Cultured Cell Lines Research Articles

Low Molecular Weight Peptides Derived from Iranian Scorpion (Odontobuthus bidentatus) Venom Induces Apoptosis in the Hepatocellular Carcinoma Cell Line (HepG2) in 3D Cell Culture

Low Molecular Weight Peptides Derived from Iranian Scorpion (Odontobuthus bidentatus) Venom Induces Apoptosis in the Hepatocellular Carcinoma Cell Line (HepG2) in 3D Cell Culture

Environment-friendly Synthesis of AgNPs from Fimbristylis miliacea Flower Extract: Characterization and its Applications as an Antioxidant Activity, Anticancerous Activity and in the Reduction of 4-Nitrophenol

Background: Silver metal is useful in a variety of fields. Different silver salts exhibit good application in the organic transformation. The use of a green methodology in AgNPs preparation has gained interest of young researchers. Plant systems are employed in the green approach of preparing AgNPs, serving as both a stabilizing and reducing agent. Silver nanoparticles (AgNPs) made from silver salts exhibit a variety of biological activities and may be useful in organic transformation. Method: AgNPs were synthesized with the use of flower extract from Fimbristylis miliacea, which acts as a stabilizing and reducing agent. Result: Fimbristylis miliacea flower extract is used as a reducing and stabilising agent for preparation of AgNPs. These synthesised AgNPs were characterised by using FTIR, XRD, FESEM/ EDS, HR-TEM, TGA, and ICP-AES. Fimbristylis milliciea flower extract was subjected to FTIR and HR-LCMS analysis in order to identify the phytoconstituents that are responsible for the reduction of AgNO3. The HR-TEM shows particle size between 8.98434+0.69669nm and 19.07464+1.4384nm. The size of AgNPs determined by using HR-TEM shows good agreement with XRD particle size. The synthesized AgNPs show excellent antioxidant properties with IC50=56.45 μg/mL. The present study confers significant cytotoxic activity against MDA-MB-231 human breast cancer cell line culture (IC50= 61.97 μg/ml). The AgNPs show a faster reduction of 4-nitrophenol. Conclusion: In conclusion, we have prepared AgNPs by using Fimbristylis miliacea flower extract. The study further concludes that green synthesis of AgNPs has many advantages as compared to chemical methods, such as cost-effectiveness, rapid, and environment-friendly methods. The results indicated that the biosynthesized AgNPs have significant cytotoxic activity against the MDA-MB-231 breast cancer cell line. Further, it indicates that biosynthesized AgNPs can be a potential alternative agent for human breast cancer therapy. The cytotoxic potential can be used for treatments and provides a new method to develop molecules for cancer therapy. The catalytic activity of AgNPs was examined by using the reduction of 4-nitrophenol. It shows that the reduction of 4-nitrophenol in 6 minutes is confirmed by using 1H NMR analysis.

Stearoyl CoA desaturase inhibition can effectively induce apoptosis in bladder cancer stem cells

Bladder cancer stands as one of the most prevalent cancers worldwide. While our previous research confirmed the significant role of stearoyl-CoA desaturase (SCD) in bladder cancer, the underlying reasons for its abnormal overexpression remain largely unknown. Moreover, the distinct response to SCD inhibitors between cancer stem cells (CSCs) and adherent cultured cell lines lacks clear elucidation. Therefore, in this experiment, we aim to conduct an analysis and screening of the SCD transcription start site, further seeking critical transcription factors involved. Simultaneously, through experimental validation, we aim to explore the pivotal role of endoplasmic reticulum stress/unfolded protein response in drug sensitivity among cancer stem cells. Additionally, our RNA-seq and lipid metabolism analysis revealed the significant impact of nervonic acid on altering the proliferative capacity of bladder cancer cell lines.

STRAIN SELECTION AND CULTIVATION OF FOOT-AND-MOUTH DISEASE VIRUSES FOR THE DEVELOPMENT OF A TRIVALENT INACTIVATED VACCINE

Foot-and-mouth disease is a well-known transboundary animal disease and has been recognised as a priority disease by the Steering Committee of the Global Framework for the Progressive Control of Transboundary Animal Diseases in Europe. The territory of the Republic of Kazakhstan is generally free of foot-and-mouth disease, however, sporadic cases of foot-and-mouth disease occur in various regions of the country as a result of importation from unfavorable countries. Epizootic safety in the Republic of Kazakhstan is maintained by vaccinating animals in the border areas of the southern and eastern regions. Inactivated vaccines against serotypes A, O, Asia produced by the All-Russian Research Institute of Animal Health (Russia) are used for vaccination. Continuous seromonitoring is carried out to maintain the level of post-vaccination antibodies at a high level, ensuring the immunity of animals to foot-and-mouth disease. However, despite all the measures taken, sporadic outbreaks of foot-and-mouth disease cause damage to livestock farming in the Republic of Kazakhstan. The effectiveness of preventive measures using vaccination depends on the strain used in the vaccine. The closer the strain used in the vaccine is genetically to the viruses circulating in that area, the more effective the preventive measures will be and the research conducted to create the most immunogenic domestic anti-foot-and-mouth disease vaccines have scientific and practical significance. This article presents the results of the selection of foot-and-mouth disease virus strains previously circulating in the territory of the Republic of Kazakhstan and studies their cultural and biological properties. Studies of the above-mentioned strains on adaptation to a transplantable cell culture line were conducted, the stability of cultural properties was studied over 10 generations and the stability was confirmed by the PCR method. Also, based on the studies, it was found that the maximum accumulation of the virus in cell culture occurs at a multiplicity of infection of 0.01-0.1 TCID50/cell. The reproductive activity of the studied isolates was within 6.5-7.25 lg TCID50/cm3 when cultivated for 16 hours.

Physiological liver microtissue 384-well microplate system for preclinical hepatotoxicity assessment of therapeutic small molecule drugs.

Hepatotoxicity can lead to the discontinuation of approved or investigational drugs. The evaluation of the potential hepatoxicity of drugs in development is challenging because current models assessing this adverse effect are not always predictive of the outcome in human beings. Cell lines are routinely used for early hepatotoxicity screening, but to improve the detection of potential hepatotoxicity, in vitro models that better reflect liver morphology and function are needed. One such promising model is human liver microtissues. These are spheroids made of primary human parenchymal and nonparenchymal liver cells, which are amenable to high throughput screening. To test the predictivity of this model, the cytotoxicity of 152 FDA (US Food & Drug Administration)-approved small molecule drugs was measured as per changes in ATP content in human liver microtissues incubated in 384-well microplates. The results were analyzed with respect to drug label information, drug-induced liver injury (DILI) concern class, and drug class. The threshold IC50ATP-to-Cmax ratio of 176 was used to discriminate between safe and hepatotoxic drugs. "vMost-DILI-concern" drugs were detected with a sensitivity of 72% and a specificity of 89%, and "vMost-DILI-concern" drugs affecting the nervous system were detected with a sensitivity of 92% and a specificity of 91%. The robustness and relevance of this evaluation were assessed using a 5-fold cross-validation. The good predictivity, together with the in vivo-like morphology of the liver microtissues and scalability to a 384-well microplate, makes this method a promising and practical in vitro alternative to 2D cell line cultures for the early hepatotoxicity screening of drug candidates.

Multiplexed, image-based pooled screens in primary cells and tissues with PerturbView.

Optical pooled screening (OPS) is a scalable method for linking image-based phenotypes with cellular perturbations. However, it has thus far been restricted to relatively low-plex phenotypic readouts in cancer cell lines in culture due to limitations associated with in situ sequencing of perturbation barcodes. Here, we develop PerturbView, an OPS technology that leverages in vitro transcription to amplify barcodes before in situ sequencing, enabling screens with highly multiplexed phenotypic readouts across diverse systems, including primary cells and tissues. We demonstrate PerturbView in induced pluripotent stem cell-derived neurons, primary immune cells and tumor tissue sections from animal models. In a screen of immune signaling pathways in primary bone marrow-derived macrophages, PerturbView uncovered both known and novel regulators of NF-κB signaling. Furthermore, we combine PerturbView with spatial transcriptomics in tissue sections from a mouse xenograft model, paving the way to in situ screens with rich optical and transcriptomic phenotypes. PerturbView broadens the scope of OPS to a wide range of models and applications.

High-Throughput Assessment of Metabolism-Mediated Neurotoxicity by Co-Culture of Neurospheres and Liver Spheroids.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC50 values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity Basic Protocol 3: Image acquisition, processing, and data analysis.

Combined additive effects of neuronal membrane glycoprotein GPM6a and the intercellular cell adhesion molecule ICAM5 on neuronal morphogenesis.

The mechanisms underlying neuronal development and synaptic formation in the brain depend on intricate cellular and molecular processes. The neuronal membrane glycoprotein GPM6a promotes neurite elongation, filopodia/spine formation, and synapse development, yet its molecular mechanisms remain unknown. Since the extracellular domains of GPM6a (ECs) command its function, we investigated the interaction between ICAM5, the neuronal member of the intercellular adhesion molecule (ICAM) family, and GPM6a's ECs. Our study aimed to explore the functional relationship between GPM6a and ICAM5 in hippocampal culture neurons and cell lines. Immunostaining of 15 days invitro (DIV) neurons revealed significant co-localization between endogenous GPM6a clusters and ICAM5 clusters in the dendritic shaft. These results were further corroborated by overexpressing GPM6a and ICAM5 in N2a cells and hippocampal neurons at 5 DIV. Moreover, results from the co-immunoprecipitations and cell aggregation assays prove the cis and trans interaction between both proteins in GPM6a/ICAM5 overexpressing HEK293 cells. Additionally, GPM6a and ICAM5 overexpression additively enhanced neurite length, the number of neurites in N2a cells, and filopodia formation in 5 DIV neurons, indicating their cooperative role. These findings highlight the dynamic association between GPM6a and ICAM5 during neuronal development, offering insights into their contributions to neurite outgrowth, filopodia formation, and cell-cell interactions.

Distinct chemical structures inhibit the CEMIP hyaluronidase and promote oligodendrocyte progenitor cell maturation

Growing evidence supports pathogenic roles for chronically elevated hyaluronidase activity in numerous conditions. Elevated expression of one such hyaluronidase, the Cell Migration Inducing and hyaluronan binding Protein (CEMIP), has been implicated in the pathogenesis and progression of several cancers as well as demyelinating diseases in the central nervous system (CNS). Developing effective and selective CEMIP inhibitors could therefore have efficacy in treating a variety of conditions where CEMIP is chronically elevated. Using two distinct screens for novel hyaluronidase inhibitors, we identified two synthetic thiocarbamates and one plant-derived flavonoid, sulfuretin, that effectively blocked CEMIP activity in live cells, including a tumorigenic cell line and primary cultures of oligodendrocyte progenitor cells (OPCs). None of these agents influenced cell proliferation, but they had differential dose-dependent and cell type specific effects on cell survival. Furthermore, we found that each of these agents could promote oligodendrocyte maturation by OPCs in the presence of high molecular weight (>2 Mda) hyaluronan, the accumulation of which is linked to the inhibition of OPC maturation and remyelination failure in demyelinating diseases. These findings indicate that CEMIP can be inhibited through distinct chemical interactions, and that CEMIP inhibitors have potential efficacy for treating demyelinating diseases or other conditions where CEMIP is elevated.

Biological active compounds from edible mushroom: Effect on hela cells

Background: Mushrooms are unique organisms rich in proteins, carbohydrates, vitamins, and minerals, which allows them to be used not only as a source of nutrition but also in cosmetics and medicine. The fungus P. ostreatus possesses two strong enzymatic complexes – lignin-decaying and cellulose-decaying. Objective: The research aimed to study the antitumor activity of treated mm-wave mycelial extracts of mushrooms on the HeLa cell line. Research on edible mushrooms against tumors is very actual and important for the field of functional food science and bioactive compounds field. Methods: The effect of the fungal culture extract on the cyto-photometric and morphometric indices of the HeLa cell line culture was studied according to the nature of proliferation and transcription of malignant tissues. Using high-resolution liquid chromatography, the amino acid composition of acid-soluble proteins in mycelial extracts of a mushroom culture treated with mm waves was studied. Results: Extracts of P. ostreatus mycelial culture significantly suppressed the growth of proliferative activity of human tumor cultures and completely suppressed the mitotic activity of continuous HeLa cell cultures to 72 hours. Our study revealed a significant increase in glutamic and aspartic acids in mycelial culture extracts treated by mm-waves. Conclusion: Based on HPLC analysis, we suggest polyfunctional peroxidase as the candidate for the role of biologically active compound in the composition of aqueous extracts of the mycelial culture. So, the polyfunctional peroxidase plays an active role in the therapeutic properties of the oyster mushroom. Keywords: anticancer activity, ant-inflammation activity, mm-waves, mushroom`s, mycelial extracts, HPLC of acid-soluble proteins, HeLa cell line.

Cell-type-specific mapping of enhancers and target genes from single-cell multimodal data.

Mapping enhancers and target genes in disease-related cell types has provided critical insights into the functional mechanisms of genetic variants identified by genome-wide association studies (GWAS). However, most existing analyses rely on bulk data or cultured cell lines, which may fail to identify cell-type-specific enhancers and target genes. Recently, single-cell multimodal data measuring both gene expression and chromatin accessibility within the same cells have enabled the inference of enhancer-gene pairs in a cell-type-specific and context-specific manner. However, this task is challenged by the data's high sparsity, sequencing depth variation, and the computational burden of analyzing a large number of enhancer-gene pairs. To address these challenges, we propose scMultiMap, a statistical method that infers enhancer-gene association from sparse multimodal counts using a joint latent-variable model. It adjusts for technical confounding, permits fast moment-based estimation and provides analytically derived -values. In systematic analyses of blood and brain data, scMultiMap shows appropriate type I error control, high statistical power with greater reproducibility across independent datasets and stronger consistency with orthogonal data modalities. Meanwhile, its computational cost is less than 1% of existing methods. When applied to single-cell multimodal data from postmortem brain samples from Alzheimer's disease (AD) patients and controls, scMultiMap gave the highest heritability enrichment in microglia and revealed new insights into the regulatory mechanisms of AD GWAS variants in microglia.

The P(III)-Amidite Based Synthesis of Stable Isotope Labeled mRNA-Cap-Structures Enables their Sensitive Quantitation from Brain Tissue.

The 5' cap structure is crucial to mRNA function, with its diverse methylation patterns depending on the cellular state. Sensitive analytical methods are sought after to quantify this cap variety also referred to as cap epitranscriptome. To address a bottleneck for accurate and precise quantitation, we report a facile and fast access to high-quality synthetic standards via a new route, involving P(III)-amidite chemistry. A range of cap nucleotides and their stable heavy isotopic labeled analogues were derived from nucleoside diphosphates, which themselves were directly prepared in a one-step reaction sequence starting from unprotected nucleosides using a triphosphorylating reagent in combination with ethylenediamine. Considering a wider scope, the route also enables direct access to magic spot nucleotides and diphosphates of isoprenyl-alcohols. Stable-isotope labeled cap nucleotides derived from this route paved the way for the development of a highly sensitive LC-MS/MS method, applied to the characterization of mouse brain cap epitranscriptomes, which turned out to be very different from those of cultured cell lines of widespread use in the life sciences.

Ethosuximide-loaded bismuth ferrite nanoparticles as a potential drug delivery system for the treatment of epilepsy disease.

Encapsulating antiepileptic drugs (AEDs), including ethosuximide (Etho), into nanoparticles shows promise in treating epilepsy. Nanomedicine may be the most significant contributor to addressing this issue. It presents several advantages compared to traditional drug delivery methods and is currently a prominent area of focus in cancer research. Incorporating Etho into bismuth ferrite (BFO) nanoparticles within diverse controlled drug delivery systems is explored to enhance drug efficacy. This approach is primarily desired to aid in targeted drug delivery to the brain's deepest regions while limiting transplacental permeability, reducing fetal exposure, and mitigating associated adverse effects. In this investigation, we explored Etho, an antiepileptic drug commonly employed for treating absence seizures, as the active ingredient in BFO nanoparticles at varying concentrations (10 and 15 mg). Characterization of the drug-containing BFO nanoparticles involved scanning electron microscopy (SEM) and elemental analysis. The thermal properties of the drug-containing BFO nanoparticles were evaluated via differential scanning calorimetry (DSC) analysis. Cytotoxicity evaluations using the MTT assay were conducted on all nanoparticles, and human neuroblastoma cell line cultures (SH-SY5Y) were treated with each particle over multiple time intervals. Cell viability remained at 135% after 7 days when exposed to 15 mg of Etho in BFO nanoparticles. Additionally, in vitro drug release kinetics for Etho revealed sustained release lasting up to 5 hours with a drug concentration of 15 mg.

Insight into the emerging insect to human pathogen Photorhabdus revealing geographic differences in immune cell tropism.

Photorhabdus asymbiotica is a species of the insect pathogenic Photorhabdus genus that has been isolated as an etiological agent in human infections. Since then, multiple isolates have been identified worldwide; however, actual clinical infections have so far only been identified in North America, Australia, and Nepal. Previous research on the clinical isolates had shown that the strains differed in their behaviour when infecting cultured human cells. In this study, we investigate the differences between the pathogenic activities of P. asymbiotica isolates from different geographic locations. Pathogenicity was analysed using infection assays with both cultured cell lines (THP-1, CHO, and HEK cells) and primary immune cells, and peripheral blood mononuclear cells (PBMCs) isolated from human blood. Here, we present the findings from the Australian (Kingscliff) and North American (ATCC43949) clinical isolates, and non-clinical soilborne nematode isolates from Thailand (PB68) and Northern Europe (HIT and JUN) of P. asymbiotica. We also show the first findings from a new clinical isolate of P. luminescens (Texas), the first non-asymbiotica species to cause a human infection, confirming its ability to infect and survive inside human immune cells. Here for the first time, we show how P. asymbiotica selectively infects certain immune cells while avoiding others and that infectivity varies depending on growth temperature. We also show that the tropism varies depending on the geographic location a strain is isolated from, with only the European HIT and JUN strains lack the ability to survive within mammalian cells in tissue culture.

Y-box binding protein 1/cyclin A1 axis specifically promotes cell cycle progression at G2/M phase in ovarian cancer

Y-box binding protein 1 (YBX1) promotes oncogenic transformation and tumor growth. YBX1 plays a role in regulation of cell cycle promotion via upregulation of cell cycle-related genes. In ovarian cancer, YBX1 also promotes tumor growth, but the mechanisms of YBX1 in cell growth and cell cycle in ovarian cancer remain not to be fully understood. Here, we investigated whether YBX1-dependent cancer cell proliferation was specifically associated with expression of cell cycle related genes in ovarian cancer. Protein and mRNA expression levels of YBX1 and cell cycle-related genes in ovarian cancer cell lines and tissues were determined by western blot analysis, immunohistochemical analysis and reverse transcription-quantitative PCR. Cell cycle analysis was performed by flow cytometry. Luciferase assay and Chromatin immunoprecipitation assay were used to investigate a transcriptional function of YBX1. YBX1 silencing induced marked growth suppression in 4 cell lines (group A), moderate suppression in 5 cell lines (group B), and no suppression in 3 cell lines (group C) among 12 ovarian cancer cell lines in culture. The YBX1 silencing induced cell cycle arrest at G2/M phase and suppressed expression of cyclin A1 gene in group A and B cell lines, but not in group C cell lines. Cyclin A1 silencing specifically suppressed cell proliferation in group A cell lines and partially in group B cell lines, but not at all in group C cell lines. YBX1 mRNA levels were significantly correlated with cyclin A1 mRNA levels in patients with high-grade serous carcinoma. Augmented YBX1 expression plays a key role in tumor growth promotion in ovarian cancer in its close association with cyclin A1.

3D Modeling: Insights into the Metabolic Reprogramming of Cholangiocarcinoma Cells.

Developing accurate in vitro models that replicate the in vivo tumor environment is essential for advancing cancer research and therapeutic development. Traditional 2D cell cultures often fail to capture the complex structural and functional heterogeneity of tumors, limiting the translational relevance of findings. In contrast, 3D culture systems, such as spheroids, provide a more physiologically relevant context by replicating key aspects of the tumor microenvironment. This study aimed to compare the metabolism of three intrahepatic cholangiocarcinoma cell lines in 2D and 3D cultures to identify metabolic shifts associated with spheroid formation. Cells were cultured in 2D on adhesion plates and in 3D using ultra-low attachment plates. Metabolic exchange rates were measured using NMR, and intracellular metabolites were analyzed using LC-MS. Significant metabolic differences were observed between 2D and 3D cultures, with notable changes in central carbon and glutathione metabolism in 3D spheroids. The results suggest that 3D cultures, which more closely mimic the in vivo environment, may offer a more accurate platform for cancer research and drug testing.

Selenium and Triple Negative Breast Cancer

Background. The deadliest, most dangerous subtype of breast cancer is triple-negative, which lacks treatment targets and accounts for 30% of all breast cancer-related deaths worldwide. TNBC is characterized by the expression of no estrogen, proges- terone, or human epidermal growth factor 2 receptors. This suggests that new treatment modalities with fewer adverse effects are required. Objective. The aim of the present study was to investigate the therapeutic potential of selenium compounds as an adjuvant therapy for Triple Negative Breast Cancer (TNBC), either on their own or in conjunction with nutritional supplements and chemotherapy medications. Methods. Using the keywords “selenium” and “triple negative breast cancer”, a thorough search was conducted in the PubMed database, yielding 23 articles. The following factors were taken into consideration for inclusion: studies using TNBC cell culture lines or in vivo tumors/specimens; full-text articles from the PubMed database; studies published in the English language; experiments with statistically significant results; and selenium used alone or in combination with other antioxidants or chemotherapy. This led to the evaluation of 13 articles in this review. Results. The results show that selenium therapy increased the anti-cancer drug’s effects and produced tumor cytotoxicity, while reducing the cellular features of the cancer (hyperproliferation, growth, and metastasis). Discussion. This study evaluated the various selenium compounds tested, the cell lines and model organisms used, the assays performed, and the cellular pathways affected. Conclusion. Examining the possible benefits of selenium in TNBC treatment highlights the need for more studies to confirm selenium compounds as viable co-therapeutic agents.

Reversible chemoresistance of pancreatic cancer grown as spheroids

Better in vitro models are needed to identify active drugs to treat pancreatic adenocarcinoma (PAC) patients. We used 3D hanging drop cultures to produce spheroids from five PAC cell lines and tested nine FDA-approved drugs in clinical use. All PAC cell lines in 2D culture were sensitive to three drugs (gemcitabine, docetaxel and nab-paclitaxel), however most PAC (4/5) 3D spheroids acquired profound chemoresistance even at 10 µM. In contrast, spheroids retained sensitivity to the investigational drug triptolide, which induced apoptosis. The acquired chemoresistance was also transiently retained when cells were placed back into 2D culture and six genes potentially associated with chemoresistance were identified by microarray and confirmed using quantitative RT-PCR. We demonstrate the additive effect of gemcitabine and erlotinib, from the 12 different combinations of nine drugs tested. This comprehensive study shows spheroids as a useful multicellular model of PAC for drug screening and elucidating the mechanism of chemoresistance.

Enhancing Drug Delivery of Glatiramer Acetate Through In Vitro Development of Controlled-Release Nanoliposomes: Investigating Drug Release Kinetics and Cytotoxic Effects

Background: Multiple sclerosis (MS) damages the myelin sheath covering nerve fibers. This condition affects 400,000 individuals in the United States and 2.5 million people globally, with a higher rate of diagnosis in women aged 20 - 40, with a ratio of 2:1. Liposomes facilitate drug dispersion, making them valuable in biomedicine. They enhance the stability of therapeutic medications, improve cellular and tissue absorption, and increase chemical bioactivity at specific locations. This technique delivers encapsulated compounds with high precision while minimizing negative consequences observed in laboratory settings. Objectives: This study aims to develop an optimal nanoliposome formulation using glatiramer acetate (GA) as the active pharmaceutical ingredient by examining drug release and associated adverse effects to achieve the most effective nanoliposomal system. Methods: The thin layer hydration method was used to prepare nanoliposomes. A comprehensive array of analytical techniques, including FE-SEM, FTIR, XRD, HPLC, and DLS, were employed to examine the physicochemical characteristics of the product. Additionally, the biosafety of the nanoliposomes was assessed using the MTT assay on the 1321N1 human astrocytoma cell line. The release profile of GA from the carrier was studied using the dialysis diffusion method, and the stability of the nanoliposomes was also inspected. Results: The size, Polydispersity Index (PDI), and zeta potential of the nanoliposomes were 91.2 ± 1.3 nm, 0.34 ± 0.03, and -27.3 ± 1.2 mV, respectively. The drug entrapment efficiency (DEE%) in nanoliposomes was approximately 70.2 ± 1.7%. The results from XRD and FTIR revealed no chemical interaction between the drug and carrier, and the nanoliposomes were safe for the cultured cell line. The nanoliposomes were stable under storage conditions and exhibited a sustained release profile. Conclusions: Nanoliposomes, as drug carriers, are known to be a potent drug delivery system due to their numerous advantages. Based on the results, GA-nanoliposomes show promise as a strategy for treating MS patients, pending further in vivo experiments and clinical trials.

DNA replication in primary hepatocytes without the six-subunit ORC.

The six subunit ORC is essential for initiation of DNA replication in eukaryotes. Cancer cell-lines in culture can survive and replicate DNA replication after genetic inactivation of individual ORC subunits, ORC1, ORC2 or ORC5. In primary cells, ORC1 was dispensable in the mouse liver for endo-reduplication, but this could be explained by the ORC1 homolog, CDC6, substituting for ORC1 to restore functional ORC. Here, we have created mice with a conditional deletion of ORC2, which does not have a homolog. Although mouse embryo fibroblasts require ORC2 for proliferation, mouse hepatocytes synthesize DNA in cell culture and endo-reduplicate in vivo without ORC2. Mouse livers endo-reduplicate after simultaneous deletion of ORC1 and ORC2 both during normal development and after partial hepatectomy. Since endo-reduplication initiates DNA synthesis like normal S phase replication these results unequivocally indicate that primary cells, like cancer cell lines, can load MCM2-7 and initiate replication without ORC.