Identification of novel RANKL inhibitors through in silico analysis.

Receptor activator of nuclear factor-κB ligand (RANKL) is considered the principal regulator of osteoclast differentiation. Therefore, strategies interfering with the RANKL-RANK signaling pathway may effectively inhibit osteoclast differentiation and mitigate bone resorption. Consequently, RANKL has become a promising target for new drug design strategies. Despite extensive research on specific drugs and antibodies, only a few have shown efficacy in treating osteoporosis. To address this challenge, we aimed to explore new approaches for designing drugs for osteoporosis. In this study, a 3D quantitative structure-activity relationship (QSAR) pharmacophore model was built for RANKL with reference to known inhibitor IC50 values. The optimal pharmacophore model was then employed as a 3D query to screen databases for novel lead compounds. The obtained compounds were subjected to ADMET and TOPKAT analyses to predict drug pharmacokinetics and toxicity. Molecular docking and de novo evolution approaches were applied to verify the docking binding affinities of the compounds. Five candidate compounds were subjected to further in vitro analyses to assess their anti-osteoporotic effects, among which compound 4 demonstrated significant inhibitory activity, achieving an inhibitory rate of 92.6 % on osteoclastogenesis at a concentration of 10 µM. Subsequent molecular dynamics (MD) simulations to assess the stability and behavior of compound 4 and its evolved variant, ZINC00059014397_Evo, within the RANKL binding site revealed that the variant is a potential therapeutic agent for targeting osteoclasts. This study offers valuable insights for developing next generation RANKL inhibitors for osteoporosis treatments.

Geranylgeraniol: Bio-based platform for teprenone, menaquinone-4, and α-tocotrienol synthesis.

By utilizing the conformational selectivity of biosynthesis and the flexibility of chemical synthesis, researchers have formulated metabolic engineering-based semi-synthetic approaches that initiate with the final product's structure and identify key biosynthesis intermediates. Nonetheless, these tailored semi-synthetic routes focused on end-products, neglecting the possibility of biobased intermediates as a platform for derivatization. To address this challenge, this studyproposed a novel strategy resembling chemosynthesis-style divergent exploration to amplify the significance of biobased intermediates, in the case of geranylgeraniol (GGOH). Using the novel bifunctional terpene synthase PTTC066 and systematic metabolic engineering modifications, the engineered yeast straindemonstrated high GGOH production levels (3.32 g/L, 0.039 g/L/h). This platformenabled the semi-synthesis of various pharmaceuticals, including the anti-ulcer drug teprenone, the osteoporosis treatment drug menaquinone-4, and introduced a novel route for synthesizingα-tocotrienol. This study offers a fresh outlook on semi-synthetic approaches, opening avenues for improvements, substitutions, and innovations in industrial production processes.

Analyzing the correlation between low proportion of hobnail features in papillary thyroid carcinoma and clinical aggressiveness risk.

PURPOSE: Hobnail features may enhance the clinical aggressiveness of papillary thyroid carcinoma (PTC). However, whether a low proportion (<30%) of these features contributes to increased PTC aggressiveness remains unclear. This study investigated whether PTC cases with a low proportion hobnail features (<30%) exhibit clinical invasiveness and pathological features of aggressiveness. METHODS: Pathological specimens from patients with postoperatively diagnosed PTC were retrospectively analyzed. Among them, 29 PTC cases with a low proportion of hobnail features (<30%) were compared with 173 consecutive classical PTC (cPTC) cases. Data regarding age at presentation, sex, tumor size, number of tumors, and histological characteristics were obtained by reviewing electronic medical records. Postoperative information was obtained during follow-up visits and telephone interviews. RESULTS: Twenty-nine patients with PTC with a low proportion of hobnail features (<30%) were identified, exhibiting a median age of 34 years. At a median follow-up of 31 (IQR, 23-37) months, two patients had recurrent disease in the PTC with a low proportion of hobnail features (<30%) group, whereas there was no recurrence in the cPTC group. No distant metastasis and postoperative mortality were observed in either group. Compared with the cPTC group, patients with PTC and a low proportion of hobnail features exhibited larger tumor volumes and higher susceptibility to capsular invasion and lymph node metastasis. Tumor size and hobnail features emerged as independent risk factors for lymph node metastasis. CONCLUSION: PTC with a low proportion hobnail features (<30%) and larger tumor volumes are associated with the occurrence of lymph node metastasis. A low proportion of hobnail features (<30%) in PTC may heighten invasiveness, elevating the risk of recurrence.

Metasurface array for single-shot spectroscopic ellipsometry.

Spectroscopic ellipsometry is a potent method that is widely adopted for the measurement of thin film thickness and refractive index. Most conventional ellipsometers utilize mechanically rotating polarizers and grating-based spectrometers for spectropolarimetric detection. Here, we demonstrated a compact metasurface array-based spectroscopic ellipsometry system that allows single-shot spectropolarimetric detection and accurate determination of thin film properties without any mechanical movement. The silicon-based metasurface array with a highly anisotropic and diverse spectral response is combined with iterative optimization to reconstruct the full Stokes polarization spectrum of the light reflected by the thin film with high fidelity. Subsequently, the film thickness and refractive index can be determined by fitting the measurement results to a proper material model with high accuracy. Our approach opens up a new pathway towards a compact and robust spectroscopic ellipsometry system for the high throughput measurement of thin film properties.

Dual Nickel- and Photoredox-Catalyzed Asymmetric Reductive Cross-Coupling To Access Chiral Secondary Benzylic Alcohols.

Transition-metal-catalyzed asymmetric cross-coupling represents a powerful strategy for C-C bond formation and the synthesis of enantiomerically pure molecules. Here, we report a dual nickel/photoredox-catalyzed enantioselective reductive cross-coupling of aryl halides with α-bromobenzoates, readily generated from aliphatic aldehydes, to provide diverse chiral secondary benzylic alcohols that are important motifs in bioactive natural products and pharmaceuticals. This dual catalytic system features mild conditions, good functional group tolerance, broad substrate scope, excellent enantiocontrol, and avoidance of stoichiometric metal reductants, presenting great potential for late-stage functionalization of complex molecules.

Nickel-Catalyzed Reductive anti-Arylative Cyclization of Alkynyl Enones with Aryl Halides.

A nickel-catalyzed reductive anti-arylative cyclization of alkynyl enones with aryl halides has been developed. The reaction avoids the use of stoichiometric organometallic reagents and has a broad reaction scope and high functional group tolerance. This method offers an efficient way to access a variety of synthetically useful carbocycles that are widely found in many natural products and biologically active molecules.

Single-shot deterministic complex amplitude imaging with a single-layer metalens.

Conventional imaging systems can only capture light intensity. Meanwhile, the lost phase information may be critical for a variety of applications such as label-free microscopy and optical metrology. Existing phase retrieval techniques typically require a bulky setup, multiframe measurements, or prior information of the target scene. Here, we proposed an extremely compact system for complex amplitude imaging, leveraging the extreme versatility of a single-layer metalens to generate spatially multiplexed and polarization phase-shifted point spread functions. Combining the metalens with a polarization camera, the system can simultaneously record four polarization shearing interference patterns along both in-plane directions, thus allowing the deterministic reconstruction of the complex amplitude light field in a single shot. Using an incoherent light-emitting diode as the illumination, we experimentally demonstrated speckle-noise-free complex amplitude imaging for both static and moving objects with tailored magnification ratio and field of view. The miniaturized and robust system may open the door for complex amplitude imaging in portable devices for point-of-care applications.

Adjuvant activity of cordycepin, a natural derivative of adenosine from Cordyceps militaris, on an inactivated rabies vaccine in an animal model.

Vaccination is the most feasible way of preventing rabies, an ancient zoonosis that remains a major public health concern globally. However, administration of inactivated rabies vaccination without adjuvants is always inefficient and necessitates four to five injections. In the current study, we explored the adjuvant characteristics of cordycepin, a major bioactive component of Cordyceps militaris, to boost immune responses against a commercially available rabies vaccine. We found that cordycepin could stimulate stronger phenotypic and functional maturation of dendritic cells (DCs). For animal experiments, mice were immunized 3 times with rabies vaccine in the presence or absence of cordycepin at 1-week interval. Analysis of T cell differentiation and serum antibody isotypes showed that humoral immunity was dominant with a Th2 biased immune response. These results were also supported by the raised ratio of follicular helper T cells (TFH) and germinal center B cells (GCB). Thus, titer of rabies virus neutralizing antibody (RVNAb) and rabies virus-specific memory B cells were both raised as a result. Furthermore, administration of cordycepin did not cause pathological phenomena or body weight loss. The findings indicate that cordycepin could be used as a promising adjuvant for rabies vaccines to get a higher range of protection without any side effects.

Quantitative comparison of three thyroidectomy approaches in neck muscles, voice, and swallowing functions.

OBJECTIVE: This study compares endoscopic thyroidectomy by gasless unilateral axillary approach (ETGUA) and sternocleidomastoid leading-edge approach (SLEA) with conventional open thyroidectomy (COT) in hemithyroidectomy. The main focus is on the protection of neck muscles (sternocleidomastoid, omohyoid, sternothyroid) and the postoperative function of voice and swallowing yielded through these common approaches. METHODS: A total of 302 patients who underwent hemithyroidectomy were enrolled and divided into three groups: ETGUA (n = 101), SLEA (n = 100), and COT (n = 101). Ultrasound was used to measure the thickness of bilateral neck muscles, including the sternocleidomastoid, omohyoid, and sternothyroid. The changes in thickness on the surgical side compared to the non-surgical side. Analyzed factors included muscle thickness changes, Swallowing Impairment Score (SIS), Voice Handicap Index (VHI), Scar Cosmesis Assessment and Rating (SCAR), Neck Injury Index (NII), surgery duration, drainage volume, hospitalization, and number of lymph nodes. RESULTS: The clinical characteristics among the three groups were consistent except for differences in sex, age, and BMI. Metrics such as sternocleidomastoid muscle, NII, hypocalcemia, postoperative PTH, transient hoarseness, and number of lymph nodes showed no significant differences among the three groups. However, significant differences were found in the duration of surgery, drainage volume, hospitalization period omohyoid muscle, Sternohyoid muscle, VHI, SIS, and SCAR (all p < 0.001). CONCLUSION: In comparison to COT, ETGUA and SLEA demonstrate superiority in protecting neck muscles and preserving voice and swallowing function without compromising surgical safety or radicality.

Metalens for Accelerated Optoelectronic Edge Detection under Ambient Illumination.

Analog systems may allow image processing, such as edge detection, with low computational power. However, most demonstrated analog systems, based on either conventional 4-f imaging systems or nanophotonic structures, rely on coherent laser sources for illumination, which significantly restricts their use in routine imaging tasks with ambient, incoherent illumination. Here, we demonstrated a metalens-assisted imaging system that can allow optoelectronic edge detection under ambient illumination conditions. The metalens was designed to generate polarization-dependent optical transfer functions (OTFs), resulting in a synthetic OTF with an isotropic high-pass frequency response after digital subtraction. We integrated the polarization-multiplexed metalens with a polarization camera and experimentally demonstrated single-shot edge detection of indoor and outdoor scenes, including a flying airplane, under ambient sunlight illumination. The proposed system showcased the potential of using polarization multiplexing for the construction of complex optical convolution kernels toward accelerated machine vision tasks such as object detection and classification under ambient illumination.

Rhoifolin protects cecal ligation and puncture induced sepsis mice model by regulating inflammatory pathway.

Sepsis is a systemic infection affects several organs, which needs novel therapy for the management of it, thus protective effect of Rhoifolin was estimated against sepsis. Cecal ligation and puncture (CLP) method was used to induce sepsis and thereafter mice were treated with rhoifolin (20 and 40 mg/kg, i.p.) for one week. Food intake and survival rate was determined sepsis mice, moreover liver function test and cytokines was estimated in the serum of sepsis mice. In the lung tissue homogenate, oxidative stress parameters were determined, histopathological analysis was performed in liver and lung tissue of sepsis mice. Food intake and percentage of survival was improved in rhoifolin treated group than sham group. Level of liver function enzyme and cytokine was reduced significantly in the serum of rhoifolin treated sepsis mice. Treatment with rhoifolin ameliorates the altered oxidative stress parameters, and mRNA expression of Toll-like receptor 4 (TLR-4) in lung tissue of sepsis mice. Histopathological changes were also reverse in rhoifolin treated group than sham group of mice. In conclusion, result of report indicates Rhoifolin treatment reduces oxidative stress and inflammation in CLP induced sepsis mice, as it regulates TLR4/MyD88/NF-κB pathway.

Metasurface-Based Solid Poincaré Sphere Polarizer.

The combination of conventional polarization optical elements, such as linear polarizers and waveplates, is widely adopted to tailor light's state of polarization (SOP). Meanwhile, less attention has been given to the manipulation of light's degree of polarization (DOP). Here, we propose metasurface-based polarizers that can filter unpolarized incident light to light with any prescribed SOP and DOP, corresponding to arbitrary points located both at the surface and within the solid Poincaré sphere. The Jones matrix elements of the metasurface are inverse-designed via the adjoint method. As prototypes, we experimentally demonstrated metasurface-based polarizers in near-infrared frequencies that can convert unpolarized light into linear, elliptical, or circular polarizations with varying DOPs of 1, 0.7, and 0.4, respectively. Our Letter unlocks a new degree of freedom for metasurface polarization optics and may break new ground for a variety of DOP-related applications, such as polarization calibration and quantum state tomography.

Carbamazepine-induced DRESS Syndrome: A Rare Delayed Hypersensitivity Reaction.

Drug rash with eosinophilia and systemic symptoms (DRESS) syndrome is a rare but severe cutaneous drug hypersensitivity reaction. Delays in making a diagnosis of DRESS syndrome and lack of timely treatment may result in morbidity and mortality. However, the presentation can be misinterpreted as other pathologies because of a broad spectrum of clinical presentations, delayed reactions, and prolonged course. Despite the discontinuation of the medication that is the culprit, relapses of drug reactions frequently occur weeks to months later. Several drugs that are associated with DRESS syndrome are prescribed as psychotropic medications. This report describes the case of a patient with DRESS syndrome who was evaluated with the RegiSCAR scoring system as a "definite case," which was possibly induced by carbamazepine prescribed to treat bipolar I disorder. The young female patient was successfully treated with steroid medication after carbamazepine was discontinued. She was prescribed aripiprazole for mood stabilization without a subsequent recurrence of DRESS syndrome. We recommend that, in cases such as described here, clinicians take DRESS syndrome into consideration and provide proper timely management, particularly for patients receiving psychotropic drugs. A brief review of the literature concerning DRESS syndrome associated with psychotropic drugs and its pathogenesis are outlined and discussed.

Pseudomembranous laryngotracheobronchitis due to coinfection with human bocavirus 1 and Mycoplasma pneumoniae: a case report.

Pseudomembranous laryngotracheobronchitis is rarely reported yet potentially life-threatening infectious cause of airway obstruction in children. The causative organisms of this condition are often considered to promote bacterial superinfection following viral infection. We report a case of pseudomembranous laryngotracheobronchitis in a patient caused by human bocavirus 1 and Mycoplasma pneumoniae (M. pneumoniae). A 2-year-old child was admitted to our hospital presenting with cough, hoarseness, and labored breathing. Computed tomography of the chest revealed atelectasis of the right middle lobe of the lung with bronchostenosis and occlusion. Laryngeal edema, pseudomembrane formation and ulceration of the trachea were found during bronchoscopy. Chronic inflammation of the mucosa and local cellulose exudation with acute and chronic inflammatory cell infiltration were confirmed by hematoxylin-eosin staining. Human bocavirus 1 and M. pneumoniae were detected in the bronchoalveolar lavage fluid by next-generation sequencing. The patient tested positive for IgM antibodies against M. pneumoniae. Bronchoscopy was performed three times to clear the secretions in the airway, and azithromycin, ceftriaxone, methylprednisolone, budesonide inhalation, and ambroxol were administered as treatment. The patient's condition improved and she was discharged 21 days after admission. Clinicians should be aware of the potential involvement of human bocavirus 1 and M. pneumoniae in pseudomembranous laryngotracheobronchitis for accurate diagnosis and timely antibiotic administration, and to lower mortality and morbidity rates.

Synergistically Enhanced Inhibitory Effects of Pullulan Nanoparticle-Mediated Co-Delivery of Lovastatin and Doxorubicin to Triple-Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC) is a subtype of breast cancer that is prone to drug resistance and difficult to treat. In this study, we grafted water-soluble pullulan with lovastatin (LV) to develop a novel amphiphilic conjugate, pullulan-encapsulated LV (PLV). The PLV conjugate was synthesized with three different ratios of pullulan to LV and characterized by Fourier transform infrared (FTIR). The degree of substitution (DS) of LV in terms of molar ratio was 7.87%, 3.58%, and 3.06% for PLV (1/2), PLV (1/3), and PLV (1/4), respectively, by proton NMR analysis. We selected the PLV (1/2) conjugate to prepare doxorubicin (DXR)-loaded PLV nanoparticles (PLV/DXR NPs) because of its superior properties. The average size and zeta potential for PLV (1/2) NPs were 177.6 nm and - 11.66 mV, respectively, determined by dynamic light scattering, and those for PLV/DXR NPs were 225.6 nm and - 10.51 mV, respectively. In vitro drug release profiling showed that PLV/DXR NPs sustainably released DXR within 72 h, which was more robust at pH 5.4 (97.90%) than pH 7.4 (76.15%). In the cytotoxicity study, PLV/DXR NPs showed greater inhibition of proliferation of TNBC MDA-MB-231 than non-TNBC MDA-MB-453 cells (IC50 0.60 vs 11.05 µM). FITC-loaded PLV/DXR NPs were prepared to investigate cellular uptake: both cell lines showed a time-dependent uptake of NPs, but the number of NPs entering MDA-MB-231 cells was greater than that entering the MDA-MB-453 cells. Pullulan-based NP co-delivery of LV and DXR could efficiently inhibit TNBC cells, which may help in designing a powerful drug delivery system for treating TNBC.

Safety and Efficacy of Laparoscopic Radiofrequency Ablation for Hepatic Hemangiomas: A Multicenter Retrospective Study.

INTRODUCTION: Radiofrequency ablation (RFA) is an effective and minimally invasive technique for the management of hepatic hemangiomas (HHs). This study aims to assess the safety and efficacy of laparoscopic RFA for HHs. MATERIAL AND METHODS: Forty-four patients with 50 hepatic hemangiomas (5-10 cm in diameter) undergoing laparoscopic RFA from January 2012 to May 2015 at three tertiary hospitals in China were retrospectively analyzed. RESULTS: Thirty-three patients with subcapsular hemangiomas were treated with a laparoscopic approach, and 11 patients with lesions in the liver parenchyma were treated with a combined laparoscopy and an ultrasound-guided percutaneous approach. No conversion to open surgery or two-step surgery occurred during the study period. Patients with small hemangiomas (< 7 cm) required a significantly shorter operating time (71.1 ± 20.18 min vs. 106 ± 23.55 min, p = 0.000) and fewer punctures compared with patients with large hemangiomas (> 7 cm) (4.61 ± 1.09 vs. 6.73 ±1.01, P < 0.05). According to the Dindo-Clavien classification, 15 patients experienced 34 Grade 1 complications, and two had complications of Grade 3a. All complications were resolved by conservative treatment. Forty-three (86.0%) HHs in 38 patients were completely ablated after RFA, and 7 (14.0%) HHs in 6 patients were incompletely ablated. All patients were followed up for 6-24 months (mean 15 ± 6 months). CONCLUSION: The data showed that laparoscopic RFA is an effective treatment for small (< 10 cm) HHs. While the incidence of postoperative complications remains high, the majority of complications are minor. Patients undergoing laparoscopic RFA for HHs, even for the small ones, should be carefully selected.

TNF-α increases Staphylococcus aureus-induced death of human alveolar epithelial cell line A549 associated with RIP3-mediated necroptosis.

AIM: To explore the role of tumor necrosis factor-alpha (TNF-α) on Staphylococcus aureus-induced necroptosis in alveolar epithelial cells. MAIN METHODS: The A549 alveolar epithelial cell line was pretreated with small interfering RNA (siRNA) against receptor interacting protein-3 (RIP3) and then stimulated by S. aureus, where some cells were pretreated with TNF-α or TNF-α with anti-TNF-α antibody simultaneously. A549 cell death was assessed using lactate dehydrogenase (LDH) leakage and flow cytometry analyses. The protein expressions of RIP1, RIP3, cleaved caspase-1, and cleaved caspase-8 were analyzed by western blot. KEY FINDINGS: S. aureus-induced LDH release was increased significantly by TNF-α. In addition, flow cytometry showed that TNF-α increased A549 cell apoptosis and necrosis in S. aureus-infected cell cultures. Levels of RIP3 and cleaved caspase-1 protein in A549 cells infected with S. aureus increased at 12 h post-infection, as shown by western blot. Significant additional increases in RIP3 expression were observed following the addition of TNF-α. Decreasing RIP3 levels by siRNA significantly suppressed the release of LDH induced by TNF-α and S. aureus. RIP3 siRNA also significantly suppressed A549 cell necrosis induced by S. aureus and TNF-α at 6 and 12 h post-infection as shown by flow cytometry analysis. SIGNIFICANCE: TNF-α enhances the damage of S. aureus on lung epithelial cells, and its mechanism is associated with RIP3 mediated necroptosis.

Appropriate Patient Selection Is Essential for the Success of Primary Closure After Laparoscopic Common Bile Duct Exploration.

BACKGROUND: Laparoscopic common bile duct exploration (LCBDE) is being increasingly used for management of common bile duct (CBD) stones. Primary CBD closure has been reported to have better short-term outcomes compared to T-tube placement. However, primary CBD closure cannot be performed in all patients. AIM: This study aims to evaluate the short- and long-term outcomes of LCBDE with primary CBD closure in appropriately selected patients and compare them with T-tube drainage. METHODS: Retrospective analysis of patients undergoing LCBDE in our department from June 2011 to October 2014 was performed. Primary closure was performed in 52 patients (group A), and a T-tube was placed in 33 patients (group B). Patient demographics, intraoperative findings, postoperative stay, complications, and long-term follow-up data were recorded and compared. RESULTS: The mean operating time was much longer in group A compared to group B (113.92 vs. 95.92 min, p = 0.032). The overall complication rate (9.6 vs. 6.3%, p = 0.701) and hospital stay (4 vs. 5.11 days, p = 0.088) were similar in both groups. No patient required conversion to the open procedure. Bile leakage was more frequent in group A (5.78 vs. 0%, p = 0.279), but this was not statistically significant. All three patients with bile leakage were treated successfully by conservative measures and gradual drain withdrawal. On long-term follow-up, recurrent stones were detected in two patients in group A. No patient was found to develop CBD stricture. CONCLUSION: LCBDE and primary CBD closure has excellent short- and long-term outcomes when performed in appropriately selected patients.

Identification of differentially expressed genes implicated in peel color (red and green) of Dimocarpus confinis.

Nowadays, there are few reports about regulatory genes implicated in peel color of longan. The basic genetic research of longan has been in stagnation for a long time as a lack of transcriptomic and genetic information. To predict candidate genes associated with peel color, Gene Functional Annotation and Coding Sequence prediction were used to perform functional annotation for our assembled unigenes and investigate differentially expressed genes (DEGs) of fruitlet peels from Longli (Dimocarpus confinis). Finally, a total of 24,044 (44.19 %) unigenes were annotated at least in one database after BLAST search to NCBI non-redundant protein sequence, NCBI non-redundant nucleotide sequences, Kyoto Encyclopedia of Genes and Genomes (KEGG) Ortholog, manually annotated and reviewed protein sequence database (Swiss-Prot), Protein family, Gene Ontology, euKaryotic Ortholog Groups databases. After searching against the KEGG-GENE protein database, a result of 6228 (11.45 %) unigenes were assigned to 245 KEGG pathways. Via comparing the distributions of expression value of all corresponding unigenes from red peel and green peel fruit, it could be intuitively concluded that high similarity was existed in the two distributions; however, on the whole, between two distributions of log RPKM expression value, some differences indicated that expression level in green-peel fruit group is slightly higher than values in red-peel fruit group. Finally, a total of 1349 unigenes were identified as DEGs after blasting the DEGs to public sequence databases, and 32 peel-color-related genes were identified in longan. Our results suggest that a number of unigenes involved in longan metabolic process, including anthocyanin biosynthesis. In addition, DRF, F3H, ANS, CYP75A1 and C1 may be the key ones. The study on key genes related to peel color will be contributed to revealing the molecular mechanisms of regulating peel color in woody plants.

Antiproliferative effects of cinobufacini on human hepatocellular carcinoma HepG2 cells detected by atomic force microscopy.

AIM: To investigate the antiproliferative activity of cinobufacini on human hepatocellular carcinoma HepG2 cells and the possible mechanism of its action. METHODS: HepG2 cells were treated with different concentrations of cinobufacini. Cell viability was measured by methylthiazolyl tetrazolium (MTT) assay. Cell cycle distribution was analyzed by flow cytometry (FCM). Cytoskeletal and nuclear alterations were observed by fluorescein isothiocyanate-phalloidin and DAPI staining under a laser scanning confocal microscope. Changes in morphology and ultrastructure of cells were detected by atomic force microscopy (AFM) at the nanoscale level. RESULTS: MTT assay indicated that cinobufacini significantly inhibited the viability of HepG2 cells in a dose-dependent manner. With the concentration of cinobufacini increasing from 0 to 0.10 mg/mL, the cell viability decreased from 74.9% ± 2.7% to 49.41% ± 2.2% and 39.24% ± 2.1% (P < 0.05). FCM analysis demonstrated cell cycle arrest at S phase induced by cinobufacini. The immunofluorescence studies of cytoskeletal and nuclear morphology showed that after cinobufacini treatment, the regular reorganization of actin filaments in HepG2 cells become chaotic, while the nuclei were not damaged seriously. Additionally, high-resolution AFM imaging revealed that cell morphology and ultrastructure changed a lot after treatment with cinobufacini. It appeared as significant shrinkage and deep pores in the cell membrane, with larger particles and a rougher cell surface. CONCLUSION: Cinobufacini inhibits the viability of HepG2 cells via cytoskeletal destruction and cell membrane toxicity.