The PCR assay was used to screen 42 strains of SOP (most without published genome sequence) representing the recognized diversity of this guild. For 13 of these strains dsrAB was detected and the respective PCR product was sequenced. Interestingly, most dsrAB-encoding SOP are capable of forming sulfur storage compounds. Phylogenetic analysis demonstrated largely congruent rDSR and 16S rRNA consensus tree topologies, indicating that lateral transfer events did not play an important role in the evolutionary history of known rDSR. Thus, this enzyme represents a suitable phylogenetic marker for diversity analyses of sulfur storage compound-exploiting SOP in the environment. The potential of this new functional gene approach was demonstrated by comparative sequence analyses of all dsrAB present in published metagenomes and by applying it for a SOP census in selected marine worms and an alkaline lake sediment.Sulfur-oxidizing prokaryotes (SOP) catalyse a central step in the global S-cycle and are of major functional importance for a variety of natural and engineered systems, but our knowledge on their actual diversity and environmental distribution patterns is still rather limited. In this study we developed a specific PCR assay for the detection of  Bacteria and Archaea employ reduced sulfur compounds as electron donors for growth and exploit this capability to successfully compete with other prokaryotes in various ecosystems. Surprisingly, a variety of sulfur oxidation pathways does exist in members of this guild (see aprBA (apsBA) (soxB (dsrAB coding for reversely operating sirohaem dissimilatory sulfite reductase (rDSR). This enzyme is homologous to, but phylogenetically clearly distinguishable from the dissimilatory (bi)sulfite reductase that catalyses the energy-conserving reduction of sulfite to sulfide in anaerobic sulfite/sulfate-reducing prokaryotes (SRP)   and soxBdsr gene cluster  and carries a thioredoxin motif typical for disulfide reductases, is a candidate for catalysing reductive release of sulfide from the carrier in the cytoplasm. rDSR could then oxidize sulfide to sulfite, followed by further oxidation to sulfate catalysed by other enzymes  to the database which now consists of diverse alpha-, beta- and gammaproteobacterial SOP, including many, but not all of the tested members of the families Chromatiaceae and Ectothiorhodospiraceae and most of the green sulfur bacteria (Chlorobiaceae)  . Regardil sulfur . Slow ox tepidum .A. vinosum (dsrAB-containing SOP is their ability to build up periplasmic (e.g. Chromatiaceae)   by blast analysis of all available SOP genomes .In agreement with the essential function of rDSR for oxidation of sulfur globule deposits in tiaceae)  or extrabiaceae)  reservesdsrAB among SOP. Under the explicit assumption that 16S rRNA genes of the analysed species were not influenced by LGT  and within the Chlorobiaceae (Chlorobium phaeobacteroides BS1), potentially indicating genetic exchange among members of the same class and family respectively . No evidence for additional dsrAB copies was obtained in cases where multiple dsrAB clones per species were sequenced. Out of those SOP whose genomes are completely sequenced  that are unexpectedly not linked with a corresponding dsrB . Initially, we surveyed the 7.7 million sequence reads from the Global Ocean Sampling expedition  representing different marine surface environments. These 83 sequences showed more than 90% amino acid identity to each other and to the rDSR sequence encoded on the BAC clone MED13k9 from the Mediterranean Sea . Elucidating the phylogeny and physiological function of the dsrAB-containing microorganisms in the anaerobic enrichment remains subject of further study.We also identified several partial t length . While sdsrAB sequences of SOP in metagenomic data sets, a considerable number of all currently available dsrAB sequences stem from sulfur-oxidizing bacterial symbionts of eukaryotic hosts , rDSR sequences from the Paracatenula and the Stilbonema worm symbionts were affiliated with the Alpha- and the Gammaproteobacteria respectively . This habitat was selected, because presence of SOP in similar ecosystems has been reported previously  sediment of the lake Herrnsee. However, given that (i) rDSR is potentially involved in oxidation of polysulfides/elemental sulfur and (ii) polysulfides are stable at high pH and therefore considered important substrates for SOP under these conditions . Although the number of database entries is still relatively low, their generally 16S rRNA-conform phylogeny suggests that dsrAB was mainly inherited vertically among SOP and is thus a useful phylogenetic marker for those SOP which contain these genes. Equipped with the newly developed primer set for amplification of dsrAB from phylogenetically diverse SOP (e.g. Proteobacteria and Chlorobi), microbial ecologists are now able to retrieve such dsrAB sequences from the environment and to assign them to known phyla and classes or to novel lineages, as demonstrated by the described proof-of-principle experiments. This study thus extends the functional gene toolbox for monitoring members of defined microbial guilds in the environment and will help to better understand the biogeography and ecology of rDSR-containing SOP.In this study a Stilbonema sp. and the two flatworms Paracatenula spp. were collected in shallow subtidal sand at Carrie Bow Cay (Belize) 16°48′N, 88°05′W and were immediately stored in absolute ethanol at 4°C.Cultures of reference organisms where obtained from the Deutsche Sammlung von Mikroorganismen und Zellkulturen  or from colleagues at other institutes . SedimenPure culture DNA was isolated by using the DNeasy Blood and Tissue Kit . Briefly, harvested cells were incubated at 37°C with proteinase K for 3 h and the DNA was extracted according to the protocol for Gram-negative bacteria. Genomic DNA from the sediment sample was extracted by using the PowerSoil™ DNA Isolation Kit , according to the manufacturer's instructions.dsrAB fragment was performed with mixtures of the rDSR1F and rDSR4R primers. The concentrations of primers rDSR1Fa, rDSR1Fb, rDSR1Fc, rDSR4Ra and rDSR4Rb were adjusted to achieve an equimolar concentration of 100 nM for each primer variant in the PCR  and 20 mM tetramethylammonium chloride  in a total volume of 50 μl. Standard thermal cycling was carried out by an initial denaturation step at 94°C for 1 min, followed by 35 cycles of denaturation at 94°C for 40 s, annealing at 48°C for 40 s and elongation at 72°C for 2 min. Cycling was completed by a final elongation step at 72°C for 10 min. A relatively low annealing temperature of 48°C was chosen in order to potentially allow for amplification of novel dsrAB from SOP with mismatches in the primer binding sites  as described previously  was additionally designed for sequencing of the internal dsrAB sequence part from all SOP.For subsequent sequencing, dsrAB-containing reference organisms was checked by 16S rRNA gene amplification and direct sequencing.The pure culture status of A. vinosum (Accession No. U84760) were used for blast searches of homologues in the genome database  at GenBank (Candidatus K. stuttgartiensis (blastx. Gene sequences of dsrAB in the metagenomic databases at the Joint Genome Institute (blast using the dsrAB sequences of A. vinosum and the Mediterranean Sea BAC clone MED13k9 (DQ068067) as query.DsrAB protein sequences from arb program package (arb neighbour joining with Jukes-Cantor correction) trees. Individual trees were calculated based on 16S rRNA alignment positions (n = 1283) conserved in more than 50% of the Bacteria.Phylogenetic analyses were performed by using the dsrAB sequences were imported into a dsrAB/DsrAB-ARB database for SRP maintained at the Department of Microbial Ecology, University of Vienna (dsrAB locus) and 551 amino acid positions (representing the dsrAB fragment that is amplified by the rDSR1F and rDSR4R primers) were used for phylogenetic inference of DsrAB sequences from SOP. For paralogous rooting, DsrA sequences were aligned against DsrB sequences by clustalx and subsequent manual adjustment. Paralogous trees were calculated using indel filters for full-length (226 amino acid positions) and partial sequences (132 amino acid positions). Dsr protein trees were inferred by using maximum-likelihood , maximum-parsimony  and distance-matrix  methods. Consensus trees were based on maximum-likelihood , maximum-parsimony and distance-matrix trees and were drawn by using established protocols .All dsrAB-associated reads’ and the relative abundance of ‘dsrAB read equivalents’ among sequences from the Global Ocean Sampling expedition were calculated as outlined previously , gyrA (encoding DNA gyrase subunit A) and rpoB , were used for normalization.The number of ‘Bacteria and Archaea (TOBA Release 7.7) (International Journal of Systematic and Evolutionary Microbiology.Names of bacterial and archaeal taxa were used according to the Taxonomic Outline of the dsrAB sequences were deposited at GenBank under the Accession Nos EU155020–EU155055.Newly determined


Research demonstrating rapid viral replication in mucosal tissue and subsequent dissemination throughout the body suggested that research priorities should shift towards a mucosal vaccine. Findings reported in Track C indicated that, in addition to reducing vertical transmission, antiretroviral therapy (ART) also lowers the risk of prematurity, stillbirth and abortion.Challenging concerns about the potential “disinhibiting” effect of ART as prevention, a Kenyan study found that widespread ART encourages greater use of condoms and does not increase the rate of risky sex. Another Kenyan study found that pregnancy increases the risk of HIV transmission in a cohort of serodiscordant couples. Although three randomized trials have conclusively demonstrated that circumcision reduces HIV transmission among heterosexual men, research presented at IAS 2009 found no evidence of a preventive impact for women. IAS 2009 included no major reports on three biomedical prevention fronts , although important Phase III microbicide and PrEP trials are under way and evidence is mounting regarding the impact of ART rollout on reducing HIV transmission ; an observational study of 3273 pregnant women in Malawi and Mozambique showed that it also lowers the risk of prematurity, abortion and stillbirth among African women, providing additional insights into the benefits of ART [Leonardo Palombi  reported that rates of abortion or stillbirth were 4.3% in the ART group and 25.7% in the no-ART group (P <0.001). Regardless of CD4 cell stratum, the prematurity rate was 70.8% lower in women who took antiretrovirals.A prospective study of 898 female bar workers in Mombasa, Kenya, found no evidence supporting the hypothesis that widespread ART will encourage more risky sex in low-income countries. Indeed, women in this cohort reported safer behaviour after they began ART  than befPregnancy doubled the risk of HIV transmission in a two-year study of more than 500 HIV-discordant couples  in Kisumu, Kenya [Those rates translated into a 1.8 times higher risk of HIV infection in couples who had a child (P=0.046). About 30% of new HIV infections happened six months before conception, another 35% six months after conception, and the remaining 35% more than six months from conception. The presenting author noted that the data did not determine intentionality to conceive and that – if this is indeed the case – couples may be risking a greater probability of HIV transmission in order to conceive.Three randomized trials conclusively demonstrate that circumcision lowers risk of HIV acquisition in heterosexual men, and circumcision programmes are being rolled out in a number of countries in sub-Saharan Africa  and uncircumcised men (12%) . The invTwo previously published placebo-controlled trials of daily acyclovir found that this antiherpes drug does not prevent HIV acquisition in women and men infected with herpes simplex virus type 2 (HSV-2) ,12. A trConnie Celum  reported that after 24 months of follow up, 84 new HIV-1 infections could be linked to study partners, 41 of them taking acyclovir and 43 taking placebo. Daily acyclovir significantly lowered the frequency of HSV-2-positive genital ulcers and reduced HIV load by an average 0.25 log, but those benefits were insufficient to protect partners from HIV transmission.3. Jairam R Lingappa  found that 284 people taking acyclovir and 325 taking placebo reached a composite endpoint of a CD4 count below 200 cells/mm3, ART initiation, or death from causes other than trauma. Those numbers translated into a 27% lower risk of reaching the composite endpoint (P=0.03).Although the modest decline in viral load recorded in this trial did not prevent coinfected partners from transmitting HIV to their partners, even a small decline may confer clinical benefits, according to secondary results of another African study . This plThe 3000-woman analysis demonstrating lower rates of prematurity, abortion and stillbirth in women taking a triple-antiretroviral combination , a findiThe study finding a higher risk of HIV transmission in HIV-discordant couples who have a child  indicateTwo studies failed to establish a lower risk of HIV infection in female partners of circumcised versus uncircumcised men ,10. The The acyclovir trials show that this antiherpes agent has no role in preventing HIV transmission , but it Track C lead rapporteur Sinead Delany-Moretlwe drew four lessons from prevention studies presented at the conference :• Treatment has progressed; prevention remains challenging.• Biomedical interventions still require behavioural change; risk compensation needs to be monitored.• The concept of treatment for prevention brings the treatment and prevention worlds closer than ever before.• New interventions for prevention are most likely to be effective when delivered in combination.Mark Mascolini and Rodney Kort are independent consultants contracted by the International AIDS Society for the purpose of drafting the IAS 2009 Impact Report: Summary of Key Research and Implications for Policy and Practice.MM drafted the initial text and RA provided editorial input and advice. RK adapted the text for publication in a peer-reviewed journal. Both authors have approved the manuscript for publication.Click here for file


Pancreatic ductal adenocarcinoma (PDA) is a lethal disease with a characteristic pattern of early metastasis, which is driving a search for biomarkers that can be used to detect the cancer at an early stage. Recently, the actin-associated protein palladin was identified as a candidate biomarker when it was shown that palladin is mutated in a rare inherited form of PDA, and overexpressed in many sporadic pancreas tumors and premalignant precursors. In this study, we analyzed the expression of palladin isoforms in murine and human PDA and explored palladin's potential use in diagnosing PDA. We performed immunohistochemistry and immunoblot analyses on patient samples and tumor-derived cells using an isoform-selective monoclonal antibody and a pan-palladin polyclonal antibody. Immunoblot and real-time quantitative reverse transcription-PCR were used to quantify palladin mRNA levels in human samples. We show that there are two major palladin isoforms expressed in pancreas: 65 and 85–90 kDa. The 65 kDa isoform is expressed in both normal and neoplastic ductal epithelial cells. The 85–90 kDa palladin isoform is highly overexpressed in tumor-associated fibroblasts (TAFs) in both primary and metastatic tumors compared to normal pancreas, in samples obtained from either human patients or genetically engineered mice. In tumor-derived cultured cells, expression of palladin isoforms follows cell-type specific patterns, with the 85–90 kDa isoform in TAFs, and the 65 kDa isoform predominating in normal and neoplastic epithelial cells. These results suggest that upregulation of 85–90 kDa palladin isoform may play a role in the establishment of the TAF phenotype, and thus in the formation of a desmoplastic tumor microenvironment. Thus, palladin may have a potential use in the early diagnosis of PDA and may have much broader significance in understanding metastatic behavior. Pancreatic adenocarcinoma is the fourth leading cause of cancer death in the United States Palladin is a cytoskeleton-associated scaffold protein that has received attention recently in the pancreas cancer field Palladin exists in all vertebrates as multiple size variants generated from a single gene that possesses alternative promoters, i.e. a “nested gene”. There are three major palladin isoforms that arise from alternative start sites  and multiple minor isoforms that result from alternative splicing PALLD). The palladin gene is unusually large and complex, spanning ∼400 kb and containing at least 25 exons, and it produces multiple palladin isoforms, including three well-described size variants that resolve at 85–90, 140 and 200 kDa http://beta.uniprot.org/uniprot/Q8WX93), and alignment of their sequences is shown in Palladin is a highly conserved protein that is found in all vertebrate species, and it arises from a single gene in mice and humans , and a second isoform-selective monoclonal antibody (4D10) were used to stain multiple sections of pancreas . It should be noted that the COM antibody was used in a previous study to for IHC staining of pancreas tumors (6). The results obtained with the COM antibody were very similar to those generated with the polyclonal 622, and the staining pattern obtained with 4D10 was virtually indistinguishable from that obtained with monoclonal 1E6 . NegativOur IHC results are in agreement with an earlier study, which reported that palladin is strongly overexpressed in the stroma of 171 of 177 sections (96.6%) of PDA tumors Two previous reports have suggested a potential role for palladin in the metastasis of breast cancer by showing that palladin levels are elevated in a highly invasive subpopulation of human breast cancer cells PDA tumor-associated fibroblasts in both primary and metastatic tumors. To extend our observations to other human cancers we performed immunoblot analysis of tumor-associated fibroblasts isolated from five different tumors obtained from breast, lung, ovary, kidney and skin. Pancreas was included as a control. We used both the polyclonal antibody 622 and the monoclonal 1E6 to compare the pattern of isoforms detected in tumor-associated fibroblasts. As expected, The results shown so far indicate that the 85–90 kDa palladin isoform is upregulated in G12DKras) targeted to the pancreas Important insights into the mechanisms of pancreatic adenocarcinoma pathogenesis have been gained from the use of genetically engineered mice that express an activating mutation in the Kras gene  is strongly upregulated in tumor-associated fibroblasts of pancreatic adenocarcinomas. It is well established that when compared to quiescent fibroblasts, TAFs display characteristic changes in gene expression and cell behavior, and are more contractile, motile and secretory Previously, palladin was shown to be rapidly upregulated in activated myofibroblasts that participate in wound closure in the skin These results generate new questions regarding palladin's contribution to TAF behavior. Previous studies using a variety of cultured cell models have established that palladin plays a critical role in the formation of actin-based structures involved in cell motility and contractility, such as stress fibers, dorsal ruffles and podosomes It is important to place our results within the context of earlier findings by multiple groups that have explored palladin expression in the context of tumorigenesis. Palladin was previously shown to contribute to the invasive motility of human breast cancer cell lines, in a study that focused exclusively on the expression of 85–90 kDa palladin The high mortality of pancreatic cancer is attributable to a lack of screening tests, inaccessibility of the pancreas, and late cancer stage at diagnosis This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Institutional Review Board of UNC Hospital (#07-2046). All patients provided written informed consent for the collection of samples and subsequent analysis. All animals were handled in strict accordance with good animal practice as defined by the relevant national and/or local animal welfare bodies, and all animal work was approved by the “Fred Hutchinson Cancer Research Center Institutional Animal Care and Use Committee” .Four different antibodies against palladin were used: two rabbit polyclonals, 622 and commercial (ProteinTech Group) and two monoclonals, 1E6 and 4D10. The commercial antibody was utilized previously for IHC and immunoblot studies Human cell types used in this study include: pancreatic ductal epithelial cells (HPDE) Cells were cultured on 100 mm tissue culture dishes, rinsed briefly with phosphate-buffered saline, and then scraped into a lysis buffer containing 50 mM Tris (pH 7.0), 150 mM NaCl, 1% Triton X-100, and a protease inhibitor cocktail for mammalian tissues. The supernatant was collected after centrifugation at 14,000 rpm for 15 min. The cell lysates were analyzed by immunoblot or frozen with liquid nitrogen and stored at −80°C for future use. For the immunoblot, lysates were boiled in 2X Laemmli buffer, and 20 µg of protein were resolved by SDS-PAGE in each lane of a 4–12% gel. The proteins were transferred to nitrocellulose and immunoblotted. Immunocomplexes were visualized using the Western Lights Chemiluminescence Detection kit from Perkin-Elmer.De-identified patient samples of primary and metastatic tumors were obtained through an IRB-approved study supported through the tissue procurement facility of the Lineberger Comprehensive Cancer Center. The protocol supports the procurement of malignant and non-malignant tissue for cancer-related research, and informed consent is obtained from patients who agree to participate. Five micron-thick, formalin-fixed paraffin-embedded (FFPE) tissue sections were collected on a coated glass slide, dried vertically overnight at room temperature and heated in a 60°C drying oven for one hour. The tissue sections were de-paraffinized in two changes of xylene, hydrated using descending grades of ethanol, and rinsed in a Tris buffer containing Tween 20 . Endogenous peroxidase activity was blocked by incubating in a 3% hydrogen peroxide/methanol solution for 10 minutes. . Antigen retrieval was performed by heating with steam in a citra buffer  for 30 minutes, cooling for 15 minutes at room temperature and placing in Tris buffered solution with Tween for 5 minutes. Immunostaining was performed on the Dako Autostainer platform (DakoCytomation) at room temperature. Briefly, each tissue section was incubated in normal horse serum (Vector Laboratories Elite kit 6102) for 15 minutes; the primary antibody was applied for 60 minutes, and detection was completed by incubating with a biotinylated link. An avidin-biotin complex (Vectastain Elite Kit 6102) was applied for 30 minutes followed by diaminobenzidine (Innovex NB314SB) chromogen for 2 minutes. Signal contrast was maximized by counterstaining in hematoxylin (DakoCytomation Mayer's Hematoxylin S3309) for 1 minute, rinsing in deionized water and finally in a bluing solution (Richard-Allan Scientific 7301) for 30 seconds. Samples were then rinsed in Tris buffer for 5 minutes, dehydrated in ethanol and placed in xylene, and mounted using Permount (Fisher Scientific Permount SP15).Pea-sized pieces of fresh tissue were snap-frozen in liquid nitrogen, ground in a chilled mortar and pestle, and extracted in lysis buffer containing 50 mM Tris pH 7.5, 8 M urea, 5% SDS, 10 mM EDTA, and protease inhibitor cocktail for mammalian tissues. Samples were then centrifuged at 15,000×g to remove any unsolubilized particulates, and the supernatant was resolved by SDS-PAGE, using 15 µg protein loaded per lane.Kras is targeted to progenitor cells of the developing pancreas Primary pancreatic ductal adenocarcinomas and metastases were isolated from genetically engineered mice in which an activating mutation in SYBR@ Green Master Mix (Applied Biosystem). Because the coding sequence of the 85–90 kDa isoform of palladin is contained and/or overlaps with the coding sequence of other palladin isoforms, RT-PCR primers were designed to amplify mRNA of the Ig3 domain containing isoforms . The primers used in the experiment were as follows: sense 5′ – AAC CGA GCA GGA CAG AAC- 3′ and antisense 5′- TGG TGG CAC TCC CAA TAC-3′. The thermal cycling conditions were 50°C for 2 min, then 95°C for 10 min followed by 40 cycles of initial denaturation step of 95°C/15 s and 60°C/60 s. No-template reactions were included as negative controls in every plate. Sequence Detection Software  results were imported into Microsoft Excel for further analysis. Raw expression levels were calculated from the average of the triplicate ddCT (RQ) values. Standard curve obtained for the primer and PCR product from a pool of samples was used as template. Each PCR reaction was carried out in triplicates.De-identified patient samples were collected after approval by each individual IRB and use of all samples for this study was approved by the UNC IRB. Samples of matched normal pancreas and pancreatic cancer were obtained at the time of operation and flash frozen in liquid nitrogen. Total RNA was extracted from the snap-frozen tumor samples using Allprep Kits (Qiagen) and quantified using nanodrop spectrophotometry (ThermoScientific). Reverse transcriptase reactions were performed using the commercial kit High-Capacity cDNA Reverse Transcription (Applied Biosystem) and contained up to 2 µg of total RNA. Real-time quantitative PCR was performed using an Applied Biosystems 7500 Fast Real Time PCR System in a a total volume of 25 µl mixture containing 2.5 µl of 10-fold diluted cDNA, 0.2 pM of sense and antisense primers and the Power Figure S1Immunoblot analysis of pancreatic tumor-derived cell lines using palladin 1E6 Ab. Normal fibroblasts were used as a positive control. Pancreatic cells: normal human pancreatic ductal epithelial cells (HPDE) and three tumor cell lines: PANC1, MiaPaCA, and ASPC1. Whole cell lysates were analyzed by western blot using the monoclonal antibody 1E6. Blots were subjected to long exposure times and also stained for tubulin as a control for equal loading.(0.24 MB TIF)Click here for additional data file.Figure S2Immunohistochemistry of paraffin-embedded patient specimens. IHC staining was performed using standard antigen-retrieval protocols, and counter-stained with hematoxylin. Tissue sections were stained for palladin using two palladin antibodies: polyclonal COM from ProteinTech group, and monoclonal 4d10.(8.42 MB TIF)Click here for additional data file.Figure S3Control staining for IHC of paraffin-embedded human and mouse pancreatic tissues. IHC staining was performed as in (10.34 MB TIF)Click here for additional data file.


In this study, we investigated the correlation between microsatellite instability (MSI) status and the incidence of metachronous recurrence of gastric cancer. According to the genetic/molecular background determined with MSI status and expression levels of hMLH1 and p53 tumour suppressor, 110 EGCs removed with ESD were subclassified into three groups: the mutator/MSI-type (8%), suppressor/p53-type (45%) and unclassified type (47%). Interestingly, patients with the mutator/MSI-type tumour had a high incidence (67%) of metachronous recurrence of gastric cancer within a 3-year observation after initial ESD, which was significantly higher than those with the suppressor/p53-type and unclassified type tumours (P<0.01). Although we investigated mucin phenotypes, there was no correlation between mucin phenotype and the recurrence of EGC. These findings suggest that subclassification of molecular pathological pathways in EGCs is required for the assessment of patients with a high risk of recurrent gastric cancer. The information delivered from our investigation is expected to be of value for decisions about therapy and surveillance after ESD.The technique of endoscopic submucosal dissection (ESD) has been developed for  In partn of ECG .Based on the tendency of gland formation, gastric carcinomas have been divided into two main histological types: diffuse-type carcinomas, which may arise from native gastric epithelial cells, and intestinal-type carcinomas, which were thought to arise from gastric epithelial cells that have undergone intestinal metaplasia . Currentp53 is a tumour suppressor that is frequently mutated in human cancer and is believed to play an important role in defending against cancer  with ESD. After initial dissection, we subclassified these patients into three groups according to molecular findings and followed them endoscopically for several years to monitor the recurrence of gastric cancer.The series consisted of 110 patients who had undergone curative resections for EGC with ESD at the Kobe University Hospital from 2000 to 2004. Their clinical and histological characteristics are summarised in μm-thick slices for DNA extraction and immunohistochemical analysis. For DNA extraction, the deparaffinised specimens were stained with haematoxylin and eosin (H&E). The tumour areas and corresponding non-neoplastic gastric mucosa were scraped using a sterile needle and placed in a microtube containing extraction buffer before being incubated at 55°C for 12 h as described elsewhere , hMLH1 , CD10 , MUC2 , MUC5AC (Novocastra Laboratories) and MUC6 (Novocastra Laboratories). Dewaxed and rehydrated specimens were autoclaved in citrate buffer for the antigen retrieval. Endogenous peroxidase activity and nonspecific binding were blocked by 0.03% hydrogen peroxidase in methanol and blocking reagent in LSAB2 kit (DakoCytomation), respectively. The slides were then incubated with the primary monoclonal antibody and incubated sequentially with a biotinylated secondary antibody, streptavidin, labelled with peroxidase and 3, 3′-diaminobenzidine. Slides were counterstained with Mayer's haematoxylin. Immunoreactivity of p53 and hMLH1 was graded as follows: almost no positive cells; +, <25% of the tumour cells showed positive immunoreactivity; ++, 25–50% of the tumour cells showed positive immunoreactivity; +++, >50% of the tumour cells showed positive immunoreactivity . After the initial Taq DNA polymerase activation step, the PCR amplification consisted of 45 cycles  followed by a final extension for 10 min at 72°C. Polymerase chain reaction products were electrophoresed in ABI PRISM 310 Genetic Analyzer along with GeneScan-500 (ROX) molecular weight standard (Applied Biosystems). The size of the PCR product was analysed using GeneScan software (Applied Biosystems). The status of MSI in each tumour was evaluated according to the criterion of Nine microsatellite markers  were analysed ; +, some positive cells (5–30%); ++, well-defined areas of positive cells (30–60%); +++, extensive areas of positive cells (>60%). According to the sums of scores of gastric markers  and intestinal markers , each case was phenotypically classified into three phenotypes: the gastric phenotype, mixed phenotype and intestinal phenotype .Statistical comparisons were performed using the Fisher's protected least significant difference test, the p53 locus (D17S250) was detected in eight (7%) of 110 cases , but no correlation was found in the other clinicopathological parameters  . The data suggest that chronic atrophic gastritis with or without intestinal metaplasia, due to H. pylori infection, certainly increases the incidence of gastric cancer, irrespective of the mutator/MSI-type or the suppressor/p53-type. However, further genetic changes may finally decide the carcinogenesis pathway for epithelial cells to develop gastric cancer. As reported previously, an MSI-positive genetic background is a risk for developing multiple cancers, including synchronous and metachronous cancer ( pylori)  and exce pylori)  are susp


Recent work indicates that allelic incompatibility in the mouse PRDM9 (Meisetz) gene can cause hybrid male sterility, contributing to genetic isolation and potentially speciation. The only phenotype of mouse PRDM9 knockouts is a meiosis I block that causes sterility in both sexes. The PRDM9 gene encodes a protein with histone H3(K4) trimethyltransferase activity, a KRAB domain, and a DNA-binding domain consisting of multiple tandem C2H2 zinc finger (ZF) domains. We have analyzed human coding polymorphism and interspecies evolutionary changes in the PRDM9 gene. The ZF domains of PRDM9 are evolving very rapidly, with compelling evidence of positive selection in primates. Positively selected amino acids are predominantly those known to make nucleotide specific contacts in C2H2 zinc fingers. These results suggest that PRDM9 is subject to recurrent selection to change DNA-binding specificity. The human PRDM9 protein is highly polymorphic in its ZF domains and nearly all polymorphisms affect the same nucleotide contact residues that are subject to positive selection. ZF domain nucleotide sequences are strongly homogenized within species, indicating that interfinger recombination contributes to their evolution. PRDM9 has previously been assumed to be a transcription factor required to induce meiosis specific genes, a role that is inconsistent with its molecular evolution. We suggest instead that PRDM9 is involved in some aspect of centromere segregation conflict and that rapidly evolving centromeric DNA drives changes in PRDM9 DNA-binding domains. Allelic incompatibility at the mouse PRDM9 (Meisetz) locus can cause hybrid male sterility due to failure in spermatogenesis The PRDM9 gene in human and mouse encodes a protein with KRAB The structure of ZF domains of the type found in PRDM9 bound to DNA is well-established We report analysis of molecular evolution of the PRDM9 gene based on sequence comparisons between primate species and on human polymorphisms.N/dS value (frequency of nonsynonymous change relative to synonymous change). However 32 codons had a high estimated dN/dS value that reached statistical significance for positive selection (P>0.95). Of these 32 codons, 26 encode major nucleotide contact residues in ZF domains and all 6 other codons are immediately adjacent to major nucleotide contact residues. Several additional codons in DNA-binding turn-helix regions also had high estimated dN/dS values that did not reach statistical significance. Divergence in the DNA-binding turn-helix regions is obvious by simple inspection of the protein multiple alignment . The remainder of the PRDM9 sequence is conserved throughout mammals and even in more basal lineages including non-mammalian chordates and echinoderms We assessed single-nucleotide polymorphisms (SNPs) in the coding region of human PRDM9 reported in dbSNP130, from 16 individual exome sequences generated by next-generation sequencing : 28 of 3We noticed that zinc fingers within the PRDM9 gene in each primate tended to be similar in sequence, despite the rapid divergence among species. We investigated the generality of this pattern by identifying putative PRDM9 sequences from 19 sequenced mammalian genomes  trimethylation) suggest a role in transcriptional regulation. However transcription factors are generally characterized by highly conserved DNA-binding domains, yet these are the regions where PRDM9 is most rapidly evolving. In contrast to transcriptional regulation, centromere structure and function is associated both with regulated histone modification states Drosophila centromeric chromatin is hypoacetylated on histones H3 and H4 The internal structure and boundaries of functional centromeres are strongly associated with histone modifications and with centromere-specific classes of histones Centromere drive is expected to be limited to the germ line, with the most obvious site of action being meiosis in the female. The only phenotype of PRDM9 knockout in the mouse is arrest at prophase of meiosis I in both sexes, consistent with various specific roles including recombination and chromatin condensation in preparation for metaphase. Our hypothesis for PRDM9 function clearly predicts that the PRDM9 protein will be physically associated with the centromere during meiosis I and that it will function there to moderate centromere drive via histone modification.N/dS estimates came from the codeml “rst” output file and P-values came from the standard codeml output (both with unconstrained model 8).Complete or nearly complete PRDM9 coding sequence was obtained either from available gene predictions  or from a genewise Human SNPs were ascertained in three ways. First, a publication provides extensive information on human coding-sequence alleles and frequencies in a Japanese population Microcebus murinus), dolphin (Tursiops truncatus), and bat (Pteropus vampyrus) genes have an upstream exon on the same contig that matches the next upstream human exon from PRDM9. Finally, all the sequences except tenrec show clear homogenization of their ZF domain sequences, a very unusual character among tandem ZF domain genes (data not shown). The tenrec sequence is included based on the protein tree, but it should be regarded as a questionable ortholog assignment because it shows no other shared PRDM9 characters. Higher primates have a partial duplicate of the PRDM9 gene (PRDM7) but it completely lacks the tandem ZF domains of PRDM9 and is thus easily distinguished.We wanted to obtain the PRDM9 ZF coding exon from available mammalian genome assemblies. Divergence in PRDM9 ZF sequences combined with large C2H2 zinc finger gene families throughout mammals made PRDM9 identification based on these sequences impossible (data not shown). For low coverage assemblies, the unique upstream coding exons were likewise useless because they were often not present on the same contig as the ZF exon. However, there is a conserved unique protein sequence upstream of the ZF domains in the ZF-containing exon see  that appFigure S1Generation of 6 SNPs by recombination. Alignment of the overlapping regions 18489–18657 and 18405–18573 of PRDM9 (numbering is genomic position relative to the ATG start codon). Colored dots indicate nucleotide differences between the two stretches. Gene conversion of PRDM9 18489–18657 (top line) with sequence from 18405–18573 (bottom line) could result in changes in one or any combination of the highlighted residues. In fact, each of these six changes appears as a SNP in the Japanese population (0.73 MB TIF)Click here for additional data file.Figure S2Generation of other SNPs by recombination. Alignment of all 12 ZF domains from human PRDM9, with arrows indicating the possible source (arrow tail) and target (arrow head) sites for generation of 20 SNPs by recombination. The arrow head is numbered to indicate the allele correspondence as given in (5.61 MB TIF)Click here for additional data file.Table S1List of human polymorphisms in PRDM9. Summary of positions, nucleotide changes, data source, and other information for human SNPs analyzed. NOTE - single large table on multiple PDF pages.(0.14 MB PDF)Click here for additional data file.


The function of the tumor suppressor RE1 silencing transcription factor (REST) is lost in colon and small cell lung cancers and is known to induce anchorage-independent growth in human mammary epithelial cells. However, nothing is currently known about the role of this tumor suppressor in breast cancer. Here, we test the hypothesis that loss of REST function plays a role in breast cancer. To assay breast tumors for REST function, we developed a 24-gene signature composed of direct targets of the transcriptional repressor. Using the 24- gene signature, we identified a previously undefined RESTless breast tumor subtype. Using gene set enrichment analysis, we confirmed the aberrant expression of REST target genes in the REST–less tumors, including neuronal gene targets of REST that are normally not expressed outside the nervous system. Examination of REST mRNA identified a truncated splice variant of REST present in the REST–less tumor population, but not other tumors. Histological analysis of 182 outcome-associated breast tumor tissues also identified a subpopulation of tumors that lack full-length, functional REST and over-express the neuroendocrine marker and REST target gene Chromogranin A. Importantly, patients whose tumors were found to be REST–less using either the 24-gene signature or histology had significantly poorer prognosis and were more than twice as likely to undergo disease recurrence within the first 3 years after diagnosis. We show here that REST function is lost in breast cancer, at least in part via an alternative splicing mechanism. Patients with REST–less breast cancer undergo significantly more early disease recurrence than those with fully functional REST, regardless of estrogen receptor or HER2 status. Importantly, REST status may serve as a predictor of poor prognosis, helping to untangle the heterogeneity inherent in disease course and response to treatment. Additionally, the alternative splicing observed in REST–less breast cancer is an attractive therapeutic target. Breast cancer is a heterogeneous disease, with highly variable disease outcomes and responses to treatment for otherwise indistinguishable tumors. Understanding this heterogeneity holds the key to better determining disease prognosis and tailoring treatments to the tumors for which they will be most efficacious. Some of the most successful work dissecting the differences between histologically identical tumors with differing disease outcomes has come from profiling the array of protein-coding transcripts present in every tumor and dividing the breast cancer profiles into multiple subtypes of varying aggressiveness. Importantly, these profiles are now being used in the clinic to predict disease outcome and plan treatment. Using a similar molecular-profiling strategy, we have identified a previously unrecognized subset of breast cancers in which the tumor suppressor gene REST is lost, which display a highly aggressive disease course. Intriguingly, we have traced the loss of the tumor suppressor to the presence of a variant of the REST protein normally found in the brain following seizures, which represents a unique and attractive therapeutic target. Additionally, the gene signature used to identify REST–less tumors shows no overlap with the profiles currently used in the clinic to assess tumor aggressiveness and may be an important new diagnostic tool. REST Enterez GeneID 5978), also known as Neuron Restrictive Silencing Factor (NRSF), and the identification of this signature in breast cancer.Identification of tumor suppressors is hampered by the fact that their loss of function can occur through any one of many mechanisms including inactivating mutations, aberrant splicing and copy number aberration. Transcription factor tumor suppressors control many downstream genes in a given cell; however, using the absence or presence of a downstream gene as a proxy for loss of tumor suppressor function is problematic because each gene is likely to be regulated by multiple transcription factors. Genome-wide transcription profiling has opened up the possibility of simultaneously measuring expression levels of multiple, if not all, downstream target genes of a tumor suppressor. In this report we describe the generation of such a signature for the tumor suppressor RE1 Silencing Transcription Factor , resulting in the overexpression of REST target genes and imparting a neuroendocrine phenotype on the cells To determine whether the tumor suppressor REST is lost in breast cancer, we developed a gene signature-based approach to screen for loss of REST function by measuring the expression of a cohort of REST target genes. Using this gene signature as well as an immuno-histochemical screen, we show here that an aggressive subset of breast cancers lack functional REST (“REST–less tumors”), instead, often expressing the truncated REST splice variant REST4.Given that loss of REST induces anchorage-independent growth in mammary epithelial cells, and is lost in colon There are approximately 2,000 RE1-bearing genes in the mouse and human genomes; however, not all of these genes are repressed by REST in every cell type −7), 63 (87.5%) either contain perfect consensus RE1 sites, are upregulated two-fold or greater upon REST knockdown, or were bound by REST in a genome-wide ChIP-Seq screen performed by Johnson et al.Using our 24-gene signature, we screened a microarray dataset containing 129 breast tumors To further test whether REST function was lost in REST–less tumors, we performed Gene Set Enrichment Analysis (GSEA) on the above breast tumors using the 24-gene signature . This meWe tested the possibility that the abrogation of REST function may be due to a loss of REST mRNA by comparing transcript levels in REST–less and RESTfl tumor samples. We saw no difference in REST transcript levels between the two tumor types . IntriguAlternative splicing of REST to generate the truncated REST4 variant results in a protein with diminished DNA binding ability and loss of repressive function, and has been identified in the nervous system and in SCLC We then used the 24-gene signature to classify the breast tumor superseries GSE6532 into REST–less and RESTfl tumors and determined how REST status associated with patient outcome  [26]. ThTo determine the frequency of REST protein loss in breast cancer, we developed an immunohistochemical (IHC) screen using an antibody directed to the C-terminus of REST . To test the specificity of the REST antibody, we utilized the REST-expressing breast cancer cell line MCF7. Cells were stably infected with lentiviruses bearing either non-targeting or REST-targeting shRNA , then paCHGA Enterez GeneID 1113), consistent with a loss of REST repression (. Patients with REST–less triple negative (TN) tumors (Estrogen Receptor (ER)/Progesterone Receptor/HER2-) endure significantly greater disease recurrence within 2 years than TN/RESTfl patients   REST predicted by the REST4 splicing in 37 (20.3%) tumor samples . Additiopression , p<0.001 n = 32) . PatientIn this study we demonstrate that REST function is lost in a fraction of breast tumors. Loss of REST is defined by either the presence of a 24 gene signature in tumors or by loss of immunohistochemical stain with a antibody raised to the C-terminus of REST. REST–less tumors (as determined by either IHC or 24-gene signature) are associated with an aggressive, rapid recurrence prognosis. At this time it is not known whether REST–less tumors defined by gene signature represent a subset of those determined to be REST–less by IHC or are a distinct group of malignancies. Answering this question will allow for the elucidation of the suite of mechanisms by which REST function can be lost in breast cancer and will require the analysis of patient cohorts that have associated patient outcome data, tissue micro-arrays as well as gene expression data.We have developed two complementary methods of screening tissue and tumors for their levels of functional REST. For samples associated with microarray data, a signature of 24 genes indicates loss of functional REST. Histological sections can be stained for the C-terminus of REST in combination with the REST target gene, chromogranin-A.in vitro allowed for the identification of a class of REST–less breast tumors. Analysis of tumors so characterized showed that REST was aberrantly spliced to generate the REST4 truncated variant. REST4 was originally identified in the hippocampii of mice after kainic acid induced seizures It is known that post-transcriptional regulation of REST occurs during neuronal differentiation and during oncogenic transformation such that protein levels are significantly reduced in the absence of altered mRNA levels Importantly, REST4 splicing is regulated by cell signaling pathways and in the case of seizure models, the alternative splicing of REST occurs within a limited time window before full-length, functional REST transcript splicing is restored. Indeed, in SCLC cell lines reintroduction of full-length REST induces apoptosis. Similarly, reintroduction of full-length REST into REST–less colon cancer cell lines restores anoikis, blocking anchorage-independent cell growth To better understand the disease course taken by REST–less tumors we took advantage of the fact that REST4 lacks the C-terminus. This allowed us to verify loss of full-length REST via an immunohistochemical screen utilizing an antibody raised to the C-terminus of REST, which will not detect REST4, degraded REST, or any previously characterized truncated mutant of REST. IHC on a panel of 182 tumor samples with associated outcome data showed that patients with REST–less tumors experience a 20% reduction in disease free survival when compared to their RESTfl counterparts over 10 years . The majRemarkably, a subset of tumors from all histological classes of breast cancer stained negative for REST C-terminus, and all classes showed a worse prognosis without REST. REST–less triple negative tumors showed a particularly aggressive disease course. Of the 32 triple negative tumors screened, 13 were found to be REST–less, six (46%) of which recurred in the first 12 months post-diagnosis, compared to just 1 of the 19 (5%) RESTfl triple negative tumors (p = 0.003). However, no triple negative REST–less tumor recurred after 12 months in 10 years of patient outcome data. ER+ REST–less tumors showed a similar pattern of early recurrence, wherein 8 of 21 (38%) patients saw disease recurrence in the first 36 months, compared to just 11% of ER+ RESTfl patients (p = 0.003). Thereafter, none of the remaining 13 disease-free patients with ER+ REST–less tumors experienced recurrence, compared to 12 of the 102 remaining disease-free ER+ RESTfl patients. These data suggest that REST–less tumors represent a distinct, aggressive subset of breast tumors with a unique disease course.et al. examine REST levels in 19 samples of normal and neoplastic breast tissues. Using 3 control tissue samples and 16 tumors spread among 3 tumor stages, they suggest that REST protein and mRNA levels undergo a stepwise, statistically significant decrease with each increasing tumor stage et al. reported that they did not observe any alternate splice variants in the tumor samples in which they find REST to be lost . However, we have found alternative REST splicing in tumors where REST function is lost . T47D cells were grown in RPMI with L-glutamine, 10 ug/mL insulin, and 10% fetal bovine serum. MCF10a cells were grown in DMEM/F12 with 5% horse serum, 10 mg/mL insulin, 20 ng/mL epidermal growth factor, and 0.5 mg/mL hydrocortisone.All cells were grown in 5% COGCAAACACCTCAATCGCCA, Non-Targeting SMARTvector shRNA Lentiviral particles  were used as an infection control.Stable REST knockdown in HEK-293, T47D and MCF10a cells for microarray analysis was achieved using a Dharmacon SMARTvector lentiviral shRNA delivery system as per manufacturer's instructions. Briefly, cells were infected in the presence of 8 mg/mL polybrene at an MOI of 5 with virus expressing a non-targeting control or REST shRNA. Puromycin selection was begun 48 hours after infection and maintained during cell expansion and experimentation. SMARTvector Lentiviral Particles  towards REST targeted the sequence RNA was extracted using TRIzol (Invitrogen) as per the manufacturer's instructions from four independent plates of each cell line T47D, HEK-293 and MCF10a, with two biological replicates of cells expressing REST shRNA and another two biological replicates expressing a non-targeting control shRNA.http://biotech.wisc.edu/GEC/). RNA integrity and quality were assessed by comparing 28S/18S rRNA ratio using Agilent RNANano6000 chips on an Agilent 2100 Bioanalyzer. First and second strand cDNA synthesis steps, followed by in vitro transcription, were performed using the Ambion Amino Allyl Messageamp II kit. Cy3 and Cy5 (Amersham) dyes were coupled to the aRNA, with each fluorophore labeling a separate biological replicate, before fragmentation and dual hybridization to Nimblegen HG18 60mer 385k Gene Expression Arrays . For dual hybridization, shControl and shREST samples from the same cell line were competitively hybridized. Arrays were scanned on an Axon4000B and the gene expression data was extracted, and RMA normalized using software provided by Nimblegen.All RNA reverse transcription, amplification and hybridizations were performed by the University of Wisconsin-Madison Gene Expression Center  and MultiExperiment Viewer 4.5.1. Tumor gene expression data were obtained from the NCBI Gene Expression Omnibus, and are identified by their GEO dataset record number. Dataset E-TABM-276 was downloaded from the Ensembl ArrayExpress. Analysis of dataset GSE6532 was performed to determine the aggressiveness of tumors identified as being REST–less using the gene signature method. All samples from this dataset that included information on duration of relapse free survival as well as relapse event information were included in our analysis, for a total of 206 outcome-associated tumors. Hierarchical clustering was performed using a one-minus correlation metric with average linkage over centered genes. Cluster diagrams were produced using BRB Arraytools, Cluster 3.0 and TreeView software. Class comparison analysis between REST–less and RESTfl tumors was performed using a random variance model for univariate tests with a significance threshold of p<10et al. ChIP-Seq were chosen on the basis of their enrichment in both experiments 1 and 2 in a region carrying an RE1 site with a p-value of <10−4. Genesets used in GSEA analysis are available as Gene Set Enrichment Analysis (GSEA) was performed using the GSEA program provided by the Broad Institute. GSEA compares the expression of a set of experimentally defined REST target genes (termed “S”) between REST–less and RESTfl tumors, and assesses the relative enrichment of S in either tumor group. The positive enrichment scores obtained from these analyses are indicative of high level enrichment of REST target gene expression in the REST–less tumor subset. To determine whether this enrichment could arise by random chance, 1,000 permutations of the above analysis were performed with each permutation randomly assigning tumors as being either REST–less or RESTfl and then assessing enrichment in each group for S. The fraction of permutations that result in an enrichment for REST target genes in the randomly assigned “REST–less” tumor group serves as an estimate of statistical significance and is presented as a nominal p-value . Similarly, the false discovery rate (FDR) q-value is a measure of the likelihood that a geneset S may be a false positive, but this statistic is controlled for gene set size and may therefore be compared between multiple genesets with normalized enrichment scores. The list of REST target genes identified in the Johnson https://www.affymetrix.com/support/downloads/manuals/expression_s21_manual.pdf). Briefly, a first strand of cDNA was generated from 100 ng of tumor RNA using T7-oligo(dT) primers, incubated at 70°C for 10 minutes, followed by 2 minutes on ice. A first-strand reaction mix with dNTPs, DTT and SuperScript III Reverse Transcriptase (Invitrogen) was added to each sample and incubated for an hour at 42°C, then transferred to ice. The second strand of cDNA synthesis was performed using a second-strand reaction mix, dNTPs, E. coli DNA ligase, E. coli DNA polymerase, and RNase H, and was incubated at 16°C for 2 hours. T4 DNA polymerase was added for the final 5 minutes of this incubation period, generating full-length double-stranded cDNA. The cDNA was purified on MinElute PCR purification columns (Qiagen) as per manufacturer's instructions. The Megascript T7 RNA polymerase kit (Ambion) was used to generate aRNA from the tumor cDNA as per the manufacturer's instructions. The aRNA was purified from the reaction mix using Trizol (Invitrogen) as per the manufacturer's instructions, and 500 ng of tumor aRNA was used to generate tumor cDNA using SuperScript III RT Kit (Invitrogen) as per manufacturer's instructions. This cDNA then served as a template for the PCR amplification of REST using Pfx Platinum high fidelity polymerase (Invitrogen) as per manufacturer's instructions.The work with human tumor RNA was found to be exempt from institutional review by the UW institutional review board (IRB). Tumor RNA was amplified in duplicate according to the Affymetrix manual on Eukaryotic Target Preparation, Section 2.1  and hREST4 Forward and hREST SV Region Reverse primers over 35 cycles.Primers used to amplify the exon junctions surrounding introns 1 and 2:GAGCGAGTATCACTGGAGGAAACATTThREST SV region forward: ATAGTCACATACAGGGCAATTGAACTGChREST SV region reverse: Primers used to amplify REST4:CATTCAGTGGGGTATGGATACChREST4 forward (Used with hREST SV reverse): GCTTCTCACCCATCTAGATCAChREST4 reverse (Used with hREST SV forward): Four-micron sections of previously characterized tissue microarrays, which contain duplicate tissue cores from 207 human breast carcinomas, were used for the labeling experiments Kaplan Meier plots were generated using SigmaStat 3.0, and Kaplan Meier curves were compared using the logrank test. We report as significant only those curves that have reached a level of p≤0.05. Correlations between REST status and disease parameters were tested using Pearsons Chi-squared test, and are reported as significant p≤0.05.Table S1This table lists genes that show a >2 fold upregulation following REST knockdown, as determined by oligonucleotide microarray in 3 cell lines: HEK-293, MCF10a, and T47D cells.(0.10 MB XLS)Click here for additional data file.Table S2This table describes the gene list determined to be associated with RESTless tumors in dataset GSE5460 by class comparison. This list of genes represents all of the genes identified as more highly expressed in RESTless tumors with respect to RESTfl tumors in the GSE5460 dataset (p<e-7) by using a random variance model for univariate tests in BRB Arraytools v3.7. Of the 72 genes in this list, 63 are putative REST targets, as defined by a consensus RE1, positive ChIP Seq signal, or a two-fold change upon REST knockdown in at least one of three cell lines.(0.08 MB XLS)Click here for additional data file.Table S3This table provides the gene sets used in gene set enrichment analysis of dataset GSE5460.(0.06 MB XLS)Click here for additional data file.


Sema6A mutants provide a unique model to test current ideas on the interactions between subcortical and cortical guidance mechanisms and cortical regionalization. In these mutants, axons from the dorsal lateral geniculate nucleus (dLGN) are misrouted in the ventral telencephalon. This leads to invasion of presumptive visual cortex by somatosensory thalamic axons at embryonic stages. Remarkably, the misrouted dLGN axons are able to find their way to the visual cortex via alternate routes at postnatal stages and reestablish a normal pattern of thalamocortical connectivity. These findings emphasize the importance and specificity of cortical cues in establishing thalamocortical connectivity and the spectacular capacity of the early postnatal cortex for remapping initial sensory representations.The establishment of connectivity between specific thalamic nuclei and cortical areas involves a dynamic interplay between the guidance of thalamocortical axons and the elaboration of cortical areas in response to appropriate innervation. We show here that  Semaphorin-6A to test this model. In these mutants, Semaphorin-6A axons from the visual part of the thalamus are subcortically misrouted and fail to innervate the presumptive visual cortex, which is instead invaded by somatosensory thalamic axons. Despite this major disruption in initial connectivity, many visual thalamic axons find their way specifically to visual cortex, arriving several days later than usual. These late-arriving axons often follow alternate routes, and upon arrival are able to out-compete earlier-arriving somatosensory axons to reestablish grossly normal thalamocortical connectivity. These results argue strongly against an essential role for early subcortical targeting in the establishment of thalamocortical connectivity patterns and suggest instead the existence of highly specific target-selection mechanisms that match thalamic axons with appropriate cortical areas.During brain development, the emergence of distinct areas in the cerebral cortex involves an interplay between patterning of the cortical sheet in the early embryo and later influences of incoming connections made from other brain areas, namely the thalamus. Connectivity between the thalamus and the cortex is initially smooth and graded, and a prominent model for how thalamocortical connectivity is established proposes thalamic axons are topographically sorted as they course through subcortical regions and then passively delivered to appropriate areas of the cortical sheet. We have used mutant mice lacking the guidance molecule  Initial misrouting and subsequent recovery of thalamocortical axon projections in Semaphorin-6A mutant mice highlights the importance of specific target-selection mechanisms that match thalamic axons with appropriate cortical areas. A dynamic interplay exists between the processes of cortical arealization and those controlling the guidance and targeting of thalamocortical projections –5. EarlyEbf1 or in the Dlx1/Dlx2 double mutants, a subset of thalamic axons is misrouted ventrally, resulting in a caudal shift of the remaining axons within the ventral telencephalon ), misrouted axons can be seen to project deep into the ventral telencephalon and project along its most superficial aspect (red arrowheads). Scale bars indicate 500 μm.(A–D) Rostral–caudal consecutive sections of PLAP-stained (1.44 MB TIF)Click here for additional data file.Figure S2Sections are at 45° to the coronal plane to encompass the internal capsule and dorsal thalamus.(A and B) A DiA crystal placed in the internal capsule of wild-type brains at E17.5 broadly labels cells throughout the dorsal thalamus.(C) In these same animals, a DiI crystal in the ventral telencephalon fails to label any cells in the dorsal thalamus, indicating that all TCAs project through the internal capsule in these animals.−/−Sema6A brains at the same age (D and E), a DiA crystal in the internal capsule labels cell bodies in the VB but specifically not in the dLGN, whereas a DiI crystal in the ventral telencephalon does label dLGN neurons specifically, indicating that dLGN axons in these animals do not project at all to the internal capsule at this age (F).(D–F) In Scale bars in (A–D) indicate 500 μm.(1.90 MB TIF)Click here for additional data file.Figure S3EphA7 (A and C) and EphrinA5 (B and D) on sagittal sections of wild-type (A and B) and −/−Sema6A (C and D) brains. EphA7 is expressed in the rostral and occipital neocortex (NCx), but is absent from the putative somatosensory cortex. In contrast, EphrinA5 is expressed is highly expressed in the somatosensory cortex. dTh, dorsal thalamus. Scale bars in (A–D) indicate 500 μm.(A and D) In situ hybridization with DIG-labeled probes for (1.17 MB TIF)Click here for additional data file.


A widespread mechanism of bacterial signaling occurs through two-component systems, comprised of a sensor histidine kinase (SHK) and a transcriptional response regulator (RR). The SHK activates RR by phosphorylation. The most common two-component system structure involves expression from a single operon, the transcription of which is activated by its own phosphorylated RR. The role of this feedback is poorly understood, but it has been associated with an overshooting kinetic response and with fast recovery of previous interrupted signaling events in different systems. Mathematical models show that overshoot is only attainable with negative feedback that also improves response time. Our models also predict that fast recovery of previous interrupted signaling depends on high accumulation of SHK and RR, which is more likely in a positive feedback regime. We use Monte Carlo sampling of the parameter space to explore the range of attainable model behaviors. The model predicts that the effective feedback sign can change from negative to positive depending on the signal level. Variations in two-component system architectures and parameters may therefore have evolved to optimize responses in different bacterial lifestyles. We propose a conceptual model where low signal conditions result in a responsive system with effectively negative feedback while high signal conditions with positive feedback favor persistence of system output. Bacteria have evolved various mechanisms for surviving unpredictable changes and stresses in the environment, such as nutrient limitation. One common survival mechanism is the two-component system, where a sensor protein responds to a particular type of stress by activating a regulator in the cell. These regulators can in turn activate genes that produce proteins for stress-appropriate responses. The activated regulator often positively regulates transcription of its own operon containing the sensor and regulator genes leading to a feedback loop. This is interesting, because positive feedback is usually associated with a slower response time than negative feedback and therefore negative feedback would often be selected for by evolution. Here we analyze a mathematical model to study the interplay of this feedback and postranslational mechanisms regulating two-component system signaling. We found that modulation of regulator activity by its operon partner can lead to overall negative feedback to result from autoactivation. This happens if (1) the sensor can both activate and deactivate the regulator, and (2) there is some reaction resulting in regulator activation independently of its cognate sensor. As a result our model predicts that two-component systems may be capable of flexibly switching between positive and negative feedback depending on different circumstances, allowing for appropriate responses in a variety of conditions. Unpredictably changing environments necessitate appropriate responses for successful survival by bacteria. Bacterial two-component system (TCS) signaling shifts transcriptional programs in response to a variety of external cues affecting bacterial growth such as nutrient availability, osmolarity, redox state, temperature, and concentrations of other important extracellular molecules Environmental signals are routed through conformational changes in SHK homodimers, which may participate in up to three biochemical processes. First, each subunit hydrolyzes ATP to trans-phosphorylate a His residue in the other subunit. Second, the phosphorylated SHK subunit transfers its phosphate to an Asp residue on unphosphorylated RR bound to the SHK. Third, in bifunctional TCSs, unphosphorylated SHK can catalyze dephosphorylation of RRP with release of inorganic phosphate. Modulation of phosphatase and/or kinase activities of SHK may therefore induce system responses by shifting the dynamic equilibrium between active (phosphorylated) and inactive (unphosphorylated) forms of RR. Interactions between the RR and exogenous phosphodonors  may also contribute to TCS activation.Activated RR may induce expression of multiple operons. This regulon often contains the operon encoding the RR and SHK. Such autoregulation  is obserE. coli have shown widespread positive and negative transcriptional autoregulation E. coli TCSs as examples of positive feedback loops. However, we suggest that the effective sign of feedback in TCSs may depend on the biochemical interactions of the autoregulated proteins. Induction of bifunctional SHK in the same operon as RR may affect the phosphorylation equilibrium, and therefore have either positive or negative effects on the amount of transcriptionally active RR. That is, the signal increases RR phosphorylation, but resultant increases in gene expression may in turn positively or negatively change the amount of phosphorylated RR amplifying or attenuating the original signal. The resulting sign of feedback can be related to the transient dynamics of TCS activation: overshoot kinetics often result from underdamped negative feedback Salmonella enterica serovar Typhimurium  in mice Previous genomic studies in We used a mathematical model of a generic TCS to demonstrate that post-translational kinetics of SHK-RR interactions can determine the effective sign of feedback. The model explains disparate results relating to transcriptional autoregulation in TCSs including the “learning” effect To determine the role of transcriptional feedback in TCSs, we constructed an ordinary differential equation model extending previous work apk in Reaction 10) or phosphatase (phk in Reaction 18) activity of SHKs Salmonella PhoPQ A signal may modulate the rate of kinase . Intuitively, the feedback increases total RR and SHK concentrations. However, increases in these concentrations may have a positive or negative effect on the phosphorylated fraction of RR. These conditions respectively correspond to effectively positive and negative feedback. The open-loop gain is notably different from the steady-state signal-response gain of the system. In all cases considered, system response (level of activated RR) increased with increased signal (decreased phk). Nevertheless, the sign of open-loop gain can be either positive and negative for the cases in which transcriptional feedback respectively amplifies or attenuates RRP concentration.To study TCS induction dynamics with the model, we chose a Monte Carlo parameter sampling approach because no general analytical solution of transient response is possible. Signal level was determined by parameter i.e., JE/(JE+JS)>10−3 where JE is flux through Reaction 19 and JS is flux through reaction 16 in Parameter sampling returns both negative and positive open-loop gains corresponding to overall negative and positive sign of feedback . In eachSalmonellaSalmonella PhoPQ compared with experimental results of downstream promoter binding from 2 at the activated steady state. A common dynamical characteristic of negative feedback is overshoot kinetics, which have been shown to occur in PhoPQ of In simple inducible genetic systems with autoregulation, negative feedback is associated with faster attainment of a given steady state as compared to positive feedback E. coli PhoBR, where responses to a second stimulus after a transient signal interruption are faster than the response to the first signal p∼0 with double machine precision). High induction capacity attainable by most positive feedback cases amplifies the “learning” effect; it does not arise from bistability or slow response times usually associated with positive feedback Another kinetic effect of feedback in TCSs is “learning” exhibited in phk). The majority of Monte Carlo parameter samples that predict negative feedback for one signaling level exhibit positive feedback for a different signal level , open-loop gain and response times of downstream protein induction from the basal state (phk = 10/s) using the default parameter set  or both kinase and phosphatase activity (apk and phk). We scanned the 2-dimensional apk × phk signal space and found that modulation of either activity may result in feedback tuning , the response time is typically slow . When RRB. subtilis DegS-DegU system TCSs are responsible for diverse, specialized, large-scale reprogramming of bacterial transcription in response to many possible signals. Nevertheless, while orthologous TCSs in different species often have drastically different regulons E. coliDespite the fact that autoregulation is almost uniformly positive in TCSs −[RRP2](kph = 10) >1 molecule/cell.sufficient induction of transcriptionally active RR after signaling: [RRP2](kph = 10) and [RRP2] ≤1 molecule/celllow transcriptional activity of RR when signal is absent: [RRPkph = 10) and [DS protein] differ by less than 10%low downstream gene expression when signal is absent: [DS protein] or 95% (τ95) of the activated steady state. The former is reported for responses where post-transcriptional kinetics dominate a response while the latter is reported when transcriptional induction dominated response times. In practice, qualitative differences between these measures are minimal.Response times were calculated as elevation time for attaining either 50%  from a high, resting state (10 s−1) to a low level (1 s−1). To determine the effect of feedback, we used an open-loop version of the model in which an exogenous regulator R0 rather than (RRP)2 controls SK/RRP production. The effective sign of feedback is determined by the sign of the gain defined as the derivative 2 overshoot without mRNA overshoot. This phenomenon is not dependent on transcriptional feedback and is not of interest here. Therefore we do not analyze these parameter sets.We used Monte Carlo sampling of parameter values to numerically determine the range of realizable dynamic behaviors in the autoregulated TCSs focusing on the sign of autoregulation and possibility of overshoot behavior. Each parameter set was used to simulate activation kinetics after a signal at 2 at high and low signal limits, normalized to the case without feedback: Induction range  is definTo determine the range of behaviors attainable by the model, we evolved examples conforming to specific criteria using a simple genetic algorithm. Using a set of seed parameter sets, the algorithm randomly perturbs parameters and selects sets with the highest value for a fitness function. This function depends on the desired criteria.Salmonella PhoPQ after a signal, the desired characteristics include at least 2-fold induction of (1) [RRP2] over the resting level (f1); (2) RRtot over the resting level (f2); and (3) peak RRP2 concentration over activated steady state RRP2 (f3). The following fitness function selects for these criteria, with each criterion noted: To select a parameter set that conforms to known important characteristics of Text S1Alternative models of two-component system kinetics(0.07 MB PDF)Click here for additional data file.Text S2Partitioned Monte Carlo sampling in a simplified model(0.03 MB PDF)Click here for additional data file.Text S3Non-steady state open loop gains and response times(0.03 MB PDF)Click here for additional data file.Text S4Relating overshoot kinetics to steady state response(0.03 MB PDF)Click here for additional data file.Figure S1Characteristics of two-component system model calibrated to resemble Salmonella PhoPQ. Dynamics of RRP2 (A), total RR (B), total SHK (C), and a downstream protein upregulated by RRP2 (D).(0.08 MB PDF)Click here for additional data file.Figure S2Schematic of a two-component system model with sensor histidine kinase (SHK) maturation. This model is identical to the main text, except that SHK-independent phosphorylation of response regulator (RR) is disallowed, and newly produced SHK enters a temporary state where it is capable of binding and phosphorylating RR, but cannot catalyze the phosphatase reaction. Newly translated SHK_0 matures at a rate kconf.(0.05 MB PDF)Click here for additional data file.Figure S3Sampling of partitioned parameter space in a simplified two-component system model. (A) The model was simplified by eliminating as many variables as possible while retaining the capability for negative open-loop gain. (B) Distribution of negative and positive open-loop gain cases for fraction of exogenous phosphorylation flux JE/(JE + JS). Histogram bins containing more than 105 members were cut off for clarity. (C) Distribution of cases with feedback-induced overshoot >10% over the activated steady state.(0.08 MB PDF)Click here for additional data file.Figure S4Two-component system kinetics with non-steady state open loop gain that switches between positive and negative.(0.09 MB PDF)Click here for additional data file.Figure S5Relationship between steady state dose-response and overshoot kinetics in two-component systems.(0.15 MB PDF)Click here for additional data file.Figure S6Altered mRNA stability in a simulated SHK knockout changes total RR concentrations. Wildtype concentrations at the default degradation rate for various signal levels are shown for reference. All simulations use the default parameter set .(0.05 MB PDF)Click here for additional data file.Figure S7Predicted steady-state effects of perturbing translational efficiency.(0.04 MB PDF)Click here for additional data file.Table S1Intervals for Monte Carlo sampling and reference parameter set(0.02 MB PDF)Click here for additional data file.Table S2Reaction mechanisms for a generalized two-component system model(0.02 MB PDF)Click here for additional data file.


Retraction of nerve terminals is a characteristic feature of development, injury and insult and may herald many neurodegenerative diseases. Although morphological events have been well characterized, we know relatively little about the nature of the underlying cellular machinery. Evidence suggests a strong local component in determining which neuronal branches and synapses are lost, but a greater understanding of this basic neurological process is required. Here we test the hypothesis that nerve terminals are semi-autonomous and able to rapidly respond to local stimuli in the absence of communication with their parent cell body.We used an isolated preparation consisting of distal peripheral nerve stumps, associated nerve terminals and post-synaptic muscle fibres, maintained in-vitro for up to 3 hrs. In this system synapses are intact but the presynaptic nerve terminal is disconnected from its cell soma. In control preparations synapses were stable for extended periods and did not undergo Wallerian degneration. In contrast, addition of purines triggers rapid changes at synapses. Using fluorescence and electron microscopy we observe ultrastructural and gross morphological events consistent with nerve terminal retraction. We find no evidence of Wallerian or Wallerian-like degeneration in these preparations. Pharmacological experiments implicate pre-synaptic P2X7 receptor subunits as key mediators of these events.The data presented suggest; first that isolated nerve terminals are able to regulate connectivity independent of signals from the cell body, second that synapses exist in a dynamic state, poised to shift from stability to loss by activating intrinsic mechanisms and molecules, and third that local purines acting at purinergic receptors can trigger these events. A role for ATP receptors in this is not surprising since they are frequently activated during cellular injury, when adenosine tri-phosphate is released from damaged cells. Local control demands that the elements necessary to drive retraction are constitutively present. We hypothesize that pre-existing scaffolds of molecular motors and cytoskeletal proteins could provide the dynamism required to drive such structural changes in nerve terminals in the absence of the cell body. Retraction of nerve terminals is a fundamental event in the development and maintenance of the nervous system ,2, occurin vitro can assemble new growth cones at lesion sites [Available data points to a degree of synaptic independence in the retraction of individual synaptic connections. Observations of single motor units during developmental synapse elimination reveal three quite separate populations of nerve terminals: stable, actively withdrawing and actively enlarging ,12 and ion sites -24 and ton sites . Taken ton sites , where sIn this study we show that nerve terminal retraction can be triggered in the absence of neuronal cell bodies by the extracellular application of an ATP analogue. The nature of the nerve terminal retraction does not resemble Wallerian or Wallerian-like degeneration; rather it resembles 'dying back' neuropathies and synapse elimination.ATP is released following injury, signals local damage  and low-Quantitative data taken from the preparations described above are shown in figure During the course of these experiments we occasionally found intact nerve terminals associated with fragmented endplates Fig . We alsoATP acts at a range of purinoceptors, though our previous studies have only been able to demonstrate P2X7 receptor subunits (P2X7RS) on motor nerve terminals . HoweverThe failure to propagate compound action potentials  and to rTo further investigate ultrastructural events during the rapid-onset retraction events observed following application of BzATP we carried out immuno-electron microscopy. Isolated peripheral nerve and skeletal muscle preparations were incubated in BzATP (100 μM) for 30 min, immediately fixed and immunostained for P2X7 receptors. Nerve terminal boutons were immunopositive for P2X7 receptors and exhibited evidence consistent with retraction from post-synaptic sites. This included: dilation of synaptic clefts and appearance of mega-omega profiles in the pre-junctional, pre-synaptic membrane  mice [We have demonstrated that BzATP triggers a controlled retraction of nerve terminals from post-synaptic sites. Importantly, this occurs in isolated nerve-muscle preparations where all connectivity with the parent motor neurone cell bodies has been abolished. The heterogeneity of synaptic responses recorded is similar to events described in single motor units during developmental synapse elimination ,35 and fs) mice . These os) mice ,6, suppos) mice , each cos) mice ,58 and ws) mice . Here a s) mice  regulates) mice . Proteoms) mice  and one s) mice  which coIn summary, we provide evidence that following disconnection from their parent cell bodies, activation of P2X7 receptors on nerve terminals by BzATP triggers synaptic retraction. This retraction closely resembles similar events occurring during synapse elimination and motor neurone disease. We suggest that molecules and mechanisms constitutively present in the distal axon and terminal are sufficient to drive this retraction response. Further, we speculate that signals from the parent cell soma may not be necessary to control retraction during synaptic plasticity.2 intoxication, and intact preparations of lumbrical muscles, plantaris tendon and sciatic/tibial nerves were dissected and pinned out at resting length in Sylgard-lined Petri dishes containing oxygenated (95%O2/5%CO2), calcium and magnesium free physiological saline  at room temperature. These were transferred to oxygenated physiological saline as above, and any muscles which failed to twitch in response to nerve stimulation were excluded from further assay. Preparations were either maintained throughout in oxygenated physiological saline or alternatively in the same saline supplemented with several different purinergic agonist and antagonists. Purine receptor agonists and antagonists used were as follows: 2'-3'-O-(4-benzoylbenzoyl)-adenosine 5'-triphosphate (BzATP: 100 μM), Brilliant blue G (BBG: 1 μM), Reactive Blue 2 (RB2: 100 μM), Suramin (100 μM). All experiments began within 60 min of animal sacrifice.Adult C57BL/6 mice were killed by COAfter the experimental period (3 hrs), preparations were fixed in 4% paraformaldehyde for 30 mins and stained for immunofluorescence microscopy. Synaptic vesicles and neurofilaments were labelled with a combination of SV2 (1:500) and NF165 , and visualised with Cy2-  secondary antibody. Post-synaptic acetylcholine receptors were labelled with tetramethyl rhodamine isothiocyanate conjugated ãbungarotoxin . Slides were coverslipped in 2.5% n-propyl gallate in glycerol and viewed with epifluorescence illumination. In preliminary experiments we determined that a 15 min pulse of BzATP was the optimum trigger for nerve terminal retraction.Mice were killed and handled as above, but in this case intact preparations of flexor digitorum brevis muscles and associated sciatic/tibial nerve were dissected and placed Immediately after dissection into freshly prepared and oxygenated physiological saline. Addition of chemicals began within 60 minutes of animal sacrifice. BzATP (100 μM) was added for 30 min and preparations were fixed in buffered 4% paraformaldehyde/0.2% glutaraldehyde, pH 7.2 either immediately or subsequent to a 60 min washout period. Muscles were fixed and labelled for electron microscopic localisation of P2X7 receptor subunits (P2X7RS) as previously described .Preparations were treated identically to those above, except at the end of the 3 hr period, they were vitally labeled and imaged. Preparations were loaded with the vital styryl dye RH414  in physiological saline solution by nerve stimulation , followed by at least 1 hr washing in oxygenated physiological saline. Preparations were transferred to the microscope stage and terminals were imaged.25 sequentially identified endplates from randomly oriented immunostained lumbrical muscles were identified and quantified for the degree of congruence between overlying nerve terminal and muscle endplate from four muscles per hindfoot. Three categories were used; 'occupied', denoting complete occupancy; 'intermediate', where either endplate was partially occupied by nerve terminal or was fragmented; 'unoccupied', where no nerve terminal was present at the endplate. Occasionally, damaged muscle fibres or unusually deep endplates were difficult to accurately assess, and were discarded from the data set. Data are presented as mean ± SEM, N = number of mice, n = number of endplates. GraphPad Prism (Graphpad software) was used to carry out statistical tests.NLB carried out and analysed the fluorescence microscopy data and drafted the manuscript, TSM carried out the electron microscopy, BB carried out and analysed additional fluorescence microscopy, JD participated in experimental design and helped draft the manuscript, SHP carried out preliminary experiments, conceived the study and drafted the manuscript.