The Protective Role of 1,8-Dihydroxynaphthalene-Melanin on Conidia of the Opportunistic Human Pathogen Aspergillus fumigatus Revisited: No Role in Protection against Hydrogen Peroxide and Superoxides.

Previously, 1,8-dihydroxynaphthalene (DHN)-melanin was described to protect Aspergillus fumigatus against hydrogen peroxide (H2O2), thereby protecting this opportunistic human pathogen from reactive oxygen species generated by the immune system. This was based on the finding that the ATCC 46645 mutant with mutations in the pksP gene of the DHN-melanin synthesis pathway showed increased sensitivity to reactive oxygen species compared to the wild type. Here, it is shown that deletion of the pksP gene in A. fumigatus strain CEA10 did not affect sensitivity for H2O2 and superoxide in a plate stress assay. In addition, direct exposure of the dormant white conidia of the pksP deletion strains to H2O2 did not result in increased sensitivity. Moreover, complementation of the ATCC 46645 pksP mutant strain with the wild-type pksP gene did result in pigmented conidia but did not rescue the H2O2-sensitive phenotype observed in the plate stress assay. Genome sequencing of the ATCC 46645 pksP mutant strain and its complemented strain revealed a mutation in the cat1 gene, likely due to the UV mutagenesis procedure used previously, which could explain the increased sensitivity toward H2O2. In summary, DHN-melanin is not involved in protection against H2O2 or superoxide and, thus, has no role in survival of conidia when attacked by these reactive oxygen species. IMPORTANCE Opportunistic pathogens like Aspergillus fumigatus have strategies to protect themselves against reactive oxygen species like hydrogen peroxides and superoxides that are produced by immune cells. DHN-melanin is the green pigment on conidia of Aspergillus fumigatus and more than 2 decades ago was reported to protect conidia against hydrogen peroxide. Here, we correct this misinterpretation by showing that DHN-melanin actually is not involved in protection of conidia against hydrogen peroxide. We show that UV mutagenesis that was previously used to select a pksP mutant generated many more genome-wide mutations. We discovered that a mutation in the mycelial catalase gene cat1 could explain the observed phenotype of increased hydrogen peroxide sensitivity. Our work shows that UV mutagenesis is not the preferred methodology to be used for generating mutants. It requires genome sequencing with single-nucleotide polymorphism analysis as well as additional validations to discard unwanted and confirm correct phenotypes.

Correction: Variation of virulence of five Aspergillus fumigatus isolates in four different infection models.

[This corrects the article DOI: 10.1371/journal.pone.0252948.].

Variation of virulence of five Aspergillus fumigatus isolates in four different infection models.

Conidia of Aspergillus fumigatus are inhaled by humans on daily basis. As a consequence, these conidia can cause infections that differ in severity ranging from allergic bronchopulmonary aspergillosis to invasive aspergillosis. In this study we compared virulence of five A. fumigatus isolates in four different infection models to address the predictive value of different model systems. Two of the A. fumigatus strains were isolated from dogs with a non-invasive sino-nasal aspergillosis (DTO271-B5 and DTO303-F3), while three strains were isolated from human patients with invasive aspergillosis (Af293, ATCC46645 and CEA10). Infection models used encompassed cultured type II A549 lung epithelial cells, Protostelium aurantium amoeba, Galleria melonella larvae and zebrafish embryos. No major differences in virulence between these five strains were observed in the lung epithelial cell model. In contrast, strain ATCC46645 was most virulent in the amoeba and zebrafish model, whereas it was much less virulent in the Galleria infection model. DTO303-F3 was most virulent in the latter model. In general, reference strain Af293 was less virulent as compared to the other strains. Genome sequence analysis showed that this latter strain differed from the other four strains in 136 SNPs in virulence-related genes. Together, our results show that virulence of individual A. fumigatus strains show significant differences between infection models. We conclude that the predictive value of different model systems varies since the relative virulence across fungal strains does not hold up across different infection model systems.

The sino-nasal warzone: transcriptomic and genomic studies on sino-nasal aspergillosis in dogs.

We previously showed that each dog with chronic non-invasive sino-nasal aspergillosis (SNA) was infected with a single genotype of Aspergillus fumigatus. Here, we studied the transcriptome of this fungal pathogen and the canine host within the biofilm resulting from the infection. We describe here transcriptomes resulting from natural infections in animal species with A. fumigatus. The host transcriptome showed high expression of IL-8 and alarmins, uncontrolled inflammatory reaction and dysregulation of the Th17 response. The fungal transcriptome showed in particular expression of genes involved in secondary metabolites and nutrient acquisition. Single-nucleotide polymorphism analysis of fungal isolates from the biofilms showed large genetic variability and changes related with adaptation to host environmental factors. This was accompanied with large phenotypic variability in in vitro stress assays, even between isolates from the same canine patient. Our analysis provides insights in genetic and phenotypic variability of Aspergillus fumigatus in biofilms of naturally infected dogs reflecting in-host adaptation. Absence of a Th17 response and dampening of the Th1 response contributes to the formation of a chronic sino-nasal warzone.

Bmi1 limits dilated cardiomyopathy and heart failure by inhibiting cardiac senescence.

Dilated cardiomyopathy (DCM) is the most frequent cause of heart failure and the leading indication for heart transplantation. Here we show that epigenetic regulator and central transcriptional instructor in adult stem cells, Bmi1, protects against DCM by repressing cardiac senescence. Cardiac-specific Bmi1 deletion induces the development of DCM, which progresses to lung congestion and heart failure. In contrast, Bmi1 overexpression in the heart protects from hypertrophic stimuli. Transcriptome analysis of mouse and human DCM samples indicates that p16(INK4a) derepression, accompanied by a senescence-associated secretory phenotype (SASP), is linked to severely impaired ventricular dimensions and contractility. Genetic reduction of p16(INK4a) levels reverses the pathology of Bmi1-deficient hearts. In parabiosis assays, the paracrine senescence response underlying the DCM phenotype does not transmit to healthy mice. As senescence is implicated in tissue repair and the loss of regenerative potential in aging tissues, these findings suggest a source for cardiac rejuvenation.

Desempeño lingüistico, cognitivo y auditivo de un grupo de escolares con trastorno específico del lenguaje / Linguistic, cognitive and auditory performance of a group of school children with specific language impairment

El Trastorno Específico del Lenguaje (TEL) se define como una limitación significativa en la capacidad lingüística que no evidencia daño cerebral, desempeños cognitivos descendidos no déficit motor. En nuestro medio, los escolares con TEL han sido poco estudiados. El propósito de este trabajo es entregar información sobre aspectos lingüísticos, cognitivos y auditivos de escolares con TEL que cursan 1° básico. Se evaluó a 25 niños con TEL y 23 escolares sin TEL con las siguientes pruebas: Test para evaluar la gramática Española de A. Toronto, Evaluación del discurso narrativo (EDNA), test de matrices progresivas coloreadas de Raven y un barrido audiométrico. Los resultados más importantes muestran que la mayoría de los niños con TEL presentan alguna alteración en las áreas evaluadas. En especial, se advierte que un gran porcentaje del grupo en estudio evidencia dificultades en los aspectos lingüísticos y en particular en el discurso narrativo. Por el contrario, un bajo porcentaje de ellos manifestó problemas en las habilidades cognitivas no verbales o un descenso en el umbral auditivo.

Specific language impairment (SLI) is defined as a significant limitation in linguistic abilities that cannot be explained by brain damage, cognitive or motor disabilities. In our context, school children with SLI have not been fully investigated. The aim of this study is to obtain information about linguistic, cognitive and auditory aspects in 25 children with SLI attending year 1 of primary school and in 23 control children without SLI. The evaluation procedures included the Test para evaluar la Gramática Española de A. Toronto, the Evaluación del Discurso Narrativo (EDNA), the Raven's coloured progressive matrices and an audiometric screening. The results indicate that most of the children with SLI show deficits in the linguistic aspects assessed, specially in narrative speech. On the other hand, a few number of children with SLI presented with difficulties in non-verbal cognitive abilities and/or with hearing deficits.

Genetic characterization of Streptococcus phocae strains isolated from Atlantic salmon, Salmo salar L., in Chile.

Streptococcus phocae is a beta-haemolytic bacterium frequently involved in disease outbreaks in seals causing pneumonia or respiratory infection. Since 1999, this pathogen has been isolated from diseased Atlantic salmon, Salmo salar, causing serious economic losses in the salmon industry in Chile. In this study, we used different molecular typing methods, such as pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR), repetitive extragenic palindromic PCR (REP-PCR) and restriction of 16S-23S rDNA intergenic spacer regions to evaluate the genetic diversity in S. phocae. Thirty-four strains isolated in different years were analysed. The S. phocae type strain ATCC 51973(T) was included for comparative purposes. The results demonstrated genetic homogeneity within the S. phocae strains isolated in Chile over several years, suggesting the existence of clonal relationships among S. phocae isolated from Atlantic salmon. The type strain ATCC 51973(T) presented a different genetic pattern with the PFGE, RAPD, ERIC-PCR and REP-PCR methods. However, the fingerprint patterns of two seal isolates were distinct from those of the type strain.

Brain barrier dysfunction in Cuban epidemic optic neuropathy.

BACKGROUND AND PURPOSE: There are practically no references to cerebrospinal fluid (CSF) studies in tropical or nutritional neuropathies. In the present paper we present the results of CSF studies in patients with Cuban Epidemic Optic Neuropathy (CEON) during epidemic and endemic periods, with an appraisal as to the contribution of brain barriers' function in the pathophysiology of this disease. METHODS: Two hundred and five patients with CEON were studied during the epidemic period (1992-1993) and 12 patients outside the outbreak (1995-1997). CSF protein determination and electrophoresis were carried out, as well as serum and CSF albumin and immunoglobulin G (IgG) quantitation for calculating IgG and Q(alb) indexes, in order to evaluate intrathecal IgG synthesis and the permeability of the blood-CSF barrier (B-CSF B). RESULTS: One fourth of the patients had increased permeability of the B-CSF B, but damage was more frequent between 16 and 60 days from onset of disease, disappearing after 120 days. B-CSF B dysfunction was more prevalent in patients with severe neurological impairment, although it was not related to the severity of ophthalmological damage. The group of patients studied outside of the outbreak (endemic period) showed similar results. DISCUSSION: The possible association of increased permeability of the B-CSF B with oxidative stress, which lies on the basis of this epidemic outbreak, is discussed.

Neumonectomía en atelectasia masiva post infección por adenovirus / Pneumonectomy in massive atelectasis post adenovirus infection

We described the clinical and radiological evolution and pulmonary function tests in three patients aging 8 to 14 years old at present time. All of them suffered severe pneumonia caused by adenovirus infection during the infant period. During the acute viral disease, massive lung atelectasis refractory to treatment, determined the indication of pneumonectomy. All of the removed lungs presented bronchiectasis, atelectasis and fibrosis. In two children the medical condition improved after surgery, allowing to discontinue oxygen therapy. Wotwith standing, both of them had severe restrictive and obstructive limitation and signs of chronic damage at CTscan obstructive exacerbations decreased and the patients have been to carry out a normal life. The third patient has evolved with progressive clinical deterioration and pulmonary hypertension. The is under continuous oxygen therapy, because of extensive damage in the remaining lung. Being the pneumonectomy an exceptional procedure in children, in these patients the indication was due to medical treatment failure and progressive deterioration. Notwithstanding two children have shown, up to now, a satisfactory adaptation to this condition.

Se describen las características clínicas, la imagenología torácica y función pulmonar de tres pacientes (edades actuales: 8-14 años). Todos ellos cursaron con una neumonía grave por adenovirus en el período de lactante y cuya evolución se agravó por una atelectasia pulmonar masiva refractaria al tratamiento que determinó la indicación de neumonectomía. En los tres casos, el pulmón resecado presentaba atelectasia, bronquiectasias y fibrosis. Dos niños mejoraron su condición clínica posterior a la neumonectomía, logrando suspender el oxígeno adicional. A pesar de presentar una limitación ventilatoria restrictiva y obstructiva severa y una tomografía computarizada de pulmón con signos de daño crónico, han disminuido las exacerbaciones obstructivas y hacen vida normal. El tercer paciente ha evolucionado con deterioro clínico progresivo, requiere oxígeno adicional a permanencia y presenta hipertensión pulmonar, determinado por el extenso daño del pulmón remanente. Siendo la neumonectomía un procedimiento extremo en pediatría, a estos pacientes se les indicó por fracaso del tratamiento médico y empeoramiento del estado general. Dos niños han mostrado hasta ahora una adaptación satisfactoria a esta condición.

[Electrophoresis of cerebrospinal fluid proteins in patients with ischemic cerebrovascular disease]. / Electroforesis de proteínas del líquido cefalorraquídeo en pacientes con enfermedad cerebrovascular isquémica.

INTRODUCTION: Cerebral infarction (CI) increases vascular permeability because of a torrent of molecular events that take place. It frequently leads to oedema, haemorrhage and neuronal death. Free radicals and proteases are also formed, which cause lesions in the blood vessels, and microvascular integrity is lost through degradation of the basal lamina and the extracellular matrix. As a result rupture of the blood brain barrier takes place. AIMS. To compare the electrophoretograms of patients with ischemic cerebrovascular disease (ICVD) with those of controls and to link the alterations in the proteinogram with the ICVD subtypes. PATIENTS AND METHODS: The CSF of 55 controls and 136 patients with ICVD was examined. The total protein (TP) concentration was determined and a polyacrylamide gel electrophoretogram was produced using Coomassie blue stain. Parallel to this, serum was prepared for haptoglobin staining. RESULTS: The TP in patients CSF rose to a significantly higher level than that of controls. Blood brain barrier damage (BBBD) was observed in 28.7% of the patients studied with CI and in 10.3% in transient ischemic attacks (TIA), while 16.2% presented oligoclonal bands. There was a difference between the two sexes: men were found to have higher TP levels, lower percentages of prealbumin 1 and more BBBD than women. CONCLUSIONS: BBBD is more frequent in infarctions than in TIA, and is predominant in men with thrombotic cerebral infarction.

Electroforesis de proteínas del líquido cefalorraquídeo en pacientes con enfermedad cerebrovascular isquémica / Electrophoresis of cerebrospinal fluid proteins in patients with ischemic cerebrovascular disease

Introducción. El infarto cerebral (IC) aumenta la permeabilidad vascular al ocurrir toda una cascada de eventos moleculares, y aparece con frecuencia edema, hemorragia y muerte neuronal. También ocurre la formación de radicales libres y proteasas que causan lesiones a los vasos sanguíneos, y se pierde la integridad microvascular por degradación de la lámina basal y la matriz extracelular; todo esto conduce a la ruptura de la barrera hematoencefálica. Objetivos. Comparar el electroforetograma de pacientes con enfermedad cerebrovascular isquémica (ECVI) con los controles y asociar las alteraciones del proteinograma con los subtipos de ECVI. Pacientes y métodos. Se estudió el LCR de 55 controles y 136 pacientes con ECVI. Se determinó la concentración de proteínas totales (PT) y el electroforetograma en geles de disco de poliacrilamida con tinción de Coomassie; el suero se procesó en paralelo para tinción de haptoglobinas. Resultados. Las PT en el LCR de los pacientes se elevaron significativamente más que en los controles. El daño de barrera hematoencefálica (DBHE) se observó en el 28,7 por ciento de los pacientes estudiados con IC y en el 10,3 por ciento en los ataques isquémicos transitorios, mientras que el 16,2 por ciento presentaron bandas oligoclonales. Hubo diferencia entre ambos sexos: los hombres tuvieron los niveles de PT elevados, menor porcentaje de prealbúmina-1 y más DBHE que las mujeres. Conclusión. El DBHE es más frecuente en los infartos que en los AIT, y predominan en los hombres con infarto cerebral trombótico (AU)

[Immunological study of cerebrospinal fluid using a human IgM antiserum produced in Cuba. A preliminary evaluation]. / Estudio inmunológico del líquido cefalorraquídeo con el empleo de un antisuero de IgG humano producido en Cuba. Evaluación preliminar.

INTRODUCTION: The immunological study of cerebrospinal fluid (CSF) is an essential diagnostic tool for evaluating patients with neurological diseases. The quantitative determination of the albumen and immunoglobulin G (IgG) in blood serum and in CSF by single radial immunodiffusion (SRID), together with the calculation of the IgG index to evaluate the presence of intrathecal synthesis of IgG and of the albumen quotient in order to evaluate the state of functioning of the blood brain barrier are essential elements to be evaluated for diagnosis and research in neurological clinical practice, as well as in the follow up of certain neurological diseases such as multiple sclerosis. Specific antiserums from commercial firms such as Boehring, SIGMA, etc. are used for the quantitative determination of IgG and albumen both in blood serum and in CSF by SRID. The high cost and the difficulty in acquiring these immunodiagnostic kits have had an important effect on the diagnostic and research opportunities throughout the country. MATERIALS AND METHODS: In this work we present the preliminary findings of the evaluation of the human IgG antiserum obtained from a ram, by Labex laboratories, for the quantitative determination of IgG in CSF by SRID, in order to find out whether this antiserum is efficient in the quantitative determination of IgG in CSF. RESULTS AND CONCLUSIONS: The studies conducted so far show that this antiserum may be a good candidate for use in immunological studies of CSF. Further work needs to be carried out on its validation in order to resolve the problems involved in immunological studies of CSF that we highlighted above. This would be achieved with an antiserum that is cheaper than those used up to now.

A monoclonal antibody that recognizes a potential coeliac-toxic repetitive pentapeptide epitope in gliadins.

OBJECTIVES: Antibodies that detect coeliac-toxic prolamins from wheat, barley and rye are important tools for controlling the diet of coeliac disease patients. Recently, a monoclonal antibody R5 that recognizes wheat gliadin, barley hordein and rye secalin equally was described. In this study, the epitope recognized by R5 was investigated. METHODS: Both a phage-displayed heptapeptide library and overlapping peptides spanning the sequence of alpha- and gamma-type gliadins (pepscan) were screened for binding of R5. RESULTS: Both techniques yielded comparable pentapeptide consensus sequences (phage display QXPW/FP; pepscan QQPFP). According to recent observations, this peptide stretch may be of key importance in the pathogenicity of coeliac disease. This sequence occurs repetitively in prolamins (in gamma- and omega-type prolamins more frequently than in alpha-type prolamins) together with several homologous peptide stretches, which are recognized less strongly. CONCLUSIONS: R5 seems to be a good candidate for the specific detection of putative coeliac disease-active sequences in prolamins and thus represents a valuable tool for the quality control of gluten-free food.

Succinylation of cyclodextrin glycosyltransferase from Thermoanaerobacter sp. 501 enhances its transferase activity using starch as donor.

A simple modification procedure, the succinylation of amino groups, was suitable to increase the transferase (disproportionation) activity of cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. 501 using different linear oligosaccharides as acceptors. On the contrary, the synthesis of cyclodextrins (CDs), the coupling of CDs with oligosaccharides, and the hydrolysis of starch decreased after chemical modification. The degree of succinylation of amino groups (45%) was accurately determined by MALDI-TOF mass spectrometry. The formation of CDs under industrial conditions was analyzed for native and succinylated CGTases, showing similar selectivity to alpha-, beta-, gamma-CD. The acceptor reaction with D-glucose using soluble starch as glucosyl donor was studied at 60 degrees C and pH 5.5. Malto-oligosaccharides (MOS) production was notably higher using the semisynthetic enzyme at different ratios (w/w) starch:D-glucose. Thus, more than 90% of the initial starch was converted into MOS (G2-G7) in 48 h employing a ratio donor:acceptor 1:2 (w/w).

Novel procedure for the identification of proteins by mass fingerprinting combining two-dimensional electrophoresis with fluorescent SYPRO red staining.

The fluorescent sensitive SYPRO Red dye was successfully employed to stain proteins in two-dimensional gels for protein identification by peptide mass fingerprinting. Proteins which are not chemically modified during the SYPRO Red staining process are well digested enzymatically in the gel and hence the resulting peptides can be efficiently eluted and analysed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). A SYPRO Red two-dimensional gel of a complex protein extract from Candida albicans was analysed by MALDI-TOF MS. The validity of SYPRO Red staining was demonstrated by identifying, via peptide mass fingerprinting, 10 different C. albicans proteins from a total of 31 selected protein spots. The peptide mass signal intensity, the number of matched peptides and the percentage of coverage of protein sequences from SYPRO Red-stained proteins were similar to or greater than those obtained in parallel with the modified silver protein gel staining. This work demonstrates that fluorescent SYPRO Red staining is compatible with the identification of proteins separated on polyacrylamide gel and that it can be used as an alternative to silver staining. As far as we know, this is the first report in which C. albicans proteins separated using 2-D gels have been identified by peptide mass fingerprinting. The improved technique described here should be very useful for carrying out proteomic studies.

An innovative sandwich ELISA system based on an antibody cocktail for gluten analysis.

A cocktail sandwich ELISA based on the employ of two monoclonal antibodies (MAbs) as coating antibodies and a third MAb conjugated to horseradish peroxidase has been developed for the analysis of gluten in foods. Given that each MAb displays a wide specificity spectrum for wheat, barley, rye and oats prolamins, their combination for ELISA ensures a high crossreactivity with most of the potentially toxic gliadin, hordein, secalin and avenin protein family. One of the unprecedented features of the cocktail sandwich ELISA is that it permits for the first time analysis of barley hordeins in foods, which is unattainable using conventional or commercial ELISA kits. Besides, gliadins, hordeins and secalins are recognised to the same extent. The system provides a high detection sensitivity for gliadins, hordeins, secalins and avenins (1.5, 0.05, 0.15 and 12 ng/ml, respectively). The working linear range comprises 3-100 ng/ml with a gliadin detection limit of 1.5 ppm. This limit of detection is even better than that demanded in the latest Codex recommendation, 10 ppm. Cocktail ELISA data were contrasted with those of commercial ELISA kits and confirmed by mass spectrometry, a non-immunological technique which provides evidence for the occurrence of false positive results with the commercial kits.