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The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis
AM Havens1,
Y Jung1,
YX Sun1,5,
J Wang1,
RB Shah2,
HJ Bühring3,
KJ Pienta4 &
RS Taichman1
The chemokine stromal derived factor-1 (SDF-1 or CXCL12) and its receptor CXCR4 have been demonstrated to be crucial for the homing of stem cells and prostate cancers to the marrow. While screening prostate cancers for CXCL12-responsive adhesion molecules, we identified CD164 (MGC-24) as a potential regulator of homing. CD164 is known to function as a receptor that regulates stem cell localization to the bone marrow.
Using prostate cancer cell lines, it was demonstrated that CXCL12 induced both the expression of CD164 mRNA and protein. Functional studies demonstrated that blocking CD164 on prostate cancer cell lines reduced the ability of these cells to adhere to human bone marrow endothelial cells, and invade into extracellular matrices. Human tissue microarrays stained for CD164 demonstrated a positive correlation with prostate-specific antigen levels, while its expression was negatively correlated with the expression of androgen receptor.
Our findings suggest that CD164 may participate in the localization of prostate cancer cells to the marrow and is further evidence that tumor metastasis and hematopoietic stem cell trafficking may involve similar processes.
Bone metastases are present in approximately 85% of patients who die due to advanced prostate cancer. Tumor cells metastasize to bone via the venous plexus and spread to the axial skeleton, pelvis and spine. Although the pattern of metastasis of some cancers may be explained by anatomy of the efferent venous and lymphatic drainages, anatomy alone does not account for the metastatic pattern of most cancers [1]. Stephen Paget proposed the 'seed and soil' hypothesis in which seeds that represent metastatic cancer cells grow preferentially in the soil of bone matrix as an explanation for the clinical observations that prostate cancers preferentially localize to specific organs [2, 3]. These observations suggest that factors in addition to those that arise within the tumor itself contribute to the metastatic fate of cancer cells.
As for metastasis, hematopoietic stem cells also 'home' to the bone marrow. Here the molecular determinants of stem cell homing have recently been determined to be largely dependent on a CXC motif chemokine, stromal-derived factor-1 (SDF-1 or CXCL12), and its receptor, CXCR4. Studies of animals that lack the CXCL12 or CXCR4 genes (knockouts) are born with marrows devoid of hematopoietic activities, despite the fact that normal fetal liver hematopoiesis occurs [4, 5]. Further evidence of the importance of CXCL12 and CXCR4 to stem cell homing includes the observations that progenitor cells and other blood cells move towards gradients of CXCL12, progenitor cell engraftment into nude mice is blocked by anti-CXCR4 antibodies [6] and the migration of hematopoietic progenitor cells from the blood to the marrow is enhanced by the expression of elevated CXCR4 levels [7].
Based upon these observations, we hypothesized that prostate cancer (CaP) cells may use the CXCL12/CXCR4 axis to home to bone marrow [8–11]. We reported previously that CXCR4 expression is correlated with tumor grade [8], and that CXCL12 signaling through CXCR4 triggers the adhesion of prostate cancer cells to bone marrow endothelial cells [11]. Furthermore, antibodies that recognize CXCR4 block the metastatic spread of the prostate cancer cells to bone in an in vivo animal model [9]. Similar findings by others suggest that the CXCL12/CXCR4 axis may play a similar role in the metastasis of other tumors types to the marrow [12, 13], including breast cancers [13], melanoma [14], pancreatic, neuroblastoma and renal carcinomas [15, 16]. Importantly, our observations in prostate cancer have largely been substantiated by others [17, 18].
It is not clear how the CXCL12/CXCR4 axis promotes metastasis. One possibility is that the binding of CXCL12 to CXCR4 activates adhesion molecules that mediate the binding of prostate cancer cells to the bone marrow endothelium. To further elucidate the parallels between metastasis and hematopoietic stem cell homing, secondary data analysis of a large set of DNA microarray data derived from human primary and metastatic human prostate cancer cells published by Dhanasekaran et al [19] was performed to identify known and unknown receptors that might regulate stem cell homing. We found that the level of expression of CD164/endolyn/MGC-24v mRNA was greater in bone metastases than in primary tumors.
CD164 was first identified as MGC-24 and cloned as a carrier of a peanut agglutinin-binding site, a tumor-associated carbohydrate marker expressed in human gastric carcinoma cells [20]. The human CD164 gene is comprised of six exons (E1-6) that through alternative splicing are expressed as three distinct isoforms; a full-length isoform or CD164(E1-6), the originally characterized form lacking exon 5 (CD164(EΔ5)) and the CD164(EΔ4) variant where exon 4 is deleted [20]. Disulfide-linked 80–85 kDa homodimers are observed, which extracellularly are comprised of mucin domains linked by cysteine-rich motifs [21]. The remainder of the protein is comprised of a transmembrane domain and a 13-amino-acid intracellular region [22]. Like other sialomucins, CD164 is highly glycosylated by both O- and N-linked glycans [23]. Transmembrane forms of CD164/MGC-24 are believed to mediate the adhesive functions [21, 24], and are thought to regulate hematopoiesis by facilitating the adhesion of human CD34+ cells to bone marrow stroma [25].
In this report, we demonstrate that the full-length CD164 is expressed by human prostate cancer cell lines under basal conditions. CXCL12 enhances the expression of CD164 mRNA along with alterations in the expression of CD164 protein. Functionally, CXCL12 induced binding to bone marrow endothelial cells or invasion into extracellular matricies can be blocked with monoclonal antibodies that target CD164. Using prostate cancer tissue microarrays, it was noted that the staining intensity for CD164 correlated with increased prostate-specific antigen (PSA) expression. Importantly, CD164 expression in osseous metastasis was greater than that in soft tissue metastasis, most notably in comparison to those found in the liver and lymph nodes. These data suggest that CD164 may play an important role in localizing tumors not only to sites where there are high levels of CXCL12 expression, but also to specific tissue locales. The results of this study suggest that CD164 may play a central role in prostate cancer metastasis.
Primary and secondary antibodies
Antibodies targeting CD164 were generated in the laboratory of Dr. H J Bühring (Tübingen, Germany) and were described in detail previously [23]. These include antibodies targeting the class I CD164 epitope (clone 105A5, mIgM), the class II epitope of CD164 (clone 103B2, mIgG3) and the CD164 class III epitopes (Clones 67D2, mIgG1 and N6B6, IgG2a). In some experiments antibody from the N6B6 clone was purchased from BD PharMingen (San Jose, CA, USA) as well as murine isotype matched monoclonal controls. The control antibodies for these investigations included IgM clone A57H (Dako, Denmark) IgG3 clone 133316 (BD Pharmigen), IgG1 clone 11711, IgG2a clone 20102 (R&D Systems).
Prostate cancer cell lines (PC3, LNCaP C4-2B, and LNCaP), and bone marrow endothelial cells (HBME) were cultured in RPMI medium 1640, supplemented with 10% FBS, 1% penicillin-streptomycin, and 1% l-glutamine (Invitrogen, Carlsbad CA). PC3 cells were originally isolated from a vertebral metastasis and were obtained from American Type Culture Collection (Rockville, MD). LNCaP cells were originally isolated from a lymph node of a patient with disseminated bony and lymph node involvement. The LNCaP C4-2B cells were derived from the parental LNCaP cell line by serial passage in mice to obtain a more metastatic cell line [30]. The HBME cells were isolated from a Caucasian male and immortalized with SV40 large T-antigen [23]. All cultures were maintained at 37°C, 5% C02, and 100% humidity.
Secondary data analysis and DNA micro arrays
Secondary data analysis was performed on data published by Dhanasekaran et al. [19] as previously reported [8]. Using a 10 K human cDNA micoarray which included ~5,520 known, named genes and 4,464 ESTs, the authors determined the gene-expression profiles of RNA obtained from more than 50 normal and neoplastic prostate specimens [19]. Supplementary information from Dhanasekaran et. al. were imported into a Microsoft Excel database and data clustered into normal associated tissues (NAP), benign hyperplasia (BPH), localized cancer (PCa) and metastasis (Met) [19]. The normalized mean and standard deviation values for CD164 was evaluated for significance with Instat 4.0 (GraphPAD software) using one-way analysis of variance (ANOVA), with the level of significance set at p < 0.05.
To determine which of these genes are responsive to CXCL12 and represent known adhesion molecules, RNA from three independent studies, carried out in triplicate and pooled, was analyzed from CXCL12 treated LNCaP and LNCaP C4-2B cells (200 ng/ml, 2 hours,). The RNA was compared in triplicate against non-treated cells by DNA micro array using the Human genome U133 gene chips (Affymetrix Corp. Santa Clara, CA). Protocols and instrumentation setups, including total RNA samples, hybridization to the human micro arrays, washing, staining, and scanning were performed as recommended (Gene Chip Micro arrays; Affymetrix, Santa Clara, CA) by the University of Michigan Dental School Microarray Facility. The resulting arrays were scanned (HP Gene Array Scanner; Hewlett Packard, Palo Alto, CA), and data analysis performed first with the accompanying software to obtain average difference intensities (Gene Chip Expression Analysis Software, ver. 3.3; Affymetrix). Later, average gene intensity values were determined using a software package (DNA-Chip (dChip version 1.1)) [26] in consultation with the University of UMCCC Microarray Core Facility.
RT- and QRT-PCR
The prostate cancer cell lines at confluence were transferred to serum free media, and stimulated with CXCL12 at 0 or 200 ng/ml for 0.5–8 h. RNA was recovered using TrizolR according to the directions of the manufacturer (Invitrogen, Carlsbad, CA). RNA integrity and purity was evaluated by electrophoresis with ethidium bromide and absorbance at A260/A280. 1.0 μg RNA, 10X RT buffer (1X RT buffer: 50 mM Tris, pH 8.3, 50 mM KCl, 8.0 mM MgCl2, and 10 mM dithiothreitol), 25 mM dXTP mix (25 mM of each dXTP (ACGT)), 3.0 μg oligo d(T), and 2.5 U Reverse Transcriptase (M-MLV Reverse Transcriptase, Invitrogen, Carlsbad, CA) were incubated at 38°C for one hour. Amplification primers were designed using PrimerExpress™ software (Applied Biosystems, Foster City, CA) to cross intron/exon boundaries. Cross-reactivity was determined using BLAST [27], those with the lowest potential were synthesized. One-fifth of the double stranded product was then mixed with 10X Taq/RT buffer (1X Taq/RT buffer: of 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, and 2.0 mM dithiothreitol), 1 mM dXTP mix, 500 ng of each sense and antisense oligonucleotide, and 2.5 U Taq polymerase (AmpliTaq DNA Polymerase; Perkin Elmer Cetus, Norwalk, CT). Five pairs of forward and reverse primers were designed to identify the alternatively spliced transcripts of the CD164 gene and are presented in Table 1[20]. The samples underwent thermal cycling at 94°C for 1 min, 60°C for 1 min, and 72°C for 2 minutes for 35 cycles, followed by a 10-min extension at 72°C (Perkin Elmer Cetus DNA thermal cycler). The PCR product of PC3, LNCaP C4-2B, and LNCaP cell lines were analyzed on 12% polyacrylamide gels and ethidium bromide stained.
Table 1 CD164 primers and coding regions detected. Five set of oligonucleotide primers were designed to identify differential spliced transcripts derived from of CD164.
Table 2 Coding regions detected in human CD164. Primer sets and regions identified in human CD164.
Real time-PCR was also performed using random hexamers and 15.0 μl of SYBR® Green PCR Master for quantitative, two step real-time RT-PCR (Applied Biosystems, Foster City, CA) with 100 nM of the E1 and E2 primers (Table 2) and 2 μl of the RT product in a total volume of 30 μl. The 2nd step PCR reaction (95°C for 30 seconds, 60°C) was run for 40 cycles after an initial single cycle of 95°C for 15 minutes to activate the Taq polymerase. The PCR product was detected as an increase in fluorescence using an ABI PRISM 7700 instrument (Applied Biosystems, Foster City, CA). The mRNA levels were expressed as relative copies (% control) normalized against 18S mRNA (Ambion QuantumRNA™ 18S Universal Primers) and a standard curve constructed from serial dilutions of a purified CD164 cDNA fragment cloned by classical PCR.
Tissue microarray and immunostaining
High-density tissue micro arrays were constructed from clinical samples obtained from a cohort of over 600 patients, who underwent radical retro pubic prostatectomy at the University of Michigan as a primary therapy (i.e., no preceding hormonal or radiation therapy) for prostate cancer and from materials obtained from the University of Michigan Rapid Autopsy Program. The arrays were provided from the University of Michigan Comprehensive Cancer Center Tissue Core of the Prostate Specialized Program of Research Excellence program as detail previously [28]. Tumors were graded using the Gleason grading system and examined to identify areas of benign prostate, prostate cancer and bone metastasis. The formalin-fixed, paraffin-embedded tissues were deparaffinized and placed in a pressure cooker containing 0.01 M buffered sodium citrate solution (pH 6.0), boiled and chilled to room temp for antigen retrieval. The slides were incubated overnight at room temperature with anti-human CD164 antibody (BD Biosciences, San Jose, CA) diluted 1:100 (10 mg/ml), IgG2a (Clone 20102, R&D Systems, Minneapolis, MN). For PSA levels, antigen retrieval was performed in citrate buffer at pH 6.0. For AR staining, antigen retrieval was performed with EDTA at pH 8.0 in a pressure cooker. Polyclonal antibodies to PSA (1:2000 dilution, Dako Cytomation, Carpenteria, CA) and a monoclonal AR-clone AR 441 (1:50 dilution, NeoMarkers, Fremont, CA) were used for staining as previously reported [29]. A streptavidin/biotin detection method with 3,3'-Diaminobenzidine Tetrahydrochloride (DAB) was employed for signal detection and Harris hematoxylin was used as a counter-stain. Digital images were then acquired with the BLISS Imaging System (Bacus Laboratory, Lombard, IL). Immunostaining intensity was scored by a genito-urinary pathologist: as absent (1), weak (2), moderate (3) or strong (4). Scoring was performed in a blinded fashion using the Profile web based telepathology system without knowledge of overall the tumor grade, tumor size or clinical outcome [30, 31].
Cell-cell adhesion assays
Prostate cancer cell lines were labeled with Vybrant CFDA SE Cell Tracer Kit, (Molecular Probes, Inc., Eugene, Oregon) for 30 min in RPMI according to the recommendations of the manufacturer and washed, and rested for 30 min. CXCL12 pretreatment was performed by incubating the CaP cells with PBS or 200 ng/ml CXCL12 (or 200 ng/ml CXCL12 that had been boiled for 15 min as a negative control), for 30 min at 37°C. Anti-CD164 mAb (N6B6) or an IgG2a isotype matched control mAb (R&D Systems) were added at 0 and 50 μg/ml to block adhesion. The prostate cancer cells (1 × 105) were then directly deposited onto confluent HBME monolayers and cell-to-cell adhesion was preformed for 30 min at 37°C. Adherence was quantified in a 96-well fluorescent plate reader (IDEXX Research Products, Westbrook, ME), and compared to input fluorescent levels. Data are presented as percent change +/- standard deviation compared to non-treated controls.
Invasion of CaP cells
Cell invasion into a reconstituted extracellular matrices costing of Matrigel™ overlaid on 8 μM pore sized in polyethylene terephthalate membranes was performed in dual chambered invasion plates (BD Biosciences, San Jose, CA). Test cells were placed in the upper chamber (1 × 105cells/well) in serum free RPMI containing 0.1% BSA, and PBS or 200 ng/ml CXCL12 was added to the top or bottom chambers. Spontaneous invasion was compared to invasion supported by a CXCL12 gradient [32]. Anti-CD164 mAb (N6B6) (BD PharMingen, San Jose, CA, USA) or an IgG (R&D Systems. Minneapolis, MN, USA) control was added at 50 μg/ml to both chambers to block invasion. At the termination of the assay (24 h), the chambers were removed and 40 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; 5 mg/ml, Sigma, St. Louis, MS) was added to the top well and 80 μl of MTT added to the bottom wells, and incubated for a further 4 h at 37°C. After completely removing residual medium or cells from the top chamber, the purple residues attached to the bottom of the invasion chambers and those residues in the invasion matrix and adherent to the bottom of the upper chamber were released with 1 ml isopropanol (Sigma). The invasion chambers were rocked for 30 min at a medium speed, and 100 μl from each well read on a multi-well scanning spectrophotometer (Molecular Devices Corp. Sunnyvale, CA.) at OD450.
PC3 and LNCaP C4-2B cells were cultured in Lab-Tek II 4-chamber slides (Nalge Nunc International, Naperville, IL, USA) at 5 × 104 cells/chamber. After 24 h the cells were incubated with PBS or CXCL12 (200 ng/ml) for 2 h, then rinsed three times with ice cold-PBS, fixed in 4% paraformaldehyde for 25 min at room temperature, washed and endogenous peroxidase activity quenched with 75 mM NH4Cl and 20 mM Glycine in PBS at room temperature for 10 minutes. Thereafter, the cells were rinsed with PBS three times. Primary antibody incubations at a 1:50 dilution in PBS using the N6B6 clone or an IgG2a matched isotype control (clone 20102, R&D Systems) for 1 h at room temperature. Antibody detection was performed using an HRP-AEC staining kit using anti-mouse biotinylated antibodies (R&D Systems), and counter stained with hematoxylin (Sigma).
Statistical differences between the means for the different groups were evaluated with Instat 4.0 (GraphPad Software, Inc. San Diego, CA) using one-way analysis of variance (ANOVA), with the level of significance at P < 0.05. All in vitro experiments were repeated two to three times with triplicate samples.
To identify those molecules that are responsible for the CXCL12-induced changes in adhesion and invasion, we examined the patterns of prostate cancer gene expression in human tissues. A secondary data analysis was performed on data published by Dhanasekaran et al. [19], as reported previously focusing on known adhesion molecules [8]. For our investigations, a three-step query was used to select genes that were expressed differentially between the benign hyperplasic and normal associated tissues (BHP/NAP) vs. prostate cancer tissue, BHP/NAP vs. metastatic tissue, and prostate cancer tissue vs. metastatic tissue. Discrimination among the selected genes [33] initially produced ~200 candidates that exhibited a significant (at least 3-fold) change in expression in all three queries (Table 3). To determine which of these adhesion genes were responsive to CXCL12, RNA from LNCaP and LNCaP C4-2B cells treated with CXCL12 (200 ng/ml for 2 h) were compared to RNA from untreated cells using a microarray analysis. Adhesion molecules that exhibited a significant change in mRNA expression in response to CXCL12 treatment included the genes that encode CD164 and αv integrin (Table 3). Since the expression of CD164 and the αv integrin were both elevated in human CaP disease, and increased in response to CXCL12 stimulation, they were chosen for further investigation. The role of CD164 is described herein, where as the regulation of the αv integrin in association with the β3 integrin in prostate cancers is described elsewhere (Sun et al., submitted).
Table 3 Secondary data analysis and DNA micro arrays. Secondary data analysis was performed on data published by Dhanasekaran et al. [19] to identify adhesion molecules the expression that are altered during prostate cancer progression. Comparisons were made between normal (norm) tissue/benign prostate hyperplasia (BPH) and cancer (CaP) and metastasis (Met). The normalized mean and standard deviation values were evaluated for significance with Instat 4.0 (GraphPAD software) using one-way analysis of variance (ANOVA) are presented, with the level of significance set at p < 0.05 (*). To determine which of the genes are responsive to CXCL12, RNA was analyzed from CXCL12 treated LNCaP and LNCaP C4-2B cells (200 ng/ml, 2 hours) were compared against non-treated cells by DNA micro array using the Human genome U133 gene chips (Affymetrix Corp. Santa Clara, CA). Later, fold change values were determined using DNA-Chip (dChip version 1.1) [26].
Several alternatively spliced variants of CD164 are known to be derived from six transcribed CD164 exons. To determine which of the CD164 transcripts are made by CaP cells, and to verify the microarray data, RNA was collected from PC3, LNCaP, and LNCaP C4-2B cells that were cultured in the presence or absence of CXCL12. For these investigations, all 6 exons of the full-length transcript were examined using reverse transcription-polymerase chain reaction (RT-PCR). Sequencing of the PCR-amplified products verified that the amplicons were derived from full-length transcripts under basal conditions (Figure 2). All of the prostate cancer cells expressed mRNA coding for the full length CD164, and none of the alternatively spliced forms were observed. Cells stimulated with CXCL12 also expressed mRNA for CD164, but once again, none of the alternatively spliced transcripts were observed (Figure 2).
Cartoon of CD164 genomic structure and PCR primer sets.
CaP cells express full length CD164 mRNA. RT-PCR detection of CD164 expression by LNCaP C4-2B, LNCaP (L) and PC3 (P) cells under basal (-) and CXCL12 (+) stimulated conditions (CXCL12 200 ng/ml at 2 h). Primers F1-R1 were used to identify the CD164 coding exons 1-2, F2-R2 identify exons 2-3 and F3-R3 identify exons 2-5. After RT-PCR, the amplicons were identified using polyacrylamide gels. A molecular weight ladder is shown to the left of each figure that corresponds to differences in 100 base pairs. The data demonstrate that full length CD164 mRNA is express by CaP cells.
To evaluate the effect of CXCL12 on CD164, prostate cancer cell lines were treated with CXCL12 and mRNA expression was evaluated using real-time RT-PCR. As before, CD164 mRNA was readily detected under basal conditions in each of the cell lines examined (Figure 3). Under basal conditions the LNCaP C4-2B cells expressed nearly 2 fold more CD164 than PC3 and LNCaP cell lines (not shown). As early as 30 min after exposure to CXCL12, the expression of CD164 mRNA increased in each of the cell lines (Figure 3). In PC3 cells, the enhanced expression of CD164 mRNA at 30 min represented the peak of the response to CXCL12, and the level of CD164 mRNA expression declined thereafter. By contrast, in the LNCaP and LNCaP C4-2B cells, the expression of CD164 mRNA peaked 2 h after exposure to CXCL12 (Figure 3). To determine if the prostate cancer cells express CD164 protein, and whether its expression was altered by CXCL12, PC3 and LNCaP C4-2B cells were treated with PBS or 200 ng/ml of CXCL12 for 2 h. Thereafter, the cells were examined by immunohistochemistry. The number of cancer cells exhibiting perinuclear and cytoplasmic expression of CD164 ranged from 90–100% in PC3 and LNCaP C4-2B cells (Figure 4). Abundant CD164 expression was detected in the cytoplasm and on the cell surface of the PC3 and LNCaP C4-2B cells following CXCL12 stimulation (Figure 4).
Time course of CD164 mRNA expression by CaPs. QRT-PCR was used to detect changes in CD164 expression by CaP cells following CXCL12 stimulation over time for LNCaP, LNCaP C4-2B, and PC3 cells. The data show that LNCaP and LNCaP C4-2B respond to high concentrations of CXCL12 by increasing CD164 mRNA expression. PC3 cells increase CD164 expression to moderate concentrations of CXCL12 (20 ng/ml), but at higher CXCL12 doses (200 ng/ml) down regulate expression.
Expression of CD164 in prostate cancer cell lines is responsive to CXCL12. To determine if the prostate cancer cells express CD164 protein, and whether its expression was altered by CXCL12, PC3 and LNCaP C4-2B cells were treated with PBS or 200 ng/ml CXCL12 for 2 h, fixed and stained with anti-CD164 mAb or an IgG isotype matched control, and photographed at 40X. Scale bars = 100 μm. The cancer cells demonstatrate perinuclear and cytoplasmic expression of CD164 in nearly all of the cells under basal conditions, and abundantly express CD164 following CXCL12 stimulation.
We demonstrated previously that activation of CXCR4 by CXCL12 enhanced the adhesion of prostate cancer cells to bone marrow endothelial cells. To determine whether CD164 is involved in CXCL12-induced adhesion, PC3 cells were treated with CXCL12. Binding assays were performed either in the presence or absence of a variety of neutralizing antibodies that recognize various epitopes of CD164 (Figure 5A shown in Figure 5B). Compared to non-stimulated cells, CXCL12 significantly enhanced the adhesion of PC3 cells to marrow endothelium by 13 ± 2.4 % and LNCaP C4-2B by 15 ± 1.1 %, while the adhesion of CXCL12 stimulated cells was inhibited by the class III epitope monoclonal antibody (clone N6B6) (Figure 5A). Similar investigations using the N6B6 antibody on CXCL12 stimulated, or non-stimulated CaPs (PC3 and LNCaP C4-2B cells), demonstrated that neutralization of CD164 decreased the binding of prostate cancer cell lines to human bone marrow endothelium; further suggesting that CD164 plays a role in prostate cancer localization to the marrow.
CD164 regulates CaP cell adhesion to HBME. PC3 and LNCaP C4-2B cells were labeled with Vybrant CFDA SE Cell Tracer Kit, (Molecular Probes, Inc., Eugene, Oregon) for 30 min and directly deposited on to human bone marrow derived endothelial (HBME) monolayers. CXCL12 pretreatment was performed by incubating the CaP cells with 200 ng/ml CXCL12 for 2 hours. In (A) 50 μg/mL anti-CD164 mAb antibodies (or Ig controls) were added to each chamber. Cell-to-cell adhesion for PC3 cells was preformed for 30 min at 37°C and quantified using a 96-well fluorescent plate reader (IDEXX Research Products, Westbrook, ME). Data are presented as percent change +/- standard deviation from non-treated controls for n = 4. In (B) the CD164 antibody (N6B6) was used in an adhesion assay with PC3 and LNCaP C4-2B cells treated with either PBS or CXCL12 (200 ng/ml) to block adhesion. * Indicates significant difference from vehicle treated cells (no CXCL12), and ** indicates significant difference from Ig treated controls at p < 0.05. The data demonstrates that CD164 blockade regulates PC3 and LNCaP C4-2B binding to HBME.
For metastatic prostate cancer cells to establish metastases, they must migrate through the subendothelial matrix after attaching to the endothelium. Migration depends on the availability of chemotactic factors that direct cell movement. To mimic this phenomenon in vitro, we explored the effect of CD164 on the ability of cancer cells to invade reconstituted extracellular matrix in response to CXCL12. Migrating cells were introduced into the upper well of an invasion chamber within which a gradient of CXCL12 (0 or 200 ng/ml) was established by placing the chemokine in the lower portion of the chamber. The role that CD164 plays in invasion was probed using a neutralizing anti-CD164 antibody (clone N6B6). As shown in Figure 6, CXCL12 enhanced the invasion of PC3 cells from 21,260 ± 140 to 37,545 ± 459, and LNCaP C4-2B cells from 19,970 ± 639 to 27014 ± 601 cells. In both cases, ant-CD164 antibody blocked invasion of the cells under both basal conditions and in the presence of CXCL12.
CD164 regulates CaP cell invasion. Cell invasion into a reconstituted extracellular matrix was performed using two chambered invasion plates (BD Biosciences, San Jose, CA). PC3 and LNCaP C4-2B cells were placed in the upper chamber (1 × 105 cells/well) in serum free medium. Spontaneous invasion was compared to invasion supported by a CXCL12 200 ng/ml gradient [32]. Where indicated, IgG control or mouse anti-human CD164 monoclonal antibody (50 μg/ml, Clone N6B6, BD PharMingen) was added at the start of the investigation to each chamber. At the termination of the assay (24 h), the invasion was quantified by MTT. * Indicates significant difference from vehicle treated cells (no CXCL12), and ** indicates significant difference from IgG treated controls at p < 0.05. The data demonstrate that anti-CD164 inhibits invasion of CaP cells
To determine at which stage CD164 is expressed during the progression of prostate cancer, we used an immunohistochemical analysis of clinical specimens of prostate cancer tumors. Staining of microarrays with CD164 monoclonal antibodies revealed that CD164 protein expression was localized to the cytoplasm and that the level of expression ranged from moderate to high in clinically benign and localized prostate cancer tumors (Figure 7A,7B). The level of CD164 expression in malignant epithelia was slightly higher than that in surrounding benign epithelia, while expression in normal epithelium was predominately weak in the cytoplasm and strong within the nucleus. High-grade (Gleason score = 4 + 4 = 8) and low-grade prostate cancer tumors (not shown) exhibited a high level of cytoplasmic expression. Similar staining was observed in bone metastases (Figure 7C). As shown in Figure 7D and 7E, the expression of CD164 was correlated with the expression of prostate specific antigen (PSA) expression but was related inversely to the level of androgen receptor expression (AR). Figure 7F presents data that illustrate the correlation of the intensity of CD164 immunoreactivity and tumor location. Sites that exhibited a high propensity for tumor metastasis and high levels of CXCL12 [9] exhibited intense CD164 immunoreactivity.
Human prostate tissue samples stained with CD164. High-density tissue micro arrays of human CaP tumors were immunostained for CD164, AR and PSA as described in the methods. Immunostaining intensity was scored by a genito-urinary pathologist: as absent (1), weak (2), moderate (3) or strong (4). Scoring was performed blindly using a telepathology system without knowledge of overall the tumor grade, tumor size or clinical outcome [30]. Representative staining of CD164 staining for (A) benign prostate hyperplasia (B) localized prostate cancer (C) metastatic prostate cancer to bone marrow. Correlation of CD164 staining with prostate specific antigen (PSA) staining intensity (D), androgen receptor (E) and tissue type (F). The data demonstrate that CD164 staining intensity increases with increasing tumor grade.
Previously, we determined that the CXCL12/CXCR4 chemokine axis is activated in prostate cancers that metastasize to bone [8, 11]. To identify which molecules mediate CXCL12-stimulated changes in adhesion and invasion of bone by metastatic cells, we first examined patterns of gene expression in human tissues and cell lines that were stimulated with CXCL12. Our work focused on two genes that play an important role in stem cell localization and the physiology of bone, namely integrin alpha V (CD51) and CD164. Together with CD61, CD51 forms the heterodimer arginine-glycine-aspartic (RGD) motif-dependent vitronectin receptor αvβ3, which is the focus of a parallel report (Sun et al., in press); in the present report, we focus on CD164. We present data that demonstrate that CD164 mRNA and protein is expressed by prostate cancer cell lines which are responsive to CXCL12 stimulation. Functionally, antibody blocking of CD164 prevents the binding and migration of prostate cancer cells to bone marrow endothelium and invasion via the extracellular matrix. Importantly, CD164 is expressed in human prostate cancer tissues paralleling the PSA expression, and negatively correlating with AR expression. Notably, CD164 expression in osseous metastasis was greater than that in soft tissue metastasis, in comparison to those found in the liver and lymph nodes – all sites that express high CXCL12 levels[9]. These data suggest that CD164 may play an important role in localizing tumors not only to sites where there are high levels of CXCL12 expression, but also to specific tissue locales.
We found that adhesion and invasion of prostate cancer cells requires the class III epitope, which is recognized by the clone N6B6 antibody. Class I and II epitopes are conformationally independent and are located within the mucin N-terminal domain I [34]. The class I epitope is associated with long-chain sialylated O-linked glycans while the class II epitope is associated with both N- and O-linked glycans. Class III epitopes are conformationally dependent and encompass the cysteine-rich subdomain that links mucin domains I and II [34]. The identity of individual class III epitopes likely depends on the peptide backbone of the protein rather than carbohydrate modifications. Monoclonal antibodies that recognize the carbohydrate-dependent mucin domains of CD164 affect the adhesion and proliferation of hematopoietic precursor cells, yet the CD164 class III epitopes are expressed widely on both hematopoietic and non-hematopoietic cell types [34]. The threeclasses of CD164 epitopes are expressed by a subset of CD34+ hematopoietic progenitor cells and stromal reticular cells from adult bone marrow [35]. In our studies, only monoclonal antibody N6B6 blocked the binding and invasion to bone by prostate cancer cells.
Prostate tumors that develop independence from androgens (hormone-refractory prostate cancer) reflect a transition from a stable to a progressive state of the disease. The development of skeletal metastases may be associated with the progression of prostate cancer towards androgen independence, yet metastasis to bone usually occurs during the late stage of the disease. Recent work revealed that androgen receptor (AR) expression is heterogeneous in cancer patients: individual patients can exhibit both AR-positive and AR-negative tumor populations. Overall, AR expression is down-regulated in hormone-refractory prostate cancer but nearly 41.5% of tumor samples examined expressed some, albeit low (<10%) levels. Previous studies have demonstrated the value of using PSA immunohistochemistry to diagnose metastatic prostate cancer [36, 37]. Stein et al. [36] demonstrated that most end-stage prostate cancers retain PSA expression. However, AR expression is not correlated with PSA expression, which suggests that PSA expression in late-stage prostate cancers may be driven by mechanisms that are independent of the AR [36]. Our observation that CD164 expression is correlated negatively with AR expression but correlated positively with PSA expression concurs with the aforementioned conclusion and may be useful as a prognostic indicator of androgen-independent tumor growth [38]. Furthermore, the correlation between staining intensity and sites of frequent metastasis and CXCL12 staining is further evidence that CD164 may be involved in tumor localization.
The adhesion of cancer cells to the end-organ vasculature is a crucial step in the metastatic cascade. When combined with organ-specific growth factors, adhesion may have a substantial effect on which site(s) are the target of metastasis [39]. The binding of cancer cells to the microvascular endothelium is believed to require two distinct steps [40]. Initial localization of cancer cells (including prostate cancer cells) to the endothelium appears to be mediated largely by lectins and mucins such as CD164 [41]. Thereafter, activation of integrin receptors and the subsequent development of secure contacts between the cancer cells and endothelium are required. Antibodies that recognize galectin-3 and RGD motif peptides reduce the ability of prostate cancer cells to bind to endothelial tissue [42]. Similar inhibition of binding by antibodies that recognize β1 integrins and CD44 isoforms suggests that integrins participate in the adhesion of cancer cells to endothelial cells [43]. In addition, a mucin-type disaccharide that is expressed on most types of cancer cell (including breast and prostate cancer cells) contributes to adhesion of cancer cells to human bone marrow endothelium [43]. Finally, the expression of hyaluronan, a high-molecular-weight glycosaminoglycan component of extracellular and cell-associated matrices, has been correlated with cancer [44].
As was the case for CD164, we observed that CXCL12 enhanced the transcription and activation of αvβ3. Moreover, the level of αvβ3 expression appeared to be correlated with an increase in tumor grade and malignancy. Unlike CD164, the constitutive expression of this receptor complex represents an inactive state (Sun et al., submitted). However, short-term exposure of cancer cells to CXCL12 activates the affinity of prostate cancer cells for αvβ3 ligands in addition to increasing the level of receptor expression. Our experiments revealed that antibodies that recognize CD164 inhibited the binding of prostate cancer cells to human bone marrow endothelium and reduced invasion of the extracellular matrix by prostate cancer cells, irrespective of whether the cancer cells were stimulated by exposure to CXCL12. The abovementioned findings are consistent with the view that metastatic cascades involve several crucial steps, including multiple adhesive events such as initial adhesion and localization of cancer cells to vascular sites and subsequent stabilization between cancer cells and endothelial cells in the face of shear stress. The findings presented in this report suggest that CD164 participates in the initial 'locking' of prostate cancer cells to the endothelium and thereby facilitates the invasion of tissue.
As noted previously, CD164 is both an adhesion receptor on human CD34+ cell subsets in bone marrow and a potent negative regulator of CD34+ hematopoietic progenitor cell proliferation [46]. This is particularly interesting in the light of the results of other studies in our laboratory that revealed that long-term survival of stem cells on osteoblasts in vitro (which may be the major source of CXCL12 in marrow) depends on stable adhesion. We demonstrated previously that neutralization of CXCL12 in vitro or following intratibial injection in vivo inhibited cell growth [45] demonstrated recently that expression of CD164 in myoblast cell lines increased the expression of biochemical markers of differentiation and enhanced the formation of multinucleate myotubes. Similarly, the expression of antisense CD164 or soluble extracellular regions of CD164 inhibited myogenesis. Treatment of cultured C2C12 myoblasts with the enzymes sialidase or O-sialoglycoprotease (both of which destroy functional epitopes on CD164) also inhibited differentiation [45]. These data are consistent with the hypothesis that CD164 may play a rate-limiting role in myogenic differentiation in vitro [45]. We noted previously that in vitro proliferation and intratibial growth of prostate cancers depends on CXCL12 [8, 9]. At present, we are in the process of evaluating whether CD164 plays a similar role in prostate cancer cells. If CD164 does regulate the growth of prostate cancer cells, this would be a particularly attractive therapeutic target for two reasons. First, because CXCL12 up regulates CD164 expression and stimulates prostate cancer cell proliferation and adhesion, therapies designed to disrupt CD164 or the CXCL12/CXCR4 axis could be used to alter tumor progression via several mechanisms. Second, because stem cell homing and niche localization likely depends on multiple and redundant adhesive mechanisms, therapies that target CD164 might have little or no side effects on the host.
In summary, we demonstrated that full-length CD164 is expressed by human prostate cancer cell lines and human prostate cancer tumors. CXCL12 enhanced the binding of prostate cancer cells to bone marrow endothelial cells, and CXCL12 up regulated the expression of CD164 mRNA and protein. Using monoclonal antibodies, we determined that the neutralization of CD164 with antibodies blocked the adhesion of prostate cancer cells to human bone marrow endothelial cells and inhibited subsequent invasion. Collectively, our data suggest that CD164 plays an important role in prostate cancer metastasis and the infiltration of bone marrow by cancer cells.
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Vaday GG, Hua SB, Peehl DM, Pauling MH, Lin YH, Zhu L, Lawrence DM, Foda HD, Zucker S: CXCR4 and CXCL12 (SDF-1) in prostate cancer: Inhibitory effects of human single chain Fv antibodies. Clin Cancer Res. 2004, 10: 5630-5639. 10.1158/1078-0432.CCR-03-0633.
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These investigations were supported in part by the National Institutes of Health with awards DK067688 (R.S.T), R01 DE13701 (R.S.T), P01 CA46952 (K.J.P, R.S.T), P50 CA69568 (K.J.P), and the Department of Defense DAMD17-02-1-0100 (R.S.T). The authors also wish to thank the University of Michigan Comprehensive Cancer Center Histology and Immunoperoxidase Core.
Department of Periodontics and Oral Medicine, University of Michigan School of Dentistry, 1011 North University Ave, Ann Arbor, Michigan, 48109-1078, USA
AM Havens
, Y Jung
, YX Sun
, J Wang
& RS Taichman
Department of Pathology, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, Michigan, 48109-0692, USA
RB Shah
Department of Internal Medicine II, Division for Hematology, Immunology, Oncology and Rheumatology, University Hospital, Tübingen, Germany
HJ Bühring
Department of Urology, University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, Michigan, 48109-0692, USA
KJ Pienta
Department of Ophthalmology, Affiliated hospital of Jiangsu University, Zhenjiang, Jiangsu, 212001, China
YX Sun
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Correspondence to RS Taichman.
A.M.H. carried out the molecular and cell culture studies, genetic studies, participated in the sequence alignment and drafted the manuscript. J.Y., designed and performed the adhesion studies, Y-X.S., designed and performed some of the invasion assays, J.W. performed the immunohistochemical studies, R.B.S., directed and evaluated the tissue microarray studies, H.J. B. provided antibodies, K.J.P and R.S.T. participated in the studies design and coordination.
Havens, A., Jung, Y., Sun, Y. et al. The role of sialomucin CD164 (MGC-24v or endolyn) in prostate cancer metastasis. BMC Cancer 6, 195 (2006) doi:10.1186/1471-2407-6-195
Prostate Cancer Cell
Prostate Cancer Cell Line
CD164 mRNA


Sea, Sand & Sky: Hello!
I'm a recent graduate of Oberlin College in northern Ohio and super excited to be in Boston this summer. I grew up in Dallas, Texas where I learned to sail with my father when I was young. This summer I'm teaching sailing classes to kids at Community Boating in the morning and will be starting an environmental education program in partnership with Save the Harbor/ Save the Bay in the afternoon. While learning to sail on the Charles river, the kids at Community Boating will find out about the what lives beneath the boats and why it is so important to take care of our river.
If you don't know about community boating you should check it out. http://community-boating.org/. It is a wonderful program with the simple mission of 'sailing for all'. For those who qualify a summer of sailing only costs 1$.
We'll fish and trap to get an up close view of what lives in the Charles . Also we'll think about where the water in the Charles is coming from and where it is going.
I also love gardening, so I have a compost pile in the works and maybe we'll even grow some food the with the compost we make at the end of the summer! Keep an eye out for me on the Charles sailing or pulling up eel traps!


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Home Actress Shannon Lee
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Shannon Emery Lee (born April 19, 1969) is an American actress, martial artist and businesswoman. She is the daughter of late martial arts film star Bruce Lee and retired martial arts teacher Linda Lee Cadwell, the granddaughter of Cantonese opera singer Lee Hoi-Chuen, and the younger sister of Brandon Lee.
She was born on April 19, 1969, in Los Angeles, to martial arts film star Bruce Lee and Linda Emery. In her youth she studied Jeet Kune Do, the martial art created by her father, under Richard Bustillo, one of her father's students. However, her serious studies did not begin until the late 1990s. To train for parts in action movies, she studied Jeet Kune Do with Ted Wong.
She studied Taekwondo under Dung Doa Liang and Wushu under Eric Chen. She also studied under the tutelage of the director of Enter the Eagles, Yuen De, Jackie Chan's Chinese opera brother. Because the film Enter the Eagles required her to fight Benny Urquidez, Urquidez himself taught her in kickboxing.
Lee made her acting debut playing a cameo in her father's 1993 biopic film Dragon: The Bruce Lee Story as the party singer of the song "California Dreamin.'" She went on to appear in the direct-to-video films Cage II (1994) with bodybuilding veteran and actor Lou Ferrigno and High Voltage (1997) with Antonio Sabato Jr. In 1998, she starred in the Hong Kong action film Enter the Eagles, directed by Corey Yuen, co-starring Michael Wong and Anita Yuen.
On television, she guest-starred in an episode of the television series Martial Law alongside Sammo Hung in 1998 and appeared in the sci-fi television film Epoch, which first aired on the Sci Fi Channel in 2001. That same year, she played the leading role in the action film Lessons for an Assassin. She was also the host of the first season of the game show WMAC Masters. She sang on the band Medicine's album The Mechanical Forces of Love in 2003. She sang a cover of "I'm in the Mood for Love" for the movie China Strike Force (2000), which starred Leehom Wang and Aaron Kwok.
Lee is president of the Bruce Lee Foundation. She was the executive producer of the 2008 television series The Legend of Bruce Lee, based on her father's life, and the 2009 documentary film How Bruce Lee Changed the World.
In 2015, Perfect Storm Entertainment and Shannon Lee announced that the series The Warrior, based an original idea by Bruce Lee, would be produced and would air on the Cinemax and the filmmaker Justin Lin was chosen to direct the series, scheduled to debut in 2019.
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Originally posted by GKHiryu:. Last edited by Matti ; 13 May, Enter the code "Keyori" and you will get a bunch of stuff. The Keyori code only gives a 3 day version of the mount, it gives other things too but the mount is a 3 day: Secks View Profile View Posts.
Originally posted by Charles Alejandro:. Last edited by Secks ; 1 Jun, Pyke View Profile View Posts. Open the Account Items tab of your Item Claim and remove the item.
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Click the Enter Code button on the right side of your account overview page. Topic Replies Last Reply. Xilokz Feb 1st , 8: Tadeshi - Feb 1st , 8: Hi I would personally like to see these changes for PvP for the classic server: Can i trade my characters from live to classic?
Will all the new costumes and mounts be available on classic? Do you guys plan to keep it permanent? Petition for PvP classic server. Players who want to avoid PvP can easily do it on channel Since classic server is all about PvP in my opinion there is nothing fun for players who dont like PvP to at least some extent we should keep things transparent I appeal to all guild leaders to….
Synd - Feb 1st , 8: Xilokz - Feb 1st , 8: Rare to see serious gamers. Borsuc - Feb 1st , 8: Turns out there is more, the whole armor set was patched to the uncensored….
Neylya Feb 1st , 5: Originally posted by GKHiryu:. Last edited by Matti ; 13 May, Enter the code "Keyori" and you will get a bunch of stuff.
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Kenji Kuramitsu is a Master of Divinity student at McCormick Theological Seminary and a Master of Social Work candidate at the University of Chicago. He has served on the boards of the Reformation Project and the Japanese American Citizens League. Follow him on Twitter @afreshmind.
Can a Digital Sacrament Still Impart Divine Grace?
As Shamika and I called upon our own experiences in church and seminary, we became especially concerned with providing a resource for those who historically have been barred from participation at the Lord's Table: the divorced, Christians of color, LGBTQ believers, those living far from physical community, or far from a church that is physically accessible. While we're not trying to replace "brick and mortar" community, we believe God calls us beyond a spirit of fear in the face of innovations in technology.
Can Christians observe Lent in a way that ties social justice consciousness to spiritual Lenten practices of repentance, resistance, and solidarity? The curators of four social media campaigns — #LentenLament, #LentLite, #EmbodiedSolidarity, and #DetoxifyChristianity — are exploring what this looks like in public. "What does it mean to come together, to lock arms together, to really just stand in the gap for people?" Alicia Crosby, executive director of the Center for Inclusivity and co-curator of last year's #EmbodiedSolidarity campaign, asked.
We can hope, work, and pray for the day when Christians of all colors might be reconciled to one another in peace. In the meantime, Get Out reminds us that white people, especially those who claim to love us, must do better.
Japanese American families form a line outside Civil Control Station located in the Japanese American Citizens League Auditorium at 2031 Bush Street in San Francisco to appear for "processing" in response to Civilian Exclusion Order Number 20 in 1942.
On Feb. 19, 1942, President Franklin D. Roosevelt empowered military commanders to execute the "forced evacuation" of anyone with Japanese ancestry from the West Coast of the United States. This order set in motion a mass incarceration that would sweep tens of thousands of people, most of them American citizens, into concentration camps throughout the country's inland regions.


The items in this line of products ease discomforts caused by a big toe that is misaligned in the metacarpophalangeal joint. It comprises products for decreasing pain from rubbing and special bandages for gently correcting misalignments.
The Valex-D day bandage corrects the misalignment of the big toe. A Perlon cover over the toe gently corrects the misalignment via an elastic strap. The splayfoot bandage with pressure pad supports this effect, while the ball of the foot is protected from pressure and rubbing with the bunion protection pad.
The Valex-N night bandage corrects the misalignment of the big toe while the foot is not subject to any strain. It does this by fixing the big toe with a softly padded toe support and a Velcro strip, thus pressing it gently into its normal position.
The toe separator is made of foam and prevents pain from pressure or rubbing between the toes, for example due to toe malpositions. This product can also be used in case of ingrown or injured nails or fungal infections of the toenails.
The toe separator is made of soft para rubber and prevents pain from pressure or rubbing between the toes, which may be caused for example by toe misalignments. The support areas above and below the toes keep the toe separator from slipping, while the rubber layers ensure highest elasticity.
The ball footbed is made of perforated foam and gently encloses the ball of the foot, thus protecting it from painful pressure or rubbing. In addition, the integrated toe separator gently returns the big toe to its natural position.
With this two-component material, you can create many types of individual pressure protection ortheses. After setting, the material retains its shape, yet at the same time stays elastic and durable.


text:
package io.grpc.transport.netty;

import com.google.common.base.Preconditions;
import com.google.common.base.Throwables;

import io.grpc.transport.AbstractReadableBuffer;
import io.netty.buffer.ByteBuf;

import java.io.IOException;
import java.io.OutputStream;
import java.nio.ByteBuffer;

/**
 * A {@link Buffer} implementation that is backed by a Netty {@link ByteBuf}. This class does not
 * call {@link ByteBuf#retain}, so if that is needed it should be called prior to creating this
 * buffer.
 */
class NettyReadableBuffer extends AbstractReadableBuffer {
  private final ByteBuf buffer;
  private boolean closed;

  NettyReadableBuffer(ByteBuf buffer) {
    this.buffer = Preconditions.checkNotNull(buffer, "buffer");
  }

  ByteBuf buffer() {
    return buffer;
  }

  @Override
  public int readableBytes() {
    return buffer.readableBytes();
  }

  @Override
  public void skipBytes(int length) {
    buffer.skipBytes(length);
  }

  @Override
  public int readUnsignedByte() {
    return buffer.readUnsignedByte();
  }

  @Override
  public void readBytes(byte[] dest, int index, int length) {
    buffer.readBytes(dest, index, length);
  }

  @Override
  public void readBytes(ByteBuffer dest) {
    buffer.readBytes(dest);
  }

  @Override
  public void readBytes(OutputStream dest, int length) {
    try {
      buffer.readBytes(dest, length);
    } catch (IOException e) {
      throw Throwables.propagate(e);
    }
  }

  @Override
  public NettyReadableBuffer readBytes(int length) {
    // The ByteBuf returned by readSlice() stores a reference to buffer but does not call retain().
    return new NettyReadableBuffer(buffer.readSlice(length).retain());
  }

  @Override
  public boolean hasArray() {
    return buffer.hasArray();
  }

  @Override
  public byte[] array() {
    return buffer.array();
  }

  @Override
  public int arrayOffset() {
    return buffer.arrayOffset() + buffer.readerIndex();
  }

  /**
   * If the first call to close, calls {@link ByteBuf#release} to release the internal Netty buffer.
   */
  @Override
  public void close() {
    // Don't allow slices to close. Also, only allow close to be called once.
    if (!closed) {
      closed = true;
      buffer.release();
    }
  }
}

redpajama_set_name: RedPajamaGithub


Transmission: Automatic Color:Red Interior Color: Black Average Vehicle Review: (4.75 reviews) Safety features like 'city safety' and dynamic cruise control are excellent and the main reasons for purchasing the car. But it is also extremely comfortable, high quality leather, good internal design, simple to use, good power delivery, European style automatic gearbox. All in all a great car.
Transmission: Automatic Color:Silver Interior Color: Black Average Vehicle Review: (5 reviews) The Volvo XC60 drives and rides like a dream! When i first started looking for a car, I wasn't sure what make I was looking for but knew I wanted something safe and spacious. I got those things and more when I purchased this car. The seats are comfortable and the car handles well. I knew Volvos were quality but I didn't know how much more I was getting for the price! I love having Bluetooth capability, the Sirius radio and went ahead and renewed it when the 3 month trial ran out. I had an EXCELLENT experience purchasing my Volvo at Car Max- the sales person Lovie Pulliam was the best!
Transmission: Automatic Color:Black Interior Color: Black Average Vehicle Review: (5 reviews) I've owned Volvos in the past and this has restored my faith in the brand. Not the cheapest, but clearly a well built car. My other car is a Lexus, which I love, but the Volvo is every bit as good.
The Volvo XC60 drives and rides like a dream! When i first started looking for a car, I wasn't sure what make I was looking for but knew I wanted something safe and spacious. I got those things and more when I purchased this car. The seats are comfortable and the car handles well. I knew Volvos were quality but I didn't know how much more I was getting for the price! I love having Bluetooth capability, the Sirius radio and went ahead and renewed it when the 3 month trial ran out. I had an EXCELLENT experience purchasing my Volvo at Car Max- the sales person Lovie Pulliam was the best!
I've owned Volvos in the past and this has restored my faith in the brand. Not the cheapest, but clearly a well built car. My other car is a Lexus, which I love, but the Volvo is every bit as good.
Safety features like 'city safety' and dynamic cruise control are excellent and the main reasons for purchasing the car. But it is also extremely comfortable, high quality leather, good internal design, simple to use, good power delivery, European style automatic gearbox. All in all a great car.
A compact luxury SUV gives you the added passenger and cargo space of an SUV with the extravagance of a luxury vehicle. CarMax's list of the top seven best luxury compact SUVs is a great place to begin your search.
Henry Ford once famously told customers that his Model T came in any color they wanted, so long as that color was black. That was over 100 years ago. Today, the vehicles available to us come in a variety of colors, shapes, and sizes, with a profusion of available options and features. Whether you're shopping for a quality used vehicle, or you want to take the plunge with a new car, 2017 is a great time to see what's available.


text:
import logging
import re

from telemetry.core import exceptions
from telemetry.core import util
from telemetry.internal.backends import browser_backend
from telemetry.internal.backends.chrome import tab_list_backend
from telemetry.internal.backends.chrome_inspector import devtools_client_backend
from telemetry.internal import forwarders
from telemetry.util import wpr_modes

class MandolineBrowserBackend(browser_backend.BrowserBackend):
  """An abstract class for mandoline browser backends. Provides basic
  functionality once a remote-debugger port has been established."""
  # It is OK to have abstract methods. pylint: disable=abstract-method

  def __init__(self, platform_backend, browser_options):
    super(MandolineBrowserBackend, self).__init__(
        platform_backend=platform_backend,
        supports_extensions=False,
        browser_options=browser_options,
        tab_list_backend=tab_list_backend.TabListBackend)
    self._port = None
    self._devtools_client = None

    if browser_options.netsim:
      self.wpr_port_pairs = forwarders.PortPairs(
          http=forwarders.PortPair(80, 80),
          https=forwarders.PortPair(443, 443),
          dns=forwarders.PortPair(53, 53))
    else:
      self.wpr_port_pairs = forwarders.PortPairs(
          http=forwarders.PortPair(0, 0),
          https=forwarders.PortPair(0, 0),
          dns=None)

    # Some of the browser options are not supported by mandoline yet.
    self._CheckUnsupportedBrowserOptions(browser_options)

  @property
  def devtools_client(self):
    return self._devtools_client

  def GetBrowserStartupArgs(self):
    args = []
    args.extend(self.browser_options.extra_browser_args)
    args.extend(self.GetReplayBrowserStartupArgs())
    # Currently the bots that run mojo perf tests use such an old kernel that
    # it doesn't support the namespace sandboxing primitives. Disable it until
    # infra fixes it. (crbug.com/546644)
    args.append('--no-sandbox')
    return args

  def _UseHostResolverRules(self):
    """Returns True to add --host-resolver-rules to send requests to replay."""
    if self._platform_backend.forwarder_factory.does_forwarder_override_dns:
      # Avoid --host-resolver-rules when the forwarder will map DNS requests
      # from the target platform to replay (on the host platform).
      # This allows the browser to exercise DNS requests.
      return False
    if self.browser_options.netsim and self.platform_backend.is_host_platform:
      # Avoid --host-resolver-rules when replay will configure the platform to
      # resolve hosts to replay.
      # This allows the browser to exercise DNS requests.
      return False
    return True

  def GetReplayBrowserStartupArgs(self):
    if self.browser_options.wpr_mode == wpr_modes.WPR_OFF:
      return []
    replay_args = []
    if self.should_ignore_certificate_errors:
      # Ignore certificate errors if the platform backend has not created
      # and installed a root certificate.
      replay_args.append('--ignore-certificate-errors')
    if self._UseHostResolverRules():
      # Force hostnames to resolve to the replay's host_ip.
      replay_args.append('--host-resolver-rules=MAP * %s,EXCLUDE localhost,'
                         #'EXCLUDE *.google.com' %
                         % self._platform_backend.forwarder_factory.host_ip)
    # Force the browser to send HTTP/HTTPS requests to fixed ports if they
    # are not the standard HTTP/HTTPS ports.
    http_port = self.platform_backend.wpr_http_device_port
    https_port = self.platform_backend.wpr_https_device_port
    if http_port != 80:
      replay_args.append('--testing-fixed-http-port=%s' % http_port)
    if https_port != 443:
      replay_args.append('--testing-fixed-https-port=%s' % https_port)
    return replay_args

  def HasBrowserFinishedLaunching(self):
    assert self._port, 'No DevTools port info available.'
    return devtools_client_backend.IsDevToolsAgentAvailable(self._port, self)

  def _InitDevtoolsClientBackend(self, remote_devtools_port=None):
    """ Initiates the devtool client backend which allows browser connection
    through browser' devtool.

    Args:
      remote_devtools_port: The remote devtools port, if any. Otherwise assumed
          to be the same as self._port.
    """
    assert not self._devtools_client, (
        'Devtool client backend cannot be init twice')
    self._devtools_client = devtools_client_backend.DevToolsClientBackend(
        self._port, remote_devtools_port or self._port, self)

  def _WaitForBrowserToComeUp(self):
    """ Waits for browser to come up. """
    try:
      timeout = self.browser_options.browser_startup_timeout
      util.WaitFor(self.HasBrowserFinishedLaunching, timeout=timeout)
    except (exceptions.TimeoutException, exceptions.ProcessGoneException) as e:
      if not self.IsBrowserRunning():
        raise exceptions.BrowserGoneException(self.browser, e)
      raise exceptions.BrowserConnectionGoneException(self.browser, e)

  @property
  def browser_directory(self):
    raise NotImplementedError()

  @property
  def profile_directory(self):
    raise NotImplementedError()

  @property
  def supports_tab_control(self):
    return False

  @property
  def supports_tracing(self):
    return False

  @property
  def supports_system_info(self):
    return False

  @property
  def supports_cpu_metrics(self):
    return False

  @property
  def supports_memory_metrics(self):
    return False

  @property
  def supports_power_metrics(self):
    return False

  def GetProcessName(self, cmd_line):
    """Returns a user-friendly name for the process of the given |cmd_line|."""
    if not cmd_line:
      return 'unknown'
    m = re.search(r'\s--child-process(\s.*)?$', cmd_line)
    if not m:
      return 'browser'
    return 'child-process'

  def Close(self):
    if self._devtools_client:
      self._devtools_client.Close()
      self._devtools_client = None

  def _CheckUnsupportedBrowserOptions(self, browser_options):
    def _GetMessage(name):
      return ('BrowserOptions.%s is ignored. Value: %r'
                  % (name, getattr(browser_options, name)))

    def _RaiseForUnsupportedOption(name):
      raise Exception(_GetMessage(name))

    def _WarnForUnsupportedOption(name):
      logging.warning(_GetMessage(name))

    if browser_options.dont_override_profile:
      _RaiseForUnsupportedOption('dont_override_profile')

    if browser_options.profile_dir:
      _RaiseForUnsupportedOption('profile_dir')

    if browser_options.profile_type and browser_options.profile_type != 'clean':
      _RaiseForUnsupportedOption('profile_type')

    if browser_options.extra_wpr_args:
      _RaiseForUnsupportedOption('extra_wpr_args')

    if not browser_options.disable_background_networking:
      _RaiseForUnsupportedOption('disable_background_networking')

    if browser_options.no_proxy_server:
      _RaiseForUnsupportedOption('no_proxy_server')

    if browser_options.browser_user_agent_type:
      _WarnForUnsupportedOption('browser_user_agent_type')


text:
Return of the Cicadas
Rebecca Katzman
OUT AND ABOUT Brood 10 cicadas last emerged across the eastern United States in 2004. RICHARD ELLIS—GETTY IMAGES
Parts of the eastern United States and Midwest will witness an extraordinary natural event this spring. Trillions of Brood 10 cicadas are expected to emerge in mid-May after 17 years underground. They'll likely be around until late June or July.
They come out to mate. Across 15 states, the cicadas will create a buzz as loud as a lawn mower—their mating call. It's the male cicada that's making all that noise, Michael Raupp told TIME for Kids. Raupp is an entomology
entomology SANDRA STANDBRIDGE—GETTY IMAGES the study of insects (noun) Jack's fascination with grasshoppers got him thinking about a career in entomology. professor at the University of Maryland, College Park. "It's going to be a big boy band up in the treetops," he says.
Cicadas are believed to come out in huge numbers to overwhelm predators. Within a few weeks, females will lay hundreds of eggs, and then, as suddenly as they appeared, the cicadas will die. Their offspring, having tunneled into the ground, won't come out for another 17 years.
"This unique event happens nowhere else on the planet," Raupp says. "The abundance of these cicadas is going to be amazing."
Brood 10 is a family of what are known as periodical cicadas, meaning they come out once in a while instead of every year or every few years. Their rare appearance in such huge numbers is what makes them fascinating, Raupp says. "I think that's part of the mystery, and what makes them so magical."
The cicadas emerge when soil temperatures reach 64°F. They crawl out from under the ground and fly up into trees. If you're standing outside, one might land on you. But don't worry: Cicadas aren't dangerous. The insects don't bite or sting. They might just give you a friendly little poke.
Up in the trees, female cicadas lay their eggs. After their mission is accomplished, the insects fall to the ground and die.
About six weeks later, the eggs develop into nymphs, or young insects, which tumble out of the trees and dig themselves into the earth. That's where they'll spend the next 17 years, feeding on root sap until they're ready to come out again.
Raupp says it's a drama you just can't miss: "There's going to be birth, there's going to be death, there's going to be predation
predation STEPHEN FRINK—GETTY IMAGES the process of one living thing killing another for food (noun) Seals are vulnerable to predation by sharks. , there's going to be romance. There are going to be songs. Just go out and enjoy these things. This is only going to happen a handful of times in your lifetime."
California Water Laws
New rules in California put limits on outdoor water usage. They're meant to combat drought. The State Water Resources Control Board voted for the restrictions on January 4. The rules include no lawn-
watering for 48 hours after rain. They could…
A Disrupted Reopening
Last week, many kids in the United States returned to school after winter break. They did so just as COVID-19 cases were reaching record levels. School districts are scrambling to protect their students. The surge in COVID-19 infections has been…
Buses Go Electric
Thomas Built Buses is a company in High Point, North Carolina. Workers at its school-bus factory are thrilled about a new plan by the United States government: the infrastructure bill, which President Joe Biden signed into law on November 15.…
A worker studies a burned-out sequoia on November 19. The tree stands in Redwood Mountain Grove, in California's Kings Canyon National Park. The grove was dev…


TILDA found that older adults had an increased risk of over 75 per cent in developing depression if they had a vitamin D deficiency.
The Irish Longitudinal Study on Ageing (TILDA) at Trinity has shown that deficiency in vitamin D is associated with an increased risk of depression of over 75 per cent among older adults.
TILDA has published new data showing, for the first time in Ireland, that a vitamin D deficiency increases the risk of developing depression. The data was collected over a four-year follow-up period and was published in the Journal of Post-Acute and Long-Term Care Medicine. The study marks the discovery of another risk factor for depression, which will help improve prevention strategies and reduce the number of undiagnosed cases of depression.
Vitamin D status is relatively easy and inexpensive to modify through supplementation or fortification. However, In Ireland, fortification of food products with vitamin D is voluntary and few manufacturers do this. This is compounded by the lack of any vitamin D guidelines from the government.
These findings are significant, as the TILDA team reported last year that one in eight older Irish adults are deficient in vitamin D. Vitamin D is essential for bone health and deficiency, and has recently been linked with other non-bone health outcomes, such as inflammation and diabetes.
The authors suggested that the findings could be due to the potential direct effect of vitamin D on the brain. Given the structural and functional brain changes seen in late-life depression, vitamin D may have a protective effect in reducing the effect of these changes. Similarly, other studies have shown that vitamin D status has also been linked other degenerative diseases that affect the brain such as dementia, Parkinson's disease and Multiple Sclerosis.
"TILDA has consistently assisted policy makers by providing strong evidence-based data on which to make recommendations but also by assisting with information of most vulnerable people and therefore those who should be targeted", she said.
Kenny said that it was TILDA's responsibility "to now ascertain whether supplementation will influence depression".
TILDA is funded by the Department of Health, Irish Life and The Atlantic Philanthropies.


We are Milwaukee's leading chiropractic firm looking for practices in greater Milwaukee that want to sell or be part of our ongoing success.
You handle the patient care and we'll handle the rest.
Want to sell your chiropractic clinic?
We will buy your practice quickly and at a fair price.
We buy now, you remain Clinic Director until you retire.
Want Help Growing Your Chiropractic Practice?
Most chiropractor are passionate about the health and wellness of their patients and provide excellent care. Unfortunately, the many demands of running a practice often take time away from patient care leaving doctors conflicted.
Chiropractic Company has grown steadily over the past 30 years and have perfected the business of chiropractic.
Joining Chiropractic Company will let you focus solely on what you love - caring for patients. We will provide the training, expertise and support you need to be a success!
Chiropractors who have joined us found they focus more on patient care and less on business mangement allowing them to take more vacation time and enjoy being part of a winning team.
Ready to Sell Your Chiropractic Clinic?
No matter what your reasons are for wanting to sell your chiropractic business, we will work with you to accomodate your needs.
Planning Retirement and Looking for a Chiropractic Exit Strategy?
We also offer several purchase options to meet your specific needs.
See what other chiropractors had to say about their experience with Chiropractic Company.
I was about five years from retirement when it hit me - what am I going to do with my practice when I retire. I wanted to make sure my long-time patients were taken care of properly, get a good price for my business, and work for another five years. I talked to business brokers who wanted a large fee and then found Chiropractic Company. I sold them my practice and joined their company. I got a fair price, the process only took about two months, and I continue to see patients on a schedule that I am very satisfied with.
I ran my chiropractic practice for 21 years. Although it did well, I felt like there had to be a better, easier way. That's when I found Chiropractic Company. We entered a trial merger that allowed either party to end the relationship at the end of one year. During that time I improved operations and grew faster than ever before. After the trial, we both agreed to finalize the merger, I was given a fair value for my clinic which was converted into shares of Chiropractic Company. I have continued to manage my office and treat patients and been a happy shareholder ever since!
I was ready to retire and for two years I talked to every chiropractor I knew and even employed the help of a business broker but I was unable to sell my business at the price I wanted especially after the large broker fees. Due to family medical issues it became imperative that I sell my business quickly. Luckily, I came across Chiropractic Company who bought my business at a good price and the entire process was completed within one month! They were a true godsend.
We make the merger or acquisition process fast and easy.
Are you interested in learning more about selling your chiropractic practice or joining Chiropractic Company? Please provide the following information and we will be in touch within 24 hours.
For more information, contact us or visit our website.


Sometimes you just want to know where to buy olive leaf extract. You need it ASAP and can't wait for that express next day delivery to show up!!
Vabori Olive Leaf Extract products are sold at select Natural Health Food Stores, Grocery Stores and Pharmacies within Australia. Please note that not all stores carry the entire Vabori range, so please get in touch with the store to confirm they carry the product you are looking for.
If you would like your nearest store to stock Vabori products, ask them to call us and in most cases they can receive delivery for you by the following business day.
Alternatively, you can view all the Vabori Olive Leaf Extract products on our website.
Are you a Vabori Olive Leaf Extract Stockist? Did we forget to add your store location to the list above or do you need high resolution stock images for your website or social media? Then please contact us.


text:
Symantec's Norton Internet Security 2004
By Patricia Fusco | Posted September 10, 2003
Symantec, McAfee Offer New Versions of Virus Protection
How Small Businesses Can Tackle Big Viruses
Network Associates Debuts Netasyst for SMBs
Symantec is preparing to unleash an updated version of its online security and privacy suite for home and small office PC users next week. Norton Internet Security 2004 is designed to provide comprehensive protection against today's most prevalent Internet threats, offering expanded protection against spyware and spam. The security software suite includes Symantec's best-of-breed anti-virus, firewall, intrusion detection and privacy protection, as well as spam and content filtering systems.
"Users are at risk from an increasing number of Internet threats such as the recent Blaster and SoBig worms. This increase in attacks is driven in part by the growing use of always-on broadband services and laptop computers," said Steve Cullen, senior vice president of Consumer and Client Product Delivery at Symantec. "With the release of our simple-to-use Internet security suite, Symantec is making it easier for all users — from novices to professionals — to secure their systems."
Dodging Viruses and Spyware
Norton Internet Security 2004 includes Norton AntiVirus 2004, which introduces expanded threat detection. This new technology identifies certain emerging non-virus threats such as spyware, adware, and keystroke logging programs. These rogue applications can compromise system security, pry into confidential data, and track online behavior.
Norton AntiVirus 2004 will also proactively protect users of file-exchange services such as peer-to-peer networks by scanning for viruses in compressed file archives. Norton AntiVirus scans both incoming and outgoing e-mail and automatically removes viruses, worms, and Trojans as well as viruses hidden in e-mail and instant message attachments. In addition, worm- and script-blocking technologies automatically defend users against known and unknown threats without the need for virus definitions. Consequently, Norton AntiVirus effectively protects users against an expansive range of malicious codes.
Cleaning E-mail Messages
Norton Internet Security 2004 also includes new Norton AntiSpam 2004 technologies that automatically detect and filter out unwanted commercial e-mail at the desktop. Spam is a growing threat both to employee productivity and PC security. It can clog e-mail systems and proliferate online scams. Norton AntiSpam 2004 immediately starts to filter e-mail once it is installed and turned on. It automatically intercepts and analyzes e-mail in standard POP3 connections and marking spam as it comes into the user's in-box.
Norton AntiSpam 2004 integrates tightly with the latest versions of Microsoft Outlook, Microsoft Outlook Express and Eudora, automatically creating a spam folder and using it to collect all e-mail identified as spam. It will even filter spam from Hotmail and MSN Mail when messages are retrieved through Microsoft Outlook. Norton AntiSpam 2004 also keeps banner ads, pop-up windows, and other Web distractions at bay.
Norton AntiSpam 2004 provides multiple layers of filters to reduce false positives that typically rob users of desirable e-mail messages. An "Allowed List" capability protects friendly e-mail messaged from being accidentally tagged as spam. A configuration wizard makes it relatively easy to import the user's address book into their "Allowed List." A "Trusted Mode" feature further enables users to set up to receive e-mail only from approved senders, while a "Blocked List" deftly blackballs e-mail addresses sent from known spammers.
Unlike many other spam filters, the trainable filtering engine in Norton AntiSpam 2004 learns what is and isn't spam by analyzing a user's outgoing e-mail messages. An intuitive user interface, automatic setup and updating, and effective ad-blocking technology ensures that users are protected from unwanted e-mail and invasive ads — right out of the box.
"Spam already accounts for the bulk of e-mail messages many people receive today, and it continues to proliferate. Symantec has brought its broad expertise to bear on this serious problem by developing a new solution to help users regain control of their e-mail in-box," said Steve Cullen, senior vice president, Consumer and Client Product Delivery at Symantec.
Securing Wanderers
Norton Internet Security 2004 also includes Norton Personal Firewall 2004. This new version of firewall software introduces innovative technology to help laptop users connect to different networks. When a laptop connects to a new or previously saved network, Norton Personal Firewall 2004 adjusts its settings accordingly. This technology ensures that users who connect to multiple networks in different locations will still have adequate online protection wherever they go. Users can control their firewall settings from their Internet Explorer Web browser, which provides the flexibility required to connect to more than one network.
Norton Personal Firewall 2004 features robust intrusion detection technology designed to thwart serious threats from worms and viruses, such as Bugbear, Nimda and Code Red. This system adds another full layer of security to the perimeter protection of the firewall. The intrusion detection system inspects the actual content of the Internet traffic — automatically blocking Internet attacks.
Norton Internet Security 2004 also includes powerful privacy protection technology. This feature prevents confidential information from being sent without user authorization to unsecured websites or through e-mail, Microsoft Office attachments and instant messaging services. For additional security, Norton Internet Security provides tamper-proof password protection that prevents security settings from being turned off manually or being overridden by malicious code.
Site Blocking
Norton Internet Security 2004 also keeps employees out of inappropriate material while online. Of course, the same technology helps parents keep kiddies out of pornography sites and other content they deem detrimental to the eyes of youth. For increased flexibility, Norton "Parental Control" enables users to set up different Internet access privileges for individuals using a single PC.
Symantec's automatic "LiveUpdate" ensures that all products in the suite are kept current with new protection updates to guard against currently unknown Internet threats. In addition, to protect users from pirated or counterfeit software, Norton Internet Security 2004 includes a product activation component. This thwarts software pirates from forging Norton Internet Security products and trapping unwitting users in a Web of wide-open access to their PCs.
Norton Internet Security 2004, Norton Internet Security 2004 Professional, and Norton Personal Firewall 2004 are available now for preorders for about $70, $100 and $50, respectively. Norton Internet Security 2004 Professional 5- and 10-user packs are priced just under $450 and $800, respectively.
Norton AntiSpam 2004 is also available as a standalone product. The software comes with 12 months of protection updates and is also available now for pre-orders for about $40. Five- and 10-user packs of Norton AntiSpam 2004 are just under $180 and $320, respectively.
All products will be available in mid-September at various retail locations as well as other online retailers. Pre-orders are available through SymantecStore.com.


He received his Doctor of Dental Surgery from the University of the Pacific in 1976- now known as Arthur A. Dugoni School of Dentistry. Dr. Russo has been practicing in San Bruno since 1982. He undergone extensive training for implants and is certified in placing Nobel Biocare Hiossen Implants. He is also a certified Invisalign provider.
When he's not working in your mouth, he enjoys a round of golf and spending quality time with his Grand-baby Eve. If you come toward the end of the day, you might just catch a glimpse of her. He enjoys traveling abroad with his girlfriend- they toured Europe and Southeast Asia this year alone! Just ask him about it!
He received his Doctor of Dental Medicine from the University of Pennsylvania and completed his General Practice Residency at Lehigh Valley Health Network Education. He comes with 8 years of experience- ranging from restorative, endodontic, and surgical treatments. With his perpetual calm demeanor, patients feel effortlessly at ease. Dr. Summerfelt provides nothing less than a positive experience.


Longer Life:Heated water attacks all metals, but not porcelain. That's why Suburban uses a porcelain-lined, steel water heater tank with an anode rod to "absorb" the electrolytic action – just like your water heater at home.
Faster Recovery:Fast recovery means more hot water and fewer cool water cycles. Suburban uses a copolymer insulation jacket specially moulded to fit the tank which does a better job of retaining heat than some other models. For higher recovery, both the gas and electric element may be used simultaneously.
More Convenience: All Suburban water heaters are designed for easy installation, operation and maintenance. The limit switch, reset button and on/off electric element rocker switch are conveniently located in a housing behind the removable outer door. The electric heat element and combination drain plug/anode rod are also located there.


One of the major innovations introduced in 2015, which will be repeated again this year on the 13th edition, was MED Fado. In the cloisters of the Convent, a Fado House was recreated, where local singers will perform. This is intended to be a tribute to Fado as Intangible Cultural Heritage of Humanity (2011). More than a musical style, Fado is part of the Portuguese soul and a key element of our tradition, so its presence in the MED will be another way to promote Portuguese culture.