Search Results (201)

Extracellular vesicles (EVs), nanoscale membrane-enclosed particles, are natural carriers of proteins and nucleic acids. Microalgae are widely used as a source of bioactive substances in the food and cosmetic industries and definitely have a potential to be used as the producers of EVs for biomedical applications. In this study, the extracellular vesicles isolated from the culture medium of two unicellular microalgae, Chlamydomonas reinhardtii (Chlamy-EVs) and Parachlorella kessleri (Chlore-EVs), were characterized by atomic force microscopy (AFM), cryo-electronic microscopy (cryo-EM), and nanoparticle tracking analysis (NTA). The biocompatibility with human cells in vitro (HEK-293T, DF-2 and A172) and biodistribution in mouse organs and tissues in vivo were tested for both microalgal EVs. An exogenous therapeutic protein, human heat shock protein 70 (HSP70), was successfully loaded to Chlamy- and Chlore-EVs, and its efficient delivery to human glioma and colon carcinoma cell lines has been confirmed. Additionally, in order to search for potential therapeutic biomolecules within the EVs, their proteomes have been characterized. A total of 105 proteins were identified for Chlamy-EVs and 33 for Chlore-EVs. The presence of superoxide dismutase and catalase in the Chlamy-EV constituents allows for considering them as antioxidant agents. The effective delivery of exogenous cargo to human cells and the possibility of the particle yield optimization by varying the microalgae growth conditions make them favorable producers of EVs for biotechnology and biomedical application. Full article

Plant flowers are recognized as a rich source of bioactive phenolic compounds. In this study, for the first time, the recovery of antioxidant phenolic compounds from Cytisus striatus flowers (CF) was optimized using microwave-assisted extraction (MAE). The variables (% of ethanol, temperature, and time) were studied using a response surface methodology (RSM). Extraction efficiency was assessed by total phenol content, total flavonoid content, and the antioxidant capacity through DPPH, ABTS, FRAP, and CUPRAC assays. Additionally, cytotoxicity and anti-inflammatory properties were evaluated in different cell lines. The optimal extraction conditions (87.6% ethanol, 160.8 °C and 8.76 min) yielded extracts rich in phenolics (85.9 mg GAE/g CF) and flavonoids (120.3 mg RE/g CF), with strong antioxidant capacity. LC-MS/MS analysis identified 27 phenolic compounds, including chrysin, apigenin, and quercetin derivatives. Cytotoxicity tests showed that CF extract maintained high viability (>80%) in human embryonic kidney (HEK293T) and human lung adenocarcinoma (A549) cells up to 2000 µg/mL, indicating low cytotoxicity. The anti-inflammatory potential was evidenced by a decrease in IL-1β levels and an increase in IL-10 cytokine production in LPS-stimulated macrophages. These results highlight the great potential of CF as a promising bioresource to obtain value-added compounds for the development of functional foods, nutraceuticals, and cosmetic products. Full article

Dysferlinopathy is caused by over 500 mutations in the gene encoding dysferlin, including close to 300 point mutations. One option to cure the disease is to use a gene therapy to correct these mutations at the root. Prime editing is a technique which can replace the mutated nucleotide with the wild-type nucleotide. In this article, prime editing is used to correct several point mutations in the DYSF gene responsible for dysferlinopathy. In vitro editing of HEK293T cells reaches up to 31%. Notably, editing was more efficient in myoblasts than in patient-derived fibroblasts. The prime editing technique was also used to create a new myoblast clone containing a patient mutation from a healthy myoblast cell line. Full article

Background and Objectives: Myricetin, a flavonoid compound, was demonstrated to effectively arrest the cell-to-cell fusion and syncytial development triggered by Nipah virus (NiV) fusion (F) and attachment (G) envelope glycoproteins in vitro involving two permissive mammalian cell lines. Methods: Time-of-addition assays were carried out using codon-optimized NiV wild type (WT) F and G plasmids followed by a challenge with the addition of myricetin 1 h and 6 h post-transfection in HEK 293T and Vero cells. Results: Upon evaluating different myricetin concentrations, it was determined that a 100 μM concentration of myricetin effectively inhibited 64–80% of syncytia in HEK and Vero cells. Interpretation & Conclusions: In this study, we concluded that myricetin mitigated the syncytial development in HEK and Vero cell lines. Given the flavonoid attributes of myricetin which is widely present in fruits, vegetables, tea, and wine, it may be regarded as a phytonutrient and a safer antiviral alternative against Nipah virus infections. Due to the BSL-4 nature of the virus, further research involving live virus culture is necessary to confirm myricetin as a potential antiviral compound for the mitigation of pathological effects of NiV infections. Full article

The phytochemical screening of extracts of Tacca leontopetaloides tubers has afforded the isolation of two novel chalcones, tarkalynins A and B, along with taccalonolide A and its 12-propanoate. The screening of Zanthoxylum zanthoxyloides stem bark yielded taraxerol acetate, dihydrochelerythrin and fagaramide. These compounds were obtained through column and thin-layer chromatography and identified using NMR and LC-HRMS. The compounds were tested against Trypanosoma brucei brucei s427 and its multi-drug-resistant clone B48, against Trypanosoma evansi, Trypanosoma equiperdum and Trypanosoma congolense, and against Leishmania mexicana. Cytotoxicity was tested against the human HEK293 cell line. The highest activities were observed with dihydrochelerythrin and fagaramide against T. b. brucei s427 and B48, T. evansi, and L. mexicana, with EC₅₀ values of 1.37, 2.559, 1.09, and 5.44 µM and 17.8, 10.9, 10.9, and 13.3 µM, respectively. In addition, tarkalynin A and taraxerol acetate displayed promising activity against T. equiperdum (EC₅₀ = 21.4 and 21.3 µM, respectively). None of these compounds showed significant cross-resistance with existing trypanocides (RF ≈ 1; p > 0.05). The compounds displayed low toxicity to human cells, with most exhibiting no growth inhibition at concentrations of 100, or even 300 µM. This report provides further evidence of the potential use of natural products for combating parasitic diseases. Full article

The NOS1AP gene encodes the nitric oxide synthase 1 adaptor protein (NOS1AP), which binds to neuronal nitric oxide synthase (nNOS) and regulates nitric oxide (NO) production by dissociating nNOS from NMDA receptors (NMDARs). Notably, NOS1AP expression is upregulated upon NMDAR activation; however, there is no available data regarding its production under the receptor inhibition. The NOS1AP gene is also 1 among more than 1000 genes that are presumed to be associated with the development of schizophrenia. Various animal models of this disorder have been developed, some of which are based on the use of NMDAR antagonists such as dizocilpine (MK-801). In this study, we investigated the expression and production of NOS1AP in rats injected with a low dose of dizocilpine (0.1 mg/kg), as well as in SH-SY5Y and HEK293T cell lines treated with varying concentrations of the same compound (10–200 µM). According to our results, neither the expression of the NOS1AP gene nor the production of NOS1AP protein was affected by dizocilpine treatment. Full article

Human T cell leukemia virus type 1 (HTLV 1) is an oncogenic retrovirus responsible for the development of adult T cell leukemia (ATL). The minus strand of HTLV-1 provirus encodes an oncoprotein named HTLV-1 bZIP factor (HBZ), which plays a pivotal role in viral replication and T cell proliferation. Of particular interest is the spliced HBZ isoform (sHBZ), which is predominantly expressed in ATL cells and localizes within the nucleolus, conferring immortalizing properties to T cells. Our previous study has shown that sHBZ colocalizes and associates with Nucleophosmin/B23, a nucleolar phosphoprotein with multiple functions. In this study, through an optimized nucleolar isolation method, we first confirmed sHBZ’s nucleolar localization via Western blotting in transfected HEK293T cells, chronically HTLV-1-infected T cell lines, and freshly infected HeLa cells. We further demonstrated that the sHBZ/B23 association predominantly occurs in the nucleolus by co-immunoprecipitation of cell fractions. Our study highlights the nucleolar localization of sHBZ and its possibly essential interaction with this nucleolar-residing protein, leading to cell immortalization. Full article

For steady transgenic expression, lentiviral vector-mediated gene delivery is a commonly used technique. One question that needs to be explored is how external lentiviral vectors and overexpressed genes perturb cellular homeostasis, potentially altering transcriptional networks. In this study, two Human Embryonic Kidney 293T (HEK293T)-derived cell lines were established via lentiviral transduction, one overexpressing green fluorescent protein (GFP) and the other co-overexpressing GFP and ADORA3 following puromycin selection to ensure stable genomic integration. Genes with differentially transcript utilization (gDTUs) and differentially expressed genes (DEGs) across cell lines were identified after short-read and long-read RNA-seq. Only 31 genes were discovered to have changed in expression when GFP was expressed, although hundreds of genes showed variations in transcript use. In contrast, even when co-overexpression of GFP and ADORA3 alters the expression of more than 1000 genes, there are still less than 1000 gDTUs. Moreover, DEGs linked to ADORA3 overexpression play a major role in RNA splicing, whereas gDTUs are highly linked to a number of malignancies and the molecular mechanisms that underlie them. For the analysis of gene expression data from stable cell lines derived from HEK293T, our findings provide important insights into changes in gene expression and alternative splicing. Full article

This study aims to enhance membrane protein production in HEK293T cells through genetic modification. HEK293T cells are used for recombinant protein and viral vector production due to their human origin and post-translational modification capabilities. This study explores enhancing membrane protein production in these cells by deleting the C-terminal of the ATF6B gene using CRISPR-Cas9 technology. The objective of this research is to investigate the effect of C-terminal deletion of the ATF6B gene on membrane protein production in HEK293T cells using CRISPR-Cas9 technology. To identify effective gene targets, sgRNAs were initially designed against multiple UPR-related genes, including ATF6A, IRE1A, IRE1B, PERK, and ATF6B. Among them, ATF6B was selected as the primary target for further investigation due to its superior editing efficiency. The efficiency of sgRNAs was evaluated using the T7E1 assay, and sequencing was performed to verify gene editing patterns. Membrane proteins were extracted from both ATF6B C-terminally deleted (ATF6B-ΔC) and wild-type (WT) cell lines for comparison. Flow cytometry was employed to assess membrane protein production by analyzing GFP expression in Membrane-GFP-expressing cells. HEK293T cells with C-terminally deleted ATF6B (ATF6B-ΔC) significantly increased membrane protein production by approximately 40 ± 17.6% compared to WT cells (p < 0.05). Sequencing revealed 11, 14, 1, and 10 bp deletions in the ATF6B-ΔC edited cells, which disrupted exon sequences, induced exon skipping, and introduced premature stop codons, suppressing normal protein expression. Flow cytometry confirmed a 23.9 ± 4.2% increase in GFP intensity in ATF6B-ΔC cells, corroborating the enhanced membrane protein production. These findings suggest that CRISPR-Cas9-mediated C-terminal deletion of the ATF6B gene can effectively enhance membrane protein production in HEK293T cells by activating the unfolded protein response pathway and improving the cell’s capacity to manage misfolded proteins. This strategy presents significant potential for the biotechnology and pharmaceutical industries, where efficient membrane protein production is essential for drug development and various applications. Full article

Background: Staphylococcus aureus is a highly lethal Gram-positive bacterium that is responsible for over one million deaths annually. As a member of the ESKAPE pathogens, its methicillin-resistant strains (MRSA) are prevalent worldwide and exhibit significant antimicrobial resistance (AMR). Bacterial efflux pumps play a pivotal role in the development of AMR by facilitating the expulsion of a range of antimicrobial agents. Methods: The S. aureus strain SA-1199B, which overexpresses NorA and carries a GrlA mutation, was utilized to comprehensively profile the mechanism of the compounds PQQ16P and PQK4F. To assess the toxicity and genotoxicity of these compounds, RAW macrophages, HEK 293T, and HepG2 cell lines were utilized. Female BALB/c mice were utilized to assess the in vivo synergism of EPIs with CPX, Results: NorA efflux pump inhibitors (EPIs), PQQ16P and PQK4F, enhanced the efficacy of the antibacterial ciprofloxacin (CPX) against resistant S. aureus strains. The mechanism of EPIs involved the inhibition of NorA efflux pump, without compromising bacterial membrane permeability, ATP levels, or mammalian calcium channels. Moreover, the EPIs significantly augmented the bactericidal and post-antibiotic effects of CPX, elevating its mutation prevention concentration without manifesting substantial toxicity to human cells. Furthermore, the EPIs reduced S. aureus invasiveness in macrophages, indicating a role for NorA in bacterial virulence. Notably, the in vivo synergism of these EPIs with CPX was observed in a mouse infection model. Conclusions: This study provides substantial evidence for the potential of employing EPIs in a combination with CPX to counteract AMR, both in vitro and in vivo. Full article

A novel series of oleanolic acid (OA, 1) derivatives incorporating phenolic and coumarin moieties were synthesized. This acid was extracted from olive pomace (Olea europaea L.) using an ultrasound-assisted method. The structures of these novel derivatives of OA were characterized through the utilization of ¹H-NMR, ¹³C-NMR and ESI-HRMS analyses. An evaluation of some biological activities of the prepared derivatives was conducted. The evaluation focused principally on the capacity of these structures to inhibit 15-lipoxygenase and α-glucosidase, as well as their anticancer properties when tested against tumour cell lines (HCT-116 and LS-174T) and a non-tumour cell line (HEK-293). In terms of their cytotoxic activity, the majority of the compounds exhibited notable inhibitory effects compared to the starting molecule, OA. Derivatives 4d, 4k and 4m exhibited particularly strong inhibitory effects against the HCT-116 cell line, with IC₅₀ values of 38.5, 39.3, 40.0 µM, respectively. Derivatives 4l, 4e and 5d demonstrated the most effective inhibition against the LS-174T cell line, with IC₅₀ values of 44.0, 44.3, 38.0 µM, respectively. However, compound 2a was the most effective, exhibiting the most potent inhibition of 15-lipoxygenase and α-glucosidase, with IC₅₀ values of 52.4 and 59.5 µM, respectively. Furthermore, molecular docking studies supported in vitro cytotoxic activity, revealing that the most potent compounds exhibited low binding energies and interacted effectively within the EGFR enzyme’s active pocket (PDB: 1M17). These findings highlight the potential of these derivatives as anticancer agents and enzymatic inhibitors, warranting further investigation. Full article

To develop a cell-based in vitro thyroid-stimulating hormone (TSH) biological activity assay that can simulate in vivo pharmacodynamic mechanisms, we constructed two HEK293-TSHR cell lines based on two main cell signaling pathways (Gαs-cAMP-PKA and Gαq/11-PLC-Ca²⁺) that TSH depends on for its in vivo physiological function. These cell lines stably expressed the luciferase reporter driven by the cAMP response element (CRE) and nuclear factor of activated T cells (NFAT) response element, and two reporter-gene assays (RGAs) were correspondingly established and validated. The two transgenic genes could measure signals produced from the simulation of the in vivo effects of TSH from the Gαs-cAMP and Gαq/11-PLC pathways after TSH activation. TSH showed a good dose–response relationship in these two cell lines and conformed to the four-parameter model. We optimized the critical experimental parameters of these two methods and performed comprehensive methodological validation according to the International Council for Harmonization (ICH) Q2 (R1) guidelines, the Chinese Pharmacopoeia, and the United States Pharmacopoeia. The two methods showed good specificity, accuracy, precision, and linearity and can be used to aid in assessments of the biological activity of TSH drugs, product characterization, final product release, stability studies, and comparability studies for biosimilar applications. Full article

We previously reported on HIV vaccines that elicited autologous Tier 2 neutralizing antibodies (nAbs) in rabbits. In the current study, we sought to establish a proof of concept that HIV vaccines using identical designs elicit Tier 2 nAbs in arhesus macaque (RM) model. DNA and MVA vaccines expressing SIV Gag and HIV-1 Env antigens were constructed, and in vitro expression was confirmed. A soluble envelope protein (gp140 Env) was expressed from a stable HEK293 cell line and purified using lectin affinity and size exclusion chromatography. The expression and secretion of SIV Gag and HIV-1 Env by the DNA and MVA vaccines was verified in vitro. Five RMs were inoculated with two DNA, followed by two MVA, and finally with two gp140 Env vaccines at weeks 0, 4, 8, 12, 20 and 28. Vaccine-induced T cell immunity was measured by IFN-γ ELISpot while nAbs were evaluated against MW965 (Tier 1A), 6644 (Tier 1B), autologous ZM109.5A and a closely-related ZM109.B4 (Tier 2) pseudovirions. Vaccinated RMs were challenged intrarectally with simian-human immunodeficiency virus (SHIV), four weeks after the final vaccination, as was an unvaccinated control group (n = 4). Following vaccination, all the animals developed moderate IFN-γ ELISpot responses after the DNA vaccinations which were boosted by the MVA vaccine. After the gp140 Env boost, all animals developed nAbs with peak median titres at 762 (MW965) and 263 (ZM109.5A). The vaccinated animals became infected after a similar number of challenges to the unvaccinated controls, and the resultant number of viral copies in the blood and the lymphoid tissues were similar. However, the duration of detectable viraemia in the vaccinated animals (median: 2 weeks) was shorter than the controls (median: 8.5 weeks). These data show that the vaccines elicited robust cellular and functional humoral immune responses that resulted in a quicker control of viraemia. Full article

Neutralization assays have become an important tool since the beginning of the COVID-19 pandemic for testing vaccine responses and therapeutic antibodies as well as for monitoring humoral immunity to SARS-CoV-2 in epidemiological studies. The spike glycoprotein (S) present on the viral surface contains a receptor binding domain (RBD) that recognizes the angiotensin-converting enzyme 2 receptor (ACE2) in host cells, allowing virus entry. The gold standard for determining SARS-CoV-2 neutralizing antibodies is the plaque reduction neutralization test (PRNT), which relies on live-virus replication performed exclusively in biosafety level 3 (BSL-3) laboratories. Here, we report the development of a surrogate live-cell imaging-based fluorescent SARS-CoV-2 neutralization assay, applicable to BSL-1 or BSL-2 laboratories, by antibody-mediated blockage of the interaction between recombinant RBD with overexpressed ACE2 receptor in a genetically modified HEK 293T stable cell line. Our approach was able to detect neutralizing antibodies both in COVID-19-positive human serum samples and polyclonal equine formulations against SARS-CoV-2. This new cell-based surrogate neutralization assay represents a virus-free fluorescence imaging alternative to the reported approaches, which can be used to detect antibody-neutralizing capabilities toward SARS-CoV-2. This assay could also be extrapolated in the future to other established and emergent viral agents. Full article

The aim of this study was to investigate the usefulness of human embryonic kidney HEK293 cells as a model of normal cells in biotin-mediated therapy. The expression and role of sodium multivitamin transporter (SMVT) in the uptake and accumulation of free biotin, as well as cationic and neutral biotinylated PAMAM dendrimers of the fourth generation synthesized in our laboratory, were assessed in HEK293 cells in comparison to other immortalized (HaCaT) and cancer cells (HepG2, U-118 MG). The obtained data showed that a higher level of SMVT in HEK293 cells was not associated with a stronger uptake of biotin and biotinylated PAMAM dendrimers. Biotinylation increased the selective uptake of neutral dendrimers in an inversely proportional manner to the concentration used; however, the accumulation in HEK293 cells was lower than that in cells of other cell lines. The time-dependent biotin and biotinylated dendrimers uptake profiles differed significantly. Therefore, it should be assumed that the efficiency of biotinylated nanoparticles’ uptake depends on multiple cellular transport mechanisms. Toxicity tests showed significantly higher sensitivity to PAMAM conjugates for HEK293 cells than for HepG2 and HaCaT cells. Molecular modeling studies and the profile of biotin uptake suggest that not only SMVT but also monocarboxylate transporter 1 (MCT-1) may play an important role in the selective transport of biotin and biotinylated nanoparticles into cells. Due to the complexity of the problem, further studies are necessary. In summary, HEK293 cells can be considered a valuable model of normal cells in the study of biotin- targeted therapy using nanoparticles based on PAMAM dendrimers. Full article