HEK293T Cell Line Research Articles

Efficacy of Chitosan-N-Arginine Chitosomes in mRNA Delivery and Cell Viability Enhancement.

Cationic lipid-based carriers are recognized for their ability to complex with mRNA and effectively deliver the mRNA for vaccination and therapeutic purposes. However, the significant cytotoxicity of these carriers often restricts their practical application. In the present study, polymer-lipid hybrid nanoparticles, termed chitosomes, incorporating chitosan-N-arginine (CSA) with the DOTAP cationic lipid and the DOPE helper lipid, were synthesized and evaluated. The addition of CSA to the lipid formulations improved their physicochemical stability and enhanced mRNA complexation, resulting in high transfection rates in the HeLa and HEK293T cell lines. However, the transfection efficiency was low in the NIH-3T3 cell line, indicating a cell type-specific response to chitosomes. Importantly, CSA significantly reduced the cytotoxicity typically associated with DOTAP. Overall, the present study indicated that optimizing the ratio of CSA to DOTAP is crucial for developing mRNA nanocarriers to achieve high transfection efficiency and reduce cytotoxicity across different cell lines.

Construction of HEK293T cell line stably expressing TRPM2 channel based on PiggyBac transposition system and its application in drug screening for cerebral ischemia and other diseases

To establish a cell line stably expressing the transient receptor potential melastatin 2 (TRPM2) channel for screening TRPM2 inhibitors based on PiggyBac transposition system. A plasmid PiggyBac-human TRPM2 (pPB-hTRPM2) eukaryotic expression vector was constructed using PiggyBac transposition system. The plasmid and a helper plasmid were co-transfected into HEK293T cells to express TRPM2, which was identified by fluorescence and patch-clamp assays. The high throughput screening performance was assessed with the Z ´ factor. Calcium imaging and patch clamp techniques were employed to assess the initial activity of eleven compound molecules, confirming the inhibitory effects of the primary molecules on TRPM2. The protective effect of the screened compounds on damaged cells was validated using the oxygen-glucose deprivation/reperfusion (OGD/R) injury model and CCK-8 kit. The level of cellular reactive oxygen species (ROS) was detected by flow cytometry. The neuroprotective effects of the compounds were evaluated using a transient middle cerebral artery occlusion (tMCAO) mouse model. The HEK293T cells transfected with pPB-hTRPM2-EGFP showed high TRPM2 expression. Puromycin-resistant cells, selected through screening, exhibited robust fluorescence. Whole-cell patch results revealed that induced cells displayed classical TRPM2 current characteristics comparable to the control group, showing no significant differences (P>0.05). With a Z ´ factor of 0.5416 in calcium imaging, the model demonstrated suitability for high-throughput screening of TRPM2 inhibitors. Calcium imaging and electrophysiological experiments indicated that compound 6 significantly inhibited the TRPM2 channel. Further experiments showed that 1.0 μmol/L of compound 6 enhanced cell viability (P<0.05) and reduced the level of ROS (P<0.05) of SH-SY5Y under OGD/R injury. 0.3 and 1.0 mg/kg of compound 6 reduced the cerebral infarction volume in tMCAO mice (both P<0.05). A stable TRPM2 gene expressing cell line has been successfully established using PiggyBac gene editing in this study. TRPM2 channel inhibitors were screened through calcium imaging and patch clamp techniques, and an inhibitor compound 6 was identified. This compound can alleviate cell damage after OGD/R by reducing cellular ROS levels and has a protective effect against cerebral ischemia-reperfusion injury in mice.

Site-Specific Integration by Circular Donor Improves CRISPR/Cas9-Mediated Homologous Recombination in Human Cell Lines

The technology for obtaining the high-efficiency expression of target proteins through site-specific recombination has made progress. However, using the CRISPR/Cas9 system for site-specific integration of long fragments and the expression of active proteins remains a challenge. This study optimized the linear DNA circularization system, eliminated the prokaryotic plasmid backbone on the traditional foreign gene vector, and generated a homologous arm-free circular donor template with a single guide RNA target site (sgRNA TS). This strategy significantly increased the co-transfection efficiency of the 1.6 kb template and Cas9 plasmid by 1.15-fold, and the average knock-in (KI) efficiency of the 4.7 kb long-fragment template for the two target gene sites increased by 1.3-fold. Subsequently, we used rhBCHE as a reporter gene to efficiently integrate the 5.4 kb fragment containing the gene of interest (GOI) into specific sites in the HEK293T cell line to detect the expression of the circular template at different target sites. Overall, this study further verifies that the length of the circular donor is more conducive to non-homologous integration, and more importantly, we provide a simple and optimized strategy for the construction of long-fragment site integration cell lines.

Novel bioassays based on 3D-printed device for sensing of hypoxia and p53 pathway in 3D cell models.

Cell-based assays are widely exploited for drug screening and biosensing, providing useful information about bioactivity of target analytes and complex biological samples. It is well recognized that 3D cell models are required to achieve highly valuable information, also from the perspective of replacing animal models. However, bioassays relying on 3D cell models are generally highly demanding in terms of facilities, equipment, and skilled personnel requirements. To reduce cost, increase sustainability, and provide a flexible 3D cell-based platform for bioassays, we here report a novel approach based on a 3D-printed microtissue device. To assess the suitability of this strategy for reporter gene technology, we selected to monitor two molecular pathways which were of interest in several applications, hypoxia signaling and the p53 pathway. The investigation of such pathways is highly relevant in fields spanning from drug screening to bioactivity monitoring for industrial by-product valorization. Microtissues of human hepatocarcinoma (HepG2) and human embryonic kidney (Hek293T) cell lines were obtained with a low-cost and sustainable chip platform and bioassays were developed to monitor the hypoxia-inducible factors (HIFs) and the p53 tumor suppressor pathway. HepG2 and Hek293T 3D cell models were genetically engineered to express the Luc2P from Photinus pyralis firefly either under the regulation of p53 or HIF response elements. The bioassays allowed quantitative assessment of hypoxia and tumoral activity with 1,10-phenanthroline for HIF and with doxorubicin for p53 pathway activation, respectively, showing good potential for applications of this sustainable and low-cost 3D-printed microfluidic platform for bioactivity analyses, drug screening, and precision medicine.

CRISPR-Mediated Gene Editing Tool Development for Patients with Usher Syndrome Type II

Usher syndrome, a rare genetic disease affecting hearing and vision, is an autosomal recessive condition affecting 4 to 17 per 100,000 people and accounting for about 50 percent of hereditary deaf-blindness cases. Usher syndrome type II is caused by mutations of the USH2A gene, a 15.7 kb gene encoding the protein usherin, which localizes to photoreceptor cilium and cochlear hair cells. This subtype of Usher syndrome includes hearing loss from birth and progressive loss of vision, prompting retinitis pigmentosa (RP). Prime editing is a viable option for addressing USH2A mutations and restoring usherin expression and function. To begin, a HEK293T cell line needs to be established for subsequent testing of prime editors. Since prime editing to make the cell line was unsuccessful, homology-directed repair was done and successfully created a cell population with the mutation of interest (single base-pair deletion). Now that this cell population has been created, prime editors will be tested and optimized to address the mutation in a HEK293T cell line and potentially patient-derived induced pluripotent stem cells that are differentiated into photoreceptor precursor cells.

Predicting the effect of CRISPR-Cas9-based epigenome editing.

Epigenetic regulation orchestrates mammalian transcription, but functional links between them remain elusive. To tackle this problem, we use epigenomic and transcriptomic data from 13 ENCODE cell types to train machine learning models to predict gene expression from histone post-translational modifications (PTMs), achieving transcriptome-wide correlations of ~ 0.70 - 0.79 for most cell types. Our models recapitulate known associations between histone PTMs and expression patterns, including predicting that acetylation of histone subunit H3 lysine residue 27 (H3K27ac) near the transcription start site (TSS) significantly increases expression levels. To validate this prediction experimentally and investigate how natural vs. engineered deposition of H3K27ac might differentially affect expression, we apply the synthetic dCas9-p300 histone acetyltransferase system to 8 genes in the HEK293T cell line and to 5 genes in the K562 cell line. Further, to facilitate model building, we perform MNase-seq to map genome-wide nucleosome occupancy levels in HEK293T. We observe that our models perform well in accurately ranking relative fold-changes among genes in response to the dCas9-p300 system; however, their ability to rank fold-changes within individual genes is noticeably diminished compared to predicting expression across cell types from their native epigenetic signatures. Our findings highlight the need for more comprehensive genome-scale epigenome editing datasets, better understanding of the actual modifications made by epigenome editing tools, and improved causal models that transfer better from endogenous cellular measurements to perturbation experiments. Together these improvements would facilitate the ability to understand and predictably control the dynamic human epigenome with consequences for human health.

Engineering of HEK293T Cell Factory for Lentiviral Production by High-Throughput Selected Genes.

Lentiviral vectors (LVs) are crucial tools in gene therapy and bioproduction, but high-yield LV production systems are urgently needed. Using clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 high-throughput screening, we identified nine critical genes (LDAH, GBP3, BPIFC, NHLRC1, NHLRC3, ZNF425, TTC37, LRRC4B, and SPINK6) from 17,501 genes that limit LV packaging and formation. Knocking out these genes in HEK293T cells significantly increased virus production, with LDAH knockout exhibiting a 6.63-fold increase. Studies on multigene knockouts demonstrated that the cumulative effects of different gene knockouts can significantly enhance lentivirus production in HEK293T cells. Triple knockout of GBP3, BPIFC, and LDAH increased LV titer by ∼8.33-fold, and knockout (or knockdown) of GBP3, NHLRC1, and NHLRC3 increased LV titer by ∼6.53-fold. This study established HEK293T cell lines with multiple genes knockout for efficient LV production, providing reliable technical support for LV production and application and offering new perspectives for studying LV packaging mechanisms and related virus research.

IgG reactivity to different desmoglein-3 ectodomains in pemphigus vulgaris: novel panels for assessing disease severity.

Pemphigus vulgaris (PV) is an autoimmune disease characterized by IgG autoantibodies targeting desmoglein-3 (Dsg3), leading to blistering of mucous membranes and skin. Although commercial ELISA kits effectively diagnose PV, correlation with clinical phenotype remains unclear. This study assesses multiple panels for monitoring disease severity and activity by profiling IgG autoantibodies against Dsg3's various extracellular ectodomains. We designed and expressed different extracellular domains of Dsg3 in HEK293T cell line and developed 15 different ELISA panels, each using a single or multi ectodomains encompassing the entire extracellular region of Dsg3 to detect specific autoantibodies against the particular part of Dsg3. To validate our approach, we compared our ELISA panel for the full Dsg3 (EC1-5) against a commercial kit using 154 random serum samples from PV patients, demonstrating a strong correlation. For evaluation of IgG autoantibody profiles in our panels, 59 PV patients were included, along with 11 bullous pemphigoid patients, and 49 healthy controls. For all the included subjects, 15 predefined ELISA panels were tested. The IgG autoantibodies against EC1 were detected in 86% of patients with a positive full Dsg3 ectodomain (EC1-5) ELISA, with 26% against EC2, 14% for EC3, 29% for EC4, and 23% for EC5. Among the panels with multiple Dsg3 ectodomains, EC1-3 and EC1-4 were representative of the entire Dsg3 ectodomain in terms of ELISA positivity across all included patients. A significant correlation (P<0.05) was observed between ELISA optical density (OD) and Pemphigus Disease Area Index (PDAI) scores in five panels, EC1, EC2-3, EC2-5, and EC3-4 in addition to the full ectodomain. It suggests an association with disease severity. Interestingly, while the ELISA panel for the entire Dsg3 extracellular ectodomains did not differentiate disease phases, in three of our panels, including EC1, EC3-5, and EC2-5, ANOVA analysis showed a statistically significant difference between the groups of patients in remission, partial remission or persistent lesions, and those with active disease (new cases or relapse). Among these three panels, EC1 was the only one that showed a significant difference in the multiple comparisons analysis; patients in the active phase had higher levels of autoantibodies than those in 'partial remission or persistent lesions' and 'complete remission' groups. The level of autoantibodies against EC1 was not only correlated with the full ectodomain but also associated with higher disease severity and active disease phase. This study indicates that a detailed autoantibody profile against Dsg3 ectodomains could serve as a marker for PV severity and activity which may potentially enhance early treatment initiation.

Hyperthermia and biological investigation of a novel magnetic nanobiocomposite based on acacia gum-silk fibroin hydrogel embedded with poly vinyl alcohol

The design and synthesis of biocompatible nanostructures for biomedical applications are considered vital challenges. Herein, a nanobiocomposite based on acacia hydrogel, natural silk fibroin protein, and synthetic protein fibers of polyvinyl alcohol was fabricated and magnetized with iron oxide nanoparticles (Fe3O4 MNPs). The structural properties of the hybrid nanobiocomposite were investigated by essential analyses such as Fourier Transform Infrared Spectrometer (FTIR), Field emission scanning electron microscopy (FE-SEM), and X-ray powder diffraction)XRD(analyses, Thermogravimetric and Differential thermogravimetric analysis (TGA-DTG), Vibrating-sample magnetometry (VSM), and Energy Dispersive X-Ray Analysis (EDX). The biological activities and functional properties of the prepared magnetic nanobiocomposite were studied. Results proved that this nanobiocomposite is non-toxic to the healthy HEK293T cell line. In addition, the synthesized nanobiocomposite showed an approximately 22 % reduction in cell viability of BT549 cells after 72 h. All results confirmed the anti-cancer properties of nanobiocomposite against breast cancer cell lines. Therefore, the prepared nanobiocomposite is an excellent material that can use for in-vivo application. Finally, the hyperthermia application was evaluated for this nanobiocomposite. The SAR was measured 93.08 (W/g) at 100 kHz.

ANT-Mediated Inhibition of the Permeability Transition Pore Alleviates Palmitate-Induced Mitochondrial Dysfunction and Lipotoxicity.

Hyperlipidemia is a major risk factor for vascular lesions in diabetes mellitus and other metabolic disorders, although its basis remains poorly understood. One of the key pathogenetic events in this condition is mitochondrial dysfunction associated with the opening of the mitochondrial permeability transition (MPT) pore, a drop in the membrane potential, and ROS overproduction. Here, we investigated the effects of bongkrekic acid and carboxyatractyloside, a potent blocker and activator of the MPT pore opening, respectively, acting through direct interaction with the adenine nucleotide translocator, on the progression of mitochondrial dysfunction in mouse primary lung endothelial cells exposed to elevated levels of palmitic acid. Palmitate treatment (0.75 mM palmitate/BSA for 6 days) resulted in an 80% decrease in the viability index of endothelial cells, which was accompanied by mitochondrial depolarization, ROS hyperproduction, and increased colocalization of mitochondria with lysosomes. Bongkrekic acid (25 µM) attenuated palmitate-induced lipotoxicity and all the signs of mitochondrial damage, including increased spontaneous formation of the MPT pore. In contrast, carboxyatractyloside (10 μM) stimulated cell death and failed to prevent the progression of mitochondrial dysfunction under hyperlipidemic stress conditions. Silencing of gene expression of the predominate isoform ANT2, similar to the action of carboxyatractyloside, led to increased ROS generation and cell death under conditions of palmitate-induced lipotoxicity in a stably transfected HEK293T cell line. Altogether, these results suggest that targeted manipulation of the permeability transition pore through inhibition of ANT may represent an alternative approach to alleviate mitochondrial dysfunction and cell death in cell culture models of fatty acid overload.

Green synthesis, characterization and biological activity analysis of silver nanoparticles from commercially available root powder of Picrorhiza kurroa Royle ex Benth.

Picrorhiza kurroa Royle ex Benth., a medicinal plant native to the alpine regions of the Himalayas, is renowned for its diverse therapeutic properties. Recently, green-synthesized silver nanoparticles have gained attention for their potential in pharmaceutical and industrial applications. In this study, silver nanoparticles (Pk-AgNPs) were synthesized using commercially available root powder of P. kurroa. The objective was to evaluate their potential biomedical applications by assessing their antioxidant properties and cytotoxicity. The Pk-AgNPs were characterized using Scanning Electron Microscopy (SEM) and Fourier-Transform Infrared Spectroscopy (FT-IR), which revealed their morphology and functional groups. The Total Antioxidant Capacity (TAC) of methanolic root extract of P. kurroa (PkRE) and the Pk-AgNPs was determined using the Phosphomolybdenum method. The results indicated that the antioxidant activity of PkRE was higher than that of the Pk-AgNPs. Moreover, the activity increased with concentration, ranging from 20 µg/mL to 120 µg/mL, demonstrating their capability to eliminate harmful free radicals. The cytotoxicity of PkRE and Pk-AgNPs was evaluated using the MTT assay against the SV40 T-antigen-containing HEK293T cell line, derived from human embryonic kidney 293 cells and the L929 cell line, a mouse fibroblast cell line. The results demonstrated dose-dependent cytotoxicity. L929 cells remained 100 % viable at concentration up to 200 µg/mL for PkRE and 100 µg/mL Pk-AgNPs. In contrast, HEK293T cells didn’t show 100 % viability even at a concentration of 50 µg/mL for both samples. This study highlights the potential use of PkRE and Pk-AgNPs as antioxidants. However, the cytotoxicity findings suggests that they may be harmful to humans if consumed orally, though they could be suitable for use in other organisms. This paves the way for further research into the application of AgNPs synthesized from P. kurroa in biomedical and pharmaceutical domains as well as in veterinary sciences.

Anticancer potential of NSAID-derived tris(pyrazolyl)methane ligands in iron(II) sandwich complexes.

Tris(pyrazolyl)methane (tpm), 2,2,2-tris(pyrazolyl)ethanol (tpmOH) and its esterification derivatives with ibuprofen and flurbiprofen (tpmIBU and tpmFLU) were used as ligands to obtain complexes of the type [Fe(tpmX)2]Cl2 (1-4). The tpmIBU and tpmFLU ligands and corresponding complexes 3 and 4 were characterized by IR and multinuclear NMR spectroscopy, and the structure of tpmIBU was elucidated by single crystal X-ray diffraction. Complexes 1-4 were also assessed for their behaviour in aqueous media (solubility in D2O, octanol/water partition coefficient, stability in physiological-like conditions). The antiproliferative activity of ligands and complexes was determined on A2780, A2780cis and A549 cancer cell lines and the non-cancerous HEK 293T and BJ cell lines. The ligands and complexes were investigated for their ability to inhibit COX-2 (cyclooxygenase) and HNE (4-hydroxynonenal) enzymes. Complexes 3 and 4 exhibited cytotoxicity that may be attributed predominantly to their bioactive fragments, while DNA binding and enhancement of ROS production do not appear to play any significant role.

Novel BRICHOS-related defensin-like antimicrobial peptide from the marine polychaeta &lt;i&gt;Arenicola marina&lt;/i&gt;

To date, polychaetes remain a poorly studied class of invertebrate animals in the context of clarification of their immune system functioning and, in particular, of antimicrobial peptides (AMPs) biodiversity. AMPs, also known as host defense peptides, play a key role in host protection from various pathogens and regulation of the species composition of symbiotic microbes. The study of biosynthesis of AMPs in polychaetes has revealed an interesting pattern, namely so-called BRICHOS domain in the precursor proteins of a number of such peptides. The conserved structure of this domain allows to perform a bioinformatic search for AMP precursors in polychaete transcriptomes. In this work, we found and studied a new BRICHOS-associated AMP from the lugworm Arenicola marina, which represents a structural family of defensin-like peptides stabilized by four disulfide bonds, not previously identified in marine worms. The peptide, designated as AmBRI-44a, contained 44 amino acid residues and was obtained by heterologous expression in Escherichia coli. AmBRI-44a was shown to have a specific activity against a narrow spectrum of Gram-positive bacteria and did not exhibit pronounced cytotoxic effects on eukaryotic cell line HEK293T. A potential mechanism of the antibacterial action of this peptide may be associated with inhibition of bacterial cell wall biosynthesis, as indicated by genetic and phenotypic analysis of selected AmBRI-44a-resistant bacteria Bacillus licheniformis B-511. The results obtained allow us to consider the novel peptide AmBRI-44a as a candidate compound for the development of an antibiotic agent that could potentially be effective in the treatment of infectious diseases mediated by multidrug-resistant Gram-positive bacteria.

SEC16A Variants Predispose to Chronic Pancreatitis by Impairing ER-to-Golgi Transport and Inducing ER Stress.

Chronic pancreatitis (CP) is a complex disease with genetic and environmental factors at play. Through trio exome sequencing, a de novo SEC16A frameshift variant in a Chinese teenage CP patient is identified. Subsequent targeted next-generation sequencing of the SEC16A gene in 1,061 Chinese CP patients and 1,196 controls reveals a higher allele frequency of rare nonsynonymous SEC16A variants in patients (4.90%vs 2.93%; odds ratio [OR], 1.71; 95% confidence interval [CI], 1.26-2.33). Similar enrichments are noted in a French cohort (OR, 2.74; 95% CI, 1.67-4.50) and in a biobank meta-analysis (OR, 1.16; 95% CI, 1.04-1.31). Notably, Chinese CP patients with SEC16A variants exhibit a median onset age 5 years earlier than those without (40.0vs 45.0; p=0.012). Functional studies using three CRISPR/Cas9-edited HEK293T cell lines show that loss-of-function SEC16A variants disrupt coat protein complex II (COPII) formation, impede secretory protein vesicles trafficking, and induce endoplasmic reticulum (ER) stress due to protein overload. Sec16a+/- mice, which demonstrate impaired zymogen secretion and exacerbated ER stress compared to Sec16a+/+, are further generated. In cerulein-stimulated pancreatitis models, Sec16a+/- mice display heightened pancreatic inflammation and fibrosis compared to wild-type mice. These findings implicate a novel pathogenic mechanism predisposing to CP.

Jingzhi Niuhuangjiedu Tablet Ameliorates Oral Mucositis via the AKT/NFκB/NLRP3 Signaling Pathway: A Network Pharmacology and Experimental Validation.

Oral Mucositis (OM) is a common and highly symptomatic complication of cancer therapy that affects patient function and quality of life. Jingzhi Niuhuangjiedu Tablet (JNT) is derived from the famous Chinese herbal formulas Huanglian Jiedu and Fangfeng Tongsheng decoctions, which have been widely used to treat heat toxin syndrome diseases, such as acute pharyngitis, periodontitis, oral ulcers, and oral mucositis (OM), but the underlying mechanism remains unclear. This study validated the efficacy and explored the potential mechanisms of JNT in the treatment of OM by integrating network pharmacological analyses and experimental verification. Network pharmacology and molecular docking techniques were used to predict the active components, key targets, and potential mechanisms of action of JNT against OM. The rat OM model was established by administering 5-Fluorouracil (5-FU) and acetic acid to the rat oral mucosa. Lipopolysaccharide (LPS)-treated human gingival fibroblasts (HGFs) were used as an inflammatory cell model. The GFP-NFκB HEK293T cell line was transfected to evaluate the anti-NFκB activity of JNT. A total of 236 Chinese herbal components and 201 corresponding targets were predicted for OM treatment using JNT. Bicuculine, luteolin, wogonin, and naringenin were identified as the important active compounds, while AKT1, ALB, IL6, MAPK3, and VEGFA were considered to be the major targets. Molecular docking revealed that these active compounds exhibited strong binding interactions with their targets. In vivo and in vitro experiments demonstrated that the anti-OM effect of JNT might be closely related to AKT1, NFκB, caspase-1, and NLRP3, as well as biological processes, such as inflammatory response and oxidative stress. Network pharmacological and experimental evidence indicates that JNT has a potential therapeutic effect on OM by regulating the Akt/NFκB/NLRP3 pathway.

Characterization of a Novel Pathogenic PLCG2 Variant Leading to APLAIDSyndrome Responsive to a TNF Inhibitor.

Autoinflammation and phospholipase C (PLC) γ2-associated antibody deficiency and immune dysregulation (APLAID) syndrome is an autoinflammatory disease caused by gain-of-function variants in PLCG2. This study investigates the pathogenic mechanism of a novel variant of PLCG2 in a patient with APLAID syndrome. Whole-exome sequencing and Sanger sequencing were used to identify the pathogenic variant in the patient. Single-cell RNA sequencing, immunoblotting, luciferase assay, inositol monophosphate enzyme-linked immunosorbent assay, calcium flux assay, quantitative PCR, and immunoprecipitation were used to define inflammatory signatures and evaluate the effects of the PLCG2 variant on protein functionality and immune signaling. We identified a novel de novo variant, PLCG2 p.D993Y, in a patient with colitis, pansinusitis, skin rash, edema, recurrent respiratory infections, B-cell deficiencies, and hypogammaglobulinemia. The single-cell transcriptome revealed exacerbated inflammatory responses in the patient's peripheral blood mononuclear cells. Expression of the D993Y variant in HEK293T, COS-7, and PLCG2 knock-out THP-1 cell lines showed heightened PLCγ2 phosphorylation; elevated inositol-1,4,5-trisphosphate production and intracellular Ca2+ release; and activation of the MAPK, NF-κB, and NFAT signaling pathways compared with control-transfected cells. In vitro experiments indicated that the D993Y variant altered amino acid properties, disrupting the interaction between the catalytic and autoinhibitory domains of PLCγ2, resulting in PLCγ2 autoactivation. Our findings demonstrated that the PLCG2 D993Y variant is a gain-of-function mutation via impairing its autoinhibition, activating multiple inflammatory signaling pathways, thus leading to APLAID syndrome. This study further broadens the molecular underpinnings and phenotypic spectrum of PLCγ2-related disorders.

Exploring 4-morpholinopyrrolo-[2,3-d]-pyrimidine derivatives as novel anti-cancer agents: Design, synthesis and biological studies

Despite extensive research efforts, cancer continues to pose a significant global health challenge. Continuing the anti-cancer research, twenty-four 4-Morpholinopyrrolo-[2,3-d]-pyrimidine derivatives were synthesized and characterized via HRMS, 1H, 13C NMR, and FTIR. A single crystal for one compound, PGT4, was developed for further confirmation. All synthesized compounds were assessed for their anti-cancer activity in three cancer cell lines, SCC-25 (oral squamous cell carcinoma), U87-MG (glioblastoma) and MCF-7 (breast cancer) and one normal cell line HEK-293T (human embryonic kidney) using WST assay. Fourteen compounds demonstrated IC50 values ranging from 2 to 10 µM against the SCC-25 cell line. PTT3 and PGT7 were the most effective compounds against the SCC-25 cell line, showing an IC50 of 2 µM. Cell cycle analysis was also performed using propidium iodide staining, and apoptosis studies were carried out using caspase 3/7 activity assay and Annexin V-FITC isothiocyanate assay. PTT3 and PGT7 arrested the cell cycle at the G1 phase and enhanced the apoptosis by 9.1 and 13.8-folds in SCC-25 cells, respectively. Treatment with PTT3 and PGT7 also resulted in a dose-dependent decrease in the colony formation of SCC-25 cells. We carried out the molecular docking and dynamic studies to find the insights on binding mechanisms of PTT3 and PGT7 within the active region of the PI3Kγ target protein (PDB ID:3IBE, 2.80Å). Additionally, in-silico ADME properties were predicted for the most active molecules, PTT3 and PGT7, aiding in assessing their pharmacokinetic profiles.

Construction of Cell Membrane Chromatography Screening Materials Based on Avi-Tag Fused G Protein-Coupled Receptors.

Mas-related G protein-coupled receptor X2 (MrgprX2) plays a crucial role in anaphylactoid reactions and allergic diseases. Some antagonists with reasonable potency and selectivity have been reported. Cell membrane chromatography (CMC) is effective for discovering ligands. Protein-tag-based CMC models (e.g., SNAP tags and HALO tags) have enhanced performance but also increased nonspecific adsorption of small molecules. The Avi tag, a short peptide sequence, binds biotin specifically via BirA catalysis. Our study showed that 2-iminobiotin (IB) can be a BirA substrate, enabling the development of a new cell membrane stationary phase (CMSP) based on the chemical properties (modifying carboxyl silica gel and specifically labeling the Avi tag) of IB. First, we constructed the MrgprX2-Avi-tag HEK293T cell line. Next, we synthesized IB-modified silica gel (SiO2-IB) stepwise. Finally, we immobilized Avi-tagged MrgprX2 cell membranes on SiO2-IB under BirA catalysis. We characterized the developed CMSP and used it to establish a MrgprX2-Avi-tag/CMC-HPLC/MS two-dimensional screening platform, successfully screening vitexicarpin fromViticis Fructus extract via a 2D/CMC platform. In vitro and in vivo experiments confirmed that vitexicarpin targets the MrgprX2 receptor, demonstrating antiallergic effects. Our IB-Avi tag-based CMC approach effectively decreased nonspecific adsorption of the screening materials. The Avi-tag-based 2D/CMC platform is suitable for screening potential drug candidates.

Circ_0043314 Modulates Proliferation and Apoptosis of Ovarian Granulosa Cells in Polycystic Ovarian Syndrome via the MicroRNA-146b-3p/Apelin 13 Axis.

Polycystic ovary syndrome (PCOS) is a common endocrine and metabolic disorder in women. At present, the pathogenesis has not been clarified, and the clinical application of drugs and lifestyle intervention may not prevent disease progression. This study aimed to investigate how circ_0043314 regulates ovarian granulosa cell biological functions to provide a theoretical basis for the treatment of patients with PCOS. MicroRNA (miR)-146b-3p/Apelin 13 axis was used to investigate the mechanism by which circ_0043314 regulated ovarian granulosa cell proliferation and apoptosis in PCOS via miR-146b-3p/Apelin 13 axis. Participants/Materials, Methods: Ovarian tissues (cortical tissues) from 35 PCOS patients and 35 normal controls, as well as HEK293T and human ovarian granulosa cell line (KGN, COV434), were included in this study. We examined the expression levels of circ_0043314, miR-146b-3p, and Apelin 13 in PCOS tissues. Ovarian granulosa cells were transfected with corresponding plasmids to clarify the influence of circ_0043314, miR-146b-3p, or Apelin 13 on proliferation and apoptosis of ovarian granulosa cells through MTT and flow cytometry assays. Moreover, the relationships among circ_0043314, miR-146b-3p, and Apelin 13 were analyzed through dual-luciferase and RNA immunoprecipitation assays. Circ_0043314 and Apelin 13 were highly expressed and miR-146b-3p was lowly expressed in ovarian tissues of PCOS compared with non-PCOS controls. Downregulation of circ_0043314 or upregulation of miR-146b-3p hindered ovarian granulosa cell proliferation and advanced its apoptosis. Downregulation of miR-146b-3p reversed the impacts of downregulation of circ_0043314, and overexpression of Apelin 13 counteracted the influences of upregulation of miR-146b-3p in ovarian granulosa cells. Mechanically, circ_0043314 could bind to miR-146b-3p, and miR-146b-3p directly targeted and modulated Apelin 13 expression. This study was limited by the lack of animal experiments. Our data demonstrated that circ_0043314 enhances ovarian granulosa cell proliferation and suppresses its apoptosis via miR-146b-3p/Apelin 13 axis.

CRISPR Base Editing to Create Potential Charcot-Marie-Tooth Disease Models with High Editing Efficiency: Human Induced Pluripotent Stem Cell Harboring SH3TC2 Variants.

Human induced pluripotent stem cells (hiPSCs) represent a powerful tool to investigate neuropathological disorders in which the cells of interest are inaccessible, such as in the Charcot-Marie-Tooth disease (CMT), the most common inherited peripheral neuropathy. Developing appropriate cellular models becomes crucial in order to both study the disease's pathophysiology and test new therapeutic approaches. The generation of hiPS cellular models for disorders caused by a single nucleotide variation has been significantly improved following the development of CRISPR-based editing tools. In this study, we efficiently and quickly generated, by CRISPR editing, the two first hiPSCs cellular models carrying alterations involved in CMT4C, also called AR-CMTde-SH3TC2. This subtype of CMT is associated with alterations in the SH3TC2 gene and represents the most prevalent form of autosomal recessive demyelinating CMT. We aimed to develop models for two different SH3TC2 nonsense variants, c.211C>T, p.Gln71* and the most common AR-CMTde-SH3TC2 alteration, c.2860C>T, p.Arg954*. First, in order to determine the best CRISPR strategy to adopt on hiPSCs, we first tested a variety of sgRNAs combined with a selection of recent base editors using the conveniently cultivable and transfectable HEK-293T cell line. The chosen CRISPR base-editing strategy was then applied to hiPSCs derived from healthy individuals to generate isogenic CMT disease models with up to 93% editing efficiency. For point mutation generation, we first recommend to test your strategies on alternative cell line such as HEK-293T before hiPSCs to evaluate a variety of sgRNA-BE combinations, thus boosting the chance of achieving edited cellular clones with the hard-to-culture and to transfect hiPSCs.