Concept: HEK cell
There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.
C.RF-Tshr(hyt/hyt) mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshr(hyt/wild) heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshr(hyt/wild) mice are smaller than those from wild-type mice, and (125)I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained (125)I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshr(hyt/wild) mice.
Cardiac potassium channels encoded byhuman Ether-à-go-go Related Gene (hERG)are major targets for structurally diverse drugs associated with acquired long QT syndrome. This study characterized hERG channel inhibition by a minimally structured high affinity hERG inhibitor, Cavalli-2, composed of three phenyl groups linked by polymethylene spacers around a central amino group, chosen to probe the spatial arrangement of side chain groups in the high-affinity drug binding site of the hERG pore. hERG current (IhERG) recorded at physiological temperature from HEK 293 cells was inhibited with an IC50of 35.6 nM, with time- and voltage-dependence characteristic of blockade contingent upon channel gating. Potency of Cavalli-2 action was markedly reduced for attenuated-inactivation mutants located near (S620T; 54-fold) and remote from (N588K; 15-fold) the channel pore. The S6 Y652A and F656A mutations decreased inhibitory potency 17- and 75-fold respectively, whilst T623A and S624A at the base of the selectivity filter also decreased potency (16- and 7-fold respectively). The S5 helix F557L mutation decreased potency 10-fold and both F557L and Y652A mutations eliminated voltage dependence of inhibition. Computational docking using the recent cryo-EM structure of an open channel hERG construct could only partially recapitulate experimental data, and the high dependence of Cavalli-2 block on F656 is not readily explainable in that structure. A small clockwise rotation of the inner (S6) helix of the hERG pore from its configuration in the cryo-EM structure may be required to optimize F656 side chain orientations compatible with high affinity block.
Patch-clamp recording has enabled single-cell electrical, morphological and genetic studies at unparalleled resolution. Yet it remains a laborious and low-throughput technique, making it largely impractical for large-scale measurements such as cell type and connectivity characterization of neurons in the brain. Specifically, the technique is critically limited by the ubiquitous practice of manually replacing patch-clamp pipettes after each recording. To circumvent this limitation, we developed a simple, fast, and automated method for cleaning glass pipette electrodes that enables their reuse within one minute. By immersing pipette tips into Alconox, a commercially-available detergent, followed by rinsing, we were able to reuse pipettes 10 times with no degradation in signal fidelity, in experimental preparations ranging from human embryonic kidney cells to neurons in culture, slices, and in vivo. Undetectable trace amounts of Alconox remaining in the pipette after cleaning did not affect ion channel pharmacology. We demonstrate the utility of pipette cleaning by developing the first robot to perform sequential patch-clamp recordings in cell culture and in vivo without a human operator.
Auto-antibodies against the paranodal proteins neurofascin-155 and contactin-1 have recently been described in patients with chronic inflammatory demyelinating polyradiculoneuropathy and are associated with a distinct clinical phenotype and response to treatment. Contactin-associated protein 1 (Caspr, encoded by CNTNAP1) is a paranodal protein that is attached to neurofascin-155 and contactin-1 (CNTN1) but has not yet been identified as a sole antigen in patients with inflammatory neuropathies. In the present study, we screened a cohort of 35 patients with chronic inflammatory demyelinating polyradiculoneuropathy (age range 20-80, 10 female, 25 male) and 22 patients with Guillain-Barré syndrome (age range 17-86, eight female, 14 male) for autoantibodies against paranodal antigens. We identified two patients, one with chronic inflammatory demyelinating polyradiculoneuropathy and one with Guillain-Barré syndrome, with autoantibodies against Caspr by binding assays using Caspr transfected human embryonic kidney cells and murine teased fibres. IgG3 was the predominant autoantibody subclass in the patient with Guillain-Barré syndrome, IgG4 was predominant in the patient with chronic inflammatory demyelinating polyradiculoneuropathy. Accordingly, complement deposition after binding to HEK293 cells was detectable in the patient with IgG3 autoantibodies only, not in the patient with IgG4. Severe disruption of the paranodal and nodal architecture was detectable in teased fibres of the sural nerve biopsy and in dermal myelinated fibres, supporting the notion of the paranodes being the site of pathology. Deposition of IgG at the paranodes was detected in teased fibre preparations of the sural nerve, further supporting the pathogenicity of anti-Caspr autoantibodies. Pain was one of the predominant findings in both patients, possibly reflected by binding of patients' IgG to TRPV1 immunoreactive dorsal root ganglia neurons. Our results demonstrate that the paranodal protein Caspr constitutes a new antigen that leads to autoantibody generation as part of the novel entity of neuropathies associated with autoantibodies against paranodal proteins.
Our previous studies using HeLa and HEK 293 cells demonstrated that selenomethionine, SeMet, exerts more of an antagonistic effect on arsenic than other selenium species. These studies attributed the antagonistic effect of SeMet to decreased levels of reactive oxygen species, ROS, changes in protein phosphorylation and possible incorporation of SeMet into proteins. The present study employs a metallomics approach to identify the selenium-containing proteins in HEK 293 cells raised with SeMet. The proteins were screened and separated using two dimensional high performance liquid chromatography (HPLC)-inductively coupled plasma mass spectrometry (ICPMS), size exclusion chromatography (SEC) and reversed-phase chromatography (RPC). The Se-containing proteins were identified by peptide mapping using nano-HPLC-Chip-electrospray ionization mass spectrometry (ESIMS).
A new biologically compatible Zn(II) sensor was fabricated by embedding Zn(II) sensing spiropyran within the surface of a liposome derived from E.coli lipids (LSP2). Solution-based experiments with increasing Zn(II) concentrations show improved aqueous solubility and sensitivity compared to the isolated spiropyran molecule (SP2). LSP2 is capable of sensing Zn(II) efflux from apoptotic cells with preliminary data indicating that sensing is localized near the surface membrane of HEK 293 cells. Finally, LSP2 is suitable for development into a nanoliter scale, dip-sensor for Zn(II) using microstructured optical fiber as the sensing platform to detect Zn(II) in the range of 100 ρM with no signs of photobleaching. Existing spiropyran based sensing molecules can thus be made biologically compatible, with an ability to operate with improved sensitivity using nanoscale liquid sample volumes. This work represents the first instance where photochromic spiropyran molecules and liposomes are assembled to create a new and multifunctional sensing entity for Zn(II).
Here, we report on a novel protocol for determining the viability of individual cells in an adherent cell culture, without adversely affecting the remaining cells in the sample. This is facilitated using a freestanding microfluidic perfusion device, the Multifunctional Pipette (MFP), which generates a virtual flow cell around selected single cells. We investigated the utility on four different cell lines, NG108-15, HEK 293, PC12, and CHO, and combined the assay with a cell poration experiment, in which we apply the pore-forming agent digitonin, followed by fluorescein diphosphate, a pre-fluorescent substrate for alkaline phosphatase, in order to monitor intracellular enzyme activity. The cell viability was instantly assessed through simultaneous perfusion with fluorescein diacetate (FDA) and propidium iodide (PI), both being dispensed through the same superfusion device used to porate and deliver the enzyme substrate. In this fluorescence assay, viable and non-viable cells were distinguished by their green and red emission, respectively, within 10 s. In addition, the enzyme activity was monitored over time as a secondary test for cellular activity. Our findings demonstrate that this microfluidic technology-assisted approach is a facile, rapid, and reliable means to determine the viability in single-cell experiments and that viability studies can be performed routinely alongside typical substrate delivery protocols. This approach would remove the need for global cell viability testing and would enable viability studies of only the cells under experimental analysis.
Background: Cells release a mixture of extracellular vesicles, amongst these exosomes, that differ in size, density and composition. The standard isolation method for exosomes is centrifugation of fluid samples, typically at 100,000×g or above. Knowledge of the effect of discrete ultracentrifugation speeds on the purification from different cell types, however, is limited. Methods: We examined the effect of applying differential centrifugation g-forces ranging from 33,000×g to 200,000×g on exosome yield and purity, using 2 unrelated human cell lines, embryonic kidney HEK293 cells and bladder carcinoma FL3 cells. The fractions were evaluated by nanoparticle tracking analysis (NTA), total protein quantification and immunoblotting for CD81, TSG101, syntenin, VDAC1 and calreticulin. Results: NTA revealed the lowest background particle count in Dulbecco’s Modified Eagle’s Medium media devoid of phenol red and cleared by 200,000×g overnight centrifugation. The centrifugation tube fill level impacted the sedimentation efficacy. Comparative analysis by NTA, protein quantification, and detection of exosomal and contamination markers identified differences in vesicle size, concentration and composition of the obtained fractions. In addition, HEK293 and FL3 vesicles displayed marked differences in sedimentation characteristics. Exosomes were pelleted already at 33,000×g, a g-force which also removed most contaminating microsomes. Optimal vesicle-to-protein yield was obtained at 67,000×g for HEK293 cells but 100,000×g for FL3 cells. Relative expression of exosomal markers (TSG101, CD81, syntenin) suggested presence of exosome subpopulations with variable sedimentation characteristics. Conclusions: Specific g-force/k factor usage during differential centrifugation greatly influences the purity and yield of exosomes. The vesicle sedimentation profile differed between the 2 cell lines.
Culturing of bone marrow cells in serum-free RPMI-1640 medium for 24 h was accompanied by a decrease in the rate of [3H]-thymidine incorporation into DNA. Addition of native apolipoprotein A-I (apoA-I) or plasma LDL and HDL to the culture medium increased this parameter. In contrast to native apoA-I, its modified form decelerated DNA synthesis in bone marrow cells. A similar inhibitory effect of modified protein was observed in cultures of human embryonic kidney cells (HEK293) and in rapidly proliferating mouse macrophage cell line ANA-1. The only exclusion was human myeloid cell line U937: neither native nor modified apoA-I affected DNA synthesis in these cells. Thus, the regulatory effects of apoA-I are tissue-specific; this protein can produce either stimulatory or inhibitory effect on DNA biosynthesis in cells depending on its conformation.