Potent Neutralizing Activity of Polyclonal Equine Antibodies Against Severe Acute Respiratory Syndrome Coronavirus 2 Variants of Concern.

Several anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) monoclonal antibodies (mAbs) have received emergency authorization for coronavirus disease 2019 (COVID-19) treatment. However, most of these mAbs are not active against the highly mutated Omicron SARS-CoV-2 subvariants. We have tested a polyclonal approach of equine anti-SARS-CoV-2 F(ab')2 antibodies that achieved a high level of neutralizing potency against all SARS-CoV-2 variants of concern tested including Omicron BA.1, BA.2, BA.2.12 and BA.4/5. A repertoire of antibodies targeting conserved epitopes in different regions of the spike protein could plausibly account for this remarkable breadth of neutralization. These results warrant the clinical investigation of equine polyclonal F(ab')2 antibodies as a novel therapeutic strategy against COVID-19.

Chemical targeting of NEET proteins reveals their function in mitochondrial morphodynamics.

Several human pathologies including neurological, cardiac, infectious, cancerous, and metabolic diseases have been associated with altered mitochondria morphodynamics. Here, we identify a small organic molecule, which we named Mito-C. Mito-C is targeted to mitochondria and rapidly provokes mitochondrial network fragmentation. Biochemical analyses reveal that Mito-C is a member of a new class of heterocyclic compounds that target the NEET protein family, previously reported to regulate mitochondrial iron and ROS homeostasis. One of the NEET proteins, NAF-1, is identified as an important regulator of mitochondria morphodynamics that facilitates recruitment of DRP1 to the ER-mitochondria interface. Consistent with the observation that certain viruses modulate mitochondrial morphogenesis as a necessary part of their replication cycle, Mito-C counteracts dengue virus-induced mitochondrial network hyperfusion and represses viral replication. The newly identified chemical class including Mito-C is of therapeutic relevance for pathologies where altered mitochondria dynamics is part of disease etiology and NEET proteins are highlighted as important therapeutic targets in anti-viral research.

Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.

Hepatitis B virus (HBV) and bile salt metabolism seem tightly connected. HBV enters hepatocytes by binding to sodium taurocholate cotransporting polypeptide (NTCP), the genome of which contains 2 active farnesoid X receptor (FXR) α response elements that participate in HBV transcriptional activity. We investigated in differentiated HepaRG cells and in primary human hepatocytes (PHHs) effects of FXR activation on HBV replication and of infection on the FXR pathway. In HepaRG cells, FXR agonists (6-ethyl chenodeoxycholic acid and GW4064), but no antagonist, and an FXR-unrelated bile salt inhibited viral mRNA, DNA, and protein production (IC50, 0.1-0.5 µM) and reduced covalently closed circular DNA pool size. These effects were independent of the NTCP inhibitor cyclosporine-A, which suggests inhibition occurred at a postentry step. Similar results were obtained in PHHs with GW4064. Infection of these cells increased expression of FXR and modified expression of FXR-regulated genes SHP, APOA1, NTCP, CYP7A1, and CYP8B1 with a more pronounced effect in PHHs than in HepaRG cells. FXR agonists reversed all but one of the HBV-induced FXR gene profile modifications. HBV replication and FXR regulation seem to be interdependent, and altered bile salt metabolism homeostasis might contribute to the persistence of HBV infection.-Radreau, P., Porcherot, M., Ramière, C., Mouzannar, K., Lotteau, V., André, P. Reciprocal regulation of farnesoid X receptor α activity and hepatitis B virus replication in differentiated HepaRG cells and primary human hepatocytes.

The metabolic sensors FXRα, PGC-1α, and SIRT1 cooperatively regulate hepatitis B virus transcription.

Hepatitis B virus (HBV) genome transcription is highly dependent on liver-enriched, metabolic nuclear receptors (NRs). Among others, NR farnesoid X receptor α (FXRα) enhances HBV core promoter activity and pregenomic RNA synthesis. Interestingly, two food-withdrawal-induced FXRα modulators, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) and deacetylase SIRT1, have been found to be associated with HBV genomes ex vivo. Whereas PGC-1α induction was shown to increase HBV replication, the effect of SIRT1 on HBV transcription remains unknown. Here, we showed that, in hepatocarcinoma-derived Huh-7 cells, combined activation of FXRα by GW4064 and SIRT1 by activator 3 increased HBV core promoter-controlled luciferase expression by 25-fold, compared with a 10-fold increase with GW4064 alone. Using cell lines differentially expressing FXRα in overexpression and silencing experiments, we demonstrated that SIRT1 activated the core promoter in an FXRα- and PGC-1α-dependent manner. Maximal activation (>150-fold) was observed in FXRα- and PGC-1α-overexpressing Huh-7 cells treated with FXRα and SIRT1 activators. Similarly, in cells transfected with full-length HBV genomes, maximal induction (3.5-fold) of core promoter-controlled synthesis of 3.5-kb RNA was observed in the same conditions of transfection and treatments. Thus, we identified a subnetwork of metabolic factors regulating HBV replication, strengthening the hypothesis that transcription of HBV and metabolic genes is similarly controlled.

Hypoxia-inducible factor-1 (HIF-1) pathway activation by quercetin in human lens epithelial cells.

Quercetin is a dietary bioflavonoid which has been shown to inhibit lens opacification in a number of models of cataract. The objectives of this study were to determine gene expression changes in human lens epithelial cells in response to quercetin and to investigate in detail the mechanisms underlying the responses. FHL-124 cells were treated with quercetin (10 microM) and changes in gene expression were measured by microarray. It was found that 65% of the genes with increased expression were regulated by the hypoxia-inducible factor-1 (HIF-1) pathway. Quercetin (10 and 30 microM) induced a time-dependent increase in HIF-1alpha protein levels. Quercetin (30 microM) was also responsible for a rapid and long-lasting translocation of HIF-1alpha from the cytoplasm to the nucleus. Activation of HIF-1 signaling by quercetin was confirmed by qRT-PCR which showed upregulation of the HIF-1 regulated genes EPO, VEGF, PGK1 and BNIP3. Analysis of medium taken from FHL-124 cells showed a sustained dose-dependent increase in VEGF secretion following quercetin treatment. The quercetin-induced increase and nuclear translocation of HIF-1alpha was reversed by addition of excess iron (100 microM). These results demonstrate that quercetin activates the HIF-1 signaling pathway in human lens epithelial cells.

Processing blackcurrants dramatically reduces the content and does not enhance the urinary yield of anthocyanins in human subjects.

Blackcurrant (BC) fruits are a rich source of biologically active anthocyanins but little is known of the anthocyanin content of commonly consumed BC products or how processing affects the absorption. We report that whereas fresh and frozen whole BC were a rich source of anthocyanins, containing 897 and 642mg (100gFW)(-1) of total anthocyanins the levels in all other products were substantially lower (0.05-10.3% of the levels in fresh fruit). Further, when the absorption and excretion of BC was assessed in volunteers consuming a portion (100g) of frozen whole BC (642mg total anthocyanins) and, 300g of a BC drink made by diluting concentrated syrup (33.6mg total anthocyanins), only small quantities of BC anthocyanins were excreted in urine (fruit, 0.053±0.022%; drink, 0.036±0.043%; mean percent urinary yield±SD) and they were not detected in plasma. These data indicate that fresh and frozen BC, but not processed products, are rich sources of anthocyanins but, regardless of the food source, these anthocyanins are poorly bioavailable.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of proteins in dry-cured hams: data registration and multivariate analysis across multiple gels.

This study investigates whether dry-cured hams from two European countries can be distinguished using SDS-PAGE. Thirty-seven commercial hams (19 Spanish, 18 French) were used in the study. Four protein fractions were extracted from each sample, with sufficient material prepared to allow each fraction to be analysed in triplicate lanes. The complete extraction process was carried out in duplicate. The 24 specimens originating from each ham sample were randomly allocated to different lane positions and gels, as were at least two reference lanes (for reference proteins). In total, 118 gels were prepared. Mathematical routines were developed using a matrix language to process the gel image files. Procedures were written to carry out 'within-gel' image correction, lane extraction and normalization, 'between-gel' data registration and linear discriminant analysis (LDA) of each fraction's data to establish whether the provenance could be systematically distinguished. The between-gel registration was carried out using a genetic algorithm (GA). Feature selection was also performed using a GA, to pass subsets of features to the LDA routine. Cross-validated classification success rates were 84, 91, 81 and 85%, respectively, for the four fractions. We conclude that SDS-PAGE can be conducted in a sufficiently quantitative manner and can potentially verify the provenance of regional speciality dry-cured hams.