However, the mechanisms of PTEN regulation still need further investigation. We here report that a combination of four microRNAs  promotes prostate cell proliferation by regulating PTEN and its downstream signals We found that the four microRNAs (miRNAs) could effectively suppress PTEN expression by directly interacting with its 3’ UTR in prostate epithelial and cancer cells. Under-expression of the four miRNAs by antisense neutralization up-regulates PTEN expression, while overexpression of the four miRNAs accelerates epithelial and prostate cancer cell proliferation. Furthermore, the expression of the four miRNAs could, singly or jointly, alter the expression of the key components in the phosphoinositide 3-kinase (PI3K)/Akt pathway, including PIK3CA, PIK3CD, PIK3R1 and Akt, along with their downstream signal, cyclin D1.invitro. These findings increase understanding of the molecular mechanisms of prostate carcinogenesis and progression, even provide valuable insights into the diagnosis, prognosis, and rational design of novel therapeutics for prostate cancer.These results suggested that the four miRNAs could promote prostate cancer cell proliferation by co-regulating the expression of PTEN, PI3K/Akt pathway and cyclin D1  Prostate cancer is one of the most common malignant tumors in men, with the morbidity and mortality significantly increasing in China in recent years, nevertheless the incidence of prostate cancer remains much lower compared to western countries such as Europe and the United States -3. The cIncreasing evidences have demonstrated that microRNAs (miRNAs) play vital roles in the initiation, progression and metastasis of various human cancers by regulation of proto-oncogenes or tumor-suppressor genes ,8. MoreoIn recent years, studies have also increased to investigate the roles of miRNAs in PTEN regulation ,15. MengIn addition, recent studies have also shown that miR-26a , miR-221The study was performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all participants involved in this study. This study was approved by the Institutional Review Board of the School of Life Sciences, Fudan University and Shanghai Jiao tong University School of Medicine, and Hospital Authority of Shanghai First People’s Hospital and Renji Hospital, Shanghai. All the cell lines used in this study were purchased from a commercial source or gifted from other researchers with published references for the origin.ACTGGTCAGTTTTGCATGGATTTGCACATGTG GCAACAGGAGTACAACTAGGTAATCCCTGGCAATGTGATGATATTATTAAGTGCAGCAGCAAGCCTATCCTGGATTACTTAAGGCATCACTTTTTTAATGTTTAACAGGCCGGGACAAGTGCAATATGTTCGATATCAGATCTCAC-3’) ended with BglII, NheI and EcoRV sites for subcloning. The reporter plasmid phEW-luc/PC (positive control) was generated by inserting the BglII-digested fragment above into the phEFW-luc plasmid [5’-GTTGCTAGCCTGATCCAGAGAATGAACC T-3’ and 5’-CTCGATATCCCCACACAATGACAAGAATG-3’. The 3’ UTR was double-digested with NheI/EcoRV and inserted into the plasmid phEW-luc/PC to replace the intact artificial target site, and the resulting reporter plasmid was designated as phEW-luc/PU. Similarly, the reporter plasmids phEW-luc/PUA, phEW-luc/PUB, phEW-luc/PUC, and phEW-luc/PUD were constructed by replacing the intact artificial target site with relevant truncated PTEN 3’ UTR fragment A , fragment B , fragment C , or fragment D  respectively. Accordingly, the mutant counterpart, of which the corresponding miRNA-binding seed region (6 bp) was replaced with a random hexanucleotide (GTCAGT), was synthesized by Shanghai Office of Life Technologies Co. . The reporter plasmids containing the mutant counterpart  were also constructed in similar ways described above. These truncated PTEN 3’ UTR fragments and their mutant counterparts were amplified by PCR using the relevant primers . This artificial target site  were used as vector control and non-effective control (containing a 304 bp mmu-let7a-1 precursor sequence) respectively. The relevant pri-miRNA fragment was first amplified by PCR using the relevant primers , and sub2 using Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (GIBCO). On the other hand, DU145 and PC-3 cell lines were cultured in F12 medium, while 22RV1 cell line was cultured in RP1640 medium, both supplemented with 10% FBS. All the cells were seeded on 6-well plates and transfected 24 hrs later using Lipofectamine 2000 (Invitrogen) with 4~5 μg DNA plasmids or a final concentration of 100 nM RNA.Human prostate normal tissues cell line PNT1B ,30, humaRenilla luciferase activity in all samples.All cells were seeded on 24-well plates and transfected 24 hr later using Lipofectamine 2000. Anti-miR miRNA inhibitor  (Applied Biosystems) for miR-19b , miR-23b , miR-26a , miR-92a  or negative control  was co-transfected with 100 ng of luciferase reporter plasmids described above and 2 ng of internal control plasmid pRL-CMV (Promega). Twenty-four hours after transfection, cells were lysed and luciferase activities analyzed using the Dual-Luciferase Assay Reporter System (Promega) according to the manufacturer’s instructions. Reporter luciferase activity was normalized to the internal control 3 cells/well and the cell numbers were counted after seeded once a day for one week. The cell density was photographed 4 days after seeding.After transfection with miRNA expression plasmids, cells were seeded in 24-well plate at 5×10-ΔΔCt as previously described [Total RNA was extracted using TRIZOL Reagent (Invitrogen) for reverse transcription. Then cDNAs were synthesized and amplified using relevant cDNA-specific primers , SYBR-Grescribed .-ΔΔCt as previously described [For miRNA expression assay, reverse transcription and qRT-PCR were carried out using TaqMan miRNA assay kits (Applied Biosystems) with hsa-miR-19b , hsa-miR-23b , hsa-miR-26a , hsa-miR-92a  or U44   -specific reverse transcription primers, according to the manufacturer’s instructions. The relative expression level of target genes was defined as fold changes by 2escribed .TM Western HRP substrate kit (Millipore). The relative quantification was measured by densitometry (Bio-Rad). These antibodies were purchased either from (i) Santa Cruz Biotechnology: antibodies to p110α (Cat. No. sc-7174) and p110δ (Cat. No. sc-7176); or (ii) Cell Signaling Technology: antibodies to p85 (Cat. No. 4292), PTEN (Cat. No. 9188), phospho-PTEN (Cat. No. 9554), AKT (Cat. No. 9272), phospho-AKT (Cat. No. 4060), cyclin D1 (Cat. No. 2978), GAPDH (Cat. No. 2118) and HRP-linked secondary antibodies (Cat. No. 7074).Cells were harvested and lysed, then subjected to SDS-PAGE electrophoresis and transferred to a polyvinylidene difluoride membrane (Amersham Biosciences) as previously described . The memt tests were used. Statistical significance was determined by the logrank test. Values of p<0.01 were considered to be statistically significant. Error bars represent the standard error (SE) unless otherwise indicated.For comparison of differences between groups, analysis of variance (ANOVA) and unpaired student’s http://www.targetscan.org/) [Post-transcriptional regulation via interaction between miRNAs and their target site(s) in 3’ UTRs results in translational repression or mRNA cleavage , so putaan.org/) , we predan.org/) , and twoan.org/) . These pAnti-miR microRNA inhibitor for each miRNA or anti-miR control was co-transfected with the above-mentioned reporter vectors into the human prostate epithelial cell line PNT1B to assess the relative luciferase activity . We founNext, we cloned 4 truncated fragments  from different parts of the PTEN 3’ UTR , each ofWe first investigated the endogenous expression of the four miRNAs in PNT1B, a normal prostate epithelial cell line, as well as in several prostate cancer cell lines such as BPH-1, LNCaP, PC-3, DU145, and 22RV1 . We founWe proceeded to transfect the miRNA expression vectors into DU145 and found that the total expression of relevant miRNA increased by more than 2-fold . We furtWe investigated the interaction between the four miRNAs and the PI3K/Akt pathway, which is a key cell proliferation pathway regulated by PTEN . SpecifiWe transiently transfected miR-19b, miR-23b, miR-26a or miR-92a expression vectors in DU145 and PNT1B cells, then measured expression levels of p110α, p110δ, p85 and Akt mRNA by qRT-PCR . Given tIn order to validate the observations above, we further employed PTEN inhibitor and PTEN siRNA#2 to repeat the experiments . The resIn addition to overexpression of the four miRNAs, we also neutralized these miRNAs using anti-miR inhibitors and subsequently analyzed the effects on the PI3K/Akt pathway components . For p11Cyclin D1 is an important downstream component regulated by PI3K/Akt signaling  and is uin vitro. The cell numbers were counted once a day for one week and cell growth observed by micrograph on Day 4 after cell seeding , PIK3R1 (p85) and Akt , and cycin vitro. These findings help to better understand the molecular mechanism of prostate carcinogenesis and progression with regard to miRNA regulation of PTEN expression, and may provide insights for better diagnosis, therapy and prognosis of prostate cancer in the future.In conclusion, we herein proposed for the first time that the four miRNAs, i.e. miR-19b, miR-23b, miR-26a and miR-92a, co-regulate PTEN expression in prostate cancer cells. The four miRNAs regulate PTEN expression post-transcriptionally, and affect the downstream PI3K/Akt pathway via PIK3CA (p110α), PIK3CD (p110δ), PIK3R1 (p85), Akt as well as cyclin D1, thus promote prostate cancer cell proliferation Table S1Primers for amplifying truncated PTEN 3’ UTR fragments and their mutant counterparts.(DOC)Click here for additional data file.Table S2Primers for amplifying pri-miRNA fragments.(DOC)Click here for additional data file.Table S3Primers for qRT-PCR.(DOC)Click here for additional data file.Table S4Target sites of the four miRNAs in the 3’UTR of the key components of PI3K/Akt pathway.(DOC)Click here for additional data file.Figure S1Sequences of RNA-target sites for the four miRNAs in the PTEN 3’ UTR.Sequences of RNA-target sites for miR-19b (A), miR-23b (B), miR-26a (C) and miR-92a (D) in the PTEN 3’ UTR are shown paired with specific miRNA sequences. The seed region of each miRNA is underlined.(DOC)Click here for additional data file.Figure S2Validation of miRNA targets in the PTEN 3’ UTR.(A) PNT1B cells were co-transfected the luciferase reporter vectors, which harbored either miR-target positive control (PC), full-length PTEN 3’ UTR (PU), or its mutant counterpart (mPU) respectively, with relevant anti-miRNA inhibitors for the luciferase reporter assay. (B-E) Luciferase reporter vectors containing relevant truncated fragment PUA (B), PUB (C), PUC (D) or PUD (E) of PTEN 3’ UTR with (MT) or without (WT) mutant(s) in its relevant miRNA binding site(s) were co-transfected with specific anti-miRNA inhibitors respectively for the luciferase reporter assay. *indicates a significant difference from the control (p<0.01).(DOC)Click here for additional data file.Figure S3TaqMan microRNA assay for the specific miRNA expression in DU145.(A) The four miRNAs were overexpressed after transient transfection of the specific miRNA expression vectors. (B) The four miRNAs were neutralized after the specific anti-miRNA inhibitors were imposed.(DOC)Click here for additional data file.Figure S4TaqMan microTNA assay for specific miRNA expression in PNT1B.(A) The four miRNAs were overexpressed after transient transfection of specific miRNA expression vectors. (B) The four miRNAs were neutralized after the specific anti-miRNA inhibitors were imposed.(DOC)Click here for additional data file.Figure S5Up-regulation of PTEN expression by neutralizing the specific miRNAs in PNT1B.The expression of PTEN was up-regulated after two (A) or three (B) of four miRNAs were neutralized simultaneously in PNT1B. The relative quantification of PTEN protein was measured by densitometry.(DOC)Click here for additional data file.Figure S6Repression of PTEN by PTEN-specific siRNA interference.PTEN expression was repressed by PTEN-specific siRNAs in DU145 (A) and PNT1B (B), and among them PTEN siRNA#2 was identified as the most effective silencer for the following experiments and to be used as a positive control. The relative quantification of PTEN protein was measured by densitometry.(DOC)Click here for additional data file.Figure S7Prediction diagram of miRNA targeting site in the PIK3CA (p110α), PIK3CD (p110δ), PIK3R1 (p85) and Akt mRNA 3’ UTR.PIK3CA 3’ UTR harbors a miR-19b targeting site, while PIK3CD 3’ UTR harbors a miR-26a and a miR-92a targeting site. In the PIK3R1 3’ UTR, multiple miR-23b, miR-26a and miR-92a targeting sites can be found. In Akt 3’ UTR, a miR-26a targeting site is located.(DOC)Click here for additional data file.Figure S8The protein expression level of p110α, p110δ, p85 and Akt was altered after the relevant miRNA was overexpressed in DU145 or PNT1B.The relative quantification of these four proteins was measured by densitometry.(DOC)Click here for additional data file.Figure S9The expression of p110α, p110δ, p85 and Akt increased after either PTEN inhibitor VO-OHpic trihydrate or PTEN siRNA#2 was imposed in DU145 or PNT1B.(A) The mRNA expression of these four genes increased (mRNA / actin mRNA) after DU145 or PNT1B cells were treated with the PTEN inhibitor. (B) The protein expression of these four genes increased after DU145 or PNT1B cells were treated with PTEN inhibitor. (C) The mRNA expression of these four genes increased (mRNA / actin mRNA) after PTEN siRNA#2 was imposed in DU145 or PNT1B. (D) The protein expression of these four genes increased after PTEN siRNA#2 was imposed in DU145 or PNT1B. The relative quantification of these four proteins was measured by densitometry.(DOC)Click here for additional data file.Figure S10The protein expression level of p110α, p110δ, p85 and Akt was altered after the relevant miRNA was neutralized in DU145 or PNT1B.The relative quantification of these four proteins was measured by densitometry.(DOC)Click here for additional data file.Figure S11Cyclin D1 was co-regulated by miR-19b, miR-23b and miR-92a at the post-transcriptional level.(A) Prediction diagram of miRNA-binding site in the CCND1 (cyclin D1) mRNA 3’ UTR. There exsit three miR-19b binding sites, a miR-23b and a miR-92a binding site in its 3’ UTR. (B) Cyclin D1 was overexpressed in prostate cancer cell line DU145 compared with the PNT1B control. The relative quantification of cyclin D1 was measured by densitometry.(DOC)Click here for additional data file.Figure S12Cyclin D1 expression increased upon treatment with PTEN inhibitor or siRNA interference in prostate cells.Cyclin D1 expression increased upon treatment for DU145 (A) and PNT1B (B) cells with PTEN inhibitor or PTEN siRNA#2. The relative quantification of cyclin D1 was measured by densitometry.(DOC)Click here for additional data file.Figure S13Overexpression of miR-19b, miR-23b, miR-26a or miR-92a stimulated cell proliferation of DU145 cells.Cell growth was observed by daily counting for one week. Microphotographs of the cells were taken on day 4 after the cells were seeded. Original magnification: 100×.(DOC)Click here for additional data file.Figure S14Cell growth was promoted after PTEN inhibitor or PTEN siRNA#2 was added to DU145 cells.Cell growth was promoted after imposing PTEN inhibitor (A) or PTEN siRNA#2 (B) in DU145 cells. Cell growth was observed by daily counting for one week. Microphotographs of the cells were taken on day 4 after the cells were seeded. Original magnification: 100×. *indicates a significant difference from the control (p < 0.01).(DOC)Click here for additional data file.Figure S15Overexpression of miR-19b, miR-23b, miR-26a or miR-92a stimulated the cell proliferation of PNT1B cells.Cell growth was observed by daily counting for one week. Microphotographs of the cells were taken on day 4 after the cells were seeded. Original magnification: 100×.(DOC)Click here for additional data file.Figure S16Cell growth was promoted after PTEN inhibitor or PTEN siRNA#2 was added to PNT1B cells.Cell growth was promoted after imposing PTEN inhibitor (A) or PTEN siRNA#2 (B) in PNT1B cells. Cell growth was observed by daily counting for one week. Microphotographs of the cells were taken on day 4 after the cells were seeded. Original magnification: 100×. *indicates a significant difference from the control (p < 0.01).(DOC)Click here for additional data file.Figure S17Overexpression of miR-26a alone or combination with miR-19b, miR-23b, or miR-92a stimulated cell proliferation in prostate cells.MiR-26a alone or combination with miR-19b, miR-23b, or miR-92a was overexpressed in DU145 cells (A) or PNT1B cells (B). Cell growth was observed by daily counting for one week. Microphotographs of the cells were taken on day 4 after the cells were seeded. Original magnification: 100×. *indicates a significant difference from the control (p < 0.01).(DOC)Click here for additional data file.


Body composition was assessed by dual energy x-ray absorptiometry. SWAT 25(OH)D concentrations were 5.8 ± 2.6 nmol/kg tissue and 6.2 ± 2.7 nmol/kg tissue pre- and post-intervention SWAT, respectively. There was a significant positive association between SWAT 25(OH)D concentration and serum 25(OH)D concentration . Both SWAT and serum 25(OH)D concentrations did not significantly change after a twelve-week period of energy restriction with approximately 5 kg of fat loss. In conclusion, we have demonstrated our LC-MS/MS method can detect 25(OH)D3 in human subcutaneous fat tissue from overweight and obese individuals and is consistent with previously reported concentrations in swine. Additionally, our findings of no significant changes in SWAT 25(OH)D3 or serum 25(OH)D after a 6% loss of total body weight and 13% reduction in total fat provides the first human evidence that adipose 25(OH)D does not likely contribute to serum 25(OH)D with moderate weight loss; whether this is also the case with larger amounts of weight loss is unknown. Weight loss alone is not sufficient to increase serum 25(OH)D and increases in dietary or dermal biosynthesis of vitamin D appear to be the most critical contributors to in vitamin D status.Cholecalciferol is known to be deposited in human adipose tissue, but it is not known whether 25-hydroxyvitamin D (25(OH)D) is found in detectable concentrations. Therefore, our objective was to determine whether 25(OH)D is detectable in subcutaneous white adipose tissue (SWAT) in overweight and obese persons enrolled in a twelve week energy restricted diet. Baseline and post-intervention gluteal SWAT biopsies were collected from 20 subjects participating in a larger clinical weight loss intervention. LC-MS/MS was utilized to determine SWAT 25(OH)D concentrations. Serum 25(OH)D and 1,25(OH) The analysis of vitamin D metabolites in adipose tissue is rare and likely due to difficulties in extracting and separating the metabolites from tissue matrices . Nonetheet al.  were analyzed using standard radioimmunoassay (RIA) procedures . Participants’ baseline and post-intervention samples were run simultaneously in duplicate and processed in accordance with manufacturer’s instructions. Interassay CVs were 6.0% and 6.3% for 25(OH)D and 1,25(OH)SWAT biopsies were obtained at the UC Davis Medical Center Clinical and Translation Science Center at “Run-In” and at the end of the 12-week intervention following a 12-h overnight fast. Biopsy samples from “Run-In” and at the end of the 12-week intervention were collected from opposite sides of the buttocks. Each sample was immediately rinsed in ice cold phosphate buffered saline (PBS) to minimize blood contamination. Samples were additionally rinsed in fresh ice cold PBS and aliquots were frozen in liquid nitrogen. Frozen samples were than stored at −80 °C until analyzed.SWAT biopsies from participants enrolled in cohorts beginning in November through January were chosen for analysis to minimize potential influences by endogenous vitamin D synthesis. Fourteen subjects from winter cohorts had complete SWAT biopsy sets (both baseline and post-intervention) available, therefore, six subjects were randomly chosen from spring cohorts (enrollment between February and April) to bring the total number of SWAT samples analyzed to twenty pairs. Biopsies used for this study were shipped overnight on dry ice to the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University for analysis.3-d6 standards , each at 111 ng/mL. The mixture was further homogenized with a spatula and transferred to a 15 mL reservoir tube. A 20 μm frit was placed on top of the reservoir. The reservoir was tightly compressed with a 10 cc syringe plunger and 8 mL of water was added. The contents were collected and reduced to dryness using a Vac-Elute set to “Collect” mode. Residues were solubilized in acetone (4 mL), with supernatants being isolated, centrifuged at 3000 g for 11 min and filtered through a 0.2 μm PTFE filter. The extract volume was reduced under N2 at 45 °C for 60 min and 200 μL of the remaining extract was transferred to an autosampler vial insert. A 50 μL aliquot was injected on a LC-MS/MS (Agilent 1100 HPLC-ABSciex 5500 QTRAP tandem mass spectrometer). The HPLC was equipped with a 4.6 × 250 mm, 5-μm ProntoSIL 200-5-C30  C-30 column. The column was maintained at room temperature during the analyses. The mobile phase flow rate was 0.9 mL/min. Solvent A was methanol and solvent B was methylene chloride. The gradient was: 0–15 min, 100% solvent A, followed by 50% solvent A from 10.0 to 25.0 min and a 10 min re-equilibrated at 100% A. Post-column solvent flow was diverted to waste during the final 13 min. The mass spectrometer employed an atmospheric pressure chemical ionization source operated in positive ion mode. The ion source temperature was 400 °C. The triple quadrupole MS/MS was operated in multiple reaction monitoring (MRM) mode to detect the mass transitions of the parent molecules after in source loss of water  for 25-hydroxy vitamin D3m/z 383.4 > 211.3 and 25-hydroxy vitamin D3-d6m/z 389.4 > 211.3, respectively. The collision gas was nitrogen and the pressure was set to medium. The collision energy for all MRM transitions was 40 V and the dwell time for each MRM transition was 1000 ms. As described above, we utilized isotope dilution procedures to internalize standards within samples [3-d6 to each sample, the isotope ratio of the resulting solution can be deconvoluted using the known isotope ratio of the labeled compounds and the natural deuterium abundance in the sample (<0.1%), to calculated the D3 metabolite concentrations in the samples. The average recovery was 72%.Subsamples of SWAT tissue (0.1–0.5 g) were added to a glass mortar filled with 2 g of 40 μm Bondesil-C18. Tissue was blended with a glass pestle and spiked with 0.2 mL mixture of 25-OH deuterated vitamin D samples . By addiBased on the average variance of low abundance calibration standards (2.5 nmol/kg), method limit of detection and limit of quantification were estimated at 1.0 and 2.9 nmol/kg, respectively. All reported data were greater than 2x the limit of detection estimate.t-tests were used to determine differences between baseline and post-intervention measurements. All P-values ≤ 0.05 were considered statistically significant. Graphical plots were constructed using the R package, ggplot2 [All statistical analyses were performed using R version 2.15.1 (R Development Core Team 2012). Univariate analysis, including mean, range and standard deviation, was used for baseline and post-intervention physical and clinical characteristics. Associations among variables were determined using Pearson’s correlation coefficients. Paired  ggplot2 .To minimize any potential effects of sun exposure, biopsy samples were specifically chosen from winter and spring cohorts. Consequently, the treatment assignment became unbalanced with 7 samples assigned to the AD group and 13 samples to the LD group. Additionally, by chance, 2 samples within the AD group had undetectable 25(OH)D, which reduced the number of paired baseline and post-intervention for the AD group to 5. Thus, treatment effect was not analyzed due to unequal and small sample sizes in each treatment group.Physical characteristics of the subjects at pre- and post-intervention are shown in 2D are shown in Serum and SWAT concentrations of 25(OH)D and 1,25(OH)3 concentrations were detected in 18 baseline samples and 19 post-intervention samples. Concentrations ranged from 2.3 to 12.8 nmol/kg SWAT (0.9–4.9 ng/g) at baseline and 2.8–13.1 nmol/kg SWAT (1.1–5.03 ng/g) post-intervention. There was no statistical difference between baseline and post-intervention SWAT 25(OH)D3 concentrations (SWAT 25(OH)Dtrations .3 concentrations varied  and SWAT and serum 25(OH)D concentrations were significantly correlated at baseline  and post-intervention  respectively . Additionally, serum 1,25(OH)2D concentrations and SWAT 25(OH)D3 concentrations were not significantly correlated .Individual changes in SWAT 25(OH)Ds varied  and rangectively . The abset al. found mean subcutaneous fat concentrations of 25(OH)D between 4.8 nmol/kg and 6.3 nmol/kg depending on the inclusion of vitamin D or 25(OH)D in their feed [3 concentrations of 5.8 nmol/kg at baseline and 6.2 nmol/kg post-intervention are in agreement with Jakobsen et al. and add to the few papers that have found measureable SWAT 25(OH)D concentrations [3 using LC-MS/MS and acknowledge that at the time of our study there was no reference method to which our data could be compared. Considering the controversy regarding accurately assessing serum 25(OH)D concentrations [Little is known about the content of 25(OH)D in human adipose tissue and whether weight loss or other conditions that impact adiposity influence fat tissue vitamin D metabolites. In swine, Jakobsen trations . This cotrations ,11,12,133 concentrations remained unchanged after a significant decrease in adipose mass would suggest a release of 25(OH)D from adipose tissue. However, there were no changes in serum 25(OH)D, suggesting little contribution of released SWAT 25(OH)D to serum 25(OH)D concentrations. Drincic et al., [The majority of the subjects in our study had insufficient serum 25(OH)D concentrations according to the current IOM guidelines. This finding is consistent with other studies reporting an inverse relationship between measures of adiposity and serum 25(OH)D ,16,17,18 et al.,  recentlyi.e., renal disease). Therefore, we would not expect any perturbations in serum 1,25(OH)2D concentrations. Regulation of serum 1,25(OH)2D is tightly controlled and is generally not a good marker of vitamin D status [2D is thought to be produced by renal 1-alpha-hydroxylase enzymes, but many non-renal tissues also contain 1-alpha-hydroxylase that convert 25(OH)D to 1,25(OH)2D for local gene regulation [In vitro evidence suggests that 1,25(OH)2D may regulate adipogenesis and lipid metabolism [The subjects in our study were overweight and obese, but did not have metabolic disorders associated with excess adiposity such as type 2 diabetes, metabolic syndrome and other metabolic diseases D concentrations. The correlation between serum and SWAT 25(OH)D was similar at baseline and at post-intervention, suggesting an equilibrium between the two compartments. However, our results are based on alternative analytical methods for adipose tissue and serum 25(OH)D measurements which likely would affect the statistical interpretation. LC-MS/MS methods and the DiaSorin RIA generally produce consistent results in serum [2 and 25(OH)D3, while the LC-MS/MS adipose method was specific for only 25(OH)D3. Thus, we run the risk of under-reporting our SWAT 25(OH)D in comparison to serum concentrations. Serum concentrations of 25(OH)D2 are substantially lower than 25(OH)D3 [2 or consuming plant sources of vitamin D2. Based on the low 25(OH)D concentrations seen in our subjects, we would expect very little vitamin D2 consumption from supplements and, subsequently, very little 25(OH)D2 in adipose depots. Still, future studies should utilize analytical methods that can distinguish between both isoforms to accurately identify the relationship between the serum and adipose compartments.We found positive and statistically significant relationships between serum 25(OH)D and SWAT 25(OH)D concentrations at baseline and post-intervention, suggesting that the deposition of 25(OH)D in SWAT could be dependent on serum 25(OH)D concentrations. Serum and adipose cholecalciferol concentrations have been previously reported to be positively correlated , but to in serum ,25, but 25(OH)D3 ,27,28 un3 measurement and removal of this datum results in non-significant correlations. It is unclear whether this participant is truly an outlier or would have been in the range of observed data from a larger sample. It should be noted that that our sample size is consistent with other studies that have measured cholecalciferol in humans [It is important to recognize that the statistical significance of these correlations should be interpreted carefully due to the limited number of samples in this exploratory study. The statistical significance of each correlation appear to be leveraged by the participant with the greatest SWAT 25(OH)Dn humans ,29,30, hThe changes in SWAT concentrations of vitamin D metabolites during energy restriction have not been reported. Our subjects lost an average of 5 kg of total fat mass after twelve weeks of reducing their energy requirements by 500 kcal, but had no significant change in SWAT, or serum, 25(OH)D concentrations. In contrast, serum cholecalciferol concentrations were shown to increase in fasted rats after the termination of a 14-day large daily vitamin D supplementation, , suggestet al. hypothesized that a total of 114.5 nmol 25(OH)D was distributed in 24.5 kg of total fat in a hypothetical 70 kg woman [As discussed earlier, Heaney kg woman ,30 and tThe strengths of our study were the longitudinal collection of SWAT, and thus measurement of within subject changes. To our knowledge, the current study is the first to report 25(OH)D concentrations in SWAT pre- and post-weight loss in humans. Additionally, we analyzed samples across a broad range of body weight, adiposity, and serum 25(OH)D concentrations. By leveraging samples from a larger controlled feeding trial, it enabled us to accurately control and assess vitamin D intake, caloric restriction, and other parameters.3 measurements are likely attributed to seasonal differences. Also, serum analysis of 25(OH)D had previously been conducted for the parent study. Therefore, differences in methodological analyses could impact the accuracy of our measurements. Our subjects were considered overweight and obese yet generally healthy, so our findings may not apply to other overweight and obese individuals diagnosed with metabolic syndrome or other metabolic disturbances. Lastly, it is acknowledged that despite quite thorough biopsy washing steps, our samples may have minor contamination with blood, which could theoretically add variability to the outcome measurements and influence serum vs. SWAT correlations. Regardless, the current work provides a solid reference framework for understanding physiological concentrations of adipose 25(OH)D concentrations in humans.Due to the exploratory, proof-of-principle nature of this investigation and the cost associated with the analyses, we restricted our first study to a limited number of samples. Additionally, our subjects were derived from a larger study that did not control for factors that could introduce inter-subject variability in our analysis. The parent project introduced dairy products that are fortified with vitamin D, therefore subjects had variable intakes of vitamin D which could contribute to the variability in post-intervention measurements. We selected samples from winter and spring cohorts to minimize the possible effect of sun exposure, but inter-subject variability in serum and SWAT 25(OH)Di.e., ≥15% loss). Therefore, dietary intake and dermal biosynthesis should be paramount when devising strategies to improve serum 25(OH)D in obese individuals.In summary, we have demonstrated a LC-MS/MS method that can detect 25(OH)D in human adipose tissue collected from overweight and obese individuals enrolled in a clinical weight loss intervention. We found mean SWAT 25(OH)D concentrations of 5.8 ± 2.6 nmol/kg tissue and 6.2 ± 2.7 nmol/kg tissue pre- and post-intervention, respectively. Obese persons are thought to be at risk for vitamin D deficiency, which could contribute to the development or exacerbate other comorbidities associated with excessive adipose tissue. Although there are very little human data, the accumulation and release of vitamin D metabolites in excess adipose tissue is the mechanism often cited behind this relationship. Aside from the small 25(OH)D concentrations detected in SWAT, our results suggest that 25(OH)D is released from adipose tissue after a loss of 6% in body weight and a 13% reduction in total fat loss, but this release contributes little to serum 25(OH)D concentrations. Perhaps a greater loss of body weight or fat is needed to observe a significant contribution to serum 25(OH)D concentrations (


Mycobacterium bovis bacille Calmette–Guèrin (rBCG) expressing three T cell epitopes of Mycobacterium tuberculosis (MTB) Ag85B antigen  fused to the Mtb8.4 protein (rBCG018) or a combination of these antigens fused to B cell epitopes from ESAT-6, CFP-10 and MTP40 proteins (rBCG032) were used to immunize Balb/c mice. Total IgG responses were determined against Mtb8.4 antigen and ESAT-6 and CFP-10 B cell epitopes after immunization with rBCG032. Mice immunized with rBCG032 showed a significant increase in IgG1 and IgG2a antibodies against ESAT-6 and MTP40 (P1) B cell epitopes and IgG3 against both P1 and P2 B cell epitopes of MPT40. Splenocytes from mice immunized with rBCG018 proliferated against Ag85B P2 and P3 T cell epitopes and Mtb8.4 protein whereas those from mice-immunized with rBCG032 responded against all Ag85B epitopes and the ESAT-6 B cell epitope. CD4+ and CD8+ lymphocytes from mice immunized with rBCG018 produced primarily Th1 type cytokines in response to the T cell epitopes. Similar pattern of recognition against the T cell epitopes were obtained with rBCG032 with the additional recognition of ESAT-6, CFP-10 and one of the MTP40 B cell epitopes with the same pattern of cytokines. This study demonstrates that rBCG constructs expressing either T or T and B cell epitopes of MTB induced appropriate immunogenicity against MTB.Recombinant  Mycobacterium tuberculosis, M. bovis bacille Calmette–Guèrin (BCG), is the most widely used vaccine in preventing TB disease especially in childhood[Tuberculosis (TB) is one of the most prevalent diseases in developing countries. WHO estimates that 8.7 million new cases and approximately 1.6 million deaths occur annually. The cuhildhood,4. Howehildhood. Thus, It has been reported that there was only a slight decrease in the protective efficacy of BCG after 50 years. The inDespite the dominant dogma that the cellular immune mechanism is primarily responsible for protection against TB, recent evidences suggest a protective role of specific antibodies in the defense against intracellular microorganisms such as MTB-14. TheIn this study, T and B cell epitopes of MTB were cloned into BCG. The immunogenicity of the rBCG expressing three T cell epitopes of the Ag85B antigen of MTB fused to the entire sequence of MTB8.4 protein and the same construct fused to B cell epitopes of ESAT-6, CFP-10 and MTP-40 proteins of MTB were evaluated to determine its ability to induce specific humoral and cellular immune responses against these epitopes.The sequences of the MTB proteins were obtained from the UNIPROT database . Linear Two multiepitopic constructs were designed: one containing three T cell epitopes of Ag85B protein of MTB fused to the Mtb8.4 protein, cloned into the shuttle plasmid pNMN018 and another with the addition of the selected B cell epitopes upstream to the previous construct, contained in the shuttle plasmid pNMN032. Both constructs were generated using preferred mycobacterium codon usage.n = 5 per group) were immunized intraperitoneally (i.p.) with 2 x 106 CFU of either rBCG018 or rBCG032 in 200 μl PBS containing 0.1% Tween 80 (PBS-T80). A control group of mice were injected with 2 x 106 CFU of the parental BCG strain in 200 μl PBS-T80. After 30 and 60 days, the same amount of either rBCG or parental BCG was injected i.p. as boosters. Sera were collected before each injection from the tail vein and kept at -20 °C for IgG antibodies measurement. Subsequently, three weeks after the last immunization the mice were sacrificed and the spleens were asseptically removed to assess the cellular immune response.Balb/c mice  and the Mtb8.4 antigen was produced in p values less than 0.05 were considered statistically significant.Total IgG and IgG1, IgG2a, IgG2b and IgG3 (only against B cell epitopes) were measured from sera of immunized mice by ELISA as described elsewhere. The results were analysed using One-way ANOVA test and the differences between pairs were determined by the Multiple Range test. T and B cell epitopes or recombinant Mtb8.4 protein were added to the cultures at a final concentration of 10 μg/ml. RPMI medium was used as negative control and the T cell mitogen, concanavalin A (10 μg/ml) was used as positive control. Results were calculated as stimulation index (SI) (SI = cpm of stimulated culture/cpm of non-stimulated culture). SI of more than 2 was considered as positive.6 spleen cells/ml. The cells were then stained with 20 μl (20 μg /ml) anti-mouse IL-2, IL-4 or IFN-γ labelled with PE (BD Pharmingen). The increase in the percentage of intracellular cytokine levels in lymphocytes of rBCG-immunized mice was compared to those of BCG-immunized mice and scored as high, moderate, or weak/absent corresponding to ≥ 3 fold, ≥ 2 fold, < 2 fold increase respectively.10 μg/ml of each peptide corresponding to the T and B cell epitopes or recombinant Mtb8.4 protein were cultured with 2 × 10Significant increase in the level of total IgG against ESAT-6 and CFP-10 B cell epitopes was observed after the third immunization of mice with rBCG032 compared to other groups. Increased levels of specific IgG1 were detected against ESAT-6 (p < 0.05); IgG2a against ESAT-6 (p < 0.01) and MTP40 (P1) (p < 0.01); and IgG3 against MTP40 (P1) (p < 0.01) and MTP40 (P2) (p < 0.05) in mice immunized with rBCG032 compared to other groups  epitope (SI = 4.9); (P3) epitope (SI = 2.5) as well as with Mtb8.4 protein (SI = 3.2), whereas no response was observed against Ag85B (P1).Splenocytes from rBCG032-immunized mice showed strong proliferative response to all the Ag85B T cell epitopes  with SI of 3.9, 5.0 and 5.3 respectively. There was no stimulation with Mtb8.4 protein. A moderate proliferative response was detected against the ESAT-6 B cell epitope (SI = 2.4) while all of the other B cell epitopes were not stimulatory (Fig. Table We can conclude that some T cell epitopes may induce the production of Th2 instead of Th1 cytokines in Balb/c mice, and that the construction of a multi-epitope vaccine need to ensure that the overall potential “protection” that was intended is not compromised. In addition, it would be necessary to test the candidate vaccines in other strains of mice to ensure a more “universal” conclusion to be drawn.. The two vaccine candidates studied in the present work fulfilled this characteristic. In general, both rBCG018 and rBCG032 stimulated strong cellular response to the T cell epitopes and for the latter strong humoral immune response as well. Our findings suggest that co-expression of T and B cell epitopes of MTB in BCG stimulated both arms of the immune response although the protective capability of rBCG expressing both T and B cell epitopes needs further investigation.The ability to elicit a predominant Th1 immune response is a desirable characteristic for any vaccine against MTBThe authors declare that they have no competing financial interests.All authors have read and approved the final manuscript. RM and MA participated in recombinant strain production, immunogenicity studies, data analyses and in writing of the manuscript. DY and NA performed the bioinformatics studies. MES, AA and MNN conceived the study, the experimental design, bioinformatics studies, data analyses and in writing and finalizing of the manuscript.


Objective: To investigate the frequency and effects of vitamin D  deficiency in children with type 1 diabetes (T1D) in a region which is known to have a high rate of vitamin D deficiency among adolescents. Methods: In this prospective cross-sectional study, 120 children and adolescents with T1D (55 girls and 65 boys) aged 3-20 years were  evaluated. Serum 25-hydroxyvitamin D [25(OH)D], parathormone (PTH), and alkaline phosphatase (ALP) levels were measured. Hemoglobin A1c levels and daily insulin requirement were also evaluated.  Classification of vitamin D status was made according to the American Academy of Pediatrics (AAP)/LWEPS’s recommendations. The patients were divided into 2 groups according to their vitamin D status and also according to the season of the year in which 25(OH)D sampling was done.  Results: Serum 25(OH)D levels revealed vitamin D deficiency or  insufficiency in 38% of the patients. Higher PTH levels were found in the patient group whose mean 25(OH)D level was <20 ng/mL as compared to the group whose mean 25(OH)D level was >20 ng/mL (p<0.05). Only 11% of patients had secondary hyperparathyroidism. The 25(OH)D levels of patients whose serum samples were taken in summer  and spring months were significantly different (p<0.05). There were no significant correlationsbetween 25(OH)D level and daily insulin dose.   Conclusion: Although we could not show a significant association between vitamin D deficiency and metabolic parameters, the frequency of vitamin D deficiency in T1D children is substantial. Vitamin D status should be assessed also in patients who do not have signs of rickets.Conflict of interest:None declared. There is a growing interest in the non-skeletal effects of vitamin D in recent years . There aThe study group consisted of one hundred twenty patients with T1D who were being followed in our pediatric endocrinology outpatient clinic. The mean age of patients was 12.7±3.8 years (range: 2.9-20.1 years). Fifty four percent of the patients (n=65) were boys and 46% (n=55) were girls.  None of the patients were taking a vitamin D supplement. Serum 25-hydroxyvitamin D [25(OH)D], parathormone (PTH), and alkaline phosphatase (ALP) levels were measured. Hemoglobin A1c (HbA1c) levels (mean of the measurements of the preceding year) and daily insulin requirement of the patients were evaluated. The season in which 25(OH)D samples were taken was also recorded. The participants were divided into two groups according to their pubertal stage. Group 1 consisted of prepubertal children (n=29) and group 2 - of children who had reached puberty (n=91). Because vitamin D status is associated with sunlight exposure, a variable which changes with the season of the year, the participants were categorized according to 25(OH)D sampling season: winter (December 22-March 21), spring (March 22-June 21), summer (June 22-September 21), and fall (September 22-December 21). Vitamin D status was classified according to the American Academy of Pediatrics (AAP)/LWEPS’s recommendations on cut-off levels for states of vitamin D. A 25(OH)D level of <5 ng/mL (<12.5 nmol/L) was considered as severe deficiency, a level between 5 and 15 ng/mL (12.5-37.5 nmol/L) as deficiency, a level of 15-20 ng/mL (37.5-50 nmol/L) as insufficiency, and a level of 20-100 ng/mL (50-250 nmol/L) as normal (sufficient) .  Our paThe demographic and biochemical features of the patients are shown in In recent years, studies suggesting that vitamin D deficiency correlates with the severity and frequency of T1D and that vitamin D supplementation reduces the risk of developing T1D have been reported  17,18). On the other hand, although the number of studies on frequency of vitamin D deficiency in children and adolescents with T1D is limited, this frequency was found to be highly variable and may be as low as 15% or as high as 90.6% ,13,19,20. On the


Cyclin-dependent kinases are most extensively studied targets for cancer chemotherapy since the tumor cells exhibit false checkpoints and can proliferate even if the genome is compromised. Cyclin-dependent kinases ensure the tight regulation of the cell cycle execution by mediating phosphorylation of cellular proteins. Deregulation of the cyclin-dependent kinase 2 activity by cellular and external factors leads to many diseases like cancers. Different methods like radiolabeled, fluorescence and luminescence are available for screening of library of compounds against kinases. However, bioluminescent methods offer several advantages like low background and no effect of fluorescent compound interference. Present study is focused on development, optimization and validation of cyclin-dependent kinase 2 assay which is suitable for identification potent and selective, ATP competitive and non-competitive inhibitors of cyclin-dependent kinase 2. The aim of present investigation was to optimize the assay for cyclin-dependent kinase 2/cylin A and cyclin-dependent kinase 2/cyclin E with use of bioluminescence based biochemical reaction. Both cyclin-dependent kinase 2 which are cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E complexes, have different affinity for ATP. Therefore, both isoform analogs of cyclin-dependent kinase 2 were optimized separately. Optimum cyclin-dependent kinase 2/cyclin A and cyclin-dependent kinase 2/cyclin E concentration were found to be 250 ng/well and 200 ng/well, respectively. Optimum substrate (histone H1) concentration was found to be 2.5 mg/ml for both cyclin-dependent kinase 2 analogs. Optimum reaction time was found to be 20 min for both cyclin-dependent kinase 2/cyclin complexes. Protein kinases are the validated drug targets for cancer and extensive efforts to develop kinase inhibitor for variety of cancers has lead to more than 40 inhibitors in clinical trials2. CurrenImportance of the CDKs in cell cycle has driven interest in development of selective and potent inhibitors for overall blockade of cell cycle to achieve growth arrest. Small mThere are a number of assay technologies available for measuring kinase activity. Bioluminescent, fluorescent, and radiolabeled assays are examples of different types of technologies available for high-throughput applications, each offering their own advantages and disadvantages. BiolumiThe aim of present investigation was to optimize the assay for CDK2/cylin A and CDK2/cyclin E with use of bioluminescence-based biochemical reaction for quantification of residual ATP, hence extent of phosphorylation and inhibition of phosphorylation.3 (S6508), EGTA (E0936), β-glycerophosphate (G6376), MOPS (M3183), DMSO (472301), histone H1 (H4524) and ATP(A9062) were purchased from Sigma Aldrich, USA. Kinase Glo® Plus Kit (V3774) was provided by Promega, USA. All other chemicals and reagents used in the study were of Molecular biology grade and were used as procured. All reactions were performed in 96-well plates. Enzymes CDK2/cyclin A  and CDK2/cyclin E  were purchased from. DTT (D9779), NaVO3) was added to all wells to bring the contents to volume of 25 μl. Content was mixed using shaker and incubated for different time interval  for both CDK2/cyclin A and CDK2/cyclin E) at room temperature. To each well, 25 μl Kinase-Glo® plus reagent was added  at different time intervals . Content of plate was mixed and incubated at room temperature for 1 min to stabilize luminescence signal prior to measurement with luminescence plate reader   were prepared in duplicate across the plate, using fixed amount of ATP (100 μM) and histone H1 (10 mg/ml) running from well 10 to well 1 in decreasing concentration. Assay dilution buffer , was histone H1. Optimal kinase amount (250 ng/well CDK2/cyclin A and 125 ng/well CDK2/cyclin E) and reaction time (20 min for CDK2/cyclin A and CDK2/cyclin E) from the previous experiment were used to determine optimal substrate concentration. Two-fold serial dilutions of kinase substrate (histone H1) were made across the plate using 250 ng/well CDK2/cyclin A or 125 ng/well CDK2/cyclin E with 100 μM ATP. For a control, same titration was performed without CDK2. The contents of plate were mixed and incubated at room temperature for 20 min for CDK2/cyclin A and CDK2/cyclin E. To each well, 25 μl of Kinase-Glo® plus reagent was added. Luminescence was recorded after 1 min of incubation, for stabilization .seleciclib) ranging from 100 μM to 0.005 μM were made across the plate using 250 ng/well CDK2 /cyclin A or 125 ng/well CDK2/cyclin E. To each well 2.5 mg/ml histone H1 was added. Reaction was started by addition of 100 μM ATP. Contents were mixed and incubated for 20 min for both CDK2/cyclin A and CDK2/cyclin E. To each well, 25 μl of Kinase-Glo® plus reagent was added. The plate was mixed and incubated at room temperature 1min to stabilize the luminescent signal. Luminescence was recorded in a plate reader /Msignal–Mbackground..(1), where S/B= signal/background, SD= standard deviation, CV= co-efficient of variance and M= mean.For Z’ determination three plates were prepared with enzyme (250 ng/well CDK2 /cyclin A or 125 ng/well CDK2/cyclin E) and three plates were prepared without enzyme to obtain the average of signal and average of background. Following equation was used to calculate the Z’ of assay. Total 48 wells with enzyme (250 ng/well CDK2/cyclin A and 125 ng/well CDK2/cyclin E) and 48 wells without enzyme were prepared. Substrate histone H1 (2.5mg/ml) was added to each well. Five microlitres of assay dilution buffer was added to each well. Five microlitres of ATP (100 μM) was added to each well to initiate the kinase reaction. Plates were incubated according to the predetermined reaction times (20 min for CDK2/cyclin A and CDK2/cyclin E). Twenty microlitres of Kinase-Glo® Plus was added to each well. Luminescence was recorded with the plate reader figs.  and 7. F50 of CYC202 was observed for water and EDB as a diluent. Difference in IC50 of CYC202 was not negligible in case of CDK2/cyclin A optimized assay, however, difference was considerably higher for CDK2/cyclin E assay (Linear activity profile was observed for both CDK2 complexes, with RLUs ranging from 0 to 670312. CDK2/Cyclin E at a concentration of 125 ng/well gave about 33586 RLU. Moreover, the results of 125 ng/well and 250 ng/well concentration gave a good dynamic range of the RLUs for CDK2/cyclin E. While for CDK2/cyclin A, both enzyme concentration of 250 ng/well and 500 ng/well obtained good dynamic range of RLU. Data obtained from titration of both of the enzyme, it can be concluded that the optimum concentration of CDK2/cyclin A is 250 ng/well and optimum concentration of CDK2/cyclin E is 125 ng/well. Optimal reaction time was 20 min for both CDK2/cyclin A and CDK2/cyclin E. For both enzymes CDK2/cyclin A and CDK2/cyclin E optimal substrate concentration was 2.5 mg/ml histone H1. Difference in IC E assay .signal = 42392.7, SDbackground = 452.3, Msignal = 2651028 and Mbackground = 1836.1. Therefore, Z’ for CDK2/Cyclin A assay was found as 0.802007. As per the Eqn. 1, Z’ of assay was calculated and that was 0.802 which was considered good. For CDK2/cyclin E assay, SDsignal = 46551.09, SDbackground = 467, Msignal = 628198.3 and Mbackground = 1894.4. Therefore, Z’ for CDK2/cyclin A assay was found as 0.774 as per Eqn. 1. With Z’ factor higher than 0.5 both assays are suitable for screening of inhibitors at different ATP concentrations using the Kinase-Glo® Assay technology. This can help to determine selectivity and potency of CDK2 inhibitors.For CDK2/cyclin A assay, SD


Data on the burden of the 2009 influenza pandemic in Asia are limited. Influenza A(H1N1)pdm09 was first reported in Thailand in May 2009. We assessed incidence and epidemiology of influenza-associated hospitalizations during 2009–2010.We conducted active, population-based surveillance for hospitalized cases of acute lower respiratory infection (ALRI) in all 20 hospitals in two rural provinces. ALRI patients were sampled 1∶2 for participation in an etiology study in which nasopharyngeal swabs were collected for influenza virus testing by PCR.Of 7,207 patients tested, 902 (12.5%) were influenza-positive, including 190 (7.8%) of 2,436 children aged <5 years; 86% were influenza A virus (46% A(H1N1)pdm09, 30% H3N2, 6.5% H1N1, 3.5% not subtyped) and 13% were influenza B virus. Cases of influenza A(H1N1)pdm09 first peaked in August 2009 when 17% of tested patients were positive. Subsequent peaks during 2009 and 2010 represented a mix of influenza A(H1N1)pdm09, H3N2, and influenza B viruses. The estimated annual incidence of hospitalized influenza cases was 136 per 100,000, highest in ages <5 years  and >75 years . The incidence of influenza A(H1N1)pdm09 was 62 per 100,000 . Eleven influenza-infected patients required mechanical ventilation, and four patients died, all adults with influenza A(H1N1)pdm09 (1) or H3N2 (3).Influenza-associated hospitalization rates in Thailand during 2009–10 were substantial and exceeded rates described in western countries. Influenza A(H1N1)pdm09 predominated, but H3N2 also caused notable morbidity. Expanded influenza vaccination coverage could have considerable public health impact, especially in young children. Increasing evidence supports influenza viruses as an important cause of disease in tropical and subtropical regions, particularly in young children and older adults The first cases of influenza A(H1N1)pdm09 infection were detected in the United States in April 2009. The ensuing pandemic resulted in heightened influenza surveillance worldwide. However, few surveillance systems included established catchment populations, meaning that population-based estimates of influenza A(H1N1)pdm09 incidence are limited, especially in tropical and resource limited settings. Robust incidence data are needed to estimate the burden of the 2009 pandemic and are important to inform policy discussions about prevention and control strategies for influenza in general.The first cases of influenza A(H1N1)pdm09 were reported in Thailand in May 2009. Although influenza A(H1N1)pdm09 quickly became the predominant circulating influenza subtype, circulation of seasonal influenza viruses continued. We used ongoing active, population-based surveillance in two Thailand provinces nd with a per capita GDP of $1290 while Sa Kaeo ranked 51st with a per capita GDP of $2,303 We have conducted active, population-based surveillance for community-acquired pneumonia in all twenty hospitals in two Thailand provinces since 2002–2003 A case of acute lower respiratory infection (ALRI) was defined as evidence of both active infection  and lower respiratory tract disease  in a hospitalized patient. Every other ALRI case-patient was systematically sampled for possible participation in an etiology study. Sampling was done by ward  and chronologically based on admission time. Chest radiographs, if performed, were digitized and interpreted by a panel of radiologists using standard criteria as previously described All adult participants and guardians of children aged <18 years provided written consent for study enrollment. A CDC Institutional Review Board and the Ethical Review Committee of the Thailand Ministry of Public Health approved this study.Streptococcus pneumoniae (pneumococcus) by antigen detection using the Binax NOW® rapid immunochromatographic assay . Blood cultures were performed as clinically indicated Nasopharyngeal swab specimens were collected from enrolled patients using a polyester swab . In Nakhon Phanom, nasopharyngeal specimens were collected using flocked swabs  starting in July 2010. Specimens were collected at the time of enrollment and were stored at 4–8°C for <24 hours before being frozen at −70°C. Specimens were transported weekly on dry ice to the Thailand National Institute of Health. All specimens were tested for influenza A and B viruses by real-time reverse transcription polymerase chain reaction (rRT-PCR) As part of the response to the 2009 influenza pandemic, additional information on underlying conditions, influenza vaccination status, and antiviral treatment was collected using a standard questionnaire on a non-systematic sample of study patients from October 2009 through August 2010.The study period included January 2009 through December 2010. Clinical and demographic characteristics were compared among patients infected with influenza A virus subtypes and those infected with influenza B virus. Dichotomous variables were compared by chi-square or Fisher’s exact test and continuous variables by t-test or Kruskal-Wallis. The crude age-specific incidence of influenza-associated ALRI hospitalizations was calculated for the 2-year study period by dividing the number of rRT-PCR-confirmed influenza cases by the combined population of the two provinces During the study period, 28,076 patients were hospitalized with ALRI . Of thosOf the 7,207 enrolled patients, 902 (12.5%) tested positive for an influenza virus, including 190 (7.8%) of 2,436 children aged <5 years. Compared to influenza-negative patients, influenza-positive patients were less likely to be age <5 years (21% vs. 36%) and less likely to be ≥65 years (14% vs. 23%) (p<0.01). After adjusting for age, influenza-positive patients were more likely than influenza-negative patients to have documented fever >38.0°C , but less likely to die , require intubation , need oxygen therapy , or to have chest radiographs consistent with pneumonia . Nasopharyngeal specimens were collected from influenza-positive patients a mean of 0.86 days after admission compared to 1.04 days for influenza-negative patients (p<0.01).Among 902 influenza cases, 779 (86%) were influenza A, 120 (13%) were influenza B, and three were positive for both influenza A (one each of influenza A(H1N1)pdm09, H1N1, and H3N2) and B. Among influenza A cases, 417 (54%) were influenza A(H1N1)pdm09, 270 (35%) were H3N2, one was A(H1N1)pdm09 and H3N2, 59 (7.6%) were H1N1, and 32 (4.1%) were not subtyped.Patients infected with influenza A(H1N1)pdm09 virus were similar in age to those with H1N1 and influenza B but younger than those with H3N2 . The preStaphylococcus aureus in a 49 year-old female with influenza A(H1N1)pdm09). In comparison, 2336 influenza-negative patients had blood cultured; 146 had a positive blood culture, including seven positive for S. aureus and 12 positive for S. pneumoniae. Among those with a blood culture, 25% had received antibiotic treatment before culture collection, which is consistent with previous descriptions from these sites Co-infection with RSV occurred in 30 (7.2%) patients with influenza A(H1N1)pdm09, 29 (11%) with H3N2, and 8 (6.7%) with influenza B virus . Of 68 influenza-RSV co-infections, 56% occurred among children aged <5 years with little variation by influenza subtype. No patients co-infected with influenza and RSV died or required mechanical ventilation, and mean hospital stay did not differ significantly between influenza-RSV-co-infected and influenza-only-infected patients . Among 445 influenza-infected patients aged >17 years tested for pneumococcus by urine antigen assay, 12 (2.7%) were positive compared to 140 (4.4%) of influenza-negative patients. One (25%) of four fatalities in influenza-infected patients was co-infected with pneumococcus compared to 11 (2.5%) of 440 non-fatalities (Fisher’s exact p = 0.10). Patients co-infected with pneumococcus were more likely to require oxygen therapy than those who tested negative for pneumococcus  but were not more likely to need mechanical ventilation; co-infected patients had longer hospital stays , though not significantly so. Three (0.34%) influenza-infected patients tested positive for adenovirus compared to 145 (2.3%) influenza-negative patients. Blood cultures were performed in 282 (31%) of influenza-infected patients, and only one was positive for a potential pathogen . Adjusted rates were highest in children aged <5 years at 477 per 100,000 person-years  and adults aged ≥75 years at 407 per 100,000 person-years  . Rates aTo account for the fact that non-enrolled ALRI patients were more likely to have severe disease  than enrolled patients and that a lower proportion of severely ill patients tested positive for influenza viruses, we re-calculated the adjusted incidence using influenza-positive percentages specific to severe vs. non-severe disease. The adjusted incidence accounting for severity was 135 per 100,000 person-years, almost identical to the incidence not accounting for severity .From October 2009 through August 2010, supplemental data were collected for 475 (14%) study patients, including 31% (82/261) of patients who tested positive for an influenza virus compared to 13% (393/3126) of those who tested negative (p<0.01). Among these 475 patients, 443 (93%) received antiviral treatment ; 50% received treatment within 2 days of symptom onset and 97% within 2 days of admission. Influenza-negative patients were as likely to receive therapy as those who were influenza-positive . Influenza vaccination was documented for 32 (7.2%) patients, but only one patient was vaccinated after the monovalent influenza A(H1N1)pdm09 vaccine was available. Co-morbid conditions were identified among 23% of influenza-positive versus 38% of influenza-negative patients (p = 0.01); asthma was most common (16% of influenza-positive).We documented the substantial incidence and the seasonality of influenza in rural Thailand for a 2-year period that included the 2009 pandemic using ongoing active, population-based surveillance for hospitalized cases of influenza-associated ALRI. Influenza incidence was largely attributable to influenza A(H1N1)pdm09 virus but with important contributions from influenza A(H3N2), and from influenza B. Our results add to the limited but growing literature on the high burden of influenza in tropical settings The incidence of hospitalized influenza cases overall was highest in children aged <5 years and adults 75 years and older , which is typical of rates documented previously in Thailand Few data on influenza incidence during the pandemic are available from tropical regions. A preliminary report from Guatemala found influenza A(H1N1)pdm09 hospitalization rates of 7.1–8.8 per 100,000 persons during 8 months when circulation was peaking The co-circulation of seasonal and pandemic influenza viruses during the 2-year period allowed us to compare characteristics of patients infected with different subtypes during the same season. Patients infected with H3N2 virus were significantly older than those infected with influenza A(H1N1)pdm09, H1N1, or influenza B, although the proportion of cases aged <5 years was not significantly different. We found no difference in clinical severity in patients infected with influenza A(H1N1)pdm09 and other influenza viruses. A comparison of patients with pandemic and other influenza viruses in Guatemala found similar characteristics, although patients in the Guatemala study had more severe disease overall Our findings likely underestimate the rate of severe influenza cases and deaths by 2–3 times, due to challenges enrolling patients with severe illness. ALRI surveillance in these provinces identified 32 deaths among 9,778 children during 2009–10, but only six were enrolled in our study and tested for influenza viruses. An early report of the first 272 hospitalized influenza A(H1N1)pdm09 cases in the U.S. found that 25% required intensive care, 15% needed mechanical ventilation, and 7% died The low case fatality proportion in our sites should be considered in light of the availability and use of oseltamivir in Thailand, where national guidelines during the pandemic recommended treatment of influenza-infected patients with complicated illness , failure to improve within 48 hours, and high-risk conditions . Data on a subset of our patients showed that >90% of those hospitalized with ALRI received oseltamivir, 50% within two days of symptom onset. Oseltamivir access was likely improved by domestic production of a generic formulation and by prepositioning the drug at government hospitals. Further, hospital access in Thailand is very good, allowing patients to be treated before illness progression. Our lower case fatality proportion may also have been related to the low prevalence of co-morbid conditions (23%) compared to other studies Similar to trends seen in the U.S. and other parts of the world, Thailand had a rapid disappearance of the H1N1 virus and relatively lower infection rates from influenza A(H1N1)pdm09 virus in older adults compared to previous influenza seasons. However, in contrast to the U.S. and most temperate regions where influenza A(H1N1)pdm09 accounted for >99% of influenza infections Few studies have systematically assessed the prevalence of other pathogens among influenza-infected patients by means other than blood culture Strengths of our analysis include systematic sampling from active surveillance for ALRI, lack of reliance on clinical reporting or testing, and a known catchment area. However, the findings must be interpreted in light of several limitations. We lacked data on some of the known or potential risk factors for severe influenza infection, including pregnancy and obesity. Our incidence estimates relied on statistical adjustments that assumed enrolled and non-enrolled patients had identical prevalences of influenza positivity. Patients admitted on nights and weekends were under-represented in our sample and may have differed from patients admitted on weekdays in terms of demographics, disease severity, and influenza prevalence. Finally, data on co-infections with bacteria other than pneumococcus relied on blood culture as the single diagnostic modality, and <30% of patients had cultures, 25% of whom were pre-treated with antibiotics.Using active, population-based surveillance, we found a high incidence of hospitalized influenza ALRI infections in Thailand, exceeding estimates from most other nations and confirming influenza’s substantial contribution to severe respiratory illness, including pneumonia, in Thailand and likely in Southeast Asia. The high disease burden in young children is particularly noteworthy and underscores the role that influenza vaccination should play in strategies to prevent childhood pneumonia globally


Histone modifications play an important role in chromatin organization and gene regulation, and their interpretation is referred to as epigenetic control. The methylation levels of several lysine residues in histone tails are tightly controlled, and JmjC domain-containing proteins are one class of broadly expressed enzymes catalyzing methyl group removal. However, several JmjC proteins remain uncharacterized, gaps persist in understanding substrate recognition, and the integration of JmjC proteins into signaling pathways is just emerging. The KDM3 subfamily is an evolutionarily conserved group of histone demethylase proteins, thought to share lysine substrate specificity. Here we use a systematic approach to compare KDM3 subfamily members. We show that full-length KDM3A and KDM3B are H3K9me1/2 histone demethylases whereas we fail to observe histone demethylase activity for JMJD1C using immunocytochemical and biochemical approaches. Structure-function analyses revealed the importance of a single amino acid in KDM3A implicated in the catalytic activity towards H3K9me1/2 that is not conserved in JMJD1C. Moreover, we use quantitative proteomic analyses to identify subsets of the interactomes of the 3 proteins. Specific interactor candidates were identified for each of the three KDM3 subfamily members. Importantly, we find that SCAI, a known transcriptional repressor, interacts specifically with KDM3B. Taken together, we identify substantial differences in the biology of KDM3 histone demethylases, namely enzymatic activity and protein-protein interactions. Such comparative approaches pave the way to a better understanding of histone demethylase specificity and protein function at a systems level and are instrumental in identifying the more subtle differences between closely related proteins. Histones are the main building block of nucleosomes that structure DNA in the nucleus and regulate local accessibility to DNA Within euchromatin, the specific status of post-translationally modified histone tails orchestrates gene regulation by rendering a locus transcriptionally active or repressed Many cell types express a plethora of different JmjC domain containing proteins, and several of these proteins actually catalyze methyl group removal on the same lysine residues. However, the system is not overly redundant as individual demethylases are recruited to specific locations in the genome, affecting only a certain set of target genes. It is becoming clear that JmjC proteins are recruited to many genomic loci but their influence on specific gene expression levels is often relatively minor; indeed, they more likely act by fine-tuning gene expression Arabidopsis thalianaC. elegans lacks a KDM3 homologue, Drosophila melanogaster has a single KDM3 homologue, CG8165; its loss of function phenotype is not known but there is some evidence that it genetically interacts with Notch signaling To address the functional specificity of different JmjC proteins, we decided to compare a whole subfamily of HDM's in the same cellular environment. To do so, we choose the KDM3  proteins KDM3A, KDM3B and JMJD1C. As compared to other HDM subfamilies, where many members are characterized, relatively little is known about the KDM3 members Here we compare and contrast enzymatic activities and cellular interaction partner candidates of the three human KDM3 subfamily members in a common cellular environment. We show that wild-type KDM3A and KDM3B are H3K9me1/2 demethylases, report absence of enzymatic activity of JMJD1C and establish Suppressor of cancer cell invasion (SCAI) as a novel interaction partner of KDM3B.We set out to identify the specificity of the three KDM3 subfamily members towards histone lysine residues. KDM3A was among the first JmjC domain-containing enzymes described with H3K9me1 and -me2 specificity JmjC containing proteins function in an iron and α-ketoglutarate dependent mechanism It has previously been suggested that a short version of mouse Jmjd1c is an active H3K9me1/2 demethylase enzyme per se, it could still be important for KDM3B targeting or be involved in signaling. We did not identify additional phosphorylation sites which are conserved in KDM3A and KDM3B but not in JMJD1C and which could explain the loss of enzymatic activity of the latter.Second, there was a recent report indicating that another JmjC-containing enzyme, PHF2, is only active upon phosphorylation by PKA Third, we generated hybrid constructs in which we exchanged the JmjC domains among the three KDM3 proteins K9me1/2/3 peptide substrates, despite significant attempts at reaction buffer optimization  in insect and mammalian cells, truncated JMJD1C(1696–2540) in insect cells, and the JmjC domain of JMJD1C(2131–2540) in mization . In addimization . We alsoinactive .Taken together, we report here that KDM3A and KDM3B are active H3K9me1/2 histone demethylases, whereas we found no evidence for enzymatic activity of JMJD1C towards H3K9me1/2/3.JmjC domain proteins generally demethylate two of the possible three methylation states on a particular lysine residue. However, it is not well understood how substrate recognition and specificity between the different methylation states is achieved. In several cases, though, it has been shown that the JmjC domain alone is not sufficient to catalyze the demethylation reaction The JmjC domain swap experiments  suggesteMulti-protein complexes are involved in the precise modulation of gene expression, and several HDM's have been shown to be integral members of such complexes in certain cell types. Apart from interactions with nuclear hormone receptors KDM3A, KDM3B and JMJD1C, respectively. Despite significant efforts, we did not identify si- or shRNA reagents that reduced JMJD1C mRNA levels below 25% of control levels (data not shown). We then tested by qRT-PCR if knockdown of individual subfamily members affected the expression of the other subfamily members. We found this not to be the case, suggesting that the three genes do not influence each others expression , each, in HEK293T cells. As controls, the same amount of empty plasmid containing IRES-BirA was transfected in parallel into HEK293T cells. 72 hours following transfection, cell lysates were prepared, and protein complexes were immunoprecipitated using streptavidin-coupled beads. Following SDS gel electrophoresis, proteins were visualized using coomassie staining . We thenAs expected, KDM3A, KDM3B and JMJD1C were among the most enriched proteins in each purification, respectively. For this analysis, we defined interactor candidates as proteins enriched on KDM3A or KDM3B by at least one standard deviation compared to the negative control, each, in two independent quantitative AP-MS experiments, respectively. Comparing the resulting interactomes with each other, we identified only a couple of common interaction candidates among KDM3 subfamily members (Table S1). Interestingly, we retrieved several interaction partner candidates specific for a particular KDM3 subfamily member. For example, the procollagen-lysine dioxygenases PLOD1, PLOD2 and PLOD3 were specifically retrieved on KDM3B. On the other hand, the suppressor of G2 allele of SKP1 homolog (SUGT1) was specifically retrieved on KDM3A. Most notably, SCAI was another protein which co-purified with KDM3B. SCAI was identified by multiple peptides covering more than 50% of the whole protein sequence . We chosBoth cell-based and biochemical approaches failed to detect enzymatic activity of JMJD1C  and 2. TArabidopsis thaliana has 6 members of the KDM3 subfamily where two have lost conserved iron- and α-KG-binding amino acids While our data suggest that JMJD1C does not act directly as a H3K9 HDM, it nevertheless might be involved in regulating transcription and/or other cellular processes. Firstly, JMJD1C could, unexpectedly, act on a different lysine residue than H3K9. While we tested if JMJD1C demethylates other commonly methylated histone lysine residues, including H3K4, H2K27 and H3K36, there remain additional residues that are poorly characterized or where methyl-specific antibodies are not currently available. Secondly, JMJD1C might require an additional co-factor(s) that, if not co-expressed, cannot generate HDM activity, as judged by global assessment of H3K9 demethylation. For example, PHF2 has been reported to lack enzymatic activity upon overexpression unless PKA is artificially activated and in turn phosphorylates PHF2 Structural studies have started to unravel the catalytic mechanism and the substrate specificity of certain JmjC proteins In general, chromatin modifying enzymes act in large protein complexes bound to chromatin to regulate transcriptional events. Individual protein complex members execute distinct functions as part of the whole chromatin modifying protein complexes. Until now, very few protein interaction partners of KDM3 subfamily proteins have been identified. JMJD1C was initially identified using yeast two-hybrid screens as a thyroid hormone receptor-interacting protein TRIP8 Another unresolved question is how the KDM3 subfamily members are recruited to chromatin. For example, we identified certain ARID proteins known to bind AT rich DNA sequences vice versa. SCAI is a highly conserved protein ranging from mammals to D. melanogaster and plants. In mammals SCAI acts as a transcriptional repressor in the RhoA-Dia1 signal transduction pathway, where it has been shown to regulate cell invasiveness through upregulation of β-integrin Importantly, we have identified SCAI as a specific interactor of KDM3B . In indeHere, we started to unravel the complex cellular functions and specific interaction partners of the KDM3 subfamily of HDM's. We showed that KDM3A and KDM3B harbor H3K9me1/2 HDM activities, while JMJD1C did not. Indeed, while we were finishing this study, a manuscript has been published describing a short version of KDM3B as a H3K9 me1/2 HDM HEK293T cells (ATCC CRL-11268) were cultured in DMEM GlutaMAX (GIBCO) containing 10% FBS (GIBCO). U-2 OS cells were cultured in DMEM/F12 (GIBCO) containing 10% FBS.Individual KDM3 constructs were cloned into a N- or C-terminal Avi-tag expression vector containing an IRES-BirA using the Gateway cloning system (Invitrogen). The sequences cloned correspond to the coding regions of NM_018433.5 for KDM3A (Full-length KDM3A and JMJD1C cDNAs in pENTR221 were Gateway-cloned into pDEST10 and pDEST26 (Invitrogen). Truncated KDM3A(aa511-1321), KDM3B(aa879-1761) and JMJD1C(aa1696-2540) were cloned into pFastBacHT_B vector (Invitrogen). Baculoviruses were generated using the Bac-to-Bac method from pDEST10 or pFastBac plasmids. For mammalian expression systems, HEK293-freestyle cells (Invitrogen) were used for transient expression of full-length JMJD1C proteins. Cell pellets containing recombinant proteins were lysed and cleared before loading onto affinity columns, purifications were achieved using His- or Flag-tag purifications followed by a desalting step prior to buffer exchange. The final buffer for protein was 25 mM Tris pH 7.5, 150 mM NaCl, 1 mM TCEP and 10% glycerol.Methylated H3K9me1, H3K9me2, H3K9me3 peptides were purchased from AnaSpec. The assay buffer contained 1 µM methylated peptide, 10–100 nM of the respective KDM3 enzyme, 20 mM HEPES pH pH 7.5, 1 mM α -ketoglutarate, 2 mM ascorbic acid, 40 µM FeSO4, 3 mM MgCl, 0.1% BSA and 0.01% Tween. Reactions were quenched with an equal volume of 20% acetic acid at different time-points between 0–120 minutes. LC-MS was used to follow both the depletion of substrate and generation of product.Sub-confluent cells were split 1:10 into poly-L-Lysine (Cultrex)-coated 96-well plates. On the next day, cells were transfected with 0.2 µg of the corresponding DNA using Lipofectamine 2000 (Invitrogen), according to the manufacturer's protocol. For Avi-tagged constructs, cells were treated with 225 nM biotin (Sigma). 24 hours later, cells were washed with PBS and fixed with 4% formaldehyde in PBS for 10 minutes. Cells were washed twice with PBS, then permeabilized and blocked for 1 hour with 0.2% triton X-100, 10% FBS in PBS. Cells were then incubated with the respective primary antibodies in 0.1% triton X-100, 5% FBS in PBS for 2 hours. Secondary Cy3-linked α-mouse and α-rabbit antibodies  were used at 1:750 dilutions during a 2 hour incubation. Streptavidin-coupled to AlexaFluor-488  identified cells containing the Avi-tag expression constructs. After one PBS wash, cells were incubated for 10 minutes with DAPI (PromoKine) before they were washed again 2 times with PBS. The following primary antibodies were used: H3K9me1: Abcam ab9045; H3K9me2: Abcam ab1220; H3K9me3: Cell Signaling Technology 9754S. Images were taken on an Olympus microscope and processed using ImageJ .2. In the morning of the following day, the transfection media was replaced with fresh media containing 225 nM biotin, and cells were incubated in 5% CO2 for another 48 hours. Cells were then washed twice with ice-cold PBS and scraped off before being snap-frozen in liquid nitrogen. Cells were incubated in lysis buffer  and 0.2 mg/ml DNAseI (Sigma) for 30 minutes at 4°C. Lysates were first cleared by centrifugation and then incubated with high capacity streptavidin agarose (Thermo Scientific) for 2 hours. Beads were washed in lysis buffer without DNAse and eluted by boiling for 10 minutes in 2X LDS loading buffer (Invitrogen) supplemented with β-Mercaptoethanol. Appropriate amounts of eluates were then loaded onto 4–12% NuPage Gels (Invitrogen), and gels were stained with commassie brilliant blue G (Sigma). Lanes were cut into 16 consecutive pieces, proteins in each gel band trypsinized and labeled with the iTRAQ reagent. Corresponding samples from lanes of control and KDM3 purifications were then pooled. Tryptic peptides were separated by online nano-high pressure liquid chromatography  on a C18 reversed phase column , using an acetonitrile/water system at a flow rate of 200 nl/min, prior to analysis on an LTQ Orbitrap Velos analyzer . Tandem mass spectra were acquired in a data-dependent manner. Typically, 10 MS/MS measurements were performed after each high accuracy spectral acquisition range survey, and both HCD and CID tandem spectra were acquired. RAW MS files were converted to peak lists using Mascot Distiller (version 2.4.0.0), with spectrum merging enabled. The human portion (taxonomy ID: 9606) of the IPI data base version 3.87 (91′491 sequences of which 810 are common contaminants) was interrogated using the Mascot search algorithm Individual KDM3 subfamily members were overexpressed in HEK293T cells using an adapted version of the calcium phosphate method HEK293T cells were cotransfected with Avi-tagged KDM3A or B and V5-tagged SCAI using the calcium phosphate method described above. Cells were treated and lysed as described for AP-MS experiments and split for incubation with either Streptavidin- or V5-agarose beads (Sigma). Co-immunoprecipitation reactions were eluted in 2X LDS loading buffer (Invitrogen) and subjected to standard SDS-PAGE and subsequent Western Blot analyses. Immunodetection reagents used were α-V5 (Invitrogen) in conjunction with α-mouse-HRP  to detect V5-SCAI, and Streptavidin-HRP (Pierce) to detect Avi-KDM3A or B. Protein bands were visualized using ECL+ .Figure S1Amino acid alignment of KDM3 subfamily members.(TIF)Click here for additional data file.Figure S2Analysis of additional methyl marks upon overexpression of JMJD1C.(TIF)Click here for additional data file.Figure S3Enzymatic activity of full-length KDM3 subfamily members towards H3K9 methylation in HEK293T, HeLa, TM3 and NIH3T3 cell lines.(TIF)Click here for additional data file.Figure S4Enzymatic activity of mJmjd1c, as well as KDM3A and hJMJD1C deletion constructs towards H3K9 methylation.(TIF)Click here for additional data file.Figure S5Avi-KDM3A, -KDM3B and –JMJD1C levels, including certain deletion constructs, upon overexpression in HEK293T cells.(TIF)Click here for additional data file.Figure S6Sub-cellular localization of JMJD1C deletion constructs.(TIF)Click here for additional data file.Figure S7Lack of enzymatic activity of JMJD1C overexpression upon treatment with kinase activators forskolin and PMA.(TIF)Click here for additional data file.Figure S8Detection of phosphorylation events in KDM3 subfamily members.(TIF)Click here for additional data file.Figure S9Enzymatic activity of mutated KDM3 subfamily members towards methylated H3K9.(TIF)Click here for additional data file.Figure S10Lack of enzymatic activity of additional JMJD1C constructs in the biochemical assay.(TIF)Click here for additional data file.Figure S11No effect on KDM3 subfamily member gene expression upon reciprocal subfamily member gene knockdown.(TIF)Click here for additional data file.Table S1Protein interaction candidates of KDM3 subfamily members as identified using quantitative AP-MS.(XLSX)Click here for additional data file.


Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition. SPARC, RGEIII, S100A10, and collagen type I Gastric cancer is one of the most common malignancies in Japan and other Asian countries. The patient prognosis of scirrhous gastric carcinoma is particularly poor. Scirrhous gastric carcinoma is often accompanied by peritoneal dissemination and metastasis to the lymph nodes and liver, which are serious problems that have to be controlled. Gene expression profile revealed gene amplifications of K-sam and c-Met in 30–40% of scirrhous gastric cancers, and that the overexpression of various growth factors, such as transforming growth factor-β (TGF-β), platelet-derived growth factor (PDGF), insulin-like growth factor (IGF) and fibroblast growth factor-2 (FGF-2) et al.Animal models that reflect the characteristics of cancerous peritonitis and metastasis to the peritoneum are important in understanding the development of malignant progression by gastric cancer. Yanagihara In our present study, we used these highly metastatic cell lines to investigate how human gastric cancer cells could acquire metastatic potential. We first compared gene expression profiles between highly metastatic and low metastatic parental cell lines by microarray analysis. We focused on one gene named N-myc downstream regulated gene 1 (NDRG1) because it was found to be markedly upregulated in the highly metastatic gastric cancer cell lines compared to their counterpart cells. It was also previously reported that NDRG1 expression was a predicative marker for malignant progression and poor prognosis in gastric patients Consistent with previous studies CDH1), P-cadherin (CDH3) and β-catenin (CTNNB1), was downregulated. By contrast, only MMP1 (MMP1) expression was markedly upregulated; the expression of cathepsin L (CTSL1) was increased, but that of cathepsin B (CTSB) was not. The expression of mesenchyme-specific genes, vimentin (VIM) and Snail (SNAI1), a key transcription factor for the suppression of E-cadherin expression, was upregulated.We next compared gene expression profiles between 58As1 cells and their parental counterpart, HSC-58, using DNA microarray analysis. Of the 41,000 RNA transcripts and variants, we identified 3691 differentially expressed genes that were upregulated to 2-fold, and 3536 genes that were downregulated to 0.5-fold or less in 58As1 cells compared to HSC-58 cells. We further compared the protein expression of several genes in HSC-58, 58As1 and 58As9 cells . The expImmunocytochemical analysis revealed that localization of E-cadherin both in cytoplasm and membrane, and β-catenin was mostly localized in cytoplasm in both HSC-58 and 58As9 cells . Immunoc*p<0.01)  .CDH1) and P-cadherin (CDH3) but decreased expression of the vimentin (VIM) and Snail (SNAI1). We next performed western blot and qRT-RCR analyses for several molecules that were modulated by NDRG1 knockdown in the microarray analysis  enhancement of E-cadherin promoter activity by NDRG1 knockdown.Of various transcription factors that suppress expression of E-cadherin including Snail, Slug, Twist, TCF4 and SIP1 GSK-3β is a key enzyme for activation of EMT pathway Comparing tumor growth rates of As1/Mock3, As1/Sic50 and As1/Sic54 cells in a xenograft model revealed slower tumor growth rates of both As1/Sic50 and As1/Sic54 by NDRG1 knockdown . NDRG1 kp = 0.21) (p<0.01) increase in the survival rate of mice inoculated with As1/Sic50 cells (51±16 days) compared with 35±14 days for mice inoculated with As1/Mock3 cells, indicating that NDRG1 knockdown prolongs survival  . However = 0.21) . The acc = 0.21) . Furthersurvival .We have previously reported that NDRG1 is closely correlated with tumor angiogenesis and poor survival in patients with gastric cancer, suggesting that NDRG1 is a predictive biomarker for malignant progression of gastric cancer KAI1 gene, is involved in NDRG1 mediated metastasis suppression of prostate cancer through ATF3-NF-κB pathway In the present study, NDRG1 knockdown resulted in the suppression of metastasis by highly metastatic gastric cancer cells , 7, suggin vitro and in vivo. NDRG1 may play a key role in the switch from a polarized epithelial phenotype to a highly motile mesenchymal phenotype. Partial or complete loss of E-cadherin is often observed during the progression toward malignancy in numerous tumors, including gastric cancer EMT is a recent highlight that could be closely associated with cancer malignant progression including acquirement of highly metastatic potential Wnt signaling has pivotal roles in the malignant progression and metastasis by lung and breast cancer cells On the other hand, GSK-3β is also known to play a role in the control of EMT Snail is a representative transcription factor that controls expression of E-cadherin In highly metastatic gastric cancer cell lines, expression of Snail was upregulated as compared to their low metastatic counterpart, HSC-58 . Both NDIn conclusion, NDRG1 knockdown induced the upregulation of E-cadherin and downregulation of vimentin and Snail, and suppressed the invasion, metastasis and accumulation of ascites by the highly metastatic gastric cancer cells. This NDRG1 mediated regulation of E-cadherin and/or vimentin expression affected epithelial mesenchymal transition of gastric cancer cells. This NDRG1 induced modification of EMT seems to be at least in part attributable to the transcriptional factor Snail. NDRG1, in its close context with EMT-related genes, might participate in the acquisition of a high metastatic potential by progressive gastric cancer cells.HSC-58 was established from human scirrhous gastric carcinomas and 58As1 and 58As9 were independently established by repeated orthotopic implantation of HSC-58 cells into the gastric wall of athymic mice as described previously Complementary RNA was amplified, labeled, and hybridized to a 44K Agilent 60-mer oligomicroarray according to the manufacturer’s instructions (Agilent Technologies). All hybridized microarray slides were scanned by an Agilent scanner. Relative hybridization intensities and background hybridization values were calculated using Agilent Feature Extraction Software. To identify up or down-regulated genes, we calculated ratios  from the normalized signal intensities of each probe for comparison between control and experimental samples. We then established criteria for regulated genes: up-regulated genes, ratio ≥2-fold; down-regulated genes, ratio ≤0.5.HindIII and BamHI cloning sites) was amplified from human genomic DNA with the primer pair 5′-AGATCTGAATTCCCCAGTGGAAAGACGCGCAGGC and 5′-AGATCTAAGCTTCTCGAGGATCCCGCGTCCTTTCCACAAGATATATAAACCCAAG, and ligated into the BglII site of pcDNA3. The partial DNA fragments of human NDRG1 DNA were chemically synthesized and cloned into pcDNA3-hU6siRNA cleaved with HindIII and BamHI to produce pcDNA3shNDRG1. The synthesized DNAs for pcDNA3shNDRG1 were: 5′-GATCCGCGTGAACCCTTGTGCGGAATTCAAGAGATTCCGCACAAGGGTTCACGTTTTTTGGAAA and: 5′-AGCTTTTCCAAAAAACGTGAACCCTTGTGCGGAATCTCTTGAATTCCGCACAAGGGTTCACGCG. The snail cDNA was prepared as described previously To obtain human a U6 siRNA vector based on pcDNA3 , the human U6 promoter , respectively, siRNA duplexes were tranfected using Lipofectamine RNAiMAX and Opti-MEM medium (Invitrogen) according to the manufacture’s recommendation.AGATCTTAGTGAGCCACCGGCGGGGC-3′5′-  and AAGCTTGGCCGGGGACGCCGAGCGAGGG-3′5′- . Underlines indicate restriction enzyme cleavage sites. The amplified fragment was ligated into the pGEM-T easy vector (Promega) and transferred to the pGL3-basic vector (Promega) in BglII and HindIII sites. E-cadherin-luc and pcDNA3-Snail were transfected using Lipofectamine LTX and Opti-MEM medium (Invitrogen) according to the manufacture’s recommendation. After 24 hr, the luciferase activity was measured according to the manufacturer’s instructions (Promega). Furthermore, we also examined luciferase activity driven by β-catenin using TopFlash reporter vector as described previously To obtain E-cadherin-Luc vector, E-cadhein promoter (−262 to +120) was amplified by PCR using the following primer pairs: 3 cells were plated in 1 ml of culture medium containing 0.36% (w/v) top agar layered over a basal layer of 0.72% (w/v) agar in 6-well plates and allowed to grow for 3–4 weeks. Colonies were photographed and counted in ten random fields of view at 50X magnification using light microscopy. Each experiment was done in triplicate.4×10Cells were lysed in buffer containing 50 mM Tris-HCl, 350 mM NaCl, 0.1% NP40, 5 mM EDTA, 50 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 1 mM Na3VO4. Total cell lysates were subjected to SDS-PAGE and blotted onto Immobilon membranes  as described previously Total RNA was isolated from cell culture using ISOGEN reagent  according to the manufacture’s instructions, as described previously ad libitum. Animals were observed for signs of tumor growth, activity, feeding, and pain in accordance with the guidelines of the Harvard Medical Area Standing Committee on Animals.All animal experiments were approved by the Ethics of Animal Experiments Committee at Kyushu University Graduate School of Medical Sciences. All mice were purchased from Charles River Laboratories and housed in microisolator cages maintained under a 12-hr light/dark cycle. Water and food were supplied 7 cells suspended in 0.2 mL RPMI1640 medium into 6-week-old female BALB/c nude mice. Mice were examined twice weekly for tumor development. The tumor mass was measured in two dimensions with calipers, and the tumor volume was calculated according to the equation (L×W2)/2 .Tumor growth rate was tested by the subcutaneous injection of 1×10et al6 cells in 0.05-mL volume of RPMI medium were inoculated into the middle wall of the greater curvature of the glandular portion of the stomach using a 30-gauge needle . The stomach was then returned into the peritoneal cavity, and the abdominal wall and skin were closed with an AUTOCLIP applier . The mice were killed when moribund, and peritoneal dissemination was evaluated by counting the number of tumor nodules in the mesenterium. We also assayed for peritoneal dissemination by direct implantation into the peritoneal cavity. We injected 1×106 cells in 0.5-mL volume of RPMI medium into 6-week-old female BALB/c nude mice. On day 55, we calculated the number of tumor nodules on the mesentery and the volume of ascites.Orthotopic implantation was performed according to Yanagihara Cells were seeded on glass coverslips, and then fixed with precooled methanol (–30°C) for 20 min. After washing with PBS, cells were stained with E-cadherin and β-catenin antibody. The anti- E-cadherin antibody was detected with a Cy3-labeled, anti-mous secondary antibody (Invitrogen). The anti-β-catenin antibody was detected with a 488-labeled, anti-rabbit secondary antibody . The nuclei were stained with DAPI. The cells were examined with a confocal microscope.IHC analysis of tumors in the xenograft animal model was performed as described previously