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Chinese hamster ovary (CHO) cells are widely used for biopharmaceutical protein production. One challenge limiting CHO cell productivity is apoptosis stemming from cellular stress during protein production. Here we applied CRISPR interference (CRISPRi) to downregulate the endogenous expression of apoptotic genes Bak, Bax, and Casp3 in CHO cells. In addition to reduced apoptosis, mitochondrial membrane integrity was improved and the caspase activity was reduced. Moreover, we optimized the CRISPRi system to enhance the gene repression efficiency in CHO cells by testing different repressor fusion types. An improved Cas9 repressor has been identified by applying C-terminal fusion of a bipartite repressor domain, KRAB-MeCP2, to nuclease-deficient Cas9. These results collectively demonstrate that CHO cells can be rescued from cell apoptosis by targeted gene repression using the CRISPRi system.

The domestication process has resulted in profound changes in the reproductive physiology of the animals that might have affected the sperm characteristics and thus their sensitivity to handling and cryopreservation procedures. This work assesses the response of the sperm of domestic and wild ungulates to a cooling storage at 15°C for 20 h followed by incubation at 38.5°C, 5% CO2, for 2 h. In addition, this paper examines the most representative sperm traits to assess their responsiveness to these stress conditions. Sperm samples were collected from domestic and their wild ancestor species: ram, mouflon, buck, Iberian ibex, domestic boar, and wild boar. Sperm motility, viability, mitochondrial membrane status, DNA fragmentation, and reactive oxygen species production were evaluated at the beginning of the experiment, after 20 h of refrigeration at 15°C, and, finally, at 2 h of incubation at 38.5°C. Sperm from all domestic species (ram, buck, and domestic boar) suffered more stress than their wild relatives (mouflon, Iberian Ibex, and wild boar). In pigs, the percentage of intact mitochondria was lower in the domestic species compared to wild boar. In sheep, we found a higher reactive oxygen species production in rams, while in goats, the curvilinear velocity was lower in the domestic species. The PCA (principal components analysis) showed that the motility and their kinetic variables were the most represented variables in the principal components of all species, indicating that they are essential biomarkers for evaluating the stress response. Sperm viability was highlighted as a representative variable for evaluating the stress response in domestic boar, mouflon, ram, and ibex.

Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas’ leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5–12 µM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects.

Tea is an interesting source of antioxidants capable of counteracting the oxidative stress implicated in liver diseases. We investigated the impact of antioxidant molecules provided by a mixture of teas' leaves (green, oolong, pu-erh) after different infusion durations in the prevention of oxidative stress in isolated rat hepatocytes, by comparison with pure epigallocatechin-3-gallate (EGCG), the main representative of tea catechins. Dried aqueous tea extracts (ATE) obtained after 5, 15 and 30 min infusion time were characterized for total polyphenols (gallic acid equivalent), catechins, gallic acid and caffeine (HPLC-DAD/ESI-MS) contents, and for scavenging ability against 2,2-diphenyl-1-picrylhydrazyl free radical. Hepatoprotection was evaluated through hepatocyte viability tests using tert-butyl hydroperoxide as a stress inducer, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red uptake, real-time cellular impedance) and mitochondrial function tests. We showed that a 5-min incubation time is sufficient for an optimal bioaccessibility of tea compounds with the highest antioxidative ability, which decreases for longer durations. A 4-h pretreatment of cells with ATE significantly prevented cell death by regulating reactive oxygen species production and maintaining mitochondrial integrity. Pure EGCG, at doses similar in ATE (5–12 μM), was inefficient, suggesting a plausible synergy of several water-soluble tea compounds to explain the ATE beneficial effects. [ABSTRACT FROM AUTHOR]

Background. Sodium palmitate causes apoptosis of β-cells, and the anti-apoptotic protein Bcl-2 has been shown to counteract this event. However, the exact mechanisms that underlie palmitate-induced pancreatic β-cell apoptosis and through which pathway Bcl-2 executes the protective effect are still unclear. Methods. A stable Bcl-2-overexpressing RINm5F cell clone (BMG) and its negative control (B45) were exposed to palmitate for up to 8 h, and cell viability, mitochondrial membrane potential (Δψm), reactive oxygen species (ROS) generation, endoplasmic reticulum (ER) stress, and NF-κB activation were studied in time course experiments. Results. Palmitate exposure for 8 h resulted in increased cell death rates, and this event was partially counteracted by Bcl-2. Bcl-2 overexpression promoted in parallel also a delayed induction of GADD153/CHOP and a weaker phosphorylation of BimEL in palmitate-exposed cells. At earlier time points (2-4 h) palmitate exposure resulted in increased generation of ROS, a decrease in mitochondrial membrane potential (Δψm), and a modest increase in the phosphorylation of eIF2α and IRE1α. BMG cells produced similar amounts of ROS and displayed the same eIF2α and IRE1α phosphorylation rates as B45 cells. However, the palmitate-induced dissipation of Δψm was partially counteracted by Bcl-2. In addition, basal NF-κB activity was increased in BMG cells. Conclusions. Our results indicate that Bcl-2 counteracts palmitate-induced β-cell death by maintaining mitochondrial membrane integrity and augmenting NF-κB activity, but not by affecting ROS production and ER stress. [ABSTRACT FROM AUTHOR]

The domestication process has resulted in profound changes in the reproductive physiology of the animals that might have affected the sperm characteristics and thus their sensitivity to handling and cryopreservation procedures. This work assesses the response of the sperm of domestic and wild ungulates to a cooling storage at 15°C for 20 h followed by incubation at 38.5°C, 5% CO 2 , for 2 h. In addition, this paper examines the most representative sperm traits to assess their responsiveness to these stress conditions. Sperm samples were collected from domestic and their wild ancestor species: ram, mouflon, buck, Iberian ibex, domestic boar, and wild boar. Sperm motility, viability, mitochondrial membrane status, DNA fragmentation, and reactive oxygen species production were evaluated at the beginning of the experiment, after 20 h of refrigeration at 15°C, and, finally, at 2 h of incubation at 38.5°C. Sperm from all domestic species (ram, buck, and domestic boar) suffered more stress than their wild relatives (mouflon, Iberian Ibex, and wild boar). In pigs, the percentage of intact mitochondria was lower in the domestic species compared to wild boar. In sheep, we found a higher reactive oxygen species production in rams, while in goats, the curvilinear velocity was lower in the domestic species. The PCA (principal components analysis) showed that the motility and their kinetic variables were the most represented variables in the principal components of all species, indicating that they are essential biomarkers for evaluating the stress response. Sperm viability was highlighted as a representative variable for evaluating the stress response in domestic boar, mouflon, ram, and ibex., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 O'Brien, García-Casado, Castaño, Toledano-Díaz, Bóveda and Santiago-Moreno.)

We investigated the effect of Wnt11 on mitochondrial membrane integrity in cardiomyocytes (CMs) and the underlying mechanism of Wnt11-mediated CM protection against hypoxic injury. A rat mesenchymal stem cell (MSC) line that overexpresses Wnt11 (MSC Wnt11 ) and a control cell line transduced with empty vector (MSC Null ) were established to determine the cardioprotective role of Wnt11 in response to hypoxia. Mitochondrial membrane integrity in MSC Wnt11 cells was assessed using fluorescence assays. The role of paracrine signaling mediated by vascular endothelial growth factor (VEGF), basic fibroblast growth factor (b-FGF), and insulin-like growth factor 1 (IGF-1) in protecting CMs against hypoxia were investigated using cocultures of primary CMs from neonatal rats with conditioned medium (CdM) from MSC Wnt11 . MSC Wnt11 cells exposed to hypoxia reduced lactate dehydrogenase release from CMs and increased CM survival under hypoxia. In addition, CMs cocultured with CdM that were exposed to hypoxia showed reduced CM apoptosis and necrosis. There was significantly higher VEGF and IGF-1 release in the MSC Wnt11 group compared with the MSC Null group, and the addition of anti-VEGF and anti-IGF-1 antibodies inhibited secretion. Moreover, mitochondrial membrane integrity was maintained in the MSC Wnt11 cell line. In conclusion, overexpression of Wnt11 in MSCs promotes IGF-1 and VEGF release, thereby protecting CMs against hypoxia., (© 2019 Wiley Periodicals, Inc.)

High-content flow (HCF) screening systems, such as the iQue Screener and HTFC Screening System from IntelliCyt, have facilitated the implementation of flow cytometry assays for high-throughput screening. HCF screening systems enable the use of smaller sample volumes and multiplexed assays to simultaneously assess different cellular parameters from a single well. This becomes invaluable when working with cells or compounds that are available in limited quantities or when conducting large-scale screens. When assays can be miniaturized to a 384- or 1536-well microplate format, it is possible to implement dose-response-based high-throughput screens, also known as quantitative HTS or qHTS. This article describes how qHTS at the new National Center for Advancing Translational Science (NCATS) has been systematically coupled with the HTFC Screening System and Multimetric Apoptosis Screening Kit from IntelliCyt to biologically validate active compounds from primary cell proliferation screens using a model of diffuse large B cell lymphoma (DLBCL).