Human Embryonic Kidney Cell Line Research Articles

Quinic acid enhances kanamycin efficacy against methicillin-resistant Staphylococcus aureus biofilms.

Methicillin-resistant Staphylococcus aureus (MRSA) form biofilms that contribute to increased antimicrobial resistance, leading to treatment failure and/or relapse. It is, therefore, necessary to develop new antibiofilm strategies to eradicate MRSA biofilms. This study was aimed to evaluate the effect of the combination of quinic acid and kanamycin against the preformed biofilms of methicillin-resistant Staphylococcus aureus. Broth microdilution method was deployed to evaluate antibacterial activity. Antibiofilm activity was evaluated by crystal violet staining, 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium-bromide (MTT) assay, and scanning electron microscopy (SEM). The checkerboard method was adopted to assess the combination effects. Quantification of exopolysaccharides was determined by the phenol-sulfuric acid method. The eDNA was quantified by spectrophotometry. Cytotoxic activity was evaluated by the MTT assay on the human embryonic kidney (HEK 293) cell line. Quinic acid, combined with kanamycin, effectively eradicated the methicillin-resistant S. aureus biofilms by affecting biofilm biomass and cell viability. Scanning electron microscopy demonstrated a less adherence of S. aureus cells was observed after treatment with quinic acid combined with kanamycin, as compared to each drug alone. The combination of quinic acid and kanamycin thus demonstrated the ability to destroy the exopolysaccharides and eDNA of biofilm matrix without any toxic effect on HEK 293 cells. Our results clearly demonstrate the possibility of using quinic acid in combination therapy, with an antibiotic, for combating infections caused by MRSA strains.

Synthesis and Evaluation of 3,5-Disubstituted-1,2,4-Oxadiazolyl Benzamides as Potential Anti-Breast Cancer Agents: In Vitro and In Silico Studies.

Herein, the synthesis, anti-cancer evaluation, and in silico studies of a series of 1,2,4-oxadiazole compounds (8-15) are disclosed. The synthesized molecules were tested in vitro for anti-cancer activity against MCF-7, MDA-MB-231, HeLa, Ishikawa cell lines and human embryonic kidney (HEK-293) cell lines. Among the synthesized compounds, 9 and 15 exhibited significant cytotoxicity, with IC50 values of 7.82 μM and 6.02 μM, respectively, against MCF-7 cell line, better than that of anti-breast cancer drug, tamoxifen (IC50 = 11.92 μM), used as control. Significantly, both 9 and 15 exhibited very low toxicity (IC50 > 20 µM) against normal HEK-293 cells. This suggests them as potentially effective anti-cancer lead molecules. The in vitro anti-cancer data was supported by in silico studies which also identified compounds 9 and 15 as potent inhibitors of the 17β-hydroxysteroid dehydrogenase1 (17β-HSD1) proteins, demonstrating strong interactions and stability The atom-based QSAR model exhibited high accuracy, significant regression, and predictive reliability, aiding in understanding and optimizing biological activity. The drug-likeness study of compounds 9 and 15 indicated favorable pharmacokinetics, with in silico toxicity predictions showing compound 15 to be non-toxic. These findings suggest compounds 9 and 15 as potential lead molecules against breast cancer.

Polydopamine-Mediated Antimicrobial Lipopeptide Surface Coating for Medical Devices.

Biofilm formation on medical implants such as catheters is a major issue which needs to be addressed as it leads to severe health care associated infections. This study explored the design and synthesis of a polydopamine-lipopeptide based antimicrobial coating. The coating was used to modify the surface of Ultrathane Catheters. The lipopeptide SL1.15 with an N-terminal cysteine was covalently conjugated to the polydopamine modified catheters via a Michael addition reaction between the thiol moiety in the peptide and the aromatic ring in the polydopamine layer. The immobilization of the peptide on the polydopamine coated catheters was confirmed using water contact angle, X-ray photoelectron spectroscopy, atomic force microscopy, and scanning electron microscopy (SEM). The antimicrobial activity of the coated catheters investigated using drug resistant and clinical strains of Gram-positive (MRSA and S. aureus) and Gram-negative (E. coli, A. baumannii, and P. aeruginosa) bacteria revealed that lipopeptide immobilization inhibited >90% bacterial adhesion to the catheter surface. Additionally, biofilm assays against MRSA and E. coli revealed that the lipopeptide immobilized catheters inhibited >85% bacterial growth after 1 week incubation. Finally, the cytotoxicity profile of the catheters using the human dermal fibroblast, and the human embryonic kidney cell lines demonstrated that the polydopamine-lipopeptide coating was not toxic after 72 h incubation.

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.

What is the real value of omics data? Enhancing research outcomes and securing long-term data excellence.

A wealth of high-throughput biological data, of which omics constitute a significant fraction, has been made publicly available in repositories over the past decades. These data come in various formats and cover a range of species and research areas providing insights into the complexities of biological systems; the public repositories hosting these data serve as multifaceted resources. The potentially greater value of these data lies in their secondary utilization as the deployment of data science and artificial intelligence in biology advances. Here, we critically evaluate challenges in secondary data use, focusing on omics data of human embryonic kidney cell lines available in public repositories. The emerging issues are obstacles faced by secondary data users across diverse domains as they concern platforms and repositories, which accept deposition of data irrespective of their species type. The evolving landscape of data-driven research in biology prompts re-evaluation of open access data curation and submission procedures to ensure that these challenges do not impede novel research opportunities through data exploitation. This paper aims to draw attention to widespread issues with data reporting and encourages data owners to meticulously curate submissions to maximize not only their immediate research impact but also the long-term legacy of datasets.

7-(Substituted amino)-5-methylthioazolo[1,5-a]pyrimidines: Synthesis, cytotoxic properties in vitro and molecular docking

A method for the synthesis of new 7-(substituted amino)-5-methylthioazolo[1,5-a]pyrimidines has been developed. Based on the MTT test, IC50 values were calculated for the obtained compounds against lung carcinoma (A549), liver carcinoma (HepG2), embryonal rhabdomyosarcoma (Rd) and human embryonic kidney (HEK 293) cell lines. Some compounds from the series demonstrated activity close to the reference drug, but with a certain selectivity. Based on the results of MTT assay and molecular docking studies for the catalytic subunits of PI3K, two isoforms (PI3Kβ and PI3Kδ) were assumed as the targets for the new series of azolo[1,5-a]pyrimidines with cytotoxic effect on the rhabdomyosarcoma cell line.

Optimization of Exon-Skipping Riboswitches and Their Applications to Control Mammalian Cell Fate.

Mammalian riboswitches that can regulate transgene expression via RNA-small molecule interaction have promising applications in medicine and biotechnology, as they involve no protein factors that can induce immunogenic reactions and are not dependent on specially engineered promoters. However, the lack of cell-permeable and low-toxicity small molecules and cognate aptamers that can be exploited as riboswitches and the modest switching performance of mammalian riboswitches have limited their applications. In this study, we systematically optimized the design of a riboswitch that regulates exon skipping via an RNA aptamer that binds ASP2905. We examined two design strategies to modulate the stability of the aptamer base stem that blocks the 5' splice site to fine-tune the riboswitch characteristics. Furthermore, an optimized riboswitch was used to generate a mouse embryonic stem cell line that can be chemically induced to differentiate into myogenic cells by activating Myod1 expression and a human embryonic kidney cell line that can be induced to trigger apoptosis by activating BAX expression. The results demonstrate the tight chemical regulation of transgenes in mammalian cells to control their phenotype without exogenous protein factors.

Salmonella Detection in Food Using a HEK-hTLR5 Reporter Cell-Based Sensor.

The development of a rapid, sensitive, specific method for detecting foodborne pathogens is paramount for supplying safe food to enhance public health safety. Despite the significant improvement in pathogen detection methods, key issues are still associated with rapid methods, such as distinguishing living cells from dead, the pathogenic potential or health risk of the analyte at the time of consumption, the detection limit, and the sample-to-result. Mammalian cell-based assays analyze pathogens' interaction with host cells and are responsive only to live pathogens or active toxins. In this study, a human embryonic kidney (HEK293) cell line expressing Toll-Like Receptor 5 (TLR-5) and chromogenic reporter system (HEK dual hTLR5) was used for the detection of viable Salmonella in a 96-well tissue culture plate. This cell line responds to low concentrations of TLR5 agonist flagellin. Stimulation of TLR5 ligand activates nuclear factor-kB (NF-κB)-linked alkaline phosphatase (AP-1) signaling cascade inducing the production of secreted embryonic alkaline phosphatase (SEAP). With the addition of a ρ-nitrophenyl phosphate as a substrate, a colored end product representing a positive signal is quantified. The assay's specificity was validated with the top 20 Salmonella enterica serovars and 19 non-Salmonella spp. The performance of the assay was also validated with spiked food samples. The total detection time (sample-to-result), including shortened pre-enrichment (4 h) and selective enrichment (4 h) steps with artificially inoculated outbreak-implicated food samples (chicken, peanut kernel, peanut butter, black pepper, mayonnaise, and peach), was 15 h when inoculated at 1-100 CFU/25 g sample. These results show the potential of HEK-DualTM hTLR5 cell-based functional biosensors for the rapid screening of Salmonella.

Production of a magnetic nanocomposite for biological and hyperthermia applications based on chitosan-silk fibroin hydrogel incorporated with carbon nitride

Hydrogels based on natural polymers have lightened the path of novel drug delivery systems, wound healing, and tissue engineering fields because they are renewable, non-toxic, biocompatible, and biodegradable. Furthermore, applying modified hydrogels can upgrade their biological activity. Herein, Chitosan (CS) was used to create a hydrogel using terephthaloyl thiourea as a cross-linker. Silk fibroin (SF) and carbon nitride (CN) were added to the hydrogel to enhance its strength and biocompatibility. Finally, CS hydrogel/SF/CN was in situ magnetized using Fe3O4 magnetic nanoparticles (MNPs) and manufactured as a nanobiocomposite for improved hyperthermia. The structural properties of the nanobiocomposite were assessed using several analytical techniques, including VSM, FTIR, TGA, EDX, XRD, and FESEM. The saturation magnetization of this magnetic nanocomposite was 23.94 emu/g. The hemolytic experiment on the nanobiocomposite resulted in ca. 98 % cell survival, with a hemolysis rate of 1.69 %. Anticancer property is confirmed by a 20.0 % reduction in cell viability of BT549 cells at 1.75 mg/mL concentration compared to 0.015 mg/mL. The nanocomposite is non-toxic to the human embryonic kidney cell line (HEK293T), indicating its potential for biomedical applications. Finally, the magnetic nanocomposite's hyperthermia behavior was examined using a specific absorption rate (SAR), achieving the highest value of 47.44 W/g at 200.0 kHz. When subjected to an alternating magnetic field, the nanobiocomposite may perform well in hyperthermia therapy. These results indicate that the magnetic nanobiocomposite has the potential to perform well in hyperthermia therapy when subjected to an alternating magnetic field.

Effect of silver nanochitosan on control of seed-borne pathogens and maintaining seed quality of wheat

Seeds, considered as the foundation of agriculture, are invaded by a broad spectrum of seed-borne pathogens. The current study aimed to control seed-borne fungal pathogens of wheat, Aspergillus flavus and A. niger, by using Ag+ nanochitosan (Ag-NC) for nano-priming of seeds and enhancing seed quality. Nanochitosan (NC) and Ag-NC were synthesized using the gelation method and characterized by UV–vis spectrophotometry, FESEM, EDXS, and HRTEM. NC and Ag-NC showed irregular surface topography with an average particle size of 275 and 325 nm, respectively. Antifungal activity of both the nanoparticles at 0.1, 0.2, 0.3, 0.4, and 0.5 mg/mL revealed that Ag-NC at 0.5 mg/mL has completely terminated the mycelial growth of both pathogens. Malonaldehyde content increased to 77.77% in A. flavus and 82.66% in A. niger when exposed to 0.5 mg/mL Ag-NC. High-intensity fluorescence due to oxidative stress was observed in Ag-NC-treated pathogens. Ultra-structural changes in Ag-NC treated pathogenic spores under SEM displayed pronounced membrane damages. Wheat seeds were nano-primed with NC and Ag-NC at 0.5 mg/mL, and fungal load was examined to evaluate the mitigation of pathogenic stress and its effect on seedling growth promotion activity. Ag-NC priming reduced the fungal load and allowed successful seed germination. Ag-NC priming increased the albumin, gliadin, gluten, and glutenin content along with total phenol, reducing sugar and starch levels. Ag-NC priming increased the overall protein levels traced through SDS-PAGE. Seed priming with Ag-NC promotes seed germination, mean germination time, stress tolerance index, vigour, etc. NC and Ag-NC at 0.5 mg/mL showed no cytotoxic effect on the Human Embryonic Kidney (HEK293) cell line that ensures the nanoparticles are non-toxic. Thus, the synthesized nanoparticles exhibit a dual role in antifungal activity and plant growth promotion.

Recombinant human epidermal growth factor-loaded liposomes and transferosomes for dermal delivery: Development, characterization, and cytotoxicity evaluation.

Recombinant human epidermal growth factor (rhEGF) is widely utilized as an antiaging compound in wound-healing therapies and cosmetic purposes. However, topical administration of rhEGF has limited treatment outcomes because of its poor percutaneous penetration and rapid proteinase degradation. To overcome these obstacles, this study aims to develop and characterize rhEGF-containing conventional liposomes (rhEGF-CLs) and transferosomes (rhEGF-TFs) as efficient dermal carriers. Physicochemical characterization such as particle size, zeta potential (ZP), morphology, encapsulation efficiency (EE%), and release properties of nanocarriers as well as in vitro cytotoxicity in human dermal fibroblast (HDF) and human embryonic kidney (HEK293) cell lines were investigated. rhEGF-TFs at the rhEGF concentration ranging from 0.05 to 1.0 μg/mL were chosen as the optimum formulation due to the desired release profile, acceptable EE%, optimal cell proliferation, and minimal cytotoxicity compared to the control and free rhEGF. However, higher concentrations caused a decrease in cell viability. The ratio 20:80 of Tween 80 to lipid was optimal for rhEGF-TFs-2, which had an average diameter of 233.23 ± 2.64 nm, polydispersity index of 0.33 ± 0.05, ZP of -15.46 ± 0.29 mV, and EE% of 60.50 ± 1.91. The formulations remained stable at 5°C for at least 1 month. TEM and SEM microscopy revealed that rhEGF-TFs-2 had a regular shape and unilamellar structure. In vitro drug release studies confirmed the superiority of rhEGF-TFs-2 in terms of optimal cumulative release of rhEGF approximately 82% within 24 h. Franz diffusion cell study showed higher rhEGF-TFs-2 skin permeation compared to free rhEGF solution. Taken together, we concluded that rhEGF-TFs can be used as a promising formulation for wound healing and skin regeneration products.

DETERMINATION OF THE CIRCADIAN OSCILLATION PATTERN OF UNFOLDED PROTEIN RESPONSE SIGNALING COMPONENTS IN HUMAN EMBRYONIC KIDNEY HEK293 CELLS

Objective: The circadian rhythm is one of the primary regulatory systems with near 24-hour oscillations. It has a crucial role in regulating physiological conditions in the human body, including body temperature and the secretion of hormones. Numerous disorders, such as cancer and diabetes, have been linked to disruptions of the cellular circadian rhythm. Herein, we aimed to investigate the relationship between the circadian rhythm and unfolded protein response (UPR) signaling, which is one of the important physiological mechanisms in mammalian cells and has recently been associated with drug resistance, invasion and metastasis in cancer. Material and Method: Human embryonic kidney cell line HEK293 was provided from the American Type Culture Collection and propagated in DMEM containing 10% FBS and growth ingredients. For in vitro circadian synchronization, cells were exposed to 50% and then the oscillation pattern of gene and protein expression of UPR-related target genes was analyzed by agarose gel electrophoresis and immunoblotting, respectively. The oscillation pattern was commented on through curve-fitting analysis. Result and Discussion: Our findings demonstrated that UPR components, including IRE1α, XBP-1s, eIF2α, phospho(Ser51)-eIF2α, PERK, ATF4, GADD34 and ATF6, tightly exhibit oscillation patterns under a circadian rhythm on a 48-hour time scale like the PER1 gene that is a core component of the circadian rhythm. Moreover, endoplasmic reticulum (ER) stress genes, BiP/GRP78 and CHOP, were similar to UPR components under the circadian rhythm. Additionally, we found the activation of UPR signaling harmoniously modulated with the circadian rhythm. Present data indicated that the expression level of UPR components exhibited strict oscillation under the circadian rhythm. Our findings may guide experimental studies of new-generation UPR-targeted drugs to be developed to treat various pathologies in accordance with the circadian rhythm.

Synthesis, spectral, in silico/molecular docking and pharmacological studies of biologically potent triorganotin(IV) carboxylates

A series of compounds containing a glutaric anhydride-based carboxylate ligand (LH) and three triorganotin derivatives R3SnL for R = Me (1), nBu (2), and pH (3) are described in this article. The synthesis of the compounds was confirmed by microelemental CHN analysis, FT-IR, NMR and in the case of LH, 1 and 2 by single crystal X-ray crystallography, revealing the formation of one-dimensional coordination polymers for 1 and 2 with trans-C3O2 trigonal-bipyramidal geometries for the tin(IV) atoms. The anti-microbial activity was explored with the MIC values of 2 (≤ 0.25 µg/mL against the bacterium S. aureus and fungal strains C. albicans and C. neoformans) being less than observed for the anti-bacterial drug vancomycin-HCl (1 μg/mL) and anti-fungal drug fluconazole (≤ 0.25 and 8 μg/mL, respectively). The maximum cytotoxicity was observed for 2, against the human embryonic kidney (HEC239) cell line, with CC50 ≤ 25 μg/mL. An evaluation of the compound-DNA interactions was performed through UV–Visible spectroscopic and viscometric experiments, with the observation of hypochromic along with slight bathochromic shifts and an increase in viscosity, respectively, revealing an intercalative mode of interaction. In comparison with the standard anti-oxidant ascorbic acid, the greatest scavenging of DPPH was recorded for 3 followed by 2, 1 and LH. The human malignant glioma cell line (MG-U87) was used to explore the anti-cancer potential of the synthesised compounds. Compounds 3 and 2 (IC50 = 5 and 21 µM, respectively) showed encouraging results when compared to cisplatin (IC50 = 133 µM). The anti-leishmanial data shows that at a 1000 µg/mL dose, the greatest percentage inhibition was observed for 2 (87.15 ± 0.01 %) which is almost equivalent to that for amphotericin B (90.67 ± 0.15 %). The docking results with SS-DNA revealed the compounds undergo an intercalative mode of interaction with the nitrogenous bases adenine, guanine and cytosine, with docking score order: 3 (-8.11) > 2 (-8.02) > 1 (-6.99) > LH (-6.38). Similarly, the docking results against the Brest cancer receptor BRCA2, indicate the highest binding score was observed for 3. The anti-leishmanial docking data indicates 2 as having maximum docking score (-7.79). The in silico study, performed to reveal the drug-likeness behaviour of the news compounds, suggest that they possess the properties of orally active drugs.

NanoTrans: an integrated computational framework for comprehensive transcriptome analysis with nanopore direct RNA sequencing

Nanopore direct RNA sequencing (DRS) provides the direct access to native RNA strands with full-length information, shedding light on rich qualitative and quantitative properties of gene expression profiles. Here with NanoTrans, we present an integrated computational framework that comprehensively covers all major DRS-based application scopes, including isoform clustering and quantification, poly(A) tail length estimation, RNA modification profiling, and fusion gene detection. In addition to its merit in providing such a streamlined one-stop solution, NanoTrans also shines in its workflow-orientated modular design, batch processing capability, all-in-one tabular and graphic report output, as well as automatic installation and configuration supports. Finally, by applying NanoTrans to real DRS datasets of yeast, Arabidopsis, as well as human embryonic kidney and cancer cell lines, we further demonstrate its utility, effectiveness, and efficacy across a wide range of DRS-based application settings.

Investigation of Anti-cancer Potential of <i>Solanum virginianum</i> L. Fruit and Leaf Extracts by <i>In Vitro</i> and <i>In Silico</i> Studies

Background: Solanum virginianum L. is used as an ancient medicinal plant in Ayurveda. Aim: The present study aimed to elucidate the anticancer properties of S. virginianum through in vitro and in silico studies. Methods: The plant’s fruits and leaves were subjected to hot extraction using methanol and water as a solvent. The cytotoxicity assay was carried out on MCF-7 (Adenocarcinoma breast cancer cell line) and HEK-293 (Human embryonic kidney cell line—a normal cell line). Cell migration inhibition assay and colony formation assay were performed conform on the MCF-7 cell line. The leaf aqueous extract was subjected to HR-LCMS/MS to evaluate different phytocompounds. The in silico study was performed by checking the binding affinity between NINE-selected phytocompounds and specific apoptotic target proteins. Results: Amongst all extracts, the leaf aqueous extracts showed promising results, as at the 72-h incubation period, the IC50 concentration was 10.4 ± 1.13 μg/mL. Leaf aqueous extract also showed significant results in cell migration inhibition assay and colony-forming assay as compared to fruits. Finding metabolites in aqueous leaf extract was done using HR-LCMS/MS, which revealed the presence of more than 30 phytocompounds. Nine selected phytocompounds were characterized for their ability to bind to specific apoptotic target proteins. Out of the selected compounds docked, ritterazine A showed very strong binding with a binding energy of above -9kcal/mol with all the target proteins. Conclusion: Based on this study, we can conclude that the leaf of S. virginianum has potent anti-cancer activities. Furthermore, there is potential for doing comprehensive research on the therapeutic applications of this plant in breast cancer treatment.

Discovery of colchicine aryne cycloadduct as a potent molecule for the abrogation of epithelial to mesenchymal transition via modulating cell cycle regulatory CDK-2 and CDK-4 kinases in breast cancer cells

In this study, we synthesized a new-generation library of colchicine derivatives via cycloaddition of colchicine utilizing position C-8 and C-12 diene system regioselectivity with aryne precursor to generate a small, focused library of derivatives. We assessed their anticancer activity against various cancer cell lines like MCF-7, MDA-MB-231, MDA-MB-453, and PC-3. Normal human embryonic kidney cell line HEK-293 was used to determine the toxicity. Among these derivatives, silicon-tethered compound B-4a demonstrated the highest potency against breast cancer cells. Subsequent mechanistic studies revealed that B-4a effectively modulates cell cycle regulatory kinases (CDK-2 and CDK-4) and their associated cyclins (cyclin-B1, cyclin-D1), inducing apoptosis. Additionally, B-4a displayed a noteworthy impact on tubulin polymerization, compared to positive control flavopiridol hydrochloride in a dose-dependent manner, and significantly disrupted the vimentin cytoskeleton, contributing to G1 arrest in breast cancer cells. Moreover, B-4a exhibited substantial anti-metastatic properties by inhibiting breast cancer cell migration and invasion. These effects are attributed to the down-regulation of major epithelial to mesenchymal transition (EMT) factors, including vimentin and Twist-1, and the upregulation of the epithelial marker E-cadherin in an apoptosis-dependent manner.

Corylus avellana cultivar “Tonda di Giffoni”, source of antioxidant diarylheptanoids: Biological evaluation and molecular docking analysis

Hazelnut “Tonda di Giffoni” (Corylus avellana L.) is a PGI (Protected Geographical Indication) product of the Campania region, Southern Italy, marked for its processing quality which guarantees origin, typical features, high physicochemical quality, and organoleptic properties. Previous investigations on hazelnut and its byproducts revealed the presence of diarylheptanoid derivatives, natural compounds with antioxidant properties. With the aim of exploring the nutraceutical properties of hazelnut diarylheptanoids, the antioxidant properties of giffonins E (1), H (2), and J (3), three cyclic diaryletherheptanoids, and carpinontriol B (4), a cyclic diarylheptanoid, were investigated. Tested compounds showed significant protection against hydrogen peroxide (H2O2)-induced cytotoxic damage in human neuroblastoma and embryonic kidney cell lines. Compounds 1–4 were also able to prevent oxidative stress by decreasing reactive oxygen species (ROS) production and levels of lipid peroxidation, and attenuating cell apoptosis (caspase-3 activity).Moreover, considering that the nuclear factor erythroid 2-related factor 2 (Nrf2) is responsible for regulating an extensive panel of antioxidant enzymes, the capability of these natural products to disrupt the interaction between Kelch-like ECH-associated protein 1 (Keap1), thereby activating the Nrf2-mediated pathway, was evaluated by molecular docking experiments and in vitro tests.The Western blot analysis revealed that giffonin H and carpinontriol B had an indirect antioxidant action, as evidenced by a significant rise in the translocated protein following their administration.

Evaluating HIV-1 Infectivity and Virion Maturation across Varied Producer Cells with a Novel FRET-Based Detection and Quantification Assay.

The maturation of HIV-1 virions is a crucial process in viral replication. Although T-cells are a primary source of virus production, much of our understanding of virion maturation comes from studies using the HEK293T human embryonic kidney cell line. Notably, there is a lack of comparative analyses between T-cells and HEK293T cells in terms of virion maturation efficiency in existing literature. We previously developed an advanced virion visualization system based on the FRET principle, enabling the effective distinction between immature and mature virions via fluorescence microscopy. In this study, we utilized pseudotyped, single-round infectious viruses tagged with FRET labels (HIV-1 Gag-iFRET∆Env) derived from Jurkat (a human T-lymphocyte cell line) and HEK293T cells to evaluate their virion maturation rates. HEK293T-derived virions demonstrated a maturity rate of 81.79%, consistent with other studies and our previous findings. However, virions originating from Jurkat cells demonstrated a significantly reduced maturation rate of 68.67% (p < 0.0001). Correspondingly, viruses produced from Jurkat cells exhibited significantly reduced infectivity compared to those derived from HEK293T cells, with the relative infectivity measured at 65.3%. This finding is consistent with the observed relative maturation rate of viruses produced by Jurkat cells. These findings suggest that initiation of virion maturation directly correlates with viral infectivity. Our observation highlights the dynamic nature of virus-host interactions and their implications for virion production and infectivity.

Elemental, phytochemical analysis and determination of antibacterial, antioxidant, and cytotoxic activities of leaves extracts of eleven Eucalyptus species

The genus Eucalyptus has attracted considerable attention from natural product specialists exploring new phytochemicals for therapeutic purposes due to their possible biological activities. This was stimulated by current findings that Eucalyptus species interrupt the propagation of the recently tormenting COVID-19 virus. Eleven Eucalyptus leaf extracts were subjected to in vitro antimicrobial studies against four bacterial strains: Staphylococcus aureus, Streptococcus pyogenes, Staphylococcus lugdunensis and Staphylococcus epidermis using the broth serial dilution assay. The potential of the extracts to inhibit free radicals 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was investigated, indicating antioxidant properties. Furthermore, the cytotoxicity test was performed against the human embryonic kidney (HEK293-T) cell line using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Phytochemicals were identified and characterized using Fourier transform infrared spectroscopy (FTIR) and one-dimensional gas chromatography mass spectrometry (GC–MS). Moreover, the microwave plasma atomic emission spectroscopy (MP-AES) coupled with inductively coupled plasma optical emission spectroscopy (ICP-OES) was used to evaluate the multielemental composition of the extracts. The MP-AES and ICP-OES techniques in this work displayed accuracy and sensitivity for multielement determination by identifying P, B, Ca, Zn, Cu, Mg, Fe, Mn, Na, and K as major constituents in Eucalyptus extracts. The extracts generally had strong antioxidant and antibacterial properties, with E. viminalis (IC50 = 2.91 ± 0.22 µg/mL) demonstrating strong action against the ABTS radical. E. nitens and E. dunnii also showed highest activity against DPPH and ABTS, with IC50 values < 10 µg/mL. Additionally, with IC50 values greater than 20 µg/mL, the extracts demonstrated relative non-toxicity against HEK293-T cells. The existence of the bioactive chemicals found by FTIR and GC–MS may be the cause of the observed biological activity. Because Eucalyptus plant species have the highest antioxidant activity and the most inhibitory action against the majority of Streptococcus bacterial strains, they can be employed as a therapeutic treatment to treat infections caused by Streptococcus.

#2062 Protein interactome and trafficking of uromodulin in HEK and MDCK cell model

Abstract Background and Aims Uromodulin (UMOD) is a kidney-specific glycoprotein and the most abundant urinary protein in healthy individuals. Genome-wide association studies have shown an association between UMOD variants, kidney diseases, and hypertension. UMOD exhibits bidirectional secretion as it releases into the renal interstitium and circulation. However, the molecular trafficking of UMOD and its interaction remains poorly defined. We aimed to establish a stable Human Embryonic Kidney (HEK293) cell line expressing human UMOD to identify the potential regulators of UMOD expression or secretion. This study will provide significant implications for conditions such as hypertension, chronic kidney diseases, acute kidney injury, or UMOD-autosomal dominant tubulointerstitial kidney disease (UMOD-ADTKD). Method A HEK-UMOD-expressing cell line was generated by introducing human UMOD transcript 1 expression and using a tetracycline-inducible expression vector (pcDNA™FRT⁄TO vector) with yellow fluorescent protein tag at the C-terminal and previously established MDCK-UMOD cell line was also used. An untargeted proteomic approach was used to identify proteins, using GFP trap co-immunoprecipitation assay followed by mass spectrometry analysis. Results We identified 1367 and 1140 UMOD interacting proteins in HEK and MDCK cell models respectively. There were 620 proteins common to both these models. Through gene ontology, we unravel the molecular mechanisms involved in the biosynthesis, folding, and transport of UMOD. We observed several proteins associated with clathrin-mediated transporting. To study the role of clathrin in UMOD transport, we treated the polarised MDCK-UMOD cells with pitstop 2. There was significantly more secretion of UMOD with pitstop 2 treatment in the apical compartment compared to untreated cells however there was no significant difference in UMOD secretion in the basolateral compartment. Conclusion These results suggested that clathrin-coated vesicles are involved in transporting and sorting UMOD. Further studies need to investigate the functional importance of these proteins in UMOD trafficking and their implications in different physiological processes.