Combining metagenomics, metatranscriptomics and viromics to explore novel microbial interactions: towards a systems-level understanding of human microbiome

AbstractThe advances in experimental methods and the development of high performance bioinformatic tools have substantially improved our understanding of microbial communities associated with human niches. Many studies have documented that changes in microbial abundance and composition of the human microbiome is associated with human health and diseased state. The majority of research on human microbiome is typically focused in the analysis of one level of biological information, i.e., metagenomics or metatranscriptomics. In this review, we describe some of the different experimental and bioinformatic strategies applied to analyze the 16S rRNA gene profiling and shotgun sequencing data of the human microbiome. We also discuss how some of the recent insights in the combination of metagenomics, metatranscriptomics and viromics can provide more detailed description on the interactions between microorganisms and viruses in oral and gut microbiomes. Recent studies on viromics have begun to gain importance due to the potential involvement of viruses in microbial dysbiosis. In addition, metatranscriptomic combined with metagenomic analysis have shown that a substantial fraction of microbial transcripts can be differentially regulated relative to their microbial genomic abundances. Thus, understanding the molecular interactions in the microbiome using the combination of metagenomics, metatranscriptomics and viromics is one of the main challenges towards a system level understanding of human microbiome

Mango (Mangifera indica L.) cv. Kent fruit mesocarp de novo transcriptome assembly identifies gene families important for ripening

"Fruit ripening is a physiological and biochemical process genetically programmed to regulate fruit quality parameters like firmness, flavor, odor and color, as well as production of ethylene in climacteric fruit. In this study, a transcriptomic analysis of mango (Mangifera indica L.) mesocarp cv. "Kent" was done to identify key genes associated with fruit ripening. Using the Illumina sequencing platform, 67,682,269 clean reads were obtained and a transcriptome of 4.8 Gb. A total of 33,142 coding sequences were predicted and after functional annotation, 25,154 protein sequences were assigned with a product according to Swiss-Prot database and 32,560 according to non-redundant database. Differential expression analysis identified 2,306 genes with significant differences in expression between mature-green and ripe mango [1,178 up-regulated and 1,128 down-regulated (FDR <= 0.05)1. The expression of 10 genes evaluated by both gRT-PCR and RNA-seq data was highly correlated (R = 0.97), validating the differential expression data from RNA-seq alone. Gene Ontology enrichment analysis, showed significantly represented terms associated to fruit ripening like "cell wall," "carbohydrate catabolic process" and "starch and sucrose metabolic process" among others. Mango genes were assigned to 327 metabolic pathways according to Kyoto Encyclopedia of Genes and Genomes database, among them those involved in fruit ripening such as plant hormone signal transduction, starch and sucrose metabolism, galactose metabolism, terpenoid backbone, and carotenoid biosynthesis. This study provides a mango transcriptome that will be very helpful to identify genes for expression studies in early and late flowering mangos during fruit ripening.

The CDR1 and other regions of immunoglobulin light chains are hot spots for amyloid aggregation

Immunoglobulin light chain-derived (AL) amyloidosis is a debilitating disease without known cure. Almost nothing is known about the structural factors driving the amyloidogenesis of the light chains. This study aimed to identify the fibrillogenic hotspots of the model protein 6aJL2 and in pursuing this goal, two complementary approaches were applied. One of them was based on several web-based computational tools optimized to predict fibrillogenic/aggregation-prone sequences based on different structural and biophysical properties of the polypeptide chain. Then, the predictions were confirmed with an ad-hoc synthetic peptide library. In the second approach, 6aJL2 protein was proteolyzed with trypsin, and the products incubated in aggregation-promoting conditions. Then, the aggregation-prone fragments were identified by combining standard proteomic methods, and the results validated with a set of synthetic peptides with the sequence of the tryptic fragments. Both strategies coincided to identify a fibrillogenic hotspot located at the CDR1 and β-strand C of the protein, which was confirmed by scanning proline mutagenesis analysis. However, only the proteolysis-based strategy revealed additional fibrillogenic hotspots in two other regions of the protein. It was shown that a fibrillogenic hotspot associated to the CDR1 is also encoded by several κ and λ germline variable domain gene segments. Some parts of this study have been included in the chapter “The Structural Determinants of the Immunoglobulin Light Chain Amyloid Aggregation”, published in Physical Biology of Proteins and Peptides, Springer 2015 (ISBN 978-3-319-21687-4)

De novo assembly and transcriptome characterization of the freshwater prawn Palaemonetes argentinus: Implications for a detoxification response

Palaemonetes argentinus, an abundant freshwater prawn species in the northern and central region of Argentina, has been used as a bioindicator of environmental pollutants as it displays a very high sensitivity to pollutants exposure. Despite their extraordinary ecological relevance, a lack of genomic information has hindered a more thorough understanding of the molecular mechanisms potentially involved in detoxification processes of this species. Thus, transcriptomic profiling studies represent a promising approach to overcome the limitations imposed by the lack of extensive genomic resources for P. argentinus, and may improve the understanding of its physiological and molecular response triggered by pollutants. This work represents the first comprehensive transcriptome-based characterization of the non-model species P. argentinus to generate functional genomic annotations and provides valuable resources for future genetic studies. Trinity de novo assembly consisted of 24,738 transcripts with high representation of detoxification (phase I and II), anti-oxidation, osmoregulation pathways and DNA replication and bioenergetics. This crustacean transcriptome provides valuable molecular information about detoxification and biochemical processes that could be applied as biomarkers in further ecotoxicology studies.Instituto de Investigaciones Bioquímicas de La PlataInstituto de Limnología "Dr. Raúl A. Ringuelet

Pneumomediastinum secondary to Macklin effect: a case report

Introducción: el neumomediastino es la presencia de aire en el espacio mediastinal procedente de bronquios, alvéolos o de una ruptura esofágica, que viaja a través de las vainas vasculares y los planos tisulares hacia el espacio mediastinal; puede ser categorizado como espontáneo, traumático o secundario.Objetivo: describir un paciente con neumomediastino secundario al efecto Macklin.Caso clínico: se trata de un paciente masculino de 32 años sin antecedentes médicos, que acude a urgencias refiriendo tos seca, dolor de garganta, dolor al ingerir alimentos, malestar general, dolores articulares a predominio de rodillas y espalda baja y fiebre de 39°C acompañada de escalofríos. El día posterior a su ingreso, tras un acceso de tos, presenta de forma súbita aumento de volumen del cuello y porción superior del tórax, acompañado de dolor en dicha zona, dificultad para respirar y hablar. Al examen en este momento se constata aumento de volumen y crepitación en cuello, fosa supraclavicular, y porción anterosuperior y posterosuperior del tórax (enfisema subcutáneo) así como estertores roncos y sibilantes diseminados en ambos campos pulmonares. Se realiza radiografía de tórax de urgencia que muestra signos sugestivos de enfisema subcutáneo y neumomediastino. Se solicita valoración por servicio de cirugía General y otorrino, los cuales sugieren tratamiento conservador.Conlusiones: el neumomediastino es una enfermedad poco frecuente y benigna, sus características clínicas son dolor torácico y enfisema subcutáneo luego de un acceso de tos y evoluciona de forma satisfactoria entre dos y 15 días. Introduction: pneumomediastinum is the presence of air in the mediastinal space coming from bronchi, alveoli or esophageal rupture, which travels through vascular sheaths and tissue planes to the mediastinal space; it can be categorized as spontaneous, traumatic or secondary.Objective: to describe a patient with pneumomediastinum secondary to the Macklin effect.Case report: a 32-year-old male patient with no medical history came to the emergency room with a dry cough, sore throat, pain when eating and general malaise, joint pain predominantly in the knees and lower back, and fever of 39°C with chills. The day after his admission, after an attack of coughing, he suddenly presented an increase in volume of the neck and upper chest, accompanied by pain in this area, difficulty in breathing and speaking. On examination at this time, there was an increase in volume and crepitus in the neck, supraclavicular fossa, and anterosuperior and posterosuperior portion of the thorax (subcutaneous emphysema) as well as hoarse and wheezing rales disseminated in both lung fields. An emergency chest X-ray was performed showing signs suggestive of subcutaneous emphysema and pneumomediastinum. An evaluation by the General Surgery and Otorhinolaryngology Departments was requested, which suggested conservative treatment.Conclusions: pneumomediastinum is a rare and benign disease, its clinical characteristics are chest pain and subcutaneous emphysema after a coughing fit and it evolves satisfactorily between 2 and 15 days

Alternative Splice Variants in TIM Barrel Proteins from Human Genome Correlate with the Structural and Evolutionary Modularity of this Versatile Protein Fold

<div><p>After the surprisingly low number of genes identified in the human genome, alternative splicing emerged as a major mechanism to generate protein diversity in higher eukaryotes. However, it is still not known if its prevalence along the genome evolution has contributed to the overall functional protein diversity or if it simply reflects splicing noise. The (βα)₈ barrel or TIM barrel is one of the most frequent, versatile, and ancient fold encountered among enzymes. Here, we analyze the structural modifications present in TIM barrel proteins from the human genome product of alternative splicing events. We found that 87% of all splicing events involved deletions; most of these events resulted in protein fragments that corresponded to the (βα)₂, (βα)₄, (βα)₅, (βα)₆, and (βα)₇ subdomains of TIM barrels. Because approximately 7% of all the splicing events involved internal β-strand substitutions, we decided, based on the genomic data, to design β-strand and α-helix substitutions in a well-studied TIM barrel enzyme. The biochemical characterization of one of the chimeric variants suggests that some of the splice variants in the human genome with β-strand substitutions may be evolving novel functions via either the oligomeric state or substrate specificity. We provide results of how the splice variants represent subdomains that correlate with the independently folding and evolving structural units previously reported. This work is the first to observe a link between the structural features of the barrel and a recurrent genetic mechanism. Our results suggest that it is reasonable to expect that a sizeable fraction of splice variants found in the human genome represent structurally viable functional proteins. Our data provide additional support for the hypothesis of the origin of the TIM barrel fold through the assembly of smaller subdomains. We suggest a model of how nature explores new proteins through alternative splicing as a mechanism to diversify the proteins encoded in the human genome.</p></div

Pipeline of bioinformatics analysis to identify the (βα)₈ barrel proteins with splice variants in the human genome.

<p>Summary of our bioinformatics data flow to extract the 135 experimentally confirmed (βα)₈ barrel splice variants in the human genome.</p

Biochemical characterization of a chimeric variant.

<p>The overall standard errors of enzyme kinetic parameters are less than 20%.</p>a<p>The apparent thermal melting temperature (°C).</p>b<p>Data obtained from Ochoa-Leyva et al., 2011 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070582#pone.0070582-OchoaLeyva1" target="_blank">[25]</a>.</p