Search Results (142)

Controlled delivery using nanoparticle-based systems has attracted considerable attention; however, achieving cell-type specificity remains a major challenge. To address this issue, we focused on the intrinsic cell tropism of viruses. The hepatocyte tropism of hepatitis B virus (HBV) is mediated by interactions between its large envelope protein (L protein) and host factors, including the sodium taurocholate cotransporting polypeptide (NTCP). In this study, we explored viral-like secretory vesicles (VLSVs) displaying HBV spike proteins as a virus-inspired vesicle platform for hepatocyte targeting. We previously established a method for producing VLSVs from HBV L- and S-expressing HEK293T cells. In the present study, we developed an improved protocol using exosome-depleted fetal calf serum and optimized ultracentrifugation, resulting in VLSVs with comparable particle numbers and sizes but approximately tenfold higher protein content per particle. VLSVs were concentrated using a two-layer sucrose cushion, labeled with DiI, and purified by sucrose density gradient ultracentrifugation. We evaluated DiI uptake in hepatocyte-derived cells (HepG2 and Huh7), non-hepatic cells (MDA-MB231, H1299, HeLa, and Vero), and NTCP-overexpressing HepG2 cells. VLSVs showed preferential uptake in the following order: NTCP-overexpressing HepG2 > HepG2 > Huh7 > non-hepatic cells. Furthermore, removal of the N-terminal Flag tag from the L protein enhanced hepatocyte-associated uptake, suggesting the importance of preserving the native structure of the preS1 domain. While vesicle characterization and mechanistic validation remain to be further investigated, these findings provide a proof-of-concept for a virus-inspired vesicle platform exhibiting preferential uptake in hepatocyte-derived cells. Full article

The sodium-taurocholate cotransporting polypeptide (NTCP) plays a critical dual role in liver function: maintaining bile acid (BA) enterohepatic circulation and acting as a receptor for the entry of hepatitis B and D viruses into hepatocytes. This review outlines the impact of various post-translational modifications (PTMs) of NTCP—including phosphorylation, oligomerization, ubiquitination, and glycosylation—on its dynamic regulatory network. These modifications coordinate the modulation of NTCP’s membrane localization, stability, conformational state, and protein interactions, precisely controlling its functions in BA uptake and viral invasion. Targeting this PTM network presents a promising strategy for next-generation therapies that selectively inhibit viral infection while preserving BA transport, overcoming the limitations of conventional inhibitors that indiscriminately disrupt virus–NTCP interactions. By synthesizing recent insights into NTCP PTM research, this article highlights its role as a central regulator of its bifunctional properties and reveals potential avenues for precision therapies in viral hepatitis, cholestasis, and related liver diseases. However, most existing evidence is derived from in vitro or cell-based models, whereas in vivo studies and clinical validation remain limited; thus, the translational feasibility of strategies targeting post-translational modifications of NTCP still requires further investigation. Full article

This retrospective planning study evaluated how arc number (AN) and control-point density (CP) affect VMAT quality, radiobiological endpoints, and workflow efficiency for locally advanced cervical cancer in a resource-conscious setting. Twenty-one patients (FIGO IIB–IIIB) were replanned in Monaco v5.51 (Monte Carlo) for 46 Gy using 6-MV beams (Elekta) with 1–4 coplanar arcs, and dual-arc plans were further analyzed using ≈250, 300, 350, and 400 CP per arc. Target coverage (D98/D95/V95/V98), conformity and homogeneity (CI, HI), and organs-at-risk (OARs) DVH metrics (including D2cc and Vx) were compared alongside monitor units, planning time, and delivery time. Increasing AN improved dose conformity and OAR sparing relative to single-arc plans, whereas increasing CP produced only modest dosimetric changes but substantially increased planning and treatment times. Radiobiological modeling using BED/EQD2 and EUD-based LKB NTCP indicated negligible bladder risk (<0.01%) and low rectal risk (<0.2%), but a higher small-bowel NTCP (~26%) driven by hotspot-sensitive descriptors; Niemierko TCP estimates were similar between leading dual-arc CP settings. Overall, a dual-arc strategy with ~250 CP per arc provided the most practical balance between plan quality, estimated biological effect, and deliverability. Full article

Hepatitis B virus (HBV) remains a major global health threat because covalently closed circular DNA (cccDNA) persists in hepatocytes and limits the efficacy of current antiviral therapies. Effective HBV research and drug screening require culture models that recapitulate the complete viral life cycle and allow for quantitative monitoring of replication. In this study, an 11-amino acid luminescent reporter, HiBiT, was inserted at multiple sites within the preS1 region of a genotype D HBV genome, and the C terminus of preS1 was identified as optimal for maintaining robust replication. We then established HepG2-B4 cells stably replicating HiBiT-HBV with HiBiT at the preS1 C terminus. Extracellular HiBiT activity and supernatant levels of HBV-DNA, HBsAg, and HBcAg increased continuously until day 42 and were reduced by nucleos(t)ide analog treatment, and cccDNA was confirmed by Southern blot analysis. Supernatants from HepG2-B4 cells infected naïve HepG2-NTCP cells and primary human hepatocytes, as shown by extracellular HiBiT activity. Transcriptome analysis revealed distinct gene expression changes in HepG2-B4 cells compared with parental HepG2 cells. These findings indicate that the HepG2-B4 system provides a rapid, quantitative, and scalable platform for HBV replication and infection studies and is suitable for mechanistic investigations and high-throughput antiviral screening. Full article

We recently reported that onnamide A, a marine-derived natural compound isolated from the sponge Theonella sp., inhibits the entry process of SARS-CoV-2 infection. However, its antiviral activity against other viruses remains largely unexplored. Here, we investigated the effects of onnamide A and its structurally related analog, onnamide B, on hepatitis B virus (HBV) infection. Using iNTCP cells, a hepatoblastoma-derived cell line permissive to HBV infection, we found that onnamides A and B exhibited cytotoxicity, with CC₅₀ values of 0.53 ± 0.10 μM and 2.37 ± 0.25 μM, respectively. Following HBV infection, the levels of total HBV RNA were significantly reduced by onnamide A (IC₅₀ = 0.06 ± 0.01 μM) and onnamide B (IC₅₀ = 0.23 ± 0.06 μM). Notably, both compounds markedly decreased the levels of HBV pregenomic RNA. Furthermore, significant inhibition was particularly evident when onnamide treatment was initiated after HBV infection. Consistent with these observations, onnamides did not affect HBV binding, entry, or covalently closed circular DNA formation, but they significantly suppressed HBV RNA transcription. In particular, the transcriptional activities driven by the core and X promoters were markedly inhibited by onnamide treatment. Taken together, our findings demonstrate that onnamides possess potent anti-HBV activity and highlight their potential as candidate compounds targeting HBV RNA transcription. Full article

(1) Background: Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of viruses that interact with hepatocytes. HBV infection is a major global health problem. Most adults clear the infection quickly after being infected with HBV, while a few people develop chronic HBV infection. It is well-known that the early innate immune response of host cells plays an important role in the fight against virus infection. However, the interactions between HBV and the intrinsic innate immune system of hepatocytes are still not fully understood. The aim of this study was to confirm the interaction between HBV and hepatocytes, and to identify the interferon-stimulated genes (ISGs) regulated by HBx and their expression in association with HBV-associated HCC (HBV-HCC), so that we can refine our understanding of the interaction between HBV and ISGs and its potential influence on HBV-HCC. (2) Methods: We analyzed data concerning the stimulation of IFN-dependent genes in primary human hepatocytes (PHHs) transfected with pathogen DNA mimetics or infected with HBV in the GSE69590 database. Bioinformatic methods, such as GSEA, GO, and KEGG, were used to analyze the differentially expressed innate immunity genes and their related pathways to identify candidate intrinsic innate immune factors. qPCR on HepG2 and Huh7 cells, which highly express HBx, was used to detect relevant intrinsic innate immune factors. qPCR, RNAi, and Elisa methods were used to identify intrinsic innate immune factors in HBV-integrated HepG2.2.15 cells, and bioinformatics analysis was conducted on the HBV-infected tissues and cells in the GEO database. (3) Results: Inhibition of the JAK-STAT pathway enhanced HBV replication in HepG2 cells transfected with HBV plasmid and HepG2-NTCP cells infected with HBV. GSEA analysis of the GSE69590 data revealed significant changes in intrinsic innate immune pathways during HBV infection with PHH for 40 h. A total of 84 differentially expressed, candidate innate immunity genes were identified in GSE69590. Validation showed that TRIM22 and TRIM56 were down-regulated when HBx was expressed. Consistently, TRIM22 and TRIM56 were up-regulated following inhibition of HBx by transfection of HBx siRNA into HepG2.2.15 cells, and HBV pgRNA was up-regulated following down-regulated expression of TRIM22 and TRIM56 in HEK293 cells. Receiver operating characteristics (ROC) and overall survival (OS) analysis of 204 HBV-HCC patients showed that expression of TRIM22 was closely associated with HBV-HCC, and high expression of TRIM22 was associated with longer survival. (4) Conclusions: Innate immunity genes TRIM22 and TRIM56 are regulated by HBx, and higher expression of TRIM22 is closely related to longer survival of HBV-HCC patients. Full article

Background/Objectives: Treatment planning for stage III non–small cell lung cancer (NSCLC) presents dosimetric challenges due to the proximity of critical structures. RapidPlan (RP), a knowledge-based planning (KBP) system, offers the potential for improved plan consistency and organ-at-risk (OAR) sparing. The objective of this study was to compare dosimetric and clinical outcomes of RP-generated plans versus manually optimized plans in patients with stage III NSCLC undergoing IMRT or VMAT. Methods: In this retrospective analysis, 50 patients treated with concurrent chemoradiation for stage III NSCLC at Hadassah Medical Center (2015–2021) were analyzed. RP plans were generated using a lung-specific model in the Eclipse treatment planning system and compared with the original clinical manual plans. Dosimetric parameters for target volumes and OARs were evaluated, and subgroup analyses were performed by technique (IMRT vs. VMAT). Toxicity and survival outcomes were analyzed, and Normal Tissue Complication Probability (NTCP) modeling was conducted. Results: RP significantly reduced mean heart dose (Δ = −2.54 Gy, p < 0.001), spinal cord maximum dose (Δ = −4.08 Gy, p < 0.001), and esophageal mean dose (Δ = −3.89 Gy, p < 0.001) compared with manual plans. Lung doses were slightly higher in RP plans (V20 Δ = +2.12%, p < 0.001). VMAT-RP plans demonstrated greater cardiac and esophageal sparing than VMAT-manual plans. RP yielded significant NTCP reductions for the heart (0.34% → 0.20%) and esophagus (16.6% → 11.5%), but no improvement for lung or spinal cord. Lung toxicity ≥ grade 2 was associated with reduced overall survival (16.2 vs. 51.8 months, p < 0.001). Conclusions: RapidPlan-based knowledge-based planning enhances OAR sparing while maintaining target coverage in locally advanced NSCLC. Slight increases in lung dose highlight the need for ongoing model refinement. An association between lung toxicity and reduced survival was observed, underscoring the impact of treatment-related morbidity on outcomes. Full article

Background/Objectives: Sepsis-induced cholestasis is caused by the release of inflammatory cytokines from lipopolysaccharide (LPS), a component of Gram-negative bacteria. No established therapy exists for this condition. We ascertained the anticholestatic potential of obeticholic acid (OCA), a potent FXR agonist, in a rat model of LPS-induced cholestasis. Methods: Male Wistar rats were randomized into Control, OCA (20 mg/kg/day, i.p., 6 days), LPS (total dose of 6.5 mg/kg, i.p., in the last 2 days, respectively), and OCA + LPS groups. Then, we assessed the serum cholestasis marker, alkaline phosphatase (ALP), and taurocholate-stimulated bile salt output. mRNA/protein levels of the main apical and sinusoidal uptake and efflux carriers were assessed by either or both RT-qPCR and Western blot. Bsep and Mrp2 localization was assessed by immunohistochemistry followed by confocal microscopy and image analysis. Inflammatory cytokines were measured in serum by ELISA. Results: OCA significantly attenuates inflammatory cytokine release and normalizes serum ALP in LPS-treated rats. OCA also increased the biliary output of the Bsep substrate, taurocholate, and partially improved total Bsep at both mRNA and protein levels. Furthermore, OCA fully normalizes Bsep in the canalicular plasma membrane fraction, suggesting improved membrane localization, a finding further confirmed by confocal microscopy. OCA sustained the beneficial downregulation of uptake transporters Ntcp and Oatp2 or the upregulation of the efflux pump Mrp3, both of which serve to minimize hepatocellular bile-salt accumulation. Conclusions: OCA prevents bile-salt accumulation in LPS-induced cholestasis by enhancing Bsep expression and localization, and by mitigating inflammation. This makes OCA a promising therapeutic candidate for sepsis-induced cholestasis. Full article

Core fucose is one of the most important glycans in HBV infection. In this study, we investigated whether Pholiota squarrosa lectin (PhoSL), a lectin that specifically binds to core fucose, exerts an inhibitory effect in an HBV infection model of normal human hepatocytes. Similarly to previous studies using hepatocellular carcinoma cells (HepG2-C4), the coexistence of PhoSL during HBV infection inhibited HBe antigen production and HBV cccDNA in normal human hepatocytes in a PhoSL concentration-dependent manner. Furthermore, this effect of PhoSL was found to be able to suppress HBe antigen production in a treatment period-dependent manner, even when PhoSL was administered after HBV infection. Our previous research has revealed that the mechanism by which PhoSL inhibits HBV infection is through physical inhibition by binding to the HBV receptor and inhibition of HBV entry into cells by inhibiting the phosphorylation of EGFR, a co-receptor for NTCP. Furthermore, this study suggested that PhoSL may also inhibit HBV proliferation in cells through other mechanisms that require further investigation. PhoSL is a lectin, derived from edible Pholiota squarrosa (shaggy scalycap) mushrooms, that is resistant to acid and heat. In addition, it has a low molecular weight and can be chemically synthesized, so it is expected to be used clinically as a new carbohydrate therapy for HBV in the future. Full article

Background: We have previously demonstrated that the function and expression of the Na⁺/taurocholate cotransporting polypeptide (NTCP) and the organic cation transporter 1 (OCT1) are affected by increasing free or unesterified cholesterol in the plasma membrane by an acute incubation with cholesterol for 30 min. In the current study we wanted to extend these findings to a more chronic condition to mimic what would be seen in obese patients. Methods: We incubated HEK293 cells that stably express NTCP or OCT1 for 24 h with 0.05 mM cholesterol and determined their function by measuring uptake of radioactive taurocholate or MPP⁺. Expression at the plasma membrane was quantified with a biotinylation assay combined with Western blots. Results: Incubation with cholesterol increased the cholesterol content of the cells by about 2-fold. Transport mediated by NTCP and OCT1 was decreased. Membrane expression for both transporters showed a slight decrease, and when kinetics were normalized for the membrane expression, the V_max for NTCP-mediated taurocholate uptake slightly decreased, but the V_max and the capacity (V_max/K_m) for OCT1-mediated MPP⁺ uptake increased by 2.5-fold and 3-fold, respectively. Acyl-Coenzyme A acyltransferase inhibitors enhanced the decrease in transport function, potentially due to retention of more free cholesterol in the plasma membrane. Conclusions: Chronic increases in free cholesterol in the plasma membrane can result in increased or decreased transporter function and expression. In the case of OCT1, which is involved in the uptake of the anti-diabetic drug metformin into hepatocytes, the 3-fold increase in transport capacity might affect drug therapy. Full article

Background: Chronic hepatitis B (CHB) remains being a major public health threat, and currently existing CHB therapies have limited efficacy and side effects. We have recently developed a vaccine termed VVX001 based on a recombinant fusion protein consisting of the preS domain of the large surface protein of hepatitis B virus (HBV) fused to grass pollen allergen peptides. VVX001 has been shown to induce preS-specific antibodies in grass pollen allergic patients, and sera of immunized subjects inhibited HBV infection in vitro. Methods: In this study we investigated if immunization with VVX001 can induce preS-specific antibodies in CHB using the adeno-associated virus (AAV)-HBV murine model of CHB. Six groups of C57BL/6 female mice (n = 6) were transduced with AAV-HBV or AAV-Empty, and after six weeks, they were immunized five times with 20 µg of aluminum hydroxide-adsorbed VVX001 or preS or vehicle (Alum alone). Serum samples were taken continuously. Two weeks after the last immunization, spleen and liver mononuclear cells were collected. Serum reactivity to preS and preS-derived peptides was assessed by ELISA. B-cell responses were measured by ELISPOT assay, and intrahepatic lymphocyte (ILH) counts were determined by FACS. HBV DNA, HBsAg, HBeAg, ALT, and AST were assessed using commercial kits. Results: Our results show that VVX001 induces preS-specific IgG antibodies that cross-react with different HBV genotypes A-H and are directed against the sodium taurocholate co-transporting polypeptide (NTCP) receptor binding site of preS both in mice with and without HBV. Actively immunized AAV-HBV-treated mice had a higher number of intrahepatic lymphocytes than vehicle-vaccinated and mock-transduced animals. Conclusions: These findings encourage performing further trials to study the potential of VVX001 for therapeutic vaccination against CHB. Full article

Introduction: Decision-making regarding radiotherapy techniques for patients with nasopharyngeal cancer requires a comparison of photon and proton plans generated using planning software, which requires time and expertise. We developed a fully automated decision tool to select patients for proton therapy that predicts proton therapy (XT) and photon therapy (PT) dose distributions using only patient CT image data, predicts xerostomia and dysphagia probability using predicted critical organ mean doses, and makes decisions based on the Netherlands’ National Indication Protocol Proton therapy (NIPP) to select patients likely to benefit from proton therapy. Methods: This study used 48 nasopharyngeal patients treated at the Cancer Hospital of the Chinese Academy of Medical Sciences. We manually generated a photon plan and a proton plan for each patient. Based on this dose distribution, photon and proton dose prediction models were trained using deep learning (DL) models. We used the NIPP model to measure xerostomia levels 2 and 3, dysphagia levels 2 and 3, and decisions were made according to the thresholds given by this protocol. Results: The predicted doses for both photon and proton groups were comparable to those for manual plan (MP). The Mean Absolute Error (MAE) for each organ at risk in the photon and proton plans did not exceed 5% and showed a good performance of the dose prediction model. For proton, the normal tissue complication probability (NTCP) of xerostomia and dysphagia performed well, p > 0.05. There was no statistically significant difference. For photon, the NTCP of dysphagia performed well, p > 0.05. For xerostomia p < 0.05 but the absolute deviation was 0.85% and 0.75%, which would not have a great impact on the prediction result. Among the 48 patients’ decisions, 3 were wrong, and the correct rate was 93.8%. The area under curve (AUC) of operating characteristic curve (ROC) was 0.86, showing the good performance of the decision-making tool in this study. Conclusions: The decision tool based on DL and NTCP models can accurately select nasopharyngeal cancer patients who will benefit from proton therapy. The time spent generating comparison plans is reduced and the diagnostic efficiency of doctors is improved, and the tool can be shared with centers that do not have proton expertise. Trial registration: This study was a retrospective study, so it was exempt from registration. Full article

The global burden of hepatitis B virus (HBV) remains high, with ongoing concerted efforts to eliminate viral hepatitis as a public health concern by 2030. The absence of curative treatment against HBV makes it an active area of research to further study HBV pathogenesis. In vitro cell culture systems are essential in exploration of molecular mechanisms for HBV propagation and the development of therapeutic targets for antiviral agents. The lack of an efficient cell culture system is one of the challenges limiting the development and study of novel antiviral strategies for HBV infection. However, the evolution of cell culture systems to study HBV pathogenesis and treatment susceptibility in vitro has made a significant contribution to public health. The currently available cell culture systems to grow HBV have their advantages and limitations, requiring further optimization. The discovery of sodium taurocholate co-transporting polypeptide (NTCP) as a receptor for HBV was a major breakthrough for the development of a robust cell model, allowing the study of de novo HBV infection through NTCP expression in the HepG2 hepatoma cell line. This review is aimed at highlighting the evolution of cell culture systems to study HBV pathogenesis and in vitro treatment susceptibility. Full article

Tan sheep outperform Dorper sheep in meat-quality traits, including muscle fiber characteristics and fatty acid composition, while Dorper sheep excel in carcass weight. However, the molecular mechanisms underlying these breed-specific traits, especially gut microbiota–bile acid (BA) interactions, remain poorly understood. As host–microbiota co-metabolites, BAs are converted by colonic microbiota via bile salt hydrolase (BSH) and dehydroxylases into secondary BAs, which activate BA receptors to regulate host lipid and glucose metabolism. This study analyzed colonic BA profiles in 8-month-old Tan and Dorper sheep, integrating microbiome and longissimus dorsi muscle transcriptome data to investigate the gut–muscle axis in meat-quality and carcass trait regulation. Results showed that Tan sheep had 1.6-fold higher secondary BA deoxycholic acid (DHCA) levels than Dorper sheep (p < 0.05), whereas Dorper sheep accumulated conjugated primary BAs glycocholic acid (GCA) and tauro-α-muricholic acid (p < 0.05). Tan sheep exhibited downregulated hepatic BA synthesis genes, including cholesterol 7α-hydroxylase (CYP7A1) and 27-hydroxylase (CYP27A1), alongside upregulated transport genes such as bile salt export pump (BSEP), sodium taurocholate cotransporting polypeptide (NTCP), and ATP-binding cassette subfamily B member 4 (ABCB4), with elevated gut BSH activity (p < 0.05). DHCA was strongly correlated with g_Ruminococcaceae_UCG-014, ENSOARG00000001393, and ENSOARG00000016726, muscle fiber density, diameter, and linoleic acid (C18:2n6t) (|r| > 0.5, p < 0.05). In contrast, GCA was significantly associated with g_Lachnoclostridium_10, g_Rikenellaceae_RC9_gut_group, ENSOARG0000001232, carcass weight, and net meat weight (|r| > 0.5, p < 0.05). In conclusion, breed-specific colonic BA profiles were shaped by host–microbiota interactions, with DHCA potentially promoting meat quality in Tan sheep via regulation of muscle fiber development and fatty acid deposition, and GCA influencing carcass traits in Dorper sheep. This study provides novel insights into the gut microbiota–bile acid axis in modulating ruminant phenotypic traits. Full article

Background/Objectives: Adjuvant radiation for gynecologic malignancies often exposes organs at risk (OARs), such as the bone marrow, bowel, rectum, and bladder, to radiation, leading to toxicities that impact treatment tolerance and patient quality of life. Scanning proton beam therapy, particularly with Individual Field Simultaneous Optimization (IFSO), may offer dosimetric and biological advantages over volumetric modulated arc therapy (VMAT). This study evaluates the clinical impact of IFSO-based proton planning in post-operative gynecologic cancer patients. Materials and Methods: Fourteen patients receiving adjuvant proton therapy to 45 Gy in 25 fractions were retrospectively analyzed. Comparison VMAT plans were generated on the same datasets. Dose–volume metrics for key OARs and normal tissue complication probabilities (NTCPs) were compared using paired statistical tests. Robustness evaluations accounted for setup and range uncertainties. Results: Proton plans significantly reduced dose to bone marrow (V10Gy: 58% vs. 86%, p < 0.00001; V20Gy: 47% vs. 58%, p < 0.00001), small bowel (V20Gy: 21% vs. 56%, p < 0.00001), and femoral heads (left femoral head mean: 11Gy vs. 13Gy, p = 0.032; right femoral head mean: 11Gy vs. 13Gy, p = 0.022). NTCP modeling predicted significantly lower rates of bowel urgency (9.4% vs. 3.3%, p < 0.001) and hematologic toxicity (10.2% vs. 4.9%, p < 0.001) with proton therapy. Plans remained robust across uncertainty scenarios. Conclusions: IFSO-based scanning proton therapy provides clinically meaningful sparing of bone marrow and bowel, with the potential to reduce hematologic and gastrointestinal toxicities. These findings support its use in patients receiving adjuvant pelvic radiotherapy, particularly those undergoing extended field treatment or chemotherapy. Full article