Formal total synthesis of amphidinolide E

A formal total synthesis of the cytotoxic macrolide amphidinolide E is reported. The strategic steps are three Julia-Kocienski reactions (J-K), for the formation of the C5-C6, C9-C10, and C17-C18 double bonds, a Suzuki-Molander C21-C22 bond formation reaction, and a Kita-Trost macrolactonization. The 'instability' of the two dienic systems and of the stereocenter at C2 (allylic methine, alpha to the carboxy group) and the protecting groups at C17-OH and C18-OH have posed difficult challenges. Each Julia-Kocienski olefination has been systematically optimized to provide the highest possible E/Z ratios

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

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

<span style="color:rgb( 33 , 33 , 33 )">Comparison of Extracellular Vesicle Isolation Methods for miRNA Sequencing</span>

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