HEK293 Cell Lines Research Articles

Transcriptional regulation in the absence of inositol trisphosphate receptor calcium signaling

The activation of IP3 receptor (IP3R) Ca2+ channels generates agonist-mediated Ca2+ signals that are critical for the regulation of a wide range of biological processes. It is therefore surprising that CRISPR induced loss of all three IP3R isoforms (TKO) in HEK293 and HeLa cell lines yields cells that can survive, grow and divide, albeit more slowly than wild-type cells. In an effort to understand the adaptive mechanisms involved, we have examined the activity of key Ca2+ dependent transcription factors (NFAT, CREB and AP-1) and signaling pathways using luciferase-reporter assays, phosphoprotein immunoblots and whole genome transcriptomic studies. In addition, the diacylglycerol arm of the signaling pathway was investigated with protein kinase C (PKC) inhibitors and siRNA knockdown. The data showed that agonist-mediated NFAT activation was lost but CREB activation was maintained in IP3R TKO cells. Under base-line conditions transcriptome analysis indicated the differential expression of 828 and 311 genes in IP3R TKO HEK293 or HeLa cells, respectively, with only 18 genes being in common. Three main adaptations in TKO cells were identified in this study: 1) increased basal activity of NFAT, CREB and AP-1; 2) an increased reliance on Ca2+- insensitive PKC isoforms; and 3) increased production of reactive oxygen species and upregulation of antioxidant defense enzymes. We suggest that whereas wild-type cells rely on a Ca2+ and DAG signal to respond to stimuli, the TKO cells utilize the adaptations to allow key signaling pathways (e.g., PKC, Ras/MAPK, CREB) to transition to the activated state using a DAG signal alone.

Chemically Synthesized 1,2,3,4,6-Pentakis-O-Galloyl-β-D-Glucopyranoside Blocks SARS-CoV-2 Spike Interaction with Host ACE-2 Receptor.

In the search for anti-COVID-19 therapy, 1,2,3,4,6-pentakis-O-galloyl-βD-glucopyranoside, a natural polyphenolic compound isolated from many traditional medicinal herbs, has been reported as an RBD-ACE2 binding inhibitor and as a broad-spectrum anticoronaviral inhibitor targeting the main protease and RNA-dependent RNA polymerase of SARSCoV-2. To facilitate the structure-activity relationship studies of 1,2,3,4,6-pentakis-O-galloyl-β-Dglucopyranoside, we describe its chemical synthesis and characterization, as well as its activity towards the SARS-CoV-2 spike interaction with host ACE2 receptor. 1,2,3,4,6-Pentakis-O-galloyl-β-D-glucopyranoside was synthesized in two quantitative steps from 3,4,5-tribenzyloxybenzoic acid and β-D-glucopyranoside: DCC-mediated esterification and palladium-catalyzed per-debenzylation. The synthesized molecule was evaluated using a SARS-CoV-2 spike trimer (S1 + S2) ACE2 inhibitor screening colorimetric assay kit, SARS-CoV2 spike S1 RBD ACE2 inhibitor screening colorimetric assay kit, and a cellular neutralization assay using the Spike (SARS-CoV-2) Pseudotyped Lentivirus, ACE2-HEK293 recombinant cell line. The chemically synthesized product blocked the binding of the spike trimer of SARSCoV-2 to the human ACE2 receptor with IC50=22±2 µM. It also blocked ACE2:spike RBD binding with IC50=27±3 µM. Importantly, it inhibited the infectivity of SARS2-CoV2-Spike pseudotyped lentivirus on the ACE2 HEK293 cell line with IC50=20±2 µM. Overall, the chemically synthesized 1,2,3,4,6-pentakis-O-galloyl-β-D-glucopyranoside represents a lead molecule to develop anti-SARS-CoV-2 therapies that block the initial stage of the viral infection by blocking the virus entry to the host cell.

Novel Pyrrolo-Pyrimidine Derivatives Bearing Amide Functionality as Potential Anticancer Agents: Synthesis and Molecular Docking Studies

Current investigation presents the synthesis, molecular docking and anticancer evaluation of novel pyrrolo-pyrimidine derivatives designed to target Janus kinase 1 (JAK1), Janus kinase 2 (JAK2) and cyclin-dependent kinase 4 (CDK4). The synthesis route commenced with 2-amino-4,6-dichloropyrimidine-5-carbaldehyde, proceeding through sequential steps involving intermediate formations and coupling reactions to yield a diverse array of pyrrolo-pyrimidine derivatives. Characterization using spectroscopic techniques (1H NMR, 13C NMR and HRMS) confirmed the chemical structures of the synthesized compounds. Molecular docking studies against JAK1, JAK2 and CDK4 demonstrated substantial binding affinities, remarkably compounds 7k, 7l and 7d, which displayed promising interactions within the active sites of the proteins. Anticancer evaluation against breast cancer (MCF-7), chronic myeloid leukemia (SET-2), colorectal carcinoma (HCT-116) and HEK-293 cell lines revealed diverse cytotoxic profiles. Specifically, compounds 7k and 7f exhibited potent activity against breast cancer (MCF-7), 7k and 7f showed efficacy against chronic myeloid leukemia (SET-2) and compounds 7k and 7l demonstrated effectiveness against colorectal carcinoma (HCT-116). These findings underscore the potential of these pyrrolo-pyrimidine derivatives as selective and effective anticancer agents, prompting further exploration for therapeutic development.

Development of polymeric composite scaffolds for defective bone repair and regeneration

Bones are vital components of the body that provide soft internal organs and tissues with structure, motion, and safety. Damage to bones can make life challenging. In repairing and regenerating damaged bone, polymeric composite materials significantly contribute to bone tissue engineering. In this article, we described developing porous scaffolds using the freeze-drying process from sodium alginate (SA), polyvinyl alcohol (PVA), and graphene oxide (GO) incorporated with zinc (Zn). These scaffolds were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), scanning electron microscopy (SEM), and universal testing machine (UTM) to investigate their structural analysis, surface morphology, and mechanical behavior, respectively. These scaffolds also exhibited less swelling in phosphate buffer saline (34.73–64.27%) than in aqueous media (42.36–75.92%) with controlled degradation. These scaffolds have potential biomineralization activities, hemocompatibility, and antibacterial activities, which were increased by increasing the Zn amount. All the scaffolds have biocompatibility as they have shown cell viability with mature cell morphology against preosteoblast and HEK-293 cell lines under standard in vitro conditions. The enhanced biological activities of the scaffolds were found by increasing the Zn amount. Thus, newly designed polymeric composite scaffolds would be promising materials to repair and regenerate fractured bone tissue.

Knockout of RIG-I in HEK293 cells by CRISPR/Cas9

We knocked out the retinoic acid-inducible gene I (RIG-I) in HEK293 cells via CRISPR/Cas9 to reveal the effects of RIG-I knockout on the key factors in the type I interferon signaling pathway. Three single guide RNAs (sgRNAs) targeting RIG-I were designed, and the recombination vectors were constructed on the basis of the pX459 vector and used to transfect HEK293 cells, which were screened by puromycin subsequently. Furthermore, a mimic of virus, poly I: C, was used to transfect the cells screened out. RIG-I knockout was checked by sequencing, real-time quantitative PCR, Western blotting, and immunofluorescence assay. Meanwhile, the expression levels of key factors of type I interferon signaling pathway such as melanoma differentiation-associated gene 5 (MDA5), interferonβ1 (IFNβ1), and nuclear factor-kappa B p65 [NF-κB(p65)], as well as cell viability, were determined. The results showed that two HEK293 cell lines (S1 and S3) with RIG-I knockout were obtained, which exhibited lower mRNA and protein levels of RIG-I than the wild type HEK293 cells (P < 0.05). The mRNA levels of MDA5 and IFNβ1 in S1 and S3 cells and the protein level of NF-κB(p65) in S3 cells were lower than those in the wild type (P < 0.05). More extranuclear NF-κB(p65) protein was detected in S1 cells than in the wild type after transfection with poly I: C. Plus, the wild-type and S1 cells transfected with poly I: C for 48 h showcased reduced viability (P < 0.05), while S3 cells did not display the reduction in cell viability. In summary, the present study obtained two HEK293 cell lines with RIG-I knockout via CRISPR/Cas9, which provided a stable cell model for exploring the mechanism of type I interferon signaling pathway.

Unveiling the insecticidal efficiency of Viola ignobilis against Macrosiphum rosae and Agonoscena pistaciae: From chemical composition to cytotoxicity analysis

Currently, there is a growing preference for eco-friendly bioinsecticides over chemical insecticides due to their safety. Plant extracts have emerged as a promising solution for this purpose. Therefore, this study aimed to evaluate the insecticidal effectiveness of Viola ignobilis extract against two key pests of rose aphid (Macrosiphum rosae) and pistachio psylla (Agonoscena pistaciae). Significant compounds were identified using GC-MS and MALDI-TOF MS. Three bioassay methods were employed to assess the extract's insecticidal potential, and its cytotoxicity was tested on HEK293 cells. Results revealed that the highest insecticidal efficacy occurred at a concentration of 20 mg/mL after a 72 h exposure. The contact bioassay method displayed greater efficiency against M. rosae than A. pistaciae, while the oral bioassay demonstrated the highest efficiency against A. pistaciae. The extract also acted as a feeding deterrent, with indices of 77.47 ± 7.98 % and 87.98 ± 3.84 % for A. pistaciae and M. rosae, respectively. Furthermore, the insecticidal potency of the extract was assessed, resulting in LC50 values of 3.58 mg/mL and 6.77 mg/mL for the contact bioassay, and 0.87 mg/mL and 0.61 mg/mL for the oral bioassay against M. rosae and A. pistaciae, respectively. Importantly, the extract showed no detrimental cytotoxic effects on the HEK293 cell line within the tested concentration range, indicating its potential safety as a bioinsecticide. Overall, these findings highlight the potential of V. ignobilis extract as a promising candidate for further development in pest control.

Anti-Inflammatory and Anti-proliferative Role of Essential Oil of Leaves of Cleistocalyx operculatus (Roxb.) Merr. & Perry.

Phytochemicals have long remained an essential component of the traditional medicine system worldwide. Advancement of research in phytochemicals has led to the identification of novel constituents and metabolites from phytochemicals, performing various vital functions ranging from antimicrobial properties to anticarcinogenic roles. This plant is traditionally used by local people to manage inflammation. In this study, we aim to extract and chemically profile the essential oil from the leaves of Cleistocalyx operculatus (Roxb.) Merr. & Perry and study of the anti-inflammatory and anti-proliferative role of essential oil. The hydro distillation method was used for the extraction of essential oil, and the GC-MS was applied for the chemical profiling. The percentage of cell viability was calculated using a crystal violet assay, colony formation assay was performed using Semiquantitative PCR, Propodium iodite staining was used for cell death assay, and Western blotting was used to determine antibodies and proteins. Schrodinger 2015 software was used for molecular docking. Myrcene, a monoterpene, constitutes 56% of the oil and could be attributed to its anti-inflammatory potential. Treatment of LPS-challenged mouse macrophages RAW264.7 cells with essential oil resulted in a decline in the inflammatory markers, such as IL-1β, TNFα, iNOS, COX-2, and NFκB. Further, essential oil inhibited cancer PC-3, A431, A549, and MCF-7 cell lines at concentrations lower than normal PNT2 and HEK-293 cell lines. This decline in proliferative potential can be attributed to a decline in anti-apoptotic proteins, such as procaspase 3 and PARP, an increase in CKIs, such as p21, and a decline in the Akt signaling responsible for survival. The essential oil of the plant Cleistocalyx operculatus may be a potential lead for anti-inflammatory and anti-proliferative function.

Protein trans-splicing: optimization of intein-mediated GFP assembly as a model for the development of gene therapy.

Adeno-associated virus (AAV)-based gene therapy has become one of the key directions of modern translational medicine geared towards treatment of hereditary disorders by means of gene replacement. At the moment, about 5,000 different syndromes are associated with mutations in large genes, which presents a great problem due to the AAV packaging capacity of 5 kilobases. The main strategies for overcoming this obstacle were the creation of truncated gene versions, overloading the viral vector, and separate delivery of partial genetic material to restore the whole gene at the level of DNA, RNA, or protein. At present, genome editing via prime editors, most effectively delivered by AAV, relies on the intein pair used to restore the protein complex. The amazing integration speed of intein-based protein trans splicing technology makes it a versatile tool for a variety of applications, albeit not always successful on the first attempt. This study discusses the key points of working with Ssp, Npu, and Ava inteins of the DnaE group, known as the most effective for assembly of large proteins. Using green fluorescent protein (GFP) as a model, we demonstrate that the successful protein assembly requires not only cysteine at position C+1 but also certain aminoacid residues on either side in its immediate environment. Furthermore, the conformation of extein-intein composition, difficult to predict by computer modeling, has an additional effect, as demonstrated by experimental tests of the three split sites optimal in amino acid composition. The NpuDnaE variant demonstrated the highest kinetics of interaction between the N and C parts in the DnaE group of inteins. Optimization of conditions using NpuDnaE intein led to GFP assembly in 80% of transfected HEK293 cells and in 55% of AAV5-transduced cells, as demonstrated by flow cytometry. The efficiency of GFP assembly post-plasmid DNA transfection or AAV transduction of the HEK293 cell line was 15% higher than that of the ARPE19 cell line. We hope that the obtained data will facilitate the development of gene therapies for the treatment of hereditary disorders caused by mutations in large genes.

Identification of antimalarial phytoconstituents from Tinospora sinensis (Lour.) Merr. Stem by in vitro whole cell assay and multiple targets directed in silico screening against Plasmodium falciparum

Ethnopharmacological relevanceTinospora sinensis (Lour.) Merr., from the family Menispermaceae, is widely used in Indian folk and Ayurvedic medicine. Indigenous tribes such as the Tea-tribe and Chorei-tribe of Assam use its bark and stem as a herbal remedy to treat malaria and it is also traditionally employed for conditions such as dyspepsia, inflammation, fever, ulcers, jaundice, diabetes and various urinary, skin, and liver diseases. Aim of the studyThis study aims to identify and characterize antimalarial phytoconstituents from the active extract of T. sinensis stem by in vitro screening against both the Chloroquine-sensitive (Pf3D7) as well as Chloroquine-resistant (PfRKL-9) strains of Plasmodium falciparum, along with exploring potential targets and mechanisms using molecular docking and dynamics simulation studies. Materials and methodsT. sinensis stems were collected from Assam, India, and authenticated by the Botanical Survey of India. The plant materials were initially extracted with non-polar to polar solvents and screened for in vitro antimalarial potency against Pf3D7 and PfRKL-9. Then, the methanol extract was selected for bioassay-guided isolation of phytoconstituent(s). The isolated phytoconstituent(s) were screened for antimalarial potential and active compounds were further evaluated for cytotoxicity using the HEK-293 cell line. Structural characterization of the active compounds involved the use of UV–VIS, IR, NMR and HRMS analyses. Molecular docking and dynamics simulation studies were performed on selected targets from P. falciparum to predict binding affinities and mechanisms of action. ResultsFrom the methanol extract of T. sinensis stem, five phytoconstituents were isolated, including isoquinoline alkaloids Berberine (NG1) and Palmatine (NG2) showed the best antimalarial activity (IC50 < 1 μg/ml) against both Pf3D7 and PfRKL-9. Cytotoxicity assays confirmed their safety and selectivity. Molecular docking and dynamic simulation studies revealed that Berberine and Palmatine formed stable complexes with P. falciparum lysyl-tRNA synthetase and P. falciparum aminopeptidase N, respectively, indicating their potential as antimalarial leads. ConclusionThis study identifies two potent antimalarial phytoconstituents in the stem of T. sinensis, validating its traditional use and demonstrating its safety and efficacy for potential global application in malaria treatment.

SelectRepair Knockout: Efficient PTC-Free Gene Knockout Through Selectable Homology-Directed DNA Repair.

Generating nonessential gene knockouts using CRISPR/Cas9 technology is becoming increasingly common in biological research. In a typical workflow, the Cas9 endonuclease is used to induce a DNA double-strand break that relies on nonhomologous end-joining (NHEJ) to introduce a premature termination codon (PTC) in the target gene. The goal is to isolate clones in which the gene produces PTC-containing mRNA transcripts that are degraded via nonsense-mediated mRNA decay (NMD) to cause loss of gene function. Unfortunately, this approach is laborious, and not all PTCs trigger NMD. More importantly, mounting evidence suggest that PTC mutations can also result in a transcriptional adaptation response that can mask the effects of a PTC-mediated gene knockout. In this chapter, we present a PTC-free gene knockout strategy that implements homology-directed DNA repair (HDR) with selectable markers to substantially reduce the complexity of the screening and validation of genome edits in cells containing more than one gene copy as in the case of the commonly used hypotriploid HEK293 cell line. We describe how to obtain a complete knockout of the Ligase IV protein (LIG4) and provide considerations for the application of this SelectRepair Knockout method to other genes.

Specific Activation of the Expression of Growth Factor Genes in Expi293F Human Cells Using CRISPR/Cas9-SAM Technology Increases Their Proliferation.

Human cell lines play an important role in biotechnology and pharmacology. For them to grow, they need complex nutrient media containing signaling proteins - growth factors. We have tested a new approach that reduces the need of cultured human cell lines for exogenous growth factors. This approach is based on the generation of a modified cell with a selectively activated gene expression of one of the endogenous growth factors: IGF-1, FGF-2, or EIF3I. We modified the Expi293F cell line, a HEK293 cell line variant widely used in the production of recombinant proteins. Gene expression of the selected growth factors in these cells was activated using CRISPR/Cas9 technology with the synergistic activation mediators CRISPR/Cas9-SAM, which increased the expression of the selected genes at both mRNA and protein levels. Upon culturing under standard conditions, the modified lines exhibited increased proliferation. A synergistic effect was observed in co-culture of the three modified lines. In our opinion, these results indicate that this approach is promising for efficient modification of cell lines used in biotechnology.

Eco-conscious synthesis of novel 1,2,4-triazolo[1,5-a]pyrimidine derivatives as potent Anti-microbial agent and comparative study of cell viability and cytotoxicity in HEK-293 cell line utilizing Indian gooseberry (Phyllanthus emblica) fruit extract

Antimicrobial resistance (AMR) is a pressing global health challenge that necessitates the search for novel antimicrobial agents and synthetic methodologies. This study investigates the synthesis and antimicrobial efficacy of 25 novel 1,2,4-triazolo[1,5-a]pyrimidine derivatives, catalyzed by Amla (Phyllanthus emblica) fruit juice, which is rich in organic acids and polyphenolic compounds, thus serving as an environmentally sustainable catalyst, in adherence to green chemistry principles. The synthesis is achieved through a one-pot, solvent-free process that yields high quantities of the desired compounds in significantly less time compared to traditional methods. Comprehensive antimicrobial evaluation was conducted against a range of clinically relevant microorganisms, including Chromobacterium violaceum, Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans, Cryptococcus neoformans and Aspergillus niger. Concurrently, cytotoxic assays were performed on HEK-293 cells, to assess safety profiles, revealing that compounds B-1, B-6, B-7, B-14 and B-15 exhibited potent antimicrobial activity while maintaining low cytotoxicity and high cell viability. These findings underscore the therapeutic potential of the synthesized compounds in combatting infectious diseases and addressing the challenges posed by AMR, highlighting the critical importance of dose optimization in therapeutic applications. This study combats contagious diseases, mitigates AMR challenges and contributes significantly to the field of antimicrobial drug discovery, emphasizing the need for sustainable synthetic strategies that align with future pharmaceutical endeavors. Our research not only advances the understanding of these novel compounds but also supports ongoing efforts to develop safe and effective therapies against resistant pathogens.

Metabolic activation of WHO-congeners PCB28, 52, and 101 by human CYP2A6: evidence from in vitro and in vivo experiments.

Despite extensive research on the metabolism of polychlorinated biphenyls (PCBs), knowledge gaps persist regarding their isoform-specific biotransformation pathways. This study aimed to elucidate the role of different cytochrome P450 enzymes in PCB metabolism, focusing on WHO-congeners 2,4,4'-trichlorobiphenyl (PCB28), 2,2',5,5'-tetrachlorobiphenyl (PCB52), and 2,2',4,5,5'-pentachlorobiphenyl (PCB101). Utilizing engineered HEK293 cell lines, we investigated the in vitro metabolism of these PCBs by CYP1A2, CYP2C8, CYP2C9, CYP3A4, CYP2A6, and CYP2E1, revealing robust production of hydroxylated metabolites. Our results show that CYP2A6 plays a major role in the metabolism of these congeners responsible for predominant formation of para-position hydroxylated metabolites, with concentrations reaching up to 1.61µg/L (5,89nM) for PCB28, 316.98µg/L (1,03µM) for PCB52, and 151.1µg/L (441nM) for PCB101 from a 20µM parent PCB concentration. Moreover, concentration-dependent cytotoxic and cytostatic effects induced by reactive intermediates of the PCB hydroxylation pathway were observed in HEK293CYP2A6 cells, for all three congeners tested. CYP2A6 was specifically capable of activating PCBs 28 and 101 to genotoxic metabolites which produced genetic defects which were propagated to subsequent generations, potentially contributing to carcinogenesis. In a clinical study examining CYP2A6 enzyme activity in formerly exposed individuals with elevated internal PCB levels, a participant with increased enzyme activity showed a direct association between the phenotypic activity of CYP2A6 and the metabolism of PCB28, confirming the role of CYP2A6 in the in vivo metabolism of PCB28 also in humans. These results altogether reinforce the concept that CYP2A6 plays a pivotal role in PCB congener metabolism and suggest its significance in human health, particularly in the metabolism of lower chlorinated, volatile PCB congeners.

Selective Inhibition of P2Y1 and P2Y12 Receptor Signal Pathways in Platelet Aggregation in Transgenic Cell Lines and Rats by Potassium 2-(1-Hydroxypentyl)-Benzoate, Puerarin and Salvianolic Acid B.

Potassium 2-(1-hydroxypentyl)-benzoate (dl-PHPB), puerarin and salvianolic acid B are three natural products or derivatives that can inhibit platelet aggregation. However, the mechanisms of dl-PHPB, puerarin and salvianolic acid B to inhibit platelet aggregation are still not clear. Here, 2-methylthioadenosine diphosphate (2-MeSADP) was used as an inducer to confirm the effects of three drugs on platelet aggregation and illustrate the corresponding mechanisms. The results indicated that dl-PHPB, puerarin and salvianolic acid B significantly inhibited platelet aggregation both invivo and invitro. In addition, the content of IP3, cAMP and intracellular [Ca2+]i were measured in HEK293 cell lines overexpressing P2Y1 and P2Y12. Dl-PHPB and puerarin could obviously reduce 2-MeSADP-induced IP3 increase, but salvianolic acid B showed no effects. Unlike dl-PHPB and puerarin, which had no effects on 2-MeSADP-induced cAMP decrease, salvianolic acid B significantly reversed the reduction of cAMP. Both dl-PHPB and puerarin could decrease the enhanced intracellular [Ca2+]i induced by 2-MeSADP; however, salvianolic acid B showed no effect on intracellular [Ca2+]i elevation. These results suggested that dl-PHPB and puerarin inhibited platelet aggregation via targeting at P2Y1 receptor and P2Y1-Gq-IP3-Ca2+ signal pathway. Differently, salvianolic acid B inhibited platelet aggregation via targeting at P2Y12 receptor and via Gi-AC-cAMP signal pathway.

Chemotherapeutic Drug Delivery Nanoplatform Development: From Physicochemical to Preclinical Evaluation.

Through this study, the synergistic behavior of small-molecular-weight, amphiphilic surfactant molecules and the triblock copolymer Pluronic 188 was extensively evaluated based on their ability to formulate nanocarriers with novel properties for the delivery of class II and IV (biopharmaceutical classification system) chemotherapeutic compounds. The combination of four different surfactants at multiple weight ratios and twelve initially formulated nanosystems resulted in four hybrid delivery platforms, which were further studied in terms of multiple physicochemical characteristics, as well as their stability in protein-rich media (fetal bovine serum/phosphate-buffer saline). Finally, we obtained a single final nanoformulation that exhibited a high loading capacity (%EE ≥ 75%) and a sustained drug release profile under physiological conditions (model drug methotrexate), without altering the original physicochemical characteristics of the carrier. With a mean hydrodynamic radius (Rh) of less than 70 nm, a polydispersity index of 0.219, and no protein complexation, the system is a suitable candidate for in vivo, intravenous, and/or intramuscular administration. The cytotoxicity and genotoxicity of both loaded and unloaded carriers were evaluated through the examination of the upregulation or downregulation of apoptosis-related pathways. Multiple conventional 2D and 3D spheroidal conformations were used for these assessments, including HEK293, HCT-116, and MCF-7 cell lines, the results of which stressed the safety and biocompatibility of the empty nanocarrier. Additionally, experiments on Caenorhabditis elegans were conducted to evaluate the system's in vivo toxicity, focusing on developmental stages, egg-laying behavior, and locomotion. Nanosystems studied in terms of chemotherapeutic encapsulation have mostly focused on the physiochemical aspect of the development of such novel delivery platforms, with only few exceptions proceeding step-by-step from cellular 2D to 3D to in vivo experimentation. The present study offers a holistic view of the behavior of such a novel system, advancing our understanding of the capabilities of polymeric/surfactant-based nanodelivery platforms.

Anti-cancer Activity Evaluation of Naphthalenonic and Chromanonic Spiroisoxazoline Derivatives as Selective COX-2 Inhibitors on HT29 Cell Lines

Background: Cyclooxygenase-2 (COX-2) is induced in response to proinflammatory conditions, and it is not only a key enzyme in the inflammatory process, but also seems to be highly expressed in various types of cancer cells. On the other hand, it is well documented that chemical compounds with spiro scaffolds in their structure could be effective chemical agents against cancer types. Objective: In this study, the cytotoxicity effects of spiroisoxazoline derivatives containing naphthalinone and chromanone spiro-bridge were investigated. Methods: The cytotoxicity effects of compounds 7a-7h were evaluated by performing the MTT assay on the HT-29 (colorectal cancer), MCF-7 (breast cancer), and HEK-293 (normal kidney) cell lines. After that, a compound with high yield and remarkable cytotoxic activity was selected to analyze the cell cycle and apoptosis mechanism. Results: The most effective cytotoxic activity was observed on HT-29 and MCF-7 cell lines of compounds 7b (IC50 value: 1.07±0.28 µM) and 7f (IC50 value: 11.92±1.07 µM). None of the compounds had a toxic effect on normal HEK-293 cells, except for compound 7g with an IC50 value of 21.30±16.14 µM, whose effect was much lower than that of cisplatin and doxorubicin, known as anti-cancer agents. Subsequently, compound 7e with significant yield and cytotoxic activity was investigated to evaluate cell cycle and apoptosis. The result showed that compound 7e induced significant G0/G1 cell cycle arrest and apoptosis in HT-29 cells Conclusion: The selective COX-2 inhibitor compounds with spiroisoxazoline core structure could be suitable scaffolds for cytotoxic effects.

Discovery of a novel benzimidazole conjugated quinazolinone derivative as a promising SARS-CoV-2 3CL protease inhibitor.

This report presents the design and synthesis of quinazolinone-based derivatives as promising SARS-CoV-2 3CL protease inhibitors. Two novel series, namely, febrifugine analogues 4a-i and quinazolinone conjugated benzimidazoles 9a-c, were successfully synthesized starting from isatoic anhydride. The synthesized quinazolinone derivatives were evaluated for their cytotoxicity against cancer cell lines and SARS-CoV-2 3CL inhibitory activity. The results showed that the synthesized compounds did not have significant toxicity for the non-cancer HEK293 cell line and MCF-7, MDA-MB-231, HEPG2 and HEPG2.2.15 cancer cell lines. Notably, compound 9b exhibited anti-3CL enzymatic activity in a dose-dependent manner, with the calculated IC50 value of 10.73 ± 1.17 μM. Docking results highlighted the interaction between 9b and 3CL protease through hydrogen bonding with key amino acids, including His41, Met49, Cys145, Met165, Arg188, His164, and Glu166, at the active site of the protease. Pharmacokinetic studies and ADME analyses provide valuable insights into the potential of compound 9b as a drug candidate. These findings support the new scaffold as a candidate for 3CLpro inhibition and advanced anti-coronavirus drug research.

Assessment the Efficacy of the CRISPR System for Inducing Mutations in the AIMP2 Gene to Create a Cell Line Model of HLD17 Disease.

Hypomyelinating leukodystrophy-17 is a neurodevelopmental disorder caused by autosomal recessive mutations in the AIMP2 gene, resulting in a lack of myelin deposition during brain development, leading to variable neurological symptoms. Research on brain function in these disorders is challenging due to the lack of access to brain tissue. To overcome this problem, researchers have utilized different cell and animal models. The CRISPR-Cas9 system is considered the most optimal and effective method for genetic modification and developing cell models. We studied the efficacy of the CRISPR-Cas9 technology in inducing mutations in the AIMP2 gene in HEK293 cell lines. The study involved transfecting HEK293 cells with recombinant PX458 plasmids targeting spCas-9 and AIMP2 sgRNA. The cells were evaluated using fluorescent microscopy and enriched using serial dilution. The CRISPR/Cas9 plasmids were validated through PCR and Sanger sequencing. After serial dilution, AS-PCR, Sanger sequencing, and TIDE program analysis showed the construct successfully induces an indel mutation in HEK cells. Our findings demonstrated the great efficacy of the CRISPR system and produced a construct for inducing mutations in the AIMP2 gene, which can be utilized to edit the AIMP2 gene in nerve cells and create a cellular model of the HLD17 disease.

GATA1 transcription factor targets the gene expression of B19 virus in HEK293 cell line.

B19 virus (B19V) is a single-strand DNA virus that has specific tropism to erythroid progenitor cells (EPCs). The virus enters the cells via P antigen and coreceptors and induces infection and cell apoptosis. GATA1 has a high expression in EPC and is a critical transcription factor for the cells development and differentiation. As human EPCs are the main target of the virus infection that have high expression of GATA-1 as the critical transcription factor, the aim of this study was to investigate the effect of GATA1 cotransfection with B19V genome on the expression of the viral mRNAs in HEK293 as nonpermissive cell line to the virus that had no mRNA expression of GATA-1. HEK293 cells were transfected with pHI0 plasmid containing the B19V genome and the plasmid of the GATA1 genome. The quantity of B19V mRNAs (NS1, 7.5 kDa, and 11 kDa) expression was evaluated after 24h of transfection. The results showed a statistically significant increase in fold change expression of (NS1 ∽12.3, VP1 ∽27.6, 11kb protein ∽38) in cotransfected cells with GATA1 and B19 plasmids compare to control group (P<0.05). This research showed transfected cells with GATA1 had elevation in the expression of the B19V genes mRNAs in a nonpermissive cell. This result may show the role of GATA1 as a critical transcription factor in support of the virus infection in EPCs. This suggests that GATA1 may potentially sport B19V replication or gene expression.

Physico-chemical properties and biological evaluation of graphene quantum dots for anticancer drug susceptibility

Graphene quantum dots (GQDs) possess unique optical and biocompatible properties, making them suitable candidates for biomedical and pharmaceutical applications. This study reports the hydrothermal synthesis of pristine-GQD and doped variants: Nitrogen-GQD and Sulfur-GQD. The materials underwent thorough characterization techniques such as UV–vis, fluorescence, XRD, FE-TEM/SEM, EDX, and Raman spectroscopy. The particle sizes of these GQDs range from 2 to 5 nm. We conducted a comprehensive study through MTT assays to evaluate the potential cytotoxic effect of GQD and the doped variants. This study demonstrated their synergistic interactions with an anti-cancer drug, methotrexate (MTX), and also improvement of cytocompatibility in the presence of folic acid (FA). Systematic MD simulations revealed a compacting effect on the dynamic behavior of GQD and its variants in the presence of drugs. Fluorescence spectroscopy and computational modeling suggest non-intercalative surface interactions between GQDs and the drugs. The cytotoxic activity of pristine GQD on HeLa cervical cancer cells is higher than that of N-GQD and S-GQD. When treated with GQD-IC50–MTX-IC50, only 5.6 % of HeLa cells remained viable. The doped variants exhibited bio-compatibility when tested on normal HEK cell lines. Overall, this study emphasizes the potential of GQDs for targeted cancer therapy through an interdisciplinary approach involving material characterization, computational modeling, and biological assays.