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Published in Oncotarget V8I40, Sep 15, 2017
A regulatory role for the co-chaperone FKBP51s in PD-L1 expression in glioma
Paolo D'Arrigo, Michele Russo, Anna Rea, Martina Tufano, Elia Guadagno, Maria Laura Del Basso De Caro, Roberto Pacelli, Felix Hausch, Stefania Staibano, Gennaro Ilardi, Silvia Parisi, Maria Fiammetta Romano _ and Simona Romano
Paolo D'Arrigo1,*, Michele Russo1,*, Anna Rea1, Martina Tufano1, Elia Guadagno2, Maria Laura Del Basso De Caro2, Roberto Pacelli2, Felix Hausch3, Stefania Staibano2, Gennaro Ilardi2, Silvia Parisi1, Maria Fiammetta Romano1 and Simona Romano1
1Department of Molecular Medicine and Medical Biotechnologies, Federico II University, Naples, Italy
2Department of Advanced Biomedical Sciences, Federico II University, Naples, Italy
3Technical University Darmstadt Institute of Organic Chemistry and Biochemistry, Darmstadt, Germany
*These authors have contributed equally to this work
Maria Fiammetta Romano, email: [email protected], [email protected]
Keywords: PD-L1, glioblastoma multiforme, FKBP51, ionizing radiation, SAFit
Received: March 10, 2017 Accepted: June 11, 2017 Published: July 17, 2017
Background: FKBP51 is a co-chaperone with isomerase activity, abundantly expressed in glioma. We previously identified a spliced isoform (FKBP51s) and highlighted a role for this protein in the upregulation of Programmed Death Ligand 1 (PD-L1) expression in melanoma. Because gliomas can express PD-L1 causing a defective host anti-tumoral immunity, we investigated whether FKBP51s was expressed in glioma and played a role in PD-L1 regulation in this tumour.
Methods: We used D54 and U251 glioblastoma cell lines that constitutively expressed PD-L1. FKBP51s was measured by immunoblot, flow cytometry and microscopy. In patient tumours, IHC and qPCR were used to measure protein and mRNA levels respectively. FKBP51s depletion was achieved by siRNAs, and its enzymatic function was inhibited using selective inhibitors (SAFit). We investigated protein maturation using N-glycosidase and cell fractionation approaches.
Results: FKBP51s was expressed at high levels in glioma cells. Glycosylated-PD-L1 was increased and reduced by FKBP51s overexpression or silencing, respectively. Naïve PD-L1 was found in the endoplasmic reticulum (ER) of glioma cells complexed with FKBP51s, whereas the glycosylated form was measured in the Golgi apparatus. SAFit reduced PD-L1 levels (constitutively expressed and ionizing radiation-induced). SAFit reduced cell death of PBMC co-cultured with glioma.
Conclusions: Here we addressed the mechanism of post-translational regulation of PD-L1 protein in glioma. FKBP51s upregulated PD-L1 expression on the plasma membrane by catalysing the protein folding required for subsequent glycosylation. Inhibition of FKBP51s isomerase activity by SAFit decreased PD-L1 levels. These findings suggest that FKBP51s is a potential target of immunomodulatory strategies for glioblastoma treatment.
Glioblastoma is the most dangerous and aggressive form of brain cancer. Currently, the curative attempt is not possible in the majority of glioblastoma patients. However, there are some long-term survivors. The use of targeting molecules that neutralize the so-called "checkpoint inhibitors" of the immune system is a promising strategy for glioblastoma treatment [1]. Similarly to many other tumours, gliomas express Programmed Cell Death-Ligand 1 (PD-L1) [2–4], encoded by the PDCD1L1 gene. Although PD-L1 is not an oncogenic driver of the tumour per se, it nevertheless protects the tumour from its immune microenvironment. Engagement of PD-L1, with its cognate receptor PD1, expressed by activated lymphocytes, inhibits T-cell activation via phosphatase SHP2-mediated inhibition of kinases [5]. PD-L1 can also exert an inhibitory effect on T cells through B7-1 [6]. Monoclonal antibodies targeting PD-1/PD-L1 are being extensively used in a wide range of solid tumour types, including melanoma, non-small cell lung cancer, squamous cell cancer of head and neck, bladder cancer, renal cell carcinoma, as well as haematological malignancies, particularly, aggressive B-cell lymphomas and multiple myeloma [7]. The predictive value of PD-L1 as a biomarker of response is under continuous investigation, especially because of the numerous technical issues associated with IHC. The use of various PD-L1 antibodies and diverse IHC cut-offs across clinical trials has made it difficult to assess the usefulness of PD-L1 as a biomarker of response. However, some available data suggest that patients with higher levels of PD-L1 expression have an improved response rate to immunotherapy and progression-free survival [8]. PD-L1 expression in glioblastoma tumours is considerably higher than that observed in melanoma and lung cancer [3]. Also, PD-L1 expression in glioma is sustained and increased by standard radiation treatments [9, 10]. Based on their encouraging efficacy in an increasing variety of human neoplasias, several clinical studies are currently under way to evaluate checkpoint inhibitors in patients with glioblastoma [11–13]. A major concern of the immune checkpoint blockade remains immunotoxicity, with related adverse events that are sometimes life-threatening and prevent the completion of the treatment. In addition, the occurrence of primary or acquired resistance to the inhibitory immune checkpoint-targeted therapy represents a further limitation of this anti-cancer approach. A better understanding of the specific mechanisms that regulate PD-L1 expression may provide new tools for circumventing the tumour's ability to suppress its immune microenvironment. Parsa et al. demonstrated the dependence of PD-L1 expression on the PI3k/Akt pathway in human glioma and showed a post-transcriptional increase of such immune-inhibitory ligands after the loss of PTEN [14]. Similarly to several other membrane proteins, PD-L1 expression is also regulated at a post-translational level. PD-L1 undergoes heavy glycosylation that greatly increases the affinity for PD1 [15]. The targeting of mechanisms that control post-translational modification can help in neutralizing the pro-tumoral effect of PD-L1. Our research has previously identified a role for a spliced variant of the FKBP5 gene in the regulation of PD-L1 expression in melanoma [16]. FKBP5 encodes a large immunophilin which was originally cloned in lymphocytes [17] but also found abundantly expressed in several tumours [18–23], including glioblastoma [24]. The FKBP51 protein structure includes a C-terminal TPR three tandem repeat domain, responsible for protein-protein interaction, and two N-terminal FK506 binding domains (FKBP), of which the most N-terminal one exerts a peptidyl-prolyl isomerase (PPIase) activity [25]. The spliced isoform (isoform 2 or FKBP51s) [16] lacks a TPR domain and has a distinct C-terminus compared to isoform 1. Isoform 2 resulted induced, bidirectionally, in melanoma and lymphocytes following their interaction through the PD-L1/PD1 ligand/receptor system [16]. Classically, the PPIase activity of FKBPs is inhibited by the immunosuppressant agents rapamycin and FK506. More recently, potent and specific inhibitors of FKBP51 have been created and named SAFit 1 and 2 because of their selectivity through an "induced fit" mechanism [26]. Here we aimed to investigate the role of FKBP51s in PD-L1 regulation in glioma. FKBP51s showed varying degrees of expression in glioma cancer cell lines and tumours from patients. Silencing of FKBP51s reduced the expression levels of glycosylated PD-L1 forms. FKBP51s physically interacted with the naïve PD-L1 protein in the endoplasmic reticulum (ER), suggesting it served as a catalyst of PD-L1 glycosylation. We also show that SAFit1 and, especially, SAFit2 reduced expression of PD-L1 and impaired ionizing radiation (IR)-induced PD-L1 up-regulation.
Expression of FKBP51s in glioma cells
We measured the expression of FKBP51 isoforms by immunoblot in D54 and U251 glioma cells, using A375 and SAN melanoma cell lines for comparison. As shown in Figure 1A, both the canonical and the spliced FKBP51 isoforms were expressed in the glioblastoma cells. Whereas in melanoma, FKBP51s was barely detected under basal conditions [16]. FKBP51s expression in glioblastoma cells was confirmed by fluorescent microscopy (Figure 1B), showing a localization in both nuclear and cytoplasmic compartments (Figure 1B). A co-staining of both isoforms showed that the canonical and the spliced FKBP51 are coexpressed, even if the canonical isoform seems to have a brighter nuclear signal than the short isoform (Figure 1C). The co-staining also suggested that, in some glioma cells, the short isoform appears as the only detectable form in the nucleus. Figure 1C also suggests the specificity of the FKBP51s signal, as it was barely detectable in another cell line (iPSCs, undifferentiated human induced Pluripotent Stem Cell) that was found to express the canonical isoform.
Figure 1: FKBP51s expression in glioma cells. (A) Immunoblot assay of FKBP51 and FKBP51s expression in D54 and U251 glioma cell lines. Lysates from A375 and SAN melanoma cell lines were run for comparison. γ-Tubulin was used as loading control. (B) FKBP51s (green) immunostaining of D54 and U251. DAPI (blue) was used to counterstain nuclei; FKBP51s/DAPI merge was shown. Scale bar 50μm. Image was captured with an inverted microscope (DMI4000; Leica Microsystems, Heidelberg, Germany) and Leica Application Suite Advanced Fluorescence (LAS AF) software (Leica Microsystems). (C) D54 glioma cell and the undifferentiated human iPSC were stained with DAPI (blue) to counterstain nuclei; FKBP51 (red); FKBP51s (green); FKBP51/FKBP51s merge was shown. Scale bar 50μm. Confocal images were captured with Leica TCSSMD FLIM (Leica Microsystems) microscopes.
FKBP51s regulates PD-L1 expression
To investigate the role of FKBP51s on PD-L1 expression regulation in glioma, we first measured PD-L1 levels in U251 and D54 glioblastoma cell lines by flow cytometry (Figure 2A) and immunoblot (Figure 2B). Both cell lines expressed high levels of PD-L1 on the plasma membrane (Figure 2A). Several PD-L1 isoforms were observed by immunoblot (Figure 2B). A lower band (at ~37 kDa) corresponding to the naïve protein and two upper bands at ~50 and ~68 kDa could be accounted for by post-translationally modified isoforms [15, 16]. However, as other authors and we noted [32], PD-L1 expression has a highly variable pattern and banding, even using the same antibody for detection. Such variability is mostly because of post-translationally modified forms or different degrees of protein maturation [32]. Consistently, in our study, additional bands between 37–50 kDa or under 37 kDa were sometimes detected. Next, we used different FKBP51s siRNAs and a PD-L1 siRNA for protein knockdown and evaluated modulation of PD-L1 levels by immunoblot. Figure 2C shows that both FKBP51s siRNAs #1 and #2 (but especially the siRNA #2 and the siRNA combination) were effective at reducing PD-L1 levels in U251 glioma cells. The intensity of the 68-kDa band was reduced with both FKBP51s siRNA and PD-L1 siRNA. The 50-kDa band was reduced when cells were treated with FKBP51s siRNAs and especially the mix of siRNAs, which strongly reduced FKBP51s levels. A decrease of the 37 kDa PD-L1 band was observed with PD-L1 siRNA and, surprisingly, also under FKBP51s silencing. The level of the PD-L1 transcript of U251 and D54 cells was reduced by PD-L1 siRNA but not FKBP51s silencing (under the latter condition, PD-L1 transcript appeared to be slightly increased) (Supplementary Figure 1). Apparently, this result excluded a transcriptional regulation by FKBP51s and, thus, the decrease in the band at 37 kDa could reflect a condition of ER stress related to possible imbalance of ER-proteins. The observation that intensity of the highest kDa bands was reduced by FKBP51s siRNA more than PD-L1 siRNA was consistent with our hypothesis that FKBP51s is involved in PD-L1 post-translational control. The effect of FKBP51s knockdown on PD-L1 was also evaluated by flow cytometry. Analysis of mean fluorescence intensities (MFI) of PD-L1 expression on the plasma membrane of U251 cells suggested a significant decrease in PD-L1 expression following FKBP51s silencing (Figure 2D). Representative PD-L1 histograms in the overlay, from U251 cells, are shown in Figure 2E. Finally, to confirm that FKBP51s positively regulated PD-L1 expression in glioma, we overexpressed this FKBP51 isoform in D54 and U251 cells and measured PD-L1 level by immunoblotting. Figure 2F shows that the ~50 and 68 kDa PD-L1 bands were increased by FKBP51s-overexpression compared to control cells transfected with empty vector. Supplementary Figure 1B shows the mRNA levels of PD-L1 and FKBP51s in transfected D54 and U251 cells.
Figure 2: Modulation of PD-L1 expression in glioblastoma cells. (A) Flow cytometric histograms of PD-L1 expression in U251 and D54 glioblastoma cells. (B) Immunoblot of lysates prepared from the same cells shows 3 isoforms ascribable to the naïve (37 kDa) and post-translational modified protein (~50 and ~68 kDa). (C) Immunoblot of cell lysates obtained from the glioblastoma cell line U251, transfected with FKBP51s siRNAs, PD-L1 siRNA or a non-silencing (NS) RNA. G3PDH was used as loading control. (D) U251 cells treated as in C, was analysed by flow cytometry for PD-L1 expression. The graph represents MFI (mean and standard deviation) of PD-L1 expression. P values are indicated (N=6). (E) Flow cytometric histograms of PD-L1 expression in U251 glioma cells, silenced or not for FKBP51s, or PD-L1. Histograms are shown in overlay. (F) Immunoblot of cell lysates obtained from the D54 and U251 glioblastoma cell lines over expressing FKBP51s or transfected with the void vector (EV). Expression of PD-L1 resulted increased by FKBP51s but not EV transfection. γ-Tubulin was used as loading control.
FKBP51s serves as a PD-L1 co-chaperone in ER
The addition of carbohydrates is the principal chemical modification to most plasma membrane proteins. PD-L1 is N-glycosylated (http://www.uniprot.org/uniprot/Q9NZQ7#ptm_processing). Because glycosylation can occur in the lumen of the ER and/or in the lumina of the Golgi cisternae, to address the role of FKBP51s in PD-L1 glycosylation, we performed subcellular fractionation of D54 cells and obtained protein extracts from these cell compartments. As shown in Figure 3A, the naïve 37 kDa PD-L1 was detectable in the ER, and scarcely in the Golgi. The 68-kDa band was expressed mostly in the Golgi. In the Golgi, the band at 50 kDa was also appreciable. FKBP51s, but not FKBP51, was found in the ER. The naïve PD-L1, FKBP51 and FKBP51s were all found in the nucleus. By using an N-glycosidase, we could confirm that the 68-kDa band was glycosylated. As shown in Figure 3B (right), treatment of the immunoprecipitated PD-L1 protein with PNGase F produced a decrease of the 68-kDa band (indicated by the right arrow) and the appearance of an additional band at around 37 kDa (indicated by the left arrow). These findings suggest that FKBP51s has a role in catalysing PD-L1 folding, an essential step of glycosylation. To address this issue, we performed co-IP of protein from purified ER extract. As shown in Figure 3C, pull-down of either FKBP51s or PD-L1 confirmed that the two proteins interact with each other in the ER.
Figure 3: FKBP51s is associated with PD-L1 in ER. (A) Immunoblot of D54 lysates obtained from sub-cellular compartments. PD-L1, FKBP51 and FKBP51s levels are shown along with relative organelle markers. (B) Whole D54 lysates immunoprecipitated with anti-PD-L1 and subjected, or not, to PNGase F treatment; the arrows indicate the higher (- PNGase F) and lower (+ PNGase F) PD-L1 band. Fetuin, on the left of the panel, was used as positive control of PNGase digestion. (C) Co-IP of PD-L1 and FKBP51s in ER fraction. ER lysate was immunoprecipitated with anti-PD-L1 and anti-FKBP51s and recognized for each protein by immunoblot.
SAFits, the selective inhibitors of FKBP51, reduce PD-L1 expression
Because SAFit compounds selectively inhibit the catalytic activity of FKBP51, we investigated their ability to modulate expression of PD-L1 in glioma cells. Figure 4A shows a dose response assay of PD-L1 expression levels, measured by flow cytometry, after 12 h of culture in the presence of one of three doses of SAFit1 and SAFit2 (5, 25 or 50 nM). Both compounds (but especially SAFit2) produced a decrease in PD-L1 expression on the plasma membrane, as measured by flow cytometry. SAFit2 significantly inhibited PD-L1 activity at 12 h, but activity returned to the baseline after 48 h (Figure 4B). Figure 4C shows a representative flow cytometry histogram of PD-L1 down modulation by SAFit2 (left) and an immunoblot (right) showing that the post-translational modified forms of PD-L1 (indicated by the arrows) are especially affected by SAFit2. Because ionizing radiation increases tumour cell expression of PD-L1 [10], we investigated whether SAFit was able to counteract such an IR effect. Using different IR doses, we detected a significant increase in PD-L1 expression at 4 and 8 Gy (p=0.05), compared with non-irradiated glioma cells (Figure 4D). Upregulation of PD-L1 after IR was also measured at the mRNA level (Supplementary Figure 2). SAFit2 produced a decrease in PD-L1 expression. In the presence of SAFit2, PD-L1 expression following 4 Gy irradiation was similar to the untreated control cells (Figure 4D). An immunoblot of D54 cell extracts prepared at 0, 3, 12 and 24 h following irradiation showed an increase of the intensity of the band at 68 kDa (indicated by the arrow) after 12 and 24 h (Figure 4E). Such an increase appeared to be counteracted by SAFit2 (Figure 4E). The blot also shows two bands of around 37 kDa. The lower of these bands was especially induced at 12 h after irradiation. This approximately 34-kDa band might correspond to a cleavage product or further isoform of PD-L1, which is increased by IR. The blot also shows a decrease of the 37 kDa band after 24 h of SAFit plus IR, which suggests that SAFit might affect protein synthesis. Analysis of extracts obtained by cell fractionation (Figure 4F) shows that, in the ER, IR induces the expression of PD-L1 forms of between 37 and 50 kDa (at 12 h after irradiation) but that these are decreased at 24 h. Interestingly, FKBP51s disappeared from the ER at 3 h after IR, reappearing at 12 h after IR. Golgi extracts showed that the levels of the PD-L1 upper band changed in accordance with the upper band of whole extracts. To address the effect of SAFit on IR-induced PD-L1 upregulation, we irradiated the cells in the absence or the presence of 50 nM SAFit2 and recovered extracts after 12 h. By this approach, we confirmed that IR induces the expression of PD-L1 forms of up to 37 kDa and that this effect is attenuated by SAFit2 (Figure 4G). The role of FKBP51s in IR-induced PD-L1 increase was further investigated using a siRNA for FKBP51s in irradiated cells. As shown in Supplementary Figure 3, FKBP51s silencing impaired IR-induced PD-L1 upregulation.
Figure 4: SAFit reduces the expression of PD-L1. (A) Dose response assay of SAFit on PD-L1 expression. D54 glioblastoma cells were incubated with SAFit1 and 2 at different concentrations. After 12 h of incubation, cells were harvested and analysed by flow cytometry for PD-L1 expression. Graph columns represent mean fluorescence intensities and bars the standard deviation of PD-L1 expression (N=3). (B) Kinetic of PD-L1 expression. Glioblastoma cells were incubated with SAFit2 at different times, then, cells were harvested and analysed by flow cytometry for PD-L1 expression. Graph represents mean fluorescence intensities of PD-L1 expression (N=3). (C) Flow cytometry (left) and immunoblot (right) of SAFit-modulated PD-L1 expression. Cells were cultured, in the absence or the presence of 50 nM SAFit2 for 12h. (D) Dose response assay of IR-induced PD-L1 expression and modulation by SAFit2. D54 glioblastoma cells were irradiated at different Gy doses (1, 4 and 8Gy), in the absence or the presence of 50 nM SAFit2. After 12 h incubation, cells were harvested and analysed by flow cytometry for PD-L1 expression. The graph represents mean fluorescence intensities (mean and standard deviation, N=3) of PD-L1 expression. (E) Kinetic of IR-induced PD-L1 expression levels. D54 cells were harvested 3, 12 and 24h after 4 Gy IR. The band at 68 kDa (indicated by the arrow) resulted increased after 12 and 24 h from IR. The addition of SAFit prevented such an increase. (F) Immunoblot of lysates obtained from sub-cellular compartments of D54 cells after a 4 Gy IR. PD-L1 and FKBP51s levels are shown along with relative organelle markers. (G) Immunoblot of D54 lysates obtained from sub-cellular compartments. D54 cells cultured in the presence or not of SAFit2, was harvested 12h after IR. PD-L1 is shown along with relative organelle markers. SAFit2 reduced ER and Golgi PD-L1 bands. (H) pAkt expression levels in D54 lysates obtained from whole cell and ER compartment, shown along with ER marker.
In addition, we thought to investigate the levels of pAkt during IR, given that Akt is the master regulator of PD-L1 expression in glioma [11]. Phospho-Akt levels were increased at 3 h after IR, remained high at 12 h after IR, and decreased at 24 h after IR (Figure 4H). An analysis of pAkt levels in ER extracts found that these levels were particularly high in this cell compartment (Figure 4H), which is consistent with the role of Akt in the ER membrane [33]. The observation that the PD-L1 maturation forms appeared in the ER only upon restoration of the FKBP51s level, is a further element in support of the important FKBP51s function as a PD-L1 molecular chaperone in ER.
SAFits reduce PD-L1-induced cell death of PBMC cocultured with glioma cells
The PD1 receptor was identified as a gene upregulated in a T-cell hybridoma undergoing cell death [34]. Accordingly, triggering PD1 through PD-L1 induces cell death [35]. We performed co-cultures of glioblastoma and healthy donor PBMCs, as previously described [16], to address whether SAFit-mediated PD-L1 down modulation on glioma cells could modulate death of PBMC via tumour-associated PD-L1. To this end, we used different glioma cell lines, D54, U251 and SF767, the latter of which expresses very low levels of PD-L1 (Figure 5A) and performed PBMC/glioma co-cultures. PBMCs were harvested after 6 h and analysed by annexin V staining in double fluorescence with CD45, by flow cytometry. A gate was placed on CD45 positive cells to exclude non-PBMC cells from the analysis (Supplementary Figure 4). Figure 5B shows box plots of cell death values of PBMC not co-cultured (no CC) or co-cultured with glioma cells (CC). We measured a significant increase in the cell death of PBMCs co-cultured with glioma cells, compared to cell death of no CC PBMCs. The addition of anti-PD1, but not an unrelated humanized antibody (abciximab) [27], reduced cell death in D54 and U251 but not in SF767 co-cultures (Figure 5B). These results suggest that glioma cells can kill PBMCs through PD-L1. However, the observation that CC/SF767 PBMC also undergoes cell death suggests a tumoral-induced cytotoxicity independent of PD-L1, possibly involving other factors (e.g. Fas ligand [36] or TGF-β [37]). A further detailed analysis for comparison of apoptosis of each experimental condition is shown in Supplementary Table 1. Use of SAFit1 and SAFit2 also resulted in protection of CC/D54- and CC/U251-PBMC from cell death, in accordance with the PD-L1 down modulation effect by SAFit. Additionally, the use of PD-L1 siRNA, to down modulate PD-L1 on glioma cell lines, reduced CD45+ cell death in D54- and U251- but not SF767- co-cultures (Figure 5C), thus supporting the causative role of PD-L1 in glioma killing of immune cells.
Figure 5: PD-L1-induced PBMC death is reduced by SAFit. (A) PD-L1 expression in D54, U251 and SF767 glioblastoma cells analysed by flow cytometry (upper) and immunoblot (lower). (B) Graphical representation of cell death values of PBMC not cocultured (no CC) or cocultured (CC/) for 6 h with D54, U251 and SF767 glioblastoma cells, in the absence or presence of anti-PD1, SAFit1, SAFit2 or a humanized antibody (abciximab) as control. PBMC was harvested and assayed in flow cytometry for Annexin-V binding in double fluorescence with CD45. Values measured for each cell line are reported as box plots. Differences (p-values) are shown (N=6). (C) Graphical representation of cell death values of PBMC no CC or cocultured for 6 h with D54, U251 and SF767 glioblastoma cells, previously silenced or not for PD-L1. PBMC was harvested and assayed by flow cytometry. Values are reported as box plots. Differences are shown (N=6).
FKBP51s is expressed in glioblastoma specimens from patients
Expression of FKBP51s and PD-L1 was investigated by immunohistochemistry in 29 glioblastoma specimens. Details on IHC semi quantitative evaluation are reported in Supplementary Table 2. PD-L1 staining was observed in glioblastoma tissues to a variable extent and intensity. In the majority of the cases, 22/29 (75.86%), a prominent cytoplasmic diffuse/fibrillary expression pattern was observed. In 3 of these 22 cases (3/29; 10.34%), we found interspersed singular or focally aggregated epithelioid cells, with membranous immunostaining for PD-L1. Very faint and focal immunoreactivity was observed in perilesional brain tissue, when present. In Figure 6A, 6B and 6C, hematoxylin and eosin staining, expression of the glial fibrillary acidic protein and PD-L1 expression of case #28 are shown. Two further PD-L1 IHC results are shown in Supplementary Figure 5. Cytoplasmic and nuclear FKBP51s expressions of tumour tissue were observed (Figure 6D–6F). Both of these were scored. The semi quantitative distribution of cytoplasmic FKBP51s was as follows: score 0 in 10 of 29 cases (35%), score 1 in 6 of 29 cases (21%), score 2 in 1 of 29 cases (3%), score 3 in 5 of 29 cases (18%), score 4 in 3 of 29 cases (10%), score 6 in 3 of 29 cases (10%) and score 9 in 1 of 29 cases (3%). Nuclear FKBP51s scores were 0 in 2 of 29 cases (7%), score 1 in 3 of 29 cases (10%), score 2 in 8 of 29 cases (28%), score 3 in 1 of 29 cases (3%), score 4 in 6 of 29 cases (21%), score 6 in 6 of 29 cases (21%), score 9 in 3 of 29 cases (10%). Statistical analysis revealed that the scores of FKBP51s were different when comparing specimens with absent/low-PD-L1 (arbitrarily defined as a score >=0, <=3) and high-PD-L1 (score >=4, <=9). As shown in Figure 6G, the levels of FKBP51s, both cytoplasmic and nuclear, are likely to be increased in high-PD-L1 samples, which is in line with in vitro findings that FKBP51s drives PD-L1 expression. FKBP51s expression was confirmed at the mRNA level in eight glioblastoma specimens (Figure 6H).
Figure 6: FKBP51s and PD-L1 immunohistochemistry of glioblastoma samples. Case #28, (A) hematoxylin and eosin staining; (B) anti-GFAP; (C) diffuse/fibrillary cytoplasmic PDL1 expression with a membranous signal in more than 5% of neoplastic cells. Original magnification x400. (D, E, F) FKBP51 immunostaining: cytoplasmic signal was strong, moderate and mild in case #12 (D), #14 (E) and #19 (F), respectively. In the nucleus, the reactivity was moderate in case #12 (D) and case #19 (F) and strong in case #14 (E). Original magnification x400. (G) box plots of FKBP51s scores (c=cytoplasmic and n=nuclear) in PD-L1low and PD-L1high expressing glioblastomas. (H) quantitative FKBP51s expression of 8 glioblastoma samples. QPCR was performed by using a coamplified β-actin internal control for sample normalization. FKBP51s value of a PBMC has been also run for comparison. Columns and bars are representative of means and standard deviations of values for each sample as individual data point.
Here, we address the expression and function in glioma of FKBP51s [10], a protein isoform that is generated by an alternative splicing of the FKBP5 gene. FKBP51s was first identified in melanoma [16]. Unlike in melanoma, in which FKBP51s was upregulated by contact with lymphocytes [16], this spliced isoform appeared to be constitutively expressed in glioblastoma cell lines at high levels. Our results show that, in glioma cells, FKBP51s is complexed with PD-L1 in the ER and its down modulation decreased the level glycosylated-PD-L1, suggesting that the isomerase activity of FKBP51s was relevant for PD-L1 folding and processing. It is worth noting that post-translational modifications of surface receptor/ligand antigens greatly influence the affinity for the cognate molecules. In particular, PD-L1 has an affinity for PD1 of 0.77 μM when glycosylated and in dimers, while the monomeric nonglycosylated protein has an affinity of 26.6 μM [15]. In accordance with its proposed role as a PD-L1 foldase, use of selective inhibitors of FKBP51 PPIase activity significantly down modulated PD-L1 expression. Most interestingly, SAFit2 was able to counteract the increase in PD-L1 levels in glioblastoma cells treated with IR. This result might have an impact in clinical practice, considering that combining IR and anti-PD-L1 treatment led to tumour regression and prevented the growth of secondary tumours in a mouse tumour xenograft model [38]. Given the above findings, FKBP51s may contribute to an efficient inhibitory checkpoint signal, and thus to glioma-edited immune suppression. Consistent with such a hypothesis, the addition of SAFit to co-cultures of PBMCs and glioma cells reduced apoptosis of immune cells induced through PD-L1/PD1. Future in vivo studies with syngeneic mouse models are now needed to validate the efficacy of selective FKBP51 inhibitors in modulating tumour PD-L1 expression and, most importantly, to verify whether these small molecules are able to positively affect tumour microenvironment and ameliorate immune defences against glioma. Besides the tumour, immune accessory cells in the microenvironment can express PD-L1 and suppress CD8-mediated tumour killing. Particularly, in glioblastomas, a very recent study [39] identified a tumour-infiltrating myeloid cell (TIM) population that expands in response to dendritic cell vaccine treatment and greatly contributes to PD-L1 expression in the glioblastoma environment. Whether or not this TIM expresses FKBP51s is unknown. Interestingly, we identified a myeloid-derived suppressor cell CD14+PD-L1+FKBP51s+ in peripheral blood of melanoma patients, expressing high TGF-β levels and apparently associated with resistance to ipilimumab [40]. Immunohistochemistry of FKBP51s performed on 29 tumoral samples showed an expression pattern (with either a nuclear and cytoplasmic localization) similar to that observed in cancer cell lines, thus reinforcing the translational implication of our findings. Regarding PD-L1 expression, in our series, the majority of cases showed a cytoplasmic pattern of reactivity, while a membranous signal was faintly evident only in a few tumour cells showing epithelioid morphologic features, as it was previously observed [3]. A possible explanation for this diffuse/fibrillary cytoplasmic pattern has been attributed to the membrane-bound PD-L1, within the delicate tumour cell process forming the pathognomic neurofibrillary matrix of gliomas [3]. Interestingly, we found that the scores of FKBP51s tended to be increased in accordance with PD-L1 scores, which generates the hypothesis (to be addressed with a larger sample size) that the two variables (FKBP51s and PD-L1) might be associated. We observed that 6 out of 22 PD-L1 positive tumour samples expressed only nuclear FKBP51s, which is in disagreement with our finding that the PD-L1 maturation occurs in the ER. An active FKBP51s shuttling within cell compartments, to convey information on nuclear and cytoplasmic activities, as it occurs for several proteins [41], might explain this result. Currently, the biological significance for the proposed shuttling, as well as the function of FKBP51s inside the nucleus, remain obscure.
Given the above, we propose that FKBP51s is a novel element that regulates PD-L1 expression in glioma cells. This co-chaperone functions as a catalyst and foldase in PD-L1 post-translational modifications, occurring during protein maturation and expression on the plasma membrane. In vitro inhibition of this protein, either by silencing or chemical agent results in PD-L1 down modulation in glioblastoma cells lines and, as a consequence, the suppressive effect exerted by the tumour on immune cells is relieved. Moreover, targeting of FKBP51s counteracts the PD-L1-increasing effect of ionizing radiation. Our findings on patient tumours show that the spliced FKBP51 is widely expressed in glioblastomas. It is worth noting that factors other than FKBP51s control the expression of PD-L1 at the transcriptional and translational level. Nevertheless, this foldase appears to be important for PD-L1 glycosylation, which increases the affinity for its cognate molecule PD1 [15]. It is noticeable that even if targeting the PD1/PD-L1 inhibitory checkpoint is, to date, the most effective immunotherapy used in the treatment of cancers, primary resistance occurs in almost 60% of patients [42]. In addition, a proportion of the responder patients develop acquired resistance. In general, very little is known about the mechanisms of resistance. A number of factors account for primary resistance, such as poor tumour immunogenicity, limited intra-tumoral T-cell infiltration, defective antigen presentation and naive T-cell priming [42]. The release of adenosine or indoleamine 2,3-dioxygenase in the microenvironment or mutations of the genes encoding JAK1 or JAK2 (key intracellular signalling mediating tumour sensitivity to interferon) or the gene encoding β-2-microglobulin (involved in folding and transport of MHC class I molecules) are recognized causes of acquired resistance [42]. We do not have sufficient knowledge on the spliced FKBP51 to speculate on potential effects of its inhibitors on circumventing resistance to anti-PD1. Our data support the conclusion that SAFit acts in the same direction of immune checkpoint blockade (i.e., contrasting PD-L1). In this view, used in combination, SAFit can reduce both the doses and side effects of the immunomodulatory treatment. In addition, a previous study on melanoma [30] showed that SAFit efficiently suppresses the pro-oncogenic activity of canonical FKBP51, namely NF-κB transcription factor activation and cyclin D upregulation. Other authors have identified that canonical FKBP51 exerts a relevant role in glioma aggressiveness [24]. Based on these considerations, SAFit could, at the same time, contrast some autonomous tumour cell functions and PD-L1-mediated T-cell impairment, in glioma tumours, thus integrating two important approaches for cancer treatment. Future studies will address this hypothesis. Finally, the low molecular weight of SAFit allows the agent to penetrate through the brain barrier, which is an important requirement for drugs candidate to glioma treatment [26]. In conclusion, our finding provides the basis for a novel molecular player in glioma immune evasion. In vivo studies with a syngeneic mouse glioma model are needed to confirm that FKBP51s is a useful target for control of glioma immune evasion, thus offering a new tool in the immunotherapy armamentarium against cancer.
Human glioma cell lines D54 and U251, from the CEINGE cell bank (Cellular Technology Platform) at the Advanced Biotechnology Institute (Naples, Italy), were cultured at 37°C and 5% CO2 in DMEM-F12 media (Biowest, Nuaillé, France) containing 10% foetal bovine serum (FBS; Biowest). In accordance with the international guidelines established by the United Kingdom Coordination Committee on Cancer Research (UKCCCR) for the good use of continued cell lines, the cell bank of CEINGE guarantees cell lines for identity, cross-contamination and microorganism contamination. Pluripotent Stem Cells (iPSC, iPS DF19-9-7T; WiCell, Madison, WI, USA) were grown on Matrigel (BD Biosciences) coated dishes in mTeSR1 medium (Stem Cell Technologies). Human glioma cell line SF767 was kindly provided by Prof. Gerolama Condorelli (University of Naples, Federico II) and cultured at 37°C and 5% CO2 in DMEM medium (Biowest, Nuaillé, France) containing 5% foetal bovine serum (FBS; Biowest). Human melanoma cell lines SAN and A375 were obtained and cultured as previously described [21]. All cell lines used are mycoplasma free. To create FKBP51s overexpressing glioma cells, a True-ORF-Myc-DDK-tagged expression vector was used (OriGene Technologies, Rockville, MD, USA). This vector carried the cDNA of the human FKBP5 transcript variant 4. The relative void vector was also transfected to generate control cells. Transfection was performed using a K2 Transfection System (Biontex, Munich, Germany), according to the manufacturer's recommendations. For knockdown experiments, cells were transfected with specific short-interfering oligoribonucleotide (siRNA) (or with a nonsilencing oligoribonucleotide [NS RNA] as control) at a final concentration of 50 nM using the K2 Transfection System. NS RNA and siRNA for PD-L1 were purchased from Novus Biological (Littleton, CO, USA). For FKBP51s siRNA, siRNA #1 and #2 were synthesized using target sequences at the 3′-coding region (between 700 and 1100 bp). In addition to siRNA #1 and #2, the siRNA mix contained siRNA #3 (target sequence in the 3′-UTR region, of between 5200 and 5800 bp). Twenty-four hours before transfection, cells were seeded into six-well plates at a concentration of 4 × 105 cells/ml to obtain 80–90% confluence at the time of transfection. Cells were treated 24 h after transfection. A 6 MV X-ray of a linear accelerator (Primus, Siemens, München, Deutschland) was used for the ionizing radiation (IR) experiments. Selective inhibitors of the FK506-binding protein 51 by induced fit (SAFits) were from the laboratory of Prof. Felix Hausch [21]. The anti-PD1 used in the co-cultures was nivolumab (BMS-936558; Bristol-Myers Squibb, Princeton, NJ, USA). For experiments with SAFit compounds (50 μM in DMSO stock solution), 1:1000 DMSO:DMEM-F12 media was used for control cells.
Immunoblot and immunoprecipitation
Whole cell lysates were homogenized in modified RIPA buffer [15] and assayed by immunoblot. The primary antibody against FKBP51s (rabbit polyclonal of our production, raised against protein C-terminus), CD274/PD-L1 (rabbit polyclonal; Novus Biological) and FKBP51 (rabbit polyclonal; Novus Biologicals) were used diluted 1:2500. For PD-L1 detection, a further rabbit polyclonal antibody Pdcd-1L1 (H-130; Santa Cruz Biotechnology, CA, USA) was used diluted 1:1000. The M2-Flag (mouse monoclonal; Sigma-Aldrich, St. Louis, MO, USA) and γ-tubulin (mouse monoclonal; Sigma-Aldrich) antibodies were used diluted 1:5000. Anti-G3PDH (rabbit monoclonal; Cell Signalling, Danvers, MA, USA) was used at 1:1000. Anti-TGN46 (1:1000; rabbit polyclonal antibody; Sigma-Aldrich); anti-Calnexin (1:1000; AF18, mouse monoclonal; Invitrogen, Carlsbad, CA, USA) and anti-Histone H1 (1:500; AE-4, mouse monoclonal; Santa Cruz Biotechnology) were used as Trans Golgi Network (TGN), endoplasmic reticulum (ER) and nucleus markers, respectively. For co-immunoprecipitation, D54 was used for endogenous pull-down of PD-L1 and FKBP51s. Five-hundred micrograms of total lysate was pre-cleared for 1 h at 4°C, with rotation. Then, 1 μg of each specific antibody was added to the lysate and kept in rotation at 4°C overnight. Twenty microliters of the Protein A-Agarose (Santa Cruz) was added to the mixture and precipitation took place for 2 h, with rotation at 4°C. Samples were then washed in modified RIPA buffer and separated by SDS-PAGE. PD-L1 and FKBP51s antibodies were recognized by immunoblot.
Immunostaining and microscopy
D54 and U251 were fixed in 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 in 1× PBS for 5 min at room temperature. The non-specific block was performed by treating cells with 10% FBS (Biowest), 1% BSA (Biorad), 0.1% in PBS for 30 min at room temperature. Then, the samples were incubated with primary and secondary antibodies. Rabbit polyclonal, FKBP51s and FKBP51 primary antibodies (see Immunoblot and immunoprecipitation section) were used at 1:2000 and 1:50 dilutions, respectively. Alexa Fluor 594 or 488 secondary antibodies were used (1:400; Invitrogen). Nuclei were counterstained with DAPI (Calbiochem-EMD Biosciences, 1:5000). Images were captured with an inverted microscope (DMI4000; Leica Microsystems, Heidelberg, Germany) with Leica Application Suite Advanced Fluorescence (LASAF) software (Leica Microsystems). When required, the brightness, contrast and colour balance of the images were adjusted in Photoshop CS2 (Adobe Systems, San Jose, CA, USA). This adjustment was applied to every pixel in each image.
Glycosidase digestion
Lysates from D54 cells were subjected to PD-L1 immunoprecipitation. Briefly, 500 μg of total lysate were incubated with 800 ng of anti-PD-L1, or rabbit control IgG (rabbit polyclonal; Santa Cruz Biotechnology), and kept in rotation at 4°C overnight. After 24 h, 20 μl of Protein A-Agarose (Santa Cruz) was added to the mixture and precipitation took place for 2 h, at 4°C in rotation. Samples were then washed thrice in modified RIPA buffer and resuspended in appropriate glycosidase buffer. The N-Glycosidase F (PGNase F) digestion was performed according to the manufacturer's instructions (CalBiochem Deglycosylation Kit, Merck Millipore, Temecula, CA) to allow N-linked oligosaccharides cleavage of PD-L1, in native conditions. Digested samples were analysed by immunoblot.
Sub-cellular fractionation
Fractionation was performed according to Sarnataro et al. [28], with little modifications. Briefly, D54 glioblastoma cells were homogenized in 800 μl of Buffer F (0.25 M sucrose, 10 mM HEPES-NaOH, pH 7.2, 10 mM KAc, 1.5 mM MgAc) by pipetting the solution up and down 8–10 times through a 22-gauge needle. The nuclear fraction was sedimented by centrifugation for 5 min at 600×g and then resuspended in RIPA modified buffer. The post-nuclear supernatant was adjusted to 0.75 M sucrose, and the ER fraction was sedimented by ultracentrifugation for 12 min at 16,000 rpm at 4°C, in a Beckman with an SW 50.1 Ti rotor and resuspended in RIPA modified buffer. The supernatant, containing ER fraction, was subjected to immunoprecipitation for PD-L1 as above described. Immunoprecipitated fractions and relative fractionated lysates were analysed by immunoblot (see Immunoblot and immunoprecipitation section for antibodies specification).
Serial sections of 10 μm from routinely Formalin-fixed, paraffin-embedded blocks were cut, and total RNA was isolated from eight paraffinized tumours, chosen among those analysed by IHC, by using the High Pure RNA Paraffin Kit (Sigma) according to the manufacturer's instructions. One microgram of each RNA was used for cDNA synthesis with iScriptTM Reverse Transcription (Bio-Rad, CA, USA). Quantitative gene expression was quantified by qPCR with the 2-ΔCT comparative method [29], using the SsoAdvancedTM SYBR Green Supermix (Bio-Rad) and specific qPCR primers to analyse each transcript. To detect FKBP51, validated QuantiTec primers from Qiagen (Valencia, CA, USA) were used. Relative quantitation of the transcript was performed using co-amplified ribosomal 18S [16] and β-Actin [30] as internal controls for normalization.
Co-cultures of glioblastoma cells and peripheral blood mononuclear cells (PBMCs)
Co-culture was performed as described previously [16]. Briefly, 24 h before starting the co-culture, D54 glioblastoma cells were plated at a concentration of 4 × 105 cells/35 mm plate. After initial plating, the culture medium was aspirated, and adherent cells were washed three times. PBMCs, isolated from heparinized blood by a Ficoll-Hypaque density gradient, were collected and washed by centrifugation. PBMCs were pre-activated with anti-CD3 (eBioscience, San Diego, CA, USA) at the concentration of 5 μg/ml. After 16 h, PBMCs were washed and resuspended in 1% FBS RPMI-1640 medium (Lonza, Belgium) at 1 × 106/ml and 2 ml suspension was added into each glioma cells-precoated plate. Cells were co-cultured at 37°C in a 5% CO2 humidified atmosphere in the presence of anti-PD1 (5 μg/ml) or Abciximab (5 μg/ml). For co-culture with SAFit, glioma cells were preincubated with SAFit1 (40 nM) or SAFit2 (60 nM) for 12 h before adding PBMC. For co-culture with PD-L1 silencing, glioma cells were transfected as described in the Cell culture and transfection section and, after 24 h, PBMC suspension was added to each glioma cell-precoated plate. After 6 h of co-culture, cells in suspension were collected and separated by centrifugation of supernatants. In each experiment, PBMC was also maintained in mono-cultures as a control. PBMC apoptosis was assayed by flow cytometry.
Expression of PD-L1 was assessed using anti-B7H1-phycoerythrin (PE) (R&D Systems, Minneapolis, MN, USA) at a concentration of 0.05 μg/ml. A relative Ig isotype PE-conjugated antibody was used as a control of non-specific binding. Samples were analysed using a BD Accuri™ C6 Cytometer (BD Biosciences, New Jersey, USA). Analysis of apoptosis was performed by annexin-V staining in double fluorescence with CD45-Peridinin Chlorophyll Protein Complex (PerCP) conjugated. Briefly, 1 × 105 cells were harvested 6 h after co-culture (see co-culture paragraph) and resuspended in 100 μl of binding buffer (10 μM Hepes/NaOH pH 7.5, 140 μM NaCl, and 2.5 μM CaCl2) containing 1 μl of annexin-V-FITC (Pharmingen/Becton Dickinson, San Diego, CA, USA) and 5 μl of CD45 for 15 min at room temperature in the dark. Then, 100 μl of the same buffer was added to each sample and analysed with BD Accuri™ C6 Cytometer (BD Biosciences).
Tissues were formalin fixed and paraffin embedded. For each case, the most representative block was selected, and two 4 μm thick sections were cut to perform immunohistochemical staining with the anti-FKBP51s primary antibody (see the Immunoblot and immunoprecipitation section) at a 1:100 dilution and anti-PD-L1 (Novus; see Immunoblot and immunoprecipitation section) at a 1:200 dilution. Sections were dewaxed in xylene, hydrated in a graded series of alcohol and subjected to heat-induced antigen retrieval. After blocking endogenous peroxidase activity, the tissue was incubated with anti-FKBP51s for 60 min and anti-PD-L1 overnight. Subsequently, the slices were rinsed and incubated with the biotinylated secondary antibody at room temperature for 30 min. The bound antibody complexes were stained for 3–5 min, or until appropriate, for microscopic examination with diaminobenzidine, and then counterstained with hematoxylin (30 s) and mounted. Appropriate positive controls for each staining were chosen: normal skin tissue, comprehensive of appendages, for FKBP51s and normal placental tissue for PD-L1. Negative controls were given by sections of glioblastomas treated only with the antibody diluent. The expression of both proteins (FKBP51 and PD-L1) was recorded according to their localization in the cells. Afterwards, a score was established as the product of a proportion score (1: ≤10%; 2: >10% and <50%; 3: ≥50% of tumour cells) and an intensity score (1.weak: the signal was evident only at 40× magnification; 2.moderate: the signal was evident at 20× magnification; 3.strong: the signal was evident at 10× magnification). The presence of epithelioid tumour cells showing a distinct PD-L1 membranous signal was recorded only if they represented ≥5% of all tumour cells (in accordance with the cut-off used a previous publication) [31].
Student's t-test was used to analyse differences between means of values. ANOVA served to compare different experimental groups. A p-value of ≤ 0.05 was considered statistically significant.
FKBP51, FK506 binding protein 51; FKBP51s, FK506 binding protein 51 short; PD-L1, programmed cell death protein-ligand 1; PD1, programmed cell death protein; PPIase, peptidyl-prolyl isomerase; TPR, tetratricopeptiderepeat; IR, ionizing radiation; ER, endoplasmic reticulum; PNGase F, N-glycosidase F; PBMCs, peripheral blood mononuclear cells.
SR and MFR made substantial contributions to conception and design, analysed and interpreted data and wrote the manuscript. FH revised it critically for important intellectual content, provided SAFit and contributed to experiments with SAFit. PD and MR conceived most experiments and carried out experiments work; EG, MLDBDC, SS and GI provided clinical information and human tissues and performed pathological analysis; RP contributed to radiobiology experiments; SP contributed to microscopy experiments; AR and MT contributed to experimental work. All authors discussed the results and commented on the manuscript.
We thank Dr. Mario Capasso for helping us with statistical analysis.
No conflicts of interest is declared.
We thank the Cardiovascular Service SRL for supporting our research.
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The former Gator running back has agreed with the Tampa Bay Bucs on a four-year deal worth $11-million, ending any possibility of a training camp holdout.
TAMPA � Earnest Graham�s patience finally paid off.
The Bucs running back has agreed to a four-year deal worth $11-million, ending any possibility of a training camp holdout.
The agreement was reported Sunday night by NFL.com. Graham, 28, did not participate in voluntary workouts or the offseason workout program, presumably because he wanted a contract extension. He did take part in mandatory minicamp.
Terms of the deal have not been released, so it�s not known how much of that $11-million is guaranteed or if it includes a signing bonus.
Graham could not be reached Sunday night. But he had expressed optimism that an agreement would be reached before training camp. He downplayed his absence from the offseason workout program which has been his routine for most of his five-year pro career.
The former Florida Gator emerged as a starter for the first time last season after injuries to Cadillac Williams and Michael Pittman. Graham, whose rushing high was 83 yards in 2005, totaled 898 yards and 10 touchdowns.
He will be an integral part of a backfield that includes Warrick Dunn, 33, and Michael Bennett, 29.
While Graham took the silent approach, quarterback Jeff Garcia has been vocal about his disgust over the lack of progress in his talks with the Bucs on a contract extension. With Graham in the fold, the team could turn its attention to reaching an accord with the 38-year-old.
Graham, who was entering the final year of his contract that would have paid him a base salary of about $600,000, has said others deserve to be rewarded.
There�s still plenty of time before the team reports to training camp July 25.


text:
Your Downtown Danceteria Since 1970
Downtown Minneapolis, MN
info@first-avenue.com
Depot Tavern
src="https://www.facebook.com/tr?id=1140702495954171&ev=PageView&noscript=1"
With a style influenced as much by John Mayer as Macklemore, singer Jake Miller emerged while still a student at the University of Miami. Miller got his start in high school, posting videos online from his Weston, FL home. In 2011, he self-released his debut EP, Summer Session, and performed his first live gig, opening for Snoop Dogg, and from there he released an EP per year. His second EP, Spotlight, arrived in 2012. He signed a deal with E1 Music in 2013 & released the Road Less Traveled EP & his debut LP, Us Against Them.
Miller unveiled Dazed and Confused in 2014 with features from Nikki Flores & Travie McCoy. The feel-good party EP Rumors released 2015. In 2016 he released the Overnight EP, which he took on tour opening for Fifth Harmony. In 2017, the singer self-released his sophomore LP, 2:00am in LA, after parting ways with Warner Bros. Less than a year later, Miller issued his third full-length, Silver Lining.
His 2018 release with RED MUSIC "Wait For You" is a bonafide smash. Produced exclusively by Miller, the track's growing success is a testament to his fearless approach to songwriting & his ability to push his own boundaries in the studio. Matched with his signature brand of pop, Jake uses heavier electronic influences on "Wait For You," while the lyrics tackle the complexity of relationships. "Wait For You is a song written about a situation someone I know was going through," Jake explained. "It's all about the right person, wrong time."
Events Featuring this Performer
JAKE MILLER — Wait For You Tour
with LOGAN HENDERSON and JUST SECONDS APART
$20.00 advance
day of show
at Fine Line
JAKE MILLER - Hit and Run Tour
with DEVIN HAYES
7th St Entry
with THE STOLEN
at The Cedar Cultural Center
JAKE MILLER - CANCELED
with MAX and SPENCER SUTHERLAND
Mainroom
© 1970-2013 701 Ventures Inc. All right reserved. Website by ThinkShout

redpajama_set_name: RedPajamaCommonCrawl


text:
First Time Leaders
Experienced Leaders
Executives and Teams
Customize Your Program
Leadership Philosophy Samples
Jim Emerick
Graduate Leadership Course
Leadership Excellence Course
Jim Emerick is a successful hi-technology entrepreneur who provides strategic management consulting and certified leadership facilitation & executive coaching worldwide.
Jim is the founder and president of Choinque LLC, leading advanced technology teams within start-up firms, government organizations, and large corporations. He is a trustee at Academy at The Lakes private school in Land O' Lakes, Florida, and a volunteer Executive Leadership Coach at Tampa's Lowry Park Zoo. Jim is an international leadership partner with CMCS, a fully integrated Project Portfolio Management solutions provider in Beirut, Lebanon. His Project Management Institute (PMI) alliances include Mile-Hi (Denver), Tampa, Pikes Peak (Colorado Springs), and Santiago, Chile chapters.
A 1987 graduate of the US Air Force Academy, Jim led an innovative government and contractor team, which developed a next generation spacecraft computer implemented into over fifty platforms including the Cassini-Huygens probe. In 1992, Jim continued his leadership journey by co-founding insyte, a pioneering integrated circuit design services company in Tampa, Florida. As President and CEO, Jim ushered in a decade of growth, from a handful of employees to a network of engineering sites in the United States, Scotland and Canada. He forged strategic alliances with industry leaders such as IBM, launched a Defense and Intelligence Advisory Board, and coordinated the sale of insyte to ITT in 2006. While at ITT, Jim established an information assurance team in Tempe, Arizona, while directing development and delivery of next generation electronics communications and encryption capabilities.
Jim earned an MBA with a marketing concentration from the University of New Mexico, and is a lifetime member of the Air Force Academy Association of Graduates and the Air Force Academy Sabre Society. He is an invited guest speaker at numerous industry trade group events. Jim, his wonderful wife Cheryl and their two awesome children live in Tampa, Florida and Pine, Colorado.
"Highly valuable at providing real life scenarios and exchange of information that can provide huge benefit for those looking to make the jump from a manager to a true leader. Can't say enough of Jim and his style of presenting the information. Makes it highly interactive and gets the best out of everyone."
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"Jim is the best facilitator in the business. I went to his leadership seminar with 2 of my colleagues and we left with several immediately actionable items to improve our organization and I also left with several action items for self improvement as well. The information from the Academy Leadership program is fantastic and Jim demonstrated a true mastery of the course. I would recommend the course and him to small to medium enterprises."
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"Tuve el placer de participar en el "The Leadership Excellence Course", que se realiza por primera vez en Santiago-Chile. Jim fue nuestro instructor.
Me complace decir que es un gran facilitador de ideas, de gram conocimiento en los temas presentados y con gran capacidad de dirigirnos y sacar el mejor provecho del tiempo asignado. Logró enseñarnos la importancia de nuestro auto-conocimiento para poder aplicarlo en el liderazgo hacia nuestro equipo.
Vale la pena vivir la experiencia"
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"I had the opportunity to participate in a Leadership Excellence Course that Jim facilitated in London, UK. Over the course of three days I was able to learn more about my own personality and also better understand how to adapt my leadership style to teams. I found Jim to be very inspirational and thought provoking. He shared several tips and tricks that work well for him that I can easily adapt in my professional and personal life. Most importantly, Jim is authentic – he gave several examples in some cases quite personal – that really brought various topics/theories to life. I took a lot away from the course and look forward to our future executive coaching sessions."
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If I cannot feel it there is simply nothing there.
when we have a destination in mind.
Even if we never reach it before the end of the line.
A little love here, a little love there, doesn't seem to matter where, you drop the line, cause who is to care, just positive feelings radiating out somewhere. Yeah, right. Try playing that tune with a jealous soul, or a person with no self esteem, try it with the reverend of the church, his take on it will pertain to his flock. Not enough nuances to cover the word, not enough adjectives to describe it, without looking into the eyes. Not enough crawling out from under the shell, too many take things verbatim; go to hell! If one cannot think out side of their box, I will still love them but not how they thought !
I know that chivalry is not dead! If it was, I would have been the one who killed it !


text:
Q: NavigationLink inside LazyVGrid cycles all entries on back, SwiftUI I have an image grid. Each image on tap should push a view on the NavigationView with the image details.
The navigation link works as intended, but when I press the back button it opens the next image and so on until it has cycled all the images. What is going on?
This is the View:
struct ImageGrid: View {

    @ObservedObject var part: Part
    @State private var showingImagePicker = false
    @State private var inputImage: UIImage?
    var body: some View {
        Button("add images"){
            self.showingImagePicker = true
        }
        LazyVGrid(columns: [GridItem(.adaptive(minimum:100))]){
            ForEach(part.images){ image in
                ZStack {
                    Image(uiImage: image.thumb)
                        .resizable()
                        .scaledToFit()
                    NavigationLink (
                        destination: ImageDetail(image:image),
                        label: {
                            EmptyView()
                        }
                    ).buttonStyle(PlainButtonStyle())
                }
            }
        }
        .sheet(isPresented: $showingImagePicker, onDismiss: loadImage) {
            ImagePicker(image: self.$inputImage)
        }

    }
    // other functions ...
    ...
}

and this is the detail View
struct ImageDetail: View {

    @ObservedObject var image: TrainingImage
    var body: some View {
        VStack {
            Image(uiImage: image.content)
                .resizable()
                .scaledToFit()
        }
    }
}

EDIT:
This is a self-contained example isolating the behaviour. It seems to stop working correctly when the grid is inside a form section. Eliminating the form and the section the navigation link works correctly

import SwiftUI

extension String: Identifiable {
    public var id:Int {
        self.hashValue
    }
}

struct ImageDetail: View {

    var image: String
    var body: some View {
        VStack {
            Image(uiImage: UIImage(systemName: image)!)
                .resizable()
                .scaledToFit()
        }
    }
}

struct ContentView: View {
    @State var images:[String] = ["plus", "minus"]
    var body: some View {
        NavigationView {
            Form {
                Section {
                    LazyVGrid(columns: [GridItem(.adaptive(minimum:100))]){
                        ForEach(images){ image in
                            NavigationLink (
                                destination: ImageDetail(image: image),
                                label: {
                                    Image(uiImage: UIImage(systemName: image)!)
                                        .resizable()
                                        .scaledToFit()
                                }
                            ).buttonStyle(PlainButtonStyle())
                        }
                    }
                }
            }
        }
    }
}

struct ContentView_Previews: PreviewProvider {
    static var previews: some View {
        ContentView()
    }
}

A: It is Form/List feature to auto-detect links in rows, but you have several in row, so the effect. The solution would be to separate cell view and hide link from auto-detection.
Tested with Xcode 12.0 / iOS 14
struct ContentView: View {
    @State var images:[String] = ["plus", "minus"]
    var body: some View {
        NavigationView {
            Form {
                Section {
                    LazyVGrid(columns: [GridItem(.adaptive(minimum:100))]){
                        ForEach(images){
                                    ImageCellView(image: $0)
                        }
                    }
                }
            }
        }
    }
}

struct ImageCellView: View {
    var image: String
    @State private var isActive = false
    var body: some View {
        Image(uiImage: UIImage(systemName: image)!)
             .resizable()
             .scaledToFit()
            .onTapGesture {
                self.isActive = true
            }
        .background(
             NavigationLink (
                  destination: ImageDetail(image: image), isActive: $isActive,
                  label: {
                    EmptyView()
                  }
             ))
    }
}

redpajama_set_name: RedPajamaStackExchange


Saturday's state tennis championships turned into a Forecaster Country coronation.
Once again, Falmouth swept the Class A crowns and the Yachtsmen had company as Greely repeated as Class B girls' champion and North Yarmouth Academy's girls captured their first title in five seasons.
Falmouth's girls produced another dominant, perfect season and continue to raise the bar of excellence to a level that no other program will likely ever reach.
The Yachtsmen went 12-0 in the regular season, earned the No. 2 seed in Western A and dispatched No. 10 Deering and No. 3 Portland by 5-0 scores in the quarterfinal and semifinal rounds, respectively. Last Wednesday, Falmouth met No. 4 Scarborough in the regional final and advanced with a 4-1 win, as Olivia Leavitt, Julia Brogan and Caroline Ray all swept their singles matches and Kate Kelley and Amanda Watson were triumphant at first doubles. Only Mary Hyland and Meg Pierce at No. 2 doubles lost in a close match.
That sent Falmouth to Lewiston High School Saturday to meet Eastern A champion Lewiston in the state match and the Yachtsmen made it 125 straight match victories with a 5-0 win, capping a 16-0 season and making it two straight years as the best team in Class A, eight straight championship seasons overall and 10 titles in 11 years (only in 2007 did Falmouth lose in the playoffs in that span, to York in the Western B semifinals).
Leavitt downed Maddi Roy at first singles, 6-0, 6-0. At second singles, Brogan was a 6-1, 6-1 winner over Kyleigh Letourneau. Ray also won, 6-1, 6-1 at third singles over Alanna Taylor. Kelley and Watson prevailed by 6-1, 6-1 scores over Claudia Dionne and Maddie LeBlond at first doubles and Hyland and Pierce were 6-3, 6-3 winners over Lizzy Michaud and Chloe Morin at second doubles.
Falmouth's 13th overall title ties Cape Elizabeth and South Portland for first all-time.
The Yachtsmen will say goodbye to Stone, who is stepping down.
"It was a great last year in terms of such a strong finale as a coach," Stone said. "We had an all-Falmouth final in the Western Maine Conference doubles tournament for the fourth consecutive year, we won our third state singles title in a row and the eighth consecutive state team championship. I've enjoyed working with the players who are wonderful young ladies, fun loving and mutually respectful and caring of each other. There's been good team spirit and bonding again this year. I look forward to reading about their continued strong tennis play, hopefully deep into the playoffs in future years as well. I will think upon these past 13 years fondly as some of my best.
Anyone hoping the dynasty will come to an end next season, will likely be disappointed.
Falmouth's boys were ranked third after an 11-1 season and downed No. 6 Scarborough and No. 7 Windham by 5-0 scores in the first two rounds. Last Wednesday in the regional final, the Yachtsmen got pushed by No. 1 Thornton Academy, but advanced with a 3-2 victory, as Peter Stegemann won at third singles, Jordan Bruce and Trey Fallon prevailed in straight sets at first doubles and Grayson Cohen and Matt Adomowicz won in three sets at second doubles. Aidan McGrory and Alex Klemperer lost their singles matches.
Saturday, against Mt. Ararat in the state match, Falmouth capped their 15-1 campaign with a 4-1 victory to repeat as Class A champions and make it four crowns in five years. Klemperer defeated Peter Mao at second singles (6-3, 6-0). Stegemann downed Trevor Mayo at third singles (6-3, 6-1). Bruce and Fallon were 6-2, 6-2 winners over Mike Crawford and Jon Roux at first doubles and Adamowicz and Cohen downed Joey Reed and Leonardo Medeiros (6-1, 6-1) to win second doubles. McGrory fell, 6-7 (1), 5-7, to Nick Mathieu at first singles.
The Yachtsmen return a lot in 2016 and should make a run at more hardware.
Greely's girls' tennis team had never won a championship prior to last spring and the Rangers enjoyed it so much, they went out and did it again. Greely was ranked second after a 10-2 regular season and advanced with a 5-0 quarterfinal round win over No. 10 Lake Region and a 3-2 victory over No. 6 Yarmouth in the semifinals. In last Wednesday's Western B Final against top-ranked Lincoln Academy, the Rangers won, 4-1, as Izzy Evans and Kathryn Pare won singles matches and Mia Lambert and Jessie Hoffman and Sam Kennedy and Madeline Day both won doubles matches.
Saturday, in the state match versus Mt. Desert Island, Greely won, 5-0, to finish 14-2. Evans won at first singles, 6-0, 6-0, over Sarah Phelps. Pare took her second singles match, 6-1, 6-4, over Ivy Wallace. Alex Tebbs was taken to three sets at third singles, but she prevailed, 3-6, 6-4, 7-5, over Maeve Geary. Lamberg and Hoffman won in straight sets at first singles, 6-3, 6-0, over Ellie Bridgers and India Janes. Kennedy and Day got taken to three sets at second doubles, but downed Emily McCarthy and Olivia McCarthy, 6-2, 3-6, 6-0.
Greely has holes to fill going into next season, but could make a run at a three-peat.
NYA won four straight titles between 2007-10, but hadn't been back to the state match since. After a 9-3 regular season, the fourth-ranked Panthers ousted No. 5 Carrabec (5-0) and top-ranked Hall-Dale (5-0) in their first two rounds, then dethroned four-time champion Waynflete in last Wednesday's regional final, 4-1 behind a singles sweep from Lena Rich, Mina Stam and Mary Morrison and a win at first doubles from Hannah Chapman and Corinne Poitras. Livy Stam and Hannah Hungerford lost at second doubles.
Saturday, NYA met up with Van Buren and rolled to a 5-0 victory, dropping just five points along the way, to return to the Class C pinnacle after a 13-3 campaign. Rich was a 6-0, 6-2 winner over Courtney Parent at first singles. Mina Stam took care of Felisha Bouchard, 6-3, 6-0, at second singles and Morrison (over Isabel Parent at third singles), Chapman and Poitras (over Alexis Ouellette and Danika Deschaine at first doubles) and Livy Stam and Hungerford (over Lindsay Gendreau and Kaila Roy at second doubles) all won by 6-0, 6-0 scores.
Falmouth's boys' and girls' tennis teams repeated as Class A champions Saturday and were are smiles afterwards.
NYA's girls' tennis team shows off its Class C championship trophy, the Panthers' first since 2010.
Greely's girls' tennis team shows off its trophy after repeating as Class B champion Saturday.


This post was update Sept. 25, 2008, to correct an error in the name of the Coast Guard's national Security Cutter.
"We're going to see very shortly in the Coast guard a revolution on how we deal with information management in the new social media, or as some people would call it the Web 2.0," Allen announced. "We need to understand that this is a permanent feature of our environment and we need to understand how to operate in it."


text:
On the Political Systems of the New Age
POLITICAL ENTROPY (Chapter 2)
Digital technologies and the globalisation of disorder
As a series of events sent shockwaves globally, a large-scale process proceeded in the background imperceptibly, with the creation of new forms and methods to manage social phenomena, to all intents and purposes representing the birth of a new type of political system.
It has been clear to everyone that political systems in Western countries have started showing signs of strain during the past decades — and have been malfunctioning. Externally, this was manifested in the inability of political systems to effectively counter manipulation by forces and factional groups extraneous to the previous balance of interests.
THE PANDEMIC AND POLITICS
INFORMATION OCHLOCRACY
THE NEW AGE ECONOMY
MASS PROTESTS AND THE MODERN WORLD
WHAT SHOULD BE DONE? INSTITUTIONALISATION OF VALUES
It goes without saying that fringe politicians with authoritarian views have always existed. However, now they have started assuming far more substantial roles in their own countries and have already started forming coalitions between each other. The key factor uniting these politicians is a rejection of the significance of and even need for the European-style political system that appeared after World War II, and repeated attempts to present this system as "wrong", unfair and unviable. There have been more than enough examples: Donald Trump in the USA, Boris Johnson in Great Britain, Vladimir Putin in Russia, Recep Erdogan in Turkey, Andrzej Duda in Poland, Viktor Orban in Hungary, and Jair Bolsonaro in Brazil.
Time Magazine cover, May 2018: President of Russia Vladimir Putin, President of Turkey Recep Erdogan, Prime Minister of Hungary Viktor Orban and President of Philippines Rodrigo Duterte
Prime Minister of Hungary Viktor Orba (from the back) welcomes resident of Russia Vladimir Putin in Beijing, May 2017. President of Turkey Recep Erdogan on the left, President of Belarus Alexander Lukashenko in the back row
It does not always involve outright rejection — frequently these ideas are veiled under calls for "reform" of the political system. In most cases, this is manifested in the form of populism — calls for the restoration of the primacy of the interests of the people (for example, "Brexit"), juxtaposed against a corrupt and egoistical "elite", and promises to make this happen if elected. At the same time, in most instances this does not concern the entire population, but instead the so—called "real" people ("true", "with core beliefs"), who are proponents of the "right" traditional values. Moreover, such people oppose not only the elite, but also other "foreign" elements and values. It is implied that they are not satisfying the demands of the whole population, where you will find diverse minorities and special groups, but only those of the "indigenous" population defining state identity.
It is notable that in such cases as a rule the changes imply not so much the establishment of some new, more progressive and just order as the restoration of some old order that had allegedly been lost for good. In this sense the new populism (incidentally like its forerunner) addresses the past, some more natural, but lost order which had allegedly ensured justice and greatness at some point in time. And this represents the core of the explicit anti-globalism of the new populism, its focus on national historical values, calls for the restoration of former glories, which are as a rule dubious from the perspective of the values of humanism, and to a large extent mythical ("again policy")1.
However, populism (based on the above understanding) is not the only phenomenon reflected in the increasing rejection in Western countries of the values of the political structure of society which gained a foothold after World War II in developed parts of the world. Another key symptom of today's political about-turn has been the negation of the values of private life, disregard for privacy and autonomy as an indelible right (and to a certain extent attribute) of the individual.
As such, it is namely the separation of private life from public life, independence from public dictates and control which represented the most important element of the concept of political and social progress throughout the 20th century. However, in the past decade the freedom of private life as a value has become a target of contempt, if not actual repudiation. Against the backdrop of the unprecedented and rapid expansion of opportunities for official and unofficial structures (and also the specific parties standing behind them) to control the actions and thoughts of individuals, resistance to such processes turned out to be surprisingly weak and impermanent. Most of the population was subconsciously already prepared for an intensification of controls — both physical, and also cultural and spiritual — in exchange for home comforts and social prestige, which is frequently illusory or confined to a narrow group. Moreover, opportunities for the integration of the spiritual lives of individuals on large-scale single platforms, which are comparatively susceptible to control and external influences, began to be perceived as a public achievement and form of progress.
The greatest vulnerability of private life — facilitating outside interference — is paralysing to a large extent the functioning of political competition and institutional controls over such competition, and increasing the risks of the emergence of political monopolies and distortions of the actual picture of public interest.
Finally, another key aspect of Western political structure of the second half of the 20th century, which has been significantly weakened over the past decade, was the hierarchical nature of the organisational system for running society. The "networking" of society2 so typical of today, accompanied by a wide distribution of forms of influence, and accordingly a decrease in individual responsibility, has ambivalent consequences. On the one hand, traditional leaders who have aspired to leading roles in a vertically organised society lose the opportunity to influence public moods and political decisions to the extent that they could in the past. On the other hand, utterly random people are promoted to the role of leaders (often impromptu individuals or so-called "men of the people"), who just happened to be at the crossroads of different networking platforms.
As a result, there has been an abrupt increase in the number of random outcomes in political systems unrelated to the actual balance of interests of public groups. More and more opportunities appear for fringe players and irrational actions, for virtually inexplicable and at times strange political decisions attracting unexpected support and a chance at implementation. Presented by enthusiasts as a tool democratising governance, in reality, "networking" generates chaos and irrationality and fosters growth in random movements frequently initiated by the beneficiaries of such destabilisation, whose goals are diametrically opposed to public interest.
Why have we witnessed all these phenomena over the past decades? There is no unambiguous response to this question if only because too little time has elapsed to draw final conclusions both on the substance of this process and also on its causes. Nevertheless, some explanations can be found, which are worthy of consideration.
All these developments can be attributed to processes frequently called the fourth industrial revolution, specifically the impact of new generation information and communications technologies on the social and political sector related to the expansion of the internet, the opportunity to collect and process large arrays of data, and use elements of artificial intelligence. These technologies, adapted for use in mass communications, triggered the dilution of the foundations of the "classical" Western model of political organisation of society (See «Information ochlocracy»).
Presentation of Self Artist: Koren Shadmi
Firstly, information and communications technologies made it possible to "network" social communications in society: without the internet and its potential, there would be no social networks where anyone can announce their political ambitions and address a mass audience without any public mandate to engage in a political career. For example, today an unknown young ambitious activist can access an audience running into millions: if in the right place at the right time, such an individual just needs to make a little effort and be lucky. These days there is no need to spend countless years climbing the rungs of a party ladder or embark on a painstaking administrative career, meticulously establishing vertical and horizontal ties, and proving leadership skills and business qualities. All an individual needs to do is simply attract the attention of a group of active users through a striking image — and the viral dissemination of content will transform any such random individual into a political figure and a public opinion leader. Previous filters operating on the basis of financial control and selection criteria no longer work.
Secondly, information and communications technologies eliminated the tried-and-tested age-old system for influencing the public mood of using informal leaders of local and professional communities — or to put it more simply, authoritative figures for different population groups. Online any informal leadership is diluted and gives way to the political and behavioural patterns of a loosely organised crowd. Meanwhile, everybody knows that a crowd does not tend to a react in an orderly manner to significant political events and is unable to support rationally planned protracted campaigns, but is by contrast a pushover when it comes to incidental issues. Naturally, such communities can also be successful targeted. However, the tools available to achieve such an impact have little to do with the logic and structure of a party representative system.
Thirdly, another attribute of the new forms of communication concerns the disappearance of any hierarchy regarding the significance of events. I am not talking about the so-called gutter-like journalism spewed out for the mainstream audience, but rather the fact that this agenda is incapable of ranking events by their degree of importance. Data flows are being transformed into anarchic batches of messages/reporting and interpretations of such reports where news of utter irrelevance from a social perspective is mixed with events that could have significant implications for millions of people. Both online, and also offline in the traditional mass media in new information formats (for example, socio-political talk shows or continuous "news" broadcasting on TV channels), political events are presented in a cynical diverting or overtly perverse form — on the same level as standard entertainment; propaganda is jumbled up with advertising that debases its own content; gimmicks deployed to attract the attention of the average consumer (increase ratings, offline and online traffic) openly take precedence over the substance of the information. As a result, comparatively streamlined and ranked knowledge on the life of society is replaced by an endless entertainment spool of words and images, interfering with real news and at times rendering any understanding of developments and their potential consequences impossible.
Fourthly, the virtualisation of social interaction releases the negative energy previously suppressed to a significant extent by physical personal contacts between different sections of society. It goes without saying that such phenomena as "hate speech", "trolling" and others are nothing new. However, they are becoming far more common and hostile in the absence of direct physical interaction. For the time being the political consequences of this phenomenon are not entirely clear, but there is no doubt that this will undermine the effectiveness of tools used in traditional party and parliamentary systems. The radicalisation of political conflicts, perception of such conflicts as an uncompromising fight between "us" and "them", incompatible with the substance and intended purpose of such systems, render these institutions dysfunctional and meaningless.
Incidentally, it could be assumed that the development of these technologies merely act as a trigger, while the outcome of any scenario is determined by the lag of human consciousness behind the achievements of the fourth industrial revolution.
For example, this is expressed in the generation gap — when the older generation is incapable of fully recognising and accepting a world that is changing so rapidly, while the young take everything for granted. However, instead of looking for answers to the questions "What is the point?", "Why?, "Is this actually needed?" and "Could things be done differently?", the new generation is preoccupied on how best to leverage the "rules of the game" which are already equated to the insurmountable laws of nature.
In addition, the new technologies continue to assume the role of a provocative alternative that facilitates the destruction of institutions.
People believe that algorithms are replacing institutions and bringing happiness to mankind; that there will be no need for politicians, scientists, doctors, teachers, institutions and systems; that their places will be taken by technologies which will be available to everyone: given that everyone has a gadget that can be used to download apps, this means everyone can use the algorithm, and that everyone in future will be able to print whatever they want on a ЗD printer…
In any case, all these factors demonstrate that the crisis of party and parliamentary systems in leading countries in the West is not some accidental short-term aberration. It is the natural consequence of the expansion of new information and communications technologies in the political sector, and accordingly represents a serious challenge for the future. The Western political system was also simultaneously a representation system of the boundaries of what is permissible in politics, in other words more or less sustainable boundaries that an individual could not transgress without being cast out of the system.
These boundaries have expanded significantly with the emergence of the current generation of populist politicians. At the same time, however, today the system is not discarding the people who have been expanding them. And most importantly, nobody knows how widely the boundaries can be expanded right now. Can any information be disregarded just because it has been called fake? Can a law be declared wrong/unjust and not be implemented? Can we simply refuse to recognise the results of elections? Can rules actually be changed unilaterally, with total disregard for previously established procedures?
For the time being nobody knows the answers to these questions. However, if all the above is possible, then this will already be a different system. Fundamentally different, even though formally all previous institutions will be retained: parliament, the judiciary, etc.
And this in turn brings us to an important question: what will come after Trump? Will America remain the same as it was 10-15 years ago? For if the boundaries of what is permissible have already expanded, who will move them back and when? Will this be done by the new elite — the "geniuses" of high-tech and Fintech? Will they actually want to spend their money, time and efforts to bring back the old times? Or will they be inclined to think that new times are coming when the world will be run by the creators and owners of artificial intelligence who dispose of an unbelievable quantity of information on people and their money and boast unimaginable opportunities to process such information, and certainly not "archaic" parliaments and the "antiquated" press? And will there be any need for law and order in its traditional form when information technologies can be used to directly control mass behaviour? Will the people holding themselves to be omnipotent actually perceive any need for checks and balances?
Incidentally, when present-day populist politicians like Donald Trump expand the boundaries of what is permissible in politics to fit their needs, objectively they also expand the boundaries for their opponents. And after Joe Biden's victory in the US presidential elections in 2020, it is highly unlikely that they will move these boundaries back voluntarily. No individual would voluntarily forego such opportunities — other than some maverick idealist, but this is highly unlikely. Society will also not demand any narrowing of the boundaries of what is permissible, as it is splintered and is no longer capable to engineer a hierarchy-based organisation of society's proactive and engaged participants, because all their energy has been squandered online, freeing up in the process the offline space for asocial elements. Meanwhile senior management of modern businesses has no interest in any boundaries, because they are not run by individuals like Rockefeller or Morgan, but instead by people like Zuckerberg and Bezos who do not believe in checks and balances for politicians, and instead worship the magic of technologies and social engineering. Against this backdrop, the disintegration of the previous political system is likely to continue, regardless of the outcome of the US presidential elections in November 2020.
We have all the grounds for assuming that the world in actual fact will no longer resemble what it has been in the past. And this has nothing to do with the coronavirus epidemic.
Posted: December 4th, 2020 under Economy, Elections, Foreign policy, Governance, History, Human Rights, Russia-Belarus Relations, Russia-China Relations, Russia-Eu relations, Russia-Ukraine relations, Russia-US Relations, Без рубрики.


Description "37 [age] ; Bricklayer ; Mexico [place of birth]."
Source Includes data from the 1880 Census Population Schedules, Town of Brownsville, ED 31, Cameron County, Texas, Series: T9, Roll: 1294, p. 393 (stamped), p. 71 (handwritten), entry 827.
Artisan or Artist Ramirez, Jesus.


CB Antoine Winfield just told me he's agreed in principal to a 1-year deal with the Seattle #Seahawks.
Let the Vikings to Seahawks pipeline continue.
The fit would be interesting since the Seahawks seem to prefer tall CBs.
Interesting, wonder if this move is in response to SF signing Asomugha. Need to one up them.
He would be a perfect fit for the Seahawks nickle corner spot.
Well we do need a guy who can play in the slot and Thurmond III is always hurt.
I'm just gonna say it... Seattle has already won the Super Bowl if they get Winfield, too.
How much does Winfield have left in the tank? Don't recall hearing about him or seeing him at all last year. But ya, the Seahawks need someone else at Nickle. Thurmond is AWESOME when on the field (better than Browner IMO) but simply can't stay on it. A real pity.
Don't see 49ers taking the division. I think we get one of the wildcards.
Winfield is a popular option as a nickel corner in the Seahawks forum. He fits the criteria to the dot.
Cos Russell Wilson is immune to a Sophomore Slump? Sherman will maintain his consistency like all CB's? All the FA's will work out and not create a 'Dream Team' scenario? Cos the Seahawks will stay healthy all season? Cos they always have elite draft classes?
Yeah, too many unanswered questions to crown them just yet. Remember, the Eagles were going to win the SB in 2012.


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