Human Embryonic Kidney 293T Research Articles

Development and Validation of a Novel Dual Luciferase Reporter Gene Assay to Quantify Ebola Virus VP24 Inhibition of IFN Signaling.

The interferon (IFN) system is the first line of defense against viral infections. Evasion of IFN signaling by Ebola viral protein 24 (VP24) is a critical event in the pathogenesis of the infection and, hence, VP24 is a potential target for drug development. Since no drugs target VP24, the identification of molecules able to inhibit VP24, restoring and possibly enhancing the IFN response, is a goal of concern. Accordingly, we developed a dual signal firefly and Renilla luciferase cell-based drug screening assay able to quantify IFN-mediated induction of Interferon Stimulated Genes (ISGs) and its inhibition by VP24. Human Embryonic Kidney 293T (HEK293T) cells were transiently transfected with a luciferase reporter gene construct driven by the promoter of ISGs, Interferon-Stimulated Response Element (ISRE). Stimulation of cells with IFN-α activated the IFN cascade leading to the expression of ISRE. Cotransfection of cells with a plasmid expressing VP24 cloned from a virus isolated during the last 2014 outbreak led to the inhibition of ISRE transcription, quantified by a luminescent signal. To adapt this system to test a large number of compounds, we performed it in 96-well plates; optimized the assay analyzing different parameters; and validated the system by calculating the Z′- and Z-factor, which showed values of 0.62 and 0.53 for IFN-α stimulation assay and VP24 inhibition assay, respectively, indicative of robust assay performance.

Fluorescence Imaging of Huntingtin mRNA Knockdown.

Huntington's disease (HD) is an autosomal-dominant neurodegenerative genetic disorder caused by CAG repeat expansion in exon 1 of the HTT gene. Expression of the mutant gene results in the production of a neurotoxic polyglutamine (polyQ)-expanded huntingtin (Htt) protein. Clinical trials of knockdown therapy of mutant polyglutamine-encoding HTT mRNA in Huntington's disease (HD) have begun. To measure HTT mRNA knockdown effectiveness in human cells, we utilized a fluorescent hybridization imaging agent specific to the region encompassing the human HTT mRNA initiation codon. We designed, synthesized, purified, and characterized Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides. The human HTT PNA 12mer complement was CATGGCGGTCTC, while the rat htt equivalent 12mer contained the sequence CATGaCGGcCTC, with two bases differing from the human sequence. The cyclized IGF1 tetrapeptide fragment d(CSKC) that promotes IGF1 receptor-mediated endocytosis was bonded to the C-terminus. We tested the reliability of HTT mRNA imaging with Cal560-spacer-peptide nucleic acid (PNA)-spacer-IGF1 tetrapeptides in human embryonic kidney (HEK) 293T cells that express endogenous HTT and IGF1 receptor. By qPCR, we quantitated HTT mRNA in HEK293T cells with and without HTT mRNA knockdown by three different siRNAs. By confocal fluorescence imaging, we quantitated the accumulation of fluorescent HTT hybridization agent in the same cells. A rat homologue differing from the human sequence by two bases showed negligible fluorescence. qPCR indicated 86 ± 5% knockdown of HTT mRNA by the most effective siRNA. Similarly, Cal560- HTT PNA-peptide fluorescence intensity indicated 69 ± 6% reduction in HTT mRNA. We concluded that the fluorescence hybridization method correlates with the established qPCR method for quantitating HTT mRNA knockdown by siRNA in HEK293T cells, with a Pearson correlation coefficient of 0.865 for all three siRNA sequences. These results will enable real time imaging and quantitation of HTT mRNA in animal models of HD.

A novel role of the antitumor agent tricyclodecan-9-yl-xanthogenate as an open channel blocker of KCNQ1/KCNE1

Tricyclodecan-9-yl-xanthogenate (D609) is widely known for its antitumor and antiviral properties via the inhibition of phosphatidylcholine-specific phospholipase C and sphingomyelin synthase. Previously, we found that chronic application of D609 suppressed the K+ channel, KCNQ1/KCNE1, more drastically than expected from its actions on the enzymes, suggesting a direct action of D609 on the channel. Here, we aimed to test this possibility by studying the affinity, specificity, and mechanisms of D609 on KCNQ1/KCNE1. The effect of D609 on KCNQ1/KCNE1 was studied using an in vitro expression system and in native cells, using electrophysiological techniques. We found that D609 rapidly and reversibly inhibited KCNQ1/KCNE1 channels expressed in human embryonic kidney 293 T (HEK293T) cells, in a concentration-dependent manner with a high affinity. D609 neither suppressed endogenous K+ currents in HEK293T cells, nor inhibited the sustained and transient K+ currents of mouse neostriatal neurons, but blocked a KCNQ1/KCNE1-like current in neostriatal neurons. D609 potently blocked IKs, the cardiac KCNQ1/KCNE1 channel, in guinea pig cardiac muscle cells. The action of D609 on KCNQ1/KCNE1 depended on the usage of the channel, suggesting that D609 binds to the channel in the open state. We identified D609 as a potent and specific open channel blocker of KCNQ1/KCNE1. Because KCNQ1/KCNE1 is highly expressed in the heart, the inner ear and the pancreas, D609, when used as an antitumor or antiviral drug, may affect the function of a number of organs in vivo even when used at low concentrations.

Distinct differences in prion-like seeding and aggregation between Tau protein variants provide mechanistic insights into tauopathies

The accumulation of aberrantly aggregated MAPT (microtubule-associated protein Tau) defines a spectrum of tauopathies, including Alzheimer's disease. Mutations in the MAPT gene cause frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), characterized by neuronal pathological Tau inclusions in the form of neurofibrillary tangles and Pick bodies and in some cases glial Tau pathology. Increasing evidence points to the importance of prion-like seeding as a mechanism for the pathological spread in tauopathy and other neurodegenerative diseases. Herein, using a cell culture model, we examined a multitude of genetic FTDP-17 Tau variants for their ability to be seeded by exogenous Tau fibrils. Our findings revealed stark differences between FTDP-17 Tau variants in their ability to be seeded, with variants at Pro301 and Ser320 showing robust aggregation with seeding. Similarly, we elucidated the importance of certain Tau protein regions and unique residues, including the role of Pro301 in inhibiting Tau aggregation. We also revealed potential barriers in cross-seeding between three-repeat and four-repeat Tau isoforms. Overall, these differences alluded to potential mechanistic differences between wildtype and FTDP-17 Tau variants, as well as different Tau isoforms, in influencing Tau aggregation. Furthermore, by combining two FTDP-17 Tau variants (either P301L or P301S with S320F), we generated aggressive models of tauopathy that do not require exogenous seeding. These models will allow for rapid screening of potential therapeutics to alleviate Tau aggregation without the need for exogenous Tau fibrils. Together, these studies provide novel insights in the molecular determinants that modulate Tau aggregation.

Genetic screening and functional analysis of CASP9 mutations in a Chinese cohort with neural tube defects.

Neural tube defects (NTDs) are birth defects of the nervous system and are the second most frequent cause of birth defects worldwide. The etiology of NTDs is complicated and involves both genetic and environmental factors. CASP9 is an initiator caspase in the intrinsic apoptosis pathway, which in Casp9-/- mice has been shown to result in NTDs because of decreased apoptosis. The aim of this study was to evaluate the potential genetic contribution of the CASP9 gene in human NTDs. High-throughput sequencing was performed to screen genetic variants of CASP9 genes in 355 NTD cases and 225 matched controls. Apoptosis-relevant assays were performed on transiently transfected E9 neuroepithelial cells or human embryonic kidney 293T cells, to determine the functional characteristics of NTD-specific rare variants under complete or low folic acid (FA) status. We found significant expression of CASP9 rare variants in NTDs and identified 4 NTD-specific missense variants. Functional assays demonstrated that a p.Y251C variant attenuates apoptosis by reducing CASP9 protein expression and decreasing activity of the intrinsic apoptosis pathway. From this, we conclude that this variant may represent a loss-of-function mutation. A 4-time recurrent p.R191G variant did not affect intrinsic apoptosis in complete medium, while it completely inhibited apoptosis induced by low FA medium. Our findings identify a genetic link for apoptosis in human NTDs and highlight the effect of gene-environment interactions in a complex disease.

Graphene oxide regulates cox2 in human embryonic kidney 293T cells via epigenetic mechanisms: dynamic chromosomal interactions

To extend the applications of engineered nanomaterials, such as graphene oxide (GO), it is necessary to minimize cytotoxicity. However, the mechanisms underlying this cytotoxicity are unclear. Dynamic chromosomal interactions have been used to illustrate the molecular bases of gene expression, which offers a more sensitive and cutting-edge technology to elucidate complex biological processes associated with epigenetic regulations. In this study, the role of GO-triggered chromatin interactions in the activation of cox2, a hallmark of inflammation, was investigated in normal human cells. Using chromosome conformation capture technology, we showed that GO triggers physical interactions between the downstream enhancer and the cox2 promoter in human embryonic kidney 293T (293T) via p65 and p300 complex-mediated dynamic chromatin looping, which was required for high cox2 expression. Moreover, tumor necrosis factor-α (TNF-α), located upstream of the p65 signaling pathway, contributed to the regulation of cox2 activation through dynamic chromatin architecture. Compared with pristine GO and aminated GO (GO-NH2), poly (acrylic acid)-functionalized GO (GO-PAA) induced a weaker inflammatory response and a weaker effect on chromatin architecture. Our results mechanistically link GO-mediated chromatin interactions with the regulation of cox2 and suggest that GO derivatives may minimize toxicity in practical applications.

Identification and functional characterization of Toll-like receptor 13 from orange-spotted grouper (Epinephelus coioides)

Toll-like receptors (TLRs) are one of the most important innate immune receptors, which recognize various pathogen-associated molecular patterns and activate the downstream immune response. Mouse TLR13 has been found to recognize a highly conserved sequence from bacterial or viral RNA and activate the myeloid differentiation primary response gene 88-dependent signaling response. The function of teleost tlr13 is still not fully understood, especially its relationship with bacterial RNA. In our study, we identified and characterized a tlr13 from Epinephelus coioides (orange-spotted grouper). The full-length cDNA of Eco. tlr13 contained a 2844 bp open reading frame, encoding 947 amino acids. The polypeptide was constitutive of a signal peptide, 13 leucine-rich repeats domains, a C-terminal leucine-rich repeats, a transmembrane domain and a conserved Toll/interleukin (IL)-1 receptor domain, indicating that Eco. Tlr13 exhibited a typical TLR structure. Multiple alignments showed that the Toll/IL-1 receptor domain of Eco. Tlr13 was identical with other homologues, and the phylogenetic tree suggested that Eco. Tlr13 was clustered with other TLR13s and had the closest relationship with predicted Lates calcarifer (sea bass) Tlr13. Subcellular localization analysis revealed that Eco. Tlr13 colocalized with the endoplasmic reticulum and early endosome. Moreover, Eco. tlr13 was broadly observed in all tested tissues with the relatively high expressions in the brain and immune-related tissues. After challenged with 19-mer Staphylococcus aureus 23S ribosomal RNA-derived oligoribonucleotide (ORN Sa19), the expression of Eco. tlr13 was significantly up-regulated in grouper spleen cells. Also, the luciferase assay further revealed that with the overexpression of Eco. Tlr13 in human embryonic kidney 293T cells, ORN Sa19 activated the promoter activity of interferon-β in a dose-dependent pattern. These results indicate that Eco. tlr13 may involve in the recognition of bacterial RNA.

In vitro Inhibitory Effect of Lanostane Triterpenoids of Kadsura coccinea on the Human Immunodeficiency Virus Type-1 Protease

Human immunodeficiency virus type-1 is the causative pathogen of acquired immunodeficiency syndrome and its protease is one of the primary targets for human immunodeficiency virus/acquired immune deficiency syndrome therapy. In this study, two seco-lanostane triterpenoids, 3,4-seco-9βH-lanost-4(28),7,24-trien-3-oic acid and 24(E)-3,4-seco-9βH-lanost-4(28),7,24-trien-3,26-dioic acid isolated from the roots of Kadsura coccinea, were found to significantly inhibit human immunodeficiency virus-1 protease, with IC50 values of 1.0±0.03 µM and 0.05±0.009 µM, respectively. Neither compound was toxic to human embryonic kidney 293T cells at concentrations effective against human immunodeficiency virus-1 protease. Our findings indicate that these triterpenoids are potential candidates for development of antihuman immunodeficiency virus/acquired immune deficiency syndrome drugs.

Display of human and rabbit monocyte chemoattractant protein-1 on human embryonic kidney 293T cell surface.

Monocyte chemoattractant protein-1 (MCP-1/CCL2) is a protein that is secreted immediately upon endothelial injury, and thereby it plays a key role in inflammation via recruitment of leucocytes to the site of inflammation at the beginning and throughout the inflammatory processes. Aim of this study was to develop two separate cell lines displaying either human MCP-1 (HMCP-1) or rabbit MCP-1 (RMCP-1) on their surface. A DNA fragment containing HMCP-1- or RMCP-1-encoding sequence was inserted into a pcDNA plasmid. Escherichia coli cells strain TOP 10F’ was separately transformed with the pcDNA/RMCP-1 or /HMCP-1 ligation mixture. Following the cloning and construct verification, human embryonic kidney cell line (HEK 293T) was transfected with either of the linearized plasmids. Plasmid integration into the genomic DNA of HEK 293T cells was verified by polymerase chain reaction (PCR). HMCP-1 and RMCP-1 expression was evaluated at RNA and protein levels by real-time PCR and flow cytometry, respectively. PCR products of the expected sizes were amplified from the chromosomal DNA of transfected HEK 293T cells, i.e. 644 bp for H-MCP1 and 737 bp for RMCP-1 constructs. Real-time PCR revealed that the copy numbers of RMCP1 and HMCP1 mRNA per cell were 294 and 500, respectively. Flow cytometry analysis indicated 85% for RMCP-1 and 87% for HMCP-1 expression levels on the surface of transfected cells, when compared with an isotype control. The experiments thus confirmed that the MCP-1 genes were integrated into the HEK 293T genomic DNA and the encoded proteins were stably expressed on the cell surface.

Characterization of Interactions Among CYP1A2, CYP2B4, and NADPH-cytochrome P450 Reductase: Identification of Specific Protein Complexes.

Cytochromes P450s (P450s) catalyze oxygenation reactions via interactions with their redox partners. However, other proteins, particularly other P450s, also have been shown to form complexes that modulate P450 function. Previous studies showed that CYP1A2 and CYP2B4 form a complex when reconstituted into phospholipid vesicles; however, details of the interactions among the P450s and NADPH-cytochrome P450 reductase (POR) have not been fully characterized. The goal of this study was to examine P450 complex formation in living cells, using bioluminescence resonance energy transfer (BRET). Various pairs of P450 and POR constructs were tagged with either green fluorescent protein or Renilla luciferase, and transfected into human embryonic kidney 293T cells. Complexes were demonstrated by measuring energy transfer between the tags, and disruption of the complex was verified by cotransfection with unlabeled P450-system proteins. CYP1A2 and CYP2B4 formed a stable complex that could not be disrupted by cotransfection of untagged POR. Interactions of both P450s with POR were detected, with untagged CYP1A2 disrupting the POR-CYP2B4 interaction. In contrast, untagged CYP2B4 did not affect the POR-CYP1A2 interaction. These data are consistent with POR preferentially binding to the CYP1A2 moiety of CYP1A2-CYP2B4. BRET-detectable homomeric CYP1A2-CYP1A2 also was detected, and was disrupted by cotransfection of either POR or CYP2B4. Both CYP1A2 and CYP2B4 activities were affected by their coexpression in a manner consistent with formation of the high-affinity POR-CYP1A2-CYP2B4 complex. These findings demonstrate that CYP1A2 and CYP2B4 form a heteromeric POR-CYP1A2-CYP2B4 complex in living cells that has altered catalytic activities relative to the homomeric enzymes.

Absence of anti-hypocretin receptor 2 autoantibodies in post pandemrix narcolepsy cases.

BackgroundA recent publication suggested molecular mimicry of a nucleoprotein (NP) sequence from A/Puerto Rico/8/1934 (PR8) strain, the backbone used in the construction of the reassortant strain X-179A that was used in Pandemrix® vaccine, and reported on anti-hypocretin (HCRT) receptor 2 (anti-HCRTR2) autoantibodies in narcolepsy, mostly in post Pandemrix® narcolepsy cases (17 of 20 sera). In this study, we re-examined this hypothesis through mass spectrometry (MS) characterization of Pandemrix®, and two other pandemic H1N1 (pH1N1)-2009 vaccines, Arepanrix® and Focetria®, and analyzed anti-HCRTR2 autoantibodies in narcolepsy patients and controls using three independent strategies.MethodsMS characterization of Pandemrix® (2 batches), Arepanrix® (4 batches) and Focetria® (1 batch) was conducted with mapping of NP 116I or 116M spectrogram. Two sets of narcolepsy cases and controls were used: 40 post Pandemrix® narcolepsy (PP-N) cases and 18 age-matched post Pandemrix® controls (PP-C), and 48 recent (≤6 months) early onset narcolepsy (EO-N) cases and 70 age-matched other controls (O-C). Anti-HCRTR2 autoantibodies were detected using three strategies: (1) Human embryonic kidney (HEK) 293T cells with transient expression of HCRTR2 were stained with human sera and then analyzed by flow cytometer; (2) In vitro translation of [35S]-radiolabelled HCRTR2 was incubated with human sera and immune complexes of autoantibody and [35S]-radiolabelled HCRTR2 were quantified using a radioligand-binding assay; (3) Optical density (OD) at 450 nm (OD450) of human serum immunoglobulin G (IgG) binding to HCRTR2 stably expressed in Chinese hamster ovary (CHO)-K1 cell line was measured using an in-cell enzyme-linked immunosorbent assay (ELISA).ResultsNP 116M mutations were predominantly present in all batches of Pandemrix®, Arepanrix® and Focetria®. The wild-type NP109-123 (ILYDKEEIRRIWRQA), a mimic to HCRTR234-45 (YDDEEFLRYLWR), was not found to bind to DQ0602. Three or four subjects were found positive for anti-HCRTR2 autoantibodies using two strategies or the third one, respectively. None of the post Pandemrix® narcolepsy cases (0 of 40 sera) was found positive with all three strategies.ConclusionAnti-HCRTR2 autoantibody is not a significant biological feature of narcolepsy or of post Pandemrix® autoimmune responses.

Aptamer-based fluorogenic sensing of interferon-gamma probed with ReS2 and TiS2 nanosheets

Abstract The fluorogenic aptamer sensing of interferon-gamma (IFN-γ) was scrutinized using two-dimensional (2D) ReS2 and TiS2 nanosheets (NSs) as a platform. The IFN-γ, an important cytokine, functions as a bio-indicator to detect infectious diseases such as tuberculosis and human immunodeficiency virus. This 2D NSs based aptamer sensor was implemented to induce the fluorescence off/on resulting from an aptamer, in the absence or presence of a target to be probed. The fluorescence emitting from the aptamer is quenched by interacting with NSs, while the ensuing fluorescence is recovered upon addition of target. Such a fluorescence off/on mechanism was proposed based on the behavior of fluorescence resonance energy transfer (FRET) between the aptamer and NSs. The fluorescence response exhibits linearity as a function of target, and the detection limit of IFN-γ was evaluated to be 57.6 and 82.7 pM for ReS2 and TiS2 NSs, respectively, being comparable to or even better than those methods adopted for probing IFN-γ. The selectivity property was also characterized with various targets, exhibiting a very specific selectivity for IFN-γ. The findings reveal that the aptamer-transition metal dichalcogenides (TMD) NSs will be a great sensing pair to the development of aptamer-based biosensors. Moreover, the biocompatibility and sensing capability of IFN-γ was implemented in human embryonic kidney 293T (HEK) live cells. This is the first report to emerging fluorogenic sensing of IFN-γ aptamer with 2D TMD, showing a promising trend for future design of biosensors.

Exome Sequencing Identifies a Novel MAP3K14 Mutation in Recessive Atypical Combined Immunodeficiency.

Primary immunodeficiency disorders (PIDs) render patients vulnerable to infection with a wide range of microorganisms and thus provide good in vivo models for the assessment of immune responses during infectious challenges. Priming of the immune system, especially in infancy, depends on different environmental exposures and medical practices. This may determine the timing and phenotype of clinical appearance of immune deficits as exemplified with early exposure to Bacillus Calmette-Guérin (BCG) vaccination and dissemination in combined immunodeficiencies. Varied phenotype expression poses a challenge to identification of the putative immune deficit. Without the availability of genomic diagnosis and data analysis resources and with limited capacity for functional definition of immune pathways, it is difficult to establish a definitive diagnosis and to decide on appropriate treatment. This study describes the use of exome sequencing to identify a homozygous recessive variant in MAP3K14, NIKVal345Met, in a patient with combined immunodeficiency, disseminated BCG-osis, and paradoxically elevated lymphocytes. Laboratory testing confirmed hypogammaglobulinemia with normal CD19, but failed to confirm a definitive diagnosis for targeted treatment decisions. NIKVal345Met is predicted to be deleterious and pathogenic by two in silico prediction tools and is situated in a gene crucial for effective functioning of the non-canonical nuclear factor-kappa B signaling pathway. Functional analysis of NIKVal345Met- versus NIKWT-transfected human embryonic kidney-293T cells showed that this mutation significantly affects the kinase activity of NIK leading to decreased levels of phosphorylated IkappaB kinase-alpha (IKKα), the target of NIK. BCG-stimulated RAW264.7 cells transfected with NIKVal345Met also presented with reduced levels of phosphorylated IKKα, significantly increased p100 levels and significantly decreased p52 levels compared to cells transfected with NIKWT. Ideally, these experiments would have been conducted in patient-derived immune cells, but we were unable to source these cells from the patient. The functional analysis described in this paper supports previous illustrations of the importance of NIK in human immune responses and demonstrates the involvement of function-altering mutations in MAP3K14 in PIDs. The genomic approach used for this patient demonstrates its value in the diagnosis of an unusual PID and as a tool for detecting rarer mutations to help guide treatment approaches.

Unc93b1 is essential for cytokine activation of five PAMPs in the orange-spotted grouper (Epinephelus coioides)

Toll-like receptors (TLRs) are important innate immune receptors that recognize multiple pathogen-associated molecular patterns (PAMPs) and activate the immune responses to resist the invasion of pathogens. Many TLRs need assistance from trafficking chaperones to transport to the specific cell compartments and then are processed before they are activated. In this study, we identified an important trafficking chaperone, Unc-93 homolog B1 (unc93b1), from the Epinephelus coioides (orange-spotted grouper). The deduced protein sequence of Eco.unc93b1 was 632 amino acids, containing 12 transmembrane domains, consistent with other UNC93B1 proteins from other species. Phylogenetic analysis showed that Eco.Unc93b1 was clustered with teleost Unc93b1 and had the closest relationship with Larimichthys crocea (large yellow croaker) Unc93b1. Eco.unc93b1 was expressed the highest in the spleen, and its protein was co-localized with the endoplasmic reticulum and early endosomes in both human embryonic kidney 293T cells and grouper spleen cells (GS cells). Moreover, the stimulation of lipopolysaccharide (LPS), high-molecular-weight poly (I:C) (HMW), imidazoquinoline (R848), polyadenylic-polyuridylic acid (poly AU), and 19-mer Staphylococcus aureus 23S rRNA-derived oligoribonucleotide (ORN Sa 19) promoted the mRNA expression of unc93b1 in GS cells with different patterns. Furthermore, the cytokine expression induced by these PAMPs was suppressed, while Eco.unc93b1 was knocked down, by small interfering RNA. In conclusion, these results suggest that Eco.unc93b1 plays an essential role in several PAMP-induced immune responses.

Binding and Signaling Studies Disclose a Potential Allosteric Site for Cannabidiol in Cannabinoid CB2 Receptors.

The mechanism of action of cannabidiol (CBD), the main non-psychotropic component of Cannabis sativa L., is not completely understood. First assumed that the compound was acting via cannabinoid CB2 receptors (CB2Rs) it is now suggested that it interacts with non-cannabinoid G-protein-coupled receptors (GPCRs); however, CBD does not bind with high affinity to the orthosteric site of any GPCR. To search for alternative explanations, we tested CBD as a potential allosteric ligand of CB2R. Radioligand and non-radioactive homogeneous binding, intracellular cAMP determination and ERK1/2 phosphorylation assays were undertaken in heterologous systems expressing the human version of CB2R. Using membrane preparations from CB2R-expressing HEK-293T (human embryonic kidney 293T) cells, we confirmed that CBD does not bind with high affinity to the orthosteric site of the human CB2R where the synthetic cannabinoid, [3H]-WIN 55,212-2, binds. CBD was, however, able to produce minor but consistent reduction in the homogeneous binding assays in living cells using the fluorophore-conjugated CB2R-selective compound, CM-157. The effect on binding to CB2R-expressing living cells was different to that exerted by the orthosteric antagonist, SR144528, which decreased the maximum binding without changing the KD. CBD at nanomolar concentrations was also able to significantly reduce the effect of the selective CB2R agonist, JWH133, on forskolin-induced intracellular cAMP levels and on activation of the MAP kinase pathway. These results may help to understand CBD mode of action and may serve to revisit its therapeutic possibilities.

Molecularly imprinted polymers synthesized via template immobilization on fumed silica nanoparticles for the enrichment of phosphopeptides.

Phosphorylation is a protein post-translational modification (PTM) that plays an important role in cell signaling, cell differentiation, and metabolism. The hyper phosphorylated forms of certain proteins have been appointed as biomarkers for neurodegenerative diseases, and phosphorylation-related mutations are important for detecting cancer pathways. Due to the low abundance of phosphorylated proteins in biological fluids, sample enrichment is beneficial prior to detection. Thus, a need to find new strategies for enriching phosphopeptides has emerged. Molecularly imprinted polymers (MIPs) are synthetic polymeric materials manufactured to exhibit affinity for a target molecule. In this study, MIPs have been synthesized using a new approach based on the use of fumed silica as sacrificial support acting as solid porogen with the template (phosphotyrosine) immobilized on its surface. Phosphotyrosine MIPs were tested against a mixture of peptides and phosphopeptides by performing micro-solid phase extraction using MIPs (μMISPE) packed in a pipette tip. First, the capability of the materials to preferentially enrich phosphopeptides was evaluated. In a next step, the enrichment of phosphopeptides from a whole-cell lysate of human embryonic kidney (HEK) 293T cells was performed. The eluates were analyzed using MALDI-MS in the first case and with nano-HPLC-ESI-MS/MS in the second case. The results showed that the MIPs provided affinity for phosphopeptides, binding preferentially to multi-site phosphorylated peptides. The MIPs could enrich phosphopeptides in over 10-fold compared with the number of phosphopeptides found in a cell lysate without enrichment.

A new chemical complex can rapidly concentrate lentivirus and significantly enhance gene transduction.

In this study, we developed a new purification method using chondroitin sulfate C (CSC) and protamine sulfate (PS) to concentrate lentivirus. To evaluate the efficiency of this new method, we compared it with several previously described purification protocols, including virus concentrated by ultracentrifugation (Ultra), precipitated by polyethylene glycol (PEG), and sedimented by CSC combined with polybrene (PB). After using the different methods to purify and concentrate equivalent amounts of lentivirus supernatant, the virus pellets precipitated by the different methods were resuspended using the equivalent volumes of DMEM. Subsequently, 10μl of each lentivirus stock carrying EGFP gene was used to transduce two types of cells, human embryonic kidney 293T (HEK293T) cells and mouse mesenchymal stem cells (mMSC). It was obvious that HEK293T and mMSC appeared much intensiver green fluorescence through virus transduction from PS method than from other methods. To quantitate the transduction efficiency of the viruses, we examined virus titer in the cells after transduction using a real-time PCR-based analysis. Accordingly, we verified that PS precipitation could generate virus with a higher titer (4.39×108IU/ml) than PB (2.43×108IU/ml), Ultra (1.16×108IU/ml), and PEG (0.56×108IU/ml) in HEK293T cells. As for HEK293T cellsin mMSC, the PS method also generated virus with a higher titer (4.66×108IU/ml) than the Ultra method (2.36×108IU/ml), and a much higher titer than those of the other chemical-based precipitation methods using PB (4.82×106IU/ml) and PEG (8.98×104IU/ml). Furthermore, the HEK293T cells and mMSC transduced by PS(1X)-virus appeared to have higher cell growth ratios, respectively, than the HEK293T cells and mMSC transduced by lentivirus using the other methods. We conclude that our new method for purifying lentivirus is cost-effective, time-saving, and highly efficient, and that lentivirus purification by this means could possibly be used to transduce a variety of cells, including stem cells.

MAS1 Receptor Trafficking Involves ERK1/2 Activation Through a β-Arrestin2-Dependent Pathway.

The MAS1 receptor (R) exerts protective effects in the brain, heart, vessels, and kidney. R trafficking plays a critical function in signal termination and propagation and in R resensitization. We examined MAS1R internalization and trafficking on agonist stimulation and the role of β-arrestin2 in the activation of ERK1/2 (extracellular signal-regulated kinase 1/2) and Akt after MAS1R stimulation. Human embryonic kidney 293T cells were transfected with the coding sequence for MAS1R-YFP (MAS1R fused to yellow fluorescent protein). MAS1R internalization was evaluated by measuring the MAS1R present in the plasma membrane after agonist stimulation using a ligand-binding assay. MAS1R trafficking was evaluated by its colocalization with trafficking markers. MAS1R internalization was blocked in the presence of shRNAcaveolin-1 and with dominant negatives for Eps15 (a protein involved in endocytosed Rs by clathrin-coated pits) and for dynamin. After stimulation, MAS1R colocalized with Rab11-a slow recycling vesicle marker-and not with Rab4-a fast recycling vesicle marker-or LysoTracker-a lysosome marker. Cells transfected with MAS1R showed an increase in Akt and ERK1/2 activation on angiotensin-(1-7) stimulation, which was blocked when the clathrin-coated pits pathway was blocked. Suppression of β-arrestin2 by shRNA reduced the angiotensin-(1-7)-induced ERK1/2 activation, whereas Akt activation was not modified. We conclude that on agonist stimulation, MAS1R is internalized through clathrin-coated pits and caveolae in a dynamin-dependent manner and is then slowly recycled back to the plasma membrane. MAS1R induced Akt and ERK1/2 activation from early endosomes, and the activation of ERK1/2 was mediated by β-arrestin2. Thus, MAS1R activity and density may be tightly controlled by the cell.

TNFR2 Stimulation Promotes Mitochondrial Fusion via Stat3- and NF-kB-Dependent Activation of OPA1 Expression.

Mitochondria are important cellular organelles and play essential roles in maintaining cell structure and function. Emerging evidence indicates that in addition to having proinflammatory and proapoptotic effects, TNFα (tumor necrosis factor α) can, under certain circumstances, promote improvements in mitochondrial integrity and function, phenomena that can be ascribed to the existence of TNFR2 (TNFα receptor 2). The present study aimed to investigate whether and how TNFR2 activation mediates the effects of TNFα on mitochondria. Freshly isolated neonatal mouse cardiac myocytes treated with shRNA targeting TNFR1 were used to study the effects of TNFR2 activation on mitochondrial function. Neonatal mouse cardiac myocytes exhibited increases in mitochondrial fusion, a change that was associated with increases in mitochondrial membrane potential, intracellular ATP levels, and oxygen consumption capacity. Importantly, TNFR2 activation-induced increases in OPA1 (optic atrophy 1) protein expression were responsible for the above enhancements, and these changes could be attenuated using siRNA targeting OPA1. Moreover, both Stat3 and RelA bound to the promoter region of OPA1 and their interactions synergistically upregulated OPA1 expression at the transcriptional level. Stat3 acetylation at lysine 370 or lysine 383 played a key role in the ability of Stat3 to form a supercomplex with RelA. Meanwhile, p300 modulated Stat3 acetylation in HEK293T (human embryonic kidney 293T) cells, and p300-mediated Stat3/RelA interactions played an indispensable role in OPA1 upregulation. Finally, TNFR2 activation exerted beneficial effects on OPA1 expression in an in vivo transverse aortic constriction model, whereby TNFR1-knockout mice exhibited better outcomes than in mice with both TNFR1 and TNFR2 knocked out. TNFR2 activation protects cardiac myocytes against stress by upregulating OPA1 expression. This process was facilitated by p300-mediated Stat3 acetylation and Stat3/RelA interactions, leading to improvements in mitochondrial morphology and function.

Functional analysis of human aromatic amino acid transporter MCT10/TAT1 using the yeast Saccharomyces cerevisiae

Tryptophan is an essential amino acid in humans and an important serotonin and melatonin precursor. Monocarboxylate transporter MCT10 is a member of the SLC16A family proteins that mediates low-affinity tryptophan transport across basolateral membranes of kidney, small intestine, and liver epithelial cells, although the precise transport mechanism remains unclear. Here we developed a simple functional assay to analyze tryptophan transport by human MCT10 using a deletion mutant for the high-affinity tryptophan permease Tat2 in Saccharomyces cerevisiae. tat2Δtrp1 cells are defective in growth in YPD medium because tyrosine present in the medium competes for the low-affinity tryptophan permease Tat1 with tryptophan. MCT10 appeared to allow growth of tat2Δtrp1 cells in YPD medium, and accumulate in cells deficient for Rsp5 ubiquitin ligase. These results suggest that MCT10 is functional in yeast, and is subject to ubiquitin-dependent quality control. Whereas growth of Tat2-expressing cells was significantly impaired by neutral pH, that of MCT10-expressing cells was nearly unaffected. This property is consistent with the transport mechanism of MCT10 via facilitated diffusion without a need for pH gradient across the plasma membrane. Single-nucleotide polymorphisms (SNPs) are known to occur in the human MCT10 coding region. Among eight SNP amino acid changes in MCT10, the N81K mutation completely abrogated tryptophan import without any abnormalities in the expression or localization. In the MCT10 modeled structure, N81 appeared to protrude into the putative trajectory of tryptophan. Plasma membrane localization of MCT10 and the variant proteins was also verified in human embryonic kidney 293T cells.