Embryonic Kidney Cell Line Research Articles

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.

Investigation of Newly Synthesized Fluorinated Isatin-Hydrazones by In Vitro Antiproliferative Activity, Molecular Docking, ADME Analysis, and e-Pharmacophore Modeling.

In this study, we investigated the in vitro antiproliferative activities and performed computational studies of newly synthesized fluorinated isatin-hydrazones. The chemical structures of the synthesized compounds were confirmed by FT-IR, 1D NMR (1H- and 13C NMR and APT), 2D NMR (HETCOR and HMBC), and elemental analysis. All compounds (1-15) were tested in human lung (A549) and liver (HepG2) cancer cell lines for 72 h. The compounds were screened against a healthy embryonic kidney cell line (HEK-293T) under the same conditions to determine their toxic effects. According to the results obtained, one of the compounds, in particular, compound 8 was effective at inhibiting the growth of cancerous cells, and its effects on both cancer cell lines were similar to IC50 values of 42.43 and 48.43 μM for A549 and HepG2, respectively. Compound 8, which was determined to be the best anticancer agent in vitro, was chosen to interact with the target via molecular docking. This selected ligand (compound 8) interacted with the targets 4HJO, 4ASD, 3POZ, and 7TZ7, and docked into the active sites. The docking score, Glide energy, and Glide emodel values were calculated and determined to be lower than those of the reference compound cisplatin. The pharmacokinetic properties, stability, and drug-likeness parameters of all designed compounds were estimated using SwissADME. Finally, the binding affinities of compound 8 for all four targets were calculated using the MM-GBSA method.

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.

Antiviral properties of titanium nanoparticles and features of their influence on the morphology of virions

The search for new antiviral substances and the development of modern antiviral preparations and disinfectants is an important area of modern science. Nanoparticles (NPs) and nanomaterials (NMs) have received increasing attention from scientists as promising antiviral substances recently. Our study aimed to investigate the antiviral activity of citrate-stabilized titanium nanoparticles (Ti NPs) against Teschovirus A (TV-A) and Potato virus Y (PVY). The biological activity of PVY has been tested using cultivated tobacco plants (Nicotiana tabacum L.) and bell pepper plants (Capsicum annum L.). It has been found that injection of plants with the virus has led to the appearance of PVY infection symptoms with further death of the plants. The threshold limit value (TLV) for Ti NPs in embryonic pig kidney cell line culture is 12.5 µg/cm3. According to the prophylactic and treatment plots, Ti NPs have slight antiviral activity against TV-A strain Dniprovskyi 34, decreasing its titer according to both plots by 0.5 lg TCD50/cm3. However, according to the virucidal plot at TLV concentration, Ti NPs show high antiviral activity against TV-A strain Dniprovskyi 34, significantly decreasing its titer in embryonic pig kidney cell line culture by 4.46 lg TCD50/cm3. Ti NPs do not have antiviral activity against PVY, according to the prophylactic and treatment plots. Nevertheless, according to the virucidal plot Ti NPs showed high antiviral activity against PVY, inactivating the virus in the reaction mixture in vitro. According to the results of TEM, Ti NPs bind to virus particles. Virus particles with NPs attached to them, deformed and partially destroyed virus particles have been found in samples of virus-containing suspension, which were incubated with Ti NPs. We suggest that Ti NPs are useful as disinfectants to control viral infections, particularly TV-A and PVY.

#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.

IL-2RG as a possible immunotherapeutic target in CRC predicting poor prognosis and regulated by miR-7-5p and miR-26b-5p

Despite advances in treatment strategies, colorectal cancer (CRC) continues to cause significant morbidity and mortality, with mounting evidence a close link between immune system dysfunctions issued. Interleukin-2 receptor gamma (IL-2RG) plays a pivotal role as a common subunit receptor in the IL-2 family cytokines and activates the JAK-STAT pathway. This study delves into the role of Interleukin-2 receptor gamma (IL-2RG) within the tumor microenvironment and investigates potential microRNAs (miRNAs) that directly inhibit IL-2RG, aiming to discern their impact on CRC clinical outcomes. Bioinformatics analysis revealed a significant upregulation of IL-2RG mRNA in TCGA-COAD samples and showed strong correlations with the infiltration of various lymphocytes. Single-cell analysis corroborated these findings, highlighting IL-2RG expression in critical immune cell subsets. To explore miRNA involvement in IL-2RG dysregulation, mRNA was isolated from the tumor tissues and lymphocytes of 258 CRC patients and 30 healthy controls, and IL-2RG was cloned into the pcDNA3.1/CT-GFP-TOPO vector. Human embryonic kidney cell lines (HEK-293T) were transfected with this construct. Our research involved a comprehensive analysis of miRPathDB, miRWalk, and Targetscan databases to identify the miRNAs associated with the 3′ UTR of human IL-2RG. The human microRNA (miRNA) molecules, hsa-miR-7-5p and hsa-miR-26b-5p, have been identified as potent suppressors of IL-2RG expression in CRC patients. Specifically, the downregulation of hsa-miR-7-5p and hsa-miR-26b-5p has been shown to result in the upregulation of IL-2RG mRNA expression in these patients. Prognostic evaluation of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p, using TCGA-COAD data and patient samples, established that higher IL-2RG expression and lower expression of both miRNAs were associated with poorer outcomes. Additionally, this study identified several long non-coding RNAs (LncRNAs), such as ZFAS1, SOX21-AS1, SNHG11, SNHG16, SNHG1, DLX6-AS1, GAS5, SNHG6, and MALAT1, which may act as competing endogenous RNA molecules for IL2RG by sequestering shared hsa-miR-7-5p and hsa-miR-26b-5p. In summary, this investigation underscores the potential utility of IL-2RG, hsa-miR-7-5p, and hsa-miR-26b-5p as serum and tissue biomarkers for predicting CRC patient prognosis while also offering promise as targets for immunotherapy in CRC management.Graphical

Methylene blue treatment with zero-valent iron/pyrite/H2O2 system under static and continuous flow conditions: Reaction mechanism and toxicity evaluation

Laboratory batch and continuous flow studies, coupled with surface and toxicity analysis, were performed to evaluate methylene blue treatment by heterogeneous Fenton process using zero-valent iron (ZVI) and pyrite as co-catalyst. The use of pyrite in batch reactors significantly enhanced methylene blue treatment by the ZVI/H2O2 system because of improved iron redox cycling. The continuous-flow experiments revealed that the reactor performance increased in the order of: ZVI/H2O2 < pyrite/H2O2 < ZVI/pyrite/H2O2 under dynamic flow conditions. The methylene blue treatment by the ternary ZVI/pyrite/H2O2 system was described by an initial degradation of methylene blue with *OH radicals, followed by the adsorption and/or co-precipitation of degradation intermediates with some spherical particles. The surface analysis showed that these spherical particles formed, even at pH less than 4. The genotoxicity and cytotoxicity tests performed on mouse embryonic fibroblast (3 T3-L1) and human embryonic kidney (HEK293) cell lines showed that the Fenton treatment of methylene blue using the ZVI/pyrite/H2O2 system resulted in the formation of degradation species with much lower toxicity levels relative to methylene blue. Moreover, the Fenton degradation species of methylene blue significantly enhanced the metabolic activity of several bacterial strains, including E. coli ATCC 8739 and P. aeruginosa PAO1.

Design and Synthesis of Potent Anticancer Agents Using Coumarin‐Quinazolinone Based Scaffolds: Exploration of ADME Properties and Molecular Docking Studies

AbstractCancer remains a complex global threat causing widespread deaths. Developing effective anticancer agents despite advancements still remained challenging. In this work it is designed and synthesised therapeutically active anticancer agents based on coumarin‐quinazolinone 5(a–j) through nucleophilic substitution reaction of 4‐bromomethyl coumarin derivatives with 6,7‐bis(2‐methoxyethoxy)quinazolin‐4(3H)‐one and characterised by using FTIR, 1HNMR, 13C NMR and LCMS analysis. The compounds 5(a–j) were screened for in vitro anticancer activity against A‐549 lung cancer cell lines and MDA‐MB‐231 cell lines. Compound 5i emerged as the most promising antiproliferative candidate demonstrating an IC50 value of 7.11 μM against A‐549 cell lines. Meanwhile compound 5g found as a hit candidate in the case of MDA‐MB‐231, exhibiting an IC50 of 5.12 μM, surpassing the potency of the standard drug doxorubicin (IC50 5.89 μM). Further, the safety profile of coumarin‐quinazolinone scafolds (5a–j) were examined for their cytotoxicity towards human embryonic kidney cell lines (HEK293). It was found that the most active compounds exhibited good safety profile towards human embryonic cell lines. Additionally, molecular docking investigations confirmed that compounds 5(a–j) exhibited elevated docking scores and demonstrated favourable interactions with the HER2 (PDB:3PP0) and VEGFR2 (PDB:4ASE). Furthermore, we investigated the ADME properties using the SwissADME tool.

Design, synthesis, biological assessment and molecular modeling studies of novel imidazothiazole-thiazolidinone hybrids as potential anticancer and anti-inflammatory agents

A new series of imidazothiazole derivatives bearing thiazolidinone moiety (4a-g and 5a-d) were designed, synthesized and evaluated for potential epidermal growth factor receptor (EGFR) kinase inhibition, anticancer and anti-inflammatory activity, cardiomyopathy toxicity and hepatotoxicity. Compound 4c inhibited EGFR kinase at a concentration of 18.35 ± 1.25 µM, whereas standard drug erlotinib showed IC50 value of 06.12 ± 0.92 µM. The molecular docking, dynamics simulation and MM-GBSA binding energy calculations revealed strong interaction of compound 4c with binding site of EGFR. The synthesized compounds were evaluated for their anticancer activity by MTT assay against three human cancer cell lines A549 (Lung), MCF-7 (Breast), HCT116 (Colon), one normal human embryonic kidney cell line HEK293 and also for their EGFR kinase inhibitory activity. Few compounds of the series (4a, 4b, 4c) showed promising growth inhibition against all the tested cancer cell lines and against EGFR kinase. Among these, compound 4c was found to be most active and displayed IC50 value of 10.74 ± 0.40, 18.73 ± 0.88 against cancer cell lines A549 and MCF7 respectively whereas it showed an IC50 value of 96.38 ± 1.79 against HEK293 cell line indicating lesser cytotoxicity for healthy cell. Compounds 4a, 4b and 4c were also examined for their apoptosis inducing potential through AO/EB dual staining assay and it was observed that their antiproliferative activity against A549 cells is mediated via induction of apoptosis. Cardiomyopathy studies showed normal cardiomyocytes with no marked sign of pyknotic nucleus of compounds 4b and 4c. Hepatotoxicity studies of compounds 4b and 4c also showed normal architecture of hepatocytes. Compounds 4a-g and 5a-d were also evaluated for their in-vitro anti-inflammatory activity by protein albumin denaturation assay. Among the tested compounds 4a-d and 5a-b showed promising activity and were selected for in-vivo inflammatory activity against carrageenan rat paw edema test. Among these compounds, 4b was found to be most active in the series showing 84.94% inhibition, whereas the standard drug diclofenac sodium showed 84.57% inhibition. Compound 4b also showed low ulcerogenic potential and lipid peroxidation. Thus, compounds 4c and 4b could be a promising lead compounds for developing anticancer and anti-inflammatory agents with low toxicity and selectivity.

In vitro antifungal activity, cytotoxicity and binding analysis of imidazolium based ionic liquids with fluconazole: DFT and spectroscopic study

Fungal infections represent a significant challenge in various living organisms, including humans, animals, and plants. Fungi are versatile organisms capable of thriving in diverse environments, which contributes to their widespread presence and potential to cause infections. Recently, imidazolium-based ionic liquids emerged as good antimicrobial agent in either monomeric or polymeric form. Therefore, herein, we have reporting the in-vitro antifungal activity, cytotoxicity, and binding analysis of 1-decyl-3-methylimidazolium chloride, [C10Mim][Cl] and 1-hexyl-3-methylimidazolium chloride, [C6Mim][Cl] ionic liquids with fluconazole (Flz). The antifungal activity of ILs and ILs with Flz against candidiasis species (C. albicans) in terms of minimum inhibitory concentration (MIC) was assessed. The antifungal activity results showed that [C10Mim][Cl]-Flz exhibit good antifungal activity then [C10Mim][Cl] and Flz alone. The cytotoxicity of ILs with Flz was checked against human embryonic kidney (HEK293) cell line regarding cell viability and result showed minute cytotoxicity of the combinations at higher concentrations. Further, the binding of ILs with Flz was also studied using various techniques such as UV–visible, fluorescence, Fourier-transform infrared (FTIR) spectroscopy, isothermal titration calorimetry (ITC), and conductivity method. It was found that [C10Mim][Cl] shows more binding affinity then [C6Mim][Cl] with Flz. In-silico molecular docking analysis of Flz-[C10Mim][Cl] complex show a higher binding affinity with CYP51 which in turn enhanced its ability to inhibit the ergosterol biosynthesis as compared to alone [C10Mim][Cl] and Flz. The classical density functional theory (DFT) model was also utilized which further supports the spectroscopic results.

Amino acid driven synthesis of gold nanoparticles: A comparative study on their biocompatibility

Gold nanoparticles (GNps) were successfully synthesized via 17 different l-Amino acids (AA-GNps) as reducing agents, in a one-pot reaction. Comparative characterization of AA-GNps revealed variations in their size, morphology, and structure, highly dependent on the nature of the employed amino acid. Aromatic amino acids proved to be fast-reducing agents directing the formation of GNps. Hydroxylic and basic amino acids were the slowest reducing agents for the synthesis of GNps. Aliphatic amino acids produced GNps of similar behavior, except Isoleucine which led to the formation of hollow nanospheres. GNps formulated by aspartic acid and asparagine have comparable properties to citrate-synthesized GNps, which is the golden standard in plasmonic nanoparticles. A new protocol was established for the determination of gold content in GNps’ colloidal solutions via a simple and cost-effective standard curve method employing UV–Vis spectroscopy. Most AA-GNps are colloidally stable, in both aqueous dispersion and simulated body fluid, posing as excellent candidates for in vitro experimentation. Comparative screening for their biocompatibility demonstrated the non-hemolytic and cytocompatible profile of most AA-GNps, via different in vitro assay protocols, performed on blood samples and the healthy cell line of Human Embryonic Kidney cells (HEK-293).

Unraveling the mechanism of antifungal action of encapsulated eugenol/chitosan nanoparticles against Aspergillus fumigatus

Aspergillus fumigatus can cause an invasive life-threatening infection in people who have weakened immune systems, underlying diseases or respiratory infections such as COVID-19. Azole-resistant A. fumigatus infections are difficult to treat and have high mortality. This study aimed to investigate the efficacy of encapsulated eugenol in chitosan biopolymer nanoparticles (NPs) to increase its antifungal activity against A. fumigatus. The physical properties of eugenol-encapsulated chitosan nanoparticles (EECNPs), size, polydispersity and the release profile of eugenol were examined by DLS, XRD and FTRI techniques. The antifungal activity of EECNPs against A. fumigatus was examined using CLSI-M38 microbroth dilution assay, time killing assay, reactive oxygen species (ROS) activity, destruction of cell membrane integrity/permeability, and cell wall morphological damage by electron microscopy. The eugenol/chitosan ratio of 1.00:1.00 was considered the optimal ratio with the highest encapsulation efficiency (43.1%) and loading capacity (30.8%). EECNPs were characterized by a hydrodynamic radius of 330.9 nm, a polydispersity index (PDI) of 0.298 and the zeta potential of 18.9 mv. The average size of the EECNPs was 300 nm according to TEM micrographs. The antifungal activities were determined at 150–300 μg/mL as MIC concentrations. The time killing assay showed that the nanoparticles could inhibit the fungal growth after 12 h. The toxicity assay revealed more than 60% survival of human embryonic kidney (HEK293) cell line in 2 × MIC concentration. The release of reactive oxygen species (ROS), and extracellular potassium in A. fumigatus caused more damaging on cell membrane integrity, through increasing 600 μg/mL concentration of nanoparticles. Our results revealed that the encapsulation of eugenol in chitosan biopolymer to produce EECNPs not only increased the half-life of eugenol but also improved its antifungal activity against A. fumigatus by a combined mechanism of damaging cell surfaces, cell membrane integrity, and the induction of ROS activity. This study is the first to report that EECNPs has the potency to use as a natural antimicrobial agent due to embracing the destructive properties on cell membrane integrity in A. fumigatus.

Novel method of measurement of in vitro drug uptake in OATP1B3 overexpressing cells in the presence of dextran.

In predictions about hepatic clearance (CLH), a number of studies explored the role of albumin and transporters in drug uptake by liver cells, challenging the traditional free-drug theory. It was proposed that liver uptake can occur for transporter substrate compounds not only from the drug's unbound form but also directly from the drug-albumin complex, aphenomenon known as uptake facilitated by albumin. In contrast to albumin, dextran does not exhibit binding properties for compounds. However, as a result of its inherent capacity for stabilization, it is widely used to mimic conditions within cells. The uptake of eight known substrates of the organic anion-transporting polypeptide 1B3 (OATP1B3) was assessed using a human embryonic kidney cell line (HEK293), which stably overexpresses this transporter. An inert polymer, dextran, was used to simulate cellular conditions, and the results were compared with experiments involving human plasma and human serum albumin (HSA). This study is the first to demonstrate that dextran increases compound uptake in cells with overexpression of the OATP1B3 transporter. Contrary to the common theory that highly protein-bound ligands interact with hepatocytes to increase drug uptake, the results indicate that dextran's interaction with test compounds does not significantly increase concentrations near the cell membrane surface. We evaluated the effect of dextran on the uptake of known substrates using OATP1B3 overexpressed in the HEK293 cell line, and we suggest that its impact on drug concentrations in liver cells may differ from the traditional role of plasma proteins and albumin.

Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance(NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the β subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 μM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

Studies on the cytotoxicity of leaf protein concentrate from Diplazium esculentum, pasta preparation and assessment of its quality during storage

Cytotoxicity studies performed with the cell lines viz., HEK 293 (Human embryonic kidney cell line), HEP G2 (Hepatoblastoma cell line) and PBMC (Peripheral blood mononuclear cells) by leaf protein concentrate (LPC) of Diplazium esculentum were analysed with an aim to possibly determine the safety of leaf protein concentrate. Results revealed the safety from the cytotoxic point of view at various levels of incorporation into pasta such as 5 %, 10 %, 15 % and 20 %. Scanning electron microscopy revealed that gluten network in which starch granules were embedded, control pasta had a smooth surface whereas leaf protein concentrate incorporated pasta had a more open structure is apparent showing the protein matrix with the majority of the starch granules absent and it was further supported by textural analysis. The proximate composition, cooking quality parameters chronologically increased with increase in percentage of leaf protein concentrate in the pasta. Aerobic plate count, yeast and mold count of pasta were within the permissible limit. Significant variations in microbial load were seen throughout the course of the same storage period when LPC was included at various quantities. After the first 30 days of storage, yeast and mold started to grow, but they did so within the allowed limits. Pasta with a 20 % addition of protein concentrate was optimised for consumption and may be safely consumed for up to 30 days. According to this study, leaf protein concentrate may be delivered in pasta-like goods to increase their nutraceutical potential without compromising the products' sensory quality or safety.

Antimicrobial properties of triazolato terpyridine Pd(II) and Pt(II) complexes formed by [3+2] cycloaddition coupling reaction

Modern classes of antimicrobials are crucial because most drugs in development today are basically antibiotic derivatives. Even though a large number of metal-based compounds have been studied as antimicrobial agents, relatively few studies have examined the antimicrobial properties of Pd(II) and Pt(II) compounds. The [3+2] cycloaddition reactions of [M(N3)L]PF6 (M = Pd(II) and Pt(II); L = 4′-(2-pyridyl)-2,2′:6′,2″-terpyridine) with 4,4,4-trifluoro-2-butynoic acid ethyl ester gave the corresponding triazolate complexes. The reaction products were fully characterized with a variety of analytical and spectroscopic tools including X-ray crystallographic analysis. The crystal structure of [Pd(triazolatoCF3,COOCH2CH3)L]PF6 provided cut-off evidence that the kinetically formed N1-triazolato isomer favoured the isomerization to the thermodynamically stable N2-analogue. The experimental work was complemented with computational work to get an insight into the nature of the predominant triazolate isomer. The lysozyme binding affinity of the triazolate complexes was examined by mass spectrometry. An analysis of the lysozyme Pd(II) adducts suggests a coordinative covalent mode of binding via the loss of the triazolato ligand. The free ligand and its triazolate complexes displayed selective toxicity against Candida albicans and Cryptococcus neoformans, while no cytotoxicity was observed against the normal human embryonic kidney cell line.

Microwave-assisted synthesis, spectral characterization, and biological activities of complexes of a Schiff base derived from S-benzyl dithiocarbazate

Co(II), Ni(II), Zn(II), and Cd(II) complexes were synthesized from a Schiff base ligand, benzyl-2-(1-(thiophen-2-yl)propylidene)hydrazine-1-carbodithioate (BTPHC), by conventional as well as microwave irradiation methods. The ligand and its metal complexes were characterized by spectroscopic techniques (FT-IR, UV-vis, mass spectrometry, and 1H NMR) and powder X-ray diffraction. Based on magnetic measurements and spectral data, octahedral environments for Co(II) and Ni(II) complexes and tetrahedral environments for Zn(II) and Cd(II) complexes are proposed. The metal(II) complexes were active toward bacterial and fungal strains. Antidiabetic studies showed that the Cd(II) complex possesses greater potential as an antidiabetic agent than the other metal complexes and the ligand. Antioxidant studies indicate that the Zn(II) and Cd(II) complexes possess high radical scavenging activities. The synthesized ligand and metal(II) complexes were screened for their anticancer properties against two human cancer cell lines MCF-7 and HeLa using an MTT cell viability assay. The Ni(II) complex excelled in halting the proliferation of HeLa and MCF-7 cancer cell lines with IC50 values of 46.63 µM and 80.90 µM, respectively, whereas it displayed insignificant cytotoxicity against the human normal embryonic kidney cell line (HEK293) with IC50 = 300.41 µM.

Design, synthesis, and biological evaluation of novel quinoxaline aryl ethers as anticancer agents.

We designed and synthesized thirty novel quinoxaline aryl ethers as anticancer agents, and the structures of final compounds were confirmed with various analytical techniques like Mass, 1 H NMR, 13 C NMR, FTIR, and elemental analyses. The compounds were tested against three cancer cell lines: colon cancer (HCT-116), breast cancer (MDA-MB-231), prostate cancer (DU-145), and one normal cell line: human embryonic kidney cell line (HEK-293). The obtained results indicate that two compounds, FQ and MQ, with IC50 values < 16 μM, were the most active compounds. Molecular docking studies revealed the binding of FQ and MQ molecules in the active site of the c-Met kinase (PDB ID: 3F66, 1.40 Å). Furthermore, QikProp ADME prediction and the MDS analysis preserved those critical docking data of both compounds, FQ and MQ. Western blotting was used to confirm the impact of the compounds FQ and MQ on the inhibition of the c-Met kinase receptor. The apoptosis assays were performed to investigate the mechanism of cell death for the most active compounds, FQ and MQ. The Annexin V/7-AAD assay indicated apoptosis in MDA-MB-231 cells treated with FQ and MQ, with FQ (21.4%) showing a higher efficacy in killing MDA-MB-231 cells than MQ (14.25%). The Caspase 3/7 7-AAD assay further supported these findings, revealing higher percentages of apoptotic cells for FQ-treated MDA-MB-231 cells (41.8%). The results obtained from the apoptosis assay conclude that FQ exhibits better anticancer activity against MDA-MB-231 cells than MQ.

Functional analyses of TRAF6 gene in Argopecten scallops

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family has been reported to be involved in many immune pathways. In a previous study, we identified 5 TRAF genes, including TRAF2, 3, 4, 6, and 7, in the bay scallop (Argopecten irradians, Air) and the Peruvian scallop (Argopecten purpuratus, Apu). Since TRAF6 is a key molecular link in the TNF superfamily, we conducted a series of studies targeting the TRAF6 gene in the Air and Apu scallops as well as their hybrid progeny, Aip (Air ♀ × Apu ♂) and Api (Apu ♀ × Air ♂). Subcellular localization assay showed that the Air-, Aip-, and Api-TRAF6 were widely distributed in the cytoplasm of the human embryonic kidney cell line (HEK293T). Additionally, dual-luciferase reporter assay revealed that among TRAF3, TRAF4, and TRAF6, only the overexpression of TRAF6 significantly activated NF-κB activity in the HEK293T cells in a dose-dependent manner. These results suggest a crucial role of TRAF6 in the immune response in Argopecten scallops. To investigate the specific immune mechanism of TRAF6 in Argopecten scallops, we conducted TRAF6 knockdown using RNA interference. Transcriptomic analyses of the TRAF6 RNAi and control groups identified 1194, 2403, and 1099 differentially expressed genes (DEGs) in the Air, Aip, and Api scallops, respectively. KEGG enrichment analyses revealed that these DEGs were primarily enriched in transport and catabolism, amino acid metabolism, peroxisome, lysosome, and phagosome pathways. Expression profiles of 28 key DEGs were confirmed by qRT-PCR assays. The results of this study may provide insights into the immune mechanisms of TRAF in Argopecten scallops and ultimately benefit scallop breeding.