IGF2 and CDKN1C, in the imprinted gene cluster on chromosome 11p15.5. Most cases are sporadic and result from epimutations at either of the two 11p15.5 imprinting centres (IC1 and IC2). However, rare familial cases may be associated with germline 11p15.5 deletions causing abnormal imprinting in cis. We report a family with BWS and an IC2 epimutation in which affected siblings had inherited different parental 11p15.5 alleles excluding an in cis mechanism. Using a positional-candidate gene approach, we found that the mother was homozygous for a frameshift mutation in exon 6 of NLRP2. While germline mutations in NLRP7 have previously been associated with familial hydatidiform mole, this is the first description of NLRP2 mutation in human disease and the first report of a trans mechanism for disordered imprinting in BWS. These observations are consistent with the hypothesis that NLRP2 has a previously unrecognised role in establishing or maintaining genomic imprinting in humans.Beckwith-Wiedemann syndrome (BWS) is a fetal overgrowth and human imprinting disorder resulting from the deregulation of a number of genes, including  NLRP2 and NLRP7 genes) in the mother. Homozygous inactivating mutations in NLRP7 in women have been associated previously with abnormal imprinting and recurrent hydatidiform moles. We identified a homozygous frameshift mutation in NLRP2 in the mother of the two children with BWS implicating NLRP2 in the establishment and/or maintenance of genomic imprinting/methylation.A small set of genes (imprinted genes) are expressed in a “parent-of-origin” manner, a phenomenon known as genomic imprinting. Research in human disorders associated with aberrant genomic imprinting provided insights into the molecular mechanisms of genomic imprinting and the role of imprinted genes in normal growth and development. Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome associated with developmental abnormalities and a predisposition to embryonic tumours. BWS results from alterations in expression or function of imprinted genes in the imprinted gene cluster at chromosome 11p15. Although BWS may be caused by a variety of molecular mechanisms, to date, all the genetic and epigenetic defects associated with BWS have been limited to 11p15.5. We report a family with two children affected with BWS and an epigenetic defect at 11p15.5 in which the primary genetic defect mapped outside the imprinted gene cluster. Using autozygosity mapping, we found an extended homozygous region on chromosome 19q13.4 (containing  IGF2, KCNQ1OT1 (LIT1) genes and the maternally expressed H19 and CDKN1C (P57KIP2) genes. KCNQ1OT1 and H19 transcripts are not translated but the IGF2 gene product is an important prenatal growth factor and the CDKN1C protein is a candidate tumour suppressor that negatively regulates the cell cycle H19 that has an “insulator function” regulated by the zinc finger transcription factor, CTCF. The insulator is methylation sensitive, such that when CTCF binds to the unmethylated maternal allele, the IGF2 promoters do not have access to (are insulated from) enhancers downstream of H19. Methylation on the paternal allele prevents CTCF from binding, thus permitting interaction between the IGF2 promoters and the enhancers H19 DMR and in these cases IGF2 shows loss of imprinting (LOI) and biallelic expression KCNQ1 gene and is known as KvDMR1. The unmethylated paternal allele permits transcription of the antisense transcript KCNQ1OT1  and silencing of genes including KCNQ1 and CDKN1C. Maternal methylation at the KvDMR1 is thought to prevent transcription of the KCNQ1OT1 gene and enable expression of CDKN1C. Loss of methylation (LOM) at the KvDMR1 is seen in up to 50% of sporadic BWS and is associated with biallelic expression (loss of imprinting) of KCNQ1OT1 and silencing of maternal CDKN1C expression trans mechanism.Genomic imprinting is an epigenetic modification that causes genes to be expressed according to their parent of origin. Although less than 100 imprinted genes have been identified in human and mice, many imprinted genes appear to have a critical role in prenatal growth and development This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the South Birmingham Research Ethics Committee  reference number CA/5175. All patients provided written informed consent for the collection of samples and subsequent analysis.NLRP2 mutations in this family, a further 11 BWS families, each with a single case of BWS (mean age 10.8 years) with KvDMR1 loss of methylation were analysed for NLRP2 mutations. This cohort included 10 patients who also had loss of methylation at other imprinted loci. Ethnically matched laboratory control samples were analysed to evaluate the significance of novel sequence variants.A consanguineous family of Pakistani origin with two affected children with BWS due to loss of methylation at KvDMR1 were investigated in the first instance. Following the identification of Genomic DNA was extracted from peripheral lymphocytes by standard techniques.In preliminary examinations chromosomal abnormalities were excluded and the methylation status of IC1 and IC2 of imprinting region 11p15.5 were determined.BstU1 yielding different sized fragments which is separated using ABI377 or 3730 (PEG1) and the Angelman/Prader-Willi locus at 15q13 (SNRPN) as described previously Methylation analysis of KvDMR1 was performed as described previously, with PCR amplification of bisulphite modified DNA and digestion with restriction enzyme JMJD2D, ZFP57, NLRP7 and NLRP2 was carried out by direct sequencing. The genomic DNA sequence of these genes was taken from Ensembl (http://www.ensembl.org/index.html) and primer pairs for the translated exons were designed using primer3 software (http://fokker.wi.mit.edu/primer3/input.htm). Amplification was performed according to standard protocols with Bio Mix Red provided by Bioline. PCR products were directly sequenced by the Big Dye Terminator Cycle Sequencing System with the use of an ABI PRISM 3730 DNA Analyzer (Applied Biosystem). DNA sequences were analyzed using Chromas software.For linkage studies a genome wide linkage scan was undertaken using the Affymetrix 250k SNP microarray. Mutation analysis of trans imprinting defect.A family with complex consanguinity  was asceNLRP2 and NLRP7 at 19q13.4. NLRP7 is a homologue of the mouse NLRP2 gene (NLRP7 is not present in the mouse) and the human NLRP2 gene. Sequencing of NLRP2 in the mother identified a homozygous frameshift mutation . Genetic linkage studies were undertaken by genotyping the two children and both parents on an Affymetrix 250k SNP array platform. Five regions of homozygosity (>2Mbases) were shared by the two children but these did not contain a gene known to be implicated in the establishment or maintenance of genomic imprinting. However inspection of the maternal genotypes revealed an ∼8 Mbase homozygous region containing trans imprinting defect extended beyond KvDMR1, we analysed methylation levels at the TND (6q24), SNRPN (15q13) and PEG1 (7q32) DMRs. Both affected siblings  had normal methylation levels at the TND and SNRPN DMRs but Child 2 demonstrated partial loss of methylation at the PEG1 DMR . The major cause of TNDM is aberrant expression of imprinted genes at chromosome 6q24 and about 20% of cases have LOM at the TND differentially methylated region (DMR). Patients with homozygous ZFP57 mutations have LOM at the TND DMR, but also at other imprinted loci including KvDMR1 ZFP57 mutations in our family. Germline NLRP7 mutations are associated with familial recurrent biparental complete hydatidiform mole (FHM) in which there is epigenetic abnormalities at DMRs in multiple imprinting regions NLRP7 mutations is inherited in an autosomal recessive manner, in contrast to ZFP57 mutation, homozygotes have normal genomic methylation but in female homozygotes there is a failure to establish methylation imprints in their germ cells leading to hydatidiform moles and reproductive wastage . The methylation defects in FHM are specific for imprinted loci, and DNA methylation at non-imprinted genes and genes subject to X-inactivation is unaffected NLRP2 and NLRP7 genes are highly homologous and the two proteins consist of 1,062 and 1,009 amino acids respectively and have identical structure and about 64% amino acid identity. Only one of the two affected children with BWS was homozygous for a NLRP2 mutation and, by analogy with FHM caused by NLRP7 mutations, the BWS phenotype most likely results from homozygosity in the mother, such that familial BWS associated with NLRP2 mutations is inherited in a similar manner to NLRP7 associated FHM and not in a conventional autosomal recessive manner.We identified a homozygous frameshift mutation in the mother of two children with BWS caused by epimutations at IC2 (KvDMR1). Most cases of BWS due to loss of methylation of KvDMR1 are sporadic, but a handful of familial cases have been described with maternally inherited germline IC2 deletions ZFP57–TNDM are associated with imprinting aberrations at multiple loci, and we identified partial loss of methylation at the PEG1 DMR in one of the affected children. Nevertheless it seems that NLRP2 mutations have a less severe effect on imprinting than NLRP7 or ZFP57 inactivation. A subset of children with BWS and an IC2 epimutation display hypomethylation at multiple imprinting centres (DMRs) (in our series these children are more likely to have been conceived by assisted reproductive technologies) NLRP2 mutation in the sporadic cases we studied. It appears that the establishment (or maintenance) of methylation at KvDMR1 is particularly sensitive to genetic and/or environmental insults. We note that one of the affected children demonstrated a partial loss of methylation at PEG1 DMR1 both by bisulphite sequencing and MS-PCR suggesting that NLRP2 mutations may be associated with an incomplete failure of imprinting establishment and/or a partial failure of maintenance methylation at this DMR. Interestingly, investigation of a mouse knockout of ZFP57 has suggested a role in both the establishment of germline methylation imprints and in the postfertilisation maintenance of methylation imprints Both FHM and NLRP2 and NLRP7 encode members of the NLRP  family of CATERPILLER proteins. NLRP family of cytoplasmic proteins comprises 14 members of similar structure that are principally encoded by two gene clusters on chromosome 11p15  and 19q13.4 . Most of the family members are well conserved from C. elegans, D. melanogaster, rat, and mouse to human but there is no rodent homologue for NLRP7 and the gene is found in only a few genomes . Some NLRP proteins are components of the inflammasome that is implicated in the sensing of, and inflammatory reaction to, extracellular pathogens and intracellular noxious compounds NLRP3 and NLRP12 are associated with familial cold autoinflammatory syndrome NLRP2 truncating mutation did not show any evidence of an immune or autoinflammatory disorder. Nevertheless most NLRP family proteins are widely expressed and not restricted to the immune system. In addition, many are expressed in human oocytes and embryos at an early stage of development. Thus Zhang et al. have reported that NLRP4, 5, 8, 9, 11, 12, 13, and 14 were highly expressed in oocytes and then gradually decreased in embryos with a very low level in day 5 embryos, whilst NLRP2 and NLRP7 progressively decreased from oocytes to day 3 embryos then showed a sharp increase on day 5 NLRP2 and NLRP7 having a similar role in early development/imprinting establishment. Although it has been suggested that FHM might result from an immune-related defect in oogenesis or early embryo development (with methylation changes being a secondary phenomenon) the specific association of the methylation defects with imprinted DMRs suggests a more direct role in the establishment or maintenance of imprinting marks. Such a view is supported by the identification of germline NLRP2 mutations in BWS and should prompt further investigation of the role of NLRP2 and NLRP7 in genomic imprinting. The apparent involvement of NLRP proteins in genome methylation and the sensing and inflammatory response to extracellular pathogens and intracellular noxious compounds is intriguing given the suggestion that cytosine methylation may have evolved as a host response to transposons NLRP7 mutations have recurrent molar pregnancies only, but at least three families have been reported in which affected women had liveborn offspring  it might be predicted that clinical heterogeneity/incomplete penetrance would be a feature of maternal NLRP2 inactivation. Although maternal NLRP2 mutations appear to be a rare cause of familial BWS, the identification of these cases is important, as the inheritance pattern differs from the autosomal dominant inheritance (with parent of origin effects) associated with other inherited forms of BWS. The inheritance of NLRP2-associated BWS has similarities to other autosomal recessive disorders in which homozygous mothers are well, but there is a high risk to their offspring .


The Infectious Diseases Society of America published in 2000 practical guidelines for the management of cryptococcosis. However, treatment strategies have not been fully validated in the various clinical settings due to exclusion criteria during therapeutic trials. We assessed here the optimal therapeutic strategies for severe cryptococcosis using the observational prospective CryptoA/D study after analyzing routine clinical care of cryptococcosis in university or tertiary care hospitals.Cryptococcus neoformans. Control of sterilization was warranted 2 weeks (Wk2) and 3 months (Mo3) after antifungal therapy onset. 208 HIV-positive or -negative adult patients were analyzed. Treatment failure  at Wk2 and Mo3 was the main outcome measured. Combination of amphotericin B+flucytosine (AMB+5FC) was the best regimen for induction therapy in patients with meningoencephalitis and in all patients with high fungal burden and abnormal neurology. In those patients, treatment failure at Wk2 was 26% in the AMB+5FC group vs. 56% with any other treatments (p<0.001). In patients treated with AMB+5FC, factors independently associated with Wk2 mycological failure were high serum antigen titer  and abnormal brain imaging  at baseline. Haematological malignancy , abnormal neurology at baseline  and prescription of 5FC for less than 14 days  were independently associated with treatment failure at Mo3.Patients were enrolled if at least one culture grew positive with Our results support the conclusion that induction therapy with AMB+5FC for at least 14 days should be prescribed rather than any other induction treatments in all patients with high fungal burden at baseline regardless of their HIV serostatus and of the presence of proven meningoencephalitis. Major information on the best therapeutic strategies for cryptococcal meningoencephalitis derives from therapeutic trials involving HIV-positive Using data from a prospective observational cohort of HIV-positive and -negative patients treated in France for cryptococcosis with or without CNS involvement, we previously showed that lack of 5FC was an independent factor of mycological failure at Wk2 whatever the HIV status and the clinical presentation at baseline The CryptoA/D study enrolled HIV-positive or -negative adults experiencing a first episode of culture-proven cryptococcosis and treated in French university hospitals or tertiary care centers C. neoformans from at least one body site. Cases were classified as meningoencephalitis  or as extrameningeal cryptococcosis. A threshold of ≥1∶512 for serum or CSF antigen titer was selected since it is linked to prognosis A case was defined by isolation of Mycological outcome was only evaluated in patients for whom at least one body site was sampled at the time of workup. Mycological failure meant that at least one of the cultured samples contained viable yeasts. Mycological failure or death up to 4 days after Wk2 workup was recorded as treatment failure at Wk2. Mycological failure or later death was recorded as treatment failure at Mo3.We first described and compared the major characteristics of the patients and the Wk2 outcome according to the induction therapy strategies. We then assessed how the current IDSA guidelines Clinical presentation and outcome were compared according to therapeutic regimens with the χ2 test for qualitative parameters (or the Fisher's exact test when necessary) or the Kruskal-Wallis's or the Mann-Whitney's test for quantitative parameters. Multinomial logistic regression Induction therapy was assessable for 208 patients including a majority of HIV-positive male patients with meningoencephalitis . At the Induction therapies included AMB  mostly associated with 5FC  or FCZ  mostly alone  . AMB wasDespite more severe infections based on our criteria, mycological failure at Wk2 was significantly less frequent among patients treated with AMB+5FC than any other regimen (20/86 (23%) vs. 47/100 (47%), p<0.001) . This waThe majority of patients (n = 174) were treated with AMB or FCZ alone, or AMB+5FC. Patients treated with AMB did not significantly differ from those treated with the combination therapy except for the lower percentage of patients with high CSF antigen titer in the AMB-treated group . By contIn HIV-infected patients with meningoencephalitis who received as induction therapy either AMB or FCZ alone or the combination AMB+5FC (n = 120), factors which significantly discriminated between combination and FCZ alone in the univariate analysis were the presence of fungemia, high serum or CSF antigen titer, and hospitalization in university hospitals with a trend towards modification of prescription in older patients, while only high CSF antigen titer was identified as a factor associated with the initiation of combination therapy vs. AMB alone . In the Among the 86 patients given AMB+5FC and assessable at Wk2, the proportion of men, and high serum or CSF antigen titer was significantly higher than with other treatments. There was also a trend towards more disseminated infections and infection by serotype A among those who experienced mycological failure at Wk2 than among those who were cured. An optimal duration and dosage of each drug in the combination had no impact. Factors independently associated with mycological failure at Wk2 were high serum antigen titer  and abnormal brain imaging  at baseline .(1)FCZ(2)), AMB followed by FCZ (AMB(1)FCZ(2)), AMB+5FC followed by FCZ (AMB+5FC(1)FCZ(2)) and “miscellaneous” corresponding to other induction therapies and/or multiple changes preventing further description.Of 189 patients surviving the Wk2 workup by more than 4 days, 168 were eligible for analysis of Mo3 outcome . The ind(1)FCZ(2) group had significantly the more severe infections, and those in the FCZ(1)FCZ(2) group the less severe ones ((1)FCZ(2) group (20%) than in the AMB(1)FCZ(2) group (52%) despite no significant difference in the cumulative dose and duration of AMB. Of note, direct examination of the Wk2 CSF samples was still positive in almost 3/4 and 1/2 of the cases in AMB+5FC(1)FCZ(2) and AMB(1)FCZ(2) treated patients, respectively. There was a trend toward less treatment failures recorded at Mo3 in the AMB+5FC(1)FCZ(2) group than in the others.Considering the first 3 treatment groups, patients in the AMB+5FCere ones . MycologIn the univariate analysis, the proportion of abnormal neurology at baseline, haematological malignancies and 5FC prescription lasting less than 14 days was significantly higher in patients recorded as treatment failures at Mo3 than in the others . There wCryptococcal meningoencephalitis is still a major health problem in developed countries with the burden of the AIDS epidemics reaching a prevalence of up to 18% in the most severely immunocompromised patients Evaluation of cryptococcosis severity represents a major factor for the management of the patients. Many parameters are predictive of treatment failure including underlying malignancy, cranial hypertension, dissemination, and high antigen titer Initial antifungal treatment strategy is a critical determinant of outcome. Major studies supporting the prescription of 5FC in combination with AMB for the induction treatment of cryptococcal meningoencephalitis in HIV-positive We indeed showed that outcome at Wk2 was statistically better with AMB+5FC compared to any other regimen including AMB alone despite more severe infections in the former group. Furthermore, the proportion of Wk2 mycological failure did not increase in the most severe cases treated with AMB+5FC in contrast to what occurred in all other treatment groups except FCZ+5FC. The drastic difference in outcome between the two groups is probably due to the fact that more severe cases than those usually enrolled in the therapeutic trials were considered here with 40% of abnormal neurology compared to less than 20% in previous studies In the setting of a therapeutical trial on cryptococcal meningoencephalitis in HIV-positive patients, AMB compared favourably with FCZ, the latter being associated with more frequent early deaths and delayed CSF sterilization When considering the other strategies, our results demonstrate that FCZ alone was associated with 42% mycological failure at Wk2 despite the fact that 40% of the patients in that group did not have meningoencephalitis and that only 15% had high antigen titers. Keeping in mind the small number of patients in each of the FCZ+5FC, AMB+FCZ or AMB+FCZ+5FC groups, none of the combination did better than AMB+5FC. Of note, Bicanic and colleagues recently underlined in South Africa the high relapse rates after treatment with FCZ alone at conventional dosages Another issue in the optimization of treatment strategies for cryptococcosis is the potential utility of protracting induction regimen. Indeed, current recommendations are based on results obtained in a therapeutic trial where switch from AMB±5FC to azoles was mandatory at Wk2 whatever the culture results at that time Thus, this observational study with its limitation compared to a randomized therapeutic trial but the assets of the “real life” confirms that the AMB+5FC combination is the optimal antifungal strategy for the induction treatment of severe cryptococcosis Table S1(0.02 MB XLS)Click here for additional data file.Table S2(0.02 MB XLS)Click here for additional data file.Appendix S1(0.03 MB DOC)Click here for additional data file.


This study investigated expression of the SART3 antigen in breast cancer to explore an appropriate molecule for use in specific immunotherapy of breast cancer patients. The SART3 antigen was detected in all of the breast cancer cell lines tested, 30 of 40 (75%) breast cancer tissue samples, and 0 of 3 non-tumourous breast tissue samples. SART3 derived peptides at positions 109–118 and 315–323 induced HLA-A24 restricted CTLs that reacted to breast cancer cells from the peripheral blood mononuclear cells (PBMCs) of breast cancer patients. Therefore, the SART3 antigen and its peptides could be an appropriate molecule for use in specific immunotherapy of the majority of HLA-A24-positive breast cancer patients. © 2001 Cancer Research Campaign


The average number of nucleolar organiser regions per cell has previously been shown to correlate well with histological grading techniques for a variety of neoplasms in man, and may thus be of value as an aid to post-surgical prognosis. In this study 50 spontaneously arising, subcutaneous canine mast cell tumours were graded and the histological grade compared with the mean AgNOR count. For well differentiated neoplasms the mean count was 1.4 per cell compared with 6.3 for poorly differentiated neoplasms, while tumours of intermediate differentiation had a mean count of 3.2 per cell. Subsequent follow up studies revealed that the AgNOR count was an accurate prognostic indicator, 73% of dogs with a high mean count (greater than 4.9) being destroyed from tumour related disease compared with 33% with an intermediate count (1.7-4.8). No dog with a count of less than 1.7 has been destroyed because of tumour recurrence to date and the AgNOR count has proved to be a better and more objective prognostic indicator than either histological tumour grade or mitotic index. Since most dogs which develop recurrent mast cell tumours do so within 6 months of initial surgery, an assessment of the predictive value of AgNORs can be obtained more quickly in canine tumours than for comparable human neoplasms.


Characterisation and quantitation of epidermal growth factor receptors (EGFR) have been carried out on eight human squamous carcinoma cell lines and the results compared with those from simian virus transformed keratinocytes and normal keratinocytes grown under similar conditions. All cells tested possess both high and low affinity receptors with dissociation constants ranging from 2.4 X 10(-10) M to 5.4 X 10(-9) M. When epidermal growth factor (EGF) binds to its receptor it is internalised and degraded and the receptor is down regulated. Malignant cells and virally transformed cells possess 5-50 times more EGF receptors than normal keratinocytes and one cell line LICR-LON-HN-5 possesses up to 1.4 X 10(7) receptors per cell, which is the highest number yet reported for a cell line. These results are discussed in the context of recent data that suggest that the increased expression of EGF receptors in epidermoid malignancies may be an important component of the malignant phenotype in these tumours.


Reserpine was found to enhance the cytotoxicity of ACNU on ACNU-resistant C6 glioma (C6/ACNU) cells in vitro. When reserpine was added along with ACNU to the C6/ACNU cells in vitro. When reserpine was added along with ACNU to the C6/ACNU culture in vitro at a concentration of 10 microM, the IC50 of ACNU for C6/ACNU cells decreased to the level of that for C6 cells and ACNU resistance was completely overcome in vitro. Furthermore, intracellular uptake of ACNU increased in both sensitive (C6) and resistant (C6/ACNU) glioma cells when 20 microM reserpine was added to the culture medium. Reserpine (20 microM) enhanced the cellular level of ACNU in C6 cells 1.5-fold and enhanced the level of ACNU in C6/ACNU cells 4-fold. The amount of ACNU incorporated into C6/ACNU cells reached the same level as that incorporated into C6 cells. The enhanced cytotoxicity of ACNU in vitro could be explained by the effective intracellular accumulation of ACNU resulting from the increase of intracellular uptake of ACNU in C6/ACNU cells by reserpine.


SLC11A1 is a membrane transporter that is expressed in late endosomes of antigen presenting cells involved in the immunopathogenic events leading to T1DM. Mycobacterium avium subsp. paratuberculosis (MAP) has been reported to be a possible trigger in the development of T1DM.The etiology of type 1 diabetes mellitus (T1DM) is still unknown; numerous studies are performed to unravel the environmental factors involved in triggering the disease. SLC11A1 gene, and screened for the presence of MAP by PCR. Differences in genotype frequency were evaluated for both T1DM patients and controls. We found a polymorphism in the SLC11A1 gene (274C/T) associated to type 1 diabetic patients and not to controls. The presence of MAP DNA was also significantly associated with T1DM patients and not with controls.Fifty nine T1DM patients and 79 healthy controls were genotyped for 9 polymorphisms of SCL11A1 polymorphism was found to be associated with T1DM as well as the presence of MAP DNA in blood. Since MAP persists within macrophages and it is also processed by dendritic cells, further studies are necessary to evaluate if mutant forms of SLC11A1 alter the processing or presentation of MAP antigens triggering thereby an autoimmune response in T1DM patients.The 274C/T  Type 1 diabetes mellitus (T1DM) is a multifactorial autoimmune disease in which T-lymphocytes infiltrate the islets of the pancreas and destroy the insulin-producing beta cell populations Mycobacterium avium subsp. paratuberculosis (MAP) in the development of T1DM as an environmental trigger Accumulating line of evidence points to role for SLC11A1 gene (previously known as NRAMP1) is a functional and a positional candidate gene that associates with T1DM as well as susceptibility to mycobacterial infections et al. SLC11A1 gene silencing using RNAi approach in mice reduced the frequency of T1DM and protected against experimental autoimmune encephalomyelitis, advocating thereby for a role for SLC11A1 in autoimmunity. Moreover, it was recently demonstrated that association of variants of the gene encoding SLC11A1 with T1DM may reflect its function in processing and presentation of islet cell self-antigens to dendritic cells (DCs) Regarding genetic susceptibility, the Thus, non-MHC genes could affect the MHC-restricted T-cell response through altered antigen processing and presentation.SLC11A1 locus have been associated with susceptibility to infectious agents and to autoimmune disorders SLC11A1 gene seems to be of particular interest, since it has been shown to affect the levels of gene expression In vitro studies of this polymorphism suggested direct contribution of particular alleles either to autoimmune  or to infectious  disease susceptibility SLC11A1 located within the coding region, the introns, and the 3′-UTR have been shown to influence susceptibility to autoimmune disorders and T1DM To date, a number of polymorphisms at the SLC11A1 polymorphisms in relation to the presence of MAP infection, with T1DM in patients from Sardinia.Our study aimed at examining the association of the SLC11A1 polymorphisms and the presence of MAP specific IS900 signature using total DNA extracted from peripheral blood mononuclear cells collected at the Institute of Diabetology, medical clinic of Sassari University, Italy. Informed written consents from patients including other necessary clearances were obtained before blood samples were drawn. Institutional review board of the University of Sassari approved the study.A total of 131 participants comprising of 59 T1DM patients  and 76 healthy controls  were tesBriefly, 5 ml blood from patients was centrifuged and plasma supernatant used in ELISA. Remaining plasma samples were aliquoted and stored frozen at −20°C for short-term storage (<6 months) and −80°C for long term storage (>6 months).To extract MAP DNA, samples were processed by using the lysor instrument - 500 µl of glass beads (Sigma) were added, followed by standard phenol-chloroform extraction and DNA precipitation with absolute ethanol and 10M ammonium acetate; the pellet was washed with 70% ethanol and resuspended in 70 µl of TE buffer.SCL11A1 gene amplification was performed as previously published Human genomic DNA extraction for SCL11A1 polymorphisms, 100 ng of template (genomic) DNA, obtained from blood cells, was amplified. The primer sequences and PCR cycling parameters that we used were previously reported MAP DNA detection was performed by PCR as previously reported NRAMP1Nine polymorphisms  were genotyped across Statistical analysis was performed by using the Chi square test with Yate's correction. Inference was aided by GraphPad InStat MAP DNA was amplified from the blood of 33 out of 59 T1DM patients (55.9%) whereas MAP was detected only in 18 out of 79 healthy controls (22.7%) indicating a statistically highly significant difference .SLC11A1 allele frequencies for 274 C/T polymorphism differed significantly between T1DM patients and control group, this polymorphism was significantly associated with diabetes type 1, in particular allele 1 and 2 generated a P value of <0.0005  is associated with tuberculosis susceptibility in Chinese children SCLA11 polymorphisms were analyzed also by stratification by sex and no differences were observed (data not shown). It may be possible that polymorphisms in different loci of the SCL11A1 gene may confer susceptibility to different intracellular pathogens although genetically very close. Further studies are necessary in order to elucidate the role of SCL11A1 polymorphisms and how it may influence MAP infection in humans.In this study we report the novel association of T1DM with the 274 C/T polymorphism within the


MYOC) and forkhead box protein C1 (FOXC1) genes for sequence variations in primary congenital glaucoma (PCG).To screen the myocilin (MYOC variations and 54 cases (negative or heterozygous for cytochrome P4501B1 mutations) for FOXC1 mutations by polymerase chain reaction (PCR) and DNA sequencing.Seventy five PCG patients were screened for MYOC and two sequence variations (GGC375ins and GGC447ins) in FOXC1. No pathogenic variations were identified in MYOC and FOXC1 in our patients.Five single nucleotide polymorphisms  were identified in MYOC and FOXC1 mutations are not involved in pathogenesis of primary congenital glaucoma in our patients. Thus, it is important to screen other loci for involvement in congenital glaucoma in cases which are negative or heterozygous for CYP1B1 mutations to have a better insight in to disease pathogenesis. The north India ,20, thesMYOC and FOXC1 in the pathogenesis of primary congenital glaucoma. Thus, it is important to screen other loci for involvement in congenital glaucoma in cases which are either negative or heterozygous for CYP1B1 mutations to have a better insight in disease pathogenesis.This is the first study from north India showing non-involvement of


The 249I and 280M alleles were             associated with PAH . In conclusion,             the increased frequencies of 249I and 280M CX3CR1 alleles in a subgroup of             patients with SSc-associated PAH suggest a role for the fractalkine system in             the pathogenesis of this             condition. Further, the 249I allele might be associated with susceptibility to SSc.Fractalkine (FKN) and its receptor CX3CR1 are critical mediators in the             vascular and tissue damage of several chronic diseases, including systemic             sclerosis (SSc) and pulmonary arterial hypertension (PAH). Interestingly, the V249I             and T280M genetic polymorphisms influence CX3CR1 expression and function. We             investigated whether these polymorphisms are associated with PAH secondary to             SSc. CX3CR1 genotypes were analyzed by PCR and sequencing in 76 patients with             limited SSc and 204 healthy controls. PAH was defined by colorDoppler echocardiography.             Homozygosity for 249II as well as the combined presence of 249II and 280MM were             significantly more frequent in patients with SSc compared to controls (17 vs 6%,


BRCA1 and BRCA2 are tumour suppressor genes the alleles of which have to be inactivated before tumour development occurs. Hereditary breast cancers linked to germ-line mutations of BRCA1 and BRCA2 genes almost invariably show allelic imbalance (AI) at the respective loci. BRCA1 and BRCA2 are believed to take part in a common pathway in maintenance of genomic integrity in cells. We carried out AI and fluorescence in situ hybridization (FISH) analyses of BRCA2 in breast tumours from germ-line BRCA1 mutation carriers and vice versa. For comparison, 14 sporadic breast tumours were also studied. 8 of the 11 (73%) informative BRCA1 mutation tumours showed AI at the BRCA2 locus. 53% of these tumours showed a copy number loss of the BRCA2 gene by FISH. 5 of the 6 (83%) informative BRCA2 mutation tumours showed AI at the BRCA1 locus. Half of the tumours (4/8) showed a physical deletion of the BRCA1 gene by FISH. Combined allelic loss of both BRCA1 and BRCA2 gene was seen in 12 of the 17 (71%) informative hereditary tumours, whereas copy number losses of both BRCA genes was seen in only 4/14 (29%) sporadic control tumours studied by FISH. In conclusion, the high prevalence of AI at BRCA1 in BRCA2 mutation tumours and vice versa suggests that somatic events occurring at the other breast cancer susceptibility gene locus may be selected in the cancer development. The mechanism resulting in AI at these loci seems more complex than a physical deletion.   http://www.bjcancer.com © 2001 Cancer Research CampaignBreast cancer susceptibility genes


Percutaneous endoscopic lumbar discectomy is a relatively new technique. Very few studies have reported the clinical outcome of percutaneous endoscopic discectomy in terms of quality of life and return to work.55 patients with percutaneous endoscopic lumbar discectomy done from 2002 to 2006 had their clinical outcomes reviewed in terms of the North American Spine Score (NASS), Medical Outcomes Study Short Form-36 scores (SF-36) and Pain Visual Analogue Scale (VAS) and return to work.The mean age was 35.6 years, the mean operative time was 55.8 minutes and the mean length of follow-up was 3.4 years. The mean hospital stay for endoscopic discectomy was 17.3 hours. There was significant reduction in the severity of back pain and lower limb symptoms  at 6 months and 2 years. There was significant improvement in all aspects of the Quality of Life  scores except for general health at 6 months and 2 years postoperation. The recurrence rate was 5% (3 patients). 5% (3 patients) subsequently underwent lumbar fusion for persistent back pain. All patients returned to their previous occupation after surgery at a mean time of 24.3 days.Percutaneous endoscopic lumbar discectomy is associated with improvement in back pain and lower limb symptoms postoperation which translates to improvement in quality of life. It has the advantage that it can be performed on a day case basis with short length of hospitalization and early return to work thus improving quality of life earlier. The surgical treatment of lumbar disc herniation constitutes a large part of orthopedic practice and it has evolved considerably in terms of surgical technique and instrumentation.Percutaneous endoscopic discectomy is a relatively new technique for removing lumbar disc herniation. It involves using an endoscope to visualize the disc removal. The discectomy is performed through a posterolateral approach using specially developed instruments. The advantage of percutaneous endoscopic discectomy is that the disc is approached posterolaterally through the triangle of Kambin ,2 withouAlthough many studies -8 have sThe purpose of this study is to determine the outcome of percutaneous endoscopic discectomy in terms of the North American Spine Score (NASS) , MedicalFrom 2002 to 2006, 55 patients with percutaneous endoscopic discectomy performed for herniated intervertebral disc at our instituition had data collected prospectively. All the operations were performed by two surgeons.Inclusion criteria were patients who had radicular symptoms due to discogenic lumbar nerve root compression and failed conservative therapy. The diagnosis of lumbar disc herniation was made on MRI and/or CT scans. Patients with calcified discs shown on CT scans were excluded. Patients who met the inclusion criteria were counseled that percutaneous endoscopic lumbar discectomy was a relatively new technique and offered the alternative of open discectomy as well. 55 patients agreed to have percutaneous endoscopic discectomy.Data on patient demographics, operative time, length of hospitalisation, postoperative complications and how soon they returned to work were obtained. In our institution, all patients who underwent spinal surgery had routine preoperative assessment and 6 month and 2 year postoperative assessments done; the patients were assessed based on the North American Spine Score (NASS – Disease specific questionnaire) , MedicalStatistical analysis was performed with the use of SPSS version 10.0. Categorical data were compared with the use of chi-square test. Non-parametric statistics were used for the analysis of continuous variables when data were not normally distributed. Significance was defined as p < 0.05.Preoperatively, all patients received one gram of cefazolin intravenously as antibiotic prophylaxis and if the patient is allergic to cefazolin, one gram of intravenous vancomycin was given instead. The patients were placed prone on a radiolucent operative table on a Wilson frame.36 of the 55 patients (66%) were done under local anesthesia. The skin, subcutaneous tissue, fascia and muscle layers were infiltrated with 1 per cent lidocaine. For relaxation and comfort of the patient, sedation with intravenous midazolam or Fentanyl was administered by the anesthetist. 19 patients (34%) were uneasy about having the operation performed under local anaesthesia and so the operation was done under general anaesthesia.Using the C-arm oriented in the postero-anterior imaging position, the midline longitudinal line is marked on the skin surface using a narrow metal rod. The metal rod is then placed transversely across the center of the target disc. A horizontal line is drawn, bisecting the disc under evaluation. The anatomic disc center is located where the transverse line crosses the longitudinal midline. The C-arm is rotated to the lateral projection. The metal rod is held along the side of the patient in the parasagittal orientation at the level of the index disc. While the metal rod is held in this position, the length from the center of that disc to the plane of the posterior skin is recorded. This length is used for the lateral distance of the skin entry point from the posterior midline and this is usually about 12 to 14 cm from the midline. [A guidewire is then inserted through the needle into the disc and the needle removed. A small stab incision is made at the entry site of the guidewire and a tapered cannulated obturator is slid over the guide wire and introduced gently into the foramen and into the disc. A beveled working cannula is then introduced over the obturator which is then withdrawn. An endoscope (Yeung Endoscopic Spine System – Y.E.S.S. endoscope)  is then The mean age of the patients was 35.6 years (range 15 – 68 years). There were 23 (41.8%) female: 32 (58.2%) male. The mean operative time was 55.8 min (30–100 min). The mean length of hospitalization was 17.3 hours (range 6 to 24 hours). The mean follow-up period was 3.4 years (range 2.0 – 6.5 years). All patients who were working preoperatively returned to work. The mean time to return to work was 24.3 days (10 – 60 days). All returned to their previous occupation (Table 39 (70.9%) patients had L4L5 discectomy done, 12 (21.8%) had L5S1, 2 (3.6%) had L3L4 and 2 (3.6%) had two levels L4L5 and L5S1 done. There were 44 (80%) disc protrusions, 10 (18.2%) extrusions and 1 (1.8%) sequestrated disc.Figure 3 patients (5%) subsequently underwent lumbar fusion for increasing back pain despite good relief of radicular symptoms after endoscopic discectomy. One of these patients was a 25 year old male who presented initially with left L4 radicular symptoms. MRI showed L4L5 and L5S1 degenerate discs with a left L4L5 prolapsed intervetebral disc. A left L4L5 endoscopic discectomy was initially performed for him but on followup, he complained of increasing back pain and had L4L5, L5S1 transforaminal lumbar interbody fusion done 3 months after endoscopic discectomy. Another was a 45 year old male who had right L5 radicular symptoms and back pain preoperatively. MRI showed a right L5S1 posterolateral disc prolapse. He underwent right L5S1 endoscopic discectomy but also had increasing back pain on followup. He eventually had L5S1 posterior lumbar interbody fusion done 7 months post-edoscopic discectomy. For both of these patients, their initial radicular symptoms resolved after endoscopic discectomy. The third patient was a 36 year old female with left L4 radicular pain. MRI showed a L4L5 prolapsed disc. Post- endoscopic discectomy, her radicular sumptoms resolved. However 3 years postoperation, she complained of back pain and left L4 radicular pain again. Postoperation MRI showed diffuse L45 disc bulge and central and lateral recess stenosis. She subsequently underwent transforaminal lumbar interbody fusion.th post-operative day, he complained of severe back pain associated with mild fever. Blood tests showed raised total white count, ESR and CRP. An MRI with contrast showed mainly granulation tissue. He underwent endoscopic washout of the disc space. Tissue cultures from the disc space grew Staphylococcus aureus. His symptoms resolved after the washout. He was treated with intravenous Augmentin for 2 weeks followed by a further 4 weeks of oral Augmentin. He had occasional back pain at 2 years follow-up. There were no complications associated with any of the subsequent surgeries performed after endoscopic discectomy.1 patient developed discitis 4 days post-endoscopic discectomy. This is a 37 year old male who underwent left L45 percutaneous endoscopic discectomy. He was discharged well 1 day post-operation. However, on the 4Based on the SF-36 questionnaire , of which 2 had open discectomy. 1 patient was found to have a sequestrated disc post-endoscopic discectomy and had open microdiscectomy subsequently. Recurrence rate of lumbar disc herniation after open discectomy has been reported as 5–11% and most have been treated with a repeated discectomy through the same approach as the initial surgery.,19 Howev3 patients subsequently underwent lumbar intervertebral body fusion for increasing low back pain despite resolution of their initial radicular symptoms. Thus while percutaneous endoscopic discectomy is effective in relieving leg symptoms, it is less effective in treating back pain and this has to be communicated to the patients.In this study, the complications for endoscopic discectomy include 1 case of discitis and 1 case of sequestrated disc, giving a complication rate of 3.6%. It has been reported that the overall complication rate for this kind of surgical procedure averages 2.6%.,22 The cPercutaneous endoscopic lumbar discectomy is a safe and efficacious technique to relieve symptoms of herniated discs and this improvement in back pain and leg symptoms translates to improvement in quality of life. It has the advantage that it can be performed on a day case basis with shorter length of hospitalization and early return to work thus improving quality of life earlier. This is important because patients become candidates for lumbar disc herniation surgery to obtain immediate pain relief and to improve their quality of life.The authors declare that they have no competing interests.CWBP performed case collection, data analysis, literature review and wrote article. WY performed case and data collection. SBT supervised and helped in manuscript preparation. All authors read and approved the final manuscript.


The homeobox genes are a large and diverse group of genes, many of which play important roles in the embryonic development of animals. Increasingly, homeobox genes are being compared between genomes in an attempt to understand the evolution of animal development. Despite their importance, the full diversity of human homeobox genes has not previously been described.DUX1 to DUX5 homeobox sequences of which we identified 35 probable pseudogenes, with many more expected in heterochromatic regions. Nomenclature is established for approximately 40 formerly unnamed loci, reflecting their evolutionary relationships to other loci in human and other species, and nomenclature revisions are proposed for around 30 other loci. We use a classification that recognizes 11 homeobox gene 'classes' subdivided into 102 homeobox gene 'families'.We have identified all homeobox genes and pseudogenes in the euchromatic regions of the human genome, finding many unannotated, incorrectly annotated, unnamed, misnamed or misclassified genes and pseudogenes. We describe 300 human homeobox loci, which we divide into 235 probable functional genes and 65 probable pseudogenes. These totals include 3 genes with partial homeoboxes and 13 pseudogenes that lack homeoboxes but are clearly derived from homeobox genes. These figures exclude the repetitive We have conducted a comprehensive survey of homeobox genes and pseudogenes in the human genome, described many new loci, and revised the classification and nomenclature of homeobox genes. The classification scheme may be widely applicable to homeobox genes in other animal genomes and will facilitate comparative genomics of this important gene superclass. Homeobox genes are characterized by the possession of a particular DNA sequence, the homeobox, which encodes a recognizable although very variable protein domain, the homeodomain ,2. Most The homeobox genes have been variously subdivided into superclasses, classes, subclasses or groups, although there has been much inconsistency in the use of these terms. The most commonly recognized groupings are the ANTP, PRD, LIM, POU, HNF, SINE, TALE, CUT, PROS and ZF groups (or variants of these names), although these are not always given equal rank in classification schemes ,2,4-8. TThe initial analyses of the draft human genome sequence published in 2001 included estimates of the number of human homeobox genes. Venter et al  found 16Using exhaustive database screening, followed by manual examination of sequences, we identified 300 homeobox loci in the human genome. Distinguishing which of these loci are functional genes and which are non-functional pseudogenes was difficult in some cases. Most loci classified as pseudogenes in this study are integrated reverse-transcribed transcripts, readily recognized by their dispersed genomic location, complete lack of intron sequences, and (in some cases) 3' homopolymeric run of adenine residues. A small minority are duplicated copies of genes, recognized by physical linkage to their functional counterparts and the same (or similar) exon-intron arrangement. In general, retrotransposed gene copies are non-functional (and therefore pseudogenes) from the moment of integration because they lack 5' promoter regions necessary for transcription. However, such sequences can occasionally acquire new promoters and become functional as 'retrogenes'. Duplicated gene copies often possess 5'promoter regions (as they are often encompassed by the duplication event); most degenerate to pseudogenes due to redundancy in a process known as non-functionalization, however some can be preserved as functional genes through sub- or neo-functionalization. Thus, in both instances, reliable indicators of non-functionality were sought in order to assign pseudogene status, notably frameshift mutations, premature stop codons and non-synonymous substitutions at otherwise conserved sites in the original coding region.PAX2, PAX5 and PAX8) and retrotransposed pseudogenes that correspond to only part of the original transcript, whether or not it includes the homeobox region or indeed any of the original coding region. Consequently, 13 retrotransposed pseudogenes that lack homeobox sequences are included . We do not include PAX1, PAX9 and CERS1; these are functional genes without homeobox motifs, albeit closely related to true homeobox genes (the other PAX and CERS genes).We currently estimate that the 300 human homeobox loci comprise 235 functional genes and 65 pseudogenes Table . These fDUX4 (DUX1 to DUX5), which have become part of 3.3 kb repetitive DNA elements present in multiple copies in the genome  is the second of two human members of the Nk3 gene family. This gene was previously known as BAPX1; we rename it NKX3-2 to show that it is a member of the Nk3 gene family.∘ NKX6-3 [Entrez Gene ID: 157848] is the third of three human members of the Nk6 gene family. We designate this previously unnamed gene NKX6-3 on the basis of clear orthology to mouse Nkx6-3, inferred from sequence identity and synteny.∘ VENTX [Entrez Gene ID: 27287] is the only functional member of the Ventx gene family in the human genome. This gene was previously known as VENTX2. We remove the numerical suffix from this gene symbol because we discovered that the sequence formerly known as VENTX1 is actually a retrotransposed pseudogene derived from this gene. Accordingly, we also replace the VENTX1 symbol with VENTXP7 (see below).∘ NANOG gene have been described previously . A short cDNA sequence [EMBL: X74862] related to the Msx gene family was reported previously . The pseudogene shares 91% sequence identity with MSX2 mRNA, lacks intronic sequence, and has remnants of a 3' poly(A) tail. It is intriguing, but probably coincidental, that the MSX2P1 pseudogene has integrated at 17q23.2, close to several ANTP-class genes .∘ eviously ; the foruse Msx3 . It is nNANOGP1 [Entrez Gene ID: 404635]. We follow Booth and Holland . We follow Booth and Holland , VENTXP2 [Entrez Gene ID: 347975], VENTXP3 [Entrez Gene ID: 349814] and VENTXP4 [Entrez Gene ID: 152101]. These four VENTX retrotransposed pseudogenes have been reported previously, and were originally known as VENTX2P1 to VENTX2P4. The correction of the VENTX2 gene symbol to simply VENTX (see above) means that each of the pseudogene names should also change; we rename them VENTXP1 to VENTXP4. VENTXP1 is transcribed but due to mutations it can no longer encode a homeodomain protein; it can however encode an antigenic peptide (NA88A) responsible for T-cell stimulation in response to melanoma [∘ VENTXP5 [Entrez Gene ID: 442384]. We designate this previously unnamed sequence VENTXP5 because it is clearly a retrotransposed pseudogene of VENTX. The genomic sequence of VENTXP5 can now be accessed via the Reference Sequence collection [RefSeq: NG_005091]. The pseudogene shares 83% identity with VENTX mRNA , lacks intronic sequence, and has remnants of a 3' poly(A) tail.∘ VENTXP6 [Entrez Gene ID: 552879]. We designate this previously unannotated sequence VENTXP6 because it is clearly a retrotransposed pseudogene of VENTX. Its lack of annotation may reflect the fact that it is located within an intron of an unrelated and well characterized gene, STAU2. The genomic sequence of VENTXP6 can now be accessed via the Reference Sequence collection [RefSeq: NG_005090]. The pseudogene shares 87% identity with VENTX mRNA  and lacks intronic sequence.∘ VENTXP7 [Entrez Gene ID: 391518]. A short cDNA sequence [EMBL: X74864] was reported previously and named HPX42 . The pseudogene shares 86% identity with VENTX mRNA , lacks intronic sequence, and has remnants of a 3' poly(A) tail.∘ ed HPX42 . This waAY151139], has been annotated as a homeobox gene and is located just 20 kb from NANOG. However, no homeodomain was detected when the deduced protein was analyzed for conserved domains. Also, secondary structure prediction did not predict the expected organisation of alpha helices. Alignment with the NANOG homeodomain reveals identity of the KQ and WF motifs, either side of the same intron position (44/45), but few other shared residues. It is possible, but unproven, that the locus arose by tandem duplication of part, or all, of the NANOG homeobox gene. This gene has generated two retrotransposed pseudogenes: one at 2q11.2 and another at 12q24.33.One other gene could conceivably be included in the ANTP class, but is excluded from our survey. This gene  is the first of three human members of the Alx gene family. This gene was previously known as CART1; we rename it ALX1 because it is related to ALX3 and ALX4; all three genes were formed by duplication from a single ancestral invertebrate gene [∘ ate gene .DRGX [Entrez Gene ID: 117065] is the only member of the newly defined Drgx gene family in the human genome. This gene was previously known as PRRXL1 and DRG11, and there is a clear mouse ortholog (Prrxl1). The symbol PRRXL1 is misleading because it infers membership of the Prrx gene family, containing PRRX1 and PRRX2 in the human genome. Several lines of evidence suggest it belongs to a different gene family. First, this gene (at 10q11.23) is not located in the same paralogon as PRRX1 (1q24.3) and PRRX2 (9q34.11) so they are not three paralogs generated during genome duplication in early vertebrate evolution. Second, it has a completely different exon-intron structure from the Prrx-family genes, and it does not contain a Prrx domain or an OAR domain  incorporates the root of the former symbol DRG11, referring to expression of the rodent ortholog in dorsal root ganglia neurons  is a human member of the Dux (double homeobox) gene family. As previously discussed, most members of this gene family are intronless and are probably derived by retrotransposition of an mRNA transcript from a functional intron-containing Dux gene (or duplication of such an integrant). Booth and Holland  is the second of two human members of the Gsc gene family. This gene was previously known as GSCL; we rename it GSC2 to remove the inadvertent implication that it is not a true gene, and also to reflect the clear orthology to chick Gsc2 as inferred by phylogenetic analysis and synteny.∘ HOPX [Entrez Gene ID: 84525] is the only member of the newly defined Hopx gene family in the human genome. The mouse version of the gene was first identified first and named Hop (homeodomain only protein) because the encoded protein is just 73 amino acids long, with 61 of these making up the homeodomain  is the only member of the newly defined Leutx gene family in the human genome. We designate this previously unnamed gene LEUTX (leucine twenty homeobox) to reflect the presence of a leucine residue at the otherwise highly conserved homeodomain position 20; other PRD-class homeodomains have a phenylalanine at this position  whilst avoiding inadvertent equivalence to specific genes within the cluster.∘ RHOXF2B [Entrez Gene ID: 727940] is the third human member of the Rhox gene family. This locus was referred to in previous studies as PEPP2b  is the only member of the Sebox gene family in the human genome. The human gene is the ortholog of mouse Sebox based on their locations in syntenic chromosomal regions (17q11.2 and 11B5 respectively) and presence of the same intron positions. However, sequence identity is lower than normal for orthologous genes in mouse and human (78% amino acid identity over the homeodomain) and there is evidence that the human gene has undergone divergence. Most surprisingly, the human sequence has two unusual substitutions in the homeodomain  is the only member of the Uncx gene family in the human genome. This gene was previously known as UNCX4.1; we remove the numerals to give UNCX as these do not denote a series within a gene family.∘ VSX2 [Entrez Gene ID: 338917] is the second of two human members of the Vsx gene family. This gene was previously known as CHX10; we rename it VSX2 to better reflect its paralogous relationship to VSX1. VSX2 has been used as an alias for this gene in other vertebrate species and the gene symbol CHX10 has the disadvantage of implicitly suggesting presence of at least nine paralogs in human (CHX1 to CHX9), which do not exist.∘ DUXA gene . We designate this previously undescribed sequence OTX2P1 because it is clearly a retrotransposed pseudogene of OTX2. The genomic DNA sequence of OTX2P1 shares significant homology with OTX2 transcript variant 2 [RefSeq: NM_172337]. There is an Alu element (AluSx subfamily) insertion, a Made1 (Mariner derived element 1) insertion, and a 1182-nucleotide deletion in OTX2P1 compared to OTX2. The OTX2P1 sequence lacks introns, ends with a poly(A) tail, and harbors critical sequence alterations (including a three-nucleotide insertion introducing a stop codon into the deduced homeodomain).∘ DUX1 [EMBL: AJ001481], DUX2 [GenBank: AF068744], DUX3 [GenBank: AF133130] and DUX5 [GenBank: AF133131]. These sequences have been cloned in previous studies . This sequence has been extensively studied as some of its multiple copies exist within the 3.3 kb repetitive elements of the D4Z4 locus at 4q35 . This gene was previously known as CXorf43. The gene encodes a highly divergent atypical (68-amino-acid) homeodomain but not a POU-specific domain, and thus it is debatable whether it should be placed within the POU class. Phylogenetic analyses of homeodomains place it basally in a clade with the POU class  [GeneID: 5461], POU5F1P4 [GeneID: 100009666], POU5F1P5 [GeneID: 100009667], POU5F1P6 [GeneID: 100009668], POU5F1P7 [GeneID: 100009669] and POU5F1P8 [GeneID: 100009670]. Prior to this study, a single retrotransposed pseudogene of the POU5F1 gene had been annotated and designated POU5F1P1 [Entrez Gene ID: 5462]. Another POU5F1-related sequence of unknown status had been annotated and designated POUF5F1L [GeneID: 5461]. We replace the gene symbol POUF5F1L with POU5F1P3 as this sequence is a retrotransposed pseudogene of POUF51. Our analyses of the human genome sequence identified a further six pseudogenes of POU5F1, which we name sequentially POU5F1P2, POU5F1P4 through to POU5F1P8. Each clearly aligns to the mRNA sequence of POU5F1 but with sequence alterations, indicating origin by retrotransposition. POU5F1P2 and POU5F1P6 have frameshift mutations in the homeobox. POU5F1P5 and POU5F1P6 have stop codons in the homeobox. POU5F1P7 and POU5F1P8 are partial integrants of POU5F1 mRNA excluding the homeobox – POU5F1P7 covers part of the 3' untranslated region and POU5F1P8 a short region around the start codon.∘ Hnf1) encodes proteins with a POU-like domain N-terminal to a highly atypical homeodomain. The POU-like domain, as its name indicates, is weakly similar in sequence to the POU-specific domain . This sequence was previously known as IRXA1; we rename it IRX1P1 because it is clearly a retrotransposed pseudogene of IRX1 and not a functional gene. The IRX1P1 sequence aligns to the mRNA of IRX1 but has a frameshift mutation and two stop codons in the homeobox.∘ IRX4P1 [Entrez Gene ID: 100009671]. We designate this previously unannotated sequence IRX4P1 because it is clearly a retrotransposed pseudogene of IRX4. The IRX4P1 sequence is a partial integrant derived from a region of the IRX4 mRNA around the stop codon; it lacks the homeobox.∘ PBX2P1 [Entrez Gene ID: 5088]. This sequence was previously known as PBXP1; we rename it PBX2P1 because it is clearly a retrotransposed pseudogene of PBX2. The former name of PBXP1 did not indicate its transcript of origin. The PBX2P1 sequence aligns to the mRNA of PBX2 but has a frameshift mutation in the coding region.∘ TGIF1P1 [Entrez Gene ID: 126052]. We designate this previously unannotated sequence TGIF1P1 because it is clearly a retrotransposed pseudogene of TGIF1. The locus has many sequence alterations when compared to TGIF1 mRNA, including a 48 nucleotide insertion within the homeobox.∘ TGIF2P1 [GeneID: 126826], TGIF2P2 [GeneID: 100009674], TGIF2P3 [GeneID: 100009672] and TGIF2P4 [GeneID: 100009673]. These four sequences were unannotated prior to this study. We designate them TGIF2P1 to TGIF2P4 because they are clearly pseudogenes of TGIF2. Each aligns to the mRNA sequence of TGIF2 but with sequence alterations, indicating origin by retrotransposition. TGIF2P1 has many sequence alterations, including a frameshift mutation in the homeobox. TGIF2P2 and TGIF2P3 are very similar neighboring loci that must have originated by tandem duplication of a retrotransposed TGIF2 mRNA; neither includes the homeobox. TGIF2P4 is a short partial integrant derived from part of the 3' untranslated region of TGIF2 mRNA.∘ Drosophila gene cut) generally encodes proteins with one or more CUT domains N-terminal to a typical homeodomain. The CUT domain is a DNA-binding domain of approximately 75 amino acids [The CUT class (named after the no acids ). A fourno acids  have proCUX1 and CUX2). We have also identified a total of three CUT-class pseudogenes in the human genome .We have identified a total of seven CUT-class homeobox genes in the human genome Tables  and 4. ACUX1 [Entrez Gene ID: 1523] and CUX2 [Entrez Gene ID: 23316]. These genes were previously known as CUTL1 and CUTL2 respectively. We rename them CUX1 and CUX2 in accordance with homeobox gene nomenclature convention.∘ CUX2P1 and CUX2P2. These sequences were unannotated prior to this study. We designate them CUX2P1 and CUX2P2 because they are clearly retrotransposed pseudogenes of CUX2. Both are short partial integrants derived from CUX2 mRNA, excluding the homeobox – CUX2P1 covers part of the coding region at the 5' end and CUX2P2 part of the 3' untranslated region.∘ SATB1P1 [Entrez Gene ID: 100033410]. We designate this previously unannotated sequence SATB1P1 because it is clearly a retrotransposed pseudogene of SATB1. SATB1P1 is a short partial integrant derived from part of the 3' untranslated region of SATB1 mRNA; it does not encompass the homeobox.∘ Drosophila gene pros) encodes proteins with a PROS domain C-terminal to an atypical homeodomain. The PROS domain is a DNA-binding domain of approximately 100 amino acids . This gene was previously known as ATBF1; we rename it ZFHX3 to reflect its close relationship to ZFHX2 and ZFHX4; indeed ZFHX3 was a synonym for this gene.∘ LAG1. There are six CERS-class genes in the human genome (CERS1 to CERS6) and five of these (CERS2 to CERS6) encode proteins with a homeodomain sequence [CERS2 to CERS6 in our comprehensive compilation of human homeobox genes, as CERS1 lacks a homeobox motif.The highly unusual CERS (ceramide synthase) class, also known as the LASS (longevity assurance) class, comprises a single gene family that is highly conserved amongst eukaryotes and includes the yeast gene and original member sequence ,82. Thessequence ,82. It iDUX1 to DUX5 sequences are also not shown, as these have undergone secondary amplification and are also most likely non-functional (see above).The chromosomal locations of genes can give clues to evolutionary ancestry, including patterns of gene duplication, and the possible existence of gene clusters. In Figure SHOX (short stature homeobox) in the PAR1 pseudoautosomal region at the tip of the short arms of X and Y being the best known. Haploinsufficiency of SHOX is implicated in the short stature phenotype of Turner syndrome patients who lack one copy of the X chromosome [TGIF2LX, has a distinct homolog on the Y chromosome, called TGIF2LY. These genes map to the largest homology block shared by the unique regions of the X and Y chromosomes, spanning 3.5 Mb. It has been proposed that the ancestor of these two genes arose by retrotransposition of TGIF2 mRNA [The first observation is that there are homeobox genes on every human chromosome. Even the two sex chromosomes harbor homeobox genes, with romosome . There aIF2 mRNA .PKNOX1) and 22 (with GSC2 and ISX). Examination of the remaining autosomes reveals that homeobox genes are quite dispersed with some interesting regional accumulations. The best known examples of close linkage between homeobox genes are the four Hox clusters on human chromosomes 2, 7, 12 and 17, comprising 9, 11, 9 and 10 genes respectively; each of these is shown as just a single line on each ideogram for simplicity  on chromosome 13, and four sets of NKL-subclass genes on 2p/8p (split), 4p, 5q and 10q, hypothesized to be derived from an ancestral array by duplication [There are other concentrations of ANTP-class genes away from the Hox clusters. These are the ParaHox cluster  was subjected to a series of tBLASTn searches ,86 usingDrosophila homeodomains were first combined with all human homeodomains in maximum likelihood and neighbor-joining analyses to enable divergent Drosophila genes to be identified and removed. These include genes lost from human, as well genes known to have undergone unusually rapid evolution in Drosophila. For the Hox3 family the rapidly evolving Drosophila genes bcd, zen and zen2 were then replaced by an ortholog from centipede (Sm Hox3b), and for the Nk4 family the rapid evolving Drosophila gene tin was replaced by an ortholog from annelid (Pd NK4). In addition, six genes from other protostome or cnidarian genomes were added to represent gene families known to be missing from Drosophila . Only 100 bootstrap resamplings were performed on this dataset because of its large size (354 homeodomains). Trees were displayed using TreeExplorer [Phylogenetic analyses were performed with homeodomain sequences, after each had been edited to an alignment of 60 amino acids  method. Bootstrap values supporting gene family designations are shown. Homeodomain sequences derived from pseudogenes are excluded. This ML tree should be compared with the neighbor-joining (NJ) tree shown in Additional file Drosophila melanogaster homeodomains, plus selected additional homeodomains from other protostomes or cnidarians when the gene family is divergent or absent in Drosophila. Divergent Drosophila genes that do not group with human genes were identified by construction of a preliminary, non-bootstrapped ML and NJ trees, and subsequently removed from the dataset. These include genes lost from human, as well genes known to have undergone unusually rapid evolution in Drosophila. For the Hox3 family the rapidly evolving Drosophila genes bcd, zen and zen2 were replaced with Sm Hox3b, and for the Nk4 family the rapid evolving Drosophila gene tin was replaced with Pd NK4. In addition, six genes from other protostome or cnidarian genomes were added to represent gene families known to be missing from Drosophila . Species abbreviations: Am, Apis mellifera (honeybee); Dm, Drosophila melanogaster (fruitfly); Hv, Hydra vulgaris (hydrozoan); Nv, Nematostella vectensis (starlet sea anemone); Pd, Platynereis dumerilii (annelid worm); Ps, Phascolion strombus (sipunculan worm); Sm, Strigamia maritima (centipede). ML performed more poorly than NJ in recovering several well known gene families, notably Hox4, Hox5, Nk4 and Alx. In contrast, ML did recover PROP1 and CG32532 as a true gene family; NJ did not. The invertebrate gene does not always lie as a strict outgroup to all human genes in a family; this effect is expected when using a short alignment. Instead, distinct grouping of invertebrate and human genes is taken as evidence of ancestry from a single gene. A few ambiguous cases were encountered, notably divergence of Drosophila H2.0 in the proposed Hlx gene family, and resolution within the Pax4/6 gene family, which is recovered as two families in NJ but one in ML. As explained in the text, several human gene families contain 'orphan' genes without invertebrate orthologs; these are Barx, Nanog, Noto, Vax, Ventx, Argfx, Dprx, Dux, Esx, Hesx, Hopx, Isx, Leutx, Mix, Nobox, Rhox, Sebox, Tprx, Hdx, Pou5, Hmbox, Satb, Adnp and Zhx/Homez. Zeb and Mkx would be placed in this category based on our ML and NJ trees, although other data suggest that Drosophila zfh1 and CG11617 respectively may be the protostome orthologs [Drosophila ortholog simply lacks the homeobox [rthologs ,94. Tshzhomeobox ,96. PhylClick here for fileNeighbor-joining phylogenetic tree of all human plus selected protostome and cnidarian homeodomains for identification of gene families. Arbitrarily rooted phylogenetic tree of all human plus selected protostome and cnidarian homeodomains constructed using the neighbor-joining (NJ) method. Bootstrap values supporting gene family designations are shown. Homeodomain sequences derived from pseudogenes are excluded. Comparison of NJ and ML trees, and description of the dataset used, is given in the legend to Additional file HOPX.Click here for fileNeighbor-joining phylogenetic tree of human ANTP-class homeodomains, for comparison to maximum likelihood tree. Arbitrarily rooted phylogenetic tree of human ANTP-class homeodomains constructed using the neighbor-joining method. Bootstrap values supporting internal nodes with over 70% are shown. Homeodomain sequences derived from pseudogenes are excluded. The proposed division between the HOXL and NKL subclasses is indicated. The position of EN1 and EN2 is unstable; this tree places them close to the base of the HOXL/NKL divergence, whereas maximum likelihood analysis of the same dataset places them firmly in the NKL subclass  was derived from a compilation of 247 human homeodomain sequences. The three horizontal lines indicate the positions of the three alpha-helices. The numbering scheme refers to amino acid position in the canonical 60-amino-acid homeodomain; insertions relative to this sequence are shown when present. Black shaded resides are invariant between all human homeodomains within each class (or family in the case of the ZF homeodomains). Sequence accession numbers are shown. For each gene family designation, maximum likelihood and neighbor-joining bootstrap support values are indicated (see Additional files Click here for filePhylogenetic input file. All human and invertebrate homeodomains used in phylogenetic analyses are shown, after alignment and removal of insertions to give a uniform 60-amino-acid alignment.Click here for file


With a median follow-up of 4 years and 9 months a significant reduction in odds of death  was observed for those with adjuvant treatment (65% at 5 year) compared to the observation group (55%). Improved relative survival was present in stage III (56% vs 44%), and in stage II patients (78% vs 70%). In rectal cancer a non-significant difference in disease-free or overall survival was observed. Distant metastases developed in 76%, while local recurrence alone occurred in 14%. An early start of adjuvant treatment (< 4 weeks) did not affect results. Compliance to 5FU plus levamisole was 69%. Severe toxicity did not occur. In conclusion, one year 5FU plus levamisole was of benefit in stage II and III colonic cancer; in rectal cancer a significant positive effect could not be demonstrated. © 2001 Cancer Research Campaign  http://www.bjcancer.comBased on the first favourable results of adjuvant therapy of 5FU plus levamisole in Dukes C colonic cancer in 1990, we conducted a prospective trial. 1029 patients were randomised to receive one year 5FU plus levamisole or no further treatment following curative surgery for stage II or III colon (


BMP4 and Wnt3a were required between differentiation day (d) 1.5 and 3 to most effectively induce Mixl1, whilst Activin A induced Mixl1 expression in ESC when added between d2 and d4, indicating a subtle difference in the requirement for Activin receptor signalling in this process. Stimulation of ESCs with these factors at earlier or later times resulted in little Mixl1 induction, suggesting that the differentiating ESCs passed through ‘temporal windows’ in which they sequentially gained and lost competence to respond to each growth factor. Inhibition of either Activin or Wnt signalling blocked Mixl1 induction by any of the three mesendoderm-inducing factors. Mixing experiments in which chimeric EBs were formed between growth factor-treated and untreated ESCs revealed that BMP, Activin and Wnt signalling all contributed to the propagation of paracrine mesendoderm inducing signals between adjacent cells. Finally, we demonstrated that the differentiating cells passed through ‘exit gates’ after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression.We identified the periods during which Bone Morphogenetic Protein (BMP) 4, Wnt3a or Activin A were able to induce expression of the mesendoderm marker, These studies suggest that differentiating ESCs are directed by an interconnected network of growth factors similar to those present in early embryos and that the timing of growth factor activity is critical for mesendoderm induction. Oct4 and Sox2 (inner cell mass), Fgf5 (epiblast) and Brachyury, Mixl1 and Gsc (primitive streak) Pdx1 (foregut endoderm), Nkx2-5 (cardiac mesoderm) and βH1 globin (yolk sac erythroid cells) The in vitro differentiation of embryonic stem cells (ESCs) represents an accessible system for analyzing parameters influencing the early stages of lineage specification and commitment. During differentiation, ESCs pass through a series of developmental milestones that mirror those traversed by cells within the embryo Brachyury, Mixl1 and GoosecoidNot only is there a correspondence between the developmental pathways followed by cells in vitro and in vivo, but there is a similar concordance between the factors that induce and pattern ESCs and the embryo during differentiation. For example, induction of the primitive streak, the structural harbinger of mesendoderm formation in the embryo, requires the activity of a number of secreted growth factors . Finally, we observed that the differentiating cells passed through ‘exit gates’ after which point they were no longer dependent on signalling from inducing molecules for Mixl1 expression. Overall, our study suggests that growth factor regulatory loops similar to those present in early embryos also exist within EBs. The timing of growth factor activity is critical for the initiation of mesendoderm formation from ESCs and paracrine signalling contributes to mesendoderm development.In this study we determined the periods within which BMP4, Wnt3a and Activin A induced mesendoderm in differentiating mouse ESCs. These factors acted during discrete ‘temporal windows’ to induce expression of a GFP reporter gene targeted to the locus of the primitive streak gene, Mixl1GFP/wMixl1, a gene whose expression is restricted to the mesendodermal precursors of the primitive streak We utilised a genetically modified mESC line, Mixl1GFP/w ESCs were differentiated in a chemically defined medium (CDM) + cells were observed on this day, and expression rapidly waned thereafter. This was consistent with observations that GFP maturation and fluorescence lagged behind the peak of Mixl1 mRNA expression that was maximal at d4 of differentiation Mixl1 locus (denoted Mixl1GFP) when present in the cultures from d1–2  and d2–3 (44.2±9.6%) (+ cells was observed when BMP4 was added from d1.5–2.5 (55.8±4.6%). A lower frequency of GFP+ cells was seen in d5 cultures stimulated between d2.5 and d3.5 (21.2±7.4%)  . Experim.2±9.6%)  indicate.2±7.4%) . FinallyMixl1 induction window for each growth factor. These experiments confirmed that BMP4 and Wnt3a were most effective at inducing GFP+ mesendoderm precursors when present in the cultures between d1.5 and d3 . On the basis of these preliminary studies, further differentiation experiments were performed in which the growth factors were included prior to, during and after the hypothesized optimal 5 and d3 . In cont5 and d3 . The incMixl1GFP/w reporter ESC line, (clone Mix 114) Mixl1GFP/w clones were derived from the same parental ESC line, we cannot rule out the possibility that different strains of ESCs might vary in their propensity to differentiate. However, in our experience, ESC lines generally respond to growth factors in a similar manner, with the main differences being in the concentration of factors required for the development of specific cell types .Similar outcomes in experiments performed with an independently targeted Mixl1 locus, and that the variable fluorescence intensity observed indicated that not all Mixl1GFP expressing cells were identical. Interestingly, we have previously observed that this heterogeneity for Mixl1GFP expression is more evident within individual EBs than between EBs Even in our relatively well defined, short-term differentiation experiments, we observed that, at best, 60–70% of cells expressed GFP from the Mixl1GFP/w cells. In the first instance, we examined the effects of adding inhibitors for Wnt and TGF-beta pathways on the ability of each ligand to induce MixlGFP expressing cells. ESCs were treated with BMP4 or Wnt3a from d1 or with Activin A from d2, because preliminary experiments showed that addition of this factor prior to d2 inhibited ESC mesendoderm differentiation (data not shown). Inhibitors of BMP4- (noggin), Activin receptor- (SB 431542) or canonical Wnt- (Dkk-1) signalling were added to the cultures at the same time as the growth factors. Cultures were analysed at differentiation d5 for expression of GFP by flow cytometry.We sought to determine whether all three growth factor signalling pathways were required for GFP induction in differentiating + cells observed in d5 cultures which had been treated with Activin A  fell outside the window of optimal responsiveness of the EBs to direct BMP4 or Wnt3a induction of Mixl1GFP+ cells.These results implied that endogenously produced BMP, Wnt and Activin-like molecules might play an important role during mesendoderm induction during ESC differentiation in vitro by complementing the actions of exogenously applied growth factors. To directly assay the paracrine mesendoderm inducing ability of these endogenous factors, we performed mixing experiments in which wild type eric EBs . These c+ cells were observed in the chimeric EBs at d5  was assessed. In the absence of inhibitors, wild type ‘stimulator’ ESCs treated with BMP4 or Wnt3a between d1–d3 effectively induced GFP expression in ‘responder’ /w cells . Inclusirequired . In addiP/w ESCs . This arP/w ESCs .Mixl1GFP/w cells closed soon after d3, consistent with the observation that addition of these growth factors after this time did not recruit many new cells into the mesendoderm differentiation program. However, this scenario did not exclude an ongoing requirement for active signaling past d3 for maximal GFP induction and/or maintenance in cells that had already committed to mesendoderm formation, a possibility raised by the effects of signaling pathway inhibitors on paracrine mesendoderm signals shown in Results of studies presented thus far suggested that the window during which BMP4 and Wnt3a efficiently induced GFP expression in Mixl1GFP/w cells were differentiated until d3 in the presence of BMP4 or Wnt3a (both added at d1) or Activin A (added d2). At d3, the factors were removed and cells differentiated for a further two days in the presence or absence of inhibitors affecting each pathway  whilst Dkk-1 and SB 431542 reduced the proportion of GFP expressing cells by ∼70% (from 67.8±2.0% to 22.3±6.9%) and ∼80% (from 67.8±2.0% to 11.7±2.3%) respectively (+ cells was seen following addition of Dkk-1 and SB 431542 (∼75% and ∼90% respectively), whilst a lesser reduction in the proportion of GFP+ cells was observed in response to treatment of cells at d3 with noggin (∼35%)  . Interesells see . This inMixl1 RNA had begun to wane in cells stimulated by BMP4 or Wnt3a  than that seen with either ligand alone (2.0±0.7% for Wnt3a and 29.6±6.3% for Activin A) and higher than that predicted by addition of contributions representing the individual factors  comprise mixtures of many different adult tissue types, whilst their malignant counterparts (teratocarcinomas) also include persistent undifferentiated stem cell components, termed embryonal carcinoma cells (ECCs) The link between normal embryos and teratocarcinomas had been made when Solter Mixl1, a homeobox gene that marks the primitive streak of the mammalian embryo, was linked to a fluorescent reporter Mixl1 expression.We have built on these earlier observations though our investigations of the induction of mesendoderm precursors by exogenously acting growth factors in differentiating mouse ESCs. Whilst early studies proved that ECCs (and later ESCs) could differentiate to form derivatives of the germ layers, the signals initiating differentiation were provided by serum and a specific dissection of the control mechanisms was not possible. We have used a suspension, embryoid body differentiation system, in which a serum free defined medium enabled us to objectively assess the influence of specific growth factors. Our studies were also aided by the use of a genetically modified ES cell line in which the induction of FGF5+Oct4+ ESC phenotype to a Rex1-Oct4+ epiblast-like state Mixl1GFP/w cells (data not shown), were consistent with the hypothesized anti-differentiative role for nodal  during the earliest stages of differentiation Following the removal of the anti-differentiative signal, LIF, cells gained responsiveness to BMP4 and Wnt3a as mesendoderm inducing signals between d1.5 and d3, at a time corresponding to their upregulation of epiblast associated genes, such as Brachyury and Mixl1 at d3, they only weakly up-regulated the anterior mesendoderm/early endoderm markers Goosecoid, Foxa2, and Sox17. Conversely, Activin A induced higher levels of these genes at d5 of differentiation , by Wnt3a and Activin A. This synergy is also consistent with results reported by Hansson and colleagues who noted the late requirement for Wnt signalling in Activin A induction of Sox17+ definitive endoderm Our data demonstrating that BMP4 propagates mesendoderm-inducing signals in differentiating EBs via the induction of endogenous TGF and Wnt growth factors are also consistent with the autoregulatory induction loops proposed to initiate and maintain gastrulation in mouse embryos + cells observed in cultures where growth factors were removed at d3 compared with cultures that were also treated with inhibitors, suggested that mesendoderm formation remained dependent upon a growth factor for a short period even after it was removed from the culture. Experiments in which we evaluated the effects of adding inhibitors to growth factor induced cultures after d3 confirmed that the requirement for BMP4 was lost earlier than dependence upon Wnt or nodal signalling. This was evidenced by the higher percentages of d5 Mixl1GFP+ cells in noggin treated cultures compared to cultures in which Wnt or Activin signalling was inhibited  Mixl1GFP/w (clone Mix147 and Mix114) 4 cells/ml in non-adherent 6 cm bacteriological dishes  or by spin EB formation with 5×102 cells seeded per well in non-adherent round bottomed 96 well plates (Nunc) in a chemically defined medium (CDM) Mixl1GFP/w cells. Growth factors were removed from the cultures by pelleting EBs by centrifugation (480×g), aspirating the media, washing once with PBS, and resuspending the EBs in fresh CDM. The EBs were then transferred to a non-adherent bacteriological dish and returned to a humidified 37°C incubator (8% CO2 in air). GFP expression was analysed by flow cytometry at day 5 of differentiation unless otherwise stated.The Mixl1w/w and Mixl1GFP/w cells were differentiated as EBs in parallel cultures for 3d. Mixl1w/w EBs were differentiated in the absence of growth factors (control cells) or in the presence of BMP4 (10 ng/ml), Wnt3a (100 ng/ml) or Activin A (100 ng/ml) (stimulator cells). BMP4 and Wnt3a were included from the onset of differentiation whilst Activin A was added at d2. For the first 3d, Mixl1GFP/w cells (responder cells) were differentiated in the absence of added growth factors. After 3d, EBs from both lines were harvested, washed in PBS, and trypsinised to form a single cell suspension. The disaggregated Mixl1w/w and Mixl1GFP/w cells were combined at a ratio of 1∶1 in CDM. Two thousand cells were placed into each well of low adherent round bottomed 96 well trays (Nunc) and chimeric spin EBs were formed by aggregation of the cells following centrifugation RNA was extracted using an RNeasy Mini Kit (Qiagen) and cDNA synthesized using SuperScript III (Invitrogen Corporation) according to the manufacturer's instructions. PCR gene expression analysis was performed as described previously Figure S1BMP4, Wnt3a and Activin A display a similar pattern of mesendoderm inducing activity in differentiating Mixl1GFP/w clone Mix 114. (A) Flow cytometry analysis of d5 Mixl1GFP/w clone 114 ESCs differentiated in cultures supplemented with 10ng/ml BMP4, 100ng/ml Wnt3a or 100ng/ml Activin A for the given time intervals, indicating the proportion of GFP+ cells. The growth factor treatment for each experiment is indicated and the corresponding no growth factor (-GF) control flow cytometry profiles are shown to the left of each series. (B) Representative brightfield and epifluorescence images of differentiating EBs. The growth factors and period of addition are indicated to the left of each row and day of differentiation when the image was taken in the top right hand corner of each panel. .(12.87 MB TIF)Click here for additional data file.Figure S2Kinetics of mesendoderm gene expression in EBs differentiated in BMP4, Wnt3a and Activin A. Real time PCR analysis for the indicated genes at (A) d3 and (B) d5 in ESCs differentiated in the absence of growth factors (-GF) or presence of BMP4, Wnt3a or Activin A for the indicated periods .(2.19 MB TIF)Click here for additional data file.Table S1Sequences of primers used for PCR analysis shown in (0.04 MB DOC)Click here for additional data file.Table S2Probe sets for real-time PCR analysis shown in (0.03 MB DOC)Click here for additional data file.


MTUS1) has been recently identified as a candidate tumor suppressor gene which resides in a genomic region (8p22) that shows frequent loss of heterozygosity (LOH) in several tumor types. It has been suggested that multiple gene promoters and alternative splicing lead to the expression of 5 known MTUS1 transcript variants.Mitochondrial tumor suppressor gene 1 (MTUS1 transcript variants.Here, we characterized the 5' untranslated regions of the different transcript variants. We also cloned and functionally tested the alternatively utilized gene promoters that contribute to the production of different MTUS1 gene are driven by multiple gene promoters.Our results confirmed the early hypothesis that the transcript variants of  MTUS1 gene is located in a region (8p22) that shows frequent loss of heterozygosity (LOH) in several tumor types, including oral cancer [MTUS1 variants comes from the study on Xenopus Icis gene, a homolog of MTUS1 variants 1 and 2, which regulates microtubule growth and spindle formation prior to anaphase [MTUS1 gene and functionally cloned the alternatively utilized gene promoters that control the production of these MTUS1 transcript variants. This will enhance our understanding on the regulation of this candidate tumor suppressor gene.The region 8p2 that shl cancer . It reprl cancer . Evidencanaphase . Here, wMTUS1 transcript variants, 5'-RACE assays were carried out using human brain reference mRNA (Ambion Inc) and a FirstChoice RLM-RACE kit from Ambion, with primers specific for various transcript variants .To characterize the 5' untranslated regions (5'-UTR) of the . The predicted transcription elements of the putative promoters were listed in Additional file MTUS1 transcript variants, the following 4 fragments were PCR amplified using specific primers  a 773 bp fragment (P1') located at the 5' flanking region of exon 1; 3) a 529 bp fragment (P2) located at 5' flanking region of exon 5; and 4) a 733 bp fragment (P3) located at 5' flanking region of exon 8. The PCR products were then cloned into the KpnI/XhoI sites of pGL4.10 vector. After verification by DNA sequencing, the constructs were transiently transfected into cells using lipofectamine 2000 (Invitrogen). The pGL4.74 vector (Promega) was co-transfected as internal control for normalization of the transfection efficiency. After 48 hours, transfected cells were harvested with ice-cold phosphate-buffered saline, and dual luciferase assay were performed according to the manufacturer's protocol (Promega) using a Lumat LB 9507 Luminometer (Berthold Technologies).The gene promoter prediction was carried out using MatInspector Professional MTUS1 transcription variants that are controlled by 3 different gene promoters, 5' RACE assays were performed as described in Materials and Methods section. The 5'RACE assay designed for the long forms of MTUS1 transcription variants  leads to the cloning of the 3 alternatively utilized exons of 91 bp, 169 bp, and 410 bp, respectively . RT-PCR assays confirm the existence of these non-coding exons (data not shown). The 5'RACE assays designed for transcription variant 4 and 5 confirm the existence of these variants and also refined the 5' untranslated region for variant 5  may be regulated by both P1 and P1' promoters. It is possible that these 2 gene promoters may serve a cooperated control circuit in vivo to regulate the expression of this gene. This interesting phenomenon has been observed in a number of other genes, including gene for bradykinin B1 receptor [Based on sequence conservation and cap-analysis gene expression (CAGE) database, 3 gene promoters were predicted: P1, located at the 5'-flanking region of the gene; P2, located in intron region 5'-flanking exon 5; and P3, located in intron region 5'-flanking exon 8. These gene promoters were supported by sequence conservation  and CAGE database . The genomic locations of these putative promoters support the existence of multiple transcript variants of e Figure . Interesreceptor , and proreceptor . Future MTUS1 gene and demonstrated the presence of multiple gene promoters. Characterization of this gene will now facilitate studies on the physiological and pathophysiological function and transcriptional regulation of the MTUS1.In summary, our results refined the genomic structure of the The authors declare that they have no competing interests.HY and XZ conceived the idea for the project. JY and XL performed the laboratory analyses. JY, HY and XZ drafted the manuscript. All authors read and approved the final manuscript.Table S1. Gene-specific Primers used for the 5'-RACE assay.Click here for fileTable S2. Predicted transcription elements in MTUS1 promoters.Click here for fileTable S3. Primers used to clone promoter regions of the MTUS1 gene.Click here for file


AAVS1 . Integration at AAVS1 requires the AAV2 replication (Rep) proteins and a DNA sequence within AAVS1 containing a 16 bp Rep recognition sequence (RRS) and closely spaced Rep nicking site . The AAV2 genome is flanked by inverted terminal repeats (ITRs). Each ITR contains an RRS and closely spaced trs, but the sequences differ from those in AAVS1. These ITR sequences are required for replication and packaging.Adeno-associated virus type 2 (AAV2) preferentially integrates its DNA at a ~2 kb region of human chromosome 19, designated AAVS1 RRS and trs can function in AAV2 replication, packaging and integration by replacing a 61 bp region of the AAV2 ITR with a 49 bp segment of AAVS1 DNA. Modifying one or both ITRs did not have a large effect on the overall virus titers. These modifications did not detectably affect integration at AAVS1, as measured by semi-quantitative nested PCR assays. Sequencing of integration junctions shows the joining of the modified ITRs to AAVS1 sequences.In this study we demonstrate that the AAVS1 sequences to substitute for the AAV2 RRS and trs provides indirect evidence that the stable secondary structure encompassing the trs is part of the AAV2 packaging signal.The ability of these  AAVS1 dCTP and oligolabeling beads . Hybrisol  was used for the hybridization. A 1 Kb DNA ladder  and pSub201(-) were used as DNA markers. Several of the bands in the 1 Kb DNA ladder contain DNA that is in pSub201(-) and hybridize to the probe. The pSub201(-) marker was made by combining PvuII and XbaI digests of pSub201(-). The digestions were stopped with 5 mM EDTA before combining.Total DNA from HEK293 cells transfected in parallel with those used for the preparation of virus supernatants was isolated using a DNeasy tissue kit  24 and 48 hours post transfection. Some samples were pre-treated with Dpn I to degrade input plasmid. Two micrograms of DNA from each sample was resolved on a 1% agarose gel and transferred to positively charged nylon membrane  for Southern blotting analysis. Briefly, the DNA was first fragmented by soaking the gel in several volumes of 0.25 M HCl for 10 minutes. The gel was washed for several minutes with water and DNA was denatured by soaking the gel in 1.5 M NaCl, 0.5 N NaOH for 30 minutes. The gel was placed front down on a solid support covered by a piece of Whatman 3 mm paper long enough to drape into a reservoir of 10× SSC . The positively charged nylon membrane was placed on the back of the gel below a stack of paper towels. After a 16 hour transfer the membrane was washed several times with 5× SSC and dried in bright light. The membrane was probed with random primer labeled pSub201(-). The pSub201(-) probe was made using [α-2 flask of ~25% confluent HeLa cells was infected for 2 hours in medium without fetal bovine serum (FBS). After 2 hours the medium was replaced with medium containing 10% FBS. Cells were harvested 48 h after infection, and genomic DNA was isolated using a DNeasy tissue kit . Several combinations of AAV2 and AAVS1 primer pairs were used to detect integration by nested PCR. For the nested PCR assay 50 ng of genomic DNA and 100 ng of each primer in a 50-μl reaction volume were used in the first round of PCR amplification. After an initial incubation for 4 min at 94°C, the reaction mixture was subjected to 28 cycles of PCR amplification for 1 min at 94°C, 1 min of annealing at 63°C, and 3 min at 72°C, using FastStart DNA polymerase (Roche). One percent of the amplification product was diluted into a new reaction mixture containing the second pair of primers. The PCR parameters were the same as those for the first amplification. The following primer sets were used. In each set the first primer listed was used in the first amplification and the second primer was used in the second amplification. AAV2 rep 5'-CAC CCA GTT CAC AAA GCT GTC AGA AAT G-3' and 5'-TCG CTG GGG ACC TTA ATC ACA ATC TC-3', AAV2 cap 5'-CAG GAC AGA GAT GTG TAC CTT CAG GG-3' and 5'-TGG ACA CTA ATG GCG TGT ATT CAG AGC-3', AAV2 ITR 5'-GCC TCA GTG AGC GAG CGA G-3' and 5'-GCA GAG AGG GAG TGG CCA-3', AAVS1 5'-AGG CAG ATA GAC CAG ACT GAG CTA TGG-3' and 5'-CAG GGA AGG AGA CAA AGT CCA GGA-3'. PCR products were resolved on a 1% agarose gel and stained with ethidium bromide. Cloning and sequencing of PCR-amplified junctions were performed as described previously [A 25 cmeviously .R.A.O. is a co-inventor on several patents involving AAV vectors. To the extent that this work will increase the value of those patents, he has a competing interest.VJM was the primary contributor to project conception, overall experimental design, plasmid construction, virus production, cell infection, integration assays, data analysis and writing of manuscript. He also performed all experiments.RAO was overall project coordinator, and contributed to experimental design, data analysis and writing of the manuscript.All authors read and approved the final manuscript.