Transfected CHO Cells Research Articles

Development of an indirect ELISA for the serologic detection of bovine viral diarrhea virus based on E2 antigen sub-genotypes 1b, 1e, and 1d.

Bovine viral diarrhea virus (BVDV) causes ongoing economic losses to cattle industries, directly through reduced herd performance or indirectly through control program costs. ELISA assays, one of the most widely used techniques due to their ease of implementation, have been a valuable tool for mass surveillance and detection of BVDV. In this study, we developed a new indirect ELISA (rE2-ELISA) for serologic detection of BVDV. The assay considers three recombinant E2 protein subtypes as antigens, allowing serologic diagnosis of BVDV-1b (high prevalence worldwide), BVDV-1d and 1e (high prevalence in southern Chile) sub-genotypes. Recombinant E2 (rE2) proteins were successfully expressed in stably transfected CHO cells. Conditions for rE2 ELISAs were established after determining appropriate concentrations of antigen, blocking agent, secondary antibody, and serum dilutions to achieve maximum discrimination between positive and negative serum samples. The developed rE2-ELISA showed a sensitivity of 92.86% and a specificity of 98.33%. Clinical testing of 180 serum samples from herds in southern Chile showed high accuracy (kappa > 0.8) compared to the commercial BVDV Total Ab kit (IDEXX), with 95.37% positive and 87.5% negative predictive value. In addition, the rE2 ELISA has shown the capability to detect anti-BVDV antibodies from naturally infected animals with sub-genotypes 1b, 1e, or undetermined. These results indicate that the developed indirect ELISA could serve as a valid, and efficient alternative for identifying BVDV-infected animals, thus contributing to the success of disease control and eradication programs.

Abstract P211: Angiotensin-(2-7): A novel antihypertensive peptide of the renin-angiotensin-system

Recently, it has been described that acetyl-angiotensin (2-7)-amide (Ac-Ang-(2-7)-NH2) is a potent ACE inhibitor. In addition, the putative endogenous peptide angiotensin-(2-7) was also reported to be an ACE inhibitor. Here we tested the effect of Ang-(2-7) in blood pressure (BP) and isolated aortic rings of Wistar and SHR. In addition, we evaluated whether this hexapeptide stimulates receptors from the alternative renin-angiotensin system (RAS), including Mas, MrgD, and AT2R using CHO-transfected cells. To record BP and administer drugs, the femoral arterial and vein were cannulated. In the baseline period, BP was recorded for 1 hour. In bolus intravenous injection of Ang-(2-7) was performed at doses of 30, 6, and 2.4 μg/kg, in a volume of 0,1 ml/100g. After the injection BP and heart rate (HR) were recorded for six hours. Additionally, we evaluated the effect of Ang-(2-7) in isolated aortic rings from SHR and Wistar rats, pre-constricted with phenylephrine (10 -7 mol.L -1 ). The peptide effect was tested in the range of 10 -12 to 10 -6 molar. The effect of the peptide was also evaluated in endothelium-denuded rings. Furthermore, the effect of Ang-(2-7) was tested in rings pre-treated with the Mas/MrgD antagonist D-Pro 7 -Ang-(1-7) (10-6 μmol.L -1 ). Finally, we used NO intracellular measurements to test for the Ang-(2-7) activation of Mas, MrgD, or AT2 receptors using transfected-CHO cells. To confirm the effectiveness of Ang-(2-7) for NO production we used 4,5-diaminofluorescein-diacetate (DAF-FM diacetate) and the human umbilical vein cell line EA.hy926. Our data showed a progressive and marked reduction in BP reaching -40 mmHg (123±12mmHg;n=3) with 6 hours after 30ug/kg administration of injection of Ang-(2-7). There was no significant effect on Blood pressure in Wistar rats after injection. The aortic rings had a vasorelaxing effect of the Ang-(2-7) in both Wistar and SHR, which was abolished after endothelium was removed. The vasorelaxant effect of Ang-(2-7) was abolished by pre-treatment with D-Pro 7 -Ang-(1-7). Accordingly, Ang-(2-7) induced NO production in EA.hy926 and MrgD-transfected CHO cells. A slight effect on NO production was also observed in AT2-transfected cells. In conclusion, we have identified a new biologically active component of the RAS with a potent antihypertensive effect in SHR. Our results suggest an important contribution of MrgD/NO to the Ang-(2-7) cardiovascular effects.

Interplay between Electric Field Strength and Number of Short-Duration Pulses for Efficient Gene Electrotransfer.

Electroporation is a method that shows great promise as a non-viral approach for delivering genes by using high-voltage electric pulses to introduce DNA into cells to induce transient gene expression. This research aimed to evaluate the interplay between electric pulse intensity and 100 µs-duration pulse numbers as an outcome of gene electrotransfer efficacy and cell viability. Our results indicated a close relationship between pulse number and electric field strength regarding gene electrotransfer efficacy; higher electric pulse intensity resulted in fewer pulses needed to achieve the same gene electrotransfer efficacy. Subsequently, an increase in pulse number had a more negative impact on overall gene electrotransfer by significantly reducing cell viability. Based on our data, the best pulse parameters to transfect CHO cells with the pMax-GFP plasmid were using 5 HV square wave pulses of 1000 V/cm and 2 HV of 1600 V/cm, correspondingly resulting in 55 and 71% of transfected cells and maintaining 79 and 54% proliferating cells. This shows ESOPE-like 100 µs-duration pulse protocols can be used simultaneously to deliver cytotoxic drugs as well as immune response regulating genetically encoded cytokines.

Potentially Pathogenic SORL1 Mutations Observed in Autosomal-Dominant Cases of Alzheimer's Disease Do Not Modulate APP Physiopathological Processing.

The SORL1 gene encodes LR11/SorLA, a protein that binds β-amyloid precursor protein (APP) and drives its intracellular trafficking. SORL1 mutations, occurring frequently in a subset of familial cases of Alzheimer's disease (AD), have been documented, but their pathogenic potential is not yet clear and questions remain concerning their putative influence on the physiopathological processing of APP. We have assessed the influence of two SORL1 mutations that were described as likely disease-causing and that were associated with either benign (SorLA924) or severe (SorLA511) AD phenotypes. We examined the influence of wild-type and mutants SorLA in transiently transfected HEK293 cells expressing either wild-type or Swedish mutated APP on APP expression, secreted Aβ and sAPPα levels, intracellular Aβ 40 and Aβ42 peptides, APP-CTFs (C99 and C83) expressions, α-, β- and γ-secretases expressions and activities as well as Aβ and CTFs-degrading enzymes. These paradigms were studied in control conditions or after pharmacological proteasomal modulation. We also established stably transfected CHO cells expressing wild-type SorLA and established the colocalization of APP and either wild-type or mutant SorLA. SorLA mutations partially disrupt co-localization of wild-type sorLA with APP. Overall, although we mostly confirmed previous data concerning the influence of wild-type SorLA on APP processing, we were unable to evidence significant alterations triggered by our set of SorLA mutants, whatever the cells or pharmacological conditions examined. Our study , however, does not rule out the possibility that other AD-linked SORL1 mutations could indeed affect APP processing, and that pathogenic mutations examined in the present study could interfere with other cellular pathways/triggers in AD.

A novel KCNC1 gain-of-function variant causing developmental and epileptic encephalopathy: "Precision medicine" approach with fluoxetine.

Variable phenotypes, including developmental encephalopathy with (DEE) or without seizures and myoclonic epilepsy and ataxia due to potassium channel mutation, are caused by pathogenetic variants in KCNC1, encoding for Kv3.1 channel subunits. In vitro, channels carrying most KCNC1 pathogenic variants display loss-of-function features. Here, we describe a child affected by DEE with fever-triggered seizures, caused by a novel de novo heterozygous missense KCNC1 variant (c.1273G>A; V425M). Patch-clamp recordings in transiently transfected CHO cells revealed that, compared to wild-type, Kv3.1 V425M currents (1) were larger, with membrane potentials between -40 and +40 mV; (2) displayed a hyperpolarizing shift in activation gating; (3) failed to inactivate; and (4) had slower activation and deactivation kinetics, consistent with a mixed functional pattern with prevalent gain-of-function effects. Exposure to the antidepressant drug fluoxetine inhibited currents expressed by both wild-type and mutant Kv3.1 channels. Treatment of the proband with fluoxetine led to a rapid and prolonged clinical amelioration, with the disappearance of seizures and an improvement in balance, gross motor skills, and oculomotor coordination. These results suggest that drug repurposing based on the specific genetic defect may provide an effective personalized treatment for KCNC1-related DEEs.

CACNA1S mutation-associated dental anomalies: A calcium channelopathy.

To identify the molecular etiology of distinct dental anomalies found in eight Thai patients and explore the mutational effects on cellular functions. Clinical and radiographic examinations were performed for eight patients. Whole exome sequencing, mutant protein modelling, qPCR, western blot analysis, scratch assays, immunofluorescence, confocal analysis, in situ hybridization, and scanning electron micrography of teeth were done. All patients had molars with multiple supernumerary cusps, single-cusped premolars, and a reduction in root number. Mutation analysis highlighted a heterozygous c.865A>G; p.Ile289Val mutation in CACNA1S in the patients. CACNA1S is a component of the slowly inactivating L-type voltage-dependent calcium channel. Mutant protein modeling suggested that the mutation might allow leakage of Ca2+ or other cations, or a tightening, to restrict calcium flow. Immunohistochemistry analysis showed expression of Cacna1s in the developing murine tooth epithelium during stages of crown and root morphogenesis. In cell culture, the mutation resulted in abnormal cell migration of transfected CHO cells compared to wildtype CACNA1S, with changes to the cytoskeleton and markers of focal adhesion. The malformations observed in our patients suggest a role for calcium signaling in organization of both cusps and roots, affecting cell dynamics within the dental epithelium.

A preliminary study to establish the transfected CHO cell lines which highly express Trastuzumab - A biosimilar product of Herceptin

Human epidermal growth factor receptor 2 (HER2) has been identified as a molecular target for breast cancer therapy, such as Trastuzumab (Herceptin®). This has been shown to improve patient survival substantially. The current study is aiming to locally produce an anti-HER2 monoclonal antibody (named Trastuzumab) which has an equivalent biological properties in comparison with the original version, Herceptin®). In silico design and construction of recombinant vectors, as well as the establishment of transfected cell lines with high expression of Trastuzumab were performed. Based on the protein sequences obtained from the Drugbank, the DNA sequences encoding for the light chain (Tras-Lc) and heavy chain (Tras-Hc) of Trastuzumab were optimized and integrated into pNanogen-Hygro and pNanogen-Puro vectors, respectively. The Neon Transfection System was used to co-transfect the pNanogen-Tras-Lc-Hygro and pNanogen-Tras-Hc-Puro constructs into CHO cells. Different co-transfected single-cell-colonies selected on media supplemented with hygromycin and puromycin were used for ELISA and SDS-PAGE assays to identify the CHO cell lines which highly express Trastuzumab. Based on the present results, 30μg of both constructs were suitable for DNA co-transfection. After 07 days of culture, the highest amount of Trastuzumab (561 µg/ml) was obtained from the H06LD68 cell line.

Intoxicating effects of alcohol depend on acid-sensing ion channels.

Persons at risk for developing alcohol use disorder (AUD) differ in their sensitivity to acute alcohol intoxication. Alcohol effects are complex and thought to depend on multiple mechanisms. Here, we explored whether acid-sensing ion channels (ASICs) might play a role. We tested ASIC function in transfected CHO cells and amygdala principal neurons, and found alcohol potentiated currents mediated by ASIC1A homomeric channels, but not ASIC1A/2 A heteromeric channels. Supporting a role for ASIC1A in the intoxicating effects of alcohol in vivo, we observed marked alcohol-induced changes on local field potentials in basolateral amygdala, which differed significantly in Asic1a-/- mice, particularly in the gamma, delta, and theta frequency ranges. Altered electrophysiological responses to alcohol in mice lacking ASIC1A, were accompanied by changes in multiple behavioral measures. Alcohol administration during amygdala-dependent fear conditioning dramatically diminished context and cue-evoked memory on subsequent days after the alcohol had cleared. There was a significant alcohol by genotype interaction. Context- and cue-evoked memory were notably worse in Asic1a-/- mice. We further examined acute stimulating and sedating effects of alcohol on locomotor activity, loss of righting reflex, and in an acute intoxication severity scale. We found loss of ASIC1A increased the stimulating effects of alcohol and reduced the sedating effects compared to wild-type mice, despite similar blood alcohol levels. Together these observations suggest a novel role for ASIC1A in the acute intoxicating effects of alcohol in mice. They further suggest that ASICs might contribute to intoxicating effects of alcohol and AUD in humans.

An emerging role for DPP6: reciprocal regulation of INa-Ito and implications for arrhythmogenesis

Abstract Background Since the association of a chromosomal risk haplotype harboring dipeptidyl peptidase-like protein-6 (DPP6) to familial idiopathic ventricular fibrillation (iVF), a growing number of DPP6 missense variants has been reported in patients with ventricular tachyarrhythmias. The mechanisms underlying DPP6 mediated-arrhythmogenesis are not yet fully elucidated. DPP6 is a subunit of the transient outward potassium (Ito) channel complex in Purkinje cells (PC) and ventricular myocytes (VM). Purpose Since other Ito-channel subunits (Navβ1, KChIP2, KCNE4 and DPP10) are also known to antagonize INa, we examined whether DPP6 could play a broader role in the inter-regulation of Kv4.3 and Nav1.5 channels. We identified two novel DPP6 variants (p.Arg274His and p.His213Tyr), each segregating in families with QT/QU prolongation. DPP6 p.Arg274His carriers suffered from iVF, ectopic beats from the conduction system, and mitral valve prolapse. Other DPP6 variants (p.Ala751Val identified in this study; p.Gln526His and DPP6-T p.His332Arg published) are associated with Brugada syndrome (BrS). We hypothesized that DPP6 has opposing effects on INa and Ito displaying a reciprocal regulation of these currents. Methods and results First, we determined the effect of the DPP6 variants on INa and Ito in transfected CHO cells. Ito density was significantly reduced only when PC subunits were co-expressed with the DPP6 p.Arg274His or p.His213Tyr variants. Indeed, DPP6 modulates Nav1.5 channels in CHO cells by reducing INa Peak and INa Late, whereas DPP6 mutants p.Arg274His or p.His213Tyr resulted in an increase of both components compared to WT. Co-immunoprecipitation experiments in human endocardium confirmed an interaction between DPP6 and Nav1.5 channels. Computing of mutant DPP6-driven Ito-INa changes in a published human PC model led to significant prolongation of the action potential duration, mainly caused by increased INa Late. On the other hand, the DPP6 p.Gln526His and p.Ala751Val variants, linked to BrS, led to a decreased INa Peak compared to the WT, while there was a tendency towards increased Ito density in both PC and VM molecular setups. DPP6 (p.Arg274His and p.Ala751Val) transfection experiments in hiPSC cardiomyocytes, expressing endogenous INa and Ito, confirmed the reciprocal results obtained in CHO cells. Conclusions DPP6 regulates INa and Ito in a reciprocal manner. The cardiac phenotype of DPP6 variants could encompass a spectrum between two opposite poles: 1) QT/QU prolongation by DPP6 variants causing loss of Ito and gain of INa, like p.Arg274His and p.His213Tyr versus 2) BrS by DPP6 variants leading to gain of Ito and loss of INa, like p.Gln526His and p.Ala751Val. Funding Acknowledgement Type of funding sources: Private grant(s) and/or Sponsorship. Main funding source(s): ESC Personal research grant, obtained in 2019

PET imaging studies to investigate functional expression of mGluR2 using [11C]mG2P001

Metabotropic glutamate receptor 2 (mGluR2) has been extensively studied for the treatment of various neurological and psychiatric disorders. Understanding of the mGluR2 function is pivotal in supporting the drug discovery targeting mGluR2. Herein, the positive allosteric modulation of mGluR2 was investigated via the in vivo positron emission tomography (PET) imaging using 2-((4-(2-[11C]methoxy-4-(trifluoromethyl)phenyl)piperidin-1-yl)methyl)-1-methyl-1H-imidazo[4,5-b]pyridine ([11C]mG2P001). Distinct from the orthosteric compounds, pretreatment with the unlabeled mG2P001, a potent mGluR2 positive allosteric modulator (PAM), resulted in a significant increase instead of decrease of the [11C]mG2P001 accumulation in rat brain detected by PET imaging. Subsequent in vitro studies with [3H]mG2P001 revealed the cooperative binding mechanism of mG2P001 with glutamate and its pharmacological effect that contributed to the enhanced binding of [3H]mG2P001 in transfected CHO cells expressing mGluR2. The in vivo PET imaging and quantitative analysis of [11C]mG2P001 in non-human primates (NHPs) further validated the characteristics of [11C]mG2P001 as an imaging ligand for mGluR2. Self-blocking studies in primates enhanced accumulation of [11C]mG2P001. Altogether, these studies show that [11C]mG2P001 is a sensitive biomarker for mGluR2 expression and the binding is affected by the tissue glutamate concentration.

A fully automated high-throughput plasmid purification workstation for the generation of mammalian cell expression-quality DNA.

Early-stage antibody discovery and engineering typically require the cloning, expression, and screening of large numbers of proteins. Normally, DNA fragments encoding proteins of interest are cloned into extra-chromosomal plasmids that are amplified in Escherichia coli. Following purification from the bacteria, the plasmids are introduced into appropriate cells, and the expressed recombinant proteins screened for desired binding or function in a high-throughput manner. Even in a 96-well plate format, plasmid purification from E. coli is typically a labor intensive and time-consuming process. To further accelerate our existing biotherapeutic discovery workflows we designed, qualified, and enabled a fully integrated high-throughput plasmid purification and quantification workstation which we have termed AMPS (Automated Miniprep Plasmid Station). Using components from a commercially available kit, AMPS can purify plasmid preparations from twenty 96-deep-well plates of E. coli cultures, measure DNA absorbance at 260 nm, calculate plasmid concentrations, and prepare 96-deep-well plates for mammalian expression in an operator-independent manner. Plasmid yields and concentrations are equivalent to those obtained off-line. Furthermore, the quality of the DNA purified on the AMPS is equivalent to that obtained off-line in terms of DNA topology, and absence of contaminating bacterial chromosomal DNA and RNA. Most importantly, plasmids purified on the AMPS provide similar antibody titers following transfection in CHO cells as plasmids purified off-line. The AMPS bridges high-throughput E. coli colony picking capabilities typically available in an automation lab with downstream CHO expression needs and will facilitate screening of large numbers of biotherapeutics in binding and cell assay screens.

Modular Cloning of MUC1 Recombinant Antibodies by Assembly of Synthetic Domain Genes

Antibody immunotherapy has emerged as a constructive way to fight cancer. New immunotherapies require the identification of a tumor‐specific antigen. The protein mucin 1 (MUC1) has long been recognized as an important specific antigen and a potential immunotherapy target. In healthy human tissues, MUC1 is responsible for protecting the epithelial lining. However, in cancer cells, MUC1 displays truncated glycosylation, revealing tumor‐specific epitopes. Recombinant antibodies are preferred for therapeutic development, and in some cases, it is desirable to produce different antibody formats, including intact IgG or Fab fragments. Using the MUC1 specific antibody 4H5 as a model, we have developed a modular cloning approach that can rapidly produce different antibody modalities to facilitate the rapid production of both IgG and Fab antibody formats. The 4H5 variable domains were PCR amplified to contain 20 base‐pair (bp) overlaps with the pcDNA3.1 vector on the 5’ end, and 20 bp overlaps to either the human CH1 or Ck domains on the 3’end of the genes. To produce the Fab, a His‐tag and a 20 base‐pair (bp) overlap to the vector was added to the 3’ end of a human CH1 domain gene sequence. For IgG production, human CH1‐CH2‐CH3 domains were PCR amplified, and the same vector overlap sequence was added to the 3’ end of the sequence. Finally, a 20 bp overlap to the vector was added to the 3’ end of a human Ck domain. The PCR products were purified, and the constructs were assembled using the NEBuilder®HiFi DNA Assembly Cloning Kit. DNA sequencing confirmed the successful assembly of the coding sequence, and the 4H5 Fab fragments and IgG were produced by transient transfection in CHO cells. The antibodies (Fab and IgG) were purified by affinity chromatography and binding to recombinant MUC1 antigens confirmed by ELISA. The cloning method presented here simultaneously produces multiple antibody formats. This approach will permit rapid assembly and screening of different antibody formats and isotypes for therapeutic antibody discovery and analysis.

Cell Engineering and Cultivation of Chinese Hamster Ovary Cells for the Development of Orthogonal Eukaryotic Cell-free Translation Systems.

The investigation of protein structures, functions and interactions often requires modifications to adapt protein properties to the specific application. Among many possible methods to equip proteins with new chemical groups, the utilization of orthogonal aminoacyl-tRNA synthetase/tRNA pairs enables the site-specific incorporation of non-canonical amino acids at defined positions in the protein. The open nature of cell-free protein synthesis reactions provides an optimal environment, as the orthogonal components do not need to be transported across the cell membrane and the impact on cell viability is negligible. In the present work, it was shown that the expression of orthogonal aminoacyl-tRNA synthetases in CHO cells prior to cell disruption enhanced the modification of the pharmaceutically relevant adenosine A2a receptor. For this purpose, in complement to transient transfection of CHO cells, an approach based on CRISPR/Cas9 technology was selected to generate a translationally active cell lysate harboring endogenous orthogonal aminoacyl-tRNA synthetase.

Sphingosine-1-phosphate, a novel TREM2 ligand, promotes microglial phagocytosis to protect against ischemic brain injury

The mechanism of sphingosine-1-phosphate (S1P)-mediated phagocytosis remains unknown. Here, we found that S1P or FTY720 (an analog of S1P) promoted microglial phagocytosis in stroke independent of S1PRs. First, we used computer simulation of molecular docking to predict that S1P might be a ligand for triggering receptor expressed on myeloid cells 2 (TREM2). Next, microscale thermophoresis (MST), surface plasmon resonance (SPR) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) were performed to reveal that S1P was a novel TREM2 ligand. Then, we confirmed the pro-phagocytosis of S1P targeting in Trem2-Dap12 transfected CHO cells and TREM2 knockdown microglia. Point mutation analysis showed that D104 was the critical binding residue. Trem2−/− mice were used to demonstrate the role of S1P-induced phagocytosis targeting on TREM2 in protecting against ischemic brain injury. Finally, further studies revealed that apolipoprotein E (APOE) loaded with S1P was released by microglia and bound to apoptotic neurons via LDL receptor related protein 1B (LRP1B) and thereby induced microglia to phagocytose apoptotic neurons. Overall, the present work reveals for the first time that S1P acts as a novel endogenous ligand of TREM2 to effectively promote microglial phagocytosis. Our findings provide a new lead compound for developing immunomodulator targeting on TREM2.

Abstract P250: A High-throughput Nitric Oxide Measurement Assay Reveals That Angiotensin-(1-5) Is An AT2 Receptor Agonist

The angiotensin AT2-receptor (AT2R) is a key component within the protective arm of the renin-angiotensin system (RAS), being involved in nitric oxide (NO) production and vasodilation. To this date, no quantitative high-throughput assay is available to identify AT2R agonists in vitro , which may be a reason for the low number of AT2R selective ligands in drug development programs. Objective: To design and validate a high-throughput method for detection of AT2R activation in vitro . Methods and Results: NO release was selected as readout for AT2R activation in AT2R transfected (CHO-AT2) and non-transfected (CHO-NT) CHO cells using DAF-FM (5x10-6 mol/L) as NO probe. Cells were seeded on 96-well plates and stimulated for 15 minutes with C21 or Ang II (established AT2R agonists, 10-6mol/L), Ang-(1-5) (molecule with unknown biological status, 10-6 mol/L) or Ang-(1-7) (Mas-receptor agonist, 10-7 mol/L). After fixation of cells, fluorescence signals were captured by fluorescence microscopy using an automated imaging system (ImageXpress Pico, Molecular Devices, San Jose, USA) and image analysis by ImageJ. In CHO-AT2, C21 (+34.78 ± 12.09%), Ang II (+28.76 ± 17.65%) and Ang-(1-5) (+78.00 ± 23.82%) increased NO release (one-way ANOVA, p < 0.05 vs control, at least 3 independent experiments), while Ang-(1-7) had no effect (+ 0.13 ± 4.74%). In CHO-NT, none of the compounds stimulated release of NO (C21 -4.91 ±10.24%; Ang II -4.79 ± 12.44%; Ang (1-5) -8.88 ± 18.16%; Ang-(1-7) -11.57 ± 19.95%) indicating that the responses in CHO-AT2 were AT2R specific. Conclusion: Measurement of NO release from AT2R transfected CHO cells by DAF-FM fluorescence in an automated way is suitable as high-throughput assay for the identification of AT2R-agonistic compounds in vitro , Application of the assay revealed that Ang-(1-5), which is commonly regarded as an inactive metabolite of Ang II, has AT2R agonistic properties.

Plasmodium vivax Gametocytes Adherence to Bone Marrow Endothelial Cells.

In a Plasmodium vivax infection, it was shown a proportionally increased on gametocyte distribution within the bone marrow aspirant, suggesting a role of this organ as a reservoir for this parasite stage. Here, we evaluated the ex vivo cytoadhesive capacity of P. vivax gametocytes to bone marrow endothelial cells (HBMEC) and investigated the involvement of some receptors in the cytoadhesion process by using transfected CHO cells (CHO-ICAM1, CHO-CD36 and CHO-VCAM), wild type (CHO-K1) or deficient in heparan and chondroitin sulfate (CHO-745). Ex-vivo cytoadhesion assays were performed using a total of 44 P. vivax isolates enriched in gametocyte stages by Percoll gradient in the different cell lines. The majority of isolates (88.9%) were able to adhere to HBMEC monolayer. ICAM1 seemed to be the sole receptor significantly involved. CD-36 was the receptor with higher adhesion rate, despite no significance was noticed when compared to CHO-745. We demonstrated that gametocyte P. vivax adheres ex vivo to bone marrow endothelial cells. Moreover, P. vivax gametocytes display the ability to adhere to all CHO cells investigated, especially to CHO-ICAM1. These findings bring insights to the comprehension of the role of the bone marrow as a P. vivax reservoir and the potential impact on parasite transmission to the vector.

STEP® vectors for rapid generation of stable transfected CHO cell pools and clones with high expression levels and product quality homogeneity of difficult-to-express proteins

Many proteins produced in CHO cells need evaluation for their clinical and commercial potential. Traditional methods based on stable clone generation are slow and unsuitable for screening larger numbers of proteins, while transient expression technologies are fast but unpredictable regarding product quality and lacking an optional path to subcloning. The STEP® vector technology introduced here combines the best properties of both methods. STEP® vectors contain a strong transcriptional cassette driving expression of a bicistronic mRNA. The gene-of-interest (GOI) is cloned upstream of a functionally impaired zeocin resistance gene (FI-Zeo) whose translation is coupled to that of the GOI through an IRES. Stable transfected cells surviving zeocin selection produce high levels of FI-Zeo and thus, high levels of the GOI-encoded protein. By using different spacers, the translational coupling efficiency and selection strength can be controlled allowing maximization of expression of any GOI. Production of laronidase and factor VII (FVII) is presented as examples of unrelated, difficult-to-express (DTE) proteins. First step is rapid generation of transfected pools with the STEP® vectors. All high expressing surviving pools showed high product quality homogeneity as did monoclonal cell lines obtained from the top pools. Up to 500 μg/mL laronidase was obtained with virtually identical glycosylation profile as reference product. For FVII, cell specific productivity of 0.45 pg/cell/day with 50 IU/μg protein matched highest reported levels of reference product even before process development. Taken together, STEP® vector technology is ideally suited for rapid, small to large-scale production of DTE proteins compared to traditional methods.

Dexmedetomidine Inhibits ASIC Activity via Activation of α2A Adrenergic Receptors in Rat Dorsal Root Ganglion Neurons.

Dexmedetomidine (DEX), a selective α2 adrenergic receptor (α2-AR) agonist, has been shown to have peripheral analgesic effects in a variety of pain conditions. However, the precise molecular mechanisms have not yet been fully elucidated. Acid sensing ion channels (ASICs) are the major player in pain associated with tissue acidosis. Given that both α2-ARs and ASICs exist in dorsal root ganglia (DRG) neurons, we therefore investigated the effects of DEX on the functional activity of ASICs. Herein, whole-cell patch-clamp recordings demonstrated that DEX suppressed ASIC-mediated and acid-evoked currents and action potentials in dissociated rat DRG neurons. DEX shifted downwards concentration-response curve to protons, with a decrease of 35.83 ± 3.91% in the maximal current response to pH 4.5. DEX-induced inhibition of ASIC currents was blocked by the α2A-AR antagonist BRL44408 in DRG neurons. DEX also inhibited ASIC3 currents in CHO cells co-expressing ASIC3 and α2A-ARs, but not in ASIC3 transfected CHO cells without α2A-ARs expression. DEX-induced inhibition of ASIC currents was mimicked by the protein kinase A inhibitor H-89, and blocked by intracellular application of the Gi/o protein inhibitor pertussis toxin and the cAMP analog 8-Br-cAMP. In addition, peripherally administration of DEX dose-dependently relieved nociceptive responses to intraplantar injection of acetic acid in rats through local α2A-ARs. Our results indicated that DEX inhibited the functional activity of ASICs via α2A-ARs and intracellular Gi/o proteins and cAMP/protein kinase A signaling pathway in rat DRG neurons, which was a novel potential mechanism that probably mediated peripheral analgesia of DEX.

High-level expression of the monomeric SARS-CoV-2 S protein RBD 320-537 in stably transfected CHO cells by the EEF1A1-based plasmid vector.

The spike (S) protein is one of the three proteins forming the coronaviruses' viral envelope. The S protein of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has a spatial structure similar to the S proteins of other mammalian coronaviruses, except for a unique receptor-binding domain (RBD), which is a significant inducer of host immune response. Recombinant SARS-CoV-2 RBD is widely used as a highly specific minimal antigen for serological tests. Correct exposure of antigenic determinants has a significant impact on the accuracy of such tests-the antigen has to be correctly folded, contain no potentially antigenic non-vertebrate glycans, and, preferably, should have a glycosylation pattern similar to the native S protein. Based on the previously developed p1.1 vector, containing the regulatory sequences of the Eukaryotic translation elongation factor 1 alpha gene (EEF1A1) from Chinese hamster, we created two expression constructs encoding SARS-CoV-2 RBD with C-terminal c-myc and polyhistidine tags. RBDv1 contained a native viral signal peptide, RBDv2 -human tPA signal peptide. We transfected a CHO DG44 cell line, selected stably transfected cells, and performed a few rounds of methotrexate-driven amplification of the genetic cassette in the genome. For the RBDv2 variant, a high-yield clonal producer cell line was obtained. We developed a simple purification scheme that consistently yielded up to 30 mg of RBD protein per liter of the simple shake flask cell culture. Purified proteins were analyzed by polyacrylamide gel electrophoresis in reducing and non-reducing conditions and gel filtration; for RBDv2 protein, the monomeric form content exceeded 90% for several series. Deglycosylation with PNGase F and mass spectrometry confirmed the presence of N-glycosylation. The antigen produced by the described technique is suitable for serological tests and subunit vaccine studies.

Biophysical Characterization of a Novel SCN5A Mutation Associated With an Atypical Phenotype of Atrial and Ventricular Arrhythmias and Sudden Death.

BackgroundSudden cardiac death (SCD) is an unexpected death that occurs within an hour of the onset of symptoms. Hereditary primary electrical disorders account for up to 1/3 of all SCD cases in younger individuals and include conditions such as catecholaminergic polymorphic ventricular tachycardia (CPVT). These disorders are caused by mutations in the genes encoding cardiac ion channels, hence they are known as cardiac channelopathies. We identified a novel variant, T1857I, in the C-terminus of Nav1.5 (SCN5A) linked to a family with a CPVT-like phenotype characterized by atrial tachy-arrhythmias and polymorphic ventricular ectopy occurring at rest and with adrenergic stimulation, and a strong family history of SCD.ObjectiveOur goal was to functionally characterize the novel Nav1.5 variant and determine a possible link between channel gating and clinical phenotype.MethodsWe first used electrocardiogram recordings to visualize the patient cardiac electrical properties. Then, we performed voltage-clamp of transiently transfected CHO cells. Lastly, we used the ventricular/atrial models to visualize gating defects on cardiac excitability.ResultsVoltage-dependences of both activation and inactivation were right-shifted, the overlap between activation and inactivation predicted increased window currents, the recovery from fast inactivation was slowed, there was no significant difference in late currents, and there was no difference in use-dependent inactivation. The O’Hara-Rudy model suggests ventricular after depolarizations and atrial Grandi-based model suggests a slight prolongation of atrial action potential duration.ConclusionWe conclude that T1857I likely causes a net gain-of-function in Nav1.5 gating, which may in turn lead to ventricular after depolarization, predisposing carriers to tachy-arrhythmias.