Cell Assay Research Articles

Blockage of αvβ3 integrin in 3D culture of triple-negative breast cancer and endothelial cells inhibits migration and discourages endothelial-to-mesenchymal plasticity

Breast cancer is a relevant cause of mortality in women and its triple-negative subtype (TNBC) is usually associated with poor prognosis. During tumor progression to metastasis, angiogenesis is triggered by the sprouting of endothelial cells from pre-existing vessels by a dynamic chain of events including VE-cadherin downregulation, actin protrusion, and integrin-mediated adhesion, allowing for migration and proliferation. The binding of tumoral and tumor-associated stromal cells with the extracellular matrix through integrins mediates angiogenic processes and certain integrin subtypes, such as the αvβ3 integrin, are upregulated in hypoxic TNBC models. Integrin αvβ3 inhibition by the high-affinity binding disintegrin DisBa-01 was previously demonstrated to induce anti-tumoral and anti-angiogenic responses in traditional 2D cell assays. Here, we investigate the effects of integrin αvβ3 blockage in endothelial and TNBC cells by DisBa-01 in 3D cultures under two oxygen conditions (1% and 20%). 3D cultures created using non-adhesive micromolds with Matrigel were submitted to migration assay in Boyden chambers and fluorescence analysis. DisBa-01 inhibited cell migration in normoxia and hypoxia in both MDA-MB-231 and HUVEC spheroids. Protein levels of integrin αvβ3 were overexpressed in HUVEC spheroids compared to MDA-MB-231 spheroids. In HUVEC 3D cultures, sprouting assays in collagen type I were decreased in normoxia upon DisBa-01 treatment, and VE-cadherin levels were diminished in HUVEC spheroids in hypoxia and upon DisBa-01 treatment. In conclusion, the blockage of integrin αvβ3 by DisBa-01 inhibits cell migration in 3D culture and interferes with tumor-derived responses in different oxygen settings, implicating its crucial role in angiogenesis and tumor progression.

Immune cells at the feto-maternal interface: Comprehensive characterization and insights into term labor

Immune cells at the feto-maternal interface play an important role in pregnancy; starting at implantation, maintenance of pregnancy, and parturition. The role of decidual immune cells in induction of labor still needs to be understood. Published reports on this topic show heterogeneity in methods of cell isolation, assay, analysis and cellular characterization making it difficult to collate available information in order to understand the contribution of immune cells at term leading to parturition. In the present study, available literature was reviewed to study the differences in immune cells between the decidua basalis and decidua parietalis, as well as between immune cells in term and preterm labor. Additionally, immune cells at the decidua parietalis were isolated from term not in labor (TNL) or term in labor (TL) samples and characterized via flow cytometry using a comprehensive, high-dimensional antibody panel. This allowed a full view of immune cell differences without combining multiple studies, which must include variation in isolation and analysis methods, for more conclusive data. The ratio of cells found in decidua parietalis in this study generally matched those reported in the literature, although we report a lower percentage of natural killer (NK) cells at term. We report that CD4 expression on CD8- NK cells decreased in term labor compared to not in labor samples, suggesting that natural killer cells may be migrating to other sites during labor. Also, we report a decrease in CD38 expression on CD8+ CD57+ T cells in labor, indicative of cytotoxic T cell senescence. Our study provides a comprehensive status of immune cells at the decidua-chorion interface at term.

Toxicity removal from contaminated water by constructed wetlands assessed using multiple biomarkers in human stem cell assays

Constructed wetlands (CWs) have been developed rapidly as a sustainable water treatment technique. However, the capability of CWs for remediating the contaminated water based on toxicity assessment remains largely unknown. Four surface flow CWs and two integrated surface-subsurface flow CWs, from five cities in central and eastern region of China were evaluated, concerning the adverse effects of effluents and the toxicity reduction efficiency. Human bone marrow mesenchymal stem cells (hBMSCs) were employed as a human relevant in vitro model. The influent extractions caused cytotoxicity in a dose-dependent manner. The non-cytotoxic dilutions of the influents enhanced the genotoxicity marker γ-H2AX and reactive oxygen species levels. In addition, the influent repressed the osteogenic and neurogenic differentiation, and stimulated the adipogenic differentiation. Cytotoxicity of the contaminated water was reduced by 54 %–86 % after treatment with CWs. CWs were effective to remove part of the sub-lethal effects, with lower reduction than cytotoxicity. The integrated biomarker response (IBR) value of the effluents from the six CWs is lower than that of four secondary and one tertiary wastewater treatment plants. The IBR of the six CWs influents were in the range of 8.6–10.6, with a reduction of 15–50 % after the pollution restoration in CWs. The two integrated surface-subsurface flow CWs achieved higher IBR removal than the four surface flow CWs, possibly due to improved treatment effects by the combined systems. Cytotoxic and genotoxic effects of polar fractions in the CW effluents were stronger than the medium-polar and the non-polar fractions. Besides, PPARγ agonists present in the effluents played crucial roles and ERα agonists may make modest contributions. The present study enhances understanding of the role of CWs in achieving safe wastewater reclamation and provides evidence for further improving toxicity reduction in CWs performance.

β-D-N4-hydroxycytidine (NHC, EIDD-1931) inhibits chikungunya virus replication in mosquito cells and ex vivo Aedes aegypti guts, but not when ingested during blood-feeding

Chikungunya virus (CHIKV) is a mosquito-borne virus transmitted by Aedes mosquitoes. While there are no antiviral therapies currently available to treat CHIKV infections, several licensed oral drugs have shown significant anti-CHIKV activity in cells and in mouse models. However, the efficacy in mosquitoes has not yet been assessed. Such cross-species antiviral activity could be favorable, since virus inhibition in the mosquito vector might prevent further transmission to vertebrate hosts. Here, we explored the antiviral effect of β-d-N4-hydroxycytidine (NHC, EIDD-1931), the active metabolite of molnupiravir, on CHIKV replication in Aedes aegypti mosquitoes.Antiviral assays in mosquito cells and in ex vivo cultured mosquito guts showed that NHC had significant antiviral activity against CHIKV. Exposure to a clinically relevant concentration of NHC did not affect Ae. aegypti lifespan when delivered via a bloodmeal, but it slightly reduced the number of eggs developed in the ovaries. When mosquitoes were exposed to a blood meal containing both CHIKV and NHC, the compound did not significantly reduce virus infection and dissemination in the mosquitoes. This was confirmed by modelling and could be explained by pharmacokinetic analysis, which revealed that by 6 h post-blood-feeding, 90% of NHC had been cleared from the mosquito bodies.Our data show that NHC inhibited CHIKV replication in mosquito cells and gut tissue, but not in vivo when mosquitoes were provided with a CHIKV-infectious bloodmeal spiked with NHC. The pipeline presented in this study offers a suitable approach to identify anti-arboviral drugs that may impede replication in mosquitoes.

Design, synthesis, and antiviral activity of 1-aryl-4-arylmethylpiperazine derivatives as Zika virus inhibitors with broad antiviral spectrum

Zika virus (ZIKV) disease has been given attention due to the risk of congenital microcephaly and neurodevelopmental disorders after ZIKV infection in pregnancy, but no vaccine or antiviral drug is available. Based on a previously reported ZIKV inhibitor ZK22, a series of novel 1-aryl-4-arylmethylpiperazine derivatives was designed, synthesized, and investigated for antiviral activity by quantify cellular ZIKV RNA amount using RT-qPCR method in ZIKV-infected human venous endothelial cells (HUVECs) assay. Structure-activity relationship (SAR) analysis demonstrated that anti-ZIKV activity of 1-aryl-4-arylmethylpiperazine derivatives is not correlated with molecular hydrophobicity, multiple new derivatives with pyridine group to replace the benzonitrile moiety of ZK22 showed stronger antiviral activity, higher ligand lipophilicity efficiency as well as lower cytotoxicity. Two active compounds 13 and 33 were further identified as novel ZIKV entry inhibitors with the potential of oral available. Moreover, both ZK22 and newly active derivatives also possess of obvious inhibition on the viral replication of coronavirus and influenza A virus at low micromolar level. In summary, this work provided better candidates of ZIKV inhibitor for preclinical study and revealed the promise of 1-aryl-4-arylmethylpiperazine chemotype in the development of broad-spectrum antiviral agents.

Quantification of Thermal Acclimation in Immune Functions in Ectothermic Animals.

This short review focuses on current experimental designs to quantify immune acclimation in animals. Especially in the face of rapidly changing thermal regimes, thermal acclimation of immune function has the potential to impact host-pathogen relationships and the fitness of hosts. While much of the field of ecoimmunology has focused on vertebrates and insects, broad interest in how animals can acclimate to temperatures spans taxa. The literature shows a recent increase in thermal acclimation studies in the past six years. I categorized studies as focusing on (1) natural thermal variation in the environment (e.g., seasonal), (2) in vivo manipulation of animals in captive conditions, and (3) in vitro assays using biological samples taken from wild or captive animals. I detail the strengths and weaknesses of these approaches, with an emphasis on mechanisms of acclimation at different levels of organization (organismal and cellular). These two mechanisms are not mutually exclusive, and a greater combination of the three techniques listed above will increase our knowledge of the diversity of mechanisms used by animals to acclimate to changing thermal regimes. Finally, I suggest that functional assays of immune system cells (such as quantification of phagocytosis) are an accessible and non-taxa-specific way to tease apart the effects of animals upregulating quantities of immune effectors (cells) and changes in the function of immune effectors (cellular performance) due to structural changes in cells such as those of membranes and enzymes.

Mechanisms of microbial photoinactivation by curcumin’s micellar delivery

Introduction: Microbial photoinactivation using UV light can be enhanced by the addition of food-grade photosensitizers (PSs), such as curcumin. Micellization of curcumin can improve its stability and antimicrobial activity. The objective of this study was to investigate the potential mechanisms that contribute to the photoinactivation of Escherichia coli O157: H7 and Listeria innocua by curcumin-loaded surfactant solutions produced with Surfynol 465 (S465) or Tween 80 (T80) below, near, and above their critical micelle concentration (CMC).Methods: Stock curcumin-surfactant solutions were produced with S465 or T80 (5 mM sodium citrate buffer, pH 3.5). Mixtures of each bacterial suspension (initial inoculum = 6 LogCFU/mL), 1 µM curcumin, and surfactants were irradiated with UV-A light (λ = 365 nm) for 5 min. Microbial recovery after treatments was assessed by monitoring the growth of the treated E. coli O157: H7 or L. innocua using an oCelloscope™. The growth curves were characterized using a modified logistic model.Results and Discussion: Both gram-positive and gram-negative bacteria showed less and slower recovery when treated with curcumin-S465 (near or at CMC) than curcumin-T80 solutions after irradiation. FLIM micrographs suggested that curcumin was preferentially localized at the cell membrane when S465 was present, as evidenced by its longer lifetimes in samples treated with curcumin-S465 solutions. Washing after treatment resulted in the removal of loosely bound or unbound S465-curcumin micelles; hence, both E. coli O157: H7 and L. innocua recovery was faster. This suggested that curcumin partitioning has a significant role in microbial photoinactivation, possibly due to the production of reactive oxygen species (ROS) closer to/within the membrane. The permeability of the membrane of E. coli O157: H7, as inferred from the Live/Dead cell assay, increased when S465 was present, suggesting that S465 can also facilitate inactivation by disrupting the membrane and by favoring the localization of curcumin adjacent to the cell membrane. Therefore, a synergistic antimicrobial effect is observed when curcumin is present alongside S465 at concentrations below or near its CMC due to the disruption of the cell membrane by S465.

Next generation risk assessment: an ab initio case study to assess the systemic safety of the cosmetic ingredient, benzyl salicylate, after dermal exposure.

We performed an ab initio next-generation risk assessment (NGRA) for a fragrance ingredient, benzyl salicylate (BSal), to demonstrate how cosmetic ingredients can be evaluated for systemic toxicity endpoints based on non-animal approaches. New approach methodologies (NAMs) used to predict the internal exposure included skin absorption assays, hepatocyte metabolism, and physiologically based pharmacokinetic (PBPK) modeling, and potential toxicodynamic effects were assessed using pharmacology profiling, ToxProfiler cell stress assay, transcriptomics in HepG2 and MCF-7 cells, ReproTracker developmental and reproductive toxicology (DART) assays, and cytotoxicity assays in human kidney cells. The outcome of the NGRA was compared to that of the traditional risk assessment approach based on animal data. The identification of the toxicologically critical entity was a critical step that directed the workflow and the selection of chemicals for PBPK modeling and testing in bioassays. The traditional risk assessment and NGRA identified salicylic acid (SA) as the "toxdriver." A deterministic PBPK model for a single-day application of 1.54g face cream containing 0.5% BSal estimated the Cmax for BSal (1nM) to be much lower than that of its major in vitro metabolite, SA (93.2nM). Therefore, SA was tested using toxicodynamics bioassays. The lowest points of departure (PoDs) were obtained from the toxicogenomics assays. The interpretation of these results by two companies and methods were similar (SA only results in significant gene deregulation in HepG2 cells), but PoD differed (213μM and 10.6µM). A probabilistic PBPK model for repeated applications of the face cream estimated the highest Cmax of SA to be 630nM. The resulting margins of internal exposure (MoIE) using the PoDs were 338 and 16, which were more conservative than those derived from external exposure and in vivo PoDs (margin of safety values were 9,705). In conclusion, both traditional and ab initio NGRA approaches concluded that the daily application of BSal in a cosmetic leave-on face cream at 0.5% is safe for humans. The processing and interpretation of toxicogenomics data can lead to different PoDs, which can subsequently affect the calculation of the MoIE. This case study supports the use of NAMs in a tiered NGRA ab initio approach.

Chemopreventive and immunomodulatory effects of phenolic-rich extract of Commiphora leptophloeos against inflammatory bowel disease: Preclinical evidence

Ethnopharmacology relevanceCommiphora leptophloeos (Mart.) J.B. Gillet (Burseraceae) is a medicinal plant native to Brazil, popularly known as “imburana”. Homemade leaf decoction and maceration were used to treat general inflammatory problems in the Brazilian Northeast population. Our previous research confirmed the anti-inflammatory activity of the C. leptophloeos hydroalcoholic leaf extract. Aim of the studyInflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gut with no ideal treatment to maintain the remissive status. This work aimed to characterize the phytochemical composition and physicochemical properties of the C. leptophloeos hydroalcoholic leaf extract and its efficacy in chemopreventive and immunomodulatory responses in inflammatory bowel disease in non-clinical models. Materials and methodsMass spectrometry and physicochemical tests determined the phytochemical profile and physicochemical characteristics of the Commiphora leptophloeos (CL) extract. The chemopreventive and immunomodulatory effects of CL extract (50 and 125 μg/mL) were evaluated in vitro in the RAW 264.7 lipopolysaccharide (LPS) induced cell assay and in vivo in the model of intestinal inflammation induced by 2,4-Dinitrobenzenesulfonic acid (DNBS) in mice when they were treated with CL extract by intragastric gavage (i.g.) at doses of 300, 400 and 500 mg/kg. ResultsPhytochemical annotation of CL extract showed a complex phenolic composition, characterized as phenolic acids and flavonoids, and satisfactory physicochemical characteristics. In addition, CL extract maintained the viability of RAW macrophages, reduced ROS and NO production, and negatively regulated COX-2, iNOS, TNF-α, IL-1β, IL-6, and IL-17 (p < 0.05). In the intestinal inflammation model, CL extract was able to downregulate NF-κB p65/COX-2, mTOR, iNOS, IL-17, decrease levels of malondialdehyde and myeloperoxidase and cytokines TNF-α, IL-1β and IL-6 (p < 0.05). ConclusionBased on these findings, CL extract reduced inflammatory responses by down-regulating pro-inflammatory markers in macrophages induced by LPS and DNBS-induced colitis in mice through NF-κB p65/COX-2 signaling. CL leaf extract requires further investigation as a candidate for treating inflammatory bowel disease.

Effect of Gracilaria changii extract and its fraction in reverse cholesterol transport by targeting SR-BI gene

This present study aimed to investigate the effects of Gracilaria changii (Red seaweed) extract and its fraction on scavenger receptor class B type I (SR-BI) gene expressions. In vitro studies of the activity of G. changii extract and its fraction against the therapeutic target for hypocholesterolemia were conducted using high-density lipoprotein (HDL) receptor SR-BI via luciferase assay in HepG2 cells. In the current study, methanol crude extract (MCE) and Fraction-2 of G. changii showed the highest expression levels of the SR-BI gene via luciferase assay and increased SR-BI mRNA expression compared to the negative control. Metabolite profiling of the MCE of G. changii by GC-MS revealed nine major compounds, comprising various fatty acids, particularly hexadecenoic acid. Therefore, the MCE and Fraction-2 of G. changii have the potential to be explored in the treatment of hypercholesterolaemia to prevent or reduce the severity of atherosclerosis development.

Abstract B009: Dual PARP and EHMT1/2 inhibition induces an interferon response and anti-tumor immunity in ovarian cancer

Abstract Ovarian cancer has a high propensity to develop therapy resistance. Epigenetic therapies can sensitize ovarian cancer cells to first-line treatments such as platinum-based chemotherapy and Poly (ADP-ribose) polymerase inhibitors (PARPi). Particularly, the reduction of histone 3 lysine 9 dimethylation (H3K9me2) via a euchromatin histone methyltransferase inhibitor (EHMTi) has been shown to sensitize PARPi-resistant cells to PARPi therapy. Additionally, cell growth defects were observed in PARPi-resistant cells treated with dual PARPi/EHMTi treatment compared to DMSO or single agent treatments. To determine the mechanism by which dual therapy sensitizes cells and reduces cell viability, bulk RNA-sequencing was performed on several PARPi-resistant cell lines treated with DMSO, PARPi, EHMTi, or dual PARPi/EHMTi. Transcriptomic analyses revealed that dual therapy increased interferon (IFN) signaling-related genes compared to DMSO or single agent treatments. Additionally, expression of transposable element (TE) transcripts, which can form double-stranded RNA (dsRNA) and induce an IFN response, was also increased in dual treated cells compared to controls. To determine if the reduction in cell viability is dependent on TEs and IFN signaling (i.e., viral mimicry), CRISPRi technology was used to silence intracellular sensors of dsRNA, RIGI and MDA5. Cell growth assays of these silenced cells showed that the reduction in cell growth observed with dual treatment was partially dependent on MDA5 expression. Based on the observed increase in IFN signaling and T cell recruiting cytokines, we performed an animal study using an immunogenic, PARPi-resistant model of ovarian cancer to determine if this dual therapy has any anti-tumor effects. Indeed, we observed that single and dual EHMTi therapy significantly reduced tumor burden and that this reduction was partially dependent on CD8 T cells. Lastly, to determine if dual therapy also induces a viral mimicry response in the human tumor microenvironment, we created ex vivo cultures of tumors resected from patients with ovarian cancer. We then treated the ex vivo tissues with DMSO, PARPi, EHMTi, and dual PARPi/EHMTi, and performed bulk RNA-sequencing and multispectral immunohistochemistry. From these analyses, we found that dual therapy upregulated TE transcripts and multiple immune-related pathways, and increased T cell activity in the tumor microenvironment. Altogether, our data suggest that dual therapy activates viral mimicry and promotes anti-tumor immunity in both PARPi-resistant and -sensitive ovarian cancer. Citation Format: Lily Nguyen, Zachary L. Watson, Elizabeth R. Woodruff, Kimberly R. Jordan, Edward Chuong, Benjamin G. Bitler. Dual PARP and EHMT1/2 inhibition induces an interferon response and anti-tumor immunity in ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B009.

Abstract B007: NAD+ metabolism drives PARPi resistance in ovarian cancer cells

Abstract NAD+ is a coenzyme for redox reactions and a cofactor for enzymes such as PARPs and Sirtuins. The nuclear enzyme PARP1 is a major consumer of NAD+, which is cleaved during the catalytic activation of PARP1 in response to DNA damage. PARP1 inhibitors compete with NAD+ for the catalytic site of PARP1 and reduce the catalytic activity of PARP1 and impair DNA damage repair. Recently PARP inhibitors (PARPi) have shown efficacy in the treatment of ovarian cancers and most patients with defects in homologous repair of DNA damage are now on PARPi maintenance. But, heterogeneous responses to PARPi is limiting their clinical efficacy. To identify the mechanisms of PARPi resistance, we generated a panel of ovarian cancer cells with acquired resistance to niraparib (PARPiR), an FDA approved PARP inhibitor. We demonstrated that the PARPiR cells are also resistant to other FDA approved PARP inhibitors rucaparib and olaparib. This data suggested that a common mechanism of resistance underlies poor response to the FDA approved PARP inhibitors. Since PARP1 activity is tightly controlled by the cellular NAD+ levels, we next investigated the role of NAD+ biosynthesis in PARPi resistance. Metabolomic profiling of the parental and PARPiR cells demonstrated that PARPiR cells have elevated levels of NAD+ metabolism. Indeed, treating the PARPiR cells with inhibitors of NAD+ synthesis restored the sensitivity to PARP inhibitors. Moreover, culturing the previously sensitive parental ovarian cancer cells in NAD+ precursors decreased their sensitivity to PARP inhibitors. These data collectively demonstrated that high levels of NAD+ metabolism causes PARP inhibitor resistance in ovarian cancer cells. Complementary to the metabolomic analysis, results from phenotypic assays in PARPiR cells revealed that high levels of NAD+ maintain cellular quiescence in PARPiR cells. In summary, our results demonstrated that high levels of NAD+ biosynthesis causes PARPi resistance by maintaining cellular quiescence. There is an urgent need to identify the mechanisms of resistance to PARP inhibitors to increase their clinical efficacy. Our study identified that maintenance of quiescence is a fundamental mechanism of PARPi resistance. We also demonstrated that inhibiting NAD+ biosynthesis reverses PARP inhibitor resistance. Citation Format: Subin Myong, Hardik Shah, Benedikt Nagele, Reshma Rajesh, Sridevi Challa. NAD+ metabolism drives PARPi resistance in ovarian cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B007.

Association of tumour mutation burden with prognosis and its clinical significance in stage III gastric cancer

Background: To explore the correlation between the tumour mutation burden (TMB) and prognosis and its clinical significance among patients with stage III gastric cancer (GC). Methods: Patients with stage III GC were divided into a high TMB and low TMB group in both a study cohort of 38 patients and the Cancer Genome Atlas (TCGA) cohort of 173 patients. In the study cohort, next-generation sequencing was used to detect mutated GC genes and obtain TMB data. In the TCGA cohort, gene set enrichment analysis was performed, and the relationship between TMB, prognosis and clinicopathologic factors was analysed. Western blot and quantitative real-time polymerase chain reaction were used to detect the expression levels of both proteins and genes. Cell viability was measured using methyl thiazolyl tetrazolium and transwell cell assays. Results: Patients in the high TMB group had better overall survival (OS) rates than patients in the low TMB group for both cohorts and TMB was associated with age, mutation signature 1 and mutation signature 17. The Cox regression analysis revealed that age, not TMB, was an independent prognosis factor. Furthermore, genes with high-frequency mutations were significantly enriched in the Notch and RTK-RAS signalling pathways. The activation of these pathways was lower in the high TMB compared with the low TMB group, and the proliferation and migration abilities of GC cells showed a similar pattern in both TMB groups. Conclusion: Patients in the high TMB group had better OS rates than patients in the low TMB group. Genes with high-frequency mutations were significantly enriched in the RTK-RAS and Notch pathways. Hence, TMB could serve as a prognosis biomarker with potential clinical significance.

A miniaturized culture platform for control of the metabolic environment.

The heart is a metabolic "omnivore" and adjusts its energy source depending on the circulating metabolites. Human cardiac organoids, a three-dimensional in vitro model of the heart wall, are a useful tool to study cardiac physiology and pathology. However, cardiac tissue naturally experiences shear stress and nutrient fluctuations via blood flow in vivo, whilst in vitro models are conventionally cultivated in a static medium. This necessitates the regular refreshing of culture media, which creates acute cellular disturbances and large metabolic fluxes. To culture human cardiac organoids in a more physiological manner, we have developed a perfused bioreactor for cultures in a 96-well plate format. The designed bioreactor is easy to fabricate using a common culture plate and a 3D printer. Its open system allows for the use of traditional molecular biology techniques, prevents flow blockage issues, and provides easy access for sampling and cell assays. We hypothesized that a perfused culture would create more stable environment improving cardiac function and maturation. We found that lactate is rapidly produced by human cardiac organoids, resulting in large fluctuations in this metabolite under static culture. Despite this, neither medium perfusion in bioreactor culture nor lactate supplementation improved cardiac function or maturation. In fact, RNA sequencing revealed little change across the transcriptome. This demonstrates that cardiac organoids are robust in response to fluctuating environmental conditions under normal physiological conditions. Together, we provide a framework for establishing an easily accessible perfusion system that can be adapted to a range of miniaturized cell culture systems.

Discovery of first-in-class DOT1L inhibitors against the R231Q gain-of-function mutation in the catalytic domain with therapeutic potential of lung cancer

Recent research certified that DOT1L and its mutations represented by R231Q were potential targets for the treatment of lung cancer. Herein, a series of adenosine-containing derivatives were identified with DOT1LR231Q inhibition through antiproliferation assay and Western blot analysis in the H460R231Q cell. The most promising compound 37 significantly reduced DOT1LR231Q mediated H3K79 methylation and effectively inhibited the proliferation, self-renewal, migration, and invasion of lung cancer cell lines at low micromolar concentrations. The cell permeability and cellular target engagement of 37 were verified by both CETSA and DARTS assays. In the H460R231Q OE cell-derived xenograft (CDX) model, 37 displayed pronounced tumor growth inhibition after intraperitoneal administration at 20 mg/kg dose for 3 weeks (TGI = 54.38%), without obvious toxicities. A pharmacokinetic study revealed that 37 possessed tolerable properties (t1/2 = 1.93 ± 0.91 h, F = 97.2%) after intraperitoneal administration in rats. Mechanism study confirmed that 37 suppressed malignant phenotypes of lung cancer carrying R231Q gain-of-function mutation via the MAPK/ERK signaling pathway. Moreover, analysis of the binding modes between molecules and DOT1LWT/R231Q proteins put forward the “Induced-fit” allosteric model in favor to the discovery of potent DOT1L candidates.

The antiviral potential of the antiandrogen enzalutamide and the viral-androgen signaling interplay in seasonal coronaviruses.

The sex disparity in COVID-19 outcomes with males generally faring worse than females has been associated with the androgen-regulated expression of the protease TMPRSS2 and the cell receptor ACE2 in the lung and fueled interest in antiandrogens as potential antivirals. In this study, we explored enzalutamide, an antiandrogen used commonly to treat prostate cancer, as a potential antiviral against the human coronaviruses which cause seasonal respiratory infections (HCoV-NL63, -229E, and -OC43). Using lentivirus-pseudotyped and authentic HCoV, we report that enzalutamide reduced 229E and NL63 entry and infection in both TMPRSS2- and nonexpressing immortalized cells, suggesting a TMPRSS2-independent mechanism. However, no effect was observed against OC43. To decipher this distinction, we performed RNA-sequencing analysis on 229E- and OC43-infected primary human airway cells. Our results show a significant induction of androgen-responsive genes by 229E compared to OC43 at 24and 72 h postinfection. The virus-mediated effect on AR-signaling was further confirmed with a consensus androgen response element-driven luciferase assay in androgen-depleted MRC-5 cells. Specifically, 229E induced luciferase-reporter activity in the presence and absence of the synthetic androgen mibolerone, while OC43 inhibited induction. These findings highlight a complex interplay between viral infections and androgen-signaling, offering insights for disparities in viral outcomes and antiviral interventions.

Protective Effect of Salvia cadmica on Fibroblast Cells from t-bhp-induced Oxidative Damage

Background:: Salvia species known as "Sage" are among the important aromatic plants used in the world. This study, it was investigated the antioxidant capacity of Salvia cadmica and to investigate its protective effect on oxidative damage in t-BHP-induced fibroblast cells. Methods:: Antioxidant activity and phenolic characterization of the extract were evaluated using DPPH, TPC, TFC, FRAP, and HPLC, respectively.TAS, TOS, MDA and 8-oxo-guanine, CAT, SOD, and GPx values were examined using enzyme-linked immunosorbent assay (ELISA). The antiproliferative and apoptosis effects of Salvia cadmica ethanolic extract were determined using XTT assay and fluorescent probes in fibroblast cells. Results:: As a result of GC-MS analysis of Salvia cadmica ethanolic extract, carvacrol content was found to be high. The IC50 value of the DPPH antioxidant assay of the Salvia cadmica ethanolic extract was 80 ± 0.51 μg/mL. TPC, TFC, and FRAP values were found to be 18.25 ± 0.64 (mg gallic acid/g powder), 1.691±0.314 (mg quercetin /g powder), 31.5 ± 0.10 (mg Trolox/g powder), respectively. Total antioxidant and TOS values were found to be 0.383±0.033 (mmol Trolox Equ L-1), 16.31±0.71 (μmol H2O2 L-1) for 0.25 mg/mL, and 0.725±0.05 (mmol Trolox Equ L-1), 12.02 ±0.56 (μmol H2O2 L-1) for 0.5 mg/mL. In addition, while CAT and GPx significantly decreased enzyme activities, no significant change was observed in SOD enzyme activity. Ethanolic Salvia cadmica extract exhibited apoptotic features compared to only the t-BHP group. Conclusion:: These results suggest that Salvica cadmica extract works through a free radical mechanism.

Identification and functional analysis of rare HECTD1 missense variants in human neural tube defects

Neural tube defects (NTDs) are severe malformations of the central nervous system that arise from failure of neural tube closure. HECTD1 is an E3 ubiquitin ligase required for cranial neural tube closure in mouse models. NTDs in the Hectd1 mutant mouse model are due to the failure of cranial mesenchyme morphogenesis during neural fold elevation. Our earlier research has linked increased extracellular heat shock protein 90 (eHSP90) secretion to aberrant cranial mesenchyme morphogenesis in the Hectd1 model. Furthermore, overexpression of HECTD1 suppresses stress-induced eHSP90 secretion in cell lines. In this study, we report the identification of five rare HECTD1 missense sequence variants in NTD cases. The variants were found through targeted next-generation sequencing in a Chinese cohort of 352 NTD cases and 224 ethnically matched controls. We present data showing that HECTD1 is a highly conserved gene, extremely intolerant to loss-of-function mutations and missense changes. To evaluate the functional consequences of NTD-associated missense variants, functional assays in HEK293T cells were performed to examine protein expression and the ability of HECTD1 sequence variants to suppress eHSP90 secretion. One NTD-associated variant (A1084T) had significantly reduced expression in HEK293T cells. All five NTD-associated variants (p.M392V, p.T801I, p.I906V, p.A1084T, and p.P1835L) reduced regulation of eHSP90 secretion by HECTD1, while a putative benign variant (p.P2474L) did not. These findings are the first association of HECTD1 sequence variation with NTDs in humans.

TRMT13 inhibits the growth of papillary thyroid cancer by targeting ANAPC4.

The recently discovered gene TRMT13 encodes a type of RNA methylase and is a member of the CCDC family (also called CCDC76). Here, we delineate its role in papillary thyroid cancer (PTC). Bioinformatics analysis shows significant TRMT13 and ANAPC4 downregulation in PTC and reveals that the expression levels of both genes are linearly correlated. Subsequent analyses confirm that both TRMT13 and ANAPC4 expressions are downregulated in PTC tissues and that this change in expression has a significant impact on cancer diagnosis. We conduct assays on PTC cells subjected to TRMT13 and ANAPC4 silencing or overexpression to assess the biological effects of these genes. We also perform rescue experiments to validate the regulatory effects of TRMT13 on ANAPC4. A nude mouse tumor model is used to evaluate the effects of TRMT13 and ANAPC4 on PTC tumorigenesis. TRMT13 expression is decreased in PTC tissues and cell lines and is positively correlated with that of ANAPC4. Cell assays reveal that TRMT13/ANAPC4 attenuates the malignancy of PTC cells by restraining cell proliferation, migration and invasion, while rescue experiments corroborate that ANAPC4 is a downstream target of TRMT13. In the nude mouse xenograft model, both TRMT13 and ANAPC4 inhibit tumor growth, and TRMT13 and ANAPC4 expression levels are significantly associated with survival. Taken together, these findings lead to the conclusion that TRMT13 inhibits PTC growth via ANAPC4, indicating a new role of TRMT13 and providing insights into the tRNA methyltransferase and coiled-coil domain-containing protein families.

M6A methyltransferase METTL14‑mediated RP1‑228H13.5 promotes the occurrence of liver cancer by targeting hsa‑miR‑205/ZIK1.

The aim of the present study was to explore the association between N6‑methyladenosine (m6A) modification regulatory gene‑related long noncoding (lnc)RNA RP1‑228H13.5 and cancer prognosis through bioinformatics analysis, as well as the impact of RP1‑228H13.5 on cell biology‑related behaviors and specific molecular mechanisms. Bioinformatics analysis was used to construct a risk model consisting of nine genes. This model can reflect the survival time and differentiation degree of cancer. Subsequently, a competing endogenous RNA network consisting of 3 m6A‑related lncRNAs, six microRNAs (miRs) and 201 mRNAs was constructed. A cell assay confirmed that RP1‑228H13.5 is significantly upregulated in liver cancer cells, which can promote liver cancer cell proliferation, migration and invasion, and inhibit liver cancer cell apoptosis. The specific molecular mechanism may be the regulation of the expression of zinc finger protein interacting with K protein 1 (ZIK1) by targeting the downstream hsa‑miR‑205. Further experiments found that the m6A methyltransferase 14, N6‑adenosine‑methyltransferase subunit mediates the regulation of miR‑205‑5p expression by RP1‑228H13.5. m6A methylation regulatory factor‑related lncRNA has an important role in cancer. The targeting of hsa‑miR‑205 by RP1‑228H13.5 to regulate ZIK1 may serve as a potential mechanism in the occurrence and development of liver cancer.