Chinese Hamster Ovary Research Articles

Mammalian perfusion cultivation at high L-Arginine concentration for efficient production of recombinant protein by increasing perfusion filter transmission

Cultivations of Chinese Hamster Ovary (CHO) cells in a perfusion setup were conducted in the presence of super physiological concentrations of L-Arginine to investigate the impact on transmission through the perfusion filter for production of a recombinant domain antibody. Our study revealed that the presence of L-Arginine within the range of 30–50 mM had a positive impact on transmission. However, the higher concentrations were found to have a negative correlation with cell viability, and an optimal concentration of approximately 40 mM was identified. The supplementation of L-Arginine improved overall cultivation performance and enhanced product quality attributes. As a result, our findings demonstrate that the supplementation of L-Arginine to mammalian perfusion cultivations stands as an effective method to address transmission issues, exerting a broad impact on process and production of recombinant proteins.

Replication of the Venezuelan Equine Encephalitis Vaccine from a Synthetic PCR Fragment.

There is no approved human vaccine for Venezuelan equine encephalitis (VEE), a life-threatening disease caused by the VEE virus (VEEV). In previous studies, plasmid DNA encoding the full-length RNA genome of the VEE V4020 vaccine was used for the preparation of experimental live virus VEE vaccines in the plasmid-transfected cell culture. Here, we used the high-fidelity polymerase chain reaction (PCR) to prepare synthetic, transcriptionally active PCR (TAP) fragments encoding the V4020 genome. TAP fragment initiated the replication of the V4020 live virus vaccine in TAP fragment-transfected cells. A transfection of less than 1 ug of TAP fragment resulted in the replication of the V4020 vaccine virus in CHO cells. We conclude that not only plasmid DNA but also synthetic PCR-generated DNA fragments can be used for the manufacturing of live vaccines for VEEV and, potentially, other viruses.

Truncated mini LRP1 transports cargo from luminal to basolateral side across the blood brain barrier

BackgroundThe most crucial area to focus on when thinking of novel pathways for drug delivery into the CNS is the blood brain barrier (BBB). A number of nanoparticulate formulations have been shown in earlier research to target receptors at the BBB and transport therapeutics into the CNS. However, no mechanism for CNS entrance and movement throughout the CNS parenchyma has been proposed yet. Here, the truncated mini low-density lipoprotein receptor-related protein 1 mLRP1_DIV* was presented as blood to brain transport carrier, exemplified by antibodies and immunoliposomes using a systematic approach to screen the receptor and its ligands’ route across endothelial cells in vitro.MethodsThe use of mLRP1_DIV* as liposomal carrier into the CNS was validated based on internalization and transport assays across an in vitro model of the BBB using hcMEC/D3 and bEnd.3 cells. Trafficking routes of mLRP1_DIV* and corresponding cargo across endothelial cells were analyzed using immunofluorescence. Modulation of γ-secretase activity by immunoliposomes loaded with the γ-secretase modulator BB25 was investigated in co-cultures of bEnd.3 mLRP1_DIV* cells and CHO cells overexpressing human amyloid precursor protein (APP) and presenilin 1 (PSEN1).ResultsWe showed that while expressed in vitro, mLRP1_DIV* transports both, antibodies and functionalized immunoliposomes from luminal to basolateral side across an in vitro model of the BBB, followed by their mLRP1_DIV* dependent release of the cargo. Importantly, functionalized liposomes loaded with the γ-secretase modulator BB25 were demonstrated to effectively reduce toxic Aß42 peptide levels after mLRP1_DIV* mediated transport across a co-cultured endothelial monolayer.ConclusionTogether, the data strongly suggest mLRP1_DIV* as a promising tool for drug delivery into the CNS, as it allows a straight transport of cargo from luminal to abluminal side across an endothelial monolayer and it’s release into brain parenchyma in vitro, where it exhibits its intended therapeutic effect.

Identification of pyrimidine structure-based compounds as allosteric ligands of the dopamine transporter as therapeutic agents for NeuroHIV.

The disruption of dopamine neurotransmission by the HIV-1 Transactivator of transcription (Tat) during HIV-1 infection has been linked to the development of neurocognitive disorders, even under combined antiretroviral therapy (cART) treatment. We have demonstrated that SRI-32742, a novel allosteric modulator of dopamine (DA) transporter (DAT), attenuates cocaine- and Tat-binding to DAT, alleviates Tat-induced cognitive deficits and potentiation of cocaine reward in inducible Tat transgenic mice. The current study determined the in vitro pharmacological profile of SRI-32743 and its optimized second-generation analogs and their effects as allosteric modulators. Through structure-activity relationship studies of SRI-32743, 170 compounds were synthesized and evaluated for their ability to modulate DAT function. We identified 21 analogs as atypical competitors of DAT (Emax {less than or equal to}60%). Four compounds, SRI-46564, SRI-47056, SRI-46286 and SRI-47867, displayed IC50 values for [3H]DA uptake inhibition from 9.33 {plus minus} 0.50 to 0.96 {plus minus} 0.05 µM and from 3.96 {plus minus} 1.36 to 1.29 {plus minus} 0.19 for DAT binding, respectively. The four analogs also displayed high potency at two different concentrations (0.5 nM and 0.05 nM) to attenuate Tat-induced inhibition of [3H]DA uptake and cocaine-mediated dissociation of [3H]WIN35,428 binding in CHO cells expressing hDAT, suggesting that the effects occur through an allosteric mechanism. In further ex vivo studies using Fast-Scan Cyclic Voltammetry, we demonstrated that the analogs do not disrupt the baseline phasic-like DA release. These findings provide a new insight into the potential for development of novel therapeutic agents to attenuate DAT-Tat interactions to normalize DA neurotransmission in NeuroHIV. Significance Statement The allosteric inhibition of the dopamine (DA) transporter by the HIV-1 Transactivator of transcription (Tat) disrupts dopamine homeostasis, leading to HIV-associated neurocognitive disorders (HANDs). Analogs of SRI-32743, a novel allosteric modulator of the Tat-DAT interaction, were evaluated in the current study and characterized as atypical ligands of DA uptake. Four novel lead compounds demonstrated high potency to attenuate Tat-induced inhibition of hDAT-mediated DA uptake in an allosteric modulatory manner with no effects on the dynamics of DA uptake-release in DAT.

Selective Transfection of a Transferrin Receptor-Expressing Cell Line with DNA-Lipid Nanoparticles.

Despite considerable progress in using lipid nanoparticle (LNP) vehicles for gene delivery, achieving selective transfection of specific cell types remains a significant challenge, hindering the advancement of new gene or gene-editing therapies. Although LNPs have been equipped with ligands aimed at targeting specific cellular receptors, achieving complete selectivity continues to be elusive. The exact reasons for this limited selectivity are not fully understood, as cell targeting involves a complex interplay of various cellular factors. Assessing how much ligand/receptor binding contributes to selectivity is challenging due to these additional influencing factors. Nonetheless, such data are important for developing new nanocarriers and setting realistic expectations for selectivity. Here, we have quantified the selective, targeted transfection using two uniquely engineered cell lines that eliminate unpredictable and interfering cellular influences. We have compared the targeted transfection of Chinese ovary hamster (CHO) cells engineered to express the human transferrin receptor 1 (hTfR1), CHO-TRVb-hTfR1, with CHO cells that completely lack any transferrin receptor, CHO-TRVb-neo cells (negative control). Thus, the two cell lines differ only in the presence/absence of hTfR1. The transfection was performed with pDNA-encapsulating LNPs equipped with the DT7 peptide ligand that specifically binds to hTfR1 and enables targeted transfection. The LNP's pDNA encoded for the monomeric GreenLantern (mGL) reporter protein, whose fluorescence was used to quantify transfection. We report a novel LNP composition designed to achieve an optimal particle size and ζ-potential, efficient pDNA encapsulation, hTfR1-targeting capability, and sufficient polyethylene glycol sheltering to minimize random cell targeting. The transfection efficiency was quantified in both cell lines separately through flow cytometry based on the expression of the fluorescent gene product. Our results demonstrated an LNP dose-dependent mGL expression, with a 5-fold preference for the CHO-TRVb-hTfR1 when compared to CHO-TRVb-neo. In another experiment, when both cell lines were mixed at a 1:1 ratio, the DT7-decorated LNP achieved a 3-fold higher transfection of the CHO-TRVb-hTfR1 over the CHO-TRVb-neo cells. Based on the low-level transfection of the CHO-TRVb-neo cells in both experiments, our results suggest that 17-25% of the transfection occurred in a nonspecific manner. The observed transfection selectivity for the CHO-TRVb-hTfR1 cells was based entirely on the hTfR1/DT7 interaction. This work showed that the platform of two engineered cell lines which differ only in the hTfR1 can greatly facilitate the development of LNPs with hTfR1-targeting ligands.

Quantitation of Host Cell Proteins by Capillary LC/IMS/MS/MS in Combination with Rapid Digestion on Immobilized Trypsin Column Under Native Conditions.

Host cell protein (HCP) impurities are considered a critical quality attribute of biopharmaceuticals because of their potential to compromise safety and efficacy, and LC/MS-based analytical methods have been developed to identify and quantify individual proteins instead of employing enzyme-linked immunosorbent assay to assess total HCP levels. Native digestion enables highly sensitive detection of HCPs but requires overnight incubation to generate peptides, limiting the throughput of sample preparation. In this study, we developed an approach employing native digestion on a trypsin-immobilized column to improve the sensitivity and throughput. We examined suitable databases for the identification of HCPs derived from Chinese hamster ovary (CHO) cells and selected RefSeq's Chinese Hamster as the optimal database. Then, we investigated methods to identify HCPs with greater efficiency than that of denatured in-solution digestion. Native in-column digestion not only reduced the digestion time from overnight to 10 min but also increased the number of quantified HCPs from 154 to 226. In addition to this rapid digestion methodology, we developed high-throughput LC/MS/MS with a monolithic silica column and parallel reaction monitoring-parallel accumulation-serial fragmentation. The optimized system was validated with synthetic peptides derived from high-risk HCPs, confirming excellent linearity, precision, accuracy, and low limit of detection (LOD) and limit of quantification (LOQ) (1-3 ppm). The optimized digestion and analysis method enabled high-throughput quantification of HCPs, and is expected to be useful for quality control and characterization of HCPs in antibody drugs.

Nanoencapsulation of quinoa oil enhanced the antioxidant potential and inhibited digestive enzymes

Quinoa oil is rich in unsaturated fatty acids and vitamin E, but its instability limits its application in food, pharmaceutical, and cosmetic products. Nanoencapsulation emerges as a promising strategy to promote water dispersibility, preserve and enhance functional properties, and increase the bioavailability of bioactive compounds. This study encapsulated quinoa oil through O/W emulsification, using porcine gelatin (OG) and isolated whey protein (OWG) as encapsulating agents. The particles were characterized by different physical and chemical methods and evaluated in vitro for cytotoxicity using Chinese hamster ovary (CHO) cells, human hepatocarcinoma cells (HepG2) and epithelial cells, and bioactive potential through the determination of Total Antioxidant Capacity (CAT) (acidic and neutral media) and iron chelation, and inhibition of digestive enzymes (α-amylase and amyloglucosidase). OG and OWG particles presented smooth surfaces, with an average size between 161 ± 7 and 264 ± 6 nm, with a polydispersity index of 0.11 ± 0.03 and 0.130 ± 0.04, encapsulation efficiency of 74 ± 1.47 % and 83 ± 2.92 %, and water dispersibility >70 %, respectively. Free and nanoencapsulated quinoa oil did not show cytotoxic effects (cell viability >70 %). Nanoencapsulation promoted the enhancement of the antioxidant activity of quinoa oil in the range of 50–63 % in a neutral medium and 96–153 % in an acidic medium than free oil (p < 0.05). OG and OWG also enhanced the inhibition of the enzymes α-amylase (by 5–7 %) and amyloglucosidase (6–9 times more) than free oil (p < 0.05). The results showed that nanoencapsulation increased the potential for quinoa oil application, enabling the development of innovative products.

Quantification of the interaction forces between dengue virus and dopamine type-2 receptor using optical tweezers

BackgroundDengue virus (DENV) causes the most significant mosquito-borne viral disease with a wide spectrum of clinical manifestation, including neurological symptoms associated with lethal dengue diseases. Dopamine receptors are expressed in central nervous system, and dopamine antagonists have been reported to exhibit antiviral activity against DENV infection in vivo and in vitro. Although identification of host-cell receptor is critical to understand dengue neuropathogenesis and neurotropism, the involvement of dopamine receptors in DENV infection remains unclear.ResultsWe exploited the sensitivity and precision of force spectroscopy to address whether dopamine type-2 receptors (D2R) directly interact with DENV particles at the first step of infection. Using optical tweezers, we quantified and characterized DENV binding to D2R expressed on Chinese hamster ovary (CHO) cells. Our finding suggested that the binding was D2R- and DENV-dependent, and that the binding force was in the range of 50–60 pN. We showed that dopamine antagonists prochlorperazine (PCZ) and trifluoperazine (TFP), previously reported to inhibit dengue infection, interrupt the DENV-D2R specific binding.ConclusionsThis study demonstrates that D2R could specifically recognize DENV particles and function as an attachment factor on cell surfaces for DENV. We propose D2R as a host receptor for DENV and as a potential therapeutic target for anti-DENV drugs.Graphical abstract

A novel approach for perfusion process design based on a "Grey-Box" kinetic model.

Perfusion cell-culture mode has caught industrial interest in the field of biomanufacturing in recent years. Thanks to new technology, perfusion-culture processes can support higher cell densities, higher productivities and longer process times. However, due to the inherent operational complexity and high running costs, the development and design of perfusion-culture processes remain challenging. Here, we present a model-based approach to design optimized perfusion cultures of Chinese Hamster Ovary cells. Initially, four batches of bench-top reactor continuous-perfusion-culture data were used to fit the model parameters. Then, we proposed the model-based process design approach, aiming to quickly find out the "theoretically optimal" operational parameters combinations (perfusion rate and the proportion of feed medium in perfusion medium) which could achieve the target steady-state VCD while minimizing both medium cost and perfusion rate during steady state. Meanwhile, we proposed a model-based dynamic operational parameters-adjustment strategy to address the issue of cell-growth inhibition due to the high osmolality of concentrated perfusion medium. In addition, we employed a dynamic feedback control method to aid this strategy in preventing potential nutrient depletion scenarios. Finally, we test the feasibility of the model-based process design approach in both shake flask semi-perfusion culture (targeted at 5 × 107 cells/ml) and bench-top reactor continuous perfusion culture (targeted at 1.1 × 108 cells/ml). This approach significantly reduces the number of experiments needed for process design and development, thereby accelerating the advancement of perfusion-mode cell-culture processes.

Novel and effective screening system for recombinant protein production in CHO cells

At present, biopharmaceuticals have received extensive attention from the society, among which recombinant proteins have a good growth trend and a large market share. Chinese hamster ovary (CHO) cells are the preferred mammalian system to produce glycosylated recombinant protein drugs. A highly efficient and stable cell screening method needs to be developed to obtain more and useful recombinant proteins. Limited dilution method, cell sorting, and semi-solid medium screening are currently the commonly used cell cloning methods. These methods are time-consuming and labor-intensive, and they have the disadvantage of low clone survival rate. Here, a method based on semi-solid medium was developed to screen out high-yielding and stable cell line within 3 weeks to improve the screening efficiency. The semi-solid medium was combined with an expression vector containing red fluorescent protein (RFP) for early cell line development. In accordance with the fluorescence intensity of RFP, the expression of upstream target gene could be indicated, and the fluorescence intensity was in direct proportion to the expression of upstream target gene. In conclusion, semi-solid medium combined with bicistronic expression vector provides an efficient method for screening stable and highly expressed cell lines.

FcRider: a recombinant Fc nanoparticle with endogenous adjuvant activities for hybrid immunization.

Active immunization (vaccination) induces long-lasting immunity with memory, which takes weeks to months to develop. Passive immunization (transfer of neutralizing antibodies) provides immediate protection, yet with high cost and effects being comparatively short-lived. No currently approved adjuvants are compatible with formulations to combine active and passive immunizations, not to mention their huge disparities in administration routes and dosage. To solve this, we engineered the Fc fragment of human IgG1 into a hexamer nanoparticle and expressed its afucosylated form in Fut8-/- CHO cells, naming it "FcRider." FcRider is highly soluble with long-term stability, easily produced at high levels equivalent to those of therapeutic antibodies, and is amenable to conventional antibody purification schemes. Most importantly, FcRider possesses endogenous adjuvant activities. Using SWHEL B cell receptor (BCR) transgenic mice, we found that HEL-FcRider induced GL7+ germinal center B cells and HEL-specific IgG. Similarly, immunizing mice with UFO-BG-FcRider, a fusion containing the stabilized human immunodeficiency virus-1 (HIV-1) Env protein as immunogen, promoted somatic hypermutation and generation of long CDR3 of the IgG heavy chains. Intramuscular injection of (Fba+Met6)3-FcRider, a fusion with two peptide epitopes from Candida albicans cell surface, stimulated strong antigen-specific IgG titers. In three different models, we showed that afucosylated FcRider functions as a multivalent immunogen displayer and stimulates antigen-specific B cells without any exogenous adjuvant. As an antibody derivative, afucosylated FcRider could be a novel platform combining vaccines and therapeutic antibodies, integrating active and passive immunizations into single-modality "hybrid immunization" to provide complete and long-lasting protection against infections, and may open new avenues in cancer immunotherapy as well.

Enhancing the Antibody Production Efficiency of Chinese Hamster Ovary Cells through Improvement of Disulfide Bond Folding Ability and Apoptosis Resistance.

The complex structure of monoclonal antibodies (mAbs) expressed in Chinese hamster ovary (CHO) cells may result in the accumulation of unfolded proteins, triggering endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). If the protein folding ability cannot maintain ER homeostasis, the cell will shut down protein translation and ultimately induce apoptosis. We co-overexpressed HsQSOX1b and survivin proteins in the antibody-producing cell line CHO-PAb to obtain a new cell line, CHO-PAb-QS. Compared with CHO-PAb cells, the survival time of CHO-PAb-QS cells in batch culture was extended by 2 days, and the antibody accumulation and productivity were increased by 52% and 45%, respectively. The proportion of (HC-LC)2 was approximately doubled in the CHO-PAb-QS cells, which adapted to the accelerated disulfide bond folding capacity by upregulating the UPR's strength and increasing the ER content. The results of the apoptosis assays indicated that the CHO-PAb-QS cell line exhibited more excellent resistance to apoptosis induced by ER stress. Finally, CHO-PAb-QS cells exhibited mild oxidative stress but did not significantly alter the redox status. This study demonstrated that strategies based on HsQSOX1b and survivin co-overexpression could facilitate protein disulfide bond folding and anti-apoptosis ability, enhancing antibody production efficiency in CHO cell lines.

Analysis of magnetic resonance contrast agent entrapment following reversible electroporation in vitro.

Administering gadolinium-based contrast agent before electroporation allows the contrast agent to enter the cells and enables MRI assessment of reversibly electroporated regions. The aim of this study was evaluation of contrast agent entrapment in Chinese hamster ovary (CHO) cells and comparison of these results with those determined by standard in vitro methods for assessing cell membrane permeability, cell membrane integrity and cell survival following electroporation. Cell membrane permeabilization and cell membrane integrity experiments were performed using YO-PRO-1 dye and propidium iodide, respectively. Cell survival experiments were performed by assessing metabolic activity of cells using MTS assay. The entrapment of gadolinium-based contrast agent gadobutrol inside the cells was evaluated using T1 relaxometry of cell suspensions 25 min and 24 h after electroporation and confirmed by inductively coupled plasma mass spectrometry. Contrast agent was detected 25 min and 24 h after the delivery of electric pulses in cells that were reversibly electroporated. In addition, contrast agent was present in irreversibly electroporated cells 25 min after the delivery of electric pulses but was no longer detected in irreversibly electroporated cells after 24 h. Inductively coupled plasma mass spectrometry showed a proportional decrease in gadolinium content per cell with shortening of T1 relaxation time (R 2 = 0.88 and p = 0.0191). Our results demonstrate that the contrast agent is entrapped in cells exposed to reversible electroporation but exits from cells exposed to irreversible electroporation within 24 h, thus confirming the hypothesis on which detection experiments in vivo were based.

A thiourea-bridged 99mTc(CO)3-dipicolylamine-2-nitroimidazole complex for targeting tumor hypoxia: Utilizing metabolizable thiourea-bridge to improve pharmacokinetics.

The 2-nitroimidazole based 99mTc-radiopharmaceuticals are widely explored for imaging tumor hypoxia. Radiopharmaceuticals for targeting hypoxia are often lipophilic and therefore, show significant uptake in liver and other vital organs. In this context, lipophilic radiopharmaceuticals with design features enabling faster clearance from liver may be more desirable. A dipicolylamine-NCS bifunctional chelator that could generate a thiourea-bridge up on conjugation to primary amine bearing molecule was used to synthesize a 2-nitroimidazole-dipicolyl amine ligand for radiolabeling with 99mTc(CO)3 core. Corresponding Re(CO)3-analogue was prepared to establish the structure of 2-nitroimidazole-99mTc(CO)3 complex prepared in trace level. The 2-nitroimidazole-99mTc(CO)3 complex showed a hypoxic to normoxic ratio of ~2.5 in CHO cells at 3 h. In vivo, the complex showed accumulation and retention in tumor with high tumor to blood and tumor to muscle ratio. The study demonstrated the utility of metabolizable thiourea-bridge in 2-nitroimidazole-99mTc(CO)3 complex in inducing faster clearance of the radiotracer from liver. The dipicolylamine-NCS bifunctional chelator reported herein can also be used for radiolabeling other class of target specific molecules with 99mTc(CO)3 core.

Safety, reactogenicity, and immunogenicity of ZR-202-CoV and ZR-202a-CoV recombinant vaccines compared with ComirnatyⓇ: A randomized, observer-blind, controlled, phase 1 study

ZR-202-CoV and ZR-202a-CoV are novel recombinant vaccines containing 25 µg of the prototype (Wuhan strain) or B.1.351 strain (Beta variant) SARS-CoV-2 S-protein expressed in CHO cells, respectively, adjuvanted with Al(OH)3 and CpG-ODN. We assessed their safety and immunogenicity in this Phase I, randomized, observer-blind, controlled study in Mali. Sixty healthy 18-55-year-old adults randomized 1:1:1 received two doses of ZR-202-CoV, ZR-202a-CoV, or ComirnatyⓇ 28 days apart. Primary outcome measures were solicited and unsolicited adverse events (AEs) including AESI (Adverse Events of Special Interest); secondary outcome was immunogenicity measured as SARS-CoV-2 specific neutralizing antibodies. Participants were followed up for 1 year. Injection site pain and headache were the most frequent solicited local and systemic AEs, respectively. No unsolicited AEs or SAEs related to vaccination were reported during the study period. Although most participants had detectable neutralizing antibodies at baseline robust immune responses were observed in all vaccine groups after the first dose with no further increase after the second dose. Cross-neutralizing antibody responses against Beta, Delta, and Omicron BA.5 variants were similar in magnitude after ZR-202-CoV, ZR-202a-CoV and ComirnatyⓇ. Similar reactogenicity and immunogenicity profiles of ZR-202-CoV, ZR-202a-CoV and ComirnatyⓇ support further clinical investigation in a wider population.

A neural ordinary differential equation model for predicting the growth of Chinese Hamster Ovary cell in a bioreactor system

A neural ordinary differential equation model for predicting the growth of Chinese Hamster Ovary cell in a bioreactor system

FABP4 Enhances Lipidic and Fibrotic Cardiac Structural and Ca2+ Dynamic Changes.

Adipocyte FABP4 (fatty acid-binding protein 4) is augmented in the epicardial stroma of patients with long-standing persistent atrial fibrillation. Because this molecule is released mainly by adipocytes, our objective was to study its role in atrial cardiomyopathy, focusing our attention on fibrosis, metabolism, and electrophysiological changes. These results might clarify the role of adiposity as a mediator of atrial cardiomyopathy. We used several preclinical cellular models, epicardial and subcutaneous stroma primary cell cultures from patients undergoing open heart surgery, human atrial fibroblasts, atrial cardiomyocytes derived from human induced pluripotent stem cells and isolated from adult mice, and Nav1.5 transfected Chinese hamster ovary cells. Fibrosis, glucose, mitochondrial and adipogenesis activity, gene expression, and proteomics were determined by wound healing, enzymatic, colorimetric, fluorescence assays, real-time quantitative polymerase chain reaction, and TripleTOF proteomics. Molecular changes were analyzed by Raman confocal microspectroscopy, calcium dynamics by confocal microscopy, and ion currents by patch clamp. Epicardial, subcutaneous, and atrial fibroblasts and cardiomyocytes were incubated with FABP4 at 100 ng/mL. Our results showed that FABP4 induced fibrosis, glucose metabolism, and lipid accumulation on epicardial and subcutaneous stroma cells and atrial fibroblasts. Besides, it modified lipid content and calcium dynamics in atrial cardiomyocytes without effects on INa. FABP4 exerts fibrotic and metabolic changes on epicardial stroma and modifies lipid content and calcium dynamic on atrial cardiomyocytes. These results suggest its possible role as an atrial cardiomyopathy mediator.

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.

Strategies for improving expression of recombinant human chorionic gonadotropin in Chinese Hamster Ovary (CHO) cells

Optimizations of the gene expression cassette combined with the selection of an appropriate signal peptide are important factors that must be considered to enhance heterologous protein expression in Chinese Hamster Ovary (CHO) cells. In this study, we investigated the effectiveness of different signal peptides on the production of recombinant human chorionic gonadotropin (r-hCG) in CHO-K1 cells. Four optimized expression constructs containing four promising signal peptides were stably transfected into CHO-K1 cells. The generated CHO-K1 stable pool was then evaluated for r-hCG protein production. Interestingly, human serum albumin and human interleukin-2 signal peptides exhibited relatively greater extracellular secretion of the r-hCG with an average yield of (16.59 ± 0.02 μg/ml) and (14.80 ± 0.13 μg/ml) respectively compared to the native and murine IgGκ light chain signal peptides. The stably transfected CHO pool was further used as the cell substrate to develop an optimized upstream process followed by a downstream phase of the r-hCG. Finally, the biological activity of the purified r-hCG was assessed using in vitro bioassays. The combined data highlight that the choice of signal peptide can be imperative to ensure an optimal secretion of a recombinant protein in CHO cells. In addition, the stable pool technology was a viable approach for the production of biologically active r-hCG at a research scale with acceptable bioprocess performances and consistent product quality.

BMP2 Binds Non-Specifically to PEG-Passivated Biomaterials and Induces pSMAD 1/5/9 Signalling.

Biomaterials are widely employed across diverse biomedical applications and represent an attractive strategy to explore how extracellular matrix components influence cellular response. In this study, the previously developed streptavidin platforms is aimed to use to investigate the role of glycosaminoglycans (GAGs) in bone morphogenetic protein 2 (BMP2) signaling. However, it is observed that the interpretation of findings is skewed due to the GAG-unrelated, non-specific binding of BMP2 on components of biomaterials. Non-specific adsorption of proteins is a recurrent and challenging issue for biomaterial studies. Despite the initial incorporation of anti-fouling polyethylene glycol (PEG) chains within biomaterials, the residual non-specific BMP2 adsorption still triggered BMP2 signaling within the same range as conditions of interest. The various options are explored to prevent BMP2 non-specific adsorption and a successful blocking condition involving a combination of bovine serum albumin and trehalose are identified. Furthermore, the effect of this blocking step improved when using gold platforms instead of glass, particularly with Chinese hamster ovary (CHO) cells. With this specific example, it is suggested that non-specific adsorption of BMPs on biomaterials may be a general concern - often undetected by classical surface-sensitive techniques - that needs to be addressed to better interpret cellular responses.