Viral Hepatitis, Cholesterol Metabolism, and Cholesterol-Lowering Natural Compounds.

Hepatitis is defined as inflammation of the liver; it can be acute or chronic. In chronic cases, the prolonged inflammation gradually damages the liver, resulting in liver fibrosis, cirrhosis, and sometimes liver failure or cancer. Hepatitis is often caused by viral infections. The most common causes of viral hepatitis are the five hepatitis viruses-hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), and hepatitis E virus (HEV). While HAV and HEV rarely (or do not) cause chronic hepatitis, a considerable proportion of acute hepatitis cases caused by HBV (sometimes co-infected with HDV) and HCV infections become chronic. Thus, many medical researchers have focused on the treatment of HBV and HCV. It has been documented that host lipid metabolism, particularly cholesterol metabolism, is required for the hepatitis viral infection and life cycle. Thus, manipulating host cholesterol metabolism-related genes and proteins is a strategy used in fighting the viral infections. Efforts have been made to evaluate the efficacy of cholesterol-lowering drugs, particularly 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors, in the treatment of hepatitis viral infections; promising results have been obtained. This review provides information on the relationships between hepatitis viruses and host cholesterol metabolism/homeostasis, as well as the discovery/development of cholesterol-lowering natural phytochemicals that could potentially be applied in the treatment of viral hepatitis.

Angiopoietin-Like Protein 3 (ANGPTL3) Modulates Lipoprotein Metabolism and Dyslipidemia.

Dyslipidemia is characterized by increasing plasma levels of low-density lipoprotein-cholesterol (LDL-C), triglycerides (TGs) and TG-rich lipoproteins (TGRLs) and is a major risk factor for the development of atherosclerotic cardiovascular disorders (ASCVDs). It is important to understand the metabolic mechanisms underlying dyslipidemia to develop effective strategies against ASCVDs. Angiopoietin-like 3 (ANGPTL3), a member of the angiopoietin-like protein family exclusively synthesized in the liver, has been demonstrated to be a critical regulator of lipoprotein metabolism to inhibit lipoprotein lipase (LPL) activity. Genetic, biochemical, and clinical studies in animals and humans have shown that loss of function, inactivation, or downregulated expression of ANGPTL3 is associated with an obvious reduction in plasma levels of TGs, LDL-C, and high-density lipoprotein-cholesterol (HDL-C), atherosclerotic lesions, and the risk of cardiovascular events. Therefore, ANGPTL3 is considered an alternative target for lipid-lowering therapy. Emerging studies have focused on ANGPTL3 inhibition via antisense oligonucleotides (ASOs) and monoclonal antibody-based therapies, which have been carried out in mouse or monkey models and in human clinical studies for the management of dyslipidemia and ASCVDs. This review will summarize the current literature on the important role of ANGPTL3 in controlling lipoprotein metabolism and dyslipidemia, with an emphasis on anti-ANGPTL3 therapies as a potential strategy for the treatment of dyslipidemia and ASCVDs.

Ribosome Protein L4 is essential for Epstein-Barr Virus Nuclear Antigen 1 function.

Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1)-mediated origin of plasmid replication (oriP) DNA episome maintenance is essential for EBV-mediated tumorigenesis. We have now found that EBNA1 binds to Ribosome Protein L4 (RPL4). RPL4 shRNA knockdown decreased EBNA1 activation of an oriP luciferase reporter, EBNA1 DNA binding in lymphoblastoid cell lines, and EBV genome number per lymphoblastoid cell line. EBV infection increased RPL4 expression and redistributed RPL4 to cell nuclei. RPL4 and Nucleolin (NCL) were a scaffold for an EBNA1-induced oriP complex. The RPL4 N terminus cooperated with NCL-K429 to support EBNA1 and oriP-mediated episome binding and maintenance, whereas the NCL C-terminal K380 and K393 induced oriP DNA H3K4me2 modification and promoted EBNA1 activation of oriP-dependent transcription. These observations provide new insights into the mechanisms by which EBV uses NCL and RPL4 to establish persistent B-lymphoblastoid cell infection.

Altered susceptibility to air sampling stress by filtration is related to colistin resistance development in Acinetobacter baumannii.

The accurate quantification of antibiotic-resistant bacteria in indoor air has recently attracted increasing attention. Here, we investigated whether the susceptibility of a nosocomial infection-related microbe, Acinetobacter baumannii, to strong sampling stress caused by Nuclepore filter changes as it develops resistance to a drug called colistin. Both colistin-sensitive A. baumannii (CSAB) and colistin-resistant A. baumannii (CRAB) are generally desiccation-resistant strains that can be collected by filter sampling. However, the resistance of CRAB to the three combined stresses (aerosolization, impaction, and desiccation) caused by filter sampling was 1.8 times lower than that of CSAB (P < 0.05). The sampling stresses caused by filter sampling not only reduced the culturability of A. baumannii but also destroyed proteins to result in cellular protein leakage. CRAB released 17%-38% more extracellular protein than did CSAB when they were both subjected to desiccation stress for 240 minutes (P < 0.01). The combination of using a sampling flow rate of 20 L/min and sampling for 60 minutes with a Nuclepore filter with open-face cassettes (OFCs) is recommended for collecting airborne A. baumannii. A Nuclepore filter operated with closed-face cassettes (CFCs) significantly decreased the culturability of CRAB due to desiccation effects.

Altered susceptibility to the bactericidal effect of photocatalytic oxidation by TiO2 is related to colistin resistance development in Acinetobacter baumannii.

Multidrug-resistant Acinetobacter baumannii is a well-documented pathogen associated with hospital-acquired infections. In addition to multidrug resistance, A. baumannii can also become resistant to colistin, the antibiotic treatment of last resort, by the loss of the lipopolysaccharide from its outer membrane. Here, we demonstrate that the development of colistin resistance also increases the resistance of A. baumannii to titanium dioxide (TiO2) photocatalysis. Both colistin-sensitive A. baumannii (CSAB) and colistin-resistant A. baumannii (CRAB) were inactivated by TiO2 when irradiated by ultraviolet A (UV-A). The resistance of CRAB to TiO2 photocatalysis was 1.5 times higher than that of CSAB, as determined by either culture assay or quantification of leaked proteins after photocatalysis (p < 0.05). The results of two-dimensional gel electrophoresis led to the speculation that the high resistance of CRAB may be associated with a lack of sensitive targets and oxidative enzymes. This hypothesis was confirmed by antimicrobial assays with 25 mM hydrogen peroxide (H2O2) and 1.07 mM sodium hypochlorite (NaClO). CRAB was significantly more resistant to H2O2 and NaClO treatment than CSAB (p < 0.01), consistent with the results of the TiO2 inactivation experiment. Therefore, the antibiotic resistance profiles of bacterial strains should be considered before the use of strains as indicators to represent sanitary quality after TiO2 photocatalysis.

beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation.

ß-Naphthoflavone (ß-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of ß-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with ß-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, ß-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by ß-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by ß-NF treatment. Our findings show that ß-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that ß-NF might prevent TNF-α-induced inflammation.

Natural phytochemicals as small-molecule proprotein convertase subtilisin/kexin type 9 inhibitors.

A decrease in the levels of low-density lipoprotein receptors (LDLRs) leads to the accumulation of LDL cholesterol (LDL-C) in the bloodstream, resulting in hypercholesterolemia and atherosclerotic cardiovascular diseases. Increasing the expression level or inducing the activity of LDLR in hepatocytes can effectively control hypercholesterolemia. Proprotein convertase subtilisin/kexin type 9 (PCSK9) protein, primarily produced in the liver, promotes the degradation of LDLR. Inhibiting the expression and/or function of PCSK9 can increase the levels of LDLR on the surface of hepatocytes and promote LDL-C clearance from the plasma. Thus, targeting PCSK9 represents a new strategy for developing preventive and therapeutic interventions for hypercholesterolemia. Currently, monoclonal antibodies are used as PCSK9 inhibitors in clinical practice. However, the need for oral and affordable anti-PCSK9 medications limits the perspective of choosing PCSK9 inhibitors for clinical usage. Emerging research reports have demonstrated that natural phytochemicals have efficacy in maintaining cholesterol stability and regulating lipid metabolism. Developing novel natural phytochemical PCSK9 inhibitors can serve as a starting point for developing small-molecule drugs to reduce plasma LDL-C levels in patients. In this review, we summarize the current literature on the critical role of PCSK9 in controlling LDLR degradation and hypercholesterolemia, and we discuss the results of studies attempting to develop PCSK9 inhibitors, with an emphasis on the inhibitory effects of natural phytochemicals on PCSK9. Furthermore, we provide insight into the mechanisms of action by which the reported phytochemicals exert their potential PCSK9 inhibitory effects against hypercholesterolemia.

C-X-C motif chemokine ligand 12-C-X-C chemokine receptor type 4 signaling axis in cancer and the development of chemotherapeutic molecules.

Chemokines are small, secreted cytokines crucial in the regulation of a variety of cell functions. The binding of chemokine C-X-C motif chemokine ligand 12 (CXCL12) (stromal cell-derived factor 1) to a G-protein-coupled receptor C-X-C chemokine receptor type 4 (CXCR4) triggers downstream signaling pathways with effects on cell survival, proliferation, chemotaxis, migration, and gene expression. Intensive and extensive investigations have provided evidence suggesting that the CXCL12-CXCR4 axis plays a pivotal role in tumor development, survival, angiogenesis, metastasis, as well as in creating tumor microenvironment, thus implying that this axis is a potential target for the development of cancer therapies. The structures of CXCL12 and CXCR4 have been resolved with experimental methods such as X-ray crystallography, NMR, or cryo-EM. Therefore, it is possible to apply structure-based computational approaches to discover, design, and modify therapeutic molecules for cancer treatments. Here, we summarize the current understanding of the roles played by the CXCL12-CXCR4 signaling axis in cellular functions linking to cancer progression and metastasis. This review also provides an introduction to protein structures of CXCL12 and CXCR4 and the application of computer simulation and analysis in understanding CXCR4 activation and antagonist binding. Furthermore, examples of strategies and current progress in CXCL12-CXCR4 axis-targeted development of therapeutic anticancer inhibitors are discussed.

Xanthohumol, a prenylated chalcone, regulates lipid metabolism by modulating the LXRα/RXR-ANGPTL3-LPL axis in hepatic cell lines and high-fat diet-fed zebrafish models.

Angiopoietin-like 3 (ANGPTL3) acts as an inhibitor of lipoprotein lipase (LPL), impeding the breakdown of triglyceride-rich lipoproteins (TGRLs) in circulation. Targeting ANGPTL3 is considered a novel strategy for improving dyslipidemia and atherosclerotic cardiovascular diseases (ASCVD). Hops (Humulus lupulus L.) contain several bioactive prenylflavonoids, including xanthohumol (Xan), isoxanthohumol (Isoxan), 6-prenylnaringenin (6-PN), and 8-prenylnaringenin (8-PN), with the potential to manage lipid metabolism. The aim of this study was to investigate the lipid-lowering effects of Xan, the effective prenylated chalcone in attenuating ANGPTL3 transcriptional activity, both in vitro using hepatic cells and in vivo using zebrafish models, along with exploring the underlying mechanisms. Xan (10 and 20 µM) significantly reduced ANGPTL3 mRNA and protein expression in HepG2 and Huh7 cells, leading to a marked decrease in secreted ANGPTL3 proteins via hepatic cells. In animal studies, orally administered Xan significantly alleviated plasma triglyceride (TG) and cholesterol levels in zebrafish fed a high-fat diet. Furthermore, it reduced hepatic ANGPTL3 protein levels and increased LPL activity in zebrafish models, indicating its potential to modulate lipid profiles in circulation. Furthermore, molecular docking results predicted that Xan exhibits a higher binding affinity to interact with liver X receptor α (LXRα) and retinoic acid X receptor (RXR) than their respective agonists, T0901317 and 9-Cis-retinoic acid (9-Cis-RA). We observed that Xan suppressed hepatic ANGPTL3 expression by antagonizing the LXRα/RXR-mediated transcription. These findings suggest that Xan ameliorates dyslipidemia by modulating the LXRα/RXR-ANGPTL3-LPL axis. Xan represents a novel potential inhibitor of ANGPTL3 for the prevention or treatment of ASCVD.

Overview of clinical status, treatment, and long-term outcomes of pediatric autosomal-dominant polycystic kidney disease: a nationwide survey in Taiwan.

This retrospective study investigated the incidence, medication use, and outcomes in pediatric autosomal-dominant polycystic kidney disease (ADPKD) using Taiwan's National Health Insurance Research Database (NHIRD). A 1:4 matched control group of individuals included in the NHIRD during the same period was used for comparative analyses. A total of 621 pediatric patients were identified from 2009 to 2019 (mean age, 9.51 ± 6.43 years), and ADPKD incidence ranged from 2.32 to 4.45 per 100,000 individuals (cumulative incidence, 1.26-1.57%). The incidence of newly developed hypertension, anti-hypertensive agent use, nephrolithiasis, and proteinuria were significantly higher in the ADPKD group than the non-ADPKD group (0.7 vs. 0.04, 2.26 vs. 0.30, 0.4 vs. 0.02, and 0.73 vs. 0.05 per 100 person-years, respectively). The adjusted hazard ratios for developing hypertension, proteinuria, nephrolithiasis and anti-hypertensive agent use in cases of newly-diagnosed pediatric ADPKD were 12.36 (95% CI 4.92-31.0), 13.49 (95% CI 5.23-34.79), 13.17 (95% CI 2.48-69.98), and 6.38 (95% CI 4.12-9.89), respectively. The incidence of congenital cardiac defects, hematuria, urinary tract infections, gastrointestinal diverticulosis, dyslipidemia, and hyperuricemia were also higher in the ADPKD group. Our study offers valuable insights into the epidemiology of pediatric ADPKD in Taiwan and could help in formulating guidelines for its appropriate management.

Shikonin, a natural naphthoquinone phytochemical, exerts anti-leukemia effects in human CBF-AML cell lines and zebrafish xenograft models.

Core binding factor acute myeloid leukemia (CBF-AML) stands out as the most common type of adult AML, characterized by specific chromosomal rearrangements involving CBF genes, particularly t(8;21). Shikonin (SHK), a naphthoquinone phytochemical widely employed as a food colorant and traditional Chinese herbal medicine, exhibits antioxidant, anti-inflammatory, and anti-cancer activities. In this study, we aim to investigate the antileukemic effects of SHK and its underlying mechanisms in human CBF-AML cells and zebrafish xenograft models. Our study revealed that SHK reduced the viability of CBF-AML cells. SHK induced cell cycle arrest, promoted cell apoptosis, and induced differentiation in Kasumi-1 cells. Additionally, SHK downregulated the gene expression of AML1-ETO and c-KIT in Kasumi-1 cells. In animal studies, SHK showed no toxic effects in zebrafish and markedly inhibited the growth of leukemia cells in zebrafish xenografts. Transcriptomic analysis showed that differentially expressed genes (DEGs) altered by SHK are linked to key biological processes like DNA repair, replication, cell cycle regulation, apoptosis, and division. Furthermore, KEGG pathways associated with cell growth, such as the cell cycle and p53 signaling pathway, were significantly enriched by DEGs. Analysis of AML-associated genes in response to SHK treatment using DisGeNET and the STRING database indicated that SHK downregulates the expression of cell division regulators regarding AML progression. Finally, we found that SHK combined with cytarabine synergistically reduced the viability of Kasumi-1 cells. In conclusion, our findings provide novel insights into the mechanisms of SHK in suppressing leukemia cell growth, suggesting its potential as a chemotherapeutic agent for human CBF-AML.

Optimization of sequence and chiral content enhances therapeutic potential of tilapia piscidin peptides.

Because antimicrobial peptides (AMPs) often exhibit broad-spectrum bactericidal potency, we sought to develop peptide-based antimicrobials for potential clinical use against drug-resistant pathogens. To accomplish this goal, we first optimized the amino acid sequence of a broad-spectrum AMP known as Tilapia Piscidin 4 (TP4). Then, we used the optimized sequence to create a pair of heterochiral variants (TP4-α and TP4-ß) with different percentages of D-enantiomers, as poly-L peptides often exhibit poor pharmacokinetic profiles. The conformations of the peptide pair exhibited inverted chirality according to CD and NMR spectroscopic analyses. Both heterochiral peptides displayed enhanced stability and low hemolysis activities. Irrespective of their different d-enantiomer contents, both heterochiral peptides exhibited bactericidal activities in the presence of human serum or physiological enzymes. However, the peptide with higher d-amino acid content (TP4-ß) caused better bacterial clearance when tested in mice infected with NDM-1 K. pneumoniae. In addition, we observed a relatively higher hydrogen bonding affinity in a simulation of the interaction between TP4-ß and a model bacterial membrane. In sum, our results demonstrate that the current design strategy may be applicable for development of new molecules with enhanced stability and in vivo antimicrobial activity.

Sequential rearrangement and stereochemical reorganization to design an antimicrobial peptide with enhanced stability.

Antimicrobial peptides (AMPs) are natural molecules that function within the innate immune system to counteract pathogenic invasion and minimize the detrimental consequences of infection. However, utilizing these molecules for medical applications has been challenging. In this study, we selected a model AMP with poor stability, Tilapia Piscidin 4 (TP4), and modified its sequence and chirality (TP4-γ) to improve its potential for clinical application. The strategy of chirality inversion was inspired by the cereulide peptide, which has a DDLL enantiomer pattern and exhibits exceptional stability. Sequential substitution of key residues and selective chirality inversion yielded a less toxic peptide with enhanced stability and notable antimicrobial activity. In addition to its superior stability profile and antimicrobial activity, TP4-γ treatment reduced the level of LPS-induced nitric oxide (NO) release in a macrophage cell line. This reduction in NO release may reflect anti-inflammatory properties, as NO is widely known to promote inflammatory processes. Hence, our heterochiral peptide construct shows a more suitable pharmacokinetic profile than its parental compound, and further studies are warranted to develop the molecule for potential clinical application.

Electrochemically synthesized green fluorescent carbon dots for quantitation of hypochlorite and carbendazim.

Green emission carbon dots (CDs) electrochemically prepared from 2,6-pyridinedicarboxylic acid and o-phenyl-enediamine were applied separately for the quantitation of hypochlorite and carbendazim. The characteristic and optical properties of the CDs were studied through fluorescence, UV-vis absorption, X-ray photoelectron spectroscopy, and transmission electron microscopy. The synthesized CDs were mainly 0.8-2.2 nm in size, with an average size of 1.5 nm. The CDs exhibited green luminescence centered at 520 nm when excited by 420 nm light. The green emission of the CDs is quenched after the addition of hypochlorite, mainly through the redox reaction between hypochlorite and hydroxyl groups on the CDs surface. Furthermore, the hypochlorite-induced fluorescence quenched can be prevented in the presence of carbendazim. The sensing approaches exhibit good linear ranges of 1-50 µM and 0.05-5 µM for hypochlorite and carbendazim, respectively, with low detection limits of 0.096 and 0.005 µM, respectively. Practicalities of the luminescent probes were separately validated by the quantitation of the two analytes in real sample matrix with recoveries ranging from 96.3 to 108.9% and the relative standard deviation values below 5.51%. Our results show the potential of the sensitive, selective, and simple CD probe for water and food quality control.

Pinostrobin modulates FOXO3 expression, nuclear localization, and exerts antileukemic effects in AML cells and zebrafish xenografts.

Acute myeloid leukemia (AML) is a disease characterized by abnormal cell proliferation in the bone marrow and is the most common quickly progressive leukemia in adults. Pinostrobin, a flavonoid phytochemical, has been reported to exhibit antioxidant, anti-inflammatory, and anticancer properties. In this study, we aimed to investigate the antileukemic effects of pinostrobin and its molecular mechanisms in human AML cells. Our study found that pinostrobin (0-80 µM) significantly reduced the viability of human AML cells, with the pronounced cytotoxic effects observed in MV4-11 > MOLM-13 > HL-60 > U-937 > THP-1 cells. Pinostrobin was found to suppress leukemia cell proliferation, modulate cell cycle progression, promote cell apoptosis, and induce monocytic differentiation in MV4-11 cells. In animal studies, pinostrobin significantly suppressed the growth of leukemia cells in a zebrafish xenograft model. Microarray-based transcriptome analysis showed that the differentially expressed genes (DEGs) in pinostrobin-treated cells were strongly associated with enriched Gene Ontology (GO) terms related to apoptotic process, cell death, cell differentiation, cell cycle progression, and cell division. Combining DisGeNET and STRING database analysis revealed that pinostrobin upregulates forkhead box 3 (FOXO3), a tumor suppressor in cancer development, and plays an essential role in controlling AML cell viability. Our study demonstrated that pinostrobin increases FOXO3 gene expression and promotes its nuclear translocation, leading to the inhibition of cell growth. Finally, the study found that pinostrobin, when combined with cytarabine, synergistically reduces the viability of AML cells. Our current findings shed light on pinostrobin's mechanisms in inhibiting leukemia cell growth, highlighting its potential as a chemotherapeutic agent or nutraceutical supplement for AML prevention or treatment.

Self-assembly of gelatin and collagen in the polyvinyl alcohol substrate and its influence on cell adhesion, proliferation, shape, spreading and differentiation.

Culture substrates display profound influence on biological and developmental characteristic of cells cultured in vitro. This study investigates the influence of polyvinyl alcohol (PVA) substrates blended with different concentration of collagen or/and gelatin on the cell adhesion, proliferation, shape, spreading, and differentiation of stem cells. The collagen/gelatin blended PVA substrates were prepared by air drying. During drying, blended collagen or/and gelatin can self-assemble into macro-scale nucleated particles or branched fibrils in the PVA substrates that can be observed under the optical microscope. These collagen/gelatin blended substrates revealed different surface topography, z-average, roughness, surface adhesion and Young's modulus as examined by the atomic force microscope (AFM). The results of Fourier transform infrared spectroscopy (FTIR) analysis indicated that the absorption of amide I (1,600-1,700 cm-1) and amide II (1,500-1,600 cm-1) groups increased with increasing collagen and gelatin concentration blended and the potential of fibril formation. These collagen or/and gelatin blended PVA substrates showed enhanced NIH-3T3 fibroblast adhesion as comparing with the pure PVA, control tissue culture polystyrene, conventional collagen-coated and gelatin-coated wells. These highly adhesive PVA substrates also exhibit inhibited cell spreading and proliferation. It is also found that the shape of NIH-3T3 fibroblasts can be switched between oval, spindle and flattened shapes depending on the concentration of collagen or/and gelatin blended. For inductive differentiation of stem cells, it is found that number and ration of neural differentiation of rat cerebral cortical neural stem cells increase with the decreasing collagen concentration in the collagen-blended PVA substrates. Moreover, the PVA substrates blended with collagen or collagen and gelatin can efficiently support and conduct human pluripotent stem cells to differentiate into Oil-Red-O- and UCP-1-positive brown-adipocyte-like cells via ectodermal lineage without the addition of mitogenic factors. These results provide a useful and alternative platform for controlling cell behavior in vitro and may be helpful for future application in the field of regenerative medicine and tissue engineering.

Comparison, Analysis, and Molecular Dynamics Simulations of Structures of a Viral Protein Modeled Using Various Computational Tools.

The structural analysis of proteins is a major domain of biomedical research. Such analysis requires resolved three-dimensional structures of proteins. Advancements in computer technology have led to progress in biomedical research. In silico prediction and modeling approaches have facilitated the construction of protein structures, with or without structural templates. In this study, we used three neural network-based de novo modeling approaches-AlphaFold2 (AF2), Robetta-RoseTTAFold (Robetta), and transform-restrained Rosetta (trRosetta)-and two template-based tools-the Molecular Operating Environment (MOE) and iterative threading assembly refinement (I-TASSER)-to construct the structure of a viral capsid protein, hepatitis C virus core protein (HCVcp), whose structure have not been fully resolved by laboratory techniques. Templates with sufficient sequence identity for the homology modeling of complete HCVcp are currently unavailable. Therefore, we performed domain-based homology modeling for MOE simulations. The templates for each domain were obtained through sequence-based searches on NCBI and the Protein Data Bank. Then, the modeled domains were assembled to construct the complete structure of HCVcp. The full-length structure and two truncated forms modeled using various computational tools were compared. Molecular dynamics (MD) simulations were performed to refine the structures. The root mean square deviation of backbone atoms, root mean square fluctuation of Cα atoms, and radius of gyration were calculated to monitor structural changes and convergence in the simulations. The model quality was evaluated through ERRAT and phi-psi plot analysis. In terms of the initial prediction for protein modeling, Robetta and trRosetta outperformed AF2. Regarding template-based tools, MOE outperformed I-TASSER. MD simulations resulted in compactly folded protein structures, which were of good quality and theoretically accurate. Thus, the predicted structures of certain proteins must be refined to obtain reliable structural models. MD simulation is a promising tool for this purpose.

The Neuroprotective Effects of Flavonoid Fisetin against Corticosterone-Induced Cell Death through Modulation of ERK, p38, and PI3K/Akt/FOXO3a-Dependent Pathways in PC12 Cells.

The overactive hypothalamic-pituitary-adrenal (HPA) axis is believed to trigger the overproduction of corticosterone, leading to neurotoxicity in the brain. Fisetin is a flavonoid commonly found in fruits and vegetables. It has been suggested to possess various biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. This study aims to explore the potential neuroprotective properties of fisetin against corticosterone-induced cell death and its underlying molecular mechanism in PC12 cells. Our results indicate that fisetin, at concentrations ranging from 5 to 40 µM, significantly protected PC12 cells against corticosterone-induced cell death. Fisetin effectively reduced the corticosterone-mediated generation of reactive oxygen species (ROS) in PC12 cells. Fisetin treatments also showed potential in inhibiting the corticosterone-induced apoptosis of PC12 cells. Moreover, inhibitors targeting MAPK/ERK kinase 1/2 (MEK1/2), p38 MAPK, and phosphatidylinositol 3-kinase (PI3K) were found to significantly block the increase in cell viability induced by fisetin in corticosterone-treated cells. Consistently, fisetin enhanced the phosphorylation levels of ERK, p38, Akt, and c-AMP response element-binding protein (CREB) in PC12 cells. Additionally, it was found that the diminished levels of p-CREB and p-ERK by corticosterone can be restored by fisetin treatment. Furthermore, the investigation of crosstalk between ERK and CREB revealed that p-CREB activation by fisetin occurred through the ERK-independent pathway. Moreover, we demonstrated that fisetin effectively counteracted the corticosterone-induced nuclear accumulation of FOXO3a, an apoptosis-triggering transcription factor, and concurrently promoted FOXO3a phosphorylation and its subsequent cytoplasmic localization through the PI3K/Akt pathway. In conclusion, our findings indicate that fisetin exerts its neuroprotective effect against corticosterone-induced cell death by modulating ERK, p38, and the PI3K/Akt/FOXO3a-dependent pathways in PC12 cells. Fisetin emerges as a promising phytochemical for neuroprotection.

A peptide derived from interleukin-10 exhibits potential anticancer activity and can facilitate cell targeting of gold nanoparticles loaded with anticancer therapeutics.

Human interleukin-10 (IL-10) is an immunosuppressive and anti-inflammatory cytokine, and its expression is upregulated in tumor tissues and serum samples of patients with various cancers. Because of its immunosuppressive nature, IL-10 has also been suggested to be a factor leading to tumor cells' evasion of immune surveillance and clearance by the host immune system. In this study, we refined a peptide with 20 amino acids, named NK20a, derived from the binding region of IL-10 on the basis of in silico analysis of the complex structure of IL-10 with IL-10Ra, the ligand binding subunit of the IL-10 receptor. The binding ability of the peptide was confirmed through in vitro biophysical biolayer interferometry and cellular experiments. The IL-10 inhibitory peptide exerted anticancer effects on lymphoma B cells and could abolish the suppression effect of IL-10 on macrophages. NK20a was also conjugated with gold nanoparticles to target the chemotherapeutic 5-fluorouracil (5-FU)-loaded nanoparticles to enhance the anticancer efficacy of 5-FU against the breast cancer cell line BT-474. Our study demonstrated that NK20a designed in silico with improved binding affinity to the IL-10 receptor can be used as a tool in developing anticancer strategies.

Computer-aided discovery, design, and investigation of COVID-19 therapeutics.

Coronavirus disease 2019 (COVID-19) pandemic is currently the most serious public health threat faced by mankind. Thus, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes COVID-19, is being intensively investigated. Several vaccines are now available for clinical use. However, owing to the highly mutated nature of RNA viruses, the SARS-CoV-2 is changing at a rapid speed. Breakthrough infections by SARS-CoV-2 variants have been seen in vaccinated individuals. As a result, effective therapeutics for treating COVID-19 patients is urgently required. With the advance of computer technology, computational methods have become increasingly powerful in the biomedical research and pharmaceutical drug discovery. The applications of these techniques have largely reduced the costs and simplified processes of pharmaceutical drug developments. Intensive and extensive studies on SARS-CoV-2 proteins have been carried out and three-dimensional structures of the major SARS-CoV-2 proteins have been resolved and deposited in the Protein Data Bank. These structures provide the foundations for drug discovery and design using the structure-based computations, such as molecular docking and molecular dynamics simulations. In this review, introduction to the applications of computational methods in the discovery and design of novel drugs and repurposing of existing drugs for the treatments of COVID-19 is given. The examples of computer-aided investigations and screening of COVID-19 effective therapeutic compounds, functional peptides, as well as effective molecules from the herb medicines are discussed.