Transcription Factors Tec1 and Tec2 Play Key Roles in the Hyphal Growth and Virulence of Mucor lusitanicus Through Increased Mitochondrial Oxidative Metabolism.

Mucormycosis is a lethal and difficult-to-treat fungal infection caused by fungi of the order Mucorales. Mucor lusitanicus, a member of Mucorales, is commonly used as a model to understand disease pathogenesis. However, transcriptional control of hyphal growth and virulence in Mucorales is poorly understood. This study aimed to investigate the role of Tec proteins, which belong to the TEA/ATTS transcription factor family, in the hyphal development and virulence of M. lusitanicus. Unlike in the genome of Ascomycetes and Basidiomycetes, which have a single Tec homologue, in the genome of Mucorales, two Tec homologues, Tec1 and Tec2, were found, except in that of Phycomyces blakesleeanus, with only one Tec homologue. tec1 and tec2 overexpression in M. lusitanicus increased mycelial growth, mitochondrial content and activity, expression of the rhizoferrin synthetase-encoding gene rfs, and virulence in nematodes and wax moth larvae but decreased cAMP levels and protein kinase A (PKA) activity. Furthermore, tec1- and tec2-overexpressing strains required adequate mitochondrial metabolism to promote the virulent phenotype. The heterotrimeric G beta subunit 1-encoding gene deletant strain (Δgpb1) increased cAMP-PKA activity, downregulation of both tec genes, decreased both virulence and hyphal development, but tec1 and tec2 overexpression restored these defects. Overexpression of allele-mutated variants of Tec1(S332A) and Tec2(S168A) in the putative phosphorylation sites for PKA increased both virulence and hyphal growth of Δgpb1. These findings suggest that Tec homologues promote mycelial development and virulence by enhancing mitochondrial metabolism and rhizoferrin accumulation, providing new information for the rational control of the virulent phenotype of M. lusitanicus.

Role of the nonribosomal peptide synthetase siderophore enzyme (Rfs) of Mucor lusitanicus in controlling the growth of fungal phytopathogens.

Dimorphic species of Mucor, which are cosmopolitan fungi belonging to subphylum Mucoromycotina, are metabolically versatile. Some species of Mucor are sources of biotechnological products, such as biodiesel from Mucor circinelloides and expression of heterologous proteins from Mucor lusitanicus. Furthermore, Mucor lusitanicus has been described as a model for understanding mucormycosis infections. However, little is known regarding the relationship between Mucor lusitanicus and other soil inhabitants. In this study, we investigated the potential use of Mucor lusitanicus as a biocontrol agent against fungal phytopathogens, namely Fusarium oxysporum f. sp. lycopersici, Fusarium solani, and Alternaria solani, which destroy economically important crops. Results showed that aerobic cell-free supernatants of the culture broth (SS) from Mucor lusitanicus inhibited the growth of the fungal phytopathogens in culture, soil, and tomato fruits. The SS obtained from a strain of Mucor lusitanicus carrying the deletion of rfs gene, which encodes an enzyme involved in the synthesis of siderophore rhizoferrin, had a decreased inhibitory effect against the growth of the phytopathogens. Contrarily, this inhibitory effect was more evident with the SS from an rfs-overexpressing strain compared to the wild-type. This study provides a framework for the potential biotechnological use of the molecules secreted from Mucor lusitanicus in the biocontrol of fungal phytopathogens.

Cytotoxicity of extracts and compounds isolated from Croton echioides in animal tumor cell (HTC).

The search for compounds with anticancer effects is of paramount importance today due to the high incidence of the disease. The Euphorbiaceae family is known for having compounds with therapeutic properties, one of its genera being Croton. It has several species, which contain compounds already known for their biological activities, presenting anti-inflammatory, antimicrobial and anticancer properties. Thus, the cytotoxicity/antiproliferative activity of semi-purified fractions and compounds isolated from Croton echioides in liver tumor cells of Rattus norvegicus (HTC) was evaluated by the MTT test. The semi-purified fractions showed cytotoxicity at concentrations above 200 µg/mL, at 24, 48 and 72 hours, reaching cell viability of 24.78% [400 µg/mL] at 24 hours, 12.79% [500 µg/mL] at 48 hours and 10.57% [300 µg/mL] at 72 hours. For the isolated compounds, lupeol had a cytotoxic effect in all concentrations (1, 5, 10, 15, 20, 40, 60, 80 and 100 µg/mL) and tested times (24, 48 and 72 hours), reaching minimum viability of 4.37% [100 µg/mL], within 72 hours. The clerodan diterpenes CEH-1 and CEH-4 also showed antiproliferative activity, with minimum viability of 36.19% [100 µg/mL] over 72 hours and 21.33% [100 µg/mL] over 48 hours, respectively. However, the clerodan diterpenes CEH-2 and CEH-3 did not shows a cytotoxic effect for HTC cells. Thus, there is a cytotoxic/antiproliferative potential of C. echioides against tumor cells, with targeted to mitochondrial enzymes, associated with cell proliferation, indicating that this species deserves prominence in the search for new molecules for the treatment of cancer.

Impacto de la salud oral en la calidad de vida en relación a la caries dental y los determinantes sociales en migrantes venezolanos Lima-2019 / Impact of Oral Health on Quality of Life in Relation to Dental Caries and Social Determinants in Venezuelan Migrants Lima-2019

Resumen Evaluar los resultados del impacto de la salud oral en la calidad de vida mediante el OHIP-14 en relación a la caries dental y los determinantes sociales en migrantes venezolanos. Se realizó un estudio transversal en una población de 350 migrantes venezolanos (30.1±7,4 años; 52% Varones). Donde para evaluar el impacto de la salud oral en la calidad de vida se utilizó el cuestionario Oral Health Impact Profile spanish versión Lima (OHIP-14sp Lima), contrastándose tanto con factores clínicos (índice CPOD) como sociodemográficos, para poder determinar la influencia de estos sobre el instrumento. Se determinó el impacto de la salud oral en la calidad de vida (OHIP-14), según el promedio de puntaje general y por dimensiones. Posteriormente, se realizó un examen clínico evaluando la presencia de lesiones de caries dental (Índice CPOD) y se recolectaron datos sociodemográficos por medio de una encuesta. El promedio del OHIP-14sp Lima generó un impacto negativo del 10.6% en la población migrante. Donde la dimensión de Malestar Psicológico fue la que demostró una mayor frecuencia de impacto negativo con un porcentaje de 38,3%. Los resultados del OHIP-14 sp Lima en el total de migrantes evaluados, fueron bajos en términos porcentuales, generando en su mayoría un promedio de un impacto positivo. Sin embargo, no se puede descartar que factores sociodemográficos como el sexo y niveles de escolaridad; fueron factores que sí generaron un impacto negativo, afectando significativamente los resultados observados en una o varias de las dimensiones evaluadas mediante el instrumento OHIP-14 en el presente estudio.

Abstract To evaluate the results of the impact of oral health on quality of life through the OHIP-14 in relation to dental caries and social determinants in Venezuelan migrants. A cross-sectional study was carried out in a population of 350 Venezuelan migrants (30.1±7.4 years; 52% Men). Where to evaluate the impact of oral health on quality of life, the Oral Health Impact Profile Spanish version Lima questionnaire (OHIP-14sp Lima) was used, contrasting with both clinical (DMFT index) and sociodemographic factors, in order to determine the influence of these on the instrument. The impact of oral health on quality of life (OHIP-14) was determined, according to the average general score and by dimensions. Subsequently, a clinical examination was carried out evaluating the presence of dental caries lesions (DMFT Index) and sociodemographic data were collected through a survey. The average of the OHIP-14sp Lima generated a negative impact of 10.6% on the migrant population. The dimension of "Psychological Discomfort" was the one that showed a higher frequency of negative impact with a percentage of 38.3%. The results of the OHIP-14 sp Lima in the total of evaluated migrants were low in percentage terms, generating mostly an average of a positive impact. However, it cannot be ruled out that sociodemographic factors such as sex and levels of education; they were factors that did generate a negative impact, significantly affecting the results observed in one or more of the dimensions evaluated using the OHIP-14 instrument in the present study.

Real Time Water-In-Oil Emulsion Size Measurement in Optofluidic Channels.

In this work, we investigated a platform for real-time emulsion droplet detection and size measurement in optofluidic platforms. An 8.2 µm core diameter input optical fiber and a multi-mode Gradient Refractive Index (GRIN) output fiber were integrated into an acrylic microfluidic channel platform consisting of three layers. Water-in-oil emulsions were investigated, since relevant applications have emerged in the recent past for these types of emulsions, such as drug encapsulation as well as droplet-based Polymerase Chain Reaction (PCR) amplification of DNA, among others. The main contribution of this work is in understanding the main physical phenomena (i.e., total internal reflection, refraction, and interference) behind the complex transmittance pattern obtained for these droplets. For this purpose, a frequency domain electromagnetic wave propagation modelling of the structure using the Finite Element Method (FEM) was used along with experimental measurements.

Secretion of the siderophore rhizoferrin is regulated by the cAMP-PKA pathway and is involved in the virulence of Mucor lusitanicus.

Mucormycosis is a fungal infection caused by Mucorales, with a high mortality rate. However, only a few virulence factors have been described in these organisms. This study showed that deletion of rfs, which encodes the enzyme for the biosynthesis of rhizoferrin, a siderophore, in Mucor lusitanicus, led to a lower virulence in diabetic mice and nematodes. Upregulation of rfs correlated with the increased toxicity of the cell-free supernatants of the culture broth (SS) obtained under growing conditions that favor oxidative metabolism, such as low glucose levels or the presence of H2O2 in the culture, suggesting that oxidative metabolism enhances virulence through rhizoferrin production. Meanwhile, growing M. lusitanicus in the presence of potassium cyanide, N-acetylcysteine, a higher concentration of glucose, or exogenous cAMP, or the deletion of the gene encoding the regulatory subunit of PKA (pkaR1), correlated with a decrease in the toxicity of SS, downregulation of rfs, and reduction in rhizoferrin production. These observations indicate the involvement of the cAMP-PKA pathway in the regulation of rhizoferrin production and virulence in M. lusitanicus. Moreover, rfs upregulation was observed upon macrophage interaction or during infection with spores in mice, suggesting a pivotal role of rfs in M. lusitanicus infection.

Cytotoxicity of extracts and compounds isolated from Croton echioides in animal tumor cell (HTC) / Citotoxicidade de extratos e compostos isolados de Croton echioides em célula tumoral animal (HTC)

The search for compounds with anticancer effects is of paramount importance today due to the high incidence of the disease. The Euphorbiaceae family is known for having compounds with therapeutic properties, one of its genera being Croton. It has several species, which contain compounds already known for their biological activities, presenting anti-inflammatory, antimicrobial and anticancer properties. Thus, the cytotoxicity/antiproliferative activity of semi-purified fractions and compounds isolated from Croton echioides in liver tumor cells of Rattus norvegicus (HTC) was evaluated by the MTT test. The semi-purified fractions showed cytotoxicity at concentrations above 200 µg/mL, at 24, 48 and 72 hours, reaching cell viability of 24.78% [400 µg/mL] at 24 hours, 12.79% [500 µg/mL] at 48 hours and 10.57% [300 µg/mL] at 72 hours. For the isolated compounds, lupeol had a cytotoxic effect in all concentrations (1, 5, 10, 15, 20, 40, 60, 80 and 100 µg/mL) and tested times (24, 48 and 72 hours), reaching minimum viability of 4.37% [100 µg/mL], within 72 hours. The clerodan diterpenes CEH-1 and CEH-4 also showed antiproliferative activity, with minimum viability of 36.19% [100 µg/mL] over 72 hours and 21.33% [100 µg/mL] over 48 hours, respectively. However, the clerodan diterpenes CEH-2 and CEH-3 did not shows a cytotoxic effect for HTC cells. Thus, there is a cytotoxic/antiproliferative potential of C. echioides against tumor cells, with targeted to mitochondrial enzymes, associated with cell proliferation, indicating that this species deserves prominence in the search for new molecules for the treatment of cancer.

A busca por compostos com efeitos anticâncer é de suma importância nos dias atuais devido à alta incidência desta doença. A família Euphorbiaceae é conhecida por possuir compostos com propriedades terapêuticas, sendo um de seus gêneros o Croton. Este possui diversas espécies, que contêm compostos já conhecidos por suas atividades biológicas, apresentando propriedades anti-inflamatórias, antimicrobianas e anticancerígenas. Assim, a citotoxicidade/atividade antiproliferativa de frações semipurificadas e compostos isolados de Croton echioides em células tumorais hepáticas de Rattus norvegicus (HTC) foi avaliada pelo teste MTT. As frações semipurificadas apresentaram citotoxicidade em concentrações acima de 200 µg/mL, em 24, 48 e 72 horas, atingindo viabilidade celular de 24,78% [400 µg/mL] em 24 horas, 12,79% [500 µg/mL] em 48 horas e 10,57% [300 µg/mL] às 72 horas. Para os compostos isolados, o lupeol teve efeito citotóxico em todas as concentrações (1, 5, 10, 15, 20, 40, 60, 80 e 100 µg/mL) e tempos testados (24, 48 e 72 horas), atingindo a viabilidade mínima de 4,37% [100 µg/mL], em 72 horas. Os diterpenos clerodan CEH-1 e CEH-4 também apresentaram atividade antiproliferativa, com viabilidade mínima de 36,19% [100 µg/mL] em 72 horas e 21,33% [100 µg/mL] em 48 horas, respectivamente. No entanto, os diterpenos clerodanos CEH-2 e CEH-3 não apresentaram efeito citotóxico para células HTC. Assim, existe um potencial citotóxico/antiproliferativo de C. echioides contra células tumorais, com enzimas direcionadas a enzimas mitocondriais, associadas à proliferação celular, indicando que esta espécie merece destaque na busca de novas moléculas para o tratamento do câncer.

Transcript pattern analysis of Arf-family genes in the phytopathogen Fusarium oxysporum f. sp. lycopersici reveals the role of Arl3 in the virulence.

Fusarium oxysporum f. sp. lycopersici is an important plant pathogen that has been used to understand the virulence mechanisms that soil inhabiting fungi exhibit during the infection process. In F. oxysporum many of the virulence factors are secreted, and the secretion process requires the formation of vesicles. Arf family members, represented by Arf (ADP- Ribosylation Factor), Arl (Arf-like), and Sar (Secretion-associated and Ras-related) proteins, are involved in the vesicle creation process. In this study we identified the Arf family members in F. oxysporum f. sp. lycopersici, which includes seven putative proteins: Arf1, Arf3, Arl1 through Arl3, Arl8B, and Sar1. Quantification of the mRNA levels of each arf encoding gene revealed that the highest expression corresponds to arf1 in all tested conditions. The phylogenetic analysis revealed that no other Arf1 paralogue, such as Arf2 from yeast, is present in F. oxysporum f. sp. lycopersici. The essential function suggested of Arf1 in F. oxysporum f. sp. lycopersici was corroborated experimentally when, after several attempts, it was impossible to obtain a knockout mutant in arf1. Moreover, arl3 mRNA levels increased significantly when plant tissue was added as a sole carbon source, suggesting that the product of these genes could play pivotal roles during plant infection, the corresponding mutant ∆arl3 was less virulent compared to the wild-type strain. These results describe the role of arl3 as a critical regulator of the virulence in F. oxysporum f. sp. lycopersici and stablish a framework for the arf family members to be studied in deeper details in this phytopathogen.

Virulence Conferred by PumA Toxin from the Plasmid-Encoded PumAB Toxin-Antitoxin System is Regulated by Quorum System.

Toxin-antitoxin (TA) systems are small genetic elements composed of a toxin gene and its cognate antitoxin that are important for plasmid stabilization (plasmid-encoded) and bacterial virulence (chromosome-encoded). These systems are also related to biofilm and persister cell formations. Pseudomonas aeruginosa is an antibiotic-resistant human pathogen that produces virulence factors modulated by quorum sensing (QS) and can form biofilms. The type II PumAB TA system of pUM505, isolated from a clinical strain of P. aeruginosa, confers plasmid stability. Additionally, the PumA toxin increases P. aeruginosa virulence and is neutralized by the PumB antitoxin. In this study, we determined whether virulence conferred by PumA toxin is regulated by QS. The pumA gene was transferred to P. aeruginosa lasI/rhlI, a mutant strain in the LasI and RhlI QS systems, to analyze the effect on virulence of the transformants. pumA transfer did not increase bacterial virulence in lettuce and Caenorhabditis elegans, suggesting that the virulence conferred by PumA requires QS modulation. pumA mRNA levels drastically decreased in the P. aeruginosa lasI/rhlI (pUC_pumA) strain, suggesting positive regulation of pumA gene expression by QS. Supplementation of the growth medium of P. aeruginosa lasI/rhlI (pUC_pumA) with C4-AHL and 3-oxo-C12-AHL autoinducers increased pumA mRNA levels and restored bacterial virulence, suggesting that both autoinducers complemented the mutations and positively regulated the toxic effects of PumA. This strengthened the hypothesis that QS regulates bacterial virulence conferred by the PumA toxin. Thus, this report establishes an important function of QS in the virulence conferred by plasmid-encoded TA systems in bacterial pathogens.

Arf-like proteins (Arl1 and Arl2) are involved in mitochondrial homeostasis in Mucor circinelloides.

Mucor circinelloides is an opportunistic dimorphic pathogen, with the dimorphic process controlled in parts by fermentative and oxidative metabolisms, which lead to yeast or mycelial growth, respectively. Dimorphic transition is important for pathogenesis since the mycelium represents the virulent morphology. We previously reported that the deletion of arl1 or arl2 stimulate anaerobic germination in M. circinelloides, suggesting an augmented fermentative metabolism. In the present study, we demonstrate that the heterokaryon Δarl1(+)(-) and homokaryon Δarl2 strains contain low number of mitochondria, which possibly results in a dysfunctional oxidative metabolism, marked by a low oxygen consumption in glucose and poor growth in glycerol as the unique carbon source. This dysfunction is compensated for by an increase in the glycolysis and fermentation in aerobic conditions, demonstrating growth kinetics similar to that in the wild-type strain. Moreover, as a consequence a high fermentative activity, the Δarl1(+)(-) and Δarl2 strains possibly increased the yeast cell growth during low oxygen concentrations in presence of glucose. To the best of our knowledge, this is the first study to demonstrate the control of members of Arf family on the mitochondrial population in a Mucor species.

Chagas vectors Panstrongylus chinai (Del Ponte, 1929) and Panstrongylus howardi (Neiva, 1911): chromatic forms or true species?

BACKGROUND: Chagas disease is a parasitic infection transmitted by "kissing bugs" (Hemiptera: Reduviidae: Triatominae) that has a huge economic impact in Latin American countries. The vector species with the upmost epidemiological importance in Ecuador are Rhodnius ecuadoriensis (Lent & Leon, 1958) and Triatoma dimidiata (Latreille, 1811). However, other species such as Panstrongylus howardi (Neiva, 1911) and Panstrongylus chinai (Del Ponte, 1929) act as secondary vectors due to their growing adaptation to domestic structures and their ability to transmit the parasite to humans. The latter two taxa are distributed in two different regions, they are allopatric and differ mainly by their general color. Their relative morphological similarity led some authors to suspect that P. chinai is a melanic form of P. howardi. METHODS: The present study explored this question using different approaches: antennal phenotype; geometric morphometrics of heads, wings and eggs; cytogenetics; molecular genetics; experimental crosses; and ecological niche modeling. RESULTS: The antennal morphology, geometric morphometrics of head and wing shape and cytogenetic analysis were unable to show distinct differences between the two taxa. However, geometric morphometrics of the eggs, molecular genetics, ecological niche modeling and experimental crosses including chromosomal analyses of the F1 hybrids, in addition to their coloration and current distribution support the hypothesis that P. chinai and P. howardi are separate species. CONCLUSIONS: Based on the evidence provided here, P. howardi and P. chinai should not be synonymized. They represent two valid, closely related species.

Sporulation on blood serum increases the virulence of Mucor circinelloides.

Mucor circinelloides is an opportunistic human pathogen that is used to study mucormycosis, a rare but lethal infection in susceptible immunosuppressed patients. However, the virulence characteristics of this pathogen have not been fully elucidated. In this study, sporangiospores (spores) produced on YPG medium supplemented with native blood serum increased the virulence of M. circinelloides compared with spores produced on YPG supplemented with denatured blood serum or on YPG alone. The spores produced from YPG supplemented with native blood serum increased nematode death and led to significant increases in interleukin (IL)-6, IL-1ß, macrophage inhibitory protein-2, and tumour necrosis factor α mRNA levels in liver and lung tissues from infected diabetic mice compared with those in tissues from animals infected with spores produced in the presence of YPG supplemented with denatured blood serum or of YPG alone. Moreover, spores produced from cultures supplemented with native blood serum showed increased germination rates and longer hyphae compared with other spores. The spores produced in YPG supplemented with native blood serum also enhanced resistance to stress factors and H2O2 and increased thermotolerance compared with spores produced under other conditions. In addition, spores produced in presence of blood serum increased the ability of the pathogen to survive in the presence of macrophages. Taken together, our results showed that these factors were important features for fungal virulence in humans and suggested that thermolabile components in the blood serum may induce M. circinelloides virulence.

Alcohol dehydrogenase 1 participates in the Crabtree effect and connects fermentative and oxidative metabolism in the Zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic Zygomycete fungus that produces ethanol under aerobic conditions in the presence of glucose, which indicates that it is a Crabtree-positive fungus. To determine the physiological role of the alcohol dehydrogenase (ADH) activity elicited under these conditions, we obtained and characterized an allyl alcohol-resistant mutant that was defective in ADH activity, and examined the effect of adh mutation on physiological parameters related to carbon and energy metabolism. Compared to the Adh+ strain R7B, the ADH-defective (Adh-) strain M5 was unable to grow under anaerobic conditions, exhibited a considerable reduction in ethanol production in aerobic cultures when incubated with glucose, had markedly reduced growth capacity in the presence of oxygen when ethanol was the sole carbon source, and exhibited very low levels of NAD+-dependent alcohol de-hydrogenase activity in the cytosolic fraction. Further characterization of the M5 strain showed that it contains a 10-bp deletion that interrupts the coding region of the adhl gene. Complementation with the wild-type allele adh1+ by transformation of M5 remedied all the defects caused by the adh1 mutation. These findings indicate that in M. circinelloides, the product of the adh1 gene mediates the Crabtree effect, and can act as either a fermentative or an oxidative enzyme, depending on the nutritional conditions, thereby participating in the association between fermentative and oxidative metabolism. It was found that the spores of M. circinelloides possess low mRNA levels of the ethanol assimilation genes (adl2 and acs2), which could explain their inability to grow in the alcohol.

Role of Arf-like proteins (Arl1 and Arl2) of Mucor circinelloides in virulence and antifungal susceptibility.

Mucor circinelloides is an etiologic agent of mucormycosis, a fungal infection produced by Mucorales often associated with mortality due to unavailability of antifungal drugs. Arl proteins belong to the Arf family and are involved in vesicle trafficking and tubulin assembly. This study identified two Arl (Arf-like)-encoding genes, arl1 and arl2, in M. circinelloides and explored their function in morphogenesis, virulence, and antifungal susceptibility. Although Arl1 and Arl2 proteins shared 55% amino acid sequence identity, arl1 and arl2 genes showed distinct transcriptional expression patterns. arl1 was expressed at higher levels than arl2 and induced in mycelia, suggesting a role in morphological transitions. Disruption of the arl1 and arl2 genes led to heterokaryon (Δarl1(+)(-)) and homokaryon (Δarl2) genotypes, respectively. The incapacity to generate homokaryon mutants for arl1 suggested that it is essential for growth of M. circinelloides. Deletion of each gene reduced the expression of the other, suggesting the existence of a positive cross-regulation between them. Thus, deletion of arl2 resulted in a ~60% reduction of arl1 expression, whereas the Δarl1(+)(-) showed ∼90% reduction of arl1 expression. Mutation of arl2 showed no phenotype or a mild phenotype between Δarl1(+)(-) and wild-type (WT), suggesting that all observed phenotypes in both mutant strains corresponded to arl1 low expression. The Δarl1(+)(-) produced a small amount of spores that showed increased sensitivity to dodecyl-sulfate and azoles, suggesting a defect in the cell wall that was further supported by decrease in saccharide content. These defects in the cell wall were possibly originated by abnormal vesicle trafficking since FM4-64 staining of both mutants Δarl1(+)(-) and Δarl2 revealed less well-localized endosomes compared to the WT. Moreover, aberrant vesicle trafficking may be responsible for the secretion of specific virulence-related proteins since cell-free medium from Δarl1(+)(-) were found to increase killing of Caenorhabditis elegans compared to WT.

Control of morphology and virulence by ADP-ribosylation factors (Arf) in Mucor circinelloides.

Mucor circinelloides is a dimorphic fungus used to study cell differentiation that has emerged as a model to characterize mucormycosis. In this work, we identified four ADP-ribosylation factor (Arf)-encoding genes (arf1-arf4) and study their role in the morphogenesis and virulence. Arfs are key regulators of the vesicular trafficking process and are associated with both growth and virulence in fungi. Arf1 and Arf2 share 96% identity and Arf3 and Arf4 share 89% identity, which suggests that the genes arose through gene-duplication events in M. circinelloides. Transcription analysis revealed that certain arf genes are affected by dimorphism of M. circinelloides, such as the arf2 transcript, which was accumulated during yeast development. Therefore, we created knockout mutants of four arf genes to evaluate their function in dimorphism and virulence. We found that both arf1 and arf2 are required for sporulation, but these genes also perform distinct functions; arf2 participates in yeast development, whereas arf1 is involved in aerobic growth. Conversely, arf3 and arf4 play only minor roles during aerobic growth. Moreover, we observed that all single arf-mutant strains are more virulent than the wild-type strain in mouse and nematode models, with the arf3 mutant being most virulent. Lastly, arf1/arf2 and arf3/arf4 double mutations produced heterokaryon strains that did not reach the homokaryotic state, indicating that these genes participate in essential and redundant functions. Overall, this work reveals that Arfs proteins regulate important cellular processes in M. circinelloides such as morphogenesis and virulence, laying the foundation to characterize the molecular networks underlying this regulation.

Erratum: The Bethe-Slater curve revisited; new insights from electronic structure theory.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

The Bethe-Slater curve revisited; new insights from electronic structure theory.

The Bethe-Slater (BS) curve describes the relation between the exchange coupling and interatomic distance. Based on a simple argument of orbital overlaps, it successfully predicts the transition from antiferromagnetism to ferromagnetism, when traversing the 3d series. In a previous article [Phys. Rev. Lett. 116, 217202 (2016)] we reported that the dominant nearestneighbour (NN) interaction for 3d metals in the bcc structure indeed follows the BS curve, but the trends through the series showed a richer underlying physics than was initially assumed. The orbital decomposition of the inter-site exchange couplings revealed that various orbitals contribute to the exchange interactions in a highly non-trivial and sometimes competitive way. In this communication we perform a deeper analysis by comparing 3d metals in the bcc and fcc structures. We find that there is no coupling between the E g orbitals of one atom and T 2g orbitals of its NNs, for both cubic phases. We demonstrate that these couplings are forbidden by symmetry and formulate a general rule allowing to predict when a similar situation is going to happen. In γ-Fe, as in α-Fe, we find a strong competition in the symmetry-resolved orbital contributions and analyse the differences between the high-spin and low-spin solutions.

Selection of reference genes for quantitative real time RT-PCR during dimorphism in the zygomycete Mucor circinelloides.

Mucor circinelloides is a dimorphic fungal model for studying several biological processes including cell differentiation (yeast-mold transitions) as well as biodiesel and carotene production. The recent release of the first draft sequence of the M. circinelloides genome, combined with the availability of analytical methods to determine patterns of gene expression, such as quantitative Reverse transcription-Polymerase chain reaction (qRT-PCR), and the development of molecular genetic tools for the manipulation of the fungus, may help identify M. circinelloides gene products and analyze their relevance in different biological processes. However, no information is available on M. circinelloides genes of stable expression that could serve as internal references in qRT-PCR analyses. One approach to solve this problem consists in the use of housekeeping genes as internal references. However, validation of the usability of these reference genes is a fundamental step prior to initiating qRT-PCR assays. This work evaluates expression of several constitutive genes by qRT-PCR throughout the morphological differentiation stages of M. circinelloides; our results indicate that tfc-1 and ef-1 are the most stable genes for qRT-PCR assays during differentiation studies and they are proposed as reference genes to carry out gene expression studies in this fungus.

Evaluation of pulmonary function in European land tortoises using whole-body plethysmography.

The aim of this study was to evaluate the use of whole-body plethysmography as a non-invasive method to determine the respiratory parameters and profiles in two tortoise species belonging to the genus Testudo. Pulmonary functions and volumetric parameters were determined in 10 adults of Testudo hermanni and in seven Testudo marginata animals, using whole-body plethysmography. A profile pattern was regularly observed: an inspiratory flow peak, an expiratory peak, an apnoea phase and a second expiratory peak, previous to the beginning of the next respiratory cycle. Positive and significant correlation was observed between the inspiratory time, weight and length of the tortoises. Larger tortoises showed a higher time of inhalation. The peak of inspiratory flow was correlated with the sex, being longer in the females. T. marginata had an inspiratory time longer than that of T. hermanii. In T. hermanii, differences related to the sex were observed in the tidal volume, peak inspiratory flow, peak expiratory flow, expiratory flow of 50 per cent and enhanced pause, which could be related to the smaller size of males. The results suggest that additional information on new technologies currently used in pet medicine or even in human medicine should be developed and adjusted as alternative ways to support the rehabilitation of turtles and tortoises.