APOE, APP, ATP5A1, CALM1, CASP3, GSK3B, IL1B, MAPT, PSEN2 and TNF—underlie the enrichment of the Kyoto Encyclopedia of Genes and Genomes (KEGG) AD pathway. With AD, the six E2-responsive genes are NRF1 target genes: APBB2, DPYSL2, EIF2S1, ENO1, MAPT, and PAXIP1. These genes are also responsive to the following EEDs: ethinyl estradiol , BPA , dibutyl phthalate , diethylhexyl phthalate (DPYSL2 and MAPT). To validate findings from Comparative Toxicogenomics Database (CTD) curated data, we used Bayesian network (BN) analysis on microarray data of AD patients. We observed that both gender and NRF1 were associated with AD. The female NRF1 gene network is completely different from male human AD patients. AD-associated NRF1 target genes—APLP1, APP, GRIN1, GRIN2B, MAPT, PSEN2, PEN2, and IDE—are also regulated by E2. NRF1 regulates targets genes with diverse functions, including cell growth, apoptosis/autophagy, mitochondrial biogenesis, genomic instability, neurogenesis, neuroplasticity, synaptogenesis, and senescence. By activating or repressing the genes involved in cell proliferation, growth suppression, DNA damage/repair, apoptosis/autophagy, angiogenesis, estrogen signaling, neurogenesis, synaptogenesis, and senescence, and inducing a wide range of DNA damage, genomic instability and DNA methylation and transcriptional repression, NRF1 may act as a major regulator of EEDs-induced brain health deficits. In summary, estrogenic endocrine disrupting chemicals-modified genes in brain health deficits are part of both estrogen and NRF1 signaling pathways. Our findings suggest that in addition to estrogen signaling, EEDs influencing NRF1 regulated communities of genes across genomic and epigenomic multiple networks may contribute in the development of complex chronic human brain health disorders.During the development of an individual from a single cell to prenatal stages to adolescence to adulthood and through the complete life span, humans are exposed to countless environmental and stochastic factors, including estrogenic endocrine disrupting chemicals. Brain cells and neural circuits are likely to be influenced by estrogenic endocrine disruptors (EEDs) because they strongly dependent on estrogens. In this review, we discuss both environmental, epidemiological, and experimental evidence on brain health with exposure to oral contraceptives, hormonal therapy, and EEDs such as bisphenol-A (BPA), polychlorinated biphenyls (PCBs), phthalates, and metalloestrogens, such as, arsenic, cadmium, and manganese. Also we discuss the brain health effects associated from exposure to EEDs including the promotion of neurodegeneration, protection against neurodegeneration, and involvement in various neurological deficits; changes in rearing behavior, locomotion, anxiety, learning difficulties, memory issues, and neuronal abnormalities. The effects of EEDs on the brain are varied during the entire life span and far-reaching with many different mechanisms. To understand endocrine disrupting chemicals mechanisms, we use bioinformatics, molecular, and epidemiologic approaches. Through those approaches, we learn how the effects of EEDs on the brain go beyond known mechanism to disrupt the circulatory and neural estrogen function and estrogen-mediated signaling. Effects on EEDs-modified estrogen and nuclear respiratory factor 1 (NRF1) signaling genes with exposure to natural estrogen, pharmacological estrogen-ethinyl estradiol, PCBs, phthalates, BPA, and metalloestrogens are presented here. Bioinformatics analysis of gene-EEDs interactions and brain disease associations identified hundreds of genes that were altered by exposure to estrogen, phthalate, PCBs, BPA or metalloestrogens. Many genes modified by EEDs are common targets of both 17 β-estradiol (E2) and NRF1. Some of these genes are involved with brain diseases, such as Alzheimer’s Disease (AD), Parkinson’s Disease, Huntington’s Disease, Amyotrophic Lateral Sclerosis, Autism Spectrum Disorder, and Brain Neoplasms. For example, the search of enriched pathways showed that top ten E2 interacting genes in AD— Endocrine disruptors (EDs) are defined as exogenous substances or mixtures that alter functions of the endocrine system and consequently cause adverse health effects in an organism, its progeny, or populations . EDs encThe human brain, a jelly-like mass of tissue weighing around 1.4 kg (3 pounds), is the most complex biological structure in the universe. Each individual brain originates from a single neural stem cell, which becomes approximately 100 billion neurons at birth. During some stage of the embryo’s development, 250,000 to 500,000 neurons per minute are produced. We also now know new brain cells are being generated throughout our lives—a process called neurogenesis. The brain has bursts of growth and then periods of consolidation, when excess connections are pruned. The most bursts are observed in the first two or three years of life, during puberty, and also a final burst in young adulthood. All these processes are strongly dependent on estrogens . For thoThere is emerging consensus that susceptibility to many of the complex and chronic brain diseases that individuals develop occur as a result of multiple interactions between biologically unique and inherited individual DNA sequence variation, epigenetic individuality, and genetic and epigenetic variation. Exposure to environmental and stochastic factors occur in both germ and somatic cells during intrauterine, postnatal, childhood, and adult life. Low DNA sequence variation across unrelated individuals and our closest related species are not sufficient enough to account for all major differences in physiological and chronic brain disease phenotypic outcomes. Nuclear respiratory factor 1 (NRF1) is one of the sequence-specific DNA-binding redox sensitive transcription factors that constitutes the most important and diverse gene-regulatory mechanism. Context specific NRF1 proteins binding to DNA response elements depends on the variation in DNA binding sequences and the local cell-dependent chromatin alterations, including histone modification, DNA methylation status, and accessibility of regulatory elements . RecentlEstrogen has a complex role in the body, and been tied to cardiovascular, metabolic, endocrine, reproductive, and neurodegenerative diseases, as well as roles in gene expression and protein synthesis ,12. EstrWhen assessing the effects of endogenous estrogens on brain health, we observed endogenous estrogen levels were quantified through blood and/or serum levels, or through the use of a surrogate of exposure to estrogen. The surrogates take into account the times at which the human female body is exposed in a lifetime, from the start of menarche to the end of menopause. We examined studies that took into account exogenous estrogen use through either controlling it as a confounder, or using it as an exclusionary criterion, since exogenous estrogen can modify the effects of endogenous estrogens.Our review identified cross-sectional epidemiological studies that assessed the effects of endogenous estrogen on brain health ,30,31,32p-value = 0.001, 95% CI, 0.008–0.02) and the mini-mental state exam scores  in a cross-sectional study of 11,094 postmenopausal women [p-value < 0.02) [p-value = 0.032) in a cross-sectional study of 579 females [A study of 393 postmenopausal women did not find any significant associations between endogenous estradiol levels and neuropsychological test performance . Anotheral women . In a cr < 0.02) . Cereda  females .We identified case-control studies that assessed the effects of endogenous estrogen exposure on brain health ,34, withp-value = 0.0235, 96% CI 0.9907–0.9993) [p-value = 0.00754, 96% CI 0.004118–0.005289) [We identified cohort studies that assessed the effects of endogenous estrogen exposure on brain health ,36,37. OOral contraceptives (OC) containing specifically ethinyl estradiol has been shown to reduce the amount of available endogenous estrogen in the body . OCs hasA cross-sectional study examined OC effects on spatial and verbal abilities found OC users to perform better on some cognitive test, but not others and between homogenous groups of specific OC medications . AnotherHormone replacement therapy (HRT) has been shown to be associated with the onset of neurodegenerative disease, although evidence points to a timeframe dependent response, depending on when the therapy is initiated. As summarized in Maki and Henderson, initial observation studies and analyses with women indicated the use of HRT containing estrogen to be associated with a reduced risk of AD . StudiesSeveral analytical studies, however, showed associations between the use of HRT and neurodegenerative disease. In the Women’s Health Initiative Memory Study (WHIMS), a randomized, double-blind, placebo-controlled clinical trial, it was found that an HRT treatment of estrogen plus progestin increased the risk for dementia in postmenopausal women and did not prevent cognitive impairment . In anotAn ancillary to the WHIMS study, the Women’s Health Initiative Study of Cognitive Aging, or WHISCA, further supports HRT’s association with neurodegenerative disease , howeverNew studies and reviews give mixed results. A newer observational study conducted by Shao et al. (2012), showed increased AD risk amongst women who used HRT more than five years after menopause, but observed a decreased risk of AD if used within five years of menopause . AnotherBPA, an EED, is widely used to make polycarbonate plastics and resin ,60. BPA Numerous studies highlight the link between BPA and neurotoxicity. Synaptogenesis, the formation of synapses between neurons , and neuCaenorhabditis elegans to examine early embryogenesis exposure to BPA and BPS into adulthood, it was shown that changes in behavior and learning were followed into adulthood [BPA has been found to cause hyperactivity in rats, compared to its derivatives, possibly due to the longer-lasting effect of the parent compound in the brain . A singldulthood . BPA wasdulthood .Animal studies have indicated BPA to affect various aspects of memory at lower than the US EPA’s reference safe daily limit of 50 µg/kg/day . The typPrenatal BPA exposure has been shown to produce more aggressive and hyperactive behavior in offspring when compared to mothers with lower BPA levels . This huN-ras expression in a K. marmortatus model, which shows exposure to BPA can predispose the host to environmental carcinogenesis [Studies have demonstrated evidence of BPA exposure and associations with brain cancer. In particular, meningioma, which account for 20% of intracranial tumors, may be linked to estrogen sensitivity and genetic predisposition . BPA expogenesis . BPA mayogenesis . In rat ogenesis .In summary, besides diethylstilbestrol (DES), BPA is one of the widely researched EEDs. BPA is detected in many human fluids and tissues including saliva, serum, urine, amniotic fluid, follicular fluid, placental tissue and breast milk . From thN-butyl phthalate, found in carpets, paints, glue, insect repellents, hair spray, nail polish, and rocket fuel [N-octyl phthalate (DNOP), found in carpet back coating, packaging films, medical tubing and blood storage bags, floor tile, wire, cables, adhesives, cosmetics, and pesticides [Phthalates, a group of endocrine-disrupting chemicals commonly used to , are useket fuel ; di-N-ocsticides ; and Diesticides . Phthalasticides ,115,116.sticides . Phthalasticides ,119.Phthalates have been found to disrupt neuroplasticity. A recent studies using a rat model suggests exposure to phthalates reduces dendritic spine density, neurogenesis, and synaptogenesis in male rats . A recenb = −2.69, 95% CI −4.33 to −1.05 and b = −2.69, 95% CI −4.22 to −1.16) [Phthalate exposure has been associated with various cognitive deficits. Animal studies indicate that specific learning deficits include decreased fear conditioning , impaireo −1.16) .There are a group of studies that have found possible links between phthalates and brain behavioral disorders such as ASD and attention deficit hyperactive disorder (ADHD). One study, examining exposure to estrogenic endocrine disrupting chemicals (EEDCs) and autism and ADHD found intracranial exposure to several classes of EEDCs, which include phthalates, caused significant hyperactivity in neonatal rats . AlteredPhthalates are suspected in the development of neurodegenerative diseases . In an aPCBs are synthetic organic chemicals composed in different combinations of 209 different chlorinated compounds, or congeners . PCBs haRecent animal and cell studies have shown PCBs to adversely affect brain health. Animal studies have shown exposure to PCBs affect brain health by affecting social behavior , increasIn a nested case-control study in Finland, serum PCBs were not associated with PD development . In a reThere are a number of studies that support the hypothesis that exposure to EEDs represent risk factors for ADHD. Lead and PCB exposures have been implicated with ADHD in children . There aCadmium is a naturally occurring metal in the earth’s crust and a natural constituent of ocean water. Populations are exposed through food, cigarettes, smoke, drinking water, and air. Cadmium is introduced into the food chain through soil and food contact surfaces, and through foods such as leafy vegetables, grains, legumes, and organ meats. Occupation exposures are highest in occupations involving cadmium-containing products, and occupations involved in alloy, battery, plastics, and coloring production. Current evidence supports cadmium being cytotoxic at high concentrations. At low doses cadmium mimics estrogen (E2) effects both in vitro and in vivo .Cell models have shown cadmium to have adverse effects on brain health. In a study using hippocampal slides of CA1 neurons, cadmium was shown to affect synaptic transmission and short-term neural plasticity . In anotStudies on human populations have also shown cadmium’s adverse effects on adult brain function. A cross-sectional study using NHANES III data from 1988 to 1994 compared neurocognitive test scores and urinary cadmium concentrations found individuals with no smoking history, or known occupational cadmium exposure were found to have lower attention/perception scores with increasing urinary cadmium levels . In a coAt low doses arsenic acts as a xenoestrogen and modifies estrogen signaling . Human pIn animal studies using rats, arsenic exposure through ingest water has been shown to impair neurogenesis, worsened spatial memory, and promote abnormal neural synapses. In addition, animal studies in rats have shown arsenic exposure to affect synaptic plasticity, by affecting the expression of NDMA receptors ,165 and In a cohort study consisting of 133 men and 201 women from the Project FRONTIER, a rural healthcare study, long-term low level exposure to arsenic from groundwater was found to be associated with poorer scores in language, visuospatial skills, and executive functioning, global cognition, processing speed, and immediate memory . In anotManganese (Mn) is a naturally abundant environmental trace mineral that is an essential nutrient required as a cofactor for various enzymes and is found naturally in grains and fruit. Manganese, which is found in various industrial processes and products, exposure can occur through inhalation, oral, dermal, and occupational routes. Recently, prepubertal exposure to environmental levels of Mn has been shown to produce precocious mammary gland development through inducing gonadotropin-releasing hormone (GnRH) regulated increases in levels of serum LH and E2 by activating the hypothalamic-pituitary-ovarian axis . MnCl2 , working memory , and perceptual memory  [In animals chronic exposure of Mn has been shown to produce subtle deficits in spatial working memory, spontaneous activity, manual dexterity, and compulsive-like behavior . A small < 0.05) . A cross < 0.05)  examinin < 0.05) . A study < 0.05) .p-value < 0.05). Koc et al. [Manganese has been shown to produce Parkinsonism ,185. Recc et al.  found hic et al. , assessic et al.  found elc et al.  found elc et al.  found hiIn summary, experimental and epidemiologic studies suggest that exposure to metalloestrogens—Cd, As and Mn may influence brain health and they may be associated in the development of some of the chronic complex brain disorders.Estrogenic Mechanisms: It is clear through the studies described above that physiologic and pharmacologic forms of estrogen and estrogenic active environmental chemicals affect brain health . EstrogeOxidative Stress Mechanisms: There is emerging evidence that exposure to estrogen and EEDs-PCBs, BPA, phthalate produce oxidative stress. Oxidative stress may be one of the mechanisms associated with the adverse effect of EEDs on brain health . PhysiolNRF-1 network genes interacting with individual EED impacting Brain Health: NRF1 , a mammalian homolog to the P3A2 sea urchin and erect wing (ewg) Drosophila protein, is a redox sensitive transcription factor ,210. NRF17-β Estradiol interacting genes which are also NRF1 target genes: The CTD search results showed that there were 6695 genes related to 17-β estradiol (E2) . The 724Ethinyl Estradiol Interacting genes common to both E2 and NRF1 target genes: The CTD search revealed that there were 6049 genes related to the active estrogenic chemical in oral contraceptives—ethinyl estradiol (EE) . Out of GPR37, MAPT and PSENEN observed interacting with all three BPA, E2, and NRF1 are associated with neurodegenerative disease [BPA and Interactions with NRF1 Target Genes: The CTD search revealed there are 19,113 genes related to BPA. Out of the 19,113 interacting genes, 2113 are NRF1 target genes, and 673 are target genes of both BPA and E2 . Three oPhthalates and Interactions with NRF1 Target Genes: The CTD search revealed there were 4816 genes interacting with dibutyl phthalate (DBP) and 1344 genes with diethylhexyl phthalate (DEHP). The 756 DBP interacting genes are NRF1 target genes, whereas 149 DEHP interacting genes are NRF1 target genes. Among DBP responsive genes, there are 309 genes common E2 and NRF1 target genes and 86 DEHP associated genes are common E2 and NRF1 target genes . MAPT, aPolychlorinated Biphenyls and Interactions with NRF1 Target Genes: There were 648 genes interacting with polychlorinated biphenyls (PCBs). The 433 PCBs genes are NRF1 target genes, and the 53 PCBs genes are common E2 and NRF1 target genes . EnricheGPR37 gene, a common Cd/E2/NRF1 gene, is associated with neurodegenerative disease [Cadmium and Interactions with NRF1 Target Genes: Using CTD search we found 2458 genes interacting with cadmium (Cd). The 263 Cd interacting genes are NRF1 target genes and 143 interacting genes are common E2 and NRF1 target genes . GPR37 g disease . EnricheArsenic and Interactions with NRF1 Target Genes: The CTD search revealed there were 4037 genes related to arsenic (As). The 520 As interacting genes are NRF1 target genes and the 190 as interacting genes are common E2 and NRF1 target genes . EnricheManganese and Interactions with NRF1 Target Genes: Using CTD search we found 462 genes interacting with manganese (Mn). The 50 Mn interacting genes are NRF1 target genes and the 30 Mn interacting genes are common E2 and NRF1 target genes . EnricheAssociation of EED interacting genes common to both E2 and NRF1 targets with Neurodegenerative Diseases: ACE, ACHE, AMFR, APBB2, APOE, APP, ARC, ATP5A1, BAX, BCHE, BCL2, BDNF, BIN1, CALM1, CASP3, CHRNA7, CHRNB2, CLU, CRH, CYP46A1, DHCR24, DPYSL2, EIF2S1, ENO1, EPHA1, ESR1, F2, GAPDHS, GSK3B, HFE, HMOX1, IGF1, IGF1R, IGF2, IGF2R, IL1B, INS, INSR, LEP, MAOB, MAPT, MIR146A, MPO, NOS3, NPY, PAXIP1, PICALM, PLAU, PPARG, PRNP, PSEN2, SLC2A4, SOD2, SORL1, TF, TFAM, TNF, TPI1, TREM2, VEGFA, VSNL. The bold indicates E2 interacting genes which are NRF1 target genes. With AD, the six E2-responsive genes are NRF1 target genes: APBB2, DPYSL2, EIF2S1, ENO1, MAPT, and PAXIP1. These genes are also responsive to the following EEDCs: ethinyl estradiol , BPA , dibutyl phthalate , diethylhexyl phthalate (DPYSL2 and MAPT), cadmium (ENO1), arsenic (ENO1 and MAPT), and manganese (MAPT)  .GAK, HSPA9, MAPT, PARK2, PARK7, PINK1, RPL14, AND VPS35. These genes are also responsive to the following EEDCs: ethinyl estradiol , BPA , dibutyl phthalate , diethylhexyl phthalate , cadmium (RPL14), and arsenic   . The CTDAIFM1 and IP6K2. These genes are also responsive to the following EEDCs: ethinyl estradiol (AIFM1), BPA (AIFM1 and IP6K2), dibutyl phthalate (IP6K2), cadmium (AIFM1), and arsenic (IP6K2)  .CHMP2B and GSR. These genes are also responsive to the following EEDCs: ethinyl estradiol (GSR), BPA (GSR and CHMP2B), dibutyl phthalate (GSR), diethylhexyl phthalate (GSR), cadmium (GSR), and arsenic (GSR)  .CIRBP, PCDH9, and GTF2I. These genes are also responsive to the following EEDCs: ethinyl estradiol (CIRBP and GTF2I), BPA , polychlorinated biphenyls (CIRBP), cadmium (CIRBP), and arsenic (PCDH9)  .PCNA, PTCH1, and RELA. These genes are also responsive to the following EEDCs: ethinyl estradiol (PCNA and PTCH1), BPA (PCNA and RELA), dibutyl phthalate (PCNA and PTCH1), cadmium (RELA), arsenic (PCNA and PTCH1), and manganese (RELA)  .2+ and redox homoeostasis through modulating mitochondria biogenesis genes as well as neuroplasticity regulating genes, NRF1 may play important roles in controlling fundamental processes in neuroplasticity, including neural differentiation, neurite outgrowth, neurotransmitter release and dendritic remodeling. Since mitochondrial dysfunction has been implicated as a possible pathway for neurodegenerative diseases [Mitochondria are known as the powerhouses of cells. They are important for cell viability and function . They codiseases , gene tadiseases . The PGCdiseases ,221. In diseases . These odiseases . PGC-1, diseases ,224,225.diseases ,224. Sindiseases . When mtdiseases . Increasdiseases . Considediseases ,229,230.MAPRE3, NPDC1, SUV39H2, SKA3, transport-related genes—RAB3IP, TRAPPC3, signal transduction-related genes—SMAD5, PIP5K1A, USP10, SPRY4, transcription-related genes—GTF2F2, NR1D1, and regulation of GTPase activity-related genes—RHOA, RAPGEF6, SMAP1, have been reported to contain NRF1 binding motif(s) in their promoters and mRNA levels of 12 of these genes are regulated by NRF-1 [FAM134C, C3orf10, and ENOX1 involved in neurite outgrowth are regulated by NRF1. FAM134C positively regulates and C3orf10 negatively regulates neurite outgrowth [Estrogen has been demonstrated to have a significant role in controlling neural progenitors in the developing embryonic brain. Estrogen has been shown to induce proliferation and differentiation of neural progenitor ,232,233.by NRF-1 . Overexputgrowth . In summGRIN2B gene which encodes NMDA receptor subunit NR2B has been suspected to play a role in the development of AD [NMDA receptors are very important for regulating neuronal plasticity including learning and memory. Soluble β-amyloid (Aβ) oligomers (Aβ), considered to be involved in the pathogenesis of Alzheimer’s disease (AD), have been reported to influence synaptic dysfunction through interacting with glutamatergic receptors of the NMDA type responsible for maintaining glutamate homeostasis such as uptake and release . There int of AD  which mant of AD , which mnt of AD . NRF1 hant of AD . NRF1 alnt of AD . This suIn vitro and in vivo BPA exposure via disrupting a chloride ion transporter Kcc2 gene expression in neurons through epigenetic mechanisms involving NRF1 target gene- Methyl-CpG binding protein 2 (MeCp2) is considered to be involved neurodevelopmental toxicity. This could possibly help to explain a NRF1 role in BPA-associated pathogenesis of human neurodevelopmental disorders .In a study examining the effects of manganese exposure with HT disease pathology, manganese exposure adversely affects medium spiny neuron dendritic length and spine density which is associated with a decrease brain-derived neurotrophic factor which caused alterations in the Htt gene which causes HT . In studThe accumulation of the β-amyloid protein outside neurons and an abnormal form of the protein tau inside neurons are considered to contribute to the development of Alzheimer’s. During the progression of Alzheimer’s disease, chemical transfer at synapses begins to fail, the number of synapses declines, and neurons eventually die. All these processes seem to be regulated by nuclear respiratory factor 1 (NRF1) target network genes. In addition to the regulation of neuroplasticity controlling genes, PSENEN (PEN2), a component of the γ-secretase complex, APLP1 (an isoform of APP) and MAPT (tau) are three principal NRF1 target genes directly associated with the development Alzheimer’s . All thrSex differences in nervous system diseases, such as multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease, exist in prevalence, severity, and progression of pathologies and consequently has become a major obstacle for the treatment. Cognitive disturbances are frequent in brain health deficit-related disease. Men show greater cognitive impairment in schizophrenia, whereas women show more severe dementia and cognitive decline with Alzheimer’s disease. Low dose of raloxifene exposure to BPA alters the expression of hypothalamic ERα and ERβ (Esr1 and Esr2) expression in a sex-specific manner in the developing brain .Menopausal hormone therapy  therapy alone (CEE-Alone) and in combination with 2.5 mg/day medroxyprogesterone acetate (CEE + MPA)) has recently been shown to produce significant Gray Matter (GM) losses compared to the placebo groups in the anterior cingulate and the adjacent medial frontal gyrus, and the orbitofrontal cortex and risk for cognitive impairment and dementia in older women .Alzheimer’s disease (AD) disproportionally affects women (F:M = 2:1) and the total number of Americans aged 65 y with AD is projected to increase from 5.1 million to 13.8 million by 2050. Despite tremendous progress in understanding the pathogenesis of AD, the molecular basis underlying the sex-dependent differences in AD remains largely unknown and consequently has become a major obstacle for the development of new sex-based molecular targets of AD. Hormone replacement therapy (HRT) and anti-estrogen therapy  are focused on circulating estrogens, as measured by blood levels, but not based on brain estrogen levels. This is one of the main reasons why such endocrine therapy is unable to produce a significant beneficial effect in human AD . There iAPOE, APP, ATP5A1, CALM1, CASP3, GSK3B, IL1B, MAPT, PSEN2 and TNF underlie the enrichment of the Kyoto Encyclopedia of Genes and Genomes (KEGG) Alzheimer’s disease pathway. With AD, the six E2-responsive genes are NRF1 target genes: APBB2, DPYSL2, EIF2S1, ENO1, MAPT, and PAXIP1. These genes are also responsive to the following EEDs: ethinyl estradiol  BPA , dibutyl phthalate , diethylhexyl phthalate (DPYSL2 and MAPT), dibutyl phthalate  (Many genes modified by EEDs are common targets of both (E2) and NRF1 and some of these genes are involved with the specified brain diseases. Therefore, using NRF1-EDC interacting genes identified from CTD , we focu1, ENO1) , Table 51, ENO1) . Other t1, ENO1) . To vali1, ENO1) . For BN We used Banjo  to identAPP, APLP1, MAPT, PEN2, PSEN2, PARH2, PINK1, PARK7, VDAC2, NR1 and 2b). EEDs -modified genes in brain health deficits are part of estrogen and NRF1 signaling pathways. NRF1 regulates targets genes with diverse functions, including involved in cell growth , apoptosis/autophagy , mitochondrial biogenesis/respiration , and neurogenesis/neuroplasticity/synaptogenesis  (A large number of genes (approximately 300) are considered to be altered during the development of complex chronic brain diseases. For example, a minimum of six to eight gene mutations and gene activation by genetic/epigenetic mechanisms is needed for the development of glioblastoma . Similard ENOX1) . Both ph2O2 to activate AKT may be attributed to the inactivation of cysteine-based phosphatases by ROS [This, in turn, may contribute to the susceptibility of normal brain cells to develop disease phenotype. NRF-1 is phosphorylated by cyclin D1-dependent kinase, p38MAPK, ERK and AKT (reviewed in . AKT con


Telomerase extends the repetitive DNA at the ends of linear chromosomes, and it is normally active in stem cells. When expressed in somatic diploid cells, it can lead to cellular immortalization. Human papillomaviruses (HPVs) are associated with and high-risk for cancer activate telomerase through the catalytic subunit of telomerase, human telomerase reverse transcriptase (hTERT). The expression of hTERT is affected by both high-risk HPVs, E6 and E7. Seminal studies over the last two decades have identified the transcriptional, epigenetic, and post-transcriptional roles high-risk E6 and E7 have in telomerase induction. This review will summarize these findings during infection and highlight the importance of telomerase activation as an oncogenic pathway in HPV-associated cancer development and progression. All HPVs complete their life cycle in stratified squamous epithelium, either cutaneous or mucosal dependent on their tropism (reviewed in [Human papillomaviruses (HPVs) are small, non-envelope, double stranded DNA viruses, and there are more than 200 papillomavirus types identified to date (see curated list at Papillomavirus Episteme (PaVE); iewed in ,2). The iewed in . There iiewed in ,5. WhethE) viral genes are expressed. E1 and E2 support HPV DNA replication and the measured expression of E6 and E7 [In the basal layer, the HPV genome escapes its viral capsid and is maintained at 50–60 copies per cells (ranging from 10 to 200 copies) [6 and E7 ,9. EvadiL) gene expression is activated. L1 and L2 form the protein shell of HPV, and they incorporate the HPV episomal DNA into new infectious virions. Those released as epithelial cells are sloughed off at the top of stratified squamous epithelium, completing the viral life cycle.As cells leave the basal layer, they rise through the suprabasal and spinous layers and progress through differentiation. HPV requires this cellular differentiation program to complete its own viral life cycle. Without it, HPV has an abortive infection. Therefore, as HPV-infected host cells rise through the differentiating layers, HPV progresses through its own life cycle. In these differentiating layers, HPV DNA becomes amplified to several thousand copies per cell , and latE6 and E7 genes. E6 and E7 drive cells to continue to grow and divide when they otherwise would not, and, to that end, E6 and E7 are expressed in the differentiating layers of stratified squamous epithelium [Although HPV requires its host cell to differentiate in order to complete its life cycle, it also requires it host cell to continue to grow when normally it would not. HPV dysregulates the balance of growth and differentiation found in stratified squamous epithelium to do so. In low-risk HPV types, this is manifest as warts. In high-risk HPV types, this leads to dysplasias and carcinoma in situ . This dyithelium ,10. By EE6 and E7 genes, thus are considered to be HPV’s viral oncogenes. If HR E6 and E7 are introduced into normal diploid cells, they become immortalized [E6 and E7 genes required for oncogenesis, but they are also required for the maintenance of malignant phenotype. There are critical oncogenic pathways that HR E6 and E7 affect. HR E7 targets the retinoblastoma protein (Rb) for degradation in epithelial cells [PSD-Dlg-ZO-1/2 (PDZ) containing proteins for degradation, disrupting cellular apicobasal orientation and cell-to-cell adhesion in the epithelium, leading to hyperplasia [There are at least 15 HPV types that are defined as high-risk (HR) by their association with cervical cancer . HPV-assrtalized ,13. If Hrtalized ,15. Thisal cells ,17. Thisal cells ,19. It serplasia ,22,23,24erplasia ,13.In this review article, we will describe the roles E6 and E7 have in telomerase induction during HPV infection and in oncogenesis. We will first define telomeric DNA, its role in DNA protection, and the enzymatic function of telomerase. Then, we will highlight the multiple ways HR E6 and E7 derepress hTERT to activate and accelerate telomerase activity. Finally, we will discuss how E6, E7, and hTERT expression changes during oncogenesis.Telomeric DNA caps the ends of linear chromosomes, is repetitive, and is approximately 5000 to 15,000 nucleotides in length in humans ,26. No gWithout telomerase activity, the linear chromosomes of cellular DNA are serially shortened with every cell cycle and division by 100 to 200 nucleotides . This DNStudies in the late 1980s defined the roles HR E6 and E7 played in cellular immortalization, cancer development, and cancer progression ,13. In aRecent studies have confirmed that low-risk (LR) E6 does not activate telomerase  while HRThe E3 Ubiquitin Ligase E6 Associated Protein (E6AP) is important for the activation of hTERT expression and telomerase activity by HR E6 ,54,55. EhTERT gene is constitutively repressed in somatic epithelial cells; this repression occurs at its promoter. The hTERT promoter is approximately 1100 nucleotides in length, with its core promoter being only 200 to 300 nucleotides long [cis elements in its core promoter, blocking transcription [cis elements are E boxes, GC-rich sites, and X boxes.Most research on telomerase regulation has focused on the expression of its catalytic subunit, hTERT. The des long ,61,62. Ncription ,67,68,69cis elements flank the transcriptional start site of hTERT [cis E boxes within the hTERT promoter are required for its transcriptional activation with or without HR E6.Two E box of hTERT ,62,66, aof hTERT ,54. Thesof hTERT ,71,72. Uof hTERT ,73,74 Alof hTERT ,72,75. Eof hTERT ,54,74,75of hTERT . The reqof hTERT ,76,77, bcis elements in the hTERT promoter 5′ of the transcriptional start site [cis elements leads to loss of hTERT promoter-driven transcriptional activation [There are five GC-rich art site ,71,74. Sart site ,71. Maz tivation .Finally, there are two X boxes in the hTERT promoter . One is cis elements and the transcriptional proteins that bind those elements, epigenetic studies of the hTERT promoter demonstrate important structural chromatin changes that affect transcriptional activation of hTERT [cis element but also binds SIN3 transcription regulator family member A (mSin3A), a transcriptional co-repressor that recruits HDACs to promoters [Beyond studies of the hTERT promoter of hTERT . Severalof hTERT ,78. The romoters .DNA methylation patterns at the hTERT promoter also shift during an HPV infection and in tissue culture studies of HPV positive cells. Specific regions of the promoter become hypermethylated, while other regions become hypomethylated, during long-term tissue culture of cells with HR E6 and E7 ,80,81,82Post-transcriptional regulation of hTERT by alternative mRNA splicing and mRNA stabilization is important for telomerase activity ,85,86. INFX1 gene  [We found that hTERT and telomerase activity are upregulated post-transcriptionally by HR E6 through the host cellular protein NFX1-123 . NFX1-12variant) ,89. Greavariant) ,89. The variant) .NFX1-123 contains two protein motifs important for binding, stabilizing, and augmenting hTERT expression. The R3H domain of NFX1-123 has putative single-stranded nucleic acid binding capabilities ,89,90, aCytoplasmic poly(A) binding proteins themselves are important in hTERT expression and telomerase activity in HR E6 positive cells. When PABPC types 1 and 4 are knocked down, hTERT and telomerase activity are reduced . ConversCollectively, these research findings highlight multiple ways hTERT mRNA is post-transcriptionally regulated. Again, the duplicative mechanisms, from DNA, chromatin, and RNA regulation, that HR E6 uses to increase hTERT and telomerase emphasizes its importance to HPV and oncogenesis.E6 genes drive hTERT expression and telomerase activity.Most studies examining the regulation of telomerase by HPV have focused on HR HPVs from the α genus. More recent work has examined the role β genus HPVs play in nonmelanomatous squamous cell carcinoma, and how the beta E6 and E7 proteins may also activate oncogenic pathways, whether similar or disparate to α HR E6 and E7 proteins. Work by Galloway et al. has determined the oncogenic potential of β HPV types through direct analysis of their E6 and E7 protein functionality, and specifically how different E6 types activate hTERT expression, telomerase activity, and immortalization ,93. β E6During cervical cancer initiation and progression, the expression of hTERT and the activity of telomerase parallels worsening disease ,95,96,97E1 and E2 genes are often lost. Even in non-integrated HPV driven cancers, the binding sites for E2 often become methylated. These changes allow for greater expression of E6 and E7, as E2 moderates the expression level of these viral oncogenes [Interestingly, during the transition from HR HPV infection, to dysplasia, to frank cancer, HPV DNA typically no longer remains episomal. It becomes integrated in the cell’s chromosomes. This happens within the context of genomic instability, created and supported by the functions of HR E6 and E7 themselves. This, by definition, means HPV can no longer form infectious virions; it also means HPV gene expression itself is dysregulated. With HPV DNA integration, the HR E6 and E7 genes are universally preserved, but the regulatory ncogenes ,101. Altncogenes . During High-risk HPV infections are associated with cancers in other sites besides the cervix . These icis and trans elements, the chromatin structure, the mRNA product, and associated RNA regulatory proteins. There are still many unanswered questions in the dysregulation of telomerase activation by HPV during infection and oncogenesis. However, its universality implies it is critical to the core function of HR HPV types and to induction of cancers caused by HPV.High-risk E6 hijacks host cell proteins from their usual function  to activate hTERT and telomerase activity. This supports cellular immortalization. These viral-cellular protein partnerships primarily control the derepression of telomerase’s catalytic subunit, hTERT. They increase hTERT through the promoter’s


Curiously, the role of endothelial Ca2+ signaling in NVC has been rather neglected, although endothelial cells are known to control the vascular tone in a Ca2+-dependent manner throughout peripheral vasculature. Methods: We analyzed the literature in search of the most recent updates on the potential role of endothelial Ca2+ signaling in NVC. Results: We found that several neurotransmitters  and neuromodulators  can induce dilation of cerebral vessels by inducing an increase in endothelial Ca2+ concentration. This, in turn, results in nitric oxide or prostaglandin E2 release or activate intermediate and small-conductance Ca2+-activated K+ channels, which are responsible for endothelial-dependent hyperpolarization (EDH). In addition, brain endothelial cells express multiple transient receptor potential (TRP) channels , which induce vasodilation by activating EDH. Conclusions: It is possible to conclude that endothelial Ca2+ signaling is an emerging pathway in the control of NVC.Background: Neurovascular coupling (NVC) is the mechanism whereby an increase in neuronal activity (NA) leads to local elevation in cerebral blood flow (CBF) to match the metabolic requirements of firing neurons. Following synaptic activity, an increase in neuronal and/or astrocyte Ca Accordingly, the brain receives up to 20% of cardiac output and consumes ≈20% and ≈25% of total body’s O2 and glucose i) in brain microvascular endothelial cells.The brain comprises only 2% of the total body mass, yet it accounts for 20% of the overall energy metabolism . As the  glucose ,4. Brain glucose . Cerebra glucose . Through glucose ,8. NVC i glucose ,9. Synap glucose ,12,13,1420-HETE) . This is20-HETE) ,16,17. Eoxane A2 ,19,20,21dilation ,23. Heretunica intima), which is separated from an intermediate layer of smooth muscle cells (tunica media) by the internal elastic lamina. An outermost layer mainly comprised of collagen fibers and fibroblasts and enriched in perivascular nerves, known as tunica adventitia, is separated from the brain by the Virchow–Robin space, which is an extension of the sub-arachnoid space i could, in turn, drive the synthesis of Ca2+-dependent vasoactive mediators. At the same time, brain microvascular endothelial cells have the potential to regulate local CBF independently of NA by detecting changes in blood flow variations and in the concentration of blood-borne agonists through distinct patterns of intracellular Ca2+ elevation.The concept of NVU was first introduced during the first Stroke Progress Review Group meeting of the National Institute of Neurological Disorders and Stroke of the NIH July 2001) to highlight the relevance to CBF regulation of the intimate association between neurons and cerebral vessels . The NVU001 to hiid space ,6. Penetid space . While pid space ,6. Relevid space ,26,27,28id space . Finallyid space ,31. Astric waste ,33. Ther2+ concentration ([Ca2+]i) within the dendritic tree is the crucial signal that triggers the synthesis and release of vasoactive messengers in response to excitatory synaptic inputs i within perisynaptic astrocytic processes  1 and 5 , which stimulate phospholipase Cβ (PLCβ) through Gqα monomer and induce inositol-1,4,5-trisphosphate (InsP3)-dependent Ca2+ release from the endoplasmic reticulum (ER) i in NVC i and recruiting the Ca2+-dependent PLA2 .,4,9.2,4,c inputs . For insrneurons ,28,33,35rneurons ,35,36,37on VSMCs ,28. Intron VSMCs ,38. Longrocesses , which llum (ER) . Howevervent NVC ,39. Nevevent NVC ,40,41. Fchannels ,39. We ri in NVC ,30,39,42i in NVC ,44, whicent PLA2 . In addi vessels .2 supply to local cerebral demand i evoked by either chemical or mechanical inputs i delivers the crucial signal to induce the synthesis of multiple vasoactive mediators i) refill the ER in a sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)-mediated fashion by releasing Ca2+ though the mitochondrial Na+/Ca2+ exchanger i in rat brain microvascular endothelial cells ER) and Orai1-2 channels, which provide the Ca2+-permeable channel-forming subunits on the plasma membrane [2+]ER, drives resting Ca2+ entry and maintains basal Ca2+ levels in endothelial cells , a, a2+ con2+ release  production , thereby inducing VSMC hyperpolarization and vessel dilation i both locally and ≈1200 μm upstream along the same vessel  i, which stimulates IKCa (KCa3.1) and SKCa (KCa2.3) channels to hyperpolarize the endothelial cell membrane  i induced by either mechanical or chemical stimuli may stimulate the Ca2+-dependent phospholipase A2 (PLA2), which cleaves AA from membrane phospholipids i engages PLA2 to liberate AA, thereby resulting in EET synthesis, either within astrocytes or adjacent smooth muscle cells I, which results in robust NO release i that activated KCa channels to induce rapidly-conducting vasodilation at distances >1 mm and travelled along the endothelium as an intercellular Ca2+ wave for 300–400 μm. As shown in previous studies i in vivo. It will also be important to decipher the role played by IKCa and SKCa channels in the hemodynamic response to NA. Earlier work showed that EDH is involved in local and retrograde vasodilation in parenchymal arterioles and middle cerebral arteries, but is absent in capillaries, where functional hyperemia is likely to initiate in response to sensory stimulation. The local vasodilation induced by NA increases the strength of laminar shear stress acting on vascular endothelium, a mechanism that leads to TRPV4-dependent NO release and EDH in peripheral circulation i in rat cerebral arteries endothelial cells. This mitochondrial-derived Ca2+ signal, in turn, induced eNOS activation, NO release and endothelium-dependent vasodilation [2+-dependent NO release by directly activating TRPV4 in several types of endothelial cells [2+ signals in human brain microvascular endothelial cells [2+ signals may also contribute to NVC.Neurovascular coupling is the crucial process to adjust local CBF to the metabolic requirements of active neurons and maintain brain function. Although functional magnetic resonance imaging is routinely employed to monitor the changes in neuronal spiking activity, BOLD signals actually reflect the increases in CBF induced by NA. Therefore, understanding the cellular and molecular bases of NVC is mandatory to interpret the complex relationship between neuronal firing, metabolism and blood flow in both physiological and pathological conditions ,7,8. Intal cells ,174. Theculation ,228. Theculation ,230,231.culation ,212. Forculation ,232,233. peptide , whose adilation . The phyal cells ,235. Somal cells . Given t


Humans have a recently duplicated GLUT member, GLUT14. Avians express the majority of GLUT members. The arrangement of multiple GLUTs across all somatic tissues signifies the important role of glucose across all organisms. Defects in glucose transport have been linked to metabolic disorders, insulin resistance and diabetes. Despite the essential importance of these transporters, our knowledge regarding GLUT members in avians is fragmented. It is clear that there are no chicken orthologs of mammalian GLUT4 and GLUT7. Our examination of GLUT members in the chicken revealed that some chicken GLUT members do not have corresponding orthologs in mammals. We review the information regarding GLUT orthologs and their function and expression in mammals and birds, with emphasis on chickens and humans.The GLUT members belong to a family of glucose transporter proteins that facilitate glucose transport across the cell membrane. The mammalian GLUT family consists of thirteen members (GLUTs 1–12 and H GLUTs are composed of 12 membrane-spanning helices with regions in the extracellular matrix and cytoplasm and contain several functionally conserved motifs [+-myo-inositol transporter (HMIT). HMIT is also known as GLUT13 [The GLUTs are a family of glucose transporter proteins that transport glucose bidirectionally across cell membranes by way of facilitative diffusion ,2. They d motifs ,4. In hus GLUT13 . Humans s GLUT13 . Based os GLUT13 . It is ws GLUT13 ,7. All es GLUT13 , while ss GLUT13 . Defectss GLUT13  and hypes GLUT13 .GLUT expression patterns are complex features. Much attention has been focused on characterizing mammalian GLUT members and elucidating their specific physiological roles. Several studies have also examined the role of GLUTs among avian species, which have provided a basis for understanding GLUT expression patterns in various tissues during different stages of development. However, due to the underlying complex nature of GLUTs, despite the progress made in GLUT research, our knowledge about avian GLUTs is fragmented. For example, the exact physiological role is still not clear for several GLUTs, and even the tissue specificity of GLUTs is not fully examined. In this article, we review what is known about facilitative GLUT family members and their function and expression in birds, with particular emphasis on chickens, and the mammalian  GLUTs as a starting point for a better understanding.After the breakdown of dietary polysaccharides, glucose, fructose and galactose are taken up by enterocytes lining the microvilli of the small intestine. GLUT5 on the lumenal surface of the small intestine mediates fructose uptake. Sodium-dependent glucose cotransporters (members of the sodium-glucose cotransporter (SGLT) protein family) mediate the uptake of glucose and galactose. GLUT2 on the basolateral surface of enterocytes facilitates the release of hexoses into the circulatory system for reuptake by other cells . When moglut1/slc2a1 gene encodes the major GLUT protein of the blood–brain barrier [glut1/slc2a1 orthologs are conserved in 124 organisms including human, chicken, chimpanzee, cow, mouse, rat, Rhesus monkey, zebrafish and Eremothecium gossypii (fungus).GLUTs 1–4, and GLUT14 in human, make up the Class I family of glucose transporters. In mammalian species, the  barrier . The enc barrier ,17. On t barrier ,19, hypo barrier ,21, cryo barrier , paroxys barrier , episodi barrier , hemiple barrier ,25, spas barrier . Overexp barrier  and to h barrier . Suppres barrier . Chicken barrier . Chicken barrier . Accordi barrier , glut1/sglut2/slc2a2 encodes a glycoprotein. The encoded protein regulates bidirectional glucose transport across liver cells, pancreatic islet beta cells that store and release insulin, epithelial kidney cells and intestines. Similar to mammalian species, chickens have abundant GLUT2 expression in the liver [glut2/slc2a2 gene mutations in humans are associated with increased disease susceptibility, including noninsulin-dependent diabetes mellitus and Fanconi–Bickel syndrome. Mutations in glut2/slc2a2 were also found to increase risk of cardiovascular disease in patients with type 2 diabetes [glut2/slc2a2 orthologs have been found in 168 organisms including human, chicken, dog, chimpanzee, cow, Rhesus monkey, rat, Xenopus tropicalis (western clawed frog), Xenopus laevis (African clawed frog) and zebrafish.In mammals, he liver , pancreahe liver . Due to diabetes ,36. Altediabetes ,37, glutglut3/slc2a3 are preserved across 70 organisms so far, including chicken, dog, cow, chimpanzee, mouse, rat, Rhesus monkey, X. tropicalis, X. laevis, zebrafish, fruit fly, mosquito, Caenorhabditis elegans (non-parasitic roundworm), Saccharomyces cerevisiae (yeast), Kluyveromyces lactis (yeast), rice, Magnaporthe oryzae (rice blast fungus), Neurosporra crassa (red bread mold) and Arabidopsis thaliana (flowering plant), according to the NCBI Gene Database [Mammalian GLUT3 facilitates the uptake of glucose, 2-deoxyglucose, galactose, mannose, xylose, fucose and other monosaccharides across the cell membrane. GLUT3 does not mediate fructose transport ,38. GLUTDatabase ,37.glut4 gene mutations in humans are associated with type 2 diabetes mellitus [glut4/slc2a4 orthologs are found in 114 organisms including dog, cow, chimpanzee, mouse, rat and Rhesus monkey [glut4 expression, and chickens are known to be naturally hyperglycemic with adipose tissue [It is well known that GLUT4 is the major insulin sensitive glucose transporter in mammals. The mechanism by which insulin regulates GLUT4 activity has been well studied. Upon stimulation by insulin, intracellular GLUT4 translocates to the plasma membrane, where GLUT4 facilitates cellular glucose uptake. This constitutes the major portion of insulin-stimulated glucose uptake, especially in adipose tissue, skeletal muscle and cardiac muscle tissues. Humans and most mammals rely on normal protein expression of GLUT4 for blood glucose homeostasis . glut4 gmellitus . Accordis monkey ,37. Chice tissue ,44 and se tissue  that is slc2a14 to having a possible role in the development of late-onset Alzheimer’s disease in a Han Chinese population [slc2a14 orthologs are present in humans and Western gorillas [Oryctolagus cuniculus (rabbit), slc2a14 is known as proteins GLUT3 and SLC2A14. In Rhesus monkey, the LOC715795 gene is known as proteins SLC2A3 and SLC2A14. slc2a3b orthologs are also present in zebrafish. UniProt lists slc2a1 as the gene that encodes the GLUT14 protein in X. tropicalis, inferred from database entries.GLUT14, a duplicon of GLUT3, has been shown to have messenger RNA (mRNA) expression in the human testis  and, accpulation . High GLpulation . Accordigorillas ,37. In Oglut5/slc2a5 are found so far in 123 organisms across chicken, dog, cow, chimpanzee, Rhesus monkey, mouse, rat and X. tropicalis. Chicken GLUT5 has been shown to have mRNA expression in the small intestine [Class II consists of GLUTs 5, 7, 9 and 11. GLUT5 is a fructose transporter protein with expression across many species . Accordintestine  and may ntestine .slc2a7 during evolution. Orthologs of slc2a7 are conserved in 55 organisms across mouse, rat, chimpanzee and Rhesus monkey, according to the NCBI Gene Database.GLUT7 has been identified as a high affinity transporter for glucose and fructose. GLUT7 does not transport galactose, 2-deoxyglucose or xylose . Human Gglut9/slc2a9. Orthologs of glut9/slc2a9 are present in 153 organisms including chicken, dog, cow, mouse, rat, chimpanzee, X. tropicalis and X. laevis.GLUT9 is a known transporter of fructose and urate and can transport glucose at a low rate. Mammalian GLUT9 plays a regulatory role in the development and survival of cartilage chondrocytes and may have a role in urate reabsorption by proximal tubules ,58. One glut11/slc2a11 orthologs are present in 111 organisms and conserved across chicken, dog, cow, chimpanzee, Rhesus monkey, zebrafish and X. tropicalis, based on the NCBI Gene Database [glut11/slc2a11 gene [According to the NCBI Gene Database, GLUT11 is also known as GLUT10. GLUT11 is a transporter of glucose and fructose, but does not transport galactose in humans. GLUT11 has roughly 42% amino acid sequence similarity to GLUT5 and 35% similarity to GLUT1 . AlternaDatabase . Rats ana11 gene .slc2a6 has alias GLUT6 and GLUT9 proteins in humans, mice and X. tropicalis. GLUT6 is a hexose transporter protein. Mammalian GLUT6 is highly expressed in the brain, spleen and leukocytes [GLUT6/SLC2A6 orthologs are present in 169 organisms including chicken, dog, cow, mouse, chimpanzee, Rhesus monkey, zebrafish, fruit fly, mosquito, X. tropicalis and X. laevis.Class III contains GLUTs 6, 8, 10, 12 and HMIT/GLUT13. According to NCBI, ukocytes . One stuukocytes . Based oukocytes ,37, GLUTglut8/slc2a8 orthologs are conserved across 171 organisms including chicken, dog, mouse, rat, cow, chimpanzee, Rhesus monkey, X. tropicalis, zebrafish, fruit fly, A. thaliana and rice, according to NCBI. Similar to mammals, chicken GLUT8 is a known insulin-responsive glucose transporter with ubiquitous expression in cells and higher mRNA concentrations in adipose tissue and kidney [Based on sequence similarity, GLUT8 has been identified as an insulin-regulated glucose transporter. According to NCBI, GLUT8 binds cytochalasin β in a glucose-inhibitable manner. Mammalian GLUT8 may be dual-specific and is inhibitable by fructose. A recent study on the mouse atria suggests that GLUT8 has a role in glucose uptake in the mammalian heart, along with GLUT4 . glut8/sglut10/slc2a10 are associated with arterial tortuosity syndrome, a rare connective tissue disorder [glut10/slc2a10 orthologs are conserved across 166 organisms including chicken, dog, mouse, rat, chimpanzee, Rhesus monkey, cow, X. tropicalis, X. laevis and zebrafish [According to the NCBI Gene Database, GLUT10 plays a role in glucose homeostasis regulation. Human GLUT10 has highest mRNA expression in the liver and pancreas . In humadisorder . Based oebrafish ,37.slc2a12 encoded protein contains alias GLUT8 and GLUT12 in humans. GLUT12 can facilitate transport of a variety of hexoses [glut12/slc2a12 are conserved across 177 organisms including chicken, dog, mouse, rat, chimpanzee, Rhesus monkey, cow, X. tropicalis, X. laevis, zebrafish, A. thaliana and rice, based on the NCBI Gene Database [According to the Gene Database at NCBI, the  hexoses . Human G hexoses . A recen hexoses . The GLU hexoses . A recen hexoses . OrtholoDatabase ,37.Xenopus oocytes have helped identify GLUT13 as a proton (H+) myo-inositol cotransporter with specificity for the transport of myo-inositol, inositol triphosphate and related stereoisomers [glut13/slc2a13 orthologs are conserved across 151 organisms including chicken, dog, cow, chimpanzee, Rhesus monkey, mouse, rat, X. tropicalis, X. laevis, zebrafish, C. elegans, S. cerevisiae, K. lactis, E. gossypii, Schizosaccharomyces pombe (fission yeast), A. thaliana and rice.Studies on oisomers ,74. Mammoisomers . Intraceoisomers  and Parkoisomers . Accordioisomers ,37, glutglut/slc2a genes across different species. Through these methods, avian species Struthio camelus australis and Anas platyrhynchos have been shown to contain orthologs of gluts 1, 2, 3, 5, 6, 8, 9, 10, 11, 12 and hmit/glut13. The turkey  contains gluts 1, 2, 5, 6, 8, 9, 10, 12 and hmit/glut13, but glut3 and glut11 have not yet been identified in turkey. From UniProt analysis, the duckbill mammal platypus (Ornithorhynchus anatinus) contains glut members 1, 2, 3, 4, 6, 8, 9, 10, 11, 12 and hmit/glut13. However, these GLUT proteins remain uncharacterized in the species. From our analysis, glut5 or glut7 genes have not been identified in platypus.Automated computational analysis using genomic sequencing prediction method and contig reference sequencing has assisted in identifying slc2a6 encoded protein as alias proteins GLUT6 and GLUT9 in human, mouse and X. tropicalis. Human slc2a11 encoded protein was annotated as alias proteins GLUT10 and GLUT11. Clearly, slc2a6 and slc2a9 in human, mouse and X. tropicalis are discrete genes that encode discrete GLUT proteins; slc2a10 and slc2a11 in human are discrete genes which encode discrete GLUT proteins; and slc2a8 and slc2a12 in human are also discrete genes which encode discrete GLUT proteins.It is not surprising that some GLUT members are annotated confusingly in public databases. Melga) and X. tropicalis. The tree was calibrated with GLUT12, assuming the time of separation between mammals and birds was 300 million years. The human GLUT11-A was included in the phylogenetic analysis. This analysis was conducted using the Neighbor–Joining bootstrap method with 50 replicates. Topological branching point divergence times were calculated with maximum likelihood based on the Jones–Taylor–Thornton matrix-based method and are based on units of the number of amino acid substitutions per site.GLUT amino acid sequences were downloaded from UniProt or NCBI and derived from evidence at the transcript level, protein level or homology. Dataset for the timetree contained 63 amino acid sequences with a total of 426 positions included in the final dataset. Positions with fewer than 95% site coverage were eliminated. Less than 5% alignment gaps, missing data and ambiguous bases were allowed at any position. The timetree is drawn to scale with the relative number of substitutions per site.According to this timetree, Class III GLUTs separated from Class I and Class II approximately 2000 million years ago. Class I and Class II GLUTs separated about 1700 million years ago, around the time when multicellular life began. GLUTs 1 and 3 separated approximately 800 million years ago. Evidently, GLUT13/HMIT orthologs could not be resolved well with this tree construction method, which can be seen from the distance between mammals and birds being not reflecting the species tree. GLUT8 and GLUT10 orthologs had the least constraints among the GLUT family members.Based on the phylogenetic analysis as well as the result of experiments conducted in our laboratory, we have recognized that accession number gg5L_X1_XP_426528.4, which is annotated as GLUT member 5-like isoform X1 in chickens, is a gene product that is separate from the true GLUT5 member of chicken and other species and has more similarity to the GLUT9. Several GLUT11-like members were also found in chickens, each of them being the product of a discrete gene .glut2 was cloned by screening a chicken liver cDNA library [Characteristics of human and chicken GLUT members are summarized in  library  and exam library ,57. GLUT library ,30,33. G library ,57. GLUT library ,2. GLUT9 library , but litGLUT expression patterns vary across species, and glucose transport may be regulated by different factors in those species. One such example is GLUT4, which is required for normal cellular metabolism in mammalian species, but is lacking in avian species.A study from 1994 showed GLUT2 to be predominantly expressed in the chicken liver, but absent in the chicken brain and heart. Similar to mammalian species, this early study revealed how multiple GLUTs coexisted in various tissues in chickens. A 2001 study examined chicken mRNA and protein expression during different stages of embryonic development. GLUT1, GLUT3 and GLUT4 were examined in several tissues during embryogenesis. In the chicken brain, GLUT1 mRNA levels were high throughout development, although GLUT1 protein expression was highest during early development. GLUT3 mRNA expression in the brain was highest during the last half of development, with high protein expression very early and very late in development. In skeletal muscle, mRNA and protein expression of GLUT1 and GLUT3 were high during early development, but decreased by mid-development. GLUT1 mRNA and protein expression were also highest during early development, then declined steadily throughout development. GLUT1 mRNA levels were high in the liver, but protein expression of GLUT1 was not detectable. This study determined that GLUT1 is developmentally regulated in the chicken brain, heart and skeletal muscle. GLUT3 in the brain increased throughout the stages of development, but was absent in skeletal muscle. This study also revealed that GLUT4 was absent in chicken heart and skeletal muscle .Because GLUT4 homologs were found to be lacking in chickens, GLUT8, an insulin-responsive GLUT in mammalian blastocytes , was tesIn a 2005 follow up study by the same team of researchers, GLUT1, GLUT2, GLUT3 and GLUT8 were examined for the extent of mRNA expression across different chicken tissues, this time using real-time quantitative polymerase chain reaction (RT-qPCR) with SYBR Green I fluorescence. In this study, GLUT1 mRNA expression was detected across most of the tissues examined, with highest concentrations in adipose tissue and brain. GLUT2 mRNA expression was detectable only in the liver and kidneys. GLUT3 was found to be highly expressed in the brain, and GLUT8 mRNA was ubiquitously expressed across all chicken tissues with higher expression in adipose tissue and kidneys [A 2014 study found that high bodyweight chickens are generally compulsive feeders with a different food intake and blood glucose threshold sensitivity to insulin . In thisRelative comparison methods were used for comparing GLUT mRNA expression across different genes. Our laboratory has developed an absolute quantification protocol to study a panel of different GLUTs across multiple chicken tissues. The absolute quantification method has allowed us to compare mRNA levels across different GLUTs  and different tissues. By calculating absolute copy number values for absolute quantification, we were able to compare GLUT mRNA expression patterns across a tissue panel of different GLUTs. Results showed that the mRNA levels of different GLUTs have clear tissue specificity; many GLUTs are expressed in the same tissues.glut genes in chickens, including a member very similar to human glut9 and several members similar to mammalian glut11. Analysis of these paralogs revealed their distinct expression pattern.With sequence analysis and RT-qPCR, we have also found new As is the case for many protein families containing multiple paralogs in a species, the GLUT family of proteins exerts essential physiological functions that need be delicately regulated. Multiple proteins with overlapping molecular functions provide a cushion for genetic, physiological and environmental disturbance. At the same time, tissue specific distribution of these transporters allows the fine tuning of expression to the tissue specific condition. Studies on the details of this fine regulation have just started. Much remains to be investigated. It would not be surprising if some day findings indicate that a specific GLUT is a limiting factor to the development of a specific cell type. Physiological and genomic evidence indicate that the regulation of glucose homeostasis in avians is distinct from the mammalian counterpart. Further examination of GLUT members in birds will shed new light on the function of these family.


Trametes versicolor F21a during the algicidal process. The obtained results showed that a total of 193, 332, 545, and 742 differentially expressed genes were identified at 0, 6, 12, and 30 h during the algicidal process, respectively. The gene ontology terms were enriched into glucan 1,4-α-glucosidase activity, hydrolase activity, lipase activity, and endopeptidase activity. The KEGG pathways were enriched in degradation and metabolism pathways including Glycolysis/Gluconeogenesis, Pyruvate metabolism, the Biosynthesis of amino acids, etc. The total expression levels of all Carbohydrate-Active enZYmes (CAZyme) genes for the saccharide metabolism were increased by two folds relative to the control. AA5, GH18, GH5, GH79, GH128, and PL8 were the top six significantly up-regulated modules among 43 detected CAZyme modules. Four available homologous decomposition enzymes of other species could partially inhibit the growth of algal cells. The facts suggest that the algicidal mode of T. versicolor F21a might be associated with decomposition enzymes and several metabolic pathways. The obtained results provide a new candidate way to control algal bloom by application of decomposition enzymes in the future.The molecular mechanism underlying the elimination of algal cells by fungal mycelia has not been fully understood. Here, we applied transcriptomic analysis to investigate the gene expression and regulation at time courses of  Algae, the food of aquatic animals, play a vital role in water ecosystems Sigee, . HoweverTrichaptum abietinum 1302BG could directly eliminate four tested algal species when fungal mycelia were co-cultured with algal cells , while β-glucosidase, protease, laccase, and manganese peroxidase could be involved in the late stages (24 to 60 h). In a recent study, our group detected 30 fungal enzymes with endo- or exo-glycosidase activities such as β-1,3-glucanase, α-galactosidase, α-glucosidase, alginate lyase, chondroitin lyase, peptidase, exonuclease, and manganese peroxidase, which should be involved in the algicidal process of fungus T. versicolor F21a .The previously isolated fungus −2 s−1 of photons in BG-11 medium . Then, the crude RNA was digested via 10 U DNase I  at 37°C for 30 min. mRNA was isolated from the crude RNA via the Dynabeads® Oligo (dT) 25  according to manufacturer's instructions. 100 ng mRNA of each sample was used to construct the sequencing library with NEBNext® UltraTM RNA Library Prep Kit . Paired-end sequencing of cDNA fragments (~300 bp) were conducted at the Illumina HiSeq 4000 platform by BGI-shenzhen, China.http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The paired-end raw reads from RNA-Seq were trimmed, thus removing low quality base-calls (Q < 30) and adaptor sequences with pipeline Fastq_clean (v2.0)   . Genes encoding lignocellulose-active enzymes were further annotated via dbCAN  , β-glucanase , Trypsase , Pepsin  were prepared according to the instructions and added to 4 mL algal solutions to assess the effects of decomposition on algal cells, respectively.T. versicolor F21a and algal cells was monitored via spectrophotometer. Figure p < 0.05).The interaction between SRP127790) showed that an overwhelming majority of the reads had quality scores above Q30  were detected via RNA-Seq, and ~23% (115 lignocellulose genes) were differentially expressed genes Table . The totThe 312 genes presented above were further divided into 46 modules, 43 of which could be detected via RNA-Seq Table . The expTo test the roles of the decomposition enzymes in the results mined by our bioinformatics analysis, several identified enzymes with endo-glycosidase activities and endopeptidase activity were purchased and applied to test their inhibitory abilities on algal cells. Figure In this study, ~66% of the predicted fungal genes could be detected during the algicidal process via RNA-Seq. The number of detected genes far exceeded that obtained via proteomic techniques, which was also observed in many other studies . Enzymes of the GH18 module might play a central role during the initial algicidal stage by disrupting cynobacterial cell walls. They could possibly also play important roles in decomposing the cell walls of eukaryotic algae.Eight genes in the module GH18 were significantly up-regulated during the algicidal process , β-1,4-cellobiosidase (EC 3.2.1.91), glucan β-1,3-glucosidase (EC 3.2.1.58), endo-β-1,4-xylanase (EC 3.2.1.8), and endo-β-1,4-mannosidase (EC 3.2.1.78) of the GH5 module can decompose cellulose, cellobioside, β-1,3-glucans, xylans, and galactomannans into glucose, xylose, galactose, and mannose  to inhibit algal bloom. Interestingly, it is a potential to use algal bloom materials as a medium for growing white rot fungi (Jia et al., T. versicolor F21a were significantly increased during the algicidal process (Du et al., As suggested above, enzymes with endo-glycosidase activity and endopeptidase activity should play a key role in the first step to break algal cells. We tested our hypothesis by assaying several available commercial decomposition enzymes that target the components of algal cell walls. GH18 member cellulase and endo-β-glucanase were purified from s Figure . This sus Figure , but oths Figure . CombiniT. versicolor F21a might be associated with decomposition enzymes and metabolic pathways.In this study, ~66% of predicted fungal coding genes during the algicidal process could be detected via RNA-Seq. The total expression levels of all CAZyme genes during the algicidal process were increased by two folds compared with the control. Four tested homologous CAZymes such as cellulase, β-glucanase, trypsase, and pepsin were approved to partially inhibit the growth of algae. The algicidal mode of WD, XC, and XW carried out majority of the experiment and bioinformatic studies. ZX and XG performed some experiments. WD, XC, and XW wrote the manuscript. CJ and RD helped draft the manuscript. GH conceived and directed the study. All authors read and approved the final 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.


Abies sibirica) terpenes, on normal and cancer cell lines. We used early passaged and senescent none-immortalized fibroblasts as cellular aging models. It was revealed that in normal fibroblasts, terpenes induced genes of stress response, apoptosis regulation and tissue regeneration. The restoration of the expression level of some prolongevity genes after fir extract treatment was shown in old cells. In Caco-2 and AsPC-1 cancer cell lines, Abisil induced expression of both onco-suppressors , and proto-oncogenes . Thus, the study demonstrates the potential anti-aging and anti-cancer effects of Abisil on senescent and cancer cell lines.A large number of terpenoids exhibit potential geroprotector and anti-cancer properties. Here, we studied whole transcriptomic effects of Abisil, the extract of fir ( Ginkgo biloba [Rosa damascena [Nymphaea root [Alpinia zerumbet leaf [Rhodiola rosea [Stachys lavandulifolia [Rosa damascene, that is rich in such terpenoid as citronellol, have been shown to increase lifespan of Drosophila by protecting against iron toxicity and enhancing flies resistance to oxidative stress [Betula spp.), have demonstrated the anti-bacterial, antimalarial, and anti-inflammatory properties, activity against the human immunodeficiency virus (HIV), and cytotoxicity towards cancer cells [It is known that certain plant extracts have geroprotector properties. The ability to prolong the life in animal models observed for extracts of hawthorn , Ginkgo o biloba , bluebero biloba , Rosa daamascena , ginsengamascena , cranberamascena , green tamascena , Nymphaeaea root , Alpiniabet leaf , natto [bet leaf , Rhodiolla rosea , black rla rosea , garlic la rosea , apple [la rosea , Stachysulifolia , and othulifolia , 17. Speulifolia –20. Theyulifolia –23. Planulifolia . Most ofulifolia . Howeverulifolia , 27. Terulifolia , 29. A wulifolia –32. For e stress , 33, 34.er cells –37. Moreer cells , 38–41. Abies sibirica terpenes (10% bornyl acetate), on human cell lines of colon adenocarcinoma (Caco-2), pancreas adenocarcinoma (AsPC-1) and human none-immortalized fibroblasts of the 6th and 13th passages from the point of view of potential geroprotector and anti-cancer properties.We studied whole transcriptomic effects of Abisil, the extract of HSPA1B, HSPA1A), heme oxygenase-1, metallothionein 1X, and dual specificity phosphatase 2 (DUSP2).Differentially expressed genes that were affected by drug treatment in different cell lines were identified . Abisil DUSP2 dephosphorylates MAPKs are involved in cellular proliferation, apoptosis, differentiation, and stress responses . It is kBHLHE40 [IFIT2 [Egr-3 is a transactivator of genes in fibroblasts, associated with tissue remodeling and wound healing [There are also genes associated with the immune response, such as BHLHE40  and IFIT0 [IFIT2 , and fac0 [IFIT2  and TRIB0 [IFIT2 . In addi0 [IFIT2 . The upr healing .th and 13th passages Abisil suppressed the expression of proapoptotic gene BMF [In fibroblasts of both the 6gene BMF  as well gene BMF  and cellgene BMF .It should be noted, that among the most represented molecular pathways induced by Abisil treatment in normal fibroblasts, a significant portion is related to longevity, including MAPK-, FOXO- and HIF-1 signaling pathways  in the cells of the 6th passage, Abisil activated 21 genes and repressed 16 genes, whereas in cells of the 13th passage, the expression level of 43 and 67 genes were affected, respectively. This result may reflect the greater randomness of the expression response in old cells compared to younger ones. Some authors have mentioned the age-dependent increase of cell-to-cell variation in gene expression, so called increased transcriptional noise [th passage, the most represented genes are of apoptosis (12 genes) and MAPK signaling pathway (5 genes). Among the 62 downregulated genes in fibroblasts of the 13th passage, the BTG2 gene is the one most associated with cellular aging in our opinion. Its expression has been shown to be important in negatively regulating cell proliferation [It is noteworthy that in normal fibroblasts of the 13al noise , 59. Amoferation . Thus, tth passage exhibit various external signs of cellular aging, such as inhibition of proliferation as compared with the 6th passage (control). When comparing the expression of old and young fibroblasts 5804 differentially expressed genes were revealed generally.In this study, fibroblast passaging was considered as one of the models of aging. Fibroblasts of the 13The following aging-associated features of the expression profiles in fibroblasts should be noted: reduced expression of various cell adhesion molecules, chemokines expression, cyclin-dependent kinases, lamin, GADD45 family members, cAMP-dependent transcription factors (CREB), and PI3K/Akt overexpression. GSEA analysis allowed us to identify a number of biological processes, with the strongest changes in gene expression being (as part of this aging model): the suppression of angiogenesis, cell differentiation, MAPK cascade, chemotaxis, response to hypoxia, tissue regeneration and other processes, as well as modulation of intercellular communication, adhesion, migration, and ion exchange.The potential geroprotector may be able to restore the expression level of genes in old cells, and transform them into younger cell gene expression profiles . The posth passage compared with the 6th passage. At the same time, Abisil treatment caused a 2-fold increase of expression level of GADD45B/G genes and 1.5-fold increase – of GADD45A.GADD45 (growth arrest and DNA damage inducible protein) gene family is associated with both tumor suppression and with the longevity. The expression level of GADD45 was reduced by 2-fold in fibroblasts of the 13HSPA1B, HSPA1A, DNAJB9 (Hsp40 B9), DNAJB4 (Hsp40 B4), HSPH1, DNAJB1 (Hsp40 B1), HSPA9, and others.Abisil treatment is accompanied by a 1.5 to3-fold increase in the expression of heat shock proteins genes Modulation of the cell cycle, in particular the MAPK signaling pathway.Modulation of NF-κB signaling pathway.Modulation of Toll-like receptor signaling pathway.Modulation of TGF-beta signaling pathway.Also worthy of attention is the induction of compensatory changes for the following genes:DUSP5, DUSP1, and DUSP6  decreases with fibroblasts aging (in the 13th passage compared with the 6th). Abisil treatment restores it to a level above the previous level.Expression of tumor-suppressive regulators of MAPK-signaling cascade MYC , JUN (jun proto-oncogene), FOSB , FOSL1 (FOS-like antigen 1) protooncogenes decreases during fibroblasts aging. Abisil treatment restores it to a level of younger cells.The expression level of SOCS3  decreases with aging. Abisil treatment restores it to a level of early passages.The expression level of CREB5 (cAMP responsive element binding protein 5) gene expression is decreased during aging and restored to the previous level after Abisil supplementation.DDIT3 (DNA damage inducible transcript 3) gene expression is decreased during aging and restored to the previous level after Abisil supplementation.KLF2 and KLF4 (Kruppel-like factors 2 and 4) expression is decreased during aging and restored to the previous level after Abisil supplementation.BMF (Bcl2 modifying factor), TRIB3 (tribbles pseudokinase 3), BHLHE40 (basic helix-loop-helix family member e40), TLR4 (toll-like receptor 4), RGS4 , GDF15 (growth differentiation factor 15), NGFR (nerve growth factor receptor), and CTGF (connective tissue growth factor) gene expression is increased during aging and restored to the previous level after Abisil supplementation.th place, and colon cancer takes 3th place [Caco-2) and pancreas adenocarcinoma (AsPC-1).Among global mortality rates for cancer, pancreas cancer takes 6th place , 63. We GADD45A, GADD45B, and GADD45G) related to the GADD45 family - both in normal fibroblasts and tumor cell lines (AsPC-1 and Caco-2). GADD45 family proteins are stress sensors and involved in the intersection of several cell signaling pathways, including apoptosis, DNA repair, and cell cycle arrest [GADD45 genes often accompany the initiation and progression of malignancies, and GADD45 mediates the effects of multiple chemotherapeutic drugs [GADD45A, but lack of GADD45 expression, however, can cause inefficiencies of chemotherapy [GADD45 activity can have anti-aging effects as well [It is noteworthy the increased expression level of all three genes , and proto-oncogenes . Data indicate that Abisil exposure was associated with the modulation of key signaling pathways responsible for cell cycle control, proliferation, differentiation, apoptosis , cell-cell signaling, stress response, cAMP-dependent signaling and protein refolding.GADD45 family genes which may serve as tumor suppressors. Chemotherapeutic drugs induced up-regulation of these genes is one of the factors, that determines the effectiveness of chemotherapy. Another important effect is the overexpression of most of the DUSP family genes responsible for the inhibition of the MAPK cascade, which plays a role in the response to chemotherapy as well.Of particular note is the increase in the expression of all three members of the Caco-2 and AsPC-1 the situation is reversed.The results of GSEA-Gene Ontology analysis allows one to make the assumption that the biological processes responsible for the negative regulation of apoptosis, prevail over the processes of induction of programmed cell death in normal human fibroblasts. In the cell lines GADD45 gene family, and the family of heat shock proteins HSPA1A/A1B/A9, Hsp40 B1/B4/B9, and HSPH1.The potential geroprotector properties of Abisil may be conditioned by induced overexpression of both ® is a complex of terpenoids obtained from capsule extract Abies sibirica of Penaceae family enriched with monoterpenoids. Chemical and quantitative composition of the pharmaceutical composition of Abisil was studied with gas-liquid chromatography (GLC) by LCM-7A chromatograph  using a thermal conductivity detector (TCD), packed column , and stationary phase “Apiezon L”  on polychrome. The consumption of carrier gas (helium) was 30 ml/min, with a column temperature of 125°C, and a vaporization chamber temperature of 180°C. Identification of the main components was carried on the relative retention times and “bystander” compounds. The revealed Abisil composition is a standardized terpenoid substance derived from capsule extract of Abies sibirica , a saponification number (100-130 mg), an ester number (10-60 mg), and an index of refraction .Caco-2 (ATCC - HTB-37) were kindly provided by Dr. Peter Chumakov . Cells were maintained in Dulbecco's modified Eagle's medium  supplemented with 10% and 20% fetal bovine serum  accordingly, 100 U penicillin per ml and 100 mkg streptomycin per ml . Cells were cultured at 37°C in a 5% CO2 atmosphere and passaged every 2-3 days by dissociation with trypsin .Human pancreas adenocarcinoma cell line AsPC-1 (ATCC - CRL-1682) and colorectal adenocarcinoma cell line th and 10th passages. Effects were studied on the 6th and 13th passages.Primary fibroblasts were provided by The Laboratory of Cell Cultures of the Institute of Medical Cell Technologies . Cells were maintained in the medium described above (with 10% FBS). Cells were passaged, when the culture had reached approximately 80% confluence. In order to save unique properties of the model, cells were frozen in DMEM with 7% DMSO and 30% FBS after the 4Cells in 70% confluence were treated for 6 hours with dilution of Abisil (1.2 mg/ml) in DMEM with 2% FBS. After that, the culture medium was replaced with a fresh medium and in 18 hours cell viability was analyzed using MTS test .All cells including control cell lines were plated in triplicate. RNA extraction for further analysis was performed right after treatment with active substance dilution.Caco-2, AsPC-1, and primary fibroblasts at the 6th and 13th passages) and corresponding control cells . RNA quality and quantity was determined with the Agilent 2100 Bioanalyzer  and the Qubit 2.0 Fluorimeter , respectively. RNA samples with an RNA integrity number (RIN) higher than 8.0 were used for downstream analysis.Total RNA was extracted from 24 samples using RNeasy Mini kit , including 4 cell lines after treatment  from each sample was used for mRNA library preparation with a TruSeq RNA Sample Preparation Kit v2 Low Sample (LS) protocol  according to the manufacturer's instructions. The quality and concentration of cDNA library was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies) and a Qubit 2.0 fluorometer (Invitrogen), respectively, before sequencing. To optimizing cluster densities, the libraries were quantified by qPCR. cDNA libraries were sequenced  on an Illumina NextSeq 500 platform .http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and Trimmomatic (http://www.usadellab.org/cms/index.php?page=trimmomatic) tools with default parameters. The clean reads were separately aligned to the human reference genome (GRCh38) in the Ensembl (release 80) using TopHat2 software (http://ccb.jhu.edu/software/tophat/index.shtml). The read counting was performed with HTSeq-count (http://www-huber.embl.de/HTSeq/doc/overview.html). The differentially expressed genes were determined with the p-value ≤ 0.01 using the edgeR, lme4, biomaRt, Rgraphviz, topGO, ggplot2, pathview, and clusterProfiler analysis packages in R.The raw single end reads were quality controlled and trimmed using FastQC (


The effects of recombinant goldfish leptin-AI and leptin-AII on PRL mRNA expression in the pituitary were further analysed, and the mechanisms underlying signal transduction for leptin-induced PRL expression were determined by pharmacological approaches. Our results showed that goldfish lepR is abundantly expressed in the brain-pituitary regions, with highly overlapping PRL transcripts within the pituitary. Recombinant goldfish leptin-AI and leptin-AII proteins could stimulate PRL mRNA expression in dose- and time-dependent manners in the goldfish pituitary, by both intraperitoneal injection and primary cell incubation approaches. Moreover, the PI3K/Akt/mTOR, MKK3/6/p38MAPK, and MEK1/2/ERK1/2—but not JAK2/STAT 1, 3 and 5 cascades—were involved in leptin-induced PRL mRNA expression in the goldfish pituitary.Leptin actions at the pituitary level have been extensively investigated in mammalian species, but remain insufficiently characterized in lower vertebrates, especially in teleost fish. Prolactin (PRL) is a pituitary hormone of central importance to osmoregulation in fish. Using goldfish as a model, we examined the global and brain-pituitary distribution of a leptin receptor (lepR) and examined the relationship between expression of  Add in 1994 , metabol in 1994 , immunit in 1994 , and pit in 1994 .Fugu rubripes by gene synteny analysis [leptin mRNA is abundantly expressed in the liver, but is lacking in the adipose tissue [Fish leptin was first identified in analysis , followeanalysis , medaka analysis , zebrafianalysis , salmon analysis , tilapiaanalysis ,15, and analysis . The fise tissue ,18. Lepte tissue ,18. Howediabetes (db) gene. Structurally, lepR can be divided into three parts: extracellular domain, transmembrane domain, and cytoplasmic domain. To date, six isoforms of lepR have been observed in humans, including a long-form receptor (lepRb), four short-form receptors , and a soluble-form receptor (lepRe) [ob/ob mice, but not in db/db mice [38MAPK (MKK3/6 and p38MAPK) and p42/44MAPK  cascades [lepR that correspond to human lepRb have been cloned in 19 species of teleost fish [The actions of leptin are mediated by its receptor, namely, leptin receptor (lepR). LepR is a single-transmembrane receptor encoded by the  (lepRe) . All of  (lepRe) . The JAK/db mice . Other S/db mice . In addi/db mice . In speccascades . The cDNost fish ,25, but lepR mRNAs are widely found in different types of pituitary cells [GH mRNA expression, but not GH release by in vitro incubation [TSH mRNA [Leptin exerts its appetite-inhibiting effects by acting on the appetite control centres of the brain, particularly those within the hypothalamus. Leptin regulates a group of hypothalamic neuropeptides, and subsequently induces the response of pituitary hormone production and secretion via the hypothalamus-pituitary axis . Based ory cells ,28,29, sry cells . Howevercubation . In Eurocubation  and somacubation  release cubation . In bighTSH mRNA . HoweverTSH mRNA , and theTSH mRNA .1/2 signal pathway during in vitro incubation [Prolactin is an important pituitary hormone that promotes lactation in mammals, whereas in fish, PRL plays an important role in freshwater osmoregulation . The impcubation . HoweverCarassiusauratus) are a freshwater fish belonging to the family Cyprinidae, under the order Cypriniformes. Goldfish are a well-established model for the regulation of pituitary hormone production and secretion [leptin genes (GenBank: FJ534535.1 and FJ854572.1) have been reported as leptin-AI and leptin-AII [leptin-AI and leptin-AII have been generated in our lab by the methylotrophic yeast expression system, and have shown effects on the inhibition of feeding behaviours and the reduction of food consumption, via a mediation of specific central appetite regulators [leptin-AI and leptin-AII expression have also been investigated [lepR in goldfish were examined, and the expression profiles of leptins, lepR and other major hormones in different parts of the pituitary were characterized. The in vivo and in vitro effects of goldfish recombinant leptins on PRL mRNA expression, and the involved signal transduction mechanisms, were also investigated. To the best of our knowledge, the present study is the first to examine PRL regulation by leptin in a Cyprinidae fish, and it may reveal the potential roles of leptin in osmoregulation.Goldfish  mRNA expression was mainly detected in the anterior pituitary. SL-α and SL-β mRNA expression was mainly distributed in the posterior pituitary, and leptin-AI and leptin-AII mRNA was mainly expressed in the medium pituitary and posterior pituitary. However, lepR mRNA was evenly distributed in the anterior pituitary, medium pituitary, and posterior pituitary, with the highest expression level in the anterior pituitary, indicating that the expression profile of lepR highly overlapped with that of PRL within the goldfish pituitary.As shown in 38MAPK and p42/44MAPK) cascades are the major intracellular signal pathways activated by leptin, we used the corresponding pathway inhibitors to understand the roles of these pathways in leptin-mediated PRL mRNA expression. As shown in PRL mRNA were significantly stimulated by leptin-AI or leptin-AII, in the absence of signalling inhibitors. For the JAK/STAT cascades, as shown in PRL mRNA expression could not be blocked by any inhibitors for JAK/STAT cascades, including the JAK2 inhibitor AG490 (50 μM), the STAT1 inhibitor fludarabine , the STAT3 inhibitor 5,15-Diphenyl-21H,23H-porphine  or the STAT5 inhibitor IQDMA (20 μM). In contrast, as shown in PRL mRNA expression could be totally abolished by co-treatment with the PI3K inhibitors LY294002 (10 μM) and wortmannin (100 nM), the Akt inhibitor API2 (100 nM) or the mTOR inhibitor rapamycin (20 nM). Furthermore, as shown in PRL mRNA expression could be totally abolished by co-treatment with the p38MAPK inhibitors PD169316 (100 nM), or the MEK1/2 inhibitors PD98059 (10 μM) and U0126 (200nM). However, the p38MAPK inhibitor SB02190 (100 nM) could only block the leptin-AI-, but not the leptin-AII-induced PRL expression. Taken as a whole, these results indicate that the PI3K/Akt/mTOR, MKK3/6/p38MAPK, and MEK1/2/ERK1/2 cascades were involved in the regulation of PRL mRNA expression by leptin in the goldfish pituitary.Given that JAK/STAT , PI3K/Akt/mTOR and MAPK , testosterone (T), TRH, GH, LH, insulin-like growth factor (IGF), and PACAP, and reduced by DA, cortisol, somatostatin (SS), urotensin-II (U-II), and vasoactive intestinal peptide (VIP) [PRL mRNA in the tilapia pituitary increased after a low dosage (0.5 μg/g bwt), but decreased after a high dosage (5 μg/g bwt) of tilapia leptin-A administration [PRL mRNA expression were 100 ng/g bwt , prolactin-releasing peptide (PRP), pituitary adenylate cyclase-activating peptide (PACAP) and prolactin-releasing peptide (PRP), stimulate PRL secretion in the anterior pituitary, while other factors, such as dopamine (DA) and somatostatin (SS), inhibit the secretion of PRL . In telede (VIP) ,54,55. Ide (VIP)  and bovide (VIP) . In an ide (VIP) . By usinstration . The resng/g bwt A and 1 nng/g bwt C in in vo 3.9 nM , indicat similar .leptin-AI and leptin-AII mRNAs are widely expressed in the central nervous system and peripheral tissues, including the pituitary [lepR mRNA was ubiquitously expressed, with high levels being detected in the hypothalamus and pituitary  [leptin is only expressed in a few pituitary cells (5–7%) and mainly colocalized with TSH cells (24-31%), while lepR is widely found in the normal pituitary and pituitary cell lines, including FS, GH3 and aT3-1 cells [PRL and lepR mRNA expression was observed, with the highest expression levels in the anterior region of the pituitary in goldfish  could only block the effects of leptin-AI, but not leptin-AII. The blockage capacity of the chemical inhibitors is dependent on their dosage, which is different between the mammalian and fish experiments.Based on the identification of  species ,57,59,60 species , whereas species , and lep species . In addius hasta , and enhus hasta . Howevergoldfish . In the gnalling , which iPRL mRNA in dose- and time-dependent manners in the pituitary, by both in vivo and in vitro approaches. We also demonstrated that the stimulatory effect was mediated through a functional coupling of PI3K/Akt/mTOR, MKK3/6/P38MAPK and MEK1/2/ERK1/2-dependent cascades. In the present working model , prior to being sacrificed by spinosectomy according to procedures approved by the Ethics Committees of Foshan University .Goldfish leptin-AI and leptin-AII recombinant protein were previously expressed in yeast by our lab, and the protein activities were also tested . For thelepR mRNA in goldfish was assessed by quantitative PCR (qPCR) in selected tissues. Three male and three female fish were used as individuals in this case, and the selected tissues included the brain, gill, heart, stomach, intestine, liver, spleen, kidney, muscle, fat, testis, and ovary. In addition, the distribution of lepR mRNA in the brain-pituitary regions, such as the olfactory bulb, telencephalon, optic tectum, cerebellum, medulla oblongata, spinal cord, hypothalamus, and pituitary, were also investigated.The tissue distribution of GH, PRL, proopiomelanocortin (POMC), GTH-α, SL-α, SL-β, leptin-AI, leptin-AII and lepR in different parts of the pituitary were examined by the qPCR technique. Goldfish pituitaries from the six individuals were each divided into three parts, including anterior pituitary ), medium pituitary ), and posterior pituitary (neurointermediate lobe (NIL), including pars intermedia (PI) and neurohypophysis (NHP)). The samples were frozen in liquid nitrogen and stored at −80 °C for RNA extraction and reverse transcription.Expression profiles of PRL mRNA expression in the goldfish pituitary were analysed with an IP injection approach, as previously described [The in vivo effects of leptin-AI and leptin-AII on escribed . Brieflyescribed ,47 dissoescribed ) was injPRL transcript levels by leptin was further examined in goldfish pituitary primary cells. After anaesthesia with MS222, the goldfish were decapitated, then the pituitaries were removed and cut into 0.5 mm thick sections and digested with trypsin (2.5 mg/mL) at 28 °C for 30 minutes. Next, pituitary fragments of the digestion were suspended in a minimum essential medium (MEM) without Ca2+, and mechanically dispersed into single cells by gentle pipetting. The dispersed pituitary cells were then separated from the remaining fragments by filtration, through a sterile 30 μm mesh, and harvested by centrifugation at 1,000 × g for 10 min at 4 °C. The viability of the cells was assessed using a Trypan blue exclusion assay, and only preparations with more than 95% viability were used in subsequent experiments. The resulting pituitary cells were seeded onto a 24-well plate at a density of 2.5 × 106 cells per well, in MEM containing 5% foetal bovine serum (FBS), then incubated at 25 °C overnight in 5% CO2 for recovery. On the second day after cell preparation, test substances prepared in MEM were gently overlaid onto pituitary cells, after the removal of the old culture medium. The cells were incubated with the test substances for another 3–48 h for time-course studies, or for 24 h for dose-dependent or pharmacological studies. Finally, the cells were harvested by dissolving them in TRIzol reagent .The regulation of PRL, leptin-AI, leptin-AII, lepR, GH, POMC, GTH-α, SL-α, SL-β, and β-actin) was detected using SYBR Premix ExTaq™ II , in a Rotor-GeneRG-3000 Real-time PCR System , with primers and PCR conditions as previously reported  and reverse transcribed using PrimeScript™ RT kit . Transcriptional expression of target genes , and analysed by using one-way ANOVA followed by Fisher’s least significant difference (LSD) test with SPSS (IBM Software). Differences were considered significant at p < 0.05.For qPCR, the raw data of target gene expression were expressed as fmol per tube, and routinely normalized as a ratio of