Statistical Evaluation of Metaproteomics and 16S rRNA Amplicon Sequencing Techniques for Study of Gut Microbiota Establishment in Infants with Cystic Fibrosis.

Newborn screening for cystic fibrosis (CF) can identify affected but asymptomatic infants. The selection of omic technique for gut microbiota study is crucial due to both the small amount of feces available and the low microorganism load. Our aims were to compare the agreement between 16S rRNA amplicon sequencing and metaproteomics by a robust statistical analysis, including both presence and abundance of taxa, to describe the sequential establishment of the gut microbiota during the first year of life in a small size sample (8 infants and 28 fecal samples). The taxonomic assignations by the two techniques were similar, whereas certain discrepancies were observed in the abundance detection, mostly the lower predicted relative abundance of Bifidobacterium and the higher predicted relative abundance of certain Firmicutes and Proteobacteria by amplicon sequencing. During the first months of life, the CF gut microbiota is characterized by a significant enrichment of Ruminococcus gnavus, the expression of certain virulent bacterial traits, and the detection of human inflammation-related proteins. Metaproteomics provides information on composition and functionality, as well as data on host-microbiome interactions. Its strength is the identification and quantification of Actinobacteria and certain classes of Firmicutes, but alpha diversity indices are not comparable to those of amplicon sequencing. Both techniques detected an aberrant microbiota in our small cohort of infants with CF during their first year of life, dominated by the enrichment of R. gnavus within a human inflammatory environment. IMPORTANCE In recent years, some techniques have been incorporated for the study of microbial ecosystems, being 16S rRNA gene sequencing being the most widely used. Metaproteomics provides the advantage of identifying the interaction between microorganisms and human cells, but the available databases are less extensive as well as imprecise. Few studies compare the statistical differences between the two techniques to define the composition of an ecosystem. Our work shows that the two methods are comparable in terms of microorganism identification but provide different results in alpha diversity analysis. On the other hand, we have studied newborns with cystic fibrosis, for whom we have described the establishment of an intestinal ecosystem marked by the inflammatory response of the host and the enrichment of Ruminococcus gnavus.

Distinct Human Gut Microbial Taxonomic Signatures Uncovered With Different Sample Processing and Microbial Cell Disruption Methods for Metaproteomic Analysis.

The use of metaproteomics for studying the human gut microbiota can shed light on the taxonomic profile and the functional role of the microbial community. Nevertheless, methods for extracting proteins from stool samples continue to evolve, in the pursuit of optimal protocols for moistening and dispersing the stool sample and for disrupting microbial cells, which are two critical steps for ensuring good protein recovery. Here, we evaluated different stool sample processing (SSP) and microbial cell disruption methods (CDMs). The combination of a longer disintegration period of the stool sample in a tube rotator with sonication increased the overall number of identified peptides and proteins. Proteobacteria, Bacteroidetes, Planctomycetes, and Euryarchaeota identification was favored by mechanical cell disruption with glass beads. In contrast, the relative abundance of Firmicutes, Actinobacteria, and Fusobacteria was improved when sonication was performed before bead beating. Tenericutes and Apicomplexa identification was enhanced by moistening the stool samples during processing and by disrupting cells with medium-sized glass beads combined with or without sonication. Human protein identifications were affected by sonication. To test the reproducibility of these gut metaproteomic analyses, we examined samples from six healthy individuals using a protocol that had shown a good taxonomic diversity and identification of proteins from Proteobacteria and humans. We also detected proteins involved in microbial functions relevant to the host and related mostly to specific taxa, such as B12 biosynthesis and short chain fatty acid (SCFA) production carried out mainly by members in the Prevotella genus and the Firmicutes phylum, respectively. The taxonomic and functional profiles obtained with the different protocols described in this work provides the researcher with valuable information when choosing the most adequate protocol for the study of certain pathologies under suspicion of being related to a specific taxon from the gut microbiota.

Combined methods of optical spectroscopy and artificial intelligence in the assessment of experimentally induced non-alcoholic fatty liver.

BACKGROUND AND OBJECTIVE: Due to the existing prevalence of nonalcoholic fatty liver disease (NAFLD) and its relation to the epidemic of obesity in the general population, it is imperative to develop detection and evaluation methods of the early stages of the disease with improved efficacy over the current diagnostic approaches. We aimed to obtain an improved diagnosis, combining methods of optical spectroscopy -diffuse reflectance and fluorescence- with statistical data analysis applied to detect early stages of NAFLD. METHODS: Statistical analysis scheme based on quadratic discriminant analysis followed by canonical discriminant analysis were applied to the diffuse reflectance data combined with endogenous fluorescence spectral data excited at one of these wavelengths: 330, 365, 385, 405 or 415 nm. The statistical scheme was also applied to the combinations of fluorescence spectrum (405 nm) with each one of the other fluorescence spectra. Details of the developed software, including the application of machine learning algorithms to the combination of spectral data followed by classification statistical schemes, are discussed. RESULTS: Steatosis progression was differentiated with little classification error (≤1.3%) by using diffuse reflectance and endogenous fluorescence at different wavelengths. Similar results were obtained using fluorescence at 405 nm and one of the other fluorescence spectra (classification error ≤1.0%). Adding the corresponding areas under the curves to the above combinations of spectra diminished errors to 0.6% and 0.3% or less, respectively. The best results for the compounded reflectance-plus-fluorescence spectra were obtained with fluorescence spectra excited at 415 nm with a total classification error of 0.2%; for the combination of the 405nm-excited fluorescence spectrum with another fluorescence spectrum, the best results were achieved for 385 nm, for which total relative classification error amounted 0.4%. The consideration of the area under the spectral curves further improved both classifiers, reducing the error to 0.0% in both cases. CONCLUSION: Spectrometric techniques combined with statistical processing are a promising tool to improve steatosis classification through a label free approach. However, statistical schemes here applied, might result complex for the everyday medical practice, the designed software including machine learning algorithms is able to render automatic classification of samples according to their steatosis grade with low error.

Study of Methionine Choline Deficient Diet-Induced Steatosis in Mice Using Endogenous Fluorescence Spectroscopy.

Non-alcoholic fatty liver disease is a highly prevalent condition worldwide that increases the risk to develop liver fibrosis, cirrhosis, and hepatocellular carcinoma. Thus, it is imperative to develop novel diagnostic tools that together with liver biopsy help to differentiate mild and advanced degrees of steatosis. Ex-vivo liver samples were collected from mice fed a methionine-choline deficient diet for two or eight weeks, and from a control group. The degree of hepatic steatosis was histologically evaluated, and fat content was assessed by Oil-Red O staining. On the other hand, fluorescence spectroscopy was used for the assessment of the steatosis progression. Fluorescence spectra were recorded at excitation wavelengths of 330, 365, 385, 405, and 415 nm by establishing surface contact of the fiber optic probe with the liver specimens. A multi-variate statistical approach based on principal component analysis followed by quadratic discriminant analysis was applied to spectral data to obtain classifiers able to distinguish mild and moderate stages of steatosis at the different excitation wavelengths. Receiver Operating Characteristic (ROC) curves were computed to compare classifier's performances for each one of the five excitation wavelengths and steatosis stages. Optimal sensitivity and specificity were calculated from the corresponding ROC curves using the Youden index. Intensity in the endogenous fluorescence spectra at the given wavelengths progressively increased according to the time of exposure to diet. The area under the curve of the spectra was able to discriminate control liver samples from those with steatosis and differentiate among the time of exposure to the diet for most of the used excitation wavelengths. High specificities and sensitivities were obtained for every case; however, fluorescence spectra obtained by exciting with 405 nm yielded the best results distinguishing between the mentioned classes with a total classification error of 1.5% and optimal sensitivities and specificities better than 98.6% and 99.3%, respectively.

Diffuse reflectance spectroscopy accurately discriminates early and advanced grades of fatty liver in mice.

Nonalcoholic fatty liver disease (NAFLD) ranges from steatosis to nonalcoholic steatohepatitis and cirrhosis. Liver biopsy, considered the gold standard to diagnose NAFLD, shows significantly high rates of interobserver variability. Thus there is a need to develop tools that accurately categorize mild and advanced grades of steatosis in order to identify patients at higher risk of developing chronic liver disease. Diffuse reflectance spectroscopy (DRS) has proved to be useful in grading liver fibrosis and cirrhosis, without having been implemented for steatosis. We aim to categorize early and advanced stages of liver steatosis in a methionine-choline deficient (MCD) mouse model. C57bl/6 mice are fed either methionine-choline control or MCD diet during 2 or 8 weeks to induce mild and advanced steatosis. Liver samples are obtained and steatosis is evaluated by oil red O staining. Diffuse reflectance spectra are directly measured on ex vivo liver specimens, in a wavelength range of 400 to 800 nm. DRS is able to discriminate between early or advanced steatosis and healthy hepatic tissue with negligible error while showing high average sensitivity and specificity (0.94 and 0.95, respectively). Our results suggest that liver steatosis can be accurately evaluated by DRS, highlighting the importance of applied spectroscopic methods in assessing NAFLD.

Pet-Compton system. Comparative evaluation with PET system using Monte Carlo simulation / Sistema Pet-compton. Evaluación comparativa con el sistema PET usando la simulación por Monte Carlo

Positron Emission Tomography (PET) in small animals has actually achieved spatial resolution round about 1 mm and currently there are under study different approaches to improve this spatial resolution. One of them combines PET technology with Compton Cameras. This paper presents the idea of the so called "PET-Compton" systems and has included comparative evaluation of spatial resolution and global efficiency in both PET and PET-Compton system by means of Monte Carlo simulations using Geant4 code. Simulation was done on a PET-Compton system made-up of LYSO-LuYAP scintillating detectors of particular small animal PET scanner named "Clear-PET"and for Compton detectors based on CdZnTe semiconductor. A group of radionuclides that emits a positron (e+) and Y quantum almost simultaneously and fulfills some selection criteria for their possible use in PET-Compton systems for medical and biological applications were studied under simulation conditions. By means of analytical reconstruction using SSRB (Single Slide Rebinning) method were obtained superior spatial resolution in PET-Compton system for all tested radionuclides (reaching sub-millimeter values of for 22Na source). However this analysis done by simulation have shown limited global efficiency values in "PET-Compton" system (in the order of 10-5 - 10-5 %) instead of values around 5-10-1% % that have been achieved in PET system.

En la actualidad la tomografía por emisión de positrones (PET en pequeños animales ha alcanzado valores de resolución espacial cercanos a mm y en estos momentos se encuentran bajo estudio diferentes aproximaciones para mejorar dicha resolución espacial. Una de ellas combina la tecnología PET con las cámaras Compton. Este trabajo presenta la idea del denominado Sistema "PET-Compton" e incluye una evaluación comparativa de la resolución espacial y la eficiencia global de los sistemas PET y PET-Compton por medio de la simulación por Monte Carlo, utilizando el código Geant4. La simulación fue realizada en un sistema PET-Compton compuesto por detectores centellantes de LYSO-LUYAP de un específico y pequeño escáner PET denominado "Clear-PET" y para detectores Compton en base al semiconductor CdZnTe. Se estudiaron bajo las condiciones de simulación un grupo de radionúclidos que emiten un positrón (e+) y un cuanto gamma casi simultáneamente y cumplen ciertos criterios de selección para su posible utilización en aplicaciones médicas y biomédicas de los sistemas PET-Compton. Por medio de la reconstrucción analítica, empleando el método de reordenamiento de cortes simples (SSRB) se obtuvo una resolución espacial superior para el sistema PET-Compton en todos los radionúclidos de prueba, que alcanzó valores por debajo del milímetro para la fuente de 22Na. Sin embargo, el análisis realizado por medio de la simulación demostró valores limitados de eficiencia global para el sistema PET-Compton (del orden de 10-5-10-5%) en contraposición a los valores cercanos a 5-10-1 % que se alcanzaron para el sistema PET.