The Microbiome in Bronchial Biopsies from Smokers and Ex-Smokers with Stable COPD - A Metatranscriptomic Approach.

Current knowledge about the respiratory microbiome is mainly based on 16S ribosomal RNA gene sequencing. Newer sequencing approaches, such as metatranscriptomics, offer the technical ability to measure the viable microbiome response to environmental conditions such as smoking as well as to explore its functional role by investigating host-microbiome interactions. However, knowledge about its feasibility in respiratory microbiome research, especially in lung biopsies, is still very limited. RNA sequencing was performed in bronchial biopsies from clinically stable smokers (n = 5) and ex-smokers (n = 6) with COPD not using (inhaled) steroids. The Trinity assembler was used to assemble non-human reads in order to allow unbiased taxonomical and microbial transcriptional analyses. Subsequently, host-microbiome interactions were analyzed based on associations with host transcriptomic data. Ultra-low levels of microbial mass (0.009%) were identified in the RNA-seq data. Overall, no differences were identified in microbiome diversity or transcriptional profiles of microbial communities or individual microbes between COPD smokers and ex-smokers in the initial test dataset as well as a larger replication dataset. We identified an upregulated host gene set, related to the simultaneous presence of Bradyrhizobium, Roseomonas, Brevibacterium.spp., which were related to PERK-mediated unfolded protein response (UPR) and expression of the microRNA-155-5p. Our results show that metatranscriptomic profiling in bronchial biopsy samples from stable COPD patients yields ultra-low levels of microbial mass. Further, this study illustrates the potential of using transcriptional profiling of the host and microbiome to gain more insight into their interaction in the airways.

Quantitative proteomic comparison of salt stress in Chlamydomonas reinhardtii and the snow alga Chlamydomonas nivalis reveals mechanisms for salt-triggered fatty acid accumulation via reallocation of carbon resources.

BACKGROUND: Chlamydomonas reinhardtii is a model green alga strain for molecular studies; its fully sequenced genome has enabled omic-based analyses that have been applied to better understand its metabolic responses to stress. Here, we characterised physiological and proteomic changes between a low-starch C. reinhardtii strain and the snow alga Chlamydomonas nivalis, to reveal insights into their contrasting responses to salinity stress. RESULTS: Each strain was grown in conditions tailored to their growth requirements to encourage maximal fatty acid (as a proxy measure of lipid) production, with internal controls to allow comparison points. In 0.2 M NaCl, C. nivalis accumulates carbohydrates up to 10.4% DCW at 80 h, and fatty acids up to 52.0% dry cell weight (DCW) over 12 days, however, C. reinhardtii does not show fatty acid accumulation over time, and shows limited carbohydrate accumulation up to 5.5% DCW. Analysis of the C. nivalis fatty acid profiles showed that salt stress improved the biofuel qualities over time. Photosynthesis and respiration rates are reduced in C. reinhardtii relative to C. nivalis in response to 0.2 M NaCl. De novo sequencing and homology matching was used in conjunction with iTRAQ-based quantitative analysis to identify and relatively quantify proteomic alterations in cells exposed to salt stress. There were abundance differences in proteins associated with stress, photosynthesis, carbohydrate and lipid metabolism proteins. In terms of lipid synthesis, salt stress induced an increase in dihydrolipoyl dehydrogenase in C. nivalis (1.1-fold change), whilst levels in C. reinhardtii remained unaffected; this enzyme is involved in acetyl CoA production and has been linked to TAG accumulation in microalgae. In salt-stressed C. nivalis there were decreases in the abundance of UDP-sulfoquinovose (- 1.77-fold change), which is involved in sulfoquinovosyl diacylglycerol metabolism, and in citrate synthase (- 2.7-fold change), also involved in the TCA cycle. Decreases in these enzymes have been shown to lead to increased TAG production as fatty acid biosynthesis is favoured. Data are available via ProteomeXchange with identifier PXD018148. CONCLUSIONS: These differences in protein abundance have given greater understanding of the mechanism by which salt stress promotes fatty acid accumulation in the un-sequenced microalga C. nivalis as it switches to a non-growth state, whereas C. reinhardtii does not have this response.

Flavonoids from the Amazon plant Brosimum acutifolium induce C6 glioma cell line apoptosis by disrupting mitochondrial membrane potential and reducing AKT phosphorylation.

Glioblastoma, which is highly invasive and has a poor patient prognosis, is the most common type of brain tumor. Flavonoids have known antiproliferative and antineoplastic effects, such as apoptosis induction and tumor growth inhibition. We investigated the effects of treatment with three flavonoids (BAS-1, BAS-4, and BAS-6) isolated from the Amazon plant Brosimum acutifolium on the proliferation and migration of the C6 glioma cell line. Cytotoxicity was evaluated by MTT assay, and morphological changes were evaluated by phase-contrast microscopy and by transmission electron microscopy. Apoptosis was determined using Annexin V-FITC-propidium iodide (PI) staining. A hemolysis assay was used to evaluate plasma membrane injury. Antiproliferative effects were assessed by wound migration and colony formation assays. Mitochondrial transmembrane potential (ΔΨm) was determined using JC-1 dye and flow cytometry. To identify the flavonoid targets, western blotting was performed. BAS-1 and BAS-4 reduced C6 cell proliferation in a dose-dependent manner. BAS-6 showed no effect. Due to its high toxicity toward primary glial cells and its high hemolytic index, BAS-1 was not used in the remaining experiments. BAS-4 treatment did not induce cytotoxicity in primary glial cells; however, in glioma cells, it suppressed migration and invasion and led to apoptosis through mitochondrial damage, ΔΨm loss, cell cycle arrest, and reduced AKT phosphorylation, which is a component of the main cell survival pathway. We conclude that BAS-4 showed potential activity against glioma by inducing apoptosis mediated by ΔΨm loss and AKT pathway disruption, and future studies should further evaluate BAS-4 as a promising antineoplastic agent against glioblastoma.

Elucidating vancomycin-resistant Enterococcus faecium outbreaks: the role of clonal spread and movement of mobile genetic elements.

Background: Vancomycin-resistant Enterococcus faecium (VREfm) has emerged as a nosocomial pathogen worldwide. The dissemination of VREfm is due to both clonal spread and spread of mobile genetic elements (MGEs) such as transposons. Objectives: We aimed to combine vanB-carrying transposon data with core-genome MLST (cgMLST) typing and epidemiological data to understand the pathways of transmission in nosocomial outbreaks. Methods: Retrospectively, 36 VREfm isolates obtained from 34 patients from seven VREfm outbreak investigations in 2014 were analysed. Isolates were sequenced on a MiSeq and a MinION instrument. De novo assembly was performed in CLC Genomics Workbench and the hybrid assemblies were obtained through Unicycler v0.4.1. Ridom SeqSphere+ was used to extract MLST and cgMLST data. Detailed analysis of each transposon and their integration points was performed using the Artemis Comparison Tool (ACT) and multiple blast analyses. Results: Four different vanB transposons were found among the isolates. cgMLST divided ST80 isolates into three cluster types (CTs); CT16, CT104 and CT106. ST117 isolates were divided into CT24, CT103 and CT105. Within VREfm isolates belonging to CT103, two different vanB transposons were found. In contrast, VREfm isolates belonging to CT104 and CT106 harboured an identical vanB transposon. Conclusions: cgMLST provides a high discriminatory power for the epidemiological analysis of VREfm. However, additional transposon analysis is needed to detect horizontal gene transfer. Combining these two methods allows investigation of both clonal spread as well as the spread of MGEs. This leads to new insights and thereby better understanding of the complex transmission routes in VREfm outbreaks.

Diagnosis of bloodstream infections from positive blood cultures and directly from blood samples: recent developments in molecular approaches.

BACKGROUND: Bloodstream infections are a major cause of death with increasing incidence and severity. Blood cultures are still the reference standard for microbiological diagnosis, but are rather slow. Molecular methods can be used as add-on complementary assays. They can be useful to speed up microbial identification and to predict antimicrobial susceptibility, applied to direct blood samples or positive blood cultures. AIM: To review recent developments in molecular-based diagnostic platforms used for the identification of bloodstream infections, with a focus on assays performed directly on blood samples and positive blood cultures. SOURCES: Peer reviewed articles, conference abstracts, and manufacturers' websites. CONTENT: We give an update on recent developments of molecular methods in diagnosing BSIs. We first describe the currently available molecular methods to be used for positive blood cultures including: a) in situ hybridization-based methods; b) DNA-microarray-based hybridization technology; c) nucleic acid amplification-based methods; and d) combined methods. Subsequently, molecular methods applied directly to whole blood samples are discussed, including the use of nucleic acid amplification-based methods, T2 magnetic resonance-based methods, and metagenomics for diagnosing BSIs. IMPLICATIONS: Advances in molecular-based methods complementary to conventional blood culture diagnostics and antimicrobial stewardship programmes may optimize infection management by allowing rapid identification of pathogens and relevant antimicrobial resistance genes. Rapid diagnosis of the causing microorganism and relevant resistance determinants is important for early administration and modification of appropriate antimicrobial therapy. Ultimately, this may lead to improved quality and cost-effectiveness of health care, as well as reduced antimicrobial resistance selection.

Ion chambers compliance results of Brazilian radiation therapy facilities.

The Brazilian Nuclear Energy Commission (cnen) has been making a constant effort to keep up to date with international standards and national needs to strengthen the status of radiological protection of the country. The guidelines related to radiation therapy facilities have been revised in the last five years in order to take into consideration the most relevant aspects of the growing technology as well as to mitigate the accidents or incidents observed in practice. Hence, clinical dosimeters have gained special importance in this matter. In the present work, we discuss the effectiveness of regulation and inspections to the enforcement of instrument calibration accuracy for the improvement of patient dosimetry and quality control. As a result, we observed that the number of calibrated instruments, mainly well chambers, is increasing each year. The same behavior is observed for instruments employed in technologically advanced radiation treatments such as intensity modulated radiotherapy, volumetric therapy and stereotatic radiosurgery. We ascribe this behavior to the new regulation.

Omega-3 therapeutic supplementation in a patient with metastatic adenocarcinoma of the pancreas with muscle mass depletion.

Pancreatic adenocarcinoma has an extremely poor prognosis. With the best available treatments, the median overall survival duration is still less than 1 year. Most patients develop anorexia and major muscle mass loss that interfere with chemotherapy tolerance and survival. In this paper, we present a case in which these problems were a major concern. A multidisciplinary approach with chemotherapy and close nutritional support permitted better control of the disease and longer survival. We also review the literature on nutritional interventions that show an improvement in quality of life and survival in these patients.

Characterization of Staphylococcus pseudintermedius isolated from diseased dogs in Lithuania.

The aim of this study was to characterize Staphylococcus pseudintermedius for its antimicrobial resistance and virulence factors with a special focus on methicillin-resistant (MRSP) strains isolated from sick dogs in Lithuania. Clinically sick adult dogs suffering from infections (n=214) and bitches with reproductive disorders (n=36) from kennels were selected for the study. Samples (n=192) from the 250 tested (76.8%) dogs were positive for Staphylococcus spp. Molecular profiling using the species-specific nuc gene identified 51 isolates as S. pseudintermedius (26.6% from a total number of isolated staphylococci) of which 15 isolates were identified as MRSP. Ten MRSP isolates were isolated from bitches with reproductive disorders from two large breeding kennels. Data on susceptibility of S. pseudintermedius to different antimicrobials revealed that all isolates were susceptible to vancomycin, daptomycin and linezolid. Two isolates (3.9%) were resistant to rifampicin. A high resistance was seen towards penicillin G (94.1%), tetracycline (64.7%) and macrolides (68.7%). Resistance to fluoroquinolones ranged from 25.5% (gatifloxacin) to 31.4% (ciprofloxacin). The most prevalent genes encoding resistance included blaZ, aac(6')-Ie-aph(2'')-Ia, mecA, and tet(M). The Luk-I gene encoding a leukotoxin was detected in 29% of the isolates, whereas the siet gene encoding exfoliative toxin was detected in 69% of the S. pseudintermedius isolates. This report of MRSP in companion animals represents a major challenge for veterinarians in terms of antibiotic therapy and is a concern for both animal and public health.

Risk factors for faecal colonisation with Escherichia coli producing extended-spectrum and plasmid-mediated AmpC ß-lactamases in dogs.

The aim of this study was to assess the prevalence and risk factors for faecal carriage of extended-spectrum ß-lactamase (ESBL) and plasmidic AmpC ß-lactamase (pAmpC) Escherichia coli producers in dogs. A three-month cross-sectional study was conducted and 151 rectal swabs were obtained from healthy dogs. ESBL and pAmpC genes were detected by PCR and were sequenced. Logistic regression models were used to investigate risk factors for the carriage of ESBL and pAmpC-producing E. coli. About 15 per cent of the isolates carried ESBL genes (blaCTX-M-32 n=8, blaCTX-M-15 n=5, blaCTX-M-1 n=3, blaCTX-M-9-like n=4) and 20 per cent carried pAmpC genes (blaCMY-2 n=23, blaCMY-2-like n=2). Thirteen dogs carried an E. coli isolate with both an ESBL and a pAmpC gene. One E. coli isolate harboured the human blaDHA-1 pAmpC gene, which has not been previously reported in companion animals in Europe. Dogs with a history of antimicrobial therapy in the past year had a higher risk of being carriers of ESBL-producing (P=0.003, OR =7.85) and pAmpC-producing (P=0.005, OR=6.28) E. coli. Dogs from shelter/breeders were approximately three times more likely to have an ESBL- or a pAmpC-producing E. coli than dogs from private owners. Males have a reduced risk of carrying a pAmpC-producing E. coli than females (P=0.017, OR =0.28). The knowledge of potential risk factors may help to limit the impact of resistance through implementation of effective control measures and judicious antimicrobial therapy.

First report of swine-associated methicillin-resistant Staphylococcus aureus ST398 in Lithuania.

During 2011, 160 nasal samples were taken from pigs on 8 different farms in Lithuania. Four methicillin-resistant Staphylococcus aureus (MRSA) isolates were obtained. The isolates were ST398, spa type t011 and SCCmec V and none carried the lukF/lukS genes. Strains were resistant to tetracycline, attributed to tetK and tetM genes, and to erythromycin owing to the ermB gene. One MRSA strain was resistant to trimethoprim/sulfamethoxazole and carried the dfrK gene. This is the first report on the presence and characteristics of livestock-associated MRSA isolated from pigs in Lithuania.

[Regression of tachycardiomyopathy after implant of pacemaker with antitachycardial function]. / Regressão de taquicardiomiopatia após implante de marcapasso com função antitaquicardia.

A 27-year-old woman with systemic lupus erythematosus and Wolff-Parkinson-White syndrome complicated with refractory tachycardia and class III heart failure treated with pacemaker implantation was described. She had cardiomyopathy that could be due to lupus erythematosus or tachycardia-induced. Nonpharmacologic therapeutic alternative was used and a universal DDD pulse generator with selected programming was chosen. Twenty-four months follow-up showed tachycardia control and regression of symptoms of heart failure to class I as well as improvement of left ventricular function evaluated by echocardiographic method. Thus, pacemaker implant may be an useful alternative approach in patients with tachycardiomyopathy in whose other nonpharmacologic therapeutic options could not be performed.