The microbiome role in cardiovascular diseases: A systematic review

Introdução: As Doenças Cardiovasculares são a maior causa de morbilidade e de mortalidade mundialmente. Apesar de cada vez mais estudadas, a sua complexidade tem justificado a relevância de procurar novos mecanismos fisiopatológicos associados de forma a promover estratégias terapêuticas mais eficazes. Recentemente, o papel desempenhado pela microbiota intestinal nas vias inflamatórias e metabólicas tem sido explorado e considerado relevante na progressão das doenças cardiovasculares, embora em termos mecanísticos o conhecimento seja insípido. Objetivo(s): O objetivo deste estudo é sistematizar e avaliar a relação entre o microbioma intestinal e as doenças Cardiovasculares, em estudos baseados apenas na população Humana. Métodos: A pesquisa literária foi feita através da MEDLINE e da Web of Science. De acordo com as orientações PRISMA, foram incluídos apenas estudos observacionais e experimentais, realizados em humanos, que avaliassem o microbioma intestinal em doentes com Fibrilhação Auricular (FA), Insuficiência Cardíaca (IC) e Acidente Cerebrovascular (AVC). Resultados: Globalmente, e considerando a classe de metabolitos, verificam-se níveis elevados de TMAO (N-óxido de trimetilamina) nas patologias Cardiovasculares quando doentes são comparados a controlos. Relativamente à microbiota intestinal, os filos predominantes foram as Actinobacteria, Bacteroidetes, Firmicutes e as Proteobacteria. Na FA, as amostras estavam enriquecidas com os géneros: Bacteroides, Parabacteroides, Enterococcus, Dorea, Ruminococcus, e Streptococcus. Na IC, comprovou-se um aumento de Streptococcus e Veillonella. Nos estudos relativos ao AVC, constatou-se um aumento da família Enterobacteriaceae e do seu género Enterobacter. Conclusão: Apesar da falta de informação quantitativa dos metabolitos e da microbiota intestinal por parte dos trabalhos incluídos, este estudo suporta a existência de uma relação entre os mecanismos fisiopatológicos das Doenças Cardiovasculares e o microbioma intestinal. Este trabalho demonstra também, que há um vasto conjunto de trabalhos muito heterógenos, sem poder amostral, que afetam a construção de um edifício de evidência forte entre a microbiota e algumas doenças cardiovasculares.Background: Cardiovascular Diseases (CVD) are a set of heterogeneous diseases affecting the heart and blood vessels whose underlying cause of the development is most often atherosclerosis. The basic mechanisms of atherosclerosis involve a complex interaction of vasculature, the immune system, and lipid metabolism. The gut microbiome plays a role in these mechanisms, with most of the contributions related to microbial metabolites. Therefore, it is crucial to clarify the link between the gut microbiome and cardiovascular diseases in humans to find new possible therapeutic pathways for the foreseeable future. Objectives: The purpose of this study is to systematize and evaluate the relationship between the gut microbiome and CVD, in human-based studies. Methods: The literary research was carried out at MEDLINE and Web of Science. Based on PRISMA Guidelines, were included human-based observational and experimental studies assessing gut microbiome and CVD, namely Atrial Fibrillation (AF), Heart Failure (HF) and stroke. Results: Overall, when compared with controls, higher TMAO levels were associated with CV diseases' patients. Relatively to the gut microbiota, the predominant phyla were Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. In AF, patients' samples were enriched with the genera Bacteroides, Parabacteroides, Enterococcus, Dorea, Ruminococcus, and Streptococcus. In HF patients, there was an increase in the genera Streptococcus and Veillonella. Studies with stroke patients reported the family Enterobacteriaceae and its genus Enterobacter enrichment. Conclusions: Despite the lack of quantitative data regarding metabolites and microbiota, this study supports a relationship between the pathophysiology of CVD and the gut microbiome. However, this work also demonstrates that there is a vast set of very heterogeneous studies, without sample power, that affect the construction of a strong evidence between the gut microbiome and CVD

Development and Integration of Informatic Tools for Qualitative and Quantitative Characterization of Proteomic Datasets Generated by Tandem Mass Spectrometry

Shotgun proteomic experiments provide qualitative and quantitative analytical information from biological samples ranging in complexity from simple bacterial isolates to higher eukaryotes such as plants and humans and even to communities of microbial organisms. Improvements to instrument performance, sample preparation, and informatic tools are increasing the scope and volume of data that can be analyzed by mass spectrometry (MS). To accommodate for these advances, it is becoming increasingly essential to choose and/or create tools that can not only scale well but also those that make more informed decisions using additional features within the data. Incorporating novel and existing tools into a scalable, modular workflow not only provides more accurate, contextualized perspectives of processed data, but it also generates detailed, standardized outputs that can be used for future studies dedicated to mining general analytical or biological features, anomalies, and trends. This research developed cyber-infrastructure that would allow a user to seamlessly run multiple analyses, store the results, and share processed data with other users. The work represented in this dissertation demonstrates successful implementation of an enhanced bioinformatics workflow designed to analyze raw data directly generated from MS instruments and to create fully-annotated reports of qualitative and quantitative protein information for large-scale proteomics experiments. Answering these questions requires several points of engagement between informatics and analytical understanding of the underlying biochemistry of the system under observation. Deriving meaningful information from analytical data can be achieved through linking together the concerted efforts of more focused, logistical questions. This study focuses on the following aspects of proteomics experiments: spectra to peptide matching, peptide to protein mapping, and protein quantification and differential expression. The interaction and usability of these analyses and other existing tools are also described. By constructing a workflow that allows high-throughput processing of massive datasets, data collected within the past decade can be standardized and updated with the most recent analyses

Bloodstream infections in patients with hematological malignancies

Patients with hematological malignancies have an increased risk of infectious complications. These complications can be caused by disease-specific factors or be treatment-related. Bloodstream infections increase the risk of morbidity, mortality, have a negative impact on quality of life, and may lead to reductions in treatment intensity. Surveillance studies on infectious complications and new technologies in diagnosing bloodstream infections are two important fields in improving management of patients with hematological malignancies. Paper I: This is a retrospective study of positive blood cultures from patients mainly treated with dose-intensive antitumoural treatment between 2002 and 2008. Bacterial distribution, bacterial resistance and mortality from 667 fever episodes are presented. Results are compared with historical, previous published, material from the same institution and setting. Subsequently, temporal trends from 1980 to 2008 could be analysed. In a setting with very low use of fluoroquinolone-prophylaxis it can be concluded that; the distribution of Gram-positive bacteremia is stable, the crude mortality remains low in an international perspective and acquired resistance is uncommon but a significant increase in ciprofloxacin resistance in Escherichia coli is observed. The five most common bacteria in the study are; E. coli, coagulase-negative staphylococci, viridans streptococci, Klebsiella spp., and Staphylococcus aureus. Paper II: This is a retrospective study that investigated temporal trends in bloodstream infections in patients with chronic lymphocytic leukemia between 1988-2008. We find a decrease in positive blood cultures over time and speculate if this could be due to more effective antitumoural treatment in recent years. Moreover a bloodstream infection is, as intuitively foreseen, associated with worse prognosis. Dominating pathogens in the study are; E. coli, Streptococcus pneumoniae, P. aeruginosa, S. aureus, and viridans streptococci. Coagulase-negative staphylococcus, a common skin contaminant, is the most frequently detected bacteria. Paper III: This is a prospective study of 33 patients with aggressive hematological malignancies in need of dose-intensive chemotherapy. One hundred thirty blood samples were collected at different time points during episodes with neutropenia and fever between 2013 and 2014. Conventional blood culture findings were compared with a method applicable also for unculturable bacteria, 16S rRNA amplicon sequencing. Sequencing yielded reads belonging to Proteobacteria (55.2%), Firmicutes (33.4%), Actinobacteria (8.6%), Fusobacteria (0.4%), and Bacteroidetes (0.1%). The results display a much broader diversity of bacteria in bloodstream infections than expected. Changes in the relative abundance in the sequence data after commencement of antibiotics could be suggestive for a new method for estimating antibiotic efficacy. Lastly, the results are indicative for translocation, especially of gut microbiota, playing an important etiological factor in fever episodes in the neutropenic host. Paper IV: This is a prospective study of 9 patients with acute leukemia in which we applied shotgun metagenomics for 27 blood samples collected during episodes of neutropenia and fever between 2013 and 2014. Shotgun metagenomics can characterize DNAemia and reconstruct unculturable microbial communities, resistance markers and gene ontology. The study confirms the method’s applicability in bloodstream infections demonstrating bacteria, viruses and fungi. Furthermore, the observed dynamics of microbe sequences during fever episodes as well as gene ontology makes this diagnostic approach appealing for exploring the fever episodes in this patient category

Novel Methods for Metagenomic Analysis

By sampling the genetic content of microbes at the nucleotide level, metagenomics has rapidly established itself as the standard in characterizing the taxonomic diversity and functional capacity of microbial populations throughout nature. The decreasing cost of sequencing technologies and the simultaneous increase of throughput per run has given scientists the ability to deeply sample highly diverse communities on a reasonable budget. The Human Microbiome Project is representative of the flood of sequence data that will arrive in the coming years. Despite these advancements, there remains the significant challenge of analyzing massive metagenomic datasets to make appropriate biological conclusions. This dissertation is a collection of novel methods developed for improved analysis of metagenomic data: (1) We begin with Figaro, a statistical algorithm that quickly and accurately infers and trims vector sequence from large Sanger-based read sets without prior knowledge of the vector used in library construction. (2) Next, we perform a rigorous evaluation of methodologies used to cluster environmental 16S rRNA sequences into species-level operational taxonomic units, and discover that many published studies utilize highly stringent parameters, resulting in overestimation of microbial diversity. (3) To assist in comparative metagenomics studies, we have created Metastats, a robust statistical methodology for comparing large-scale clinical datasets with up to thousands of subjects. Given a collection of annotated metagenomic features (e.g. taxa, COGs, or pathways), Metastats determines which features are differentially abundant between two populations. (4) Finally, we report on a new methodology that employs the generalized Lotka-Volterra model to infer microbe-microbe interactions from longitudinal 16S rRNA data. It is our hope that these methods will enhance standard metagenomic analysis techniques to provide better insight into the human microbiome and microbial communities throughout our world. To assist metagenomics researchers and those developing methods, all software described in this thesis is open-source and available online

Evaluating The Resistome And Microbial Composition During Food Waste Feeding And Composting On A Vermont Poultry Farm

While commonly thought of as a waste product, food scraps and residuals represent an important opportunity for energy and nutrient recapture within the food system. As demands on production continue to increase, conservation of these valuable resources has become a priority area. In the wake of new legislation in Vermont, Act 148, the Universal Recycling Law, the fate of microbial species in food waste, scraps and residuals is increasingly important. The presence of antimicrobial resistance genes in all types of foods calls for an increased need to estimate risk of antibiotic resistance transfer and maintenance across all segments of food production and distribution systems, from farm to fork. Specifically, the fate of antibiotic resistance genes (ARGs) in these co-mingled food wastes has not been sufficiently characterized; as legislative programs increase in popularity, surveillance of these materials is pressing and should be documented to assess the risk and potential measures for mitigation and management as we approach commercial scales of implementation Previous studies have relied on a combination of targeted techniques, such as 16S rRNA sequencing and qPCR on a specific subset of ARGs; however, these may not cover the full extent of resistance or microorganisms of concern in any given sample. As sequencing technologies improve and costs continue to drop, more comprehensive tools, such as shotgun metagenomic sequencing, can be applied to these problems for both surveillance and novel gene discovery. In this study, we leveraged the increased screening power of the Illumina HiSeq and shotgun metagenomic sequencing to identify and characterize ARGs, microbial communities, and associated virulence factors of food scraps, on-farm composts, and several consumer products. Isolates were also screened for antibiotic resistance to demonstrate the functionality of ARGs identified. The resistome, microbiome, and virulence genes were characterized in all samples. Fifty unique ARGs were identified that spanned 8 major drug classes. Most frequently found were genes related to aminoglycoside, macrolide, and tetracycline resistance. Additionally, 54 distinct virulence factors and 495 bacterial species were identified. Virulence factors were present across the farm setting and mainly included gene transfer mechanisms, while bacteria clustered distinctly into site and farm, as well as separate on farm niches. The relationship between these categories was also assessed by both Pearson correlation and co-inertia analysis, with the most significant relationship being between ARGs and virulence factors (P = 0.05, RV = 0.67). While limited in this study, these patterns reinforce the finding that spread of antibiotic resistance genes may be dependent on the virulence factors present enabling transfer, rather than total microbial community composition

Research Experiences for Undergraduates: Advanced Engineered Wood Composites

The aim of this program is interdisciplinary research experience for undergraduate science and engineering students. The focal point is the development of the next generation of engineered wood composites for construction applications. The disciplines involved include structural engineering, mechanics, composite materials, and wood science. The educational paradigm will be one of combining hands-on laboratory work with training in fundamental science and engineering principles. Previous experience with REU sites indicates that many students become interested in graduate research when they are able to see the fruits of their work used in some application. Thus the basis of this REU site is to provide students with projects and a research environment where they may, with reasonable diligence, complete a small research project that is a clearly defined piece of the greater research and development program. Ten students will work at U Maine for a ten-week period during the summer. Prior to arrival on site, the advisors will contact their students to discuss the nature of their projects and to provide written background material. During the summer, each student will be involved in four types of activities: their own individual project, work with others in their sub discipline, weekly group seminars, and group field trips. Faculty will work closely with their students, especially during the early part of the summer. Weekly seminars will include discussions of research techniques, ethics, graduate schools, as well as three presentations made by the students. Group field trips include trips to major field test sites, government agencies, industries, and social events. Follow-through after the students leave the site will consist of advisors working with their students on a technical paper based on the research and on applying to graduate school

Understanding host-microbe interactions in maize kernel and sweetpotato leaf metagenomic profiles.

Functional and quantitative metagenomic profiling remains challenging and limits our understanding of host-microbe interactions. This body of work aims to mediate these challenges by using a novel quantitative reduced representation sequencing strategy (OmeSeq-qRRS), development of a fully automated software for quantitative metagenomic/microbiome profiling (Qmatey: quantitative metagenomic alignment and taxonomic identification using exact-matching) and implementing these tools for understanding plant-microbe-pathogen interactions in maize and sweetpotato. The next generation sequencing-based OmeSeq-qRRS leverages the strengths of shotgun whole genome sequencing and costs lower that the more affordable amplicon sequencing method. The novel FASTQ data compression/indexing and enhanced-multithreading of the MegaBLAST in Qmatey allows for computational speeds several thousand-folds faster than typical runs. Regardless of sample number, the analytical pipeline can be completed within days for genome-wide sequence data and provides broad-spectrum taxonomic profiling (virus to eukaryotes). As a proof of concept, these protocols and novel analytical pipelines were implemented to characterize the viruses within the leaf microbiome of a sweetpotato population that represents the global genetic diversity and the kernel microbiomes of genetically modified (GMO) and nonGMO maize hybrids. The metagenome profiles and high-density SNP data were integrated to identify host genetic factors (disease resistance and intracellular transport candidate genes) that underpin sweetpotato-virus interactions Additionally, microbial community dynamics were observed in the presence of pathogens, leading to the identification of multipartite interactions that modulate disease severity through co-infection and species competition. This study highlights a low-cost, quantitative and strain/species-level metagenomic profiling approach, new tools that complement the assay’s novel features and provide fast computation, and the potential for advancing functional metagenomic studies

Bradford Non-Lethal Weapons Research Project (BNLWRP). Research Report No. 7.

yesThe length of this Bradford Non-Lethal Weapons Research Project Report No.7 again reflects the interest related to non-lethal weapons from academics, research institutes, policy makers, the police and the military. A number of reports, particularly concerning the Taser electro-shock weapon, have been published from these sectors since our last BNLWRP Report No.6 in October 2004. Some, such as the Amnesty International (U.S. and Canada) have again raised, and stressed, the concerns about the safety of the weapon and the number of deaths associated with its use. Others, such as the Joint Non-Lethal Weapons Human Effects Center of Excellence (HECOE), Human Effectiveness and Risk Characterization of the Electromuscular Incapacitation Device ¿ A Limited Analysis of the TASER. (March 2005) concluded that the Taser was relatively safe, but that further research was needed into potential bio-effects, and for continual development into a safer weapon. Reaction to these reports was mixed. Some US legislators called for limitations on the use of Tasers, more accountability, and the detailed recording of incidents in which they were used.1 Others called for a ban on their use until more testing was carried out regarding their potentially harmful effects. A number of US police forces stopped the use of Taser, slowed down the deployment and ordering of the weapons, reviewed their rules of engagement and reporting, and revisited their operational guidelines. The International Association of Chiefs of Police (IACP) published the Electro- Muscular Disruption Technology (EMDT). A Nine-Step Strategy For Effective Deployment. (April 2005) as a response to these growing concerns. Certain elements of the media, especially The Arizona Republic2 and others, took a hostile view of what they considered the scandal of the number of deaths and associated serious injuries caused by the Taser. Taser International challenged allegations that their weapon was directly responsible for these deaths and quoted reports, such as the Madison Police Department report (February 2005), the study by McDaniel, W & Stratbucker, R & Nerheim, M & Brewer, J. Cardiac Safety of Neuromuscular Incapacitating Defensive Devices (January 2005), and the U.K. DOMILL Statement (March 2005) to support their view. The controversy continues. Other than Tasers, there are still few reports of the newer non-lethal technologies actually being deployed in operations. The exception to this is the Long Range Acoustic Device (LRAD), which is now in widespread use in Iraq. Little additional information has appeared regarding the `active denial¿ weapon we have described in previous reports

QUANTITATIVE CHARACTERIZATION OF PROTEINS AND POST-TRANSLATIONAL MODIFICATIONS IN COMPLEX PROTEOMES USING HIGH-RESOLUTION MASS SPECTROMETRY-BASED PROTEOMICS

Mass spectrometry-based proteomics is focused on identifying the entire suite of proteins and their post-translational modifications (PTMs) in a cell, organism, or community. In particular, quantitative proteomics measures abundance changes of thousands of proteins among multiple samples and provides network-level insight into how biological systems respond to environmental perturbations. Various quantitative proteomics methods have been developed, including label-free, metabolic labeling, and isobaric chemical labeling. This dissertation starts with systematic comparison of these three methods, and shows that isobaric chemical labeling provides accurate, precise, and reproducible quantification for thousands of proteins. Based on these results, we applied this approach to characterizing the proteome of Arabidopsis seedlings treated with Strigolactones (SLs), a new class of plant hormones that modulate various developmental processes. Our study reveals that SLs regulate the expression of a range of proteins that have not been assigned to SL pathways, which provides novel targets for follow-up genetic and biochemical characterization of SL signaling. The same approach was also used to measure how elevated temperature impacts the physiology of individual microbial groups in an acid mine drainage (AMD) microbial community, and shows that related organisms differed in their abundance and functional responses to temperature. Elevated temperature repressed carbon fixation by two Leptospirillum genotypes, whereas carbon fixation was significantly up-regulated at higher temperature by a third member of this genus. Further, we developed a new proteomic approach that harnessed high-resolution mass spectrometry and supercomputing for direct identification and quantification of a broad range of PTMs from an AMD microbial community. We find that PTMs are extraordinarily diverse between different growth stages and highly divergent between closely related bacteria. The findings of this study motivate further investigation of the role of PTMs in the ecology and evolution of microbial communities. Finally, a computational approach has been developed to improve the sensitivity of phosphopeptide identification. Overall, the research presented in the dissertation not only reveals biological insights with existing quantitative proteomics methods, but also develops novel methodologies that open up new avenues in studying PTMs of proteins (e.g. PTM cross-talk)