Wine aroma results from the combination of numerous volatile compounds, some produced by yeast and others produced in the grapes and further metabolized by yeast. However, little is known about the consequences of the genetic variation of yeast on the production of these volatile metabolites, or on the metabolic pathways involved in the metabolism of grape compounds. As a tool to decipher how wine aroma develops, we analyzed, under two experimental conditions, the production of 44 compounds by a population of 30 segregants from a cross between a laboratory strain and an industrial strain genotyped at high density.de novo by yeast, such as nerolidol, ethyl esters and phenyl ethanol, and others derived from grape compounds such as citronellol, and cis-rose oxide. In three of these eight regions, we identified genes involved in the phenotype. Hemizygote comparison allowed the attribution of differences in the production of nerolidol and 2-phenyl ethanol to the PDR8 and ABZ1 genes, respectively. Deletion of a PLB2 gene confirmed its involvement in the production of ethyl esters. A comparison of allelic variants of PDR8 and ABZ1 in a set of available sequences revealed that both genes present a higher than expected number of non-synonymous mutations indicating possible balancing selection.We detected eight genomic regions explaining the diversity concerning 15 compounds, some produced PDR8 gene in the production of farnesyldiphosphate derivatives, of ABZ1 in the production of numerous compounds and of PLB2 in ethyl ester synthesis. This work also provides a basis for elucidating the metabolism of various grape compounds, such as citronellol and cis-rose oxide.This study illustrates the value of QTL analysis for the analysis of metabolic traits, and in particular the production of wine aromas. It also identifies the particular role of the  AMN111], was Fermentations were performed in 250 mL flasks equipped with airlocks to maintain anaerobiosis without stirring (design A) and in 1 L fermenters with constant stirring (design B). Small flask fermentations were weighed twice daily and stopped as soon as the daily loss was less than 1% of the expected total loss.-1 constant flow). The GC oven temperature was programmed without initial hold time at a rate of 2.7°C min-1 from 70°C to 235°C (hold 10 min). The injector was set to 250°C and used in pulsed splitless mode (25 psi for 0.50 min). The temperatures of the interface, MS ion source and quadrupole were 270°C, 230°C and 150°C, respectively. The mass spectrometer was operated in electron impact ionization mode  and the masses were scanned over a m/z range of 29 – 300 amu. Agilent MSD chemStation software  was used for instrument control and data processing. The mass spectra were compared with the Wiley’s library reference spectral bankWine aroma compounds were analyzed by the Stir Bar Sorptive Extraction method  adapted The following compounds were analyzed: isoamyl alcohol, isoamyl acetate, isobutanol, 2-3 butanediol, 2-phenylethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate, ethyl dodecanoate, ethyl myristate, ethyl palmytate, ethyl laurate, 2-phenyl ethanol, hexanoic acid, octanoic acid, decanoic acid, dodecanoic acid, ethyl 9-decenoate, isoamyl octanoate, 2-phenyl ethyl hexanoate, 2-phenylethyl octanoate, 2-phenylethyl decanoate, ethyl 4 hydroxy butanoate, ethyl 3-hydroxydecanoate, ethyl 3-hydroxyoctanoate, nerolidol, farnesol, 2-3 dihydro farnesol, - or - farnesol (A), - farnesol (A), farnesyl acetate, isoamyl octanoate, isoamyl decanoate, isoamyl dodecanoate, methyl oleate, trans-β-farnesene, -α-farnesene, α-bisabolene, ß-bisabolene, -α-farnesene, α-terpineol, linalol, citronellol, geraniol, nerol, citronellyl acetate, geranyl acetate, neryl acetate, cis-rose oxide, trans-rose oxide.Heritability was calculated according to the method of Brem et al. . StatistAs the distribution of most phenotypes was not normal and due to the small sample size, linkage analysis was performed using both parametric and non-parametric models to evaluate the robustness of the parametric model. The parametric model consists of a linkage analysis performed using a normal model with the Haley-Knott regression method implemented in the R/qtl package ,53. As tABZ1-S288C allele which provokes large variations in fermentation kinetics [ABZ1 marker  as an additive covariate. For these regions, a significant effect was indeed observed for both flocculation and the ABZ1-specific markers.To overcome the potential effects of flocculation  and of tkinetics , we perfFor the three models and the two datasets analyzed, logarithm of odds (LOD) scores were computed for each marker every 2.5cM. An interval estimate of the location of each QTL was obtained as the 1-LOD support interval. The LOD significance threshold was estimated after permutation tests that were replicated 1000 times. The percentage of variance explained by each QTL was estimated from a drop-one-term analysis of results in the global model.PDR8 59A, and cells were sampled when 70% of the glucose had been fermented. RNA was extracted with trizol as described previously [UBC6 and SCR1, both giving similar results.Fermentations (900 mL of MS300 medium) were performed in triplicate with strains 59A and S288C-eviously . cDNA wahttp://genome.jouy.inra.fr/genyeastrait/[The comparison of the sequences of the 59A and S288C genomes and the differences between them can be found at eastrait/.ABZ1 and PDR8, we collected their sequences from genomes available at SGD (http://www.yeastgenome.org/). All uncompleted or frameshift-containing sequences where discarded from this set. The phylogenies were inferred with MEGA [http://bioinf3.uab.cat/mkt/MKT.asp website.To infer the evolutionary history of ith MEGA  by the Mith MEGA . The treith MEGA  test wasThe list of the sequences used for the two analyses is given in supplementary data . DS wrote a first draft of the manuscript. CA produced the strain set and performed fermentations (experiment B). CB performed allelic replacement for S288c- PDR8 59A strain fermentations and QPCR expression analysis. PC, analyzed aroma compounds. PD built ABZ1 hemizygote strains and performed fermentations. IS, performed statistical analysis (QTL). JLL performed phylogenic analysis and tests. CE, BB, FK, JLL conceived the study ,designed and coordinated the research JLL wrote the manuscript. All authors analyzed the data. All authors read and approuved the final manuscript.DS: performed fermentation (experiment A), aroma and QTL analysis , candidate gene search and validation (Distribution of the different phenotypes for the population of segregants and parental strains.Click here for fileImpact of flocculation and ABZ1 on aroma production at the different QTL and variablity of fermentation length.Click here for fileEffect of the addition of p-aminobenzoic acid on the production of 2phenylethanol and 2-phenylethanolacetate.Click here for filePrimers used in this study.Click here for fileSequenced used for McDonald Kreitman test.Click here for file


Klebsiella pneumoniae RYC492, which produces the amyloid-forming and antibacterial peptide microcin E492. The sequenced genome consists of a 5,095,761-bp assembled open chromosome where the gene cluster for microcin production is located in a putative 31-kb genomic island flanked by sequence repeats and containing a putative integrase-coding gene.Here, we report the draft genome sequence of the Gram-negative strain  Klebsiella pneumoniae RYC492, a Gram-negative strain  is a bacteriocin produced by e strain  that is e family ; it aggre family , and itse family , 7. The hia coli , 9. AlthK. pneumoniae RYC492 was obtained using the AxyPrep bacterial genomic DNA miniprep kit (Axygen Biosciences), and its sequence was determined at OMICS Solutions  using an Ion Torrent personal genome machine (PGM), according to the manufacturer’s instructions . The NC018522 sequence, from K. pneumoniae strain 1084, was selected to assemble 82.1% of the reads, which were completed with segments (3.37% of the reads) identified with Mauve (K. pneumoniae RYC492 chromosome (46-fold coverage), with a length of 5,095,761 bp and a G+C content of 57.89%. The reads not included in the chromosome assembly were used to perform a de novo assembly, generating 158 contigs that contained a total of 288,021 bp. Both the chromosome and contigs were annotated using the NCBI Prokaryotic Genomes Automatic Annotation Pipeline, revealing a total of 5,095 protein-coding genes, 77 tRNAs, 6 copies of 16S rRNA, 8 copies of 23S rRNA, and 9 copies of 5S rRNA. The MccE492 gene cluster was found to be included in a putative genomic island of approximately 31 kb , inserted into an Asn-tRNA site, flanked by repeated sequences and containing an integrase-coding gene.The genomic DNA of ructions . A totalAmong the nonchromosomal genes, we found two distinct replication systems (RepA and ColE1), suggesting the existence of compatible plasmids in the RYC492 strain, as reported previously . The draK. pneumoniae RYC492 Whole-Genome Shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession no. APGM00000000. The version described in this paper is the first version, accession no. APGM01000000.This


Recent studies have reported associations of DNA repair pathway gene variants and risk of various cancers and precancerous lesions, such as chronic atrophic gastritis (CAG).APEX1, ERCC1, ERCC2/XPD, PARP1 and XRCC1), in CD3EAP/ASE-1 and PPP1R13L were analysed.A nested case-control study within the German population-based ESTHER cohort was conducted, including 533 CAG cases and 1054 controls. Polymorphisms in eleven DNA repair genes (No association was disclosed for any of the analysed polymorphisms. Nor did stratified analyses according to ages < 65 and ≥ 65 years show any significant association with CAG risk.The results of this large German case-control study do not reveal associations of DNA repair pathway polymorphisms and risk of CAG. On the basis of a large number of CAG cases, they do not support associations of DNA repair pathway SNPs with CAG risk, but suggest the need of larger studies to disclose or exclude potential weak associations, or of studies with full coverage of candidate genes. Helicobacter pylori (H. pylori), and by dietary exposure to salt and nitrate, which cause DNA damage [H. pylori may provoke a loss of genomic integrity and promote transformation, are postulated [Chronic atrophic gastritis (CAG) is a well-established precursor lesion in the aetiology of intestinal gastric cancer (GC), the most common type of GC . Unlike A damage -7. To dastulated ,7. Thesestulated ,7.The consequences of DNA damage are manifold and generally adverse. Thus, acute effects arise from a disturbed DNA metabolism, inducing cell cycle arrest or apoptosis, while long term effects from irreversible mutations may contribute to carcinogenesis . Four maAPEX1 [ERCC1 [ERCC2/XPD [PARP1 [XRCC1 [CD3EAP/ASE-1 [PPP1R13L genes [ERCC1 and 2 on chromosome 19q.13.3 [Therefore, we sought to evaluate the relationships between putative functional single nucleotide polymorphisms (SNPs) in APEX1 , ERCC1 [1 [ERCC1 ,12,16, ERCC2/XPD ,14,17,20D [PARP1 -11,19 an1 [XRCC1 ,18,19, aThe present study comprised a subsample of the German population-based ESTHER cohort study, including 533 serologically defined CAG cases without GC history and a number of 1054 age- and sex-matched controls. Details of the ESTHER study design have been described previously . BrieflyAccording to the study protocol and informed consent, serum and blood samples were obtained from all participants. Serum concentrations of pepsinogen (PG) I and II were measured by ELISA . CAG was defined by applying the most frequently used serological definition, being PG I < 70 ng/ml and PG I/PG II < 3 [PARP1 -17G > C rs907187 and V762A rs1136410, XRCC1 -77T > C rs3213245), five SNPs in NER genes , and two SNPs in CD3EAP/ASE-1 (rs735482) and PPP1R13L (rs6966) which, together with ERCC1 N118N rs11615, represent the high-risk haplotype on chromosome 19q13.3 [In line with recent epidemiologic studies that have revealed associations of BER and NER pathway gene variants with risk of glioma, colorectal, prostate, lung and gastric cancers and their precursors -20, sele® single base primer extension and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry  [Genotyping was performed with iPLEXgo, USA) , and a rP ≤ 0.01) was assessed using Pearson's chi-squared test. SNP associations were evaluated using unconditional logistic regression models to estimate sex- and age-adjusted odds ratios (ORs) and 95% confidence intervals (CIs). As CAG strongly increases with age [Genotypes of participants were used to estimate allele frequencies, and departure from Hardy-Weinberg equilibrium (HWE) in controls , and power calculations were employed with the power and sample size software PS [For reasons of statistical power, we restricted our analyses to SNPs with minor allele frequencies (MAFs) > 10%, according to dbSNPs HapMapCEU data tware PS , applyinH. pylori infection, however, was more common among cases [Of 9,444 ESTHER participants with available PG concentrations (94.9%), 533 met the serological definition of CAG and were selected for this study together with 1054 controls . Among tGenotype distributions for controls were consistent with HWE, and the average call rate for the analysed SNPs was 97.5% (range: 95.6% to 98.7%).We observed no evidence for significant associations of the eleven SNPs and CAG risk , while the three non-synonymous XRCC1 SNPs R194W, R280H and R399Q, whose functional characteristics are not determined yet, showed no association with NSCLC risk [Remarkably, CLC risk . As demoCLC risk . ContrarCLC risk . After eCLC risk , which mCLC risk .The present study has both strengths and limitations. Strengths include the well-defined and homogeneous study population. In addition, we analysed SNPs for which associations with CAG (and/or GC and other cancers) are biologically plausible and for which associations have been previously reported -20. We hAlthough the eleven investigated SNPs are strong candidates for susceptibility to cancers and their precursor lesions -16, thisOur findings, based on a large number of CAG cases, do not support associations of DNA repair pathway gene SNPs with the risk of CAG. Much larger studies are needed to reveal potential weak associations. Moreover, full coverage of candidate DNA repair genes, i.e. tagging SNP approaches should be aimed for in future studies.The authors declare that they have no competing interests.BF, HB, HM, MNW, ER were responsible for the study design. BF, NK, TI were involved in data acquisition and analysis. Statistical analyses, data interpretation and manuscript drafting were done by BF. All authors critically reviewed and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/11/440/prepubTable S1. DNA repair pathway single nucleotide polymorphisms (SNPs) and risk of chronic atrophic gastritis.Click here for fileTable S2. Associations of DNA repair pathway single nucleotide polymorphisms (SNPs) with the risk of chronic atrophic gastritis among individuals < and ≥ 65 years of age.Click here for file


Anisogramma anomala. In our approach, Illumina reads were first assembled into a draft genome, and the latter was then used in microsatellite discovery. A. anomala is an obligate biotrophic ascomycete that causes eastern filbert blight disease of commercial European hazelnut. Little is known about its population structure or diversity. Approximately 26 M 146 bp Illumina reads were generated from a paired-end library of a fungal strain from Oregon. The reads were assembled into a draft genome of 333 Mb (excluding gaps), with contig N50 of 10,384 bp and scaffold N50 of 32,987 bp. A bioinformatics pipeline identified 46,677 microsatellite motifs at 44,247 loci, including 2,430 compound loci. Primers were successfully designed for 42,923 loci (97%). After removing 2,886 loci close to assembly gaps and 676 loci in repetitive regions, a genome-wide microsatellite database of 39,361 loci was generated for the fungus. In experimental screening of 236 loci using four geographically representative strains, 228 (96.6%) were successfully amplified and 214 (90.7%) produced single PCR products. Twenty-three (9.7%) were found to be perfect polymorphic loci. A small-scale population study using 11 polymorphic loci revealed considerable gene diversity. Clustering analysis grouped isolates of this fungus into two clades in accordance with their geographic origins. Thus, the “Seq-Assembly-SSR” approach has proven to be a successful one for microsatellite discovery. High-throughput sequencing has been dramatically accelerating the discovery of microsatellite markers . Both 454 and Illumina reads have been used directly in microsatellite discovery and primer design (the “Seq-to-SSR” approach). However, constraints of this approach include: 1) many microsatellite-containing reads do not have sufficient flanking sequences to allow primer design, and 2) difficulties in removing microsatellite loci residing in longer, repetitive regions. In the current study, we applied the novel “Seq-Assembly-SSR” approach to overcome these constraints in  They are valuable tools in many research areas, such as population biology, genome mapping, and the study of genealogy, because they are multi-allelic, inherited co-dominantly, usually abundant, and cover most or all parts of the genome. The traditional method to develop microsatellite markers, which is still used by many labs today, generally involves the following steps: enrich microsatellite-containing sequences from sheared genomic DNA; clone the microsatellite-enriched DNA; extract plasmids; sequence the inserts through Sanger sequencing; design primers; and screen individual loci. The whole process can require several months of work and considerable resources. In fungi, which is the subject organism of this study, the traditional approach is even more time- and resource-consuming, because fungal species usually have lower densities of microsatellite loci and the alleles are often shorter with fewer polymorphisms, compared to many other organisms  and reverse primer, 40 nM of forward primer, 0.2 mM dNTPs, 1x reaction buffer, and 0.2 U PerfectTaq DNA polymerase (5 PRIME). The forward and reverse primers first amplify their target. After the forward primer is exhausted, FAM-M13(-21) anneals to the M13(-21) sequence fused to the 5’ end of the forward primers. It then serves as the forward primer, and together with reverse primer, amplifies the product and provides fluorescence. As a first-pass screening, PCR was performed in 96-well plates without replicates. The PCR cycle parameters were as follows: an initial denaturation at 94 °C for 5 min was followed by 30 cycles of denaturation , annealing , and extension , followed by another 8 cycles of denaturation , annealing , and extension , and a final extension at 72 °C for 10 min. The PCR fragments were analyzed on ABI 3730 sequencer. Allele sizes were determined with the aid of software Peak Scanner version 1.0 (Life Technologies).  A subset of loci was tested experimentally. Four geographically representative protocol  was usedhttp://evolution.genetics.washington.edu/phylip.html).  A collection of 30 isolates from the United States were screened using 11 polymorphic loci as described above. The data was analyzed using PowerMarker version 3.25 . SummaryTable S1A. anomala.A genome-wide database of microsatellite loci in (XLSX)Click here for additional data file.Table S2A. anomala by motif type and number of repeats.Microsatellite motifs in (XLSX)Click here for additional data file.Table S3A. anomala using four isolates from New Jersey, Ohio, Oregon, and Wisconsin.Results of experimentally screened microsatellite loci in (XLSX)Click here for additional data file.


The physiological states pregnancy and parturition are undoubtedly associated with clinical changes of most of the autoimmune diseases. An altered Th1/Th2 balance has been proposed as an underlying mechanism. A pregnancy has protective effect on Th1-mediated autoimmune diseases, and a deteriorative effect on Th2-mediated autoimmune diseases. Numerous studies, both experimental and clinical, imply that estriol and progesterone, at high doses (such as those achieved during pregnancy), have potent anti-inflammatory and neuroprotective roles. These studies, as well as a further investigation of pregnancy-associated immunomodulation, can be used to advance and develop a new therapy approaches. A third Th cell subset, a regulatory T cell (Treg), has been recently identified. Th1 cells primarily secrete pro-inflammatory cytokines as IFN (interferon)-γ, IL (interleukin)-2 and TNF (tumor necrosis factor)-α, which promote cellular immunity, whereas Th2 cells secrete a different set of anti-inflammatory cytokines IL-4, IL-5, IL-6 and IL-13, which promote humoral immunity. Treg cells produce cytokines like IL-4, IL-10 and TGF (transforming growth factor)-β, and have suppressive properties for both Th1 and Th2 cells. A Th1 and Th2 responses are mutually inhibitory, and to some extent opposing, whereas Treg prevent the derangement of the Th1/Th2 balance in either direction , corticosteroids, and numerous protein hormones  . During Estrogens and PRG induce a substantial Th2 shift, most likely at concentrations associated with pregnancy.The estrogens are known to exert opposing, bimodal, dose-specific effect to immune response. Low levels facilitate a cell-mediated pro-inflammatory immune response, whereas their relatively high levels, such as those achieved during pregnancy, promote an anti-inflammatory Th2 response . StudiesEstrogens can display a wide range of neuroprotective activities, including stabilization of neurotransmission, inhibition of apoptosis, and direct antioxidant activities which may be important for defense against oxidative stress . SimilarPRG mediates promotion of Th2 response, inducing conversion of Th0 cells into Th2 cells. At concentrations comparable to those present at the maternal-fetal interface PRG induces a development of Ag-specific CD4+ T cell lines that show enhanced ability to produce IL-4 and IL-5 . Also, PTaken together, the above studies imply that the effects of sex hormones on the immune and nervous system are clearly apparent, although imperfectly understood Table . The traThus, further randomized clinical trials are necessary. It is important to minimize the risk of treatment, and design therapies optimized for anti-inflammatory and neuroprotective efficacy.


Pancreatic cancer develops and progresses through complex, cumulative biological processes involving metabolic disorder, local inflammation, and deregulated molecular pathways. The resulting tumor aggressiveness hampers surgical intervention and renders pancreatic cancer resistant to standard chemotherapy and radiation therapy. Based on these pathologic properties, several therapeutic strategies are being developed to reverse refractory pancreatic cancer. Here, we outline molecular targeting therapies, which are primarily directed against growth factor receptor-type tyrosine kinases deregulated in tumors, but have failed to improve the survival of pancreatic cancer patients. Glycogen synthase kinase-3β (GSK3β) is a member of a serine/threonine protein kinase family that plays a critical role in various cellular pathways. GSK3β has also emerged as a mediator of pathological states, including glucose intolerance, inflammation, and various cancers . We review recent studies that demonstrate the anti-tumor effects of GSK3β inhibition alone or in combination with chemotherapy and radiation. GSK3β inhibition may exert indirect anti-tumor actions in pancreatic cancer by modulating metabolic disorder and inflammation. PancrPancreatic cancer develops through a series of complex, cumulative biological processes involving metabolic disorder and chronic local inflammation in association with stromal changes and deregulated molecular pathways . Based o2.Although systemic metabolic disorders such as obesity and type II diabetes mellitus (DM) influence the risk of developing pancreatic cancer and clinical outcomes , the2.1.The association between obesity/DM and pancreatic cancer risk has long been controversial. However, recent reviews and meta-analyses of prospective observational studies have demonstrated that obesity, defined by an increased body mass index, is significantly associated with a risk of pancreatic cancer development ,16. DM iA number of studies have reported the potential role of glucose-lowering therapies in reducing the risk of pancreatic cancer (reviewed in ). Metfor2.2.18F]-fluoro-2-deoxy-D-glucose (FDG). FDG-PET in combination with computed tomography (PET-CT) enables detection of metastatic lesions of most cancers with both sensitivity and specificity greater than 90% [The fundamental metabolic characteristics of cancer cells include increased glucose uptake, aerobic glycolysis even under normoxic condition (Warburg effect) , and impthan 90% . PharmacA glucose analogue, 2-DG, is the most attractive agent for targeting aberrant glucose metabolism in cancer cells . 2-DG ini.e., TCA cycle defects) and resistance to apoptosis is attributed to decreased mitochondrial hydrogen peroxide production and cytochrome C release [The association between the glycolytic phenotype , cyclooxygenase-2 (COX-2), peroxisome proliferator-activated receptor-γ (PPARγ), nitric oxide (NO) synthesized by inducible NO synthase (iNOS), DNA damage caused by release of proteolytic enzymes and ROS, and somatic mutations in oncogenes  and tumor suppressor genes  -49. PancHistopathologic findings of chronic pancreatitis include marked fibrosis, in which pancreatic stellate cells (PSCs) play a crucial role ,51. In t3.2.in vitro and in vivo studies suggest that cancer cells recruit PSCs to tumors, where PSCs promote cancer cell proliferation and facilitate their invasion and metastasis, and that FGF and PDGF mediate these interactions between the two cell types .Phenotypic changes also occur in cancer cells, as represented by epithelial-mesenchymal transition (EMT) at the interface between tumor and stroma, in which epithelial cells undergo morphologic changes characterized by a transition from epithelial to fibroblastic  phenotypes. Most factors involved in pancreatic cancer-stromal interactions have the potential to induce EMT in cancer cells. This process involves loss of cell-to-cell adhesion and E-cadherin expression, actin cytoskeleton reorganization, and increased expression of mesenchymal molecules . In this way, EMT facilitates the invasion and metastasis of cancer cells and renders them resistant to chemotherapy and radiation ,57. Acco4.Most cases of pancreatic cancer are resistant to conventional chemotherapy and radiation therapy -7; there4.1.Molecular studies have investigated the complex genetic mechanisms of cancer, which involve multidirectional signal transduction pathways ,59,60. A4.2.Currently available agents that target these factors include anti-EGFR antibodies , small molecule EGFR inhibitors , an anti-VEGF antibody (bevacizumab), and a small molecule VEGFR inhibitor (axitinib). A number of phase III clinical trials have tested kinase inhibitors as monotherapy or in combination therapy with gemcitabine for pancreatic cancer, but other than the combination of erlotinib and gemcitabine , these a5.GSK3β has emerged as a critical factor that plays distinct pathologic roles in glucose intolerance, inflammation, and in various cancer types . Here, we briefly review recent studies, including our own, that demonstrate the direct anticancer effects of GSK3β inhibition, alone or in combination with chemotherapy and radiation.5.1.GSK3β was first identified as a serine/threonine protein kinase that regulates glucose/glycogen metabolism under the control of insulin signaling. Unlike most protein kinases, GSK3β is active in normal cells, and its activity is controlled by subcellular localization, differential phosphorylation at serine 9 (S9) and tyrosine 216 (Y216) residues, and different binding partners. In addition to regulating its primary target, glycogen synthase, GSK3β is involved in other fundamental cellular pathways depending on its substrates and binding partners -68. GSK35.2.Under physiologic conditions, GSK3β phosphorylates several transcription factors , cell cycle regulators , and proto-oncoproteins , thereby triggering their degradation via the ubiquitin-proteasome system. GSK3β is, therefore, hypothesized to inhibit tumor development by interfering with oncogenic signaling  . Howeverin vitro and in tumor xenografts [In the last five years, we demonstrated that deregulated expression, phosphorylation of S9 and Y216, and GSK3β activity are distinct features of gastrointestinal cancers including pancreatic cancers and glioblastoma, and that GSK3β sustains the survival and proliferation of these tumor cells. A pathologic role for GSK3β is supported by observations that inhibition of its activity reduced the survival and proliferation of different cancer cell types, predisposing them to apoptosis both nografts -76. We anografts ,76 and dnografts .Kip1, cyclin-dependent kinase (CDK), cyclin D1, c-Myc, and NF-κB [Simultaneously and following our studies on the antitumor effects of GSK3β inhibition, similar observations from other laboratories were reported for various cancer types with underlying mechanisms that included regulation by GSK3β inhibition of several pathways mediated by p53, Rb, p27nd NF-κB . Althougnd NF-κB ,80, the nd NF-κB .Based on the potential involvement of GSK3β in NF-κB-mediated cell survival ,82, a nu5.3.Increasing evidence suggests that GSK3β participates in a wide range of physiological processes that determine cell fate, including cell motility, energy metabolism, and transcriptional control (reviewed in ). In add6.Here, we reviewed recent studies on the epidemiologic characteristics of pancreatic cancer and the molecular and biological mechanisms contributing to its development and progression, and highlight advances in alternatives to conventional treatments. Although molecular target-directed therapy is currently attracting considerable attention, especially for cancer refractory to standard chemotherapy and radiation, this therapy produces therapeutic effects that are still far from sufficient for most patients with advanced and recurrent pancreatic cancer. For many cancer patients, including those with pancreatic cancer, resistance to currently available therapeutics presents a major obstacle and is due to the aberrant metabolism of cancer cells, their microenvironment, their ability to invade and metastasize, and the acquisition of gene mutations. Multidisciplinary approaches directed to a more complete understanding of pancreatic cancer pathogenesis hold great promise in improving the outlook of this disease.


The signal transducers and activators of transcription 3 (STAT3) signaling pathway plays important roles in oncogenesis, angiogenesis, immunity, and tumor cell invasion. In the present study, we investigated the association of interleukin (IL)-6/STAT3 signaling pathway with T lymphocytes and clinical implication in patients with gastric cancer.+, CD4+, CD8+, CD4+/CD8+) and natural killer (NK) cells by a flow cytometry. Furthermore, the expression of IL-6, survivin, STAT3, STAT3 phosphorylation (p-STAT3), and VEGF were determined in human gastric cancer and adjacent normal mucosa through Western blot and immunohistochemistry.Seventy one patients who underwent gastrectomy due to gastric adenocarcinoma were studied. Blood samples were collected before and after surgical gastrectomy to quantify the levels of IL-6, IL-10 and VEGF using an enzyme-linked immunosorbent assay, as well as T lymphocyte subsets (CD3Postoperative levels of IL-6, IL-10 and VEGF in serum were significantly lower than preoperative levels. Percentages of T-cell subsets and NK cells in blood were significantly increased after postoperative-week 1 as compared to preoperative group, which was further augmented at 1 month after gastrectomy. In addition, the expression of IL-6, survivin, STAT3, p-STAT3, and VEGF were increased in human gastric cancer tissues as compared to adjacent normal mucosa. Their expression was associated with TNM stage of gastric cancer. The level of STAT3 activation in clinical samples was correlated with IL-6 expression. All gastric tumor samples, which expressed p-STAT3, also expressed IL-6 with weak expression detected in adjacent normal mucosa.Increased IL-6-induced activation of STAT3 was observed in neoplastic gastric tissue, which positively correlated with tumor progression. Moreover, IL-6 and STAT3 downstream signals such as IL-10 and VEGF were reduced in patients after removal of gastric cancer as compared to pre-operation. Therefore, inhibition of the IL-6/STAT3 signaling pathway may provide a new therapeutic strategy against gastric cancer. Gastric cancer is the second most common cause of cancer-related deaths, and nearly 1 million new cases are diagnosed worldwide each year [Signal transducer and activator of transcription 3 (STAT3) is a transcription factor, which can be activated by tyrosine phosphorylation in response to growth factors and cytokines . IL-6, which originally was characterized as a B cell differentiation-inducing cytokine, is a cytokine that play important roles in a plethora of biological functions including inflammation, plasmacytoma genesis, immunoglobulin production by mediating cell growth, cell proliferation, and cell survival -4. DepenSTAT3 activation contributes to growth stimulation, anti-apoptosis, and angiogenesis, which is signiﬁcantly associated with inflammation, immunity, and oncogenesis ,11,12. CThis investigation complies with the regulations stipulated by Anhui Medical University Ethical Committee, which follows the protocol outlined in WMA Declaration of Helsinki-Ethical Principles for Medical Research Involving Human Subjects. All patients were screened and treated for the purpose of the study at the Affiliated Hospital of Anhui Medical University, Hefei, China,and signed an informed consent form. The human ethics guidelines in the clinical research project No. kj2011Z210  was discussed and approved by the Human Ethics Committee in the First Affiliated Hospital of Anhui Medical University on March 25, 2011. All enrolled patients underwent total or subtotal gastrectomy. After surgery, each patient received four cycles or more of chemotherapy (chemotherapy regimens: FOLFOX4 program).9 cells/L, platelets ≥80×109 cells/L, total bilirubin ≤ 2 × Upper Limit of Normal, hemoglobin ≥9.0 g/dL and creatinine ≤170 µmol/L), and ECOG performance status between 0-2. Patients were excluded for serious disorders, peripheral neuropathy (NCI-CTC1 level and above), pregnancy, or breast-feeding. Patients that had verified distant metastases were excluded.Eligible patients were adults (18 years old to 75 years old) with biopsy-confirmed gastric adenocarcinoma, who had previously completed at least four cycles of chemotherapy. According to a random procedure (1:1 ratio) into the research, all patients had normal hepatic, renal and bone marrow function  was collected before treatment and postoperative week 1 as well as after a month. Serum was centrifuged at 2000 rpm per min for 10 minutes, and the upper fluid (serum) was collected.All blood samples without EDTA were centrifuged immediately, and then supernatants were all stored at −80°C until assayed. The serum concentrations of human IL-10, IL-6 and VEGF were quantified by an enzyme-linked immunosorbant assay (ELISA) according to the manufacturer’s instructions. The kits of IL-10, IL-6 and VEGF were provided by R&D Systems .T lymphocyte subsets in blood were determined using a flow cytometer ,27. The Sections (5 µm thick) of formalin-fixed, paraffin-embedded primary gastric specimens from gastrectomy were stained with an anti-STAT3 antibody  ,25. The Whole tissue lysates were prepared from human gastric tissues with RIPA buffer. Western blotting was performed using an anti-IL-6, anti-VEGF, anti-survivin, anti-STAT3 and phosphorylated STAT3 (Tyr705) antibodies  . ProteinP<0.05 was considered statistically significant.Data were expressed as mean±SD. All statistical analysis was performed in SPSS 15.0 software package. The comparison of multiple groups were analyzed with one-way analysis of variance (ANOVA) followed by LSD test and Dunnett test. Eight one patients were recruited for the study, and 10 cases were withdrawn due to failure of follow up and death. As shown in In this study, 71 patients were eligible. Before and at the end of each surgery, the patients’ serum samples were quantified for cytokine levels. In this study, the levels of IL-6, IL-10 and VEGF were quantified to determine any significant difference between cytokine concentrations in serum preoperatively and postoperatively. +, CD4+), and CD4+/CD8+ ratio as well as NK cells were significantly increased after postoperative-week 1, which was further augmented in postoperative-month 1 as compared to those before gastrectomy , anti-IL-6, anti-survivin, anti-VEGF antibodies, respectively. As expected, a single band was observed using each antibody , and theAs shown in + and CD4+ T lymphocytes as well as NK cells. In addition, IL-6/STAT3 signals including surviving and VEGF were significantly augmented in gastric carcinoma tissues as compared to adjacent normal mucosa. Overall, our findings provide the first evidence that increased IL-6/STAT3 activation correlates with aberrant immunity, leading to the progression and invasion of gastric cancer.IL-6 and its downstream signals, such as STAT3, play essential roles in the process of inﬂammation and aberrant immunity as well as carcinogenesis -30. STATIL-6 has been shown to enhance invasion of gastric cancer cells through sustained activation of STAT3 ,34. This+, CD4+, CD8+) as well as their correlation within tumor tissues are not known, which needs further investigation.Cancer immunesurveillance is an important process to eliminate tumor cells . This isIt has been shown that STAT3 activation in tumor cells can mediate an immune response by inhibiting the activity of immune cells through inflammatory cytokines probably release from invading tumor tissue ,45. Our In conclusion, IL-6-induced activation of STAT3 is observed in neoplastic gastric tissue, which positively correlated with tumor progression. Moreover, IL-6 and STAT3 downstream signals such as IL-10 and VEGF were reduced in patients after removal of gastric cancer as compared to pre-operation, this was associated with recovery of T lymphocytes and NK cells in peripheral circulation in these patients. Therefore, inhibition of the IL-6/STAT3 signaling pathway may provide a new therapeutic strategy against gastric cancer.


Evaluate the bone mass in a group of patients diagnosed with juvenile systemic lupus erythematosus (JSLE), which carry high risks of developing low bone mass, as well as verifying alterations on calcium metabolism.Cross-sectional study, in which were evaluated 53 patients from the Rheumatology ambulatory in HUCFF/ UFRJ, diagnosed with JSLE in their childhood or teenage years, according to ACR (American College of Rheumatology) criteria. Subjects were submitted to a single blood and urine collection for dosing of the following variables: alkaline phosphatase, serum phosphorus and calcium, urea, creatinine, erythrocyte sedimentation rate (ESR), intact PTH, free T4, TSH, 24-hour calciuria. Bone density was measured from lumbar spine, and full body and/or total femur, according to age range, using DEXA methods and Prodigy Advance equipment. Also, medical records were analyzed collecting the following data: age, sex, time coping with the disease, body mass index (BMI), disease progression with SLEDAI average for the last 6 months, number of diagnostic criteria, cumulative dose of 3 drug-induced osteoporosis classes . Finally, the collected data was submitted to statistic analysis. Patients suffering from renal insufficiency or currently on bone antiresorptive or bone formation stimulators drugs were excluded.2 ; SLEDAI – 3,7  R=1,23. Not only was the accumulative glucocorticoid dosage calculated, but also the prednisone equivalency. The maximum used dosage was 133,88g, with an average of 29,1g and standard deviation of 27,8 . The only anticoagulant used was warfarin, with maximum dosage of 39,76g, average of 1,3g and standard deviation of 5,9 . Maximum dosage for methotrexate was 2,49g, average of 0,2g and standard deviation of 0,6 . 15 patients presented densitometry alterations, characterized by Z-score lower or equal to 2 standard deviation. No clinically significant laboratorial alterations on calcium metabolism were found.92% of patients were females, being only 4 male subjects. Analyzed parameters averages, and their corresponding standard deviation (SD): Age – 21 years old ; time coping with the disease – 7,9 years ; number of ACR criteria at diagnosing moment – 5,6 ; BMI – 23,2 kg/mNo direct or inverse linear relation (correlation) was observed between the analyzed parameters and densitometry findings according to the Z-score.Ana Paula Roenick Guenka: None; Blanca Elena Rios Gomes Bica: None; Laura Maria Carvalho de Mendonça: None; Maria Lucia Fleiuss de Farias: None; Lucas Pinheiro Aversa de Escobar Velloso: None; Mario Newton Leitão de Azevedo: None.


DUOX2mutations. However, the underlying etiology of TCH is not determined in some cases. In this study, we conducted the first systematic investigation on the etiology of TCH, using screening population-based cohort in Niigata Prefecture, Japan.Several conditions have been known to causetransient congenital hypothyroidism (TCH), including transplacental passage of TSH receptor blocking antibody (TSBAb), maternal antithyroid drug usage, iodine deficiency, iodine excess, fetal prematurity and inactivatingDUOX2.Between April 2003 and March 2009, 148,100 newborns were screened for CH in Niigata prefecture, and 159 patients were considered positive for CH. We diagnosed patients as having TCH that fulfilled the following two criteria: 1) serum TSH level >30 mU/L and serum FT4 level <1.5 ng/dLat the initial examination, 2) serum TSH level <5 mU/L while investigative discontinuation of thyroxine replacement at 2 years of age. A total of 9 patients  diagnosed with TCH were evaluated. To determine the etiology of TCH, we examined the following: 1) maternal medical history, 2)gestational age and birth weight, 3) maternal anti-thyroid antibodies, 4) urinary iodine concentration at initial visit, and 5) DNA sequence of DUOX2 mutations.Among the nine TCH patients, one had extremely high maternal TSBAb level, one was exposed to propylthiouracil, and two were exposed to excessive iodine.Furthermore, we found that five had biallelicDUOX2 mutations were the majorcause of TCH in our cohort study.


ACT) is most frequently used as qRT-PCR reference gene without validation.Bivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and β-actin (Patinopectenyessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ∆Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae.In this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (This work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species. Gene expression studies are increasingly important in identifying genes, pathways, and networks underlying cellular and developmental processes. Real-time quantitative reverse transcription PCR (qRT-PCR) is frequently used technique for gene expression analysis, due to its advantage in sensitivity, speed, throughput, and speciﬁcity -3. To enACT) [GAPDH) [Ideally, reference genes used in qRT-PCR should be expressed constantly across various biological or experimental conditions, such as different tissues, developmental stages, or experimental treatments -9. So thACT) -18 and g [GAPDH) ,19,20, c [GAPDH) .ACT, but without evaluation. By far, in the limited studies on reference gene selection for bivalve species, most of them focused on single tissues, such as haemocyte in parasite attacked flat oyster (Ostreaedulis) [Mya Arenaria) [Mytilusedulis) [Nodipectensubnodosus) [Patinopectenyessoensis) with respect to starvation treatment, the reference genes were recommended for different single tissues, and those could be generally used among tissues were not evaluated or identified [Bivalves comprise around 30,000 extant species. Recently, for the importance of this class of invertebrates in ecosystems, aquaculture and evolutionary studies , more anedulis)  and bactenaria) , and gonedulis)  and liondosus) . Althougentified . Therefoentified ,38-40, wentified . On the entified ,35,36 maPyessoensis. , and over 20,000 genes were obtained [Yesso scallop which naturally distributes along the coastline of northern Japan, the Far East of Russian, and the northern Korean Peninsula, has become one of the main maricultural shellfish in the north of China since it was introduced in 1982 . For theobtained , providiAll the experiments on scallops were conducted following the institutional and national guidelines. The Yesso scallop samples used in this study were collected from Zhangzidao Fishery Group Co., Dalian, China. Tissues from six healthy adult Yesso scallops (65.6 ± 1.7 mm in shell height), including mantle, gill, gonad, kidney, striated muscle and digestive gland were dissected, immediately frozen in liquid nitrogen, and then stored at -80°C. Normal embryos and larvae of Yesso scallop, including fertilized eggs, blastulae, gastrulae, trochophore larvae, and D-shaped larvae were collected and preserved at -80°C. For each of the above embryo/larva stage, 3 sets of samples  were collected. Thus, a total of 36 tissue samples and 15 embryo/larva samples were used in the following analysis.260/OD280 ratio between 1.8 and 2.0, and OD260/OD230 ratio higher than 2.0 were used for subsequent analysis. Each RNA sample was assayed in triplicate and the average value was determined. The first-strand cDNA was synthesized according to the manufacturer’s instruction of M-MLV Reverse Transcriptase  with 2µg DNase I-treated total RNA and 2µM Oligo (dT)18 (Takara Bio) primer in a 25-µL volume , Cytochrome B (CB), elongation factor 1-β (EF-1-β), Ubiquitin (UBQ), TATA-box binding protein (TBP), 60S ribosomal protein L16 (RPL16), DEAD-box RNA helicase (HELI), β-Tubulin (TUB), Cyclophilin A (CYP), and Histone H3.3 (His3.3) were selected for expression stability analysis  was analyzed to search for the orthologs of candidate reference genes previously reported in qRT-PCR analysis. The assembled transcriptome sequences were compared against the NCBI non-redundant (Nr) protein database and Swiss-Prot database using BlastX . Gene naanalysis .Primers for qRT-PCR were designed using the Primer 5.0 software (http://www.premierbiosoft.com/primerdesign/index.html). The sizes of PCR products were between 100-200 bp. The annealing temperature for all primer-pairs was optimized to 62.8°C. Amplicons of each primer pair were tested by 2% agarose gel electrophoresis to verify the products size and specificity. Then the target amplicons were purified using MinElute Gel Extraction Kit , and sequenced in Sangon Biotech  to confirm the sequence specificity. For each primer pair, amplification products of no-RT control were run to rule out potential DNA contamination during RNA isolation, and no-template control amplification was performed to ensure the absence of other contamination or primer dimer. The information of the primers is listed in qRT-PCR reactions were conducted in a 96-well plate using ABI7500 Real-Time system . Each reaction was performed in triplicate and in a 20-µL volume containing 1× Real-time PCR Master Mix with SYBR Green dye , 0.4 µM of each primer and 2 µL cDNA, using the following thermal conditions: 50°C for 2min, 95°C for 10min, followed by 40 cycles of 95°C for 15s and 62.8°C for 1min. A melting curve analysis (60 °C to 95 °C) was performed at the end of each PCR to further confirm the specificity of amplicons. The raw fluorescence data were exported and analyzed using an online software Real-time PCR Miner (http://www.miner.ewindup.info/miner/Version2) , and thehttp://medgen.ugent.be/jvdesomp/geNorm/) [http://www.mdl.dk/publicationsnormfinder.htm) [The expression stability of the selected reference genes was evaluated using three frequently used statistical approaches ,19,49,50geNorm/) , NormFinder.htm) , and comder.htm)  to get cGeNorm is a popular algorithm to determine the most stable reference genes from the candidate ones, and the optimal number of genes needed for accurate normalization in qRT-PCR . After tNormFinder is a model-based approach for identifying stable reference genes through calculating intra- and intergroup variations which are then combined into a stability value . CandidaThe comparative ΔCt method was used to estimate the gene expression stability further. By comparing relative expression of gene pairs within each sample for all possible gene combinations, this method calculated the mean of standard deviation for each candidate genes . The expCB to 99.28% for UBQ  would be adequate in qRT-PCR normalization for scallop tissues, and the addition of the forth gene is not necessary.The expression level data of these candidate reference genes were analyzed using three algorithms, geNorm, NormFinder, and the comparative ∆Ct method. GeNorm ranked the candidate genes based on their average expression stability , and a lower M value indicated more stable expression. For different tissues, geNorm analysis showed that,  and ACT , Table 2as 0.145 , thus thHELI, UBQ, CB, RPL16, GAPDH, TBP, CC, EF-1-β, CYP, His3.3, TUB, and ACT  of the most four stable genes were the same as those identified using geNorm and NormFinder, although the ranking was different. From most to least stable, the order of the other 8 candidate genes was CC, GAPDH, CYP, TBP, EF-1-β, TUB, His3.3, and ACT. ACT was the least stable gene once again.The 12 candidate reference genes were further evaluated using comparative ∆Ct method. The results were similar to those from geNorm and NormFinder . HELI waHELI, UBQ, and RPL16 were identified as the most stable genes for qRT-PCR analysis in different tissues of Yesso scallop. The final ranking orders of all the 12 genes were listed in To get a final rank of the candidate genes, the results obtained from all the three algorithms were further analyzed using the method reported previously . For eacCC and CB were the most highly expressed genes in embryos/larvae, too, with a median Ct value of 17.8 and 18.1, respectively. The other 10 genes all had median Ct values greater than 20, and two (UBQ and TBP) of them showed Ct values greater than 30. In respect to gene expression variation among different developmental stages, UBQ, CB, CC, His3.3 and CYP exhibited more narrow ranges of Ct dispersal than other genes, while HELI, TUB, and ACT showed greater variation  was a little bit higher than the cut-off value 0.150. The V4/5 value was 0.123, thus the most four stable genes were required for accurate normalization . AccordiRPL16) and the least stable gene (ACT). But no optimal gene combination could be provided. All the 10 Vn/Vn+1 values for the 12 candidate genes generated by geNorm were much higher than the suggested cut-off value 0.150 , followed by GADPH (2 articles) and EF-1-β (2 articles). However, their suitability as normalization factors for the corresponding samples was not evaluated. In this study, for both different tissues and different embryo/larva stages of scallop, the commonly used reference gene ACT was among the least stably expressed genes, while EF-1-β was mediate stable. As for GADPH, it was ranked as the 4th most stable gene and suggested to be used as reference gene for different embryo/larva stages, while in different tissues, it was mediate stable. So, for the sample sets we examined in this study, the currently used reference genes in scallops, especially ACT and EF-1-β, were less stable than those we recommended.A literature analysis showed that, in 44 randomly selected publications describing target gene expression of scallops using qRT-PCR, the most frequently used reference gene is In addition, as an intention to find the generally appropriate reference genes for both scallop tissues and embryos/larvae, we analyzed all the expression data together. Although the three programs presented similar evaluation results , no optiIn conclusion, we reported the first systematic analysis for the selection of superior reference genes used in qRT-PCR normalization with respect to normal tissues and embryo/larva stages in scallops. Through evaluating the expression stabilities of 12 candidate genes in 36 tissue samples and 15 embryo/larva samples from Yesso scallops, a specific combination of reference genes was recommended for each of the two sample sets. These reference genes will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species.Table S1qRT-PCR Ct values for the 12 candidate reference genes obtained in Yesso scallop tissues.(DOC)Click here for additional data file.Table S2qRT-PCR Ct values for the 12 candidate reference genes obtained in Yesso scallop embryos/larvae.(DOC)Click here for additional data file.Table S3Ranks of the 12 candidate genes considering tissues and embryos/larvae together.(DOC)Click here for additional data file.Figure S1Pairwise variation analysis by geNorm to determine the number of reference genes required for qRT-PCR normalization when considering tissues and embryos/larvae together.(TIF)Click here for additional data file.


Heat shock proteins (Hsp) play critical roles in the body's self-defense under a variety of stresses, including heat shock, oxidative stress, radiation, and wounds, through the regulation of folding and functions of relevant cellular proteins. Exercise increases the levels of Hsp through elevated temperature, hormones, calcium fluxes, reactive oxygen species (ROS), or mechanical deformation of tissues. Isotonic contractions and endurance- type activities tend to increase Hsp60 and Hsp70. Eccentric muscle contractions lead to phosphorylation and translocation of Hsp25/27. Exercise-induced transient increases of Hsp inhibit the generation of inflammatory mediators and vascular inflammation. Metabolic disorders (hyperglycemia and dyslipidemia) are associated with type 1 diabetes (an autoimmune disease), type 2 diabetes , and atherosclerotic cardiovascular disease. Metabolic disorders activate HSF/Hsp pathway, which was associated with oxidative stress, increased generation of inflammatory mediators, vascular inflammation, and cell injury. Knock down of heat shock factor-1 (HSF1) reduced the activation of key inflammatory mediators in vascular cells. Accumulating lines of evidence suggest that the activation of HSF/Hsp induced by exercise or metabolic disorders may play a dual role in inflammation. The benefits of exercise on inflammation and metabolism depend on the type, intensity, and duration of physical activity.  Hsp regulate the conformation and functions of a large number of cellular proteins in order to protect the body from stress . The exp disease . This paHsp has evolved to perform multiple roles within cells, organs, and organisms . These uαA- and αB-crystallin, Hsp20, 22, 25/27, and other Hsp60, the Hsp70 and 90 families and Hsp110, and their cochaperones . HSF represents a family of transcription factors induced by both stressful and nonstressful stimuli. The fundamental structure of HSF has been well conserved from yeast to humans , 19. Fouα- and β-adrenergic receptors [Given the importance of the heat shock response, it is not surprising that there are multiple redundant pathways by which the response may be activated . Followieceptors –38 as weeceptors , 40  [Physical activity, or exercise, is known to improve overall health and protect against, delay the progress of, or ameliorate many common chronic diseases , 64, in  (PAI-1) , and cel (PAI-1) , protect (PAI-1) , 83 and  (PAI-1) . The ant (PAI-1) , 65, 78  (PAI-1) , C-react (PAI-1) , and mod (PAI-1) .Interestingly, heat shock exhibits beneficial effects on the vasculature which are similar to exercise, with reduced inflammation , reducedαB-crystallin in cardiac tissue [αB-crystallin may reciprocally assist in controlling venous tone [As throughout the rest of the body, Hsp likely play specific roles within the vasculature. The response of the vasculature to shear stress is complicated. Laminar flow, such as that associated with exercise, induces positive vascular remodeling, whereas turbulent or low flow, such as that associated with vascular inflammation and atherosclerosis, leads to adhesion of blood borne molecules and inflammation  see Fig. The incc tissue , and botc tissue –112. Indous tone . Hsp70 mous tone , and Hspous tone .Normal endothelium provides an effective barrier to foreign materials and does not interact with circulating factors. With a variety of chronic diseases, including atherosclerosis, metabolic syndrome, and diabetes, there is a subtle change in the endothelium which leads to their “activation” see . InitialκB) pathway plays a critical role in this process [κB has both anti- and proinflammatory roles; however, with progression of vascular damage, it primarily activates inflammatory pathways [κB family, including p50, p52, p65, relB, and C-Rel, form homo- or heterodimers which are found in the cytoplasm in an inactive state bound to the inhibitor IκB. Various stressors can release the IκB from NF-κB through a pathway which involves phosphorylation of IκB by the IKK complex . The phosphorylation of IκB leads to its degradation by the ubiquitin proteasome pathway. The degradation of IκB allows translocation of the NF-κB dimer to the nucleus, where depending on the NF-κB composition, recruited cofactors, and the sequence of targeted genes, variable responses may be observed [κB may also be activated via an IKKα-specific, noncanonical pathway [κB activation in vascular smooth muscle cells [Although there are a variety of pathways by which inflammation can influence this vascular dysfunction, the nuclear factor kappa light chain enhancer of activated B cells , and insulin resistance, are often associated with diabetes, metabolic syndrome, vascular inflammation, and atherosclerotic cardiovascular disease. Hyperglycemia interrupts the colocalization of Hsp90 and eNOS in endothelial cells, which may affect the production of NO and endothelium-dependent vascular relaxation . Circulaβ-cell destruction induced by an autoimmune response. Imbalance between autoreactive Th1 lymphocytes and protective Th2 lymphocytes is found in type 1 diabetes, which leads to both proinflammatory cytokines  and anti-inflammatory cytokines  [β and IFN-γ) and the receptors on membrane of β-cells may activate the caspase cascade and result in apoptosis. TNF-β and IFN-γ may also activate macrophages, which leads to the release of TNF-α, IL-1β, NO, and superoxide, which may increase oxidative stress and downregulation of Bcl-2, which activate NF-κB and β-cell apoptosis leading to insulin deficiency [β-cells may cause oxidative stress, insulin resistance, and β-cell damage [β-cells through stimulating the expression of uncoupling protein 2 (UCP2). UCP2 may inhibit electron transport in mitochondria and increase ROS production. Prolonged hyperglycemia may increase UCP2 in β-cells, which may contribute to insulin deficiency in both type 1 and type 2 diabetes [The major underlying mechanism for insulin deficiency in type 1 diabetes is d IL-13) . Interacficiency . Active l damage . Oxidatidiabetes . Relativdiabetes . Exercisdiabetes . The impBoth exercise and metabolic stress activate HSF1/Hsp pathway in the body. Transient stress responses induced by regular and moderate exercise tend to downregulate vascular inflammation and protect vessels from injury. Chronic stress responses induced by metabolic disorders upregulate inflammatory mediators, which leads to vascular inflammation, apoptosis, and injury. The HSF1/Hsp-mediated stress response to exercise and metabolic disorders play, distinguishable and possibly opposite roles in vascular inflammation, which may be related to the involvement of different types of Hsp, body temperature, or shear stress of blood flow. The consequences of stress responses induced by exercise and metabolic disorders, particularly of autoimmune diseases such as type 1 diabetes, on vascular inflammation require further investigation.


Mitogen-activated protein kinases (MAPKs) comprise a family of serine/threonine kinases that are activated by a large variety of extracellular stimuli and play integral roles in controlling many cellular processes, from the cell surface to the nucleus. The MAPK family includes four distinct MAPK cascades, that is, extracellular signal-regulated kinase 1/2 (ERK1/2), p38 MAPK, c-Jun N-terminal kinase or stress-activated protein kinase, and ERK5. These MAPKs are essentially operated through three-tiered consecutive phosphorylation events catalyzed by a MAPK kinase kinase, a MAPK kinase, and a MAPK. MAPKs lie in protein kinase cascades. The MAPK signaling pathways have been demonstrated to be associated with events regulating the expression of the steroidogenic acute regulatory protein (StAR) and steroidogenesis in steroidogenic tissues. However, it has become clear that the regulation of MAPK-dependent StAR expression and steroid synthesis is a complex process and is context dependent. This paper summarizes the current level of understanding concerning the roles of the MAPK signaling cascades in the regulation of StAR expression and steroidogenesis in different steroidogenic cell models. For the control of diverse signaling in response to the extracellular milieu, cells develop sophisticated tools to transmit the appropriate signals and thereby orchestrate the responses. The signaling mechanism of an agent involves effector-receptor coupling, production of second messengers, activation of protein kinases, and distribution of these transducers to specific intracellular targets. Mitogen-activated protein kinases (MAPKs) are the serine/threonine kinase family of conserved enzymes that are considered to be the central building blocks in the intracellular signaling networks –5. ThereThe steroidogenic acute regulatory protein (StAR) mediates the rate-limiting and regulated step in steroid hormone biosynthesis, that is, the transfer of the substrate for all steroid hormones, cholesterol, from the outer to the inner mitochondrial membrane –25. As sThe MAPK signaling cascade involves activations of several transmembrane signaling molecules, cytoplasmic protein kinases, and a network of interacting proteins, and these, in turn, regulate almost all cellular processes, from gene expression to cell death , 41, 42.The MAPK signaling pathway has long been implicated in the regulation of cell cycle progression, is used as a biochemical marker in evaluating the mitogenic effects of a variety of stimuli, and is a point of convergence for diverse signaling pathways , 51, 52.The ERK1/2 signaling cascade is the most widely studied member of the MAPKs. ERK1 p44 MAPK) and ERK2 (p42 MAPK) are highly conserved throughout eukaryotic cells and bring together transmembrane receptors and a network of various proteins and integrating signals to control many cellular processes, including cell survival, differentiation, tumor progression, and invasion 4 MAPK an, 56–58. α, and interleukin-1α (IL-1α) but decreases steroid levels [2cAMP and hCG)- and PKC-mediated (activated by PMA) regulation of steroidogenesis [β-hydroxysteroid dehydrogenase (3β-HSD) enzyme activities, as ERK1/2 inhibition had no effect on 22R-hydroxycholesterol-mediated steroid synthesis [2cAMP, and hCG-induced StAR expression and progesterone levels were inversely correlated with the levels of a negative transcription factor, namely, dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X-chromosome, gene 1 (DAX-1) [2cAMP/hCG [2cAMP and PMA can elevate the levels of scavenger receptor class B type 1  receptor that binds various lipoprotein particles and facilitates cellular cholesterol influx) and that ERK1/2 inhibition decreases SR-B1 expression [The involvement of the ERK1/2 pathway in steroidogenesis has been widely studied. This signaling pathway is involved in regulating StAR expression and steroid biosynthesis, but several seeming contradictions have been reported in different steroidogenic tissues , 60–62. d levels , 62, 63.d levels , 65. We ogenesis , 52, 66.ynthesis . Notewor (DAX-1) . While UcAMP/hCG , 66. In pression . The binpression . SR-BI ipression . Thus itpression , 71. Spe2cAMP, cholera toxin and forskolin stimulated progesterone production [in vivo or treatment with gonadotropins/cAMP in vitro is associated with down-regulation of Cyp19a1 and upregulation of StAR and Cyp11a1 mRNA expression in both mural and cumulus granulosa cells of mouse preovulatory follicles [β-HSD enzymes [α2 (PGFα2), an agent that influences luteal regression through the induction of the phospholipase C/diacylglycerol/PKC pathway, induces phosphorylation of ERK1/2, which, in contrast, decreases basal and LH/hCG-stimulated StAR expression and progesterone synthesis in human granulosa-luteal cells [α2 in the steroidogenic response can be reversed following inhibition of MEK activity by PD98059. Moreover, the inhibitory effect of PGFα2 on StAR expression and steroidogenesis, in rat luteal cells, is modulated by the negatively acting transcription factors, DAX-1 and ying yang 1 [In granulosa cell-derived rLHR-4 and rFSHR-17 cell lines, treatments with LH/hCG and FSH, respectively, increase ERK1/2 phosphorylation, StAR expression, and progesterone synthesis , 72. Conoduction , 60. Furoduction . Also, Eollicles . These e enzymes . Additio enzymes . In thec enzymes . Prostagal cells . These eg yang 1 , 79. TheIn mouse Y-1 adrenocortical tumor cells the induction of forskolin-mediated corticosterone synthesis is dependent upon the activation of ERK1/2 and is correlated with phosphorylation of SF-1 and increased StAR gene transcription . Accordiα (MAPK14), p38β (p38-2), p38γ (ERK6 or SAPK3), and p38δ (SAPK4) and share approximately 60% homology in their amino acid sequences [α) and Exip. P38 MAPKs contain a Thr-Gly-Tyr activation loop sequence (TGY). They are activated by dual phosphorylations on threonine and tyrosine residues in response to numerous stimuli, including cytokines, hormones, GPCRs, heat shock, and other stresses and play important roles in controlling many cellular functions [α is expressed in most cells; however, expression of other isoforms is tissue specific. Cellular distribution, activation, and substrate specificity of p38 MAPKs result in diverse biological functions. Once activated, p38 MAPKs either transmit the signals via a three-tier cascade or phosphorylate other regulatory molecules such as PLA2, heat shock proteins, and transcriptions factors  [The p38 MAPK signaling cascade is thought to participate in the response of cells to stress. Four members of the p38 MAPK family have been cloned and named p38equences , 88–91. unctions , 92, 93. others) , 92, 94. others) , 95, 96) others) , 98. Stu others) . The p38 others) , 101. α activates p38 MAPK and that this event is associated with StAR expression and testosterone synthesis in immature rat Leydig cells [α is also capable of phosphorylating CREB and Fos/Jun through the activation of a p38 substrate which is a RSK family member , suggesting that this process may play a role in the differentiation of immature into adult Leydig cells [α also activates ERK1/2 and inhibition of the latter by U0126 augments expression and phosphorylation of the StAR protein but decreases androgen synthesis by dissipating the mitochondrial electrochemical potential [α-mediated steroidogenesis in mouse Leydig cells. The p38 MAPK signaling cascade has been implicated in regulating steroidogenesis. It has been demonstrated that IL-1ig cells , 103. Thig cells . In addiotential . These fGonadotropins have been shown to activate both p38 and ERK1/2 MAPKs and result in varying effects on StAR expression and steroidogenesis in ovarian granulosa cells , 51, 104kip1 expression (a protein known to block the cell cycle in the G1 phase). The effects of Ang II can be reversed through inhibition of p38 activity by SB203580, suggesting that Ang II plays an important role in adrenal physiology. Accumulating evidence indicates that activation of the p38 MAPK signaling cascade is linked to the aging-induced, oxidative stress-mediated suppression of steroidogenesis in adrenal cells [In primary cultures of rat adrenal glomerulosa cells, Ang II activates the p38 and ERK1/2 signaling pathways and results in increases in StAR expression, steroidogenic enzymes, and steroid synthesis , 107. Coal cells –110. Altal cells , 112, inγ (46 kDa), JNK2/SAPKα (54 kDa), and JNK3/SAPKβ (52 kDa), are approximately 85% identical in their core catalytic domains [JNK/SAPKs are also considered as stress-activated MAPKs; however, they are different from p38 MAPKs , 114. Is domains , 116. Wh domains , 118. Se domains . However domains –119.α (TNFα) activates JNK/SAPK; however, the latter is associated with decreases in basal and cAMP-induced steroidogenesis by reducing Cyp17 gene expression in MA-10 cells [α also decreases the phosphorylation of ERK1/2 while simultaneously increasing the abundance of cJun as well as increasing AP-1 binding activity, suggesting the involvement of a number of MAPKs in TNFα signaling. Therefore, the activation of JNK/SAPK and ERK1/2 MAPKs appears to play a mutually antagonistic role in TNFα-mediated steroidogenesis. In rat R2C Leydig tumor cells, bisphenol A (BPA), an endocrine disruptor, is capable of activating JNK/SAPK and results in an elevation in aromatase activity and an attenuation of testosterone synthesis by targeting both CREB and Akt [2−) and hydrogen peroxide (H2O2), have been involved in the repression of testicular StAR expression and steroid synthesis [Several lines of evidence demonstrate the involvement of the JNK/SAPK signaling cascade in steroidogenesis. For example, tumor necrosis factor-10 cells . TNFα al and Akt . Increasynthesis –124. In In human granulosa cells, leptin activates a number of MAPKs including the JNK/SAPK signaling pathway where it decreases cAMP-induced StAR protein expression and progesterone synthesis . Likewis2O2, sorbitol, and UV irradiation) agents [The ERK5/BMK1 is the largest known MAP kinase (~110 kDa) family member. The signaling pathway leading to ERK5 activation is poorly understood in comparison with other MAPKs as a consequence of conflicting findings in the literature –130. Imp) agents , 133. Th) agents –136. The) agents , 137–139) agents . MEK5 th) agents . ERK5 ca) agents –143. Relatively little is known regarding the role of ERK5 in steroidogenesis. Recently, it has been reported that orexins A and B activate ERK5 and concomitantly increase expression of the StAR protein and steroidogenic enzyme genes in H295R cells , 84. TheERK3, ERK4, ERK7, NLK, MOK, and others are considered as atypical MAPKs . RegulatThe MAPK signaling cascades are the major components of pathways controlling a wide variety of cellular processes, including embryogenesis, gene expression, acute responses to hormones, cell survival, and apoptosis and are of critical importance in the transduction of extracellular signals in cells. Noteworthy, the MAPK cascade has been implicated in the pathogenesis of a number of human disorders, including Alzheimer's disease, Parkinson's disease, and many cancers. Indeed, the multiple effects of MAPKs could be a result of the involvement of a wide variety of substrates that include protein kinases and phosphatases, transcription factors, cytoskeletal elements, and other signal-regulated molecules. The studies summarized here have emphasized the roles of ERK1/2, p38 MAPK, JNK/SAPK, and ERK5 MAPKs in the regulation of StAR expression and steroidogenesis in different steroidogenic tissues. The StAR protein plays an indispensable role in the production of steroid hormones required for bodily homeostasis and normal reproductive development and function. Based on the results available at this time, it is obvious that the MAPK signaling cascades play diverse roles in controlling StAR expression and steroid biosynthesis in tissue-, stimulus-, and pathway-specific manners. These processes appear to be dependent on receptor-effector coupling, signaling crosstalk, and/or other factor(s) involved in steroidogenesis. Moreover, under specific circumstances, the regulation of MAPK-dependent StAR expression and steroidogenesis involves more than one MAPK signaling, and as a consequence different, and even opposing, effects of MAPKs can be seen in different steroidogenic cells. Additionally, different cells express distinct sets of transcription factors, and this diversity may account for the cell-type-dependent specificity of MAPK action. Given the physiological and pathological roles of the MAPK signaling pathways, elucidation of tissue- and disease-specific effects of each of the MAPK signaling cascades together with their downstream effectors requires better understanding. An abundance of molecular tools including high throughput genomic and proteomic technologies will undoubtedly provide valuable insights into these regulatory mechanisms.


NPHP1-12) have been identified. NPHP1 and NPHP4 control the ciliary access at the transition zone and the velocity of some intraflagellar transport (IFT)/BBS proteins in C.elegans. Recently, in a collaborative effort, we have identified, in families with isolated NPH, mutations in TTC21B as well as in WDR19, which encode the retrograde IFT-A proteins IFT139 and IFT144, respectively. By ciliome sequencing of 1600 candidate genes from 14 NPH patients followed by Sanger sequencing of a cohort of 52 patients, we have found respectively 8 and 7 patients carrying pathogenic missense mutations in genes coding IFT-A proteins, including WDR35, TTC21B and IFT140, which could partially affect their function. Together, these results indicate that IFT-A are involved in nephronophtisis. Moreover, alteration of cilia length was observed in patient kidney, Nphp4-/- mice kidney tubules and NPHP1 or NPHP4 knockdown IMCD3 cell lines. In these cells, primary cilia present swellings at the distal region accompanied by an accumulation of IFT-B at the base and the tip, similar to what was observed in IFT-A mutants, suggesting a possible alteration of retrograde transport. Additionally, ARL13B, a small GTPase required for proper cilium shape and IFT stability, is absent along the axoneme of NPHP4-KD-IMCD cells. By controlling the entry of ciliary components at the transition zone, NPHP1 and NPHP4 may modulate IFT-A cargos thus participating in the same pathway (i.e. Wnt/PCP), alteration of which would lead to renal lesions observed in nephronophthisis.Nephronophtisis (NPH) is a kidney ciliopathy often associated with extra-renal defects and for which 12 genes (


Pseudomonas resinovorans strain CA10 can grow on carbazole as its sole carbon and nitrogen source. Here, we report the complete nucleotide sequence of the CA10 genome . CA10 carries a larger number of genes that are potentially responsible for aromatic hydrocarbon metabolism than do other previously sequenced Pseudomonas spp. Pseudomonas resinovorans strain CA10 (NBRC 106553) was isolated from activated sludge and can utilize carbazole, a nitrogen heteroaromatic compound, as its sole carbon, nitrogen, and energy source  for GS FLX Titanium and a paired-end library (200 to 500 bp) for HiSeq 1000. A total of 117,465,953 base sequences 276,857 reads) were obtained with a depth of 18-fold genome coverage by the GS FLX Titanium system, and 487,867,241 base paired-end sequences  were obtained with a depth of 75-fold genome coverage by the HiSeq 1000 system. The assembly was performed using Phred , was deleted on pCAR1.3. Complete sequences of the CA10 genome were analyzed using MIGAP (http://www.migap.org/) for predicting protein-coding, tRNA, and rRNA genes. Their functional annotations were assigned using UniProt, InterPro, Hamap, and an in-house database composed of manually curated microbial genome sequences . The CA10 chromosome has 5,652 coding sequences (CDSs), five rRNA operons, and 70 tRNA genes. The anthranilate-degradating genes antABC and antR were found not only on pCAR1.3 but also on the chromosome, as was predicted previously (Pseudomonas strains using the RAST server (http://rast.nmpdr.org/)  and a circular plasmid named pCAR1.3 . pCAR1.3 is shorter than the previously reported pCAR1 and pCAR1.2 plasmids , because one of the 1,261-bp DNA regions containing eviously . Comparidr.org/)  showed tAP013068 and AP013069, respectively.The nucleotide sequences of the CA10 chromosome and pCAR1.3 were deposited in the DDBJ/EMBL/GenBank databases under the accession no.


IGF-1) gene polymorphisms are genetically linked with high-grade myopia (HM), which is a complex-trait eye disorder in which numerous candidate loci and genes are thought to play a role. We investigated whether the IGF-1 single nucleotide polymorphisms (SNPs) rs6214, rs10860860, and rs2946834 are associated with HM (≤-6.0 diopters [D]) and any myopia (≤-0.5 D) phenotype in Polish families.Recent work has suggested that insulin-like growth factor 1  and family-based Pedigree Disequilibrium Test (PDT) were used to examine the potential association of the IGF-1 SNPs rs6214, rs10860860, and rs2946834 with HM or any myopia. To determine the distribution of the HM-associated SNPs rs6214 and rs10860860, 543 unrelated individuals from the general Polish population were also analyzed.Forty-two multiplex HM Polish families, of whom 127 had HM, participated in the study. All of the family members (n=306) underwent a detailed ophthalmic examination, including axial length measurements. The IGF-1 SNPs rs6214, rs10860860, and rs2946834 and HM or any myopia phenotype in Polish HM families. In the general Polish population, the minor allele frequencies of the SNPs rs6214 and rs10860860 did not deviate significantly from the distribution reported for European populations (p=0.629). In the FBAT analysis under the dominant model, the haplotype consisted of T allele of rs10860860, with C allele of rs2946834 of IGF-1 was found less frequently transmitted to HM individuals (p=0.0065), pointing to a nonassociated or protective haplotype.We found no significant association between the IGF-1 gene with HM and any myopia phenotypes. Further replication studies involving other populations are needed to investigate the possible role of IGF-1 as a potential myopia candidate gene.Our results do not support recent studies reporting an association of the SNPs rs6214, rs10860860, and rs2946834 in the  Myopia affects 25% of the Western world, making this condition the most common eye disorder in the West and constituting a significant public health and economic problem ,2. The cMYP) have been mapped, including 12 loci linked to HM: MYP1, chromosome Xq28 (OMIM 310460) [MYP2 18p11.31 (OMIM 160700) [MYP3 12q21-q23 (OMIM 603221) [MYP4 7q36 (OMIM 608367) [MYP5 17q21-q22 (OMIM 608474) [MYP11 4q22-q27 (OMIM 609994) [MYP12 2q37.1 (OMIM 609995) [MYP13 Xq23-q25 (OMIM 300613) [MYP15 10q21.1 (OMIM 612717) [MYP16 5p15.33-p15.2 (OMIM 612554) [MYP18 14q22.1-q24.2 (OMIM 255500) [MYP19 5p15.1-p13.3 (OMIM 613969) [Myopia has a diverse etiology, with both environmental and genetic factors believed to be involved in the condition’s development and progression. The environmental factors implicated in myopia include near work, light exposure, lack of physical activity, diet, a higher level of education, and urbanization -10. Howe 310460) ,17; MYP2 160700) ; MYP3 12 603221) -22; MYP4 608474) ; MYP11 4 609994) ; MYP12 2 609995) ; MYP13 X 612554) ; MYP18 1q25 OMIM 0613 [MYPCOL1A1) [TGFB1) [TGIF) [LUM) [HGF) [MYOC) [PAX6) [UMODL1) [Candidate gene association studies have revealed several HM susceptibility genes, including: collagen, type I, alpha 1 (COL1A1) ,35, tran [TGFB1) ,37, tran) [TGIF) ,39, lumiF) [LUM) ,41, hepaM) [HGF) ,43, myoc) [MYOC) ,45, pair) [PAX6) ,47, and [UMODL1) . Howeverrs6214, rs10860860, and rs2946834 and familial myopia in a large, international cohort of myopia pedigrees of Caucasian origin, suggesting that insulin-like growth factor 1 (IGF-1) may be a candidate gene for HM. These three SNPs are located within the MYP3 locus (OMIM 603221) mapped to chromosomal region 12q21-q23. This locus was previously reported to be associated with autosomal dominant HM [rs6214  lies in the 3′-untranslated region (UTR) of IGF-1 (OMIM 147440), whereas the SNPs rs10860860  and rs2946834  are located in the noncoding sequence in close proximity to IGF-1.Recently, Metlapally et al.  reportedinant HM ,21. The IGF-1 gene encodes insulin-like growth factor (pIGF-1), which is a member of the signaling system involved in development, cellular growth, differentiation, protein translation, metabolism, apoptosis, and aging [IGF-1 with numerous human diseases, such as diabetes [IGF-1 has been also implicated in ocular diseases, including retinopathy of prematurity [IGF-1 to be considered as a candidate gene for HM, previously published findings need to be replicated.The nd aging ,51. The diabetes , cancer diabetes , and grodiabetes  has beenmaturity ,56, age-maturity , and diamaturity ,59. Howers6214, rs10860860, and rs2946834 in the IGF-1 gene with HM and any myopia phenotype in 42 Polish families. Our findings do not confirm the results reported previously for another myopic Caucasian cohort, however which was different from the Polish population [IGF-1, which previous work has suggested may be associated with any myopia and HM phenotypes.In the present study, we tested the association of pulation . To our Forty-two multigenerational Polish HM families were enrolled in the study. The subjects were classified into three groups, including: 1) affected individuals (HM), 2) individuals with an unknown status, and 3) unaffected persons. All of the affected individuals had: A) bilateral axial HM in excess of or equal to −6.0 D (≤-6.0 D) in at least one eye and in excess of or equal to −5.0 D (≤-5.0 D) in the second eye, B) a history of onset of myopia at age ≤15 years, and C) a multiplex family with affected relatives in different generations. Individuals who were classified as unknown were: A) all children ≤15 years unless they fulfilled criteria for affected status as specified above, or B) individuals who have myopia with −6.0 D< X ≤-4.0 D, or C) individuals with a refractive error of ≤-6.0 D for one eye and a refractive error >-5.0 D for the second eye, or D) individuals with late age of onset (>15 years). All of the remaining individuals were considered unaffected, including hyperopic, normal seeing, and myopic subjects with a spherical refractive error (SPH) in the range from ≤-0.5 D to >-4.0 D.rs6214, rs10860860, and rs2946834 were examined in 306 subjects, including 127 with HM, 148 unaffected, and 31 individuals with an unknown status. In addition, to determine the distribution of the genotypes possibly associated with HM (rs6214 and rs10860860) [The SNPs 0860860)  in the gAll of the individuals  who participated in this study underwent a complete ophthalmic assessment, including a visual acuity testing, best-corrected visual acuity testing, a slit lamp evaluation, intraocular pressure examination, fundoscopy, axial length determination, keratometry, and refractometry. The detailed clinical evaluation and pedigrees of Polish HM families have been previously described . The cliAppendix 1.In addition, any myopia phenotype status was established in reference to the criteria published by Metlapally et al. , who defThe research protocol was approved by the Institutional Review Board at Poznan University of Medical Sciences in Poland. In accordance with the Declaration of Helsinki, written informed consent was obtained for the genetic studies from all of the participating family members and individuals from the general Polish population group.rs6214 and rs10860860 were analyzed by PCR-RFLP and direct sequencing methods. Two pairs of primers were used to amplify DNA harboring the analyzed SNPs. The amplicons were digested by the restriction enzymes TaiI and NdeI, respectively, according to the manufacturer’s instructions . The detailed PCR-RFLP protocols used in this study are provided in Appendix 2. In addition, all homozygous rs6214 AA and rs10860860 TT samples were verified by direct sequencing using the BigDye® Terminator v3.1 Cycle Sequencing Kit  and an ABI 3730xl analyzer. The results were analyzed by Sequencher® Software 4.10.1 . Due to a lack of a restriction site for SNP rs2946834, we analyzed this polymorphism by direct sequencing as described above. The detailed PCR protocol, as well as the primer sequences, are given in Appendix 2.The SNPs Primer3 v.0.4.0 tool and synthesized by Genomed Co. .All of the primer pairs used in this study were designed with the PEDCHECK version 1.1 [sion 1.1  was usedsion 1.1  was usedHaploview [HapMap Public Release #27).aploview  was usedrs6214, rs10860860, and rs2946834 polymorphisms of the IGF-1 gene and HM and any myopia using the Family-Based Association Test . FBAT is a generalized approach derived from the transmission/disequilibrium test (TDT) [FBAT included the additive, dominant, and recessive models [FBAT analysis was performed based on the compound null hypothesis of no linkage and no association between the phenotype and the genetic variant. To analyze the haplotype using FBAT, 42 multiplex Polish families were divided into 122 nuclear families (two parents and their offspring), and they were tested using a two-stage procedure. First, the SNPs rs6214, rs10860860, and rs2946834 were examined separately. Testing was then undertaken for possible haplotypes of the specific alleles of rs6214, rs10860860, and rs2946834. Analyses in which there were less than ten informative families for a particular marker were excluded from consideration. Unaffected individuals were included in the study to increase the statistical power of the FBAT analysis.We tested for an association between the st (TDT) , which ae models ,67. The FBAT results, the genetic association between the three selected polymorphisms of the IGF-1 gene and HM and any myopia phenotypes were tested using the family-based Pedigree Disequilibrium Test  [PDT analysis program evaluates evidence of LD in general pedigree data.In addition, to verify the ion 5.1)  as descrion 5.1) . The PDTThe genotype distribution and allele frequencies in affected, unaffected, and individuals with unknown status, as well as in the general Polish population group, are shown in rs6214 A and rs10860860 T, respectively. The coexistence of two minor homozygous genotypes of rs6214 AA and rs10860860 TT was observed in 3.1% of individuals . IGF-1 polymorphisms and the haplotypes. Moreover, we found a random distribution of genotypes and alleles of the SNPs examined in the Polish HM families (Appendix 3).The rs6214, rs2946834, and rs10860860 did not reveal high LD between SNP pairs in the Polish HM families. Moreover, the r2 values for pairs of markers were similar in Polish HM families and the CEU population (p=0.629).The analysis of LD between IGF-1 may be a candidate gene for familial myopia based on a positive association between the SNPs rs6214 and rs10860860 and the HM phenotype and the SNPs rs6214, rs10860860, and rs2946834 and any myopia phenotype.Genetic association studies, including case-control and family-base studies, have been widely used to search for genetic factors involved in human diseases, including HM ,70. Recers6214 is considered to be a functional polymorphism due to its location in the 3′-UTR of the IGF-1 gene. The 3′-UTR, a noncoding sequence, contains regulatory motifs crucial for gene expression, mRNA stability, and cellular location of mRNA or the binding of microRNA [The SNP microRNA ,72. PrevmicroRNA ,74.IGF-1 in eye growth and elongation; and therefore, in myopia development and progression. Insulin and IGF-1 injected into chick eyes resulted in an increase in the rate of axial length, ocular elongation, and the anterior chamber depth [The chick model of experimental myopia suggests a possible role for er depth ,76. Howeer depth ,78.262500) who received treatment with IGF-1. However, there was no difference in axial length between IGF-1 treated LS patients and healthy controls [Cordian et al.  stated tcontrols .rs6214, rs10860860, and rs2946834 in the IGF-1 gene with familial HM or any myopia phenotypes. The SNPs selected for the analysis were not in LD in the Polish families; the r2 values for pairs of markers were similar in the CEU population and in the Polish HM families. Simultaneously, we estimated the allele distributions of the selected SNPs (rs6214 and rs10860860) in the general Polish population group. We found no significant differences in minor allele frequencies observed in the general Polish population  and the data provided by HapMap Public Release #28 for CEU: Utah residents with Northern and Western European ancestry from the Centre d”Etude du Polymorphisme Humain (CEPH) collection . In our family-based study, we found no evidence of an association between the SNPs rs6214, rs10860860, and rs2946834 in the IGF-1 gene and any myopia and HM. In contrast to previously published data [rs10860860, and allele C of rs2946834 of IGF-1 was found less frequently in HM individuals than expected (p=0.0065), pointing to a protective effect of the haplotype. These and other allele haplotypes were not examined in the Metlapally et al. [In the present study, we investigated the previously reported association of the SNPs hed data , random y et al.  study.Over the years, various studies have reported associations between numerous nucleotide variants in several genes and HM ,42,44,80IGF-1 polymorphisms in a large cohort of Caucasian HM families, but the subjects were derived from different countries, and the majority of families in their study were from the US (60%). European-Americans are often treated as a homogeneous group; however, due to historical immigration, this cohort is formed of diverse source populations [We analyzed a homogenous ethnic group. All of the participants were Polish, white Caucasian, and of European origin. Metlapally et al.  examinedulations .rs6214 and rs10860860, as well as any myopia (including low and medium myopia) and the SNPs rs6214, rs10860860, and rs2946834, out of 13 tested SNPs in IGF-1. Therefore, we performed an additional analysis using the any myopia phenotype criteria described by Metlapally et al. [FBAT and PDT analyses showed no positive association between the IGF-1 SNPs rs6214, rs10860860, and rs2946834 and any myopia phenotype.Unlike the study by Metlapally et al. , we emply et al. . Again, −5 or even p≤2×10−7 provides a ratio of a truly positive, reproducible association for candidate genes [−7) and second-class (5×10−6) associations provide some assurance of reproducibility. However, the reliability of second-class associations depends on assumptions. As these criteria are not fulfilled in the Metlapally et al. [Metlapally et al.  defined te genes . Althougte genes , irreproy et al.  study, ty et al. ,89 mightIGF-1 gene polymorphisms rs6214, rs10860860, and rs2946834 with HM and any myopia phenotypes in Polish families.In conclusion, our study revealed no evidence to support the previously reported genetic association of the