Intracellular Activity Research Articles

Frequencies and spectra of aflatoxin B1-induced mutations in liver genomes of NEIL1-deficient mice as revealed by duplex sequencing.

Increased risk for the development of hepatocellular carcinoma (HCC) is driven by a number of etiological factors including hepatitis viral infection and dietary exposures to foods contaminated with aflatoxin-producing molds. Intracellular metabolic activation of aflatoxin B1 (AFB1) to a reactive epoxide generates highly mutagenic AFB1-Fapy-dG adducts. Previously, we demonstrated that repair of AFB1-Fapy-dG adducts can be initiated by the DNA glycosylase NEIL1 and that male Neil1-/- mice were significantly more susceptible to AFB1-induced HCC relative to wild-type mice. To investigate the mechanisms underlying this enhanced carcinogenesis, WT and Neil1-/- mice were challenged with a single, 4 mg/kg dose of AFB1 and frequencies and spectra of mutations were analyzed in liver DNAs 2.5 months post-injection using duplex sequencing. The analyses of DNAs from AFB1-challenged mice revealed highly elevated mutation frequencies in the nuclear genomes of both males and females, but not the mitochondrial genomes. In both WT and Neil1-/- mice, mutation spectra were highly similar to the AFB1-specific COSMIC signature SBS24. Relative to wild-type, the NEIL1 deficiency increased AFB1-induced mutagenesis with concomitant elevated HCCs in male Neil1-/- mice. Our data establish a critical role of NEIL1 in limiting AFB1-induced mutagenesis and ultimately carcinogenesis.

Kigelia africana (Lam.) Benth. fruit inhibits iron-induced lipid peroxidation and ?-amylase enzyme activity

Insufficiencies in insulin secretion and/or action cause diabetes mellitus (DM), a complex condition characterized by abnormal blood glucose levels. Diabetes is often treated by modifying either glucose metabolism, lipids metabolism, or both. A direct correlation has been found between high levels of lipid peroxide in individuals with diabetes and lower intracellular antioxidant activity. Therefore, this study was designed to investigate the effect of the aqueous extract of Kigelia africana fruit on the carbohydrate hydrolyzing enzyme, ?-amylase, and FeSO4-induced lipid peroxidation in the rat pancreas. The in vitro antioxidant potential, including assays for (1,1 diphenyl-2-picryl hydrazyl (DPPH), ferric reducing antioxidant property (FRAP), nitric oxide (NO), hydroxyl (OH) scavenging, and Fe2+ chelating abilities, of the fruit extract was also evaluated. The effect of the fruit extract on the ?-amylase enzyme, and FeSO4-induced lipid peroxidation in the rat pancreas was also evaluated. K. africana at a concentration of 0.5 mg/mL and 1 mg/mL scavenged DPPH radicals, with the lower concentration showing significant differences compared to the control. The K. africana fruit extract reduced ferric compounds significantly compared to the standard. The OH* radical scavenging results revealed that all concentrations of K. africana fruit scavenged OH* radicals without significant differences compared to the control. K. africana fruit also chelated iron significantly compared to the control. The fruit extract inhibited iron-induced lipid peroxidation in the rat pancreas and ?-amylase activity compared to the control. K. africana fruit displayed promising potential in inhibiting ?-amylase activity and associated lipid peroxidation in diabetics. Therefore, it holds strong promise for use in diabetes treatment, validating its traditional use for managing the condition.

Effects of DDT and DDE on placental cholinergic receptors

The placental cholinergic system; known as an important factor in intracellular metabolic activities, regulation of placental vascular tone, placental development, and neurotransmission; can be affected by persistent organic pesticides, particularly organochlorine pesticides(OCPs), which can influence various epigenetic regulations and molecular pathways. Although OCPs are legally prohibited, trace amounts of the persistent dichlorodiphenyltrichloroethane(DDT) are still found in the environment, making prenatal exposure inevitable. In this study, the effects of 2,4’-DDT and 4,4’-DDT; and its breakdown product 4,4’-DDE in the environment on placental cholinergic system were evaluated with regards to cholinergic genes. 40 human placentas were screened, where 42,50% (17 samples) were found to be positive for the tested compounds. Average concentrations were 10.44 μg/kg; 15.07 μg/kg and 189,42 μg/kg for 4,4’-DDE; 2,4’-DDT and 4,4’-DDT respectively. RNA-Seq results revealed 2396 differentially expressed genes in positive samples; while an increase in CHRM1,CHRNA1,CHRNG and CHRNA2 genes at 1.28, 1.49, 1.59 and 0.4 fold change were found(p<0028). The increase for CHRM1 was also confirmed in tissue samples with immunohistochemistry. In vitro assays using HTR8/SVneo cells; revealed an increase in mRNA expression of CHRM1, CHRM3 and CHRN1 in DDT and DDE treated groups; which was also confirmed through western blot assays. An increase in the expression of CHRM1,CHRNA1, CHRNG(p<0001) and CHRNA2(p<0,05) were found from the OCPs exposed and non exposed groups.The present study reveals that intrauterine exposure to DDT affects the placental cholinergic system mainly through increased expression of muscarinic receptors. This increase in receptor expression is expected to enhance the sensitivity of the placental cholinergic system to acetylcholine.

Neutrophil Elastase Degrades Histone Deacetylases and Sirtuin 1 in Primary Human Monocyte Derived Macrophages.

Neutrophil elastase (NE) is taken up by macrophages, retains intracellular protease activity, and induces a pro-inflammatory phenotype. However, the mechanism of NE-induced pro-inflammatory polarization of macrophages is not well understood. We hypothesized that intracellular NE degrades histone deacetylases (HDAC) and Sirtuins, disrupting the balance of lysine acetylation and deacetylation and resulting in nuclear to cytoplasmic translocation of a major alarmin, High Mobility Group Box 1 (HMGB1), a pro-inflammatory response in macrophages. Human blood monocytes were obtained from healthy donors or from subjects with cystic fibrosis (CF) or chronic obstructive pulmonary disease (COPD). Monocytes were differentiated into blood monocyte derived macrophages (BMDMs) in vitro. Human BMDMs were exposed to NE or control vehicle, and the abundance of HDACs and Sirtuins was determined by Western blotting of total cell lysates or nuclear extracts or determined by ELISA. HDAC, Sirtuin, and Histone acetyltransferase (HAT) activities were measured. NE degraded most HDACs and Sirtuin (Sirt)1, resulting in decreased HDAC and sirtuin activities, with minimal change in HAT activity. We then evaluated whether the NE-induced loss of Sirt activity or loss of HDAC activities would alter the cellular localization of HMGB1. NE treatment or treatment with Trichostatin A (TSA), a global HDAC inhibitor, both increased HMGB1 translocation from the nucleus to the cytoplasm, consistent with HMGB1 activation. NE significantly degraded Class I and II HDAC family members and Sirt 1, which shifted BMDMs to a pro-inflammatory phenotype.

Allelochemicals of Alexandrium tamarense and its algicidal mechanism for Prorocentrum donghaiense and Heterosigma akashiwo

The effects of culture filtrate of Alexandrium tamarense on Prorocentrum donghaiense and Heterosigma akashiwo were investigated, including determination of algal density, photosynthesis, intracellular enzyme content and activity. The filtrate of A. tamarense had a stronger inhibitory effect on P. donghaiense than H. akashiwo, and the inhibitory effect decreased with higher temperature treatment of the filtrate. Instantaneous fluorescence (Ft) and maximum quantum yield of photosystem II (Fv/Fm) values of both kinds of target algae were reduced as exposed to the filtrate of A. tamarense, which proved that allelopathy could inhibit the normal operation of photosynthetic system. The increase of Malondialdehyde (MDA) content of the two kinds of target algae indicated that the cell membrane was seriously damaged by allelochemicals released by A. tamarense. The different responses of Superoxide Dismutase (SOD) and Catalase (CAT) activity in two kinds of target algae demonstrated the complexity and diversity of allelopathic mechanism. The filtrate of A. tamarense also influenced the metabolic function (ATPases) of P. donghaiense and H. akashiwo, and the influence on P. donghaiense was greater. Liquid-liquid extraction was used to extract and isolate allelochemicals from the filtrate of A. tamarense. It was found that only component I with molecular weight of 424.2573 and 434.2857 could inhibit the growth of P. donghaiense by HPLC-MS.

Evaluation of the EMulate Therapeutics Voyager’s ultra-low radiofrequency energy in murine model of glioblastoma

BackgroundGlioblastoma (GBM) presents as an aggressive brain cancer, notorious for its recurrence and resistance to conventional treatments. This study aimed to assess the efficacy of the EMulate Therapeutics Voyager®, a non-invasive, non-thermal, non-ionizing, battery-operated, portable experimental medical device, in treating GBM. Using ultra-low radiofrequency energy (ulRFE) to modulate intracellular activity, previous preliminary results in patients have been encouraging. Now, with a focus on murine models, our investigation seeks to elucidate the device's mechanistic impacts, further optimizing its therapeutic potential and understanding its limitations.MethodsThe device employs a silicone over molded coil to deliver oscillating magnetic fields, which are believed to interact with and disrupt cellular targets. These fields are derived from the magnetic fluctuations of solvated molecules. Xenograft and syngeneic murine models were chosen for the study. Mice were injected with U-87 MG or GL261 glioma cells in their flanks and were subsequently treated with one of two ulRFE cognates: A1A, inspired by paclitaxel, or A2, based on murine siRNA targeting CTLA4 + PD1. A separate group of untreated mice was maintained as controls.ResultsMice that underwent treatments with either A1A or A2 exhibited significantly reduced tumor sizes when compared to the untreated cohort.ConclusionThe EMulate Therapeutics Voyager® demonstrates promising potential in inhibiting glioma cells in vivo through its unique ulRFE technology and should be further studied in terms of biological effects in vitro and in vivo.

In Vitro/Vivo Mechanisms of Antibacterial Peptide NZ2114 against Staphylococcus pseudintermedius and Its Biofilms.

Staphylococcus pseudintermedius is an opportunistic pathogen commonly found in canines, and has garnered escalating interest due to its potential for zoonotic transmission and increasing antimicrobial resistance. However, the excessive use of antibiotics and the characteristic of S. pseudintermedius forming biofilms make treatment challenging. In this study, the in vivo and in vitro antimicrobial activity and mechanisms of action of NZ2114, a plectasin-derived peptide, against S. pseudintermedius were investigated. NZ2114 exhibited potent antibacterial activity towards S. pseudintermedius (minimum inhibitory concentration, MIC = 0.23 μM) with a lower probability of inducing drug-resistant mutations and efficient bactericidal action, which was superior to those of mopirucin (MIC = 0.25-0.5 μM) and lincomycin (MIC = 4.34-69.41 μM). The results of electron microscopy and flow cytometry showed that NZ2114 disrupted S. pseudintermedius' cell membrane, resulting in cellular content leakage, cytoplasmic membrane shrinkage, and, eventually, cell death. The intracellular ROS activity and Alamar Blue detection showed that NZ2114 interferes with intracellular metabolic processes. In addition, NZ2114 effectively inhibits biofilm formation, and confocal laser scanning microscopy further revealed its antibacterial and anti-biofilm activity (biofilm thickness reduced to 6.90-17.70 μm). The in vivo therapy of NZ2114 in a mouse pyoderma model showed that it was better than lincomycin in effectively decreasing the number of skin bacteria, alleviating histological damage, and reducing the skin damage area. These results demonstrated that NZ2114 may be a promising antibacterial candidate against S. pseudintermedius infections.

Does administration of hydroxychloroquine/amiodarone affect the efficacy of enzyme replacement therapy for Fabry mice?

As a standard therapy for Fabry disease, enzyme replacement therapy (ERT) with recombinant human α-galactosidase A (α-Gal) has been successfully used, and the instructions for this drug state that “it should not be co-administrated with cationic amphiphilic drugs such as hydroxychloroquine (HCQ) and amiodarone (AMI), since these drugs have the potential to inhibit intracellular α-Gal activity”. However, there would be cases in which HCQ or AMI is required for patients with Fabry disease, considering their medical efficacy and application.Thus, we examined the impact of HCQ/AMI on recombinant human α-Gal by in vitro, cellular, and animal experiments. The results revealed that HCQ/AMI affected the enzyme activity of α-Gal incorporated into cultured fibroblasts from a Fabry mouse when the cells were cultured in medium containing these drugs and the enzyme, although their direct inhibitory effect on the enzyme is not strong. These lysosomotropic drugs may be trapped and concentrated in lysosomes, followed by inhibition of α-Gal.On the other hand, no reduction of α-Gal activity incorporated into the organs and tissues, or acceleration of glycoshingolipid accumulation was observed in Fabry mice co-administered with HCQ/AMI and the enzyme, compared with in the case of usual ERT. As HCQ/AMI administered are catabolized in the liver, these drugs possibly do not affect ERT for Fabry mice, different from in the case of cultured cells in an environment isolated from the surroundings.

Granzymes in health and diseases: the good, the bad and the ugly.

Granzymes are a family of serine proteases, composed of five human members: GA, B, H, M and K. They were first discovered in the 1980s within cytotoxic granules released during NK cell- and T cell-mediated killing. Through their various proteolytic activities, granzymes can trigger different pathways within cells, all of which ultimately lead to the same result, cell death. Over the years, the initial consideration of granzymes as mere cytotoxic mediators has changed due to surprising findings demonstrating their expression in cells other than immune effectors as well as new intracellular and extracellular activities. Additional roles have been identified in the extracellular milieu, following granzyme escape from the immunological synapse or their release by specific cell types. Outside the cell, granzyme activities mediate extracellular matrix alteration via the degradation of matrix proteins or surface receptors. In certain contexts, these processes are essential for tissue homeostasis; in others, excessive matrix degradation and extensive cell death contribute to the onset of chronic diseases, inflammation, and autoimmunity. Here, we provide an overview of both the physiological and pathological roles of granzymes, highlighting their utility while also recognizing how their unregulated presence can trigger the development and/or worsening of diseases.

Association between Intracellular Calcium Signaling and Tumor Recurrence in Human Non-Functioning Pituitary Adenomas.

Clinically non-functioning pituitary adenomas (CNFPAs) are the second most frequent sellar tumor among studies on community-dwelling adults. They are characterized by the absence of hormonal hypersecretion syndrome, and patients present with compressive symptoms, such as a headache and visual field defects. Immunohistochemically, most CNFPAs are of gonadotrope differentiation, with only a few of them being truly null cell adenomas. Although these tumors express receptors for one or more hypothalamic releasing hormones, to what extent this has an impact on the biological and clinical behavior of these neoplasms remains to be defined. In this research, we evaluated the basal and hypothalamic secretagogue-stimulated intracellular calcium mobilization in 13 CNFPAs, trying to correlate this response to the phenotypic features of the patients. Our results indicate that the recurrence of a CNFPA correlates positively with cellular responsiveness, as measured by spontaneous intracellular calcium activity and the ability to respond to multiple hypothalamic secretagogues. We conclude that this finding may be a useful tool for predicting the clinicopathologic behavior of CNFPAs, by testing the variation of cellular responsiveness to hypothalamic secretagogues.

Functionalized microporous sulfonated polyetheretherketone: Osteogenesis and anti-osteoclastogenesis effects via ROS and NLRP3-mediated pyroptosis in macrophages

The treatment of osteoporotic implant failures tends to be challenging due to an imbalance in bone homeostasis. Recently, the accumulation of intracellular reactive oxygen species (ROS) and activation of the inflammasome containing nucleotide-binding oligomerization domain-like-receptor family pyrin domain-containing 3 (NLRP3) protein have been shown to indirectly affect osteogenesis and osteoclastogenesis. Owing to the release of multi-metal ions from laponite (LAP), we fabricated a multifunctional LAP coating on a three-dimensional microporous sulfonated polyetheretherketone (SPEEK) surface via polydopamine (PDA) modification (SPEEK@PDA-LAP). The balance between osteogenesis and osteoclastogenesis was examined using macrophages in an osteo-immune microenvironment. Employing in vitro assays, we showed that the SPEEK@PDA-LAP alleviated intracellular ROS accumulation and inhibited the activation of NLRP3 inflammasome-mediated pyroptosis in macrophages, creating an osteoimmunomodulatory microenvironment to stimulate osteogenesis and inhibit osteoclastogenesis. The micro-computed tomography and histological results of in vivo experiments indicated that SPEEK@PDA-LAP implants reversed the balance between osteogenesis and osteoclastogenesis to promote the formation of new bone around the implant even under osteoporotic conditions. The results indicate that inhibiting the NLRP3 inflammasome and regulating intracellular ROS in macrophages can be an effective therapy for osteoporosis. These results highlight the potential of functional PEEK materials for bone regeneration in patients with osteoporosis.

Bioinformatics analysis reveals the key factors affecting the progress of osteoporosis

The incidence rate of osteoporosis is high, and patients usually have decreased bone density and increased risk of fracture, which seriously affects their quality of life. This study aims to reveal key biomarkers that affect the progression of osteoporosis through bioinformatics methods. This study selected the GSE91033 and GSE93883 datasets for analysis, and obtained differentially expressed miRNAs using the GEO2R analysis tool; Predicting potential miRNA target factors through miRDIP and conducting pathway enrichment analysis of target factors through DAVID database; Analyze the interaction relationships between target factors through the STRING database, and construct a protein interaction network and a miRNA mRNA interaction network. The results showed that 73 and 79 differentially expressed genes were obtained in the GSE91033 and GSE93883 datasets, respectively. Common genes included hsa-miR-4508, hsa-miR-660-5p, and hsa-miR-424-5p. Pathway enrichment analysis showed that downstream target factors of differentially expressed miRNAs involved PIK/AKT, Wnt, Hippo, MAPK, and NF-κB pathway is closely related to cell proliferation and differentiation, and also involves intracellular phosphorylation activity. Protein interaction analysis revealed that CCND1, WEE1, MAP2K1, BTRC, FGF2, AXIN2, PTCH1, CCND2, KIF5C, DYNC1I1 and PAFAH1B1 are node genes involved in the progression of osteoporosis. The miRNA mRNA interaction network revealed that hsa-miR-424-5p is a key factor affecting the progression of osteoporosis.

Abstract 5245: Anti-CSPG4 CAR-Macrophages synergize with anti-CD47 in melanoma cytotoxicity

Abstract While CAR T-cells have been a success in treating liquid tumors, these successes have not been replicated in solid tumors. There are several reasons for these challenges, including physical matrix barriers and increased tumor antigen heterogeneity in solid tumors. Macrophages are frequently found in melanoma tumors, even in those that have low T-cell infiltrate, suggesting macrophages are well equipped to penetrate the physical barriers surrounding solid tumors. Macrophages are also antigen-presenting cells, thus, if macrophages phagocytose the tumor, they will likely present additional antigens to T-cells, potentially enhancing the adaptive immune response in cancer treatment. Thus, we hypothesized that macrophages expressing chimeric antigen receptors (CAR-Macrophages or CAR-Ms) are able to access solid tumors, phagocytose cancer cells, and subsequently re-educate the tumor immune environment by directly polarizing tumor-associated macrophages and by presenting novel antigens to recruit and educate adaptive immune cells. To test these hypotheses, we generated CAR-Ms with primary human macrophages using an scFV specific to chondroitin sulfate proteoglycan 4 (CSPG4), a highly expressed antigen enriched and specific to melanoma tumors, and an intracellular activation domain to promote phagocytosis. We found that anti-CSPG4 CAR-Ms phagocytosed human melanoma cells at high efficiency, however, this phagocytosis did not result in high levels of melanoma cell cytotoxicity. To improve CAR-M-mediated melanoma cell death engulfment, we blocked the anti-CD47/SIRPα (“don’t eat me”) signaling axis with an anti-CD47 blocking antibody, which resulted in additive effects for phagocytosis, and a robust effect on the melanoma cell killing. Preliminary in vivo xenograft experiments suggest that anti-CSPG4 CAR-Ms alone delay melanoma growth. We are now testing whether combining anti-CD47 and CSPG4 CAR-Ms therapies in vivo can 1) further reduce primary tumor growth and 2) prevent recurrence following surgical resection by treating remnants of the tumor. Our work shows that CSPG4 CAR-M-mediated killing of CSPG4-positive cancer cells is significantly enhanced when in combination with anti-CD47 antibody treatment. Citation Format: Daniel Greiner, Qian Xue, Trinity Waddell, Minna Roh-Johnson. Anti-CSPG4 CAR-Macrophages synergize with anti-CD47 in melanoma cytotoxicity [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 5245.

Abstract 4421: Intracellular cytomatrix actin and the Warburg effect

Abstract Introduction: The cytoplasm of living cells is dynamic and the motion arising from fluctuating forces in cells is an active process, rather than being thermally induced. Motor proteins could be the primary causative force of intracellular movement. However, which motor protein is involved in the intracellular dynamics, what process requires fluctuational intracellular activity, and where the energy source comes from is unknown. To address the impact of intracellular motion in cellular processes, we chemically separated the viscous fluid (cytosol) and elastic solid (cytomatrix) phases of cytoplasm. The multiomic analysis was used to dissect the compositional differences between the liquid and solid phases. LC-MS/MS-based proteome analysis was used to identify the composition of the HCT-15 colon cancer cell cytomatrix. The mRNA translation was measured by Ribosome footprinting. Results: Since the kinesis of the solid phase can trigger liquid phase flux, the composition of the cytomatrix was the primary focus of the study of cytoplasmic motion. The systemic analysis of the cytomatrix constituency allowed the identification of a diverse functional group of proteomes that included cytoskeletal and structural elements of organoids, metabolic enzymes, and normal- and proto-oncogenic signaling pathways. The two-phase system elucidated how diverse chemical reactions can occur simultaneously within the cell cytoplasm. Namely, the immobilization of catalytic complexes on the solid cytomatrix physically segregates biochemical reactants, thereby overcoming spatial impediments and allowing diverse metabolic reactions to coincide. Actomyosin and other actin-binding proteins of cytomatrix were the most abundant and diverse proteome, indicating that the primary machinery driving cytomatrix mobility consists of actomyosin, actin-binding, and actin-regulating proteins of cytomatrix. The cytomatrix dynamics trigger the cytosolic motion that carries substrates to immobilized catalytic complexes of the cytomatrix, thereby increasing product output, which appears to be a more intense process in cancer cells due to genetic and epigenetic aberrations. Under normal physiological conditions, mitochondrial respiration can deliver the energy requirements of cytomatrix micromechanics, but in cancer cells, additional energy of ATP is supplied by the aerobic glycolysis known as the Warburg effect to provide the ATP surplus for actomyosin mechanics. Conclusion: The intracellular motion is supported through non-muscle actomyosin action of the solid phase (cytomatrix), permitting increased glycolytic pathway activity in tumor cells. This novel concept of cancer links the Warburg effect with the bioenergetics of cytomatrix micromechanics and the dynamics of metabolic processes in tumors. Thus, two of the mysteries in cancer cell biology, how chemical reactions occur in the cytoplasm and energy is generated by tumor cells, have now been linked. Citation Format: Tattym E. Shaiken. Intracellular cytomatrix actin and the Warburg effect [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 4421.

Protective Mechanism of Rosa roxburghii Tratt Fermentation Broth against Ultraviolet-A-Induced Photoaging of Human Embryonic Skin Fibroblasts.

This study takes the fruit of Rosa roxburghii Tratt (RRT) as a fermentation substrate and carries out a quantitative visual analysis of the domestic and foreign literature on screenings of five different lactic acid bacteria to obtain a fermentation broth. Systemic anti-photoaging effects are analyzed at the biochemical, cellular, and molecular biological levels. DPPH and ABTS free radical scavenging activities are used to verify the antioxidant capacity of the RRT fruit fermentation broth in vitro. Human embryonic skin fibroblasts (HESs) are used to establish a UVA damage model, and the antioxidant capacity of the RRT fruit fermentation broth is verified in terms of intracellular reactive oxygen species (ROS) and antioxidant enzyme activity. RT-qPCR and ELISA are used to detect the expression of TGF-β/Smad, MMPs, and the MAPK/AP-1 and Nrf2/Keap-1 signaling pathways in order to explore the anti-oxidation and anti-photoaging effects of the RRT fruit fermentation broth by regulating different signaling pathways. The results show that an RRT fruit fermentation broth can effectively protect cells from oxidative stress caused by UVA and has significant anti-photoaging effects, with the co-cultured Lactobacillus Yogurt Starter LYS-20297 having the highest overall effect.

Development of a self-microemulsifying drug delivery system to deliver delamanid via a pressurized metered dose inhaler for treatment of multi-drug resistant pulmonary tuberculosis

Tuberculosis (TB) is a serious health issue that contributes to millions of deaths throughout the world and increases the threat of serious pulmonary infections in patients with respiratory illness. Delamanid is a novel drug approved in 2014 to deal with multi-drug resistant TB (MDR-TB). Despite its high efficiency in TB treatment, delamanid poses delivery challenges due to poor water solubility leading to inadequate absorption upon oral administration. This study involves the development of novel formulation-based pressurized metered dose inhalers (pMDIs) containing self-microemulsifying mixtures of delamanid for efficient delivery to the lungs. To identify the appropriate self-microemulsifying formulations, ternary diagrams were plotted using different combinations of surfactant to co-surfactant ratios (1:1, 2:1, and 3:1). The combinations used Cremophor RH40, Poly Ethylene Glycol 400 (PEG 400), and peppermint oil, and those that showed the maximum microemulsion region and rapid and stable emulsification were selected for further characterization. The diluted self-microemulsifying mixtures underwent evaluation of dose uniformity, droplet size, zeta potential, and transmission electron microscopy. The selected formulations exhibited uniform delivery of the dose throughout the canister life, along with droplet sizes and zeta potentials that ranged from 24.74 to 88.99 nm and − 19.27 to − 10.00 mV, respectively. The aerosol performance of each self-microemulsifying drug delivery system (SMEDDS)-pMDI was assessed using the Next Generation Impactor, which indicated their capability to deliver the drug to the deeper areas of the lungs. In vitro cytotoxicity testing on A549 and NCI-H358 cells revealed no significant signs of toxicity up to a concentration of 1.56 µg/mL. The antimycobacterial activity of the formulations was evaluated against Mycobacterium bovis using flow cytometry analysis, which showed complete inhibition by day 5 with a minimum bactericidal concentration of 0.313 µg/mL. Moreover, the cellular uptake studies showed efficient delivery of the formulations inside macrophage cells, which indicated the potential for intracellular antimycobacterial activity. These findings demonstrated the potential of the Delamanid-SMEDDS-pMDI for efficient pulmonary delivery of delamanid to improve its effectiveness in the treatment of multi-drug resistant pulmonary TB.

Metformin ameliorates PM2.5-induced functional impairment of placental trophoblasts by inhibiting ferroptosis

To investigate the protective effect of metformin against PM2.5-induced functional impairment of placental trophoblasts and explore the underlying mechanism. Sixteen pregnant Kunming mice were randomly assigned into two groups (n=8) for intratracheal instillation of PBS or PM2.5 suspension at 1.5, 7.5, and 12.5 days of gestation. The pregnancy outcome of the mice was observed, and placental zonal structure and vascular density of the labyrinth area were examined with HE staining, followed by detection of ferroptosis-related indexes in the placenta. In cultured human trophoblasts (HTR8/SVneo cells), the effects of PM2.5 exposure and treatment with metformin on cell viability, proliferation, migration, invasion, and tube formation ability were evaluated using CCK8 assay, EDU staining, wound healing assay, Transwell experiment, and tube formation experiment; the cellular expressions of ferroptosis-related proteins were analyzed using ELISA and Western blotting. M2.5 exposure of the mice during pregnancy resulted in significantly decreased weight and number of the fetuses and increased fetal mortality with a reduced placental weight (all P<0.001). PM2.5 exposure also caused obvious impairment of the placental structure and trophoblast ferroptosis. In cultured HTR8/SVneo cells, PM2.5 significantly inhibited proliferation, migration, invasion, and angiogenesis of the cells by causing ferroptosis. Metformin treatment obviously attenuated PM2.5-induced inhibition of proliferation, migration, invasion, and angiogenesis of the cells, and effectively reversed PM2.5-induced ferroptosis in the trophoblasts as shown by significantly increased intracellular GSH level and SOD activity, reduced MDA and Fe2+ levels, and upregulated GPX4 and SLC7A11 protein expression (P<0.05 or 0.01). PM2.5 exposure during pregnancy causes adverse pregnancy outcomes and ferroptosis and functional impairment of placental trophoblasts in mice, and metformin can effectively alleviate PM2.5-induced trophoblast impairment.

High-Fidelity Sensitive Tracing Circulating Tumor Cell Telomerase Activity.

Dynamic tracing of intracellular telomerase activity plays a crucial role in cancer cell recognition and correspondingly in earlier cancer diagnosis and personalized precision therapy. However, due to the complexity of the required reaction system and insufficient loading of reaction components into cells, achieving a high-fidelity determination of telomerase activity is still a challenge. Herein, an Aptamer-Liposome mediated Telomerase activated poly-Molecular beacon Arborescent Nanoassembly(ALTMAN) approach was described for direct high-fidelity visualization of telomerase activity. Briefly, intracellular telomerase activates molecular beacons, causing their hairpin structures to unfold and produce fluorescent signals. Furthermore, multiple molecular beacons can self-assemble, forming arborescent nanostructures and leading to exponential amplification of fluorescent signals. Integrating the enzyme-free isothermal signal amplification successfully increased the sensitivity and reduced interference by leveraging the skillful design of the molecular beacon and the extension of the telomerase-activated TTAGGG repeat sequence. The proposed approach enabled ultrasensitive visualization of activated telomerase exclusively with a prominent detection limit of 2 cells·μL-1 and realized real-time imaging of telomerase activity in living cancer cells including blood samples from breast cancer patients and urine samples from bladder cancer patients. This approach opens an avenue for establishing a telomerase activity determination and in situ monitoring technique that can facilitate both telomerase fundamental biological studies and cancer diagnostics.

A Review of Approaches to Potentiate the Activity of Temozolomide against Glioblastoma to Overcome Resistance.

A glioblastoma (GBM) is one of the most aggressive, infiltrative, and treatment-resistant malignancies of the central nervous system (CNS). The current standard of care for GBMs include maximally safe tumor resection, followed by concurrent adjuvant radiation treatment and chemotherapy with the DNA alkylating agent temozolomide (TMZ), which was approved by the FDA in 2005 based on a marginal increase (~2 months) in overall survival (OS) levels. This treatment approach, while initially successful in containing and treating GBM, almost invariably fails to prevent tumor recurrence. In addition to the limited therapeutic benefit, TMZ also causes debilitating adverse events (AEs) that significantly impact the quality of life of GBM patients. Some of the most common AEs include hematologic (e.g., thrombocytopenia, neutropenia, anemia) and non-hematologic (e.g., nausea, vomiting, constipation, dizziness) toxicities. Recurrent GBMs are often resistant to TMZ and other DNA-damaging agents. Thus, there is an urgent need to devise strategies to potentiate TMZ activity, to overcome drug resistance, and to reduce dose-dependent AEs. Here, we analyze major mechanisms of the TMZ resistance-mediated intracellular signaling activation of DNA repair pathways and the overexpression of drug transporters. We review some of the approaches investigated to counteract these mechanisms of resistance to TMZ, including the use of chemosensitizers and drug delivery strategies to enhance tumoral drug exposure.

Suppression of A-to-I RNA-editing enzyme ADAR1 sensitizes hepatocellular carcinoma cells to oxidative stress through regulating Keap1/Nrf2 pathway

BackgroundA-to-I RNA editing is an abundant post-transcriptional modification event in hepatocellular carcinoma (HCC). Evidence suggests that adenosine deaminases acting on RNA 1 (ADAR1) correlates to oxidative stress that is a crucial factor of HCC pathogenesis. The present study investigated the effect of ADAR1 on survival and oxidative stress of HCC, and underlying mechanisms.MethodsADAR1 expression was measured in fifty HCC and normal tissues via real-time quantitative PCR, and immunohistochemistry. For stable knockdown or overexpression of ADAR1, adeno-associated virus vectors carrying sh-ADAR1 or ADAR1 overexpression were transfected into HepG2 and SMMC-7721 cells. Transfected cells were exposed to oxidative stress agonist tBHP or sorafenib Bay 43-9006. Cell proliferation, apoptosis, and oxidative stress were measured, and tumor xenograft experiment was implemented.ResultsADAR1 was up-regulated in HCC and correlated to unfavorable clinical outcomes. ADAR1 deficiency attenuated proliferation of HCC cells and tumor growth and enhanced apoptosis. Moreover, its loss facilitated intracellular ROS accumulation, and elevated Keap1 and lowered Nrf2 expression. Intracellular GSH content and SOD activity were decreased and MDA content was increased in the absence of ADAR1. The opposite results were observed when ADAR1 was overexpressed. The effects of tBHP and Bay 43–9006 on survival, apoptosis, intracellular ROS accumulation, and Keap1/Nrf2 pathway were further exacerbated by simultaneous inhibition of ADAR1.ConclusionsThe current study unveils that ADAR1 is required for survival and oxidative stress of HCC cells, and targeting ADAR1 may sensitize HCC cells to oxidative stress via modulating Keap1/Nrf2 pathway.