopulmonary syndrome was obtained with Mann Whitney U test and a value of p < 0.05 was considered statistically significant. Results: Fifteen patients received ECLS. Five were veno-arterial (VA), 7 veno-venous (VV), and 3 pumpless extracorporeal lung assist (P-ELA). Demographic, cardiopulmonary variables, diagnosis, time on ECLS and outcome are described. Mechanical ventilation (MV), ICU and hospital stay were 17 (4-49), 38 (4-93) and 46 (4-102) days respectively. All patients who required VA-ECLS were affected for Hanta cardiopulmonary syndrome. Seven of fifteen patients died. Two out of five VA-ECLS suffered some degree of lower extremity (arterial cannulation) compartmental syndrome and a mild abnormal gait sequel affected them. Vascular accesses and bleeding were not a concern. Two patients died due to septic shock from gram negative bacilli after leaving ECLS. Conclusion: ECLS for catastrophic ARF in adults is useful therapeutic option to rescue patients who do not respond to conventional MV strategies. The progress in technical devices, use an algorithm to medical decision making contribute to reducing morbidity and mortality.

Resumo: Introdução: O suporte de vida extracorpórea (ECLS) é usado com sucesso em neonatos com insuficiência respiratória aguda (IRA). As dificuldades técnicas, custos e resultados decepcionantes confinaram a esta técnica como última estratégia em adultos. Nosso objetivo é relatar nossa experiência com ECLS em adultos. Métodos: Foram analisados pacientes adultos tratados com ECLS entre 2002 e 2012. Foram recolhidas as variáveis demográficas e cardiopulmonares. Os dados são expressos em mediana (desvio padrão). As diferenças entre os pacientes com IRA isolada e com síndrome cardiopulmonar por Hantavírus foi analisada com o teste U de Mann Whitney e foi considerou um valor estatisticamente significativo p < 0.05. Resultados: Quinze pacientes receberam ECLS. Cinco veno-arteriais (VA), 7 veno-venosas (V-V) e trêis asssistências pulmonares extracorpóreas sem bomba (P-ELA). Se descrevem variáveis demográficas, cardiopulmonar, diagnóstico, tempo no ECLS e os resultados. Os dias de ventilação mecânica (VM), tempo de UTI e hospitalar foram: 17 (4-49), 38 (4-93) e 46 (4-102) dias respectivamente. Sete dos quinze pacientes morreram (47%). Dois pacientes morreram com choque séptico por bacilos gram negativos depois de um ECLS exitoso. A única diferença estatisticamente significativa entre os pacientes com IRA isolada (ECLS-VV ou P-ELA) e pacientes com SCPH (ECLS-VA) foi o lactato na admissão (p < 0.05). Conclusão: O ECLS é uma estratégia útil como resgate de adultos com IRA refractária a estratégias de VM avançada. O progresso dos equipamentos e o uso de algoritmos na tomada de decisões têm ajudado a reduzir a morbimortalidade.

Overexpression of smORF YNR034W-A/EGO4 in Saccharomyces cerevisiae increases the fermentative efficiency of Agave tequilana Weber must.

Fermentative processes are widely used to produce food, beverages and biofuels. Saccharomyces cerevisiae is an efficient ethanol-producing microorganism. However, a concentration of high ethanol and other metabolites can affect yeast viability and decrease the ethanol yield. Many studies have focused on improving the fermentative efficiency, mostly through the genetic engineering of genes that have a direct impact on specific metabolic pathways. In the present study, we characterized a small open reading frame encoding a protein with an unknown function and biological role termed YNR034W-A. We analyzed the expression profile of the YNR034W-A gene during growth and glucose treatment, finding that it is expressed during the diauxic shift and stationary phase and is negatively regulated by glucose. We overexpressed the YNR034W-A gene in the BY4741 laboratory strain and a wild-type yeast strain (AR5) isolated during the Tequila fermentation process. Transformant derivatives of the AR5 strain showed an improved fermentative efficiency during fermentation of Agave tequilana Weber juice. We suggest that the improved fermentative efficiency is the result of a higher stress tolerance response in the YNR034W-A overexpressing transformant.

Safety and efficacy of coil embolization of the septal perforator for septal ablation in patients with hypertrophic obstructive cardiomyopathy.

OBJECTIVE: The objective of this study was to evaluate safety, efficacy, and durability of coil embolization of the major septal perforator of the left anterior descending coronary artery in patients with hypertrophic obstructive cardiomyopathy (HOCM). BACKGROUND: The long-term effect of coil embolization therapy in HOCM patients is not well defined. METHODS: We evaluated 24 symptomatic HOCM patients in a single center who underwent coil embolization of the septal perforator artery(ies). RESULTS: Twenty-four patients on optimal medical therapy presented with NYHA functional class III (75%) or IV (25%) underwent the procedure. The procedure was successful in 22 patients, with significant reduction in left ventricular outflow tract (LVOT) gradient. The functional class significantly improved to class I (54.2%) or II (41.7%) (P < = 0.01). The LVOT gradient was significantly lower during follow up echocardiography (21.3 ± 19 vs. 81.3 ± 41 mm Hg; P < = 0.01). Interventricular septal thickness decreased over time (16.3 ± 3 vs. 18.5 ± 2 mm, P< = 0.01). The procedure was aborted in one of the patients after the third coil prolapsed from the septal perforator in to the left anterior descending artery. The coil was effectively snared out. Three patients required additional coil placement in the second major septal perforator. New permanent pacemaker placement was required in one patient. However, three patients underwent ICD implantation at follow up due to ventricular arrhythmias. CONCLUSIONS: The results of this study suggest that the use of coil embolization for septal ablation is safe, effective, and durable in patients with symptomatic HOCM. © 2016 Wiley Periodicals, Inc.

The Adh1 gene of the fungus Metarhizium anisopliae is expressed during insect colonization and required for full virulence.

Zymography of alcohol dehydrogenase (ADH) activity in the entomopathogenic fungus Metarhizium anisopliae grown under various conditions revealed that micro-aerobic growth was associated with increased ADH activity. The major ADH protein, AdhIp, was purified to homogeneity by affinity chromatography and has an estimated molecular weight of 41kDa and an isoelectric point (pI) of 6.4. Peptide mass fingerprint analysis allowed the identification and cloning of the gene that encodes this protein, Adh1, as annotated in the M. anisopliae genome database. AdhIp is related to the medium-chain dehydrogenase/reductase (MDR)/zinc-dependent alcohol dehydrogenase-like family and contains conserved ADH sequence motifs, such as the zinc-containing ADH signature, the FAD/NAD binding domain and amino acid residues that are conserved in most microbial ADHs. Semi-quantitative RT-PCR analysis revealed that Adh1 gene expression occurs at low levels during early Plutella xylostella infection and that the Adh1 gene was primarily expressed at larval death and as mycelia emerge from the insect cuticle before conidiation. Antisense-RNA experiments indicated that NAD(+)-dependent ADH activity was diminished by 20-75% in the transformants, and the transformants that had lower ADH activity showed allyl alcohol resistance, which indicates that reduction in ADH activity also occurs in vivo. Bioassays performed using antisense adh1 transformants, which have lower ADH activity, showed that LC50 values were two to five times higher than the wild-type, indicating that AdhIp is required for full capability of the fungus to penetrate and/or colonize the insect.

Gap junction intercellular communications regulate NK cell activation and modulate NK cytotoxic capacity.

Gap junctions (GJs) mediate intercellular communication between adjacent cells. Previously, we showed that connexin 43 (Cx43), the main GJ protein in the immune system, mediates Ag transfer between human dendritic cells (DCs) and is recruited to the immunological synapse during T cell priming. This crosstalk contributed to T cell activation, intracellular Ca(2+) responses, and cytokine release. However, the role of GJs in NK cell activation by DCs and NK cell-mediated cytotoxicity against tumor cells remains unknown. In this study, we found polarization of Cx43 at the NK/DC and NK/tumor cell-contact sites, accompanied by the formation of functional GJs between NK/DCs and NK/tumor cells, respectively. Cx43-GJ-mediated intercellular communication (GJIC) between human NK and DCs was bidirectional. Blockage of Cx43-GJIC inhibited NK cell activation, though it affected neither the phenotype nor the function of DCs. Cx43 knockdown or inhibition using mimetic peptides greatly reduced CD69 and CD25 expression and IFN-γ release by DC-stimulated NK cells. Moreover, blocking Cx43 strongly inhibited the NK cell-mediated tumor cell lysis associated with inhibition of granzyme B activity and Ca(2+) influx. Our data identify a novel and active role for Cx43-GJIC in human NK cell activation and antitumor effector functions that may be important for the design of new immune therapeutic strategies.

Nutrient transfer to plants by phylogenetically diverse fungi suggests convergent evolutionary strategies in rhizospheric symbionts.

Most land plants are able to form symbiotic associations with fungi, and in many cases these associations are necessary for plant and fungal survival. These plant/fungal associations are formed with mycorrhizal (arbuscular mycorrhizal or ectomycorrhizal) or endophytic fungi, fungi from distinct phylogenetic lineages. While it has been shown that mycorrhizal fungi are able to transfer nutrients to plant roots in exchange for carbon, endophytes have been thought as asymptomatic colonizers. Recently, however, it has been shown that some insect pathogenic endophytic fungi are able to transfer insect derived nitrogen to plant roots, likely in exchange for plant sugars. Here we explore potential convergent evolutionary strategies for nutrient transfer between insect pathogenic endophytes and mycorrhizal fungus.

Differential expression of insect and plant specific adhesin genes, Mad1 and Mad2, in Metarhizium robertsii.

Metarhizium robertsii is an entomopathogenic fungus that is also plant rhizosphere competent. Two adhesin-encoding genes, Metarhizium adhesin-like protein 1 (Mad1) and Mad2, are involved in insect pathogenesis or plant root colonization, respectively. Here we examined the differential expression of the Mad genes when grown on a variety of soluble (carbohydrates and plant root exudate) and insoluble substrates (locust, tobacco hornworm, and cockroach cuticle, chitin, tomato stems, cellulose, and starch) and during insect, Plutella xylostella, infection. On insect cuticles Mad1 was up regulated, whereas bean root exudate and tomato stems resulted in the up regulation of Mad2. During the early stages of insect infection Mad1 was expressed while Mad2 was not expressed until fungal hyphae emerged and conidiated on the insect cadaver. The regulation of Mad2 was compared to that of other stress-related genes (heat shock protein (Hsp)30, Hsp70, and starvation stress gene A (ssgA)). Mad2 was generally up regulated by nutrient starvation (similar to ssgA) but not by pH, temperature, oxidative or osmotic stresses. Whereas Hsp30 and Hsp70 were generally up regulated at 37 °C or by oxidative stress even under nutrient enriched conditions. We fused the promoter of the Mad2 gene to a marker gene (green fluorescent protein (GFP)) and confirmed that Mad2 was up regulated when M. robertsii was grown in the presence of nutrient starvation. Examination of the promoter region of Mad2 revealed that it possessed two copies of a stress-response element (STRE) known to be regulated under the general stress-response pathway.

Isolation, characterization and expression analysis of the ornithine decarboxylase gene (ODC1) of the entomopathogenic fungus, Metarhizium anisopliae.

The gene ODC1, which codes for the ornithine decarboxylase enzyme, was isolated from the entomopathogenic fungus, Metarhizium anisopliae. The deduced amino acid sequence predicted a protein of 447 amino acids with a molecular weight of 49.3 kDa that contained the canonical motifs of ornithine decarboxylases. The ODC1 cDNA sequence was expressed in Escherichia coli cells; radiometric enzyme assays showed that the purified recombinant protein had ornithine decarboxylase activity. The optimum pH of the purified Odc1 protein was 8.0-8.5, and the optimum reaction temperature was 37°C. The apparent K(m) for ornithine at a pyridoxal phosphate concentration of 20mM was 22 µM. The competitive inhibitor of ODC activity, 1,4-diamino-2-butanone (DAB), at 0.25 mM inhibited 95% of ODC activity. The ODC1 mRNA showed an increase at the beginning of appressorium formation in vitro. During the M. anisopliae invasion process into Plutella xylostella larvae, the ODC1 mRNA showed a discrete increase within the germinating spore and during appressorium formation. The second expression peak was higher and prolonged during the invasion and death of the insect. The ODC1 gene complements the polyamine auxotrophy of Yarrowia lipolytica odc null mutant.

Flexible metabolism in Metarhizium anisopliae and Beauveria bassiana: role of the glyoxylate cycle during insect pathogenesis.

Insect pathogenic fungi such as Metarhizium anisopliae and Beauveria bassiana have an increasing role in the control of agricultural insect pests and vectors of human diseases. Many of the virulence factors are well studied but less is known of the metabolism of these fungi during the course of insect infection or saprobic growth. Here, we assessed enzyme activity and gene expression in the central carbon metabolic pathway, including isocitrate dehydrogenase, aconitase, citrate synthase, malate synthase (MLS) and isocitrate lyase (ICL), with particular attention to the glyoxylate cycle when M. anisopliae and B. bassiana were grown under various conditions. We observed that ICL and MLS, glyoxylate cycle intermediates, were upregulated during growth on 2-carbon compounds (acetate and ethanol) as well as in insect haemolymph. We fused the promoter of the M. anisopliae ICL gene (Ma-icl) to a marker gene (mCherry) and showed that Ma-icl was upregulated when M. anisopliae was grown in the presence of acetate. Furthermore, Ma-icl was upregulated when fungi were engulfed by insect haemocytes as well as during appressorium formation. Addition of the ICL inhibitor 3-nitroproprionate delayed conidial germination and inhibited appressorium formation. These results show that these insect pathogenic fungi have a flexible metabolism that includes the glyoxylate cycle as an integral part of germination, pathogenesis and saprobic growth.