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The concentration of free cytosolic Ca2+ is a critical second messenger in almost every cell type, with the signal often being carried by the period of oscillations, or spikes, in the cytosolic Ca2+ concentration. We have previously studied how Ca2+ influx across the plasma membrane affects the period and shape of Ca2+ oscillations in HEK293 cells. However, our theoretical work was unable to explain how the shape of Ca2+ oscillations could change qualitatively, from thin spikes to broad oscillations, during the course of a single time series. Such qualitative changes in oscillation shape are a common feature of HEK293 cells in which STIM1 and 2 have been knocked out. Here, we present an extended version of our earlier model that suggests that such time-dependent qualitative changes in oscillation shape might be the result of balanced positive and negative feedback from Ca2+ to the production and degradation of inositol trisphosphate. [ABSTRACT FROM AUTHOR]

Background: Several in silico studies have determined that quercetin, a plant flavonol, could bind with strong affinity and low free energy to SARS-CoV-2 proteins involved in viral entry and replication, suggesting it could block infection of human cells by the virus. In the present study, we examined the ex vivo ability of quercetin to inhibit of SARS-CoV-2 replication and explored the mechanisms of this inhibition. Methods: Green monkey kidney Vero E6 cells and in human colon carcinoma Caco-2 cells were infected with SARS-CoV-2 and incubated in presence of quercetin; the amount of replicated viral RNA was measured in spent media by RT-qPCR. Since the formation of syncytia is a mechanism of SARS-CoV-2 propagation, a syncytialization model was set up using human embryonic kidney HEK293 co-expressing SARS-CoV-2 Spike (S) protein and human angiotensin converting enzyme 2 (ACE2), [HEK293(S + ACE2) cells], to assess the effect of quercetin on this cytopathic event by microscopic imaging and protein immunoblotting. Results: Quercetin inhibited SARS-CoV-2 replication in Vero E6 cells and Caco-2 cells in a concentration-dependent manner with a half inhibitory concentration (IC50) of 166.6 and 145.2 µM, respectively. It also inhibited syncytialization of HEK293(S + ACE2) cells with an IC50 of 156.7 µM. Spike and ACE2 co-expression was associated with decreased expression, increased proteolytic processing of the S protein, and diminished production of the fusogenic S2' fragment of S. Furin, a proposed protease for this processing, was inhibited by quercetin in vitro with an IC50 of 116 µM. Conclusion: These findings suggest that at low 3-digit micromolar concentrations of quercetin could impair SARS-CoV-2 infection of human cells partly by blocking the fusion process that promotes its propagation. [ABSTRACT FROM AUTHOR]

Background: Discovering new cytotoxic compounds has received significant attention due to the rise in drug resistance and the adverse effects associated with chemotherapy drugs. In this study, the cytoplasmic extract of Lactobacillus casei was used to produce iron oxide nanoparticles (Fe2o3 NPs), and the cytotoxic effects of NPs were investigated on MCF-7 and human embryonic kidney 293 (HEK-293) cells. Methods: The cytoplasmic extract of L. casei was mixed with 10³M iron sulfate solution and incubated for 3 weeks at 37 °C and 5% CO2. The coprecipitation method was used to synthesize chemical Fe2o3 NPs. The synthesis of NPs was studied by electron microscopy and X-ray diffraction (XRD) analysis, and the cytotoxic effects were evaluated with dilutions (10, 100, and 1000 µg/mL) on MCF-7 and HEK cells. Results: X-ray diffraction analysis and scanning electron microscopy presented the mean of NPs synthesized by the green method to be about 15 nm and their shape to be spherical, as well as the average of chemically synthesized NPs to be about 20 nm with cubic structure. Chemical and green synthesized NPs only at a concentration of 1000 µg/mL were able to significantly reduce the survival rate of normal HEK-293 cells; chemically synthesized NPs decreased MCF-7 cell survival only at 1000 µg/mL and green synthesis at 100 µg/mL and 1000 µg/mL. Conclusion: Generating Fe2o3 NPs is biologically safe using the green synthesis method and the cytoplasmic extract of L. casei, which may be a suitable candidate for the treatment of cancer cells. [ABSTRACT FROM AUTHOR]

Bispecific antibodies (bsAbs) are some of the most promising biotherapeutics due to the versatility provided by their structure and functional features. bsAbs simultaneously bind two antigens or two epitopes on the same antigen. Moreover, they are capable of directing immune effector cells to cancer cells and delivering various compounds (radionuclides, toxins, and immunologic agents) to the target cells, thus offering a broad spectrum of clinical applications. Current review is focused on the technologies used in bsAb engineering, current progress and prospects of these antibodies, and selection of various heterologous expression systems for bsAb production. We also discuss the platforms development of bsAbs for the therapy of solid tumors. [ABSTRACT FROM AUTHOR]

Spike protein from SARS-CoV-2, the etiologic agent of the COVID-19 pandemic disease, constitutes a structural protein that proved to be the main responsible for neutralizing antibody production. Thus, its sequence is highly considered for the design of candidate vaccines. Animal cell culture represents the best option for the production of subunit vaccines based on recombinant proteins since they introduce post-translational modifications that are important to mimic the natural antigenic epitopes. Particularly, the human cell line HEK293T has been explored and used for the production of biotherapeutics since the products derived from them present human-like post-translational modifications that are important for the protein's activity and immunogenicity. The aim of this study was to produce and characterize a potential vaccine for COVID-19 based on the spike ectodomain (S-ED) of SARS-CoV-2 and two different adjuvants: aluminum hydroxide (AH) and immune-stimulating complexes (ISCOMs). The S-ED was produced in sHEK293T cells using a 1-L stirred tank bioreactor operated in perfusion mode and purified. S-ED characterization revealed the expected size and morphology. High N-glycan content was confirmed. S-ED-specific binding with the hACE2 (human angiotensin-converting enzyme 2) receptor was verified. The immunogenicity of S-ED was evaluated using AH and ISCOMs. Both formulations demonstrated the presence of anti-RBD antibodies in the plasma of immunized mice, being significantly higher for the latter adjuvant. Also, higher levels of IFN-γ and IL-4 were detected after the ex vivo immune stimulation of spleen-derived MNCs from ISCOMs immunized mice. Further analysis confirmed that S-ED/ISCOMs elicit neutralizing antibodies against SARS-CoV-2. Key points: Trimeric SARS-CoV-2 S-ED was produced in stable recombinant sHEK cells in serum-free medium. A novel S-ED vaccine formulation induced potent humoral and cellular immunity. S-ED formulated with ISCOMs adjuvant elicited a highly neutralizing antibody titer. [ABSTRACT FROM AUTHOR]

Recombinant adeno-associated virus (rAAV) is a viral vector commonly used in gene therapy. Residual host cell DNA is an impurity that has been associated with the risk of infection and oncogenicity. Thus, it needs to be monitored for quality control. We aimed to develop a droplet digital polymerase chain reaction (ddPCR) method targeting 18S ribosomal RNA (rRNA) genes to quantitate residual host cell DNA. The copy number of the 18S rRNA gene was determined using two sets of primer pairs for 116- and 247-bp amplicons sharing the C-terminus. For conversion of the copy number of the 18S rRNA gene into the mass concentration of genomic DNA, the accurate copy number of 18S rRNA genes in HEK293 genomic DNA was determined by comparison with copy numbers of three reference genes (EIF5B, DCK, and HBB). Results showed that 88.6–97.9% of HEK293 genomic DNA spiked into rAAV preparations was recovered. The ddPCR-based assay was applied to rAAV preparations to quantitate residual host cell DNA as an impurity. Our findings indicate that the assay can be used for the quantitation and size distribution of residual host cell DNA in rAAV products. [ABSTRACT FROM AUTHOR]

The cytosolic immune receptor NLRP3 (nucleotide-binding domain, leucine-rich repeat (LRR), and pyrin domain (PYD)-containing protein 3) oligomerizes into the core of a supramolecular complex termed inflammasome in response to microbes and danger signals. It is thought that NLRP3 has to bind NEK7 (NIMA (never in mitosis gene a)-related kinase 7) to form a functional inflammasome core that induces the polymerization of the adaptor protein ASC (Apoptosis-associated speck-like protein containing a CARD (caspase recruitment domain)), which is a hallmark for NLRP3 activity. We reconstituted the NLRP3 inflammasome activity in modified HEK293 (human embryonic kidney 293) cells and showed that the ASC speck polymerization is independent of NEK7 in the context of this cell system. Probing the interfaces observed in the different, existing structural models of NLRP3 oligomers, we present evidence that the NEK7-independent, constitutively active NLRP3 inflammasome core in HEK293 cells may resemble a stacked-torus-like hexamer seen for NLRP3 lacking its PYD (pyrin domain). [ABSTRACT FROM AUTHOR]

Cyanotoxins are secondary metabolites produced by different types of cyanobacteria. Among them, Cylindrospermopsin (CYN) and Microcystins (MCs) stand out due to their wide geographical distribution and toxicity in various organs, including the kidney, which is involved in their distribution and elimination. However, the renal toxicity caused by CYN and MCs has hardly been studied. The aim of this work was to assess the cytotoxicity effects caused by CYN and MC-LR in the renal cell line HEK293, and for the first time, the influence of CYN on the gene expression of selected genes in these cells by quantitative real-time PCR (qRT-PCR). CYN caused an upregulation in the gene expression after exposure to the highest concentration (5 µg/mL) and the longest time of exposure (24 h). Moreover, shotgun proteomic analysis was used to assess the molecular responses of HEK293 cells after exposure to the individuals and combinations of CYN + MC-LR. The simultaneous exposure to both cyanotoxins caused a greater number of alterations in protein expression compared to single toxins, causing changes in the cellular, lipid and protein metabolism and in protein synthesis and transport. Further studies are needed to complete the toxicity molecular mechanisms of both CYN and MC-LR at the renal level. [ABSTRACT FROM AUTHOR]

Insects are able to detect a plethora of olfactory cues using a divergent family of odorant receptors (ORs). Despite the divergent nature of this family, related species frequently express several evolutionarily conserved OR orthologues. In the largest order of insects, Coleoptera, it remains unknown whether OR orthologues have conserved or divergent functions in different species. Using HEK293 cells, we addressed this question through functional characterization of two groups of OR orthologues in three species of the Curculionidae (weevil) family, the conifer‐feeding bark beetles Ips typographus L. ("Ityp") and Dendroctonus ponderosae Hopkins ("Dpon") (Scolytinae), and the pine weevil Hylobius abietis L. ("Habi"; Molytinae). The ORs of H. abietis were annotated from antennal transcriptomes. The results show highly conserved response specificities, with one group of orthologues (HabiOR3/DponOR8/ItypOR6) responding exclusively to 2‐phenylethanol (2‐PE), and the other group (HabiOR4/DponOR9/ItypOR5) responding to angiosperm green leaf volatiles (GLVs). Both groups of orthologues belong to the coleopteran OR subfamily 2B, and share a common ancestor with OR5 in the cerambycid Megacyllene caryae, also tuned to 2‐PE, suggesting a shared evolutionary history of 2‐PE receptors across two beetle superfamilies. The detected compounds are ecologically relevant for conifer‐feeding curculionids, and are probably linked to fitness, with GLVs being used to avoid angiosperm nonhost plants, and 2‐PE being important for intraspecific communication and/or playing a putative role in beetle–microbe symbioses. To our knowledge, this study is the first to reveal evolutionary conservation of OR functions across several beetle species and hence sheds new light on the functional evolution of insect ORs. [ABSTRACT FROM AUTHOR]

At present, approximately 80% of the currently approved recombinant therapeutic proteins are produced by Chinese hamster ovary cells. However, considering that the human embryonic kidney (HEK) 293 cell line comes from human sources and has the advantages of high transfection rate, fast growth rate, good post-translational modifications, and growth rate in serum-free suspension culture, the HEK293 cell line could produce high-quality and highly biologically active recombinant therapeutic proteins. Till now, several biotherapeutic proteins are selected to be produced by HEK293 cell lines. In establishing HEK293 cell lines with high-yield performance, they must be transformed and optimized by molecular biology and high-throughput analysis. Therefore, this paper primarily introduces the currently industrialized HEK293 cell line, HEK293 cell line expression system, and genetic engineering modification methods and proposes specific transformation strategies to provide insights into the future transformation of new HEK293 cell lines and new strategies for the efficient expression of recombinant proteins by HEK293 cells in industry. • New strategies for the recombinant protein expression in HEK293 cells. • Different modification strategies of the HEK293 cell line. • The development of high-yield HEK293 cell lines. [ABSTRACT FROM AUTHOR]

Gag virus-like particles (VLPs) are promising vaccine candidates against infectious diseases. VLPs are generally produced using the insect cell/baculovirus expression vector system (BEVS), or in mammalian cells by plasmid DNA transient gene expression (TGE). However, VLPs produced with the insect cell/BEVS are difficult to purify and might not display the appropriate post-translational modifications, whereas plasmid DNA TGE approaches are expensive and have a limited scale-up capability. In this study, the production of Gag VLPs with the BacMam expression system in a suspension culture of HEK293 cells is addressed. The optimal conditions of multiplicity of infection (MOI), viable cell density (VCD) at infection, and butyric acid (BA) concentration that maximize cell transduction and VLP production are determined. In these conditions, a maximum cell transduction efficiency of 91.5 ± 1.1%, and a VLP titer of 2.8 ± 0.1 × 109 VLPs/mL are achieved. Successful VLP generation in transduced HEK293 cells is validated using super-resolution fluorescence microscopy, with VLPs produced resembling immature HIV-1 virions and with an average size comprised in the 100–200 nm range. Additionally, evidence that BacMam transduction occurs via different pathways including dynamin-mediated endocytosis and macropinocytosis is provided. This work puts the basis for future studies aiming at scaling up the BacMam baculovirus system as an alternative strategy for VLP production. [ABSTRACT FROM AUTHOR]

Objective To test the cardiac toxicity of new compound HMS-01 and evaluate the safety profile for clinical trials. Methods Manualpatch clamp method was used to measure human Ether-a-go-go-Related Gene (hERG) potassium channel currents with different concentrations of HMS-01. Cisapride was selected as the positive control drug. HMS-01 was diluted to the concentration of 0.3, 1, 3, 10 and 30 μmol/L and applied to the cells. The changes in electrical currents were recorded and the inhibition rate was calculated. Results At the highest concentration of 30μmol/L, the inhibitory rate of HMS-01 on hERG channel was less than 30%. There was no obvious inhibitory effect compared with cisapride. Conclusion Compared with the cisapride, HMS-01 has no obvious inhibitory effect on hERG channel and has no cardiotoxicity. [ABSTRACT FROM AUTHOR]

Tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) is a widely used, additive flame retardant that migrates from end-use products, leading to ubiquitous exposure of humans around the world. However, little is known about whether TDCIPP disrupts the physiology of human embryonic cells. Therefore, the objective of this study was to determine whether TDCIPP alters cell viability, cellular metabolism, cytosine methylation, and reactive oxygen species (ROS) levels within human embryonic kidney (HEK293) cells. Relative to vehicle controls, TDCIPP (0.015–0.1225 µM) resulted in a concentration-dependent increase in cell viability, a finding that was driven by an increase in relative ATP abundance. Interestingly, TDCIPP (0.061–0.98 µM) increased the rate of glycolysis – an adaptive mechanism consistent with the Warburg effect exhibited by tumorigenic cells. Moreover, relative to vehicle-treated cells, TDCIPP (0.245–15.63 µM) exposure for 48 h (but not 24 h) resulted in a significant, concentration-dependent decrease in ROS in situ, and TDCIPP (0.245 µM) exposure significantly increased carnosine within the histidine metabolism pathway. However, TDCIPP did not affect global 5-methylcytosine (5-mC) methylation (0.015–15.63 µM), cell membrane integrity (0.061–0.98 µM), nor the abundance of mitochondria (0.061–1.95 µM). Overall, our findings with TDCIPP point to a novel mechanism of action that may be relevant to human embryonic stem cells. [Display omitted] • TDCIPP is a flame retardant with ubiquitous human exposure around the world. • Little is known about TDCIPP-induced effects on human embryonic cells. • TDCIPP increases the viability and rate of glycolysis within HEK293 cells. • TDCIPP decreases mitochondrial ROS generation after 48 h of exposure. • TDCIPP increases carnosine levels within the histidine metabolism pathway. [ABSTRACT FROM AUTHOR]

BACKGROUND: Studies have found that SMARCAL1 is selectively expressed in a variety of cells in human developing and mature kidneys, indicating that it plays an important role in kidney development. OBJECTIVE: To investigate the effect of SMARCAL1 overexpression on the proliferation of embryonic kidney cells. METHODS: 293T and HEK293 cells at passage 3 were selected. pHBLV-CMV-MCS-3FLAG-EF1-ZsGreen-T2A-PURO vector was used to construct lentiviral vector carrying human SMARCAL1 gene. The human SMARCAL1 gene was over-expressed using lentiviral vector and transfected into 293T cells. There were three groups: blank group untreated HEK293 cells; negative control group, in which HEK293 cells were transfected with empty virus; SMARCAL1 overexpression group, in which HEK293 cells were transfected with SMARCAL1 overexpressing virus. Stably transfected cell lines were screened using puromycin. Virus titer was determined based on the transfection rate. The stable transfection of SMARCAL1 into embryonic kidney cells was verified by quantitative real-time PCR and western blot. The effect of SMARCAL1 overexpression on the proliferation of HEK293 cells was assessed by cell counting kit-8 and EdU assays. Fluorogenic quantitative PCR was further used to evaluate the effect of SMARCAL1 overexpression on Wnt pathway. RESULTS AND CONCLUSION: The overexpressed SMARCAL1 plasmid gene sequence was consistent with the target gene. The titer of the SMARCAL1 overexpressed lentivirus was 1×108 TU/mL and 3×108 TU/mL for the control lentivirus. The mRNA and protein levels of SMARCAL1 in HEK293 cells were significantly increased after transfection with SMARCAL1 overexpressed lentivirus (P < 0.05). Up-regulation of SMARCAL1 expression gradually inhibited the proliferation of HEK293 cells (P < 0.05). The mRNA expression levels of CTNNB1 and WNT4 genes in the Wnt pathway were also decreased (P < 0.05). The SMARCAL1 overexpressed lentiviral vector is successfully constructed and expressed in HEK293 cells. The SMARCAL1 gene may be involved in regulating the proliferation of embryonic kidney cells through the Wnt signaling pathway and may affect the development of kidney. [ABSTRACT FROM AUTHOR]

Tungsten has no known function in humans and is a relatively new contaminant, whereas molybdenum, its congener in the periodic table, is a nutritionally essential element. In addition to early studies on molybdosis in ruminants, their toxic effects in the form of tungstate and molybdate have been addressed primarily in rodents and are predominantly mediated by inducing oxidative stress in various tissues. The purpose of this study was to evaluate the differences between tungstate and molybdate in human liver (HepG2) and kidney (HEK293) cell lines in terms of retention in cells, effect on reactive oxygen species, and activities of xanthine oxidase and phosphatases. The cell lines were exposed to tungstate or molybdate (1 µM to 10 mM) for 24 h, lysed and analyzed for the above biochemical parameters. Despite the chemical similarity of the two anions, cell-specific differential effects were observed. At all concentrations, tungstate was retained more in HEK293 cells while molybdate was retained more in HepG2 cells. HepG2 cells were more sensitive to tungstate than molybdate, showing reduced viability at concentrations as low as 10 µM. Exposure to either anion resulted in the inhibition of protein tyrosine phosphatases at 1 mM and an increased production of reactive oxygen species (ROS) at 100 µM despite their inhibition of the ROS-producing molybdenum enzyme xanthine oxidase. In conclusion, the results indicate that excess of nutritionally essential molybdate or non-essential tungstate causes toxicity by affecting ROS- and phosphorylation-dependent signaling pathways and ensuing gene expression. [ABSTRACT FROM AUTHOR]

Background: The protective effects of Montelukast (Mont), as an anti-inflammatory drug, against cadmium-induced kidney cell damage have already been studied and identified. Since the significant part of cadmium nephrotoxicity is caused by oxidative stress, this in vivo and in vitro study was conducted to investigate the possible role of Montelukast antioxidant properties in the protection. Methods: In the in vivo section, 42 rats were treated in seven groups of six rats as follows: Control; Cadmium Chloride (CdCl2) control; Montelukast control; CdCl2 plus Montelukast treatment; CdCl2 with Montelukast pre-treatment; Vitamin E control; CdCl2 plus Vitamin E treatment. In the in vitro section, human embryonic kidney cells (HEK293) were treated with CdCl2; Montelukast; Combined CdCl2 and Montelukast; Vitamin E; Combined CdCl2 and Vitamin E. Results: Montelukast, in both treatment and pretreatment forms, reduced serum urea, creatinine, and potassium levels compared to CdCl2 group, in vivo. Similar to vitamin E, the pre-treatment with Montelukast was associated with a significant decrease in Nitric Oxide (NO) and Total Antioxidant Capacity (TAC) in serum and renal tissue, and a significant increase in Glutathione Peroxidase (GPX) activity in serum compared those in the CdCl2 group. In the in vitro section of the study, Montelukast significantly reduced Malondialdehyde (MDA) and NO while the TAC level, Superoxide Dismutase (SOD), and the GPX activity increased significantly. Conclusion: Overall, the antioxidant effects of Montelukast appear to play a prominent role in preventing the renal toxicity due to cadmium exposure. [ABSTRACT FROM AUTHOR]

Objective: Hemophilia-A is a common genetic abnormality resulted from decreased or lack of factor VIII (FVIII) pro-coagulant protein function caused by mutations in the F8 gene. Majority of molecular studies consider screening of mutations and their relevant impacts on the quality and expression levels of FVIII. Interestingly, some of the functions in FVIII suggest a probable involvement of small non-coding RNAs embedded within the sequence of F8 gene. Therefore, microRNAs which are encoded within the F8 gene might have a role in hemophilia development. In this study, miRNAs production in the F8 gene was investigated by bioinformatics prediction and experimental validation. Materials and Methods: In this experimental study, bioinformatics tools have been utilized to seek the novel microRNAs inserted within human F8 gene. The ability to express new microRNAs in F8 locus was studied through reliable bioinformatics databases such as SSCProfiler, RNA fold, miREval, miR-FIND, UCSC genome browser and miRBase. Then, expression and processing of the predicted microRNAs were examined based on bioinformatics methods, in the HEK293 cell lines. Results: We are unable to confirm existence of the considered mature microRNAs in the transfected cells. Conclusion: We hope that through changing experimental conditions, designing new primers or altering cell lines as well as the expression of vectors, exogenous and endogenous expressions of the predicted miRNA will be confirmed. [ABSTRACT FROM AUTHOR]

• T. phanerophlebia is a medicinal plant that is commonly found in Southern Africa. • The toxicogenic effects and molecular mechanisms of T. phanerophlebia extract was investigated. • The extract reduced cell viability with an IC 50 of 1.36 mg/ml. • T. phanerophlebia decreased initiator and executioner caspase activity. • T. phanerophlebia caused DNA fragmentation. • The extract exerted toxic effects on kidney cells via the apoptotic pathway. The study investigated the toxicogenic effects and molecular mechanisms of Terminalia phanerophlebia on human renal cells. The methylthiazol tetrazolium (MTT) assay was used to assess the cytotoxicity of a crude leaf extract of Terminalia phanerophlebia in Hek293 cells. Luminometry was used to determine caspase activity, phosphatidylserine externalization and necrosis. The comet assay evaluated DNA fragmentation and the lactate dehydrogenase (LDH) assay was used to assess membrane integrity. Western blotting measured the expression of bcl-2-like protein 4 (Bax), inhibitors of apoptosis proteins (IAP), cleaved poly (ADP-ribose) polymerase (cPARP), p53 and nuclear factor kappa B (NF-κB) in treated Hek293 cells. A reduction in cell viability was observed with a half maximal inhibitory concentration (IC 50) of 1.36 mg/ml. At this concentration, Terminalia phanerophlebia decreased initiator and executioner caspase activity. Phosphatidylserine validated apoptosis in IC 25 -treated cells, while necrosis was observed in all treated cells. Comet formation indicated that there was DNA fragmentation. High levels of p53 resulted in upregulation of Bax in IC 25 -treated cells, while the opposite was noted in IC 50 -treated cells. Upregulation of NF-κB is associated with decreased caspase-3 and cPARP and further supported by an increase in IAP indicating that Hek293 cell death was mainly through apoptosis and necrosis. The findings suggest that while Terminalia phanerophlebia is a good extract for tuberculosis therapeutic intervention, it may exert toxic effects on kidney cells via the apoptotic pathway. [ABSTRACT FROM AUTHOR]

The ribosomal protein eL38 is a component of the mammalian translation machine. The deletion of the Rpl38 locus in mice results in the Tail-short (Ts) mutant phenotype characterized by a shortened tail and other defects in the axial skeleton development. Here, using the next-generation sequencing of total RNA from HEK293 cells knocked down of eL38 mRNA by transfection with specific siRNAs, we examined the effect of reduced eL38 content on genomic transcription. An approximately 4-fold decrease in the level of eL38 was shown to cause changes in the expression of nearly 1500 genes. Among the down-regulated genes, there were those responsible for p53 activity, Ca2+ metabolism and several signaling processes, as well as genes involved in the organization and functioning of the cytoskeleton. The genes related to rRNA processing and translation, along with many others, including those whose dysregulation is associated with developmental disorders, turned out to be up-regulated. Thus, we demonstrated that the decreased RPL38 expression leads to a significant reorganization of genomic transcription. Our findings suggest a possible link between the balance of eL38 and genes implicated in osteogenesis, thereby contributing to the elucidation of the reasons for the appearance of the above Ts mutant phenotype in animals. • Reduced level of ribosomal protein eL38 in cells largely reorganizes transcription. • eL38 knockdown down-regulates genes responsible for p53 activity. • eL38 knockdown activates genes associated with rRNA processing and translation. • BMP2 and BMP6 genes involved in osteogenesis are activated at a reduced eL38 level. [ABSTRACT FROM AUTHOR]

Background: Bark beetles are major pests of conifer forests, and their behavior is primarily mediated via olfaction. Targeting the odorant receptors (ORs) may thus provide avenues towards improved pest control. Such an approach requires information on the function of ORs and their interactions with ligands, which is also essential for understanding the functional evolution of these receptors. Hence, we aimed to identify a high-quality complement of ORs from the destructive spruce bark beetle Ips typographus (Coleoptera, Curculionidae, Scolytinae) and analyze their antennal expression and phylogenetic relationships with ORs from other beetles. Using 68 biologically relevant test compounds, we next aimed to functionally characterize ecologically important ORs, using two systems for heterologous expression. Our final aim was to gain insight into the ligand-OR interaction of the functionally characterized ORs, using a combination of computational and experimental methods. Results: We annotated 73 ORs from an antennal transcriptome of I. typographus and report the functional characterization of two ORs (ItypOR46 and ItypOR49), which are responsive to single enantiomers of the common bark beetle pheromone compounds ipsenol and ipsdienol, respectively. Their responses and antennal expression correlate with the specificities, localizations, and/or abundances of olfactory sensory neurons detecting these enantiomers. We use homology modeling and molecular docking to predict their binding sites. Our models reveal a likely binding cleft lined with residues that previously have been shown to affect the responses of insect ORs. Within this cleft, the active ligands are predicted to specifically interact with residues Tyr84 and Thr205 in ItypOR46. The suggested importance of these residues in the activation by ipsenol is experimentally supported through site-directed mutagenesis and functional testing, and hydrogen bonding appears key in pheromone binding. Conclusions: The emerging insight into ligand binding in the two characterized ItypORs has a general importance for our understanding of the molecular and functional evolution of the insect OR gene family. Due to the ecological importance of the characterized receptors and widespread use of ipsenol and ipsdienol in bark beetle chemical communication, these ORs should be evaluated for their potential use in pest control and biosensors to detect bark beetle infestations. [ABSTRACT FROM AUTHOR]

Our previous work showed that there is a limitation in the use of dihydrofolate reductase (dhfr)/methotrexate (MTX)-mediated gene amplification systems in dhfr-non-deficient HEK293 cells, as endogenous dhfr may interfere with the amplification process. In the present study, we successfully generated Epstein-Barr virus nuclear antigen-1 (EBNA-1)-amplified HEK293 cells in a dhfr-non-deficient HEK293 cell background using a single-plasmid vector-based gene amplification system with shRNA targeting the 3′-UTR of endogenous dhfr. The introduction of this shRNA efficiently downregulated the expression of endogenous dhfr in the HEK293 cells without affecting exogenous dhfr expression. The downregulation of endogenous dhfr improved the efficiency of EBNA-1 amplification, as evidenced by a comparison with the amplification extent in cells lacking shRNA expression at the same MTX concentration. The EBNA-1 expression levels from the EBNA-1-amplified clones selected in this study were higher than those obtained from EBNA-1-amplified clones that were generated using the conventional amplification in our previous study. Consistent with previous studies, EBNA-1 amplification improved the production of the Fc-fusion protein through a specific protein productivity (qp)-enhancing effect, rather than by improving cell growth or transfection efficiency. In addition, the N-glycan profiles in the Fc-fusion protein produced using this transient gene expression (TGE) system were not affected by EBNA-1 amplification. These results indicate the potential utility of EBNA-1-amplified mammalian cells, developed using a single-plasmid vector-based gene amplification system, for efficient protein production. Key points: • EBNA-1-amplified HEK293 cells were established using gene amplification system. • EBNA-1 amplification in TGE system can increase the Fc-fusion protein productivity. • EBNA-1 amplification does not affect the N-glycan profile in the Fc-fusion protein. [ABSTRACT FROM AUTHOR]

In cardiomyocytes, the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA2a) is a central component of intracellular Ca2+ regulation. Several heart diseases, including heart failure, are associated with reduced myocardial contraction due to SERCA2a downregulation. Therefore, the need for developing new drugs that could improve SERCA2a function is high. We have recently identified SERCA2a modulators (Compounds 6 and 8) from our screening campaigns and confirmed activation of biochemical SERCA2a ATPase activity and Ca2+ uptake activity. In this study, confocal microscopy and in-cell Ca2+ imaging were used to characterize the effects of these SERCA2a activators on Ca2+ regulation in mouse ventricular myocytes and endoplasmic reticulum (ER) Ca2+ uptake in a HEK293 cell expressing human SERCA2a. Analysis of cytosolic Ca2+ dynamics in cardiomyocytes revealed that both Compounds (6 and 8) increase the action potential-induced Ca2+ transients and sarcoplasmic reticulum (SR) Ca2+ load. While Compound 6 induced a negligible effect on Ca2+ transients invoked by the L-type Ca2+ channel (LTCC) current, Compound 8 increased Ca2+ transients during LTCC activation, suggesting an off-target protein interaction of Compound 8. Analysis of ER Ca2+ transport by human SERCA2a in HEK cells showed that only Compound 6 increased both ER Ca2+ uptake and ER Ca2+ load significantly, whereas Compound 8 had no effect on SERCA2a Ca2+ transport. This study revealed that Compound 6 exhibits promising characteristics that can improve intracellular Ca2+ dynamics by selectively enhancing SERCA2a Ca2+ uptake. • SERCA2a pump plays a central role in intracellular Ca2+ regulation of cardiomyocytes. • Confocal Ca2+ imaging determined effects of SERCA2a modulators on SERCA2a function and Ca2+ regulation. • Compound 6 shows therapeutic promise to improve intracellular Ca2+ dynamics by increasing SERCA2a function. • Compound 6 could restore healthy intracellular Ca2+ dynamics and contraction in heart disease. [ABSTRACT FROM AUTHOR]

It is well-documented that lead (Pb) toxicity can affect almost all systems in living organisms. It can induce selective autophagy of mitochondria (mitophagy) by triggering reactive oxygen species production. Emerging evidence has suggested that Pb-induced autophagy can also be activated by the endoplasmic reticulum (ER) stress pathway. However, the interplay between ER stress and mitophagy remains to be elucidated. In this study, human embryonic kidney HEK293 cells were employed to investigate the role of ER stress in Pb-induced mitophagy. The results showed that the cell viability was decreased and cell damage was induced after exposure to Pb (0, 0.5, 1, 2, and 4 mM) for 24 h in a dose-dependent manner. Moreover, the expression of LC3-Ⅱ was significantly increased, and the expression of HSP60 was dramatically decreased after exposure to 1 mM and 2 mM Pb, indicating the induction of mitophagy following Pb exposure. Meanwhile, the expressions of activating transcription factor 6, inositol-requiring protein-1α, CCAAT/enhancer binding protein homologous protein, and glucose-regulated protein 78 were dramatically increased after Pb treatment, signifying the initiation of ER stress. Notably, the mitophagic effect was significantly compromised when ER stress was inhibited by 0.5 mM 4-phenylbutyrate, which was evidenced by lesser decreases in HSP60 expression and level of LC3-Ⅱ, suggesting Pb-induced mitophagy may be activated by the ER stress. Taken together, these findings provide a better understanding of Pb toxicity and suggest that Pb-induced ER stress may play a regulatory role in the upstream of mitophagy. [ABSTRACT FROM AUTHOR]

Post-translational hydroxylation occurs in three mammalian ribosomal proteins, uS12, uL2, and uL15, which are located in the small (S) and large (L) subunits of the ribosome near the most important decoding and peptidyltransferase functional centers. We have used cell cultures, which produce protein uL15 labeled with the 3xFLAG epitope at the C-terminus (uL153xFLAG) or mutant forms of uL153xFLAG that contain His39Ala, His39Thr, or His40Ala substitutions, to examine the role of hydroxylated His39 of uL15 in maintaining the translational activity of ribosomes. It has been found that exogenous uL153xFLAG is able to functionally replace endogenous uL15 in HEK293 cells transfected with an appropriate DNA construct. However, the translational activity of ribosomes decreases by about 35% in cells that produce the above mutant forms of uL153xFLAG compared with that in cells that produce nonmutated uL153xFLAG. Analysis of the structural model of the human ribosome has allowed us to assume that the hydroxyl group in His39 is involved in the local stabilization of the ribosome structure through the formation of a hydrogen bond between this group and the nitrogen atom of the His40 imidazole ring. Given that His39 is located near the E site of the ribosome, we believe that this stabilization of the ribosome structure ensures the maintenance of its translational activity. [ABSTRACT FROM AUTHOR]

Unique characteristics displayed by phytoconstituent conjugated nanoparticles and their crucial interactions with proteins serve to develop nanoparticle-bio-interface platform. Gold nanoparticles of 16 nm in size were generated using aqueous extracts of pine bark and further conjugated to human serum albumin. The gold nanoparticles-protein complex was characterized by surface plasmon resonance, UV–vis and emission spectroscopy techniques. Further, it was characterized for surface morphology and elemental composition, crystallographic quality, nanoparticles size, shape, stability, structural determination and the identification of capping agent. Moreover, the interaction of gold nanoparticles with human serum albumin was investigated using conventional spectroscopy techniques. Fluorescence quenching and absorption studies demonstrated an effective binding of human serum albumin with oleamide capped gold nanoparticles. The molecular docking study showed a binding affinity of -6.1 kcal/mol whereas the molecular dynamics simulation indicated that the binding of oleamide to human serum albumin. A biological evaluation of pine bark extract-gold nanoparticles showed cytotoxicity with increased cell mortality in lung cancer cells and minimal toxicity on non-cancerous human embryonic kidney cells, respectively. [ABSTRACT FROM AUTHOR]

Background: The R-Spondin proteins comprise a family of secreted proteins, known for their important roles in cell proliferation, differentiation and death, by inducing the Wnt pathway. Several studies have demonstrated the importance of RSPOs in regulation of a number of tissue-specific processes, namely: bone formation, skeletal muscle tissue development, proliferation of pancreatic β-cells and intestinal stem cells and even cancer. RSPO1 stands out among RSPOs molecules with respect to its potential therapeutic use, especially in the Regenerative Medicine field, due to its mitogenic activity in stem cells. Here, we generated a recombinant human RSPO1 (rhRSPO1) using the HEK293 cell line, obtaining a purified, characterized and biologically active protein product to be used in Cell Therapy. The hRSPO1 coding sequence was synthesized and subcloned into a mammalian cell expression vector. HEK293 cells were stably co-transfected with the recombinant expression vector containing the hRSPO1 coding sequence and a hygromycin resistance plasmid, selected for hygror and subjected to cell clones isolation. Results: rhRSPO1 was obtained, in the absence of serum, from culture supernatants of transfected HEK293 cells and purified using a novel purification strategy, involving two sequential chromatographic steps, namely: heparin affinity chromatography, followed by a molecular exclusion chromatography, designed to yield a high purity product. The purified protein was characterized by Western blotting, mass spectrometry and in vitro (C2C12 cells) and in vivo (BALB/c mice) biological activity assays, confirming the structural integrity and biological efficacy of this human cell expression system. Furthermore, rhRSPO1 glycosylation analysis allowed us to describe, for the first time, the glycan composition of this oligosaccharide chain, confirming the presence of an N-glycosylation in residue Asn137 of the polypeptide chain, as previously described. In addition, this analysis revealing the presence of glycan structures such as terminal sialic acid, N-acetylglucosamine and/or galactose. Conclusion: Therefore, a stable platform for the production and purification of recombinant hRSPO1 from HEK293 cells was generated, leading to the production of a purified, fully characterized and biologically active protein product to be applied in Tissue Engineering. [ABSTRACT FROM AUTHOR]

Human xylosyltransferases-I and –II (XT-I and XT-II) catalyze the initial and rate-limiting step in proteoglycan (PG)-biosynthesis. Because PG are major components of the extracellular matrix (ECM), an alternated XT expression is associated with the manifestation of ECM-related diseases. While Drosophila melanogaster and Caenorhabditis elegans only harbor one XT-isoform, all higher organisms contain two isoforms, which are expressed in a tissue-specific manner. The reason for the appearance of two isoenzymes remains unexplained and remarkable, as all other enzymes involved in the synthesis of the tetrasaccharid linker, which connects the PG core protein with attached glycosaminoglycans, only show one isoform. In human, mutations in the XYLT genes cause diseases affecting the homeostasis of the ECM, such as skeletal dysplasias. We investigated for the first time whether already XT-I-deficient human embryonic kidney (HEK293) cells can compensate for decreased expression levels of both XT-isoforms. A siRNA-mediated XYLT2 mRNA knockdown led to reduced cellular proliferation rates and a partially increased cellular senescence of treated HEK293 cells. These results were verified by conducting a stable CRISPR/Cas9-mediated XYLT2 knockout, which revealed that only cells expressing at least partially functional XT-II proteins remain proliferative. Our study, therefore, shows for the first time that cells lacking both XT-isoforms are not viable and clearly indicates the importance of the XT concerning the cellular metabolism. • XT-deficient human HEK293 cells show a strongly reduced proliferation capacity. • Diminished expressions of one XT-isoform are not compensated by the remaining isoform. • Our study indicates the importance of the XT concerning the cellular metabolism. [ABSTRACT FROM AUTHOR]

Zika virus (ZIKV) was first detected in Brazil in 2015 and then rapidly spread to more than 80 countries in Africa, Asia and the Americas. ZIKV infection was correlated with severe congenital malformations in newborns from infected mothers, as well as with Guillain-Barré syndrome in adults. Although the number of infected people has declined in the affected countries lately, the development of a vaccine for ZIKV is of great importance to avoid the future resurgence of the virus in endemic areas or the future spread to currently non-endemic regions. Among many different platforms currently under study, virus-like particles (VLPs) are a promising alternative for the development of vaccines, since tridimensional particles mimicking the virus – but lacking its genome – can be produced and present the antigen in a repetitive way, potentially eliciting robust immune responses. In this work, we demonstrated the generation of stably transfected HEK293 cells constitutively expressing Zika VLPs. Small-scale shake flask studies using a stable cell pool enriched by Fluorescence-Activated Cell Sorting (FACS) showed that daily medium exchange (intermittent perfusion) significantly enhances viable cell density and VLP production (∼4-fold) over batch cultures. Continuous perfusion in a controlled bioreactor coupled to an ATF-2 cell retention device resulted in maximum VLP titers similar to those obtained under small-scale intermittent perfusion. Our results show that the use of cell lines constitutively expressing Zika VLPs, cultured in stirred-tank perfusion bioreactors, represents a promising system for the production of a VLP-based Zika vaccine candidate. [ABSTRACT FROM AUTHOR]

Since the use of protein therapeutics is effective for treating intractable human diseases, the production of biologic therapeutic agents has dramatically increased over the past three decades. The Chinese hamster ovary (CHO) cell lines are the most commonly used host cell expression system for recombinant protein production. High productive and stable clonal cell lines for recombinant protein production have been established from the DHFR -deficient CHO cell using the dihydrofolate reductase/methotrexate (DHFR/MTX) selection methods. Human embryonic kidney 293 (HEK293) cells are alternative host cells widely used for protein production. In most case, however, the cells are used for the transient expression, and there is no gene amplification system in HEK293 cells. In this study, we established a DHFR -deficient HEK293 cell line for the high yield of recombinant proteins. We doubly knocked out DHFR and DHFR2 in the MAN1A1 / A2 / B1 / C1 -quadruple knockout HEK293 (QD-KO) cells, using the CRISPR/Cas9 system. The DHFR -deficient QD-KO cells were used to overexpress two proteins, lysosomal acid lipase and the constant fragment of human immunoglobulin G 1 by the DHFR/MTX gene-amplification method. This method resulted in a dramatic increase in the two protein expressions in the DHFR -deficient QD-KO cells by increasing MTX concentration. Our system could be adopted in the production of several recombinant proteins including therapeutic proteins. [ABSTRACT FROM AUTHOR]

Objective: Nigella sativa, commonly known as the black seed, has been used since ancient times in folk medicine, and its various therapeutic benefits have been mentioned in some ancient medical sources and confirmed by modern science. Thymoquinone is the major compound and essential active ingredient of Nigella sativa seed oil. Thymoquinone has been reported to have high biological activity and broad therapeutic potential through several mechanisms that affect cells including anti-oxidant, anti-inflammatory, and anti-tumor properties. Although it has been widely reported that Thymoquinone inhibits cell growth and proliferation and stimulates apoptosis in various types of cancer cells, the mechanisms and signaling pathways are not fully understood. The aim of this study was to determine the effect of Thymoquinone on apoptosis in both tumor and non-tumor cells. Methods: In this study, breast cancer cell line (MCF-7) was used as tumor cells and Human Embryonic Kidney Cell line (HEK293) was used as non-tumor cells. Cells were treated with Thymoquinone and viability test performed by MTT assay. Gene expression of apoptosis markers such as Bax, BAD, Bcl-2, and p53 was determined by Real-Time PCR. HEK293 cells were used as non-tumor control. Results and Conclusion: Results suggest that Thymoquinone has a strong effect on cell proliferation and vitality. Thymoquinone has increased the expression of BAD, Bax genes which induce apoptosis and decreased the p53 gene in breast cancer cells. Therefore Thymoquinone promotes apoptosis and enhances anti-cancer efficacy in breast cancer cells. [ABSTRACT FROM AUTHOR]

The type 2a sarco-/endoplasmic reticulum Ca2+-ATPase (SERCA2a) plays a key role in Ca2+ regulation in the heart. However, available techniques to study SERCA function are either cell destructive or lack sensitivity. The goal of this study was to develop an approach to selectively measure SERCA2a function in the cellular environment. The genetically encoded Ca2+ sensor R-CEPIA1er was used to measure the concentration of Ca2+ in the lumen of the endoplasmic reticulum (ER) ([Ca2+]ER) in HEK293 cells expressing human SERCA2a. Coexpression of the ER Ca2+ release channel ryanodine receptor (RyR2) created a Ca2+ release/ reuptake system that mimicked aspects of cardiac myocyte Ca2+ handling. SERCA2a function was quantified from the rate of [Ca2+]ER refilling after ER Ca2+ depletion; then, ER Ca2+ leak was measured after SERCA inhibition. ER Ca2+ uptake and leak were analyzed as a function of [Ca2+]ER to determine maximum ER Ca2+ uptake rate and maximum ER Ca2+ load. The sensitivity of this assay was validated by analyzing effects of SERCA inhibitors, [ATP]/ [ADP], oxidative stress, phospholamban, and a loss-of-function SERCA2a mutation. In addition, the feasibility of using R-CEPIA1er to study SERCA2a in a native system was evaluated by using in vivo gene delivery to express R-CEPIA1er in mouse hearts. After ventricular myocyte isolation, the same methodology used in HEK293 cells was applied to study endogenous SERCA2a. In conclusion, this new approach can be used as a sensitive screening tool to study the effect of different drugs, posttranslational modifications, and mutations on SERCA function. [ABSTRACT FROM AUTHOR]

Despite its exceptionally low circulating concentration, apolipoprotein (apo) A-V is a potent modulator of plasma triacylglycerol levels. The secretion efficiency of nascent apoA-V was investigated in cultured cells transfected with mRNA. Following transfection of HepG2 cells with wild type apoA-V mRNA, apoA-V protein was detectable in cell lysates by 6 h. At 24 h post transfection, evidence of apoA-V secretion into media was obtained, although most apoA-V was recovered in the cell lysate fraction. By contrast, apoA-I was efficiently secreted into the culture medium. A positive correlation between culture medium fetal bovine serum content and the percentage of apoA-V recovered in conditioned media was observed. When transfected cells were cultured in serum-free media supplemented with increasing amounts of high density lipoprotein, a positive correlation with apoA-V secretion was observed. The data indicate that, following signal sequence cleavage, the bulk of nascent apoA-V remains cell associated. Transit of nascent apoA-V out of cultured cells is enhanced by the availability of extracellular lipid particle acceptors. Image 1 • Transfection of cells with apoA-V mRNA induced protein production. • Compared to a control apolipoprotein, apoA-V secretion is inefficient. • Culture medium fetal bovine serum content altered apoA-V secretion efficiency. • In serum free medium isolated HDL enhanced apoA-V secretion from HepG2 cells. [ABSTRACT FROM AUTHOR]

Abstract Ochratoxin A (OTA) and citrinin (CTN) are important mycotoxins, which often coexist in food and feed stuff. In this study, individual and combinative cytotoxicity of OTA and CTN were tested in human embryonic kidney (HEK) 293 cells via MTT assay, and synergistic cytotoxic effects were found following co-treatment with OTA and CTN, manifested by significant accumulation of HEK293 cells in S and G2/M stages. Transcriptomic and sRNA sequencing were performed to explore molecular signatures mediating individual or combinative cytotoxicity. A total of 378 miRNAs were identified, among which 66 miRNAs targeting thousands of genes were differentially expressed in response to different treatments, and 120 differentially expressed genes (DEGs) were regulated by either individual or combinative treatments. Correlations between two representative miRNAs (hsa-miR-1-3p and hsa-miR-122–5p), and their target genes, programmed cell death 10 (PDCD10) and cyclin G1 (CCNG1), associated with apoptotic signaling and cell cycle were analyzed by luciferase assay system. Further, their expression patterns were validated by quantitative real-time PCR and western blot analysis, suggesting that both miRNA-target interactions might account for the mycotoxin-induced cell death. Taken together, these findings provide molecular evidences for synergistic cytotoxic effects of exposure to single and mixture of OTA and CTN in HEK293 cells. Highlights • Synergistic cytotoxic effects of OTA and CTN were found in HEK293 cells. • 66 DEmiRs and 120 DEGs were identified in mycotoxin-treated HEK293 cells. • hsa-miR-1-3p/hsa-miR-122–5p involved in OTA and CTN-induced apoptosis and cell cycle. [ABSTRACT FROM AUTHOR]

Abstract This study aimed to investigate the cellular disposition of liquiritigenin via the sulfonation pathway and the role of efflux transporters in liquiritigenin sulfate excretion. The sulfonation disposition of liquiritigenin was investigated using SULT1A3 overexpressed HEK293 cells (HEK-SULT1A3 cells). Liquiritigenin generated one mono-sulfate metabolite (7-O-sulfate) in HEK-SULT1A3 cell lysate. And the sulfonation followed the Michaelis-Menten kinetic (V max = 0.84 nmol/min/mg and K m = 7.12 μM). Expectedly, recombinant SULT1A3 (hSULT1A3) showed a highly similar kinetic profile with cell lysate. Furthermore, 7-O-sulfate was rapidly generated and excreted in HEK-SULT1A3 cells. Ko143 (a BCRP-selective inhibitor) at 20 μM significantly decreased the excretion rate of liquiritigenin sulfate (>42.5%, p < 0.001). Moreover, the pan-MRPs inhibitor MK-571 at 20 μM essentially abolished the liquiritigenin sulfate effluxion, resulting in the marked reduction of excretion rate (>97.4%, p < 0.001). Furthermore, knockdown of BCRP led to moderate reduction in sulfate excretion (15.9%–16.9%, p < 0.05). Silencing of MRP4 caused significant decreased in sulfate excretion (20.2%–32.5%, p < 0.01). In conclusion, one sulfate metabolite was generated from liquiritigenin in HEK-SULT1A3 cells. BCRP and MRP4 should be the key factors for the cellular excretion of liquiritigenin sulfate. Graphical abstract Unlabelled Image [ABSTRACT FROM AUTHOR]

Abstract We compared membrane permeabilization by nanosecond pulsed electric field (nsPEF) in HEK293 cells with and without assembled CaV1.3 L-type voltage-gated calcium channel (VGCC). Individual cells were subjected to one 300-ns pulse at 0 (sham exposure); 1.4; 1.8; or 2.3 kV/cm, and membrane permeabilization was evaluated by measuring whole-cell currents and by optical monitoring of cytosolic Ca2+. nsPEF had either no effect (0 and 1.4 kV/cm), or caused a lasting (>80 s) increase in the membrane conductance in about 50% of cells (1.8 kV/cm), or in all cells (2.3 kV/cm). The conductance pathway opened by nsPEF showed strong inward rectification, with maximum conductance increase for the inward current at the most negative membrane potentials. Although these potentials were below the depolarization threshold for VGCC activation, the increase in conductance in cells which expressed VGCC (VGCC+ cells) was about twofold greater than in cells which did not (VGCC− cells). Among VGCC+ cells, the nsPEF-induced increase in membrane conductance showed a positive correlation with the amplitude of VGCC current measured in the same cells prior to nsPEF exposure. These findings demonstrate that the expression of VGCC makes cells more susceptible to membrane permeabilization by nsPEF. Time-lapse imaging of nsPEF-induced Ca2+ transients confirmed permeabilization by a single 300-ns pulse at 1.8 or 2.3 kV/cm, but not at 1.4 kV/cm, and the transients were expectedly larger in VGCC+ cells. However, it remains to be established whether larger transients reflected additional Ca2+ entry through VGCC, or were a result of more severe electropermeabilization of VGCC+ cells. Graphical abstract Unlabelled Image Highlights • 300-ns pulses at 1.8 and 2.3 kV/cm caused permeabilization in HEK2913 cells. • The increase in inwardly rectifying membrane conductance persisted for over 80 s. • Effect was stronger in cells which expressed more voltage gated Ca2+ channels. • Exposure caused lasting inhibition of voltage-gated current through Ca2+ channels. [ABSTRACT FROM AUTHOR]

The MEKK1 kinase is a key regulator of stress signaling in Arabidopsis; however, little is known about the regulation of its kinase activity. Here, we found that recombinant MEKK1, expressed in both mammalian HEK293 cells and Escherichia coli, shows a mobility shift in SDS‐PAGE, and immunoblotting detected phosphorylation of serine, threonine, and tyrosine residues. N‐terminal deletions, site‐directed mutagenesis, and protein phosphatase treatment revealed that the mobility shift results from autophosphorylation of the kinase domain. We identified the tyrosine autophosphorylation sites in the N‐terminal region of MEKK1. Tyrosine to phenylalanine mutations decrease phosphorylation of the substrate MKK1, suggesting the important role of this residue in the regulation of MEKK1 kinase activity. The present study is the first to show that plant MAPKKKs are regulated by tyrosine phosphorylation. [ABSTRACT FROM AUTHOR]

Abstract The odorant receptors (ORs) of insects are crucial for host and mate recognition. In moths (Lepidoptera), specialized ORs are involved in male detection of the sex pheromone produced by females. Most moth sex pheromones are C 10 -C 18 acetates, alcohols, and aldehydes (Type I pheromones), and most pheromone receptors (PRs) characterized to date are from higher Lepidoptera (Ditrysia), responding to these types of compounds. With few exceptions, functionally characterized PRs fall into what has been called the “PR-clade”, which also contains receptors that have yet to be characterized. While it has been suggested that moth PRs have evolved from plant odor-detecting ORs, it is not known when receptors for Type I pheromones arose. This is largely due to a lack of functionally characterized PRs from non-ditrysian Lepidoptera. The currant shoot borer moth, Lampronia capitella (Prodoxidae), belongs to a non-ditrysian lineage, and uses Type I pheromone compounds. We identified 53 ORs from antennal transcriptomes of this species, and analyzed their phylogenetic relationships with known lepidopteran ORs. Using a HEK293 cell-based assay, we showed that three of the LcapORs with male-biased expression (based on FPKM values) respond to Type I pheromone compounds. Two of them responded to pheromone components of L. capitella and one to a structurally related compound. These PRs are the first from a non-ditrysian moth species reported to respond to Type I compounds. They belong to two of the more early-diverging subfamilies of the PR-clade for which a role in pheromone detection had not previously been demonstrated. Hence, our definition of the monophyletic lepidopteran PR-clade includes these receptors from a non-ditrysian species, based on functional support. Graphical abstract Image 1 Highlights • We identified 53 odorant receptors from the non-ditrysian Lampronia capitella. • Three pheromone receptors (PRs) responded to Type I moth pheromone compounds. • We extend the conserved PR clade to include additional receptor subfamilies. [ABSTRACT FROM AUTHOR]

Recombinant adenoviral (Ad) vectors are highly efficient gene transfer vectors widely used in vaccine development and immunotherapy. To promote the industrial application of Ad vectors, studies focusing on reducing the cost of manufacturing, shortening the preclinical research period, and improving the quality of products are needed. Here, we describe a highly efficient and economical process for producing Ad vector in a novel, single-use bioreactor system suitable for clinical trials. A mini-bioreactor was used for parameter optimization and development of medium replacement protocols for Ad5-GFP production before scale-up. HEK293 cell culture and virus infection were monitored in a disposable AmProtein Current Perfusion Bioreactor and Bioflo310 bioreactor using optimized parameters and medium replacement protocols. The total cell number increased from 2.0 × 109 to 3.2 × 1010 after 6 days of culture. The total number of viral particles obtained in a single batch was 1.2 × 1015. These results demonstrate the efficiency and suitability of this system for Ad vector production for research and GMP applications. [ABSTRACT FROM AUTHOR]

The functional state of δ-opioid receptor signaling cascade in live cells exposed to a therapeutic concentration of lithium for a prolonged period of time (weeks) is not known because the previous studies of Li interference with OR were oriented to µ-OR only. The same applies to the analysis of the prolonged effect of Li on oxidative stress in context with δ-OR function. HEK293 cells stably expressing δ-OR were cultivated in the presence or absence of 1 mM LiCl for 7 or 21 days, homogenized and the post-nuclear (PNS) and plasma membrane (PM) fractions prepared from all four types of cells. Level of δ-OR in PM was determined by specific radioligand [ 3 H]DADLE binding and immunoblot assays; the functional coupling between δ-OR and G proteins was determined as DADLE-stimulated high-affinity [ 35 S]GTPγS binding. In the whole cells, general oxidative stress was monitored by fluorescent dye 2′,7′-dichlorofluorescein diacetate (DCF) and results verified by analysis of PNS and isolated PM . Generation of 4-hydroxy-2-nonenal (4-HNE)-protein adducts and malondialdehyde (MDA) level were determined as products of lipid peroxidation. Li-treated cells exhibited the decreased amount of δ-OR. This was evidenced by both [ 3 H]DADLE binding and immunoblot assays. The δ-OR-G protein coupling efficiency was diminished. Simultaneously, in Li-treated cells, the highly increased oxidative stress measured as DCF fluorescence intensity was noticed. Importantly, this result was detected in live cells as well as PNS and PM. Accordingly, production of 4-HNE-protein adducts and MDA was clearly increased in Li-treated cells. The general significance of our work lies in presentation of novel data indicating that prolonged exposure of live HEK293 cells to the therapeutic concentration of Li results in down-regulation of δ-OR protein level and attenuation of δ-OR function in parallel with increased oxidative stress and increased level of lipid peroxidation products. [ABSTRACT FROM AUTHOR]

Background Sudden cardiac death (SCD) occurs in a broad spectrum of cardiac pathologies and is an important cause of mortality in the general population. Idiopathic ventricular fibrillation (IVF) is a rare but important factor resulting in SCD. It is diagnosed in a resuscitated cardiac arrest victim underlying unknown cause, with documented ventricular fibrillation. Previous studies have demonstrated that mutations in dipeptidyl aminopeptidase-like protein-6 (DPP6) and cardiac sodium channel Nav1.5 (SCN5A) are the most important genetic factors involve in IVF. Aim By using whole sequencing to identify the genetic lesion of a family with suspicious idiopathic ventricular fibrillation Design Prospective genetic study. Methods In this study, we employed whole-exome sequencing in combination with arrhythmia-related gene filtering to identify the genetic lesion for a family suffering from suspicious IVF, syncope and SCD. We then generated the plasmids of DPP6 -pcDNA3.1+ (WT and c.1578G>C/p.Q526H). Kv4.3 -pcDNA3.1+ was co-transfected together with/without DPP6 -pcDNA3.1+ (WT and/or c.1578G>C/p.Q526H) into HEK293 cells to perform the patch clamp experiments. Results A novel missense mutation (c.1578G>C/p.Q526H) of DPP6 was identified and co-segregated with affected patients in this family. Patch clamp experiments suggested that this novel mutation might result in a gain of function and disturb the efflux of potassium ion. Conclusion Our study not only reported the second missense mutation of DPP6 in heart disease and expanded the spectrum of DPP6 mutations, but also contribute to the genetic diagnosis and counseling of families with suspicious IVF, syncope and SCD. [ABSTRACT FROM AUTHOR]

Human ribosomal protein eS26 is an indispensable component of the small (40S) ribosomal subunit and, along with other ribosomal proteins, is involved in interaction with mRNAs during translation. Here, we explored the behavior of the exogenous ribosomal protein eS26 modified at the C-terminus in the events related to translation in human cells using a doxycycline-inducible HEK293-derived cell line enabling the stable production of C-terminal FLAG-tagged eS26 (eS26 FLAG ). The production of eS26 FLAG in cells was accompanied by a decrease in the endogenous eS26 content although its mRNA level did not change. Exogenous eS26 FLAG was able to replace endogenous eS26 in 40S ribosomal subunits, without affecting the assembly and translational activity of 80S ribosomes. However, eS26 FLAG -containing ribosome fractions from the respective polysome profile displayed a reduced content of nucleophosmin, a multifunctional protein, which, as is known, is involved in the formation and nuclear export of ribosomal subunits. In general, our data showed that although the appearance of the FLAG tag at the C-terminus of eS26 does not affect translation, it interferes with nucleophosmin incorporation into the 40S subunit, pointing out the importance of the C-terminus integrity of eS26 for nucleophosmin binding. In addition, with the recombinant protein, we demonstrated the binding of nucleophosmin to both isolated eS26 and 40S subunits in the presence of HeLa nuclear extract that phosphorylated the recombinant nucleophosmin. These findings suggest that for nuclear export, nucleophosmin could directly bind to pre-40S subunits in the mRNA exit site region where the C-terminus of eS26 is located. [ABSTRACT FROM AUTHOR]

A strain of embryonic human kidney cells (HEK293) was transiently co-transfected with the expression vectors coding for the α- and β-subunits of human thyroid-stimulating hormone (hTSH), and, for the first time, a human cell-derived recombinant hTSH was synthesized and extensively characterized. The purification strategy involving two steps provided an overall yield of 55% and a purity level > 90%. The purified material (hTSH-HEK) was analyzed and compared to a CHO-derived recombinant preparation (hTSH-CHO) and to a pituitary-derived (hTSH-Pit) preparation. The three preparations showed an equivalent purity (> 95%) with a hTSH-HEK molecular mass 2.1% lower than that of hTSH-CHO and 2.7% higher than that of hTSH-Pit. Remarkable differences were found in the carbohydrate moiety, the lowest sialic acid content and highest fucose content being observed in hTSH-HEK. In vivo biological activity was confirmed for the three preparations, the hTSH-HEK bioactivity being 39 and 16% lower than those of hTSH-CHO and hTSH-Pit, respectively. The hTSH-HEK circulatory half-life ( t ) was also shorter than those of hTSH-CHO (1.5-fold) and hTSH-Pit (1.2-fold). According to these findings, HEK-293-derived hTSH can be considered to be useful for clinical applications, in view as well of its human origin and particular carbohydrate composition. [ABSTRACT FROM AUTHOR]

Mammalian cells, e.g., CHO, BHK, HEK293, HT-1080, and NS0 cells, represent important manufacturing platforms in bioengineering. They are widely used for the production of recombinant therapeutic proteins, vaccines, anticancer agents, and other clinically relevant drugs. HEK293 (human embryonic kidney 293) cells and their derived cell lines provide an attractive heterologous system for the development of recombinant proteins or adenovirus productions, not least due to their human-like posttranslational modification of protein molecules to provide the desired biological activity. Secondly, they also exhibit high transfection efficiency yielding high-quality recombinant proteins. They are easy to maintain and express with high fidelity membrane proteins, such as ion channels and transporters, and thus are attractive for structural biology and electrophysiology studies. In this article, we review the literature on HEK293 cells regarding their origins but also stress their advancements into the different cell lines engineered and discuss some significant aspects which make them versatile systems for biopharmaceutical manufacturing, drug screening, structural biology research, and electrophysiology applications. [ABSTRACT FROM AUTHOR]

The importance of evaluating the toxic effects associated with the use of reclaimed water has been increasing. The purpose of this research was to investigate the cytotoxicity and molecular toxicity of reclaimed water on the human embryonic kidney 293 (HEK293) cells. The culture medium was synthesized using the reclaimed water samples. Wastewater treatment plant influent (WTI) and effluent (WTE), containing micropollutants at the nanogram per liter level, decreased cell proliferation (93.4–98.9% and 91.5–96.6% of the control, respectively) and increased cell damage (103.6–117.5% and 100.7–109% of the control, respectively) at all exposure times, except for a decrease in cell damage observed after an 8-h exposure to WTE. Membrane bioreactor permeate (MBRP) increased cell proliferation (102.1–106.7% of the control) and decreased cell damage at 8 and 12 h (92.4 and 98.4% of the control, respectively), but slightly increased cell damage at 24 h and later time points (101.1–104.9% of the control). All three water samples induced cell apoptosis (120.9–123.4% of the control). They also affected the expression of cell-cycle regulatory proteins (p16 INK4a , p27 Kip1 , cyclin-dependent kinases 2 and 4, cyclin D1, and cyclin E) and apoptosis-related regulatory proteins ( p -JNK, Bcl-2, caspase-9, and caspase-3). In conclusion, all three water samples showed cytotoxicity and molecular toxicity in the HEK293 cells, and the results of the cell-cycle and apoptosis regulatory protein expression after WTI and WTE treatments were consistent with the results of the cytotoxicity. [ABSTRACT FROM AUTHOR]

Increasing recombinant protein production while ensuring a high and consistent protein quality remains a challenge in mammalian cell culture process development. In this work, we combined a nutrient substitution approach with a metabolic engineering strategy that improves glucose utilization efficiency. This combination allowed us to tackle both lactate and ammonia accumulation and investigate on potential synergistic effects on protein production and quality. To this end, HEK293 cells overexpressing the pyruvate yeast carboxylase (PYC2) and their parental cells, both stably producing the therapeutic glycoprotein interferon α2b (IFNα2b), were cultured in media deprived of glutamine but containing chosen substitutes. Among the tested substitutes, pyruvate led to the best improvement in growth (integral of viable cell density) for both cell lines in batch cultures, whereas the culture of PYC2 cells without neither glutamine nor any substitute displayed surprisingly enhanced IFNα2b production. The drastic reduction in both lactate and ammonia in the cultures translated into extended high viability conditions and an increase in recombinant protein titer by up to 47% for the parental cells and the PYC2 cells. Product characterization performed by surface plasmon resonance biosensing using Sambucus nigra (SNA) lectin revealed that the increase in yield was however accompanied by a reduction in the degree of sialylation of the product. Supplementing cultures with glycosylation precursors and a cofactor were effective at counterbalancing the lack of glutamine and allowed improvement in IFNα2b quality as evaluated by lectin affinity. Our study provides a strategy to reconcile protein productivity and quality and highlights the advantages of PYC2-overexpressing cells in glutamine-free conditions. [ABSTRACT FROM AUTHOR]

Ochratoxin A (OTA), a common contaminant of grain and fruit and known carcinogen, has been linked to impaired antioxidant response and cellular repair. The effect of OTA on inflammation in cells has not been explored. This study investigated OTA's influence on inflammatory mediators using a range of OTA concentrations (0.5 μM (sub-IC 50 ), 1.2 μm (IC 50 ) and 2 μm (supra-IC 50 )) on human embryonic kidney (HEK293) cells over 24hr. The markers of inflammation in HEK293 cells were evaluated using the following techniques: western blotting (phosphorylated (p-)NFκB (Ser536), p-IKK (Ser176/180) and p-p53 (Ser392), total NFκB, IKK, IκBα and p53), luminometry (caspases 1, 3/7, 8, 9, ATP) and ELISA to determine IL-1β levels. The results indicate increased activation of the inflammatory pathway in the sub-IC 50 concentration, evidenced by significant increases in p-NFκB ( p = 0.0006). The IC 50 concentration indicates decreased inflammatory induction supported by decreased levels of IL-1β and caspase 1 ( p = 0.0186 and p = 0.0068 respectively) with decreased IKK and increased IκBα ( p = 0.0046 and p = 0.0006 respectively). Furthermore, a decrease in inflammatory pathway activation was seen in O3 (increased IκBα, p < 0.05) coupled with increased apoptosis via elevated caspase 3/7 ( p = 0.0002), 8 ( p = 0.0011) and 9 activity ( p = 0.0002); as well as decreased ATP levels. This data suggests a new mechanism of OTA toxicity and its involvement in inflammation, kidney disease and fibrosis. [ABSTRACT FROM AUTHOR]

Background and Objectives CD40L is a membrane protein and a member of the tumor necrosis factor super family (TNFSF) that plays an important role in transferring cell signaling in innate and adaptive immunity. Since this protein plays its role through clustering the receptors on the target cell surface, it seems that the multimeric form of this molecule can have a stronger effect than the natural trimeric form. So in this study we describe cloning and expression of dodecameric soluble CD40L through self multimerizing protein, surfactant protein D (SP-D), in the HEK293 cell line. Materials and Methods In this experimental study, the SPD-CD40L was designed in silico and cloned in pcDNA3.1(+) plasmid. HEK293 cells were transfected and used as the expression host cells. The expression of SPD-CD40L was determined at the RNA level by RT-PCR method. After the purification of the recombinant proteins by affinity chromatography, the molecular weight of the recombinant protein was determined by SDS-PAGE. To evaluate the specificity of protein, ELISA and Dot Blot were used. Results The results indicated that HEK293 cells were transfected and recombinant protein was expressed 24 and 48 hours after Transfection. Moreover, the results of ELISA and Dot Blot methods represented the specificity of recombinant SPD-CD40L chimeric protein. Conclusions Chimeric protein SPD-CD40L was expressed successfully by pcDNA3.1(+) in eukaryotic HEK293 cell line and the molecular weight of 4-trimeric (dodecameric) protein was shown to be consistent with the expected amount. [ABSTRACT FROM AUTHOR]

Background: Chemotherapeutic approaches to the control of HPV infection suffer from a lack of specificity. For most existing HPV inhibitors, the weak antiviral effects observed in cellular assays suggest that further improvements in selecting targets, in drug potency, and in bioavailability and cell uptake are required. Objective: To synthesize novel 1,3-oxazole derivatives and define their antiviral activities against the human papillomavirus (HPV) in vitro. Methods: Determination of transient replication of an HPV-11 in transfected HEK293 cells, and HPV-18 DNA amplification in an organotypic squamous epithelial raft culture of primary human keratinocytes (PHKs), and cytotoxicity assays were used. Results: Bioassays showed that the synthesized compounds 2, 4, 5, and 9 exhibited potent antiviral activity against low-risk HPV-11 (IC50 = 1.7-9.6 µM) in a transient DNA replication assay and exhibited low cytotoxicity in HEK293 cells compared to cidofovir (CDV), antiviral agent in clinical use. Selectivity indices of compounds 4 and 5 were 20-40 times greater than that of CDV. However, compounds 4 and 9 did not exhibit a significant antiviral effect against high-risk HPV-18 infections in organotypic epithelial raft cultures. Although prophylactic HPV vaccines are now available to protect against primary infections by the seven genotypes most commonly found in cervical, penile, anal and oro-pharyngeal cancers (HPV16, 18, 31, 33, 45, 52, and 58) and two genotypes (HPV6 and 11) that cause benign anogenital warts and laryngeal papillomas, they do not protect against infections by other HPV types. Moreover, individuals already infected with HPV will not benefit from the vaccines. Thus, the need for antiviral agents to treat HPV-associated diseases remains great, but few options currently exist. Conclusions: We show that substituted 1,3-oxazole derivatives are a promising structure class of chemical compounds for the development of antiviral drugs against HPV lesions. [ABSTRACT FROM AUTHOR]

The focus of this study is on a rapid and cost‐effective approach for the synthesis of silver nanoparticles (AgNPs) using Artemisia quttensis Podlech aerial parts extract and assessment of their antioxidant, antibacterial and anticancer activities. The prepared AgNPs were determined by ultraviolet–visible spectroscopy, X‐ray diffraction, Fourier transform infra‐red spectroscopy, transmission electron microscopy, scanning electron microscopy, energy‐dispersive spectroscopy, and dynamic light scattering and zeta‐potential analysis. The AgNPs and A. quttensis extract were evaluated for their antiradical scavenging activity by 2, 2‐diphenyl, 1‐picryl hydrazyl assay and anticancer activity against colon cancer (human colorectal adenocarcinoma cell line 29) compared with normal human embryonic kidney (HEK293) cells. Also, the prepared AgNPs were studied for its antibacterial activity. The AgNPs revealed a higher antioxidant activity compared with A. quttensis extract alone. The phyto‐synthesised AgNPs and A. quttensis extract showed a dose–response cytotoxicity effect against HT29 and HEK293 cells. As evidenced by Annexin V/propidium iodide staining, the number of apoptotic HT29 cells was significantly enhanced, following treatment with AgNPs as compared with untreated cells. Besides, the antibacterial property of the AgNPs indicated a significant effect against the selected pathogenic bacteria. These present obtained results show the potential applications of phyto‐synthesised AgNPs using A. quttensis aerial parts extract. [ABSTRACT FROM AUTHOR]