In vitro, UA inhibited proliferation, induced apoptosis, suppressed NF-κB activation and its regulated proliferative, metastatic, and angiogenic proteins. UA (20 μM) also enhanced gemcitabine (200 nM)-induced apoptosis and suppressed the expression of NF-κB-regulated proteins. In the nude mouse model, oral administration of UA (250 mg/kg) suppressed tumor growth and enhanced the effect of gemcitabine (25 mg/kg). Furthermore, the combination of UA and gemcitabine suppressed the metastasis of cancer cells to distant organs such as liver and spleen. Immunohistochemical analysis showed that biomarkers of proliferation (Ki-67) and microvessel density (CD31) were suppressed by the combination of UA and gemcitabine. UA inhibited the activation of NF-κB and STAT3 and the expression of tumorigenic proteins regulated by these inflammatory transcription factors in tumor tissue. Furthermore, the combination of two agents decreased the expression of miR-29a, closely linked with tumorigenesis, in the tumor tissue. UA was found to be bioavailable in animal serum and tumor tissue. These results suggest that UA can inhibit the growth of human pancreatic tumors and sensitize them to gemcitabine by suppressing inflammatory biomarkers linked to proliferation, invasion, angiogenesis, and metastasis.The development of chemoresistance in human pancreatic cancer is one reason for the poor survival rate for patients with this cancer. Because multiple gene products are linked with chemoresistance, we investigated the ability of ursolic acid (UA) to sensitize pancreatic cancer cells to gemcitabine, a standard drug used for the treatment of pancreatic cancer. These investigations were done in AsPC-1, MIA PaCa-2, and Panc-28 cells and in nude mice orthotopically implanted with Panc-28 cells.  Carcinoma of the pancreas has had a markedly increased incidence during the past several decades. Pancreatic cancer accounts for about 7% of all cancer deaths and ranks fourth as a cause of cancer death among both men and women in the US. In 2015, an estimated 48,960 new cases and 40,560 deaths from pancreatic cancer will occur in the United States . AlthougGemcitabine is the standard chemotherapeutic agent for advanced pancreatic cancer. Clinical trials of various phases have investigated combinations of gemcitabine with erlotinib [an epidermal growth factor receptor (EGFR) inhibitor], platinum analogues, bevacizumab [a vascular endothelial growth factor (VEGF) inhibitor], or celecoxib [a cyclooxygenase-2 (COX-2) inhibitor] . HoweverOne compound with potential for this use is ursolic acid (UA), a pentacyclic triterpenoid that has been identified in a large variety of medicinal plants, including rosemary and holy basil. Earlier studies have shown that UA can inhibit proliferation and induce apoptosis in leukemia, melanoma, and cancers of the breast, prostate, lung, and endometrium –10. UA hin vitro and in orthotopically implanted nude mice. Whether the inhibition in tumor growth is connected with changes in the expression level of miR-29a, a member of miR-29 family, was investigated [In the present study, we investigated whether UA can enhance the effect of gemcitabine against human pancreatic cancer cells stigated . We founin vitro and in an orthotopic nude mouse model.The aim of the present study was to determine whether UA could improve the efficacy of gemcitabine against pancreatic cancer. To determine this, the mechanism by which UA manifests its effects was investigated against human pancreatic cancer cells To determine whether UA inhibits proliferation in human pancreatic cancer cells, we treated AsPC-1, MIA PaCa-2, and Panc-28 cells with 0, 5, 10 and 20 μM UA for 1, 3, or 5 days and measured cell viability using an MTT assay. UA inhibited proliferation in all 3 cell lines in a dose- and time-dependent manner  treated cells Figure .Because NF-κB has been linked with both proliferation and chemoresistance, we next examined whether UA could inhibit constitutive NF-κB activation in Panc-28 cells. Our results showed that UA inhibited constitutive NF-κB activation in a dose-dependent manner Figure .Next we determined whether UA also suppresses NF-κB regulated gene products. Panc-28 cells were exposed to UA at different concentration and then analyzed by western blotting. Results showed that UA dose-dependently inhibited the expression of proteins linked with survival, proliferation, invasion and metastasis Figure .in vivo. As shown in Figure We also examined whether UA affects the long-term colony formation ability of pancreatic cancer cells, which more closely mirrors the situation To determine whether UA not only induces cytotoxicity rather it enhances the inhibitory effects of gemcitabine, pancreatic cancer cells were treated with different concentration of UA or gemicitabine alone or in combination. Results obtained from MTT assay showed that UA or gemicitabine alone induced moderate cytotoxicity in dose dependent manner. However, it also increased the cytotoxic activity of gemcitabine dose dependently Figure .in vitro models.To evaluate the type of drug interactions, we calculated the combination index (CI) values using CalcuSyn software based on the median effect principle . The obtTo determine whether UA enhances the effects of gemcitabine in inhibition of cell survival, proliferative and metastatic proteins, Panc-28 cells were exposed to UA and then treated with gemcitabine. Western blot analysis showed that UA inhibited the expression of proteins associated with survival , proliferation (cyclin D1 and cMyc), and invasion and metastasis  were moderately inhibited by either UA or gemcitabine alone, however it enhanced the inhibitory effects of gemcitabine. The expression levels of cIAP-2, cMyc and ICAM-1 were not affected by gemcitabine but moderately or slightly decreased by UA treatment; however, their expression levels were decreased substantially by the combination of UA and gemcitabine Figure .To determine whether UA enhances gemcitabine-induced cell death, we pretreated AsPC-1, MIA PaCa-2, and Panc-28 pancreatic cells with UA and then gemcitabine. The LIVE/DEAD assay showed that UA and gemcitabine were highly effective at doses at which UA or gemcitabine alone were minimally effective Figure .We also examined whether UA enhances the inhibitory effect of gemcitabine on long-term colony formation assay. UA or gemcitabine when administered alone had little effect on the colony-forming ability of Panc-28 cells. UA and gemcitabine alone had 30.3% and 21.5% decreased colony formation respectively compared to control. However, administration of UA and gemcitabine in combination significantly decreased (73.8%) the colony formation of Panc-28 cells Figure .in vivo studies because this cell line is stably transfected with luciferase.Figure The bioluminescence imaging  and in the tumors (228±24 ng/g) of the mice treated with UA Figure . AlthougTo determine whether UA affects pancreatic cancer growth and metastasis by modulating Ki-67 and CD31, we examined the expression of these markers of proliferation and angiogenesis, respectively. Our results showed that UA alone significantly decreased the expression of Ki-67 and CD31, but UA with gemcitabine provided the largest decrease in the expression of either marker Figure .Next examined whether UA modulates the STAT3 phosphorylation in pancreatic tumors. Western blot analysis showed that UA alone significantly suppressed the activated STAT3 by inhibiting phosphorylation at Tyr705, and the combination of UA and gemcitabine further enhanced the inhibitory activity Figure , left. TWe also investigated whether the effects of UA on tumor growth in the mice were associated with the inhibition of NF-κB activation. The results of Immunofluorescence analysis showed that treatment with UA alone and UA plus gemcitabine significantly suppressed NF-κB activation in tumor samples Figure , right.Next we examined whether UA modulates the expression of proteins involved in cell survival, invasion, and metastasis in orthotopically implanted pancreatic tumors. Western blotting revealed significant reductions in the expression of markers for cell survival (survivin and Bcl-2), proliferation (cyclin D1), inflammation (COX-2), and metastasis (ICAM-1 and MMP-9) in tumors from the groups treated with UA or gemcitabine alone compared with those from the control group. However, gemcitabine in combination with UA was more effective in suppressing the expression of these proteins than either UA alone or gemcitabine alone Figure .Immunohistochemical analysis of pancreatic tumor tissues also revealed that UA, alone or in combination with gemcitabine, significantly decreased the expression of cyclin D1, COX-2, VEGF, and MMP-9 molecules compared with the control treatment Figure .Because miR-29a has been shown to induce resistance to gemcitabine in pancreatic cancer cells , we sougCurrently, gemcitabine is the key chemotherapy for pancreatic cancer, however the drug has only 5.4% partial response rate and imparts a progression-free survival from 0.9 to 4.2 months .In the present study, we found that gemcitabine alone induced apoptosis in MIA PaCa-2, AsPC-1, and Panc-28 cell lines minimally but that gemcitabine-induced apoptosis was significantly enhanced by UA. Similar to these results, another report showed that pancreatic cancer cells BxPC-3, Capan-1, and PancTu-1 were unaffected by gemcitabine treatment , likely We also found that UA downregulated the expression of proteins associated with cell proliferation (cyclin D1 and COX-2) and survival , and this downregulation was enhanced when UA was given in combination with gemcitabine. Because these tumorigenic proteins are regulated by NF-κB and STAT3, it is likely that the inhibition of these transcription factors contributes to the downregulation of the proteins. The miR-29a family of microRNAs is aberrantly expressed in numerous cancer types . A recenIn our orthotopic mouse model, UA or gemcitabine alone were equally potent in inhibiting tumor growth, but the combination of UA and gemcitabine further suppressed the tumor growth. In agreement with these observations, a previous study showed that UA inhibited the growth of DU145 cancer xenografts in nude mice . The dosOur study, however, is the first to show that UA alone can inhibit pancreatic cancer metastasis to distant organs such as the spleen and liver. Our study also is the first of its kind to demonstrate that metastatic spread of pancreatic cancer is further suppressed by the combination of UA and gemcitabine. Further study showed that UA inhibited proteins involved in metastasis and angiogenesis, such as MMP-9 and VEGF. Suppression of these proteins could be the mechanism by which by UA and gemcitabine inhibited the metastasis of pancreatic cancer to the distant organs.Next we examined the mechanism by which UA manifests its effects against pancreatic cancer in nude mice model and found that the proliferation marker Ki-67 as well as micro vessel density indicator CD31 was down regulated by UA. The downregulation of NF-κB activity in tumor tissues may also account for inhibitory effects of UA on tumor growth in mice model. The downregulation in the expression of tumorigenic proteins such as cyclin D1, COX-2, and survivin in tumor tissue may be due to the suppression of NF-κB activity. Previously we have shown that UA inhibited the expression of cell survival proteins survivin and Bcl-2 in a model of orthotopically transplanted human colorectal cancer in nude mice , which sOldenlandia diffusa, one of the sources of UA, has also been shown in Caco-2 colon cancer cells [One of the major concerns in cancer drug development is that most drugs possess limited bioavailability. However, in this study, we found a significant presence of UA in the pancreatic tumor tissue of mice. The bioavailability of er cells . The bioer cells . These sOur finding that UA enhanced the antitumor effect of gemcitabine in an animal model by inhibiting NF-κB and its downstream targets—leading to the inhibition of proliferation, angiogenesis, and invasion—suggests that UA has significant potential for the treatment of pancreatic cancer. Whether the synthetic triterpenoid C-28 methyl ester of 2-cyano-3,12-dioxooleana-1,9,-dien-28-oic acid —which is a homologue of the UA family of compounds, is known to suppress inflammatory pathways , and is As the doses of gemcitabine (25 mg/kg) and UA (250 mg/kg) used in our animal study are safe and relevant to that in human subjects, the current study provides a basis for evaluating the efficacy of gemcitabine and UA combinations in clinical trials.UA was kindly supplied by Kingsing Guan, China. The antibodies against Bcl-2, COX-2, cyclin D1, ICAM-1, MMP-9, NF-κB p65, cIAP-1, pSTAT3, and STAT3 were purchased from Santa Cruz Biotechnology ; the antibodies against VEGF and Ki-67  were obtained from Neomarkers ; the antibodies against survivin were obtained from R&D Systems ; the antibodies against XIAP and cFLIP were obtained from Imgenex Corp  and the antibodies against β-actin were procured from Sigma . Mouse CD31 monoclonal antibody was obtained from Pharmingen . The liquid DAB+ substrate chromogen system-HRP used for immunohistochemistry was obtained from Dako . Penicillin, streptomycin, RPMI 1640, and DMEM were obtained from Mediatech Inc. . Fetal bovine serum (FBS) was purchased from Atlanta Biologicals . Gemcitabine  was stored at 4°C and dissolved in sterile PBS on the day of use. D-luciferin potassium salt  was dissolved in sterile PBS at a concentration of 40 mg/mL.The human pancreatic cancer cell lines AsPC-1 and MIA PaCa-2 were obtained from the American Type Culture Collection . The Panc-28 human pancreatic carcinoma cell line was kindly provided by Dr. Shrikanth Reddy . MIA PaCa-2 and Panc-28 cells were cultured in DMEM, and AsPC-1 cells were cultured in RPMI 1640 supplemented with 10% FBS, 100 U/mL of penicillin, and 100 μg/mL of streptomycin.The effect of UA on cell proliferation was determined by the MTT uptake method as described previously . BrieflyThe sub-G1 population of cells as indicator of apoptosis was analyzed by using PI staining as described previously . The assThe clonogenic assay determines the ability of cells in a given population to undergo unlimited division and form colonies. Panc-28 cells  were seeded in 6-well plates, incubated for 12 hours, and then treated with different concentration of UA or gemcitabine. After 24 hours medium was replaced with fresh medium and allowed to form colonies. After 9 days, colonies were washed with PBS, fixed in a solution of methanol and acetic acid (3:1), stained with 0.25% crystal violet and counted manually.To investigate whether UA could potentiate the efficacy of gemcitabine for causing apoptosis in pancreatic cancer cells, we used a LIVE/DEAD cell viability assay kit (Invitrogen), which is used to determine intracellular esterase activity and plasma membrane integrity and was described previously. AsPC-1, MIA PaCa-2, and Panc-28 cells  were incubated in chamber slides, pretreated with 20 μM of UA for 8 hours. Cell were washed with PBS to remove UA and then treated with 100 nM of gemcitabine for 24 hours. The cells were then stained with the assay reagents for 30 minutes at room temperature, and cell viability was determined by counting live (green) and dead (red) cells under a fluorescence microscope.To determine the level of NF-κB activation, we prepared nuclear extract from treated and untreated Panc-28 pancreatic cancer cells and carried out electrophoretic mobility shift assays essentially as described previously .nu/nu mice were obtained from the breeding colony of the Department of Experimental Radiation Oncology at MD Anderson. The animals were housed in standard poly(methyl methacrylate) cages (5 per cage) in a room maintained at constant temperature and humidity and in a 12-hour light/12-hour dark cycle. Their diet consisted of regular autoclave chow and water ad libitum. None of the mice exhibited any lesions, and all mice were tested pathogen free. All the mice were acclimatized to a pulverized diet for 3 days before orthotopic cancer cell implantation. This experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee at The University of Texas MD Anderson Cancer Center.Four-week-old male athymic 6) in 50 μL PBS were injected into the subcapsular region of the pancreas with a 27-gauge needle. A cotton swab was held for 1 minute over the site of injection to prevent leakage. The abdominal wound was closed with wound clips .Panc-28 cells, stably transfected with luciferase, were orthotopically implanted in the pancreas of nude mice as described previously . After aOne week after orthotopic implantation, 10 mice each were randomly assigned to the following treatment groups: (a) untreated control , (b) UA alone , (c) gemcitabine alone , or (d) UA  plus gemcitabine .in vivo bioluminescence imaging system , which includes a cryogenic cooling unit and a data acquisition computer that uses Living Image software. Imaging was performed on days 0, 7, 14, 21, and 28 of treatment as previously described.Tumor volumes were monitored weekly with the IVIS 200 3, where r is the mean of the three dimensions . Each tumor was divided in two. Half of the tumor tissue was fixed in formalin and embedded in paraffin for immunohistochemistry. The other half was snap frozen in liquid nitrogen and stored at −80°C. At autopsy, the mice were also examined for distant organ metastases, and the number of metastatic foci in liver and spleen were counted.Therapy was continued for 4 weeks. One week after treatment ended, the mice were euthanized, and the primary tumors in the pancreas were excised. The final tumor volume was measured as V = 2/3πrPancreatic tumor samples were fixed with paraformaldehyde and embedded in paraffin. After washing with PBS, the slides were blocked with protein block solution (Dako) for 20 minutes and incubated overnight with anti-human VEGF, COX-2, MMP-9, and cyclin D1 antibodies . The expression of VEGF, COX-2, MMP-9, and cyclin D1 were evaluated using an immunohistochemical method described previously .Frozen sections in slides were de-paraffinized and rehydrated through a graded alcohol series. Antigen retrieval was performed by boiling the slides for 20 minutes in antigen retrieval solution . Slides were cooled for 30 minutes at room temperature, rinsed briefly in PBS, circled with a PAP pen and blocked with 1% normal goat serum. The sections were incubated with primary antibodies followed by their respective secondary antibodies (anti-rabbit IgG Alexa Fluor 610 and anti-mouse IgG Alexa Fluor 488). Slides were mounted using Vectashield Mounting Medium with DAPI (Vector Labs) viewed with a Nikon FXA fluorescence microscope equipped with Photometrics Cool Snap CF color camera  and MetaMorph version 4.6.5 software (Molecular Devices). The stored images were processed using NIH ImageJ software and Adobe Photoshop.Pancreatic cancer (Panc-28) cells and tumor tissues (75–100 mg per mouse) from vehicle control and experimental mice were minced and incubated, and Western blot analysis was performed, as previously described .−ΔΔCT method [To examine the effects of UA, gemcitabine and the combination of two agents on miR-29a expression in tumor tissues, we performed quantitative real time PCR as described previously . Total RT method .The mice were fed UA 4 hours before being euthanized. The mice were anesthetized, blood was collected by cardiac puncture, and then the mice were killed humanely. High-performance liquid chromatography (HPLC) analysis was performed as previously described  to detert-test was used to compare means of 2 independent variables. One-way analysis of variance was used to determine the statistical differences between more than 2 groups. Statistical significance was established as P < 0.05. A significant interaction was interpreted by a subsequent median effect principle of the Chou-Talalay method.Data are presented as mean ± SD. The Student


The bromodomain containing 1 (BRD1) gene has been implicated with transcriptional regulation, brain development, and susceptibility to schizophrenia and bipolar disorder. To advance the understanding of BRD1 and its role in mental disorders, we characterized the protein and chromatin interactions of the BRD1 isoforms, BRD1-S and BRD1-L.Stable human cell lines expressing epitope tagged BRD1-S and BRD1-L were generated and used as discovery systems for identifying protein and chromatin interactions. Protein-protein interactions were identified using co-immunoprecipitation followed by mass spectrometry and chromatin interactions were identified using chromatin immunoprecipitation followed by next generation sequencing. Gene expression profiles and differentially expressed genes were identified after upregulating and downregulating BRD1 expression using microarrays. The presented functional molecular data were integrated with human genomic and transcriptomic data using available GWAS, exome-sequencing datasets as well as spatiotemporal transcriptomic datasets from the human brain.We present several novel protein interactions of BRD1, including isoform-specific interactions as well as proteins previously implicated with mental disorders. By BRD1-S and BRD1-L chromatin immunoprecipitation followed by next generation sequencing we identified binding to promoter regions of 1540 and 823 genes, respectively, and showed correlation between BRD1-S and BRD1-L binding and regulation of gene expression. The identified BRD1 interaction network was found to be predominantly co-expressed with BRD1 mRNA in the human brain and enriched for pathways involved in gene expression and brain function. By interrogation of large datasets from genome-wide association studies, we further demonstrate that the BRD1 interaction network is enriched for schizophrenia risk.Our results show that BRD1 interacts with chromatin remodeling proteins, e.g. PBRM1, as well as histone modifiers, e.g. MYST2 and SUV420H1. We find that BRD1 primarily binds in close proximity to transcription start sites and regulates expression of numerous genes, many of which are involved with brain development and susceptibility to mental disorders. Our findings indicate that BRD1 acts as a regulatory hub in a comprehensive schizophrenia risk network which plays a role in many brain regions throughout life, implicating e.g. striatum, hippocampus, and amygdala at mid-fetal stages.The online version of this article (doi:10.1186/s13073-016-0308-x) contains supplementary material, which is available to authorized users. P = 3.31 × 10–7) in the most recent schizophrenia genome-wide association study (GWAS) by the Psychiatric Genomics Consortium using conventional statistical methods ) then moved to Protein LoBind tubes  and then washed six times in Triton X-100 buffer 150 . Proteins were eluted in 100 μL Elution buffer (HENG) . Cell lysate was kept cold at all times, between 1 °C and 6 °C. The process was carried out in one continuous procedure without any freezing/thawing of the cell lysate. IP with anti-V5 antibody and IP with anti-HA antibody was performed four times with either extracts from HEK293T, stable BRD1-S-V5, or BRD1-L-V5 expressing cell lines, adding to a total of 24 IP samples prepared for mass spectrometry.IP of V5 epitope tagged proteins was performed as follows: Anti-V5 and anti-HA antibody conjugated agarose beads  were washed twice in PBS before use and blocked in 1 % BSA. Cell lysates were pre-cleared for 30 min at 4 °C under rotation using 30 μL anti-HA antibody conjugated bead solution before collecting the supernatant (400 rpm for 5 min) for IP. IP was performed using 60 μL of the bead solution per 10 mL lysate. Beads were added to the pre-cleared supernatant and incubated at 4 °C overnight before they were collected at 400 rpm for 15 min. Beads were washed twice in 10 mL Triton X-100 buffer 250  were washed twice in PBS and blocked in 1 % BSA . Pre-clearing and IP was performed in a 50/50 μL mix of blocked protein A and G bead solutions. A total of 30 μL of protein A and G bead solution was used for preclearing of cell extracts and 60 μL of protein A and G bead solution was used for IP. Anti-PBRM1 antibody  was diluted in Triton X-100 buffer 150 according to antibody specifications and incubated with blocked protein A and G bead solution at 4 °C overnight. IP control reactions were performed in parallel by the same method but without the addition of antibody. Furthermore, elution was performed with 1 % SDS instead of the V5 peptide solution as described above.Proteins eluted upon IP were precipitated in six volumes of acetone (–20 °C), incubated at –20 °C overnight, and centrifuged at 15,000 × g at 4 °C. The supernatant was cautiously removed and the protein pellet was carefully washed in ice-cold 90 % acetone, centrifuged at 15,000 g and air-dried. Disulfide bonds of the proteins were reduced in 90 μL buffer containing 50 mM tris-(2-carboxyethyl)phosphine (TCEP)  and 50 mM ammonium bicarbonate, and incubated at 60 °C for 10 min. The reduced cystein residues were blocked by 50 mM of the alkylation agent iodoacetamide . Proteins were digested into peptides by addition of 1 μg trypsin  in 100 μL 50 mM ammonium bicarbonate.The peptide mixtures were analyzed by nanoliquid chromatography  coupled to mass spectrometry (MS) , with separation on a reverse phase column  at a flow rate of 300 nL/min using a 100 min gradient (5–35 %) of acetonitrile in 0.4 % acetic acid. The MS detection constituted a full scan (m/z 400 − 2000) in Orbitrap (<3 ppm mass accuracy) followed by up to four data dependent MS/MS fragmentation scans using collision induced dissociation (CID).The resulting MS files were processed essentially as previously described . Briefly2 (sample) – log2 (control)), where control experiment is IP without proper BRD1 in the sample or without antibody against the proper BRD1 construct. The four criteria were: (1) the enrichment ratio IP:V5 BRD1-(S or L)-V5 - IP:HA BRD1-(S or L)-V5 of a protein is > cutoff value; (2) the enrichment ratio IP:V5 BRD1-(S or L)-V5 - IP:V5 HEK293T of a protein is > cutoff value; (3) the enrichment ratio IP:V5 BRD1-(S or L)-V5 - IP:HA HEK293T of a protein is > cutoff value; (4) the enrichment ratio IP:V5 BRD1-(S or L)-V5 - the mean of all controls of a protein is > cutoff value. Both two- and threefold cutoffs were applied to sort the identified protein-protein interactions according to co-IP strength  or anti-HA conjugated antibody (IP:HA) and extracts from stable BRD1-S-V5, BRD1-L-V5 expressing cells, or HEK293T cells, yielding: . Four criteria (1–4) were raised to systematically identify repeatedly co-immunoprecipitated (co-IPed) proteins by calculating the enrichment ratio  and then placed in a heating block for 5 min at 100 °C. Denatured proteins were loaded onto TGX or Tris-HCL 4–15 % linear gradient polyacrylamide gels (Bio-Rad Laboratories) and separated by electrophoresis at 100–120 V. Proteins were transferred to Hybond-P Membranes  using a wet blotting apparatus (Bio-Rad Laboratories). Membranes were activated in 99 % alcohol for 20 s, washed in redistilled water for 1 min, and transferred to transfer buffer . Membranes were blocked for 1 h in blocking buffer . Primary antibodies were: monoclonal anti-V5 antibody  diluted 1:2000 in wash buffer  and anti-BRD1 antibody (65/66 Sigma Genosys), previously described [2 slideflasks. Cells were fixed in 4 % freshly prepared paraformaldehyde, 0.1 % gluteraldehyde, 0.1 % Trition X-100 solution on ice for 30 min, followed by wash with PBS for 5 min. Slides were incubated with anti-V5 antibody (Invitrogen) for 1 h at 4 °C using a 1:200 dilution in PBS and washed 3× 5 min with PBS containing 0.1 % Triton X-100. Incubation with anti-mouse immunoglobulins/FITC F(ab’)2  was performed for 1 h at 4 °C using a 1:200 dilution in PBS supplemented with 5 % FCS. Cell nuclei were stained by Hoechst staining (Hoechst stain 1 μg/mL) for 10 min, slides were then washed 2× 5 min in PBS containing 1 % Triton X-100 and dried. Two drops of fluorescent mounting media were added to the fixed cells and stored in the dark at 4 °C until media were dried out and then viewed under a fluorescent microscope . A total of 20 μL of 5 M NaCl was added to eluates and incubated for 4 h at 65 °C for reversing cross-links. A total of 10 μL of 5 M EDTA, 20 μL of 1 M Tris-HCl pH 6.5, and 2 μL of proteinase K  was added to eluates and incubated for 1 h at 45 °C. Phenol/chloroform and isopropanol (v/v) was added, mixed, and centrifuged. A total of 2 μL of glycogen  and 0.7 × volume isopropanol was added to supernatant and incubated at –20 °C overnight. Precipitated DNA was collected by centrifugation at maximum speed for 30 min at 4 °C. Pellet was carefully washed in 70 % ethanol, air-dried, and dissolved in 50 μL redistilled water. ChIP DNA concentrations were measured on a microplate reader  by fluorescence of double-stranded DNA (dsDNA) . All ChIP DNA was diluted to the same concentration before quantitative real-time PCR.Immunoprecipitations were carried out using 20 μL of anti- V5 or anti- HA conjugated agarose beads . First, the beads were washed twice in PBS and protease inhibitors  with intermediate low g centrifugation. Beads were blocked in 10 × volume PBS, 200 μg/mL sonicated herring DNA, and 1.5 % BSA at RT and washed in ChIP dilution buffer. Extracts were pre-cleared with 20 μL of blocked anti-HA beads at rotation for 30 min before immunoprecipitation with blocked anti-V5 and anti-HA beads over night at 6 °C. Beads were collected by centrifugation and washed twice in low salt buffer , twice in high buffer , once in LiCl buffer , and twice in TE buffer. Elution was performed in 500 μL freshly made elution buffer  and sequenced on a Genome Analyzer IIx. The sequence reads were aligned to the human genome (hg19), through the Galaxy project portal , using tCultured cells were lysed using Qiashredder columns  and RNA was purified using the RNAeasy Mini kit (Qiagen) according to the manufacturer’s protocol. RNA concentrations were determined using a NanoDrop 1000 version 3.7.1 . RNA integrity was assessed by 1 % agarose gel electrophoresis. A complementary DNA (cDNA) library was generated from 1 μg of RNA using the iScript™ cDNA Synthesis Kit . All steps were performed according to the manufacturer’s protocol using a mixture of random hexamers and Oligo-dT primers provided with the kit.Microarray (Affymetrix U219) and RNA quality control was performed at Aros Biotechnology, Skejby. Further information is provided elsewhere . A thresQuantitative real-time PCR was performed on the LightCycler®480  system. A total of 96 or 384 well plates, with a respective total reaction volume of 10 μL or 5 Μl, were used, respectively. Fluorescence of dsDNA was determined by addition of SYBR®Green  to the reactions in the concentration described by the manufacturer. The standard settings on the LightCycler software were used for excitation and detection of fluorescence. Primer3  was usedWe used publicly available summary statistics from single-marker GWASs , 22–31 cData on schizophrenia, bipolar disorder, anorexia nervosa, autism, MDD, ADHD, and cross-disorders were accessed and downloaded via the PGC website . Data onwww.brainspan.org [P value for the number of tests performed (Bonferroni correction).Human brain transcriptome (RNA-seq) data were obtained from span.org . We filtspan.org . TemporaIngenuity Pathway analysis was used for the bioinformatics analysis. Simulations, statistical analysis, and graphical illustrations were conducted in R, Python, Excel , and GraphPad Prism 5.https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE62811) and GSE79255 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE79255). The protein-protein interactions from this publication have been submitted to the IMEx consortium [The ChIPseq and microarray data discussed in this publication have been deposited in NCBI's Gene Expression Omnibus  and are nsortium , 47 thronsortium  and assiYWHAE, YWHAH, and YWHAZ with schizophrenia and bipolar disorder [PBRM1 locus has surpassed genome-wide significance in both schizophrenia and bipolar disorder GWASs [In order to identify PPIs of the two BRD1 isoforms (BRD1-S and BRD1-L), we generated HEK293T cells stably expressing either V5 epitope tagged BRD1-S or BRD1-L (referred to as BRD1-S and BRD1-L cells). Both epitope tagged BRD1 isoforms localized to the nucleus of the HEK293T cells  isolated from Brd1–/– mouse embryos [To identify BRD1 chromatin interactions and target genes, we conducted chromatin immunoprecipitation of epitope tagged BRD1-S and BRD1-L from the cell lines described above  software to identify enrichment of specific pathways. Three datasets  were analyzed according to gene categories  and pathways as defined by IPA. After correction for multiple testing, we identified two, one, and 21 significant IPA categories, as well as, 14, six, and 27 significant canonical pathways among the three tested BRD1 interaction networks, respectively  supporting that BRD1-S and BRD1-L are involved in regulating expression of genes when bound in close proximity to the TSSs. Within these segments, upregulation of either BRD1-S or BRD1-L resulted in more upregulated compared to downregulated genes while downregulating BRD1 resulted in more downregulated compared to upregulated genes. Furthermore, upregulation of BRD1-L proportionally generated more downregulated genes compared to BRD1-S in these segments  is illustrated in Fig. nts Fig. . We concnts Fig. . This suIn order to investigate BRD1 gene-regulatory potential while considering all genes on the array (and not only DEGs), we segmented and calculated the average fold change of all probes within windows +/– 10 kb and +/– 200 kb from BRD1 binding sites similarly as in the previous analysis. Overexpressing BRD1-S primarily increased the overall expression fold change of genes with BRD1-S binding approximately +/– 1 kb from their TSSs while downregulating BRD1 reversibly downregulated genes within the same range  for all brain regions whereas a high BRD1 expression in the cerebellar cortex was observed throughout the timeline of the datasets  to –1 (complete anti-correlation with BRD1 expression). In total, 1241 and 672 interaction partners (PTGs + PPIs) for BRD1-S and BRD1-L, respectively, were identified in the Brainspan database ; in particular, SUV420H1, DNMT1, PBRM1, and CHD4 were highly correlated with BRD1  we investigated whether the BRD1 interaction network is enriched for mental disorder risk applying the MAGMA program . As the gion see , 468 of Padjusted = 0.03, Fig. P <0.05). These results suggest that the BRD1-S network may play a larger role in mental disorder risk compared to the BRD1-L network.In order to explore whether the interaction network might also be enriched for susceptibility to other mental disorders, we tested 12 other GWAS datasets, comprising eight psychiatric disorders and four disorders not considered related to brain function. In addition, to investigate whether the risk enrichment is dependent on the specific isoform, we stratified for BRD1-S and BRD1-L sub-networks. When adjusting for the number of GWASs tested, significant enrichment was observed solely for the entire BRD1 network in schizophrenia  [Finally, we explored whether the network was enriched for de novo mutations and rare disruptive variants observed in autism and schizophrenia –42. The SUV420H1 , 41, as In summary, we identify the BRD1 interaction network to be enriched for schizophrenia risk thereby providing supporting evidence that BRD1 plays a role in the etiology of schizophrenia. Additionally, our analyses suggest that the BRD1-S interaction network is more enriched for mental disorder risk, including schizophrenia risk, compared to the interaction network of BRD1-L.BRD1 in the human brain, we applied an approach similar to the one described here [BRD1 for each of the 32 intervals. Previously we showed that the majority of genes in the BRD1 network are positively correlated with BRD1 expression across all samples in Brainspan, including the BRD1 PPI networks. To gain insight into which spatiotemporal intervals that may be driving this positive correlation, we investigated the fraction of BRD1 co-expressed genes in the BRD1-S and BRD1-L PPI networks and compared them to the background fraction across all genes in the Brainspan dataset. A higher fraction of co-expressed genes was observed for both networks during pcw 16–26  in the prefrontal cortex regions (R2) and the hippocampus, striatum, and amygdala regions (R3)  Fig. . The BRDP value <0.05. The highest fraction of genes co-expressed with BRD1 consistent across all BRD1 networks was observed for pwc 16–26 (P2) in the hippocampus, striatum, and amygdala regions (R3)  and the BRD1 schizophrenia sub-network consisting of the 468 genes that achieved a gene-wide R3) Fig. . All BRDval Fig. . We notials Fig. .In this study we have obtained results providing novel insights into the molecular interactions of BRD1 and its relation to mental disorders.Our results provide confirming evidence that BRD1 is part of a histone acetyltransferase complex comprising ING4, ING5, MEAF6, and MYST2 , 49 alonInterestingly, we identify several novel protein interactions that are important for chromatin modulation and gene regulation, including PBRM1 which is part of the SWI/SNF chromatin remodeling complex PBAF  and the Although the identified PPI networks of BRD1-S and BRD1-L seem to comprise a common set of core proteins, we identify several isoform-specific PPIs that could indicate a significant functional difference between the two isoforms. For instance, the histone methyltransferase SUV420H1 was identified only for the BRD1-S isoform and QSER1, a relatively uncharacterized protein, was only identified for BRD1-L. In addition, BRD1-S and BRD1-L had a relatively limited overlap of PTGs as well as DEGs suggesting that these two isoforms, although very similar in amino acid sequence, target and regulate different gene sets. By integrating ChIP-seq and expression data, we were able to show that BRD1-S primarily regulate gene expression in proximity to TSSs while BRD1-L, in addition to regulating gene expression in proximity to TSSs, has the capacity to regulate gene expression further up- and downstream from its binding sites. Based on our observations, it is tempting to speculate that BRD1-L is involved in mediating chromatin loop status thereby regulating the expression of distal genes similar to the mechanisms identified for SATB1 . Convers14-3-3 genes with schizophrenia and bipolar disorder [Ywhae+/– and Ywhaz–/– mice have been shown to display neurodevelopmental and schizophrenia-associated phenotypes [PBRM1 gene has been associated with susceptibility to schizophrenia and bipolar disorder [Several studies have implicated disorder –55, 68, disorder , and Ywhenotypes , 71. Furdisorder , 72, 73.In addition to the similarities and differences of the molecular and cellular functions of BRD1-S and BRD1-L, we explored their interaction networks for enrichment of disease risk. We observed that the entire BRD1 network is enriched for schizophrenia risk and we found that the BRD1-S network was generally more enriched with schizophrenia risk and perhaps other mental disorders compared to BRD1-L. Moreover, the entire BRD1 network was more enriched with schizophrenia compared to the BRD1-S network, suggesting an enhanced effect of combining the networks from both isoforms. Our results support a recently published large-scale study of autism risk genes which demonstrates the importance of considering splice isoforms when exploring disease networks . It shouAlthough the BRD1 interaction network was not identified to be enriched with bipolar disorder risk genes, it is reasonable to speculate that this difference is based on power constraints in the original GWAS rather than on a less prominent role of BRD1 in the etiology of bipolar disorder. Future studies in larger bipolar disorder GWAS datasets are warranted to confirm this hypothesis. In general, the smaller GWAS sets may be underpowered which should be taken into consideration when interpreting the results.Further integrative analyses using spatiotemporal transcriptome data from the human brain suggested that both BRD1-S and BRD1-L interaction networks play a role in brain function and schizophrenia, especially at mid-fetal stages (pcw 16–26) in the hippocampus, amygdala, and striatum. However, the BRD1-S networks seem also to play a role in brain function and schizophrenia later in life, particularly during childhood and early adulthood. These observations are supportive of the enrichment analysis showing the highest enrichment of schizophrenia risk when analyzing the entire BRD1 network as well as more enrichment of schizophrenia risk when analyzing the BRD1-S network compared to the BRD1-L network.BRD1 gene with schizophrenia and bipolar disorder [Here we expand the molecular and functional characterization of BRD1 and provide evidence that BRD1 acts as a regulatory hub in a comprehensive schizophrenia risk network and possibly risk networks for other mental disorders as well, thereby supporting previous association studies implicating the disorder . Further


Infection of human cells by human T cell lymphotropic virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein  to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response and, therefore, represent attractive components for a HTLV-1 vaccine. Recently, we demonstrated that recombinant forms of surface glycoprotein (sRgp46) or TM are capable of eliciting polyclonal antibody responses following vaccination in a murine model. Analysis of monoclonal antibodies derived from these animals indicates that they exhibit properties that are typical of those observed in natural human infections. We now demonstrate that neutralizing antibodies appear to recognize a limited set of surface accessible neutralising epitopes. We have mapped some of these epitopes at amino acid resolution and demonstrate that several overlapping epitopes within the receptor-binding domain of SU define a hotspot for recognition by neutralising antibodies. Surprisingly, some non-neutralising monoclonal antibodies also appear to recognise surface exposed epitopes of SU; models for sensitivity to neutralisation will be discussed. Finally, we demonstrate that many HTLV-1 infected patients direct antibodies to gp21 TM, but this activity is largely unable to block viral infectivity. The implications of our findings for HTLV-1 pathogenesis and vaccine development will be discussed.


C11-Vinyl-terminated self-assembled monolayers (SAMs) on silica surfaces are successfully modified in C–C bond forming reactions with dihalocarbenes to generate SAMs, terminated with dihalo-  cyclopropane motifs with about 30% surface coverage. In the latter chemical modification of the reactive groups of the pre-coated SAM has to be efficient enough such that a reasonable conversion can be obtained, with chemical specificity and lack of surface degradation. In this respect ‘click’ reactions have become attractive including azide–alkyne cycloadditions [Self-assembled monolayers (SAMs) are increasingly being used as a means of surface modification to alter properties in a tuneable manner –3. The mdditions –10, Dieldditions –12, maledditions , thiol–edditions , and imidditions . In this2), dichloro- (:CCl2) and difluoro- (:CF2) carbenes [We recently reported the formation of high quality vinyl-terminated SAMs generated from the vapour phase by adsorption of octadecyltrichlorosilanes onto silicon wafers . With accarbenes –23. The 18-methyl (Me)-terminated SAMs, to ensure that only the vinyl group was responsible for surface reactivity. The resultant XPS analyses are shown for the vinyl-terminated SAMs for each carbene in 18-Me-terminated SAMs.After exposure to carbenes the vinyl-terminated SAMs were characterised by XPS, contact angle measurements and ellipsometry see . With XP2 generated from CHBr3. New peaks appeared at binding energies of 71 and 182 eV in all of the samples. These were assigned to Br 3d and Br 3p signals, respectively [2 generated from CHCl3. New signals at binding energies of 201 and 270 eV were detected. These were assigned to Cl 2p and Cl 2s [2 generated from TMSCF3. A new signal at binding energy of 688.7 eV was detected and assigned to F 1s, consistent with a CF2 group present on the surface [ectively –25. Figund Cl 2s . Finallygem-dibromo-, gem-dichloro- and gem-difluorocyclopropane-terminated SAMs will be formed, following the usual transformations of these carbenes with double bonds. To add further support to this expectation, model reactions were carried out under each of the reaction conditions with dec-1-ene ), where d is the film thickness (determined with ellipsometry) and λ the mean free path of the electrons. This accounts for the partial attenuation of the C3 XPS carbon signal. The water contact angles (CAs) of Br, Cl and F carbene treated surfaces were recorded and the CA values obtained of 80°, 85° and 104°, respectively, are in good agreement with the literature [3 terminated film (~118–120°) [Turning to the Cterature –30. Nota18–120°) , but thiFinally AFM images were recorded for the three dihalocyclopropane modified surfaces and they are shown in An alternative approach, also illustrated in In summary we have been able to demonstrate that vinyl-terminated SAMs can be chemically modified by a range of dihalocarbenes to generate surfaces carrying the corresponding dihalocyclopropane motifs. The reactions demonstrate that these organic chemical transformations, which have been relatively widely used in solution reactions of olefins, can be extended to surface reactions of SAMs. This opens up prospects too of modifying surfaces in this manner with carbenes carrying more elaborate functional groups, and thus a more dramatic change to the surface properties.3), chloroform (CHCl3) and the Ruppert–Prakash reagent (CF3Si(CH3)2) [3 or CHCl3 was stirred with a solution of benzyltriethylammonium chloride  in dichloromethane for 10 min at 0 °C. Pre-coated silicon wafers (1 cm × 1.5 cm) with C11-vinyl-terminated SAMs, were immersed in the reaction mixture and the liquids were stirred at room temperature for fixed periods of time 2) –34 were time see . SAMs on3) was used to prepare the gem-difluorocyclopropane-terminated SAMs by generating difluorocarbene, following the procedure of Wang et al. [3 in THF (2 mL), and then immersing the silicon wafers (1 cm × 1.5 cm) into the reaction mixture at 65 °C for a fixed period of time (see The Ruppert–Prakash reagent (TMSCFg et al.  for smaltime see . The expFile 1Synthesis protocols and additional surface analysis data.


Commercial dry food consisted of 90% of the food they provided, with 66% of them feeding once a day, with less food per cat per day than the other feeder groups. Interestingly, “heavy” feeders were usually singles, had on average fewer offspring, a clear preference for owning cats as pets, and lived in lower income neighborhoods. According to the feeders’ reports on the FRC populations they fed, 69.7% (2325/3337) cats were neutered and 11.8% (395/3337) were kittens. In addition, they reported that 1.6% (54/3337) of the cats were limping, 2% (67/3337) suffered from a systemic disease, 4% (135/3337) had skin lesions, and 3.9% (130/3337) were suffering from a chronic disability. Abundance of kittens and morbidity rate were significantly and negatively associated with neutering rate. These findings are in accordance with the suggestion that neutering may potentially improve cat welfare by reducing morbidity. Collaboration by the authorities with these heavy feeders, who represent a small number of FRC feeders and feed substantial FRC numbers, may be significant for the control and monitoring of FRC populations and their resources.Cat feeders serve as an important source of available food for free-roaming cats (FRCs) and can play a central role in providing data on FRC distribution, welfare, and health. Data on cat feeder personalities as well as a better understanding of their feeding practices offer relevance for decision making concerning FRC population control strategies. The current study surveyed 222 FRC feeders who responded to a municipal trap-neuter-return (TNR) campaign in an Israeli central urban setting. The aim of the study was to describe their personal characteristics, feeding practices, and the FRC populations they feed. Feeders were divided into four groups according to the number of cats they claimed to feed per day . Most feeders were women (81%), with a median age of 58 years (range 18–81). The feeders reported an overall feeding of 3337 cats in 342 different feeding locations. Feeders of group 4 comprised 15.31% ( Free-roaming cats (FRCs) living in close proximity to humans might constitute a potential public health risk and cause nuisances. Their potential to transmit several zoonotic diseases , has been well described in the literature –4. TheirIn Israel, active feeding of FRC provides an important food source for these cats, especially in urban settings . FeedingDespite the high abundance of cat feeders worldwide, there are only limited data available on their personal characteristics and motivation. In one study, most daily feeders were women aged 50–79 years , while iThe use of culling for controlling FRC populations in Israel was banned by the Israeli Supreme Court in 2004. Since then, the method of trap-neuter-return (TNR) is frequently used for controlling FRC populations and managing their associated nuisances. In addition, it was previously suggested that neutering might improve cat health and thereby contribute to their welfare –15. LocaHence, the study objectives were (1) to describe the feeding practices of FRC feeders, (2) to describe the personal characteristics of FRC feeders, (3) to determine possible associations between feeders’ personal characteristics and their feeding practices, and (4) to determine the association between reported neutering rates and health and welfare conditions of FRC.The study population comprised cat feeders who were registered with the municipal veterinary services of the city of Rishon-Lezion, Israel. The total population of Rishon-Lezion was 237,600 citizens at the end of 2013 . Data consisted of all requests to the municipality for FRC trap-neuter-return (TNR) by cat feeders, registered with the municipal veterinary services between February 2010 and October 2013. These requests followed municipal advertisements in the local press, encouraging citizens to report on preferred locations for municipal TNR actions to take place. The respondents were requested to provide information on location and neighborhood for the TNR requested actions, number of cats to be neutered, date of application, name of applicant, and applicant’s phone number. In addition, for each request, the applicants were asked if they consider themselves as cat feeders. Of the total request list, double applications and non-feeders were excluded from further consideration. This dataset was the basis for a cross-sectional telephone interview study, which was performed in the Hebrew language by one of the authors , between July 16 and October 20, 2013. Calls were made at different times of the day and on different days. This enabled maximal opportunity to contact the applicants, aiming to contact as much feeders listed in the dataset. At least three attempts were made to contact each applicant. Duration of interviews varied between 5 and 30 min, depending on the number of fed cats and feeding locations.n = 10). A total of 16 questions were divided into three sections related to the following issues: (1) feeding practices, (2) description of the feeding cats, and (3) personal details of the feeder. Questionnaire is provided in the supplementary information. Cat feeders were asked first for the locations and frequency of feeding, type and amount of food they provide, number of cats they feed in each feeding group, and whether they routinely also provide water to the FRC. They were then asked to provide their most accurate estimation of the number of kittens (up to 6 months old), neutered cats (marked in Israel by ear tipping), cats with systemic diseases , skin lesions, and permanent disability (such as eye absence and limb deformation or absence), among the FRC they had fed during the 2 weeks prior to filling in the questionnaire. Finally, cat feeders were asked for their year of birth, home address, marital status, number of offspring, number of offspring living with them, and for the species and number of their pets.The questionnaire was developed based on the study aims and was validated by a pilot telephone survey to cat feeders that fed cats in another city . Further data for each statistical area included education level of citizens aged 25–54 (presented as the average number of years of education), percentage of people aged 25–54 who possessed an academic degree, and average income per citizen [in New Israel Shekels (NIS)].Data on feeders’ neighborhoods were obtained from the 2008 population census of the Israeli Bureau of Statistics. For this census, the State of Israel was divided into 1616 geographic areas, called “statistical areas.” These areas were determined according to spatial features and roughly according to homogeneity of socioeconomic characteristics. The city of Rishon-Lezion was divided into a total of 63 statistical areas, of which 58 were residential areas with an average population of 3000 citizens each. The statistical areas were graded according to socioeconomic status, calculated using multiple criteria .® for Windows® or WinPepi version 4.0 (2007). In all analyses, a significance level of p < 0.05 was applied.Personal characteristics of feeders, their feeding practices, and the details of the cats they fed were summarized on an Excel datasheet. Spatial distribution of feeders’ home addresses and feeding locations were summarized using Arcmap 10.2.1. Analyses were performed using either SPSS 21.0In order to examine trend association of feeding practices and personal characteristics in relation with the number of fed FRC per feeder, cat feeders were arbitrarily divided into four groups according to the total number of cats they had claimed to feed per day: (1) fed up to 5 cats, (2) fed 6–10 cats, (3) fed 11–20 cats, and (4) fed 21 cats and more. These ranks are referred to below as feeder groups 1–4, respectively.2 for trend was performed to examine trend among feeder groups 1–4. For continuous data, we first visualized Q–Q plot for normal distribution and then performed Leven’s test for homogeneity of variance. To examine trend among groups 1–4 of variables with normal distribution and similar variances between groups, a Pearson correlation coefficient was calculated. For all other cases, a non-parametric Spearman correlation coefficient was calculated for trend. For analyzing distance between home address to feeding locations, data of five feeders whom home address were out of the city of Rishon-Lezion were omitted.For categorical data, χT-test. Gender differences between the feeder population and the general adult population of the district were compared, using chi-square test.Age distribution of the feeders was compared to age distribution of the general adult population, using independent samples Association between neutering rate of FRC per feeder and the presence of kittens and health status of the cats per feeder was determined by fitting a binary logistic regression model to each of the dependent variables separately , controlling for feeder’s group rank. This analysis was performed only for feeding groups of whose size exceeded five cats.A total of 1092 TNR requests were registered at the Veterinary Services of the city of Rishon-Lezion between February 2010 and October 2013. A total of 364 FRC feeders were identified in these requests. Of these, 142 feeders did not participate in the survey: 5 due to language barrier, 29 refused, 46 were unavailable despite three phone calls, 52 were no longer FRC feeders, 9 had left the city, and 1 was deceased. Not all respondents answered all the questions, due to differences in their willingness to share personal data.n = 34) of all feeders but fed 56% (n = 1869) of the FRC in 37.4% (n = 128) of the feeding locations  reported feeding FRC on a daily basis. The proportion of these daily feeders was significantly associated with feeder group category (Table p < 0.001). Feeder age distribution  was higher than the age of the general adult population . Overall, most feeders were married , while the rest  were single, divorced, or widows. Most feeders had offspring , but 37.7% of them (66/175) were no longer living with their offspring during the survey period. Most feeders owned pets , 65.2% (103/220) owned pet cats (in addition to the FRCs they fed), 54.4% (86/220) owned dogs, and 7.59% (12/220) owned other pets. Due to the small number of feeders who owned pets other than cats and dogs, their data were not included in further statistical analysis of pet ownership among feeders.Most feeders  were women, with almost no difference in this sex ratio among the four feeder groups. This sex ratio is much higher than the percentage of women in the general adult population in Rishon-Lezion , significantly more of the “heavy” feeders (groups 3–4) were not married, did not have offspring, did not live with their offspring, and owned pets, mainly cats. No significant differences were found in the percentage of dog ownership or the average number of pet dogs owned ; “heavy” feeders lived in neighborhoods with lower income. The number of feeders per 1000 citizens was not associated with residential neighborhood average income .No trend association was found between feeder group and socioeconomic parameters in the residential neighborhoods Table . HoweverAccording to the feeders’ reports on the FRC population they fed, 69.7% (2325/3337) of cats were neutered and 11.8% (395/3337) were kittens; in addition, they reported that 1.6% 54/3337) of the cats were limping, 2% (67/3337) suffered from systemic disease, 4% (135/3337) had skin lesions, and 3.9% (130/3337) were cats suffering from chronic disability. Neutering rate had a significant negative association with the number of kittens and with all types of illness .We found that FRC feeders varied in the number of cats they feed, from only a few up to a few hundred cats per day. Our findings reveal that in our survey, most of the cats were fed by a small number of feeders. These “heavy” feeders, especially those who belong to feeder group 4 (who fed ≥21 FRC) are characterized by unique feeding practices and personal characteristics. In comparison to “light” feeders (feeders of groups 1–2), these feeders traveled significantly longer distances in order to feed FRC, most of them were daily feeders, delivered significantly less food per cat per day, and fed the FRC mostly with commercial dry food. These data indicate that most feeders of a large number of cats (range 21–320 cats per feeder) kept to a routine, in which they walked or even traveled by car to distinctive feeding locations on a daily basis. They delivered commercial dry food, which is more economic, constantly available, and more convenient for handling. Furthermore, the percentage of leftovers used for feeding was significantly and negatively associated with feeder group rank, in which feeders in group 1 delivered the largest percentage of leftovers. Since leftovers can be considered as the most economic but least suitable food for cats, we conclude that in comparison to “heavy” feeders (groups 3–4), “light” feeders (groups 1–2) probably invest fewer resources on average in feeding each cat.ad libitum dry food per day per adult intact cat was 53.1–57.8 g, and for sterilized adult cats 52.0–72.8 g  is not reached –31. SuchTo the best of our knowledge, this is the largest study to date to characterize feeders of FRCs and the only one to address the characteristics of extremely “heavy” cat feeders. Despite their low numbers, these feeders are dominant among the cat caretakers and their contribution to the availability of vital resources to these cats is significant. Therefore, we believe that this population of feeders should be taken into consideration in any program for managing FRC populations. Such suggested program should include predefined feeding locations, in which continuing actions of TNR will be performed. The approach of feeding in predefined locations will enable authorities to control and monitor neutering rate, cat nuisances, and cat population resources. Authorities might have the aid of devoted feeders by reporting on feeding locations (other than the predefined sites), monitoring cat populations in the predefined sites, and even assist in cat capturing during TNR campaigns. The fact that heavy feeders often travel long distances in order to feed the cats may contribute to their willingness to feed at such predefined sites. Understanding the characteristics of these “heavy” feeders may aid in understanding their incentives and motivation, and may improve communication of the authorities with them.IG: planned the research, managed data collection, analyzed the data, and drafted the manuscript. TR: planned the research, reviewed, and participated in writing the manuscript. YZ: planned the research, involved in data collection, and reviewed the manuscript. YB: collected the data and reviewed the manuscript. EK: planned the research, analyzed the data, reviewed, and participated in writing the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pneumocystis jirovecii pneumonia. Disease progression of smoldering ATL along opportunistic infections was observed with very high levels of serum sIL-2R  and an increased percentage of CD4+CD25+ T cells (87.2%) in peripheral blood. In patients with suspected opportunistic infections, both HTLV-1 and HIV should be considered. In HTLV-1-positive patients, an increase in the CD4+CD25+ T cell subset may have its value as a prognostic marker.As an acquired immunodeficiency, human immunodeficiency virus (HIV) infection is primarily responsible for opportunistic infections in infected patients. However, opportunistic infections also occur in individuals with human T cell lymphotrophic virus type 1 (HTLV-1) infection. Here, we report opportunistic infections in two Japanese HTLV-1-seropositive patients. The first patient was a 67-year-old male, who had cytomegalovirus infection associated with esophagogastritis and terminal ileitis. The patient was HTLV-1-positive and was diagnosed with smoldering adult T cell leukemia (ATL). High levels of serum soluble IL-2 receptor  and an increased percentage of CD4+CD25+ T cells (75.5%) in peripheral blood were also detected. The second patient was a 78-year-old female, a known asymptomatic HTLV-1 carrier, who presented with persistent herpes zoster, followed by  Pneumocystis jirovecii pneumonia (PCP). Both patients showed significant increases in the CD4+CD25+ T cell subpopulation in peripheral blood in addition to opportunistic infections.Human T cell leukemia virus type 1 (HTLV-1) infection is associated with asymptomatic HTLV-1 carriers and with the development of inflammatory HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukemia (ATL). Furthermore, HTLV-1 is associated with the development of opportunistic infections . The risIn November 2014, a 67-year-old male  presented with epigastralgia, anorexia, and excessive weight loss (10 kg per month). Endoscopic studies revealed multiple small ulcers extending from the cardiac to the pyloric region and an irregular shaped ulcer in the angular region of the stomach; however, a biopsy was not performed and the patient was treated with a histamine H2-receptor antagonist. Two months later, the patient presented at our clinic with a high fever, epigastralgia, diarrhea, and continuous anorexia. On admission, the patient was neither anemic nor icteric. No lymphadenopathy was noted. The blood test results are summarized in  Pneumocystis jirovecii (9 × 105/μg DNA) in bronchoalveolar lavage fluid by quantitative polymerase chain reaction. She also had elevated levels of serum β-D-glucan   had tested positive for HTLV-1 with a high titer of anti-HTLV-1 antibodies of 1 : 4,096 four years earlier. During the next 4 years, the patient remained asymptomatic for HTLV-1 infection and did not undergo any specific treatment. In December 2014, the patient was hospitalized with herpes zoster in the left flank region. Laboratory data from 2010 and 2014 are shown in 0 pg/mL) . The pne0 pg/mL)  led us tAlthough HTLV-1 carriers do not necessarily develop opportunistic infections , Tashiroβ-D-glucan levels in case 2 reflected PCP activity; this is consistent with the findings of previous reports [HTLV-1-related opportunistic infections are widely recognized; indeed, CMV gastritis, gastroenterocolitis , 14, PCP reports , 21. Strongyloides hyperinfection [The precise mechanisms underlying the immunocompromised status in HTLV-1-infected individuals remain unknown. Yasunaga and coworkers  suggestenfection . Furthernfection , 7. In bTreatment of HTLV-1-associated opportunistic infections is rather difficult due to the immunodeficient status of the patient and the infiltration of various organs by HTLV-1-infected cells. Antiviral agents such as acyclovir and ganciclovir alone do not cure the disease. Aggressive chemotherapy may be required to kill HTLV-1-infected CD4+ cells, but increasing the risk of opportunistic infections and further immunosuppression. The cases described herein were not candidates for allogeneic hematopoietic stem cell transplantation  due to their age. We attempted to treat case 2 with etoposide ; however


PCGF1 encodes one of six human Polycomb RING finger homologs that are linked to transcriptional repression and developmental gene regulation. Individual PCGF proteins define discrete Polycomb Repressor Complex 1 (PRC1) multi-protein complexes with diverse subunit composition whose functions are incompletely understood. PCGF1 is a component of a variant PRC1 complex that also contains the BCL6 co-repressor BCOR and the histone demethylase KDM2B. To further investigate the role of PCGF1, we mapped the physical interactions of the protein under endogenous conditions in a cell model of neuronal differentiation. Using stringent statistical cut-offs, 83 highly enriched interacting proteins were identified, including all previously reported members of the variant PRC1 complex containing PCGF1, as well as proteins linked to diverse cellular pathways such as chromatin and cell cycle regulation. Notably, a sub-network of proteins associated with the establishment and maintenance of pluripotency  were found to independently interact with PCGF1 in a subsequent round of physical interaction mapping experiments. Furthermore, knockdown of PCGF1 results in reduced expression of DPPA4 and other subunits of the variant PRC1 complex at both mRNA and protein levels. Thus, PCGF1 represents a physical and functional link between Polycomb function and pluripotency. The regulation of gene expression through epigenetic mechanisms operates at several levels. These include modification of DNA itself, modification of the histone proteins in contact with DNA, as well as higher order regulation involving ‘remodelling’ and three-dimensional rearrangement of chromosomes to increase or decrease accessibility to the DNA by transcription factorsDrosophila, where mutants display a variety of developmental phenotypes, suggesting a critical role for Polycomb genes in the regulating of genes involved in cell fate determination and differentiation46789One family of such complexes are known as Polycomb Repressive Complexes (PRC)2Additional core components include a chromodomain-containing protein  and a Polyhomeotic protein 6101110The mechanism by which PCGF1/PRC1 is recruited to target genes is not fully understood, but one component, KDM2B  houses a Zf-CxxC domain that has affinity for CpG-rich DNA1515161719The combination of affinity purification and high resolution/high mass accuracy mass spectrometry has significantly increased the power of protein interaction mapping experiments in recent years. Exogenously expressed affinity-tagged proteins can be captured using reagents that recognize tags such as FLAG, HA, and poly-histidine. Alternatively, immunoprecipitation using antibodies specific to the target protein can be used, with the advantage that the endogenous protein is sampled in its native molecular environment. Affinity tagging approaches were used to identify interactors of PCGF1 and the five other PCGF homologs1014trans retinoic acidin situ on agarose beads to yield soluble peptides. The peptides were desalted, adsorbed onto C18 zip tips, eluted in high acetonitrile, and separated online by nano-chromatography interfaced with a Q Exactive mass spectrometer   as a negThe set of PCGF1 interacting proteins was analysed for enrichment in annotated functional properties using the BiNGO Gene Ontology network tool324We next focused on describing the physical PCGF1-containing complex(es). We estimated the relative subunit stoichiometry of PCGF1 to its interaction partners using mass spectrometry ion signal, and we used size exclusion chromatography to attempt to resolve complexes of differing size. Ion signal intensity can serve as a semi-quantitative measure of relative protein content by summing the intensities recorded for peptides unique to parent proteins, adjusting for protein size and the presence of computationally predicted ionizable peptides, and normalizing the data to the targeted proteinIn our experiment, DPPA4 showed an elution pattern distinct from that of PCGF1. DPPA4 appeared to be present in a high mass complex of ~2 MDa, a smaller complex of ~300 KDa, and in a low mass form ~50–100 KDa). The high mass complex containing DPPA4 may be similar or identical to variant PCGF1/PRC1, since it has a similar elution profile to BCOR, and is similar to the PCGF1-containing vPRC1 described by others, for example those observed when affinity-tagged PCGF proteins were stably expressed in 293TREx cells by Gao and coworkers0 KDa. ThNANOG and OCT4 and the repression of differentiation genes such as HOXA9), we reasoned that the sub-network of proteins linked to maintenance of pluripotency  may share interaction partners with PCGF1. Although we did not detect it in our initial PCGF1 physical interaction screen, the pluripotency gene OCT4 has also been functionally linked to Polycomb function2729Since the NT2 cell model displays a partial pluripotency phenotype  protein, raising the possibility of involvement of PCGF1 in the regulation of these pluripotency factors . This raises the possibility that PCGF1 is involved in the regulation of NANOG and DPPA4 at the level of transcription. However, ChIP experiments designed to investigate this did not perform in our hands using this antibody.The reduced NANOG and DPPA4 that we observed could originate from: a) reduced mRNA expression because PCGF1 is a direct or indirect regulator of the downstream genes, or b) increased degradation of NANOG and DPPA4 protein in the absence of a (direct or indirect) stabilizing interaction with PCGF1. In order to investigate these possibilities, we used qPCR to measure the expression of mRNA following knockdown of PCGF1  and founIn order to map the physical interactome of PCGF1 in a context as close as possible to its native environment, we used an endogenous immunoprecipitation approach in a cell line with neural progenitor cell properties. We detected an interaction between PCGF1 and DPPA4 that seems to be independent of other members of the vPRC1 complex. Both DPPA4 and a related family member, DPPA2 (35% amino acid sequence identity), encode SAP (Scaffold attachment factor – Acinus – Protein inhibitor of activated STATs) domains, are close together on chromosome 16, and are expressed in pluripotent cells and developing germ lines32333437DPPA4 using shRNA was previously found to decrease expression of pluripotency markers like OCT4, NANOG and REX-1 in differentiating ES cells, and this result  was interpreted as indicating a role for DPPA4 in ES cell renewal and inhibition of differentiation markersDPPA4, nor evidence for an essential role in self-renewal or pluripotencyDPPA4 null mice died late embryonically/perinatally and displayed defects in the skeletal development, which suggests that DPPA4 plays an important developmental role. However as the allele was not conditional so they were unable to test for a maternal effects which could manifest at a very early developmental stages. Interestingly, DPPA4 was also recently found in a screen for oncogenic foci-inducing genesDPPA4 led to up-regulation of transcripts for genes such as CCNB1, CCND1, E2F1, and MTBP. Variant PRC1 itself has been implicated in the regulation of cellular proliferation. The variant PRC1 protein KDM2B was found to influence the regulation of proliferation and senescence through regulation of the INK4A and INK4B loci3940Knockdown of NANOG and DPPA4, or acts indirectly, for example by repressing expression of a negative regulator of NANOG and DPPA4. The observations raise the possibility of the presence of a network of interrelated feedback loops that serve to regulate gene expression among the network. In this context, the recent finding that over- and under-expression of PCGF1 correlates with expression of pluripotency factors NANOG, OCT4 and SOX2 in P19 embryonal carcinoma cells is of interestPCGF4 at the level of mRNA expression. For example, PCGF2 as been shown to repress PCGF4 in human fibroblast cellsA notable outcome of our work is the finding that disruption of PCGF1 expression leads to decreased expression of: a) some, but not all, members of the variant PRC1 complex; b) at least one other PCGF ortholog ; and c) pluripotency factors including DPPA4 and NANOG. The presence of PCGF1 is associated with active expression of NANOG and DPPA4. It is unclear if our data indicates that PCGF1 directly stimulates transcription of PCGF4 expression. By corollary, disruption of PCGF1 activity in our shRNA experiment could lead to increased PCGF2 and therefore reduced PCGF4 expression. Further genetic and biochemical studies will hopefully shed light on the direction and reciprocity of Polycomb-Polycomb and Polycomb-Target regulatory mechanisms within pluripotency networks, the mechanisms by which they operate (i.e. direct or indirect), and the nature of the PRC1-related complexes involved.If such a mechanism operates in NT2 cells, then decreased PCGF2 expression arising from PCGF1/PRC1-mediated repression could plausibly lead to increased HEK293T cells  were cultured in 92 mm Nunclon tissue culture dishes (Fisher Scientific) in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum (Hyclone), 100 U/ml penicillin and 100 U/ml streptomycin (Gibco). Cells were passaged by trypsinizing with 0.25% Trypsin-EDTA (Invitrogen) and plated at a ratio of 1:10. NT2/D1/D1 cells  were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% (v/v) Fetal Bovine Serum (Hyclone), 100 U/ml penicillin and 100 U/ml streptomycin (Gibco). Cells were passaged by trypsinizing with 0.25% Trypsin-EDTA (Invitrogen) and plated at a ratio of 1:6. To induce neuronal differentiation, 10 μM all-trans retinoic acid (Sigma) was added to media once cells reached a density of ~50%. During the 8-day differentiation time course media was changed every 2–3 days.For total lystate analysis, cells were resuspended in RIPA buffer . Lysates were incubated for 15 min on ice and cell membranes disrupted mechanically by syringing 5 times with 23G narrow gauge needle and sonicating 3 × 2 s at high power. Lysates were incubated on ice for another 15 min and lysates pre-cleared by centrifugation at 14,000 rpm 4 °C 30 min to remove cellular debris. For analysis of nuclear fraction, lysates were resuspended in an equal volume of Buffer C  and dounced 20 times with tight pestle type B (Tight). Lysates were incubated for 45 min rotating to dissociated chromatin-bound proteins and pre-cleared by centrifugation at 14,000 rpm 4C for 30 min to remove cellular debris and intact chromatin. Immunoblotting was performed using antibodies described in 10 μg antibody was coupled to 50 μL packed Protein A beads (Sigma P9424) by incubation in 1 mL PBS (0.1% Tween-20) at 4 °C rotating overnight. Beads were collected by centrifugation at 1700 × g for 3 min and washed twice in 1 mL 0.2 M Sodium Borate pH 9.0. Antibodies were then crosslinked to beads by incubation in 1 mL 0.2 M Sodium Borate pH 9.0 (20 mM dimethyl pimelimidate dihydrochloride) at room temperature rotating for 30 min. Reaction was terminated by washing beads once in 1 mL 0.2 M Ethanolamine pH 8.0 and incubating for 2 hr at room temperature rotating in 1 mL 0.2 M Ethanolamine pH 8.0. Beads were washed twice in Buffer C100 and blocked for 1 h min 4 °C rotating in Buffer C100 , 0.2 mg/mL Chicken egg albumin (Sigma A5503), 0.1% (v/v) fish skin gelatin (Sigma G7041). Antibody-crosslinked beads were incubated with nuclear lysates, in the presence of 250 U/mL Benzonase nuclease, at 4 °C rotating overnight and washed 5 × 5 min in Buffer C100 (0.02% NP-40). After the final wash, beads were resuspended in 50 μL 2x SDS sample buffer. Immunoprecipitated material was eluted by boiling for 5 min with shaking and associated proteins were separated by SDS-PAGE and analysed by immunoblotting.Immunoprecipitations (IPs) were performed on nuclear protein lysates prepared in low salt buffer containing protease inhibitors . IPs of FLAG tagged proteins were performed using M2 anti- FLAG agarose and mouse IgG agarose (Sigma) overnight at 4 °C. Elution of FLAG tagged proteins was performed at 4 °C using 250 ug/ml of 3xFLAG peptide (Sigma) in 0.05% NP40 with horizontal shaking. Eluted protein fractions were separated by SDSPAGE and analyzed by western blot or liquid chromatography mass spectrometry.TM 6 10/300 GL gel filtration column  was equilibrated with one column volume of running buffer . 300–500 μg of total nuclear protein (prepared as described above) was injected and run through column at 0.35 mL/min. 1 mL fractions were collected and protein was concentrated by incubation with 4 μL StrataClean resin (Agilent Technologies) for 1 hour at room temperature. Resin was collected by centrifugation at 5000 rpm for 3 min and protein was eluted by boiling in 20 μL 2X SDS sample buffer for 5 min shaking at 1400 rpm. Eluted protein analysed by SDS-PAGE and immunoblotting.The SuperoseProteins were treated with trypsin as describedHomo sapiens. The interactome networks obtained from this first screening were analyzed with BiNGO, a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networksThe data network design of protein-protein interactions (interactome network) between PCGF1, and the potential interactors in NT2 cells was performed using Cytoscape software, version 3.1.1. For this purpose, we used the interactome data of Extracted RNA was used to generate cDNA by reverse transcriptase PCR using the TaqMan Reverse Transcription kit (Applied Biosytems). Relative mRNA expression levels were determined using the SYBR Green I detection chemistry on LightCycler 480II Real–Time PCR System (Roche). The ribosomal constituent RPO was used as normalizing gene. Primers are listed in PCGF1-specific shRNA sequences were purchased from Sigma .


Tuberculosis (TB) is still a major health problem. IFN-γ and TNF are critical cytokines in the control of Mycobacterium tuberculosis (Mtb) infection. It has already been demonstrated that human T cell lymphotropic virus type 1 (HTLV-1) infection leads to a spontaneous IFN- γ and TNF production, however HTLV-1 infected subjects possess a 2-4 fold increased risk of developing tuberculosis. Nevertheless, the immune mechanisms involved in this phenomenon are still unclear. The aim of this study was to evaluate immunological features of the association between HTLV-1/Mtb in order to better understand the events leading to higher susceptibility to TB observed in HTLV-1 infected subjects. This was a cross-sectional study evaluating four groups: healthy control (HC group), HTLV-1 infected subjects without TB (HTLV-1 group) or with TB (HTLV-1 + TB group), and individuals with only TB (TB group). TNF, IL-1β, and IL-17 levels were measured in supernatants of non-stimulated or PPD stimulated peripheral blood mononuclear cells (PBMCs) by ELISA. PBMCs from HTLV-1 infected individuals had a decrease (p<0.05) in TNF production following PPD stimulation  compared to non-stimulated cells . When we normalized the data to non-stimulated cells, IL-1β production following PPD stimulation by individuals in the HTLV-1 + TB group  was lower (p<0.05) than in patients with TB . Similarly, HTLV-1/Mtb co-infected individuals had lower (p<0.05) production of IL-17  when compared to TB patients . Impairment in TNF, IL-1β, and IL-17 production upon stimulation with mycobacterial antigens may contribute to the higher susceptibility to Mtb infection observed in HTLV-1 infected subjects. Financial support: CNPq, CAPES.


Elevated uric acid (UA) is commonly associated with gout and it is also a known cardiovascular disease risk factor. In contrast to such deleterious effects, UA possesses neuroprotective properties in the brain and elucidating the molecular mechanisms involved may have significant value regarding the therapeutic treatment of neurodegenerative disease. However, it is not yet fully established how UA levels are regulated in the brain. In this study, we investigated the distribution of mouse urate transporter 1 (URAT1) in the brain. URAT1 is a major reabsorptive urate transporter predominantly found in the kidney.Immunohistochemistry of wild type and URAT1 knockout mouse brain using paraffin or frozen sections and a rabbit polyclonal anti-mouse URAT1 antibody were employed.Antibody specificity was confirmed by the lack of immunostaining in brain tissue from URAT1 knockout mice. URAT1 was distributed throughout the ventricular walls of the lateral ventricle, dorsal third ventricle, ventral third ventricle, aqueduct, and fourth ventricle, but not in the non-ciliated tanycytes in the lower part of the ventral third ventricle. URAT1 was localized to the apical membrane, including the cilia, of ependymal cells lining the wall of the ventricles that separates cerebrospinal fluid (CSF) and brain tissue.In this study, we report that URAT1 is expressed on cilia and the apical surface of ventricular ependymal cells. This is the first report to demonstrate expression of the urate transporter in ventricular ependymal cells and thus raises the possibility of a novel urate transport system involving CSF. Uric acid (UA) is the end product of purine metabolism in human and hyperuricemia is a known risk factor for gout, hypertension, renal disease, and metabolic syndrome . While hPhysiologically, UA concentrations are an order of magnitude lower in cerebral tissue compared to blood UA levels . InteresMultiple urate transporters in the kidney are involved in the regulation of serum UA levels (SUA). Renal urate reabsorption is mainly mediated by urate transporter 1 (URAT1) on the apical side of the renal proximal tubular cells, and the voltage-driven urate transporter (URATv1/Glut9) on the basolateral side ,9. In adIn the present study, we investigated the distribution of URAT1 in mouse brain by immunohistochemical analysis using an anti-URAT1 antibody , the speMale C57BL/6 J mice  and homozygous URAT1 knockout mice  were mai2, pH 7.5). Brains were removed and post-fixed overnight (for paraffin embedding) or 3 h (for cryoprotection) at 4°C in the same fixative. Brains were then cut coronally or sagittally and prepared for paraffin embedding or cryoprotection. For paraffin embedding, tissues were dehydrated in a graded series of alcohols, cleared with Hemo-De , embedded in Paraplast , and sectioned at 3 μm. After deparaffinization and rehydration, endogenous peroxidase activity was blocked by 0.03% H2O2 methanol solution for 10 min. Sections were then incubated for 1 h at room temperature in blocking solution containing 2% bovine serum albumin  in 0.01 M PBS with 0.02% Tween-20, followed by incubation with primary antibodies diluted in blocking solution at 4°C overnight . Immunostaining was visualized with EnVision™ Systems and DAB+, Liquid . To prepare cryostat sections (12 μm), post-fixed brains were cryoprotected in 15% sucrose (wt/vol) in PBS for 48 h at 4°C, embedded in Tissue-Tek OCT compound , and frozen on dry ice. Cryostat sections were fixed in methanol  and acetone , incubated for 1 h at room temperature in blocking solution containing 5% normal goat serum  in 0.01 M PBS with 0.1% Triton X-100, followed by incubation with primary antibodies diluted in blocking solution at 4°C overnight. The following antibodies were used for double staining: anti-URAT1, 1:1000; anti-acetylated-α-tubulin , 1:500. Sections were then incubated with Alexa 488- or Alexa 594-conjugated secondary antibodies  for 1 h at room temperature and mounted with Vectashield containing DAPI .Nine-week-old C57BL/6 J male mice and URAT1 knockout male mice were anesthetized by pentobarbital injection  and perfused intra-cardially with 4% paraformaldehyde in HEPES buffer . For immunofluorescence experiments, single-plane images were captured using a Nikon A1 confocal microscope  with identical settings.To investigate the distribution of URAT1 in the mouse brain, we performed immunohistochemistry upon adult mouse brain paraffin coronal sections by using a rabbit polyclonal anti-mouse URAT1 antibody . Strong Ependymal cells form an epithelial layer that lines the cerebral ventricles and function as a barrier between the cerebrospinal fluid and the brain parenchyma. Morphologically, these cells possess cilia and microvilli on their apical surfaces. At higher magnification, dense URAT1 positive signals were detected in cilia-like protrusions  , a majorAnother possibility is that UA incorporated into the ependymal cells is further transported into the brain parenchyma. The fact that we detected strong URAT1 expression in ependymal cells may indicate a novel trans-cellular transport mechanism for UA across ependymal cells. This is reminiscent of the UA transport system in the proximal renal tubular cells, in which UA is reabsorbed by URAT1 in the apical membrane and effluxed by URATv1/Glut9 in the basolateral membrane. If urate efflux transporters exist on the basolateral side of ependymal cells, then it is possible that CSF-derived UA could pass through the brain parenchyma and be finally incorporated into neurons and/or astrocytes. One could speculate that UA transport may occur at the blood–brain barrier (BBB). However, the provision of UA from blood to brain parenchyma seems unlikely, since at this point, immunohistochemical data indicate that the urate efflux transporter ABCG2 is the sole UA transporter localized on the luminal side of brain capillaries . CollectBased on epidemiological and clinical data showing that higher UA levels are associated with reduced risk and slower progression of PD, UA-elevating strategies have emerged as a potential therapy for PD . FurtherOur data suggests that the urate transporter URAT1 is expressed on cilia and the apical surface of ventricular ependymal cells in mouse brain. Ependymal URAT1 may thus be involved in a novel UA transport mechanism between CSF and brain parenchyma.CSF: Cerebrospinal fluid; UA: Uric acid; PD: Parkinson’s disease; SUA: Serum UA levels.The authors declare that they have no competing interests.NHT carried out immunohistochemical studies and drafted the manuscript. MN, KI, and TM carried out studies on URAT1 knockout mice. MD and YD designed the study. MH conceived the study, participated in its design and coordination, and helped draft the manuscript. All authors read and approved the final manuscript.


Mycobacterium tuberculosis infection but up to now it is unknown if HTLV-1 influences the severity of tuberculosis and if tuberculosis may influence the outcome of HTLV-1. The aims of this study were to determine the prevalence and severity of tuberculosis (TB) in HTLV-1 infected patients and analyze whether TB influences the outcome of HTLV-1 infection. This is a cross-sectional study, in which the prevalence of tuberculosis was analyzed in 166 HTLV-1 infected individuals. Cytokine productions were determined by ELISA, the proviral load was evaluated by PCR. Tuberculosis occurred in 33 (22%) of cases and 70 (42%) had a positive tuberculin skin test (TST) (latent tuberculosis). The majority of the cases did not have severe tuberculosis. There was no difference in the proviral load, but there was a higher frequency of HAM/TSP in the group with tuberculosis than in HTLV-1 infected subjects without TB (P<0.05). This study shows that the prevalence of M.tuberculosis infection is 6 fold higher in HTLV-1 infected individuals than that described in the Brazilian population the majority of co-infected had latent tuberculosis and HTLV-1 may influence progression from infection to HAM/TSP.HTLV-1 infects mainly T cells, leading to activation and cellular proliferation with exaggerated production of pro-inflammatory cytokines. The majority of HTLV-1 infected patients are considered as HTLV-1 carriers, but 5% will develop HTLV-1 associated myelopathy (HAM) and about 15% overactive bladder, an oligosymptomatic form of HAM. HTLV-1 is associated with increased susceptibility to National Institute of Health (NIH AI 079238) and CNPq.