Individual responses to oxaliplatin (L-OHP)-based chemotherapy remain unpredictable. Our recent proteomics studies have demonstrated that intracellular protein expression levels of S100A10 are significantly correlated with the sensitivity of colorectal cancer (CRC) cells to L-OHP, but not 5-FU, suggesting that S100A10 is a candidate predictive marker for the response to L-OHP. In this study, we investigated whether S100A10 is involved in L-OHP sensitivity or not.50) for L-OHP (P = 0.003), but did not change that for 5-FU, indicating that S100A10 is more specific to L-OHP than 5-FU. Silencing of the S100A10 gene showed no apparent effect on sensitivity to L-OHP in HT29 cells. Silencing of the annexin A2 (a binding partner of S100A10) gene alone downregulated both annexin A2 and S100A10 protein levels, with no change in S100A10 gene expression. However, original levels of intact S100A10 protein in CRC cells positively correlated with S100A10 mRNA levels .Forced expression of S100A10 in COLO-320 CRC cells significantly increased the 50% inhibitory concentration (ICThe present results have shown that protein expression of S100A10 was associated with resistance to L-OHP, but not 5-FU, supporting the hypothesis that S100A10 expression may predict L-OHP sensitivity. Thus, our present study provides basic findings to support that S100A10 expression can be used as a predictive marker for tumor sensitivity to L-OHP. ERCC1), xeroderma pigmentosum group D , glutathione-S-transferase-P1 (GSTP1), or other candidate biomarkers that have previously shown promise [Oxaliplatin (L-OHP) is a key drug used for the treatment of colorectal cancer (CRC) . L-OHP a promise .2-(annexin A2)2 heterotetramer [Recently, our proteomics studies have demonstrated that intracellular S100A10 protein expression levels are significantly correlated with the sensitivity of CRC cells to L-OHP, but not 5-FU, providing a new candidate predictive markers for the response to L-OHP -14, inditetramer -25. Annetetramer -29. HoweIn this study, we investigated whether alterations in S100A10 and/or annexin A2 expression were involved in mediating chemosensitivity to L-OHP by using forced overexpression of S100A10 in stably transfected cells and RNA interference. Our results have demonstrated the potential contribution of S100A10 to resistance to L-OHP.50 values  compared to that for the COLO-320/vector (2.3 ± 2.0 μM) and untreated COLO-320 cells  of cells for 5-FU were as follows: COLO-320/S100A10, 3.4 ± 1.8; COLO-320/vector, 3.6 ± 2.6; and untreated COLO-320 cells, 4.1 ± 3.2  showed strong expression of S100A10  as a 44-kDa protein . The level of expression was similar to that of endogenous S100A10 levels in HT29 cells, a line which shows high S100A10 protein expression. Annexin A2, the binding partner of S100A10, was not expressed in COLO-320/S100A10 cells  exhibited slightly lower cell proliferation rates at day 3 compared to those for cells treated with transfection reagent alone (mock), cells transfected with nontargeting control siRNA (control), and cells transfected with S100A10 siRNA (HT-29/siS100A10)  between the 2 targets .In DLD-1 cells, annexin A2 appeared as 2 bands around 36 kDa  in clinical setting, and Cmax is thought to be important for efficacy, considering that antitumor activity of L-OHP is concentration and time dependent [Forced expression of S100A10 in COLO-320 CRC cells, which do not express endogenous S100A10, significantly reduced the sensitivity of these cells to L-OHP. In particular, substantial changes in cell viability were observed between 1 and 10 μM of L-OHP exposure, corresponding approximately to the blood concentration of L-OHP in clinical use . A roughependent  and infuependent ,34,35.On the other hand, forced expression of S100A10 did not alter sensitivity to 5-FU, suggesting that, as a predictive marker, S100A10 is more specific to L-OHP sensitivity than to 5-FU sensitivity. These observations strongly support our recent findings that CRC cells with higher S100A10 protein expression levels exhibit lower sensitivity to L-OHP, but not to 5-FU . S100A10The differences in the antitumor mechanisms and/or the opposite effects exerted on NF-κB by L-OHP or 5-FU -46 may b2-(annexin A2)2 heterotetramer [2 phase [The interaction between S100A10 and annexin A2 may be partly involved in the mechanisms of sensitivity to L-OHP. Most of the S100A10 is tightly associated with annexin A2 dimers, forming an (S100A10)tetramer -25. S100tetramer , and anntetramer -49. Convtetramer ,50, alsotetramer . In this[2 phase , is unli[2 phase . These fIntracellular S100A10 protein expression may also be mediated by post-translational suppression possibly due to the instability of S100A10 protein induced by annexin A2 suppression, considering that annexin A2 stabilizes intracellular S100A10 via binding, which masks the S100A10 polyubiquitination signal leading to proteasomal degradation, as previously reported -58. Howe1B receptor and modulates its function [Additionally, it cannot be excluded that intracellular S100A10 is a surrogate of other active molecules related to cell survival after exposure to antitumor drugs since S100A10 has been shown to interact with a variety of proteins, including plasma membrane-resident receptors and channels -14. Serofunction , which hfunction ,61. Downfunction ,63, redufunction .Thus, the molecular backgrounds of S100A10 described in previous reports are partly consistent with our hypothesis that S100A10 protein expression levels may reflect cell sensitivity to L-OHP. In addition, we demonstrated that protein expression of S100A10 was involved in mediating sensitivity to L-OHP, at least in part, by using stably transfected cells expressing S100A10. However, the mechanisms of S100A10 involvement in chemoresistance are still unknown, and the suppression of S100A10 protein expression induced by knockdown of annexin A2 makes it difficult to differentiate between the effects of annexin A2 and those of S100A10 on chemosensitivity. In the present study, we have demonstrated that protein expression of S100A10 was associated with resistance to L-OHP, but not 5-FU. However, further studies are required in order to fully elucidate the molecular mechanisms through which S100A10 acts as a predictive biomarker of the response to L-OHP.We have shown that protein expression of S100A10 was associated with resistance to L-OHP, but not 5-FU, using forced expression of S100A10 in CRC cells. Our results provide basic findings for S100A10 as a predictive marker of the response to L-OHP. Further clinical validation and functional analysis are required to confirm this hypothesis and to elucidate the underlying biological mechanisms.L-OHP and 5-FU were purchased from WAKO Chemicals  and Sigma-Aldrich , respectively. Purified mouse anti-annexin II Light Chain (S100A10) monoclonal antibodies (mAbs) and purified mouse anti-human annexin II (annexin A2) mAbs were obtained from BD Biosciences . Anti-human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mAbs were obtained from Life Technologies . All other chemicals and reagents were of the highest purity available.2. Cells were used when in the exponential growth phase.DLD-1, LOVO, HT29, SW480, SW1116, WiDR, and HCT116 cells were purchased from the European Collection of Cell Cultures , and SW620 cells were purchased from the American Type Culture Collection . COLO205, HCT-15, and LS174T cells were provided by the Cell Resource Center for Biomedical Research, Tohoku University . COLO201 cells were provided by the Japanese Collection of Research Bioresource , and COLO-320 cells were provided by RIKEN Bio-Resource . The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine at 37°C in humidified air containing 5% CO5 cells per 35-mm dish 24 h prior to transfection. Cells were transfected with either S100A10 in the pFN21AB8860 (COLO-320/S100A10) or with the vector control (COLO-320/vector) using TransIT-LT1 transfection reagent  according to the manufacturer’s instructions. Cells stably expressing S100A10 were selected with 0.8 mg/mL of geneticine  and subsequent subcloning. COLO-320/S100A10 cells were maintained in medium containing 0.4 mg/mL G418 disulfate. Stable expression of S100A10 in the cells was verified using western blot analysis.The Flexi HaloTag clone pFN21AB8860, an S100A10 expression vector capable of producing N-terminally HaloTag-fused recombinant S100A10 protein, was obtained from Kazusa DNA Research Institute . The S100A10-nonexpressing CRC cells, that is, COLO-320 cells, were plated at a density of 3 × 103 cells/well in 96-well plates for cell proliferation assays, chemosensitivity tests, and mRNA expression analyses and were plated at 1.5 × 105 cells/well in 6-well plates, scaling up proportionally to the relative surface area of culture vessels, to prepare cell lysates for western blot analysis. Cells were transfected with appropriate siRNAs for 24 h by using Lipofectamine RNAiMAX (Life Technologies) as described in the manufacturer’s standard protocol. The final siRNA concentration was 10 nM for single-knockdown experiments and 20 nM for double-knockdown experiments (concentration of each siRNA: 10 nM). After transfection, cells were processed for subsequent chemosensitivity test. siRNA-mediated knockdown of target molecules was confirmed by quantitative real-time reverse transcription (qRT)-PCR and western blot analysis at 48 h after transfection.S100A10 siRNA , annexin A2 siRNA , and nontargeting control siRNA (Silencer Select Negative Control #1) were purchased from Life Technologies. Twenty-four hours prior to transfection, HT29 cells were plated at a density of 5 × 103 cells/well in 96-well plates. For chemosensitivity tests, cells were plated at a density of 0.5 × 103 cells/well in 96-well plates 24 h prior to exposure to L-OHP  or 5-FU . Cell viability was assessed after 48 h exposure to L-OHP or 72 h of exposure to 5-FU, and the 50% inhibitory concentration (IC50) was calculated from graphical plots.The viability of cells in the cell proliferation assay and chemosensitivity test in stably transfected COLO-320 cells was assessed by using the CellTiter96 AQueous One Solution Cell Proliferation Assay  according to the manufacturer’s protocol. Cell proliferation was measured 4 days after plating cells at a density of 0.5 × 1050 values were calculated.Crystal violet staining (CVS) was used to determine cell numbers for assessing the cell viability of HT29 cells in siRNA-mediated knockdown experiments. Cell proliferation was measured at day 0 (just before transfection), day 3 and day 5, that is, 48 and 96 h after the end of transfection, respectively. For chemosensitivity tests, cells transfected with appropriate siRNAs for 24 h were subsequently exposed to L-OHP  for 24 h. The medium was then changed, and cell survival was assessed by CVS after 24 h of incubation. The CVS was performed by the modified method of Sergent et al. . Briefly50 values for L-OHP or 5-FU were log transformed for normal distribution, and the log10 IC50 values were used for further statistical analysis.The ICg for 30 min at 4°C, and protein concentrations were determined by the DC protein assay . In experiments using stably transfected COLO-320 cells or RNA interference, cell lysates were prepared by using M-PER Mammalian Protein Extraction Reagent  with 1 mM DTT, 0.1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Sigma-Aldrich) according to the manufacturer’s protocol. After centrifugation at 15,000 × g for 10 min at 4°C to remove cellular debris, protein concentrations were determined by the Pierce BCA protein assay (Thermo Fisher Scientific Inc.), and aliquots were quickly frozen in liquid nitrogen and stored at −80°C until analysis in all experiments.To investigate the relationship between S100A10 and annexin A2 protein expression, 8 CRC cell lines  were used. Cells were scraped off 100-mm dishes into a lysis buffer containing 9 M urea, 2% CHAPS, 1 mM dithiothreitol (DTT), and a protease inhibitor cocktail (Sigma-Aldrich). After incubation on ice for 10 min followed by sonication on ice, the lysates were cleared by centrifugation at 15,000 × Cell lysates were separated by SDS-polyacryamide gel electrophoresis. The separated proteins were transferred electrophoretically to polyvinylidene difluoride membranes and probed with the respective primary antibodies and alkaline phosphatase-conjugated secondary antibodies (Life Technologies) as described previously . GAPDH w3 cells/well in 96-well plates in all experiments. Extraction of total RNA from cultured cells and synthesis of cDNA from total RNA were performed using the TaqMan Gene Expression Cells-to-CT Kit (Life Technologies) according to the manufacturer’s instructions. The real-time qRT-PCR measurement of individual cDNAs was performed using TaqMan Gene Expression Assays for S100A10 (Assay ID: Hs00237010_m1), annexin A2 (Assay ID: Hs00237010_m1), and GAPDH (Assay ID: Hs99999905_m1) on an ABI 7900 Real-Time PCR System (Life Technologies). The reactions were run in 384-well plates, using the following program: 50°C for 2 min followed by 95°C for 10 min, followed by 40 cycles of 95°C for 15 s, 60°C for 1 min. The cycling parameters were manufacturer’s specifications. The relative standard curve method, preparing serial dilution of total RNA  prepared from the pool of total RNA obtained by combining aliquots of samples for all assay, was used to quantify the results obtained by real-time qRT-PCR. Relative fold-changes were normalized to the expression of GAPDH.For quantification of mRNA expression, cells were plated at a density of 5 × 10P values of less than 0.05 were considered statistically significant.Statistical analyses were performed using SPSS software 19.0 J for Windows . Comparison between groups was performed by one-way analysis of variance (ANOVA) followed by post-hoc multiple pairwise comparison using Tukey’s test to determine statistical differences. To evaluate relationships between 2 variables, Pearson’s correlation coefficient test was used. in vitro study described in this manuscript used the cell lines commercially available. This type of study does not apply to human subject research by standards of Guidance from US-Office for Human Research Protection (OHRP). OHRP states that “OHRP does not consider the act of solely providing coded private information or specimens  to constitute involvement in the conduct of the research”.Our Furthermore, NIH Office of Extramural Research in US Department of Health & Human Service (HHS) also answers to investigators in their FAQs that “Research that proposes the use of human cell lines available from American Type Culture Collection or a similar repository is not considered human subjects research because the cells are publicly available and all of the information known about the cell lines is also publicly available”. Therefore, our study does not apply to an ethics committee.L-OHP: Oxaliplatin; CRC: Colorectal cancer; IC50: 50% inhibitory concentration; ANXA2: Annexin A2.Yusuke Tanigawara, Sayo Suzuki and Yakult Honsha Co., Ltd. hold patents on S100A10 titled as “METHOD FOR DETERMINATION OF SENSITIVITY TO ANTI-CANCER AGENT “(Patent No. 2010/05157 and 586972). This does not alter the authors’ adherence to all Proteome Science policies on sharing data and materials.YT was responsible for planning and designing the study and data interpretation. SS performed the experiments and the data analysis and wrote the manuscript. Both authors read and approved the final manuscript.


Kocuria salsicia G1 and Kocuria rhizophila G2, which were isolated from a meat chopper at a small slaughterhouse in Denmark. The two annotated genomes are 2.99 Mb and 2.88 Mb in size, respectively.We report here the draft genome sequences of  Kocuria rhizophila and Kocuria salsicia are Gram-positive, coccoid, spherical saprotrophic bacteria belonging to the family Micrococcineae. Kocuria species are ubiquitous and highly adapted to their ecological niches , according to the manufacturer’s recommendations, and then sequenced as part of the flow cell, as 2 × 250-base paired-end reads using the Illumina MiSeq (Illumina) technology. The reads were cleaned and trimmed using CLC Genomics Workbench 7 . Quality-filtered reads were assembled using SPAdes version 3.5.0 . The annK. salsicia G1 resulted in 199 contigs at 27× coverage, with an average G+C content of 70.43%. K. rhizophila G2 is assembled into 87 contigs at 126× coverage, with an average G+C content of 70.81%. The annotated results from G1 predicted 2,565 coding sequences, with an average length of 971 bp , 19 tRNA-coding genes, and 5 rRNA-coding genes. The predictions from G2 included 2,531 coding sequences, with an average length of 955 bp , 18 tRNA-coding genes, and 7 rRNA-coding genes. Both strains had single predicted copies of 16S and 23S rRNA genes, with the only difference in 5S rRNA gene copies, with 3 for G1 and 5 for G2. There are 359 and 358 predicted subsystems in the genomes of G1 and G2, respectively. Metabolic network comparisons revealed 1,774 putative protein-encoding genes (PEGs) conserved in both G1 and G2 genomes. In a function-based comparison to the genome of DC2201, the genomes of G1 had 179 unique PEGs and 147 PEGs in G2. The main differences observed in a comparison of K. salsicia G1 to K. rhizophila DC2201 and K. rhizophila G2 were the presence of sequences encoding clustered regularly interspaced short palindromic repeat (CRISPR) elements, iron acquisition, and metabolism subsystems identified in G1 only. These suggest a prominent influence of phage exposure and possible adaptation mechanisms of isolate G1 to a more densely populated environment, such as the animal gut. Further work with these genomes is expected to facilitate the identification and understanding of genes associated with adaptive mechanisms of these strains and biofilm formation.The assembly of K. salsicia G1 and K. rhizophila G2 have been deposited at the European Nucleotide Archive (ENA) under the contig accession numbers CZJU01000001 to CZJU01000199 and CZJW01000001 to CZJW01000087, respectively. The versions described in this paper are the first versions.The whole-genome sequencing (WGS) projects for


Epigenetic regulation has emerged to be the critical steps for tumorigenesis and metastasis. Multiple histone methyltransferase and demethylase have been implicated as tumor suppressors or oncogenes recently. But the key epigenomic events in cancer cell transformation still remain poorly understood.A breast cancer transformation model was established via stably expressing three oncogenes in primary breast epithelial cells. Chromatin immunoprecipitation followed by the next-generation sequencing of histone methylations was performed to determine epigenetic events during transformation. Western blot, quantitative RT-PCR, and immunostaining were used to determine gene expression in cells and tissues.Histones H3K9me2 and me3, two repressive marks of transcription, decrease in in vitro breast cancer cell model and in vivo clinical tissues. A survey of enzymes related with H3K9 methylation indicated that KDM3A/JMJD1A, a demethylase for H3K9me1 and me2, gradually increases during cancer transformation and is elevated in patient tissues. KDM3A/JMJD1A deficiency impairs the growth of tumors in nude mice and transformed cell lines. Genome-wide ChIP-seq analysis reveals that the boundaries of decreased H3K9me2 large organized chromatin K9 modifications (LOCKs) are enriched with cancer-related genes, such as MYC and PAX3. Further studies show that KDM3A/JMJD1A directly binds to these oncogenes and regulates their transcription by removing H3K9me2 mark.Our study demonstrates reduction of histones H3K9 me2 and me3, and elevation of KDM3A/JMJD1A as important events for breast cancer, and illustrates the dynamic epigenomic mechanisms during breast cancer transformation.The online version of this article (doi:10.1186/s13148-016-0201-x) contains supplementary material, which is available to authorized users. Recent advances in epigenetics and epigenomics have revealed that epigenetic abnormality is one of the critical causes for tumorigenesis –5. DNA mHistone methylation usually occurs on lysines and arginines, and each site has three different forms , 13. In TERT), and Harvey rat sarcoma viral oncogene homolog v12 mutant (RAS (V12)), the engineered primary cell will extend life span, become immortalized, and finally gain tumorigenic capacity, which is considered to represent different stages of tumor cell transformation [The early diagnosis and treatment are the most effective ways to cure cancer. But we are still lack of good markers and drugs for precision medicine. During transformation, the epigenetic programs in cells change dramatically, helping them to survive and gain growth advantage , 3. Alonormation , 34. Conormation . But dueIn this study, we took advantage of the in vitro tumor transformation model and made a series of transformed cell lines starting from human primary mammary cell (HMC). By combining biochemical and epigenomic approaches, we demonstrate that histones H3K9me2 and H3K9me3 decrease during breast cancer transformation and contribute to the process with different mechanisms. Furthermore, we identified that KDM3A/JMJD1A, an H3K9me2 demethylase, is responsible for the H3K9me2 reduction and critical for breast tumor transformation.TERT, and RAS (V12) were stably expressed in human primary mammary cell respectively via retroviral infection. Four cell lines were generated for following studies, namely HMC-p6 , HMC-L (HMC with large T stable expression), HMC-LT (HMC with large T and TERT stable expression), and HMC-LTR  stable expression) . Gene expression profiles of 100 cases of paired breast cancer and normal tissues were downloaded from the Cancer Genome Atlas (TCGA), and the DEGs (twofolds) were analyzed according to the pipeline described in experimental procedure  . The H3Ker types . All theAfter determining the reduction of H3K9 methylation as a frequent event in breast cancer, we started to investigate the underlying molecular mechanisms. We firstly examined the mRNA levels of all the known H3K9 methyltransferases and demethylases by RNA-seq and quantitative RT-PCR, but none matched the observed H3K9 methylation pattern  or chloroquine  was used to treat HMC cell line. Both drugs increased the protein level of KDM3A/JMJD1A  and found that KDM3A/JMJD1A deficiency rescued the expression of most cancer-related genes in HMC-LTR to the levels in primary cells , sodium pyruvate (Invitrogen), and nonessential amino acid (Invitrogen); T47D is a gift from Dr. Yong-Feng Shang of Tianjin Medical University and cultured in RPMI1640 with FBS and insulin. Both the primary cells and cancer cell lines were sub-cultured 1:4 on reaching confluence; each passage was considered two PD.Human primary mammary cells were purchased from Chi Scientific and cultured in DMEM/F12  at 37 °C with 5 % COAntibodies were purchased from the indicated companies: H3K9ac, hTERT, KRT14, and ACTG (Epitomics); KRT18 (ProteinTech), JMJD1B, JMJD2B, JMJD2A, H4K20me3, H3K79me2, E-cadherin, and RAS (CST); H3K4me1, H3K4me3, H3K27me3, H3K27me1, H3K27me2, and H3K36me2 (Millipore); H3, H3K9me1, H3K4me2, and H3K36me3 (Abcam); H3K9me2, H3K9me3, GAPDH, EHMT2, SUV39H1, CBX5, DNMT3A, DNMT3B, DNMT1, and KDM1A ; KDM3A/JMJD1A . The information of primers and siRNAs are listed in Additional file Transformed breast cell lines were generated as previous described. 293FRT cells were co-transfected with packaging plasmid ZV77 (psPAX and pMD2G for lentivirus) and pBabe retroviral plasmids containing desired complementary DNA (cDNA). Supernatants containing virus were harvested 48 h later and HMC was infected together with 8 μg/ml polybrene. Typically, more than 80 % of cells were infected as measured by parallel infections with a GFP-expressing construct. Drug selection was performed with 200 μg/ml G418 for neomycin, 50 μg/ml hygromycin, or 0.5 μg/ml puromycin. pBabe with large T antigen, hTERT, or RAS (V12) were purchased from Addgene.www.alenabio.com) were fixed in 10 % formalin, embedded in paraffin, and followed by standard dewaxing procedures. Cells were cultured on the cover slips and fixed with freezing methanol after washing twice in PBS. The cover slips or tumors tissue array were then washed three times by PBS and blocked in PBS with 1 % BSA for 10 min or 1 h. The cover slips or tumor tissue arrays were hybridized with first and second antibodies for 1 h, respectively. Then, the slips were mounted with prolong anti-fade kit (Invitrogen) and observed with fluorescent microscopy. The arrays used for staining are as follows: H3K9me2 - BR243K, BR243L, BR243M, BR724, BR725, BCN963a; H3K9me3 - BR243B, BR243D, BR243K, BR243L, BR243A, BC081120, BCN963a; and KDM3A - BR724, BR725.Tumors tissue array  according to manufacturer’s manual. Approximately 1 μg of total RNA was used for reverse transcription with a first-strand cDNA synthesis kit (Toyobo). The amount of mRNA was assayed by quantitative PCR. β-Actin was used to normalize the amount of each sample. Assays were repeated at least three times. Data shown were average values ± SD of one representative experiment. All primer sequences are presented in Additional file 7 cells were fixed with 1 % formaldehyde and quenched by glycine. The cells were washed three times with PBS and then harvested in ChIP lysis buffer . DNA was digested to 150–300 bp by MNase (Sigma) before extensive centrifugation. Four volumes of ChIP dilution buffer  was added to the supernatant. The resulted lysate was then incubated with protein G beads and antibodies at 4 °C over night. The beads were washed five times and DNA was eluted by Chip elution buffer . The elution was incubated at 65 °C over night and DNA was extracted with DNA purification kit (TIANGEN). The purified DNA was assayed by quantitative PCR with Biorad MyIQ. Assays were repeated at least three times. Data shown were average values ± SD of representative experiments. The sequences of primers are in Additional file ChIP assay was performed as previously described . Brieflyhttp://david.abcc.ncifcrf.gov) [mRNA-seq library was performed by using Illumina TruSeq library construction kit. A 5 μg of total RNA was used as initiation and then prepared according to the manufacturer’s instruction. mRNA-seq libraries were sequenced using HiSeq2000 for 100-bp paired-end sequencing. Quality control of mRNA-seq data was performed using Fatsqc, and then low quality bases were trimmed. After quality control, data were mapped to hg19 genome reference by Tophat2 and allow maximum 2 mismatch. Cufflinks were used to find out differential expression genes. Gene ontology analysis was performed using DAVID (crf.gov) , 45.ChIP was performed using desired antibodies. Library was prepared using Illumina TruSeq kit according to the manufacturer’s procedure. Briefly, DNA was prepared for end repair and “A” tailing, adaptor ligation, and library amplification. ChIP-seq libraries were sequenced on HiSeq 2000 for 100-bp paired-end sequencing.Quality control of ChIP-seq data was performed using Fastqc, and then low quality bases and adaptor contamination were deleted. After quality control and data filtering, data were mapped to hg19 using BWA aln algorithm. Since H3K9me2 and H3K9me3 appear large scale in chromatin, SICER software was used for peaks calling with window size 1000 and gap size 10,000. H3K9me2 and H3K9me3 enrichment region gene annotation was performed using RefSeq gene reference . Gene onhttps://tcga-data.nci.nih.gov/tcga/, the data of total 101 pairs were downloaded but that of one cancer tissue were not readable, so only 100 pairs were used). In order to figure out significant differential expression genes between cancer sample and normal tissue, genes’ expression level less than 5 FPKM in all 201 samples were deleted, and then ANOVA analysis was performed for the rest genes with P value cutoff 0.001. After ANOVA analysis, genes’ average expression level between cancer and normal tissues less than twofold change was deleted. Gene ontology analysis of differential expression genes was performed using DAVID.The gene expression data pf 100 paired breast cancer and normal tissues were downloaded from TCGA data portal for analysis of differential expressed genes  and RNase (100 μg/mL) for 30 min at 37 °C. Cell cycle analysis was performed on an Epics XL-MCL flow cytometer (Beckman Coulter) with System II (version 3.0) software (Beckman Coulter). Additional analysis of cell cycle distribution was determined using Flowjo software.3 per well in 96-well plates. Next every 24 h, the cells were added with MTT (0.25 μg) in each well for 4 h at 37 °C; the medium with the formazan sediment was dissolved in 50 % DMF and 30 % SDS (pH 4.7). The absorption was read at 570 nm.Cell viability was performed by the MTT assay as previously described . BrieflyThe bottom layer of 0.6 % agar noble in medium was first placed onto 6-well plate. Cells were seeded in 0.35 % top agar containing medium. Fresh top agar was added 1.5 weeks later, and colonies were counted 8 weeks later. For HMCs, the 50,000 cells were seeded while 5000 or 10,000 cells were seeded for T47D.The RTCA assay was done as the manufacturer’s protocol. Cells were cultured at 6000 per well in CIM-Plate wells coated with (invasion) or without (migration) matrigel. The cell index signals were read by xCELLigence RTCA DP Analyzer (ACEA Bioscience Inc.). Invasion and migration are monitored continuously over a 48-h period.5 per well in 24-well transwell plates coated with (invasion) or without (migration) matrigel. The cells were fixed in 4 % PFA and stained by crystal violet after 48 h. The positive cells were counted under microscope.The transwell assay was done as follows. Briefly, cells were split at 1 × 10Each experiment was repeated three times and results were presented as mean ± SD.All the cancer tissues are collected after obtaining the consents of the patients. All the experiments are carried out in accordance with the approved guidelines and protocols by Medical Ethics Committee of Zhongnan Hospital, Wuhan University.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE64367.The data have been uploaded to GEO database and can be found at the following URL:


In curves more than 45 Cobb degrees fusion is considered as basically the only possible treatment although studies about intensive Schroth intervention show that it may be effective in slowing curve progression of patients with Adolescent Idiophatic Scoliosis (AIS).The main purpose of this study was to evaluate the effect of an intensive, 1-year-long and individualized Schroth intervention on curve progression in patients with AIS more than 45 Cobb degrees focused on the efficiency in terms of Cobb angle, axial trunk rotation (ATR) and objective body image.Prospective case series designIn this case series we included 7 female patients with AIS at the age of 13 with primary thoracic curve, wearing TLSO brace 23 hours/day, Risser: 2-3. The mean Cobb angle at the start of treatment was 47.3° ±2.4°, while the mean ATR with Scoliometer was 14.3° ±3.4°.Patients received Schroth intervention for 12 months in an outpatient clinic. Schroth therapy was individual and personalized, 45 minutes weekly for each patient, combined with minimum 60 minutes of daily home Schroth exercises 5 times per week. Outcomes were recorded at baseline, 6 and 12 months. Patients are currently in treatment.The Cobb angle improved 9.8° in 4 patients, worsened 7° in 1 patient, remained the same 2.5°, in 2 patients. The ATR measurements improved 5.3° in 4 patients, remained the same in 3 patient. Objective body images gave a positive feedback to all patients.In conclusion the results of this study confirm the effectiveness of an intensive 1-year-long, individual Schroth intervention for patients with AIS in curves more than 45°. At high risk of progression personalized Schroth intervention appears to be more effective in reducing the progression of scoliosis with additional bracing.


DACT2 is frequently methylated in human lung, hepatic, gastric and thyroid cancers. The methylation status and function of DACT2 remain to be elucidated in human esophageal cancer. Ten esophageal cancer cell lines, 42 cases of dysplasia and 126 cases of primary esophageal cancer samples were analyzed in this study. The expression of DACT2 was detected in YES2 cells, while reduced DACT2 expression levels were found in TE8 and KYSE70 cells, and complete loss of DACT2 expression was found in KYSE30, KYSE140, KYSE150, KYSE410, KYSE450, TE3 and TE7 cells. Loss of expression or reduced expression of DACT2 correlated with promoter region hypermethylation in esophageal cancer cells. Restoration of DACT2 expression was induced by 5-aza-2′-deoxycytidine. In human primary esophageal squamous carcinoma, 69% (87/126) of samples were methylated. Methylation of DACT2 was significantly associated with tumor stage and metastasis . DACT2 suppressed colony formation, cell migration and invasion in esophageal cancer cells, and it also suppressed esophageal cancer cell xenograft growth. DACT2 inhibited Wnt signaling in human esophageal cancer cells. In conclusion, DACT2 is frequently methylated in human esophageal cancer and its expression is regulated by promoter region methylation. DACT2 suppresses esophageal cancer growth by inhibiting Wnt signaling.Esophageal cancer is one of the most common malignancies worldwide.  Helicobacter pylori infection [Esophageal cancer is the sixth leading cause of cancer-related mortality and the eighth most common cancer worldwide. The 5-year overall survival ranges from 15% to 25% . In the nfection –9. Both nfection –12.DACT2 was identified by Katoh et al., and it is located on human chromosome 6q27 [DACT2 silencing by promoter region hypermethylation was detected in many tumor types including lung, gastric, hepatocellular and thyroid cancers [DACT2 in human esophageal cancer remain to be elucidated. Therefore, in this study, we analyzed the epigenetic changes and functions of DACT2 in human esophageal ESCC.The Wnt signaling pathway is reported to be involved in many phases of vertebrate embryonic development and the initiation and progression of human esophageal cancer –15. Actiome 6q27 . DACT2 s cancers –23. The The expression of DACT2 was detected by semi-quantitative RT-PCR in human esophageal cancer cells. DACT2 expression was detected in YES2 cells, while expression of DACT2 was reduced in TE8 and KYSE70 cells, and no expression was detected in KYSE30, KYSE140, KYSE150, KYSE410, KYSE450, TE3 and TE7 cells Figure . The proDACT2 in KYSE150 and KYSE450 cells, while partial methylation was detected in KYSE70 cells and unmethylation was found in YES2 cells  of primary esophageal cancer samples, 35.7% (15/42) of dysplasia samples were methylated and no methylation (0/27) was found in normal esophageal mucosa. The frequency of DACT2 methylation was increased in progression tendency during esophageal development (P < 0.001) , while no association was found between DACT2 methylation and gender, age, differentiation and tumor size . The expression of DACT2 was evaluated by immunohistochemistry in 50 cases of available matched esophageal ESCC and adjacent tissue samples. The staining intensity and extent of the stained area were scored using the German semi-quantitative scoring system. DACT2 staining was existed in both the nucleus and cytoplasm . Reduced expression was found in 39 cases of cancer tissue. Among the 39 cases in which reduced levels of DACT2 were detected, 31 cases were methylated. The reduced expression of DACT2 was significantly associated with promoter region hypermethylation . These results indicate that the expression of DACT2 is regulated by promoter region methylation in primary esophageal cancer.The methylation status of ) Figure . Methylam Figure . The expvs. 0.326 ± 0.012 (P < 0.001) and 0.658 ± 0.037 vs. 0.56 ± 0.016 (P < 0.001) before and after restoration of DACT2 expression in KYSE150 cells and KYSE450 cells, respectively. Cell viability was reduced after restoration of DACT2 expression in KYSE150 and KYSE450 cells. The effect of DACT2 on cell growth was further validated by knocking down DACT2 in YES2 cells. The OD values were 0.787 ± 0.022 vs. 0.876 ± 0.033 (P < 0.001) before and after knockdown DACT2 in YES2 cells  in KYSE150 cells and 201.3 ± 24.2 vs. 63.7 ± 13.1 (P < 0.001) in KYSE450 cells before and after restoration of DACT2 expression. The number of clones was reduced after re-expression of DACT2 in KYSE150 and KYSE450 cell lines. The effect of DACT2 on clonogenicity was further validated by knocking down DACT2 in YES2 cells. The clone number was 129.3 ± 17.0 vs. 200.3 ± 4.5 (P < 0.01) before and after knockdown of DACT2 in YES2 cells . In KYSE450 cells, the cell phase distribution was 50.54 ± 1.65% vs. 41.76% ± 1.37% in G0/G1 phase, 35.37 ± 1.95% vs. 38.75 ± 2.47% in S phase, and 14.74 ± 1.42% vs. 19.59 ± 1.62% in G2/M phase before and after restoration of DACT2 expression. The G2/M phase was significantly different before and after re-expression of DACT2 in KYSE450 cells (P < 0.05). The effect of DACT2 on cell cycle was further validated by knocking down DACT2 in DACT2 highly expressed YES2 cells. The distribution of cell phases was 65.54 ± 1.96% vs. 68.49 ± 0.40% in G0/G1 phase, 24.33 ± 0.98% vs. 23.13 ± 0.17% in S phase, and 10.13 ± 0.97% vs. 8.37 ± 0.30% in G2/M phase. The G2/M phase was significantly reduced by knocking down DACT2 in YES2 cells  [Dapper was first identified as a Dishevelled associated antagonist of Wnt signaling in way(JNK) . In zebrway(JNK) . Su et away(JNK) . Our preway(JNK) , 23, 26.DACT2 is methylated in 35.7% of esophageal squamous dysplasia and 69% of human primary esophageal squamous cancer samples tested, and the expression of DACT2 is regulated by promoter region methylation. These data suggest that DACT2 methylation may serve as an esophageal cancer early detection marker, and methylation of DACT2 is involved in esophageal cancer development. The association of DACT2 methylation with tumor stage suggests that methylation of DACT2 is related to esophageal cancer progression. As DACT2 methylation is associated to metastasis, it suggests that DACT2 methylation may serve as a prognostic marker in ESCC. Our further investigation found that DACT2 suppresses esophageal cancer growth both in vitro and in vivo, indicating that it acts as a tumor suppressor in human esophageal cancer. In vitro study suggests that DACT2 induced G2/M phase arrest. To further understand the mechanism of DACT2 on cell cycle, the key regulators in G2/M checkpoint were analyzed in human esophageal cancer cells. The cyclin B1-Cdk1  complex is a key regulator of mitotic entry [c-Myc proto-oncogene (Myc), a Wnt/β-catenin target gene, may promote the cell cycle [Our present study mainly focused on the epigenetic regulation and function of DACT2. We found that ic entry . A largeic entry –30. The ic entry . In contic entry . Our stuic entry –35. Our ic entry –38. The ll cycle , 40. In ll cycle . We concDACT2 is frequently methylated in human esophageal cancer. The expression of DACT2 is regulated by promoter region methylation. Methylation of DACT2 is significantly associated with tumor stage and metastasis. DACT2 suppresses esophageal cancer development by inhibiting the Wnt signaling pathway.In conclusion, n =4), stage II (n = 79), stage III (n = 42), and stage IV (n = 1). Ten esophageal cancer cell lines  were included in this study. All esophageal cancer cell lines were previously established from primary esophageal cancer and maintained in 90% RPMI media 1640  supplemented with 10% fetal bovine serum. Cells were passaged 1:3 when total confluence (~106 cells) was reached in a 75 cm2 culture flask .A total of 126 cases of esophageal cancer, 42 cases of dysplasia samples and 27 cases of normal esophageal mucosa from patients without cancer were collected from the Chinese PLA General Hospital in Beijing. Snap-frozen fresh tissue samples were collected by surgery resection. All samples were collected under the guidelines approved by the institutional review board at the Chinese PLA General Hospital. Among the patient cases, 91: cases were male and 35 cases were female. The median age was 62.5 years old (range 46–87 years old). All cancer samples were classified according to the TNM staging system (AJCC2010), which included tumor stage I  12 hours before drug treatment. Cells were treated with 5-aza-2′-deoxycytidine  at a concentration of 2 μM. Growth medium, which included 5-aza-2′-deoxycytidine at 2 μM, was exchanged every 24 hours for total of 96 hours of treatment.Total RNA was isolated by Trizol reagent . Agarose gel electrophoresis and spectrophotometric analysis were used to check RNA quality and quantity. Total RNA (5 μg) was used to synthesize first strand cDNA according to the manufacturer's instructions . The reaction mixture was diluted to 100 μl with water, and 2.5 μl of diluted cDNA mixture was added to each 25 μl PCR reaction. The DACT2 PCR primer sequences were as follows: 5′-GGC TGA GAC AAC AGG ACA TCG-3′ (F) and 5′-GAC CGT CGC TCA TCT CGT AAAA-3′ (R). Products were amplified for 35 cycles. GAPDH was amplified for 25 cycles as an internal control. The primers for GAPDH were as follows: 5′-GAC CAC AGT CCA TGC CAT CAC-3′ (F), and 5′-GTC CAC CAC CCT GTT GCT GTA-3′ (R). The amplified PCR products were examined by 1.5% agarose gels.Genomic DNA was extracted by the proteinase K method. The bisulfite modification assay was performed as previously described . MSP priImmunohistochemistry (IHC) was performed in primary esophageal cancer and paired adjacent tissue samples. DACT2 antibody was diluted to 1/400 dilution . The staining intensity and extent of the stained area were scored using the German semi-quantitative scoring system. The staining intensity of DACT2 expression was quantified as follows: no staining = 0; weak staining = 1; moderate staining = 2; strong staining = 3. The extent of DACT2 expression was quantified as follows: 0% = 0, 1–24% = 1, 25–49% = 2, 50–74% = 3, 75–100% = 4 , 45. TheDACT2 cDNA (GenBank accession number NM_214462) was cloned into the pLenti6-GFP vector [6 per 100 mm dish) using Lipofectamine 2000 Reagent  at a ratio of 1:3 (DNA mass : Lipo mass). Viral supernatant was collected and filtered after 48 hours. KYSE150 and KYSE450 cell lines were then infected with viral supernatant. Cells stably expressing DACT2 were selected with Blasticidin  at concentrations of 0.2 μg/ml (KYSE150) and 0.4 μg/ml (KYSE450) for 2 weeks.The human full length P vector . Primers3 cells/well. 5 × 103 cells were plated into 96-well plates before and after knockdown of DACT2 in YES2 cells. The cell viability was measured by the MTT assay at 0, 24, 48 and 72 h . Absorbance was measured on a microplate reader  at a wave length of 490 nm. The results were plotted as means ± SD.DACT2 stably expressed and unexpressed KYSE150 and KYSE450 cells were plated into 96-well plates at a density of 3 × 10DACT2 stably expressed and unexpressed KYSE150 and KYSE450 cell lines were seeded in 6-well plates at a density of 1000 cells per well. YES2 cells before and after knockdown of DACT2 were seeded in 6-well plates at a density of 1000 cells per well. Growth medium, which included blasticidin at 0.2 μg/ml (KYSE150) or 0.4 μg/ml (KYSE450), was exchanged every 24 hours. Cells were fixed with 75% ethanol for 30 min and stained with 0.2% crystal violet after 14 days. The number of clones was then counted. Each experiment was repeated three times.DACT2 stably re-expressed and unexpressed KYSE150 and KYSE450 cells were starved 12 hours for synchronization, and cells were re-stimulated with 10% FBS for 24 hours. Cells were fixed with 70% ethanol and treated using the Cell Cycle Detection Kit . Cells were then detected using a FACS Caliber flow cytometer . YES2 cells with or without knockdown of DACT2 were analyzed the cell cycle as well. Cells phase distribution was analyzed using the Modfit software .5 DACT2 unexpressed and re- expressed KYSE150 and KYSE450 cells, were suspended in 200 μl serum-free RPMI 1640 media and added to the upper chamber of 8.0 μm pore size transwell apparatus . Cells that migrated to the lower surface of the membrane were stained with crystal violet and counted in three independent high-power fields (×100) after incubating for 20 hours. 8 × 104 YES2 cells before and after knockdown of DACT2 were added to the upper chamber of 8.0 μm pore size transwell apparatus. Cells were migrated to the lower surface of the membrane after incubating for 36 hours.Migration: 1 × 105) were seeded to the upper chamber of a transwell apparatus coated with Matrigel  and incubated for 36 hours. Cells that invaded to the lower membrane surface were stained with crystal violet and counted in three independent high-power fields (×100). 1 × 105 YES2 cells before and after knockdown of DACT2 were added to the upper chamber of a transwell apparatus coated with Matrigel. Cells were invaded to the lower membrane surface after incubating for 48 hours.Invasion: the top chamber was coated with a layer of extracellular matrix. Cells ; RNAi negative control duplex . RNAi oligonucleotide or RNAi negative control duplex  were transfected into DACT2 highly expressed YES2 cells.®  and β-actin .Protein samples from unexpressed and stably re-expressed KYSE150 and KYSE450 cells were collected and western blots were performed as described previously . Antibod6 cells in 0.15 ml phosphate-buffered saline) were injected subcutaneously into the dorsal left side of 4–week-old male Balb/c nude mice (n = 5). Tumor volume was measured every 4 days for 24 days starting 8 days after implantation. Tumor volume was calculated according to the formula: V = L × W2/2, where V represents volume (mm3), L represents biggest diameter (mm), and W represents smallest diameter (mm). All procedures were approved by the Animal Ethics Committee of the Chinese PLA General Hospital.Stably transfected KYSE150 cell line with pLenti6-GFP vector or pLenti6-DACT2 vector (4 × 102 test for independent dichotomous variables. All data were presented as means ± standard deviation (SD) of at least three independent experiments and analyzed using the student's t test. Results were reported to be statistically significant at P < 0.05(*).SPSS 17.0 software  software was applied using χ


The overgrowth-associated Beckwith-Wiedemann syndrome (BWS) and the undergrowth-associated Silver-Russell syndrome (SRS) are characterized by heterogeneous molecular defects affecting a large imprinted gene cluster at chromosome 11p15.5-p15.4. While maternal and paternal duplications of the entire cluster consistently result in SRS and BWS, respectively, the phenotypes associated with smaller duplications are difficult to predict due to the complexity of imprinting regulation. Here, we describe two cases with novel inherited partial duplications of the centromeric domain on chromosome 11p15 associated with contrasting growth phenotypes.in cis maternally inherited duplication of 0.88 Mb including the CDKN1C gene that was significantly up-regulated. The duplication did not include the long non-coding RNA KCNQ1OT1 nor the imprinting control region of the centromeric domain (KCNQ1OT1:TSS-DMR or ICR2) in which methylation was normal. In the mother, also referring a growth restriction phenotype in her infancy, the duplication was de novo and present on her paternal chromosome. A different in cis maternal duplication, 1.13 Mb long and including the abovementioned duplication, was observed in a child affected by Tetralogy of Fallot but with normal growth. In this case, the rearrangement also included most of the KCNQ1OT1 gene and resulted in ICR2 loss of methylation (LOM). In this second family, the mother carried the duplication on her paternal chromosome and showed a normal growth phenotype as well.In a male patient affected by intrauterine growth restriction and postnatal short stature, we identified an in cis microduplications encompassing part of the centromeric domain of the 11p15.5-p15.4 imprinted gene cluster and both including the growth inhibitor CDKN1C gene. Likely, as a consequence of the differential involvement of the regulatory KCNQ1OT1 RNA and ICR2, the smaller duplication is associated with growth restriction on both maternal and paternal transmissions, while the larger duplication, although it includes the smaller one, does not result in any growth anomaly.We report two novel Our study provides further insights into the phenotypes associated with imprinted gene alterations and highlights the importance of carefully evaluating the affected genes and regulatory elements for accurate genetic counselling of the 11p15 chromosomal rearrangements.The online version of this article (doi:10.1186/s13148-016-0236-z) contains supplementary material, which is available to authorized users. The ICRs are 2–4-kb long genomic sequences characterized by repressive and permissive epigenetic marks on the opposite parental alleles. A large cluster of imprinted genes that is located on chromosome 11p15.5-p15.4 harbors two independent ICRs, H19/IGF2:IG (Intergenic)-DMR , and KCNQ1OT1:TSS (transcription start site)-DMR . ICR2 controls the imprinting of the centromeric domain. This region corresponds to the promoter of KCNQ1OT1, a long non-coding RNA that is transcribed antisense to KCNQ1 and represses in cis the flanking imprinted genes on the paternal chromosome. These include KCNQ1, a member of the potassium channel KQT-family, and two genes with growth inhibitory properties, CDKN1C and PHLDA2 [Less than 1 % of human genes are imprinted, that is, their expression is monoallelic and parent of origin-dependent as a result of epigenetic modifications acquired during gametogenesis [d PHLDA2 .CDKN1C variations affecting CDKN1C function can also cause these diseases. Maternally inherited loss-of-function mutations have been described in 5 % of the BWS patients  while gain-of-function mutations have been reported in the intrauterine growth restriction (IUGR)-associated IMAGe syndrome and in a single familial case of SRS [Opposite genetic and epigenetic anomalies of the 11p15.5-p15.4 region result in the overgrowth-associated Beckwith-Wiedemann syndrome  [e of SRS , 8.IGF2 in the telomeric domain and CDKN1C and PHLDA2 in the centromeric domain [Deletions/duplications of chromosome 11p15.5-p15.4 have generally been reported in only 2–6 % of BWS and SRS patients , but a min cis duplications including part of the centromeric domain of the 11p15 imprinted gene cluster. The duplications extend 0.88 and 1.13 Mb from the middle of the KCNQ1 gene toward the centromere, respectively. Despite both chromosome aberrations involve the CDKN1C gene and two out of the three putative enhancers [KCNQ1OT1 provides a possible explanation for the associated contrasting growth phenotypes.Here, we describe two novel submicroscopic nhancers , we findKCNQ1, about 20-kb centromeric to ICR2, and the centromeric breakpoint between chr11:3,632,246 and chr11:3,632,370 bp in an intergenic region among the pseudogenes LOC650368 and TRPC2 (p15.5-p15.4) ).Two unrelated children with rare submicroscopic imbalances in the centromeric domain of the 11p15 imprinted gene cluster were identified and subjected to further laboratory analyses and clinical examination. In the proband of the first family (family 1), a duplication of about 0.88 Mb of chromosome 11p15.5-p15.4 was identified by comparative genomic hybridization (CGH) and single nucleotide polymorphism (SNP) array analyses. These methods allowed locating the telomeric breakpoint between chr11:2,739,336 and chr11:2,742,159 bp (GRCh37/hg19) within the intron 10 of The proband of family 1 was the third son of three children from unrelated parents. Pes equinovarus was observed by ultrasound scan by gestational age 16 + 2 weeks and an amniocentesis was obtained. The maternally inherited 11p15 duplication was identified. Due to this finding several ultrasound scans were performed during the pregnancy and IUGR was observed: −18 % at 30 + 6 weeks of gestation, −25 % at 32 + 5 weeks, −27 % at 34 + 5 weeks, and −30 % at 36 + 5 weeks. Due to IUGR induction of delivery was performed. He was born small for gestational age (SGA) at gestational age (GA) 37 + 6 weeks. His birth weight was 2070 g, (−3 SDS), birth length 44 cm (−3 SDS), and occipital frontal circumference (OFC) 30 cm (−2.5 SDS). The placenta weight  was also reduced. Further, blood glucose was 1.4 mmol/l by delivery. He received treatment with intravenous glucose for 1 day. Afterwards he was only breast feed. The pes equinovarus was treated with plaster and tenotomy of the Achilles tendons at 45 days of age.Physical examination at 14 months confirmed the growth restriction, in particular the short stature: weight 9.0 kg (−1.5 SDS), length 73 cm (−2.5 SDS), and OFC 45 cm (−1.5 SDS). The father, aged 40 years, was referred to be of normal stature (176 cm). The mother’s height was 163 cm and her weight is 59 kg at 38 years of age. It was referred that in childhood, she was very small and the general practitioner suspected she was a kind of a dwarf at 5–6 years of age.The first son was born by GA 42 weeks with a birth weight of 3320 g (−0.5 SDS) and a birth length of 52 cm (average). By 14 years of age, his height was 168 cm (+1 SDS); the second son was born at 33 weeks of gestation. The spontaneous preterm delivery was caused by membrane rupture. His birth weight was 1575 g (−1.1 SDS), the birth length 41 cm (−1 SDS). Information on fetal growth parameters during pregnancy was not available. He was treated with intravenous glucose infusions age 1–3 days due to low blood glucose (blood glucoses at day 1: 1.9–2.6 mmol/l). By 7 years, his height was 127 cm (+1 SDS). He was affected by mild attention deficit hyperactivity disorder (ADHD).The presence of the duplication was searched in the proband relatives by SNP array performed on DNA derived from buccal swab of the brothers and from blood of the parents and the maternal grandfather. The chromosome 11 duplication was identified in the mother and in her second son but not in the first son neither in the maternal grandfather . A bacterial artificial chromosome (BAC) probe , birth length 51 cm (average), and OFC 35 cm (+0.5 SDS). Apgar scores were 8/1, 8/5, and 8/10. Neonatal plasma glucose was normal. In the medical record, it is described that there was slight cranial asymmetry with left side of parietal and frontal region a little flat. The head was described as slight narrow, the nasal bridge as slightly wide, and there was strabismus and retention testis. Further, there were described bilateral dysplastic nails on third, fourth, and fifth toes. He was affected by Steno-Fallot Tetralogy, diagnosed on day 1 by echocardiography required because of a systolic murmur. Operation was performed by age 8 month. Neonatal ultrasound scans of cerebrum and kidneys were both normal. At 1 year old, he showed slight frontal bossing, slight hypoplasia of maxilla, slightly flaccid occiput, and bilateral single palmar creases. Neither umbilical hernia nor ear lobe creases were observed. At 8 and 20 months of age, the auxological parameters were still close to the average: 8 month: weight 9.2 kg (+0.5 SDS), length 71.5 cm (+0.5 SDS) and OFC 44 cm (−0.5 SDS); 20 month: weight 11.3 kg (−0.5 SDS), length 84.5 cm (−0.5 SDS) and OFC 46.3 cm (−1.5 SDS). The psychomotor development was normal.The mother was 35 years old, with normal phenotype except for the presence of bilateral ear lobe creases. Her height was 170 cm and weight was 56.5 kg. She was born by GA 41 + 5 weeks, with the birth weight 3740 g (+0.5 SDS), birth length 53 cm (+1 SDS), and OFC 35.5 cm (+1.5 SDS).KCNQ1 exons 13–17 and slight loss of ICR2 methylation were identified in the proband and his mother, while ICR1, IGF2, H19, and control probes showed normal copy number and methylation status  and RP11-699D10 , were used. As in family 1, the results of the metaphase FISH indicated that the duplication was Maternal duplications of the centromeric domain of the 11p15 imprinted gene cluster generally result in SRS phenotype. In this study, we describe two familial cases with overlapping maternal duplications that partially affect the centromeric domain and show contrasting growth phenotypes.CDKN1C gene and two of its putative enhancers on the maternal chromosome 11p15 [KCNQ1OT1 and an unmethylated ICR2 in the 1.13 Mb but not in the 0.88 Mbp duplication  was growth restricted during her infancy. This phenotype likely results from mes Fig. . No CDKNCDKN1C enhancers . In coners Fig. . It is wKCNQ1 and was associated with ICR2 hypomethylation [KCNQ1 and included a non-methylated copy of ICR2 and the 5′ part of the KCNQ1OT1 gene [KCNQ1OT1 allele causing CDKN1C repression in cis. A complex 277 Kb rearrangement on the paternal chromosome 11p15 has been recently described associated with SRS [KCNQ1OT1 and the entire CDKN1C gene were duplicated, but these duplications were discontinuous and did not include ICR2. In this case, the SRS phenotype likely results from the unregulated expression of CDKN1C on the paternal chromosome.Only a few other duplications encompassing partially the centromeric domain of the 11p15 imprinted gene cluster have been described so far. Two of these are 50 and 160 kb long, respectively, and both were associated with BWS upon maternal transmission. The 50-kb duplication spans from intron 1 to intron 2 of hylation . The 160OT1 gene . In bothwith SRS . In thisin cis and in inverted orientation and include a duplicated incomplete but likely functional copy of KCNQ1OT1 and a hypomethylated ICR2. However, the former rearrangement is more extended toward the centromere and includes CDKN1C. The consequence is that the 160 kb duplication results in reduced CDKN1C expression and BWS, while the 1.13-Mb duplication is associated with normal growth and likely normal CDKN1C level  affecting the proband of family 2 is a severe congenital heart malformation (MIM #187500) with both environmental and genetic etiology. The genetics of TOF is complex and involves many loci, but studies performed on large cohorts have not identified any strict association with defects on chromosome 11p , 22. NevCDKN1C alterations, they can be found in BWS cases with other types of 11p15.5 molecular defect including paternal duplications. The finding of such sign in the mother of family 2 proband is intriguing and may be due to altered expression of some 11p15.5 genes or may be coincidental and have different causes.Ear lobe creases are a common sign of BWS . AlthougIn summary, our study is an example of how the analysis of the small copy number variation (CNVs) affecting the imprinted gene clusters can increase our understanding on the imprinting regulatory mechanisms and help to predict the clinical phenotypes resulting from such type of rearrangements.DNA from peripheral blood leukocytes of all the family members was extracted by an automated Chemagic Magnetic Separation Module . DNA from buccal swab samples of the relatives of the proband from family 1 was extracted by using the Maxwell 16 LEV Buccal Swab DNA kit .TM-3 . DNA from cell culture of placenta and umbilical cord and amniotic fluid cells was extracted directly using the Maxwell 16 LEV Blood DNA kit . RNA was extracted by using TRIzol reagent , according to the protocol of the manufacturer.Cells from umbilical cord and placenta of the proband from family 1 and three normal controls were cultured in BIOAMFAll the genetic analyses were performed after informed consent had been obtained. All the clinical and research have been done following the ethical rules of the Danish and Italian law.Z scores of each member under study were calculated referring to standard growth rate of Denmark.MS-MLPA. MS-MLPA was performed on genomic DNA of the proband from family 2, his parents and maternal grandparents. The SALSA MS-MLPA kit ME030-C3 for BWS–SRS  was used following the manufacturer’s instructions. The amplified products were separated by capillary electrophoresis using ABI 3130 Genetic Analyzer . Data was analyzed using the built-in MS-MLPA tool of the software Genemarker v 2.2.0 .The samples were analyzed using the SurePrint G3 Human CGH microarray 180k . Sample and reference genomic DNA (500 ng) were labelled with Cy5 (reference) or Cy3 (specimen) using the Sure Tag Complete DNA labelling Kit (Agilent Technologies Inc.) and purified as described in the manufacturer’s protocol. Labelled sample and reference DNA were pooled, and 5-μl human COT-1 DNA (1 mg/ml), 10× blocking agent, and 2× hybridization buffer were added. Hybridization was performed for 24 h at 65 °C. Scanning and image acquisition were carried out using an Agilent microarray scanner and microarray image files were analyzed using CytoGenomics, version 2.9 (Agilent Technologies Inc.). Copy number was determined using the adm-2 algorithm and profile deviations consisting of four or more neighboring oligonucleotides were considered genomic aberrations. The resolution is thus approximately 50 kb.http://dgv.tcag.ca/dgv/app/home; Decipher: http://decipher.sanger.ac.uk; ISCA: http://clinicalgenome.org/).Detected copy number gains or losses were compared with our in-house database of CNVs and with public CNV databases (Database of Genomic Variants: http://genome.ucsc.edu/cgi-bin/hgGateway), build GRCh37 (hg19).Whole-genome copy number variation (CNV) analysis was carried out using the CytoScan HD array platform . This array contains more than 2.6 million markers for copy number analysis and approximately 750,000 SNPs that fully genotype with greater than 99 percent accuracy. The CytoScan HD assay was performed starting with 250 ng DNA as previously described . Both quD11S4088 short tandem repeat (STR) marker mapping to the duplicated region and TH, D11S4046 and D11S900 STR mapping at the 11p15.5-4 region outside the duplication, were analyzed in the probands and their relatives to verify the origin of duplication and follow the segregation through the three generations. Primers specific for the STR were obtained from NCBI Genome Database together with the PCR conditions. PCR amplification of 100-ng DNA was done using forward primer end labelled with Fam or Hex. Twenty-eight cycles of PCR products were run on the fluorescent capillary system ABI 3130XL. Data were analyzed using GeneMapper Software.Microsatellites of chromosome 7  were also analyzed to exclude the UPD7 associated with the 10 % of the SRS cases (data not shown).FISH analysis was performed on metaphase or interphase nuclei spread from PHA-stimulated umbilical cord cell culture  and peripheral blood leukocytes  using standard procedures. The RP11-699D10 (red) and RP11-11A9 (green) BlueFISH probes (Illumina) targeting the 11p15.4 duplicated region were used for FISH analyses of proband of family 1. RP11-179B7 (red) on 11q22.3 served as control for chromosome 11 to exclude a translocation defect. RP11-11A9 (green) and the BAC clone RP11-81 K4 DNA labelled with red fluorophore using a non-enzymatic nucleic acid labelling method  were used as probes for the interphase FISH on proband of family 2. The probes used for the metaphase FISH were RP11-81K4  and RP11-876C12 . The chromosomes and nuclei were counterstained with DAPI. Hybridizations were analyzed using a Leica DMRB microscope  or a Nikon Eclipse-1000 epifluorescence microscope . Images captured and elaborated using the ISIS software v. 5.1  or the Genikon systemv. 3.8.5 .Two micrograms of genomic DNA extracted from cells/tissues was treated with sodium bisulfite by using the EpiTect Bisulfite kit  following the manufacturer’s protocol. The converted DNA was analyzed by COBRA and Pyrosequencing.BstUI (CGCG) and the digestion products were run on a polyacrylamide gel to separate the digested (methylated) from the undigested (non-methylated) bands. The percentage of methylation was calculated by computer quantitation of the gel following exposure to phosphorimager. Primers sequences, PCR, and restriction enzyme reaction conditions were previously described [Bisulfite-treated DNA was amplified with primers specific for CTCF target site 1 of ICR1 and ICR2. The PCR products were then digested with escribed , 28.In order to obtain more quantitative DNA methylation data, pyrosequencing was performed to assess methylation at seven CpGs within ICR2 and three CpGs within ICR1, as control. Primers and PCR conditions were previously described  . Reactions were run on ABI PRISM 7500 using the default cycling conditions. Relative expression was determined using the ΔΔCT method, and gene expression values were normalized to the expression of the GAPDH reference gene. The primers used are CDKN1C For 5′- AGAGATCAGCGCCTGAGAAG-3′ and CDKN1C Rev 5′-CACCTTGGGACCAGTGTACC-3′ [GAPDH For 5′-CACCATCTTCCAGGAGCGAG-3′ and GAPDH Rev 5′-TCACGCCACAGTTTCCCGGA-3′.About 1 μg of total RNA extracted from placenta and umbilical cord cultured cells was treated with RNase-free DNase, and first-strand complementary DNA (cDNA) was synthesized using Quantitech Reverse Transcription Kit , according to the protocol of the manufacturer. GTACC-3′ ; GAPDH FBAC, bacterial artificial chromosome; BWS, Beckwith-Wiedemann syndrome; CGH, array comparative genomic hybridization; COBRA, combined bisulfite restriction assay; Ctrl, control; Dupl, duplication; FISH, fluorescence in situ hybridization; GOM, gain of methylation; ICR, imprinting control region; ID, imprinting disorders; IG-DMR, intergenic differentially methylated region; LOM, loss of methylation; MS-MLPA, methylation-specific multiplex-ligation-dependent amplification probe assay; OFC, occipital frontal circumference; SDS, standard deviation score; SNP, single nucleotide polymorphisms; SRS, Silver-Russell syndrome; TSS-DMR, transcription start site—differentially methylated region; UPD, uniparental disomy


V2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) play key roles in neurons and neuroendocrine cells including the control of cellular excitability, neurotransmitter / hormone secretion, and gene expression. Calcium entry is precisely controlled by channel gating properties including multiple forms of inactivation. “Fast” voltage-dependent inactivation is relatively well-characterized and occurs over the tens-to- hundreds of milliseconds timeframe. Superimposed on this is the molecularly distinct, but poorly understood process of “slow” voltage-dependent inactivation, which develops / recovers over seconds-to-minutes. Protein kinases can modulate “slow” inactivation of sodium channels, but little is known about if/how second messengers control “slow” inactivation of Ca2+ channels. We investigated this using recombinant CaV2.2 channels expressed in HEK293 cells and native CaV2 channels endogenously expressed in adrenal chromaffin cells. The PKC activator phorbol 12-myristate 13-acetate (PMA) dramatically prolonged recovery from “slow” inactivation, but an inactive control (4α-PMA) had no effect. This effect of PMA was prevented by calphostin C, which targets the C1-domain on PKC, but only partially reduced by inhibitors that target the catalytic domain of PKC. The subtype of the channel β-subunit altered the kinetics of inactivation but not the magnitude of slowing produced by PMA. Intracellular GDP-β-S reduced the effect of PMA suggesting a role for G proteins in modulating “slow” inactivation. We postulate that the kinetics of recovery from “slow” inactivation could provide a molecular memory of recent cellular activity and help control CaV2 channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.Ca V2.2 (N-type) voltage-gated calcium channels (Ca2+ channels) are widely expressed in neurons and neuroendocrine cells where they control neurotransmitter / hormone secretion, gene expression, activation of Ca2+-dependent enzymes / ion channels, and a variety of other cellular functions. Calcium entry is precisely regulated by second messengers including G proteins, kinases, and lipid signaling molecules that converge to fine tune CaV2 function [2+channel inactivation also controls Ca2+ entry and thus cellular excitability and short term synaptic plasticity [V2 inactivation is mediated by distinct calcium or voltage-dependent mechanisms: calcium-dependent inactivation is triggered by “global” elevations of cytosolic [Ca2+] and transduced via calmodulin tethered to the C-terminal tail of the channel [Cafunction –8. Ca2+casticity –12. CaV2 channel –17. Volt channel , 18, 19. channel , 21, as  channel . An addi channel –25. “Slo channel –29.V2 channels is regulated. The CaV β subunit might play a role as an indirect consequence of altered “fast” inactivation [V2.2 [V2 channels from “slow” inactivation. We postulate this novel regulation could provide a basis for molecular memory of recent cellular activity and help control Ca2+channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.Interestingly, protein kinases modulate “slow” inactivation of sodium channels and thereby control neuronal excitability , 31. Muctivation , and synon [V2.2 , 32. In V2.2, β1b, and α2d subunits) kindly provided by Dr. Heidi Hamm (Vanderbilt University) [per manufacturer instructions. Cells were transfected with calcium channel subunits in a ratio of 1;1;1 . The β subunit plasmid also expressed EGFP downstream of an IRES sequence to enable visual identification of transfected cells. In some experiments cells were transfected with CaV2.1, β2a and α2δ. The specific contructs used were as follows: CaV2.1, rat α1A subunit (Genbank # M64373) and rat α2δ Genbank # M86621) both kindly provided by Dr Terry Snutch ; CaV2.2 –bovine α1B (Genbank # NM174632) and bovine β1b (Genbank # AF174415) both kindly provided by Dr Aaron Fox ; rat brain β2a (Genbank # M80545) kindly provided by Dr Roger Colbran . Patch-clamp recording was performed ~48–72 hours after transfection on cells that had been re-plated onto poly-lysine coated glass coverslips for ~12–24 hours. Transfected cells were visually identified using fluorescence of EGFP. The culture medium consisted of MEM supplemented with fetal bovine serum (10%), glutamine (2mM), penicillin/streptomycin (100 unit ml-1/100 μg ml-1), and for G1A1 cells the medium also included G418 (0.5 mg ml-1). Cells were maintained in an incubator (37 C in 95% air and 5% CO2 at ~90% humidity) and passaged every 3–5 days for up to ~25 passages.Recombinant channels were recorded from transiently transfected HEK293 cells or from G1A1 cells (HEK293 cells stably expressing Caversity) , 33, 34.6 cell /mL. Fibroblasts and other proliferating cells were effectively suppressed with cytosine arabinoside (10 μM) , leaving relatively pure chromaffin cell cultures. The culture medium for chromaffin cells consisted of Dulbecco’s modified Eagle medium \ F12 (1:1) supplemented with fetal bovine serum (10%), glutamine (2 mM), penicillin/streptomycin (100 unit mL-1/100 μg mL-1), cytosine arabinoside (10 μM) and 5-fluorodeoxyuridine (10 μM). The culture medium was replaced the day after isolation and experiments were performed 2–5 days following cell isolation. All tissue culture reagents were from Life Technologies .Adrenal chromaffin cells: Male bovine adrenal glands were obtained from a local slaughterhouse , and chromaffin cells prepared by digestion with collagenase followed by density gradient centrifugation as described previously . The celCells were placed in a recording bath (volume ~300 μL) which was continually perfused with fresh solution at a flow rate of ~3–4 ml/min from gravity-fed reservoirs, and viewed using a Nikon TE2000 inverted microscope. Patch pipette electrodes were pulled from borosilicate glass capillary tubes  using a Sutter P-97 pipette puller , coated with dental wax  and fire-polished to a final resistance of ~2 MΩ when filled with a CsCl-based internal solution. Cells were voltage-clamped in the conventional whole-cell configuration using an Axopatch 200B amplifier, Digidata 1400A interface, and PClamp10 (Clampex) acquisition software . Analog data were filtered at 2–3 kHz and digitized at 50 kHz. Series resistance was partially compensated using the Axopatch circuitry (~60–80%). Linear capacitance and leak subtraction (performed offline) used P/N protocols (P/-4 or P/-8) with the leak pulses applied following the test pulses. Some of the voltage-protocols involved very long stimulus steps or trains. For these experiments leak subtraction was not applied, in part because the protocols were designed to monitor recovery from inactivation which could be altered in a voltage-dependent manner. In these experiments only cells with high resistance seals (> 1GΩ) and low holding current (< 50 pA) that remained stable for the duration of the experiment were used. Raw data were analyzed using PClamp10 (Clampfit) and graphing / statistical analyses were performed using OriginPro software  or Prism5 software . All experiments were performed at room temperature (~ 23–25°C).2 1, HEPES 20, BAPTA 10 , Na2GTP 0.35, adenosine triphosphate (MgATP) 4, creatine phosphate  14, pH 7.3, osmolarity ~310–315 mOsm. In some experiments GTP was omitted and 0.5 mM GDP-β-S  was included to test the potential involvement of G protein signaling. In other experiments a peptide inhibitor of PKC (2μM PKC(19–36)) or a peptide inhibitor of dynamin (50 μM dynamin inhibitory peptide) were added to the patch pipette solution on the day of use.The intracellular (patch pipette) solution consisted of (in mM): CsCl 110, MgCl2 1, glucose 10, HEPES 10, CaCl2 2, pH 7.3, osmolarity approx 315 mOsm. After entering the whole-cell recording configuration the bath solution was switched. For HEK293 and G1A1 cell recordings the extracellular solution contained (in mM): tetraethylammonium Cl 155, glucose 10, HEPES 10, BaCl2 5, pH 7.3, 320-330mOsm. For chromaffin cell recording it contained (in mM): NaCl 150, KCl 2, MgCl2 2, glucose 10, HEPES 10, CaCl2 5, TTX 0.05–0.1, pH 7.3, osmolarity approximately 315 mOsm. Note barium and TEA were not used in chromaffin cells recordings because they both block potassium channels which results in depolarization of all the non-voltage-clamped cells in the recording chamber. These cells release a variety of neurotransmitters and hormones which can alter the cell being recorded from, for example via G protein coupled receptors [The extracellular NaCl-based solution used to bathe cells before and during seal formation consisted of (in mM): NaCl 145, KCl 2, MgCleceptors .Tetrodotoxin (TTX)  was prepared as a 1 mM aqueous stock and aliquots frozen until use . PMA, 4α-PMA  bisindolylmaleimide-1, Go6983 and calphostin C  were all prepared as stock solutions in DMSO (1-2mM) and aliquots diluted in extracellular solution on the day of use .0 + A(1-e-X/t) or Y = Y0 + A1(1-e-X/t1) + A2(1-e-X/t2) in which Y0 is the offset / initial amplitude at recovery time zero, A is the amplitude or span, X is the recovery time and t is the recovery time constant. For representation in figures, the mean recovery data were plotted and then fit with the above equations. The parameters from the fits to the mean data are reported in the figure legends. For statistical comparison of specific fit parameters (i.e. recovery time constants), each cell was individually fit with the exponential function to yield the relevant parameters and these were then pooled for statistical comparison. Statistical significance between two datasets was determined using paired or independent Student’s t-test, and ANOVA was used to compare multiple datasets.Statistical analyses were performed using OriginPro software  or Prism5 software . Recovery from inactivation was fit with a single or double exponential association function of the form: Y = YV2.2 Ca2+ channels and how it might be regulated by the PKC-activator PMA. To isolate voltage-dependent inactivation, we used barium rather than calcium as the extracellular divalent cation and included BAPTA (10 mM) in the intracellular patch-pipette solution to prevent calcium-dependent inactivation. G1A1 cells stably express CaV2.2, α2δ and β1b calcium channel subunits. Under our recording conditions, acute application of PMA (200 nM for 5-minutes) had no effect on the peak barium current (IBa) amplitude  was followed by a short test pulse to determine the extent of recovery  applied at increasing intervals following the prepulse . It was 2a subunit which dramatically reduces fast inactivation [V2.2, α2δ, and either β2a or β1b subunits (with EGFP as a marker). As expected, inactivation of IBa during a step depolarization was much slower in β2a than β1b containing channels  of 48.6 ± 4.7 s (n = 16), which was significantly longer compared β1b containing channels . As expected for voltage-dependent inactivation, the recovery kinetics of IBa showed no correlation with the amount of barium entry (2a subunit than in G1A1 cells (β1b expressing cells). However, when the recovery time constant was plotted against charge density, the slope of a linear fit using Deming regression was not significantly different from zero (0 = 0.75 for β1b expressing cells and p = 0.76 for β2a expressing cells). This was confirmed using Pearson’s correlation coefficient which again showed no statistically significant correlation between recovery rate and charge density: for β1b cells r = -0.13, p = 0.74; for β2a cells r = -0.08, p = 0.76. We also found that, as predicted for voltage-dependent inactivation, recovery was significantly accelerated at more hyperpolarized holding potentials  (V2.1 (P/Q-type) channel . Recovery from inactivation in CaV2.1 channels was faster than in CaV2.2 channels, but was still significantly prolonged by PMA from 14.8 ± 3.2 s to 35.5 ± 9.7 s . To compare the effects of PMA across these channels with different subunit combinations that had inherently different rates of recovery, we calculated the change in recovery time constant (i.e. tau in the presence of PMA / tau before application of PMA). This tau ratio showed that PMA prolonged recovery from inactivation to a similar extent in all cases: the tau ratio was 2.66 ± 0.35 for CaV2.2 + β2a (n = 6), 3.09 ± 0.3 for CaV2.2 + β1b (n = 9), and 2.38 ± 0.38 for CaV2.1 + β2a (n = 5) .PMA significantly prolonged the recovery time constant in β t-test) . We alsoTo more closely mimic physiologically relevant electrical activity, we stimulated cells with trains of brief depolarizations applied at 50Hz for 1s, or 5Hz for 10s . The extV2.2, β1b and α2δ, . This is consistent with the involvement of PKC, so we tested several PKC inhibitors to see if they blocked the effect of PMA on recovery from inactivation. Cells were pre-incubated for 20–30 minutes with the inhibitors and then stimulated first in the presence of the inhibitor alone and then in the presence of the inhibitor + PMA. The inhibitors used were PKC(19–36), a pseudosubstrate peptide inhibitor of PKC that was added to the intracellular patch-pipette solution (2 μM); a combination of bisindolylmaleimide-1 (500nM) + Go6983 (100nM); calphostin C (200 nM). Recovery from inactivation following a 5Hz/10s train was fit with an exponential to determine the recovery time constant   and found it was significantly reduced by bisindolylmaleimeide-1 + Go6983 and abolished by calphostin C  . In conthostin C  (p = 0.0V2.2 channels by G protein βγ subunits (Gβγ) [IBa by binding directly to the CaV2.2 subunit, and activation of PKC by phorbol ester has been shown to partially reverse this inhibition [IBa. A trademark characteristic of Gβγ-mediated inhibition is that it can be reversed by strong membrane depolarization due to transient voltage-dependent dissociation of Gβγ from the channel [Given the incomplete block of PMA by bisindolylmaleimide-I and especially PKC19–36) (see above and 9–36 (seehibition –43. We t40 (% inhibition of recovery at 40s by PMA) was not significantly altered by the Ras mutants: 36 ± 4% in controls, 27 ± 7% in S17N and 31 ± 6% in G12V . Phorbol esters / PKC have also been reported to modulate surface expression of some ion channels and transporters through dynamin dependent endocytosis [V2 channels are known to associate with endocytic complexes and are subject to internalization by various stimuli [V2.2, β1b and α2δ, from 28.9 ± 3 s to 65.5 ± 14.1 s  suggesting endocytosis is not playing a significant role under our recording conditions.PMA can also activate some additional signaling pathways, for example by binding to the C1-domain of RasGRP (Ras Guanine nucleotide Releasing Protein) which in turn leads to activation of the monomeric G protein Ras , 45. To ocytosis –49 and C stimuli –53. HoweICa from inactivation in adrenal chromaffin cells, an important neuroendocrine component of the sympathetic nervous system [V2 channels account for ~85–90% of the whole cell calcium current in a roughly 1:1 ratio of N-type and P/Q-type ICa, with the remainder due primarily to a small (~10%) CaV1 (L-type) component [ICa to enable investigation of voltage-dependent inactivation [2+ as the extracellular divalent charge carrier because Ba2+ depolarizes the surrounding cells in the recording chamber leading to exocytosis of catecholamines, ATP, and opioids that can act in a paracrine manner to modulate the ICa in the cell being recorded from [It is possible that channels in heterologous expression systems are regulated in a different manner than would be the case in a native cellular environment. Therefore, we investigated the ability of PMA to slow recovery of s system . In boviomponent , 55, 56.tivation , 57. Howded from .2a subunit [ICa both in control conditions (95 ± 1%) and in the presence of PMA . Recovery from inactivation exhibited at least two kinetic components (40 (% change in recovery at 40s) showed that PMA inhibited recovery by 35.5 ± 1.6% (n = 7). Pretreating cells with bisindolylmaleimide-1 modestly reduced R40 to 21.6 ± 7% (n = 5) although this was not statistically significant, and calphostin C significantly reduced R40 to 12.8 ± 4%   . These dV2.2, β1b and α2δ subunits stimulated with a 5Hz/10s train. In the absence of PMA, GDP-β-S increased the extent of inactivation at the end of the 5Hz train compared to control cells . The rate of recovery from inactivation under control conditions (before application of PMA) was not altered by GDP-β-S, but the slowing of recovery produced by PMA was significantly reduced, as reflected in the tau ratio  in the intracellular patch pipette solution to prevent activation of heterotrimeric and monomeric G proteins . First wau ratio . We repeau ratio . Similarau ratio . TogetheV2.2 plays an important role in controlling the temporal response of the channels to different electrical stimuli. “Fast” inactivation is relatively well-understood and thought to involve pore occlusion by the cytoplasmic I-II linker [V2 channels [V2.2 channels using both recombinant channels expressed in HEK293 cells, and endogenous channels in adrenal chromaffin cells. In both these cell types the CaV2 channels undergo Ca2+-dependent inactivation which confounds investigation of voltage-dependent inactivation. Therefore, we included 10mM BAPTA in the patch pipette solution and, for HEK293 cells, we also used Ba2+ rather than Ca2+as the charge carrier to prevent Ca2+-dependent inactivation.Voltage-dependent inactivation of CaI linker , 18, 19.I linker –29. “SloI linker , 60, 61.channels  but littV2.2 channels are mixed/complex. In some cases there is a pronounced increase in current amplitude, at least in part due to reversal of tonic Gβγ-mediated inhibition [ICa, perhaps through distinct kinase isoforms and phosphorylation sites, and its effects might be altered by the CaVβ subunit [IBa amplitude (2+ buffering (10mM BAPTA) which might preclude activation of classic (Ca2+-dependent) PKC isoforms. We also found no effect of PMA on the I-V curve, inactivation curve, or “fast” inactivation during a step depolarization  following a 10s step was faster than for the CaV2.2 channels, but still prolonged by treatment with PMA. Thus, PMA prolongs recovery from “slow” inactivation in both CaV2 channel types, and it will be interesting to determine if this extends to the CaV1 or CaV3 Ca2+channels.Although we did not systematically compare the effects of different β subunits, we did investigate the βtivation . It is pV2.2 channels . Superimposed on this is the molecularly distinct, but poorly understood process of “slow” inactivation which develops and recovers over the course of seconds-to-minutes. The slow onset favors recruitment by sustained periods of electrical activity or membrane depolarization, while the slow recovery kinetics might confer a short-term “molecular memory” for this preceding cellular activity. In this study we report the novel finding that PMA dramatically prolongs recovery of CaV2.2 channels from “slow” inactivation. This effect is likely mediated at least in part through PKC and might also involve G protein / guanosine nucleoside signaling. Regardless of the precise molecular details, this could provide a novel mechanism for dynamic, activity-dependent regulation of Ca2+channel availability, electrical excitability, and neurotransmission in the seconds-to-minutes timeframe.“Fast” voltage-dependent inactivation of Ca