Caveolae are 70–100 nm diameter plasma membrane invaginations found in abundance in adipocytes, endothelial cells, myocytes, and fibroblasts. Their bulb-shaped membrane domain is characterized and formed by specific lipid binding proteins including Caveolins, Cavins, Pacsin2, and EHD2. Likewise, an enrichment of cholesterol and other lipids makes caveolae a distinct membrane environment that supports proteins involved in cell-type specific signaling pathways. Their ability to detach from the plasma membrane and move through the cytosol has been shown to be important for lipid trafficking and metabolism. Here, we review recent concepts in caveolae trafficking and dynamics. Second, we discuss how ATP and GTP-regulated proteins including dynamin and EHD2 control caveolae behavior. Throughout, we summarize the potential physiological and cell biological roles of caveolae internalization and trafficking and highlight open questions in the field and future directions for study. Caveolae are 70–100 nm diameter sized plasma membrane invaginations that form bulb shape invaginations into the cytosol . They arIn vitro and in vivo studies have shown that Caveolin/cholesterol complexes incorporate into the plasma membrane forming elongated and rather shallow invaginations , Cavins (Cavin1-4), the BAR protein domain-containing syndapin/Pacsin2, and the dynamin-related ATPase EHD2 see . In vitrDespite the large number of caveolae in muscle and endothelial cells or adipocytes, the loss of caveolae due to Cav1 or Cavin1 deletion is not generally lethal  and Guanosine-5'-triphosphate (GTP) serve as cellular energy resources to drive, localize, and direct these actions. Both nucleosides play essential roles during intracellular trafficking to promote membrane interactions and deformations, protein-protein interactions, and conformational changes in molecular machines. Here, we summarize the state of understanding of caveolae membrane trafficking and highlight the roles of ATP and GTP-dependent processes within these specialized membrane structures.How do caveolae move? Caveolae endocytosis and trafficking has been observed in many cell types although the cellular consequences of these movements are currently not well-understood. The role and even occurrence of caveolae traffic has, indeed, been controversial  and Golgi, it was suggested that caveolae are able to migrate from the cell surface to the ER  are close to the sarcoplasmic reticulum (SR)  and its related EHD proteins belong to the dynamin protein family as they share the same overall domain organization , followed by the central bundle signaling element (BSE), the stalk region, and the pleckstrin homology (PH) domain at the C-terminus . The stain vivo studies showed the GTP dependent dynamin oligomerization and membrane scission at clathrin coated pits (Takei et al., How does dynamin facilitate membrane scission at caveolae? The scission of a phospholipid membrane requires the transition of the chemical energy gained during GTP hydrolysis into mechanical constriction to merge the lipid bilayers of the vesicle neck (Daumke and Praefcke, Some previous studies struggled to clearly localize dynamin to caveolar invaginations in different cell types. Using EM immunogold labeling, however, Dyn2 localization in caveolae was shown in kidney cells (Yao et al., Importantly, oligomerization of dynamin at the plasma membrane can only occur when thin membrane tubes are present. As previously described, EHD2 oligomerizes in rings around lipid bilayers in diameters ranging from 20 to 80 nm. Therefore, at the caveolar neck, we propose that EHD2 is key to create the necessary membrane structure for correct dynamin assembly and subsequent fission of caveolar invaginations (see model in Besides dynamin and EHD2, several other GTP/ATP dependent proteins were assigned roles in caveolae trafficking. Specifically, Rab proteins have been linked with caveolae endocytosis. Rab proteins are small Ras-like GTPases that switch between the “on,” GTP bound—and the “off”—GDP bound—state and therefore temporally and spatially regulate the recruitment of various effector proteins such as vesicle coat proteins, membrane fusion complexes or motor proteins (see review Stenmark, The characteristic lipid environment of caveolar invaginations leads to a specific set of membrane proteins including ATP handling enzymes localized to the caveolar membrane. One example is the distribution of Na/K-ATPase within different plasma membrane domains. A non-pumping Na/K-ATPase subpopulation is found in caveolae (Wang et al., Besides Src, the tyrosine kinase Abl is also able to phosphorylate Cav1 tyrosine 14 in response to oxidative or tension stress (Sanguinetti and Corley Mastick, Caveolae internalization is found in many cells and tissues. Here, we summarized current concepts in caveolae trafficking and its role in physiology. As discussed above, essential steps during the internalization and migration are dependent on ATP or GTP (see also summary Of particular interest is the development or identification of caveolae specific cargos and receptors that are only internalized via caveolar endocytosis. This would allow caveolae-specific intracellular routes to be monitored and further identify novel factors involved in these pathways. New techniques for identifying the protein-protein interactions will provide additional insights in caveolae contact sites between various organelles. Finding these caveolae contact sites will help to determine how caveolae are involved in lipid metabolism and diseases. Another largely unexplored aspect in caveolae dynamics is the influence of local ATP/GTP concentrations. These changes can occur due to hypoxia. Indeed, cells growing under low oxygen levels show altered caveolae behavior. In adipocytes, it was observed that loss of oxygen results in decreased Cav1 expression and consequently reduced caveolae number (Regazzetti et al., CM wrote the manuscript and prepared figures. JT commented on and edited the manuscript.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.


Statins, known as HMG-CoA reductase (HMGCR) inhibitors, have primarily been utilized for metabolic and angiographic medical applications because of their cholesterol-lowering effects. Similar to other drugs, statins may also induce a series of potential side effects. Statins inhibit the HMGCR (rate-limiting enzyme) activity in early stages of mevalonate pathway and then indirectly affect a number of intermediate products, including non-sterol isoprenoids , which can result in impaired functions of body organs. Recently, scores of studies have uncovered additional functional mechanisms of statins in other diseases, such as diabetes mellitus, nervous system diseases, coronary heart disease, inflammation and cancers. This review aims to summarize the positive and adverse mechanisms of statin therapy. Statin care should be taken in the treatment of many diseases including cancers. Since the underlying mechanisms are not fully elucidated, future studies should spend more time and efforts on basic research to explore the mechanisms of statins. Statins are a class of drugs that contain a naphthyl ring skeleton structure and are known as HMGCR inhibitors, which regulate the concentration of plasma lipoproteins by inhibiting cholesterol synthesis. Statins could reduce the synthesis of cholesterol, thus increasing the number and activity of low-density lipoprotein (LDL) receptors on the membrane of liver cells and enhancing the clearance of plasma cholesterol Aspergillus terreus in 1980 and marketed by Merck in 1987. Subsequently, other statin, including lovastatin and simvastatin, were discovered and studied Mevastatin was the first discovered HMG-CoA reductase inhibitor and purified by Akira Endo from the mold fermentation nutrient medium As an important component in the electron transport chain during cellular respiration, coenzyme Q10 (CoQ10) (ubiquinone) mediates electron transfer and results in the shifting of hydrogen ions into the mitochondrial intermembrane space to form a hydrogen ion concentration gradient, ultimately required for the formation of ATP Recently, clinical trials and observational studies have indicated an approximate 10% increase in new-onset diabetes mellitus among patients taking statins, but the mechanisms underlying these clinical effects are not yet fully understood. Clinical and basic studies have shown that statins can potentially increase plasma glucose levels and diabetes risk by enhancing insulin resistance or impairing β-cell function. Here, we describe the potential mechanisms underlying how statins affect glucose homeostasis. Many studies have indicated that the effects of statins in diabetes are related to the inhibition of HMG-CoA reductase activity Statin-induced cholesterol depletion can decrease GLUT4 gene expression and increase GLUT1 protein levels Figure 1). Both in vivo and in vitro experiments have proved that simvastatin reduce ATP production in a dose-dependent manner and that insulin secretion requires ATP Isoprene residues are among the important intermediate molecules of the mevalonate synthesis pathway and are also required for the synthesis of CoQ10 Our purpose is to explore possible risks underlying the effects of statins in diabetogenic clinical treatment. Relevant risk factors related to diabetes during the administration of statins, such as statin variety, treatment dose and hazards for patients with type II diabetes, are very important to be considered in the treatment of diabetic patients. Some studies have found that patients with diabetes receiving statin therapy appear to suffer more risk factors for type II diabetes, particularly in elderly age (age >70 years) Clinically, a great body of evidence indicates statins are employed to treat atherosclerosis in patients with cerebral thrombosis and Alzheimer's disease as it can significantly reduce the incidence of ischemic and hemorrhagic stroke Figure 2). Mevalonate can reverse the effects of atorvastatin in T cells and antigen-presenting cells and inhibit isoprenoid synthesis, thereby preventing intracellular signaling Studies from Yousself et al. have shown that statins decrease the infiltration of the central nervous system (TH1 cell) with inflammatory cells, reduce the expression of major histocompatibility complex II (MHC-II) and inhibit CD40, CD80, and CD86. Oral atorvastatin can prevent and reverse chronic relapses of multiple sclerosis in patients with clinical symptoms of paralysis. Atorvastatin can increase the secretion of TH2 cytokines  . One study found that among 845 cases of elderly patients (average: 80.5 years old), 20.1% had dementia, while in the 10.9% of people without dementia taking lipid-lowering medication, only 3.5% developed dementia. AD is one of the most common causes of dementia, and taking statins may reduce morbidity, improve cognitive function and delay the progression of AD; thus, statin treatment brings new hope to AD therapy The association between long-term statin use and adverse cognitive effects has not been well established. A recent review analyzed the MedWatch drug surveillance system and found statins could also cause memory loss in some patients. It was reported that 60 cases of memory loss were associated with statin use, of which 36 cases took simvastatin, 23 cases took atorvastatin, 1 took pravastatin. Memory loss occurred within 2 months after taking statins in 50% of patients. Among 25 cases taking simvastatin, 14 patients developed improved memory after stopping the drug. However, many of the studies evaluating the effects of statins on cognitive dysfunction have a relatively short follow-up, limiting the ability to assess whether there is a true caused relationship between long-term statin use and cognitive function Statins can be used to treat coronary heart disease by alleviating endothelial dysfunction, inhibiting adhesion between leukocytes and endothelial cells, resisting oxidation, stabilizing plaques, affecting blood flow, suppressing smooth muscle cell proliferation and exerting other effects To study whether statins can suppress cell proliferation via affecting DNA replication in rat aorta vascular smooth muscle cells, Bruemmer et al. selected two chromosomal proteins, MCM6 and MCM7, which played important roles in DNA replication initiation stage. Results showed that MCM6 and MCM7 expression was suppressed by atorvastatin in a dose-dependent manner, and mevalonate could completely reverse the effect. And their study also found that atorvastatin may inhibit MCM6 and MCM7 enhancer activity. Meanwhile, since the MCM6/7 enhancer contains several E2F transcription sites, adenovirus-mediated overexpression of E2F could reverse the inhibition exerted on MCM6 and MCM7 by atorvastatin. These results suggested that atorvastatin was associated with MCM6 and MCM7 mRNA transcription Figure 3). One critical issue is to evaluate the activation of Rac1 GTPase, which is a key component of cardiovascular pathologies, including cardiac hypertrophy, fibrosis and atrial fibrillation Recent advances have shown that Rac1 GTPase is a major master regulator of cell motility through regulating the generation of reactive oxygen species and cortical actin reorganization, which are induced by regulating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity Basic and clinical studies have demonstrated statins' anti-inflammatory effects Nuclear factor κB (NF-κB) is an important transcription regulatory protein and it is a homo- or heterodimeric complex formed by the Rel-like domain-containing proteins. It is involved in activation of immune cells, T and B lymphocyte development, the stress response, apoptosis and other cellular activities Figure 4).Nitric oxide (NO) synthesis is catalyzed by nitric oxide synthase (NOS) with L-arginine, NADPH and molecular oxygen. It is released from the endothelium and present in a sustained form in the vascular system and also acts as an important protective factor. NO could inhibit platelet aggregation and leukocyte adhesion and infiltration, eliminate free oxygen radicals, dilate blood vessels and affect inflammatory reactions in many other ways Geranylgeranyl pyrophosphate and isoprenoid farnesyl pyrophosphate produced by the mevalonate pathway are able to make proteins lipidation with C-terminal motifs Unlimited proliferation is one of the important characteristics of cancer cells and is an important reason why cancer is difficult to cure. Ras, RhoA and other small G-proteins play critical roles in cell proliferation, metastasis and apoptosis Signal transduction process is closely linked to cell proliferation and survival. Vosper et al. found lovastatin blocked the cell cycle in prostate cancer cells, causing cells to stay in the G1 phase, and inhibited cancer cell proliferation. Downregulation of CDKs or upregulation of cell cycle blocking factor occurred during this process. However, the addition of farnesyl pyrophosphate (FPP) or geranylgeranyl pyrophosphate (GGPP) recovered cell cycle A large number of studies have shown that cessation of the cell cycle does not depend on the lipid-lowering effects of statins, but statins inhibit cancer cell mitosis in a protease-associated manner and exert anti-proliferation effects involving the downregulation of CDK2 expression and upregulation of p21 and p27 activity in vivo and in vitro that statins can induce cancer cell apoptosis, including lovastatin and simvastatin. Apoptosis is a hallmark of cancer cells and induction of cancer cell apoptosis is a hotspot for anti-cancer drug researches. It has been confirmed both Figure 5). Researchers found low concentrations of cerivastatin and lovastatin enhanced endothelial cell proliferation, whereas high concentrations inhibited angiogenesis. Furthermore, these phenotypic variations are related to the prenylation of small G-proteins. Accumulating data show statins induce angiogenesis in a dose-dependent manner, that is, low concentration stimulating angiogenesis and high concentration suppressing angiogenesis In many cancers, angiogenesis is induced to provide nutrients and oxygen for cancer cell growth through the transfer of vessels providing transport to other organs and parts of the body, leading to tumor metastasis. A large number of experiments have shown that statins can inhibit tumor angiogenesis. In Ras-3T3 transgenic mouse tumor models, lovastatin suppresses tumor growth and angiogenesis by inhibiting tumor necrosis factor (TNF-α) Statins can cause many side effects while treating various diseases. The side effects of statins include liver toxicity Statins are primarily metabolized by the liver and excreted by the kidneys. Therefore, if there is liver and renal failure, statins should not be administered. Alcoholics should be closely followed after using statins. Cholestasis can delay the excretion of statins, and increased plasma concentrations of statins may increase the risks of statin-induced myopathy (SIM). In addition, a history of statin drug allergy in SIM patients should also preclude the use of statins Despite being originally developed to address the escalating problem of high cholesterol in cardiovascular disease, statins appear to have extensive untapped potential. What we need to do is to maximize the benefits of statins and try to eliminate the potential side effects. Based on clinical experience, the side effects of various statins are not identical. By comparing the toxicities of various statins in liver cancer cells, it was found that the rank order of cytotoxicity was cerivastatin > simvastatin > atorvastatin > lovastatin > pravastatin


Hence correcting the concerned deficiency and supplementation with S-adenosyl methionine could prove to be protective in chronic alcohol use.One of the significant consequences of alcohol consumption is cancer formation via several contributing factors such as action of alcohol metabolites, vitamin deficiencies, and oxidative stress. All these factors have been shown to cause epigenetic modifications via DNA hypomethylation, thus forming a basis for cancer development. Several published reviews and studies were systematically reviewed. Omnivores and vegetarians differ in terms of nutritional intake and deficiencies. As folate deficiency was found to be common among the omnivores, chronic alcoholism could possibly cause damage and eventually cancer in an omnivorous individual via DNA hypomethylation due to folate deficiency. Furthermore, as niacin was found to be deficient among vegetarians, damage in vegetarian chronic alcoholics could be due to increased NADH/NAD One of the significant consequences of alcohol consumption is cancer formation. Approximately 3.6% of all cancer-related cases worldwide and 3.5% of all cancer-related deaths are attributed to chronic alcohol drinking.This article focuses on a brief account of the processes of alcohol-induced epigenetic modifications and the impact it could have on omnivores and vegetarians based on various published reviews, experimental trials, and randomized control trials. Finally, it highlights some of the more recent progress toward developing therapeutic agents in this promising target space.The concept of epigenetics was understood by going through the Handbook of Epigenetics: The New Molecular and Medical Genetics, 2nd edition by Trygve Tollefsbol published by Elsevier. Several published reviews and studies were systematically reviewed from the following sources:PubmedMedGenTaylor & Francis OnlineElsevierThe search terms included “alcoholism and epigenetics,” “cancer and epigenetics,” “alcohol-induced hepatocellular carcinoma,” “vitamins and cancer,” and “nutrient deficiencies in omnivores and vegetarians,” “meat and cancer.” Reference lists of eligible papers were also searched.DNA methylation has been the center of discussion in several reviews. DNA methylation refers to donation of methyl group by S-adenosyl methionine (SAM) to a cytosine moiety of cytosine–guanine (CpG) dinucleotides in mammalian DNA catalyzed by family of enzymes called DNMTsOnce SAM donates a methyl group, it becomes S-adenosyl homocysteine (SAH), which then forms homocysteine by the action of SAH hydrolase. Homocysteine can either be remethylated to form methionine via a methyl group transfer either from N5-methyltetrahydrofolate (MTHF) by methionine synthase (MS), or from betaine by betaine homocysteine methyltransferase (BHMT) or enter transsulfuration pathway to form glutathione (GSH)Ethanol interferes with DNA methylation in the following ways.Transferase enzymes+deficiencyNADReactive oxygen species (ROS) formationFolate deficiencyTransferase EnzymesEthanol inhibits methionine adenosyl transferase (MAT) and MS, thus inhibiting the formation of SAMDecreased SAM/SAH ratio in the liver was noticed after ethanol feeding in rodent models of alcoholic liver disease (ALD)Acetaldehyde, the first product of oxidative ethanol breakdown also affects DNMT activity in vitro+/NADHNAD+) to NADH while forming acetaldehyde in cytoplasm and acetate in mitochondria by dehydrogenase enzymes , reactive oxygen species (ROS) are also generatedAnother way by which excessive ROS formation can decrease DNA methylation is by acutely depleting GSH and thus diverting homocysteine to transsulfuration pathway to form GSH, rather than producing methionine and SAMFolateDietary folate contributes to the production of 5-methyltetrahydrofolate (5-MTHF) in the DNA synthesis pathway. 5-MTHF is the initial methyl donor to the substrate homocysteine thus forming methionine by the action of Vit B12 dependent MS which in turn forms SAM with the help of MAT and ATPSeveral studies over the years have shown folate deficiency in chronic alcoholism due to several causes.Folate absorption: In chronic alcoholics coming to hospital emergency rooms for alcohol withdrawal, two studies showed a decrease in absorption of 3H-labeled folic acid in comparison to acute ingestion of alcohol.An in vitro studyFolate storage: A study involving alcohol fed rats noticed decreased folate storage enzyme in the liver called folylpolyglutamate synthetase.Folate urinary excretion: Loss of folate in the urine has been documented in chronic alcoholic subjects,Hence folate deficiency in chronic alcoholics can contribute to low levels of SAM and thus a decrease in SAM/SAH ratio+ratio, ROS-induced DNA damage and folate deficiency is reduced SAM/SAH ratio, leading to DNA hypomethylation.The net effect of all these alcohol-induced reduction in methyltransferase activity, reduced synthesis of SAM, increased NADH/NADBased on epidemiological data, it has been concluded that carcinogenesis in numerous organs, including the upper aerodigestive tract, liver, colorectum, and female breast is induced by alcohol.A study reported explains the changes in gene expression and liver injury through epigenetic modifications induced by chronic ethanol feeding and not by acute feeding. The results support the concept that chronic ethanol ingestion induces altered gene expression as a result of changes in epigenetic mechanisms, where acetylation and methylation of histones were altered.All the above-mentioned pathways lead to global DNA hypomethylation. HCC with several etiologies, including chronic alcoholism, is associated with a significant global DNA hypomethylation, which may result in malignant transformation through mechanisms such as loss of imprinting and chromosomal instability.Chronic ethanol feeding in rats alters the methylation and acetylation of histones in the liver.Another study shows that the transformation to HCC is driven by the toll-like receptor 4 (TLR4) signaling pathway, which is upregulated by combined effect of an increase in lipopolysaccharides levels in the liver that result from alcohol abuse and at epigenetic level by inhibition of DNA methylationIncreased DNA instability and aberrant DNA methylation patterns are the two main mechanisms described that may explain the cancer-promoting effects of limited folate levels. Folate deficiency alters the balance of the pool of nucleotides needed for the synthesis of new DNA molecules, leading to dUMP accumulation . As a rA study in Switzerland demonstrated that the highest prevalence for vitamin and mineral deficiencies was as follows: in the omnivorous group, for folic acid (58%); in the vegetarian group, for vitamin B6 and niacin .Studies have shown that low folate intake coupled with high alcohol intake increases the risk for colon cancerAlternatively, prospective cohort study in Australia, Sweden including a cohort from 10 European countries concluded that the consumption of fresh red meat and processed meat seemed to be associated with an increased risk of colorectal cancer.As described above, red or processed meat consumers with folate deficiency may lead to several types of cancers, especially colorectal cancer at a faster rate.+ADP ribosyltransferase and associated DNA repair play an important role in the early initiating stage of liver carcinogenesis.+concentrations in humans lead to HCC.On the other hand, the vegetarians show niacin deficiency. The dietary status of niacin (vitamin B3) has the potential to influence DNA repair, genomic stability, and the immune system, eventually having an impact on cancer risk. Niacin, in the form of NAD, participates in a wide variety of ADP-ribosylation reactions.Various studies have suggested that folate, niacin, betaine, and SAM could prevent ALD progression and tumor formation.Christensen et al+pools via dietary nicotinamide riboside in high cancer risk mice not only reduced dysplastic lesions, but also prevented tumor development.A study using rat hepatoma cells (cultured with ROS generating system) with increased invasive capacity demonstrated that niacin and trigonelline suppressed this ROS-potentiated invasive capacity.In the folate-deficient ethanol fed micropig model, experimental SAM treatment was shown to prevent ALD and its gene expressions for lipogenesis, apoptosis, and oxidative liver injury.+for alcohol oxidative metabolism by alcohol dehydrogenase,Several studies in animal models suggested the possible mechanisms of action of betaine which include increased SAM hepatic levels, reduced SAH levels by increased homocysteine metabolism thus improving SAM/SAH ratio,To summarize, dietary folate and niacin could be protective in cancer development. While there is abundant evidence that SAM and betaine are protective in the development of experimental ALD in animal models, the efficacy of SAM as a treatment modality of established ALD has not been proven conclusively in clinical trials.DNA hypomethylation in chronic alcoholism has been shown to be at the center of cancer development. It can be caused by several ways, especially folate and niacin deficiencies. With studies showing a reduced folate intake as well as reduced plasma levels in omnivores, chronic alcoholism could potentiate the process of colorectal cancer and possibly HCC. Subsequently niacin deficiency in chronic alcoholic vegetarians could result in an increased risk of tumor formation as well. Further studies are required to link cancer development in chronic alcoholics based on contrasting diets and to prove this clinically. If proven, this could very well help in therapeutic applications in the form of folate supplementation in omnivores and niacin supplementation in vegetarians to prevent cancer development or ALD progression. Upregulation of the TLR4 pathway which has been shown to drive transformation of progenitor cells to HCC could be prevented by SAM supplementation as shown in chronically fed rat models. The protective role of SAM and betaine still remains unclear in clinical trials and further studies are necessary to confirm this.


ASXL1, U2AF1, SRSF2 and ZRSR2. The understanding of the underlying biology is limited but a number of hypotheses have been developed and are currently being investigated. This review summarizes the current knowledge about sex-related differences in the clinical outcomes and genomic profile of patients with chronic myeloid neoplasms and discusses the hypothesized biologic mechanisms as an attempt to explain these observations.Chronic myeloid neoplasms are clonal diseases with variable clinical course and outcomes and despite the introduction of novel therapies, patients with high-risk disease continue to have overall poor outcomes. Different groups have highlighted that men have overall worse survival and higher incidence of transformation to acute leukemia compared to women across neoplasms such as myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap neoplasms, and CML. More recent studies evaluating the genomic profile of patients with these neoplasms demonstrated a male predominance for mutations in high-risk genes including  Chronic myeloid neoplasms represent a broad spectrum of disorders ranging from myelodysplastic syndrome (MDS) characterized by dysplasia in the marrow and cytopenias in the peripheral blood to myeloproliferative neoplasms (MPN) characterized by hyperplasia in the marrow and elevated counts in the peripheral blood. These diseases can lead to failure of normal hematopoiesis or transformation to acute myeloid leukemia (AML). Despite the variability in their pathology and clinical presentation these neoplasms arise from malignant stem and progenitor cells carrying somatic mutations . The curIt has been well described that men have overall higher incidence of cancer and higher cancer-related mortality . SimilarThe confirmation of these differences in bigger cohorts and prospective studies may allow the introduction of sex as a prognostic factor in the risk assessment tools used in the clinic. Finally, a deeper understanding of the underlying biology would be particularly valuable as it can provide scientific rationale and potentially lead to the discovery of novel oncogenic pathways implicated in the progression of these neoplasms.The aim of this review is to summarize the existing literature on sex-related differences in the clinical presentation and outcomes of patients with chronic myeloid neoplasms, discuss the recent advances in the association of these clinical observations with differences at the genomic landscape between women and men and present the current biologic hypotheses attempting to explain these findings.Patient characteristics, such as age, performance status and comorbidities, affect the clinical presentation and outcomes of chronic myeloid neoplasms and have been included in risk assessment tools used in the clinic ,10. VariClonal hematopoiesis of indeterminate potential (CHIP) is defined as the presence of at least one somatic mutation that is relevant clinically and is otherwise found in MDS (or other myeloid neoplasms) without the presence of persistent cytopenias or diagnosis of myeloid neoplasms . Male seEarly data provided evidence that MDS is overall more common in men across various age groups  but the MDS/MPN overlap syndromes is a heterogeneous group of malignancies with overlapping features of both MDS and MPN showing a male predominance which is more prominent in chronic myelomonocytic leukemia (CMML) . Wang etJAK2, CALR or MPL genes but with a notable variability in the clinical presentations and outcomes [MPN are clonal myeloid neoplasms including essential thrombocytosis (ET), polycythemia vera (PV) and myelofibrosis (MF) sharing common driver mutations in the outcomes . For ET outcomes .Sex is an important factor affecting the presentation of patients with MPN. Women have overall a higher prevalence of MPN, they are younger at diagnosis but they predominate in ET while men predominate in PV and PMF ,6,21. InThe impact of sex in the clinical outcomes of MPN patients has been studied by different groups over the last decade. Tefferi et al. analyzed 1494 patients with ET demonstrating that male sex is associated with worse survival independent of patients’ age, leukocyte count and IPSET score . Our groThe natural history of CML has been altered significantly following the introduction of tyrosine kinase inhibitors (TKI) with a tremendous improvement of patients’ survival . Given tSokal et al. in an early study highlighted that male sex is a negative prognostic indicator independent of spleen size, hemoglobin, levels, platelet counts and percentage of circulating blasts among young (<45 years) patients with CML . Berger Based on the sex-related differences in the presentation and outcomes of CML patients before the use of TKIs, it could be hypothesized that male sex may be implicated in the acquisition of secondary molecular events driving disease progression.The observed differences in the presentation and outcomes between women and men with chronic myeloid neoplasms are summarized in Chronic myeloid neoplasms are characterized by the presence of genomic alterations in stem and progenitor cells and the analysis of the genomic landscape of these neoplasms has significantly improved our understanding of their biology and has led to the introduction of novel therapeutic agents for their treatment ,19. DespDNMT3A and TET2 mutations in women with CHIP [U2AF1 and ZSRS2 mutations whereas women with MDS have higher incidence of DNMT3A and TP53 mutations [DNMT3A mutations while mutations in the ASXL1 gene were more common among men [EZH2 gene independent of the specific disease sub-type .The comparison of the frequency of specific CHIP-related somatic mutations between women and men has revealed a modest increase in the frequency of ith CHIP . De-Morgutations . Bose etmong men . We receASXL1, IDH1/2, U2AF1, SRSF2 and EZH2 [The analysis of the next generation sequencing data of 227 patients with MPN revealed that men have a higher number of non-MPN-specific somatic mutations and higher incidence of one or two mutations in genes such as and EZH2 . These gand EZH2 .Despite that the connection between the sex-related differences in the mutational profile and the worse clinical outcomes of men is still missing, these results suggest that men with chronic myeloid neoplasms have overall more mutations in genes involved in RNA splicing and epigenetic regulation and confer a higher risk for disease progression and overall worse outcomes.ASXL1, U2AF1, SRSF2, BCOR and RUNX1 genes [RUNX1, ASXL1, SRSF2, STAG2, and BCOR genes were more prevalent in men compared to women in a cohort of 664 AML patients [RUNX1, ZRSR2, SRSF2, U2AF1, ASXL1 and EZH2 mutations [The evaluation of AML cohorts has also revealed interesting findings associated with sex-related differences that can be utilized to extract useful conclusions given that a significant percentage of AMLs are derived from underlying chronic myeloid neoplasms. De-Morgan et al. demonstrated that men with AML had more often “pre-leukemic” somatic mutations which are found usually in chronic myeloid neoplasms such as mutations in X1 genes . The higpatients . Finallyutations . These rThe underlying biologic mechanisms implicated in the sex-related differences in the clinical outcomes and genomic profile of patients with chronic myeloid neoplasms have not been well studied. Men in the USA carry an age-adjusted excess risk of 20.4% of developing any cancer and there is ≥2:1 male predominance for some cancer types . These dATRX, CNKSR2, DDX3X, KDM5C, KDM6A, and MAGEC3) harbor more often loss-of-function mutations in men compared to women across 21 different cancer types [BCOR, PHF6, STAG2 and ZRSR2 are encoded by chromosome X [ZRSR2 causing either a premature truncation or a large structural change of the protein, leading to loss-of-function are found exclusively among men and not women [ZRSR2 mutations are enriched in blastic plasmacytoid dendritic cell neoplasm, a rare leukemia with a three times higher incidence among men and almost all these mutations occur in men [The “escape from X-inactivation tumor-suppressor” (EXITS) hypothesis is that biallelic expression of genes encoding tumor suppressors located in the non-pseudoautosomal region (PAR) of X-chromosome affords females enhanced cancer protection, which substantively contributes to the observed higher incidence of some tumors in men ,39,40. Der types  providinmosome X . Thus, amosome X . Consistot women . More rer in men .Xist, which is a non-coding X-linked gene and is expressed only in females. Of note, the silencing of this gene causes aberrant maturation and age-dependent-loss in hematopoietic stem cells through X chromosome re-activation. Particularly, Xist deletion in the hematopoietic compartment of mice led to the development of a highly aggressive MDS/MPN phenotype in female mice.The initiation and maintenance of the X-chromosome inactivation requires Overall, these results support that potentially X-chromosome inactivation can protect women from loss-of-function mutations in genes that are implicated in the development and progression of chronic myeloid neoplasms and particularly genes involved in splicing machinery and transcriptional regulation.An alternative hypothesis to explain the different outcomes between women and men with myeloid neoplasms is that they could have different responses to the available therapies. Particularly, the cytidine analogue drugs azacitidine and decitabine are the most commonly used FDA approved agents for the treatment of patients with MDS and MDS/MPN . These aMahfouz et al. found that men have significantly higher CDA activity compared to women associated with worse overall survival in men with MDS treated with azacitidine or decitabine compared to women . The autPossible implications of these differences in the clinic have already been studied. DeZern et al. evaluated the impact of sex on the response of MDS patients to cytidine analogues and found that women treated with decitabine had significantly better overall survival compared to women treated with azacitdine while treatment with decitabine or azacitidine did not affect the survival of men . Given tFinally, another important implication of sex-related differences in the metabolism of cytidine analogues is the assessment of response of men and women to oral cedazuridine/decitabine, which was recently approved for patients with MDS and CMML . CedazurJAK2V617F CD34+ progenitor and neutrophil allele burdens in MPN patients has been correlated with more advanced disease and MF phenotype [JAK2V617F allele burden in the neutrophils of 524 MPN patients and found that there are no significant differences between men and women across the different MPN phenotypes [JAK2V617F allele burden compared to women with PV [JAK2V617F allele burden in their neutrophils [JAK2V617F allele burden in the CD34+ cells of 121 patients we found that men have higher allele burden independent of the specific MPN phenotype with the difference being more prominent in the lower-risk phenotypes of ET and PV [The increased burden of somatic mutations in bone marrow samples has been associated with overall worse survival in patients with myeloid neoplasms ,51,52,53henotype . Our gro with PV  while amMore rapid expansion of neoplastic clones in the stem and progenitor compartments in men could be an interesting alternative hypothesis providing a rational for their overall worse outcomes and faster progression to higher-risk phenotypes and acute leukemia. However, further studies in bigger cohorts of different myeloid neoplasms should be performed to confirm these findings. Finally, if this observation is confirmed the underlying molecular biology needs to be further elucidated.Estrogen and androgen receptors  have been implicated in the development and progression of malignancies such as breast, prostate and gastric cancer . NumerouJAK2V617F mutated hematopoietic stem cells and its activation by tamoxifen suppresses the progression of JAK2V617F-mediated MPN disease in mice and increases the sensitivity of MLL-AF9+ leukemias to chemotherapy [JAK2V617F allele burden in the CD34+ cells of women [Sanchez-Aguilera et al. showed that the ER induces the apoptosis of of women ,17. GiveBased on a study by Chuang et al., AR knockout in a transgenic mouse model leads to the development of significant neutropenia due to suppression of terminal differentiation of granulocytes . The autZRSR2, U2AF1, SRSF2, and ASXL1. Despite that various hypotheses have been developed including the X-chromosome inactivation hypothesis, the different metabolism of cytidine analogues, alterations in the primitive cells’ compartment and implication of hormonal receptors, these differences remain not well understood. It is possible that more than one of these mechanisms contribute to these clinical observations. Better understanding of the underlying biology can improve the prognostication of patients with myeloid neoplasms and provide opportunities for exciting novel therapies. Finally, further confirmation of these differences could permit the addition of sex as an independent prognostic factor in the risk-assessment tools used in the clinic especially for patients with lower risk diseases.Based on data from different cohorts, men with myeloid diseases have overall worse outcomes compared to women, which tend to be more prominent among patients with lower risk phenotypes. These results have been associated with reproducible sex-related differences in the genomic landscape of patients with myeloid neoplasms demonstrating that men have higher incidence of mutations in high-risk genes such as


This finding was evident after adjustment for adherence to the protein intake guideline and abdominal obesity. Similar impacts were observed for muscle mass and 5-STS but not for handgrip strength. In conclusion, guideline adherence to MSA is related to lower sarcopenia risk in older adults who already accumulate 150 weekly minutes of MVPA, which reinforces the promotion of the MSA guideline, alongside the MVPA guideline, to fight against sarcopenia progression in ageing populations.Sarcopenia in older adults is associated with a higher risk of falls, disability, loss of independence, and mortality. Current physical activity (PA) guidelines recommend engagement in muscle-strengthening activities (MSA) in addition to aerobic moderate-to-vigorous physical activity (MVPA). However, little is known about the impact of MSA in addition to adherence to the MVPA recommendation in the guidelines. The aim of the present cross-sectional study was to determine whether or not engagement in MSA is linked to sarcopenia risk in older adults who meet the PA guidelines of 150 min of MVPA per week. A total of 193 community-dwelling older men and women (65–70 years) were included in the study. A continuous sex-specific clustered sarcopenia risk score (SRS) was created based on muscle mass assessed using bioelectrical impedance analysis, handgrip strength, and five times sit-to-stand (5STS) time, assessed using standardized procedures. Adherence to PA guidelines was assessed using the Actigraph GT3x accelerometer and the EPAQ2 questionnaire. Guideline adherence to MSA twice a week was related to a significantly ( Sarcopenia is a progressive skeletal muscle disorder clinically defined by low levels of muscle strength and muscle mass, with physical performance as an indicator of severity . Older aGiven the global ageing trend, with the estimated number of people worldwide aged over 65 years reaching nearly 1.5 billion in 2050 , it is eUnfortunately, it is currently estimated that less than 20% of older adults adhere to the current PA guidelines including the MSA recommendation ,14,15, wCurrently, there is a paucity of data on the potential health-related impacts of the MSA recommendation beyond those inferred by accumulating 150 weekly minutes of MVPA. Hence, the MSA recommendation has been referred to as the forgotten guideline . A limitTherefore, the aim of the present study was to determine whether or not engagement in MSA is linked to sarcopenia risk in older adults who meet the recommendation in the PA guidelines of 150 min of MVPA per week.n = 71) and women (n = 122), 65–70 years old, recruited through local advertisement within the frame of the EURODIET project. Inclusion criteria included the following: absence of overt disease, cardiovascular, diabetes and psychiatric conditions; disability in respect to mobility; accumulating a weekly amount of at least 150 min of MVPA. All investigations were conducted in accordance with the principles set by the Declaration of Helsinki. All participants were provided written information about the study and written consent was obtained. The study protocol was approved by the regional ethics committee of Uppsala, Sweden.The present cross-sectional study included 193 community-dwelling older men  using standard procedures. Waist circumference (WC) was measured to the nearest 0.1 cm at the midpoint between the iliac crest and lower costal margin using the Seca 203  circumference measuring tape. Body weight and skeletal muscle mass index (SMI) were assessed using bioelectrical impedance analysis . Skeletal muscle mass (SMM) was calculated according to the equation of Janssen et al. [Adherence to the PA guidelines regarding 150 weekly min of MVPA was assessed using the Actigraph GT3x  accelerometer for a week, as previously described . AccelerAdherence to recommended amounts of protein intake was assessed by a validated 84-item food frequency questionnaire (FFQ) . Daily pHandgrip strength was assessed by standardized procedures using a Jamar handheld dynamometer . A five times sit-to-stand (5STS) was performed, whereby participants were instructed to start from a standing fully upright position and to sit down in a chair and repeat this sequence 5 times.A continuous clustered sarcopenia risk score (SRS) was created in accordance with the recent operational definition of sarcopenia by the European Working Group on Sarcopenia in Older People (EWGSOP) , includiData are presented as mean and standard deviation unless otherwise stated and checked for normality using visual inspection of probability plots, as well as conducting the Kolmogorov–Smirnov normality test. Differences between males and females were determined by either independent sample t-tests or Chi-square tests. Factorial analysis of variance (ANOVA) was employed to determine differences in sex-specific SRS between those reporting MSA at least twice a week (adherence) or not (no adherence). First, ANOVA models were adjusted by potential influence of age, use of prescribed medication (yes/no) and tobacco use , waist circumference (WC) based on established metabolic risk cut-points of ≥94 cm for men and ≥80 cm for women , and adhp < 0.05) and SMI (p < 0.05) compared to women, with no differences observed in 5STS , whereas those reporting MSA twice a week had a significantly lower SRS compared to those who did not (p < 0.05) and 5STS (p < 0.05), with higher SMI and better 5STS performance in those adhering to the MSA guideline compared to those who did not (p = 0.999).We investigated the related impact of adhering to MSA twice a week on SRS and its separate components . The ANOVA revealed a significant main effect of MSA adherence on SRS , where those reporting MSA at least once a week had a better functional performance (5STS)  as compared with those who did not. These main effects remained evident after adjustment for adherence to the recommended protein intake guideline and other covariates. In contrast to results when comparing groups who reported MSA at least twice a week, no differences in SRS  and SMI  were revealed when comparing groups reporting MSA at least once a week or not. Finally, no difference in handgrip strength was observed between MSA groups . In addition, given that older adults may still receive health benefits from lower volumes of MSA than currently recommended, we re-analyzed our data to investigate whether engagement in MSA at least once instead of at least twice a week impacts on SRS and its related components. Interestingly, a significant main effect of MSA on 5STS performance was observed  directly affecting the weekly volume of MSA . Indeed, a previous systematic review concluded that training twice a week promoted superior effects on measures of muscle hypertrophy as compared with once a week [Our study findings are strengthened by the use of validated tools for assessment of adherence to PA guidelines, including accelerometery-determined MVPA time and the well-established EPAQ2 questionnaire. Another strength is the inclusion of important variables known to have an influence on measures of sarcopenia risk when determining the independent role of MSA. By adjusting for adherence to the protein intake guideline, we accounted for a powerful driver of muscle hypertrophy, as it has recently been linked to both muscle mass  and sarcThe present study shows that engagement in MSA at least twice a week is linked to sarcopenia risk, with larger muscle mass and better physical performance in older adults who already accumulate 150 weekly minutes of MVPA. This beneficial impact is independent of adherence to the recommended protein intake, which further reinforces the promotion of the recommendation of MSA at least twice a week, along with the general accumulation of weekly MVPA time to combat sarcopenia progression in older adults.


Pig pluripotent stem cells have tremendous potential because the pig is a valuable animal as both an agricultural resource and as a preclinical model of human therapy. To date, a lack of understanding of pig pluripotency has inhibited the derivation of embryonic stem cells (ESCs) and transgene-free induced pluripotent stem cells. Therefore, there has been no accessible or reliable transcriptome data for researching the genuine pig pluripotency network. Our previous study isolated authentic pig ESCs, which had teratoma-forming and direct differentiation ability, that were derived by activating the FGF2, ACTIVIN A, and WNT pathways. Here, we aimed to provide detailed information on transcriptome data of the newly derived pig ESCs and perform a comparative analysis with pig preimplantation embryo transcriptomes in a public database.The transcriptome data of ESCs derived from in vitro fertilized and parthenogenetic embryos were generated by HiSeq 2500. Then, differentially expressed genes (DEGs) from each sample were compared with fibroblasts, and gene expression profiling was carried out for comparative analysis. Our data, as the first transcriptome dataset for genuine pig pluripotent cells, could be a general reference for explaining the molecular mechanism of species-specific pluripotency and improving understanding of the embryo development of domestic animals. The pig is a valuable animal as a preclinical model of human therapy as well as an agricultural resource. Recent studies have found that pig germ cell development more closely resembles the molecular features of human germ cell specification than that of the mouse  and thatTranscriptome data of PSCs derived from embryos and somatic cells are considered to be valuable tools for the investigation of pluripotency. Transcriptome analysis of pluripotent cells provides a comprehensive biological insight into developmental cues that we couldn’t acquire before through traditional gene analysis techniques. Pluripotent cells originate during the preimplantation period of embryo development and the reprogramming process of somatic cells . AccordiPig ESCs were cultured with feeder cells made of mitotically inactivated mouse embryonic fibroblasts to maintain their pluripotency in vitro. Therefore, the feeder cells needed to be removed from the ESC samples to obtain a highly reliable RNA-seq data from a homogenous population of the undifferentiated pig ESCs. To exclude these contaminating cells, the pig ESCs were sorted by a MACS system using an antibody against SSEA1, which is highly expressed on the surface of undifferentiated pig ESCs . The RNAAs evaluated by the Agilent 2100 BioAnalyzer, all of the libraries used in this study have a uniform and constant size of approximately 380 bp and passed quality control to be used for sequencing. Then, base call quality scores were calculated by the FastQC program to assess the quality of the raw sequencing data of the cDNA libraries (Dataset 1). Low-quality reads were filtered according to the criteria as described in the Methodology (Supplementary file 1). After filtration of low-quality reads, as shown in the box plot distributions of Figure S1, the quality scores across all bases were in the high confidence range. The filtered sequence reads and previously reported data for pig preimplantation embryos  were alip value < 0.05 . Compared with fibroblasts, 2189, 2450 and 2327 genes were upregulated in PG-ES-3, PG-ES-7 and IVF-ES-11, respectively, and 1887 genes were commonly expressed in the three different ESC lines . Moreover, upregulated DEGs in the embryo samples were compared with genes commonly upregulated in ESCs. As a result, 200 genes were commonly expressed in all samples, and 936, 542, 269 and 283 genes were uniquely expressed in ESCs, day 7–8 ICM, day 10–11 epiblasts and day 12–13 epiblasts, respectively . Finally, expression of genes related to pluripotency, cellular signaling and lipid metabolism were analyzed . Pig ESCs had more similarity with epiblasts than the ICM, indicating similar patterns observed in a previous study [The R package TTC was used for differentially expressed genes (DEGs) analysis (Dataset 3). DEGs of embryos and ESCs compared with fibroblasts were measured based on cutoffs of fold change > 2.5 and a The sample size of the experimental groups was not sufficient for reliable comparative analysis. Gender and sequencing platforms were not matched between ESCs and embryo samples.


Malignant brain tumor—glioblastoma is not only difficult to treat but also hard to study and model. One of the reasons for these is their heterogeneity, i.e., individual tumors consisting of cancer cells that are unlike each other. Such diverse cells can thrive due to the simultaneous co-evolution of anatomic niches and adaption into zones with distorted homeostasis of oxygen. It dampens cytotoxic and immune therapies as the response depends on the cellular composition and its adaptation to hypoxia. We explored what transcriptome reposition strategies are used by cells in the different areas of the tumor. We created the hypoxic map by differential expression analysis between hypoxic and cellular features using RNA sequencing data cross-referenced with the tumor’s anatomic features (Ivy Glioblastoma Atlas Project). The molecular functions of genes differentially expressed in the hypoxic regions were analyzed by a systematic review of the gene ontology analysis. To put a hypoxic niche signature into a clinical context, we associated the model with patients’ survival datasets (The Cancer Genome Atlas). The most unique class of genes in the hypoxic area of the tumor was associated with the process of autophagy. Both hypoxic and cellular anatomic features were enriched in immune response genes whose, along with autophagy cluster genes, had the power to predict glioblastoma patient survival. Our analysis revealed that transcriptome responsive to hypoxia predicted worse patients’ outcomes by driving tumor cell adaptation to metabolic stress and immune escape. Malignant tumors of the brain, such as glioblastomas, are among the most feared ones. They progress fast, killing about half of the patients within a year from the diagnosis . Unlike There are many hurdles for brain tumor therapy: heterogeneity, acidic microenvironment, or macrophage clearance and immunosuppression ,7, and aTranscriptome profiling of both: glioblastoma tissue specimens  and singHIF1A/EPAS1 are currently clinically approved.Hypoxia also promotes tumor immune evasion via diverse mechanisms. These include cellular adaptation, autophagy, and immune-suppressive secretion ,15,16,17Clearly, the adaptation of cancer cells to constantly evolving conditions of their microenvironment during tumor progression requires dynamic and flexible mechanisms. Yet, there is often insufficient understanding of the intra-tumoral evolution of such adaptation and co-operation between cancer cells that result in hypoxia- and autophagy-driven survival. To shed some light on the link between response to hypoxia and cell survival or death, we analyzed drivers and effectors of hypoxic response, also in the context of a recently rapidly developing immunotherapy strategy. EPAS1 gene (EPAS1 (likewise for HIF1A) is regulated mostly by protein stabilization [To show how assessments between RNA-seq and in situ hybridization (ISH) were performed , we presAS1 gene A, bottomlization , the traDifferential gene expression analysis revealed a total of 2707 genes whose expression was enriched or depleted in the PN hypoxic niche vs. CT . The oriEPAS1 in the hypoxic zone is PRKAA2 encoding for alpha 2 subunit of AMP-activated protein kinase (AMPK) kinase complex—a conserved evolutionary sensor of energy availability [EPAS1 expression in the IT/LE feature  binding motifs. Both up and down-regulated signatures revealed significant enrichment of certain TF dependent genes, indicating considerable activation of transcriptional regulation of genes in the PN zone . It reveMSH2 (gene encoding DNA mismatch repair protein MSH2) and CD274 genes (encoding programmed death-ligand 1 (PD-L1) also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) from CT and PN anatomic features  (The genes associated with the process of autophagy D are enrotein 3) F. We queBNIP3 was MiR-210HG (r = 0.734) – (non-coding RNA MIR210 host gene) hypoxia adaptative response gene [OLFML3) is a gene the most inversely associated with BNIP3 expression (r = −0.767)  . As OLFMreceptor , lack ofPLK3) engaged in the regulation of chaperone-mediated autophagy [The analysis of transcriptome has a long history of clinical implications, and in addition to discovering new biomarkers or therapeutic targets, it can identify prognostic markers as well. To funnel our analysis into the context of clinically relevant targets, we developed an admixture model using a 2707 gene signature  queried utophagy  and pro-utophagy   transcripts are not only the most prevalent in the transcriptome, but they are often expressed in a cell- and tissue-specific manner . As a reSome experimental evidence suggests that tumor cells may cope with hypoxia by turning on the migratory phenotype to escape from metabolically stressful events/locations . As an eDecades of research on hypoxia in the brain provided ample examples of its participation in the neurodegeneration, including an inverse correlation between the likelihood of cancer and neurodegenerative diseases , which, The buildup of hypoxia is one of the hallmarks of solid tumors, including glioblastoma. The hypoxic environment that cannot be eradicated from the brain tumor environment is undoubtedly the source of intra-tumoral heterogeneity and strong selective force that leads to the emergence of exceptionally durable and resistant tumor cell subpopulations. Thus, the cell’s capability to respond and adapt to hypoxia is an essential pro-survival trait, whose nuts and bolts require a better understanding for the development of targeted therapies, including immunotherapy. Our metadata analysis-based findings identifying the selective rise of the autophagy within the region, along with the remodeled immune response, shed new light on the adaptative strengths of cancer cells withstanding the hypoxic environment. These transcriptome rearrangements point toward the repositioning of metabolism and immune evasion as primary mechanisms promoting cancer cell survival and therapy resistance due to hypoxic signaling.The median survival for a patient with glioblastoma is 12 to 15 months, and not much improvement have been achieved for decades , they rep < 0.1, Benjamini–Hochberg corrected).To create the hypoxic map, we first performed differential expression analysis using the data from Ivy Glioblastoma Atlas Project (Ivy GAP). This clinical and genomic database was created using 41 glioblastoma grade IV patient cohort and 122 RNA samples see  that wern = 528) and control specimens (n = 10) analyzed by Affymetrix HT_HG-U133A microarray gene chips were queried with selected gene sets from Ivy GAP. These data allowed us to select genes with the utmost predictive outcome using Kaplan–Meier estimator survival analysis. In the Ivy GAP dataset, only three tumors from 41-patient cohort harbored Arg132 → His mutation in isocitrate dehydrogenase 1 (IDH1); thus, there was insufficient statistical power for the analysis of this mutation by anatomic feature. Consequently, we excluded patients with this mutation from the survival analysis. To catalog the molecular function of genes differentially expressed in the hypoxic region of the tumor, we conducted a systematic review using the Gene Ontology database created in December 2019. This strategy identified genes’ sets whose expression is enriched or depleted in the hypoxic anatomic features and described a molecular/cellular function of such signatures. Genes were eligible for inclusion if the standard nomenclature identified them as human genes in the HUGO Gene Nomenclature Committee database. To put a hypoxic niche signature into a broader clinical context, we queried the list of genes with The Cancer Genome Atlas glioblastoma (TCGA GBM) data portal, as previously described . BrieflyEthical Statement: The collection of the data from TCGA GBM and the Ivy GAP was compliant with all applicable laws, regulations, and policies for the protection of human subjects.Over the past decades, transcriptome-wide studies on human malignancies have produced vast amounts of data. Yet understandable need to prioritize, along with bandwidth limitations, led to inadequate depth of analysis of these valuable resources. Thus, our goal in this study was to use the metadata toolbox to gain more insight into our area of interest, i.e., transcriptomic fundaments of adaptative capabilities possessed by brain tumor cells to cope with hallmark exposure to hypoxic stress. We analyzed various layers of acquired data to identify pathways and functional signaling modes prevalent in hypoxic areas of the tumor. We identified the “autophagy” as the most unique, and the “immune response” as diversely de-regulated classes of genes prevalent in hypoxic tumors. These findings have clinical implications, as they revealed transcriptional programs acting in tumor anatomic site- and hypoxia-dependent contexts perpetuated by tumor evolution and stress buildup enhancing tumor heterogeneity and promoting therapy resistance; thus, co-targeting of hypoxic adaptations would be beneficial for glioblastoma patients.


Pulmonary infections are common in hematopoietic cell transplant (HCT) patients of all ages and are associated with high levels of morbidity and mortality. Bacterial, viral, fungal, and parasitic pathogens are all represented as causes of infection. The lung mounts a complex immune response to infection and this response is significantly affected by the pre-HCT conditioning regimen, graft characteristics, and ongoing immunomodulatory therapy. We review the published literature, including animal models as well as human data, to describe what is known about the pulmonary immune response to infection in HCT recipients. Studies have focused on the pulmonary immune response to Aspergillus fumigatus, gram-positive and gram-negative bacteria, and viruses, and show a range of defects associated with both the innate and adaptive immune responses after HCT. There are still many open areas for research, to delineate novel therapeutic targets for pulmonary infections as well as to explore linkages to non-infectious inflammatory lung conditions. Hematopoietic cell transplantation (HCT) is being used for treatment of an increasing number of malignant and non-malignant disorders in all age groups, but is associated with numerous complications. Infections are a major complication due to immune system derangements associated with HCT. In particular, pulmonary infections make up a large portion of HCT-related infections in both autologous and allogeneic HCT patients and are associated with significant morbidity and mortality , 2. Pulmvia the innate immune response, using pattern recognition receptors (PRRs) that identify pathogen-associated molecular patterns (PAMPs) such as bacterial or fungal cell wall components or viral nucleic acids. Alveolar macrophages also have a critical function in activating immune and inflammatory responses. Airway and alveolar epithelial cells are increasingly recognized to have an important part in recognizing pathogens as well. On recognition of pathogens, production of inflammatory cytokines and chemokines leads to the recruitment of neutrophils and monocytes to the lung for phagocytosis and killing of pathogens. Antigen-presenting cells that include AMs as well as resident dendritic cells provide a bridge to activate the adaptive immune system by traveling to local lymphoid tissue and presenting antigens to T cells. The activation of T cell and B cell responses results in killing of intracellular pathogens, production of antibodies, development of immune memory, and regulation of the immune response. The T helper (Th) response can be polarized toward different pathways, such as Th1, Th2, Th17, and T regulatory (Treg), with varying effects on the predominant immune response  play an important role in initial recognition of pathogens response –6.Immune reconstitution post-HCT is a complex and prolonged process, with the timing affected by many factors including type of transplant , conditioning regimen (myeloablative vs. non-myeloablative), source of transplant , and presence of graft vs. host disease (GVHD) , 8. The Overall, ample data suggest that recipient-derived AMs are only fully ablated with the most severe total body irradiation (TBI)-based conditioning, and for patients not receiving TBI-based conditioning, reconstitution of AMs from the donor lineage can take weeks. In mice, Matute-Bello et al. found that reconstitution of AMs of donor origin was slower than reconstitution of peripheral leukocytes after total body irradiation and syngeneic HCT . At 30 d+ DCs depleted by day 3 post-irradiation and repleted with more than 90% donor cells by day 7 post irradiation. CD11b+ DCs were less sensitive, with 60% depletion by day 5 post-transplant and reconstitution with more than 90% donor derived cells at 4 weeks post-transplant  and lung tissue that provide information on immune response in the human lung. In addition, studies of genetic polymorphisms predisposing to infection in human HCT patients have provided suggestions of the importance of particular components of the immune response to the response to pulmonary infections in HCT patients. The primary areas of study have focused on the following three pathogens or groups of pathogens: Aspergillus fumigatus, gram-negative and gram-positive bacteria, and viruses (primarily herpesviruses).A substantial body of work on pulmonary immune responses to infection after HCT has focused on the ubiquitous mold Aspergillus fumigatus. Invasive aspergillosis (IA) most commonly involves the lung, is the most common invasive fungal infection in HCT patients, and has reported case fatality rates of 29–87% , 17. Immvia the innate immune response and antigen presenting cells is critical to anti-fungal immunity. The importance of PRRs in activating the innate response to IA is suggested by a number of studies associating polymorphisms in human PRRs with susceptibility to IA in HCT patients. Toll-like receptors (TLRs) comprise a family of PRRs that play a key role in the innate immune response by recognizing a range of PAMPs and signaling to produce a pro-inflammatory response. Donor-derived polymorphisms in TLR1, TLR4, and TLR6 have been associated with increased risk of IA in HCT patients  cells providing control of infection via IFN-γ and IL-10 production and Dectin-1 on hematopoietic cells driving an IL-17 mediated response , which acts as an opsonin for A. fumigatus, was described to have a donor-derived polymorphism associated with increased IA risk in allogeneic HCT patients. Individuals who received a transplant with this haplotype had decreased production of PTX3 in BAL fluid and in lung tissue in the setting of IA. Neutrophils from patients with the polymorphism demonstrated reduced phagocytosis and killing of A.fumigatus that was restored by exogenous PTX3 .Defects in anti-A. fumigatus immunity due to impaired cellular killing have also been identified. Effective clearance of A. fumigatus with appropriate control of the inflammatory response involves an autophagy process known as LC3-associated phagocytosis. IFN-γ activates a signaling response that includes death-associated protein kinase 1 (DAPK1). A polymorphism in patient-derived DAPK1 was associated with a significantly increased risk of IA in allogeneic HCT recipients. HCT recipients with the at-risk polymorphism in DAPK1 also demonstrated decreased expression of DAPK1 and increased expression of the inflammasome component NLRP3 and production of IL-1β in PBMCs in response to A. fumigatus conidia, consistent with a phenotype of increased inflammation .via its ligands CCL19 and CCL2. Hartigan et al. demonstrated, unexpectedly, that CCR7 deficient mice had improved survival and decreased lung inflammation compared to WT mice in a neutropenic model. Neutropenic mice receiving syngeneic HCT of CCR7 KO cells also had improved survival and decreased inflammation in Aspergillus infection, associated with increased dendritic cell recruitment to the lung  in a group of allogeneic HCT recipients . Taken tvia TBI as well as those that received conditioning with a combination of cyclophosphamide and busulfan , was also increased in mouse syngeneic HCT AMs secondary to PGE2 stimulation. Inhibition or knock out of PTEN increased AM phagocytosis and killing of P. aeruginosa with increased production of TNF-α  was downregulated in lung tissue, in association with increased serum C3a, in infected HCT mice. Knocking out DAF resulted in worsened survival, increased viral load, increased CD8+ T cells in lungs, and increased IFN-γ production in CD8+ cells after allogeneic HCT, suggesting an important role for the complement system in defense against viral infection , and TGF-β1 in the lungs. The defect in antiviral immunity was shown to be mediated through TGF-β1  pneumonitis, wherein elevated levels of TGF-β1 in BAL fluid appear associated with the severity of HSV pneumonia . Interes+ T cells to the lungs in allogeneic HCT mice, and was improved by adoptive transfer of CD8+ cells from un-transplanted mice recovering from Sendai virus infection. Interestingly, syngeneic HCT mice had similar results to un-transplanted mice after infection  and the presence of Th2 type cytokines in the lungs. Mouse HCT models of murine CMV showed recruitment of but also defects in cytotoxic efficacy of CD8+ T cells, as well as involvement of the complement and leukotriene pathways. Similar to mouse models of bacterial pneumonia, TGF-β1in HCT mice infected with γHV-68 mediates diminished antiviral effect and skewing toward Th17 cytokine response. TGF-β1 also appears to be associated with severity of HSV pneumonia in an allogeneic HCT mouse model. Studies of HSV pneumonitis in human HCT recipients suggest cytotoxic effects of leukocytes against vascular endothelium. CD8+ T cells were also shown to be essential in defense against two respiratory viruses (Sendai virus and adenovirus) in mouse allogeneic HCT models.To summarize, the response to viral pulmonary infections after HCT involves alterations in innate and adaptive immunity. Data on CMV infection in humans demonstrate recruitment of T cells (although there are contradictory findings on involvement of CD4via PRRs and post-PRR signaling pathways as well as polarization of the Th cell response toward Th1 and Treg responses and away from Th17 responses. Dendritic cells appear to be key in controlling inflammation and Th response polarization which has a significant effect on the downstream immune response and either control of infection with regulated inflammation or dysregulated inflammation without infection control. This dysregulated inflammation appears to be associated with lung injury and mortality. Clearance of bacterial infection post HCT is compromised by reduced capacity for phagocytosis and bacterial killing by AMs, a phenotype regulated by TGF-β1 which is produced by alveolar epithelial cells and causes increased production of PGE2, which in turn mediates defects in AM phagocytosis and killing. Neutrophils are also deficient in bacterial killing and NET production due to PGE2 effects. Finally, viral infections require the innate immune system as well as the adaptive immune system for adequate defense, including the activity of CD8+ T cells. The eicosanoid pathway also appears to have an important role in antiviral responses in the lungs post-HCT. Skewing toward Th17 responses involving signaling by TGF-β1 and dendritic cells can have detrimental effects on virus clearance and cause increased inflammatory response and result in a phenotype akin to non-infectious inflammatory HCT-related disorders of the lung such as IPS.Our review of the literature shows that there are a number of derangements in pulmonary immune responses associated with increased vulnerability to infection after HCT. Reconstitution of AMs in the lung lags behind reconstitution of peripheral leukocytes after HCT, and dendritic cells in the lung are more sensitive to radiation than AMs. The timing of reconstitution of other immune cell types such as T cells in the lung post HCT is unclear. Effective defense against A. fumigatus involves innate immune activation There are limitations to the studies described in this review. Some of the polymorphisms associated with alterations in susceptibility to infections have not had a mechanistic investigation. Studies of HCT patients are limited to BAL fluid and lung tissue biopsies. The mouse HCT models vary in strain, conditioning regimen, method of hematopoietic cell isolation, and dose of hematopoietic cells transplanted, which potentially affect the reconstitution of the immune response. In particular, the majority of the mouse models undergo syngeneic transplants which do not have the alloimmune responses seen in allogeneic human HCT. For example, syngeneic HCT mouse models do not get GVHD and are not receiving immunosuppressive medications for prevention or treatment of GVHD, which is a significant difference from much of the HCT patient population. Beyond this, one must also consider the limitations of mouse models such as genetic differences from humans, lack of underlying disease for which patients undergo HCT, and differences in conditioning and post-HCT treatment.The lungs have a unique immune response that requires direct sampling for study, as peripheral blood may have different responses. It is particularly important to understand the pulmonary immune response after HCT given the frequency of pulmonary infections and the associated morbidity and mortality despite aggressive prophylaxis and treatment with a variety of antimicrobial medications. Existing studies have identified significant immune changes post-HCT that affect vulnerability to fungal, bacterial, and viral pathogens. New methods such as next generation sequencing can produce large microbiological and transcriptional datasets that can provide large amounts of information about the host response to infection in the lung. Better understanding of these immune changes yields new targets for therapy and may help understand non-infectious lung disease.MZ performed literature review and manuscript writing and editing. JH performed literature review and manuscript writing and editing. All authors contributed to the article and approved the submitted version.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.


Astrocytes, characterized by a satellite-like morphology, are the most abundant type of glia in the central nervous system. Their main functions have been thought to be limited to providing homeostatic support for neurons, but recent studies have revealed that astrocytes actually actively interact with local neural circuits and play a crucial role in information processing and generating physiological and behavioral responses. Here, we review the emerging roles of astrocytes in many brain regions, particularly by focusing on intracellular changes in astrocytes and their interactions with neurons at the molecular and neural circuit levels. Islr in the olfactory bulb; Kcnj10, Norrin, Olig2, Lgr6, and Fndc5 in the cortex; μ-crystallin in the striatum; Myoc in the dorsal midbrain; and Gdf10 in Bergmann glia of the cerebellum [Astrocytes are the most abundant glial cells in the central nervous system. They are typically characterized by satellite-like morphology with numerous fine processes, but they also exhibit many morphological variations . A massirebellum ,8,9,10.Since astrocytes are non-excitable, they have been traditionally regarded as passive cells that support neurons in the brain . For exaThe olfactory bulb (OB) is the brain region involved in the sense of smell . There aThe prefrontal cortex (PFC) is a part of the cerebral cortex that is connected to many other brain regions, including the hippocampus, nucleus accumbens (NAc), and amygdala ,34. SeveOne of the well-known functions of astrocytes is regulating the extracellular glutamate level, mainly by reuptake of glutamate from the synapse through the glutamate transporter GLT-1 that is expressed in their plasma membranes ,37. It h3R2), which is the astrocyte-specific protein responsible for calcium release from intracellular stores. Similarly, astrocyte-selective overexpression of the dominant-negative domain of synaptobrevin 2 peptide (dnSNARE) in mice leads to a decreased extracellular ATP in PFC and depressive-like behaviors. On the other hand, chemogenetic activation of the Gq pathway in astrocytes by using transgenic mice overexpressing MrgprA1 will promote ATP release from PFC astrocytes and attenuate depressive-like behaviors such as immobility in FST and CSDS-induced social avoidance. Taken together, these results suggest that ATP released from PFC astrocytes exerts its antidepressant effects via the activation of P2 purinergic receptors.It has also been shown that astrocytes in the PFC release ATP into the extracellular space. The level of extracellular ATP in the PFC is decreased in mice susceptible to chronic social defeat stress (CSDS) but unaffected in mice resistant to this stress . IntraceA recent study has demonstrated that lipid signaling in PFC astrocytes modulates depressive-like behaviors . Produci1 adenosine receptor antagonist, suggesting that adenosine is the astrocytic mediator evoking these behavioral consequences. Although it is still necessary to further identify the type of PFC astrocytes mediating the depressive and ethanol-elicited responses, it appears that PFC astrocytes utilize different gliotransmitters and generate different behavioral outcomes in response to different types of stimuli and contexts.PFC astrocytes are also known to participate in the regulation of ethanol consumption and intoxication . Chemoge3R2 have revealed that astrocytes in the motor cortex are significantly impaired in terms of generating spontaneous and extracellular ATP-evoked intracellular calcium transients. IP3R2-knockout mice exhibit poor performance in the forelimb reaching task [It has been shown that astrocytes are necessary for motor-skill learning. Calcium imaging experiments involving brain slices prepared from mice lacking IPing task . SimilarAstrocytes in the primary somatosensory cortex (S1) have been shown to participate in the mechanical allodynia elicited by a partial sciatic nerve ligation . FollowiThe anterior cingulate cortex (ACC) is another brain region that is responsible for pain perception and accompanying emotional experiences ,43. It h1R antagonist in the hippocampus, suggesting ATP/adenosine release from hippocampal astrocytes is responsible for sleep deprivation-induced electrophysiological and cognitive dysfunction [The hippocampus is a crucial brain structure that is responsible for learning and memory . Numeroufunction .Several astrocytic molecules in the hippocampus have been found to mediate synaptic plasticity as well as learning and memory. For example, mice deleted of interleukin-1 receptor type 1 (IL-1rKO) exhibit defective long-term potentiation (LTP) and dysfunctional memory in the fear-conditioning and Morris water maze tests . These pB receptor by GABA released from MSNs is sufficient to elicit an intracellular calcium transient and upregulation of TSP-1 in astrocytes, resulting in augmentation of firing of MSNs and induction of hyperlocomotion.The striatum is a primary input area in the basal ganglia that integrates excitatory and inhibitory signals from several brain areas involved in action selection, motor function, and repetitive and habitual behaviors ,54. A re1 adenosine receptors of presynaptic neurons in the NAc and depresses excitatory synaptic transmission to MSNs. This synaptic depression can be phenocopied by elevating intracellular calcium in astrocytes with hM3Dq. Genetic deletion of IP3R2 abolishes dopamine-induced calcium transients in NAc astrocytes, impairs synaptic depression in the NAc, and reduces amphetamine-induced hyperlocomotion. These findings suggest that astrocytes are the key component in the dopaminergic signaling and mesolimbic reward system.The nucleus accumbens (NAc) of the striatum is a brain region associated with reward and addiction that receives dopaminergic input from the ventral tegmental area (VTA) in the midbrain . A recenThe amygdala is a neural structure associated with the regulation of emotions, including fear and anxiety ,60. It c1 and A2A adenosine receptors [Neuroimaging studies have revealed that patients with mood disorders exhibit a volumetric reduction in the amygdala ,68,69,70eceptors . ConsequA molecular study identified GLT-1 in the CeA as a protein responsible for the comorbidity of depression and anxiety . InhibitIn many brain regions, astrocytes are connected to each other through gap junctions. The importance of gap junctions in fear memory consolidation has been examined in the BLA . ContextAnother study has found that L-lactate acts in the BLA as a gliotransmitter that is critical for drug memory reconsolidation . BlockinOne of the Rho family of GTPases, Ras-related C3 botulinum toxin substrate 1 (Rac1), is another astrocytic protein in the BLA that is involved in conditioned fear memory . AnimalsAstrocytes in the ventrobasal complex (VB) and the lateral habenula (LHb) have been found to regulate somatosensation and depression, respectively ,79. The It has been found that the rat model of depression induced by an intraperitoneal injection of lipopolysaccharide promotes the expression of inwardly rectifying potassium channels 4.1 (Kir4.1) in LHb astrocytes. Kir4.1 is found in the astrocytic plasma membrane surrounding the neuronal soma. Overexpression of Kir4.1 in LHb astrocytes decreases the resting membrane potential in LHb neurons and increases the percentage of neurons exhibiting burst firing. Mice that overexpress Kir4.1 in LHb display depression-like behaviors in the forced swim and sucrose preference tests. In contrast, knockdown of Kir4.1 in the LHb with short hairpin RNA (shRNA) produces the opposite effect on the resting membrane potential and neuronal bursting and reduces depressive-like behaviors exhibited in the congenital learned helplessness model of rats . These rThe arcuate nucleus of the hypothalamus (ARH) is a key region controlling appetite and energy metabolism . Genetic1 receptor antagonist increases ghrelin-evoked food intake and abolishes astrocyte activation-induced inhibition of AGRP neuronal firing. These results suggest that extracellular adenosine converted from extracellular ATP released from astrocytes regulates the activity of AGRP neurons through adenosine A1 receptors and feeding behavior. ARH astrocytes are also involved in high-fat diet-evoked hypothalamic inflammation and weight gain [Yang  has showght gain . AstrocyA antagonist, either pentylenetetrazole or picrotoxin, suggesting that Bmal1 in SCN astrocytes controls the circadian rhythm of SCN neurons and circadian behavior via GABAA receptor-mediated signaling. The suprachiasmatic nucleus (SCN) of the hypothalamus is the central pacemaker responsible for controlling circadian rhythms . CircadiAnother piece of evidence showing the involvement of SCN astrocytes in circadian rhythm has been provided by a study using mice lacking both Cry1 and Cry2 . The delThe VTA is a midbrain structure that has been implicated in reward and aversion and contains diverse neuronal cell types, including dopaminergic and GABAergic neurons ,94. OptoThe periaqueductal gray (PAG) is a midbrain region surrounding the cerebral aqueduct that receives nociceptive inputs from the spinal cord and some regions of the brain and then transmits the information to other brain regions associated with pain processing . ReactivReactive astrocytes have also been observed in the PAG of mice with bone cancer pain (BCP) . A localThe preBötzinger complex (preBötC) is a region of the ventrolateral medulla oblongata that is responsible for the generation of respiratory rhythm ,102,103.The cerebellum (CB) is a brain region involved in movement and coordination, including maintaining balance, coordinating movement, and motor learning ,115. TheThe role of astrocytic GABA in regulating motor function has also been investigated by blocking tonic GABA release from BG by using knockout mice deleted of either bestrophin 1 or astrocytic GABA synthesizing monoamine oxidase B (MAOB), or by performing pharmacological inhibition of MAOB with selegiline . Such maThe potential involvement of astrocytes in chronic itch has been identified in the spinal dorsal horn, a key region involved in the processing and relaying of itch signals received from pruriceptors to the brain ,123. ReaMultiple lines of evidence indicate that astrocytes in the spinal cord participate in pain processing, particularly that of chronic pain. For example, selective genetic deletion of GLT-1 only from astrocytes in the spinal cord results in aggravated tactile allodynia in the mouse model of neuropathic pain . InteresNerve injury causes upregulation of Cx43 and also a transition from Cx43-containing gap junctions to Cx43 hemichannels in astrocytes . Astrocy1 receptors and therefore disinhibiting NK1R-expressing projection neurons. In behavioral terms, optogenetic stimulation of astrocytes is sufficient to elicit mechanical allodynia and thermal hyperalgesia in uninjured animals and augments nocifensive behavior in animals with neuropathic pain.Finally, Nam and coworkers have demonstrated that optogenetic manipulation of astrocytes in the spinal cord’s dorsal horn using ChR2 leads to ATP release . ExtraceMany recent studies have identified astrocytes as one of the pivotal components required for information processing in the central nervous system and the generation of physiological and pathological behaviors . The novA brain region typically contains heterogeneous subpopulations of neurons that contribute to generating a behavioral outcome through excitatory and inhibitory interactions. Many brain regions have also been found to contain subpopulations of neurons that mediate distinct or opposing behaviors ,142,143.One of the barriers to understating the role of astrocytes is the difficulty in determining the inputs that initiate astrocytic responses in vivo. Unlike the situation in neurons, physical or chemical interaction of astrocytes with potential communicating partners such as neurons and microglia is difficult to identify. Some studies have shown that astrocytes are activated either by neural signals passing through the neighboring neurons or by inflammation and pathological changes in the brain area ,150,151.3 receptor type 2, which is the most abundant isoform of IP3R in astrocytes, have been employed in many studies and contributed significantly to revealing novel functions of astrocytes from diverse brain regions [3R2 from the whole body throughout the lifetime of the animal and the insufficient inhibition of calcium signaling in astrocytes [3R-mediated calcium release in astrocytes by overexpressing either the IP3 ligand-binding domain of IP3R1 or the pleckstrin homology domain of phospholipase C-like protein p130 to compete with the endogenous IP3Rs for IP3 binding [Another obstacle to studying astrocytes in vivo is the limited efficacy of molecular genetic tools to perform so-called “loss-of-function” manipulations. Except for cytotoxic chemicals ablating astrocytes, most of the available tools are designed to inhibit an elevation of calcium in the cytosolic compartment of astrocytes, which is the key event necessary for astrocytic function ,153. For regions ,154,155.trocytes ,157. A f binding ,159. 3R nor those involving hPMCA2w/b allow for reversible, temporally controlled manipulation, which is required for establishing a causal relationship between a manipulation and its behavioral consequences. ChR2, the most widely used optogenetic tool, is a nonselective cation channel gated by blue light. When expressed in neurons, the opening of ChR2 causes a passive diffusion of proton, sodium, potassium, and calcium ions down their concentration gradients, which leads to depolarization of the plasma membrane and the generation of action potentials. ChR2 has been adapted in the study of astrocytes often as a “gain-of-function” tool. A recent study has shown an unanticipated finding that ChR2 in astrocytes can lead to activation of nearby neurons by elevating extracellular potassium ions [The chemogenetic and optogenetic toolbox primarily developed to study neurons has also been introduced for the study of astrocytes ,160,161.ium ions . AlthougIn vivo and ex vivo electrophysiological recording and imaging have contributed significantly to revealing neural circuits underlying behavior by establishing the correlative relationship between neural activity and behavioral outcome. They have also disclosed the molecular mechanisms regulating neural activity and synaptic transmission. However, these pieces of evidence are often lacking in the study of astrocytes and are frequently replaced by a surrogate measurement of subsequent neural activity. Recently, there have been a few studies designed to obtain electrophysiological recordings of both astrocytes and neurons in order to reveal the mechanism(s) underlying the interactions between neurons and glia at the molecular level. For example, Woo et al. found two kinetically separable glutamate release mechanisms in astrocytes by using cerebellar slice recordings . FollowiAlthough astrocytes are the most abundant cell type in the brain, for many years they have received very little attention when compared to the extensive studies that have been conducted on neurons. With the advent of novel experimental techniques in recent years, significant progress has been made in our understanding of astrocytes . Togethe