Pulmonary fibrosis is often triggered by an epithelial injury resulting in the formation of fibrotic lesions in the lung, which progress to impair gas exchange and ultimately cause death. Recent clinical trials using drugs that target either inflammation or a specific molecule have failed, suggesting that multiple pathways and cellular processes need to be attenuated for effective reversal of established and progressive fibrosis. Although activation of MAPK and PI3K pathways have been detected in human fibrotic lung samples, the therapeutic benefits of in vivo modulation of the MAPK and PI3K pathways in combination are unknown. Overexpression of TGFα in the lung epithelium of transgenic mice results in the formation of fibrotic lesions similar to those found in human pulmonary fibrosis, and previous work from our group shows that inhibitors of either the MAPK or PI3K pathway can alter the progression of fibrosis. In this study, we sought to determine whether simultaneous inhibition of the MAPK and PI3K signaling pathways is a more effective therapeutic strategy for established and progressive pulmonary fibrosis. Our results showed that inhibiting both pathways had additive effects compared to inhibiting either pathway alone in reducing fibrotic burden, including reducing lung weight, pleural thickness, and total collagen in the lungs of TGFα mice. This study demonstrates that inhibiting MEK and PI3K in combination abolishes proliferative changes associated with fibrosis and myfibroblast accumulation and thus may serve as a therapeutic option in the treatment of human fibrotic lung disease where these pathways play a role. Fibrotic lesions in the lung cause distortion of pulmonary architecture and interruption of gas exchange that ultimately can result in death Fibroblast foci are clusters of fibroblasts and myofibroblasts that lie in continuity with established fibrosis, a characteristic histologic feature of pulmonary fibrosis especially idiopathic pulmonary fibrosis (IPF), and are thought to be central in mediating the progression of lung disease Several growth factors and their downstream signaling pathways that maintain tumors are also activated in both human and mouse models of pulmonary fibrosis We previously have generated doxycycline (Dox)-regulatable transgenic mice overexpressing the EGFR ligand, transforming growth factor-alpha (TGFα), under control of the lung epithelial-specific 2.3-kb rat Clara cell secretory protein (CCSP) gene promoter The purpose of this study was to test the hypothesis that combined inhibition of MEK and PI3K signaling pathways would be more effective in treating established and progressing fibrosis in TGFα model than inhibiting either pathway alone. This study also examined transcriptional and fibroproliferative changes in the drug treated groups to determine overlapping or unique functions of each pathway in the maintenance of established and progressive fibrosis in the lungs.All mice were derived from the FVB/NJ inbred strain. TGFα-transgenic mice were generated and maintained as described previously After removal, lungs were immediately inflation fixed using 10% neutral buffered formalin. Lung tissue sections were prepared and stained with Masson Trichrome as described previously 2) that encompassed alveolar, pleural, and adventitial regions of the lung for each mouse. The proliferation index was determined by counting the total number of Ki67-staining nuclei and dividing by the total number of nuclei, in each field. The lung sections were immunostained for alpha smooth muscle actin as previously described For immunohistochemical detection of proliferation, paraffin-embedded lung sections were immunostained with anti-mouse Ki67 antigen . The total numbers of Ki67-staining nuclei were counted, as well as the total number of nuclei in ten randomly selected uniform fields  as described previously 5′-GCCCTTGACTATAATGAGTACTTCAGG-3′ (forward),5′-TCAACTTGCGCTCATCTTAGG-3′ (reverse); MYCN 5′-AGCACCTCCGGAGAGGATAC-3′ (forward), 5′-CCACATCGATTTCCTCCTCT-3′ (reverse); CDK4 5′-AGAGCTCTTAGCCGAGCGTA-3′ (forward), 5′-TTCAGCCACGGGTTCATATC-3′ (reverse).Total RNA was extracted from lung tissue and cells using the RNeasy Mini Kit from Qiagen  as described previously From each experimental group, four lung tissue samples were sequenced using an Illumina HiSeq-1000 Sequencer . An in-house analytical pipeline was created to align reads to the reference genome, perform quality control, and conduct statistical analyses to identify and cluster differentially expressed genes. Using TopHat aligner P-value cut-off of 0.05 as well as a two-fold change (up or down), resulting in 1790 genes in total. Using this set of significant genes, hierarchical clustering on the log-transformed read counts normalized for different lengths of gene-coding regions  http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE52854; accession number: GSE52854).To identify differentially expressed genes between Day 0 (untreated) and Week 8 (treated on Dox) samples, statistical analysis was performed using the DESeq Bioconductor package Lung homogenates were processed for Western blot and quantified using the PhosphorImager software volume-integration function, Imagequant 5.2  as described previously 4 cells/well) and collagen (3 mg/ml) were combined at a ratio of 2∶1 to obtain collagen gels with cells, and plated in 24 well tissue culture plates. The next day gels were gently taken out of 24 well plates and placed them in 6 well plates containing media, ARRY (0.1 µM), PX866 (0.1 µM) or both the inhibitors in combination. Culture media was changed on every day and also gel images taken to quantify the surface area of collagen gels.Collagen gel contraction was performed as described elsewhere with few modifications P values less than 0.05.All data were analyzed using Prism . One-way ANOVA with Tukey’s Multiple Comparison post-test was used to compare different experimental groups, and data were considered statistically significant for To evaluate the effects of combined therapy with ARRY and PX-866, we used TGFα transgenic mice which develop progressive and severe pulmonary fibrosis To determine whether combined therapy with ARRY and PX-866 influences the progression of fibrosis when fibrosis is already established (following 4 weeks of Dox), TGFα transgenic mice were treated with ARRY and PX-866, either alone or in combination, while remaining on Dox for an additional 4 weeks . Total lLung sections were stained with Masson Trichrome to visualize fibrotic changes in the lungs of CCSP/TGFα mice treated with either vehicle or inhibitors . OverexpTo examine mechanisms whereby inhibitors alone or in combination prevent the progression of fibrotic disease in the CCSP/TGFα model, a gel contraction assay was performed on primary fibroblasts from the lungs of CCSP/TGFα mice on Dox for 4 weeks. Gels receiving inhibitors alone or in combination demonstrated significantly less contraction than untreated gels with the greatest improvement seen in fibroblasts receiving combination treatment . One posThe excessive proliferation of mesenchymal and epithelial cells has been shown to contribute to progressive fibrosis in both human and mouse lungs. To determine the role of MEK and PI3K inhibitors in proliferation, we quantified changes in proliferation by counting Ki67-positive cells in lung sections. Overexpression of TGFα mice for 8 weeks had doubled cells staining for Ki67 compared with vehicle-treated CCSP/− mice. TGFα transgenic mice treated with either ARRY or PX-866 alone had reduced Ki67 staining compared with vehicle-treated TGFα transgenic mice . CombineTo identify unique and overlapping molecular networks by which MEK and PI3K pathways regulate proliferation of cells and fibrosis in the lung, we assessed gene-expression changes in the lungs of TGFα mice treated with vehicle or inhibitors using next-generation sequencing. As shown in The network of genes that contribute to the excessive proliferation of cells in fibrotic lesions is of major interest, since combined therapy was more effective in inhibiting proliferation of cells in the lung. Also, the gene targets regulated by both MEK and PI3K pathway-driven proliferation are largely unknown. Heat mapping was used to show the top genes that are differentially expressed and involved in cell proliferation, using ingenuity pathway analysis. As shown in Pulmonary fibrosis complicates a wide range of adult and pediatric diseases including autoimmune, inflammatory, and idiopathic disorders Cellular pathways are highly integrated webs with multiple redundancies that may be activated in response to the inhibition of a signaling pathway Although fibrosis was reduced using combined treatment compared to single-inhibitor therapy, fibrosis endpoints in the combination-treatment group remained altered compared with non-fibrotic controls. We have previously demonstrated that hydroxyproline levels and pleural thickening remain elevated up to 18 weeks following extinction of TGFα overexpression Several cancers are associated with mutations in the MAPK and/or PI3K pathways, and there are multiple pharmacologic agents that specifically target these pathways currently in advanced clinical trials. Since both MAPK and PI3K signaling pathways are elevated in fibrotic lung diseases, the findings of this study are potentially translational. Currently there are a number of clinical trials in oncology where both inhibitors are administered in combination, so safety data will be emerging on the feasibility of this approach for treating lung fibrosis Figure S1Combined inhibition of MEK and PI3K pathway inhibitors had no severe toxic effects on kidney (creatinine) and liver (serum ALT) functions during TGFα-induced fibrosis.(PDF)Click here for additional data file.Figure S2Representative photomicrograph of lung tissue sections immunostained for Ki67 for each treatment group. Scale bar, 100 µm.(PDF)Click here for additional data file.Figure S3Expression of MYCN in the lungs of individuals with interstitial lung disease. Average Mycn mRNA levels are elevated in individuals with interstitial lung disease compared to controls and COPD. Mycn expression data was extracted from mRNA expression catalogue available in the database of Lung Genomic Research Consortium (LGRC) (https://www.lung-genomics.org/research).(PDF)Click here for additional data file.Data S1(PDF)Click here for additional data file.


Humans are very different when it comes to pain. Some get painful piercings and tattoos; others can not stand even a flu shot. Interindividual variability is one of the main characteristics of human pain on every level including the processing of nociceptive impulses at the periphery, modification of pain signal in the central nervous system, perception of pain, and response to analgesic strategies. As for many other complex behaviors, the sources of this variability come from both nurture (environment) and nature (genes). Here, I will discuss how these factors contribute to human pain separately and via interplay and how epigenetic mechanisms add to the complexity of their effects. Pain perception  can be modulated and modified (enhanced or abolished) by many “environmental” factors including psychological and personality-related factors such as previous pain experiences, emotionality and cognition, somatization and catastrophizing, presence of acute and chronic stressful life events, fatigue, anxiety, fear, boredom and anticipation of more pain, as well as socioeconomic factors . In addition, pain behavior is different among genders and ethnicities, and varies with age. In addition, some clinical and medical factors correlate with risk for increased severity or chronicity of many painful conditions.The experience of pain is one of the fundamental human senses and most ancient protective survival skills. However, the ways in which people express and treat pain change across time and origin and are influenced by cultural and social factors –4. AlthoGender effects on human pain have several aspects. First of all, research showed that gender of experimenter(s) influences the results of pain assessment in laboratory studies. For example, males reported significantly less cold pressor-evoked pain in front of a female experimenter than a male experimenter, while female subjects tended to report higher pain to the male experimenter . FurtherThere is increasing recognition that aging can have a profound effect on pain. Pain sensitivity may diminish in adults of advanced age . Exampleμ-opioid receptors peripherally and centrally, respectively [Life style and habits  also contribute to human pain perception. Exercise can be both a treatment and a stimulus of pain, so that too much exercise increases pain, while too little exercise may worsen pain through multiple mechanisms . Exectively . Therefoectively , chronicectively , osteoarectively , rheumatectively . AccordiCigarette smoking and alcohol drinking also have a complex relationship with pain. On the one hand, both nicotine and alcohol have analgesic properties. In general, nicotine administration via nasal spray or transdermal patches reduces pain sensitivity in both smokers and nonsmokers likely resulting from effects at both central and peripheral nicotine acetylcholine receptors . Also smPersonality, mood, and sleep patterns profoundly contribute to pain perception and behavior. Particularly, catastrophizing as a personality trait  can heighten the intensity of pain in both experimental and clinical settings. Paraphrasing Daniel Defoe, one can say: “In pain trouble to be troubled is to have your pain doubled.” This correlation has been observed across multiple pain measures and in diverse conditions and populations, including mixed chronic pain, low back pain, rheumatoid arthritis, aversive diagnostic procedures, surgery, dental procedures, burn dressing changes, whiplash injuries, and survey samples of young adults and asymptomatic individuals participating in experimental pain procedures and varsity athletes (reviewed in ). RecentAlthough the “environmental” influence on human pain is multidimensional, the role of different groups of factors is not equally important. Research showed that clinical factors have limited power on pain modulation and/or modification. Factors such as patient's knowledge of the diagnosis, disease-related variables, treatment outcome, operative procedures, or the degree of tissue trauma have only little value predicting the risk for long-term pain severity or chronicity , 86. AddAs described above, the impact of nurture on human pain is multifactorial, and “environmental” factors may shape the effects of each other. The relationships among these factors merit close attention. Future research will uncover the exact connections between psychological and demographic influences on pain. For instance, it is possible that ethnic differences are more pronounced among females than males (or vice versa). Also, it would be interesting to know whether ethnic differences decline or increase with advancing age. While demographic factors modify the effect of other factors on human pain via interact , psycholSince “environmental” factors may explain only a small portion of overall interindividual variability in pain sensitivity and pain perception, molecular genetics of pain has been under extensive investigation for the last decades aiming to identify a “missing piece in the puzzle” . Indeed,Drosophila melanogaster) have been used in genetics studies and neuropharmacology for a long time, and they recently have become a powerful model organism for pharmacological pain research [Drosophila larvae produce a stereotypical rolling behavior, unlike the response to an unheated probe. However, Painless mutants lack this noxious heat response [2 laser beam assay, painless gene essential for nociception in Drosophila larvae also influenced heat avoidance behavior in adult Drosophila, and, as in mammals, the latency of this behavior is inversely related to stimulus intensity [Painless adult Drosophila, hundreds of genes implicated in heat nociception in the fly have been identified, 35% of which were already suspected to be pain genes, and other had no previous functional annotations [straightjacket gene, a member of the α2δ family of genes that function as subunits of voltage-gated Ca2+ channels and control the function and development of synapses [α2δ3 , as predicted, affects heat pain variance in humans: the minor allele of the single nucleotide polymorphism  rs6777055 contributed to reduced thermal pain sensitivity in healthy volunteers  were associated with less chronic pain in sciatica patients “confirming” the reported implication of thermosensitive neurons in human chronic pain. These data reinforce the extraordinary conservation of the neurobiological mechanisms of nociception from its manifestation as avoidance of damage in primitive creatures like flies to the complex sensation of pain in humans. Other genetic factors and molecular pathways related to pain may also be evolutionary conserved, and data from lower species may be easily translated to humans. By cross-referencing fly and mammal data with human genetic information from the public domain-like gene expression profiling or genetic association studies new potential therapeutic targets can be pinpointed [α2δ3 is a perfect example of such an approach, since it is a close homolog of α2δ1 which is the molecular target of gabapentin and pregabalin [α2δ3 will appear as powerful analgesic. Pain genetic research in other model organisms such as fish [Fruit flies (research . In respresponse . In the ntensity . Using gotations . One of synapses . This flvolution . α2δ3 muaffected . Most instimulus ). In addnpointed . α2δ3 isegabalin , widely egabalin . It is,  as fish  or inver as fish  can provSCN9A gene encoding the α-subunit of the voltage-gated sodium channel Nav1.7 [Genetic linkage and association studies of human pain are based on the heritability estimates for experimental pain phenotypes and clinical painful conditions that apparently are similar to what has been found in animal models . The herl Nav1.7 . SCN9A is responsible for three rare human pain disorders. Nonsense mutations cause a complete absence of pain [The gene  of pain , whereas of pain , 112. SCN9A mutations have been linked to PE [SCN9A mutations have been linked to PEPD [Primary erythromelalgia ) is an autosomal dominant disorder with symptoms typically including episodes of burning pain triggered by heat or exercise, together with erythema and mild swelling primarily in the hands and feet . The onsed to PE . All muted to PE . Penetraed to PE . Interesed to PE . Symptom to PEPD , most of to PEPD . SCN9A alleles causing CIP have been reported, all leading to loss-of-function mutations occurring in coding regions of the gene [SCN9A variation for human pain led to the hypothesis that common mutations  in the same gene may underlie the differing pain perception in general population. Indeed, from 27 common SNPs genotyped, a significant association was found between pain score and SNP rs6746030 in five tested cohorts; so that the rarer A allele was associated with increased pain scores compared to the commoner G allele in patients with lumbar discectomy, osteoarthritis, sciatica, and phantom pains [Unlike PE and PEPD, congenital insensitivity to pain (CIP) is autosomal recessive and extremely rare disorder. Although CIP patients exhibit varying degrees of deficits in terms of sensing pain and their response to painful stimuli , other sensory (nonnociceptive) modalities remain intact. To date, thirteen rare the gene , 120. Thom pains . The A aom pains . These dSPTLC1 [HSN2, an alternatively spliced exon of WNK1 gene [IKBKAP mutations [NTRK1 mutations [NGFB [SCN9A alleles, these HSN genes may influence more than one type of rare pathological pain conditions; a novel mutation in NTRK1 gene known to be responsible for HSN IV apparently causes HSN V [NTRK1 mutations and polymorphisms result in CIPA  [Another group of such disorders consists of five types of hereditary sensory neuropathies (HSNs) characterized by sensory loss, insensitivity to pain, and a variable degree of muscle weakness and wasting, as well as autonomic features . MoleculSPTLC1 ; HSN II NK1 gene ; HSN IIIutations ; HSN IV utations ; and HSNns [NGFB , 127. Hoes HSN V , and othidrosis) . CACNA1A on chromosome 19p13 (FHM1), ATP1A2 on chromosome 1q23 (FHM2), and SCN1A on chromosome 2q24 (FHM3) were identified as causing FHM [The final group of pain disorders with a very prominent genetic impact is three types of familial hemiplegic migraine (FHN), a rare monogenic dominant autosomal disease due to mutations in a single gene . Mutatiosing FHM . Besidessing FHM . Future Mendelian heritable pain disorders have provided insights into general human pain mechanisms highlighting genes encoding for sodium and calcium channels and suggesting new analgesic drug targets that may be of relevance for the treatment of both rare and common pain diseases . It is aSCN9A allele discussed above contributed to pain in four frequent chronic painful diseases suggesting that they may share the same genetically determined pain pathway [GCH1 gene, encoding for the rate-limiting enzyme for the synthesis of tetrahydrobiopterin (BH4), an essential cofactor for catecholamine, serotonin, and nitric oxide productions, was protective for persistent sciatica [KCNS1 gene, encoding for potassium channel alpha subunit involved in neuronal excitability, also affects multiple chronic pain conditions, so that the “valine risk allele,” was significantly associated with higher pain scores in five of six independent patient cohorts assayed [SLC6A4) are associated with fibromyalgia [COMT gene is a major “pain gene” contributing to a variety of pain phenotypes and particularly to “idiopathic” pain conditions. Enzyme encoded by this gene (COMT) metabolizes catechol neurotransmitters dopamine, noradrenaline, and adrenaline that are involved in various physiological functions closely related to pain [COMT variants include at least two SNPs and common haplotypes : the most studied SNP rs4680, also known as Val158Met, leading to a three- to four-fold reduced activity of the COMT enzyme [COMT P2 promoter region also affecting enzyme activity [COMT promoter region  and three in the COMT coding region at codons his62his , leu136leu , and Val158met ) leading to the largest, up to 20-fold difference in enzyme activity, via changes in the stability of the secondary mRNA structure [COMT alleles resulting in high enzyme activity will metabolize catecholamine more efficiently and presumably have reduced catecholamine-mediated neurotransmission [COMT Met158 allele showed increased sensitivity to pain and the number of pressure points compared to other genotypes [COMT Val158Met SNP showed differential allelic distribution among patients with nonfamilial migraine and healthy controls, so that Met allele was over represented in the migrainous patients [COMT functional haplotypes correlate with the risk of developing myogenous TMJD [COMT Met158 allele predicts strongest placebo effect in IBS patients [COMT pain vulnerable haplotype were more likely to report moderate-to-severe neck pain after motor vehicle collision and also longer time to physical and emotional recovery, indicating that COMT genetic variation affected both somatic and psychological responses in the allele carriers [COMT variants. Other genes may affect these disorders through other mechanisms, such as recent findings of SCN9A allele described above influencing interstitial cystitis/bladder pain syndrome [IL1RN) and melanocortin-1 receptor (MC1R) genes increasing the risk of VVS [IL-6 and αTNF- changing individual susceptibility to IBS [COMT effects are not limited to “idiopathic” pain disorders and have been found in other pain models. COMT Val158Met and haplotypes showed significant associations with the maximum postoperative pain rating  in third molar extraction model [COMT haplotype was associated with less chronic postoperative pain (greater improvement in Oswestry Disability Index scores) in patients with disk degenerative disease one year after surgery, suggesting that COMT variation may be predictive in terms of treatment outcome [Pain perception in general population is one of the most complicated measurable traits and a complex genetic trait of polygenic nature . “Common pathway . A commosciatica , advancesciatica , and a psciatica  suggesti assayed . The uni assayed . Common omyalgia , chronicomyalgia , migrainomyalgia , and TMJomyalgia . COMT ge to pain . FunctioT enzyme ; SNP rs2activity ; and thrtructure . It was smission . With reenotypes ; Brazilienotypes , and Spaenotypes . COMT Vapatients . In a lapatients , while tpatients . COMT fuous TMJD . Homozygpatients , while tcarriers . Therefosyndrome ; functiok of VVS ; or proiy to IBS . On the on model . In addi outcome .Genetic studies of certain pain conditions may be more challenging than others due to higher degree of phenotypic diversity. For example, acute and chronic low back pain (LBP) may be influenced by different genetic factors, depending on underlying primary causing pathology such as lumbar disc degeneration or other condition affecting the spine , irradiating pain from hip diseases, spine injury or just overuse of muscles, ligaments, and joints. Such a heterogeneous etiology and pathophysiology require careful consideration of all significant clinical factors as covariates in association analysis and meta-analysis of LBP genetic data . NeverthCOMT functional haplotypes contribute to variability in thermal and other evoked pain sensitivity [5HTT or SLC6A4) exhibit significantly reduced conditioned pain inhibition for pressure pain thresholds and heat pain [VR1) moderates heat/capsaicin sensitivity in healthy humans [MC1R) [COMT Met/Met genotype  showed higher sensitivity to thermal and pressure pain stimuli than patients carrying the Val alleles [Many heterozygous clinical pain phenotypes can be dissected for genetic analysis using “intermediate” pain phenotypes or “endophenotypes”—such as cytokine profiling, brain imaging, or quantitative sensory testing (QST) variables—measurable components along the pathway between the complex trait and genotype. Endophenotypes represent simpler clues to genetic underpinnings than the syndrome itself, promoting the view that such a syndrome can be decomposed or deconstructed, which can result in more straightforward and successful genetic analysis . Charactsitivity . Individeat pain . A functy humans . Increass [MC1R) . In clin alleles . A study alleles . SpecifiInvestigation of the nature of analgesia and analgesic responses is a new but rapidly developing field attracting increasing attention in light of recent studies of pharmacogenetics and pharmacogenomics of pain. Pharmacogenetics describes the effects of polymorphic genes on the enzymes that metabolize drugs, and the genetic differences in metabolic pathways can affect individual responses to drugs, both in terms of therapeutic effect as well as adverse effects . Thus, pOPRM1, has been the primary candidate in pharmacogenetic and pharmacogenomic studies. It was shown that healthy individuals who are homozygous for the 118G allele (A118G SNP) require 2–4 folds higher opioid consumption to achieve analgesia for evoked pain when compared to individuals not carrying the genotype [OPRM1 A118G SNP and cytochrome P450 3A4 (CYP3A4)*18B polymorphisms that jointly affect postoperative fentanyl analgesia so that patients with OPRM1 AA and CYP3A4*1*18B genotypes received fewer fentanyl doses compared with other genotype groups [OPRM1 and COMT common functional polymorphisms on morphine postoperative analgesia and central side-effects. The heterozygous patients with OPRM1 A118G and COMT G1947A SNPs consumed significantly less morphine in the postanesthetic recovery room and 48 hours after surgery compared with homozygous patients, and nausea and sedation scores were also significantly lower during all observed postoperative periods for heterozygous patients [The most ancient and powerful analgesics are opiates, although they have very different effects on individuals, from pain relief to secondary hyperalgesia, with a wide range of side-effects including sedation, respiratory depression, constipation, and addiction. Recently, several studies found that genetic polymorphisms greatly affect both analgesic effects of morphine and other opioid drugs and their adverse effects. Since the mu-opioid receptor is the primary site of action for both exogenous  and endogenous  opioids, its encoding gene, genotype . Similargenotype . Interesgenotype . Anothergenotype . Finallye groups . These fpatients . In addipatients  and resppatients . COMT may contribute directly to morphine efficacy. Cancer patients with the Val/Val genotype  needed more morphine compared to those with the Val/Met or Met/Met genotypes [COMT SNPs could also play a role in morphine efficacy since carriers for the most frequent haplotype constructed from ten COMT SNPs, in addition to the Met allele, need lower morphine doses than patients not carrying that haplotype [enotypes . Other Caplotype .ABCB1/MDR1 gene encoding for adenosine triphosphate-binding cassette, subfamily B, member 1, a drug efflux transporter, considered to be a major component of the blood-brain barrier and a major determinant of morphine bioavailability in the central nervous system [OPRM1 A80G SNP, Campa et al. were able to predict patients as being “strong responders,” “responder,” or “nonresponders” for morphine pain relief, with sensitivity close to 100% and specificity more than 70% [ABCB1 alleles, 1236C > T and 2677G > T/A protect against the respiratory depressive effects of fentanyl [Another gene involved in opioid pathway is s system . Its funs system . Moreovethan 70% . These gfentanyl . CYP2D6. While individuals lacking CYP2D6 function suffer from poor codeine analgesia, CYP2D6 duplication genotype carriers  may experience exaggerated and even potentially dangerous opioidergic effects due to ultrarapid codeine metabolism [CYP2D6 genotypes predicting ultrarapid metabolism resulted in about 50% higher plasma concentrations of morphine and its glucuronides compared with the extensive metabolizers. Accordingly, the drug dosage required for the same level of analgesia may differ significantly between the “poor” , “intermediate” , “extensive” , and “ultrarapid”  metabolizers. For example, a daily dose of 10–20 mg of nortriptyline may be sufficient for a patient who is a poor metabolizer; however, an “ultrarapid metabolizer” inheriting multiple copies of the gene could require as much as 500 mg a day [CYP2D6 genotype is clinically relevant for the prediction of analgesic response as well as effects of other CYP2D6-dependent drugs like beta-blockers or antidepressants. Besides morphine and fentanyl, codeine also acts predominantly on mu-opiate receptors. Its metabolizing enzyme, cytochrome P450 2D6, is encoded by gene tabolism . CYP2D6 mg a day . While oMC1R gene that encodes this receptor and nearly always causes the red hair, also influence opioid analgesic effects. Women with functional MC1R SNPs  displayed significantly greater analgesia from the kappa opioid and pentazocine than all other groups tested [It is now a well-known fact that red-heads respond differently to local anesthetic drugs or require more of these drugs to get analgesia in clinical settings such as dental office or in response to painful stimuli . The hums tested . In addis tested , 200.Overall, growing evidence suggests that multiple genetic factors influence pharmacokinetics and pharmacodynamics of analgesic drugs, and many common functional polymorphisms may affect analgesic response independently and through interaction . MajoritCOMT, OPRM1, and TRPV1 have become “gold standard” and the most popular candidates and are constantly tested in different pain models, and studies of functional variants within these genes outnumber studies of all other genes combined [TRPM8, which codes for a cold pain sensitivity; and rs2651899 (PRDM16) with yet unclear role in migraine [CCT5 and FAM173B, nearby this locus, as potential targets in the regulation of pain [To date, a relatively limited number of pain genes have been implicated as contributing to human pain phenotypes, while at least 358 genes are thought to be relevant to pain or analgesia according to the Pain Genes Database . About tcombined . Knowledmigraine . A few pmigraine  or poorlmigraine  or endommigraine . Latest  of pain ; and a m of pain . These f of pain . CurrentAVPR1A gene encoding the vasopressin-1A receptor of arginine vasopressin, but only in male subjects reporting stress at the time of testing [Inheritance may play a significant role in human pain, though most of painful disorders are triggered by known or yet unidentified environmental factors. Chronic pain provides the best illustration of the interaction between nature and nurture when it comes to pain. Inflammatory and/or nerve damages are suspected to be the etiology of most chronic pain syndromes like osteoarthritis, diabetic neuropathy, or postherpetic neuralgia, but only a small proportion of those subjected to such injuries actually develop chronic pain, and the degree of pain severity varies a lot across patients. Genes may predispose some to more intense or more chronic pain, but the environment, in turn, can shape the genetic effects. One of the most important environmental factors is stress. Stressful experiences, depending on the type, intensity, and duration of stressor can alter human pain sensitivity by either reducing pain  or exacerbating pain , and the mechanisms of both phenomena are not fully understood. Generally, acute stress induces analgesia , while the effects of chronic stress in nociception are less predictable . Epidemi testing . Moreove testing . If the BDNF Val66Met) with fibromyalgia syndrome [CYP1A1 MspI and HincII genotypes) modified the association between passive smoking and painful dysmenorrhea [β2-Adrenergic receptor genotype , ethnicity and weight contributed to labor maternal pain [OPRM1 A118G genotype were shown as independent and significant contributors to variation in pain perception and postoperative morphine use in patients undergoing cesarean delivery [TRPV1  and OPRD1  interacted with gender, ethnicity, and temperament contributing to individual variation in thermal pain and cold pain sensitivity in healthy subjects [COMT , such that individuals with met/met genotype experienced a greater decline in positive affect on days when pain was elevated compared to individuals with other genotypes [COMT effects on human pain also seem to interact with gender and ethnicity in that the Met allele contributed to variability in short duration cold pain sensitivity (cold pressor test) only in females of Caucasian origin but not in males or other ethnicities [OPRM1 A118G genotype regarding the pain intensity in patients with low back pain and sciatica after lumbar disc herniation showing that G allele increases the pain intensity in women but has a protective effect in men the first year after disc herniation [Demographic factors have been reported quite often interplaying with the genetic effects on human pain. For example, increased body mass index mediated the association of a common functional SNP in the gene of brain-derived neurotrophic factor  exert epigenetic actions. Epigenome shapes physical structure of the genome: it tightly wraps inactive genes making them unreadable and relaxes active genes making them easily accessible thus adjusting specific genes in response to rapidly changing environment. Some epigenetic processes happen during pregnancy or early life time, other changes appear to occur randomly, and they may be triggered by signals from inside the cell, from neighboring cells, or from environmental factors such as radiation, toxins , nutrition, physical activity, and stress. Importantly, histone modifications and DNA methylation are associated with the subject's age [Apparently, nature and nurture are not the only elemental forces at work in regard to pain. There is one other factor in play tied to neither nature nor nurture that in some cases serves as a bridge between the genes and environment and in others operates on its own, shaping individual's pain perception. This third factor, called epigenetics, represents a mechanism by which the environment can directly regulate the translation of the DNA information into proteins via stable and/or heritable changes in gene function that are not intrinsic to the genetic code, but affect gene expression in a tissue-specific manner, resulting in a specific phenotype . Epigenect's age . As withct's age . FinallyGDF5 demonstrated that DNA methylation can affect the allelic expression imbalance between the C and T alleles of the GDF5 SNP rs143383, such that loss of methylation of the 5′UTR leads to a significantly greater imbalance in expression between the two alleles influencing their penetrance in susceptibility to osteoarthritis [ To date, several studies reported promising findings on the role of epigenetics for pain although human data is still very limited. Histone deacetylase (HDAC) inhibitors—compounds that prevent the removal of acetyl groups from histones—can ameliorate pain symptoms in animals and humans via suppression of cytokines . Epigenerthritis . Similarrthritis , 243. Forthritis . Epigenerthritis , at leasrthritis .  Epigenetic inheritance adds another dimension to the complexity of the nature and nurture effects on human pain continually adjusting individual's gene expression to fit the changing environment without altering DNA code and translating environmental signals to the fixed genome. Epigenetic analysis may identify mechanisms critical to the development and/or persistence of painful diseases and may provide new pathways and target mechanisms for future drug development and individualized pain medicine. Current investigation of genome-wide distribution of acetylation and methylation “tags”—places along the genome where methylation changes the pattern of gene expression—in multiple human cell lines leads to epigenome sequencing, creation of DNA methylation and histone modification maps and evaluation of individual's unique epigenetic profile—an epigenetic signature that determines the level of gene expression, including pain genes. Further, exploring the epigenetic mechanisms of gene regulation in pain and other complex traits and diseases may explain some of the missing heritability identified in GWAS studies . In summary, complex traits such as pain are highly heritable, and further intensive scrutiny of DNA sequences themselves may reveal many more important genetic variants with larger or smaller effect size. Modern genetics and genomics provide with powerful tools allowing us to investigate shared biological processes that are common to all individuals  and to dissect the genetic basis for interindividual differences in pain susceptibility, behavior, and pathology . Underst


Most interestingly, we identified 52 novel myokines which have not been described before to be secreted by skeletal muscle cells. For 48 myokines we show that their release is regulated by contractile activity. This profiling study of the human skeletal muscle secretome expands the number of myokines, identifies novel contraction-regulated myokines and underlines the overlap between proteins which are adipokines as well as myokines.Proteins secreted by skeletal muscle, so called myokines, have been shown to affect muscle physiology and additionally exert systemic effects on other tissues and organs. Although recent profiling studies have identified numerous myokines, the amount of overlap from these studies indicates that the secretome of skeletal muscle is still incompletely characterized. One limitation of the models used is the lack of contraction, a central characteristic of muscle cells. Here we aimed to characterize the secretome of primary human myotubes by cytokine antibody arrays and to identify myokines regulated by contraction, which was induced by electrical pulse stimulation (EPS). In this study, we validated the regulation and release of two selected myokines, namely pigment epithelium derived factor (PEDF) and dipeptidyl peptidase 4 (DPP4), which were recently described as adipokines. This study reveals that both factors, DPP4 and PEDF, are secreted by primary human myotubes. PEDF is a contraction-regulated myokine, although PEDF serum levels from healthy young men decrease after 60 min cycling at VO Skeletal muscle is the largest organ in the human body and path-breaking work during the last decade has demonstrated that skeletal muscle is an active endocrine organ releasing a host of so-called myokines Skeletal muscle exhibits profound adaptability in response to environmental influences. Exercise training enhances muscular endurance in vitro and in vivo to stimulate a broad program of brown-fat-like development in vitro in rat myotubes in vitro to induce insulin resistance in hepatocytes Boström et al. recently published that PGC1α over-expression in mouse muscle stimulates increased expression of FNDC5, a membrane protein that is cleaved at the N-terminus thereby releasing a soluble protein, called irisin.This protein acts on white adipose tissue-derived adipocytes Previously, we have published the secretome of primary human adipocytes and found 44 novel adipokines To gain a broader view, recent efforts have focused on exploring the complete secretome of skeletal muscle by proteomic studies. Using this approach, Yoon et al. have studied the regulation of protein secretion by rat skeletal muscle cells after insulin stimulation in vitro model of human skeletal muscle cell contraction. Similarly to exercising skeletal muscle in vivo, EPS induced contractile activity in human myotubes, accompanied by formation of sarcomeres, activation of AMP-activated protein kinase and increased IL-6 secretion in vitro conditions.Recently, we have established and characterized an Currently, no biochemical technique exists that can efficiently separate and consistently detect the total protein composition of the cellular secretome, e.g. neither the rodent cell studies Reagents for SDS-PAGE were provided by GE Healthcare  and by Sigma , rotiphorese was supplied by Carl Roth . The following antibodies were used: anti-DPP4 , anti-PEDF , anti-tubulin . Horseradish peroxidase-conjugated goat anti-rabbit and anti-mouse IgG were purchased from Promega . All other chemicals were of the highest analytical grade commercially available and were purchased from Sigma.5 cells/well and cultured in α-modified Eagle’s (αMEM)/Ham’s F-12 medium containing skeletal muscle cell growth medium supplement pack  up to near-confluence. The cells were then differentiated in αMEM containing 2% horse serum  until day 5 of differentiation followed by overnight starvation in αMEM without serum after washing with PBS twice.Primary human skeletal muscle cells  supplied as proliferating myoblasts were cultured as described in our earlier study EPS was applied to fully differentiated myotubes in six-well culture dishes using a C-Dish in combination with a C-Pace pulse generator . Myotubes were stimulated with a frequency of 1 Hz, 2 ms pulse duration, an intensity of 11.5 V for 4 to 24 h. This protocol rather reflects regular exercise due to unchanged MHCIIa expression Skeletal muscle cells were treated as indicated and lysed. The immunoblotting procedure was carried out as described before Total RNA was isolated and reverse transcribed using the RNeasy Mini kit and Omniscript Reverse Transcription kit  according to the manufacturer’s instructions. Gene expression was determined by quantitative real-time PCR using QuantiTect primer assays and SYBR green reagents  with 0.016–20 ng of cDNA on a Step One Plus Cycler . Expression levels of investigated genes were normalized to actin. Gene expression was analyzed via the ΔCt method.Primary human myotubes (4 different donors) were subjected to EPS or left unstimulated, and CM was collected over a period of 24 h to assure that differences of low abundant proteins were in the range of array and ELISA sensitivity. Undiluted CMs were then analyzed by RayBio human cytokine antibody arrays  according to the manufactures instructions and signals were visualized and evaluated on a LUMI Imager  work station. Only signals that were above the background and detected in the CM of at least three donors were included in the analysis. For further validation cytokine concentration in CM was determined using DPP4 (R&D Systems) and PEDF ELISA (Biovendor), respectively, both assays used according to the manufacturer’s protocol.2, VO2max 65.5±1.7 ml/min/kg) participated in the study. A baseline blood sample was taken before start of the exercise. Subjects cycled at 70% VO2max for 60 min and then rested for 2 h. Blood was sampled immediately after the exercise session as well as 30 min and 120 min post exercise. Written informed consent was obtained from all the participants. The study was approved by the Regional Committee for Medical and Health Research Ethics, Region Sør-Øst-Norge, Norway (2011/927b).Eight well-trained healthy lean male volunteers (body mass index 23.1±0.6 kg/mData are the means ± SEM. Unpaired two-tailed Student t-test or one-way ANOVA (post hoc test Tukey’s multiple comparison test) were used to determine statistical significance. All statistical analyses were performed using Prism5  considering a P value of <0.05 as statistically significant. Corresponding significance levels are indicated in the figures.In this study, we identified the two known adipokines DDP4 and PEDF to be secreted by skeletal muscle cells. The analyses of myocyte lysates confirmed the expression of PEDF and DPP4 in both human myoblasts and myotubes . MyocytePEDF mRNA expression and protein level were induced during the first days of myogenesis  reachingUsing three different cytokine antibody arrays, we analyzed CM obtained from myotubes in order to identify novel proteins secreted by skeletal muscle cells. In total, 179 peptides were analyzed and for 116 of these a positive signal was detected after incubating the membranes with CM obtained from at least three different donors , Controlin vitro model no free fatty acids are present in the medium this might explain why we have not detected ANGPTL4 in the supernatant of contracting myotubes.In the case of 40 factors, no signal was detected  includinIn addition, 19 cytokines were only detected in the CM obtained from one or two donors and were not included in the analyses.Human cytokine antibody array analysis was additionally used as a screening method to detect novel contraction-regulated myokines by directly comparing CM obtained from control and EPS-treated myotubes . IL-6, IIn addition to the four positive controls, 44 factors were significantly regulated by contraction of skeletal muscle cells . FifteenDetailed characterization of the human skeletal muscle secretome is essential to understand the beneficial effect of exercise in the context of chronic diseases. Therefore, this study was focused on the identification of myokines released by contracting myotubes.To consistently detect the total protein composition of the cellular secretome, it is important to combine various technical approaches, since no biochemical technique can efficiently detect all proteins Secretome analysis showed that the profile of proteins secreted by skeletal muscle changes in response to the treatment with insulin Norheim et al. described the proteomic identification of 236 proteins secreted from non-contracting human skeletal muscle cells and validated the expression of candidates in response to strength training in vivo exercise might be better mimicked by short-term, high frequency EPS, as recently described by Nikolić et al. One myokine we have analyzed in this study is PEDF, which is a 50 kDa secreted glycoprotein that belongs to the non-inhibitory serpin group The second validated myokine is DPP4, a cell surface type II membrane glycoprotein. A BioGPS query revealed that DDP4 is ubiquitously expressed. DPP4 cleaves N-terminal dipeptides of polypeptides like glucagon-like peptide 1 (GLP1) and glucose dependent insulinotropic polypeptide (GIP), two post-prandial activated incretins Substantial DPP4 activity is also found in plasma because of a soluble form of DPP4 lacking the cytoplasmic tail and the transmembrane region of this protein. BioGPS query revealed that DPP4 is predominantly expressed in the kidney, smooth muscle cells, cardiac myocytes, prostate and small intestine. However, the major source of circulating DPP4 and its regulation remain unknown. By using adipose tissue explants from lean and obese subjects, Lamers et al. observed an increase in DPP4 serum levels that strongly correlated with adipocyte volume and parameters of the metabolic syndrome, while DPP4 serum levels decreased to the lean level after weight reduction Most interestingly, we compared the CM of control and contracting myotubes by cytokine antibody array analysis. Among others, C-C motif chemokine 2 (MCP-1) and somatotropin were identified as contraction-regulated myokines.in vitro data demonstrate this factor has the ability to induce insulin resistance in adipocytes and skeletal muscle cells MCP-1 resembles IL-6, which was long-time only recognized as inflammatory cytokine and then found to be an exercise factor with a positive impact on muscle physiology Somatotropin is a pleiotropic peptide hormone with an important role in the regulation of metabolism via stimulation of lipid mobilization and oxidation This study now revealed that there is a considerable overlap between the adipokines and myokines. A considerable number of secreted proteins from skeletal muscle are also secreted by adipocytes. We defined these proteins as adipo-myokines. Among others, DPP4 The present study contributes to the understanding of the endocrine function of human myotubes. Secretory proteins are part of a complex physiological network, and they exert different effects under various environmental conditions. Therefore, our current knowledge of contraction-regulated secretory proteins from skeletal muscle and their roles in distinct signaling pathways according to the organ has to be determined in future studies. For novel myokines presented in this study, additional basic research and clinical studies will provide a further understanding in exercise metabolism.Table S1Cytokines that were not detected in CM of hSkMC using cytokine antibody arrays. CM of control and EPS-treated myotubes were collected after 24 h and analysed as described. This list contains all cytokines that did not produce a signal above the background.(DOCX)Click here for additional data file.Table S2List of Myokines that were not regulated by contraction. CM of control and EPS-treated myotubes were collected after 24 h and analysed as described.(DOCX)Click here for additional data file.


While several thousand square kilometers of land area have been subject to surface mining in the Central Appalachians, no reliable estimate exists for how much coal is produced per unit landscape disturbance. We provide this estimate using regional satellite-derived mine delineations and historical county-level coal production data for the period 1985–2005, and further relate the aerial extent of mining disturbance to stream impairment and loss of ecosystem carbon sequestration potential. To meet current US coal demands, an area the size of Washington DC would need to be mined every 81 days. A one-year supply of coal would result in ∼2,300 km of stream impairment and a loss of ecosystem carbon sequestration capacity comparable to the global warming potential of >33,000 US homes. For the first time, the environmental impacts of surface coal mining can be directly scaled with coal production rates. Mountaintop removal coal mining (MTR) is a particularly invasive mining practice developed in the United States (US) capable of producing low-cost coal. The process of MTR uses explosives and heavy machinery to remove entire mountain ridges in order to access near surface coal deposits, producing vast quantities of mine spoil that fills valleys and buries streams. MTR has expanded dramatically in recent decades and is now the dominant driver of land-use change across the Central Appalachian region Growing scientific evidence demonstrates that these surface mining activities present severe, negative environmental consequences including widespread destruction of forest habitat, long-term impairment of ecosystem carbon and nutrient cycling, and regional deterioration of stream water quality During the process of mining, forests are cleared and the soil and bedrock overlying coal seams (overburden) are removed. The land surface is dramatically reengineered, leaving behind a new topography constructed of reshaped mine spoil. Post-mining soil profiles often have higher bulk densities, lower water infiltration rates, and lower nutrient contents permitted for mining, not all permitted areas are ultimately mined; single mines occupy large tracts of land (up to 88 km2) and topographic variation makes some coal difficult to access 2) While adequate data are available describing the amount of land area https://www1.nga.mil/ProductsServices). The hyperspherical direction cosine (HSDC) method Mined areas were delineated at decadal intervals  using previously published methods Surface mines have long lifecycles often exceeding 10 years lm function, Stat package, CRAN-R statistical software) Simple linear regression (SLR) was used to estimate the relationship between county-level coal production and aerial mining disturbance extent  to translate this estimate of stream impairment (m ha−1) into units of coal produced .In a previous study, we estimated the threshold value of watershed mining impacts at which a receiving stream is likely to be classified as biologically impaired (as defined by significant losses of pollution intolerant stream macroinvertebrates) turbance . We use −2 yr−1, respectively  exceeds that for reclaimed mine sites converted to grasslands (167 g C m−2 yr−1) or sites supporting forest regrowth (357 g C m−2 yr−1). Our estimate of foregone ecosystem C sequestration resulting from mining, then, ranges from 32–222 g C m−2 yr−1. Because most sites mined post-SMCRA (after 1977) have been converted to grasslands −2 yr−1). We divide this estimate of foregone ecosystem C sequestration per unit area disturbance by the regression value of coal produced per unit area disturbance to derive our final estimate of foregone ecosystem C sequestration in units of coal produced.Clearing of forests prior to mining can result in large initial losses of soil and biomass C pools. These ecosystems will re-accumulate C following mine reclamation, but likely at a slower rate because post-mining soils are often compacted with low nutrient content and reduced fertility ectively . Amichevil pools . Thus, tTo bring perspective to these per-unit-coal estimates of the environmental costs of MTR, we calculate the extent of environmental impacts across the region by multiplying the per-unit-coal values by the cumulative annual coal production data across all counties. While we could also estimate the extent of these impacts directly from the amount of land area identified as having been mined in the satellite imagery analysis, the imagery analyses are only of decadal resolution while the coal production data are reported annually. Thus, using the coal production data allow us to represent the expansion of these environmental impacts with higher temporal resolution.6, R2  = 0.79) or, inversely, for each ton of coal 0.87 m2 of landscape is disturbed  would need to be mined every 81 days. To supply one year of current US coal demand would require converting 803 km2 of Appalachian mountains to surface mines, leading the to the biological impairment of an estimated ∼2,300 km of streams and forgone ecosystem C sequestration of ∼185,000 Mg C yr−1   .severity of surface mining impacts extent of these environmental impacts and in providing the metrics necessary to compare these environmental costs to the obvious economic benefits of coal. This has been a critical shortcoming, as even the most severe impacts may be tolerable if they are believed to be sufficiently limited in extent. We show, however, that the extent of environmental impacts of these surface mining practices is staggering, particularly in terms of the amount of coal that is produced. Tremendous environmental capital is being spent to achieve what are only modest energy gains.Surface coal mining in the Central Appalachians has become an extremely contentious method for producing energy. While the scientific community has adequately demonstrated the File S1Contains Table S1 and Table S2.(DOCX)Click here for additional data file.


The transcription factor heat shock factor 1 (HSF1) is the master regulator of the heat shock response. It is crucial for cell homeostasis and implicated in aging, neurodegenerative disease and cancer . The resThe heat shock response, mediated by activation of HSF1, is an ancient, highly conserved mechanism that protects organisms against various adverse environmental and pathological conditions that damage cellular proteins . In cancIn healthy, non-stressed cells, HSF1 is located in the cytoplasm in an inhibitory complex with the molecular chaperone heat shock protein 90 (HSP90) – another key player in proteostasis and malignant progression. Upon proteotoxic stress as classically induced by heat shock, HSF1 dissociates from HSP90, trimerizes, translocates to the nucleus and associates with its cognate DNA sequence localized at the promoters of its target genes to modulate their transcription  and HSP90 inhibitors  enhance proteotoxic stress in cancer cells, causing cell cycle arrest and apoptosis. However, their efficacy is limited by activation of HSF1 and hence induction of numerous cytoprotective proteins, including chaperones like HSP90 and HSP70 family members that ameliorate the proteostatic damage, causing drug resistance -8.Accumulating evidence shows that HSF1 is critically involved in oncogenesis. Key studies using HSF1-knockout mice demonstrate the requirement for HSF1 in tumorigenesis by oncogenic RAS or mutant P53 . Furtheret al. now demonstrate that HSF1 rewires the transcriptome in cancer in a surprising new way – one that is quite distinct from the classical heat shock response.The common view is that the key effects of HSF1 in oncogenesis are mediated via increased HSP expression with oncogenic-support properties . Howeveret al demonstrate that HSF1 protein expression is up-regulated constitutively and also activated – as shown by its nuclear localization and its phosphorylation at serine 326 – in oncogene-transformed breast cancer cells that are highly tumorigenic and metastatic, as compared to their less malignant isogenic counterparts. Also, the most aggressive cancer cell lines are more dependent on HSF1 for their growth and survival, indicating a stronger ‘addiction’ and therapeutic vulnerability.First, Medillo et al. use chromatin immunoprecipitation linked to next-generation sequencing (ChIP-Seq) to identify the genes bound by HSF1. Under basal conditions HSF1 binds many more genes in the highly malignant compared to less aggressive cancer cells or immortalized but non-transformed cells. Binding occurs at both promoter regions and distal sites. Following heat shock, HSF1 binding to genes is enhanced in all the cell lines. Surprisingly, however, there are marked differences in the genes bound by HSF1 in cancer under basal conditions versus normal cells following heat shock. Many genes bound specifically in the aggressive breast cancer cells are not classical heat shock proteins and there is enrichment for genes involved in protein translation, RNA binding, metabolism and adhesion. Examples of cancer-specific genes highlighted by Medillo et al. include CKS2 encoding cyclin-dependent kinase interacting protein; LY6K coding for glycophosphatidyl-inositol-anchored membrane protein with cancer connections; and RBM23 encoding an RNA-binding protein implicated in estrogen-mediated transcription.Next, Medillo et al. use RNA interference to confirm the role of HSF1 in regulating transcription of its identified bound genes.On the other hand, another set of genes bound by HSF1 in the cancer cell lines under basal conditions are common to those binding in non-transformed cell lines after heat shock. Thus the results indicate both interesting differences and a level of overlap between the HSF1 gene signature regulated by thermal stress and that associated with malignancy – raising the possibility that different molecular mechanisms  may be preferentially involved under these two stress-activating conditions  and/or epigenetic states.In addition to the implications for basic research there are important translational consequences. Understanding the functional relevance of the epigenetic factors and post-translational modifications that modulate HSF1 activity, as well as identifying the proteins involved and the cofactors associated with HSF1 transcriptional activity, is likely to provide both new cancer biomarkers and novel ways to target more specifically and efficiently (eg Figure Thus, although HSF1 has been around for a billion years of evolution, it continues to surprise us. In the near future we will very likely see more shocks about heat shock in cancer.


Gfap promoter. This review is a summary of our current understanding of the physiological functions of OASIS in the CNS.Eukaryotic cells can adapt to endoplasmic reticulum (ER) dysfunction by producing diverse signals from the ER to the cytosol or nucleus. These signaling pathways are collectively known as the unfolded protein response (UPR). The canonical branches of the UPR are mediated by three ER membrane-bound proteins: double-stranded RNA-dependent protein kinase (PKR)-like endoplasmic reticulum kinase (PERK), inositol-requiring enzyme-1 (IRE1) and activating transcription factor 6 (ATF6). These ER stress transducers basically play important roles in cell survival after ER stress. Recently, novel types of ER stress transducers that share a region of high sequence similarity with ATF6 have been identified. They have a transmembrane domain, which allows them to associate with the ER, and possess a transcription-activation domain and a basic leucine zipper (bZIP) domain. These membrane-bound bZIP transcription factors include OASIS, BBF2H7 CREBH, CREB4 and Luman, and are collectively referred to as OASIS family members. Despite their structural similarities with ATF6, differences in activating stimuli and tissue distribution indicate specialized functions of each member on regulating UPR signaling in specific organs and tissues. One of them, OASIS, is expressed preferentially in astrocytes in the central nervous system (CNS). OASIS temporally regulates the differentiation from neural precursor cells into astrocytes to promote the expression of Glial Cell Missing 1 through dynamic interactions among OASIS family members followed by accelerating demethylation of the  The endoplasmic reticulum (ER) is an essential organelle that regulates the synthesis and post-translational modifications of secretory and membrane proteins. Nascent proteins are folded in the ER with the assistance of molecular chaperones and folding enzymes. Various pathophysiological conditions, such as ER-calcium depletion, oxidative stress, hypoglycemia, expression of mutated proteins and hypoxia, interfere with the correct folding of proteins followed by promoting the accumulation of these unfolded or misfolded proteins in the ER lumen. These conditions are collectively termed ER stress, and have the potential to induce cellular damage. The ER responds to these perturbations by activating an integrated signal transduction pathway, called the unfolded protein response (UPR) Ron . Activat Caenorhabditis  elegans. C. elegans contains three bZIP transcription factors. Although the degree of homology is less than those in the mammalian family, these proteins are related most closely to ATF6, OASIS and Luman  transcription factors  mice are born at the expected Mendelian ratios, but the mice exhibit severe osteopenia involving a decrease in bone density at all skeletal sites and spontaneous fractures —a major component of osseous tissues—was identified as one of the targets for OASIS. OASIS activates the transcription of Col1 through direct binding to a CRE-like sequence that exists in the Col1 promoter regions. Indeed, Oasis−/− osseous tissues show a significant decrease in the amounts of type I collagen. The defect of bone formation involving decreased amounts of type I collagen is rescued completely by osteoblast-specific overexpression of OASIS, indicating that osteopenia in Oasis−/− mice is caused primarily by deletion of Oasis and its target, Col1 gene in osteoblasts . In hippocampal astrocytes injured by intraperitoneal injection of KA, ER stress is induced and Oasis mRNA is strongly up-regulated  mice. The number of glial fibrillary acidic protein (GFAP)-positive reactive astrocytes, an astrocyte marker, was decreased in the hippocampi of Oasis−/− mice compared with those of WT mice after brain injury. In embryonic stages, Oasis mRNA is barely observed in the cerebral cortices of embryonic day (E) 14.5 mice; however, strong signals are detected at E16.5 and E18.5  and bone morphogenetic protein 2 (BMP2), which promote the differentiation of NPCs into astrocytes  is identified as a target of OASIS. OASIS promotes the expression of ter Fig. . The mamGcm1 (Saito et al. Gcm1 during astrocyte differentiation. Conversely, the Luman N-terminus inhibits the formation of the OASIS-CREB4 heterodimer by binding to the OASIS N-terminus and down-regulating transcription of Gcm1, leading to a cessation of astrocyte differentiation (Fig. The bZIP type transcription factors including OASIS family members are known to form homodimers or heterodimers to promote transcription (Hai and Hartman ion Fig. . Therefoion Fig. , chondroion Fig. , and plaion Fig. . During ion Fig. . Thus, pion Fig. . It is pCreb4−/− mice revealed that this molecule plays a role in differentiation of sperm cells (Adham et al. UPR signaling was originally found as a system for evading cellular damage in acute ER dysfunction. However, recent advanced studies have revealed that UPR signaling provides important signals for regulating cellular physiology. Not only canonical ER stress transducers, but also OASIS family members act as signaling centers for numerous networks originating from the ER. In particular, OASIS family members are specialized in biological regulation, including cell differentiation, maturation and maintenance of basal cellular homeostasis. Thus far, ER stress damages cells, and has been seen as a major cause of various diseases (Schroder and Kaufman Gcm1 in the CNS (Saito et al. The signaling from the ER mediated by OASIS family members is presumed to be triggered by ER perturbation. However, is ER stress actually only the stimulation for activating OASIS family members that are required for initiating physiologically important signaling events? Indeed, OASIS is weakly expressed in hippocampal neurons under control conditions, although it is not activated by KA-induced ER stress (Chihara et al.


Recent studies have shown that these factors might provide a molecular link between increased adiposity and impaired insulin sensitivity. Since hepatic insulin resistance plays the key role in the whole body insulin resistance, clarification of the regulatory processes about hepatic insulin resistance by adipokines in rodents and human would seem essential in order to understand the mechanism of type 2 diabetes and for developing novel therapeutic strategies to treat it.Obesity is a major risk factor for insulin resistance and type 2 diabetes. Adipose tissue is now considered to be an active endocrine organ that secretes various adipokines such as adiponectin, leptin, resistin, tumour necrosis factor- In many developed and developing countries, obesity has reached epidemic proportions, resulting in an increasing prevalence of type 2 diabetes characterized by insulin resistance of peripheral tissues such as liver, muscle, and fat which cannot be overcome by hypersecretion of pancreatic beta cells . One surα (TNF-α) [Adipose tissue was traditionally regarded as a passive energy reservoir. However, since the discovery of leptin and subsequent identification of other adipose tissue-derived cytokines  in the last two decades , it beca (TNF-α) . All of  (TNF-α)  previous (TNF-α) . Althoug (TNF-α) , 8, but Since the identification of adiponectin as a protein exclusively produced from adipocytes exclusively by Scherer et al. in 1995 . FollowiThe adipokine adiponectin is greatly expressed in human serum where it accounts for 0.01% of total plasma protein and is predominantly secreted by adipose tissue   agonist thiazolidinediones were diminished in ob/ob obese mice with the targeted mutation of the adiponectin gene [Recent data has exhibited more specification as most of the studies have implicated HMW oligomers in attenuation of hepatic insulin resistance. For instance, the synthesis of the HMW oligomers is necessary to mediate the insulin sensitizing effects of adiponectin on the suppression of hepatic gluconeogenesis in primary rat hepatocytes . Moreovetic mice . Animal tic mice . The roltin gene . Notablytin gene . Pioglittin gene .Plethora of animal data has been published describing the role of adiponectin in hepatic insulin resistance. Different animal models have supported this supposition widely. For example, a high scale study of adiponectin function in animal models of obesity, insulin resistance, and type 2 diabetes provided strong evidence that adiponectin promotes insulin sensitivity in muscle and in the liver . FurtherMoreover, adiponectin-deficient mice showed mild insulin resistance in the liver with administration of standard diet . SimilarClinical studies have described the similar evidences regarding the role of adiponectin in liver insulin resistance. Instantly, adiponectin levels are associated, in healthy humans, with plasma concentrations of various liver function indices . FurtherIn conclusion, adiponectin is a promising target for hepatic insulin resistance and resultantly metabolic disorder, but further clinical research is required to translate similar impacts in humans keeping in mind the pleiotropic nature of this hormone.In 1994, ob gene was first cloned on mice chromosome 6 . A year Lots of studies reported that a strong positive correlation exists between the concentration of leptin and insulin sensitivity and obesity –54, but in vivo and in vitro.In summary, leptin acts as an important target to improve the hepatic sensitive to insulin, and the precise mechanisms about leptin underlying hepatic insulin resistance should be investigated by experiments α, RELM-β, and the recently discovered RELM-γ [Resistin is a member of a family of cysteine-rich proteins referred to as resistin-like molecules, termed RELMs, which include RELM-d RELM-γ . It was d RELM-γ  and was d RELM-γ . This reIn rodents, resistin primarily expressed in and secreted from mature adipocytes, with some expression in pancreatic islets and portions of the pituitary and hypothalamus. However, resistin is expressed primarily by macrophages and seems to be involved in the recruitment of other immune cells and the secretion of proinflammatory factors in humans . The divA study in rodents showed that the infusion of resistin rapidly induced severe hepatic insulin resistance, resulting in a reduced insulin-mediated suppression of gluconeogenesis and increased glycogenolysis . ConsistIn a word, resistin could induce hepatic insulin resistance by inhibiting the phosphorylation of Akt and GSK3, and its effect on insulin sensitivity is opposite to those reported for the adipocyte-secreted hormone adiponectin, which increases insulin sensitivity of the same liver-specific functions.IL-6 is secreted by many cell types, including immune cells, fibroblasts, endothelial cells, myocytes, and a variety of endocrine cells . AdiposeThe inflammatory regulator IL-6 has also emerged as a factor that is implicated in hepatic insulin resistance. However, the role of IL-6 in the etiology of insulin resistance is not fully understood. Impaired insulin receptor signaling and insulin-dependent glycogen synthesis were found in HepG2 cells and primary mouse hepatocytes acutely pretreated with IL-6 . IL-6 caIL-6 mediates insulin sensitivity through many distinct mechanisms. Of note, there is a significant correlation between the level of IL-6 in adipose tissue and several metabolic parameters such as fasting plasma glucose, basal and insulin-stimulated glucose transport, and whole-body glucose disposal . Thus, ITo sum up, the effect of IL-6 on hepatic insulin sensitivity is uncertain; it appeared to be determined by whether it is present acutely or chronically; the latter is the setting associated with insulin resistance.α is a proinflammatory cytokine produced by various types of cells, mainly but not only inflammatory cells like macrophages and lymphocytes. The release of TNF-α in noninflammatory cells has also been reported, at lesser degrees. Since the first recognized sequencing of TNF-α in 1984, it has established itself as a prime proinflammatory cytokine [α is involved in array of pathological conditions like obesity, congestive heart failure, inflammations, and insulin resistance [TNF-cytokine , making sistance , neverthα has been proposed as a link between adiposity and the development of insulin resistance [α have been observed in obese and insulin resistance humans and animals [α-mediated pathway interference has shown protection in diet induced animal models of obesity and metabolic syndrome [α therapy [α therapy has also shown to attenuate in insulin resistance in animal studies involving fructose-red rats [α expression is increased in adipose tissue in obese rodents and humans [α signaling either by knocking TNF-α out or by infusing blocking antibodies can reduce insulin resistance in obese rodents [α and IR with parallel increase in body mass index (BMI) factor [α level accompanied by an improvement in insulin resistance after administration of rosiglitazone [α and higher doses linked directly to insulin sensitivity [α [α as the sole biomarker and predictor for insulin resistance during pregnancy [α or its receptors showed protection against developing insulin resistance [α; diet induced macrophages which subsequently release the TNF-α are found to be involved in insulin resistance in paracrine signaling.TNF-sistance , becausesistance . Elevate animals . TNF-α-msyndrome . A clini therapy . Anti-TNred rats . Furtherd humans , and red rodents . More re) factor . A recenlitazone . Anothersitivity . Moreoveivity [α . A cliniregnancy . In one sistance . Liver rα is reported to demonstrate wide range of effects in pathogenesis of insulin resistance. The precise signaling pathway is, however, not clear yet even after scores of studies and scientific data put forward on this particular subject. However, the most attention grabbing signaling pathway in liver supported by numerous evidences is through the phosphorylation of the insulin receptor substrate-1 (IRS-1) protein on serine residues. This could prevent its interaction with the insulin receptor beta subunit and stop the insulin signaling pathway [α in human insulin signaling requires extensive scientific investigation in future to clear its signaling pathway.TNF- pathway –97. Howeα antibody infusion in humans was shown not to alter insulin sensitivity, fueling lingering uncertainty about the biological relevance of this pathway in human insulin resistance states [Conversely, numerous clinical studies have not been able to support similar findings and failed to prove its role in the relationship between type 2 diabetes coexisting with insulin resistance , 99. TNFe states .α role in modulating insulin resistance with no doubt varies from species to species and depends on animal models adopted for particular study. Further, due to its release by various types of cell types involving both inflammatory and noninflammatory cells which then can be involved in insulin resistance pathogenesis both by autocrine or paracrine manner, TNF-α is the subject of uncertainty regarding its precise role in insulin resistance. Without doubt, further scientific work is required in order to unravel its exactness in insulin resistance.Conclusively, TNF-α, IL-6, and resistin. In contrast, the presence of adipose tissue is vital in the prevention of hepatic insulin resistance, at least in part, via secretion of the following cytokines: leptin and adiponectin. Indeed, adipocytokines are major regulators of hepatic insulin sensitivity potentially linking insulin resistance and obesity. Further work is needed to clearly determine how they regulate the insulin signaling in hepatocytes and influence insulin sensitivity in other tissues  in rodents and human and their contribution to glucose homeostasis in diabetes.During the last couple of years it has been shown that adipokines play an important role in physiology and pathophysiology of insulin sensitivity. Our review summarizes recent findings regarding the relationship between adipocytokines and hepatic insulin resistance . The mec


In conclusion, our data indicates that skeletal muscle Ad resistance is rapidly induced in both oxidative and glycolytic muscle, independently of altered cellular redox state.A high fat (HF) diet rapidly impairs the ability of adiponectin (Ad) to stimulate fatty acid (FA) oxidation in oxidative soleus muscle, but the underlying mechanism remains elusive. Mere days of HF feeding also increase the muscle’s production and accumulation of reactive oxygen species (ROS) and shift cellular redox to a more oxidized state. It seems plausible that this shift towards a more oxidized state might act as negative feedback to suppress the ability of Ad to stimulate FA oxidation and generate more ROS. Therefore, we sought to determine whether i) a shift towards a more oxidized redox state (reduction in GSH/2GSSG) coincided with impaired Ad-stimulated palmitate oxidation in oxidative and glycolytic rodent muscle after 5 days of HF feeding , and ii) if supplementation with the antioxidant, N-acetylcysteine (NAC) could prevent the HF-diet induced impairment in Ad-response. Globular Ad (gAd) increased palmitate oxidation in isolated soleus and EDL muscles by 42% and 34%, respectively (p<0.05) but this was attenuated with HF feeding in both muscles. HF feeding decreased total GSH  and GSH/2GSSG  in soleus, but not EDL. Supplementation with NAC prevented the HF diet-induced reductions in GSH and GSH/2GSSG in soleus, but did not prevent the loss of Ad response in either muscle. Furthermore, direct incubations with H Dysregulation of fatty acid (FA) metabolism and deposition of lipids represent one mechanism by which insulin response in skeletal muscle can be impaired Given how rapidly Ad resistance can be induced, it is conceivable that this phenomenon may actually be a protective mechanism, preventing excessive rates of FA entry into the mitochondrion. Adiponectin stimulates FA oxidation by increasing flux into the mitochondria secondary to stimulation of AMPK and a reduction in malonyl CoA production and its inhibition of CPTI. Interestingly, our findings Reactive oxygen species are a normal byproduct of energy metabolism and are an important regulatory sensor. The cellular redox environment is a reflection of the balance between ROS production and metabolism. The ratio of GSH (reduced glutathione) to GSSG (oxidized disulfide glutathione) is a reliable marker of cellular redox state The GSH/2GSSG ratio and total GSH content are decreased in muscle from obese individuals (i.e. shift towards more oxidized state), as well as in rodent muscle following a 6-week HF diet The purpose of this study was to determine i) if a brief period (i.e. 5 days) of HF feeding could result in a more oxidized redox state (oxidative stress) in rat skeletal muscle that coincided with the rapid loss of Ad’s ability to stimulate FA oxidation; ii) if the HF-diet induced Ad resistance could be prevented through supplementation with the general anti-oxidant N-acetylcysteine (NAC); and iii) to examine and compare these responses in muscles of different oxidative capacity (oxidative (soleus) and glycolytic ).All procedures were carried out in accordance with the recommendations of the Canadian Council of Animal Care, and were approved by the Animal Care Committee at the University of Guelph . All surgeries were performed under sodium pentobarbital anesthesia and all efforts made to prevent discomfort and suffering.ad libitum chow diet  while the other half were give ad libitum access to a diet high in saturated fat  with the purpose of inducing muscle Ad resistance. Within each dietary group, half of the animals were given the antioxidant, N-acetylcysteine , for a total of 4 experimental groups . Dietary/experimental treatments lasted for 5 days at which time animals were sacrificed following an 8 to 12-hour overnight fast.Female Sprague Dawley rats  were housed in groups on a 12-hour reverse-dark cycle and fed standard rodent chow. Animals were given 48 hours to acclimate to their environment before being assigned to their respective experimental groups. Animals were randomly assigned to one of two diets. Half of the animals maintained their 2O2 to determine whether there was a direct effect of altered muscle redox state on Ad’s ability to stimulate FA oxidation.Following an overnight fast, animals were anaesthetized with an intraperitoneal injection of sodium pentobarbital (6 mg/100 g body wt). Soleus (oxidative) and EDL (glycolytic) muscles from one hind limb were stripped longitudinally and excised, yielding two intact muscle strips which were used to assess basal and Ad-stimulated palmitate oxidation . Whole soleus and EDL muscles were removed from the second leg and flash frozen in liquid nitrogen for the assessment of total and oxidized glutathione content. Red and white gastrocnemius muscles were sampled and flash frozen for the assessment of protein carbonylation as a marker of oxidative damage. In a separate set of experiments, soleus and EDL muscle strips from chow-fed animals were incubated in medium containing 0, 0.5 or 2 mM H2/5%CO2) Medium 199, containing 4% BSA and 0.5 mM palmitate was used as a base for all buffers. Immediately following isolation, soleus and EDL muscle strips were placed in pre incubation buffer for 30 min. This buffer consisted of the base buffer only. Following pre-incubation, muscle strips were carefully transferred to vials containing incubation buffer for 60 min. The incubation buffer consisted of the base buffer with the addition of 0.5 uCi/mL [1-14 C] palmitate . In the Ad-stimulated conditions, gAd  was added for a final concentration of 2.5 ug/mL, which is the concentration previously used to elicit a max response of FA oxidation Pre-warmed (30°C), pre-gassed . Palmitate oxidation was interpreted as the sum of that from the benzathonium hydroxide and the aqueous extract.Muscle samples were homogenized using a Brinkman Polytron at 25 000 rpm, 2 mL of Milli Q water was added to separate aqueous and lipid soluble phases, and samples were placed on a rocker for 10 minutes. Following the extraction, 0.5 mL of the aqueous phase was sampled in duplicate and counted using a liquid-scintillation counter to measure 2O2 for 2 hours to determine whether there was a direct effect of altered muscle redox state on Ad response. In pilot experiments, we determined that the acute exposure to 0.5 mM H2O2 for 2 hours increased the content of oxidized glutathione and decreased the ratio of total to oxidized glutathione ratio by about 50%, which is similar to that achieved by 5 days of HF feeding. Palmitate oxidation was assessed immediately following the two hour incubation.In a separate set of experiments, basal and Ad-stimulated FA oxidation was measured in soleus and EDL muscle strips following incubation in medium containing 0, 0.5 or 2 mM Hg for 10 min and the supernatant was removed and analyzed using a commercially available kit . Concentrations were expressed per gram of extract protein as determined by BCA assay. In a separate set of incubations, we analyzed total and oxidized glutathione content in soleus and EDL muscle strips after a 30 min incubation in base buffer to ensure that dietary affects on muscle redox state were still present (n = 5 per group).Total and oxidized glutathione content were assessed in soleus and EDL muscles removed from fasted animals to determine the effects of diet and antioxidant supplementation. Samples were powdered under liquid nitrogen and split into two tubes containing 5% metaphosphoric acid. To assess oxidized GSH, methyl-2-vinylpyridinium triflate was added to rapidly remove reduced GSH. Following a 5 min incubation, samples were centrifuged at 1 000×g and the supernatant assayed for total protein and diluted in PBS to a final protein concentration of 10 ug/mL. Protein carbonyls were then measured using a commercially available ELISA kit .Protein carbonyls were measured in other representative oxidative and glycolytic muscle (red and white gastrocnemius), due to the fact that the smaller soleus and EDL muscles were completely utilized for the assessment of palmitate oxidation and glutathione content. Briefly, muscle was chipped under liquid nitrogen and homogenized in 200 uL of 0.1 M PBS containing a protease inhibitor cocktail . Samples were centrifuged at 1500×2O2 treatment was analyzed using a paired t-test. Statistical significance was accepted at p≤0.05.All data are reported as mean ± the standard error (SE). Ad-stimulated FA oxidation, glutathione and protein carbonyl data following HF feeding and/or NAC treatment, was analyzed using a randomized block-design two-way  ANOVA. Ad-stimulated FA oxidation data following HThere were no significant differences in terminal body weight or fasting blood glucose among the experimental groups .2O2 did not impair the ability of Ad to stimulate FA oxidation in either muscle  in control rats . There wr muscle .The content of total glutathione (GSH) in soleus muscle from animals fed a HF diet was reduced by 26% compared to that of control-fed animals p<0.05; . High faGlutathione content  was not affected in EDL with either HF feeding or NAC supplementation .High fat feeding had no significant effect on protein carbonylation in red and white gastrocnemius muscle. Supplementation with NAC also had no significant effect on protein carbonylation in either muscle type .2O2 altered redox state in both muscles, but did not impair the ability of Ad to stimulate FA oxidation. Neither HF feeding nor NAC supplementation had a discernable effect on protein carbonylation in either fiber type. Collectively, our data indicates that the rapid onset of Ad resistance occurs independently of changes in the redox state.The ability of Ad to stimulate FA oxidation is blunted in muscle from obese humans and in HF-fed rodents The insulin sensitizing effects of Ad are believed to be due in part to the stimulation of FA oxidation GSH scavenges oxidants by oxidation of the central cysteine moiety at which point it can form glutathione disulfide (GSSG) or mixed disulfides with other cysteine containing proteins. In this way GSH can also act as a substrate in the regulation of redox sensitive proteins In the current study, we did not observe a diet effect on the redox state in glycolytic skeletal muscle (EDL). Recently, ROS generation in isolated mitochondria has been shown to be elevated when respiration is supported predominantly by long chain FA Prolonged changes in redox state and depressions in oxidant buffering capacity increase the likelihood for oxidative protein damage 2O2, muscle from streptozotocin-treated mice 2O2, did not impair Ad-stimulated FA oxidation.N-acetylcysteine is an acetylated derivative of the amino acid cysteine, the rate limiting substrate in GSH production It should be recognized that while NAC is effective at preventing oxidative stress in several experimental models We have previously reported that the ability of Ad to stimulate FA oxidation in soleus is impaired within 3 days of HF feeding The results from our current study further our previous work by demonstrating that Ad resistance develops rapidly in both oxidative and glycolytic muscle fiber types. The redox state of the oxidative soleus muscle was altered within the same time frame as the development of Ad resistance; however, the redox state was unaltered in glycolytic muscle. Furthermore, while treatment with the antioxidant NAC prevents HF diet-induced alterations in redox state in the soleus, this did not result in the protection of Ad response. From these findings we must conclude that shifts in whole muscle redox state are not responsible for the rapid impairment in Ad response.