Evaluation of Lipid Extracts from the Marine Fungi Emericellopsis cladophorae and Zalerion maritima as a Source of Anti-Inflammatory, Antioxidant and Antibacterial Compounds.

Marine environments occupy more than 70% of the earth's surface, integrating very diverse habitats with specific characteristics. This heterogeneity of environments is reflected in the biochemical composition of the organisms that inhabit them. Marine organisms are a source of bioactive compounds, being increasingly studied due to their health-beneficial properties, such as antioxidant, anti-inflammatory, antibacterial, antiviral, or anticancer. In the last decades, marine fungi have stood out for their potential to produce compounds with therapeutic properties. The objective of this study was to determine the fatty acid profile of isolates from the fungi Emericellopsis cladophorae and Zalerion maritima and assess the anti-inflammatory, antioxidant, and antibacterial potential of their lipid extracts. The analysis of the fatty acid profile, using GC-MS, showed that E. cladophorae and Z. maritima possess high contents of polyunsaturated fatty acids, 50% and 34%, respectively, including the omega-3 fatty acid 18:3 n-3. Emericellopsis cladophorae and Z. maritima lipid extracts showed anti-inflammatory activity expressed by the capacity of their COX-2 inhibition which was 92% and 88% of inhibition at 200 µg lipid mL-1, respectively. Emericellopsis cladophorae lipid extracts showed a high percentage of inhibition of COX -2 activity even at low concentrations of lipids (54% of inhibition using 20 µg lipid mL-1), while a dose-dependent behaviour was observed in Z. maritima. The antioxidant activity assays of total lipid extracts demonstrated that the lipid extract from E. cladophorae did not show antioxidant activity, while Z. maritima gave an IC20 value of 116.6 ± 6.2 µg mL-1 equivalent to 92.1 ± 4.8 µmol Trolox g-1 of lipid extract in the DPPH• assay, and 101.3 ± 14.4 µg mL-1 equivalent to 106.6 ± 14.8 µmol Trolox g-1 of lipid extract in the ABTS•+ assay. The lipid extract of both fungal species did not show antibacterial properties at the concentrations tested. This study is the first step in the biochemical characterization of these marine organisms and demonstrates the bioactive potential of lipid extracts from marine fungi for biotechnological applications.

Genome and Metabolome MS-Based Mining of a Marine Strain of Aspergillus affinis.

Aspergillus section Circumdati encompasses several species that express both beneficial (e.g., biochemical transformation of steroids and alkaloids, enzymes and metabolites) and harmful compounds (e.g., production of ochratoxin A (OTA)). Given their relevance, it is important to analyze the genetic and metabolic diversity of the species of this section. We sequenced the genome of Aspergillus affinis CMG 70, isolated from sea water, and compared it with the genomes of species from section Circumdati, including A. affinis's strain type. The A. affinis genome was characterized considering secondary metabolites biosynthetic gene clusters (BGCs), carbohydrate-active enzymes (CAZymes), and transporters. To uncover the biosynthetic potential of A. affinis CMG 70, an untargeted metabolomics (LC-MS/MS) approach was used. Cultivating the fungus in the presence and absence of sea salt showed that A. affinis CMG 70 metabolite profiles are salt dependent. Analyses of the methanolic crude extract revealed the presence of both unknown and well-known Aspergillus compounds, such as ochratoxin A, anti-viral (e.g., 3,5-Di-tert-butyl-4-hydroxybenzoic acid and epigallocatechin), anti-bacterial (e.g., 3-Hydroxybenzyl alcohol, l-pyroglutamic acid, lecanoric acid), antifungal (e.g., lpyroglutamic acid, 9,12,13-Trihydroxyoctadec-10-enoic acid, hydroxyferulic acid), and chemotherapeutic (e.g., daunomycinone, mitoxantrone) related metabolites. Comparative analysis of 17 genomes from 16 Aspergillus species revealed abundant CAZymes (568 per species), secondary metabolite BGCs (73 per species), and transporters (1359 per species). Some BGCs are highly conserved in this section (e.g., pyranonigrin E and UNII-YC2Q1O94PT (ACR toxin I)), while others are incomplete or completely lost among species (e.g., bikaverin and chaetoglobosins were found exclusively in series Sclerotiorum, while asperlactone seemed completely lost). The results of this study, including genome analysis and metabolome characterization, emphasize the molecular diversity of A. affinis CMG 70, as well as of other species in the section Circumdati.

Genomic and Metabolomic Analyses of the Marine Fungus Emericellopsis cladophorae: Insights into Saltwater Adaptability Mechanisms and Its Biosynthetic Potential.

The genus Emericellopsis is found in terrestrial, but mainly in marine, environments with a worldwide distribution. Although Emericellopsis has been recognized as an important source of bioactive compounds, the range of metabolites expressed by the species of this genus, as well as the genes involved in their production are still poorly known. Untargeted metabolomics, using UPLC- QToF-MS/MS, and genome sequencing (Illumina HiSeq) was performed to unlock E. cladophorae MUM 19.33 chemical diversity. The genome of E. cladophorae is 26.9 Mb and encodes 8572 genes. A large set of genes encoding carbohydrate-active enzymes (CAZymes), secreted proteins, transporters, and secondary metabolite biosynthetic gene clusters were identified. Our analysis also revealed genomic signatures that may reflect a certain fungal adaptability to the marine environment, such as genes encoding for (1) the high-osmolarity glycerol pathway; (2) osmolytes' biosynthetic processes; (3) ion transport systems, and (4) CAZymes classes allowing the utilization of marine polysaccharides. The fungal crude extract library constructed revealed a promising source of antifungal (e.g., 9,12,13-Trihydroxyoctadec-10-enoic acid, hymeglusin), antibacterial (e.g., NovobiocinA), anticancer (e.g., daunomycinone, isoreserpin, flavopiridol), and anti-inflammatory (e.g., 2'-O-Galloylhyperin) metabolites. We also detected unknown compounds with no structural match in the databases used. The metabolites' profiles of E. cladophorae MUM 19.33 fermentations were salt dependent. The results of this study contribute to unravel aspects of the biology and ecology of this marine fungus. The genome and metabolome data are relevant for future biotechnological exploitation of the species.

Toxicity of Recombinant Necrosis and Ethylene-Inducing Proteins (NLPs) from Neofusicoccum parvum.

Neofusicoccum parvum is a fungal pathogen associated with a wide range of plant hosts. Despite being widely studied, the molecular mechanism of infection of N. parvum is still far from being understood. Analysis of N. parvum genome lead to the identification of six putative genes encoding necrosis and ethylene-inducing proteins (NLPs). The sequence of NLPs genes (NprvNep 1-6) were analyzed and four of the six NLP genes were successfully cloned, expressed in E. coli and purified by affinity chromatography. Pure recombinant proteins were characterized according to their phytotoxic and cytotoxic effects to tomato leaves and to mammalian Vero cells, respectively. These assays revealed that all NprvNeps tested are cytotoxic to Vero cells and also induce cell death in tomato leaves. NprvNep2 was the most toxic to Vero cells, followed by NprvNep1 and 3. NprvNep4 induced weaker, but, nevertheless, still significant toxic effects to Vero cells. A similar trend of toxicity was observed in tomato leaves: the most toxic was NprvNep 2 and the least toxic NprvNep 4. This study describes for the first time an overview of the NLP gene family of N. parvum and provides additional insights into its pathogenicity mechanism.

Detection of premature stop codons leading to truncated internalin A among food and clinical strains of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen responsible for outbreaks and sporadic cases of listeriosis, a severe invasive disease. Internalin A (InlA) a protein encoded by inlA has a key role in the mechanism of pathogenesis in L. monocytogenes infection, specifically in the invasion of human intestinal epithelial cells. Studies on inlA have shown that mutations leading to premature stop codons (PMSCs) occur naturally and are associated with impaired virulence of L. monocytogenes strains. Increasing evidence suggests that inlA PMSCs mutations are frequent in strains from foods, but rare among clinical isolates. In this study, 22 L. monocytogenes strains collected in Portugal from the processing environment of a bakery industry (n = 1), different food products (n = 10) and human clinical cases (n = 11) were analysed for mutations in inlA and invasion efficiency in Caco-2 cells. Sequencing revealed previously reported mutations types leading to PMSCs in three food and one clinical strain presenting different molecular serotypes (i.e., IIa, IIb and IIc). The remaining 18 isolates did not show PMSCs in inlA. The four strains with PMSCs in inlA presented lower invasiveness efficiencies in Caco-2 cells (below 8.9%) when compared to the control strain (full-length InlA). In addition, one clinical isolate showed reduced invasion efficiency but no PMSCs in inlA. This isolate showed increased inlA transcript levels to that obtained for the laboratory control strain. Our data support the hypothesis that L. monocytogenes isolated from food have attenuated invasion due to the presence of inlA PMSCs. This information would be critically needed for adequate risk-assessments of the foodborne illness burden associated with L. monocytogenes strains.

Diversity of endophytic Pseudomonas in Halimione portulacoides from metal(loid)-polluted salt marshes.

Phytoremediation assisted by bacteria is seen as a promising alternative to reduce metal contamination in the environment. The main goal of this study was to characterize endophytic Pseudomonas isolated from Halimione portulacoides, a metal-accumulator plant, in salt marshes contaminated with metal(loid)s. Phylogenetic analysis based on 16S rRNA and gyrB genes showed that isolates affiliated with P. sabulinigri (n = 16), P. koreensis (n = 10), P. simiae (n = 5), P. seleniipraecipitans (n = 2), P. guineae (n = 2), P. migulae (n = 1), P. fragi (n = 1), P. xanthomarina (n = 1), and Pseudomonas sp. (n = 1). Most of these species have never been described as endophytic. The majority of the isolates were resistant to three or more metal(loid)s. Antibiotic resistance was frequent among the isolates but most likely related to species-intrinsic features. Common acquired antibiotic resistance genes and integrons were not detected. Plasmids were detected in 43.6 % of the isolates. Isolates that affiliated with different species shared the same plasmid profile but attempts to transfer metal resistance to receptor strains were not successful. Phosphate solubilization and IAA production were the most prevalent plant growth promoting traits, and 20 % of the isolates showed activity against phytopathogenic bacteria. Most isolates produced four or more extracellular enzymes. Preliminary results showed that two selected isolates promote Arabidopsis thaliana root elongation. Results highlight the diversity of endophytic Pseudomonas in H. portulacoides from contaminated sites and their potential to assist phytoremediation by acting as plant growth promoters and as environmental detoxifiers.

Microbacterium proteolyticum sp. nov. isolated from roots of Halimione portulacoides.

An endophytic actinobacterial strain RZ36T, isolated from roots of the salt-marsh plant Halimione portulacoides, was subjected to a polyphasic taxonomic characterization. 16S rRNA gene sequence analysis revealed that this strain belonged to the genus Microbacterium. The closest phylogenetic relative was Microbacterium hominis DSM 12509T, with a pairwise 16S rRNA gene sequence similarity of 98.8 %. The DNA-DNA hybridization value between strain RZ36T and M. hominis DSM 12509T was 16 %. The affiliation to the genus Microbacterium was corroborated by phenotypic and chemotaxonomic characteristics. The cell-wall peptidoglycan type was B2ß and the diagnostic diamino acid was ornithine. Whole-cell sugars detected were galactose, glucose, rhamnose, ribose and xylose. The major fatty acids were anteiso-C15 : 0 and iso-C16 : 0 and the major menaquinone was MK-11 (64 %). Main polar lipids consisted of diphosphatidylglycerol, phosphatidylglycerol and two unknown glycolipids. The genomic DNA G+C content was 69.7 mol%. Thus, on the basis of phenotypic, genotypic and chemotaxonomic data, strain RZ36T is considered to represent a novel species of the genus Microbacterium, for which the name Microbacterium proteolyticum sp. nov. (type strain RZ36T = DSM 27100T = CECT 8356T) is proposed.

Neptunomonas phycophila sp. nov. isolated from a culture of Symbiodinium sp., a dinoflagellate symbiont of the sea anemone Aiptasia tagetes.

A Gram-stain-negative, facultatively anaerobic, oxidase- and catalase-positive, rod-shaped bacterium, strain SYM1(T), was isolated from a culture of Symbiodinium sp., an algal symbiont of the sea anemone Aiptasia tagetes collected in Puerto Rico. Growth was observed at 4-40 °C (optimum 30 °C), at pH 5.0-11.0 (optimum pH 8.0) and with 0.5-8 % (optimum 2 %) (w/v) NaCl. Phylogenetic analyses of 16S rRNA gene sequences showed that strain SYM1(T) was a member of the genus Neptunomonas with the type strain of Neptunomonas naphthovorans as the closest phylogenetic relative with a pairwise sequence similarity of 98.15 %. However, DNA-DNA relatedness between strain SYM1(T) and N. naphthovorans CIP 106451(T) was 24 %. Moreover, strain SYM1(T) could be distinguished from its closest relative by several phenotypic characteristics such as NaCl, pH and temperature tolerance, nitrate reduction and utilization of carbon substrates. The major cellular fatty acids were C16 : 0, C18 : 1ω7c and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 0 2-OH). The genomic DNA G+C content of strain SYM1(T) was 45 mol%. Ubiquinone-8 (Q-8) was the only respiratory quinone detected. Based on a polyphasic taxonomic characterization, strain SYM1(T) represents a novel species of the genus Neptunomonas, for which the name Neptunomonas phycophila sp. nov. is proposed. The type strain is SYM1(T) ( = LMG 28329(T) = CECT 8716(T)).

Effects of cadmium and resource quality on freshwater detritus processing chains: a microcosm approach with two insect species.

Detritus processing is vital for freshwater ecosystems that depend on the leaf litter from riparian vegetation and is mediated by microorganisms and aquatic invertebrates. Shredder invertebrates transform coarse particulate organic matter into fine particulate organic matter used as food by collector species. Direct and indirect effects of contaminants can impair detritus processing and thus affect the functioning of these ecosystems. Here, we assessed the combined effects of a toxic metal (cadmium) and resource quality (leaf species) on detritus processing and shredder-collector interactions. We considered two types of leaves, alder and eucalyptus that were microbially conditioned under different Cd concentrations in the laboratory. The microbial communities present on leaves were analyzed by Denaturing Gradient Gel Electrophoresis (DGGE), and we also measured microbial respiration rates. Sericostoma vittatum (a caddisfly shredder) and Chironomus riparius (a midge collector) were also exposed to Cd and allowed to consume the corresponding alder or eucalyptus leaves. We evaluated C. riparius growth and leaf mass loss in multispecies microcosms. Cadmium exposure affected leaf conditioning and fungal diversity on both leaf species, as assessed by DGGE. Cadmium exposure also affected the mass loss of alder leaves by reductions in detritivore feeding, and impaired C. riparius growth. Chironomus riparius consumed alder leaf discs in the absence of shredders, but S. vittatum appear to promote C. riparius growth in treatments containing eucalyptus. These results show that indirect effects of contaminants along detritus-processing chains can occur through effects on shredder-collector interactions such as facilitation but they also depend on the nutritional quality of detritus and on sensitivity and feeding plasticity of detritivore species.

Fibrilaçäo atrial crônica no idoso / Aspects Chronic atrial fibrillation in elderly patients

Purpose - To evaluate the different aspects of chronic atrialfibrillation (CAF) in elderly patients. Methods - Forty nine patients with CAF (18 men) aged 66 to 87 years (76,1 years on average) were studied. Their evaluation consisted of clinical examination, blood tests, electrocardiogram, chest X-Ray and echocardiogram. Results - The frequency of CAF in the outpatient Geriatric Clinic was 4.8% and it rose with aging. Cardiocirculatory diseases were observed in 39 (79.6%) patients: 25 (51.0%) had systemic arterial hypertension, 11 (22.4%) had mitral valve disease and six (11.2%) had aortic valve disease. Thirty patients had diseases of other systems, eight (16.3%) of whom were diabetic and seven (14.3%) had hyperthyroidism. Vascular brain disease was observed in four (8.2%) patients. Electrocardiographic changes other than CAF were observed in 41 (83.7%) patients. Changes in the ST-T segment were the morefrequent, being observed in 28 (57.1%) patients, followed by left ventricular hypertrophy in 15 (30.1%). Cardiomegaly was seen in 36 (78.3%) out of 46 patients who did the chest radiography. An echocardiogram was performed in 35 patients, being found normal in seven (20.0%). In the remaining cases, the abnormality morefrequently seen was the left atrial enlargment, seen in 16 (45.7%) patients. Four patients didn't show clinical, electrocardiographic, radiological or echocardiographic evidence of cardiocirculatory disease and in these, the thyroidfunction was normal. Digitalis therapy was used in 37 patients. Of the remaining 12,10 had normal ventricular frequency without the use of the drug...

A importância da calcificaçäo mitral no idoso / The importance of mitral calcification in the elderly

O estudo ecocardiográfico de 480 pacientes com idade igual ou superior a 60 anos, demonstrou que 37 eram portadores de calcificaçäo mitral (CM). Destes, 33 eram do sexo feminino e quatro do sexo masculino, com idade variável de 63 a 98 anos (média 75,4 anos). A análise dos dados clínicos, eletrocardiográficos, fonocardiográficos, ecocardiográficos e metabólicos permitiu evidenciar: 1) a CM foi obsrvada em 7,7% dos pacientes com mais de 60 anos, sendo que a incidência tende a aumentar com o progredir da idade; 2) a CM foi mais freqüente e acentuada nas mulheres; 3) afecçöes cardiocirculatórias, que elevam a pressäo sistólica ventricular esquerda, como a hipertensäo arterial (64,9%) e a estenose aórtica (10,8%) foram as mais observadas nesses pacientes; 4) näo se conseguiu evidenciar antecendentes de enfermidades que pudessem ter lesado previamente a valva mitral; 5) näo se observaram alteraçöes dos lípides e do cálcio séricos; 6) o progressivo depósito de cálcio, atingindo as lacíneas, determinou em 27,0% dos casos, insuficiência e/ou estenose mitral, passível até de correçäo cirúrgica; 7() foram freqüentes as alteraçöes no mecanismo de produçäo e/ou conduçäo do estímulo cardíaco, levando ao desenvolvimento de bradiarritmias, taquiarritmias e de bloqueios intraventriculares. A vista do acima exposto, recomenda-se que, após confirmaçäo diagnóstica, o apciente deverá ser acompanhado periódicamente para que as complicaçöes sejam detectadas e tratadas prececemente