Tissue-Specific Tumour Suppressor and Oncogenic Activities of the Polycomb-like Protein MTF2.

The Polycomb repressive complex 2 (PRC2) is a conserved chromatin-remodelling complex that catalyses the trimethylation of histone H3 lysine 27 (H3K27me3), a mark associated with gene silencing. PRC2 regulates chromatin structure and gene expression during organismal and tissue development and tissue homeostasis in the adult. PRC2 core subunits are associated with various accessory proteins that modulate its function and recruitment to target genes. The multimeric composition of accessory proteins results in two distinct variant complexes of PRC2, PRC2.1 and PRC2.2. Metal response element-binding transcription factor 2 (MTF2) is one of the Polycomb-like proteins (PCLs) that forms the PRC2.1 complex. MTF2 is highly conserved, and as an accessory subunit of PRC2, it has important roles in embryonic stem cell self-renewal and differentiation, development, and cancer progression. Here, we review the impact of MTF2 in PRC2 complex assembly, catalytic activity, and spatiotemporal function. The emerging paradoxical evidence suggesting that MTF2 has divergent roles as either a tumour suppressor or an oncogene in different tissues merits further investigations. Altogether, our review illuminates the context-dependent roles of MTF2 in Polycomb group (PcG) protein-mediated epigenetic regulation. Its impact on disease paves the way for a deeper understanding of epigenetic regulation and novel therapeutic strategies.

Locked nucleic acid (LNA): A modern approach to cancer diagnosis and treatment.

Cancer is responsible for about one in six deaths in the world. Conventional cancer treatments like chemotherapy, radiotherapy, and surgery are associated with drug poisoning and poor prognosis. Thanks to advances in RNA delivery and target selection, new cancer medicines are now conceivable to improve the quality of life and extend the lives of cancer patients. Antisense oligonucleotides (ASOs) and siRNAs are the most important tools in RNA therapies. Locked Nucleic Acids (LNAs) are one of the newest RNA analogs, exhibiting more affinity to binding, sequence specificity, thermal stability, and nuclease resistance due to their unique properties. Assays using LNA are also used in molecular diagnostic methods and provide accurate and rapid mutation detection that improves specificity and sensitivity. This study aims to review the special properties of LNA oligonucleotides that make them safe and effective antisense drugs for cancer treatment by controlling gene expression. Following that, we go over all of the molecular detection methods and cancer treatment antisense tactics that are possible with LNA technology.

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.

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.

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.

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.

The relationship between demographic factors and levels of self-care against coronavirus in pregnant women referred to maternity wards.

Background: The adverse effects of coronavirus infection on pregnant women and their infants are not apparent. The best strategies to deal with this disease is avoiding the infection and preventing its transmission. Purpose: the present study aimed to investigate the relationship between demographic factors and levels of self-care against coronavirus in pregnant women referred to maternity wards of Kerman, southeast Iran. Method: The present descriptive study was conducted on 200 pregnant women who referred to maternity wards in Kerman in 2020 and met the inclusion criteria. The required information was collected using demographic and obstetric questionnaires and a self-care checklist. Findings: The mean age of the participants was 28.89 ± 7.07. Iranian and Afghan citizens comprised 82 and 18% of the participants, respectively. The highest level of self-care measures against coronavirus in pregnant women was attributed to the use of face masks (74%), and the lowest was warning the personnel to wear masks (28%). There was a statistically significant relationship between the nationality of the participants and warning the personnel to wear facemasks (r = 0.183; p = 0.02), having a sick spouse (r=0.149;P = 0.039), and having a sick child (r = 0.191; p = 0.043), and between the husbands' job and the patients' demand for a private room (r = 0.173; p = 0.013). There was an inverse relationship between mothers' age and warning the personnel about paying attention to their hygiene (r = -0.145; p = 0.04). Conclusions: The results indicated that most pregnant women in the present study were active in self-care against coronavirus. Using face masks was more widely followed than other self-care measures; moreover, there was a relationship between personal characteristics and self-care levels.

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.

Functionalized carbon microfibers (biomass-derived) ornamented by Bi2S3 nanoparticles: an investigation on their microwave, magnetic, and optical characteristics.

The biomass-derived materials emerged as novel, low-cost, green, and light-weight microwave absorbers. On the other hand, the sulfide nanostructures due to narrow band gap demonstrated significant dielectric features. In this research, the pure carbon microfibers were prepared using Erodium cicutarium harvest and they were functionalized by a sonochemistry method. The treated microfibers were coated by Bi2S3 nanoparticles, obtained by a novel modified solvothermal route. X-ray powder diffraction, Fourier transform infrared, diffuse reflection spectroscopy, field emission scanning electron microscopy (FE-SEM), transmission electron microscopy, and vector network analyzer analyses were applied to characterize the features of the prepared structures. The obtained results manifest that the anchoring nanoparticles onto the functionalized microfibers narrowed band gap to 1.35 eV and reinforced polarizability of the nanocomposite, desirable for dielectric attenuation. In this study, the interfacial interactions were modulated using polyacrylonitrile (PAN) and polyvinylidene fluoride. Interestingly, FCMF blended in PAN demonstrated an eye-catching efficient bandwidth as wide as 8.13 GHz (RL > 10 dB) with only 2.00 mm in thickness, whereas it illustrated an outstanding reflection loss of 81.96 at 11.48 GHz with a thickness of 2.50 mm. More significantly, FCMF/Bi2S3/PAN nanocomposite promoted the efficient bandwidth to 3.07 GHz (RL > 20 dB). Noteworthy, all of the samples illustrated total electromagnetic interference shielding effectiveness (SET) more than 15 dB entire the x and ku-band frequency.

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

Calmodulin (CaM) is an important Ca2+-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 Ca2+ 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. Despite its importance in muscle, the regulation of CaM-particularly its availability to bind to and activate downstream targets-is an emerging area of research. In this minireview, we discuss recent studies revealing the importance of small IQ motif proteins that bind to CaM to either facilitate (nuclear receptor interacting protein; NRIP) its activation of downstream targets, or sequester (neurogranin, Ng; and growth-associated protein 43, GAP43) CaM away from their downstream targets. Specifically, we discuss recent studies that have begun uncovering the physiological roles of NRIP, Ng, and GAP43 in skeletal and cardiac muscle, thereby highlighting the importance of endogenously expressed CaM-binding proteins and their regulation of CaM in muscle.

A Low-Therapeutic Dose of Lithium Inhibits GSK3 and Enhances Myoblast Fusion in C2C12 Cells.

Glycogen synthase kinase 3 (GSK3) slows myogenic differentiation and myoblast fusion partly by inhibiting the Wnt/ß-catenin signaling pathway. Lithium, a common medication for bipolar disorder, inhibits GSK3 via Mg+ competition and increased Ser21 (GSK3α) or Ser9 (GSK3ß) phosphorylation, leading to enhanced myoblast fusion and myogenic differentiation. However, previous studies demonstrating the effect of lithium on GSK3 have used concentrations up to 10 mM, which greatly exceeds concentrations measured in the serum of patients being treated for bipolar disorder (0.5-1.2 mM). Here, we determined whether a low-therapeutic (0.5 mM) dose of lithium could promote myoblast fusion and myogenic differentiation in C2C12 cells. C2C12 myotubes differentiated for three days in media containing 0.5 mM lithium chloride (LiCl) had significantly higher GSK3ß (ser9) and GSK3α (ser21) phosphorylation compared with control myotubes differentiated in the same media without LiCl (+2-2.5 fold, p < 0.05), a result associated with an increase in total ß-catenin. To further demonstrate that 0.5 mM LiCl inhibited GSK3 activity, we also developed a novel GSK3-specific activity assay. Using this enzyme-linked spectrophotometric assay, we showed that 0.5 mM LiCl-treated myotubes had significantly reduced GSK3 activity (-86%, p < 0.001). Correspondingly, 0.5 mM LiCl treated myotubes had a higher myoblast fusion index compared with control (p < 0.001) and significantly higher levels of markers of myogenesis (myogenin, +3-fold, p < 0.001) and myogenic differentiation (myosin heavy chain, +10-fold, p < 0.001). These results indicate that a low-therapeutic dose of LiCl is sufficient to promote myoblast fusion and myogenic differentiation in muscle cells, which has implications for the treatment of several myopathic conditions.

Quantification of Mitochondrial Network Characteristics in Health and Disease.

The term 'mitochondrial dynamics' is commonly used to refer to ongoing fusion and fission of mitochondrial structures within a living cell. A growing number of diseases, from Charcot Marie Tooth Type 2a neuropathies to cancer, is known to be associated with the dysregulation of mitochondrial dynamics, leading to irregularities of mitochondrial network morphology that are associated with aberrant metabolism and cellular dysfunction. Studying these phenomena, and potential pharmacological interventions to correct them, in cultured cells is a powerful approach to developing treatments or cures. Appropriately designed experiments and quantitative approaches for characterizing mitochondrial morphology and function are essential for furthering our understanding. In this chapter, we discuss the importance of cell incubation conditions, choices around imaging modalities, and data analysis tools with respect to experimental outcomes and the interpretation of results from studies of mitochondrial dynamics. We focus primarily on the quantitative analysis of mitochondrial morphology, providing an overview of the available tools and approaches currently being used and discussing some of the strengths and weaknesses associated with each. Finally, we discuss how the ongoing development of imaging and analysis tools continues to improve our ability to study normal and aberrant mitochondrial physiology in vitro and in vivo.

Neurogranin is expressed in mammalian skeletal muscle and inhibits calcineurin signaling and myoblast fusion.

Calcineurin is a Ca2+/calmodulin (CaM)-dependent phosphatase that plays a critical role in promoting the slow fiber phenotype and myoblast fusion in skeletal muscle, thereby making calcineurin an attractive cellular target for enhancing fatigue resistance, muscle metabolism, and muscle repair. Neurogranin (Ng) is a CaM-binding protein thought to be expressed solely in brain and neurons, where it inhibits calcineurin signaling by sequestering CaM, thus lowering its cellular availability. Here, we demonstrate for the first time the expression of Ng protein and mRNA in mammalian skeletal muscle. Both protein and mRNA levels are greater in slow-oxidative compared with fast-glycolytic muscles. Coimmunoprecipitation of CaM with Ng in homogenates of C2C12 myotubes, mouse soleus, and human vastus lateralis suggests that these proteins physically interact. To determine whether Ng inhibits calcineurin signaling in muscle, we used Ng siRNA with C2C12 myotubes to reduce Ng protein levels by 60%. As a result of reduced Ng expression, C2C12 myotubes had enhanced CaM-calcineurin binding and calcineurin signaling as indicated by reduced phosphorylation of nuclear factor of activated T cells and increased utrophin mRNA. In addition, calcineurin signaling affects the expression of myogenin and stabilin-2, which are involved in myogenic differentiation and myoblast fusion, respectively. Here, we found that both myogenin and stabilin-2 were significantly elevated by Ng siRNA in C2C12 cells, concomitantly with an increased fusion index. Taken together, these results demonstrate the expression of Ng in mammalian skeletal muscle where it appears to be a novel regulator of calcineurin signaling.

Hypoxia inducible factors as mediators of reactive oxygen/nitrogen species homeostasis in physiological normoxia.

Although once considered by biologists almost exclusively for their toxicity, reactive oxygen (ROS) and nitrogen (RNS) species produced within normal cells under baseline physiological conditions are now appreciated as redox regulators of a wide range of protein functions. Two families of enzymes, the NADPH oxidases (NOXs) and nitric oxide synthases (NOSs), are major sources of ROS/RNS from molecular oxygen. Aquaporins (AQPs) are membrane channels capable of transporting some ROS/RNS, in particular hydrogen peroxide and perhaps nitric oxide. The activities of all these enzymes and channels are sensitive to variations in oxygen levels within the physiological range experienced by cells in the human body. Since ROS/RNS have important physiological roles and their endogenous production is affected by oxygen levels, we hypothesize that the synthesis of these proteins is increased at lower oxygen levels within the physiological range of most human cells in vivo, i.e. 2-5%, in order to facilitate the maintenance of ROS/RNS production rates. We further postulate that this is achieved, at least in part, by transcriptional stimulation mediated by the activity of hypoxia inducible factors (HIFs), which are strongly regulated by oxygen levels over the same range of oxygen. Here we survey the evidence supporting this hypothesis, including induction of expression of NOXs, NOSs, and AQPs at lower oxygen levels, presence of hypoxia response elements in the corresponding human genes, and evidence from chromatin immunoprecipitation (ChIP) experiments that HIF-1 and/or HIF-2 bind these regions. We find a significant amount of empirical data supporting the hypothesis that HIFs could function as physiological regulators of ROS/RNS homeostasis in the normoxic range in human cells.

Resveratrol integrates metabolic and growth effects in PC3 prostate cancer cells-involvement of prolyl hydroxylase and hypoxia inducible factor-1.

Resveratrol (RES) is a polyphenol produced by certain plant species that has been well studied due to its ability to slow the growth of cancer cells. In numerous cell types and tissues, RES has been demonstrated to promote mitochondrial biogenesis, fusion, and oxidative phosphorylation. The present study investigated the interaction between RES's effects on growth and metabolism in PC3 prostate cancer cells, and demonstrated that RES-mediated growth inhibition is only observed under conditions in which a metabolic shift from glucose fermentation to mitochondrial respiration can occur. When this shift was prevented by growing cells in galactose medium or by pharmacologically inhibiting prolyl hydroxylase (PHD) in order to stabilize hypoxia inducible factor-1α, RES did not effect mitochondrial fusion, biogenesis, respiration or cell growth. Similar results were observed in PC3 cells expressing a mutant HIF-1α lacking the prolines that are hydroxylated by PHD to promote its degradation. Thus, RES appears to slow PC3 cell growth by interfering with glucose fermentation and promoting respiration. Consistent with this, RES was observed to be particularly effective at inhibiting PC3 cell growth under hypoxic conditions that precluded increased reliance on oxidative phosphorylation. These observations are important in understanding how RES may affect cancer cell growth in vivo where hypoxia is common in growing tumours.

Oxygen and Glucose Levels in Cell Culture Media Determine Resveratrol's Effects on Growth, Hydrogen Peroxide Production, and Mitochondrial Dynamics.

Resveratrol is a plant-derived polyphenol that has been widely studied for its putative health promoting effects. Many of those studies have been conducted in cell culture, in supra-physiological levels of oxygen and glucose. Resveratrol interacts with reactive oxygen species (ROS) as an antioxidant or pro-oxidant. Resveratrol affects the expression and activities of ROS-producing enzymes and organelles. It is therefore important to consider how cell culture conditions might determine the effects of resveratrol on cultured cells. We determined the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics in C2C12 mouse myoblasts and PC3 human prostate cancer cells under conditions of physiological (5%) and supra-physiological (18%) oxygen, and normo- (5 mM) and hyper-glycemia (25 mM). Interestingly, most effects of resveratrol on the parameters measured here were dependent upon prevailing oxygen and glucose levels during the experiment. Many of the effects of resveratrol on cell growth, hydrogen peroxide production, and mitochondrial network characteristics that were seen in 25 mM glucose and/or 18% oxygen were absent under the physiologically relevant conditions of 5 mM glucose with 5% oxygen. These findings emphasize the importance of using physiologically meaningful starting conditions for cell-culture experiments with resveratrol and indeed any manipulation affecting ROS metabolism and mitochondria.

How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions.

Most mammalian tissue cells experience oxygen partial pressures in vivo equivalent to 1-6% O2 (i.e., physioxia). In standard cell culture, however, headspace O2 levels are usually not actively regulated and under these conditions are ~18%. This drives hyperoxia in cell culture media that can affect a wide variety of cellular activities and may compromise the ability of in vitro models to reproduce in vivo biology. Here, we review and discuss some specific O2-consuming organelles and enzymes, including mitochondria, NADPH oxidases, the transplasma membrane redox system, nitric oxide synthases, xanthine oxidase, and monoamine oxidase with respect to their sensitivities to O2 levels. Many of these produce reactive oxygen and/or nitrogen species (ROS/RNS) as either primary end products or byproducts and are acutely sensitive to O2 levels in the range from 1% to 18%. Interestingly, many of them are also transcriptional targets of hypoxia-inducible factors (HIFs) and chronic cell growth at physioxia versus 18% O2 may alter their expression. Aquaporins, which facilitate hydrogen peroxide diffusion into and out of cells, are also regulated by HIFs, indicating that O2 levels may affect intercellular communication via hydrogen peroxide. The O2 sensitivities of these important activities emphasize the importance of maintaining physioxia in culture.

The effect of hormone replacement therapy on cognitive function in postmenopausal women: An RCT.

BACKGROUND: During the reproductive age, the human brain becomes a target for gonadal steroid hormones. Estrogens influence neural function through effects on neurons and affects indirectly the oxidative stress, inflammation, the cerebral vascular and the immune system. OBJECTIVE: To evaluate the effect of the traditional hormone replacement therapy (HRT) on the cognitive function in postmenopausal women. MATERIALS AND METHODS: In this randomized clinical trial, 140 postmenopausal women, from November 2014 to February 2015, were included. Women were randomly divided into two groups. Each woman in the case group took traditional HRT (0.625mg conjugated equine estrogens+2.5mg medroxyprogesterone acetate daily) plus one Cal+D tablet (500 mg calcium+200 IU vitamin D) daily for four months. Women in the control group received only one Cal+D tablet (500 mg calcium+200 IU vitamin D) daily for four months period. The Montreal Cognitive Assessment (MoCA) and Green Climacteric Scale (GCS) questionnaires filled out after the intervention and compared between the two groups. RESULTS: The mean points of the MoCA after the intervention indicate that all MoCA domains except for the orientation improved in the case group. There was a significant difference in the memory domain after the treatment between the two groups. MoCA domains and GCS were negatively correlated after the intervention ( r = - 0 . 235 , p = 0 . 006 ). CONCLUSION: The HRT has affected some of the MoCA factors. The effects of HRT on cognitive function should be studied in a large prospective study in a group of women in their early and late menopausal ages with periodic assessment of their cognitive function during these follow-up years.