PDGF-C promotes cell proliferation partially via downregulating BOP1.

Platelet-derived growth factor C (PDGF-C) is a member of PDGF/VEGF family, which is well-known for important functions in the vascular system. It is widely reported that PDGF-C is able to modulate cell proliferation. However, it is still not very clear about this cell modulating mechanism at the molecular level. In a screening of factors regulated by PDGF-C protein, we fished out a factor called block of proliferation 1 (BOP1), which is a pivotal regulator of ribosome biogenesis and cell proliferation. In this study, we investigated the regulation of BOP1 by PDGF-C and its role in modulating cell proliferation. We found that BOP1 was downregulated at both mRNA and protein levels in cells treated with PDGF-C-containing conditioned medium. On the other hand, BOP1 was upregulated in PDGF-C deficient mice. Furthermore, we confirmed that overexpression of BOP1 inhibited HEK293A cell proliferation, whereas knockdown of BOP1 promoted cell proliferation. The mitogenic effect of PDGF-C could be attenuated by downregulation of BOP1. Our results demonstrate a clear PDGF-C-BOP1 signaling that modulates cell proliferation.

Phytochemical Compounds of Euphorbia bivonae Extract and Their Cytotoxicity Effects on the Lethality of Brine Shrimp Artemia salina and Embryonic Kidney (HEK293) Cells.

In this research article, we investigated the effect of Euphorbia bivonae extract compounds on the lethality of brine shrimp Artemia salina and on embryonic cell lines (HEK293) proliferation. Our GC/MS analysis revealed that the E. bivonae ethanolic extract contained essentially sitosterol, euphol, and lupeol. The 24-h LC50 was determined using the probit analysis method (LC50=357.11 mg l-1 ). Depending on this cytotoxicity test result, E. bivona extract induced a significant increase in Superoxide Dismutase (SOD), Catalase (CAT), Glutathione-Peroxidase (GPx) activities, and lipid peroxidation (LPO) in A. salina larvae. In addition, the cytotoxicity effect of this extract had proved against the HEK293 cell lines in vitro. We suggest that the three compounds of E. bivonae extract (sitosterol, euphol, and lupeol) are the most responsible for this cytotoxicity. The possible application of this extract as an alternative natural antiproliferative is considered.

Transcriptomic Analysis of CRISPR/Cas9-Mediated PARP1-Knockout Cells under the Influence of Topotecan and TDP1 Inhibitor.

Topoisomerase 1 (TOP1) is an enzyme that regulates DNA topology and is essential for replication, recombination, and other processes. The normal TOP1 catalytic cycle involves the formation of a short-lived covalent complex with the 3' end of DNA (TOP1 cleavage complex, TOP1cc), which can be stabilized, resulting in cell death. This fact substantiates the effectiveness of anticancer drugs-TOP1 poisons, such as topotecan, that block the relegation of DNA and fix TOP1cc. Tyrosyl-DNA phosphodiesterase 1 (TDP1) is able to eliminate TOP1cc. Thus, TDP1 interferes with the action of topotecan. Poly(ADP-ribose) polymerase 1 (PARP1) is a key regulator of many processes in the cell, such as maintaining the integrity of the genome, regulation of the cell cycle, cell death, and others. PARP1 also controls the repair of TOP1cc. We performed a transcriptomic analysis of wild type and PARP1 knockout HEK293A cells treated with topotecan and TDP1 inhibitor OL9-119 alone and in combination. The largest number of differentially expressed genes (DEGs, about 4000 both up- and down-regulated genes) was found in knockout cells. Topotecan and OL9-119 treatment elicited significantly fewer DEGs in WT cells and negligible DEGs in PARP1-KO cells. A significant part of the changes caused by PARP1-KO affected the synthesis and processing of proteins. Differences under the action of treatment with TOP1 or TDP1 inhibitors alone were found in the signaling pathways for the development of cancer, DNA repair, and the proteasome. The drug combination resulted in DEGs in the ribosome, proteasome, spliceosome, and oxidative phosphorylation pathways.

Xylosyltransferase-deficient human HEK293 cells show a strongly reduced proliferation capacity and viability.

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.

High density lipoprotein promotes nascent apolipoprotein A-V secretion from mRNA transfected cells.

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.

A reliable fluorimetric method to screen the nitric oxide synthase inhibitors in 96 well plate.

In general, 4 amino-5-methylamino-2',7'-difluorescein diacetate (DAF-FM-DA) dye is used to detect nitric oxide in biological systems through cell imaging. In this study, we have used 96 well plate format to quantify nitric oxide using DAF-FM-DA through a multimode reader (or independently using fluorospectrometer) and could be visualized in a fluorescence microscope. Similar study otherwise will require a high-end instrument. The method has been validated to screen NOS inhibitors in the HEK 293T cell lines over-expressing the NOS isoforms. We observed that the method is very simple to use, adaptive, sensitive and most importantly it saves time. REAGENTS/TOOLS: Ethanol (70% [v/v] in distilled water), Nω-Nitro-l-arginine (l-NAME), 7-Nitro-Indazole (7-NI) (Sigma, St. Louis, MO), HEK 293T cell lines (National Centre for Cell Science (NCCS), Pune, India), DMEM (Himedia laboratories Pvt), Fetal Bovine Serum (FBS) (Invitrogen, Carlsbad, CA), 100 U/mL penicillin, and 0.1 mg/mL streptomycin in a 5% CO2 atmosphere. Hank's Balanced Salt Solution (HBSS) without Phenol Red of pH 7.4 was prepared with the following composition: NaCl, 8.0g, KCl, 0.4g, CaCl2, 0.14g, MgSO4⋅7H2O, 0.1g, MgCl2·6H2O, 0.1g, Na2HPO4·2H2O, 0.06g, KH2PO4, 0.06g, glucose, 1.0g, NaHCO3, 0.35g, H2O, to 1000 ml, Sterilized and refrigerated, Calcium Ionophore A23187 (Sigma Aldrich 52665-69-7) DAF-FM Di Acetate (Molecular Probes Life Technologies), and DAF-FM Di Aceatate was prepared as a stock solution (5 mM) in DMSO, divided into aliquots and stored at -20 °C, followed by dilution to the required concentration in HBSS buffer before use. EQUIPMENT: Neubauer chamber, Microtube centrifuges (1.5 mL), Micropipettors,10,100, and 1000 mL with corresponding tips, multimode reader (Tecan, Synergy-HT), inverted fluorescence microscope (Nikon, eclipse Ti-S), black flat bottom Microplates (96-well) (Corning 3603).

Transcriptomic analysis of HEK293A cells with a CRISPR/Cas9-mediated TDP1 knockout.

Tyrosyl-DNA phosphodiesterase 1 (TDP1) is a human DNA repair protein. It is a member of the phospholipase D family based on structural similarity. TDP1 is a key enzyme of the repair of stalled topoisomerase 1 (TOP1)-DNA complexes. Previously, with the CRISPR/Cas9 method, we obtained HEK293A cells with a homozygous knockout of the TDP1 gene and used the TDP1 knockout cells as a cellular model for studying mechanisms of action of an anticancer therapy. In the present work, we hypothesized that the TDP1 knockout would alter the expression of DNA repair-related genes. By transcriptomic analysis, we investigated for the first time the effect of the TDP1 gene knockout on genes' expression changes in the human HEK293A cell line. We obtained original data implying a role of TDP1 in other processes besides the repair of the DNA-TOP1 complex. Differentially expressed gene analysis revealed that TDP1 may participate in cell adhesion and communication, spermatogenesis, mitochondrial function, neurodegeneration, a cytokine response, and the MAPK signaling pathway.

Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development.

Coxsackievirus A16 (CVA16) is one of the primary pathogens that causes hand, foot, and mouth disease (HFMD) in young children. In previous studies, CVA16 vaccine development has encountered several challenges, such as inefficient replication of the CVA16 virus in present culture systems, the induction of only mild neutralizing antibody titers, and neutralizing antibodies induced by certain vaccine candidates that are unable to protect against CVA16 viral challenge. In this study, we constructed a DNA-launched CVA16 infectious clone (CVA16ic) based on the genomic sequence of the CVA16 N5079 strain to minimize interference from viral quasispecies. The biochemical properties of this CVA16ic strain were similar to those of its parental strain. Serum-free HEK293A suspension cells, which produced higher virus titers than Vero cells, were demonstrated to improve CVA16 production yields. In addition, our study showed that inactivated EV-A71 antigens could enhance the immunogenicity of inactivated CVA16 mature/full particles (F-particles), suggesting that a bivalent CVA16 and EV-A71 vaccine may be an effective strategy for CVA16 vaccine development. These findings are expected to provide novel strategies and accelerate the development of bivalent HFMD vaccines.

Propagation and immunological characterization of coxsackievirus A10 in a serum-free HEK293A cell culture system.

Coxsackievirus A10 (CVA10) is one of enteroviral pathogens that cause the hand, foot, and mouth disease (HFMD). Since CVA10 was reported to be not easily propagated in the Vero cell culture, a feasible manufacture process for producing formalin-inactivated CVA10 vaccine is urgently needed. Several cell lines that commonly used for viral vaccine production was tested for CVA10 (M2014 strain) culture in this study, and our result showed that CVA10 could be easily propagated in the HEK293A cells. A serum-free HEK293A cell culture system was developed for CVA10 production and the yields have reached over 108 TCID50/mL. The biochemical and immunogenic properties of CVA10 particles obtained from this serum-free HEK293A culture were identical to our previous study. Two major particles of CVA10 were separated by ultracentrifugation, and only the infectious mature particles were capable of inducing CVA10 neutralizing antibody responses in the mouse immunogenicity studies. Additionally, we found that coxsackievirus A6 and enterovirus A71 could also be easily propagated using this serum-free HEK293A cell culture system. Our results provide a solution to overcome the obstacle in the propagation of CVA10 and facilitate the development of multivalent vaccines for prevention of HFMD.

Molecular signatures of cytotoxic effects in human embryonic kidney 293 cells treated with single and mixture of ochratoxin A and citrinin.

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.

Functional characterization of odorant receptors from Lampronia capitella suggests a non-ditrysian origin of the lepidopteran pheromone receptor clade.

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 C10-C18 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.

Assessment of cytotoxicity of choline chloride-based natural deep eutectic solvents against human HEK-293 cells: A QSAR analysis.

Deep eutectic solvents (DESs) are a new generation of solvents. To consider them as green solvents, investigation of their toxicity is essential. In this work, the cytotoxicity of a number of natural deep eutectic solvents (NADESs) against HEK-293 human embryonic kidney cells was evaluated by MTT assay. The NADESs were prepared with choline chloride (ChCl) as hydrogen-bond acceptor (HBA) and different sugar alcohols as hydrogen-bond donor (HBD) constituents. They showed IC50 values in the range of 3.52-75.46 mM. These results were used to evaluate the effect of structural parameters on the cytotoxicity of the studied NADESs by using quantitative structure activity relationship (QSAR) analysis. A three-parameter linear model was obtained between - log(IC50) as a dependent variable and structural descriptors as independent variables. Rotatable bond number (RBN), mean atomic van der Waals volume (Mv) and the interaction of second power carbon numbers with the molar ratio of HBA to HBD in each NADES (C2 Ratio), were three major parameters. The statistical model covered about 76.4% and 69.8% variance of data in training and leave-one-out cross-validation, respectively. This work, as the first study on the QSAR analysis of DESs, can provide a good perspective for designing greener novel DESs.

Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence.

It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the co-expression of a secreted antibody fragment and an intracellular reporter protein, enhanced yellow fluorescent protein from lentiviral shuttle plasmids by inserting a furin-2A (F2A) sequence between the two cDNAs, in two different orientations, in the expression cassette. We show that the order of these two transgenes relative to the F2A sequence affects expression levels. Reduced expression of each transgene positioned downstream of F2A, compared with upstream of F2A, was observed (p<0.05). Moreover, protein expression from double-cDNA plasmids was significantly lower than from their corresponding single transgene counterparts (p<0.05).

Co-expression of the human cannabinoid receptor coding region splice variants (hCB1) affects the function of hCB1 receptor complexes.

The human type 1 cannabinoid (hCB1) receptor is expressed at high levels in the central nervous system. mRNA variants of the coding region of this receptor, human cannabinoid hCB1a and hCB1b receptors, have been identified, their biological function remains unclear. The present study demonstrated that the three human cannabinoid hCB1 coding region variants are expressed in the human and monkey (Macaca fascicularis) brain. Western blot analyses of homogenates from different regions of the monkey brain demonstrated that proteins with the expected molecular weights of the cannabinoid CB1, CB1a and CB1b receptors were co-expressed throughout the brain. Given the co-localization of these receptors, we hypothesized that physical interactions between the three splice variants may affect cannabinoid pharmacology. The human cannabinoid hCB1, hCB1a, and hCB1b receptors formed homodimers and heterodimers, as determined by BRET in transiently transfected HEK 293A cells. We found that the co-expression of the human cannabinoid hCB1 and each of the splice variants increased cell surface expression of the human cannabinoid hCB1 receptor and increased Gi/o-dependent ERK phosphorylation in response to cannabinoid agonists. Therefore, the human cannabinoid hCB1 coding region splice variants play an important physiological role in the activity of the endocannabinoid system.