Fungal microbiota in newborn infants with and without respiratory distress syndrome.

BACKGROUND: Pneumocytis jirovecii infection in preterm newborns has recently been associated with neonatal respiratory distress syndrome and bronchopulmonary dysplasia. Changes in the bacterial microbiota of the airways have also been described in infants with bronchopulmonary dysplasia. However, until now there has been no information on the airway mycobiota in newborns. The purpose of this study was to describe the airway mycobiota in term and preterm newborns and its possible association with respiratory distress syndrome. METHODS: Twenty-six matched preterm newborns with and without respiratory distress syndrome were studied, as well as 13 term babies. The identification of the fungal microbiota was carried out using molecular procedures in aspirated nasal samples at birth. RESULTS: The ascomycota phylum was identified in 89.7% of newborns, while the basidiomycota phylum was found in 33.3%. Cladosporium was the predominant genus in both term and preterm infants 38.4% vs. 73% without statistical differences. Candida sake and Pneumocystis jirovecii were only found in preterm infants, suggesting a potential relationship with the risk of prematurity. CONCLUSIONS: This is the first report to describe the fungal microbiota of the airways in term and preterm infants with and without respiratory distress syndrome. Although no differences have been observed, the number of cases analyzed could be small to obtain conclusive results, and more studies are needed to understand the role of the fungal microbiota of the airways in neonatal respiratory pathology.

Selective modifications of lactose and N-acetyllactosamine with sulfate and aromatic bulky groups unveil unique structural insights in galectin-1-ligand recognition.

Galectins, a family of endogenous glycan-binding proteins, play crucial roles in a broad range of physiological and pathological processes. Galectin-1 (Gal-1), a proto-type member of this family, is overexpressed in several cancers and plays critical roles in tumor-immune escape, angiogenesis and metastasis. Thus, generation of high-affinity Gal-1 inhibitors emerges as an attractive therapeutic approach for a wide range of neoplastic conditions. Small-molecule carbohydrate inhibitors based on lactose (Lac) and N-acetyllactosamine (LacNAc) structures have been tested showing different results. In this study, we evaluated Lac- and LacNAc-based compounds with specific chemical modifications at key positions as Gal-1 ligands by competitive solid-phase assays (SPA) and isothermal titration calorimetry (ITC). Both assays showed excellent correlation, highlighting that lactosides bearing bulky aromatic groups at the anomeric carbon and sulfate groups at the O3' position exhibited the highest binding affinities. To dissect the atomistic determinants for preferential affinity of the different tested Gal-1 ligands, molecular docking simulations were conducted and PRODIGY-LIG structure-based method was employed to predict binding affinity in protein-ligand complexes. Notably, calculated binding free energies derived from the molecular docking were in accordance with experimental values determined by SPA and ITC, showing excellent correlation between theoretical and experimental approaches. Moreover, this analysis showed that 3'-O-sulfate groups interact with residues of the Gal-1 subsite B, mainly with Asn33, while the ester groups of the aromatic anomeric group interact with Gly69 and Thr70 at Gal-1 subsite E, extending deeper into the pocket, which could account for the enhanced binding affinity. This study contributes to the rational design of highly optimized Gal-1 inhibitors to be further studied in cancer models and other pathologic conditions.

Structural analysis of ING3 protein and histone H3 binding.

Proteins belonging to the ING family regulate the transcriptional state of chromatin by recruiting remodeling complexes to sites with histone H3 trimethylated at Lysine 4 (H3K4me3). This modification is recognized by the Plant HomeoDomain (PHD) present at the C-terminal region of the five ING proteins. ING3 facilitates acetylation of histones H2A and H4 by the NuA4-Tip60 MYST histone acetyl transferase complex, and it has been proposed to be an oncoprotein. The crystal structure of the N-terminal domain of ING3 shows that it forms homodimers with an antiparallel coiled-coil fold. The crystal structure of the PHD is similar to those of its four homologs. These structures explain the possible deleterious effects of ING3 mutations detected in tumors. The PHD binds histone H3K4me3 with low-micromolar, and binds the non-methylated histone with a 54-fold reduced affinity. Our structure explains the impact of site directed mutagenesis experiments on histone recognition. These structural features could not be confirmed for the full-length protein as solubility was insufficient for structural studies, but the structure of its folded domains suggest a conserved structural organization for the ING proteins as homodimers and bivalent readers of the histone H3K4me3 mark.

Fungal microbiota dynamics and its geographic, age and gender variability in patients with cystic fibrosis.

OBJECTIVES: In cystic fibrosis (CF), there is a predisposition to bronchial colonization by potentially pathogenic microorganisms, such as fungi. Our aims were to describe the dynamics of respiratory mycobiota in patients with CF and to evaluate the geographic, age and gender variability in its distribution. METHODS: Cohort study in which 45 patients with CF from four hospitals in three Spanish cities were followed up during a 1-year period, obtaining spontaneous sputum samples every 3 to 6 months. Fungal microbiota were characterized by Internal Transcribed Spacer sequencing and Pneumocystis jirovecii was identified by nested PCR in a total of 180 samples. RESULTS: The presence of fungi were detected in 119 (66.11%) of the 180 samples and in 44 (97.8%) of the 45 patients: 19 were positive and 1 negative throughout all follow-ups and the remaining 25 presented alternation between positive and negative results. A total of 16 different genera were identified, with Candida spp. (50/180, 27.78%) and Pneumocystis spp. (44/180, 24.44%) being the most prevalent ones. The distribution of fungal genera was different among the evaluated centres (p < 0.05), by age (non-adults aged 6-17 years vs. adults aged ≥18 years) (p < 0.05) and by gender (p < 0.05). DISCUSSION: A high prevalence of fungal respiratory microbiota in patients with CF was observed, whose dynamics are characterized by the existence of multiple cycles of clearance and colonization, reporting the existence of geographic, age and gender variability in the distribution of fungal genera in this disease.

Pulmonary embolism - An EFIM guideline critical appraisal and adaptation for practicing clinicians.

BACKGROUND: Several trials have been conducted in the last decades that challenged the management of patients with acute pulmonary embolism (PE) in terms of diagnosis and treatment. Updated international clinical practice guidelines (CPGs) endorsed the evidence from these trials. The aim of this document was to adapt recommendations from existing CPGs to assist physicians in decision making concerning specific and complex scenarios related to acute PE. METHODS: The flow for the adaptation procedure was first the identification of unsolved clinical issues in patients with acute PE (PICOs), then critically appraise the existing CPGs and choose the recommendations, which are the most applicable to these specific and complex scenarios. RESULTS: Five PICOs were identified and CPGs appraisal was performed. Concerning diagnosis of PE when computed tomographic pulmonary angiography is not available/contraindicated and d-dimer is less specific, perfusion lung scan is the preferred option in the majority of clinical scenarios. For the treatment of PE when relevant clinical conditions like pregnancy or severe renal failure are present heparin is to be used. Poor evidence and low-level recommendations exist on the best bleeding prediction rule in patients treated for PE. The duration of anticoagulation needs to be tailored concerning the presence of predisposing factors for index PE and the consequent risk for recurrence. Finally, recommendations on the opportunity to screen for cancer and thrombophilia patients without recognized thrombosis risk factors for PE are reported. Overall, 35 recommendations were endorsed and the rationale for the selection is reported in the main text. CONCLUSION: By the use of proper methodology for the adaptation process, this document offers a simple and updated guide for practicing clinicians dealing with complex patients.

Pneumocystis jirovecii among patients with cystic fibrosis and their household members.

We conducted a pilot study of patients with cystic fibrosis (CF) to assess intra-family transmission of P. jirovecii and compare it with data on other prevalent pathogens such as P. aeruginosa and S. pneumoniae, in which respiratory transmission has already been documented. Oral swab samples from 10 patients with CF and 15 household members were collected at baseline and 2 weeks later. P. aeruginosa and S. pneumoniae were assessed using standardized culture methods and PCR, and P. jirovecii was assessed using real and nested PCR, genotyping the positive samples by direct sequencing. P. aeruginosa cultures were positive for 7/10 (70%) of patients with CF at baseline and was identified by PCR in 8/10 (80%) of cases at baseline and 2 weeks later. S. pneumoniae cultures were negative for all patients, but the microorganism was identified by PCR in two cases. P. jirovecii was detected by real time and nested PCR in 5/10 (50%) of the patients at the two time points. In the household members, P. aeruginosa and P. jirovecii were identified in 7/15 (46.7%), and S. pneumoniae was identified in 8/15 (53,3%). The concordance of positive or negative pairs of patients with CF and their household members was 33.3% (5/15) for P. aeruginosa, 46.7% (7/15) for S. pneumonia and 93.3% (14/15) for P. jirovecii. The concordance for P. jirovecii genotypes among five pairs with available genotype was 100%. This study suggests for the first time the possible transmission of Pneumocystis in the home of patients with CF, indicating that patients and their household members are reservoirs and possible sources of infection. LAY SUMMARY: This study suggests for the first time the possible transmission of Pneumocystis in the family environment of patients with cystic fibrosis, indicating that patients and their household members are reservoirs and possible sources of this infection.

Changing Trends in the Epidemiology and Risk Factors of Pneumocystis Pneumonia in Spain.

Objective: The information about the epidemiology of Pneumocystis pneumonia (PcP) in Europe is scarce, and in Spain there are only data nationwide on patients with HIV infection. This study has been carried out with the aim of knowing in our country the current epidemiological spectrum and the risk factors of PcP. Methods: Observational, descriptive transversal study that included all patients admitted in Spain with diagnosis upon discharge of PcP registered in the National Health System's Hospital Discharge Records Database of Spain, between 2008 and 2012. Results: Four thousand five hundred and fifty four cases of PcP were reported, 1,204 (26.4%) in HIV-negative patients. During the study period, mean annual incidence (cases per million) was 19.4, remaining globally stable, increasing from 4.4 to 6.3 in HIV-negative patients and decreasing from 15.5 to 13.4 among HIV-infected patients. Risk factors were identified in 85.5% of HIV-negative cases, the most frequent being hematological neoplams (29%), chronic lung diseases (15.9%), and non-hematological cancers (14.9%). Mean mortality and hospitalization cost were high (25.5% and 12,000 euros, respectively). Conclusions: The results of this first nationwide study in Spain allow a change in the misconception that, after the AIDS epidemic, PcP is an infrequent disease, showing that today it is an emerging problem in patients without HIV infection. These findings underlines the need for increased efforts toward a better characterization of risk groups to improve prophylactic strategies and reduce the burden of disease.

A Potent Isoprenylcysteine Carboxylmethyltransferase (ICMT) Inhibitor Improves Survival in Ras-Driven Acute Myeloid Leukemia.

Blockade of Ras activity by inhibiting its post-translational methylation catalyzed by isoprenylcysteine carboxylmethyltransferase (ICMT) has been suggested as a promising antitumor strategy. However, the paucity of inhibitors has precluded the clinical validation of this approach. In this work we report a potent ICMT inhibitor, compound 3 [UCM-1336, IC50 = 2 µM], which is selective against the other enzymes involved in the post-translational modifications of Ras. Compound 3 significantly impairs the membrane association of the four Ras isoforms, leading to a decrease of Ras activity and to inhibition of Ras downstream signaling pathways. In addition, it induces cell death in a variety of Ras-mutated tumor cell lines and increases survival in an in vivo model of acute myeloid leukemia. Because ICMT inhibition impairs the activity of the four Ras isoforms regardless of its activating mutation, compound 3 surmounts many of the common limitations of available Ras inhibitors described so far. In addition, these results validate ICMT as a valuable target for the treatment of Ras-driven tumors.

Design-functionality relationships for adhesion/growth-regulatory galectins.

Glycan-lectin recognition is assumed to elicit its broad range of (patho)physiological functions via a combination of specific contact formation with generation of complexes of distinct signal-triggering topology on biomembranes. Faced with the challenge to understand why evolution has led to three particular modes of modular architecture for adhesion/growth-regulatory galectins in vertebrates, here we introduce protein engineering to enable design switches. The impact of changes is measured in assays on cell growth and on bridging fully synthetic nanovesicles (glycodendrimersomes) with a chemically programmable surface. Using the example of homodimeric galectin-1 and monomeric galectin-3, the mutual design conversion caused qualitative differences, i.e., from bridging effector to antagonist/from antagonist to growth inhibitor and vice versa. In addition to attaining proof-of-principle evidence for the hypothesis that chimera-type galectin-3 design makes functional antagonism possible, we underscore the value of versatile surface programming with a derivative of the pan-galectin ligand lactose. Aggregation assays with N,N'-diacetyllactosamine establishing a parasite-like surface signature revealed marked selectivity among the family of galectins and bridging potency of homodimers. These findings provide fundamental insights into design-functionality relationships of galectins. Moreover, our strategy generates the tools to identify biofunctional lattice formation on biomembranes and galectin-reagents with therapeutic potential.

Pneumocystis jirovecii colonization in patients treated with infliximab.

BACKGROUND: Infliximab, a chimeric antitumour necrosis factor (TNF) monoclonal antibody, has become an established effective therapy for inflammatory rheumatic disease. However, TNF is a critical factor in host defence, and the suppression of its biological activity may be associated with the increased risk of opportunistic infections. The frequent use of infliximab in clinical practice has identified Pneumocystis jirovecii pneumonia (PcP) as a serious complication. Individuals colonized with Pneumocystis may be at high risk of development of PcP when they have undergone immunosuppression. Hence, we addressed the question of the frequency of Pneumocystis colonization among patients treated with infliximab. DESIGN: We examined 125 oropharyngeal washes collected from 78 individuals with rheumatoid arthritis, 30 with ankylosing spondylitis and 17 with psoriatic arthritis, half of them underwent infliximab therapy, using a real-time polymerase chain reaction assay that employs specific primers from a portion of the mitochondrial large-subunit rRNA gene of P. jirovecii. RESULTS: Pneumocystis jirovecii colonization was detected in 32 (25·6%) patients. In a multivariate regression model, only duration of infliximab treatment for more than 3 years and use of corticosteroid were significantly and independently associated with risk of Pneumocystis colonization. However, the effect of corticosteroid on P. jirovecii colonization rate was not linearly dose dependent as showed other logistic regression analysis. CONCLUSIONS: There is a high rate of P. jirovecii colonization among patients with rheumatologic diseases treated with infliximab. The identification of patients colonized by P. jirovecii before starting the treatment with infliximab could be a strategy for PcP prevention.

Characterization of necrosis and ethylene-inducing proteins (NEP) in the basidiomycete Moniliophthora perniciosa, the causal agent of witches' broom in Theobroma cacao.

The hemibiotrophic basidiomycete Moniliophthora perniciosa causes witches' broom disease of Theobroma cacao. Analysis of the M. perniciosa draft genome led to the identification of three putative genes encoding necrosis and ethylene-inducing proteins (MpNEPs), which are apparently located on the same chromosome. MpNEP1 and 2 have highly similar sequences and are able to induce necrosis and ethylene emission in tobacco and cacao leaves. MpNEP1 is expressed in both biotrophic and saprotrophic mycelia, the protein behaves as an oligomer in solution and is very sensitive to temperature. MpNEP2 is expressed mainly in biotrophic mycelia, is present as a monomer in solution at low concentrations (<40 microM) and is able to recover necrosis activity after boiling. These differences indicate that similar NEPs can have distinct physical characteristics and suggest possible complementary roles during the disease development for both proteins. This is the first report of NEP1-like proteins in a basidiomycete.

Structural insights into enzyme-substrate interaction and characterization of enzymatic intermediates of organic hydroperoxide resistance protein from Xylella fastidiosa.

Organic hydroperoxide resistance proteins (Ohr) belong to a family of proteins that possess thiol-dependent peroxidase activity endowed by reactive cysteine residues able to reduce peroxides. The crystal structure of Ohr from Xylella fastidiosa in complex with polyethylene glycol, providing insights into enzyme-substrate interactions is described herein. In addition, crystallographic studies, molecular modeling and biochemical assays also indicated that peroxides derived from long chain fatty acids could be the biological substrates of Ohr. Because different oxidation states of the reactive cysteine were present in the Ohr structures from X. fastidiosa, Pseudomonas aeruginosa and Deinococcus radiodurans it was possible to envisage a set of snapshots along the coordinate of the enzyme-catalyzed reaction. The redox intermediates of X. fastidiosa Ohr observed in the crystals were further characterized in solution by electrospray ionization mass spectrometry and by biochemical approaches. In this study, the formation of an intramolecular disulfide bond and oxidative inactivation through the formation of a sulfonic acid derivative was unequivocally demonstrated for the first time. Because Ohr proteins are exclusively present in bacteria, they may represent promising targets for therapeutical drugs. In this regard, the structural and functional analyses of Ohr presented here might be very useful.