Chronic obstructive pulmonary disease (COPD) is an incurable and prevalent respiratory disorder that is characterized by chronic inflammation and emphysema. COPD is primarily caused by cigarette smoke (CS). CS alters numerous cellular processes, including the post-transcriptional regulation of mRNAs. The identification of RNA-binding proteins (RBPs), microRNAs (miRNAs), and long non-coding RNAs (lncRNAs) as main factors engaged in the regulation of RNA biology opens the door to understanding their role in coordinating physiological cellular processes. Dysregulation of post-transcriptional regulation by foreign particles in CS may lead to the development of diseases such as COPD. Here we review current knowledge about post-transcriptional events that may be involved in the pathogenesis of COPD. Human organ systems rely on the dynamics of gene expression to regulate homeostasis, cell survival, fate and differentiation, as well as responses to stress and environmental signals . EukaryoRBPs are a group of over 2000 proteins, each possessing multiple RNA binding domains, and which are known to be involved in RNA decay . RBPs asRBP-mediated post-transcriptional regulation is essential for proper cellular function, and its perturbation can lead to the development of disease. For example, the fragile X syndrome of mental retardation is caused by a defect in the RBP fragile X mental retardation protein, which is important for normal brain development . The fatDrosophila-like (ELAVL1), is also involved in the polyadenylation step. HuR is a ubiquitously-expressed RBP which is predominantly nuclear, but shuttles between the nucleus and cytoplasm. HuR blocks polyadenylation of the simian virus 40 late (SVL) poly(A) site that has U-rich sequences both upstream and downstream of the cleavage site. This leads to a decrease of SVL poly(A) site-containing mRNA, and an increase of pre-mRNA levels [Polyadenylation of pre-mRNA is an essential processing event for RNA nuclear export, stability, and translation. Polyadenylation is a maturation step in which all pre-mRNAs in eukaryotic cells, except mRNA encoding histones, receive poly(A) tails of around 200 adenine (A) nucleotides to their 3’ end by a multiprotein machinery complex ,23. ThisA levels . Splicing of pre-mRNA is a step of gene expression in which introns (noncoding sequences) are removed, and exons (coding sequences) are assembled by the spliceosome. The spliceosome is a ribonucleoprotein complex composed of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, and more than 50 protein factors, such as U2 auxiliary factor and SR (serine-arginine rich) proteins ,31. SomeRNA editing is a type of RNA modification characterized by the alteration of site-specific RNA sequences from that encoded in DNA . The RNAThe translation of mRNA is coupled with its stability and decay. RBPs that regulate mRNA stability are either mRNA decay activators or mRNA stabilizers. Activators of mRNA decay recognize the constitutive decay AU- and GU-rich elements of their target mRNAs, and affect its cellular levels by several mechanisms . For exaPTGS2 mRNA is sequestered in SGs by HuR, thereby decreasing protein levels due to a delay in translation [Localization of mRNA is critical for protein synthesis. Stress granules (SGs) and processing (P-) bodies are cytoplasmic RNA granules consisting of aggregates of ribonucleoprotein complexes. SGs and P-bodies are assembled in stressed and in unstressed cells, respectively . SGs seqnslation . SGs alsnslation . In contnslation . For insnslation . PTGS2 mRNA [The regulation of mRNA translation controls gene expression in the cytoplasm. Numerous proteins, including RBPs, regulate mRNA location and assembly into ribosomes for protein synthesis. For example, the RBP PABP that binds to stable mRNA also interacts with eukaryotic initiation factors 4E (eIF4E), whereby the 48S and 80S ribosome initiation complex are assembled and translation is initiated . AnotherGS2 mRNA . Some RBGS2 mRNA , but canGS2 mRNA . Caenorhabditis elegans [miRNA are small non-coding RNAs (~22 nucleotides) that affect gene expression ,83. In t elegans ,88. At t elegans . The imp elegans ,90.The function of miRNAs has been explored in murine knockout models, which have revealed important roles for miRNA in various biological processes, including development and immunity. For example, targeted ablation of miRNA-1-2, a muscle-specific miRNA, leads to cardiac morphogenetic and electrophysiologic defects . FurtherLncRNA are a class of ncRNA that are more than 200 nucleotides in length and do not translate into protein ,96,97. lBased on the molecular mechanisms of action, lncRNA can also be categorized as decoy, scaffold, and guide ,99. DecoCOPD is a leading cause of chronic morbidity and mortality worldwide . The WorThe main cause of COPD is CS . GloballMechanistically, the development of COPD is initiated by inflammation caused by repeated exposure to CS, which induces a pulmonary inflammatory response in several cell types, including epithelial cells, fibroblasts, and macrophages ,11,12. RNo disease-modifying therapies exist for COPD. After smoke exposure and in COPD, transcriptional regulation alters the expression of inflammatory mediators, such as IL-6, COX-2, TNF-α, IL-1β, and IL-8 . HoweverPTGS2 mRNA by preventing HuR translocation into the cytoplasm [RBPs appear to play a role in the cellular response to CS. We have previously shown that lung fibroblasts produce COX-2 in response to CS ,121, andytoplasm . It is nytoplasm ,121,122,ytoplasm . Furtherytoplasm . HRV infytoplasm ,131. ThuRbm5 loss-of-function (heterozygous) mice exposed to the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) develop more aggressive lung cancer [Another RBP that has been studied in the context of smoking is RNA-binding motif protein 5 (RBM5). The gene of RBM5, also known as H37 or Luca15, located in chromosomal region 3p21.3, is frequently deleted in heavy smokers and lung cancer patients ,133. Rbmg cancer . In cellg cancer . β-cateng cancer . EMT is g cancer . AlthougIRP2 mRNA and protein levels are elevated in lungs from COPD subjects [Elucidation of changes in the expression and the function of RBPs may suggest putative pathogenetic roles for them. Targeting RBPs could also be a novel therapeutic strategy. However, only a handful of studies directly investigate RBPs in COPD. One of these was a genome-wide association study which identified iron-responsive element binding protein 2 (IRP2 or IREB2), an RNA-binding protein that regulates cellular iron homeostasis, as a COPD susceptibility gene. subjects ,140,141,subjects . Previousubjects ,144, whiAnother RBP studied in the context of COPD is HuR, where its expression is increased in the airway epithelium from smokers with or without COPD . This suFinally, the RBP AUF1, which participates in mRNA decay, is decreased in the bronchial epithelium from COPD subjects compared to smokers without COPD. Analysis of a microarray from a primary airway epithelium of COPD revealed that AUF1 target genes are upregulated, including those associated with inflammation . AlthougTNF-α by binding to AREs present within the 3’ UTR [PTGS2, IL-6, CXCL8, and CCL2 [TNF-α mRNA [CCL2, CCL7, CXCL1, and CXCL5, is also abrogated in TTP-knockout cells [CS causes direct damage to airway and alveolar epithelial cells, which leads to the recruitment of inflammatory cells, and the release of numerous inflammatory mediators, including TNF-α ,121,148,e 3’ UTR . TTP alsand CCL2 ,152. GluF-α mRNA . Glucocout cells . OverallTNF-α. AUF1 knockout mice are susceptible to endotoxin challenge due to TNF-α and IL-1β overproduction [AUF1 is another RBP that induces the decay of target mRNAs, including oduction —these mioduction . Given toduction , and thaCCL2 and TNF-α, in pulmonary epithelial cells [CCL-11 (eotaxin) mRNA levels [While AUF1 and TTP may be important in controlling inflammation by promoting mRNA decay, other RBPs, such as HuR, stabilize mRNA associated with inflammation, such as al cells , as wellal cells . HuR itsA levels . This maA levels . Thus, tVEGF mRNA [VEGF expression [Emphysema is characterized by the loss of lung structural cells, including alveolar epithelial cells and fibroblasts ,160. MecEGF mRNA . In contpression . hnRNP Lpression ,167. Intpression , raisingMMP-9 and MMP-2 mRNAs [MMP-9 mRNA to stabilize its expression [MMP-9 mRNA [In addition to aberrant cell death, lung tissue destruction in COPD is mediated by proteases. Activated neutrophils are a potent source of proteases, such as NE, cathepsin G, proteinase-3, MMP-8, and MMP-9, all of which can contribute to the destruction of alveolar walls ,170. Mac-2 mRNAs , and HuRpression . ChangesP-9 mRNA ,175.CTSS) [CTSS mRNA, and controls its stability and expression [PDCD4 mRNA, whereas decreasing TIA-1 induces HuR binding to the PDCD4 mRNA [Dynamic interactions between RBPs may fine-tune post-transcriptional modifications of common mRNAs. RBPs can either cooperate or compete to bind target mRNAs. For example, ADAR1 cooperates with HuR, and forms an RNA-dependent complex, which regulates the stability of ADAR1 targets in human B cells . ADAR1 mCTSS) , a cysteCTSS) . HuR alspression . Interespression . HuR andCD4 mRNA . FurtherCD4 mRNA . Togethelet-7, miR-206, and miR-16 are downregulated in lung cancer. The observation of miR-16 is of interest, as overexpression of miR-16 prevents cell proliferation and invasion [Another aspect of post-transcriptional regulatory mechanisms of relevance for smoke-induced lung disease are miRNAs. Thus, miRNAs are being pursued as therapeutic targets or, through their utility, as a diagnostic tool. For example, invasion ,184,185.invasion ,187. Theinvasion ,205,206.invasion ,191. Oneinvasion . Some miinvasion , miR-199As the severity of COPD increases, miRNA expression changes. For example, miR-206, miR-133a-5p, and miR-133a-3p are upregulated in extracellular vesicles of plasma from moderate COPD patients compared to mild and severe patients—these miRNAs are involved in hundreds of biological processes that are associated with COPD features . miRNAs Experimental studies profiling miRNA expression have shed light on the possible biological significance of dysregulated miRNA to COPD pathogenesis, including their ability to regulate smoke-induced inflammation. In one such study of miRNA expression in current and never-smokers’ bronchial airway epithelium, it was found that 28 miRNAs are differentially expressed. The most downregulated miRNA in smokers was miR-218—its expression is also decreased in primary bronchial epithelial cells exposed to cigarette smoke condensate . The matOther miRNA implicated in regulating inflammation are miR-181c, miR-145, miR146a, and miR-16, all of which are downregulated in response to CS and/or in COPD ,202,210.Other miRNAs are increased by smoking, include miR-101, miR-144 , miR-135Alveolar macrophages are among the first cell types to respond to smoke inhalation, as these innate immune cells patrol the luminal surface of alveoli . AlveolaIn addition to miRNA playing fundamental roles in inflammation, many miRNAs are also implicated in regulating cell survival. Both miR-23a and miR-421, for example, repress caspase-9 and caspase-3, respectively ,217. miRPTGS2 mRNA stability by reducing miR-16 binding to the 3′UTR of PTGS2 mRNA [PTGS2 mRNA decay [STAT3 mRNA in myotubes exposed to IFN-γ and TNF-α by binding to its 3′UTR—this binding interferes with miR-330–mediated translation inhibition [MYC mRNA to repress its expression [CDKN1B; cyclin dependent kinase inhibitor 1b) mRNA, and facilitate its association with miR-221/222 to destabilize p27 expression [miRNAs and RBPs interact to further fine-tune post-transcriptional regulatory mechanisms. For example, hnRNP K increases GS2 mRNA , and oveNA decay . HuR alshibition . HuR coupression . Other epression . While tAltered expression of lncRNA is now thought to be involved in the pathogenesis of COPD. For example, a previous study found that 109 lncRNA are differentially expressed in the lungs of mice exposed to CS, of which 51 lncRNAs were significantly upregulated, and 58 were significantly downregulated. Gene ontology analysis of potential lncRNA target protein-coding genes showed enrichment in pathways involved in the cellular response to interferon-beta . FurtherDysregulation of lncRNA expression suggests they have roles in the pathogenesis of CS-related diseases. For example, lncRNA are upregulated by smoking: this includes smoke- and cancer–associated lncRNA–1 (SCAL1) , and lunLncRNAs that are involved in EMT are also increased in COPD, including MALAT1  and taurLncRNAs are also involved in inflammation, such as maternally expressed gene 3 (MEG3) , MALAT1 CDK6), which promotes G1/S transition of the cell cycle. HnRNP K also inhibits miR-16 binding to the 3′UTR of CDK6 [PTGS2 mRNA by reducing the association of miR-16 to the PTGS2 3′UTR [PTGS2 mRNA. Another example of such an association is the lncRNA functional intergenic repeating RNA element (FIRRE), which interacts with hnRNP U to stabilize vascular cell adhesion molecule 1 (VCAM1), a cell adhesion molecule that is involved in inflammation [MYC mRNA by interacting with two RBPs. Linc-RoR interacts with hnRNP I to stabilize MYC mRNA, and interacts with the destabilizing RBP AUF1 to inhibit its binding to MYC mRNA [MYC mRNA. The discovery of the interplay between RBPs and lncRNAs highlights their regulation of post-transcriptional mechanisms of target mRNAs. This would be another step in understanding the landscape of genes that are involved in the pathogenesis of chronic lung disease.LncRNA are known to interact with RBPs, a feature which might be important in understanding the functional role of RBPs and lncRNAs in the development of lung diseases. For example, the lncRNA c-Myc-upregulated (MYU) associates with hnRNP K to stabilize the mRNA of cyclin-dependent kinase 6 ( of CDK6 . AlthougS2 3′UTR , hnRNP Kammation . FinallyMYC mRNA . This sulet-7, whose exogenous delivery reduces tumor development in a mouse model of lung cancer [RBPs, miRNA, and lncRNA are examples of post-transcriptional regulons that may be involved in COPD pathogenesis. To date, many studies have largely focused on transcriptional regulatory pathways implicated in the development of COPD. However, it is increasingly apparent that post-transcriptional regulation of gene expression adds a dynamic layer of complexity to chronic diseases, as RBPs regulate polyadenylation, pre-mRNA splicing, RNA modification, nuclear export, localization, and turnover of target mRNAs. Similarly, miRNA and lncRNA typically regulate post-transcriptional repression of target mRNAs. RBPs, miRNA, and lncRNA function independently, or may operate cooperatively or competitively, adding complexity to the system. Altered function of post-transcriptional regulation may contribute to the development of chronic diseases, particularly those caused by environmental exposures, such as COPD, and future work should address these mechanisms. In this regard, both RBPs and miRNAs represent potential therapeutic targets. For instance, MS-444 is a small molecule inhibitor that interferes with the RNA binding activity of HuR , and hasg cancer . Therefo


The outcomes of pediatric acute lymphoblastic leukemia (ALL) have improved remarkably during the last five decades. Such improvements were made possible by the incorporation of new diagnostic technologies, the effective administration of conventional chemotherapeutic agents, and the provision of better supportive care. With the 5-year survival rates now exceeding 90% in high-income countries, the goal for the next decade is to improve survival further toward 100% and to minimize treatment-related adverse effects. Based on genome-wide analyses, especially RNA-sequencing analyses, ALL can be classified into more than 20 B-lineage subtypes and more than 10 T-lineage subtypes with prognostic and therapeutic implications. Response to treatment is another critical prognostic factor, and detailed analysis of minimal residual disease can detect levels as low as one ALL cell among 1 million total cells. Such detailed analysis can facilitate the rational use of molecular targeted therapy and immunotherapy, which have emerged as new treatment strategies that can replace or reduce the use of conventional chemotherapy. Approximately 6000 new cases of acute lymphoblastic leukemia (ALL) are diagnosed in the United States annually ,2,3,4. A9/L vs. ≥50 × 109/L) at diagnosis), cytogenetic and genomic analysis of ALL cells, and response evaluation with a minimal residual disease (MRD) assay. Dosage adjustment based on pharmacodynamic and pharmacogenomic studies and supportive care  have also contributed substantially to improved outcomes. Therefore, current dosages/schedules for “conventional” chemotherapy have been truly optimized.Currently, the survival of pediatric patients with ALL treated in high-income countries exceeds 90%  1,2,3,4,3,42,3,4With the current high rate of survival, further improvement in outcomes with conventional chemotherapy is challenging. In fact, there was very little improvement in 5-year overall survival (OS) between our two recent frontline ALL trials, St. Jude Total Therapy XV (5-year OS: 93.5%) and XVI (5-year OS: 94.3%)  7,8]. M,8. M7,8]Recently, several molecular targeted agents and immunotherapy approaches have been introduced, and they promise to improve outcomes. For these agents to be used optimally, detailed genetic characterization of leukemia cells and response evaluation by MRD in individual patients are critical. In this review, we will review the genetic subgroups of ALL, the evaluation of MRD, and newer treatment strategies.ETV6/RUNX1 fusion, TCF3/PBX1 fusion, BCR/ABL1 fusion, and KMT2A (MLL) rearrangement (BCR/ABL1-like ALL (Ph-like ALL), intrachromosomal amplification of chromosome 21 (iAMP21), DUX4-rearranged ALL, ZNF384-rearranged ALL, MEF2D-rearranged ALL, PAX5-altered  ALL, NUTM1-rearranged ALL, and ETV6/RUNX1-like ALL. Characterization of genetic abnormalities in ALL cells is important in order to identify unfavorable genetic abnormalities and to incorporate molecular targeted therapy to reduce the risk of relapse.The revolutionized approach to genomic analysis subdivides pediatric ALL into more than 30 genetic subgroups ,10,11. Iangement . GeneticETV6/RUNX1-rearranged ALL represents approximately 20% of pediatric ALL and is associated with excellent outcomes . V. V107]. nged ALL , hypodipnged ALL , BCR/ABLtive ALL , TCF3/HLnged ALL , and T-Anged ALL ,111. LowLL cells . VenetocL, navitoclax (a BCL-2 and BCL-XL inhibitor) was tested in combination with venetoclax and chemotherapy for pediatric and adult patients with relapsed/refractory ALL or lymphoblastic lymphoma [Phase I studies of venetoclax in combination with chemotherapy in pediatric and young adult patients with ALL have shown the regimen to be well tolerated with preliminary efficacies . As the lymphoma . Among 4Proteasome inhibitors have shown efficacy in ALL and work synergistically with chemotherapy agents such as corticosteroids and doxorubicin  115]. I. I115]. Dysregulation of the PI3K/AKT/mTOR pathway is frequently observed in ALL and is associated with resistance to chemotherapy ,118. mTOEpigenetic modification, the biochemical alteration of chromatin, has been implicated in the pathogenesis of cancer . InsteadThree major categories of immunotherapy are currently in use for pediatric ALL : bispeciCD19 exons 2–5 or alternative splicing at exon 2 produce a truncated protein with a nonfunctional or absent transmembrane domain and/or no antibody binding site [KMT2A- and ZNF384-rearranged B-ALL [Blinatumomab has bispecific single-chain Fv fragments that link CD3+ T cells to CD19+ leukemia cells and cause a cytotoxic immune response  127,128128. It iing site ,132. Sused B-ALL ,134. An ed B-ALL .Pneumocystis jirovecii pneumonia prophylaxis [CAR T cells express single-chain Fv fragments against B-lineage markers  with intracellular signaling domains such as 4-1BB or CD28 with CD3ζ . A phasephylaxis .Mechanisms of resistance to CAR T-cell therapy include the loss of CAR T-cell persistence and B-cell aplasia and antigen loss on ALL cells ,142. In For extramedullary relapse , CAR T cells can migrate and show anti-leukemia effects; therefore, they can be considered not only for isolated bone marrow relapses but also for isolated or combined extramedullary relapses, thereby avoiding radiation therapy ,149.Inotuzumab ozogamicin is an anti-CD22 antibody that is linked to calicheamicin, a cytotoxic antitumor antibiotic that causes double-strand DNA breaks . InotuzuThe diagnosis of ALL, the treatment of patients, and the evaluation of the treatment response have undergone remarkable improvement. The detailed genetic characterization of ALL cells, functional genomics and proteomics, and drug sensitivity assays with ex vivo and patient-derived xenograft (PDX) models for molecular targeted agents and immunotherapy will lead to new therapeutic strategies. Furthermore, the evaluation of germline genetics can lead to an understanding of leukemogenesis, cancer predisposition, and the differences in drug response and metabolism (pharmacogenomics). Basic, translational, and clinical research on ALL will not end until all patients can be cured without acute complications or late sequelae.


Otp expression during development.The homeodomain transcription factor Orthopedia (Otp) is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate otp orthologues in zebrafish (otp1 and otp2) and explored otp1 in the context of the morphogenetic pathways that specify neuroectodermal regions. During forebrain development, otp1 is expressed in anterior groups of diencephalic cells, positioned in the preoptic area (PO)  and the posterior tuberculum (PT) . The latter structure is characterized by Tyrosine Hydroxylase (TH)-positive cells, suggesting a role for otp1 in the lineage restriction of catecholaminergic (CA) neurons. Disruptions of Hedgehog (HH) and Fibroblast Growth Factor (FGF) pathways point to the ability of SHH protein to trigger otp1 expression in PO presumptive neuroblasts, with the attenuating effect of Dzip1 and FGF8. In addition, our data disclose otp1 as a determinant of CA neurons in the PT, where otp1 activity is strictly dependent on Nodal signaling and it is not responsive to SHH and FGF.Here, we identified two otp1 transcription factor in cell states of the diencephalon anlage and early neuronal progenitors. Furthermore, our data indicate that morphogenetic mechanisms differentially regulate otp1 expression in alar and basal plates.In this study, we pinpoint the evolutionary importance of  Parkinson's disease and addiction) .-40.syu, nts Fig. , which agulation ,40. Inhiotp1 expression after shh microinjection was selectively observed in the optic placodes  interferes with the Nodal pathway causing massive defects in the rostral brain [otp1 expression in cyc mutant embryos with intermediate phenotypes  shows midline fusion of PO-specific otp1 expression; moreover, we noticed that the domain is decreased in size   mutant embryos. This phenotype is not caused by shh deficiency since otp1 expression in the posterior basal plate of the hypothalamus is not modulated by HH signaling, but could be determined by the absence of the prosencephalic ventral structures. This issue has been addressed overexpressing ndr2 and analyzing the effects on otp1 transcription in the PT area. Increase of Ndr2 function is shown to determine the expansion of the PT-specific otp1 domain  Fig. , pointinain Fig. , in accoberculum ,82.otp1. Because both otp1- and TH-positive neurons occur in the PT, we co-labelled this area. Interestingly, otp1 and TH partially overlap in a small clade of neuronal precursors  [otp1-positive neurons   show lesons Fig. . The hypion Fig. . Altogetotp1 expression and Nodal, SHH and FGF signaling cascades in the hypothalamus. The first piece of evidence demonstrates that Nodal, in concert with fgf8 and shh, mediates otp1 expression in the preoptic area  has been isolated by means of PCR from a 16–40 hour old zebrafish embryo cDNA library using degenerate primers ort1_fw (5'-CCNGCNCAGCTSAACGA-3') and ort2_rv (5'-CKYTTYTTCCAYTTNGC-3'), corresponding to PAQLNE and AKWKKR regions of the mouse Otp homeodomain, respectively. The first round of PCR has been carried out using the ort2_rv degenerate primer with the T3 library-vector specific primer. An aliquot of this reaction has been used as template with the ort1_fw and ort2_rv primers. The 150 bp cDNA band obtained encoded a partial homeodomain identical to the corresponding residues of the mouse Otp protein. Gene- and vector-specific primers were used on the same library to isolate the 5' and 3' ends of the otp1 mRNA.The zebrafish homologue of Bos taurus, Otp XM_604218; Gg, Gallus gallus, Otp AY651764; Cf, Canis familiaris, Otp XM_546055, Otx2 XM_547830; Pt, Pan troglodytes, XM_517691; Hs, Homo sapiens, Otp NM_032109, Otx2 NM021728; Mm, Mus musculus, Otp CAA71439; Rn, Rattus norvegicus, Otp XP_215445; Dr, Danio rerio, Otp1 AF071496, Otx1 NM131250, Otx5 NM_181331; Dm, Drosophila melanogaster, Otp NM_206187; Pd, Platynereis dumerilii, Otx AJ278856; Sp, Strongylocentrotus purpuratus, Otp XM_779506, Otx NM_214588; At, Achaearanea tepidariorum, Otd AB096074; Tc, Tribolium castaneum, Otd1 NM_001039424, Otd2 NM_001039437; Ci, Ciona intestinalis, Otp AB210618; Ef, Euscorpius flavicaudis, Otd AY738138; Xt, Xenopus tropicalis, Otx1 NM_203885; Sk, Saccoglossus kowalevskii, Otp AAP79292; Pv, Patella vulgata, Otp AF440099; Lv, Lytechinus variegatus, Otp AAR17090; Pl, Paracentrotus lividus, Otp O76971; Ht, Heliocidaris tuberculata, Otp AAS00592; He, Heliocidaris erythrogramma, Otp AAS00591; Ds, Drosophila subobscura, Antp X60995; Dv, Drosophila virilis, Antp AY333070; Am, Apis mellifera, Antp NM_001011571; Sc, Sacculina carcini, Antp AF393443; Pc, Podocoryne carnea, Tbx4/5 AJ581006.A total of 47 sequences, gathered from several EST and genomic databases, were aligned using the ClustalW algorithm  as impleDugesia japonica (Dj) Otp sequence was obtained directly from Umesono and co-workers [Hydra magnipapillata (Hm), Takifugu rubripes (Tr) and Tetraodon nigroviridis (Tn) Otp sequences were generated in the course of this study and are available from the authors. The alignment was manually refined and used as the basis for all tree reconstruction methods. Maximum parsimony (MP) analysis was carried out in PAUP* 4.0b10 (Windows version) [While -workers , Hydra mversion)  using thversion)  implemenversion)  using thversion) . Four Maversion)  was calcversion) . The besotp1 expression using the forward (zotpS: 5'-ATGCTCTCTCATGCCGACCT-3') and reverse (zotpRv: 5'-TCTGTTGGTTTTGCTGGCCG-3') primers spanning the cDNA region involved in alternative splicing. The products of the PCRs were loaded and resolved onto 2% agarose gels.Total RNAs from 15 samples corresponding to 10 different developmental stage embryos  and 3 adult organs  were purified, DNase treated, and reverse-transcribed. The cDNAs obtained were tested for the presence of in vitro labelled with digoxigenin or fluorescein (Roche). For double WISH and antibody labelling, WISH was performed first, then embryos were exposed to rat anti-Tyrosine Hydroxilase (TH) (Chemicon) and mouse anti-acetylated α-tubulin (Sigma). Embryos incubated with anti-TH were treated with biotinylated secondary antibody (Vector Laboratories).WISH hybridization was carried out according to Thisse and co-workers  on embryshh, ndr2, and fgf8 mRNAs were injected repeatedly (n > 3) at concentrations of 400, 200, and 200 pg per embryo, respectively. Injections were carried out on 1- to 2-cell stage embryos. To repress otp1 mRNA translation, an ATG-targeting morpholino was designed : 5'-CCAAGAGGTCGGCATGAGAGAGCAT-3'.Synthetic capped in silico cloning procedures, the molecular genetic and functional studies, and drafted the manuscript. AP and RT carried out some of the molecular genetic and functional studies. BDB participated to the identification of otp1 and preliminary expression analysis. NA carried out sequence alignments and evolutionary analysis. FC participated in the design and coordination of the study. All authors read and approved the final manuscript.LDG and PS designed the study, carried out the wet and


A significant association was found between elevated levels and advanced (Ann Arbor stage III or IV) disease or hepatic infiltration, but not with other sites of extranodal involvement or bulky disease. Patients with normal levels at presentation had a 70% remission rate with treatment compared to 37% of those with elevated levels (P < 0.001). With median follow up of 6 years duration of remission was significantly greater in patients with normal beta-2 microglobulin at presentation . Survival overall was also better in the group with normal levels . Multivariate analyses including treatment type, age, sex, B symptoms, stage, bulk, albumin, sodium, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase and beta-2 microglobulin, placed beta-2 microglobulin among the three most influential independent variables for prediction of response rate, duration of remission and overall survival.


Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential.Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 . The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene.Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, consistent with their requirement for DNA replication. The essential genes code for two of ten Orc/Cdc6 proteins, two out of three DNA polymerases, the MCM helicase, two DNA primase subunits, the DNA polymerase sliding clamp, and the flap endonuclease.The results showed that ten out of nineteen eukaryotic-type DNA replication genes are essential for  Halobacterium sp. NRC-1 provides an excellent model system to address questions of fundamental DNA replication biology using bioinformatic, genomic, and genetic approaches . ,18. orc/p. NRC-1 . A few gp. NRC-1 .orc/cdc6 genes and a polB gene are dispensable to cells. This study shows the first in vivo evidence for genes likely to be critical for DNA replication in Archaea.With the availability of an inventory of replication factors likely acting at haloarchaeal DNA replication origins and a facile gene knockout system, we sought to answer basic questions regarding the essentiality of DNA replication gene assignments in an archaeon. The inability to recover deletion mutants indicates the requirement of genes coding two Orc/Cdc6 proteins, two different replicative DNA polymerases, a replicative helicase, a eukaryotic-type primase, a DNA polymerase sliding clamp, and the flap endonuclease. Eight of the Halobacterium sp. NRC-1, ten orc/cdc6 genes are present, with orc6, orc7, orc8, and orc10 genes located on the large chromosome, orc1-5 located on pNRC200, and orc9 located on both the pNRC100 and pNRC200 replicons. The gene products are quite diverse, ranging from 21–91% similarity (data not shown), with Orc2 and Orc4 being the most similar overall and Orc8 and Orc10 being the most similar encoded chromosomally. The haloalkaliphilic archaeon, Natronomonas pharaonis, encodes the fewest number of orc/cdc6 genes among haloarchaea (five), while Haloarcula marismortui encodes the most (seventeen). Phylogenetic reconstruction of Orc/Cdc6 protein sequences from sequenced haloarchaeal genomes and representative eukaryotes indicated the presence of five distinct haloarchaeal/archaeal clades, all distantly related to eukaryotic Orc1 and Cdc6 , or on the opposite side as the integration . In theory, for a nonessential gene, either event should be recovered with the same frequency, yielding 50% wild-type restoration and 50% deletion allele replacement .] In all these cases, between 15 and 30 % of Foar isolates of integrants were knockouts. In contrast, however, we did not find any deletions of two orc genes, orc10, located on the large chromosome, and orc2, present on pNRC200, indicating that these genes are essential, which is consistent with their involvement in DNA replication. Orc10 belongs to a clade of uniquely haloarchaeal Orc proteins along with Orc8, eight other Orc/Cdc6 members from the distantly related archaeon H. marismortui, and a single member from N. pharaonis , indicating that this gene is dispensable to the cell  . In Haloell Fig. .mcm is required in Archaea, we targeted the single mcm gene in Halobacterium sp. NRC-1 for deletion. A suicide plasmid containing ~500 bp flanking the mcm gene . For orc10, 80 Foar colonies were screened without identifying a single knockout, indicating a probability > 99.9999999 % of this gene being essential  (Table All target genes where no deletion was obtained were tested for rejection of the null hypothesis. A minimum of 40 and a maximum of 80 colonies were screened in each case. For the r) gene in Halobacterium sp. NRC-1. This replicating plasmid vector was then transformed into the respective Halobacterium sp. NRC-1 Δura3 strain containing the gene deletion plasmid which had been stably integrated into the specific targeted gene locus. Excisants of the gene deletion vector were selected using Foar while the replicating plasmids were maintained with mevinolin selection , polB1 , mcm , pri2 , or rad2  gene plus the native promoter were introduced into a Halobacterium sp. NRC-1 Δura3 strain containing the corresponding deletion plasmid, respectively, integrated into the chromosome. After selection for excisants using Foar and Mevr selection, candidate clones were screened for wild-type or deletion alleles of either polD1, polB1 , four accessory proteins, the replicative helicase protein MCM, primase proteins Pri1/Pri2, processivity clamp protein PCNA, and Okazaki fragment maturation protein Rad2. Taken together, our results provide a better view of the likely in vivo requirements for DNA replication in Halobacterium sp. NRC-1.Analysis of DNA replication components in archaeal systems has been restricted primarily to bioinformatic analysis and lication ,9. In thorc2, orc10, polD1, polD2, polB1, mcm, pri1, pri2, pcn, and rad2), we have very strong evidence for the requirement of these genes for cell viability. In five cases tested by complementation analysis , knockouts were recovered when a functional copy of the gene was present on a replicating plasmid, confirming that the genes were essential to cells and also dominant in trans. These results provide a genetic system for further analysis of essential DNA replication genes in Halobacterium sp. NRC-1.Significantly, our study has targeted the largest number of genes for deletions in any archaeon to date ,7,21-33.orc genes encoded in Halobacterium sp. NRC-1 are essential. We had previously hypothesized that orc7 and likely orc6 would be essential for viability, based upon our previous genetic work showing the requirement of orc7 for autonomous replication ability of a minichromosome plasmid replicon [S. solfataricus [Pyrococcus abyssi [orc7 gene of Halobacterium sp. NRC-1 to be dispensable under standard growth conditions. Because NRC-1 contains ten orc/cdc6 homologs, it is possible that another gene may be functionally redundant to orc7 in this archaeon. In contrast, Orc7 orthologs are found in a single gene copy in most other Archaea, with the exception of Sulfolobus spp. which have two orc7 orthologs linked to two chromosomal DNA replication origins [Interestingly, we found that only two of ten replicon . Biochemataricus  and a chs abyssi  are also origins ,16.orc6 gene ortholog in their genomes [Halobacterium sp. NRC-1. Sulfolobus spp. Orc6 proteins have been found to bind origin DNA sequences, although in partially synchronized cultures, expression of the Orc6 ortholog appears to be in G2 phase cells [Methanothermobacter thermoautotrophicus have also been shown to interact with MCM and inhibit helicase activity, with the Orc6 ortholog being a more potent inhibitor [Halobacterium sp. NRC-1, the orc6 gene is not essential for viability and no discernable phenotypes are observed when it is deleted, possibly as a result of functional redundancy.Most Archaea encode an  genomes , but ourse cells . It is pnhibitor ,35. It iorc10 on the large chromosome, and orc2 on pNRC200 are essential. Although these genes are not found to be conserved in the genomes of non-halophilic Archaea, there are likely orthologs and paralogs found in all halophilic Archaea. Orc10 shares 50% sequence similarity to the non-essential Orc8 protein from Halobacterium sp. NRC-1. It also shares sequence similarity to Cdc6-3 from N. pharaonis and eight homologs from H. marismortui, including a previously unrecognized Orc/Cdc6 homolog on the pNG500 replicon . Interestingly, the orc10 genetic locus harbors an ISH12 element 100 bp from the orc10 predicted translational start codon and is also an area of the large chromosome with extrachromosomal characteristics, e.g. an increased AT% and a higher concentration of IS elements [Halobacterium sp. NRC-1, and forms a clade with Cdc6-5 from N. pharaonis, four homologs from H. marismortui, and at least seven homologs from H. volcanii (data not shown). At this time we cannot strictly state that the orc10 and orc2 genes are essential for DNA replication, only that they are essential for viability of Halobacterium sp. NRC-1, although their homology to the other haloarchaeal, archaeal, and eukaryotic orc/cdc6 genes would strongly indicate that they are involved in some essential and thus far uniquely haloarchaeal role in DNA replication , so it would appear to be a functional DNA polymerase. A PolB2 homolog is also found in the genome of the distantly related halophile, H. marismortui, on extrachromosomal replicon pNG600. Of interest, as well, is the fact that in both Halobacterium sp. NRC-1 and H. marismortui, the polB2 gene is divergently oriented with respect to an Orc5 clade member gene [polB2 and an Orc5 clade member gene in these two haloarchaea is currently unknown, but in Halobacterium sp. NRC-1 both orc4 and polB2 are non-essential genes. While much in vitro work has been directed at determining the properties of archaeal DNA polymerases, especially since the discovery of a novel DNA polymerase family in euryarchaea [in vivo analysis had previously been performed to determine whether these DNA polymerase family members were essential, consistent with a requirement for DNA replication.The non-essentiality of ber gene . The funyarchaea , no in vmcm, pri1, pri2, pcn, and rad2 are essential for normal growth of Halobacterium sp. NRC-1. The in vitro biochemical work done on these various gene products had indicated that it was likely that they would function in an analogous manner to their eukaryotic homologs. Though no biochemical work has been done on the haloarchaeal MCM, our genetic analysis is consistent with its predicted function as a replicative helicase. With Pri1/Pri2 (homologs of the eukaryotic p48 and p58 proteins), the archaeal complex likely acts as the DNA-dependent RNA primase for DNA replication. The finding of the essential nature of the pri1 and pri2 genes in Halobacterium sp. NRC-1 is consistent with their role as a replicative primase. In contrast, the function of the bacterial-type primase, DnaG, coded by most archaeal genomes, including Halobacterium sp. NRC-1 is unknown, although in S. solfataricus it has been reported to be associated with the archaeal exosome [pcn, similar to eukaryotes, two crenarchaea, S. solfataricus and Aeropyrum pernix, are exceptions, with three pcn genes each, reminiscent of the eukaryotic 9-1-1 complex [Halobacterium sp. NRC-1, we have found that the single pcn gene is essential, consistent with PCNA acting as the homotrimeric DNA polymerase sliding clamp. Rad2 family flap endonucleases are important in both the processes of DNA replication, (during Okazaki fragment maturation), and repair (in nucleotide excision repair). Organisms can possess multiple homologs, although just a single flap endonuclease gene was detected in the genome of Halobacterium sp. NRC-1 [rad27, the rad2/FEN1 homolog in S. cerevisiae, is not essential unless a recombination gene (e.g. rad51 or exo1) is also deleted [rad2 gene is essential for viability of Halobacterium sp. NRC-1. This finding is consistent with flap endonucleases being required for DNA replication via their role in Okazaki fragment maturation in this archaeon.For the other five accessory genes examined here, whose products comprise four protein complexes, the results were as expected:  exosome . For PCN complex ,41. In Hp. NRC-1 . Genetic deleted . In the orc gene family representing a unique aspect of DNA replication in haloarchaea. In our emerging model, archaeal chromosomal DNA replication origins are comprised of a large inverted repeat flanking an AT rich DNA sequence proximal to the gene encoding an origin binding protein, an orc/cdc6 gene that is an orc7 ortholog. These large inverted repeats likely serve as binding sequences for the origin binding protein, probably Orc7, although a multimeric ORC complex or other Orc proteins, especially the orc2 and orc10 gene products cannot be ruled out. Binding of origin recognition protein(s) would lead to local DNA helix destabilization of the intervening AT rich region allowing for recruitment of the essential mcm gene-coded replicative helicase complex, potentially by the orc6 gene product, followed by association of other replisome components, such as the essential eukaryotic-type primase (pri1/pri2 gene products). Once the primase lays down an RNA primer at the origin, the essential pcn gene product may be loaded onto the primed template and essential B (polB1) and D (polD1/polD2) family replicative DNA polymerases. The rad2 gene product encodes the likely flap endonuclease which helps to mature Okazaki fragments. During the replication process, the polB1 gene product coding the B family DNA polymerase may act as the leading strand DNA polymerase and the polD1 and polD2 gene products coding the D family DNA polymerase may act as the lagging strand DNA polymerase for processive and faithful duplication of the genome.The results obtained in this and a previous investigation  are releHalobacterium sp. NRC-1, we have established a foundation on which to explore further the in vivo roles of these DNA replication genes. With facile genetics, complete genome sequence, and established post-genomic methodologies, Halobacterium sp. NRC-1 provides an excellent model system to further study the characteristics of archaeal DNA replication. In addition, the gene knockout and complementation methodology used for studying DNA replication in Halobacterium sp. NRC-1 may be applied to the investigation of many other aspects of archaeal biology [By utilizing a well developed in-frame gene knockout system in  biology .Restriction enzymes, calf intestinal phosphatase, T4 DNA polymerase, T4 polynucleotide kinase, and T4 DNA ligase were purchased from New England Biolabs, Beverly, MA. XL DNA Polymerase was purchased from Applied Biosystems, Branchburg, NJ. Oligonucleotides were purchased from Sigma-Genosys, The Woodlands, TX. Gel extraction kits and plasmid purification kits were purchased from Machery-Nagel, Easton, Pa. Uracil dropout formula and Nitrogen base were purchased from Sigma-Aldrich, St. Louis, MO.Escherichia coli DH5α was grown in Luria-Bertani medium supplemented with 100 μg of ampicillin/mL at 37°C. Halobacterium sp. NRC-1 Δura3 was cultured in CM+ medium containing 4.3 M NaCl, trace metals, and 250 μg/mL of 5-Foa at 42°C [Halobacterium sp. NRC-1 Δura3 containing integrated suicide plasmids were grown in HURA+ medium at 42°C [Halobacterium sp. NRC-1 genomic DNA  and then transformed into the Halobacterium sp. NRC-1Δura3 strain harboring an integrated copy of the original suicide vector. Subsequent selection for suicide plasmid excision (Foar) and replicating plasmid maintenance (Mevr), by plating on CM+ solid media containing 20 μg/mL of mevinolin and 250 μg/mL of 5-Foa, results in selection of chromosomal knockouts, even if the targeted gene is essential, due to complementation in trans by the plasmid borne wild-type allele of the gene.To further address the question of essential genes we developed a complementation strategy . In this0=geneX is non-essential with the probability of identifying the wild type allele PWT = 0.75, the probability of identifying 40 out of 40 wild-type alleles is P = 10-5, providing strong evidence to reject H0.Taking the null hypothesis HHomo sapiens, Drosophila melanogaster, and Saccharomyces cerevisiae were downloaded from KOG1514 and KOG2227 at NCBI. Protein sequences for Halobacterium sp. NRC-1 and Haloarcula marismortui were generated locally. Sequences for Natronomonas pharaonis and Arabidopsis thaliana were downloaded from NCBI. Protein sequences were aligned using CLUSTAL_X1.83 and alignments manually inspected. Quartet puzzling maximum likelihood phylogenic analysis was performed with TREEPUZZLE5.2 using the JTT amino acid substitution matrix.Protein sequences for The authors declare that they have no competing interests.BRB performed research, with assistance from PD, and drafted the manuscript. SD supervised the research, including design, data analysis, and finalized the manuscript, with assistance from PD.


Porcine congenital splayleg (PCS) is the most important congenital condition of piglets, associated with lameness and immobility, of unknown aetiology and pathogenesis, hence the need to better understand the condition by defining, in the first instance, its histopathology and molecular pathology.Semitendinosus, longissimus dorsi, and gastrocnemius muscles were removed from 4 sets of 2-day-old splayleg piglets, each with a corresponding normal litter mate. Based on immunohistochemistry and histological image analysis, PCS piglets showed significantly smaller fibre size without any accompanying sign of inflammation. Although there was no dramatic change in fibre type composition in affected muscles, several structural myosin heavy chain genes were significantly down-regulated. MAFbx, a major atrophy marker, was highly up-regulated in nearly all PCS muscles, in comparison with controls from normal litter mates. In contrast, P311, a novel 8 kDa protein, was relatively down-regulated in all the PCS muscles. To investigate a functional role of P311 in skeletal muscle, its full-length cDNA was over-expressed in murine C2C12 muscle cells, which resulted in enhanced cell proliferation with reduced myotube formation. Hence, reduced P311 expression in PCS piglets might contribute to atrophy through reduced muscle cell proliferation. P311, predictably, was down-regulated by the over-expression of calcineurin, a key signalling factor of muscle differentiation.MAFbx and P311 is of potential in the diagnosis of subclinical PCS.We demonstrated that PCS is a condition characterised by extensive fibre atrophy and raised fibre density, and propose that the combined differential expression of  Porcine congenital splayleg (PCS), also known as straddlers, and myofibrillar hypoplasia, is a clinical condition of newborn piglets, characterised by muscle weakness, resulting in the inability to properly stand and walk, with affected limbs extended sideways or forwards . It is aPCS can be found at or a few hours after birth in up to 2 to 3 piglets in an affected litter. Arising from long periods of recumbency, abrasions and ulceration may develop, which could predispose the affected individual to secondary arthritis, pododermatitis, and osteomyelitis of the digits. Mortality rate can reach 50% due to starvation or crushing by the sow. Affected piglets can recover after a week if supportive treatment is provided. PCS is prevalent in Landrace, Large White breed, and in other heavily muscular breeds -6. Male Fusarium (F-2) toxin  contaminated grain [PCS appears to be a multifactorial condition of unknown aetiology. The predisposing factors are thought to include genetics and environmental factors, such like nutrition, management, pharmacological administration, and mycotoxins. Pregnant sows treated with glucocorticoids , and exped grain  showed hed grain . The roled grain ,12. Admied grain , and of ed grain  have beesemitendinosus (ST), longissimus dorsi (LD) and gastrocnemius (G) muscles. Muscle Atrophy F-box (MAFbx), a marker of muscle atrophy, was more highly expressed in all PCS muscles. Conversely, expression of P311, a novel 8 kDa protein with a conserved PEST domain, was down-regulated in all PCS muscles tested.A range of pathological lesions has been described for PCS, the most common feature being the presence of myofibrillar hypoplasia, often interpreted as an immaturity of the muscle ,10. MyofMorphological and fibre type analyses were conducted on 4 sets of muscles from 4 separate litters of pigs ; each set comprised one 2-day-old affected PCS piglet and a normal litter mate. Histological examination of all PCS muscles showed no evidence of significant inflammatory change (data not shown). The average fibre cross-sectional area of affected PCS piglets was consistently smaller than their normal counterparts in all 3 muscles Fig. . ConsequMYHembryonic, MYHperinatal, MYHslow, MYH2a, MYH2x, MYH2b and α-actin. PCS piglets showed significantly lower expression of MYHslow in LD and G muscles .To further assess the phenotypic differences between muscles of normal and PCS piglets, TaqMan quantitative real-time PCR was performed on the muscles from the 4 sets of litter mates to determine the relative mRNA expression levels of les Fig. . Expressets Fig. . No signMAFbx expression with muscle atrophy, we determined the relative expression of porcine MAFbx in normal and PCS muscles  assays were performed on P311-adenovirus infected and stably transfected C2C12 cells. Both constructs promoted proliferation compared with control GFP cells  of development  consistently showed fibre atrophy with concomitant increase in fibre density Fig. . It appeene Fig. . Hence PMAFbx mRNA in all 4 atrophic PCS piglets from 4 litters   Fig. . The eleP311 expression throughout gestation, which was maintained in post-natal normal muscles  was down-regulated in late differentiation of P311-over-expressed C2C12 cells , for MYH slow used at 1:50 dilution  and for MYH fast used at 1:50 dilution , as previously described . For eacP311 cDNA and a constitutively active murine calcineurin A (Cn)  were cloned into an adenovirus vector, using the Adeno-X Expression System 2 (BD Biosciences). The starting plasmid vector pDNR-CMV for the insertion of P311 and Cn housed an expression cassette that was derived from the plasmid pAAV-IRES-hrGFP (Stratagene) which comprised a multiple cloning site for the creation of a 3'-end FLAG fusion gene and a GFP reporter gene, with an internal ribosomal entry site (IRES) between the two. For time course studies, infection with P311 and Cn-adenovirus constructs were used at multiplicity of infection (MOI) of 5. For stable transfection, full length P311 cDNA was cloned into a neomycin resistant pBK-CMV expression plasmid vector (Stratagene), where transcription was driven by a cytomegalovirus immediate early promoter.Full length porcine P311 plasmid, by the use of lipofectamine and Opti-MEM, according to manufacturer's instructions (Invitrogen). Next day, the transfection medium was replaced with PM for 1 day, followed by G418 (Invitrogen) selection at 2000 μg/ml over several days until a day after all cells in the control untransfected flask had been eliminated by G418. All subsequent experiments on stably transfected cells were conducted in the absence of G418.C2C12 cells (CRL-1772) were grown in proliferation medium (PM) . At 80% confluence, PM was replaced by differentiation medium (DM) (4% horse serum in DMEM with penicillin and streptomycin). For stable transfections, C2C12 cells at 35% confluence in T25 culture flasks were transfected with 6.0 μg P311 or GFP control adenovirus, were grown for 3 days in PM, followed by 8 days in DM. To improve P311 stability, o-phenanthorline and lactacystin were added to the DM, at final concentrations of 1.26 mM and 10 mM respectively, 2 hour prior to fixation. Cells were fixed in 4% paraformaldehyde and incubated with the primary anti-FLAG M2 monoclonal antibody (Sigma), at a dilution of 1/2000, overnight at 4°C. A goat anti-mouse-TRITC (Southern Biotech) at 1/200 was used as the secondary antibody in a 45 minute incubation at 37°C. It was mounted using Vectashield (Vector) hard set mounting medium with DAPI. Images were captured by a CCD camera mounted on an inverted Olympus fluorescence microscope.C2C12 cells, infected with P311-adenovirus and stably transfected with P311 plasmid construct, were performed as previously described [BrdU assays on C2C12 cells, infected with For fusion index assays, after 9 days in DM, cells were fixed and incubated with a primary antibody  at 4°C overnight. Secondary FITC conjugated sheep anti-mouse antibody (Sigma) was used at 1/200 dilution. For each sample, 5 fields were randomly chosen for counting. Fusion index was defined as the number of DAPI stained nuclei within myotubes in a given field divided by the total number of DAPI stained nuclei in the same field. Three independent experiments were conducted.Cell cytotoxicity assay, a resazurin-based reduction assay that measured the metabolic capacity of cells, was performed according to manufactururer's protocol, normalised to unit weight of protein .P311 adenovirus, C2C12 cells were infected in PM for 3 days (3I), followed by 3 days in DM (3I 3D) or by 6 days in DM (3I 6D). TaqMan quantitative real-time RT-PCR (Applied Biosystems) was performed on a number porcine and murine genes: MYHembryonic, MYHperinatal, MYHslow, MYH2a, MYH2x, MYH2b, β-actin, α -actin, myf-5, MAFbx and P311. Sequence details of primers and TaqMan probes of porcine genes were previously given [Total RNA was isolated from porcine muscles and C2C12 cells using the RNeasy Mini kit (Qiagen), quantitated in an Agilent 2100 bioanalyzer and converted into cDNA with random primers using a Reverse Transcription System (Promega). Two μg of total RNA were used for each cDNA conversion. Four sets of cDNA samples from PCS and normal piglets were prepared (litters 1 to litters 4). With pre-natal muscles, LD was pooled from 3 Pietrain-based pig foetuses from gestation stage of 35, 49, 63, 77 and 91 days . Additioly given . All othP311 and GFP control) as fixed effects with experiments and replicate as random effects. The standard errors of the difference between sample means were calculated using the error mean square from the ANOVA. Differences between pair-wise combinations of the least square means were tested for significance (p < 0.05). Unless specifically stated, quantitative real-time RT-PCR data were expressed as mean ± standard deviation from triplicate samples within the same experiment.SAS software  was used. Fibre size, fibre density, real-time PCR data for PCS Fig.  to 5 werPTO performed most of the experimental work and wrote the draft manuscript. NDC helped with the real time RT-PCR experiments and provided intellectual input to the work. JE helped with the cell culture procedures and project planning. KCC initiated and coordinated the study, and critically revised the manuscript. All authors read and approved the final manuscript.


A variety of diseases are caused by chromosomal abnormalities such as aneuploidies , microdeletions, microduplications, and uniparental disomy. High density single nucleotide polymorphism (SNP) microarrays provide information on chromosomal copy number changes, as well as genotype (heterozygosity and homozygosity). SNP array studies generate multiple types of data for each SNP site, some with more than 100,000 SNPs represented on each array. The identification of different classes of anomalies within SNP data has been challenging.We have developed SNPscan, a web-accessible tool to analyze and visualize high density SNP data. It enables researchers (1) to visually and quantitatively assess the quality of user-generated SNP data relative to a benchmark data set derived from a control population, (2) to display SNP intensity and allelic call data in order to detect chromosomal copy number anomalies (duplications and deletions), (3) to display uniparental isodisomy based on loss of heterozygosity (LOH) across genomic regions, (4) to compare paired samples , and (5) to generate a file type for viewing SNP data in the University of California, Santa Cruz (UCSC) Human Genome Browser. SNPscan accepts data exported from Affymetrix Copy Number Analysis Tool as its input. We validated SNPscan using data generated from patients with known deletions, duplications, and uniparental disomy. We also inspected previously generated SNP data from 90 apparently normal individuals from the Centre d'Étude du Polymorphisme Humain (CEPH) collection, and identified three cases of uniparental isodisomy, four females having an apparently mosaic X chromosome, two mislabelled SNP data sets, and one microdeletion on chromosome 2 with mosaicism from an apparently normal female. These previously unrecognized abnormalities were all detected using SNPscan. The microdeletion was independently confirmed by fluorescence in situ hybridization, and a region of homozygosity in a UPD case was confirmed by sequencing of genomic DNA..SNPscan is useful to identify chromosomal abnormalities based on SNP intensity  and heterozygosity data (including regions of LOH and some cases of UPD). The program and source code are available at the SNPscan website  A single nucleotide polymorphism (SNP) is a variation in a DNA sequence that occurs in an appreciable portion of the population. High density SNP microarrays provide information on the copy number of individual SNPs, based on measurement of the intensity of hybridized genomic DNA fragments to the microarray. These microarrays also provide genotype information on the state of heterozygosity or homozygosity at each SNP position. SNP arrays facilitate studies of chromosomal copy number change, large-scale linkage analysis, and whole genome association studies.A variety of platforms for SNP-based genotyping are commercially available . Most rep) values across one chromosome, as well as genotype scores and p values as regions of LOH. Several recent reports describe the visualization of SNP array data using this tool or similar plots of intensity ratios and/or log10 p values (y-axis) versus chromosomal position (x-axis) . BAC clones were obtained from Roswell Park Cancer Institute . 1 μg of BAC DNA was labelled by nick translation with a kit  including incorporation of Spectrum Orange dUTP. The BAC DNA was separated from the reaction mixture, co-precipitated with highly repetitive DNA, denatured, pre-annealed, then hybridized in situ to metaphase cells.Lymphoblastoid cells were obtained from the Kennedy Krieger Institute  or the Coriell Cell Repository . Cells were grown in RPMI medium, treated with colcemid (a mitotic spindle inhibitor), and metaphase spreads were incubated with chromosome-specific telomere probes  and labelled BAC clones. Cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI) to visualize nuclei. Fluorescent signals were visualized using a Zeiss AxioSkop equipped with epifluorescence, appropriate filter sets, and a computer-assisted FISH capture system and software for producing high resolution images.Genomic DNA samples from five apparently normal cases  and one case suspected to have UPD (NA12874) were purchased from Coriell Cell Repositories. Genomic DNA (100 ng) was PCR amplified  using standard conditions including 30 to 35 cycles using denaturation , annealing , and extension . The eight sets of oligonucleotide primers were as follows : 1p1, 5'-CTCTGTGCAAGGTGTGAGGA-3' and 5'-ATGGCCCAAGGTCACATAAA-3'; 1p2, 5'-TTGAAACACTTCACAAAAGATGTG-3' and 5'-GTGCTCCTGGGAGAACTCAG-3'; 1p3, 5'-CCCAGTGCCATTATTACACTCA-3' and 5'-ATAGGGGCTCTGCACCTTTC-3'; 1p4, 5'-TGTATTGTTGGATTTGGTTTGC-3' and 5'-AGTCCCAGATGGGTTCACTG-3'; 1q1, 5'-TGTCTTCCAAAACGCACTTG-3' and 5'-AGCCCATCACGTCATATTCC-3'; 1q2, 5'-GGGGGTATCAGAGGCAATTT-3' and 5'-AGTGAAGAGCTCCTGCCTTG-3'; 1q3, 5'-CATCCGTGAGAATGGAAACC-3' and 5'-ATGAGGTCCATGCAGGAAAA-3'; 1q4, 5'-CAGGCAGGCTTTGACTCTTC-3' and 5'-CCCTAGAAACAGCTCCCAAA-3'. PCR products were electrophoresed on a 1.5% agarose gel, purified , and sequenced . Eight sets of primers were designed to span 2 or 3 SNPs  in <500 base pair regions of chromosome 1p (where UPD is not expected) and 1q.Project name: SNPscanProject home page: Operating system(s): Platform independent websiteProgramming languages: Perl, HTML, R v2.1Other requirements: Data input is from CNATv2.0 or v2.1 (Affymetrix).License: GNU GPLAny restrictions to use by non-academics: none. The source code is downloadable from the website.CEPH, Centre d'Étude du Polymorphisme Humain; CGH, comparative genomic hybridization; CNAT, Copy Number Analysis Tool; DAPI, 4',6-diamidino-2-phenylindole; FISH, fluorescence in situ hybridization; GDAS, GeneChip DNA Analysis Software; LOH, loss of heterozygosity; Mb, megabase; PDF, portable document format; PNG, portable network graphics; PS, PostScript; SNP, single nucleotide polymorphism; TIFF, tagged image file format; UCSC, University of California, Santa Cruz; UPD, uniparental disomy; WIG, wiggle track.JT designed the SNPscan algorithm, developed the KKISNP tools, and implemented the SNPscan website. He identified novel chromosomal anomalies in the apparently normal 90 individuals discussed in this paper via SNPscan and performed data analysis in R. YY performed the FISH studies and sequencing of genomic DNA, and contributed to writing the manuscript. GHT made intellectual contributions to the design and interpretation of the experiments and contributed to the writing of the manuscript. IR contributed to the data analysis and software development. JP conceived of the study and contributed to data analysis, development of the SNPscan website, and writing the manuscript. All authors read and approved the final manuscript.


Many receptors on natural killer (NK) cells recognize major histocompatibility complex class I molecules in order to monitor unhealthy tissues, such as cells infected with viruses, and some tumors. Genes encoding families of NK receptors and related sequences are organized into two main clusters in humans: the natural killer complex on Chromosome 12p13.1, which encodes C-type lectin molecules, and the leukocyte receptor complex on Chromosome 19q13.4, which encodes immunoglobulin superfamily molecules. The composition of these gene clusters differs markedly between closely related species, providing evidence for rapid, lineage-specific expansions or contractions of sets of loci. The choice of NK receptor genes is polarized in the two species most studied, mouse and human. In mouse, the C-type lectin-related Ly49 gene family predominates. Conversely, the single Ly49 sequence is a pseudogene in humans, and the immunoglobulin superfamily KIR gene family is extensive. These different gene sets encode proteins that are comparable in function and genetic diversity, even though they have undergone species-specific expansions. Understanding the biological significance of this curious situation may be aided by studying which NK receptor genes are used in other vertebrates, especially in relation to species-specific differences in genes for major histocompatibility complex class I molecules. Natural killer (NK) cells destroy cells infected with certain viruses and other intracellular pathogens. They also influence immune responses through the release of cytokines. A key aspect of recognition of appropriate target cells is the ubiquitously expressed major histocompatibility complex (MHC) class I molecule, a ligand for which NK cells generally have multiple receptors . All livNK cell receptors come in two forms: inhibitory and activating. Inhibitory receptors regulate NK actions by interrupting intracellular activation signals when MHC class I molecules are correctly expressed . ActivatGenes that encode NK receptors are arranged in two main clusters: the leukocyte receptor complex (LRC) and the natural killer complex (NKC). The LRC encodes members of the immunoglobulin superfamily (IgSF), while the NKC encodes type II transmembrane, C-type lectin-like proteins . The extThe main NK receptors for MHC class I molecules in humans belong to the IgSF and are encoded in the LRC on Chromosome 19q13.4 . Of the The key LRC genes directing NK cell recognition of MHC class I are the KIRs. The KIR gene cluster exhibits marked differences in gene content and allelic polymorphism between individual haplotypes ,17, withCentromeric to the KIR family is another extensive IgSF gene family, the LILRs. This family consists of two inverted, duplicated clusters of six and seven loci, respectively . The LILAdditional genes related to those in the LRC are dispersed further centromeric of the LILR and KIR gene families in the “extended LRC” region. This region includes the receptor and transporter of the Fc fragment of IgG (FCGRT), which interestingly may have a common origin with MHC class I molecules , the siaThe other major cluster of NK receptor genes, the human NKC, is located on Chromosome 12p13.1  and encoKLRA1 , in humans, as opposed to the multiple homologous genes encoding MHC class I ligands in rodents , and NKG2H) receptors, some of which, such as NKG2B, NKG2E, and NKG2H, are products of alternative splicing [(KLRC4), that has a charged residue in the transmembrane region, an ITIM-like domain, and no C-type lectin–like domain [Members of the NKG2 g motifs . Some KLle HLA-E . This fasplicing . When bosplicing . The NKGe domain . The fun(KLRK1) shows limited sequence identity to other NKG2 molecules and is expressed on NK cells, T cells, and macrophages as a homodimer [NKG2D omodimer . For actomodimer ,34. Humaomodimer .KLRG1, also called mast cell function–associated antigen [KLRF1, which is present in humans but not mice , stimula antigen , is more antigen . Other N antigen , lectin- antigen , and CD6 antigen . Genes t antigen , oxidize antigen , CLECSF1 antigen . are best reciprocal human orthologs, cover 106 kilobases [Chlorocebus sabaeus, another Old World primate more closely related to rhesus monkeys than to the apes, possesses multiple KIR genes, namely KIR3DL, KIR2DL4, KIR2DL5, and KIR3DH, a KIR form found in rhesus and African green monkeys but not apes, and a hybrid of KIR2DL5 and KIR3DH [KIR2DL4 is the only orthologous KIR gene found in humans, chimpanzees, gorillas, rhesus macaques, and African green monkeys. Interestingly, not all gorillas appear to have KIR2DL4 [KIR genes have diverged dramatically between different primate species, consistent with rapid, species-specific expansion of the gene family . In chimilobases ,42. In tilobases . Orangutilobases . The rheilobases ,46. The d KIR3DH . KIR2DL4 KIR2DL4 , and it  KIR2DL4 . Table 1These differences in KIR gene presence accord with differences in MHC class I gene content among primate species . For exaFCAR and NCR1 [LILR genes are present in all primates studied, and although the gene content of this family shows greater differences between primate species than is observed throughout the genome, LILR genes are less variable in gene number between haplotypes than the KIRs. In chimpanzee, the nine LILR genes border the KIR sequences and are arranged in two duplicated clusters similar to human ,57. Fourand NCR1 .CD94 have been found in chimpanzees [KLRC2, for which there are two paralogs [NKG2A, NKG2B, NKG2C, NKG2D, and several splice variants [CD94, NKG2A, NKG2D, and NKG2F are present, along with a hybrid gene combining NKG2C and NKG2E midway between their expected syntenic positions [KLRA1 (Ly49L) [Ly49 sequence, which appears to be functional, has also been reported in baboons [There is limited information on NKC genes in primates. Members of the NKG2 family and mpanzees , includiparalogs . Rhesus variants . In the ositions . Orangut (Ly49L) , which i (Ly49L) ,59. A si baboons .GP6 [NCR1, RPS9, and LAIR1 [Pira1 to Pira11 and Pirb), which share sequence identity with the human LILRs, are found between Ncr1 and Rps9, [The mouse LRC is located on Chromosome 7 , althougGP6 , NCR1, Rnd LAIR1 . The Pirnd Rps9, . In addind Rps9, . Genes oPirb, Ncr1 [Fcar, which, while also present in humans, was lost in the mouse lineage [In the rat, the LRC is located on Chromosome 1 and includes an ortholog of murine rb, Ncr1 , and onerb, Ncr1 . The rat lineage .OCIL) and Nkrp1 (Klrb1) gene families [Orthologs of human NKC genes are reported in a syntenic region of murine Chromosome 6. The gene families are arranged in a similar order to the human NKC, except for expansions and interchanges of the C-type lectin related (Clr) (ortholog of human families . Interesfamilies . There afamilies . Membersfamilies .Nkrp1 [CD69 and KLRD1 (CD94); however, there are still many differences between human and rodent in the gene content of the NKC [In the rat, the NKC is located on Chromosome 4. In fact, the first NKC gene discovered in any species was rat Nkrp1 ,69. Huma the NKC  . While these genes encode an Ig domain, there is a cluster of C-type lectins encoded within this larger cluster ,104. Anonterpart . Other tchilotes  and rainchilotes , demonstAs explained above, features of NK receptor genes both within and between species are consistent with rapid evolutionary change. Inevitably, studies focus on human KIR and mouse Ly49 genes, but there are indications from the few studies of other vertebrates that variable C-type lectin and IgSF receptors for MHC class I molecules may co-exist in a species, such as horse , or may Emerging data are also consistent with rapid evolution of NKC-encoded genes. These include comparisons of KIR haplotypes in chimpanzee, rhesus macaque, and human, where, in addition to gene content differences, repeat elements in intronic regions suggest rapid evolution . AnotherPerhaps because of this “response mode” fluctuation in activating versions of inhibitory receptors, it has proved difficult to identify their role and indeed their ligands. An interesting exception is mouse Ly49h, which recognizes mCMV-infected cells by a direct interaction with the m157 mCMV gene product . So far,Why do some species expand KIR genes while others expand Ly49 genes? While these two gene families produce proteins of analogous function, they do not share a common ancestor. These expansions vary greatly even within lineages, such as between human and chimpanzee (diverged approximately 5 million years ago) or mouse and rat (diverged approximately 20 million years ago) . It is t+ αβ T cells and by distinct receptors present exclusively on each class [NK receptor gene clusters coevolve with MHC genes ,15, and ch class . The larch class ,119, altch class . Some MHch class , producech class . Ig domach class . IgC2 doch class , which, ch class , could hch class ,5 and foch class .Additional insights into the rapid coevolution of the NK receptor genes and genes encoding their MHC class I ligands can be gained by studying their levels of polymorphism. Publicly available polymorphism data, evaluated in a previous study from our laboratory , showed As more configurations of NK receptor genes are determined for different species, it will become possible to track the way groups of KIR and Ly49 loci have followed different species lineages. Were rodents the only species to lose KIR function? Are primates unique in losing Ly49? What are the intermediates on the way to “all KIR” and “all Ly49” models? And what selective advantages drove the specialization towards the KIR or Ly49 model in different species?


Rheumatoid arthritis (RA) is an autoimmune disease affecting approximately 1% of the population worldwide. Previously, we showed that human T-cell leukemia virus type I-transgenic mice and interleukin-1 receptor antagonist-knockout mice develop autoimmunity and joint-specific inflammation that resembles human RA. To identify genes involved in the pathogenesis of arthritis, we analyzed the gene expression profiles of these animal models by using high-density oligonucleotide arrays. We found 1,467 genes that were differentially expressed from the normal control mice by greater than threefold in one of these animal models. The gene expression profiles of the two models correlated well. We extracted 554 genes whose expression significantly changed in both models, assuming that pathogenically important genes at the effector phase would change in both models. Then, each of these commonly changed genes was mapped into the whole genome in a scale of the 1-megabase pairs. We found that the transcriptome map of these genes did not distribute evenly on the chromosome but formed clusters. These identified gene clusters include the major histocompatibility complex class I and class II genes, complement genes, and chemokine genes, which are well known to be involved in the pathogenesis of RA at the effector phase. The activation of these gene clusters suggests that antigen presentation and lymphocyte chemotaxisis are important for the development of arthritis. Moreover, by searching for such clusters, we could detect genes with marginal expression changes. These gene clusters include schlafen and membrane-spanning four-domains subfamily A genes whose function in arthritis has not yet been determined. Thus, by combining two etiologically different RA models, we succeeded in efficiently extracting genes functioning in the development of arthritis at the effector phase. Furthermore, we demonstrated that identification of gene clusters by transcriptome mapping is a useful way to find potentially pathogenic genes among genes whose expression change is only marginal. Rheumatoid arthritis (RA) is a systemic, chronic inflammatory disease primarily affecting the joints. The synovial inflammation leads to cartilage destruction, bone erosion, joint deformity, and loss of joint function . This dipX region that activates multiple cellular genes, including those for cytokines, cytokine receptors, and immediate early transcriptional factors, via activation of enhancers such as cAMP-responsive enhancer, nuclear factor kappa B-dependent enhancers, or serum-responsive elements /IL-3R), and Csf3r (G-CSFR/CD114), increased, expression of their ligands could not be detected by our analysis. TNF and TNF-R family genes, such as Tnfsf3 (LTβ), Tnfsf11 (RANKL/ODF), Tnfsf31 (Pglyrp), Tnfrsf5 (CD40), and Tnfrsf21 (DR6), were significantly elevated. The expression of genes encoding growth factor and growth factor-related proteins, including Mdk, Il18bp, Grn, Tnfaip6, C1qtnf6, Fgf10, Igf1, Igfbp4, Igfbp7, Pdgfrl, and Bmp1, were also enhanced. In addition to these chemokine/chemokine receptor and cytokine/cytokine receptor genes, several genes were identified that were upregulated more than 10-fold in comparison with WT mice. These genes include Igh-4 (Serum IgG1) and Mmp3 (Stromelysin-1), which are transcripts known to be increased in RA [Glipr2, Kcnj15, and Col4a2 were also elevated, no augmentation of these genes has been previously reported in patients with RA. Moreover, major histocompatibility complex (MHC) class I genes  and MHC class II genes  were also significantly detected in this data set. Using northern blot hybridization techniques, we examined the expression of some of these genes, including key cytokines and cytokine receptors , chemokines and their receptors , class I and class II MHC genes, and several selected genes, and confirmed the augmented expression of these genes , complement cluster  and MHC class I cluster . The expression of additional immune-related genes, including Psmb9 (proteasome) and Ltb (lymphotoxin β), was also increased significantly. Another peak was on chromosome 11, 82-Mb locus (#5) and neighboring 81-Mb (#4) and 83-Mb (#6) locus, which corresponds to the Slfn and Ccl gene cluster. An expanded view of this region, shown in Figure Ccl2, Ccl6, Ccl7, Ccl8, Ccl9, and Ccl5) and schlafen genes , are shown. Moreover, chromosome 6, 68- to 70-Mb locus (#7–9) and chromosome 12, 112- and 113-Mb locus  formed a cluster of immunoglobulin genes, kappa chain and heavy chain, respectively. High peaks were also detected at chromosome 6, 124-Mb locus (#12), which includes several genes of complement receptor and C-type lectin superfamily, and chromosome 15, 79-Mb locus (#13), which includes colony-stimulating factor 2 receptor beta (Csf2rb) genes, and chromosome 19, 11-Mb locus (#14), which includes membrane-spanning four-domain, subfamily A (Ms4a) genes. An expanded view of these loci, shown in Figure C1r, C3ar1) and C-type lectin superfamily members . (Clecsf8 mapped into the 125-Mb locus but was next to Clecsf6.) Chromosome 15, 79-Mb locus (#13) includes two Csf2rb genes (Csf2rb1 and Csf2rb2). Chromosome 19, 11-Mb locus (#14) corresponds to the Ms4a gene cluster . Although no gene family is detected in chromosome 2, 165-Mb locus (#15) and chromosome 19, 5-Mb locus (#18), chromosome 3, 146-Mb locus (#16) and chromosome 17, 17-Mb locus (#17) included gene clusters of guanylate nucleotide binding protein and formyl peptide receptor, respectively. Additional clusters of arthritis-related genes were identified, including a cluster of selectin genes on chromosome 1, 167-Mb locus (#20), Fc receptor cluster on chromosome 1, 174- and 175-Mb locus (#21), a paired-Ig-like receptor cluster on chromosome 7, 3-Mb locus (#26), serum amyloid A cluster on chromosome 7, 39-Mb locus, and the gene cluster of CC chemokine receptor on chromosome 9, 126-Mb region (#30).To identify individual gene clusters, significantly changed genes were mapped into the whole genome in 1-Mb scale windows. Figure b locus # and neigd 83-Mb # locus, wtax, whereas the other is caused by excessive IL-1 signaling. We extracted common genes that were involved in the pathogenesis of both models irrespective of etiology. We expected to identify genes involved in the effector phase of the disease, given that the molecular mechanisms of the initial phase are likely to be different between the two models. With RNA samples derived from the arthritic joints of either animal model, 1,467 clones on the array (approximately 4%) changed at least threefold. The SAM method demonstrated that, of these genes, 554 independent genes changed significantly in both models. These results suggest that a large proportion of the genes functioned in the pathogenesis of arthritis in both models. These common genes may function during the effector phase, whereas those specific to the individual models may function during the initiation phase in a manner dependent on the disease etiology. We analyzed those genes whose changes in expression levels were common to both models.To identify genes involved in the pathogenesis of arthritis, we used two mouse models of RA. We compared the gene expression profiles between arthritic and normal joints using these models and high-density oligonucleotide arrays, in which approximately 36,000 genes and ESTs were analyzed. We analyzed whole synovial tissues instead of using specific cell types because we are interested in not only expression level changes of a gene in a cell but also cell population changes in the synovial tissues . In thisSaa3, which encodes serum amyloid A3, were activated in these models. This gene was upregulated to the greatest extent in arthritic mice in comparison with normal mice. IL-1β induces Saa3 expression; SAA3, in conjunction with SAA1/SAA2, the expressions of which were also elevated in our RA animal models, induce the transcription of matrix metalloproteinases (MMPs) [Mmp-3 and Mmp-9, two such MMPs, were also upregulated in these models. We also observed the upregulation of chemokines, such as Cxcl5 and Cxcl1, which recruit neutrophils to inflammatory sites through interactions with their receptor, Cxcr2, which was also elevated and confirmed by northern blot analysis. Cxcr4 may be involved in the chemotaxis of naïve and memory T cells. Ccr1 and Ccr2 are expressed on memory T cells, whereas Ccr5 is expressed on Th1 cells. Cxcl12, the ligand for Cxcr4, is produced in the synovial tissues of patients with RA [Ccr6, Ccr1,Ccr2, and Ccr5 are specifically expressed by immature dendritic cells (DCs). Cxcl16, a T-cell chemoattractant, is expressed by both DCs and macrophages. Ccl9, Ccl6, and Cxcl13 are produced by macrophages, whereas Cxcl14 is produced by fibroblasts. In addition, proinflammatory cytokines and their cognate receptors, such as IL-1β, IL-1RI, TNF-α R, IL-6Rα, IL-2Rγ, and IL-17R, were also significantly elevated. Thus, the augmented expression of chemokines, cytokines, and their receptors indicates the importance of these molecules in the pathogenesis of arthritis. It should be noted, however, that the augmentation of the expression of these genes does not necessarily mean that they are actually activated in synovial tissues. The infiltrated cells to the inflammatory sites may also contribute to the expression pattern. Elevated expression of serum amyloid proteins, metalloproteinases, chemokines, and cytokines is frequently seen in the joints of patients with RA [We found that several interesting genes, including s (MMPs) . Mmp-3 a with RA . Ccr6, C with RA ,29, indiThe gene expression profiles of the well-known collagen-induced arthritis (CIA) model  and protH-2 gene cluster. Other significant peaks corresponded to chromosome 11, 81- to 83-Mb locus (#4–6), which includes members of the Ccl and Slfn families. Immunoglobulin kappa chain cluster in chromosome 6, 68- to 70-Mb locus, and heavy chain cluster in chromosome 12, 112- and 113-Mb locus were also clearly detected. Moreover, chromosome 6, 124-Mb locus, chromosome 15, 79-Mb locus, and chromosome 19, 11-Mb locus were included in those attractive gene clusters. The contribution of individual genes in these clusters was relatively small; the significance of a number of genes was recognized only after summation of genes in a specific region. These gene clusters, however, clearly contained a number of genes important in the development of arthritis. For more than two decades, the MHC gene cluster has been known to affect susceptibility to a variety of autoimmune diseases [H2-Bf gene within this cluster, was also augmented. Factor B is an essential component of the alternative pathway of the complement system. This pathway is also critical in K/BxN mice, another animal model for RA [We next mapped these arthritis-related genes into chromosomes. Working under the assumption that functionally related genes form clusters, we attempted to detect the activation of genes as a cluster, despite the fact that small individual changes were not prominent enough to be detected in our initial analysis. The most significant peak was detected at chromosome 17, 33- to 35-Mb locus (#1–3), which corresponds to the diseases ,33. Thisb locus #–6, whichl for RA . AlthougA similar study was reported using CIA and PGIA models, identifying gene clusters in chromosomes 2, 3, 11, and 17 . Their mCcl and Slfn genes. CCL2, CCL7, and CCL8, members of the chemokine family, recruit monocytes to sites of injury and infection. CCL2 influences innate immunity through this effect on monocytes and modulates adaptive immunity via control of Th2 polarization. An antagonist of CCL2 suppresses arthritis in the MRL-lpr mouse model [+ DCs and promotes osteoclast differentiation and survival [Ccl gene cluster. This region coincides with a number of autoimmune susceptibility loci, including the Idd4, Eae7, and Orch3 loci [The gene density peak on chromosome 11, 81- to 83-Mb locus (#4–6) includes the se model . CCL5, ase model . CCL6 wase model . CCL9 resurvival ,42. Thussurvival . These pch3 loci . The synch3 loci . TherefoCsf2rb1, and Csf2rb2. It is known that GM-CSF plays an important role in the effector phase of arthritis [Ms4a family molecules, CD20 (Ms4a1), and high-affinity IgE receptor β chain  tended to be upregulated in these animals, but these genes were not significantly altered in this study. Although the upregulation of Ms4a7, Ms4a4d, Ms4a6d, and Ms4a6c expression in chromosome 19, 11-Mb locus (#14) was also observed in these models of RA, the roles of these molecules in the development of arthritis are not yet known [The chromosome 6, 124-Mb (#12) locus is one of the non-MHC susceptibility loci linked to complex autoimmune diseases. The mouse 6 F2 cytoband, the syntenic rat 4q42 and human 12p13 regions, have been implicated in several inflammatory diseases, including arthritis -48, systrthritis . Ms4a faet known .MMP-8 and MMP-13 genes from significantly activated genes in this analysis. Unfortunately, MMP-1a and MMP-1b were not mounted on the GeneChip we used (Murine Genome U74v2 Set). Nonetheless, the present data are important for understanding the transcriptome change as a whole, reflecting the gene expression levels not only in a single cell but also in the cell population in the joints during arthritis.We succeeded in identifying several arthritis-related genes by transcriptome mapping, but we might have failed to detect some potentially important genes. Because we analyzed mRNA preparations from whole joints, but not from a single species of cell, we could not detect the change of gene expression in a single species of cell when the content of this single species of cell was low in the whole joint. Furthermore, we could not detect the MMP gene cluster consisting of MMP-1a, MMP-1b, MMP-3, and MMP-13 on chromosome 9, which were suggested to be involved in the pathogenesis . In thisWe have performed a comprehensive transcriptome analysis of two etiologically different RA models by using microarrays and identified many genes that were commonly activated in both models. Because the initial mechanism of the pathogenesis should be different between two models, these genes are likely to function during the effector phase of the disease. Moreover, by mapping genes with marginal expression changes into chromosomes, we succeeded in detecting several gene clusters that may be involved in the pathogenesis of arthritis. A similar approach, using microarrays, will likely be useful in detecting genes related to multiple disease processes.CIA = collagen-induced arthritis; Csf2rb = colony-stimulating factor 2 receptor beta; DC = dendritic cell; EST = expressed sequence tag; GM-CSF = granulocyte-macrophage colony-stimulating factor; HTLV-I = human T-cell leukemia virus type I; Ig = immunoglobulin; IL-1Ra = interleukin-1 receptor antagonist; KO = knockout; KS = knockout severe; MHC = major histocompatibility complex; MMP = matrix metalloproteinase; Ms4a = membrane-spanning four-domains, subfamily A; PGIA = proteoglycan-induced arthritis; RA = rheumatoid arthritis; SAM = significance analysis of microarrays; SD = standard deviation; Tg = transgenic; TS = transgenic severe.The authors declare that they have no competing interests.NF, SS, and YI participated in the design of the study. SS prepared the RNA samples and carried out northern blot hybridization with NF. NF carried out the analysis of the microarray data, developed the transcriptome mapping analysis method, and drafted this manuscript as a part of his doctoral thesis. YI planned, designed, and organized the study and finalized the manuscript. All authors read and approved the final manuscript.An Excel file containing a table that lists the first 1,467 selected genes, the expression of which changed more than three times in one of the mouse models.Click here for fileAn Excel file containing a table that lists five hundred fifty-four non-redundant genes, the expression of which significantly changed in both models.Click here for file