Revisiting reactive oxygen species production in hypoxia.

Cellular responses to hypoxia are crucial in various physiological and pathophysiological contexts and have thus been extensively studied. This has led to a comprehensive understanding of the transcriptional response to hypoxia, which is regulated by hypoxia-inducible factors (HIFs). However, the detailed molecular mechanisms of HIF regulation in hypoxia remain incompletely understood. In particular, there is controversy surrounding the production of mitochondrial reactive oxygen species (ROS) in hypoxia and how this affects the stabilization and activity of HIFs. This review examines this controversy and attempts to shed light on its origin. We discuss the role of physioxia versus normoxia as baseline conditions that can affect the subsequent cellular response to hypoxia and highlight the paucity of data on pericellular oxygen levels in most experiments, leading to variable levels of hypoxia that might progress to anoxia over time. We analyze the different outcomes reported in isolated mitochondria, versus intact cells or whole organisms, and evaluate the reliability of various ROS-detecting tools. Finally, we examine the cell-type and context specificity of oxygen's various effects. We conclude that while recent evidence suggests that the effect of hypoxia on ROS production is highly dependent on the cell type and the duration of exposure, efforts should be made to conduct experiments under carefully controlled, physiological microenvironmental conditions in order to rule out potential artifacts and improve reproducibility in research.

The effect of baseline O2 conditions on the response of prostate cancer cells to hypoxia.

The transcriptional response to hypoxia is largely regulated by the hypoxia-inducible factors (HIFs), which induce the expression of genes involved in glycolysis, angiogenesis, proliferation, and migration. Virtually all cell culture-based hypoxia experiments have used near-atmospheric (18% O2) oxygen levels as the baseline for comparison with hypoxia. However, this is hyperoxic compared with mammalian tissue microenvironments, where oxygen levels range from 2% to 9% O2 (physioxia). Thus, these experiments actually compare hyperoxia to hypoxia. To determine how the baseline O2 level affects the subsequent response to hypoxia, we cultured PC-3 prostate cancer cells in either 18% or 5% O2 for 2 wk before exposing them to hypoxia (∼1.1% pericellular O2) for 12-48 h. RNA-seq revealed that the transcriptional response to hypoxia was dependent on the baseline O2 level. Cells grown in 18% O2 before hypoxia exposure showed an enhanced induction of HIF targets, particularly genes involved in glucose metabolism, compared with cells grown in physioxia before hypoxia. Consistent with this, hypoxia significantly increased glucose consumption and metabolic activity only in cells previously cultured in 18% O2, but not in cells preadapted to 5% O2. Transcriptomic analyses also indicated effects on cell proliferation and motility, which were followed up by functional assays. Although unaffected by hypoxia, both proliferation and migration rates were greater in cells cultured in 5% O2 versus 18% O2. We conclude that an inappropriately hyperoxic starting condition affects the transcriptional and metabolic responses of PC-3 cells to hypoxia, which may compromise experiments on cancer metabolism in vitro.NEW & NOTEWORTHY Although human cell culture models have been instrumental to our understanding of the mechanisms involved in the cellular response to hypoxia, in virtually all experiments, cells are routinely cultured in near-atmospheric (∼18% O2) oxygen levels, which are hyperoxic relative to physiological conditions in vivo. Here, we show for the first time that cells cultured in physiological O2 levels (5% O2) respond differently to subsequent hypoxia than cells grown at 18%.

Characterizing the effects of muscle-specific GSK3α/ß reduction on murine muscle contractility and metabolism in female mice.

Dysregulation of skeletal muscle morphology and metabolism is associated with chronic diseases such as obesity and type 2 diabetes. The enzyme glycogen synthase kinase 3 (GSK3) is highly involved in skeletal muscle physiology and metabolism, acting as a negative regulator of muscle size, strength, adaptive thermogenesis, and glucose homeostasis. Correspondingly, we have shown that partial knockdown (∼40%) of GSK3 specifically in skeletal muscle increases lean mass, reduces fat mass, and activates muscle-based adaptive thermogenesis via sarco(endo)plasmic reticulum Ca2+ (SERCA) uncoupling in male mice. However, the effects of GSK3 knockdown in female mice have yet to be investigated. Here, we examined the effects of muscle-specific GSK3 knockdown on body composition, muscle size and strength, and whole body metabolism in female C57BL/6J mice. Our results show that GSK3 content is higher in the female soleus versus the male soleus; however, there were no differences in the extensor digitorum longus (EDL). Furthermore, muscle-specific GSK3 knockdown did not alter body composition in female mice, nor did it alter daily energy expenditure, glucose/insulin tolerance, mitochondrial respiration, or the expression of the SERCA uncouplers sarcolipin and neuronatin. We also did not find any differences in soleus muscle size, strength, or fatigue resistance. In the EDL, we found that an increase in absolute and specific force production, but there were no differences in fatigability. Therefore, our study highlights sex differences in the response to genetic reduction of gsk3, with most of the effects previously observed in male mice being absent in females.NEW & NOTEWORTHY Here we show that partial GSK3 knockdown has minimal effects on whole body metabolism and muscle contractility in female mice. This is partly inconsistent with previous results found in male mice, which reveal a potential influence of biological sex.

US FDA's Dose Optimization Postmarketing Requirements and Commitments of Oncology Approvals and the Impact on Product Labels from 2010 to 2022: An Emerging Landscape from Traditional to Novel Therapies.

BACKGROUND: Dose optimization is a focal point of many US Food and Drug Administration (FDA) drug approvals. We sought to understand the impact of the FDA's Postmarketing Commitments/Postmarketing Requirements (PMCs/PMRs) on dose optimization and prescriber labeling for oncology drugs. METHODS: Publicly available information was aggregated for all FDA oncology drug approvals between January 1, 2010, and December 31, 2022. Study completion dates were compared to product labeling before and after PMC/PMR fulfillment dates to evaluate labeling changes associated with dose-related PMCs/PMRs. Data were analyzed individually (2010-2015 and 2016-2022) due to differences in available information. RESULTS: From 2010 to 2015, 14 of 42 (33.3%) new molecular entities (NMEs) had dose-related PMCs/PMRs, with 6 of 14 (42.9%) resulting in a relevant label change. From 2016 to 2022, of the 314 new or supplemental applications approved, 21 had dose-related PMCs/PMRs (6.7%), which trended upward over time; 71.4% of dose-related PMCs/PMRs were NMEs. Kinase inhibitors (KIs) and antibody/peptide drug conjugates (ADCs/PDCs) were the most affected drug classes. Ten of the 21 approvals with dose-related PMCs/PMRs fulfilled their dosing PMCs/PMRs, and 3 of the 10 (30%) had relevant label changes. CONCLUSION: Most dose-related PMRs/PMCs were issued for NMEs. Of these, KIs and ADCs/PDCs were highly represented, reflecting their novelty and greater uncertainty around their safety profile. PMC/PMR issuance broadly increased over time. With the implementation of the FDA's Project Optimus in 2021, it remains to be seen whether fewer dose-related PMCs/PMRs emerge in future due to enhanced dose optimization in the premarketing setting.

Human Brain Microvascular Endothelial Cells Exposure to SARS-CoV-2 Leads to Inflammatory Activation through NF-κB Non-Canonical Pathway and Mitochondrial Remodeling.

Neurological effects of COVID-19 and long-COVID-19, as well as neuroinvasion by SARS-CoV-2, still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro exposure by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the blood-brain barrier. Despite the low to non-productive viral replication, SARS-CoV-2-exposed cultures displayed increased immunoreactivity for cleaved caspase-3, an indicator of apoptotic cell death, tight junction protein expression, and immunolocalization. Transcriptomic profiling of SARS-CoV-2-challenged cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Oxygen toxicity: cellular mechanisms in normobaric hyperoxia.

In clinical settings, oxygen therapy is administered to preterm neonates and to adults with acute and chronic conditions such as COVID-19, pulmonary fibrosis, sepsis, cardiac arrest, carbon monoxide poisoning, and acute heart failure. In non-clinical settings, divers and astronauts may also receive supplemental oxygen. In addition, under current standard cell culture practices, cells are maintained in atmospheric oxygen, which is several times higher than what most cells experience in vivo. In all the above scenarios, the elevated oxygen levels (hyperoxia) can lead to increased production of reactive oxygen species from mitochondria, NADPH oxidases, and other sources. This can cause cell dysfunction or death. Acute hyperoxia injury impairs various cellular functions, manifesting ultimately as physiological deficits. Chronic hyperoxia, particularly in the neonate, can disrupt development, leading to permanent deficiencies. In this review, we discuss the cellular activities and pathways affected by hyperoxia, as well as strategies that have been developed to ameliorate injury. • Hyperoxia promotes overproduction of reactive oxygen species (ROS). • Hyperoxia dysregulates a variety of signaling pathways, such as the Nrf2, NF-κB and MAPK pathways. • Hyperoxia causes cell death by multiple pathways. • Antioxidants, particularly, mitochondria-targeted antioxidants, have shown promising results as therapeutic agents against oxygen toxicity in animal models.

Culture of Cancer Cells at Physiological Oxygen Levels Affects Gene Expression in a Cell-Type Specific Manner.

Standard cell culture is routinely performed at supraphysiological oxygen levels (~18% O2). Conversely, O2 levels in most mammalian tissues range from 1-6% (physioxia). Such hyperoxic conditions in cell culture can alter reactive oxygen species (ROS) production, metabolism, mitochondrial networks, and response to drugs and hormones. The aim of this study was to investigate the transcriptional response to different O2 levels and determine whether it is similar across cell lines, or cell line-specific. Using RNA-seq, we performed differential gene expression and functional enrichment analyses in four human cancer cell lines, LNCaP, Huh-7, PC-3, and SH-SY5Y cultured at either 5% or 18% O2 for 14 days. We found that O2 levels affected transcript abundance of thousands of genes, with the affected genes having little overlap between cell lines. Functional enrichment analysis also revealed different processes and pathways being affected by O2 in each cell line. Interestingly, most of the top differentially expressed genes are involved in cancer biology, which highlights the importance of O2 levels in cancer cell research. Further, we observed several hypoxia-inducible factor (HIF) targets, HIF-2α targets particularly, upregulated at 5% O2, consistent with a role for HIFs in physioxia. O2 levels also differentially induced the transcription of mitochondria-encoded genes in most cell lines. Finally, by comparing our transcriptomic data from LNCaP and PC-3 with datasets from the Prostate Cancer Transcriptome Atlas, a correlation between genes upregulated at 5% O2 in LNCaP cells and the in vivo prostate cancer transcriptome was found. We conclude that the transcriptional response to O2 over the range from 5-18% is robust and highly cell-type specific. This latter finding indicates that the effects of O2 levels are difficult to predict and thus highlights the importance of regulating O2 in cell culture.

Low-dose lithium supplementation promotes adipose tissue browning and sarco(endo)plasmic reticulum Ca2+ ATPase uncoupling in muscle.

Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) uncoupling in skeletal muscle and mitochondrial uncoupling via uncoupling protein 1 (UCP1) in brown/beige adipose tissue are two mechanisms implicated in energy expenditure. Here, we investigated the effects of glycogen synthase kinase 3 (GSK3) inhibition via lithium chloride (LiCl) treatment on SERCA uncoupling in skeletal muscle and UCP1 expression in adipose. C2C12 and 3T3-L1 cells treated with LiCl had increased SERCA uncoupling and UCP1 protein levels, respectively, ultimately raising cellular respiration; however, this was only observed when LiCl treatment occurred throughout differentiation. In vivo, LiCl treatment (10 mg/kg/day) increased food intake in chow-fed diet and high-fat diet (HFD; 60% kcal)-fed male mice without increasing body mass-a result attributed to elevated daily energy expenditure. In soleus muscle, we determined that LiCl treatment promoted SERCA uncoupling via increased expression of SERCA uncouplers, sarcolipin and/or neuronatin, under chow-fed and HFD-fed conditions. We attribute these effects to the GSK3 inhibition observed with LiCl treatment as partial muscle-specific GSK3 knockdown produced similar effects. In adipose, LiCl treatment inhibited GSK3 in inguinal white adipose tissue (iWAT) but not in brown adipose tissue under chow-fed conditions, which led to an increase in UCP1 in iWAT and a beiging-like effect with a multilocular phenotype. We did not observe this beiging-like effect and increase in UCP1 in mice fed a HFD, as LiCl could not overcome the ensuing overactivation of GSK3. Nonetheless, our study establishes novel regulatory links between GSK3 and SERCA uncoupling in muscle and GSK3 and UCP1 and beiging in iWAT.

Supraphysiological Oxygen Levels in Mammalian Cell Culture: Current State and Future Perspectives.

Most conventional incubators used in cell culture do not regulate O2 levels, making the headspace O2 concentration ~18%. In contrast, most human tissues are exposed to 2-6% O2 (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes. In this review, we discuss how supraphysiological O2 levels affect reactive oxygen species (ROS) metabolism and redox homeostasis, gene expression, replicative lifespan, cellular respiration, and mitochondrial dynamics. Furthermore, we present evidence demonstrating how hyperoxic cell culture conditions fail to recapitulate the physiological and pathological behavior of tissues in vivo, including cases of how O2 alters the cellular response to drugs, hormones, and toxicants. We conclude that maintaining physioxia in cell culture is imperative in order to better replicate in vivo-like tissue physiology and pathology, and to avoid artifacts in research involving cell culture.

Rapid nutrient depletion to below the physiological range by cancer cells cultured in Plasmax.

Mammalian cell culture is a fundamental tool used to study living cells. Presently, the standard protocol for performing cell culture involves the use of commercial media that contain an excess of nutrients. Although this reduces the likelihood of cell starvation, it creates nonphysiologic culture conditions that have been shown to "re-wire" cellular metabolism. Recently, researchers have developed new media like Plasmax, formulated to approximate the nutrient composition of human blood plasma. Although this represents an improvement in cell culture practice, physiologic media may be vulnerable to nutrient depletion. In this study, we directly addressed this concern by measuring the rates of glucose and amino acid depletion from Plasmax in several cancer cell lines (PC-3, LNCaP, MCF-7, and SH-SY5Y) over 48 h. In all cell lines, depletion of glucose from Plasmax was rapid such that, by 48 h, cells were hypoglycemic (<2 mM glucose). Most amino acids were similarly rapidly depleted to subphysiological levels by 48 h. In contrast, glucose and most amino acids remained within the physiological range at 24 h. When the experiment was done at physiological oxygen (5%) versus standard (18%) with LNCaP cells, no effect on glucose or amino acid consumption was observed. Using RNA sequencing, we show that this nutrient depletion is associated with enrichment of starvation responses, apoptotic signaling, and endoplasmic reticulum stress. A shift from glycolytic metabolism to mitochondrial respiration at 5% O2 was also measured using Seahorse analysis. Taken together, these results exemplify the metabolic considerations for Plasmax, highlighting that cell culture in Plasmax requires daily media exchange.

SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non-productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

SARS-CoV-2 infection of human brain microvascular endothelial cells leads to inflammatory activation through NF-κB non-canonical pathway and mitochondrial remodeling.

Neurological effects of COVID-19 and long-COVID-19 as well as neuroinvasion by SARS-CoV-2 still pose several questions and are of both clinical and scientific relevance. We described the cellular and molecular effects of the human brain microvascular endothelial cells (HBMECs) in vitro infection by SARS-CoV-2 to understand the underlying mechanisms of viral transmigration through the Blood-Brain Barrier. Despite the low to non- productive viral replication, SARS-CoV-2-infected cultures displayed increased apoptotic cell death and tight junction protein expression and immunolocalization. Transcriptomic profiling of infected cultures revealed endothelial activation via NF-κB non-canonical pathway, including RELB overexpression, and mitochondrial dysfunction. Additionally, SARS-CoV-2 led to altered secretion of key angiogenic factors and to significant changes in mitochondrial dynamics, with increased mitofusin-2 expression and increased mitochondrial networks. Endothelial activation and remodeling can further contribute to neuroinflammatory processes and lead to further BBB permeability in COVID-19.

Heterozygous SOD2 deletion selectively impairs SERCA function in the soleus of female mice.

The sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) restores intracellular Ca2+ ([Ca2+ ]i ) to resting levels after muscle contraction, ultimately eliciting relaxation. SERCA pumps are highly susceptible to tyrosine (T)-nitration, impairing their ability to take up Ca2+ resulting in reduced muscle function and increased [Ca2+ ]i and cellular damage. The mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), converts superoxide radicals into less reactive H2 O2 . Heterozygous deletion of SOD2 (Sod2+/- ) in mice increases mitochondrial oxidative stress; however, the consequences of reduced SOD2 expression in skeletal and cardiac muscle, specifically the effect on SERCA pumps, has yet to be investigated. We obtained soleus, extensor digitorum longus (EDL), and left ventricle (LV) muscles from 6 to 7 month-old wild-type (WT) and Sod2+/- female C57BL/6J mice. Ca2+ -dependent SERCA activity assays were performed to assess SERCA function. Western blotting was conducted to examine the protein content of SERCA, phospholamban, and sarcolipin; and immunoprecipitation experiments were done to assess SERCA2a- and SERCA1a-specific T-nitration. Heterozygous SOD2 deletion did not alter SERCA1a or SERCA2a expression in the soleus or LV but reduced SERCA2a in the EDL compared with WT, though this was not statistically significant. Soleus muscles from Sod2+/- mice showed a significant reduction in SERCA's apparent affinity for Ca2+ when compared to WT, corresponding with significantly elevated SERCA2a T-nitration in the soleus. No effect was seen in the EDL or the LV. This is the first study to investigate the effects of SOD2 deficiency on muscle SERCA function and shows that it selectively impairs SERCA function in the soleus.

Mitochondrial and metabolic remodelling in human skin fibroblasts in response to glucose availability.

Cell culture conditions highly influence cell metabolism in vitro. This is relevant for preclinical assays, for which fibroblasts are an interesting cell model, with applications in regenerative medicine, diagnostics and therapeutic development for personalized medicine, and the validation of ingredients for cosmetics. Given these cells' short lifespan in culture, we aimed to identify the best cell culture conditions and promising markers to study mitochondrial health and stress in normal human dermal fibroblasts (NHDF). We tested the effect of reducing glucose concentration in the cell medium from high glucose (HGm) to a more physiological level [low glucose medium (LGm)], or its complete removal and replacement by galactose [medium that forces oxidative phosphorylation (OXPHOSm)], always in the presence of glutamine and pyruvate. We have demonstrated that only with OXPHOSm was it possible to observe the selective inhibition of mitochondrial adenosine triphosphate (ATP) production. This reliance on mitochondrial ATP was accompanied by changes in oxygen consumption rate and extracellular acidification rate, oxidation of citric acid cycle substrates, fatty acids, lactate, and other substrates, increased mitochondrial network extension and polarization, the increased protein content of voltage-dependent anion channel (VDAC) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha and changes in several key transcripts related to energy metabolism. LGm did not promote significant metabolic changes in NHDF, although mitochondrial network extension and VDAC protein content were increased compared to HGm-cultured cells. Our results indicate that short-term adaptation to OXPHOSm is ideal for studying mitochondrial health and stress in NHDF.

The Effect of Oxygen and Micronutrient Composition of Cell Growth Media on Cancer Cell Bioenergetics and Mitochondrial Networks.

Cancer cell culture is routinely performed under superphysiologic O2 levels and in media such as Dulbecco's Modified Eagle Medium (DMEM) with nutrient composition dissimilar to mammalian extracellular fluid. Recently developed cell culture media (e.g., Plasmax, Human Plasma-Like Medium (HPLM)), which are modeled on the metabolite composition of human blood plasma, have been shown to shift key cellular activities in several cancer cell lines. Similar effects have been reported with respect to O2 levels in cell culture. Given these observations, we investigated how media composition and O2 levels affect cellular energy metabolism and mitochondria network structure in MCF7, SaOS2, LNCaP, and Huh7 cells. Cells were cultured in physiologic (5%) or standard (18%) O2 levels, and in physiologic (Plasmax) or standard cell culture media (DMEM). We show that both O2 levels and media composition significantly affect mitochondrial abundance and network structure, concomitantly with changes in cellular bioenergetics. Extracellular acidification rate (ECAR), a proxy for glycolytic activity, was generally higher in cells cultured in DMEM while oxygen consumption rates (OCR) were lower. This effect of media on energy metabolism is an important consideration for the study of cancer drugs that target aspects of energy metabolism, including lactate dehydrogenase activity.

Media composition and O2 levels determine effects of 17ß-estradiol and selective estrogen receptor modulators on mitochondrial bioenergetics and cellular reactive oxygen species.

Estradiol (E2) and selective estrogen receptor modulators (SERMs) have broad-ranging cellular effects that include mitochondrial respiration and reactive oxygen species (ROS) metabolism. Many of these effects have been studied using cell culture models. Recent advances have revealed the extent to which cellular metabolism is affected by the culture environment. Cell culture media with metabolite composition similar to blood plasma [e.g., Plasmax, Human Plasma-Like Medium (HPLM)] alter cellular behaviors including responses to drugs. Similar effects have been observed with respect to O2 levels in cell culture. Given these observations, we investigated whether the effects of E2 and SERMs are also influenced by media composition and O2 level during cell culture experiments. We analyzed mitochondrial network characteristics, cellular oxidative metabolism, and H2O2 production in C2C12 myoblasts growing in physiological (5%) or standard cell culture (18%)O2 and in physiological (Plasmax) or standard cell culture [Dulbecco's modified Eagle's medium (DMEM)] media. Although E2 significantly lowered H2O2 production from cells growing in 18% O2/DMEM (standard cell culture), it had no effect on cells growing in Plasmax. Moreover, culture conditions significantly altered the effects of E2 and SERMs on mitochondrial abundance and network characteristics. These results indicate that the effects of E2 and SERMs on various aspects of cell physiology strongly depends on growth conditions, which in turn emphasizes the need to consider this carefully in cell culture experiments.

Neuroglobin and mitochondria: The impact on neurodegenerative diseases.

Dysfunctional mitochondria have severe consequences on cell functions including Reactive Oxygen Specie (ROS) generation, alteration of mitochondrial signaling, Ca2+ buffering, and activation of apoptotic pathway. These dysfunctions are closely linked with degenerative diseases including neurodegeneration. The discovery of neuroglobin (NGB) as an endogenous neuroprotective protein, which effects seem to depend on its mitochondrial localization, could drive new therapeutic strategies against aged-related neurodegenerative diseases. Indeed, high levels of NGB are active against several brain injuries, including neurodegeneration, hypoxia, ischemia, toxicity, and nutrient deprivation opening a new scenario in the comprehension of the relationship between neural pathologies and mitochondrial homeostasis. In this review, we provide the current understanding of the role of mitochondria in neurodegeneration and discuss structural and functional connection between NGB and mitochondria with the purpose of defining a novel mitochondrial-based neuroprotective mechanism(s).

Vertebrate cell culture as an experimental approach - limitations and solutions.

Mammalian cell culture has provided the foundation for the incredible expansion of cell biology to uncover the 'inner life of the cell'. The protocols for propagating cells in the laboratory have their origins in the mid-20th century. At that time the focus was on creating cell culture media that kept cells viable and favoured replicative growth. To the extent that oxygen level was considered as an important parameter, it was in the context of ensuring that oxygen was not depleted; the idea that environmental oxygen levels could be toxic was not widely appreciated. We increasingly understand that media composition and oxygen levels have important effects on cellular functions and that maintaining physiologically relevant conditions is necessary to maintain in vivo behaviours. We also understand much about the impact of growing cells that function in a 3D environment in 2D adherent monolayers. In this review, we examine some of the issues affecting standard cell culture approaches and new solutions that address these issues to increase the physiological accuracy of the cellular environment. We have reached a threshold in cell biology in which we know enough about the problems and their solutions to inform useful adjustments to protocols moving forward. This will increase the accuracy and translatability of this reductionist approach to understanding cell behaviours.

Exploring Transfers between Earth-Moon Halo Orbits via Multi-Objective Reinforcement Learning.

Multi-Reward Proximal Policy Optimization, a multi-objective deep reinforcement learning algorithm, is used to examine the design space of low-thrust trajectories for a SmallSat transferring between two libration point orbits in the Earth-Moon system. Using Multi-Reward Proximal Policy Optimization, multiple policies are simultaneously and efficiently trained on three distinct trajectory design scenarios. Each policy is trained to create a unique control scheme based on the trajectory design scenario and assigned reward function. Each reward function is defined using a set of objectives that are scaled via a unique combination of weights to balance guiding the spacecraft to the target mission orbit, incentivizing faster flight times, and penalizing propellant mass usage. Then, the policies are evaluated on the same set of perturbed initial conditions in each scenario to generate the propellant mass usage, flight time, and state discontinuities from a reference trajectory for each control scheme. The resulting low-thrust trajectories are used to examine a subset of the multi-objective trade space for the SmallSat trajectory design scenario. By autonomously constructing the solution space, insights into the required propellant mass, flight time, and transfer geometry are rapidly achieved.

Bioabsorbable metal zinc differentially affects mitochondria in vascular endothelial and smooth muscle cells.

Zinc is an essential trace element having various structural, catalytic and regulatory interactions with an estimated 3000 proteins. Zinc has drawn recent attention for its use, both as pure metal and alloyed, in arterial stents due to its biodegradability, biocompatibility, and low corrosion rates. Previous studies have demonstrated that zinc metal implants prevent the development of neointimal hyperplasia, which is a common cause of restenosis following coronary intervention. This suppression appears to be smooth muscle cell-specific, as reendothelization of the neointima is not inhibited. To better understand the basis of zinc's differential effects on rat aortic smooth muscle (RASMC) versus endothelial (RAENDO) cells, we conducted a transcriptomic analysis of both cell types following one-week continuous treatment with 5 µM or 50 µM zinc. This analysis indicated that genes whose protein products regulate mitochondrial functions, including oxidative phosphorylation and fusion/fission, are differentially affected by zinc in the two cell types. To better understand this, we performed Seahorse metabolic flux assays and quantitative imaging of mitochondrial networks in both cell types. Zinc treatment differently affected energy metabolism and mitochondrial structure/function in the two cell types. For example, both basal and maximal oxygen consumption rates were increased by zinc in RASMC but not in RAENDO. Zinc treatment increased apparent mitochondrial fusion in RASMC cells but increased mitochondrial fission in RAENDO cells. These results provide some insight into the mechanisms by which zinc treatment differently affects the two cell types and this information is important for understanding the role of zinc treatment in vascular cells and improving its use in biodegradable metal implants.