Altered hypoxia- and redox-related transcriptional signatures in mitochondrial-DNA-depleted PC-3 cells.

Rho 0 (ρ) cells are widely used as a tool to investigate how the absence of respiring mitochondria affects a variety of physiological and pathological processes. Prominently, ρ cells have been used to study the role of mitochondrial reactive oxygen species (ROS) production and/or mitochondrial respiration in the stabilization of the hypoxia-inducible factor (HIF) in hypoxia. In this study, we cultured ρ and WT PC-3 cells in 5% O (physioxia) and Plasmax medium for 2 weeks prior to transcriptomic and functional analyses.

Tumor microenvironment-like conditions alter pancreatic cancer cell metabolism and behavior.

The tumor microenvironment is complex and dynamic, characterized by poor vascularization, limited nutrient availability, hypoxia, and an acidic pH. This environment plays a critical role in driving cancer progression. However, standard cell culture conditions used to study cancer cell biology in vitro fail to replicate the in vivo environment of tumors.

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.

The effect of baseline O 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% O) 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% O (physioxia).

Characterizing the effects of muscle-specific GSK3α/β 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 Ca (SERCA) uncoupling in male mice.

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.

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% O). Conversely, O 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.

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

Most conventional incubators used in cell culture do not regulate O levels, making the headspace O concentration ~18%. In contrast, most human tissues are exposed to 2-6% O (physioxia) in vivo. Accumulating evidence has shown that such hyperoxic conditions in standard cell culture practices affect a variety of biological processes.

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

Sarco(endo)plasmic reticulum Ca-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.

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.

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.

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) 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.

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

The sarco(endo)plasmic reticulum Ca ATPase (SERCA) restores intracellular Ca ([Ca ] ) to resting levels after muscle contraction, ultimately eliciting relaxation. SERCA pumps are highly susceptible to tyrosine (T)-nitration, impairing their ability to take up Ca resulting in reduced muscle function and increased [Ca ] and cellular damage. The mitochondrial antioxidant enzyme, superoxide dismutase 2 (SOD2), converts superoxide radicals into less reactive H O .

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).

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 O 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.

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.

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

Estradiol (E) 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.

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, Ca 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.

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-20 century. At that time the focus was on creating cell culture media that kept cells viable and favoured replicative growth.

In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design.

The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance.

Zn-dependent suppression of vascular smooth muscle intimal hyperplasia from biodegradable zinc implants.

Biodegradable arterial implants based on zinc have been found to suppress neointimal hyperplasia, suggesting that biodegradable materials containing zinc may be used to construct vascular implants with a reduced rate of restenosis. However, the molecular mechanism has remained unclear. In this report, we show that zinc-containing materials can be used to prevent neointimal formation when implanted into the rat aorta.

Calmodulin-Binding Proteins in Muscle: A Minireview on Nuclear Receptor Interacting Protein, Neurogranin, and Growth-Associated Protein 43.

Calmodulin (CaM) is an important Ca-sensing protein with numerous downstream targets that are either CaM-dependant or CaM-regulated. In muscle, CaM-dependent proteins, which are critical regulators of dynamic Ca handling and contractility, include calcineurin (CaN), CaM-dependant kinase II (CaMKII), ryanodine receptor (RyR), and dihydropyridine receptor (DHPR). CaM-regulated targets include genes associated with oxidative metabolism, muscle plasticity, and repair.