Investigating cartilage-related diseases by polarization-resolved second harmonic generation (P-SHG) imaging.

Establishing quantitative parameters for differentiating between healthy and diseased cartilage tissues by examining collagen fibril degradation patterns facilitates the understanding of tissue characteristics during disease progression. These findings could also complement existing clinical methods used to diagnose cartilage-related diseases. In this study, cartilage samples from normal, osteoarthritis (OA), and rheumatoid arthritis (RA) tissues were prepared and analyzed using polarization-resolved second harmonic generation (P-SHG) imaging and quantitative image texture analysis. The enhanced molecular contrast obtained from this approach is expected to aid in distinguishing between healthy and diseased cartilage tissues. P-SHG image analysis revealed distinct parameters in the cartilage samples, reflecting variations in collagen fibril arrangement and organization across different pathological states. Normal tissues exhibited distinct χ33/χ31 values compared with those of OA and RA, indicating collagen type transition and cartilage erosion with chondrocyte swelling, respectively. Compared with those of normal tissues, OA samples demonstrated a higher degree of linear polarization, suggesting increased tissue birefringence due to the deposition of type-I collagen in the extracellular matrix. The distribution of the planar orientation of collagen fibrils revealed a more directional orientation in the OA samples, associated with increased type-I collagen, while the RA samples exhibited a heterogeneous molecular orientation. This study revealed that the imaging technique, the quantitative analysis of the images, and the derived parameters presented in this study could be used as a reference for disease diagnostics, providing a clear understanding of collagen fibril degradation in cartilage.

Dual Effects of 3-epi-betulin from Daphniphyllum glaucescens in Suppressing SARS-CoV-2-Induced Inflammation and Inhibiting Virus Entry.

The continuous emergence of SARS-CoV-2 variants has led to a protracted global COVID-19 pandemic with significant impacts on public health and global economy. While there are currently available SARS-CoV-2 vaccines and therapeutics, most of the FDA-approved antiviral agents directly target viral proteins. However, inflammation is the initial immune pathogenesis induced by SARS-CoV-2 infection, there is still a need to find additional agents that can control the virus in the early stages of infection to alleviate disease progression for the next pandemic. Here, we find that both the spike protein and its receptor CD147 are crucial for inducing inflammation by SARS-CoV-2 in THP-1 monocytic cells. Moreover, we find that 3-epi-betulin, isolated from Daphniphyllum glaucescens, reduces the level of proinflammatory cytokines induced by SARS-CoV-2, consequently resulting in a decreased viral RNA accumulation and plaque formation. In addition, 3-epi-betulin displays a broad-spectrum inhibition of entry of SARS-CoV-2 pseudoviruses, including Alpha (B.1.1.7), Eplison (B.1.429), Gamma (P1), Delta (B.1.617.2) and Omicron (BA.1). Moreover, 3-epi-betulin potently inhibits SARS-CoV-2 infection with an EC50 of <20 µM in Calu-3 lung epithelial cells. Bioinformatic analysis reveals the chemical interaction between the 3-epi-betulin and the spike protein, along with the critical amino acid residues in the spike protein that contribute to the inhibitory activity of 3-epi-betulin against virus entry. Taken together, our results suggest that 3-epi-betulin exhibits dual effect: it reduces SARS-CoV-2-induced inflammation and inhibits virus entry, positioning it as a potential antiviral agent against SARS-CoV-2.

Terpenoids with anti-influenza activity from the leaves of Euphorbia leucocephala.

Two rearranged terpenoids with a rare 3,4,5-trimethyl-cyclohexa-2,5-dien-1-one moiety, namely leucocephins A (1) and B (2), and a megastigmane, namely leucocephin C (3), as well as three known compounds, hollongdione (4), 3-acetoxy-lup-12,20(29)-diene (5), and ß-amyrin acetate (6) were isolated from the leaves of Euphorbia leucocephala. Their structures and absolute configurations were determined by spectroscopic methods and comparing with literature data. Compounds 4-6 exhibited potent anti-influenza A virus activity comparable to the positive control, betulinic acid.

Dual Effects of Let-7b in the Early Stage of Hepatitis C Virus Infection.

MicroRNA let-7b expression is induced by infection of hepatitis C virus (HCV) and is involved in the regulation of HCV replication by directly targeting the HCV genome. The current study demonstrated that let-7b directly targets negative regulators of type I interferon (IFN) signaling thereby limiting HCV replication in the early stage of HCV infection. Let-7b-regulated genes which are involved in host cellular responses to HCV infection were unveiled by microarray profiling and bioinformatic analyses, followed by various molecular and cellular assays using Huh7 cells expressing wild-type (WT) or the seed region-mutated let-7b. Let-7b targeted the cytokine signaling 1 (SOCS1) protein, a negative regulator of JAK/STAT signaling, which then enhanced STAT1-Y701 phosphorylation leading to increased expression of the downstream interferon-stimulated genes (ISGs). Let-7b augmented retinoic acid-inducible gene I (RIG-I) signaling, but not MDA5, to phosphorylate and nuclear translocate IRF3 leading to increased expression of IFN-ß. Let-7b directly targeted the ATG12 and IκB kinase alpha (IKKα) transcripts and reduced the interaction of the ATG5-ATG12 conjugate and RIG-I leading to increased expression of IFN, which may further stimulate JAK/STAT signaling. Let-7b induced by HCV infection elicits dual effects on IFN expression and signaling, along with targeting the coding sequences of NS5B and 5' UTR of the HCV genome, and limits HCV RNA accumulation in the early stage of HCV infection. Controlling let-7b expression is thereby crucial in the intervention of HCV infection.IMPORTANCE HCV is a leading cause of liver disease, with an estimated 71 million people infected worldwide. During HCV infection, type I interferon (IFN) signaling displays potent antiviral and immunomodulatory effects. Host factors, including microRNAs (miRNAs), play a role in upregulating IFN signaling to limit HCV replication. Let-7b is a liver-abundant miRNA that is induced by HCV infection and targets the HCV genome to suppress HCV RNA accumulation. In this study, we demonstrated that let-7b, as a positive regulator of type I IFN signaling, plays dual roles against HCV replication by increasing the expression of IFN and interferon-sensitive response element (ISRE)-driven interferon-stimulated genes (ISGs) in the early stage of HCV infection. This study sheds new insight into understanding the role of let-7b in combatting HCV infection. Clarifying IFN signaling regulated by miRNA during the early phase of HCV infection may help researchers understand the initial defense mechanisms to other RNA viruses.

Hepatitis C virus down-regulates SERPINE1/PAI-1 expression to facilitate its replication.

Identification of host factors involved in viral replication is critical for understanding the molecular mechanism of viral replication and pathogenesis. Genes differentially expressed in HuH-7 cells with or without a hepatitis C virus (HCV) sub-genomic replicon were screened by microarray analysis. SERPINE1/PAI-1 was found to be down-regulated after HCV infection in this analysis. Down-regulation of SERPINE1/PAI-1 expression at the transcriptional level was verified by the real-time reverse transcriptase (RT)-PCR assay. Reduced SERPINE1/PAI-1 protein secretion was detected in the supernatant of HCV replicon cells and in sera from HCV-infected patients. SERPINE1 gene expression was down-regulated by HCV NS3/4A and NS5A proteins through the transforming growth factor-ß (TGF-ß) signalling pathway at the transcriptional level. Down-regulated genes in HCV replicon cells could be the factors supressing HCV replication. Indeed, over-expressed PAI-1 inhibited HCV replication but the mechanism is unknown. It has been demonstrated that HCV induces the expression of TGF-ß, and TGF-ß enhances HCV replication by a not-yet-defined mechanism. SERPINE1/PAI-1 is also known to be potently induced by TGF-ß at the transcriptional level through both Smad-dependent and Smad-independent pathways. The exogenously expressed SERPINE1/PAI-1 suppressed the expression of the endogenous SERPINE1 gene at the transcriptional level through the TGF-ß signalling but not the Smad pathway. Thus, SERPINE1/PAI-1 could suppress HCV replication possibly by negatively regulating TGF-ß signalling. A model is proposed for the interplay betweenthe TGF-ß signalling pathway, HCV and SERPINE1/PAI-1 to keep the homeostasis of the cells.

Chemical Constituents from Flueggea virosa and the Structural Revision of Dehydrochebulic Acid Trimethyl Ester.

In an attempt to study the chemical constituents from the twigs and leaves of Flueggea virosa, a new terpenoid, 9(10→20)-abeo-ent-podocarpane, 3ß,10α-dihydroxy-12-methoxy-13- methyl-9(10→20)-abeo-ent-podocarpa-6,8,11,13-tetraene (1), as well as five known compounds were characterized. Their structures were elucidated on the basis of spectroscopic analysis. In addition, the structure of dehydrochebulic acid trimethyl ester was revised as (2S,3R)-4E-dehydrochebulic acid trimethyl ester based on a single-crystal X-ray diffraction study. The in vitro anti-hepatitis C virus (anti-HCV) activity and cytotoxicity against Huh7.5 cells for the isolated compounds were evaluated.

Hepatitis C virus non-structural protein 3 interacts with cytosolic 5'(3')-deoxyribonucleotidase and partially inhibits its activity.

Infection with hepatitis C virus (HCV) is etiologically involved in liver cirrhosis, hepatocellular carcinoma and B-cell lymphomas. It has been demonstrated previously that HCV non-structural protein 3 (NS3) is involved in cell transformation. In this study, a yeast two-hybrid screening experiment was conducted to identify cellular proteins interacting with HCV NS3 protein. Cytosolic 5'(3')-deoxyribonucleotidase (cdN, dNT-1) was found to interact with HCV NS3 protein. Binding domains of HCV NS3 and cellular cdN proteins were also determined using the yeast two-hybrid system. Interactions between HCV NS3 and cdN proteins were further demonstrated by co-immunoprecipitation and confocal analysis in cultured cells. The cellular cdN activity was partially repressed by NS3 protein in both the transiently-transfected and the stably-transfected systems. Furthermore, HCV partially repressed the cdN activity while had no effect on its protein expression in the systems of HCV sub-genomic replicons and infectious HCV virions. Deoxyribonucleotidases are present in most mammalian cells and involve in the regulation of intracellular deoxyribonucleotides pools by substrate cycles. Control of DNA precursor concentration is essential for the maintenance of genetic stability. Reduction of cdN activity would result in the imbalance of DNA precursor concentrations. Thus, our results suggested that HCV partially reduced the cdN activity via its NS3 protein and this may in turn cause diseases.

Let-7b is a novel regulator of hepatitis C virus replication.

The non-coding microRNA (miRNA) is involved in the regulation of hepatitis C virus (HCV) infection and offers an alternative target for developing anti-HCV agent. In this study, we aim to identify novel cellular miRNAs that directly target the HCV genome with anti-HCV therapeutic potential. Bioinformatic analyses were performed to unveil liver-abundant miRNAs with predicted target sequences on HCV genome. Various cell-based systems confirmed that let-7b plays a negative role in HCV expression. In particular, let-7b suppressed HCV replicon activity and down-regulated HCV accumulation leading to reduced infectivity of HCVcc. Mutational analysis identified let-7b binding sites at the coding sequences of NS5B and 5'-UTR of HCV genome that were conserved among various HCV genotypes. We further demonstrated that the underlying mechanism for let-7b-mediated suppression of HCV RNA accumulation was not dependent on inhibition of HCV translation. Let-7b and IFNα-2a also elicited a synergistic inhibitory effect on HCV infection. Together, let-7b represents a novel cellular miRNA that targets the HCV genome and elicits anti-HCV activity. This study thereby sheds new insight into understanding the role of host miRNAs in HCV pathogenesis and to developing a potential anti-HCV therapeutic strategy.

293 cells over-expressing human ADI1 and CD81 are permissive for serum-derived hepatitis C virus infection.

Human aci-reductone dioxygenase 1 (ADI1) is a member of the Cupin superfamily. It binds to and inhibits the activities of membrane-type 1 matrix metalloproteinase, a protein known to interact with the tight junction protein, claudin-1. Previously, a variant protein, named submergence-induced protein-like factor (Sip-L), consisting of ADI1 amino acids 64-179, was found to support hepatitis C virus (HCV) infection and replication in 293 cells. In the present study, it was discovered that over-expression of human ADI1 in 293 cells (293-ADI1 cells) also supported HCV infection and replication. Using serum-derived HCV as an infectious source, enhanced cell uptake of HCV to a Northern blot detectable level was found in 293 cells over-expressing both CD81 and ADI1 (293-ADI1-CD81 cells). The enhanced cell entry was confirmed by the use of the vesicular stomatitis virus-based HCV pseudotype particles. However, transfection of HCV replicon RNA by electroporation into naïve 293 and 293-ADI1 cells revealed no difference in replication efficiency. Using the infectious J6/JFH chimera as an infectious source, the infectivity was compared between 293-ADI1-CD81 and Huh-7.5 cells. More infection foci were formed in the 293-ADI1-CD81 cells in the first round of infection. In conclusion, human ADI1 over-expression in 293 cells enhances cell entry but not replication of HCV. 293-ADI1-CD81 cells are permissive for serum-derived HCV infection.

Hepatitis C virus infection in mouse hepatoma cells co-expressing human CD81 and Sip-L.

Although human CD81 has been shown to be essential for hepatitis C virus (HCV) infection, non-hepatic cells or transgenic animals expressing human CD81 alone did not support HCV replication. Co-expression of other cofactors was thus necessary for HCV replication. Previously, a hepatic factor named Sip-L was found to support HCV replication in an otherwise non-permissive cell line. To understand the species specificity of hepatic factors required for HCV replication, mouse hepatoma cells co-expressing human CD81 and Sip-L (Hepa1-6-CD81-Sip-L cells) were subjected for HCV infection assay. It was discovered that Hepa1-6-CD81-Sip-L cells were permissive for HCV infection and replication. An animal model was thus established by subcutaneous injection of the permissive cells into nude mice to generate tumors. Viral passages could be achieved in these animals. The antiviral effects of interferon and sodium stibogluconate administrated as a single agent or in combination were demonstrated in this animal model.