Mechanism-based Studies Research Articles

Синергическая эффективность низкоинтенсивной экстракорпоральной ударно-волновой терапии и обогащенной тромбоцитами плазмы при эректильной дисфункции

Erectile dysfunction (ED), an unusual sexual condition in which the person fails to attain or sustain an erectile penis, severely impacts personal relationships, confidence, and efficiency. To date, low-intensity extracorporeal shock wave therapy (Li-ESWT) is an option to manage ED; however, it is associated with adverse events such as bruising, redness, and pain. Hence, in this study, we applied platelet-rich plasma (PRP), a blood-derived biomaterial containing cargo of growth factors, to enhance the therapeutic efficacy of Li-ESWT on ED. We assessed the synergistic effect of PRP+Li-ESWT, in which Li-ESWT was extracorporeally applied simultaneously with PRP. They were evaluated clinically at 22 ± 2, 50 ± 2 and 78 ± 2 days. Statistical analysis was performed using a non-parametric test, Friedman repeated measures as an alternative non-parametric test of ANOVA test. The international index of erectile function (IIEF-5) and erection hardness score (EHS) were recorded. IIEF-5 score in the pre-treated group was 8.36 ± 1.44. After 22 ± 2 days of synergistic PRP+Li-ESWT treatment, the score was 14.45 ± 2.12 (p < 0.028). This score further increased to 15.45 ± 1.93 (p < 0.008) and 16.18 ± 1.48 (p < 0.001) after 50 ± 2 days and 78 ± 2 days of treatment, respectively. The mean pre-treated EHS was 1.64 ± 0.20 (p < 0.002), which increased to 2.81 ± 0.26 (p < 0.002), 3.09 ± 0.25 (p < 0.0002) and 3.18 ± 0.12 (p < 0.000) on day 22 ± 2, 50 ± 2 and 78 ± 2 days, respectively. Conclusively, our study demonstrated potent synergistic therapy of PRP+Li-ESWT in ED treatment by improving IIEF-5 and EHS scores. However, extensive mechanism-based clinical studies are needed to reach a consensus.

The association between Lifelines Diet Score (LLDS) and Polycystic Ovary Syndrome (PCOS) in Iranian women: a case-control study

BackgroundAlthough adherence to a healthy dietary pattern is one of the primary recommendations for the prevention of polycystic ovary syndrome (PCOS), there is still no conclusive evidence of which specific dietary pattern is best. The Lifelines diet score (LLDS) is a new, evidence-based scoring system to determine diet quality, and its association with PCOS has not been investigated. The present study aimed to assess the association between LLDS and PCOS in Iranian women.Materials and methodsThis frequency-matched case-control study was carried out on 108 women with PCOS and 108 women without PCOS as a control group in Yazd, Iran. Healthy controls were matched to PCOS women based on age and BMI. The validated 178-item food frequency questionnaire was used to assess the usual dietary intake. Logistic regression was used to estimate the association between LLDS and PCOS.ResultsThe findings of the present study showed women in the highest tertile of LLDS compared with the participants in the lowest tertile had 90% lower odds of PCOS (Odds Ratio (OR): 0.10; 95% Confidence Interval (CI): 0.04 to 0.21, p for trend: <0.001). This association remained significant after adjustment for energy intake, marital status, pregnancy history, WC, chronic disease history, physical activity, and BMI (Odds Ratio (OR): 0.11; 95% (CI):0.05 to 0.27, p for trend: <0.001).ConclusionAlthough the present study found a significant protective association between adherence to LLDS and PCOS, more mechanism-based studies are needed to confirm these findings in the future.

Machine Learning in Identifying Marker Genes for Congenital Heart Diseases of Different Cardiac Cell Types.

Congenital heart disease (CHD) represents a spectrum of inborn heart defects influenced by genetic and environmental factors. This study advances the field by analyzing gene expression profiles in 21,034 cardiac fibroblasts, 73,296 cardiomyocytes, and 35,673 endothelial cells, utilizing single-cell level analysis and machine learning techniques. Six CHD conditions: dilated cardiomyopathy (DCM), donor hearts (used as healthy controls), hypertrophic cardiomyopathy (HCM), heart failure with hypoplastic left heart syndrome (HF_HLHS), Neonatal Hypoplastic Left Heart Syndrome (Neo_HLHS), and Tetralogy of Fallot (TOF), were investigated for each cardiac cell type. Each cell sample was represented by 29,266 gene features. These features were first analyzed by six feature-ranking algorithms, resulting in several feature lists. Then, these lists were fed into incremental feature selection, containing two classification algorithms, to extract essential gene features and classification rules and build efficient classifiers. The identified essential genes can be potential CHD markers in different cardiac cell types. For instance, the LASSO identified key genes specific to various heart cell types in CHD subtypes. FOXO3 was found to be up-regulated in cardiac fibroblasts for both Dilated and hypertrophic cardiomyopathy. In cardiomyocytes, distinct genes such as TMTC1, ART3, ARHGAP24, SHROOM3, and XIST were linked to dilated cardiomyopathy, Neo-Hypoplastic Left Heart Syndrome, hypertrophic cardiomyopathy, HF-Hypoplastic Left Heart Syndrome, and Tetralogy of Fallot, respectively. Endothelial cell analysis further revealed COL25A1, NFIB, and KLF7 as significant genes for dilated cardiomyopathy, hypertrophic cardiomyopathy, and Tetralogy of Fallot. LightGBM, Catboost, MCFS, RF, and XGBoost further delineated key genes for specific CHD subtypes, demonstrating the efficacy of machine learning in identifying CHD-specific genes. Additionally, this study developed quantitative rules for representing the gene expression patterns related to CHDs. This research underscores the potential of machine learning in unraveling the molecular complexities of CHD and establishes a foundation for future mechanism-based studies.

Current Protocols: Alopecia Areata Mouse Models for Drug Efficacy and Mechanism Studies.

Alopecia areata is the second most common form of hair loss in humans after androgenetic alopecia. Although a variety of animal models for alopecia areata have been described, currently the C3H/HeJ mouse model is the most commonly used and accepted. Spontaneous hair loss occurs in 15%-25% of older mice in which the lesions wax and wane, similar to the human disease, with alopecia being more common and severe in female mice. Full-thickness skin grafts from mice with spontaneous alopecia areata to young, normal-haired, histocompatible mice provide a highly reproducible model with progressive lesions that makes it useful for drug efficacy and mechanism-based studies. As alopecia areata is a cell-mediated autoimmune disease, transfer of cultured lymph node cells from affected mice to unaffected, histocompatible recipients also promotes disease development and provides an alternative, nonsurgical protocol. Protocols are presented to produce these models such that they can be used to study alopecia areata and to develop novel drug therapies. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Full-thickness skin grafts to reproducibly induce alopecia areata in C3H/HeJ mice Basic Protocol 2: Adoptive transfer of cultured lymphoid cells provides a nonsurgical method to induce alopecia areata in C3H/HeJ mice.

Machine Learning Reveals Impacts of Smoking on Gene Profiles of Different Cell Types in Lung.

Smoking significantly elevates the risk of lung diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. This risk is attributed to the harmful chemicals in tobacco smoke that damage lung tissue and impair lung function. Current research on the impact of smoking on gene expression in specific lung cells is limited. This study addresses this gap by analyzing gene expression profiles at the single-cell level from 43,539 lung endothelial cells, 234,349 lung epithelial cells, 189,843 lung immune cells, and 16,031 lung stromal cells using advanced machine learning techniques. The data, categorized by different lung cell types, were classified into three smoking states: active smoker, former smoker, and never smoker. Each cell sample encompassed 28,024 feature genes. Employing an incremental feature selection method within a computational framework, several specific genes have been identified as potential markers of smoking status in different lung cell types. These include B2M, EEF1A1, and TPT1 in lung endothelial cells; FTL and MT-ATP8 in lung epithelial cells; HLA-B and HLA-C in lung immune cells; and HSP90B1 and LCN2 in lung stroma cells. Additionally, this study developed quantitative rules for representing the gene expression patterns related to smoking. This research highlights the potential of machine learning in oncology, enhancing our molecular understanding of smoking's harm and laying the groundwork for future mechanism-based studies.

Abstract 4737: The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer

Abstract Efforts to develop targeted therapies for drug resistant tumors have been relatively unsuccessful. The 5-year survival for patients with advanced pancreatic cancer is ~3%. Gemcitabine is usually combined with nab-paclitaxel as a standard of care for treatment of locally advanced or metastatic pancreatic ductal adenocarcinoma (PDAC). However, no chemotherapeutic regimens thus far evaluated have been curative. We developed the first PDAC patient-derived xenograft (PDX) models in which resistance against gemcitabine was acquired in vivo. We used RNA-seq and ingenuity pathway analysis to compare expression profiles of paired parent vs gemcitabine resistant PDX models. This comparison suggested a previously unreported mechanism for gemcitabine resistance, involving aberrations of cholesterol biosynthesis and lipid metabolism pathways. We also made the unexpected observation that gemcitabine-resistant PDAC models derived from primary tumors have high levels of 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2), an enzyme essential to cholesterol biosynthesis and rate limiting in ketogenesis. Notably, the BET bromodomain inhibitor JQ1 decreases HMGCS2 levels; and BET inhibitors + gemcitabine exert synergistic cytotoxicity in gemcitabine-resistant PDAC cells. We determined that the BET bromodomain inhibitor JQ1 + gemcitabine induces regressions of two in vivo gemcitabine-resistant tumor models which we developed directly from primary human tumor tissue. These mechanism-based studies suggest that inhibiting HMGCS2 represents a novel approach to therapy for PDAC. We propose that combining gemcitabine with agents that decrease levels of HMGCS2 comprises effective treatment for patients with drug resistant PDAC. Citation Format: Aubrey L. Miller, Samuel C. Fehling, Eric Josh Brown, Eric O. Heard, Tracy L. Gamblin, Rebecca B. Vance, Karina J. Yoon. The combination of the BET inhibitor JQ1 and gemcitabine induces regressions in gemcitabine resistant patient-derived xenograft models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4737.

The relationship between kidney disease and mitochondria: a bibliometric study

Background Due to its highly reabsorptive function, the kidney is a mitochondria-dependent organ. Research on the association between mitochondria and kidney disease has always been a serious focus of researchers, with many publications. Bibliometrics is a secondary analysis of published literature that extracts relevant information to gain insights into hotspots and trends in the field. Through bibliometric analysis, we aimed to understand the development trends and hotspots in the field of research on the association between kidney disease and mitochondria. Method Three bibliometric mapping tools (Biblimetrix R Package, VOS Viewer, CiteSpace) were used to provide an overview of the literature and analyze the co-occurrence of keywords and reference citations. Results A total of 2672 relevant research articles were included. The co-occurrence network identified three clusters related to the association between mitochondria and kidney disease, including experimental methods, research mechanisms, and disease phenotypes. We found that research in this field has shifted from disease-level studies to mechanism-based studies, with the most prominent disease being diabetic nephropathy and the most prominent pathogenic mechanism being related to mitochondrial ROS production. Conclusion The bibliometric analysis provided a comprehensive understanding of the progress of research on the role of mitochondria in kidney disease, enriching the review literature in this field.

Extending the shelf life of HLM chips through freeze-drying of human liver microsomes immobilized onto thiol-ene micropillar arrays.

Microfluidic flow reactors functionalized with immobilized human liver microsomes (HLM chips) represent a powerful tool for drug discovery and development by enabling mechanism-based enzyme inhibition studies under flow-through conditions. Additionally, HLM chips may be exploited in streamlined production of human drug metabolites for subsequent microfluidic in vitro organ models or as metabolite standards for drug safety assessment. However, the limited shelf life of the biofunctionalized microreactors generally poses a major barrier to their commercial adaptation in terms of both storage and shipping. The shelf life of the HLM chips in the wetted state is ca. 2-3 weeks only and requires cold storage at 4 °C. In this study, we developed a freeze-drying method for lyophilization of HLMs that are readily immobilized inside microfluidic pillar arrays made from off-stoichiometric thiol-ene polymer. The success of lyophilization was evaluated by monitoring the cytochrome P450 and UDP-glucuronosyltransferase enzyme activities of rehydrated HLMs for several months post-freeze-drying. By adapting the freeze-drying protocol, the HLM chips could be stored at room temperature (protected from light and moisture) for at least 9 months (n = 2 independent batches) and up to 16 months at best, with recovered enzyme activities within 60-120% of the non-freeze-dried control chips. This is a major improvement over the cold-storage requirement and the limited shelf life of the non-freeze-dried HLM chips, which can significantly ease the design of experiments, decrease energy consumption during storage, and reduce the shipping costs with a view to commercial adaptation.

Abstract C083: HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity

Abstract The proteasome functions as an essential component of immune surveillance by generating peptides from intracellular antigens that are then presented to cytotoxic T-cells. The vast majority of defined antitumor T-cell responses involves the proteasomal processing of intracellular proteins and their presentation in the context of major histocompatibility complex (MHC) class I molecules to antigenic peptide-specific CD8+ T-cells. Such antigenic peptides generated by the proteasome are generally 8-10 amino acids in length and mirror the linear sequence of the parental protein. However, immune evasion is a hallmark of cancer cells that disrupt the antigen processing and presentation machinery. Tumors develop strategies to evade host immunity and resist current immunotherapies, while others display a low tumor mutation burden limiting the presence of tumor antigenicity. Here, we postulated that pharmacologic enhancement of proteasome catalytic activity would increase MHC class I antigen presentation, modulate the myeloma immunopeptidome, and promote CD8+ T-cells anti-myeloma activity. A cell-based, high-throughput screen revealed that the histone deacetylase 6 (HDAC6) inhibitors tubastatin-A, ACY-738 and ACY-1215 increased proteasome activity in multiple myeloma (MM) cells. HDAC6 inhibitors increased pan-MHC class-I expression in MM cells as well as a panel of cancer cell lines. Treatment of MM patient tumor cells with HDAC6 inhibitors increased the cytotoxic effect of autologous patient-derived T-cells. Mechanism-based studies demonstrated that pharmacologic blockade or genetic ablation of the HDAC6 ubiquitin-binding (BUZ) domain disrupted HDAC6 association with HR23B, a substrate-shuttle factor that delivers ubiquitinylated cargo to proteasomes. HDAC6 inhibitors or HDAC6-BUZ domain deletion reduced the association of HR23B with HDAC6 and promoted binding of HR23B to proteasomes and a consequent increase in proteasome activity. The results validate the HDAC6-BUZ domain as an attractive drug discovery target and demonstrate that proteasome activators enhance antitumor immunity. Proteasome activators represent a paradigm-shifting approach to overcome mechanisms of immune escape in cancer cells. FDA-approved drugs that increase proteasome activity and boost antigen presentation and can be repositioned as cancer immunotherapeutics to overcome the existing bottlenecks in drug development. Taken together, our results highlight the breadth and magnitude of the proteasome in governing fundamental cellular processes and the immense potential of therapeutics that exploit proteasomes to treat human disease. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C083.

Diuretic Potential of Fenchyl Acetate with Its Mechanism of Action: Toxicity Study.

Hypertension has become a global threat and is one of the greatest risk factors for chronic kidney disease. Fenchyl acetate is a monoterpene that has been assessed for its various pharmacological activities in the past, but no study has evaluated its diuretic potential and the mechanism involved in the diuretic activity after prolonged administration in rats. Therefore, this study aimed to measure the safety and diuretic profile of fenchyl acetate in rats. For evaluating the acute toxicity, a single dose of 2000 mg/kg was administered as per the OECD guideline no. 425, and the rats were observed for 14 days. After 14 days, blood samples were assessed for biochemical, hematological, and oxidative stress parameters. For the acute diuretic study, fenchyl acetate was given in doses of 100, 200, and 400 mg/kg, and urine samples after 8 h were assessed for sodium, potassium, creatinine, uric acid excretion, and urinary output. A single dose of fenchyl acetate (F.A) was selected for prolonged diuretic activity, and furosemide was taken as the standard drug in a repeated dose administration for 7 days. Rats' urine was assessed for pH, sodium, potassium, creatinine, and uric acid excretion along with urinary volume excretion. Furthermore, blood was withdrawn by cardiac puncture, and selected organs like the heart, liver, kidney, and spleen were analyzed for oxidative stress biomarkers. Using pharmacological antagonists or inhibitors, the involvement of L-NAME, acetylcholine, or prostaglandin in F.A.-induced diuresis was determined. Mitochondrial respiratory chain enzyme complexes were also assessed in the kidney homogenates. The acute toxicity results showed F.A to be safe as its LD50 was greater than 2000 mg/kg and there were no signs of mortality or toxicity. The acute diuretic study showed that F.A resulted in a significant and dose-dependent increase in sodium, potassium, creatinine, and uric acid excretion along with urinary output, and these results were comparable to the standard drug furosemide. Prolonged administration with F.A (400 mg/kg) resulted in a comparable excretion of sodium, potassium, creatinine, uric acid, and urine output with furosemide (15 mg/kg). The oxidative stress parameters revealed that F.A (400 mg/kg) resulted in reducing the formation of free radicals. The results from the mechanism-based studies showed the involvement of NO in inducing diuresis. Furthermore, F.A (400 mg/kg) significantly increased the mitochondrial complexes I, II, III, IV, I + III, and II + III in the kidney homogenates, thus restoring the mitochondrial enzymes and improving the renal function. The current study suggests that F.A is safe with a significant diuretic potential with the involvement of NO in its mechanism of action.

Obesity is associated with quality of sperm parameters in men with infertility: a cross-sectional study

BackgroundPrevious studies examined the effects of obesity on sperm parameters and reported inconsistent results. Thus, the present study aimed to evaluate the association between obesity and the quality of sperm parameters in infertile men.Material and methodsThe present cross-sectional study evaluated 218 infertile men aged 20–50. To this end, the 168-item food frequency questionnaire (FFQ) was utilized to evaluate dietary intake. The anthropometric and biochemical variables were examined using standard methods. Further, the association between obesity and the quality of sperm parameters was evaluated using the controlled linear regression for potential confounders.ResultsThe normal sperm morphology had a significant inverse association with BMI [adjusted β − 0.074, CI (− 0.141 to − 0.008), P = 0.029] and WC [adjusted β − 0.026, CI (− 0.051 to − 0.001), P = 0.038]. Additionally, visceral fat had a marginal inverse association with normal sperm morphology [adjusted β − 0.065, CI (− 0.138 to 0.008), P = 0.079] and non-progressive sperm motility [adjusted β − 0.241, CI (− 0.495 to 0.014), P = 0.063].ConclusionEven though the present results indicated that obesity, abdominal obesity, and visceral fat had inverse associations with normal sperm morphology, more mechanism-based studies should be conducted to confirm these findings.

Exploring the underlying biology of cancer and potential therapeutic strategies: a special issue focused on mechanism-based studies.

Exploring the underlying biology of cancer and potential therapeutic strategies: a special issue focused on mechanism-based studies.

Identification of new modulator of DNA repairing pathways based on natural product (±)-peharmaline A

The complex heterogenic environment of tumour mass often leads to drug resistance and facilitate chemo insensitivity triggering more malignant phenotypes among cancer patients. Major DNA-damaging cancer drugs have been consistently proven unsuccessful in terms of elevating chemo-resistance. (±)-peharmaline A, a hybrid natural product isolated from seeds of Peganum harmala L. possesses significant cytotoxic activities. Herein, we have described the design, and synthesis of a novel library of close and simplified analogues around the anticancer natural product (±)-peharmaline A and investigated their cytotoxic activities, which led to the identification of three structurally simplified lead compounds exhibiting better potency than parent natural product. Among them, demethoxy analogue of peharmaline A was further investigated for its anticancer potential eliciting demethoxy analogue as potent DNA-damage inducing agent attenuating the expression of the proteins responsible for the DNA damage repair. Therefore, this demethoxy analogue warrants detailed investigations for the confirmations of the molecular mechanism-based studies responsible for its anticancer activity.______________________________________________________________________________

Facial Fracture Patterns and Severity of Associated Injuries After Motorcycle Accidents.

Motorcycle injuries remain a significant cause of motor vehicle-related morbidity and mortality in Taiwan. Besides, the facial region is a commonly fractured site under motorcycle accidents. To date, there are no trauma mechanism-based studies of facial fractures. This study aims to determine the facial fracture patterns, the severity of head trauma, and associated injuries by different motorcycle-related trauma mechanisms enabling a greater understanding of its distribution and magnitude. This is a retrospective descriptive analysis conducted over a 2-year period at Linkou Chang Gung Memorial Hospital. We focused on the population of maxillofacial injury caused by motorcycle accidents. We divided the patient cohort into 3 main trauma mechanisms: single-motorcycle collision (SM group), motorcycle-to-motorcycle collision (MM group), motorcycle-to-vehicle collision (MV group). Data, including demographics, age, fracture patterns of facial bones, and other associated injuries, were collected. A total of 881 cases were identified that involved facial fractures. Most patients were male (71%), young adult (mean age, 32.49 years), and the most common fracture region is the midfacial fracture (79.5%, 700 victims). Among the 3 groups, the MM group was less likely to sustain severe injuries by trauma score system, less head injury and mortality rate. The MV group and SM group have similar mortality rates but different fracture pattern tendencies. Lower facial fractures were more likely in the MV group, but midface fractures in the SM group. Associated injuries were higher in the MV group. Our study presents the different trends of fracture patterns and injury under 3 main mechanisms of motorcycle casualties. We document all these data in the hope of providing insights into trauma doctors dealing with motorcycle accidents.

Farnesol Protects against Cardiotoxicity Caused by Doxorubicin-Induced Stress, Inflammation, and Cell Death: An In Vivo Study in Wistar Rats

Doxorubicin (DOXO) is an antineoplastic drug that is used extensively in managing multiple cancer types. However, DOXO-induced cardiotoxicity is a limiting factor for its widespread use and considerably affects patients' quality of life. Farnesol (FSN) is a sesquiterpene with antioxidant, anti-inflammatory, and anti-tumor properties. Thus, the current study explored the cardioprotective effect of FSN against DOXO-induced cardiotoxicity. In this study, male Wistar rats were randomly divided into five groups (n = 7) and treated for 14 days. Group I (Control): normal saline, p.o. daily for 14 days; Group II (TOXIC): DOXO 2.4 mg/kg, i.p, thrice weekly for 14 days; Group III: FSN 100 mg/kg, p.o. daily for 14 days + DOXO similar to Group II; Group IV: FSN 200 mg/kg, p.o. daily for 14 days + DOXO similar to Group II; Group V (Standard): nifedipine 10 mg/kg, p.o. daily for 14 days + DOXO similar to Group II. At the end of the study, animals were weighed, blood was collected, and heart-weight was measured. The cardiac tissue was used to estimate biochemical markers and for histopathological studies. The observed results revealed that the FSN-treated group rats showed decrease in heart weight and heart weight/body weight ratio, reversed the oxidative stress, cardiac-specific injury markers, proinflammatory and proapoptotic markers and histopathological aberrations towards normal, and showed cardioprotection. In summary, the FSN reduces cardiac injuries caused by DOXO via its antioxidant, anti-inflammatory, and anti-apoptotic potential. However, more detailed mechanism-based studies are needed to bring this drug into clinical use.

The uses of transcriptomics and lipidomics indicated that direct contact with graphene oxide altered lipid homeostasis through ER stress in 3D human brain organoids

The potential uses of graphene-based nanomaterials (NMs) in various fields lead to the concern about their neurotoxicity, considering that graphene-based NMs are capable to cross blood brain barrier (BBB) and enter central nervous system (CNS). Although previous studies reported the possibility of graphene-based NM exposure to alter lipid homeostasis in animals or cultured neurons, recent studies suggested the need to use 3D human brain organoids for mechanism-based toxicological studies as this model might better recapitulate the complex human brains. Herein, we used multi-omics techniques to investigate the mechanisms of graphene oxide (GO) on lipid homeostasis in a novel 3D brain organoid model. We found that 50 μg/mL GO induced cytotoxicity but not superoxide. RNA-sequencing data showed that 50 μg/mL GO significantly up-regulated and down-regulated 80 and 121 genes, respectively. Furthermore, we found that GO exposure altered biological molecule metabolism pathways including lipid metabolism. Consistently, lipidomics data supported dose-dependent alteration of lipid profiles by GO in 3D brain organoids. Interestingly, co-exposure to GO and endoplasmic reticulum (ER) stress inhibitor 4-phenylbutyric acid (4-PBA) decreased most of the lipid classes compared with the exposure of GO only. We further verified that exposure to GO promoted ER stress marker GRP78 proteins, which in turn activated IRE1α/XBP-1 axis, and these changes were partially or completely inhibited by 4-PBA. These results proved that direct contact with GO disrupted lipid homeostasis through the activation of ER stress. As 3D brain organoids resemble human brains, these data might be better extrapolated to humans.

Succinimide Derivatives as Antioxidant Anticholinesterases, Anti-α-Amylase, and Anti-α-Glucosidase: In Vitro and In Silico Approaches.

Based on the diverse pharmacological potency and the structural features of succinimide, this research considered to synthesize succinimide derivatives. Moreover, these compounds were estimated for their biological potential in terms of anti-diabetic, anti-cholinesterase, and anti-oxidant capacities. The compounds were synthesized through Michael addition of various ketones to N-aryl maleimides. Similarly, the MOE software was used for the molecular docking study to explore the binding mode of the potent compounds against different enzymes. In the anti-cholinesterase activity, the compounds MSJ2 and MSJ10 exhibited outstanding activity against acetylcholinesterase (AChE), i.e., 91.90, 93.20%, and against butyrylcholinesterase (BChE), i.e., 97.30, 91.36% inhibitory potentials, respectively. The compounds MSJ9 and MSJ10 exhibited prominent α-glucosidase inhibitory potentials, i.e., 87.63 and 89.37 with IC50 value of 32 and 28.04 μM, respectively. Moreover, the compounds MSJ2 and MSJ10 revealed significant scavenging activity against DPPH free radicals with IC50 values of 2.59 and 2.52, while against ABTS displayed excellent scavenging potential with IC50 values 7.32 and 3.29 μM, respectively. The tentative results are added with molecular docking studies in the active sites of enzymes to predict the theoretical protein-ligand binding modes. Further detailed mechanism-based studies in animal models are essential for the in vivo evaluation of the potent compound.

Abstract 407: OTS964, a TOPK inhibitor, is susceptible to ABCG2-mediated drug resistance

Abstract The overexpression of ATP-binding cassette (ABC) transporters has known to be one of the most important mechanisms responsible for the development of multidrug resistance (MDR). OTS964 is a potent T-LAK cell-originated protein kinase (TOPK) inhibitor. Herein, we investigated the interaction of OTS964 and MDR-associated ATP-binding cassette sub-family G member 2 (ABCG2, breast cancer resistance protein/BCRP). The cell viability assay indicated that the effectiveness of OTS964 is limited in drug-resistant and gene-transfected cells overexpressing ABCG2. We found that the known ABCG2 inhibitor is able to sensitize ABCG2-overexpressing cells to OTS964. In mechanism-based studies, OTS964 shows inhibitory effect on the efflux function mediated by ABCG2, and in turn, affects the pharmacokinetic profile of other ABCG2 substrate-drugs. Furthermore, OTS964 upregulates ABCG2 protein and mRNA expression levels, resulting in enhanced resistance to ABCG2 substrate-drugs. The ATPase assay demonstrated that OTS964 stimulates ATPase activity of ABCG2 in a concentration-dependent manner, and that this stimulation can be antagonized by a verified ABCG2 ATPase inhibitor. Additionally, the computational molecular docking analysis combined with results from ATPase assay suggested that OTS964 interacts with drug-binding pocket of ABCG2 protein and has substrate-like behaviors. Thus, OTS964 is an MDR-susceptible agent due to its interactions with ABCG2, and overexpression of ABCG2 transporter may attenuate its therapeutic effect in cancer cells. Citation Format: Yuqi Yang, Zhuo-Xun Wu, Jing-Quan Wang, Qiu-Xu Teng, Zi-Ning Lei, Sabrina Lusvarghi, Suresh V. Ambudkar, Ning Ji, Zhe-Sheng Chen. OTS964, a TOPK inhibitor, is susceptible to ABCG2-mediated drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 407.

A full-scale tracing study of "ghost peaks" encountered in impurity analysis of budesonide based on experimental operation inspection-liquid chromatography/mass spectrometry fingerprint-mechanism based stress studies integrated strategy.

Budesonide is an active pharmaceutical ingredient used in various dosage forms of finished products for the treatment of asthma. During the process of drug development, unbiased analysis of related substances is of utmost significance for both pharmaceutical research and quality control purposes. In this work, the official method documented in the United States Pharmacopoeia was selected to determine the related substances of budesonide considering the pros and cons of critical chromatographic parameters, compared to the European Pharmacopoeia. In doing so, several unpredictable interference peaks, namely "ghost peaks", were observed occasionally during analysis. A strategy that integrated information derived from experimental operation inspection, liquid chromatography/mass spectrometry fingerprint analysis, and mechanism-based stress studies was then proposed for comprehensively and quickly exploring those non-degradable and degradable peaks. Some ghost peaks were found to originate from nylon syringe filter, illumination, and alkali borosilicate glass high-performance liquid chromatography vials. Besides, degradation pathways under alkaline conditions were also unraveled through liquid chromatography-mass spectrometry qualitative analysis. Overall, an optimization of the analytical methodology based on the United States Pharmacopoeia for its application in impurity analysis of budesonide and corresponding formulations was carried out with the design of experiments, by which "ghost peaks" could be suppressed or prevented. The results obtained herein are not only crucial to studies on budesonide's stability or degradation kinetics but also contribute to clarifying the impurity research of other drugs.

Induction of p53 mediated mitochondrial apoptosis and cell cycle arrest in human breast cancer cells by plant mediated synthesis of silver nanoparticles from Bergenia ligulata (Whole plant)

The biological synthesis of nanoparticles is a growing research trend because it has numerous pharmaceutical and biomedical applications. The present study describes the preparation, characterization and anti-cancer evaluation of silver nanoparticles synthesized using an aqueous extract of Bergenia ligulata whole plant as a reducing agent. The physiochemical properties of the Bergenia ligulata silver nanoparticles (BgAgNPs) were measured by ultraviolet–visible spectrophotometry, Fourier transform infrared spectrophotmetry (FTIR), X-ray powder diffraction (XRD) and Scanning electron microscopy (SEM) analysis for identifying functional groups, crystallinity, structural and morphological features, respectively. Further, BgAgNps, along with the Bergenia ligulata aqueous extract (BgAE), were investigated for their effects on cell proliferation and apoptosis through MTT, colony-forming assay, wound-healing assay and flow cytometry-based approaches. The cytotoxic effects were more pronounced in cells treated with BgAgNps in comparison to BgAE. These effects were evidenced by the decreasing cell viability, migration capacity and loss of characteristic morphological features. In addition, BgAgNps unveiled significant induction of apoptosis in human breast cancer (MCF-7) cells, possibly through oxidative stress-mediated reactive oxygen species (ROS) generation and loss of mitochondrial membrane potential (MMP). Moreover, molecular mechanism-based studies revealed that BgAgNps robustly augmented p53 levels and pro-apoptotic downstream targets of p53 like Bax and cleaved caspase 3 in MCF-7 cells. Of note, BgAgNps had little or no cytotoxic effect on p53-deficient cancer cells (Mda-mb-231 and SW-620). These findings confirm that the BgAgNPs exhibited superior anti-cancer potential and could be exploited as a promising, cost-effective, and environmentally benign strategy in treating this disease in the future.