Metabolic flux balance analysis during lactate and glucose concomitant consumption in HEK293 cell cultures.

At early stages of the exponential growth phase in HEK293 cell cultures, the tricarboxylic acid cycle is unable to process all the amount of NADH generated in the glycolysis pathway, being lactate the main by-product. However, HEK293 cells are also able to metabolize lactate depending on the environmental conditions. It has been recently observed that one of the most important modes of lactate metabolization is the cometabolism of lactate and glucose, observed even during the exponential growth phase. Extracellular lactate concentration and pH appear to be the key factors triggering the metabolic shift from glucose consumption and lactate production to lactate and glucose concomitant consumption. The hypothesis proposed for triggering this metabolic shift to lactate and glucose concomitant consumption is that HEK293 cells metabolize extracellular lactate as a response to both extracellular protons and lactate accumulation, by means of cotransporting them (extracellular protons and lactate) into the cytosol. At this point, there exists a considerable controversy about how lactate reaches the mitochondrial matrix: the first hypothesis proposes that lactate is converted into pyruvate in the cytosol, and afterward, pyruvate enters into the mitochondria; the second alternative considers that lactate enters first into the mitochondria, and then, is converted into pyruvate. In this study, lactate transport and metabolization into mitochondria is shown to be feasible, as evidenced by means of respirometry tests with isolated active mitochondria, including the depletion of lactate concentration of the respirometry assay. Although the capability of lactate metabolization by isolated mitochondria is demonstrated, the possibility of lactate being converted into pyruvate in the cytosol cannot be excluded from the discussion. For this reason, the calculation of the metabolic fluxes for an HEK293 cell line was performed for the different metabolic phases observed in batch cultures under pH controlled and noncontrolled conditions, considering both hypotheses. The main objective of this study is to evaluate the redistribution of cellular metabolism and compare the differences or similarities between the phases before and after the metabolic shift of HEK293 cells (shift observed when pH is not controlled). That is from a glucose consumption/lactate production phase to a glucose-lactate coconsumption phase. Interestingly, switching to a glucose and lactate cometabolization results in a better-balanced cell metabolism, with decreased glucose and amino acids uptake rates, affecting minimally cell growth. This behavior could be applied to further develop new approaches in terms of cell engineering and to develop improved cell culture strategies in the field of animal cell technology.

Extracellular vesicle-carried Jagged-1 inhibits HUVEC sprouting in a 3D microenvironment.

NOTCH signalling is an evolutionarily conserved juxtacrine signalling pathway that is essential in development. Jagged1 (JAG1) and Delta-like ligand 4 (DLL4) are transmembrane NOTCH ligands that regulate angiogenesis by controlling endothelial cell (EC) differentiation, vascular development and maturation. In addition, DLL4 could bypass its canonical cell-cell contact-dependent signalling to influence NOTCH signalling and angiogenesis at a distance when it is packaged into extracellular vesicles (EVs). However, it is not clear whether JAG1 could also be packaged into EVs to influence NOTCH signalling and angiogenesis. In this work, we demonstrate that JAG1 is also packaged into EVs. We present evidence that JAG1-EVs inhibit NOTCH signalling and regulate EC behaviour and function. JAG1-EVs inhibited VEGF-induced HUVEC proliferation and migration in 2D culture condition and suppressed sprouting in a 3D microfluidic microenvironment. JAG1-EV treatment of HUVECs leads to a reduction of Notch1 intracellular domain (N1-ICD), and the proteasome and the intracellular domain of JAG1 (JAG1-ICD) are both required for this reduction to occur. These findings reveal a novel mechanism of JAG1 function in NOTCH signalling and ECs through EVs.

Generation of apoptosis-resistant HEK293 cells with CRISPR/Cas mediated quadruple gene knockout for improved protein and virus production.

Apoptosis has important functions during pathophysiologic processes. However, from a biopharmaceutical point of view, active apoptosis of host cells is undesirable during viral packaging or protein expression, because it decreases the efficiency of viral or protein production. Here we used the CRISPR/Cas technique to knock out four pro-apoptotic genes, Caspase3, Caspase6, Caspase7 and AIF1, in HEK293 cells, and successfully produced an apoptosis-resistant cell line. Furthermore, this cell line showed higher expression levels of pro-apoptotic proteins and higher packaging efficiency for the virus carrying these proteins than control HEK293 cells. This study not only produced an apoptosis-resistant cell line that is useful in producing apoptosis-inducing proteins or viruses expressing these proteins, but also provides a methodology to build other apoptosis-resistant cell lines.

MiRNA mimic screen for improved expression of functional neurotensin receptor from HEK293 cells.

Obtaining adequate quantities of functional mammalian membrane proteins has been a bottleneck in their structural and functional studies because the expression of these proteins from mammalian cells is relatively low. To explore the possibility of enhancing expression of these proteins using miRNA, a stable T-REx-293 cell line expressing the neurotensin receptor type 1 (NTSR1), a hard-to-express G protein-coupled receptor (GPCR), was constructed. The cell line was then subjected to human miRNA mimic library screening. In parallel, an HEK293 cell line expressing luciferase was also screened with the same human miRNA mimic library. Five microRNA mimics: hsa-miR-22-5p, hsa-miR-18a-5p, hsa-miR-22-3p, hsa-miR-429, and hsa-miR-2110were identified from both screens. They led to 48% increase in the expression of functional NTSR1 and to 239% increase of luciferase expression. These miRNAs were also effective in enhancing the expression of secretedglypican-3 hFc-fusion protein from HEK293 cells.The results indicate that these molecules may have a wide role in enhancing the production of proteins with biomedical interest.

Drug screening using a library of human induced pluripotent stem cell-derived cardiomyocytes reveals disease-specific patterns of cardiotoxicity.

BACKGROUND: Cardiotoxicity is a leading cause for drug attrition during pharmaceutical development and has resulted in numerous preventable patient deaths. Incidents of adverse cardiac drug reactions are more common in patients with preexisting heart disease than the general population. Here we generated a library of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from patients with various hereditary cardiac disorders to model differences in cardiac drug toxicity susceptibility for patients of different genetic backgrounds. METHODS AND RESULTS: Action potential duration and drug-induced arrhythmia were measured at the single cell level in hiPSC-CMs derived from healthy subjects and patients with hereditary long QT syndrome, familial hypertrophic cardiomyopathy, and familial dilated cardiomyopathy. Disease phenotypes were verified in long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy hiPSC-CMs by immunostaining and single cell patch clamp. Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) and the human ether-a-go-go-related gene expressing human embryonic kidney cells were used as controls. Single cell PCR confirmed expression of all cardiac ion channels in patient-specific hiPSC-CMs as well as hESC-CMs, but not in human embryonic kidney cells. Disease-specific hiPSC-CMs demonstrated increased susceptibility to known cardiotoxic drugs as measured by action potential duration and quantification of drug-induced arrhythmias such as early afterdepolarizations and delayed afterdepolarizations. CONCLUSIONS: We have recapitulated drug-induced cardiotoxicity profiles for healthy subjects, long QT syndrome, hypertrophic cardiomyopathy, and dilated cardiomyopathy patients at the single cell level for the first time. Our data indicate that healthy and diseased individuals exhibit different susceptibilities to cardiotoxic drugs and that use of disease-specific hiPSC-CMs may predict adverse drug responses more accurately than the standard human ether-a-go-go-related gene test or healthy control hiPSC-CM/hESC-CM screening assays.

High-throughput measurement of gap junctional intercellular communication.

Gap junctional intercellular communication (GJIC) is a critical part of cellular activities and is necessary for electrical propagation among contacting cells. Disorders of gap junctions are a major cause for cardiac arrhythmias. Dye transfer through microinjection is a conventional technique for measuring GJIC. To overcome the limitations of manual microinjection and perform high-throughput GJIC measurement, here we present a new robotic microinjection system that is capable of injecting a large number of cells at a high speed. The highly automated system enables large-scale cell injection (thousands of cells vs. a few cells) without major operator training. GJIC of three cell lines of differing gap junction density, i.e., HeLa, HEK293, and HL-1, was evaluated. The effect of a GJIC inhibitor (18-α-glycyrrhetinic acid) was also quantified in the three cell lines. System operation speed, success rate, and cell viability rate were quantitatively evaluated based on robotic microinjection of over 4,000 cells. Injection speed was 22.7 cells per min, with 95% success for cell injection and >90% survival. Dye transfer cell counts and dye transfer distance correlated with the expected connexin expression of each cell type, and inhibition of dye transfer correlated with the concentration of GJIC inhibitor. Additionally, real-time monitoring of dye transfer enables the calculation of coefficients of molecular diffusion through gap junctions. This robotic microinjection dye transfer technique permits rapid assessment of gap junction function in confluent cell cultures.

The HCN domain couples voltage gating and cAMP response in hyperpolarization-activated cyclic nucleotide-gated channels.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels control spontaneous electrical activity in heart and brain. Binding of cAMP to the cyclic nucleotide-binding domain (CNBD) facilitates channel opening by relieving a tonic inhibition exerted by the CNBD. Despite high resolution structures of the HCN1 channel in the cAMP bound and unbound states, the structural mechanism coupling ligand binding to channel gating is unknown. Here we show that the recently identified helical HCN-domain (HCND) mechanically couples the CNBD and channel voltage sensing domain (VSD), possibly acting as a sliding crank that converts the planar rotational movement of the CNBD into a rotational upward displacement of the VSD. This mode of operation and its impact on channel gating are confirmed by computational and experimental data showing that disruption of critical contacts between the three domains affects cAMP- and voltage-dependent gating in three HCN isoforms.

Expression of recombinant surfactant protein SFTA3 in the human kidney cell line HEK 293T.

Pulmonary surfactant is broadly known to keep the lung dry, clean and open by lowering the surface tension of the fluid-film that lines the alveoli. The surfactant's protein component, the so called surfactant proteins (SPs), make up a multifunctional protein family. In addition to the four "classical" surfactant proteins (SP-A, SP-B, SP-C and SP-D), which possess immunologic as well as surfactant regulatory properties, two novel putative surfactant proteins (SFTA2 and SFTA3) have recently been described. Neither of them shows sequential nor structural similarity with the already known surfactant proteins. However, bioinformatic analyses as well as first molecular-biological studies reveal properties that have already been described for known surfactant proteins. In our present work we introduce a technique to synthesize, purify and stabilize recombinant SFTA3 derived from the human embryonic kidney cell line HEK 293T. This will provide investigators with a valuable source of further examination and characterization of this fascinating novel member of the surfactant protein family.

Congenital myopathy with "corona" fibres, selective muscle atrophy, and craniosynostosis associated with novel recessive mutations in SCN4A.

We describe two brothers with lower facial weakness, highly arched palate, scaphocephaly due to synostosis of the sagittal and metopic sutures, axial hypotonia, proximal muscle weakness, and mild scoliosis. The muscle MRI of the younger sibling revealed a selective pattern of atrophy of the gluteus maximus, adductor magnus and soleus muscles. Muscle biopsy of the younger sibling revealed myofibres with internalized nuclei, myofibrillar disarray, and "corona" fibres. Both affected siblings were found to be compound heterozygous for c.3425G>A (p.Arg1142Gln) and c.1123T>C (p.Cys375Arg) mutations in SCN4A on exome sequencing, and the parents were confirmed carriers of one of the mutations. Electrophysiological characterization of the mutations revealed the Cys375Arg confers full and Arg1142Gln mild partial loss-of-function. Loss of function of the Nav1.4 channel leads to a decrement of the action potential and subsequent reduction of muscle contraction. The unusual muscle biopsy features suggest a more complex pathomechanism, and broaden the phenotype associated with SCN4A mutations.

The hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contain multiple S-palmitoylation sites.

Expression of hyperpolarization-activated cyclic nucleotide-gated channels (HCN1-4) on distal dendrites of neurons is suggested to modify synaptic integration in the central nervous system. However, the mechanisms of dendritic localization are not fully understood. Recent studies have revealed that S-palmitoylation plays an important role in the enrichment of various molecules at the postsynaptic membrane. Thus, we performed an acyl-biotinyl exchange assay, and found that HCN1, HCN2, and HCN4, but not HCN3, were S-palmitoylated in HEK293 cells. Mutation of multiple intracellular cysteine residues at the N-terminus of HCN2 was required for complete inhibition of S-palmitoylation. However, this mutagenesis had a minimal effect on surface expression of HCN2 proteins or electrophysiological properties of HCN2 current when expressed in HEK293 cells or in Xenopus oocytes. These findings provide insight into the physiological roles of S-palmitoylation of HCN channels in native neurons.

ANTXR-1 and -2 independent modulation of a cytotoxicity mediated by anthrax toxin in human cells.

Several animal models have shown that anthrax toxin (ATX) elicits a cytotoxic effect on host cells through anthrax toxin receptor (ANTXR) function. In this study, compared with mouse cells, cells obtained from humans exhibited low sensitivity to ATX-mediated cytotoxicity, and the sensitivity was not correlated with expression levels of ANTXRs. ATX treatment also induced a cytotoxic effect in other cultured human cells, human embryonic kidney (HEK) 293 cells, that express ANTXRs at undetectable levels. Furthermore, ectopic expression of ANTXRs in HEK293 cells did not affect the sensitivity to ATX treatment. These findings suggest that there is an ANTXR-independent cytotoxic mechanism in human cells.

Automated Patch Clamp Analysis of nAChα7 and Na(V)1.7 Channels.

Automated patch clamp devices are now commonly used for studying ion channels. A useful modification of this approach is the replacement of the glass pipet with a thin planar glass layer with a small hole in the middle. Planar patch clamp devices, such as the three described in this unit, are overtaking glass pipets in popularity because they increase throughput, are easier to use, provide for the acquisition of high-quality and information-rich data, and allow for rapid perfusion and temperature control. Covered in this unit are two challenging targets in drug discovery: voltage-gated sodium subtype 1.7 (Na(V)1.7) and nicotinic acetylcholine α7 receptors (nAChα7R). Provided herein are protocols for recording activation and inactivation kinetics of Na(V)1.7, and activation and allosteric modulation of nAChα7R.

Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteins.

Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.

Dominantly inherited myotonia congenita resulting from a mutation that increases open probability of the muscle chloride channel CLC-1.

Myotonia congenita-inducing mutations in the muscle chloride channel CLC-1 normally result in reduced open probability (P (o)) of this channel. One well-accepted mechanism of the dominant inheritance of this disease involves a dominant-negative effect of the mutation on the function of the common-gate of this homodimeric, double-barreled molecule. We report here a family with myotonia congenita characterized by muscle stiffness and clinical and electrophysiologic myotonic phenomena transmitted in an autosomal dominant pattern. DNA sequencing of DMPK and ZNF9 genes for myotonic muscular dystrophy types I and II was normal, whereas sequencing of CLC-1 encoding gene, CLCN1, identified a single heterozygous missense mutation, G233S. Patch-clamp analyses of this mutant CLC-1 channel in Xenopus oocytes revealed an increased P (o) of the channel's fast-gate, from ~0.4 in the wild type to >0.9 in the mutant at -90 mV. In contrast, the mutant exhibits a minimal effect on the P (o) of the common-gate. These results are consistent with the structural prediction that the mutation site is adjacent to the fast-gate of the channel. Overall, the mutant could lead to a significantly reduced dynamic response of CLC-1 to membrane depolarization, from a fivefold increase in chloride conductance in the wild type to a twofold increase in the mutant-this might result in slower membrane repolarization during an action potential. Since expression levels of the mutant and wild-type subunits in artificial model cell systems were unable to explain the disease symptoms, the mechanism leading to dominant inheritance in this family remains to be determined.

PPARγ is an E3 ligase that induces the degradation of NFκB/p65.

Nuclear factor-κB (NFκB) and peroxisome proliferator activated receptor-γ (PPARγ) are both transcription factors that perform distinct but overlapping roles in cellular regulation. Here we report that PPARγ acts as an E3 ubiquitin ligase, physically interacting with p65 to induce its ubiquitination and degradation. The ligand-binding domain of PPARγ interacts with the Rel Homology Domain region of NFκB/p65 to undergo robust ubiquitination and degradation that was independent of PPARγ transcriptional activity. Moreover, the ligand-binding domain of PPARγ delivered Lys48-linked polyubiquitin, resulting in the ubiquitination and degradation of p65. Lys28 was found to be critically important for PPARγ-mediated ubiquitination and degradation of p65, as it terminated both NFκB/p65-mediated pro-inflammatory responses and xenograft tumours. These findings demonstrate that PPARγ E3 ubiquitin ligase activity induces Lys48-linked ubiquitination and degradation of p65, and that this function is critical to terminate NFκB signalling pathway-elicited inflammation and cancer.

Establishment of a cell line carrying single copy of an exogenous mutant reporter gene for assaying the biological activity of ZFNs.

In this study, in order to detect the genome-editing activities of ZFNs, a cell line carrying a single copy of mutant reporter eGFP or luciferase gene, with a ZFN target sequence inserted in the middle of the coding region, was built through AAVS1 ZFN mediated knock-in technique. Briefly, AAVS1 ZFN expression vector and donor vector expressing mutant eGFP or luciferase were co-transfected into HEK 293 cells followed by positive/negative selection and cloning procedure. The targeted insertion of a single copy of the exogenous gene was confirmed by PCR, sequencing and southern blot. To prove the principle, hVEGF ZFN was used to test this system. hVEGF ZFN expression vector and donor vector carrying a fragment of wild-type reporter gene corresponding to the mutation-disabled stretch were co-transfected into 293-eGFP-hVEGF-TSF or 293-luci-hVEGF-TSF cell lines. 4 days post transfection, 293-eGFP-hVEGF-TSF group showed increased eGFP positive clones with a correction efficiency of 0.11%, which was significantly higher than that of the control. Similar results were obtained for the 293-luci-hVEGF-TSF group. The results indicated that the novel system, established by taking advantage of AAVS1 ZFN mediated knock-in technique, was useful for detecting the genome-editing activities of ZFNs. AAVS1 ZFN mediated knock-in was much easier to use than the current existing FLP-in technique. In addition, our donor vector system, featuring both positive and negative selection mechanisms, made it even more efficient to set up a system for assaying the biological activity of a new assembled ZFN.

Identification and characterization of a human mitochondrial NAD kinase.

NAD kinase is the sole NADP(+) biosynthetic enzyme. Despite the great significance of NADP(+), to date no mitochondrial NAD kinase has been identified in human, and the source of human mitochondrial NADP(+) remains elusive. Here we present evidence demonstrating that a human protein of unknown function, C5orf33, is a human mitochondrial NAD kinase; this protein likely represents the missing source of human mitochondrial NADP(+). The C5orf33 protein exhibits NAD kinase activity, utilizing ATP or inorganic polyphosphate, and is localized in the mitochondria of human HEK293A cells. C5orf33 mRNA is more abundant than human cytosolic NAD kinase mRNA in almost all tissues examined. We further show by database searches that some animals and protists carry C5orf33 homologues as their sole NADP(+) biosynthetic enzyme, whereas plants and fungi possess no C5orf33 homologue. These observations provide insights into eukaryotic NADP(+) biosynthesis, which has pivotal roles in cells and organelles.

Liposome impaired the adhesion and spreading of HEK293 cells: an AFM study.

Gene transfer has been proven to be a promising approach for treatment of several diseases. The cytotoxicity of transfection reagents is one of the key factors for clinical applications. The cytotoxicity of liposome has been extensively studied. However, its effects on the adhesion and spreading of transformed cells are still unclear. In this study, the cytotoxic effects of liposome on cell viability and mitochondrial membrane potential of HEK293 cells were first evaluated. Then, an atomic force microscope (AFM) was recruited to investigate the effects of liposome on the adhesion and spreading of HEK293 cells. AFM data indicated that liposome induced a significant decrease in number of cellular pseudopodia and cell-surface particles, in cell-surface roughness, and in average adhesion force of cell membranes. The AFM data implied that liposome impaired the adhesion and spreading of HEK293 cells.