Development of pipeline to create and validate metagenomic datasets enabling microbiota associations with host traits

The microbiota can be depicted as a measurable organ consisting of microbial cells, and creating a unique ecosystem together with the host eukaryotic cells. Therefore, to understand the normal physiology and pathology of animal ecosystems, it is mandatory to tackle a comprehensive analysis of the host, the microbiota, and their interactions. Since over 99.8% of the microbes cannot be cultured, metagenomics offers a path to the study of their community structures and metabolic potential. These goals are achievable thanks to the recent advances in sequencing complex assortments of small genomes, through 16S and WGS approach. In spite of the great number of latest studies, there is not a defined and standardized pipeline to measure the microbial community; rather, significant efforts are needed to optimize sample preparation and data analysis workflows for metagenomics analysis of microbiome. In the present PhD Thesis, a major task is aimed at developing rapid and efficient workflows for metagenomics analysis. These include the choice of the sample, the extraction methods more efficient according to sample features, choice of sequencing approaches and the statistical methods. A developed workflow was successfully applied to investigate to the first time the sheep gut microbiome, to perform association studies linking gut microbiota to T1D host traits in mice and to assess dietary and immunogenetic background impact over gut microbiota in a translational murine model of NAFLD

The impact of sequence database choice on metaproteomic results in gut microbiota studies

Background: Elucidating the role of gut microbiota in physiological and pathological processes has recently emerged as a key research aim in life sciences. In this respect, metaproteomics, the study of the whole protein complement of a microbial community, can provide a unique contribution by revealing which functions are actually being expressed by specific microbial taxa. However, its wide application to gut microbiota research has been hindered by challenges in data analysis, especially related to the choice of the proper sequence databases for protein identification. Results: Here, we present a systematic investigation of variables concerning database construction and annotation and evaluate their impact on human and mouse gut metaproteomic results. We found that both publicly available and experimental metagenomic databases lead to the identification of unique peptide assortments, suggesting parallel database searches as a mean to gain more complete information. In particular, the contribution of experimental metagenomic databases was revealed to be mandatory when dealing with mouse samples. Moreover, the use of a "merged" database, containing all metagenomic sequences from the population under study, was found to be generally preferable over the use of sample-matched databases. We also observed that taxonomic and functional results are strongly database-dependent, in particular when analyzing the mouse gut microbiota. As a striking example, the Firmicutes/Bacteroidetes ratio varied up to tenfold depending on the database used. Finally, assembling reads into longer contigs provided significant advantages in terms of functional annotation yields. Conclusions: This study contributes to identify host- and database-specific biases which need to be taken into account in a metaproteomic experiment, providing meaningful insights on how to design gut microbiota studies and to perform metaproteomic data analysis. In particular, the use of multiple databases and annotation tools has to be encouraged, even though this requires appropriate bioinformatic resources

Learning mutational graphs of individual tumour evolution from single-cell and multi-region sequencing data

Background. A large number of algorithms is being developed to reconstruct evolutionary models of individual tumours from genome sequencing data. Most methods can analyze multiple samples collected either through bulk multi-region sequencing experiments or the sequencing of individual cancer cells. However, rarely the same method can support both data types. Results. We introduce TRaIT, a computational framework to infer mutational graphs that model the accumulation of multiple types of somatic alterations driving tumour evolution. Compared to other tools, TRaIT supports multi-region and single-cell sequencing data within the same statistical framework, and delivers expressive models that capture many complex evolutionary phenomena. TRaIT improves accuracy, robustness to data-specific errors and computational complexity compared to competing methods. Conclusions. We show that the application of TRaIT to single-cell and multi-region cancer datasets can produce accurate and reliable models of single-tumour evolution, quantify the extent of intra-tumour heterogeneity and generate new testable experimental hypotheses

Metaproteogenomics Reveals Taxonomic and Functional Changes between Cecal and Fecal Microbiota in Mouse

Previous studies on mouse models report that cecal and fecal microbial communities may differ in the taxonomic structure, but little is known about their respective functional activities. Here, we employed a metaproteogenomic approach, including 16S rRNA gene sequencing, shotgun metagenomics and shotgun metaproteomics, to analyze the microbiota of paired mouse cecal contents (CCs) and feces, with the aim of identifying changes in taxon-specific functions. As a result, Gram-positive anaerobes were observed as considerably higher in CCs, while several key enzymes, involved in oxalate degradation, glutamate/glutamine metabolism, and redox homeostasis, and most actively expressed by Bacteroidetes, were clearly more represented in feces. On the whole, taxon and function abundance appeared to vary consistently with environmental changes expected to occur throughout the transit from the cecum to outside the intestine, especially when considering metaproteomic data. The results of this study indicate that functional and metabolic differences exist between CC and stool samples, paving the way to further metaproteogenomic investigations aimed at elucidating the functional dynamics of the intestinal microbiota

International Coordination of Long-Term Ocean Biology Time Series Derived from Satellite Ocean Color Data

[ABSTRACT] In this paper, we will describe plans to coordinate the initial development of long-term ocean biology time series derived from global ocean color observations acquired by the United States, Japan and Europe, Specifically, we have been commissioned by the International Ocean Color Coordinating Group (IOCCG) to coordinate the development of merged products derived from the OCTS, SeaWiFS, MODIS, MERIS and GLI imagers. Each of these missions will have been launched by the year 2002 and will have produced global ocean color data products. Our goal is to develop and document the procedures to be used by each space agency (NASA, NASDA, and ESA) to merge chlorophyll, primary productivity, and other products from these missions. This coordination is required to initiate the production of long-term ocean biology time series which will be continued operationally beyond 2002. The purpose of the time series is to monitor interannual to decadal-scale variability in oceanic primary productivity and to study the effects of environmental change on upper ocean biogeochemical processes

Metaproteogenomics Reveals Taxonomic and Functional Changes between Cecal and Fecal Microbiota in Mouse

Previous studies on mouse models report that cecal and fecal microbial communities may differ in the taxonomic structure, but little is known about their respective functional activities. Here, we employed a metaproteogenomic approach, including 16S rRNA gene sequencing, shotgun metagenomics and shotgun metaproteomics, to analyze the microbiota of paired mouse cecal contents (CCs) and feces, with the aim of identifying changes in taxon-specific functions. As a result, Gram-positive anaerobes were observed as considerably higher in CCs, while several key enzymes, involved in oxalate degradation, glutamate/glutamine metabolism, and redox homeostasis, and most actively expressed by Bacteroidetes, were clearly more represented in feces. On the whole, taxon and function abundance appeared to vary consistently with environmental changes expected to occur throughout the transit from the cecum to outside the intestine, especially when considering metaproteomic data. The results of this study indicate that functional and metabolic differences exist between CC and stool samples, paving the way to further metaproteogenomic investigations aimed at elucidating the functional dynamics of the intestinal microbiota

Pain and stress after vaginal delivery: characteristics at hospital discharge and associations with parity

The objective of this study was to characterise pre-discharge maternal pain and stress severity after vaginal delivery and associations with parity. This is a descriptive analysis with 148 women in the early post-partum period (84 primiparae and 64 secondiparae) after vaginal delivery. Pain and stress were measured by McGill Pain Questionnaire (MGPQ) and by the Psychological Stress Measure (PSM). Vaginal delivery in primiparae women was associated with MGPQ, significantly higher pain scores. Sensorial, affective and mixed pain descriptive categories were also significantly higher. Pain location involved lower abdomen, vagina and perianal area. In addition, their PSM showed a significantly higher ‘Sense of effort and confusion’ subscale scores. In conclusion, this study provides important information on the quality of care implications of hospital-to-home discharge practices in puerperae after vaginal delivery, a critical time characterised by qualitatively and quantitatively high pain and stress in primiparae.Impact statement What is already known on this subject? Pain and fatigue are the most common problems reported by women in the early postpartum period. What the results of this study add? Primiparae who delivered vaginally presented at the time of hospital-to-home discharge significantly higher pain and stress, as compared to secondiparae. Pain involved lower abdomen, vagina and perianal area, whereas the stress was quantitatively higher in the ‘sense of effort and confusion’. What the implications are of these findings for clinical practice and/or further research? Awareness of problematic pain and stress associations with parity may offer the opportunity to better support puerperae to develop maternal orientation and adjust to motherhood

Potential and active functions in the gut microbiota of a healthy human cohort

Abstract Background The study of the gut microbiota (GM) is rapidly moving towards its functional characterization by means of shotgun meta-omics. In this context, there is still no consensus on which microbial functions are consistently and constitutively expressed in the human gut in physiological conditions. Here, we selected a cohort of 15 healthy subjects from a native and highly monitored Sardinian population and analyzed their GMs using shotgun metaproteomics, with the aim of investigating GM functions actually expressed in a healthy human population. In addition, shotgun metagenomics was employed to reveal GM functional potential and to compare metagenome and metaproteome profiles in a combined taxonomic and functional fashion. Results Metagenomic and metaproteomic data concerning the taxonomic structure of the GM under study were globally comparable. On the contrary, a considerable divergence between genetic potential and functional activity of the human healthy GM was observed, with the metaproteome displaying a higher plasticity, compared to the lower inter-individual variability of metagenome profiles. The taxon-specific contribution to functional activities and metabolic tasks was also examined, giving insights into the peculiar role of several GM members in carbohydrate metabolism (including polysaccharide degradation, glycan transport, glycolysis, and short-chain fatty acid production). Noteworthy, Firmicutes-driven butyrogenesis (mainly due to Faecalibacterium spp.) was shown to be the metabolic activity with the highest expression rate and the lowest inter-individual variability in the study cohort, in line with the previously reported importance of the biosynthesis of this microbial product for the gut homeostasis. Conclusions Our results provide detailed and taxon-specific information regarding functions and pathways actively working in a healthy GM. The reported discrepancy between expressed functions and functional potential suggests that caution should be used before drawing functional conclusions from metagenomic data, further supporting metaproteomics as a fundamental approach to characterize the human GM metabolic functions and activities

Gestational weight gain and eating-related disorders

This prospective cohort study took place at the Division of Perinatal Medicine of Policlinico Abano Terme, Italy, from January to November 2018. In the second day postpartum, 463 healthy at term puerperae, 122 (26.35%) with inadequate gestational weight gain (GWG), 210 (45.46%) with adequate GWG, and 131 (28.29%) with excessive GWG, were studied by EAT-26, through distinguishing three factors: ‘Dieting’, ‘Bulimia and food preoccupation’, and ‘Oral control’. EAT-26 Global score increased from inadequate, to adequate, and excessive GWG puerperae, resulting significantly higher in excessive GWG group (p = .0029, Anova’s). In addition, among EAT-26 subscales ‘Dieting’ scores significantly increased from inadequate, to adequate, and to excessive GWG category women, resulting significantly higher in excessive GWG group (p = .006, Anova’s). It was found that excessive GWG is a warning indicator of unhealthy eating and ‘Dieting’ disorders. This relationship highlights the potential for interventions directed towards psychosocial support to have salutary effects upon GWG. Excessive gestational weight gain across an uncomplicated pregnancy is a warning indicator of unhealthy eating and dieting disorders.IMPACT STATEMENT What is already known on this subject? Pregnancy represents a time of rapid trimester-specific changes in body weight and size. What do the results of this study add? Excessive gestational weight gain is a warning indicator of unhealthy eating and dieting disorders. What are the implications of these findings for clinical practice and/or further research? This relationship highlights the potential for interventions directed towards psychosocial support to have salutary effects upon gestational weight gain

Caloric restriction promotes rapid expansion and long-lasting increase of Lactobacillus in the rat fecal microbiota

Previous studies indicated that caloric restricted diet enables to lower significantly the risk of cardiovascular and metabolic diseases. In experimental animal models, life-long lasting caloric restriction (CR) was demonstrated to induce changes of the intestinal microbiota composition, regardless of fat content and/or exercise. To explore the potential impact of short and long-term CR treatment on the gut microbiota, we conducted an analysis of fecal microbiota composition in young and adult Fisher 344 rats treated with a low fat feed under ad libitum (AL) or CR conditions (70%). We report here significant changes of the rat fecal microbiota that arise rapidly in young growing animals after short-term administration of a CR diet. In particular, Lactobacillus increased significantly after 8 weeks of CR treatment and its relative abundance was significantly higher in CR vs AL fed animals after 36 weeks of dietary intervention. Taken together, our data suggest that Lactobacillus intestinal colonization is hampered in AL fed young rats compared to CR fed ones, while health-promoting CR diet intervention enables the expansion of this genus rapidly and persistently up to adulthood