Improvement strategies for transient gene expression in mammalian cells.

Mammalian cells are suitable hosts for producing recombinant therapeutic proteins, with Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells being the most commonly used cell lines. Mammalian cell expression system includes stable and transient gene expression (TGE) system, with the TGE system having the advantages of short cycles and simple operation. By optimizing the TGE system, the expression of recombinant proteins has been significantly improved. Here, the TGE system and the detailed and up-to-date improvement strategies of mammalian cells, including cell line, expression vector, culture media, culture processes, transfection conditions, and co-expression of helper genes, are reviewed. KEY POINTS: • Detailed improvement strategies of transient gene expression system of mammalian cells are reviewed • The composition of transient expression system of mammalian cell are summarized • Proposed optimization prospects for transient gene expression systems.

Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development.

Coxsackievirus A16 (CVA16) is one of the primary pathogens that causes hand, foot, and mouth disease (HFMD) in young children. In previous studies, CVA16 vaccine development has encountered several challenges, such as inefficient replication of the CVA16 virus in present culture systems, the induction of only mild neutralizing antibody titers, and neutralizing antibodies induced by certain vaccine candidates that are unable to protect against CVA16 viral challenge. In this study, we constructed a DNA-launched CVA16 infectious clone (CVA16ic) based on the genomic sequence of the CVA16 N5079 strain to minimize interference from viral quasispecies. The biochemical properties of this CVA16ic strain were similar to those of its parental strain. Serum-free HEK293A suspension cells, which produced higher virus titers than Vero cells, were demonstrated to improve CVA16 production yields. In addition, our study showed that inactivated EV-A71 antigens could enhance the immunogenicity of inactivated CVA16 mature/full particles (F-particles), suggesting that a bivalent CVA16 and EV-A71 vaccine may be an effective strategy for CVA16 vaccine development. These findings are expected to provide novel strategies and accelerate the development of bivalent HFMD vaccines.

Therapeutic delivery of recombinant glucocerebrosidase enzyme-containing extracellular vesicles to human cells from Gaucher disease patients.

BACKGROUND: Gaucher disease (GD) is one of the most common types of lysosomal storage diseases (LSDs) caused by pathogenic variants of lysosomal ß-glucocerebrosidase gene (GBA1), resulting in the impairment of Glucocerebrosidase (GCase) enzyme function and the accumulation of a glycolipid substrate, glucosylceramide (GlcCer) within lysosomes. Current therapeutic approaches such as enzyme replacement therapy and substrate reduction therapy cannot fully rescue GD pathologies, especially neurological symptoms. Meanwhile, delivery of lysosomal enzymes to the endocytic compartment of affected human cells is a promising strategy for treating neuropathic LSDs. RESULT: Here, we describe a novel approach to restore GCase enzyme in cells from neuropathic GD patients by producing extracellular vesicle (EVs)-containing GCase from cells overexpressing GBA1 gene. Lentiviral vectors containing modified GBA1 were introduced into HEK293T cells to produce a stable cell line that provides a sustainable source of functional GCase enzyme. The GBA1-overexpressing cells released EV-containing GCase enzyme, that is capable of entering into and localizing in the endocytic compartment of recipient cells, including THP-1 macrophage, SH-SY5Y neuroblastoma, and macrophages and neurons derived from induced pluripotent stem cells (iPSCs) of neuropathic GD patients. Importantly, the recipient cells exhibit higher GCase enzyme activity. CONCLUSION: This study presents a promising therapeutic strategy to treat severe types of LSDs. It involves delivering lysosomal enzymes to the endocytic compartment of human cells affected by conditions such as GDs with neurological symptoms, as well as potentially other neurological disorders impacting lysosomes.

EZH2 specifically regulates ISL1 during embryonic urinary tract formation.

Isl1 has been described as an embryonic master control gene expressed in the pericloacal mesenchyme. Deletion of Isl1 from the genital mesenchyme in mice leads to an ectopic urethral opening and epispadias-like phenotype. Using genome wide association methods, we identified ISL1 as the key susceptibility gene for classic bladder exstrophy (CBE), comprising epispadias and exstrophy of the urinary bladder. The most significant marker (rs6874700) identified in our recent GWAS meta-analysis achieved a p value of 1.48 × 10- 24 within the ISL1 region. In silico analysis of rs6874700 and all other genome-wide significant markers in Linkage Disequilibrium (LD) with rs6874700 (D' = 1.0; R2 > 0.90) revealed marker rs2303751 (p value 8.12 × 10- 20) as the marker with the highest regulatory effect predicted. Here, we describe a novel 1.2 kb intragenic promoter residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site, which is located in the reverse DNA strand and harbors a binding site for EZH2 at the exact region of marker rs2303751. We show, that EZH2 silencing in HEK cells reduces ISL1 expression. We show that ezh2-/- knockout (KO) zebrafish larvae display tissues specificity of ISL1 regulation with reduced expression of Isl1 in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2-/- ko zebrafish Tg(wt1ß:eGFP) reporter lines. Our study shows, that Ezh2 is a key regulator of Isl1 during urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.

Characterization of residual microRNAs in AAV vector batches produced in HEK293 mammalian cells and Sf9 insect cells.

With more than 130 clinical trials and 8 approved gene therapy products, adeno-associated virus (AAV) stands as one of the most popular vehicles to deliver therapeutic DNA in vivo. One critical quality attribute analyzed in AAV batches is the presence of residual DNA, as it could pose genotoxic risks or induce immune responses. Surprisingly, the presence of small cell-derived RNAs, such as microRNAs (miRNAs), has not been investigated previously. In this study, we examined the presence of miRNAs in purified AAV batches produced in mammalian or in insect cells. Our findings revealed that miRNAs were present in all batches, regardless of the production cell line or capsid serotype (2 and 8). Quantitative assays indicated that miRNAs were co-purified with the recombinant AAV particles in a proportion correlated with their abundance in the production cells. The level of residual miRNAs was reduced via an immunoaffinity chromatography purification process including a tangential flow filtration step or by RNase treatment, suggesting that most miRNA contaminants are likely non-encapsidated. In summary, we demonstrate, for the first time, that miRNAs are co-purified with AAV particles. Further investigations are required to determine whether these miRNAs could interfere with the safety or efficacy of AAV-mediated gene therapy.

Factors affecting rAAV titers during triple-plasmid transient transfection in HEK-293 cells.

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficiency utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhance fully packaged rAAV particle yield, we discovered cell-line-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.

Expression Level of Cardiac Ryanodine Receptors Dictates Properties of Cardiac Ca2+-Induced Ca2+ Release.

The type 2 ryanodine receptor (RyR2) is the major Ca2+ release channel required for Ca2+-induced Ca2+ release (CICR) and cardiac excitation-contraction coupling. The cluster organization of RyR2 at the dyad is critical for efficient CICR. Despite its central role in cardiac Ca2+ signaling, the mechanisms that control CICR are not fully understood. As a single RyR2 Ca2+ flux dictates local CICR that underlies Ca2+ spark, RyR2 density in a cluster and therefore the distance between RyR2s should have a profound impact on local CICR. Here, we studied the effect of RyR2 expression level ([RyR2]) on CICR activation, termination, and amplitude. The ER-targeted Ca2+ sensor RCEPIA-1er was used to directly measure the endoplasmic reticulum (ER) [Ca2+] (Ca2+]ER) in T-Rex-293 SERCA2a stable cell line expressing human RyR2. Cells co-expressing RyR2 and SERCA2a produced periodic [Ca2+]ER depletions in the form of spontaneous Ca2+ waves due to propagating CICR. For each studied cell, [Ca2+]ER at which Ca2+ waves are activated and terminated was analyzed as a function of [RyR2]. CICR parameters, such as [Ca2+]ER activation, termination, and amplitude, were inversely proportional to [RyR2] at low-intermediate levels. Increasing sensitivity of RyR2 to cytosolic Ca2+ lowered [Ca2+]ER at which CICR is activated and terminated. Decreasing the sensitivity of RyR2 to cytosolic Ca2+ had the opposite effect on CICR. These results suggest that RyR2 density in the release cluster should have a significant impact on local CICR activation and termination. Since SR Ca2+ load is evenly distributed throughout the SR network, clusters with higher RyR2 density would have a higher probability to initiate spontaneous CICR.

Exploring P2X receptor activity: A journey from cellular impact to electrophysiological profiling.

The development of in vitro pharmacological assays relies on creating genetically modified cell lines that overexpress the target protein of interest. However, the choice of the host cell line can significantly impact the experimental outcomes. This study explores the functional characterization of P2X7 and P2X4 receptor modulators through cellular assays and advanced electrophysiological techniques. The influence of different host cell lines (HEK-293, HEK-293FT, and 1321N1) on the activity of reference agonists and antagonists targeting human and murine P2X4 and P2X7 receptors was systematically investigated, highlighting the significant impact of the host cell on experimental results. The 1321N1 cell line was identified as the preferred host cell line when investigating the human P2X4 receptor due to more consistent agonist activities, antagonist potencies, and a more stable assay signal window. Furthermore, a patch-clamp protocol that allows for the repetitive recording of ATP-mediated inward currents from isolated human CD4+ T-cells was established, revealing that both P2X7 and P2X4 receptors are crucial for immune cell regulation, positioning them as promising therapeutic targets for managing inflammatory disorders.

Functional Characterization Supports Multiple Evolutionary Origins of Pheromone Receptors in Bark Beetles.

Chemical communication using pheromones is thought to have contributed to the diversification and speciation of insects. The species-specific pheromones are detected by specialized pheromone receptors (PRs). Whereas the evolution and function of PRs have been extensively studied in Lepidoptera, only a few PRs have been identified in beetles, which limits our understanding of their evolutionary histories and physiological functions. To shed light on these questions, we aimed to functionally characterize potential PRs in the spruce bark beetle Ips typographus ("Ityp") and explore their evolutionary origins and molecular interactions with ligands. Males of this species release an aggregation pheromone comprising 2-methyl-3-buten-2-ol and (4S)-cis-verbenol, which attracts both sexes to attacked trees. Using two systems for functional characterization, we show that the highly expressed odorant receptor (OR) ItypOR41 responds specifically to (4S)-cis-verbenol, with structurally similar compounds eliciting minor responses. We next targeted the closely related ItypOR40 and ItypOR45. Whereas ItypOR40 was unresponsive, ItypOR45 showed an overlapping response profile with ItypOR41, but a broader tuning. Our phylogenetic analysis shows that these ORs are present in a different OR clade as compared to all other known beetle PRs, suggesting multiple evolutionary origins of PRs in bark beetles. Next, using computational analyses and experimental validation, we reveal two amino acid residues (Gln179 and Trp310) that are important for ligand binding and pheromone specificity of ItypOR41 for (4S)-cis-verbenol, possibly via hydrogen bonding to Gln179. Collectively, our results shed new light on the origins, specificity, and ligand binding mechanisms of PRs in beetles.

Enhanced ER protein processing gene expression increases rAAV yield and full capsid ratio in HEK293 cells.

The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies: XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS: • Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells • The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly • The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions.

In vitro assessment of the risk of ABCB1-mediated drug-drug interaction between rivaroxaban and tacrolimus in human embryonic kidney 293 recombinant cell lines.

Background: In lung transplant patients, direct oral anticoagulants are often taken in combination with immunosuppressive drugs such as tacrolimus. Since tacrolimus is a substrate and inhibitor of the efflux protein ABCB1, also transporting direct oral anticoagulants, a possible drug-drug interaction mediated by competition for this transporter needs to be investigated. Objectives: To determine the in vitro effect of tacrolimus on ABCB1-mediated rivaroxaban transport in order to support clinician practice. Methods: Recombinant cell line models, based on human embryonic kidney 293 cells, were generated by a stable transfection process to overexpress ABCB1 or not (control cells). The impact of tacrolimus on ABCB1-mediated rivaroxaban transport was assessed by accumulation experiments. Results: ABCB1 expression decreased the cellular accumulation of rivaroxaban and tacrolimus at their respective clinically relevant concentrations when compared with control cells. This confirms the involvement of ABCB1 in the active transport of tacrolimus and rivaroxaban. However, tacrolimus had no significant influence on rivaroxaban disposition at those clinically relevant concentrations. Conclusion: Our study does not provide evidence for a possible interaction between tacrolimus and rivaroxaban when used together in practice.

α-Lipoic acid increases the viability of nephrocytes and elevates sulfane sulfur level in plasma of patients with chronic kidney disease.

BACKGROUND: Kidney diseases are a major global health problem affecting millions of people. Despite this, there is as yet no effective drug therapy improving outcome in patients with renal disease. The aim of this study was to examine the nephroprotective effect of α-lipoic acid (ALA) in vitro and to examine the effect of ALA administered in vivo on the production of reactive sulfur species (RSS), including hydrogen sulfide (H2S) and compounds containing sulfane sulfur. METHODS: The effect of ALA was studied in vitro by determining the viability of human embryonic kidney cells (HEK293) in normoxic and hypoxic conditions as well as in vivo in two groups of chronic kidney disease (CKD) patients: non-dialyzed (ND) and undergoing continuous ambulatory peritoneal dialysis (PD) after 30 days of ALA supplementation. RESULTS: The results revealed that the viability of HEK293 cells was significantly decreased by hypoxic conditions, while ALA administered during hypoxia increased the viability to the level observed in normoxic conditions. Studies performed in plasma of CKD patients after ALA supplementation suggested that ALA did not affect the parameters of oxidative stress, while significantly increased the level of reactive sulfane sulfur in both ND and PD patients suffering from CKD. The results suggest that ALA can exert nephroprotective effects which are related to sulfane sulfur production.

Cas-CLOVER-mediated knockout of STAT1: A novel approach to engineer packaging HEK-293 cell lines used for rAAV production.

In addressing the limitations of CRISPR-Cas9, including off-target effects and high licensing fees for commercial use, Cas-CLOVER, a dimeric gene editing tool activated by two guide RNAs, was recently developed. This study focused on implementing and evaluating Cas-CLOVER in HEK-293 cells used for recombinant adeno-associated virus (rAAV) production by targeting the signal transducer and activator of transcription 1 (STAT1) locus, which is crucial for cell growth regulation and might influence rAAV production yields. Cas-CLOVER demonstrated impressive efficiency in gene editing, achieving over 90% knockout (KO) success. Thirteen selected HEK-293 STAT1 KO sub-clones were subjected to extensive analytical characterization to assess their genomic stability, crucial for maintaining cell integrity and functionality. Additionally, rAAV9 productivity, Rep protein pattern profile, and potency, among others, were assessed. Clones showed significant variation in capsid and vector genome titers, with capsid titer reductions ranging from 15% to 98% and vector genome titers from 16% to 55%. Interestingly, the Cas-CLOVER-mediated STAT1 KO bulk cell population showed a better ratio of full to empty capsids. Our study also established a comprehensive analytical workflow to detect and evaluate the gene KOs generated by this innovative tool, providing a solid groundwork for future research in precise gene editing technologies.

Fed-batch strategies for intensified rVSV vector production in high cell density cultures of suspension HEK293 cells.

Vesicular stomatitis virus (VSV) has been increasingly demonstrated as a promising viral vector platform. As the interest over this modality for vaccine and gene therapy applications increases, the need for intensified processes to produce these vectors emerge. In this study, we develop fed-batch-based operations to intensify the production of a recombinant VSV-based vaccine candidate (rVSV-SARS-CoV-2) in suspension cultures of HEK293 cells. A feeding strategy, in which a commercial concentrated medium was added to cultures based on cell growth through a fixed cell specific feeding rate (CSFR), was applied for the development of two different processes using Ambr250 modular bioreactors. Cultures operated in hybrid fed-batch/perfusion (FB/P) or fed-batch (FB) were able to sustain infections performed at 8.0 × 106 cells/mL, respectively resulting in 3.9 and 5.0-fold increase in total yield (YT) and 1.7 and 5.6-fold increase in volumetric productivity (VP) when compared with a batch reference. A maximum viral titer of 4.5 × 1010 TCID50/mL was reached, which is comparable or higher than other processes for VSV production in different cell lines. Overall, our study reports efficient fed-batch options to intensify the production of a rVSV-based vaccine candidate in suspension HEK293 cells.

High-yield extracellular vesicle production from HEK293T cells encapsulated in 3D auxetic scaffolds with cyclic mechanical stimulation for effective drug carrier systems.

Extracellular vesicles (EVs) show promise in drug loading and delivery for medical applications. However, the lack of scalable manufacturing processes hinders the generation of clinically suitable quantities, thereby impeding the translation of EV-based therapies. Current EV production relies heavily on non-physiological two-dimensional (2D) cell culture or bioreactors, requiring significant resources. Additionally, EV-derived ribonucleic acid cargo in three-dimensional (3D) and 2D culture environments remains largely unknown. In this study, we optimized the biofabrication of 3D auxetic scaffolds encapsulated with human embryonic kidney 293 T (HEK293 T) cells, focusing on enhancing the mechanical properties of the scaffolds to significantly boost EV production through tensile stimulation in bioreactors. The proposed platform increased EV yields approximately 115-fold compared to conventional 2D culture, possessing properties that inhibit tumor progression. Further mechanistic examinations revealed that this effect was mediated by the mechanosensitivity of YAP/TAZ. EVs derived from tensile-stimulated HEK293 T cells on 3D auxetic scaffolds demonstrated superior capability for loading doxorubicin compared to their 2D counterparts for cancer therapy. Our results underscore the potential of this strategy for scaling up EV production and optimizing functional performance for clinical translation.

Changes in the Transcriptome and Long Non-Coding RNAs but Not the Methylome Occur in Human Cells Exposed to Borrelia burgdorferi.

Lyme disease, caused by infection with members of the Lyme borreliosis group of Borrelia spirochete bacteria, is increasing in frequency and distribution worldwide. Epigenetic interactions between the mammalian host, tick, and bacterial pathogen are poorly understood. In this study, high-throughput next-generation sequencing (NGS) allowed for the in vitro study of the transcriptome, non-coding RNAs, and methylome in human host cells in response to Borrelia burgdorferi infection. We tested the effect of the Borrelia burgdorferi strain B31 on a human primary cell line (HUVEC) and an immortalized cell line (HEK-293) for 72 h, a long-duration time that might allow for epigenetic responses in the exposed human host cells. Differential gene expression was detected in both cell models in response to B. burgdorferi. More differentially expressed genes were found in HUVECs compared to HEK-293 cells. Borrelia burgdorferi exposure significantly induced genes in the interferon, in addition to cytokine and other immune response signaling in HUVECs. In HEK-293 cells, pre-NOTCH processing in Golgi was significantly downregulated in Borrelia-exposed cells. Other significantly altered gene expressions were found in genes involved in the extracellular matrix. No significant global methylation changes were detected in HUVECs or HEK-293 cells exposed to B. burgdorferi; however, two long non-coding RNAs and a pseudogene were deregulated in response to B. burgdorferi in HUVECs, suggesting that other epigenetic mechanisms may be initiated by infection.

Characterizing ER Retention Defects of PDZ Binding Deficient Cx36 Mutants Using Confocal Microscopy.

Overexpression of proteins in transiently transfected cells is a simple way to study basic transport mechanisms and the underlying protein-protein interactions. While expression systems have obvious drawbacks compared to in vivo experiments, they allow a quick assessment of more conserved functions, for instance, ER export or sorting of proteins in the Golgi. In a previous study, our group described the formation of ER-derived removal vesicles for the gap junction protein Cx36 in transfected HEK293T cells. These removal vesicles, termed "whorls" because of their concentric structure, were formed by Cx36 channels that failed to escape the ER. In this article, we describe an imaging protocol that can be used to determine these ER retention defects for Cx36 expressed in cultured cells. The protocol we provide here employs regular confocal microscopy, which allows for sufficient resolution to reveal the characteristic shape of ER whorls.

Antioxidative and anti-cytogenotoxic potential of Lysiphyllum strychnifolium (Craib) A. Schmitz extracts against cadmium-induced toxicity in human embryonic kidney (HEK293) and dermal fibroblast (HDF) cells.

Exposure to cadmium (Cd) results in bioaccumulation and irreversible damage; this encourages an investigation of alternatives to address Cd toxicity, using natural compounds. Lysiphyllum strychnifolium, a well-known Thai medicinal plant, was investigated for its phytochemical compounds and corresponding bioactivities, including antioxidant and anti-cytogenotoxic effects against Cd toxicity in HEK293 renal and HDF dermal cell models. The crude extract of L. strychnifolium (LsCrude) was partitioned into four fractions, using sequential polarity solvents (hexane, dichloromethane, ethyl acetate, and water, denoted as LsH, LsD, LsE, and LsW, respectively). The extraction yields were 1.79 %, 5.08 %, 8.53 %, and 70.25 % (w/w), respectively. Phytochemical screening revealed the presence of tannins, alkaloids, and flavonoids in LsCrude and its fractions, except for LsH. LsE exhibited the highest concentrations of phenolics (286.83 ± 6.83 mg GAE/g extract) and flavonoids (86.36 ± 1.29 mg QE/g extract). Subsequent 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging and ferric-reducing ability of plasma (FRAP) reducing powder assays demonstrated the high antioxidant capacity of LsCrude and its fractions. The lowest IC50 value (9.11 ± 0.43 µg/mL) in the DPPH assay corresponded to LsW, whereas the highest total FRAP value (6.06 ± 0.70 mg QE Eq./g dry mass) corresponded to LsE. MTT and alkaline comet assays revealed the lack of toxicity of the extracts, which were considered safe. Upon exposure to Cd at the CC50 level, HEK293 cells treated with LsE suppressed Cd-induced damage. HDF cells treated with LsCrude, LsD, or LsE attenuated Cd-induced damage. In the pre-treatment, LsD protected the HDF cells against Cd-mediated cytogenotoxicity. These anti-cytogenotoxic potentials are likely due to the antioxidant properties of the phytochemicals. Our findings highlight the cyto-geno-protective properties of L. strychnifolium stem extracts against Cd toxicity in HEK293 and HDF cells, and provide a novel approach for combating oxidative stress and DNA damage caused by environmental pollutants.

Hypoxia decreases mitochondrial ROS production in cells.

We re-examined the reported increase in mitochondrial ROS production during acute hypoxia in cells. Using the Amplex Ultrared/horseradish peroxidase assay we found a decrease, not increase, in hydrogen peroxide release from HEK293 cells under acute hypoxia, at times ranging from 1 min to 3 h. The rates of superoxide/hydrogen peroxide production from each of the three major sites (site IQ in complex I and site IIIQo in complex III in mitochondria, and NADH oxidases (NOX) in the cytosol) were decreased to the same extent by acute hypoxia, with no change in the cells' ability to degrade added hydrogen peroxide. A similar decrease in ROS production under acute hypoxia was found using the diacetyldichlorofluorescein assay. Using a HIF1α reporter cell line we confirmed earlier observations that suppression of superoxide production by site IIIQo decreases HIF1α expression, and found similar effects of suppressing site IQ or NOX. We conclude that increased mitochondrial ROS do not drive the response of HIF1α to acute hypoxia, but suggest that cytosolic H2O2 derived from site IQ, site IIIQo and NOX in cells is necessary to permit HIF1α stabilization by other signals.

Towards cost-effective side-chain isotope labelling of proteins expressed in human cells.

Side chain isotope labelling is a powerful tool to study protein structure and interactions by NMR spectroscopy. 1H,13C labelling of side-chain methyl groups in a deuterated background allows studying large molecules, while side-chain aromatic groups are highly sensitive to the interaction with ligands, drugs, and other proteins. In E. coli, side chain labelling is performed by substituting amino acids with isotope-labelled precursors. However, proteins that can only be produced in mammalian cells require expensive isotope-labelled amino acids. Here we provide a simple and cost-effective method to label side chains in mammalian cells, which exploits the reversible reaction catalyzed by endogenous transaminases to convert isotope-labelled α-ketoacid precursors. We show by in-cell and in-lysate NMR spectroscopy that replacing an amino acid in the medium with its cognate precursor is sufficient to achieve selective labelling without scrambling, and how this approach allows monitoring conformational changes such as those arising from ligand binding.