Benchmarking microbiome transformations favors experimental quantitative approaches to address compositionality and sampling depth biases

While metagenomic sequencing has become the tool of preference to study host-associated microbial communities, downstream analyses and clinical interpretation of microbiome data remains challenging due to the sparsity and compositionality of sequence matrices. Here, we evaluate both computational and experimental approaches proposed to mitigate the impact of these outstanding issues. Generating fecal metagenomes drawn from simulated microbial communities, we benchmark the performance of thirteen commonly used analytical approaches in terms of diversity estimation, identification of taxon-taxon associations, and assessment of taxon-metadata correlations under the challenge of varying microbial ecosystem loads. We find quantitative approaches including experimental procedures to incorporate microbial load variation in downstream analyses to perform significantly better than computational strategies designed to mitigate data compositionality and sparsity, not only improving the identification of true positive associations, but also reducing false positive detection. When analyzing simulated scenarios of low microbial load dysbiosis as observed in inflammatory pathologies, quantitative methods correcting for sampling depth show higher precision compared to uncorrected scaling. Overall, our findings advocate for a wider adoption of experimental quantitative approaches in microbiome research, yet also suggest preferred transformations for specific cases where determination of microbial load of samples is not feasible

La Nova etapa pedagògica de la Mentora Alsina : documentació i divulgació del patrimoni científic i tècnic al mNACTEC

The Mentora Alsina permanent exhibition was inaugurated on 23th May 2006 at mNACTEC. Reproducing the Physics Experimental Laboratory Mentora Alsina (1906-1991) —objects, practices, spaces, educational methodologies—, we have tried to reproduce knowledge, skills, interests and amazements in the new audienc of the new century. At once, the historical trajectories of the collections have permitted to show scientific contexts and critical reviews

Meibomian Gland Morphology: The Influence of Structural Variations on Gland Function and Ocular Surface Parameters

Purpose: To objectively and quantitatively characterize meibomian gland morphology and to investigate the influence of morphological variations on gland function and ocular surface and tear film parameters. Methods: One hundred fifty subjects were enrolled. The examinations included tear osmolarity, tear meniscus height, bulbar conjunctival hyperemia, noninvasive tear film breakup time, lid margin thickness, foam secretion, meibomian gland expressibility, count of functioning glands, corneal and conjunctival staining, fluorescein breakup time, lid wiper epitheliopathy, and Schirmer test. Patient symptoms were assessed using the Ocular Surface Disease Index questionnaire. Images from noncontact meibography were analyzed using an automated method that objectively estimates dropout area, number of glands, gland length and width, and gland irregularity. Results: Gland irregularity highly correlated with dropout area (r = −0.4, P < 0.001) and showed significant partial correlations with fluorescein breakup time (r = 0.162, P = 0.049) and the Ocular Surface Disease Index questionnaire (r = −0.250, P = 0.002) Subjects with dropout area <32% were divided into 2 groups: high and low irregularity. Gland expressibility was statistically significantly different between the 2 groups (U = 319.5, P = 0.006). In the high irregularity group, gland irregularity correlated with the Schirmer test (r = 0.530, P = 0.001) and corneal fluorescein staining (r = −0.377, P = 0.021). Conclusions: Automated morphological analysis of meibomian gland structure provides additional quantitative and objective information regarding gland morphology. The link between dropout area and gland function is not clear. Assessment of gland irregularity might better predict gland function and its effects on ocular surface and tear film parameters

A Novel Automated Approach for Infrared-Based Assessment of Meibomian Gland Morphology

Purpose: We present and validate a new methodology for analyzing, in an automated and objective fashion, infrared images of the meibomian glands (MG). Methods: The developed algorithm consists of three main steps: selection of the region of interest, detection of MG, and analysis of MG morphometric parameters and dropout area (DOA). Additionally, a new approach to quantify the irregularity of MG is introduced. We recruited 149 adults from a general population. Infrared meibography, using Keratograph 5M, was performed. Images were assessed and graded subjectively (Meiboscore) by two experienced clinicians and objectively with the proposed automated method. Results: The correlation of subjective DOA assessment between the two clinicians was poor and the average percentage of DOA estimated objectively for each Meiboscore group did not lie within their limits. The objective assessment showed lower variability of meibography grading than that obtained subjectively. Additionally, a new grading scale of MG DOA that reduces intraclass variation is proposed. Reported values of MG length and width were inversely proportional to the DOA. Gland irregularity was objectively quantified. Conclusions: The proposed automatic and objective method provides accurate estimates of the DOA as well as additional morphologic parameters that could add valuable information in MG dysfunction understanding and diagnosis. Translational Relevance: This approach highlights the shortcomings of currently used subjective methods, and provides the clinicians with an objective, quantitative and less variable alternative for assessing MG in a noninvasive and automated fashion. It provides a viable alternative to more time-consuming subjective methods

Specific Plasma MicroRNA Signatures Underlying the Clinical Outcomes of Hepatitis E Virus Infection

The pathogenic mechanisms determining the diverse clinical outcomes of HEV infection (e.g., self-limiting vs chronic or symptomatic vs asymptomatic) are not yet understood. Because specific microRNA signatures during viral infection inform the cellular processes involved in virus replication and pathogenesis, we investigated plasma microRNA profiles in 44 subjects, including patients with symptomatic acute (AHE, n = 7) and chronic (CHE, n = 6) hepatitis E, blood donors with asymptomatic infection (HEV BDs, n = 9), and anti-HEV IgG+IgM- (exposed BDs, n = 10) and anti-HEV IgG-IgM- (naïve BDs, n = 12) healthy blood donors. By measuring the abundance of 179 microRNAs in AHE and naïve BDs by RT-qPCR, we identified 51 potencial HEV-regulated microRNAs (PBH < 0.05). Further analysis showed that HEV genotype 3 infection is associated with miR-122, miR-194, miR-885, and miR-30a upregulation and miR-221, miR-223, and miR-27a downregulation. AHE showed significantly higher levels of miR-122 and miR-194, and lower levels of miR-221, miR-27a, and miR-335 compared to HEV BDs. This specific microRNA signature in AHE could promote virus replication and reduce antiviral immune responses, contributing to the development of clinical symptoms. We found that mir-194, miR-335, and miR-221 can discriminate between asymptomatic HEV infections and those developing acute symptoms, whereas miR-335 correctly classify AHE and CHE. Conclusions: Our data suggest that diverse outcomes of HEV infection result from different HEV-induced microRNA dysregulations. The specific microRNA signatures described offer novel information that may serve to develop biomarkers of HEV infection outcomes and improve our understanding of HEV pathogenesis, which may facilitate the identification of antiviral targets

Randomized crossover trial of silicone hydrogel contact lenses

Purpose: The aim of the current study is to assess, using new technologies, the interaction of four monthly silicone hydrogel contact lenses on the ocular surface and the comfort over 15 days of use. Methods: Prospective cross-over, randomized and double-masked study including four materials (lotrafilcon-B, samfilcon-A , comfilcon-A and filcom-V3). Clinical examination was performed in the following order: tear meniscus height, first break-up of the tear film, the average time of all tear film breakup incidents, bulbar redness, limbal redness (Keratograph 5M ,Oculus, Germany); central corneal thickness (Pentacam, Oculus, Germany), thermography values (FLIR A325; FLIR Systems Inc., USA), and slit-lamp evaluations, including ocular surface staining. Finally, subjective comfort was obtained from Contact Lens Dry Eye Questionnaire-8. Results: The impact of contact lens wear on the ocular surface didn’t show statistically significant changes over time except for corneal and conjunctival staining grades on day 15 compared to day 1 for the comfilcon A group (P = .003 and P = .01, respectively). Contact lens stability and impact on the ocular surface during contact lens wear didn’t show statistically significant changes over time except in the case of the comfilcon A material with respect to the irritation item (P = .01). Conclusions: These results suggest that the impact of monthly silicone hydrogel contact lens materials on the ocular surface after and during contact lens wear, contact lens stability over time, and subjective comfort did not reveal any significant changes over 15 days of use for any of the materials

The influence of meibomian gland loss on ocular surface clinical parameters

Purpose: To assess the relationship between the meibomian gland loss (MGL) and relevant ocular surface clinical parameters as well as the influence of age in this relationship. Methods: A total of 161 participants (mean age; 42±17 years) were enrolled in this study. Infrared meibography was performed using Keratograph 5M (K5M; Oculus GmbH, Wetzlar). Participants were divided into five groups according to total meiboscore and the ocular surface parameters of each MGL group were studied. In addition, the relationship between MGL and the ocular surface parameters was established including age as covariant. Results: Both eyelids were taken into account since no association between the MGL from upper and lower eyelid was found (k value=0.2; p=0.3) despite they were significantly correlated (r= 0.3; p<0.001). No statistically significant differences were found in symptomatology among different MGL groups. Statistically significant differences were found among MGL groups in tear osmolarity (p=0.02), bulbar redness (p=0.04), corneal and conjunctival staining (p=0.01 and p=0.004, respectively). Despite this, only corneal staining showed a significant correlation with MGL when age was covariant (r=0.2; p=0.04). Conclusions: MGL higher than 50% seems to be accompanied by signs on the ocular surface. Furthermore, age demonstrated to be a relevant factor when assessing MGL. For this reason, future studies should compare age-matched groups in order to know the contribution of the MGL on the ocular surface and establish valid cut-off values for dry eye diagnosis

Partial restoration of immune response in Hepatitis C patients after viral clearance by direct-acting antiviral therapy

Fibrosi hepàtica; Cèl·lules T; Teràpia de citocinesFibrosis hepática; Células T; Terapia de citocinasLiver fibrosis; T cells; Cytokine therapyBackground & aims HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. Methods We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Ɣ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Ɣ) and CFSE-based proliferation assays. Results We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Ɣ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. Conclusions Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA’s therapies.This study was funded by Instituto de Salud Carlos III, cofinanced by the European Regional Development Fund (ERDF): grant numbers PI16/00337, PI18/00210 and PI19/00301. C.P. is supported by the Miguel Servet program of the Instituto de Salud Carlos III (CP14/00121 and CPII19/00001) cofinanced by the European Regional Development Fund (ERDF)

Partial restoration of immune response in Hepatitis C patients after viral clearance by direct-acting antiviral therapy

Background & aims: HCV CD4+ and CD8+ specific T cells responses are functionally impaired during chronic hepatitis C infection. DAAs therapies eradicate HCV infection in more than 95% of treated patients. However, the impact of HCV elimination on immune responses remain controversial. Here, we aimed to investigate whether HCV cure by DAAs could reverse the impaired immune response to HCV. Methods: We analyzed 27 chronic HCV infected patients undergoing DAA treatment in tertiary care hospital, and we determined the phenotypical and functional changes in both HCV CD8+ and CD4+ specific T-cells before and after viral clearance. PD-1, TIM-3 and LAG-3 cell-surface expression was assessed by flow cytometry to determine CD4+ T cell exhaustion. Functional responses to HCV were analyzed by IFN-Ɣ ELISPOT, intracellular cytokine staining (IL-2 and IFN-Ɣ) and CFSE-based proliferation assays. Results: We observed a significant decrease in the expression of PD-1 in CD4+ T-cells after 12 weeks of viral clearance in non-cirrhotic patients (p = 0.033) and in treatment-naive patients (p = 0.010), indicating a partial CD4 phenotype restoration. IFN-Ɣ and IL-2 cytokines production by HCV-specific CD4+ and CD8+ T cells remained impaired upon HCV eradication. Finally, a significant increase of the proliferation capacity of both HCV CD4+ and CD8+ specific T-cells was observed after HCV elimination by DAAs therapies. Conclusions: Our results show that in chronically infected patients HCV elimination by DAA treatment lead to partial reversion of CD4+ T cell exhaustion. Moreover, proliferative capacity of HCV-specific CD4+ and CD8+ T cells is recovered after DAA's therapies

Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing

MicroRNAs (miRNAs) encapsulated in extracellular vesicles (EVs) are potential diagnostic and prognostic biomarkers. However, discrepancies in miRNA patterns and their validation are still frequent due to differences in sample origin, EV isolation, and miRNA sequencing methods. The aim of the present study is to find a reliable EV isolation method for miRNA sequencing, adequate for clinical application. To this aim, two comparative studies were performed in parallel with the same human plasma sample: (i) isolation and characterization of EVs obtained using three procedures: size exclusion chromatography (SEC), iodixanol gradient (GRAD), and its combination (SEC+GRAD) and (ii) evaluation of the yield of miRNA sequences obtained using NextSeq 500 (Illumina) and three miRNA library preparation protocols: NEBNext, NEXTFlex, and SMARTer smRNA-seq. The conclusion of comparison (i) is that recovery of the largest amount of EVs and reproducibility were attained with SEC, but GRAD and SEC+GRAD yielded purer EV preparations. The conclusion of (ii) is that the NEBNext library showed the highest reproducibility in the number of miRNAs recovered and the highest diversity of miRNAs. These results render the combination of GRAD EV isolation and NEBNext library preparation for miRNA retrieval as adequate for clinical applications using plasma samples