Mito-LND and (E)-Akt inhibitor-IV: novel compounds inducing endoplasmic reticulum stress and ROS accumulation against hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. Although multi-kinase inhibitors can prolong the overall survival of late-stage HCC patients, the emergence of drug resistance diminishes these benefits, ultimately resulting in treatment failure. Therefore, there is an urgent need for novel and effective drugs to impede the progression of liver cancer. METHODS: This study employed a concentration gradient increment method to establish acquired sorafenib or regorafenib-resistant SNU-449 cells. Cell viability was assessed using the cell counting kit-8 assay. A library of 793 bioactive small molecules related to metabolism screened compounds targeting both parental and drug-resistant cells. The screened compounds will be added to both the HCC parental cells and the drug-resistant cells, followed by a comprehensive assessment. Intracellular adenosine triphosphate (ATP) levels were quantified using kits. Flow cytometry was applied to assess cell apoptosis and reactive oxygen species (ROS). Real-time quantitative PCR studied relative gene expression, and western blot analysis assessed protein expression changes in HCC parental and drug-resistant cells. A xenograft model in vivo evaluated Mito-LND and (E)-Akt inhibitor-IV effects on liver tumors, with hematoxylin and eosin staining for tissue structure and immunohistochemistry staining for endoplasmic reticulum stress protein expression. RESULTS: From the compound library, we screened out two novel compounds, Mito-LND and (E)-Akt inhibitor-IV, which could potently kill both parental cells and drug-resistant cells. Mito-LND could significantly suppress proliferation and induce apoptosis in HCC parental and drug-resistant cells by upregulating glycolytic intermediates and downregulating those of the tricarboxylic acid (TCA) cycle, thereby decreasing ATP production and increasing ROS. (E)-Akt inhibitor-IV achieved comparable results by reducing glycolytic intermediates, increasing TCA cycle intermediates, and decreasing ATP synthesis and ROS levels. Both compounds trigger apoptosis in HCC cells through the interplay of the AMPK/MAPK pathway and the endoplasmic reticulum stress response. In vivo assays also showed that these two compounds could significantly inhibit the growth of HCC cells and induce endoplasmic reticulum stress. CONCLUSION: Through high throughput screening, we identified that Mito-LND and (E)-Akt inhibitor-IV are two novel compounds against both parental and drug-resistant HCC cells, which could offer new strategies for HCC patients.

Modeling and Control of a Two-Axis Stabilized Gimbal Based on Kane Method.

A two-axis stabilizing gimbal is a device that ensures a sensor is working properly on a moving platform. When classical mechanics (Newton-Euler and Lagrange) is employed to model a two-axis stable gimbal, its limitations can complicate the modeling process. To address this issue, a method for establishing a dynamic model for a two-axis stabilizing platform based on the Kane method is proposed in this paper. The Kane method offers the advantage of a simple model structure and computational efficiency. Initially, utilizing a generalized coordinate system, expressions of the generalized velocities, deflection velocities and angular velocities are derived. Subsequently, the generalized active forces and inertial forces acting on the two-axis stabilized gimbal are analyzed. Finally, by combining force and velocity with the Kane equation, the dynamic model of the two-axis stable platform is obtained, demonstrating the validity of the Kane method for establishing the two-axis stable platform model. To ensure the pointing accuracy stability of the two-axis stabilizing platform, a Novel Particle Swarm Optimization Proportion Integration Differentiation (NPSO-PID) controller is designed using the PSO algorithm. It is then simulated in MATLAB/Simulink and compared with a classical PID controller. Simulation results demonstrate that NPSO-PID exhibits superior object tracking performance compared to classical PID controllers and better optimization of control parameters compared to traditional PSO-PID controllers.

Empagliflozin rescues lifespan and liver senescence in naturally aged mice.

Empagliflozin is currently known to decrease blood glucose levels, delay renal failure, and reduce the risk of cardiovascular death and all-cause mortality in patients with type 2 diabetes with cardiovascular disease. However, the effects of empagliflozin on the lifespan and health of naturally aged organisms are unclear. This study was designed to investigate the impacts and potential mechanisms of empagliflozin on lifespan and liver senescence in naturally aged mice. Our study revealed that empagliflozin improved survival and health in naturally aged mice. Empagliflozin extended the median survival of male mice by 5.9%. Meanwhile, empagliflozin improved learning memory and motor balance, decreased body weight, and downregulated the hepatic protein expression of P21, P16, α-SMA, and COL1A1. Empagliflozin modulates the structure of the intestinal flora, increasing the relative abundance of Lachnospiraceae, Ruminococcaceae, Lactobacillus, Blautia, and Muribaculaceae and decreasing the relative abundance of Erysipelotrichaceae, Turicibacter, and Dubosiella in naturally aged mice. Further exploration discovered that empagliflozin increased the concentration of SCFAs, decreased the levels of the inflammatory factors TNF-α, IL-6, and CXCL9, and regulated the PI3K/AKT/P21 and AMPK/SIRT1/NF-κB pathways, which may represent the underlying mechanisms involved in these beneficial hepatic effects. Taken together, the above results indicated that empagliflozin intervention could be considered a potential strategy for extending lifespan and slowing liver senescence in naturally aged mice.

Peptide Inhibitor Targeting the Extraterminal Domain in BRD4 Potently Suppresses Breast Cancer Both In Vitro and In Vivo.

BRD4 is associated with a variety of human diseases, including breast cancer. The crucial roles of amino-terminal bromodomains (BDs) of BRD4 in binding with acetylated histones to regulate oncogene expression make them promising drug targets. However, adverse events impede the development of the BD inhibitors. BRD4 adopts an extraterminal (ET) domain, which recruits proteins to drive oncogene expression. We discovered a peptide inhibitor PiET targeting the ET domain to disrupt BRD4/JMJD6 interaction, a protein complex critical in oncogene expression and breast cancer. The cell-permeable form of PiET, TAT-PiET, and PROTAC-modified TAT-PiET, TAT-PiET-PROTAC, potently inhibits the expression of BRD4/JMJD6 target genes and breast cancer cell growth. Combination therapy with TAT-PiET/TAT-PiET-PROTAC and JQ1, iJMJD6, or Fulvestrant exhibits synergistic effects. TAT-PiET or TAT-PiET-PROTAC treatment overcomes endocrine therapy resistance in ERα-positive breast cancer cells. Taken together, we demonstrated that targeting the ET domain is effective in suppressing breast cancer, providing a therapeutic avenue in the clinic.

The SGLT2 inhibitor empagliflozin inhibits skeletal muscle fibrosis in naturally aging male mice through the AMPKα/MMP9/TGF-ß1/Smad pathway.

ABSTACT: With advancing age, the incidence of sarcopenia increases, eventually leading to a cascade of adverse events. However, there is currently a lack of effective pharmacological treatment for sarcopenia. Sodium-glucose co-transporter 2 inhibitor (SGLT2i) empagliflozin demonstrates anti-fibrotic capabilities in various organs. This study aims to determine whether empagliflozin can improve skeletal muscle fibrosis induced by sarcopenia in naturally aging mice. A natural aging model was established by feeding male mice from 13 months of age to 19 months of age. A fibrosis model was created by stimulating skeletal muscle fibroblasts with TGF-ß1. The Forelimb grip strength test assessed skeletal muscle function, and expression levels of COL1A1, COL3A1, and α-SMA were analyzed by western blot, qPCR, and immunohistochemistry. Additionally, levels of AMPKα/MMP9/TGFß1/Smad signaling pathways were examined. In naturally aging mice, skeletal muscle function declines, expression of muscle fibrosis markers increases, AMPKα expression is downregulated, and MMP9/TGFß1/Smad signaling pathways are upregulated. However, treatment with empagliflozin reverses this phenomenon. At the cellular level, empagliflozin exhibits similar anti-fibrotic effects, and these effects are attenuated by Compound C and siAMPKα. Empagliflozin exhibits anti-fibrotic effects, possibly associated with the AMPK/MMP9/TGFß1/Smad signaling pathways.

Single-cell transcriptome and metagenome profiling reveals the genetic basis of rumen functions and convergent developmental patterns in ruminants.

The rumen undergoes developmental changes during maturation. To characterize this understudied dynamic process, we profiled single-cell transcriptomes of about 308,000 cells from the rumen tissues of sheep and goats at 17 time points. We built comprehensive transcriptome and metagenome atlases from early embryonic to rumination stages, and recapitulated histomorphometric and transcriptional features of the rumen, revealing key transitional signatures associated with the development of ruminal cells, microbiota, and core transcriptional regulatory networks. In addition, we identified and validated potential cross-talk between host cells and microbiomes and revealed their roles in modulating the spatiotemporal expression of key genes in ruminal cells. Cross-species analyses revealed convergent developmental patterns of cellular heterogeneity, gene expression, and cell-cell and microbiome-cell interactions. Finally, we uncovered how the interactions can act upon the symbiotic rumen system to modify the processes of fermentation, fiber digestion, and immune defense. These results significantly enhance understanding of the genetic basis of the unique roles of rumen.

Empagliflozin improves kidney senescence induced by D-galactose by reducing sirt1-mediated oxidative stress.

Sodium-glucose cotransporter-2 (SGLT-2) inhibitors have received widespread attention because of their significant protective effects on the kidney. Previous studies have shown that Sirt1, as which is an antiaging protein, is closely related to the maintenance of redox homeostasis. The goal of this study was to determine whether empagliflozin could ameliorate D-galactose-induced renal senescence in mice, and examine the possible mechanisms of Sirt1. We constructed a rapid ageing model in mice by administering D-galactose. An ageing model was constructed by treating cells with high glucose. Treadmill and Y-maze tests were used to assess exercise tolerance and learning memory ability. Pathologically stained sections were used to assess kidney injury. Tissue and cell senescence were evaluated by senescence-associated ß-galactosidase staining. The expression levels of P16, SOD1, SOD2 and Sirt1 were detected by immunoblotting. D-gal-treated mice exhibited significant age-related changes, as measured by behavioural tests and ageing marker protein levels. empagliflozin alleviated these ageing manifestations. In addition, Sirt1, SOD1 and SOD2 levels were downregulated in model mice and upregulated by empagliflozin treatment. Empagliflozin had similar protective effects at the cellular level, and these effects were reduced by the Sirt1 inhibitor. Empagliflozin has an antiaging effect, which may be related to reducing Sirt1-mediated oxidative stress.

A specific JMJD6 inhibitor potently suppresses multiple types of cancers both in vitro and in vivo.

Jumonji C-domain-containing protein 6 (JMJD6), an iron (Fe2+) and α-ketoglutarate (α-KG)-dependent oxygenase, is expressed at high levels, correlated with poor prognosis, and considered as a therapeutic target in multiple cancer types. However, specific JMJD6 inhibitors that are potent in suppressing tumorigenesis have not been reported so far. We herein report that iJMJD6, a specific small-molecule inhibitor of JMJD6 with favorable physiochemical properties, inhibits the enzymatic activity of JMJD6 protein both in vitro and in cultured cells. iJMJD6 is effective in suppressing cell proliferation, migration, and invasion in multiple types of cancer cells in a JMJD6-dependent manner, while it exhibits minimal toxicity in normal cells. Mechanistically, iJMJD6 represses the expression of oncogenes, including Myc and CCND1, in accordance with JMJD6 function in promoting the transcription of these genes. iJMJD6 exhibits suitable pharmacokinetic properties and suppresses tumor growth in multiple cancer cell line- and patient-derived xenograft models safely. Furthermore, combination therapy with iJMJD6 and BET protein inhibitor (BETi) JQ1 or estrogen receptor antagonist fulvestrant exhibits synergistic effects in suppressing tumor growth. Taken together, we demonstrate that inhibition of JMJD6 enzymatic activity by using iJMJD6 is effective in suppressing oncogene expression and cancer development, providing a therapeutic avenue for treating cancers that are dependent on JMJD6 in the clinic.

Zn and Ag Doping on Hydroxyapatite: Influence on the Adhesion Strength of High-Molecular Polymer Polycaprolactone.

In this study, density functional theory was employed to calculate the adsorption of polycaprolactone (PCL) by pure hydroxyapatite (HA), Zn-doped HA, and Ag-doped HA, and the interaction of PCL on the surface of HA (001) was simulated. The results show that there was significant electron transfer between the carbonyl O in PCL and the Zn, Ag, and Ca in HA, forming coordinate bonds. The binding energies of Ag-doped HA/PCL and Zn-doped HA/PCL were much higher than those of HA/PCL. HA doped with Ag had the highest binding energy to PCL. Therefore, we believe that when HA is doped with Ag atoms, its adsorption capacity for PCL can be increased. The results obtained in this study can be used as a guide for the development of HA/PCL bone graft composite material doped with appropriate metal ions to improve its adsorption capacity.

Sorption Studies of Tetracycline Antibiotics on Hydroxyapatite (001) Surface-A First-Principles Insight.

Owing to the limitations of traditional systemic drug delivery in the treatment of bone diseases with side effects on normal cells, the selection of materials with high affinities for bones, as targeting ligands to modify drug carriers, has become an important research topic. Tetracyclines (TCs) have an adsorption effect on hydroxyapatite (HAp). Thus, they can be used as bone-targeting ligands and combined with drug carriers. In this study, density functional theory is used to analyze the interaction mechanism of TC, oxytetracycline (OTC), chlortetracycline, and HAp. We calculate the electrostatic potential (ESP) and molecular orbitals to predict the possible binding sites of TCs on the HAp surface. The adsorption energy is used to compare the affinities of the three TCs to HAp. An independent gradient model analysis is performed to study the weak interaction between TCs and HAp. The coordination bond between TCs and the HAp surface is evaluated by conducting a charge density difference analysis. The results show that OTC has the highest affinity to HAp because the introduction of hydroxyl groups change the adsorption configuration of OTC. Thus, OTC adsorbed on HAp in a broken-line shape exposes more binding sites. This study provides a theoretical basis for TCs as bone-targeting ligands in treating bone diseases and in improving the safety of treatment by selecting different bone-targeting ligands.

microRNA-10b promotes the apoptosis of bovine ovarian granulosa cells by targeting plasminogen activator inhibitor-1.

Granulosa cells (GCs) are essential somatic cells in the ovaries, and apoptosis of GCs causes follicular atresia. microRNA-10b (miR-10b) is pivotal for cell apoptosis. However, currently, little is known about the role of miR-10b in bovine ovarian GCs (BGCs). In this study, the effect of miR-10b on the apoptosis of BGCs was investigated. Our results showed that the overexpression of miR-10b could increase the apoptosis rate of BGCs, which is associated with the increased expression of Caspase-3 and decreased expression ratio of Bcl-2/Bax (P < 0.05). Furthermore, plasminogen activator inhibitor-1 (PAI-1) was confirmed to be a validated target of miR-10b in BGCs using dual-luciferase reporter analysis, and transfection of miR-10b mimics decreased the expression of PAI-1 (P < 0.05). In addition, overexpression of PAI-1 significantly inhibited BGC apoptosis (P < 0.05), and PAI-1 could alleviate BGC apoptosis induced by miR-10b (P < 0.05). Subsequently, phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) was found to be the downstream pathway of PAI-1 by RNA-Seq analysis and verified by Western blot. Finally, a PI3K/AKT inhibitor (Miltefosine) was used to inhibit the PI3K/AKT pathway, which reversed the inhibitory effect of PAI-1 on the apoptosis of BGCs (P < 0.05), and enhanced the promotion effect of miR-10b on the apoptosis of BGCs (P < 0.05). Our results indicated that miR-10b promotes BGC apoptosis by targeting PAI-1 to regulate the PI3K/AKT pathway.

Bias Evaluation of the Accuracy of Two Extraoral Scanners and an Intraoral Scanner Based on ADA Standards.

The spread and application of computer-aided design/computer-aided manufacturing (CAD/CAM) technology have contributed to the rapid development of digitalization in dentistry. The accuracy of scan results is closely related to the devising subsequent treatment plans and outcomes. Professional standards for evaluating scanners are specified in the American National Standard/American Dental Association Standard 132 (ANSI/ADA No. 132). The aims of this study were to use the three samples mentioned in ANSI/ADA No. 132 and evaluate the accuracy and reproducibility of two extraoral scanners and an intraoral scanner based on the inspection standards recommended by ANSI/ADA No. 132. In this study, two trained operators used two extraoral scanners (E4, 3Shape, Denmark & SHINING DS100+, Shining, China) and an intraoral scanner (TRIOS SERIES3, 3Shape, Denmark) to perform 30 scans of each of the three samples at a temperature of 25 ± 2°C and export standard tessellation language files and used reverse engineering software to perform measurements and iterative nearest point matching experiments. The measured values obtained were compared with the reference values measured by a coordinate measuring machine (NC8107, Leader Metrology, USA). We performed a normal distribution test (Shapiro-Wilk test), the nonparametric Kruskal-Wallis test, and an independent-samples t-test to analyze the reproducibility of each scan for different models. The experimental results indicate that the trueness and precision of the two extraoral scanners and the intraoral scanner had a slight mean deviation. The trueness and precision of the three scanners on the curved surface and groove areas are poor. The accuracy and reproducibility of E4 outperformed SHINING and TRIOS. The iterative closest point matching experiment also showed good matching results. The two extraoral scanners and the intraoral scanner in this study can meet the basic clinical requirements in terms of accuracy, and we hope that digital technology will be more widely used in dentistry in the future.

In Silico Discovery of JMJD6 Inhibitors for Cancer Treatment.

The 2-oxoglutarate (2OG)-dependent oxygenase JMJD6 is emerging as a potential anticancer target, but its inhibitors have not been reported so far. In this study, we reported an in silico protocol to discover JMJD6 inhibitors targeting the druggable 2OG-binding site. Following this protocol, one compound, which we named as WL12, was found to be able to inhibit JMJD6 enzymatic activity and JMJD6-dependent cell proliferation. To our best knowledge, this is the first case in drug discovery targeting JMJD6.

Evaluation of Formalin Fixation for Tissue Biopsies Using Shear Wave Laser Speckle Imaging System.

Chemical fixation is the slowest and often the most uncontrolled step in the multi-step process of preparing tissue for histopathology. In order to reduce the time from taking a core needle biopsy to making a diagnosis, a new approach is proposed that optically monitors the common formalin fixation process. A low-cost and highly-sensitive laser speckle imaging technique is developed to measure shear wave velocity in a biospecimen as small as 0.5 mm in thickness submerged in millifluidic channels. Shear wave velocity, which is the indicator of tissue mechanical property and induced by piezoelectric-actuation, was monitored using gelatin phantom and chicken breast during fixation, as well as post-fixed liver and colon tissues from human. Fixation levels in terms of shear wave velocity increased by approximately 271.0% and 130.8% in gelatin phantom and chicken breast, respectively, before reaching the plateaus at 10.91 m/s and 7.88 m/s. Within these small specimens, the plateaus levels and times varied with location of measurement, and between gelatin and chicken breast. This optical-based approach demonstrates the feasibility of fine-tuning preanalytical variables, such as fixation time, for a rapid and accurate histopathological evaluation; provides a quality metric during the tissue preparation protocol performed in most pathology labs; and introduces the millifluidic chamber that can be engineered to be a future disposable device that automates biopsy processing and imaging.

JMJD6 Licenses ERα-Dependent Enhancer and Coding Gene Activation by Modulating the Recruitment of the CARM1/MED12 Co-activator Complex.

Whereas the actions of enhancers in gene transcriptional regulation are well established, roles of JmjC-domain-containing proteins in mediating enhancer activation remain poorly understood. Here, we report that recruitment of the JmjC-domain-containing protein 6 (JMJD6) to estrogen receptor alpha (ERα)-bound active enhancers is required for RNA polymerase II recruitment and enhancer RNA production on enhancers, resulting in transcriptional pause release of cognate estrogen target genes. JMJD6 is found to interact with MED12 in the mediator complex to regulate its recruitment. Unexpectedly, JMJD6 is necessary for MED12 to interact with CARM1, which methylates MED12 at multiple arginine sites and regulates its chromatin binding. Consistent with its role in transcriptional activation, JMJD6 is required for estrogen/ERα-induced breast cancer cell growth and tumorigenesis. Our data have uncovered a critical regulator of estrogen/ERα-induced enhancer coding gene activation and breast cancer cell potency, providing a potential therapeutic target of ER-positive breast cancers.