H. pylori, whose infection increases tumor invasiveness and metastasis, is generally labelled as the strongest risk factor for the development of gastric cancer. It appears not to be a coincidence that there is also an overexpression of CXCR4 and an obvious involvement in gastric cancer metastasis. The aim of this study attempts to investigate and further to establish a link between them. With H. pylori being a potent inducer of TNF-α, whether TNF-α, a tumor promoter, is involved in the induction of CXCR4 expression by H. pylori was also under research in this study.Expression of CXCR4, TNF-α, IL-6 and IL-1β mRNA was determined by real-time PCR. CXCR4 protein expression was detected by Western blotting. Concentrations of TNF-α, IL-6 and IL-1β in cell culture supernatants were measured using the Quantikine Elisa kit. To abrogate TNF-α expression in HGC27 cells, TNF-α RNAi plasmid was used to transfect them.+ ) or Tx30a (cag PAI- ); The induction of CXCR4 expression by H. pylori was inhibited significantly by a neutralizing TNF-α antibody, infliximab; CXCR4 expression was upregulated in MKN45 cells after treatment with exogenous TNF-α or co-culture with macrophage, and was downregulated in HGC27 cells after transfection with TNF-α RNAi plasmid. There was a significant increase in the migration of MKN45 cells treated with H. pylori 26695, and a strong inhibition when AMD 3100, a CXCR4 antagonist, or infliximab, was added.Levels of CXCR4 and TNF-α mRNA were significantly higher in H. pylori-positive gastric cancers (n = 19) compared to H. pylori-negative ones (n = 15). A subsequently Spearman's rank correlation test showed there was a positive correlation between the level of CXCR4 mRNA and that of TNF-α in 34 primary gastric cancers. Other results followed: Expression of CXCR4 and TNF-α was upregulated in gastric cancer cell MKN45 and HGC27 after infection with H. pylori 26695 (cag PAIOur findings demonstrated that H. pylori upregulates CXCR4 expression in gastric cancer through TNF-α. It is well accepted that Helicobacter pylori (H. pylori) is a strong risk factor for the development of various gastric diseases, namely chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer, and it is acknowledged that the interaction between H. pylori and epithelial cells contributes to such development. In fact, H. pylori infection induces inflammation in microenvironment of the stomach associated with induction of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and IL-6, which mH. pylori infection also increases tumor invasiveness and metastasis -6, thougOne of the key chemical mediators implicated in inflammation-associated cancers is TNF-α, and there is now substantial evidence in its involvement in promotion and progression of experimental and human cancers ,11. True2. 34 primary gastric cancer specimens were acquired from patients under operation with all their informed consent at Shengjing hospital, Chinese Medical University, and were frozen in liquid nitrogen immediately after surgical removal. Haematoxylin- and eosin-staining sections were prepared for assessment of the percentage of tumor cells, and only specimens with > 70% tumor cells were selected for analysis. This study was carried out with the approval of the ethical committee of China Medical University. All experiments were done at least three times.The human gastric cancer cell MKN45 and HGC27 were obtained from Keygen Biotech. Co. , and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum, at 37°C in a humid incubator with 5% CO2.The macrophage cell RAW 264.7 was provided by the American Type Culture Collection , and was maintained in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37°C in a humid incubator with 5% CO+ ) and Tx30a  were offered by ATCC . They were grown on sheep blood agar plates at 37°C under microaerophilic conditions. Bacteria were transferred after 48 h into Brucella broth containing 5% fetal bovine serum for 24 h. A multiplicity of infection of 100 was used in all studies.H. pylori strain 26695 , 1 μl; RNase inhibitor, 0.25 μl; RNA 1 μl; dH2O, 3.75 μl. Conditions for RT were: 30°C for 10 minutes, 42°C for 25 minutes, 99°C for 5 minutes, and 5°C for 5 minutes. Real-time PCR was performed using the LightCycler system together with the LightCycler DNA Master SYBR Green I Kit (Roche Diagnostics). The total volume is 20 μl, containing 25 mM MgCl2, 3 μl; 10 × Buffer, 5 μl; 10 μM forward Primer, 1 μl; 10 μM reverse Primer, 1 μl; LightCycler DNA Master SYBR Green I, 2 μl; cDNA, 2 μl; dH2O, 6 μl. Conditions for PCR were: 50°C for 2 minutes, 95°C for 10 minutes, and then 40 cycles of 5 seconds at 95°C and 20 seconds at 60°C. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. Gene expression was quantified by the comparative CT method, normalizing CT values to GAPDH and calculating relative expression values. Primer sequences were provided by Takala  as follows: CXCR4 forward, 5'-GAGGAAATGGGCTCAGGG-3', reverse, 5'-AGTCAGCAGGAGGGCAGGGA-3'; TNF-α forward, 5'-AGTGACAAGCCTGTAGCCC-3', reverse, 5'-GCAATGATCCCAAAGTAGACC-3'; IL-1β forward, 5'-CCACCACTACAGCAAGGG-3', reverse, 5'-GAACTGGGCAGACTCAAA-3'; IL-6 forward, 5'-CCTTCGGTCCAGTTGCCTTCT-3', reverse, 5'-GCATTTGTGGTTGGGTCA-3'; GAPDH forward, 5'-GGGAAACTGTGGCGTGAT-3', reverse, 5'-AAAGGTGGAGGAGTGGGT-3'.Total RNA was isolated from tissues and cell lines by Trizol  according to the protocol supplied by the manufacturers. cDNA was synthesized using Takara RNA PCR 3.0 Kit  in a total volume of 10 μl, containing AMV reverse transcriptase, 0.5 μl; random 9 primer, 0.5 μl; 25 mM MgCLβ-actin  was used as an internal control. Blots were visualized with FX pro plus system (Bio-Rad) and quantified using Scion Image 4.03 software.Cell lysates were prepared with sample buffer  and were subjected to a 12% sodium dodecyl sulfate (SDS)/acrylamide gel. The proteins on acrylamide gel were transferred to a nylon membrane, which was blocked overnight (4°C in PBS with 0.1% Tween and 10% milk powder). Polyclonal antibodies for CXCR4 , and the corresponding secondary antibodies  were applied before immunoblotting. The human gene 5 . On the following day, cells were transfected with TNF-α siRNA or Control siRNA using Lipofectamine 2000  according to the manufacturer's instructions.TNF-α RNAi plasmid and nonsilencing control RNAi plasmid were purchased from Takala . Cells were seeded into a 24-well plate at a density of 2 × 10Concentrations of TNF-α, IL-1β and IL-6 in cell culture supernatants were measured using the Quantikine Elisa kit  according to the manufacturer's instructions. The sensitivity of the assay was 2 pg/ml for TNF-α, 4 pg/ml for IL-1β and 4 pg/ml for IL-6.5 ) were added to the upper chamber and medium supplemented with CXCL12  was added to the lower chamber. Migration assays were incubated for 18 hours at 37°C and 5% CO2. Migrated cells on the lower surface were stained using 1% toluidine blue after fixation with 100% methanol. For each transwell, the number of migrated cells in 10 medium power fields (× 20) was counted.The migration of cultured cells was assayed using Matrigel invasion chamber . Cells . Difference was considered significant when P-value was < 0.05.Mann-Whitney GAPDH expression. The results revealed its level significantly higher in H. pylori-positive gastric cancers (n = 19) compared to H. pylori-negative ones (n = 15)  and more secretion of TNF-α protein into the culture supernatant  in MKN45 and HGC27 cells , was then used to treat MKN45 and HGC27 cells after an infection by 26695, and the induction of CXCR4 was inhibited significantly  for 6 hours in attempt to further explore the role of TNF-α in the upregulation of CXCR4 expression, and real time-PCR and Western blotting detection revealed it was upregulated significantly in a dose-dependent manner  and IL-6 (50 ng/ml) respectively, to rule out the possibility that IL-1β and IL-6 may upregulate CXCR4 expression like TNF-α. Neither IL-1β nor IL-6 was found to upregulate CXCR4 expression significantly Figure .P < 0.001, Figure P < 0.01, respectively, Figure Migration analysis was performed using the Matrigel invasion chamber in an attempt to investigate whether the upregulated CXCR4 was functional. In the initial phase of the experiment with 100 ng/ml of CXCL12 in the lower chamber, there was a significant increase (up to 4.5 folds) in the migration of MKN45 cells with 26695 treatment in the upper chamber, compared with control cells without 26695 treatment  was found to increase MKN45 cell migration significantly (H. pylori is blamed to infect about 50% of the world's population as a definitive gastric carcinogen for humans . Pathogevs 3.1 folds in MKN45 cells), which suggested H. pylori products in cag PAI may be also involved in the induction. In fact, it had been reported that cagA of H. pylori could induce TNF-α in gastric cancer biopsy specimens [Though the mechanism of induction of TNF-α by H. pylori remains relatively unclear, a protein family has been disclosed in the last decade, including Helicobacter pylori-membrane protein-1 (HP-MP1) and TNF-α inducing protein (Tipα) -24. Tipαpecimens . In addipecimens .TNF-α, a key cytokine in many chronic inflammatory diseases, was originally labelled as a serum factor for the induction of hemorrhagic necrosis of transplanted solid tumors in mice. However, presently it is commonly identified as a tumor promoter in local tumor microenvironment, and therefore the deletion or inhibition of it is supposed to reduce the incidence of experimental cancers. TNF-α/TNF-R1 knock down mice are resistant to chemical-induced carcinogenesis ,28. It iTNF-α was found not only involved in cell transformation and proliferation, but also in tumor metastasis. Such a finding was initially based on an animal model with colon cancer, in which injection of LPS enhanced the development of lung metastasis dependent on TNF-α production by host cells . The subOverexpression of CXCR4, whose involvement in various human tumors is well known, was frequently observed in gastric cancer tissues to increase gastric cancer metastasis. Some human gastric carcinoma cells also express CXCR4 mRNA and protein at high levels ,8. Our sMacrophages' involvement in the carcinogenesis and tumor invasion and metastasis ,32 generStudies have ultimately attributed the abnormal activation of NF-κB in cancer cells to the excessive secretion of TNF-α, whose role in CXCR4 upregulation is subsequently assumed to be related to pathways mediated by NF-κB. Others findings have revealed TNF-α antagonists can inhibit the upregulation of CXCR4 expression by H. pylori, and both it and CXCR4 antagonists can suppress the increased migration of gastric cancer cells in vitro. These results suggest that these antagonists alone, or in combination with other therapies, may serve as effective therapies for gastric cancer patients.TNF-α is involved in the upregulation of CXCR4 expression in gastric cancer induced by H. pylori.The authors declare that they have no competing interests.ZC designed the study, carried out PCR analysis, analyzed and interpreted the data, and drafted the manuscript. LX performed Western analysis. BM performed cell transfection. ZN was engaged in drafting the manuscript and in statistical analysis. WW performed Elisa analysis. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/10/419/prepub


Caenorhabditis elegans MBT protein LIN-61 is a member of the synMuvB chromatin-remodelling proteins that control vulval development. Here we report a new role for LIN-61: it protects the genome by promoting homologous recombination (HR) for the repair of DNA double-strand breaks (DSBs). lin-61 mutants manifest numerous problems associated with defective HR in germ and somatic cells but remain proficient in meiotic recombination. They are hypersensitive to ionizing radiation and interstrand crosslinks but not UV light. Using a novel reporter system that monitors repair of a defined DSB in C. elegans somatic cells, we show that LIN-61 contributes to HR. The involvement of this MBT protein in HR raises the possibility that MBT–deficient tumours may also have defective DSB repair.Malignant brain tumour (MBT) domain proteins are transcriptional repressors that function within Polycomb complexes. Some MBT genes are tumour suppressors, but how they prevent tumourigenesis is unknown. The  The genome is continually under threat from exogenous sources of DNA damage, as well as from sources that originate within the cell. DNA double-strand breaks (DSBs) are arguably the most problematic type of damage as they can cause dangerous chromosome rearrangements, which can lead to cancer, as well as mutation at the break site and/or cell death. A complex network of molecular pathways, collectively referred to as the DNA damage response (DDR), have evolved to protect the cell from these threats. We have discovered a new DDR factor, LIN-61, that promotes the repair of DSBs. This is a novel and unexpected role for LIN-61, which was previously known to act as a regulator of gene transcription during development. C. elegans to humans DNA is maintained in the cell as chromatin: double-stranded DNA wrapped around core histone octomers to form nucleosome subunits. Chromatin folds into higher order structures depending on how tightly DNA is wrapped around the histones and how closely the nucleosomes interact C. elegans MBT protein LIN-61 is also implicated in transcriptional regulation. It is a member of the synthetic multivulva (synMuv) class B group of proteins that act redundantly with synMuvA proteins to repress transcription of lin-3 EGF and lin-60 Ras lin-61 is also involved in maintaining genome stability. Worms depleted of lin-61 have elevated rates of germline and somatic mutation, including small DNA insertions and deletions, but how LIN-61 maintains the genome fidelity was unknown lethal(3)malignant brain tumour [l(3)mbt)] mutants of Drosophila develop malignant transformations of the adult optic neuroblast and ganglion mother cells of the larval brain L3MBTL2, L3MBTL3 and SCML2 are mutated in rare cases of medulloblastoma l(3)mbt malignancies of Drosophila ectopically express germline genes, the expression of which is required for tumour growth lin-61 mutants also express germline genes in the soma in a temperature-dependent manner Some MBT domain proteins act together with Polycomb group (PcG) repressor complexes that are best known for establishing and maintaining gene expression patterns during development lin-61 mutants. We show that LIN-61 acts within the DDR where it is needed for efficient double-strand break (DSB) repair in both the germline and somatic cells of C. elegans. LIN-61 promotes DSB repair by homologous recombination (HR), but not the competing pathways, non-homologous end joining (NHEJ) or single-strand annealing (SSA). Despite the requirement for LIN-61 in HR, it is dispensable for meiotic recombination and the DNA damage checkpoints (cell cycle arrest and apoptosis) in the germline. We also use a novel GFP-based HR reporter assay that confirms LIN-61 is needed for HR. This reporter monitors the repair of a single defined DSB and is a new tool for measuring HR in C. elegans somatic cells. This is the first report demonstrating that an MBT protein promotes DNA repair and provides an explanation for why MBT-deficient cells have genomic instability.As well as regulating transcription, an increasing number of chromatin-remodelling proteins (including PcG proteins) have been found to act within the DNA damage response (DDR). These proteins accumulate at sites of DNA damage where they locally modify chromatin to allow the recruitment of DNA repair proteins lin-61 , which is a potent inducer of DSBs  mutants with IR but found that they were not more sensitive than wild type controls , shares a high degree of sequence conservation with LIN-61 and both proteins are comprised almost entirely of four MBT domains . We wondcontrols . Therefolin-61 mutant germlines suggested that LIN-61 might be required for DSB repair during gametogenesis. We therefore investigated if LIN-61 also had a role in the repair of programmed DSBs that arise during meiosis. Meiotic DSB repair is required for the proper segregation of chromosomes to gametes and involves the repair of programmed DSBs introduced by the topoisomerase-like protein SPO-11 lin-61 meiotic cells was indistinguishable from those of wild types (lin-61 mutants. Interhomolog HR enables crossover (CO) formation, which establishes the physical connection (chiasmata) that holds homologs together until their separation at the first meiotic cell division. Diakinesis stage oocytes of lin-61 mutants contained the correct complement of six bivalents (paired homologs), which indicated that CO formation was competent in these mutants. Furthermore, lin-61 mutants produced mostly viable progeny and did not display an increased incidence of males (Him) phenotype (brc-1 and the cohesin-like mutants smc-5/-6. These mutants are IR hypersensitive due to defective DSB repair by HR that uses the sister chromatid (intersister HR) lin-61 mutants were hypersensitivity to IR suggested that LIN-61 might also contribute to intersister HR.The IR-hypersensitivity of ld types . This inhenotype . Failed brc-1, or the rad-51 paralog rfs-1 are therefore hypersensitive to ICLs lin-61 mutants were hypersensitive to nitrogen mustard (HN2), which is a potent inducer of ICLs . ICLs are particularly cytotoxic lesions that block the replication fork by covalently linking opposing strands of double-stranded DNA  of ICLs .polh-1 mutants are therefore hypersensitive to UV-C rfs-1 are not lin-61 mutants were not hypersensitive to UV-C  do not cause a DSB and do not require HR for repair. Instead, translesion synthesis (TLS) DNA polymerases such as POLH-1 bypass these lesions to allow replication to proceed  to UV-C . The senlin-61 promotes HR in somatic cells, we scored the viability of γ-irradiated early stage lin-61 embryos. These embryos were indeed hypersensitive to IR, which was indicative of an HR defect  and lin-61(pk2225) L1 larvae did not display substantial growth delay following IR, whereas most NHEJ-deficient cku-80 mutants failed to develop to the L4 stage 48 hours after irradiation , and thereby preventing interhomolog HR, it has been possible to demonstrate that BRC-1 contributes to meiotic intersister HR brc-1 mutants that were depleted of key SC components brc-1 mutants, neither the oocytes of lin-61(pk2225) or lin-61(n3809) contained chromosomal fragmentation after depletion of the core SC component, SYP-2  but are hypersensitive to IR. To confirm that LIN-61 is required for DSB repair specifically in mitotic germ cells we used an assay that directly tests whether DSBs are adequately repaired in irradiated germ cells. Completion of DSB repair can be determined in germ cells by observing chromosomes at diakinesis because chromosome fragments are present if DSBs are unrepaired lin-61 mutants contained six bivalents and were not fragmented   , which iadiation . Thus wh mutants . Moreove pathway .lin-61 mutants. The first stages of HR involve the nucleolytic processing at the DSB to expose single stranded 3′ overhangs (DNA end resection) and subsequent coating of these overhangs with RAD-51. RAD-51 foci rapidly formed in the γ-irradiated mitotic germ cells of both wild types and lin-61 mutants   . Since tlin-61 mutants was due to defective DSB repair (and not other types of damage), we developed an assay that specifically measures HR-mediated repair of a defined DSB. This assay was based on the DR-GFP reporter system, which has been used extensively to measure HR proficiency in cultured human cells C. elegans researcher. The new C. elegans reporter consisted of a gfp gene in which part of the open reading frame had been deleted and replaced by an I-SceI endonuclease recognition site, which rendered the GFP non-functional, and provided the defined location where the DSB could be introduced  was located downstream of the reporter and served as a template for synthesis-dependent strand annealing (SDSA)  . SDSA isonal GFP .elt-2 promoter as their nuclei undergo endoreplication (S phase without mitosis) at several points during post-embryonic development gfp sequences since a disabled version of the HR reporter, which lacked these sequences, was not able to express GFP , we chose to express the reporter in intestinal cells using the ress GFP . To conftivation . This cotivation . Therefold types . This inlin-61(n3809) and lin-61(pk2225) mutants showed a substantial reduction in the frequency of HR reporter activation compared with wild types , the lin-61 mutants had enlarged mitotic nuclei and a reduced number of germ cells 24 hours after γ-irradiation (Sensitivity to DNA damage can be caused by failure to activate DNA damage checkpoints adiation .lin-61(n3809) and lin-61(pk2225) animals, while cep-1 mutants failed to undergo DNA damage-dependent apoptosis  were analysed in order to increase the proportion of germ cells present in the samples, considering LIN-61 is needed for repair of DSBs in both somatic and germ cells. Microarrays were performed on two different lin-61 alleles (n3809 and pk2225) in order to control for changes in gene expression that were due by background mutations present within only one of the single strains. 58 genes were identified that, in both mutants, had a 1.5-fold or greater change in expression level  , with only 6 genes (10%) downregulated. This is consistent with LIN-61 acting as a transcriptional repressor. Importantly, none of the genes alternatively expressed in lin-61 mutants were implicated in DNA repair. The lin-61 transcript served as a positive control in the microarray analysis as we had previously shown, using qRT-PCR, that this transcript was reduced approximately 4-fold in lin-61 mutants, likely due to nonsense-mediated decay  has only a minor effect on global gene transcription. Finally, since these experiments indicated that DNA repair genes are expressed at normal levels in lin-61 mutants, it is likely that LIN-61 influences DSB repair directly and not by ensuring that other DDR genes are appropriately expressed.Since LIN-61 is a transcriptional repressor, we checked whether DDR genes were appropriately expressed in ue<0.01) . Most ofed decay . Accordilin-61 mutants: DSBs are not adequately repaired due to defective HR. Accordingly, these animals are hypersensitive to IR and nitrogen mustard and DSBs remain unrepaired in diakinesis oocytes of γ-irradiated lin-61 mutants. LIN-61 contributes to HR in mitotic cells but it is dispensable for DSB repair during meiosis. Sensitivity of lin-61 germ cells to DSBs is not due to faulty DNA damage checkpoints as both cell cycle arrest and apoptosis are functional. Moreover, DNA repair genes are not inappropriately expressed in lin-61 mutants. The role of LIN-61 in HR is not restricted to germ cells because the somatic cells of early stage embryo are also very sensitive to IR. Also, later in development, intestinal cells are HR defective, as determined by the GFP-based HR reporter system. HR is essential for genome stability, as it is the principal DSB repair route in germ cells. It is also an error-free repair pathway. Blocking HR enables mutagenic and toxic repair routes to become active, which likely contributes to genomic instability in lin-61 mutants.In this study we have identified the underlying cause of genomic instability in lin-61 mutants as they form chiasmata normally and produce nearly completely viable broods. What is more, RAD-51 foci that appear in prophase are resolved by late pachytene in both wild type and lin-61 mutants, indicative of the successful repair of programmed DSBs. The proficiency of intersister HR can be tested in meiotic cells by disrupting the SC in order to prevent interhomolog HR. In this situation, DSBs remain unrepaired if intersister HR too is defective, which manifests as chromosomal fragmentation at diakinesis. Unlike brc-1 and smc-5/-6 mutants lin-61 mutants depleted of the SC component SYP-2 do not have fragmented diakinesis chromosomes, indicating that intersister HR is proficient in the meiotic cells of these mutants. Moreover, DSBs introduced by IR into lin-61 meiotic cells, but not brc-1 meiotic cells, are efficiently repaired.LIN-61 is expressed in all nuclei, both in the germline and somatic tissues lin-61 mutants are proficient in meiotic HR, their mitotic cells are defective in HR. These cells display signs of persistent and spontaneous DNA damage. Further, γ-irradiation of mitotic germ cells causes severe chromosome fragmentation in lin-61 mutants. Finally, lin-61 mutants are also hypersensitive to ICLs and the repair of these lesions occurs in S/G2 phase using the newly synthesised sister chromatid as the HR repair template lin-61 are also hypersensitive to IR and mitotic cells exclusively use the sister chromatid for HR While lin-61 mutants cannot explain their HR defect, LIN-61 likely acts directly at sites of DNA damage to promote DSB repair. This is an attractive hypothesis considering that chromatin can act as a physical barrier that must be remodelled to allow access of DDR factors to sites of damage. In addition, many proteins that alter chromatin structure have recently been implicated in the DDR including NuRD components MTA1, MTA2, CHD4, HDAC1 and HDAC2 C. elegans counterparts of these proteins are also synMuvB proteins like LIN-61. Intriguingly, L3MBTL2, the putative human orthologue of LIN-61, is part of a PcG-like complex (PRC1L4) that shares RING1, RING2 and HP1γ as partner members C. elegans hlp-2 HP1 mutants lin-61 mutants. PRC1L4, or a related L3MBTL2-containing PcG complex, may therefore act in DSB repair like LIN-61. Using immunofluorescence, we were not able to detect a change in LIN-61 intracellular localisation upon IR (data not shown). However LIN-61 is abundantly present and localised at chromatin in all cells, which may conceal its relocalisation around sites of DNA damage. Recruitment to sites of DNA damage has also not been observed for any other C. elegans synMuvB proteins, likely for similar reasons.Considering that the transcriptional profile of It is unknown how PcG activity promotes DSB repair but it is argued that inhibiting transcription locally at the DSB may be important as the transcriptional machinery could interfere with repair proteins or with DNA repair intermediates lin-61 mutants were HR-defective was that they might have altered expression of DDR genes. But contrary to this, microarray expression analysis did not reveal any alternatively expressed DDR genes in these mutants. Some alternatively expressed genes were identified but none are implicated in DNA repair. The vast majority of the alternatively expressed genes were upregulated rather than downregulated, which is in accordance with LIN-61 being a transcriptional repressor. A previous study found that germline genes were ectopically expressed in the somatic tissues of lin-61 mutants, but only when maintained at the relatively high temperature of 26°C lin-61 mutants grown at the normal laboratory temperature of 20°C had only minor changes in gene expression and did not overexpress germline genes. Importantly, lin-61 mutants grown at 20°C displayed a profound HR defect, which further indicated that altered gene expression was not the cause of defective DNA repair. The microarrays were performed using RNA from a mixed population of germ and somatic cells. We cannot strictly exclude the possibility that a distinct population of cells had altered DDR gene expression that went undetected. This is unlikely though, as the defect in DSB repair was systemic, occurring in multiple tissues and at various stages of development, and not isolated to a small number of cells.One possible explanation we considered for why C. elegans somatic cells. The reporter confirmed that LIN-61 is needed for HR. This tool was previously unavailable for C. elegans researchers. We propose it as a method for testing candidate HR genes, for example it confirmed that both BRC-1 and RTEL-1 have roles in HR in somatic cells, analogous to their functions previously only described in meiotic germ cells. Our experiments with the HR reporter also supported previous findings that suggested DSB repair pathways are dynamic and are in competition in somatic cells In this study we introduce a novel reporter system for monitoring HR in C. elegans. Also, in its current form the HR reporter is expressed only in intestinal cells, which in contrast to most C. elegans somatic cells still cycle postembryoniccally. This choice of cell type was largely motivated by the likely need for S- and G2 phase dependent DNA end resection at DSBs for HR type of repair to occur. However, when interpreting the data it must be considered that these cells are atypical because they progress and grow through cycles of endoreduplication and not via canonical cell cycle stages including mitosis. It is thus possible that the response to the HR reporter is cell type-dependent. Finally, since formation of the DSB relies on expression of the I-SceI transgene using the heatshock promoter, any possible differences in heatshock response must be carefully controlled for as these differences may affect the level of DSB induction.Though this system is a new tool that provides for the readout of repair, probably by an SDSA mechanism, of a defined DSB, it does have limitations. For example, the HR reporter does not easily allow for dissection of the biochemical processes that underpin HR pathways. These approaches are not well suited to L3MBTL2, L3MBTL3 or SCML2This is the first report showing that an MBT protein is needed for DSB repair. Genes encoding MBT proteins have previously been linked with tumourigenesis and can act as tumour suppressor genes. However, their contribution to DNA repair and genome stability is unknown. Our finding that LIN-61 is required for efficient HR may have implications for the treatment of MBT-deficient tumours, which may also be HR defective. HR-deficient tumours, such as those with BRCA1 or 2 hypomorphic mutations, are very susceptible to poly(ADP ribose) polymerase (PARP) inhibitors lin-61(n3809)lin-61(pk2225) (this study), lin-61(tm2649)mbtr-1(n4775)cep-1(gk138)rtel-1(tm1866)brc-1(tm1145)cku-80(ok861)polh-1(lf31)lfIs129  (this study); and LG X: lfIs82  (this study). To determine brood sizes, L4 larvae were singled on 6 cm plates with OP50 E. coli and transferred each day for three days. The number of viable progeny and unhatched eggs were counted, as well as the number of males in the brood.The Bristol N2 strain was used as the wild type strain and maintained at 20°C according to standard protocols syp-2 RNAi was performed as in lin-61(tm649) for which a single germ line was scored.All γ-irradiation was performed with a dose rate of 15 Gy/minute using an electronic X-ray generator set to 200 kV 12 mA . For L4 larval IR sensitivity, three L4 animals per plate (three plates per condition) were treated with various doses of γ-irradiation. For UV-C sensitivity, young adult (24 post L4 stage) worms were exposed to UV . HN2 sensitivity assays were performed as described Germlines were dissected in egg salts, Tween, levamisole and fixed in 2% paraformaldehyde for 5 minutes at room temperature, and snap frozen on dry ice, then placed in methanol at −20°C for 10 minutes, washed three times for 10 minutes in PBS with 1% Triton X-100 and blocked in PBST (PBS with 0.1% Tween 20) and 1% BSA for 30 minutes at room temperature. Samples were incubated overnight at 4°C with rabbit anti-RAD-51 antibodies  diluted 1∶200 in PBST 1% BSA and detected with Alexa488 goat anti-rabbit antibodies (Invitrogen) diluted 1∶1000. DNA was counterstained with 0.5 µg/ml DAPI and samples were mounted with VectaShield. RAD-51 foci were imaged with a Leica DM6000 deconvolution microscope collecting 0.5 µm Z-sections. The number of foci per nucleus was counted for each of the seven zones of the germline as described Worms were synchronised as L1 larvae by bleaching and grown to the L4 stage. Total RNA was isolated with Trizol reagent (Invitrogen), and cleaned with RNeasy kit (Qiagen). Service XS  performed the Affymetrix expression analysis according to standard protocols. Data was analysed with the MAS 5.0 algorithm using Tukey's biweight estimator. Significance  was determined using Wilcoxon's rank test. Sequence of qRT-PCR primers is available in elt-2::HR and Pelt-2::SSA reporter strains are provided in Details on construction of the PFigure S1lin-61 mutants are hypersensitive to IR. L1 larvae were irradiated with the indicated dose of IR and grown to adulthood before their brood sizes was determined. The average brood size of five adults was counted for each condition. Depicted is the average brood size from two experiments, normalised to the brood size of unirradiated animals. Error bars are standard error of the mean.The primordial germ cells of (JPG)Click here for additional data file.Figure S2mbtr-1 mutants are not sensitive to IR. The percentage of viable progeny laid by irradiated L4 larvae is plotted. Error bars represent standard deviation.mbtr-1 mutants are not sensitive to IR. (A) Protein sequence alignment of LIN-61 and MBTR-1. Asterisk (*), semicolon (:) and full stop (.) denote identical residues, conserved substitutions and semi-conservative substitutions, respectively. Residues present in the four MBT domains are coloured red, blue, green and purple. (B) (PDF)Click here for additional data file.Figure S3Quantification of RAD-51 foci in lin-61 germlines. (A) Stacked histograms showing the average number of RAD-51 foci per nucleus present in each of the seven zones of the germline. (B) Diagram depicting the germline divided into seven zones. Zones one and two include the mitotic zone; zone three is the transition zone (TZ); zones four and five are early-mid pachytene; zone six is late pachytene; and zone seven is late pachytene/diplotene. DTC, distal tip cell.(PDF)Click here for additional data file.Figure S4brc-1 L1 larvae do not display developmental delay following IR. Depicted is the proportion of animals that developed to the L4 stage 48 hours after being γ-irradiated as L1 larvae with the indicated dose. Error bars are s.d.(PDF)Click here for additional data file.Figure S5HR reporter activation requires donor sequence for activation. (A) Schematic diagram of versions of the HR reporter that contain  or lack  the gfp donor cassette. These reporters are expressed using the heatshock promoter. (B) Epifluorescence and brightfield images of adult worms 24 hours after DSB induction. GFP is visible in intestinal cells in XF460, but not XF444.(PDF)Click here for additional data file.Figure S6Pelt-2::SSA reporter. (A) Schematic showing the Pelt-2::SSA reporter. The Pelt-2::SSA reporter consists of an out-of-frame LacZ gene, disrupted by an I-SceI sites and stop codons in all three frames. A region of LacZ is duplicated and located between the elt-2 promoter and the I-SceI site, and provides homologous sequences for SSA. A DSB is introduced in the centre of the reporter by expressing Pheatshock::mCherry::I-SceI. Repair of the DSB by SSA places the LacZ gene in-frame and deletes the sequences between the homologous repeats (including the I-SceI site and stop codons). (B) LacZ  activity was visualised by the conversion of X-gal to 5,5′-dibromo-4,4′-dichloro-indigo, which has an intense blue colour. Shown are representative bright field images of L4/young adult worms expressing LacZ in their intestinal cells (C) Graph showing the percentage of worms containing at least one blue intestinal cell. Induction of mCherry::I-SceI was achieved by heatshocking L1 stage worms for 30 or 60 min. These worms were stained for LacZ expression 48 hours after heatshock. Error bars represent standard deviation.(JPG)Click here for additional data file.Table S1lin-61 mutants. This table lists the genes alternatively expressed in L4 stage lin-61 mutants compared with wild types, as determined by Affymetrix expression analysis.Genes alternatively expressed in (XLSX)Click here for additional data file.Text S1lin-61 mutant alleles; construction of Pelt-2::HR reporter, Pelt-2::SSA reporter and Phsp16-41::mCherry::ISce-I; SSA reporter assay; list of qRT-PCR primers and L1 larvae IR assay.Supporting Experimental Procedures. Methods and materials are described for the following supporting experimental procedures: Description of (DOCX)Click here for additional data file.


Rp1 is a complex locus of maize, which carries a set of genes controlling race-specific resistance to the common rust fungus, Puccinia sorghi. The resistance response includes the “Hypersensitive response” (HR), a rapid response triggered by a pathogen recognition event that includes localized cell death at the point of pathogen penetration and the induction of pathogenesis associated genes. The Rp1-D21gene is an autoactive allelic variant at the Rp1 locus, causing spontaneous activation of the HR response, in the absence of pathogenesis. Previously we have shown that the severity of the phenotype conferred by Rp1-D21 is highly dependent on genetic background.Rp1-D21 is highly dependent on temperature, with lower temperatures favoring the expression of the HR lesion phenotype. This temperature effect was observed in all the 14 genetic backgrounds tested. Significant interactions between the temperature effects and genetic background were observed. When plants were grown at temperatures above 30°C, the spontaneous HR phenotype conferred by Rp1-D21 was entirely suppressed. Furthermore, this phenotype could be restored or suppressed by alternately reducing and increasing the temperature appropriately. Light was also required for the expression of this phenotype. By examining the expression of genes associated with the defense response we showed that, at temperatures above 30°C, the Rp1-D21 phenotype was suppressed at both the phenotypic and molecular level.In this study we show that the phenotype conferred by Rp1-D21 is temperature sensitive in a reversible manner, that the temperature-sensitivity phenotype interacts with genetic background and that the phenotype is light sensitive. This is the first detailed demonstration of this phenomenon in monocots and also the first demonstration of the interaction of this effect with genetic background. The use of temperature shifts to induce a massive and synchronous HR in plants carrying the Rp1-D21 genes will be valuable in identifying components of the defense response pathway.We have shown that the lesion phenotype conferred by maize autoactive resistance gene  Rp1 locus of maize carries variable numbers of tandemly-repeated genes belonging to the nucleotide binding site, leucine-rich repeat (NB-LRR) class of major disease resistance genes  and F1 progeny carrying the wild-type (i.e. non-autoactive) H95 allele at the Rp1 locus (i.e. wild type F1 progeny). Importantly the wild type and mutant F1 progeny were almost entirely isogenic outside the Rp1 locus [The l to H95 . Rp1-D21p1 locus .Rp1-D21-H95 line and crosses between the Rp1-D21-H95 line and 13 diverse lines were used for this study  and several in which it is substantially enhanced  as well as several crosses with intermediate phenotypes.The 1 crosses, together with the Rp1-D21-H95 line were grown in the greenhouse at three different temperature regimes: 22/18°C, 26/22°C, and 30/26°C higher/ lower for 12 hours each. For simplicity, these regimes are referred to here as, respectively, LowGH, MidGH, and HiGH (see Methods). Two traits associated with the severity of the Rp1-D21 phenotype were scored on all the plants, mean lesion score (LES), and mean mutant to wild type height ratio (HTR). LES is simply a score of the lesion severity  in every case regardless of genotype. However, the ranking of these two traits at 18/22°C and 22/26°C depended on the genetic background although in general the phenotype was most extreme at the lower temperature  interactions between genotype and environment, and genotype and temperature for both traits  showed that the most significant sources of variation for both LES and HTR were environment (meaning the two different occasions on which the experiment was performed in 2009 and 2010), temperature and genotype. There were also highly significant (ts Table .p < 0.001). At each temperature range the correlation was strong . The correlation showed a trend of weakening association between LES and HTR as temperature increased. This was probably because higher temperature suppressed the phenotypic expression of the Rp1-D21 phenotype.Since LES and HTR measure aspects of the same phenotype, it was not surprising that they were highly correlated. Combined data from Fall 2009 and Fall 2010 showed strong negative phenotypic correlation between LES and HTR - i.e. 4°C higher than the HiGH. After three weeks growth at 30/34°C (~four leaf stage) no lesions were visible on any of the plants and it was impossible to discern wild type from mutant segregants. In one growth chamber the temperature was then dropped to 18/22°C. After 48 hours, half the plants (i.e. presumably the plants carrying the Rp1-D21 gene) from the M37W, Tx303, M162W F1 families developed copious lesions  lesions started developing in these regions also , we wanted to determine whether exposure to light was important for lesion formation. In a separate experiment, eight seedlings of Tx303 × Rp1-D21 plants grown in bottles at ~100% humidity display characteristic lesions (data not shown). Therefore it seems that both light and a permissive temperature are required for Rp1-D21-associated lesions to form.It should be noted that wrapping a portion of the leaf in aluminum foil as well as blocking out light, may have the effect of locally increasing the humidity and possibly also the temperature of the wrapped part of the leaf. So, conceivably, altered temperature and humidity might underlie the failure to form lesions. However, we have observed in other experiments that Rp1-D21 expression is associated with other characteristics of the classical HR response including the generation of H2O2 and superoxide as well as the induction of expression of defense-related genes such as PR1, PR5, PRms and WIP1[Rp1-D21 phenotype could also be suppressed at the molecular (as opposed to the phenotypic) level at elevated temperatures, the following experiment was performed: Twelve seeds from a Tx303 × Rp1-D21-H95 F1 family were germinated and grown in a growth chamber for three weeks at 30°C. At this point all 12 plants appeared as wild type and leaf samples were taken from all of them. The temperature was then reduced to 18°C and within 48 hours, as expected, lesions were visible on the entire area of six of the plants, these were the segregants carrying the Rp1-D21 gene while the plants without lesions were their wild type, near-isogenic, siblings. Leaf samples from two wild type and three mutant plants were taken at the four week time-point (i.e. a week after reducing the temperature). Semi-quantitative RT-PCR analysis was performed on RNA extracted from the leaves of these five plants sampled at both the three- and four-week timepoints (i.e. before and after the temperature drop). The results indicated that, after three weeks of continuous growth at 30°C, PR1 and PRms were not detectably expressed while WIP1 was expressed at relatively low levels in both wild type and mutant segregants. After a week at 18°C, all three of these genes were highly induced relative to their levels at 30°C in mutant but not in wild type plants  were referred to as LowGH, MidGH, and HiGH, respectively. In each year the experiment was laid out in randomized complete block design with two replicates per year per temperature condition. Within each replicate three wild type and three mutant plants were measured per genotype in 2009 and two plants per genotype in 2010. Each replicate was arranged as a randomized complete block. The experiment was performed in fall 2009 and fall 2010. No artificial lighting was used.In each GH seeds were planted on a plastic germination tray. As soon as it was possible to distinguish wild types from mutant counterparts, seedlings were transplanted to a six inch pot with standard soil mix. Throughout the study period seedlings were watered two times a day on regular basis. Changes in growth and lesion development were photographed and documented.Rp1-D21 entirely and to determine whether the phenotype was reversible. Four F1 families: Tx303 × Rp1-D21-H95, M162W × Rp1-D21-H95, and M37W × Rp1-D21-H95, B97 × Rp1-D21-H95 were used for this experiment. In the first three of these F1 families, the Rp1-D21 phenotype is relatively enhanced; in the last it is relatively suppressed. The Rp1-D21-H95 and Rp1-D21-B73 lines were also included as standard checks for comparison. Eight plants were grown for each of the crosses in two growth chambers. Both growth chambers were initially calibrated at low temperature of 30°C and high temperature of 34°C. Illumation was fixed at 12 h dark and 12 h light and seedlings were regularly watered twice a day. In one growth chamber, after three weeks (corresponding to the four leaf stage), the temperature was changed to a low temperature of 18°C and high temperature of 22°C. After four weeks the temperature in this growth chamber was shifted back to 30/34°C. Temperature shifts took about 20 minutes to complete in every case. Light duration remained unchanged.For all the growth chamber experiments light level were maintained at approximately 325 micromoles/m/s using cool white fluorescent lights. Growth chamber experiments were performed to determine whether constant temperatures above 30°C could abrogate the expression of Rp1-D21-associated lesion formation, F1 crosses of Rp1-D21-H95 crossed to A632, B73, Mo17 and Tx303 were used. Twenty plants from each of the four F1 families cross were grown in pots (2 plants per pot) in a growth chamber under a daily photoperiod of 12 h. Initially the temperature of the growth chamber was maintained at a constant temperature of 30°C, but after 8-days of growth (V2 stage) the temperature was dropped to 26°C. Plants expressing HR lesions were counted twice every day for the next 9 days.For the experiment to measure the kinetics of Plants were evaluated for a lesion severity phenotype on a 0–10 scale with 0 being no symptoms and 10 being dead. Wild type to mutant height ratio was also measured. This was simply the average height of the mutant plants divided by the average height of the wild type plants ,14. LesiANOVA was performed using SAS version 9.2 . SAS Version 9.2 PROC CORR  was used to estimate the Pearson correlation coefficients. Heritability estimates were calculated using PROC MIXED procedure of SAS, as described previously .Total RNA was extracted from maize leaf tissue using Trizol  according to manufacturer’s instructions. RNA concentration, quality and integrity were monitored by the NanoDrop and agarose gel electrophoresis. For cDNA synthesis, 1μg of total RNA was reverse transcribed using M-MLV  following standard protocols. Briefly, total RNA was mixed with 1μl of Oligo (dT)20 primers , heat denatured at 65°C for 5 min and chilled on ice for 2 min. To each reaction 4 μl of 5× First Strand buffer, 2 μl of 0.1 M DTT, and 1μl RNaseOUT  was added. The reaction was incubated at 37°C for 2 min and then 1 μl of M-MLV was added, to a final volume of 20 μl. cDNA synthesis was performed at 37°C for 2 hours, followed by a 15 min incubation at 75°C for enzyme inactivation. The cDNA reaction was then diluted 5× using water.PR1, PRms and WIP1, respectively [EF1α and GAPDH2 were used as reference and amplified using primers EF1a-F1 (5’- ATCTGAAGCGTGGGTATGTG-3’) and EF1a-R1 (5’- GCATAGCCATTGCCAATCTG-3’) and GAPDH2-F2 (5’- GACTTCCTTGGTGACAGCAGG-3’) and GAPDH2-R2 (5’- CTGTAGCCCCACTCGTTGTC-3’), respectively. The EF1a and GAPDH2 primers were from Hachez et al. [GAPDH gene using B73 genomic DNA as positive control for the PCR.The cDNAs were used for semi- quantitative measurements of gene expression using PCR. Primers PR1-F (5′-AGGCTCGCGTGCCTCCTAGCTCTGG-3′) and PR1-R (5′-GGAGTCGCGCCACACCACCTGCGTG-3′), PRms-F (5′-ACCTGGAGCACGAAGCTGCAG-3′) and PRms-R (5′-GCAGCCGATGCTTGTAGTGGC-3′), WIP1-F (5′-TGCTGATCCTGTGCCTCCAG-3′) and WIP1-R (5′-CTCTCTGATCTAGCACTTGGGG-3′) were utilized to amplify the maize defense genes z et al.  and sligThe authors declare that they have no competing interests.AN, GJ, PBK planned the project. AN, GFW performed experiments examining the interactions between genetic background and temperature. LB planned and performed the RT-PCR experiments. CMK planned and performed the experiments for the objective quantification of the background effect on temperature. GFW, VC planned and performed the experiments on the effect of light. AN, PBK analyzed the data. AN, PBK wrote the manuscript. AN, GJ, PBK, GFW edited the manuscript. All authors read and approved the final manuscript.The scoring scale used for visual scoring of the lesions. Leaves scored from 1–8 are shown. An entirely dead leaf would score a “10” whereas a “9” would be a leaf with just a few patches, about 5%, of living tissue.Click here for file1 cross between the line indicated and Rp1-D21-H95. The red line indicates the point up to which the 5th leaf had emerged at the time of the temperature shift. While only one plant is pictured for each background, at least 3 mutant F1 plants were observed in each background and the results were essentially similar in each case.Plants in 13 different backgrounds were grown for four weeks at 22/18°C, 12 hr light/dark and then shifted to 34/30°C 12 hr light/dark. At this point at which the 5th leaves were partially emerged from the whorl. Pictures were taken from 5th leaves five days after temperature shift. In every case the genotype of the plant is an FClick here for file


Blood IL-6 and TNF-α concentrations of 5 subjects estimated from the microchip data were compared with results obtained by the conventional method, good correlations were observed between the methods according to linear regression analysis . The reproducibility of the presented assay for the determination of the blood IL-6 and TNF-α concentration was comparable to that obtained with the 96-well plate. Simultaneous detection of blood IL-6 and TNF-α was possible by the deposition and fixation of each 1st antibody on the surface of a separate microchannel. This assay enabled us to determine simultaneously blood IL-6 and TNF-α with accuracy, satisfactory sensitivity, time saving ability, and low consumption of sample and reagents, and will be applicable to clinic diagnosis.Sandwich enzyme-linked immunosorbant assay (ELISA) using a 96-well plate is frequently employed for clinical diagnosis, but is time-and sample-consuming. To overcome these drawbacks, we performed a sandwich ELISA on a microchip. The microchip was made of cyclic olefin copolymer with 4 straight microchannels. For the construction of the sandwich ELISA for interleukin-6 (IL-6) or tumor necrosis factor-α (TNF-α), we used a piezoelectric inkjet printing system for the deposition and fixation of the 1st anti-IL-6 antibody or 1st anti-TNF-α antibody on the surface of the each microchannel. After the infusion of 2 µl of sample to the microchannel and a 20 min incubation, 2 µl of biotinylated 2nd antibody for either antigen was infused and a 10 min incubation. Then 2 µl of avidin-horseradish peroxidase was infused; and after a 5 min incubation, the substrate for peroxidase was infused, and the luminescence intensity was measured. Calibration curves were obtained between the concentration and luminescence intensity over the range of 0 to 32 pg/ml (IL-6: R The expression of inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) has been associated with not only acute inflammation but also a number of chronic conditions associated with aging, such as cardiovascular disease, diabetes, physical disabilities and cognitive decline The analysis of biomarkers at a location near the patient, which analysis is called point of care testing (POCT), is a continuously expanding trend in the practice of laboratory diagnosis In the present study, we demonstrated the potential of the sandwich ELISA on a microchip, with the 1st antibody deposited by piezoelectric inkjet printing for the quantitative analysis of IL-6 and TNF-α which concentrations are very low in the blood of healthy individuals (pg/ml levels). Furthermore, simultaneous quantitative detection of IL-6 and TNF-α was performed by using the sandwich ELISA in independent microchannels on a microchip.The principle of the sandwich ELISA on a microchip was described in our earlier report Human IL-6 ELISA MAX Deluxe Sets were purchased from BioLegnd  and employed for the determination of the plasma concentration of IL-6 with a microtitration plate as the conventional method according to the supplied instructions. Briefly, 100 µl of Human IL-6 ELISA MAX™ Capture Antibody (2 µg/ml) was coated on a 96-well microtitration plate overnight at 4°C as 1st antibody, and then blocked with 200 µl of 1X Assay Diluent for 1 hr at room temperature. Then, 100 µl of standard sample or plasma sample was added to each well and incubated for 2 hr at room temperature. To construct a calibration curve we diluted Human IL-6 Standard with 1X Assay diluent A to 32, 16, 8, 4, and 2 pg/ml and used them as standard samples. Next, 100 µl of biotinylated Human IL-6 ELISA MAX™ Detection Antibody as 2nd antibody was added to each well, followed by a 1 hr incubation at room temperature. Thereafter, 100 µl of avidin-horseradish peroxidase (HRP) was subsequently added; and incubation was continued for 30 min at room temperature. After washing of the microtitration plate with wash buffer, 100 µl of substrate solution for peroxidase was added to each well; and the plate was then incubated for 30 min at room temperature. Finally, stop solution was added to each well; and the absorbance of the solution was then measured at 450 nm by using a microplate reader . The plasma concentration of IL-6 was calculated from the calibration curve and expressed as pg/ml.Human TNF-α antibody (MAB610) as 1st antibody, biotinylated anti-human TNF-α/TNFSF1A antibody (BAF210) as 2nd antibody, and recombinant human TNF-α (210-TA) as standard were purchased from R&D Systems Inc.  and employed for the determination of the plasma concentration of TNF-α with a microtitration plate as the conventional method. Briefly, 100 µl of 1st anti-TNF-α antibody was coated onto the wells of a 96-well microtitration plate, after which 100 µl of standard sample or plasma sample was added to each well. Following a 2 hr incubation at room temperature, 100 µl of 2nd anti-TNF-α antibody was added to each well; and incubation was carried out for 2 hr at room temperature. Finally, 100 µl of Streptoavidin-HRP was subsequently added to each well; and after 20 min, 100 µl of substrate solution was then added to the well. To construct a calibration curve, we diluted TNF-α with 1X Assay diluent to 32, 16, 8, 4, and 2 pg/ml and used them as standard samples. The plasma concentration of TNF-α was calculated from the calibration curve and expressed as pg/ml.For the sandwich ELISA assay on the microchip, we used the same monoclonal anti-IL-6 antibodies and anti-TNF-α antibodies that were used in the 96-well microtitration plates. For the deposition and fixation of 1 mg/ml 1st anti-IL-6 antibody and 1st anti-TNF-α antibody in spotting buffer  on the surface of the microchannel, a piezoelectric inkjet printing system  was employed. A jetting voltage of 8 V for antibody “inks” was used in this study. The volume of the drop ejected from the inkjet nozzle was 120 pl; and 12 drops were spotted onto the microchannel surface, 3 of which are depicted in Peripheral venous blood samples were collected from healthy subjects by standard venipuncture. All blood samples were collected into vacuum blood-collecting tubes containing EDTA-2Na , and the plasma samples were obtained by centrifugation at 1450×g for 10 min. The plasma was then stored at −80°C until used for determination of protein levels.This study was approved by the Institutional Review Board of the National Institute of Advanced Industrial Science and Technology for the use of human derivatives for biomedical research, and by the Ethics Committee of the University of Tokushima. All subjects provided written informed consent for the collection of samples and subsequent analysis.In the present study, we developed sandwich ELISAs on a COC microchip by depositing and fixing the 1st anti-IL-6 antibody and/or 1st anti-TNF-α antibody on the surface of the microchannels with a piezoelectric inkjet printing system . In both2 = 0.9954, TNF-α : R2 = 0.9928). These results indicated that accurate determination of plasma IL-6 and TNF-α could be achieved by use of our new method. Three different samples were analyzed repeatedly within 1 day or for several days for their IL-6 and TNF-α concentration . These results indicated that simultaneous quantitative detection of plasma IL-6 and TNF-α could be performed on a microchip.Next, we tested to see if we could determine the concentrations of plasma IL-6 and TNF-α simultaneously in separate microchannels on a microchip . Anti-ILher IL-6  or TNF-αher IL-6 ; p>0.05.We employed an inkjet printing system to develop the quantitative sandwich ELISA for measuring plasma IL-6 and TNF-α in the microchannel, because it has been shown to deliver small reagent volumes with precise alignment and high reproducibility Microchip-based multianalyte assay systems using a sandwich ELISA had already been reported earlier. In these assays, 1st-antibody-conjugated beads were introduced into the microchip and employed for the detection of several biomarkers In conclusion, we performed simultaneous analysis of IL-6 and TNF-α in human plasma with high accuracy and reproducibility. Furthermore, this assay also offers other considerable advantages, including low consumption of sample and reagents, reduced assay time, and ease of operation. This assay is thus applicable to POCT.


Moreover, TNF-α/IFN-γ, but not TNF-α plus ATP, increased dye uptake in EOC20 cells. Blockade of Cx43 and Panx1 HCs prevented dye coupling induced by TNF-α/IFN-γ, but not TNF-α plus ATP. In addition, IL-6 prevented the induction of dye coupling and HC activity induced by TNF-α/IFN-γ in EOC20 cells. Our data support the notion that extracellular ATP affects the cellular communication between microglia through autocrine and paracrine mechanisms, which might affect the timing of immune response under neuroinflammatory conditions.Microglia are the immune cells in the central nervous system. After injury microglia release bioactive molecules, including cytokines and ATP, which modify the functional state of hemichannels (HCs) and gap junction channels (GJCs), affecting the intercellular communication via extracellular and intracellular compartments, respectively. Here, we studied the role of extracellular ATP and several cytokines as modulators of the functional state of microglial HCs and GJCs using dye uptake and dye coupling techniques, respectively. In microglia and the microglia cell line EOC20, ATP advanced the TNF- Microglia are the major immune effectors in the central nervous system (CNS). Under resting conditions, surveillance microglia have a ramified morphology and monitor their local microenvironment , 2. Howeα, IL-1β, IL-6, IFN-γ, and other soluble molecules, such as glutamate and ATP i) and ATP release, through P2X7 receptors [2+]i or a calcium ionophore induce microglia activation i and extracellular ATP via P2 receptors i i –49, we dα, 1 ng/mL IFN-γ, or 1 ng/mL IL-1β showed only a slight increase in IDC, which was not statistically different from that of control cells (P > 0.05: Supporting information Table S1). However, treatment with mixes of these molecules during different time periods caused a significant and transient increase in IDC; the dye transfer data is expressed as percentage of the corresponding control condition  increased the IDC, reaching a maximum response at around 9 h after treatment  as previously described i in microglia i compared with resting microglia [α/IFN-γ, leading to release of IL-1β [α plus ATP, even after acute application of ATP, we propose that under these conditions ATP released by microglia via HCs was not required to induce IL-1β release. The latter is consistent with the prevention of TNF-α/IFN-γ-, but not TNF-α plus ATP-induced dye coupling in EOC20 cells treated with 10Panx1, a Panx1 HC blocker. In addition, we speculate that after treatment with TNF-α plus ATP P2X receptors also contribute in a Panx1 HC-independent way, as it has been proposed to occur during microglial proliferation [α/IFN-γ–induced dye coupling was confirmed using Cx43(E2) antibody, a specific Cx43 HC blocker. However, this conclusion should be taken cautiously because it was recently shown that several hours after Cx43(E2) antibody application, gap junctional communication is partially reduced [As shown in this work, the activity of microglial Cx and Panx HCs was increased by TNF-icroglia , 76, 77.icroglia . Extraceof IL-1β . Becauseferation . The rol reduced . α in presence of IFN-γ upregulates Cx43 GJCs in microglia as it was previously demonstrated [α/IL-1β increased Cx43 levels in microglia. On the other hand, IL-6 prevents the formation of GJCs induced by pro-inflammatory cytokines in dendritic cells [β release.Under control conditions microglial cells express low levels of Cxs , 26–28. nstrated , 28. In nstrated , TNF-α/Iic cells . Accordidel/del mice do not express functional GJCs [(E2) antibody prevented the pro-inflammatory-induced dye coupling, it is suggested that dye coupling induced by TNF-α plus ATP or TNF-α/IFN-γ could be due to Cx43 GJCs. To recapitulate, we propose that in presence of extracellular ATP, Panx1 HC activity is enhanced and microglia migrate toward the injured site and release cytokines, as reported previously [β release and providing a pro-inflammatory microenvironment, which promotes an early up-regulation of Cx43 and Panx1, favoring the formation of HCs and GJCs in a stimulus-dependent manner  plus ATP. Treatment with IL-6, IL-1ra, oATP, La3+, Cx43(E2) antibody or 200 µM 10Panx1 were simultaneous to cytokine treatment. We used β-GA for acute GJCs blocking.Short description for Supplemental Material: Microglia were seeded and after 48 h under control conditions were treated with 1 mM ATP or 1 ng/ml TNF-Click here for additional data file.


Hence some hasty catastrophic previsions have been neither correct nor reasonable. The stabilization of the IF 2010 on the value of 2.015 has reconfirmed that the reliance in the contents of the journal is constant and well placed. On behalf of the whole great team of people who work at this project, I express the sense of honor and the great satisfaction for the service rendered to the scientific community.The publication on June 28 2011 of the Journal Citation Reports(i)The number of citations has increased by 20% as compared to 2009(ii)The number of accepted papers has increased to 70% as compared to 2009(iii)The number of submitted papers has increased to 71% as compared to 2009(iv)The rejection rate has remained steady at the value of 55%After the JHP appearance, just 2 years ago, in the international IF ranking and the following push towards a rise, a phase of stabilization and consolidation of the results is welcomed and reassuring , 2. The Taking into account that we have fully recognized an increased quantity  of the submitted papers (iii) and it has resulted in a higher number of accepted papers (ii), making vain the point relative to the citations (i). This, however, witnessed the higher interest that the readership keeps showing towards JHP.The much debated and feared opening towards the strategy of open access to contents for everyone, without costs, has been feasible through the utilization of various grants. I thank European Headache Federation, Lifting The Burden, Sapienza University, Springer and advertisers, who made this choice possible. Consequently, the visibility of JHP has had a sudden and violent rise. In fact, in the first 4 months of this new SpringerOpen model, the rate of downloaded papers has risen up by 60%. All this has accomplished a much wider educational mission of every paper and therefore of JHP also, and lays strong basis for a positive fall-out in terms of number of citations on the next evaluation in 2012.It is now important to attract more and more high-value reviews, original papers, basic science papers or tutorials. These are the pillars of the editorial development of JHP. There is a need to attract also updated therapeutic guidelines, new classification proposals, or position papers, or RCTs, which are proved to be a great boost factor for the IF indexes. But these are field choices for the affiliated societies.Anyways, from 2012 JHP will appear with eight issues per year and the articles, now freely available online from the SpringerLink platform , will adconundrum of IF fills the month of June with hopes, doubts and expectations. JHP does not live this event with less anxiety; the Journal, beyond its possible imperfections, attributes real values to the work and the choices of all of us: Editors, Board Members, Referees, Researchers, Readers, Publisher.Every year the I think this is the staging post for every scientific journal, to serve the civil society helping it to reach its scientific, educational and health care unmet needs.Supplementary material (PDF 52 kb)Below is the link to the electronic supplementary material.


Lactobacillales based expression systems pSIP (Lactobacillus plantarum) and NICE (Lactococcus lactis) were evaluated with regard to their effictiveness to produce 2,5-DKG reductase from Corynebacterium glutamicum. Our results indicate that both systems are suitable for 2,5-DKG reductase expression. Maximum production yields were obtained with Lb. plantarum/pSIP609 by pH control at 6.5. With 262 U per litre of broth, this represents the highest heterologous expression level so far reported for 2,5-DKG reductase from C. glutamicum. Accordingly, Lb. plantarum/pSIP609 might be an interesting alternative to Escherichia coli expression systems for industrial 2,5-DKG reductase production.2,5-diketo-D-gluconic acid reductase  catalyses the reduction of 2,5-diketo-D-gluconic acid  to 2-keto-L-gulonic acid (2-KLG), a direct precursor (lactone) of L-ascorbic acid (vitamin C). This reaction is an essential step in the biocatalytic production of the food supplement vitamin C from D-glucose or D-gluconic acid. As 2,5-DKG reductase is usually produced recombinantly, it is of interest to establish an efficient process for 2,5-DKG reductase production that also satisfies food safety requirements. In the present study, three recently described food grade variants of the  Pectobacter cypripedii HEPO1 (DSM 12939) and the subsequent reduction of 2,5-DKG to 2-KLG using 2,5-DKG reductase from Corynebacterium glutamicum was previously developed in our laboratory  is an NAD(P)(H)-dependent oxidoreductase assigned to the aldo-keto reductase (AKR) family Ellis . 2,5-DKdkr), usually achieved with Escherichia coli. However, the utilization of genetically modified organisms (GMOs) to produce enzymes intended for food applications is strictly regulated  complementing corresponding gene deletions in the host chromosomes.The above mentioned processes depend on the heterologous (high-level) expression of the 2,5-DKG reductase gene  as the expression hosts. It is therefore important as well to evaluate the performance of such LAB expression systems with genes of taxonomic distant origin. The aim of the present work was to evaluate the food grade expression systems pSIP and NICE for their capacity to produce the industrially important enzyme 2,5-DKG reductase from C. glutamicum .Previous studies on the above mentioned expression systems demonstrated high expression levels with bacterial β-galactosidase genes  was used according to the instructions of the manufacturer. The PureYield™ Plasmid Miniprep System for isolation of E. coli plasmids and the Wizard® SV Gel & PCR Clean-UP kit for the purification of DNA fragments were obtained from Promega . The Quick Ligation Kit and restriction enzymes with their corresponding buffers were purchased from New England Biolabs . Phusion High-Fidelity PCR Master Mix (New England Biolabs) and a C1000 Thermal Cycler  were used to amplify DNA by PCR.Chemicals for enzyme assays, protein analysis and media components were purchased from commercial suppliers at the highest available level of purity. 2,5-DKG, the substrate for 2,5-DKG reductase assays, was produced by fermentation of glucose with C. glutamicum DSM 20301, cultivated in DSMZ medium 53 (German Collection of Microorganisms and Cell Cultures), was used as template for amplification of the 2,5-DKG reductase gene (dkr) (GenBank accession JQ407590.1). Plasmids were isolated from Lactococcus und Lactobacillus following the previously described protocol  of pTM51R was excised with BglII and SpeI, and replaced by the multiple cloning site (mcs) from pNZ8150  and SpeI. This yielded the expression plasmid pVK51dkr. Additionally, the complete dkr open reading frame (ORF) with its start codon located 73 bases upstream of the dkr translation start , following the protocol ofHolo and Nes (-1), supplemented with 1% lactose at 30°C.NICE expression vectors were based on the pTM51 vector series as presented by Maischberger et al. . The β- and Nes . Positi and Nes  with agdkr was amplified with the primer pair V1/V3 (Table sppA (pSIP603R) and PsppQ (pSIP609R)  was ligated directly to the pJet1.2 blunt-end cloning vector  and transformed into chemically competent E. coli NEB 5-α cells (New England Biolabs). The inserts were excised with SpeI and XhoI  were constructed. For dkr ORF amplification forward primer 5′-ATGTCTGTTGTGGGTACCGG-3′ and reverse primer V2 .The coding region of  et al. . CulturBatch cultivations of LAB with food grade vectors were performed in computer-controlled stirred reactors (6 × 0.5 L) of the HT-Multifors system . Comparative studies without and with pH control (pH 6.5) were performed. Culture pH was maintained by automated addition of sterile NaOH (1 M). To ensure homogenous distribution of the culture broth with limited oxygen transfer, a low agitation speed of 80 rpm was used. All experiments were performed in triplicate.L. lactis; MRS for Lb. plantarum) and incubation at 30°C without shaking. After 12 hours, the cells were transferred to the bioreactor already containing the corresponding medium to reach an optical density at 600 nm (OD600) of ∼0.1. Expression was induced at an OD600 of 0.35 ± 0.03. For the induction of Lb. plantarum harbouring pSIP alr-based vectors, the synthetic peptide pheromone SppIP  was used. To induce the NICE expression system with L. lactis NZ3900, nisin . All experiments were carried out at 30°C for 20 hours following induction.Inocula for the batch cultivations were prepared by transferring 20 μl of a frozen stock culture to 200 mL fresh medium (M17 for 600 and wet cell weight (WCW) after centrifugation at 15,000 × g for 15 min at 4°C. 2,5-DKG reductase activities and the total intracellular protein concentrations were determined in order to evaluate the expression levels. For that purpose, bacterial cells were harvested from 5 mL of culture by centrifugation at 3,220 × g for 10 min at 4°C, washed with Bis-Tris buffer  and resuspended in 500 μL of the same buffer. The cells were mechanically disrupted through bead beating with ∼1 g glass beads (average diameter of 0.5 mm) using a Precelly 24 glass bead mill . The cell-free crude extracts obtained after 10 min centrifugation at 9,000 × g (4°C) were used for 2,5-DKG reductase activity assays and determination of protein concentrations.Samples were taken in appropriate time intervals during the fermentations to monitor the growth of bacterial cultures by measuring OD+ per min .2,5-DKG reductase activity assay was performed spectrophotometrically as previously described  using 5% stacking gels and 10% separating gels. Samples were prepared according to method of Laemmli Laemmli  and loaC. glutamicum dkr gene by L. lactis subsp. cremoris MG1363 and Lb. plantarum WCFS1  and that of Lb. plantarum WCFS1 based on 3057 CDS’s (934462 codons)  were obtained 4 hours after induction. L. lactis reached a maximum OD600 of approx. 4 and a WCW of 3.6 g L-1. Lb. plantarum (with both pSIP603 and pSIP609) reached a maximum OD600 of just above 9 (WCW approx. 10 g L-1) and displayed maximum 2,5-DKG reductase activities of approximately 100 U L-1 after 8 hours of induction. The results of expressions without pH control  to 2.5 (Lb. plantarum/pSIP609) (see Table L. lactis reached a maximum OD600 of approx. 6 (WCW of 5.5 g L-1) and Lb. plantarum an OD600 of approx. 10 (WCW = 11.7 g L-1 with pSIP603 and 11.3 g L-1 with pSIP609) after 7 – 8 hours of induction. After reaching the growth maximum, volumetric activities began to drop in the Lb. plantarum cultures  was higher than with L. lactis, the induction factors did not differ significantly because of slightly higher basal expression of noninduced Lb. plantarum TLG02 cells. It can be concluded that some basal 2,5-DKG reductase expression, caused by “leakage” of the corresponding promoters, occured in noninduced Lb. plantarum TLG02 cells   (GenBank accession JQ407590.1) was used as translation start  when the complete dkr ORF (His6-tagged) was expressed with an E. coli expression system (Pacher dkr gene shows a region with a high concentration of purine bases (GAG GAA GAG), located downstream of the first initiation codon  using the third ATG codon as translational start  with all systems  and 8 codons (dkr), respectively can be considered “rare codons” (i.e. codons used in less than 20% of the cases) or “very rare codons” (i.e. codons used in less than 10% of the cases). Conversely, for Lb. plantarum there is no rare codon with a low fraction of usage within the first 50 codons of both ORF and dkr gene from C. glutamicum  were focussed on 2,5-DKG reductase optimization by site-directed mutagenesis and the kinetic characterisation of the obtained mutants after expression in E. coli, rather than the optimization of expression yields  that naturally accumulate 2,5-DKG from D-glucose have been used as expression host for dkr as well, and have been employed in the one-step production of 2-KLG  , the estimated costs for 2,5-DKG reductase production with Lb. plantarum would be at least 3 fold compared to E. coli. A strong argument to employ food grade expression systems however is that such, the costs to satisfy food safety requirements may be significantly reduced  were obtained with C. glutamicum dkr gene with Lb. plantarum , clearly indicate that the expression characteristics of the same system can be significantly influenced by the used promotor , as pSIP609 showed improved expression levels compared to pSIP603 in all cases. These data stand in contrast to the results recently published by Nguyen and co-workers  from L. lactis expressed with Lb. plantarum NC8 harbouring corresponding pSIP based vectors with erythromycin resistance  naturally uses in its highly expressed genes, the risk of tRNA depletion during translation can be minimized and hence the heterologous expression by lactic acid bacteria could be further optimized  ; NADP+: Nicotinamide adenine dinucleotide phosphate (oxidized form); NICE: Nisin controlled gene expression; ORF: Open reading frame; PepN: Aminopeptidase N; PnisA: Promoter nisin A; PsppA PsppQ: The bacteriocin promoters in the spp gene cluster.AKR: Aldo-keto reductase; Alr: Alanine racemase gene; CmThe authors declare that they have no competing interests.L. lactis NZ3900, Lb. plantarum TLG02 and Lb. plantarum WCFS1 cultivated without pH maintainance. Panel A: L. lactis NZ3900/pVK51ORFdkr; Panel B: Lb. plantarum/pSIP603ORFdkr; Panel C: Lb. plantarum/pSIP609ORFdkr. Panel A: Lane 1 and Lane 8, molecular mass standard protein; Lane 2, culture uninduced; Lane 3-7, induced culture after 2, 4, 6, 8 and 10 hours. Panel B, C: Lane 1 and Lane 8, molecular mass standard protein; Lane 2, culture uninduced; Lane 3-6, induced culture after 2, 4, 6 and 8 hours; Lane 7, wild type Lb. plantarum WCFS1. The arrows indicate the band representing heterolougously expressed complete dkr ORF.SDS-PAGE of cell free extracts of strains Click here for fileL. lactis and Lb. plantarum TLG02 after expression of the complete dkr ORF.Maximum activities of 2,5-DKG reductase measured in cell free extracts of induced and noninduced Click here for fileL. lactis NZ3900 or Lb. plantarum TLG02 cultivated without pH regulation. Panel A: L. lactis NZ3900/pVK51ORFdkr; Panel B: Lb. plantarum/pSIP603ORFdkr; Panel C: Lb. plantarum/pSIP609ORFdkr. The graph shows OD600 (triangles down), pH (crosshairs), 2,5-DKG reductase activity (units per liter of fermentation broth) (circles white) and specific activity (units per milligram protein) (circles black).Time course for growth of Click here for fileL. lactis NZ3900 or Lb. plantarum TLG02 cultivated with pH control at pH 6.5. Panel A: L. lactis NZ3900/pVK51ORFdkr; Panel B: Lb. plantarum/pSIP603ORFdkr; Panel C: Lb. plantarum/pSIP609ORFdkr. The graph shows OD600 (triangles down), 2,5-DKG reductase activity (units per liter of fermentation broth) (circles white) and specific activity (units per milligram protein) (circles black).Time course for growth of Click here for filedkr ORF (A) and dkr (B) of C. glutamicum in L. lactis subsp. cremoris MG1363. The vertical axis indicates the relative adaptiveness values (%) of triplet codons in L. lactis subsp. cremoris MG1363. The codons used in less than 20% of the cases are considered as rare and their codon usage fraction appears in grey.Codon usage analysis of the 50 first codons in complete Click here for filedkr ORF (A) and dkr (B) of C. glutamicum in Lb. plantarum WCFS1. The vertical axis indicates the relative adaptiveness values (%) of triplet codons in Lb. plantarum WCFS1. The codons used in less than 20% of the cases are considered as rare and their codon usage fraction appears in grey.Codon usage analysis of the 50 first codons in complete Click here for file


Since their appearance in the armamentarium for inflammatory bowel disease (IBD) more than a decade ago, antitumor necrosis factor (TNF) inhibitors have demonstrated beneficial activity in induction and maintenance of clinical remission, mucosal healing, improvement in quality of life, and reduction in surgeries and hospitalizations. However, more than one-third of patients present primary resistance, and another one-third become resistant over time. One of the main factors associated with loss of response is the immunogenicity of anti-TNF biologics leading to the production of antidrug antibodies (ADAbs) accelerating their clearance. In this review we present the current state of the literature on the place of TNF and its blockage in the treatment of patients with IBD and discuss the usefulness of serum trough levels and ADAb monitoring in the optimization of anti-TNF therapies. Antitumor necrosis factor (TNF) biologics appeared over a decade ago in the armamentarium for inflammatory bowel disease (IBD). Originally evaluated in Crohn's disease (CD) and thereafter in ulcerative colitis (UC), their efficacy was demonstrated in both diseases and has deeply modified the management of patients with IBD . Althougγ and IL17 (A and F), respectively, which stimulate macrophages and induce the production of other inflammatory cytokines such as IL-1β and TNFα that subsequently promote matrix metalloproteinases (MMPs) production by stroma cells and mucosal damage [α plays a strategic role in IBD pathophysiology, at the cross talk of the different inflammatory pathways involved in gut mucosal inflammation [α are known to be mediated by TNF receptor I (TNF-RI) or TNF-RII. Ligation of TNF-RI, which is expressed on a wide range of immune and nonimmune cells, results in NF-κB activation, cytotoxicity, and induction of proinflammatory cytokines and chemokines as well as antiapoptotic peptides [α with TNF-RII inducing a costimulatory signal to TCR-mediated T cell activation, thereby increasing T cell proliferation, expression of T cell activation markers , and secretion of inflammatory cytokines including IFNγ and TNFα [γ, IL-13, IL-17A, and TNFα) of both CD4+ and CD8+ T cell populations derived from UC patients [α and TNF-RII are also able to activate and expand protective CD4(+)FoxP3(+) regulatory T cells (Tregs) and seem critical for the stabilization of their phenotype and function in the inflammatory environment of the lamina propria in a mouse model of colitis [α on effector versus regulatory T cells may explain unexpected and disappointing results obtained with anti-TNF in some autoimmune diseases such as multiple sclerosis [α function via TNF-RI or TNF-RII on the course of intestinal inflammation, due to different susceptibility of epithelial cells and effector or regulatory immune cells. As an illustration, in dextran sulfate sodium- (DSS-) induced acute colitis in BALB/c mice, TNF-RI ablation led to exacerbation of the disease with increased inflammation and intestinal damage, while TNF-RII deficiency had opposite effects [α on immune and nonimmune cells through TNF-RI and TNF-RII in order to develop more specific inhibitors with potentially an increased efficacy and/or safety.While the etiology of IBD is still unknown, it is thought to involve complex interactions between genetic disposition, environmental conditions, life style, and microbial and immune factors resulting in a deregulated and excessive immune response directed against components of the normal microflora. CD and UC have been associated with exaggerated T helper (Th) type 1 and Th2 responses, respectively. More recent studies demonstrated that tissue damages result from mucosal inflammation mainly mediated by proinflammatory Th1 and Th17 lymphocyte subpopulations and their respective proinflammatory effector cytokines. In the gut of CD patients, activated Th1 and Th17 cells produce IFNammation . Accordipeptides , 10. Theand TNFα . Accordipatients . On the clerosis . Altoget effects . Nonetheα is the major target molecule of biologic treatments in CD and UC. Numerous randomized clinical trials and meta-analyses have demonstrated the efficacy of monoclonal antibodies against TNFα for both induction and maintenance of remission in both CD and UC [α, was shown to induce and maintain clinical response in patients with active moderate-to-severe UC [TNFD and UC –18. InflD and UC –25. SeveD and UC . MoreoveD and UC , 28. CerD and UC . Recentlevere UC , 31.However, although 60 to 80 percent of patients exhibit a good initial response to anti-TNF treatments (defined as a Crohn's Disease Activity Index (CDAI) decrease from baseline >70 points for CD and a decrease in the Mayo score of at least 3 points and at least 30 percent for UC), only one-third of patients are in clinical remission without steroids at one year  . ConsequDespite the high effectiveness of anti-TNF in patients with IBD, more than one-third of patients present primary resistance, and another one-third become resistant over time . Optimal ad hoc basis in practice and in 10 to 20% of patients in randomized controlled trials of maintenance therapy [Several studies assessed IFX trough levels after induction treatment or during maintenance therapy as predictors of sustained clinical response and showed a significant correlation between low IFX trough levels and decreased clinical response in CD and UC adult patients , 45–47 a therapy . ATIs we therapy , 50–52.  therapy –55. ThesThere are fewer data for adalimumab, but some studies also described a positive association between serum adalimumab concentration and clinical remission in CD –58. FurtThere is so far no data concerning trough levels and antidrug antibodies for adalimumab in children and in all patients for certolizumab and golimumab.Serum trough levels measurement to detect subtherapeutic drug concentrations and identification of ADAb (therapeutic drug monitoring or TDM) are the most relevant and useful parameters for the monitoring of anti-TNF drugs to facilitate informed decision making in IBD patients with secondary loss of response to TNF antagonists. The clinical utility of the immunomonitoring was evaluated in a retrospective study conducted on 155 patients with IBD and loss of response to IFX . They shμg/mL at 14 weeks being associated with a sustained therapeutic response [Interestingly, in a prospective study examining the course of ADAb formation and the clinical relevance of its assessment in the followup of patients with rheumatoid arthritis, Bartelds et al. showed that, among patients positive for antiadalimumab Abs, 67% developed ADAbs during the first 28 weeks and almost one-third during the first month of treatment . Howeverresponse .On the other hand, supratherapeutic anti-TNF trough levels might also be associated with paradoxical inflammatory side effects such as psoriasiform eczema or arthralgia . In suchAltogether, these data plead for the clinical and economical utility of early therapeutic drug monitoring in the management of patients receiving TNF inhibitors. In case of a loss of response with low trough level without ADAb, an intensified therapy with the same drug should be recommended by increasing doses and/or decreasing intervals and eventually adding an immunosuppressant. When low trough level is related to the presence of ADAbs, therapy should be switched within the anti-TNF class and if necessary to a drug with another mode of action . The addIn a very recent study using a decision analytic model that simulated 2 cohorts of patients with CD who become resistant to anti-TNF inhibitors, Velayos et al. compared the effectiveness of empiric dose escalation versus testing-based strategy over a 1-year time period . AlthougLarge prospective and randomized studies are still required to validate all these approaches in patients with IBD and clear dose toxicity/efficacy relationships have yet to be established for anti-TNF inhibitors.Finally, we have to keep in mind that, in the absence of standardization, the numerous assays developed for serum trough levels and ADAb measurement  exhibit α and its receptors on immune and nonimmune cells during IBD might be crucial for the development of more efficient and safe biological inhibitors.Since the advent of anti-TNF biologics more than a decade ago, they have demonstrated beneficial activity in induction and maintenance of clinical responses, mucosal healing, improvement in quality of life, reduction in surgeries and hospitalizations, and the treatment of extraintestinal manifestations of IBD. However, despite good overall initial effectiveness, a significant proportion of patients lose response over time mainly because of ADAb production and accelerated drug clearance. Although optimal treatment strategies remain controversial, therapeutic algorithms were proposed based on serum trough levels and ADAb monitoring in order to rationalize drug adjustment. For the future, a better understanding of the ambivalent protective and deleterious effects mediated by TNF


It is becoming increasingly evident that the so called “resistant” (R-type) VGCC Cav2.3 is critical in several physiologic and pathophysiologic processes in the central nervous system, vascular system and in endocrine systems. However its eponymous attribute of pharmacologic inertness initially made in depth investigation of the channel difficult. Although the identification of SNX-482 as a fairly specific inhibitor of Cav2.3 in the nanomolar range has enabled insights into the channels properties, availability of other pharmacologic modulators of Cav2.3 with different chemical, physical and biological properties are of great importance for future investigations. Therefore the literature was screened systematically for molecules that modulate Cav2.3 VGCCs.Membrane-bound voltage-gated Ca Many of-channel ,26 and a-channel ,28.v2.3-mediated inward currents as reported for structurally similar ion channels. The segments S6 participate in gating the ion channels [+ channel to a great extent [Additional elements may contribute to the kinetic properties of Cachannels ,30,31,32channels ,38,39,40channels ,42, and t extent .v1.2 L-type VGCCs have been co-crystallized with functionally auxiliary subunits [v2.3 have been reported such as with a β-subunit [v1.1 and Cav1.2 is located in a conserved region between domain I and II [v2.3 with β-subunits [Only segments of the cytosolic loops from Casubunits  or functsubunits ,46,47,48-subunit ,16 or wi-subunit ,50,51,52I and II ,54, whicsubunits ,15,16.2+-mediated modulation of the Cav2.3 voltage-gated Ca2+ channel [v2.3 [v2.3 signaling to muscarinic receptor activation [2+ current in hippocampal CA1 pyramidal neurons [v2.3 also contains the better known, carboxyterminal Ca2+/calmodulin interaction site, which was not only found for the members of the Cav2/non-L-type but also for members of the classical L-type Ca2+ channel subfamily [The II-III linker harbors a unique site located within the arginine-rich stretch, which is responsible for Ca channel . It may el [v2.3 , linkingtivation ,59,60,61 neurons . Cav2.3 ubfamily .v2.3d was originally cloned as a fetal splice variant from human brain [v2.3 from different species . Doe-1, cloned from Discopyge ommata, represented a novel Ca2+ channel type, which was insensitive towards dihydropyridines, but was antagonized rather than activated by 5 µM Bay K. This channel type was only slightly and readily reversibly inhibited by 5 µM ω-conotoxin-MVIIC, was insensitive towards ω-agatoxin-IVA, and fully reversibly blocked by ω-conotoxin-GVIA, an irreversible antagonist of N-type Ca2+ channels [2+ current component in rat cerebellar granule neurons and called the doe-1-like component “R-type current” [The first “pharmacoresistant” Ca in 1993 ,105, whi in 1993 ) and 199channels . InteresHysterocratis gigas [v2.3 with an IC50 value of 15–30 nM and was the first and still is the only Cav2.3-prevalent antagonist,. At concentrations higher than 500 nM SNX-482 also inhibits N-type Ca2+ currents [2+ currents are inhibited by about 25% at concentrations of 200 nM SNX-482 [v2.3-prevalent, but not as Cav2.3-specific or -selective.The peptide antagonist SNX-482, which was initially purified from the venom of the tarantula is gigas  blocks Ccurrents , whereas SNX-482 . Therefov2.3 isoforms could be distinguished from eachother by their varying SNX-482 IC50 values of 6 nM and 81 nM, and a third R-type Ca2+ current component by its insensitivity to SNX-482 [In cerebellar granule cells, two Ca SNX-482 .v2.3 led to knock-out mice, which in cerebellar granule cells and in DRG neurons still expressed a drug insensitive Ba2+ current. The peak inward current (IBa) was even larger than in cultured mouse neurons from contol mice (knock-out IBa 113 ± 27 pA (n = 5 ); control 85 ± 21 pA (n = 9)) [v2.3-deficient mice, leading to the conclusion that a non-Cav2.3-dependent R-type current may exist.The first gene inactivation of Ca(n = 9)) . Only thv2.3 contributes not only to the SNX-482-sensitive component of the R-type Ca2+ current, which was recorded in the presence of combination of Ca2+ channel antagonists , but also to the SNX-482-insensitive part [1/2, act) was shifted to more positive voltages in all three cell types investigated , specially in the neocortex, where it was reduced from –68 ± 2 mV to –58 ± 7 mV [2+ current may be more than only the Cav2.3-gene encoded Ca2+ channel in neuronal tissues [In murine hippocampal and neocortical neurons, Caive part . Interes8 ± 7 mV . Overall tissues ,112.2+ inward currents  or to specifially inhibit some T-type and the R-type Ca2+ current (Ni2+). Unfortunately, the half maximal concentrations for Cav2.3 and Cav3.2 are close to each other (10–30 µM), rendering Ni2+ blockade unsuitable for distinction of Cav2.3 currents in tissue in which Cav3.2 is also expressed. Physiologically, homeostasis of other divalent cations like Cu2+ and Zn2+ may play an important role [Divalent and trivalent heavy metal cations were often used to antagonize either all voltage-gated Cach other –30 µM, rant role ,114, notant role .v2.3 reported in the literature. Most drugs in the table are non-selective, in the sense that currents through other Ca2+ channel Cavα1 subunits are also antagonized with an IC50 not larger than tenfold. Many substances show inhibitory effects on Cav2.3 or on R-type Ca2+ currents. One set of drugs is related to anticonvulsive effects, others are used as anesthetic drugs. Even high concentrations of classical Ca2+ channel antagonists can inhibit Cav2.3 induced inward currents as shown for the dihydropyrdines isradipine [2+ channels, a dihydropyridine concentration of around 10 µM is chosen by electrophysiologists. Considering that such high concentrations of isradipine or nicardipine substantially block E-/R-type Ca2+ currents, lower concentrations of e.g., isradipine of 0.5 µM are more suitable, in order to observe antagonism by low concentrations of SNX-482 as shown for cardiac E-/R-type Ca2+ currents in murine myocytes [2+ current at elevated concentrations [radipine  and nicaradipine . Routinemyocytes . Howevertrations .v2.3-deficient mice, which were generated and analysed in several different laboratories .v2.3 is expressed in many regions of the CNS and also in peripheral organs and tissues, which makes it difficult to explore its full function in vivo. Cav2.3 triggers or participates in the release of several neurotransmitters such as dopamine in the substantia nigra [v2.3 contributes to fast glutamatergic transmission [v2.3 participates in basic processes related to learning and memory formation [v2.3 is an important regulator in spines: activation of Cav2.3 triggers opening of small conductance Ca2+-activated K+-channels in CA1 hippocampal pyramidal neurons [2+ currents, which were recorded as Ni2+-sensitive tail currents, are available at resting potential and contribute to after-depolarization, and thus to the initiation of burst firing in CA1 hippocampal neurons [Caia nigra . In the smission , where iormation ,120,121. neurons ,123,124, neurons . R-type  neurons .v2.3 isoform [v2.3 during neuronal development point to an important role of Cav2.3 during early prenatal stages [2+ channels are replaced by P-/Q-type Ca2+ channels during development [The existance of a fetal brain Ca isoform  and the l stages ,148,149.elopment .v2.3-deficient mice reveal altered pain response [v2.3 could be identified in rat nociceptive neurons [v2.3e, which was also detected in the cerebellum, heart and endocrine system (Caresponse , and tra neurons . The maje system , Table 2v2.3 is highly expressed in the amygdala, in which the R-type Ca2+ current represents the largest component of high-voltage gated Ca2+ currents. Cav2.3-deficient mice exhibited signs of enhanced fear assuming that Cav2.3-based R-type Ca2+ currents in the amygdala may be associated with fear [Caith fear .v2.3-deficient mice represent an important model for convulsive and non-convulsive seizures as was summarized in [v2.3 transcripts in the insulinoma cell line INS-1 [v2.3-deficient mice, which showed disturbance not only of glucose-induced insulin release [Carized in . Based one INS-1 ,73, addi release ,75, but  release , and mor release .2+ current was related to the release of gonadotropin-releasing hormone [v2.3 VGCCs, possibly by producing the global increase in cytoxolic Ca2+ required for refilling of the readily releasable pool of granules during the second phase of insulin release [SNX-482 sensitive R-type Ca hormone  and of o hormone ,77. Over release ,154.v2.3 has been shown to contribute to cerebral artery constriction i.e., vasospasm [After cerebral aneurysm rupture and subarachnoidal hemorrhage Caasospasm , a devasasospasm .v2.3 in cardiomyocytes is still under discussion: Cav2.3 protein has yet to be detected reliably in murine cardiomyocytes, but transcripts could be amplified by single cell RT-PCR from microscopically identified murine cardiomyocytes [v2.3 ablation causes cardiac arhythmia and disturbances in autonomic cardiac control, suggesting that Cav2.3 in pacemaker cells as well as in autonomic nerve endings may participate in cardiac signalling [The expression of Camyocytes ,88. Furtgnalling .v2.3 modualtors will be a key in establishing the exact role of Cav2.3 in the physiological and pathophysiological processes, that it contributes to. Furthermore, recent evidence points to Cav2.3 as a potential pharmacologic target in therapy of epilepsy, chronic pain, endocrine disturbances and vasospasms after subarchnoid hemmorhage. In this light, non-ion selective Cav2.3 inhibitors with favourable pharmakokinetics could represent new therapeutic strategies for these disorders.In future, more specific Ca