Nanosized extracellular vesicles released by Neurospora crassa hyphae.

Extracellular vesicles (EVs) are nanosized structures containing proteins, lipids, and nucleic acids, released by living cells to the surrounding medium. EVs participate in diverse processes, such as intercellular communication, virulence, and disease. In pathogenic fungi, EVs carry enzymes that allow them to invade the host or undergo environmental adaptation successfully. In Neurospora crassa, a non-pathogenic filamentous fungus widely used as a model organism, the vesicle-dependent secretory mechanisms that lead to polarized growth are well studied. In contrast, biosynthesis of EVs in this fungus has been practically unexplored. In the present work, we analyzed N. crassa culture's supernatant for the presence of EVs by dynamic light scattering (DLS), transmission electron microscopy (TEM) and proteomic analysis. We identified spherical membranous structures, with a predominant subpopulation averaging a hydrodynamic diameter (dh) of 68 nm and a particle diameter (dp) of 38 nm. EV samples stained with osmium tetroxide vapors were better resolved than those stained with uranyl acetate. Mass spectrometry analysis identified 252 proteins, including enzymes involved in carbohydrate metabolic processes, oxidative stress response, cell wall organization/remodeling, and circadian clock-regulated proteins. Some of these proteins have been previously reported in exosomes from human cells or in EVs of other fungi. In view of the results, it is suggested a putative role for EVs in cell wall biosynthesis and vegetative development in N. crassa.

F-actin dynamics following mechanical injury of Trichoderma atroviride and Neurospora crassa hyphae.

We investigated hyphae regeneration in Trichoderma atroviride and Neurospora crassa, with particular focus on determining the role of the actin cytoskeleton after mechanical injury. Filamentous actin (F-actin) dynamics was observed by live-cell confocal microscopy in both T. atroviride and N. crassa strains expressing Lifeact-GFP. In growing hyphae of both fungi, F-actin localized in three different structural forms: patches, cables and actomyosin rings. Most patches were conspicuously arranged in a collar in the hyphal subapex. A strong F-actin signal, likely actin filaments, colocalized with the core of the Spitzenkörper. Filaments and cables of F-actin were observed along the cortex throughout hyphae. Following mechanical damage at the margin of growing mycelia of T. atroviride and N. crassa, the severed hyphae lost their cytoplasmic contents, but plugging of the septal pore by a Woronin body occured, and the rest of the hyphal tube remained whole. In both fungi, patches of F-actin began accumulating next to the plugged septum. Regeneration was attained by the emergence of a new hyphal tube as an extension of the plugged septum wall. The septum wall was gradually remodeled into the apical wall of the emerging hypha. Whereas in T. atroviride the re-initiation of polarized growth took  âˆ¼ 1 h, in N. crassa, actin patch accumulation began almost immediately, and new growing hyphae were observed âˆ¼ 30 min after injury. By confocal microscopy, we found that chitin synthase 1 (CHS-1), a microvesicle (chitosome) component, accumulated next to the plugged septum in regenerating hyphae of N. crassa. We concluded that the actin cytoskeleton plays a key role in hyphal regeneration by supporting membrane remodeling, helping to facilitate transport of vesicles responsible for new wall growth and organization of the new tip-growth apparatus.

Danger signals activate a putative innate immune system during regeneration in a filamentous fungus.

The ability to respond to injury is a biological process shared by organisms of different kingdoms that can even result in complete regeneration of a part or structure that was lost. Due to their immobility, multicellular fungi are prey to various predators and are therefore constantly exposed to mechanical damage. Nevertheless, our current knowledge of how fungi respond to injury is scarce. Here we show that activation of injury responses and hyphal regeneration in the filamentous fungus Trichoderma atroviride relies on the detection of two danger or alarm signals. As an early response to injury, we detected a transient increase in cytosolic free calcium ([Ca2+]c) that was promoted by extracellular ATP, and which is likely regulated by a mechanism of calcium-induced calcium-release. In addition, we demonstrate that the mitogen activated protein kinase Tmk1 plays a key role in hyphal regeneration. Calcium- and Tmk1-mediated signaling cascades activated major transcriptional changes early following injury, including induction of a set of regeneration associated genes related to cell signaling, stress responses, transcription regulation, ribosome biogenesis/translation, replication and DNA repair. Interestingly, we uncovered the activation of a putative fungal innate immune response, including the involvement of HET domain genes, known to participate in programmed cell death. Our work shows that fungi and animals share danger-signals, signaling cascades, and the activation of the expression of genes related to immunity after injury, which are likely the result of convergent evolution.

A Trichoderma atroviride stress-activated MAPK pathway integrates stress and light signals.

Cells possess stress-activated protein kinase (SAPK) signalling pathways, which are activated practically in response to any cellular insult, regulating responses for survival and adaptation to harmful environmental changes. To understand the function of SAPK pathways in T. atroviride, mutants lacking the MAPKK Pbs2 and the MAPK Tmk3 were analysed under several cellular stresses, and in their response to light. All mutants were highly sensitive to cellular insults such as osmotic and oxidative stress, cell wall damage, high temperature, cadmium, and UV irradiation. Under oxidative stress, the Tmk3 pathway showed specific roles during development, which in conidia are essential for tolerance to oxidant agents and appear to play a minor role in mycelia. The function of this pathway was more evident in Δpbs2 and Δtmk3 mutant strains when combining oxidative stress or cell wall damage with light. Light stimulates tolerance to osmotic stress through Tmk3 independently of the photoreceptor Blr1. Strikingly, photoconidiation and expression of blue light regulated genes was severally affected in Δtmk3 and Δpbs2 strains, indicating that this pathway regulates light responses. Furthermore, Tmk3 was rapidly phosphorylated upon light exposure. Thus, our data indicate that Tmk3 signalling cooperates with the Blr photoreceptor complex in the activation of gene expression.

Socioeconomic status and asthma control in African American youth in SAGE II.

OBJECTIVE: African Americans are disproportionately burdened by asthma. We assessed the individual and joint contribution of socioeconomic status (SES) on asthma morbidity among African American youth. METHODS: We examined 686 African Americans (8-21 years) with asthma. To account for the joint effects of SES, a composite index was derived from maternal educational attainment, household income, and insurance status. Ordinal logistic regression was used to estimate the individual and joint effect of SES on asthma control. Models were adjusted for age, sex, controller medication use, in utero smoke exposure, family history of asthma, family history of rhinitis, breastfeeding, daycare attendance, and mold exposure. RESULTS: Participants were classified as Poorly Controlled Asthma (40.8%), Partially Controlled Asthma (29.7%), or Controlled Asthma (30.2%). Of the individual SES indicators, low income was the strongest predictor of poor asthma control. Children with low income had worse asthma control than those with higher income (OR 1.39; 95% CI 0.92-2.12). The SES index ranged from 4-9. SES was associated with 17% increased odds of poor asthma control with each decrease in the index (95% CI 1.05-1.32). The SES index was associated with asthma-related symptoms, nocturnal awakenings, limited activity, and missed school days. CONCLUSIONS: The negative effects of SES were observed along the entire socioeconomic gradient, and the adverse asthma outcomes observed in African American youth were not limited to the very poor. We also found that the SES index may be a more consistent and useful predictor of poor asthma outcomes than each indicator alone.

Obesity and bronchodilator response in black and Hispanic children and adolescents with asthma.

BACKGROUND: Obesity is associated with poor asthma control, increased asthma morbidity, and decreased response to inhaled corticosteroids. We hypothesized that obesity would be associated with decreased bronchodilator responsiveness in children and adolescents with asthma. In addition, we hypothesized that subjects who were obese and unresponsive to bronchodilator would have worse asthma control and would require more asthma controller medications. METHODS: In the Study of African Americans, Asthma, Genes, and Environments (SAGE II) and the Genes-environments and Admixture in Latino Americans (GALA II) study, two identical, parallel, case-control studies of asthma, we examined the association between obesity and bronchodilator response in 2,963 black and Latino subjects enrolled from 2008 to 2013 using multivariable logistic regression. Using bronchodilator responsiveness, we compared asthma symptoms, controller medication usage, and asthma exacerbations between nonobese (< 95th% BMI) and obese (≥ 95th% BMI) subjects. RESULTS: The odds of being bronchodilator unresponsive were 24% (OR, 1.24; 95% CI, 1.03-1.49) higher among obese children and adolescents compared with their not obese counterparts after adjustment for age, race/ethnicity, sex, recruitment site, baseline lung function (FEV1/FVC), and controller medication. Bronchodilator-unresponsive obese subjects were more likely to report wheezing (OR, 1.38; 95% CI, 1.13-1.70), being awakened at night (OR, 1.34; 95% CI, 1.09-1.65), using leukotriene receptor inhibitors (OR, 1.33; 95% CI, 1.05-1.70), and using inhaled corticosteroid with long-acting ß2-agonist (OR, 1.37; 95% CI, 1.05-1.78) than were their nonobese counterpart. These associations were not seen in the bronchodilator-responsive group. CONCLUSIONS: Obesity is associated with bronchodilator unresponsiveness among black and Latino children and adolescents with asthma. The findings on obesity and bronchodilator unresponsiveness represent a unique opportunity to identify factors affecting asthma control in blacks and Latinos.

Extracellular ATP activates MAPK and ROS signaling during injury response in the fungus Trichoderma atroviride.

The response to mechanical damage is crucial for the survival of multicellular organisms, enabling their adaptation to hostile environments. Trichoderma atroviride, a filamentous fungus of great importance in the biological control of plant diseases, responds to mechanical damage by activating regenerative processes and asexual reproduction (conidiation). During this response, reactive oxygen species (ROS) are produced by the NADPH oxidase complex. To understand the underlying early signaling events, we evaluated molecules such as extracellular ATP (eATP) and Ca(2+) that are known to trigger wound-induced responses in plants and animals. Concretely, we investigated the activation of mitogen-activated protein kinase (MAPK) pathways by eATP, Ca(2+), and ROS. Indeed, application of exogenous ATP and Ca(2+) triggered conidiation. Furthermore, eATP promoted the Nox1-dependent production of ROS and activated a MAPK pathway. Mutants in the MAPK-encoding genes tmk1 and tmk3 were affected in wound-induced conidiation, and phosphorylation of both Tmk1 and Tmk3 was triggered by eATP. We conclude that in this fungus, eATP acts as a damage-associated molecular pattern (DAMP). Our data indicate the existence of an eATP receptor and suggest that in fungi, eATP triggers pathways that converge to regulate asexual reproduction genes that are required for injury-induced conidiation. By contrast, Ca(2+) is more likely to act as a downstream second messenger. The early steps of mechanical damage response in T. atroviride share conserved elements with those known from plants and animals.

Análisis genético del polimorfismo del virus de la inmunodeficiencia humana en pacientes colombianos / Mutational analysis of HIV in Colombian patients

Objetivo. La resistencia a los medicamentos antirretrovirales se ha asociado con mutaciones características en los genes que codifican las enzimas que son el blanco de la terapia antirretroviral. En el presente trabajo se busca evaluar, desde el punto devista cualitativo y cuantitativo, las diferentes mutaciones reveladas por la tipificación molecular de virus procedentes de diferentes pacientes remitidos al Instituto de Referencia Andino, en Bogotá, para la genotipificación con fines terapéuticos.Diseño: Se hizo la tipificación de un total de 1.064 mutaciones diferentes en virusprocedentes de 16 pacientes no relacionados entre sí, con solicitud de genotipificación del VIH para análisis de sensibilidad a antirretrovirales. Se procedió a la tabulación de las mutaciones encontradas en cuatro categorías principales: a- mutaciones asociadas a resistencia, b- mutaciones silenciosas, c- polimorfismos genéticos por fuera de los sitios asociados a resistencia, y de mutaciones en sitios de resistencia que hasta el momento no han sido asociadas a resistencia.Metodología. Se seleccionaron, en estricto orden de llegada, 16 muestras de sangre en EDTA de pacientes con solicitud de genotipificación del VIH para análisis de sensibilidad a antirretrovirales. Se extrajo el ARN viral de cada muestra por el métodoQIAamp® y se procedió a su amplificación por medio de la PCR con transcriptasa inversa (RT-PCR). Una vez amplificado el ácido nucleico viral, se procedió a su tipificación molecular en el secuenciador LongReadTower-Opengene®, utilizando el estuche Trugene HIV-1®. Las secuencias obtenidas se transcribieron a hoja electrónica Excel®, y se hizo un cálculo de frecuencias de mutaciones por conteo directo...

Objective: Resistance to antiretroviral drugs has been associated with characteristic mutations in the genes that encode enzymes which are the target of anti-retroviral therapy. In this paper we evaluate the different mutations revealed by molecular typing of viruses from differentpatients who were referred to be genotyped at the Instituto de Referencia Andino in Bogotá fortherapeutic purposes. Design: A total number of 1064 mutations in viruses from 16 HIV infected unrelated patientswere initially genotyped for the analysis of sensitivity to antiretrovirals. Afterwards, weproceeded to the tabulation of the mutations found in four main categories: a- resistance mutations, b- silent mutations, c-genetic polymorphisms outside of sites associated with resistance, and d- mutations at sites not yet associatedwith resistance. Materials and methods: Sixteen EDTA blood samples drawn from patients with HIV genotyping application for analysis of sensitivity to antiretroviral drugs were selected in strict order of arrival. Viral RNA was extracted from each sample by the QIAamp® method, and we then proceeded to its amplification by reverse transcriptase PCR (RT-PCR). Once the viral nucleic acid was successfully amplified, we proceededto molecular typing in the sequencer LongRead-Tower-Opengene®, using the Trugene® HIV-1 kit. The sequences obtained were transcribed toan Excel® spreadsheet, to calculate the frequencies of mutations by direct counting...