Drosophila melanogaster. Endocycle progression in cells of the prothoracic gland (PG) is tightly coupled with the growth checkpoint, and block of endocycle in PG cells causes larval developmental arrest due to reduction in biosynthesis of the steroid hormone ecdysone. Moreover, inhibition of the nutrient sensor target of rapamycin (TOR) in the PG during the checkpoint period causes endocycle inhibition and developmental arrest, which can be rescued by inducing additional rounds of endocycles by Cyclin E. We propose that a TOR-mediated cell cycle checkpoint in steroidogenic tissue provides a systemic growth checkpoint for reproductive maturation.Many animals have an intrinsic growth checkpoint during juvenile development, after which an irreversible decision is made to upregulate steroidogenesis, triggering the metamorphic juvenile-to-adult transition. However, a molecular process underlying such a critical developmental decision remains obscure. Here we show that nutrient-dependent endocycling in steroidogenic cells provides the machinery necessary for irreversible activation of metamorphosis in  Onset of sexual maturation constitutes a point of no return in animals; once this life-changing decision is made, upregulation of steroidogenesis leads to irreversible juvenile-to-adult transition in humans and insects alike. While nutrient signals contributing to this decision-making process have been well studied, molecular events that ultimately determine its precise timing remain a mystery. We report here that nutrient-dependent endoreplication, the replication of genomic DNA without cell division, in steroidogenic cells functions as an intrinsic timer, whereby degree of polyploidy sets the timing of reproductive maturation (i.e. metamorphosis) in fruit flies. The cumulative and irreversible nature of endoreplication thus provides an intrinsic molecular machinery underlying the irreversible decision-making process, which may be widely leveraged as a fundamental developmental timing mechanism. Animals are heterotrophic and need to ingest nutrients from the environment during postembryonic development. Both availability and quality of food therefore are critical for timing animal growth and maturation. Since differentiation and functional maturation of each tissue are coordinated by the endocrine system, understanding how nutrient status affects hormonal states in developing animals is key to elucidating timing mechanisms responsible for juvenile-to-adult transition in animals.Drosophila melanogaster, CW is attained in the early half of the last (3rd) instar, after which starvation no longer delays the timing of metamorphosis . Tissues were washed with 0.1% PBT three times for 10 min each, washed with 1% PBT (1% Triton X-100 in PBS) for 5 min, blocked with 2% bovine serum albumin (Gemini bio-products) in 0.1% PBT for 30 min, and then incubated at 4°C overnight with primary antibodies diluted in blocking solution. Tissues were washed with 0.1% PBT three times for 10 min each, and incubated at 4°C overnight with secondary antibodies in 0.1% PBT. Together with the secondary antibody, Hoechst 33342 (Life technologies) was added at a 1:1500 dilution to detect DNA. After washing with 0.1% PBT three times for 10 min each, tissues were mounted in Vectashield mounting medium (Vector Laboratories).The following primary antibodies were used at indicated dilutions: anti-Dib, 1:500; anti-CycE , 1:500. Anti-Dib antibody was a gift from M. B. O’Connor . Alexa FEdU incorporation experiment was performed using Click-iT EdU 555 Imaging Kit (Life technologies). Larvae were dissected in Ringer’s solution, and tissues were incubated for 1 hour with 10 μM EdU in Ringer’s solution, fixed in 4% PFA in 0.1% PBT for 25 min. Fixed tissues were briefly washed twice in 0.3% PBT (0.3% Triton X-100 in PBS), washed in 0.3% PBT twice for 20 min each, blocked with 1% bovine serum albumin in 0.3% PBT for 30 min, and then incubated with Click-iT reaction cocktail including Alexa Fluor azide for 30 min. Tissues were washed briefly twice in 0.3% PBTw, washed in 0.3% PBT twice for 20 min each, and incubated at 4°C overnight with Hoechst 33342 (Life technologies) diluted at a 1:1500 in 0.1% PBT. In case of GFP staining, the primary antibody against GFP  was added at a 1:500 dilution, incubated 4°C overnight, washed with 0.1% PBT three times for 10 min each, and then incubated at 4°C overnight with Hoechst 33342 and the secondary antibody (Life technologies) in 0.1% PBT. After washing in 0.1% PBT three times for 10 min each, tissues were mounted in Vectashield (Vector laboratories).Images were taken with a Zeiss Axio Imager M2 equipped with ApoTome.2. Image acquisition settings were as follows: 14 bits image depth, 1024 x 1024 pixels for CycE and incorporated EdU staining, and 2048 x 2048 pixels for DNA quantification. A series of 2D images was taken every 0.5 μm slices. Image analysis was performed using Fiji .phm22 > +) and TORDN (phm22 > TOR.TED) larvae, UAS-mCD8::GFP transgene was introduced both in control (phm22 > mCD8::GFP) and TORDN  animals. PG cells of Oregon R were distinguished by their nuclei larger than surrounding cells in the ring gland. CycE-positive PG cells were defined as those in which nuclear CycE staining intensity was significantly stronger than cytoplasmic one. PG cells in s-phase were defined as those with pervasive EdU staining in the nucleus.To distinguish PG cells stained with anti-CycE or incorporated EdU in control  Developmental profile of Oregon R flies. Percentages of larvae and pupariated animals are shown at indicated stages. Mean percentages of three independent groups  are shown with standard errors. (B and C) Schematic diagrams of the starvation experiments. In the continuous feeding scheme (B), larvae reared on standard Drosophila medium (black line) were starved on wet filter paper (dashed lines) from indicated time points (white circles). In the discontinuous feeding scheme (C), larvae starved on wet filter paper from 72 to 120 hAEL were transferred to standard Drosophila medium, and re-starved from indicated time points (white circle). For each time point, three independent groups (10 larvae in a group) were weighed before starvation, and pupariated animals were counted during starvation. (D–G) The CW checkpoint in Oregon R. Percentages of pupariated animals after starvation at a given time point (D and E) and weight (F and G) are shown in the continuous feeding (D and F) and discontinuous feeding scheme (E and G). Mean percentages of three independent groups (10 larvae in each group) are shown with standard errors.(TIF)Click here for additional data file.S2 Fig(A) Schematic diagram of the discontinuous feeding scheme using wild-type Oregon R flies. (B) Starvation before CW attainment causes arrest of DNA content increase in the PG. The PGs  and nuclei of PG cells  were labeled for Dib (green) and DNA (white) at indicated stages of the discontinuous feeding scheme. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (C and D) The C value (C) and number (D) of PG cells at indicated stages. The CW checkpoint in control is indicated by dashed line in C. The C value is normalized against that of the PG in control at 108 hAEL (see (E) Starvation before CW attainment causes a decrease in CycE expression and EdU incorporation in the PG. The PGs were labeled for DNA (blue) and CycE or EdU (magenta) at 156 hAEL. The PGs are outlined by dashed lines. Scale bars, 50 μm. (F and G) Percentages of CycE-positive (F) and EdU-positive (G) s-phase PG cells at indicated stages. The CW checkpoint in control is indicated by dashed lines. 16–30 PGs were analyzed for each group.hAEL see . Error b(TIF)Click here for additional data file.S3 Fig(A) Schematic diagram of interaction between endocycle regulators. (B) Knockdown of endocycle regulators in the PG causes reduction in DNA content. Each gene was knocked down using two independent RNAi lines. The PGs  and nuclei of PG cells  of control (phm22 > dicer2) and RNAi  animals were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (C-P)E2F1 RNAi-1 (C) and 2 (D), CycE RNAi-1 (E) and 2 (F), Cdk2 RNAi-1 (G) and 2 (H), Cdt1 RNAi-1 (I) and 2 (J), PCNA RNAi-1 (K) and 2 (L), Cul4 RNAi-1 (M) and 2 (N), and Ddb1 RNAi-1 (O) and 2 (P) animals were reared on 20E-containing (0.5 mg/g 20E) or control medium from 72 hAEL. Percentages of pupariated animals are shown at indicated stages. Numbers of animals tested are in parentheses.(TIF)Click here for additional data file.S4 Fig(A and B) Expression of TORDN in the PG causes developmental arrest at the 3rd instar larval stage. Percentages of larvae and pupariated animals in control-1 (phm22 > +) (A) and TORDN (phm22 > TOR.TED) (B) are shown at indicated stages. Mean percentages of three independent groups (10 larvae in each group) are shown with standard errors. (C) Knockdown of TOR in the PG causes developmental arrest at the 3rd instar larval stage. Developmental profiles of control-1, TORDN, control-2 (phm22 > dicer2), TOR RNAi-1 , and TOR RNAi-2  animals are shown. Numbers of animals tested are in parentheses. (D) Pupariated control-1 and control-2 animals as compared to TORDN, TOR RNAi-1, and TOR RNAi-2 animals arrested at the 3rd instar larval stage. (E–J and E’–J’) Inhibition of TOR in the PG causes reduction in expression of ecdysone biosynthetic genes. Expression profiles of ecdysone biosynthetic genes were measured using qPCR. Average values of triplicate data sets are shown with standard errors. (K) Expression of TORDN in the PG causes decrease in ecdysteroid level. Whole-body ecdysteroid levels in control and TORDN larvae at 120 hAEL were measured using ELISA. Average values of five independent data sets are shown with standard errors. Statistical significance was calculated using Student’s t-test (* P < 0.05). (L–N) 20E feeding rescues developmental arrest in TORDN, TOR RNAi-1 and TOR RNAi-2 animals. TORDN (L), TOR RNAi-1 (M), and TOR RNAi-2 (N) animals were reared on 20E-containing (0.5 mg/g 20E) or control medium from 72 hAEL. Percentages of pupariated animals are shown at indicated stages. Numbers of animals tested are in parentheses. (O and P) The CW checkpoint in control-1. Percentages of pupariated control-1 and TORDN animals after starvation at a given time point (O) and weight (P) are shown. Mean percentages of three independent groups (10 larvae in each group) are shown with standard errors. (Q) Knockdown of TOR in the PG causes decrease in DNA content. The PGs  and nuclei of PG cells  were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (R and S) The C value (R) and number (S) of PG cells at 120 hAEL. Average C value in control-2 is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. (T) PG cells expressing CycE-1 transgene undergo endocycle. The PGs  and nuclei of PG cells  in control (phm22 > +) and CycE-1 (phm22 > CycE-1) larvae were labeled for Dib (green) and DNA (white) at 120 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (U and V) The C value (U) and number (V) of PG cells at 120 hAEL. Average C value in control is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. (W) Expression of CycE in the PG does not alter developmental timing. Percentages of pupariated animals are shown at indicated stages. Numbers of animals tested are in parentheses.(TIF)Click here for additional data file.S5 Figts  and TORDN +Gal80ts  animals. Percentages of pupariated animals after starvation at a given time point (A) and weight (B) in the continuous 18°C experiment are shown. Mean percentages of three independent groups (10 larvae in each group) are shown with standard errors.The CW checkpoint in control +Gal80(TIF)Click here for additional data file.S6 Fig(A and B) 20E feeding rescues developmental delay in RagADN and InRDN animals. RagADN (A) and InRDN (B) larvae were reared on 20E-containing (0.5 mg/g 20E) or control medium from 72 hAEL. Percentages of pupariated animals are shown at indicated stages. Numbers of animals tested are in parentheses. (C and D) The CW checkpoint in control +Gal80ts and InRCA +Gal80ts animals. Percentages of pupariated animals after starvation at a given time point (C) and weight (D) in the continuous 18°C experiment are shown. Mean percentages of three independent groups (10 larvae in each group) are shown with standard errors. (E) The PGs  and nuclei of PG cells  under nutrient-rich condition at 18°C. The PGs were labeled for Dib (green) and DNA (white) at 192 hAEL. Scale bars, 50 μm (upper panels) and 10 μm (lower panels). (F) The C value of PG cells at 120 hAEL. Average C value in control +Gal80ts is normalized to 64C. Error bars represent standard errors. Numbers of animals tested are in parentheses. (G) Developmental profile of animals fed on standard Drosophila medium continuously at 18°C. Percentages of pupariated animals are shown at indicated stages. Numbers of animals tested are in parentheses.(TIF)Click here for additional data file.S1 TableDrosophila Stock Center; VDRC, Vienna Drosophila Resource Center.*BDSC, Bloomington (PDF)Click here for additional data file.S2 Table*BDSC, Bloomington Drosophila Stock Center; VDRC, Vienna Drosophila Resource Center.(PDF)Click here for additional data file.S3 Tablew1118 were backcrossed with w1118 (BDSC #5905) three times. ** see * Flies with (PDF)Click here for additional data file.S4 Table(PDF)Click here for additional data file.


To elucidate its pathological significance, hepatic SHARPIN expression was suppressed in mice by injecting shRNA adenovirus via the tail vein. Seven days after this transduction, without additional inflammatory stimuli, substantial inflammation and fibrosis with enhanced hepatocyte apoptosis occurred in the livers. A similar but more severe phenotype was observed with suppression of HOIP, which is responsible for the E3 ligase activity of LUBAC. Furthermore, in good agreement with these in vivo results, transduction of Hepa1-6 hepatoma cells with SHARPIN, HOIL-1L, or HOIP shRNA adenovirus induced apoptosis of these cells in response to tumor necrosis factor-α (TNFα) stimulation. Thus, LUBAC is essential for the survival of hepatocytes, and it is likely that reduction of LUBAC is a factor promoting hepatocyte death in addition to the direct effect of drug toxicity.Linear ubiquitin chain assembly complex (LUBAC), composed of SHARPIN (SHANK-associated RH domain-interacting protein), HOIL-1L (longer isoform of heme-oxidized iron-regulatory protein 2 ubiquitin ligase-1), and HOIP (HOIL-1L interacting protein), forms linear ubiquitin on nuclear factor-κB (NF-κB) essential modulator (NEMO) and induces NF-κB pathway activation. SHARPIN expression and LUBAC formation were significantly reduced in the livers of mice 24 h after the injection of either carbon tetrachloride (CCl Recently, a novel head-to-tail linear type of ubiquitination was shown to play an essential role in nuclear factor-κB (NF-κB) activation ,2,3,4,5.4) or acetaminophen (APAP), and SHARPIN expression was revealed to be significantly downregulated in the livers of these acute hepatic injury models. We previously demonstrated that LUBAC formation is severely impaired mainly via markedly reduced SHARPIN expression in the NASH livers of mice fed a methionine choline deficient (MCD) diet for eight weeks [4 or APAP-induced liver injury occurred within a surprisingly short period , which accompanied a marked reduction of LUBAC formation.In this study, first, mice were treated with either carbon tetrachloride  adenoviruses. Importantly, livers deficient in SHARPIN or HOIP exhibited marked death of hepatocytes, which may have resulted in severe inflammation and fibrosis. Herein, we present data suggesting that reduction of LUBAC is involved in the development of CCl4 or APAP according to the procedure described in the literature [4 and APAP injections both induced severe liver injuries, as shown by hematoxylin and eosin (H-E) staining  treated with APAP for 24 h f. At the4 and APAP-induced hepatocyte death.These results suggest LUBAC deficiency as a possible underlying mechanism in the development of CCl4 and APAP induced acute liver injury models , both mRNA and protein levels of SHARPIN were significantly downregulated, whereas HOIL-1L expression was significantly upregulated probably as a compensatory response a,b. Gel In the livers of L-SHARPIN KD mice, immune cell infiltration was apparent d. Consis4 or APAP /body) of Ad-HOIP shRNA  via the tail vein. In mice injected with Ad-HOIP shRNA (L-HOIP KD mice), the HOIP protein level was dose-dependently decreased in the liver by about 80% in those injected with the highest dose , which covalently binds to glutathione (GSH) and depletes hepatic anti-oxidative capacity. Uncontrolled oxidative stress leads to hepatocyte cell death and the appearance of damage associated molecular patterns, a process which in turn activates Kupffer cells and results in the release of inflammatory cytokines and subsequent infiltration of neutrophils and macrophages [4 is also mediated via oxidative stress exerted by its degraded metabolites, trichloromethyl (CCl3) and trichloromethyl peroxyl (CCl3O2), both of which are unstable radicals [APAP overdosage results in the formation of rophages ,21. HepaWe speculated that not only the toxicities of these agents themselves but also impaired LUBAC formation may contribute to the aggravation of liver injury. In fact, it was clearly shown that hepatic knockdown of either SHARPIN or HOIP by adenoviral transfer of the corresponding shRNA into mice produced severe inflammation and fibrosis accompanied by hepatocyte death, features typically observed in both drug-induced hepatitis and NASH livers. Similar results were obtained using the Hepa1-6 cell line. Knockdown of any one of the LUBAC components in Hepa1-6 cells induced apoptosis in the presence of TNFα-stimulation a. One dicpdm mice (mice with a spontaneous Sharpin gene mutation), but these aggregates were absent when the Tnf gene was further knocked out in cpdm mice. In the mouse system, gut-derived bacterial endotoxins, such as lipopolysaccharide, have been regarded as an endogenous inducer of hepatic inflammation which exerts its effects by stimulating TNFα release from Kupffer cells, which is assumed to be a factor contributing to the progression from simple steatosis to NASH [4-induced hepatocyte apoptosis and fibrosis [In contrast, either SHARPIN or HOIP knockdown by adenoviral transfer of the corresponding shRNA into mouse livers was sufficient for rapid induction of massive hepatocyte death together with inflammation and fibrosis, with no additional stimulation being necessary. Gerlach et al.  reported to NASH ,23,24. I to NASH . It is nfibrosis  and is rfibrosis ,27, was l-SHARPIN KD mice showed a milder phenotype than l-HOIP KD mice, however, judging from serum ALT  or 300 mg/kg APAP  after being fasted for 20 h and were then sacrificed 24 h after the injection [C57BL6/J male mice, at seven to eight weeks of age, were fed normal chow diet. To prepare the mouse model with toxic agent-induced fulminant hepatitis, mice were intraperitoneally injected with 5 mL/kg CClnjection . To reduLivers were fixed with 10% formaldehyde and embedded in paraffin. Sections were cut and stained with hematoxylin and eosin or picrosirius red. For TUNEL staining, either the Apoptag Peroxidase In Situ Apoptosis Detection Kit  or the DeadEnd Colorimetric Apoptosis Detection System  was used according to the manufacturers’ instructions.g for 30 min. Alanine aminotransferase (ALT) was assayed with the Transaminase CII Test .Whole blood samples were obtained from the heart and serum was collected after centrifugation at 1000× 2 in air. At sub-confluence, Hepa1-6 cells were infected with LacZ, SHARPIN, HOIL-1L, or HOIP shRNA expressing adenoviruses and stimulated with 10 ng/mL TNFα for 8 or 24 h. Three days after the transduction, total lysates were prepared and then subjected to immunoblotting. Quantitative cell death assays were performed by staining dead cells with trypan blue. Two days after adenoviral infection, Hepa1-6 cells were stimulated with 10 ng/mL TNFα for 24 h. Cells were trypsinized, resuspended in culture medium, and stained by adding a 1/10 volume of 0.5% trypan blue. In the experiment designed to investigate the effect of APAP in vitro, Hepa1-6 cells were stimulated with 5 or 20 mM APAP for 24 h and total lysates were subjected to immunoblotting.Hepa1-6 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum at 37 °C in 5% COAntibodies were purchased from Cell Signaling Technology  and Santa Cruz . The antibodies against SHARPIN, HOIL-1L, and HOIP were prepared as previously described .3VO4, and 1 mM phenylmethylsulfonyl fluoride (PMSF). The lysates were incubated on ice for 30 min and then centrifuged at 15,000 rpm for 10 min and subsequently for 30 min at 4 °C. After adjusting the protein concentrations, the supernatants were mixed and boiled with sample buffer. Samples were electrophoresed with SDS (sodium dodecyl sulfate) -polyacrylamide gel, transferred to polyvinylidene difluoride (PVDF) membranes, and subjected to immunoblotting using Supersignal West Pico Substrate  or ImmunoStar LD (Wako).Livers were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM etylenediaminetetraacetic acid (EDTA), 1% Triton X-100, 1 mM NaF, 1 mM NaTM U6 RNAi Entry Vector Kit and the BLOCK-iTTM Adenoviral RNAi Expression System (Invitrogen). The top and bottom strands used to generate each of the shRNA expressing recombinant adenoviruses are as follows: SHARPIN shRNA, top strand 5′-CACCGCGGAAGCTGCAATTGATAGCCGAAGCTATCAATTGCAGCTTCCGC-3′, bottom strand 5′-AAAAGCGGAAGCTGCAATTGATAGCTTCGGCTATCAATTGCAGCTTCCGC-3′; HOIL-1L shRNA, top strand 5′-CACCGCCTATCTCTACCTGCTGTCACGAATGACAGCAGGTAGAGATAGGC-3′, bottom strand 5′-AAAAGCCTATCTCTACCTGCTGTCATTCGTGACAGCAGGTAGAGATAGGC-3′; HOIP shRNA, top strand 5′-CACCGGTCTTCTCAGCTCTCCAATACGAATATTGGAGAGCTGAGAAGACC-3′, bottom strand 5′-AAAAGGTCTTCTCAGCTCTCCAATATTCGTATTGGAGAGCTGAGAAGACC-3′.For the individual knockdowns of mouse SHARPIN, HOIL-1L, and HOIP, we generated each shRNA expressing recombinant adenoviruses using the BLOCK-iTTotal RNA from the liver was isolated using Sepazol reagent  and first-strand cDNA was obtained using the Verso cDNA Synthesis Kit (Thermo Scientific), according to the manufacturers’ instructions. Real-time PCR was performed using the CFX96 real-time PCR system  with SYBR Premix Ex Taq . The primers used are shown in 2, 1 mM PMSF, 1 mM dithiothreitol (DTT), and protease inhibitor cocktail. After adding an equal volume of lysis buffer containing 300 mM NaCl, lysates were centrifuged at 15,000 rpm for 30 min. The supernatants were subjected to gel filtration using Superdex 200 10/300 GL  and fractionated at 1 mL/min using ÄKTA explorer 10S . Proteins were precipitated with trichloroacetate and acetone from the collected samples, mixed and boiled with sample buffer, and subjected to western blotting.Gel filtration was performed to detect LUBAC components forming the LUBAC complex, which could be eluted in fractions of higher molecular weight and thereby distinguished from those existing as monomers. Livers were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1 mM MgClt-test when comparing two groups, and one-way ANOVA or the Kruskal-Wallis test for multiple comparisons. We considered p < 0.05 to indicate a statistically significant difference.Statistical analyses were performed using EZR . Values are presented as means ± S.E. We used student’s unpaired 4- or APAP-induced hepatitis. Furthermore, either SHARPIN or HOIP knockdown by adenoviral transfer of the corresponding shRNA into mouse livers rapidly induced massive hepatocyte death together with inflammation and fibrosis. Therefore, the LUBAC impairment observed in the livers of CCl4- or APAP-induced hepatitis may be a factor exacerbating hepatocyte death, in addition to the direct toxicity of the agent itself.LUBAC formation, especially the SHARPIN expression level, was reduced in the livers of mice with CCl


FABP6 and FABP9 mRNAs whose levels in all malignant cell lines were higher than those in the benign cells. Thereafter we assessed the expression status of FABP6 and FABP9 in both prostate cell lines and in human tissues. FABP6 protein was overexpressed only in 1 of the 5 malignant cell lines and its immunostaining intensities were not significantly different between benign and malignant prostate tissues. In contrast, FABP9 protein was highly expressed in highly malignant cell lines PC-3 and PC3-M, but its level in the benign PNT-2 and other malignant cell lines was not detectable. When analysed in an archival set of human prostate tissues, immunohistochemical staining intensity for FABP9 was significantly higher in carcinomas than in benign cases and the increase in FABP9 was significantly correlated with reduced patient survival times. Moreover, the increased level of staining for FABP9 was significantly associated with the increased joint Gleason scores (GS) and androgen receptor index (AR). Suppression of FABP9 expression in highly malignant PC3-M cells inhibited their invasive potential. Our results suggest that FABP9 is a valuable prognostic marker to predict the outcomes of prostate cancer patients, perhaps by playing an important role in prostate cancer cell invasion.In contrast to numerous studies conducted to investigate the crucial role of fatty acid binding protein 5 (FABP5) in prostate cancer, investigations on the possible involvement of other FABPs are rare. Here we first measured the mRNA levels of 10 FABPs in benign and malignant prostate cell lines and identified the differentially expressed  Prostate cancer is the most common male cancer in the developed countries and a worldwide male health threat , 2. The To investigate the molecular mechanisms involved in the malignant progression of prostate cancer cells and to identify possible novel markers, we characterised the role of FABP5 and demonstrated its crucial function in promoting tumorigenicity and metastasis of prostate cancer , 14. AftFABP6 and FABP9 mRNA levels in all malignant cell lines tested were higher than those in the benign cell lines. We then used Western blot and immunohistochemical staining to assess the expression status of FABP6 and FABP9 proteins in both prostate cell lines in culture and human prostatic tissues. The differences in expression profiles between benign and malignant cell lines and prostatic tissues were fully assessed and their possible value in diagnosis and prognosis have been systematically analysed.In this study, we have investigated whether FABPs other than FABP5 can be used as diagnostic or prognostic markers; these may be involved in initial development and in malignant dissemination of prostate cancer cells. We first measured mRNA levels of 10 FABPs in benign and malignant human prostate epithelial cell lines with quantitative RT-PCR and found that FABP4, FABP5, FABP6, FABP9 and FABP12 exhibited clearly higher levels in all malignant cell lines. For FABP1, FABP3, FABP2 FABP7 and FABP8, no clear and unified differences in their mRNA levels were identified when malignant were compared with benign cell lines.Messenger RNAs of 10 different FABPs were isolated; their levels in the benign PNT-2 cells and 5 malignant prostate cell lines were measured by quantitative RT-PCR and the results are shown in Figure p = 0.01). The levels of FABP6 in low malignant LNCaP cells, moderately malignant 22RV-1 cells, highly malignant PC-3 and PC3-M cells were lower than that in PNT-2 cells . FABP9 protein running as a single band at 14 kDa was highly expressed in 2 of the 5 malignant PC-3 and PC3-M cells, but its expression in other prostate cells including benign PNT-2 cells was not detectable .Differential expression of FABPs in levels of protein is shown in Figure s Figure , but thee Figure . Furthere Figure  that the2 test, p>0.08).The expression of FABP6 and FABP9 in the control, BPH and carcinoma tissues was detected by immunohistochemical (IHC) staining and representative staining patterns are shown in Figure Table 12 test, p<0.0001).The staining of FABP9 in carcinoma tissues was cytoplasmic only (identified by the arrow) Figure  and the 2 test, p<0.00001). Highly significant difference was also observed in staining intensities between low malignant cases and highly malignant cases . When the staining intensities between the moderately malignant and the highly malignant cases were compared, the difference was not statistically significant .According to their GS, carcinomas were divided into 3 groups: low (GS ≤5), moderate (GS 6-7) and high malignant (GS 8-10), as shown in Table p = 0.02).The cumulative survival of patients was plotted against the time in months using Kaplan-Meier survival graphs for different levels of the 4 parameters and the significance for each parameter was assessed by log-rank tests as shown in Figure p = 0.052).When the correlation between the patient survival time and AR index level was assessed Figure , the medp = 0.246).When the correlation between patient survival time and the blood level of PSA was assessed Figure , the medp < 0.0001).When the relationship between patient survival time and GS was assessed Figure , the medU test, p < 0.001). The staining intensity for FABP9 in cases with moderate GS (6 to 7) was significantly higher than that in cases with low GS . However, the difference in staining for FABP9 between cases with high GS and those with moderate GS was not significantly different .Assessment of correlation between the intensity of staining for FABP9 and GS, AR index and PSA level is shown in Figure U test, p = 0.03). However, the differences in AR index levels either between cases with moderate and weak FABP9 staining  or between cases with strong and weak staining for FABP9  were not statistically significant.Box plot of the correlation between staining intensity for FABP9 and AR index showed that the AR index level was significantly higher in cases with strong staining for FABP9 than in those cases with weak staining for FABP9 Figure  , between strong and moderate stains  and between moderate and weak stains for FABP9  were not significantly different.For the correlation between the staining intensity of FABP9 and PSA level Figure , box plop< 0.0001)  are shown in Figure ) Figure . TherefoFABP9 mRNA. When Western blots were used to measure the level of FABP9 from different individual colonies of transfectants, a single 14 kDa FABP9 band was detected and levels of FABP9 in scramble control PC3-M, parental PC3-M and 5 single transfectant clones are shown in Figure 2 test, p = 0.008) and 65.1% , respectively.For stable transfection, shRNAs were cloned into the psiRNA-h7SKGFPzeo plasmid and then transfected into the PC3-M cell line to knockdown stably FABPs in prostate epithelial cells to assess whether any of them were differentially expressed between benign and malignant phenotypes. The results showed that FABP4, FABP5, FABP6, FABP9 and FABP12 exhibited clearly higher levels in all tested malignant cell lines compared to their levels in the benign PNT-2 cells  , its expp < 0.02; Figure p < 0.0001; Figure The immunohistochemical analysis showed the staining intensity of FABP9 was significantly higher in carcinomas than in BPH Table  and the p = 0.052; Figure p = 0.246; Figure Both PSA and AR index (a parameter used to measure the staining intensity of AR ) are useU test, p = 0.03; Figure U test, p = 0.142) nor between strong and moderate FABP9 staining   Figure . This reTo study the biological significance of increased FABP9 in highly malignant prostate cancer cells, knockdown of FABP9 mRNA was performed via RNAi in highly-expressing PC3-M cells to establish highly- and moderately-suppressed transfectant cell lines, named PC3M-FABP9-siH and PC3M-FABP9-siM, respectively. When tested in an invasion assay, the number of invaded cells from PC3M-FABP9-siH cells, whose level of FABP9 was reduced by 91%, was reduced by 2.8-fold. In contrast that from PC3M-FABP9-siM, whose level of FABP9 was reduced by only 50%, was reduced by 1.6-fold Figure . Thus thThe following seven cell lines were used: MCF7 breast cancer cell line (the positive control for FABP6), benign prostate epithelial cell line PNT-2 , 43, weaFABP genes were used to design the real-time PCR primers. The total RNAs were extracted and purified from 6 different cell lines using RNAeasy Mini Kit. The quality and integrity of the mRNA was assessed using RNA 6000 Nano LabChip on Agilent 2100 Bio-analyser. The RNA templates were converted into a complementary DNA (cDNA) using reverse transcriptase. The real-time PCR mixtures for all FABPs and β actin were prepared with 2x brilliant SYBR Green qPCR master mix, 5μM forward primer, 5μM reverse primer, cDNAs and nuclease water. The reactions were centrifuged and placed in a real-time PCR thermocycler. The relative fold differences of FABPs mRNAs between benign and malignant cells were calculated.DNA sequence spanning exon junctions of all FABP6 and FABP9 expression in prostate cell lines was detected by Western blots using ECL system  . The polHuman prostate cancer tissues used in this work were obtained from an archival set of prostate cancer cases that were held in the archives of the original Pathology Department and our Molecular Pathology Laboratory, as described previously , 47. ThoHistological sections were cut from formalin-fixed, paraffin-embedded tissues (4-μm), de-waxed and dehydrated in xylene and ethanol, respectively . TissuesA standard light microscope (x400) was used to evaluate cytoplasmic and nuclear expression of FABP6 and FABP9. Immunoreactivity was examined independently by two qualified observers. The intensity of cytoplasmic staining was classified into 4 categories: unstained, weakly, moderately and strongly stained which were expressed as 0 (−), 1 (+), 2 (++) and 3 (+++), respectively. Nuclear staining was assessed by the intensity of staining which was expressed as 0 (−), 1 (+), 2 (++) and 3 (+++). The differences in scoring categories between 2 observers were <5%.2 test. Kaplan Meier plots were used to separate survival times of patients with differentially stained samples for FABP6 or FABP9. The graphs were labelled the significance association with Log Rank test . Correlation between FABP9 and GS, AR or PSA level was assessed via box plot and Mann Whitney U 2x3 tests. Statistical significance was defined as P < 0.05.The analysis was performed using the Statistical Package for Social Sciences (SPSS). Correlation between FABP6 and FABP9 expression and the benign and malignant prostate tissues were assessed by χBioinformatics & Research computing Software (Whitehead siRNA selection program) was used to select three different candidate sequences for FABP9 siRNA. Specificity of these sequences to FABP9 was confirmed by Blast search. Oligonucleotides made from these 3 sequences were commercially purchased :Sequence 1, sense strand 5′: GTACCTCGGTTAGTGAAACCGACAGTTCAAGAG; antisense strand 5′: AGCTTTTCCAAAAAGGTTAGTGAAACCGACAGTCTCTTGAACTGTCGGTTTCACTAACCGAG. Sequence 2, sense strand 5′: GTACCTCCTCAATGATTCACGTCCAATCAAGAGTTGGACGTGAATCATTGAGTTTTTGGAAA; antisense strand 5′: AGCTTTTCCAAAAACTCAATGATTCACGTCCAACTCTTGATTGGACGTGAATCATG. Scramble negative control, sense 5′: GTACCTCGCAGACCTTCCCATATAATTCAAGAGATTATATGGGAAGGTCTGCTTTTTGGAAA; antisense 5′: AGCTTTTCCAAAAAGCAGACCTTCCCATATAATCTCTTGAATTATATGGGAAGGTCTGCGAG.PC3-M cell line was transfected transiently with the X-tremeGENE siRNA Transfection Reagent (Roche) and the level of FABP9 was measured by Western blot analysis after the transient transfection of each of the 3 candidate sequences. The most efficient suppressor sequence was identified from the blot and was used to design short hairpin RNA (shRNA) using siRNA Wizard™ Software. Two shRNA inserts containing RNAi sequences against FABP9 were cloned into the psiRNA-h7SKGFPzeo plasmid  separately. PC3-M was stably transfected with vectors containing different FABP9 shRNAs using X-tremeGENE HP DNA Transfection Reagent . In a separate transfection, a vector containing scrambled RNA was used as a control. Transfected cells were cultured in a selective medium containing Zeocin (100μg/ml) (Life Technologies) for 3-4 weeks until the colonies were visualized. Ring cloning technique was used to isolate five single colonies to establish FABP9-supressed PC3-M transfectants and the level of FABP9 in these transfectants was measured by Western blot analysis.4 cells/ml were loaded into every upper compartment of chambers. Routine medium was loaded in the lower compartment and serum-free medium was used as negative control for each cell line. The chambers were maintained in a humidified tissue culture incubator at 37°C, 5% CO2 (v/v). After 24-hour cells in the upper compartment were removed and washed with PBS. Then, the cells, which invaded the lower part, were fixed and stained with 2% (v/v) crystal violet for 10 minutes. The number of invaded cells was counted using a light microscope.The invasiveness of the transfectants was assessed in a BD BioCoat™ Matrigel™ Invasion Chamber . After 24-hour starvation, PC3-M and transfected cells at a density of 2.5x10


ESR1 mutations have attracted attention as a potentially important marker and treatment target in endocrine therapy-resistant breast cancer patients. The E380Q mutation, which is one of the ESR1 mutations, is associated with estradiol (E2) hypersensitivity, increased DNA binding to the estrogen response element, and E2-independent constitutive trans-activation activity, but its frequency in ESR1 mutations remains unknown. The present study aimed to investigate the E380Q mutation in comparison with the other representative ESR1 mutations.ESR1 mutations  using droplet-digital polymerase chain reaction. Plasma was collected at more than two points of the clinical course, in whom changes of ESR1 mutations under treatment were investigated.We screened a total of 62 patients (66 tumor tissues and 69 plasma cell-free DNA (cfDNA)) to detect ESR1 mutations in 21% (12/57) of MBCs. The E380Q ESR1 mutation was found in 16% (2/12) and the other ESR1 LBD mutations were five (41.6%) of Y537S, and four each (33.3%) of D538G, Y537N, and Y537C, in 12 ESR1 mutant breast cancer patients. Five tumors had multiple ESR1 mutations: three had double ESR1 mutations; Y537S/E380Q, Y37S/Y537C, and Y537S/D538G, and two had triple ESR1 mutations; Y537S/Y537N/D538G. In plasma cfDNA analysis, the E380Q mutation was not detected, but increases in other ESR1 mutations were detected in 46.2% (6/13) of MBC patients under treatment.We detected ESR1 mutations in metastatic tissue and plasma. Each ESR1 mutation may have different clinical significance, and it will be necessary to investigate them all.We have shown that there are distinct populations of The online version of this article (doi: 10.1186/s12885-017-3779-2) contains supplementary material, which is available to authorized users. ESR1 ligand-binding domain (LBD) mutations that induce endocrine therapy (ET) resistance in breast cancer (BC) were first reported almost two decades ago [ESR1 LBD mutations act as drivers of ET resistance [ESR1 LBD mutations in plasma cfDNA [ESR1 mutation in plasma cfDNA, which are the representative ESR1 LBD mutations, and each of them was associated with shorter overall survival [ESR1 LBD mutation present; the D538G ESR1 mutation derived a large benefit from treatment with an mTOR inhibitor, whereas those with the Y537S mutation did not. These data suggest that each ESR1 LBD mutation may play a different role and more work is needed to confirm this.ades ago  and novesistance –8. In adsistance . It has ma cfDNA –12. In tsurvival . Interes, resulted in a requirement for less estradiol than wild-type (WT) ESR1 to achieve maximal activity and this mutation also showed high trans-activation activity in the absence of added hormone. They suggested that this ESR1 LBD mutation may be important in DNA binding and protein–protein interactions that modulate transcriptional activity of the estrogen receptor (ER). After 20 years, the presence of the E380Q ESR1 mutation came to be reported in both tumor tissue DNA (ttDNA) and plasma cfDNA [ESR1 mutant plasma samples [ESR1 mutation was found in 9.5% (6/63) in the SoFEA study for hormone receptor (HR)-positive BC patients who had demonstrated prior sensitivity to AIs, but it was found in 24.4% (22/91) in the PALOMA3 trial for HR–positive BC patients who had progressed during prior ET [ESR1 mutation may be a marker for screen of ET-resistant BC like the other representative ESR1 LBD mutations  [ESR1 mutation in Japanese BC patients. Thus, the present study screened for the presence of the ESR1 E380Q mutation in ttDNA and plasma cfDNA of 62 ER-positive Japanese BC patients using droplet digital polymerase chain reaction (ddPCR) and compared the frequency with the representative ESR1 LBD mutations . To our knowledge, this is the only precise study to use ddPCR to examine the presence of the E380Q ESR1 mutations in a series of tumor tissue and plasma samples of Japanese BC patients.Pakdel and colleagues  found thma cfDNA , 14–19.  samples . Fribbend D538G) –12, 20. A total of 62 patients (9 tumor tissue and 18 plasma samples in the primary BC (PBC) group and 57 tumor tissue and 51 plasma samples in the MBC group), treated at Kumamoto University Hospital between 2005 and 2014, were enrolled in this study. Informed consent was obtained from all patients before biopsy or surgery. The Ethics Committee of Kumamoto University Graduate School of Medicine  approved the study protocol. The treatment for PBC was carried out in accordance with the recommendations of the St. Gallen international expert consensus on the primary therapy of early BC at that time and the treatment for MBC was carried out in accordance with the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology . PatientESR1 mutations, respectively, as described elsewhere [Tumor cells and plasma were processed and each solution, which was examined for quality and quantity, was used as template ttDNA and cfDNA for the analysis of lsewhere .™ Droplet Digital™ PCR System  and PCR data were quantified using QuantaSoft™ software (Bio-Rad Laboratories) and results are expressed as fractional abundance  for each tumor tissue sample and as copies/μL of mutant DNA for each plasma sample as described previously [ESR1 LBD mutations  had already been optimized by comparative analysis of a dilution series of each synthetic ESR1 LBD mutant oligonucleotide as reported previously [ESR1 WT molecule and each ESR1 mutant molecule as positive control. A water-only (no template) control and WT normal human DNA (TaqMan Control Genomic DNA) were run in parallel for each ddPCR reaction as negative control.ddPCR assay was performed on the same sample twice using the QX200eviously . Our ddP® Probe ESR1 E380Q (#65116) as the detection probe for ESR1 E380Q  and Custom TaqMan SNP Genotyping assays  for the detection of other ESR1 LBD mutations , as described previously [We used LBxeviously .ESR1 LBD. We generated ESR1 E380Q using PrimeSTAR® GXL DNA polymerase . WT ER expression vector (pcDNA-ER) was used as a template with the following mutagenesis primers:The gene art site-directed mutagenesis system (Life Technologies) was used to generate mutations within E380Q: Forward, 5′-CTTCTACAATGTGCCTGGCTAGAGATCCT-3′’Reverse, 5′-TACTAGTCCAGGTGGAAGATGTTACACGGA -3′’ESR1 mutant molecule for ddPCR was amplified using PCR based on the ESR1 mutated plasmid, as described [The escribed .Immunohistochemical staining for ER alpha, progesterone receptor (PgR), human epidermal growth factor receptor 2 (HER2), and Ki67 was carried out on selected 4-μm thick tumor section. Primary antibodies, their visualization methods, and their evaluation were as previously described .ESR1 mutation analysis. Baseline characteristics of primary and metastatic ER-positive BC specimens are summarized in Table ESR1 LBD mutations  were previously evaluated with all samples of this study [A total of nine 14.5%) of 62 women with PBC and a total of 53 (85.5%) of 62 women with MBC were enrolled  where the total DNA content of the ddPCR reaction was 20 ng and “WT double” was 40 ng. We confirmed that this assay was able to detect the ESR1 E380Q molecule in a background of WT normal human DNA with the lowest concentration and was not able to detect any false-positives in the WT normal human DNA.Comparative ddPCR analysis of a dilution series of the indicated synthetic DNA Fig. . The dilESR1 E380Q and the other ESR1 LBD mutations . The rates of ESR1 mutations in primary and metastatic tumors from BC patients are shown in Table ESR1 mutations in 21% (12/57) of MBCs, but could not detect them in primary breast tumors. The E380Q ESR1 mutation was found in 16% (2/12) of ESR1 mutant MBC patients, with a MAF of 21.7% (Case M43) and 2.69% (Case M50), respectively. Interestingly, two patients with the E380Q ESR1 mutation had multiple metastatic sites. Other ESR1 LBD mutations were five (41.6%) of Y537S, and four each (33.3%) of D538G, Y537N, and Y537C, in 12 ESR1 mutant BC patients. We observed five cases with multiple ESR1 LBD mutations in the same tumor: three had double ESR1 mutations; Y537S/E380Q, Y37S/Y537C, and Y537S/D538G, and two had triple ESR1 mutations; Y537S/Y537N/D538G.We screened nine PBC and 57 MBC tumors to detect ESR1 mutation identified in ttDNA. Case M43 had metachronous bilateral BC. Clinical recurrence was detected at 16 months during adjuvant ET. She had previously received systemic treatment with five different therapies, not including ET, before MBC resection. A resected breast tumor had the E380Q ESR1 mutation beyond the selected cut-off level. Hormonal therapy was administered in the adjuvant setting only and was not effective in this case. In case M50, ipsilateral breast tumor recurrence was detected at 72 months after primary surgery. This patient had received eight systemic therapies including four ETs before cervical lymph node biopsy. A cervical lymph node metastasis showed double ESR1 mutations, the Y537S and the E380Q ESR1 mutation, with a MAF of 12.8 and 2.69%, respectively. Concerning the effect of ET, some ETs were initially effective, but, eventually, all ETs including AI became ineffective in this case. There were an insufficient number of samples to formally analyze a predicted association between the E380Q ESR1 mutation and patient prognosis.Table ESR1 mutation and the other ESR1 LBD mutations . Plasma was collected at more than two points of the clinical course in nine PBC and 13 MBC patients (two blood draws in 10 patients and three blood draws in three patients) and we were able to investigate the change of ESR1 LBD mutations in plasma cfDNA under treatment . Four early representative studies on ESR1 mutations in ttDNA reported a total of one (2.5%) of E380Q in comparison with a total of 14 (35.8%) of D538G, 10 (25.6%) of Y537S, four (10.2%) of Y537N, three (7.6%) of Y537C, and seven other ESR1 mutations among a total of 39 ESR1 LBD mutation-positive ER-positive MBC patients [ESR1, which consisted of 20 (21.1%) of E380Q, 34 (35.8%) of D538G in comparison with 13 (13.7%) of Y537S, 6 (6.3%) of Y537C, and 5 (5.3%) of Y537N [ESR1 mutation was found in 26% (15/57) [ESR1 mutant plasma samples whose frequency was more than that of one of the major ESR1 LBD mutations, Y537N [The raw data of E380Q of one 2.% of E380of Y537N . Meanwhi (15/57)  and 24.2s, Y537N .ESR1 mutation in our study seems to be rare among ESR1 LBD mutations. We found a total of two (16.6%) E380Q ESR1 mutation out of 12 MBC with ESR1 LBD mutations and we did not find the E380Q ESR1 mutation in plasma cfDNA  in metastatic tissue and plasma using ddPCR assay. The identification of recurrent ESR1 mutations in metastatic ER-positive BCs may provide the basis of understanding ET resistance mechanisms, which may contribute to the development of new molecular targeted therapy.We demonstrated the presence of a distinct population of


Highly pathogenic avian influenza viruses (HPAIVs) of H5 subtype have persistently caused outbreaks in domestic poultry and wild birds worldwide and sporadically infected humans. Rapid and accurate diagnosis is one of the key strategies for the control of H5 HPAIVs. However, the sensitivity of the diagnosis of H5 HPAIVs has gradually reduced due to extensive antigenic variation during their evolution. Particularly, the previously developed immunochromatographic diagnosis kit for H5 viruses, Linjudge Flu A/H5, exhibits reduced detection of H5 HPAIVs isolated in recent years. In the present study, we established a new advanced H5 rapid immunochromatographic detection kit (New Linjudge Flu A/H5) by a combination of two anti-H5 hemagglutinin monoclonal antibodies, A64/1 previously applied in the Linjudge Flu A/H5 and A32/2, a novel monoclonal antibody generated from a clade 2.3.4.4 H5 HPAIV. The new kit broadly detected all classical and recent H5 influenza viruses and showed a higher specificity and sensitivity than the original Linjudge Flu A/H5 with recently circulating H5 HPAIVs. Furthermore, the applicability of the New Linjudge Flu A/H5 was demonstrated by detecting antigens from the swabs and tissue homogenates of naturally infected birds and experimentally infected chickens with H5N6 HPAIVs belonging to the genetic clade 2.3.4.4. Our study, therefore, can provide an effective point-of-care rapid antigen detection kit for the surveillance of H5 avian influenza viruses and as a prompt countermeasure against the current widespread of the clade 2.3.4.4 H5 HPAIVs in domestic and wild birds. Influenza A virus (IAV) has been classified into different subtypes according to their surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA), in which 16 HA (H1–H16) and 9 NA (N1–N9) subtypes were recognized ,3. AfterThe typical diagnoses of IAV comprise serology or genetic identification; however, these methods are time-consuming and require appropriate facilities and biosafety . In receIn the present study, we established an improved H5 IC rapid diagnosis kit (New Linjudge Flu A/H5) using two MAbs; A64/1, originally used in the Linjudge Flu A/H5 , in addiTwo MAbs, A64/1 and A32/2, were used in the New Linjudge Flu A/H5. The MAb, A64/1, was previously produced using a hybridoma cell line against an H5 low pathogenic avian influenza virus (LPAIV), A/duck/Pennsylvania/10218/1984 (H5N2) , and a nA total of 28 strains of influenza viruses including 26 strains of IAVs, 17 strains of non-H5 viruses, 9 strains of H5 viruses, and 2 strains of influenza B viruses were used (Tables 50) value by using Madin–Darby canine kidney (MDCK) cells maintained in minimum essential medium supplemented with 0.3 mg/mL L-glutamine, 100 U/mL penicillin G, 0.1 mg/mL streptomycin, 8 mg/mL gentamicin and 10% calf serum. Ten-fold dilutions of viruses in serum-free minimal essential medium were inoculated onto confluent monolayers of cells and incubated at 35°C for 1 h. After 72 h of incubation at 35°C, the cytopathic effects of the cells were observed. TCID50 titers were calculated by the method of Read and Muench (1938). In addition to TCID50, the virus infectivity of H5 avian influenza viruses was measured as the 50% egg infectious dose (EID50) by using 10-day chicken embryos. Dilution and titer calculation were performed as described in the TCID50 method. The virus titration by EID50 and TCID50 was performed using the same original working aliquot of each virus.Virus titration was performed based on the 50% tissue culture infectious dose  , and the Linjudge Flu A/H5 kit . The tesIn addition, oropharyngeal and cloalcal swabs collected from 25 healthy chickens in commercial poultry farms were tested to examine cross reactivity of the New Linjudge Flu A/H5 with the field specimens. These samples were also confirmed to be negative with IAV by virus isolation using embryonated chicken eggs as previously described .8.4 EID50 of A/black swan/Akita/1/2016 (H5N6). Each chicken was housed in a self-contained isolator unit at the BSL3 facility in our laboratory. All chickens were monitored every 24 hours after the inoculation according to the standard protocol [10 TCID50/test.A dead black swan and a dead whooper swan suspected of having natural infections with H5 HPAIVs were transferred to our laboratory for diagnosis. Two H5N6 HPAIVs were isolated from these birds and named A/black swan/Akita/1/2016 (H5N6) and A/whooper swan/Hokkaido/X12/2017 (H5N6), respectively . Simultaprotocol . After 2http://www.dx.doi.org/10.17504/protocols.io.icmcau6.All experimental protocols in this study were available in the protocols.io as All the animal experiments were authorized by the Hokkaido University Animal Care and Use Committee  and all experiments were performed per the guidelines of the committee. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.The diagnostic specificity of the New Linjudge Flu A/H5 was compared with that of the original Linjudge Flu A/H5 kit and ImunoAce Flu (NP) by using a panel of 18 reference strains of IAVs including H1–H16 subtypes together with two strains of influenza B viruses . The NewTo demonstrate the New Linjudge Flu A/H5 had no potential to give false-positive detection, a total of 50 oropharyngeal and cloalcal swabs confirmed to be negative with IAV were used for the kit evaluation. It was evident that the New Linjudge Flu A/H5 did not yield any false positivity with the field negative samples .4.2–106.5 EID50/test against all H5 influenza viruses examined in this study. On the other hand, the Linjudge Flu A/H5 detected HA antigens from classical clade 1.1 and 2.2 H5 HPAIVs with a comparable detection limit to the New Linjudge Flu A/H5 (105.2–106.3 EID50/test). However, for recent H5 HPAIVs classified in clade 2.3.2.1c, and 2.3.4.4, the detection limit of the original Linjudge Flu A/H5 is higher and the kit could not detect several strains in these sub-clades. Furthermore, the comparable range of detection limits of the New Linjudge Flu A/H5, the Linjudge Flu A/H5 and ImunoAce Flu (NP) were observed to detect LPAIVs and HPAIVs in a clade of 2.2 viruses  strain in MDCK cells, infectivity of the virus titrated by TCID50 was much lower compared with EID50.The sensitivity of the new H5 kit was then evaluated with nine strains of H5 influenza viruses including LPAIVs and HPAIVs. Serial two-fold dilutions of each virus stock were concurrently examined by the New Linjudge Flu A/H5, the Linjudge Flu A/H5 and ImunoAce Flu (NP). The detection limit shows the lowest virus titer which was detectable by the individual tests . The res viruses . Slight The applicability of the New Linjudge Flu A/H5 for the diagnosis of H5 influenza infection was demonstrated by detecting H5 HA antigen in infected animals. Two naturally infected birds, a black swan and a whooper swan, confirmed infection with H5N6 HPAIVs, and three chickens experimentally infected with an H5N6 HPAIV, A/black swan/Akita/1/2016 (H5N6) were used for kit evaluation. A dead black swan from a local zoo was found on November 15, 2016, and stored at -20°C for shipment. Necropsy was performed on November 20, 2016 in the BSL3 facility in our laboratory. Similarly, the carcass of the whooper swan, a wild bird, was found on January 15, 2017 and transferred to our laboratory for necropsy on January 17, 2017.2.2–103.4 TCID50/test). On the other hand, all three kits evaluated in this study did not show non-specific reactivity with specimens with the absence of H5 antigen from the uninfected chicken as control  could detect the presence of H5 antigen and NP antigen, respectively in most specimens of infected birds; however, the presence of H5 antigen could not be detected with the original Linjudge Flu A/H5 kit . Interes control . All res50 and TCID50 [50 and TCID50 were observed in this study  is in the range of 103.7–105.8 EID50/test which is almost comparable to other commercial kits to detect viral NP protein [For the controls of H5 HPAI in birds and H5 virus infection in humans, rapid and accurate diagnosis of the causative viruses plays a vital role to facilitate subsequent countermeasures ,8. Multid TCID50 ,11,15,16is study . These r protein . The det protein ,14 or foPoint-of-care applicability of the New Linjudge Flu A/H5 was examined by detecting H5 antigen from clinical specimens of infected animals. A zoo bird and a wild bird, confirmed to be naturally infected with H5N6 HPAIVs, were transferred to our laboratory for further virological diagnosis. The New Linjudge Flu A/H5 could detect H5 HA antigen from most tissue homogenates tested. Owing to considerable roles of migratory birds in the transmission of the H5 HPAIVs, the surveillance of H5 HPAI and LPAI in wild birds has become a global emphasis ,28. OrdiIt was previously concluded that the point-of-care rapid antigen detection kits are appropriate for wildlife surveillance of avian influenza despite low specificity and sensitivity of the methods . In thisDevelopment of the New Linjudge Flu A/H5, in fact, was initiated as an adjunct tool for laboratory rapid diagnosis against H5 HPAI. The New Linjudge Flu A/H5 has been frequently used and its applicability was truly recognized by detecting recent multiple outbreaks of H5 HPAI in Japan . FurtherIn summary, our study is the first report to evaluate diagnostic efficacy of the point-of-care rapid antigen detection method to detect H5 HPAIVs belonging to clade 2.3.4.4 which expansively spread and cause multiple outbreaks in domestic and wild birds worldwide ,28,32. TS1 FigResult of presence/absence of antigens is indicated by +/- by appearing a line in the control judgment region (C) and presence of colored lines in both control and test judgment lines (T). Intensity of the positive test line was further recorded by a scale from + to 6+.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


Medulloblastoma is a highly aggressive pediatric brain tumor, in which sporadic expression of the pluripotency factor OCT4 has been recently correlated with poor patient survival. However the contribution of specific OCT4 isoforms to tumor aggressiveness is still poorly understood. Here, we report that medulloblastoma cells stably overexpressing the OCT4A isoform displayed enhanced clonogenic, tumorsphere generation, and invasion capabilities. Moreover, in an orthotopic metastatic model of medulloblastoma, OCT4A overexpressing cells generated more developed, aggressive and infiltrative tumors, with tumor-bearing mice attaining advanced metastatic disease and shorter survival rates. Pro-oncogenic OCT4A effects were expression-level dependent and accompanied by distinct chromosomal aberrations. OCT4A overexpression in medulloblastoma cells also induced a marked differential expression of non-coding RNAs, including poorly characterized long non-coding RNAs and small nucleolar RNAs. Altogether, our findings support the relevance of pluripotency-related factors in the aggravation of medulloblastoma traits classically associated with poor clinical outcome, and underscore the prognostic and therapeutic value of OCT4A in this challenging type of pediatric brain cancer. Primary malignant tumors of the central nervous system (CNS), although rare, are challenging to treat, often associated with severe patient morbidity and high mortality rates. This is the case of some embryonal CNS tumors occurring mostly in pediatric patients , 2. In yTo minimize the risk of secondary tumors, as well as considerable neurological, vascular, and endocrinal sequels of radiation therapies, children over three years of age with minimum residual disease (average risk group) receive adjuvant radiotherapy at lower doses than children with partial tumor resection and/or metastatic disease (high risk group) . FollowiPOU5F1/OCT4 [LIN28B [SOX2 [L1TD1 [LIN28B that was correlated with increased LIN28B expression particularly in Group 3 and Group 4 medulloblastomas [Pursuing novel therapeutic targets is also encouraged for medulloblastoma. For this particular purpose, a better knowledge of the basic biology of this important embryonal CNS tumor is crucial. Previous independent studies have shown an intriguing abnormal expression of the pluripotency-related genes 5F1/OCT4 , LIN28B 8B [SOX2  and L1TD2 [L1TD1 , in meduOCT4 expression indirectly, by binding its inhibitory microRNA let-7, and through direct binding of OCT4 transcripts, thereby enhancing their translation [OCT4 has been detected in different types of aggressive cancers [OCT4 expression was shown capable of discriminating average risk patients with poorer survival typical of high risk patients [OCT4 contribution to more aggressive traits in medulloblastoma is missing.In embryonic stem cells (ESC), LIN28 promotes nslation . Abnorma cancers –14. In mPOU5F1 gene located in chromosome 6. Alternative splicing of the POU5F1 primary transcript generates five transcript variants, encoding the isoforms OCT4A, OCT4B-190, OCT4B-265, OCT4B-164 and OCT4B1 [The OCT4 transcription factor is encoded by the d OCT4B1 –17. OCT4d OCT4B1 , while Od OCT4B1 . The ided OCT4B1 , 21, 22.OCT4 transcript variants in medulloblastoma, we found a specific correlation between OCT4A and poor survival, as well as a potent oncogenic activity for OCT4A. These findings highlight the involvement of OCT4A in a mechanism driving aggressiveness of medulloblastoma, which could be further explored not only as a prognostic indicator, but also as a therapeutic target for a precision medicine approach in neuro-oncology.In light of these recent observations, when evaluating expression of LIN28A and POU5F1 has been correlated in medulloblastoma [POU5F1 transcripts investigated, only OCT4A transcript levels significantly correlated with LIN28A expression in clinical medulloblastoma specimens  revealed that these pro-oncogenic effects are expression-level dependent . These rOCT4A overexpression significantly aggravated tumor formation and progression, as indicated by both ectopic and orthotopic models of medulloblastoma. When subcutaneously inoculated in Balb/C Nude mice, medulloblastoma cells overexpressing OCT4A generated tumors faster than respective control cells Figure . While Din vivo imaging studies of medulloblastoma-bearing mice, which showed a bimodal pattern of metastatic tumor foci in the brain and/or distant metastatic foci in the spinal cord occurring predominantly in animals injected with OCT4A-overexpressing cells . Extraneuroaxial metastases (M4) are rare and associated with more advanced disease without proper treatment [From the clinical point of view, of particular importance was the fact that OCT4A overexpression significantly enhanced medulloblastoma cell mobility properties SNORD14C have been suggested as prognostic marker of high risk squamous cell carcinomas of the larynx [GNL3  were also observed after OCT4A overexpression. Nucleostemin is a nucleolar protein implicated in self-renewal of ESC [NS induces dedifferentiation of somatic cells and reprogramming to a pluripotent stem cell state [NS expression in different cancer cells [Notably, some non-coding RNAs differentially expressed as a consequence of OCT4A overexpression in medulloblastoma cells have been associated with poor prognosis. Elevated levels of e larynx , while se larynx . Interesl of ESC , 42 and l of ESC . Ectopicll state . In cancll state  and withll state –47. Moreer cells , 48, 49.In medulloblastoma cells, OCT4A overexpression also induced chromosomal copy number aberrations, which varied in frequency and type according to the cell line. Two common mutated regions, 1p32.3 and 9p23.1, were detected in Daoy and USP-13-Med cells overexpressing OCT4A. Interestingly, trisomies of chromosomes 1 and 9 have been reported in human somatic cells after reprogramming to a pluripotent state . These f2 atmosphere at 37°C and subcultured when 80% of confluence was reached.Medulloblastoma cell lines Daoy and D283Med were purchased directly from ATCC  and cultured as previously described . USP-13-® Mini Kit (Qiagen) and genomic DNA was removed by treating with DNAse I following manufacturer recommendations. RNA samples were reverse transcribed into cDNA using SuperScript® II Reverse Transcriptase System (Life technologies) according to manufacturer's instructions. Primers were designed to specifically amplify OCT4A, OCT4B, and OCT4B1. Their specificity was determined by melting curve analysis, amplicons electrophoresis and sequencing (Nanog and Sox2 are described in [RNA was extracted from samples using RNeasyquencing . Reactioribed in .The protein extracts were submitted to western blotting standard protocol. Proteins were transferred to poly(vinylidene) difluoride (PVDF) membranes . The antibodies used were mouse anti-OCT4A antibody , anti-mouse IgG, HRP-linked antibody  and anti-beta Actin HRP-linked antibody (Abcam). The immunoblots were developed using the ImmobilonTM Western Chemiluminescent HRP Substrate .OCT4A overexpression by retroviral transduction . Briefly, HEK 293-T cells were transfected with 10% Polyethylenimine (PEI), 700 ng/mL CMV-GP plasmid, 350 ng/mL VSVG plasmid and 1 μg/mL OCT4A plasmid. Viral supernatant was collected after 48 hours, concentrated by ultracentrifugation (60.000 × g for 2 hours) and suspended in residual media overnight. Medulloblastoma cells were exposed to retroviral particles for 24 hours and expanded in culture for further analysis. OCT4A overexpression was confirmed at the transcriptional and protein levels by quantitative real time PCR and western blot, respectively.All medulloblastoma cell lines underwent stable To evaluate the proliferative profile of medulloblastoma cells after OCT4A overexpression, a cumulative population doubling level (PDL) was performed as previously described . The PDLThe cell cycle was analyzed using Cell Cycle Reagent Kit (Millipore) following manufacturer's procedure. The cell cycle was previously synchronized by serum starvation. Analyses were performed using Guava EasyCyte 5HT™ Flow Cytometer and GuavaSoft 2.1 software (Millipore).2) were seeded over a coating of 0.6% agarose solution containing supplemented media at normal conditions. The cells were allowed to set up for 10 minutes at room temperature and then covered by a 0.3% agarose solution. Plates were incubated at 37°C with 5% CO2 humidified atmosphere and media were replaced every 3-4 days. After 15 days, colonies over 50 μm were counted.Single cells at low densitiy  in DMEM/F12 supplemented with B-27, N-2, 20 ng/mL of EGF and 20 ng/mL of bFGF. The tumorspheres were counted after 7 days of incubation at 37°C with 5% CO3/mL were allowed to attach into a 24 well-plate. D283Med were plated in 96 well-plate previously coated with Poly-L-ornithine 0.01% . Non-attached cells were discarded after 45 minutes and residual cells were incubated with MTT solution  for 4 hours at 37°C. As control of total adhesion, cells were not discarded after 45 minutes. The supernatant was discarded and formazan crystals were dissolved with DMSO. The optical density was measured at 560 nm.Cells at a density of 8×10® 3D Spheroid Cell Invasion Assay  following the manufacturer's recommendation. Invasion area of the spheroids was measured as recommended by the manufacturer on days 0, 1, 3, 5 and 7 after invasion matrix addition. The D283Med cell line was not included in this assay since these cells fail to form spheroids under such experimental condition [Spheroid invasion capacity was evaluated using Cultrexondition .6 cells . Tumor growth were observed and measured weekly. Paraffin sections were subjected to standard H.E. procedures and analyzed under a light microscope.Female Balb/C Nude mice were inoculated subcutaneously in the right flank with 2×106) contained in 4-6 μL were stereotaxically injected in the right lateral ventricle of female Balb/C Nude mice in a ratio of 1 μL/min . The coordinates used were 1 mm to the right and 0.5 mm posterior of the bregma and 2.2 mm of depth. Animals were euthanized after 50 days of inoculation or after the development of neurological deficits and/or excessive body weight loss. The study was approved by the ethics committee for animal research of the University of São Paulo (CEUA protocol no. 132/2011). The brain and medulla were collected to standard H.E. procedure. A total of 10 sections were obtained randomly from each brain and medulla to analyze the presence of metastasis. Metastasis classification was based on M-Stage proposed by Chang [The model used was adapted from Studebaker et al. . Brieflyin vivo imaging using a bioluminescence-based method with the IVIS Imaging System . Medulloblastoma cells were engineered to constitutively express firefly luciferase using pLV/Luc lentiviral vector, described by Rocha et al. [Tumor development was also assessed by a et al.  and orthDNA was extracted using standard cholorophorm/phenol protocol. Array-based Comparative Genomic Hibridization (aCGH) was performed for detecting copy number alterations using 60K whole-genome platform . All procedures were carried out following the manufacturer's recommendation. Microarray scanned images were processed using the Feature Extraction Software and copy number aberrations (CNAs) were called using the statistical algorithm ADM-2 (sensitivity threshold: 6.7) in Genomic Workbench 6.9 6 Software (both from Agilent Technologies). Chromosome deletions and duplications were considered when log2 ratio of Cy3/Cy5 intensities were detected < −0.3 and > 0.3, respectively. Hybridizations were carried out using the OCT4A overexpressing cell lines as test samples and their corresponding tumor parental cells as reference sample.® Human Gene 2.0 ST whole-transcript arrays . The quality control and normalization of data were processed by Affymetrix® Expression Console Software (Affymetrix). Differentially expressed genes were identified with the One-Way ANOVA, with a p-value cutoff of 0.05, using Transcriptome Analysis Console v3.0 (Affymetrix). Functional Annotation was conducted on Ingenuity Pathway Analysis (Qiagen). Raw data is available at GEO with accession number: GSE77947.Total RNA of 2-3 clones of each cell line (Control and OCT4A overexpression) was extracted with the RNeasy kit (Qiagen), following the manufacturer's protocol. Gene expression profiling were carried out independently for each sample using Affymetrix GeneChipt test and Spearman's rank correlation coefficient were performed with Graph Pad Prism 6 . Statistical significance was established at p < 0.05 level in all analyzes. Experiments were conducted in triplicate and three independent experiments were carried out. Data are presented as the mean ± SEM.Unpaired Student's


SLC4A11 in ovarian cancer and in normal ovarian tissues, its prognostic value and the possible mechanism of its dysregulation. Bioinformatic analysis was performed by using data from the GEO datasets, the Cancer Genome Atlas-Ovarian Cancer (TCGA-OV) and the Human Protein Atlas (HPA). Results showed that SLC4A11 was upregulated in ovarian cancer compared with normal ovarian epithelial tissues. In patients with primary serous ovarian cancer in TCGA-OV, the cases with lymphatic invasion (N = 133) had significantly higher SLC4A11 expression than those without lymphatic invasion (N = 77) (p = 0.0069). High SLC4A11 expression was consistently associated with worse overall survival (OS). Univariate and multivariate analysis confirmed that high SLC4A11 expression was an independent prognostic factor for poor OS in grade 3/4 (G3/G4) tumors . 320 out of 578 (55.4%) ovarian cancer cases had SLC4A11 amplification. High methylation group had a significantly lower level of SLC4A11 expression. Based on these findings, we infer that high SLC4A11 expression is an independent predictor for poor OS in grade 3/4 serous ovarian cancer. Both DNA amplification and hypomethylation contribute to its upregulation in ovarian cancer.In this study, we aimed to examine the expression of  SLC4A1- loaders . SLC4A4,xtruders . SLC4A11nsporter . Therefo cancers . Upreguler cells . SLC4A9 ma cells .+ efflux compared with non-tumor cells [i is higher in ovarian cancer A2780 cells than in normal ovarian HOSE cells [SLC9A1 amplification is a mechanism of the high basal PHi and is associated with unfavorable overall patient survival [Ovarian cancer cells have an elevated Hor cells . One recSE cells . SLC9A1 survival . These fSLC4A11 in ovarian cancer/normal ovarian tissues and further assessed its prognostic value and the possible mechanism of its dysregulation.In this study, we examined the expression of The normalized data of one previous microarray (GDS3592)  that expSLC4A11 expression at the protein level in normal ovarian tissues and in serous ovarian cancer tissues was compared by using the immunohistochemistry (IHC) staining data provided by the Human Protein Atlas (HPA) (http://www.proteinatlas.org/) [as.org/) , 19.SLC4A11 expression, its copy number alteration and its DNA methylation in patients with ovarian cancer were examined using data from TCGA-OV. Original data downloaded from the UCSC Xena Browser (https://xenabrowser.net/) were given in SLC4A11 expression and overall survival (OS) or recurrence-free survival (RFS) was assessed by generating Kaplan-Meier survival curves, with median SLC4A11 expression as the cutoff. The analysis was performed using the UCSC Xena Browser or by using GraphPad Prism 6.0. Among 595 patients with primary serous ovarian cancer in TCGA-OV, 540 patients had SLC4A11 expression measured by AgilentG4502A_07_3. This proportion of patients was included in the univariate and multivariate analysis of the association between SLC4A11 expression and OS/RFS.SLC4A11 expression was assessed by using χ2 tests. The significance of the difference between the survival curves was assessed by log-rank test. Univariate and multivariate Cox regression models were used to evaluate prognostic significance. Welch’s t-test was conducted to compare SLC4A11 expression between patients with or without lymphatic invasion and between groups with high/low methylation. p<0.05 was considered statistically significant.The association between clinicopathological characteristics and SLC4A11 was the most significantly upregulated gene among the 10 SLC4A family members  had significantly higher SLC4A11 expression (p = 0.0069) than those without lymphatic invasion (N = 77)  .SLC4A11 expression in patients with primary serous ovarian cancer was summarized in SLC4A11 expression was significantly associated with lower grade tumors (GB/G1/G2) (p = 0.038), and a larger proportion of lymphatic invasion (p = 0.027) and deceased cases (p = 0.036)   and exon RNAseq (polyA+ IlluminaHiSeq). Among 540 patients with SLC4A11 measured by AgilentG4502A_07_3, 534 patients had OS data. Both IlluminaHiSeq and RNAseq data included 302 patients with OS data   . In addi= 0.050) . However= 0.050) . Then, K OS data . Log-ranrable OS . In univctively) . Howevers of RFS .SLC4A11 dysregulation. Among 578 patients with copy number measured, 320 (55.4%) cases had DNA amplification   compared with the low SLC4A11 group. Actually, G1/G2 serous ovarian carcinomas belong to type I tumors, while G3/G4 serous ovarian carcinomas belong to type II tumors, which have different molecular and biological profiles [In this study, we found that sporters , 26, SLCsporters , 6. In tprofiles , 21. We SLC4A11 expression was robustly associated with poor OS. Following univariate and multivariate analysis confirmed that high SLC4A11 expression was an independent prognostic factor for poor OS in G3/G4 tumors. These findings suggest that SLC4A11 might be a potential clinical marker in this group of patients. Although the association between SLC4A11 expression and OS in GB/G1/G2 patients was not significant, it showed a trend toward significance (p = 0.055). Considering the small number of GB/G1/G2 patients included in this study (N = 76), it is meaningful to further explore its prognostic value with a large sample base in the future.Currently, a series of predictors such as clinical stage, CA125 levels, age, response to chemotherapy and residual disease after debulking surgery are used to predict prognosis of ovarian cancer patients. However, there is still some discrepancy in the prognosis of similar tumors characterized by these clinical variables . OvarianSLC4A11 locates in chromosome 20p in the human genome, a region with a relatively high frequency of genetic amplification in ovarian cancer [SLC4A11 amplification. Therefore, DNA amplification is one important mechanism of upregulated SLC4A11 in ovarian cancer. Epigenetic regulation, such as methylation and histone modification are also important mechanisms of dysregulated genes in ovarian cancer [SLC4A11 expression. By using the results from Illumina 27k methylation array, we found that the group with high DNA methylation had significantly lower SLC4A11 expression, suggesting that epigenetic alterations also contribute to SLC4A11 upregulation in ovarian cancer.n cancer , 31. By n cancer , 33. ThuSLC4A11 expression is an independent predictor for poor OS in grade 3/4 serous ovarian cancer. Both DNA amplification and hypomethylation contribute to its upregulation in ovarian cancer.Based on findings above, we infer that high S1 Table(XLSX)Click here for additional data file.


NR4A1) promoter was hypermethylated in patients with T2D and in a mouse model of T2D. Moreover, DNA hypermethylation of the NR4A1 promoter reduced NR4A1 mRNA expression. Transient transfection of human NR4A1 into RIN-m5F and 293T cells caused DNMT1 inhibition and induced insulin receptor activation. NR4A1knockdown by shRNA resulted in overexpression of DNMT1 and inhibition of insulin receptor, suggesting that the NR4A1 gene is involved in the epigenetics pathway. Furthermore, T2D model mice treated with the DNMT1 inhibitor aurintricarboxylic acid (ATA) showed reduced activation of DNMT1 in pancreatic β cells; this effect reversed the changes in NR4A1 expression and decreased blood glucose in T2D model mice. Thus, our results showed for the first time that DNMT1 caused NR4A1 DNA hypermethylation and blocked insulin signaling in patients with T2D. Importantly, ATA therapy may be useful for decreasing blood glucose levels by reversing NR4A1-dependent insulin signaling. These findings improve our understanding of the crucial roles of these regulatory elements in human T2D.Our previous genome-wide association studies showed that DNA methyltransferase 1 (DNMT1) is associated with increased susceptibility to type 2 diabetes (T2D) in Han Chinese individuals. Here, we aimed to further evaluate the role of DNMT1 in T2D. We performed a genome-wide DNA methylation array and found that the nuclear receptor subfamily 4 group A member 1 ( DNMT1) gene as the allele having the strongest association with T2D in Han Chinese individuals by epigenetic regulation of the protein tyrosine phosphatase receptor type delta (PTPRD) gene in the insulin signaling pathway [Type 2 diabetes (T2D) is a complex disease that is characterized by hyperglycemia, pancreatic β-cell dysfunction, decreased insulin signaling, and increased hepatic glucose production –4. We pr pathway –7. Addit pathway , 9. ReceNR4A1) gene, which has been shown to be associated with insulin in cell models [Nuclear receptor subfamily 4 group A (NR4A) family receptors are expressed as early response genes of physiological and pathological stimuli, including fatty acids, stress, prostaglandins, growth factors, calcium and inflammatory cytokines, peptide hormones, and neurotransmitters , and arel models , encodesl models –15.Genome-wide DNA methylation arrays have become important in evaluation of epigenetic changes associated with disease progression. Here, we performed a pilot study using a DNA methylation array with samples from patients with T2D in order to identify biomarkers associated with the onset and development of T2D in Taiwan. Moreover, to gain insights into the functions and mechanisms through which DNMT1 affects the insulin signaling pathway, we examined epigenetic changes in patients with T2D and in a mouse model of T2D.http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE81868). Analysis of DNA methylation status, as indicated by model-based analysis of tiling-arrays (MAT) scores (range: 5.03684-8.45898), showed that the following genes had the highest methylation scores: ABR, ERICHI, WDFY2, GUSBP1, NR4A1, AFF2, PIPK1B, LOC100288637, APBA1, and PTPRN2 , accession number GSE81868  was constructed and transiently transfected into 293T and RIN-m5F cells. The results indicated that expression of human NR4A1 inhibited the activity of DNMT1, but induced insulin receptor overexpression in cells . The results showed that ATA induced NR4A1 expression in not only RIN-m5F cells and 293T cells but also NR4A1-knockdown cells. In addition, the insulin receptor was induced in RIN-m5F and 293T cells Figure .P < 0.05; Figure yKK mice, which were used in this study, insulin resistance is associated with hypertrophy of pancreatic islets and degranulation of β-cells. After ATA treatment, pancreas islets showed decreased mass  has been shown to be correlated with susceptibility to T2D, and DNMT1 has a preference for hemimethylated DNA [In a previous study, we found that the controls . Moreoveated DNA –22.y mice carry the lethal yellow obese (Ay) mutation and develop diabetes of polygenic origin, showing severe obesity, hypertriglyceridemia, hyperglycemia, hyperinsulinemia, and insulin resistance by 16 weeks of age [yKK mice with ATA, which has been shown to inhibit DNMT1 [KK-As of age , 24. In it DNMT1 . Importain vivo and is increased in diabetic mice exhibiting elevated gluconeogenesis [Hepatic expression of NR4A1 is induced by the cAMP axis in response to glucagon and fasting ogenesis . Our preogenesis . Moreoveogenesis , and druogenesis . Anotherogenesis . Thus, oIn summary, we found that NR4A1 expression was significantly lower in patients with T2D. Additionally, NR4A1 was involved in the insulin signaling pathway and could be epigenetically silenced by DNA methylation; epigenetic recovery of NR4A1 was observed following DNMT1 inhibition. We also found that the DNMT1 inhibitor ATA functioned to regulate blood glucose in T2D. These findings improve our understanding of the crucial roles of various regulatory elements in human T2D susceptibility.20.A MethylMiner Methylated DNA Enrichment Kit  was used to isolate methylated DNA. Genomic DNA was extracted from peripheral blood leukocytes using a Genomic DNA extraction kit , according to the manufacturer's instructions. One microgram of genomic DNA was sonicated to produce fragments ranging from 100 to 500 bp. Then, 3.5 mg (7 mL) of MBD-Biotin Protein was added to 10 mL Dynabeads M-280 Streptavidin according to the manufacturer's instructions. The MBD-magnetic bead conjugates were washed three times and suspended in 1 volume of 13 bind/wash buffer. The capture reaction was performed by adding 1 mg sonicated DNA to the MBD-magnetic beads on a rotating mixer for 1 h at room temperature. All capture reactions were carried out in duplicate. The beads were washed three times with 13 bind/wash buffer. The methylated DNA was eluted in one of the following two ways: (1) as a single fraction with a high-salt elution buffer (2000 mM NaCl), denoted as MBD-SF; or (2) as distinct subpopulations based on the degree of methylation using an increasing NaCl concentration of the elution buffer, from 200 to 2000 mM in a stepwise gradient . Each fraction was concentrated by ethanol precipitation using 1 mL glycogen (20 mg/mL), 1/10th volume of 3 M sodium acetate (pH 5.2), and two sample volumes of 100% ethanol and was subsequently resuspended in 60 mL HImmunoprecipitated DNA and input DNA from MeDIP immunoprecipitations and MBD-Capture reactions were amplified with a GenomePlex Single Cell Whole Genome Amplification Kit , according to the manufacturer's instructions. Fifty nanograms of DNA was used in each amplification reaction. The reactions were cleaned up using cDNA cleanup columns , and 7.5 μg of amplified DNA was fragmented and labeled according to Affymetrix Chromatin Immunoprecipitation Assay Protocol P/N 702238 Rev. 3. Affymetrix GeneChip Human Promoter 1.0R arrays (P/N 900777) were hybridized using a GeneChip Hybridization wash and stain kit (P/N 900720).Hybridization was performed with an Affymetrix GeneChip Promoter 1.0R array. The arrays were hybridized for 17 h at 45°C and 60 rpm. Arrays were subsequently washed (Affymetrix Fluidics Station 450), stained with streptavidin-phycoerythrin , and scanned on an Affymetrix GeneChip Scanner 3000.NR4A1 sense: 5′-TCTATGTCCTCGCCTTGGTT-3′, antisense: 5′-ATTATCCCGTCTGCCTTCAG-3′; GAPDH sense: 5′-CAGCCTCAAGATC ATCAGCA-3′, antisense: 5′-TGTGGTCATGAGTCCTTCCA-3′. This study was approved by the Human Studies Committee of China Medical University Hospital, and informed consent was obtained from either the participants or their parents.A total of 192 blood samples  were collected. All individuals attended the China Medical University Hospital in Taichung, and patients fulfilled the diagnostic criteria for T2D. Total RNA was isolated from human blood using a High Pure RNA Isolation Kit  according to the manufacturer's instructions. cDNA was synthesized from 1 μg total RNA using a High Capacity cDNA reverse transcription kit , in a 20-μL reaction volume, according to the manufacturer's instructions. cDNA was diluted to 10 ng/L, and 1 μL cDNA was used for each qRT-PCR assay in a final reaction volume of 10 μL. For quantification of gene expression with the ABI ViiA 7 Real-Time PCR System (Applied Biosystems), FastStart Universal SYBR Green Master mix (Roche) was used. Primer sequences were as follows: RIN-m5F rat pancreatic β-cells and 293T cells were purchased from Food Industry Research and Development Institute . Cells were seeded at 150,000 cells per well in six-well culture plates and incubated until the culture reached 50-80% confluence. Cells were then transfected with either the empty pcDNA3.0 vector, pcDNA3.0-NR4A1 construct, or lentiviral expression system for NR4A1 shRNA  using Xfect Transfection Reagent , according to the manufacturer's instructions. At 48 h after transfection, total protein was isolated from the cells.g for 30 min at 4°C. The supernatant was used for western blotting. Proteins in a 40-μg sample of the supernatant fraction were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 10% acrylamide resolving gels and transferred to polyvinylidene difluoride membranes. Membranes were incubated in blocking solution containing 5% nonfat dry milk and 0.1% Tween-20 in Tris-buffered saline, followed by incubation with rabbit anti-NR4A1 , anti-DNMT1 , anti-insulin receptor , anti-PTPRD , and anti-actin  polyclonal primary antibodies. Membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG  secondary antibodies. Proteins were visualized using SuperSignal West Pico Chemiluminescent Substrate or SuperSignal West Femto Chemiluminescent Substrate .The cells were homogenized in ice-cold RIPA lysis buffer  with freshly added protease inhibitor and phosphatase inhibitor . The homogenate was incubated on ice for 30 min and centrifuged at 13,000 × yA/J mice were obtained from Jackson Laboratories . Sixteen-week-old mice were treated intraperitoneally with ATA (50 mg/kg/day) for 2 weeks. Animals were housed in individual cages and given lab chow ad libitum . The animals were housed in a room with a constant temperature (22-25°C), relative humidity (50-70%), and photoperiod (12-h light/12-h dark). This study was approved by the Institutional Animal Care and Use Committee (IACUC) of China Medical University (IACUC: 102-217).Four-week-old male KK and KK.Cg-2O2 in distilled water for 30 min at room temperature, followed by antigen retrieval by boiling the sections in 0.01 M citrate buffer for 20 min for deparaffinization and rehydration. Sections were then washed in 50 mM Tris-HCl (pH 7.6) with 0.05% Tween for 2 min. To block nonspecific binding, all sections were incubated with 5% nonfat dry milk for 30 min at room temperature. The slides were then incubated with anti-DNMT1 antibodies (1:250) for 1 h at room temperature. The reaction was stopped by rinsing the sections with 0.01 M PBS. The sections were then incubated with biotinylated anti-mouse/rabbit IgG serum (secondary antibody), followed by treatment with a peroxidase-labeled streptavidin-biotin complex and diaminobenzidine substrate to visualize the positive cells. Finally, sections were counterstained with hematoxylin, prior to being mounted for examination by light microscopy. DNMT1-positive cells per area in the mouse pancreas were counted using a Q500MC Image Analysis System . DNMT1-positive cells were quantified in 20 fields from the cortex and 15 fields from the medulla at a magnification of 200×.DNMT1 was performed using rabbit anti-DNMT1 antibodies (LifeSpan BioSciences) and a streptavidin-biotin-peroxidase complex for immunohistochemistry staining. Formalin-fixed paraffin-embedded tissue sections were incubated in 3% HP < 0.05 (two-tailed).Data were organized using Microsoft Excel software and analyzed using SPSS 15.0  or GraphPad Prism version 3 . All values were expressed as means ± standard deviations. Normality of the data was tested using the Kolmogorov-Smirnov test. Hierarchical gene analysis and heat maps were determined using a Pearson correlation matrix. Depending on the probability distribution pattern and the total number of subjects, parametric  or nonparametric tests (skewed distribution or < 50 subjects) were used. The level of significance was set at