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Experimental Therapeutics, Molecular Targets, and Chemical Biology
Resistance to an Irreversible Epidermal Growth Factor Receptor (EGFR) Inhibitor in EGFR-Mutant Lung Cancer Reveals Novel Treatment Strategies
Zhiwei Yu, Titus J. Boggon, Susumu Kobayashi, Cheng Jin, Patrick C. Ma, Afshin Dowlati, Jeffrey A. Kern, Daniel G. Tenen and Balázs Halmos
Zhiwei Yu
Titus J. Boggon
Susumu Kobayashi
Cheng Jin
Patrick C. Ma
Afshin Dowlati
Jeffrey A. Kern
Daniel G. Tenen
Balázs Halmos
DOI: 10.1158/0008-5472.CAN-07-1248 Published November 2007
Patients with epidermal growth factor receptor (EGFR)–mutant non–small cell lung cancer derive significant clinical benefit from treatment with the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Secondary EGFR mutations such as EGFR T790M commonly lead to resistance to these agents, limiting their long-term efficacy. Irreversible EGFR inhibitors such as CL-387,785 can overcome resistance and are in clinical development, yet acquired resistance against these agents is anticipated. We carried out a cell-based, in vitro random mutagenesis screen to identify EGFR mutations that confer resistance to CL-387,785 using T790M-mutant H1975 lung adenocarcinoma cells. Mutations at several residues occurred repeatedly leading to functional resistance to CL-387,785. These variants showed uninhibited cell growth, reduced apoptosis, and persistent EGFR activation in the presence of CL-387,785 as compared with parental H1975 cells, thus confirming their role in resistance. A screen of alternative agents showed that both an alternative EGFR inhibitor and a cyclin-dependent kinase 4 inhibitor led to significant inhibition of cell growth of the resistant mutants, suggestive of potential alternative treatment strategies. These results identify novel mutations mediating resistance to irreversible EGFR inhibitors and reveal alternative strategies to overcome or prevent the development of resistance in EGFR-mutant non–small cell lung cancers. [Cancer Res 2007;67(21):10417–27]
epidermal growth factor receptor
CDKs and CDK inhibitors
Growth factor receptors and other surface molecules as targets for therapy
The epidermal growth factor receptor (EGFR) ATP-competitive tyrosine kinase inhibitors erlotinib and gefitinib showed success in the treatment of advanced non–small cell lung cancers following the failure of front-line chemotherapy ( 1). Approximately 10% to 25% of patients will have a major response to these agents, and erlotinib treatment leads to an overall survival advantage as compared with placebo in patients with chemorefractory non–small cell lung cancer ( 1). Most responding patients' tumors harbor recurrent somatic EGFR mutations ( 2– 4). The most common of the EGFR mutations, L858R and exon 19 deletions affecting an LRE amino acid sequence, represent 85% to 90% of all mutations and are clear-cut predictors of major responses to this class of drugs ( 5– 8). Whereas some responses are dramatic and durable, ultimately all patients seem to progress on continued EGFR tyrosine kinase inhibitor therapy. Secondary EGFR mutations, most commonly the EGFR T790M mutation, were identified as the mechanism of resistance to this class of agents ( 9– 11). Resistance mediated by T790M can be overcome by irreversible EGFR inhibitors such as CL-387,785, HKI-272, and EKB-569 ( 11– 13). Whereas such inhibitors are in clinical development and early signs of success have been reported in erlotinib-refractory lung cancers, it is clear that resistance develops against this class of inhibitors as well ( 14). We developed a cell-based random mutagenesis screen to identify resistance mechanisms to the model irreversible tyrosine kinase inhibitor CL-387,785 using T790M-mutant H1975 cells. Clinically relevant imatinib-resistant mutations of BCR-ABL and KIT almost exclusively occur in the exons encoding the tyrosine kinase domain ( 15, 16). Our current model system therefore focused on the kinase domain of EGFR to identify potentially clinically relevant resistance mutations. We also pursued further studies for the identification of alternative treatment strategies to overcome such resistance.
Cell culture and reagents. NIH-H1975 lung adenocarcinoma cells, which contain mutant EGFR-L858R-T790M ( 10), were propagated in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and 100 units/mL penicillin. All cells were grown at 37°C in a humidified atmosphere with 5% CO2 and were in the logarithmic growth phase at the initiation of all experiments. CL-387,785, EGFR/ErbB2/ErbB4 inhibitor, cyclin-dependent kinase (Cdk)-4 inhibitor, LY294002, AG1478, and U0126 were purchased from Calbiochem (EMD Biosciences, Inc.). GW583340 was purchased from Sigma. Erlotinib was obtained from a commercial source. Drugs were dissolved in DMSO to give a 10 mmol/L stock solution and then stored at −20°C and the final DMSO concentration in all experiments was <0.1% in medium.
EGFR mutagenesis, library generation, and drug resistance screen. The plasmid construct pCDNA 3.1(−)-EGFR-L858R-T790M-HA was generated as previously described ( 9). This construct was further modified by mutating the GAGACG sequence to GTGACG through site-directed mutagenesis using the QuickChange mutagenesis kit (Stratagene), thereby destroying one of the two BsmBI sites at position 1,011 (human EGFR, GeneBank ID NM005228) without changing the amino acid sequence. A 1,308-bp region of EGFR containing the entire tyrosine kinase domain of EGFR was amplified by PCR using the primers 5′-GAATGTCAGCCGAGGCAGGGAATG-3′ (sense) and 5′-TCACGGAACTTTGGGCGACTATCT-3′ (antisense) in the presence of 50 μmol/L MnCl2, which reduces the fidelity of Taq and thereby promotes the introduction of errors leading to random point mutations ( 17). Subcloning into p-GEM-T Easy vector and sequencing of individual colonies showed that essentially all of the PCR products contained mutations, with 50% of the products containing a 1-bp change, showing the success of the mutagenesis procedure. The pool of amplified DNA fragments was digested with BsmBI and ClaI, gel purified after dephosphorylation with calf intestinal phosphatase at 37°C for 1 h to avoid self-ligation, and religated into the backbone pCDNA 3.1(−)-EGFR-L858R-T790M-HA vector. Parental NIH-H1975 cells were transfected with the mutagenized libraries by the use of Fugene 6 reagent (Roche). Culture supernatants were replaced by fresh medium containing 500 μg/mL neomycin and 1 μmol/L CL-387,785 after 24 h. Culture medium was changed every 2 to 3 days with addition of G418 and fresh CL-387,785. Nearly 1 month later, isolated cell colonies that became visible were picked and amplified in liquid culture in the presence of both neomycin and CL-387,785.
Genomic DNA from the neomycin and CL-387,785–resistant cells was isolated. Relevant regions of the transfected EGFR DNA were recovered by PCR amplification with the use of primers 5′-GGTCTGCCATGCCTTGTGCTC-3′ (sense) and 5′-ATCCATCAGGGCACGGTAGAAGTT-3′ (antisense) and the PCR products were sequenced to identify the mutations that may lead to drug resistance. For sequencing, the following primers were used: primer set 1, 5′-GGTCTGCCATGCCTTGTGCTC-3′ (sense) and 5′-GTGCGCTTCCGAACGATGTA-3′ (antisense); primer set 2, 5′-GATGGTGGGGGCCCTCCTCTT-3′ (sense) and 5′-CTTTCTCTTCCGCACCCAGCAGTT-3′ (antisense); and primer set 3, 5′-TCCGGGAACACAAAGACAATA-3′ (sense) 5′-ATCCATCAGGGCACGGTAGAAGTT-3′ (antisense). Of note is that the obtained sequences were plasmid derived because the sequencing primers spanned several exons; therefore, genomic EGFR would not be expected to be amplified with the use of these primers. Sequence alignment and analysis was done with DNASTAR and Mutation SURVEYOR software (SoftGenetics).
Generation of EGFR mutants. Four of the mutations identified repeatedly in the isolated clones were regenerated by site-directed mutagenesis (QuickChange) of the previously listed pCDNA3.1(−)-EGFR-L858R-T790M-HA plasmid construct. The site of mutations and primers used for site-directed mutagenesis were L655H, 5′-GGGGGCCCTCCACTTGCTGCTGG-3′ (sense) and 5′-CCAGCAGCAAGTGGAGGGCCCCC-3′ (antisense); L658P, 5′-CCTCCTCTTGCTGCCGGTGGTGGCCCTGG-3′ (sense) and 5′-CCAGGGCCACCACCGGCAGCAAGAGGAGG-3′ (antisense); H773L, 5′-GGACAACCCCCTCGTGTG-3′ (sense) and 5′-CACACGAGGGGGTTGTCC-3′ (antisense); and E931G, 5′-CCATCCTGGAGAAAGGAGGACGCCTCCCTCAGC-3′ (sense) and 5′-GCTGAGGGAGGCGTCCTCCTTTCTCCAGGATGG-3′ (antisense). Two of these mutations, E931G and L658P, were similarly reengineered into the pCDNA3.1(−)-EGFR-L858R-HA plasmid (non-T790M). The proper sequence of all generated constructs was confirmed by resequencing. Transient transfection studies were done using Cos-7 cells with these reconstructed plasmids. Western blot analyses were used to confirm the success of transfection with the use of an anti-hemagglutinin tag antibody.
The irreversible inhibitor CL-387,785 blocks the EGFR kinase enzymatic function by covalently binding to Cys797 of EGFR ( 18– 21). We generated the C797S mutation in our pCDNA3.1(−)-EGFR-L858R-T790M-HA construct by site-directed mutagenesis using primers 5′-GCTCATGCCCTTCGGCTCCCTCCTGGACTATGTCCG-3′ (sense) and 5′-CGGACATAGTCCAGGAGGGAGCCGAAGGGCATGAGC-3′ (antisense), and then transfected it onto NIH-H1975 cells. G418 (500 μg/mL) was used to select stably transfected cells and isolated colonies were picked using cloning rings and continued to be cultured in the presence of G418. Sequencing was done to confirm the presence of the mutant C797S DNA in the cells with the primers as listed earlier. Expression of the EGFR-C797S mutant was confirmed by the detection of the expression of the hemagglutinin tag as above.
Protein immunoblotting. Cells were serum starved overnight and then incubated with various concentrations of different EGFR inhibitors for 3 h or Cdk4 inhibitor for 24 h before EGF (100 ng/mL; Sigma) stimulation (37°C, 15 min). After treatment, cells were lysed with 10% TCA lysis buffer and lysates were clarified by centrifugation. Proteins were separated by 7.5% SDS-PAGE gel and transferred onto nitrocellulose membranes (Bio-Rad) for Western blot analysis. Antibodies to EGFR (SC-3), anti-EGFR-phycoerythrin, and hemagglutinin tag were purchased from Santa Cruz Biotechnology. Antibodies directed against phosphorylated EGFR (pY1068), total extracellular signal–regulated kinase (ERK) and phosphorylated ERK, and total Akt and phosphorylated Akt, and Rb (Ser780, Ser795, Ser807/811) Antibody Kit were purchased from Cell Signaling. The β-actin antibody was purchased from Sigma-Aldrich. After washing and incubation with secondary antibodies, blots were developed with a chemiluminescence system (Perkin-Elmer).
Cell viability assay to determine IC50. NIH-1975 cells (6 × 103) and its subclones expressing tyrosine kinase inhibitor–resistant mutant EGFR proteins were plated in each well of 96-well plates in RPMI 1640 containing 10% FBS. The following day, dilutions of compound were added to the media in increasing concentrations (final concentration: 0, 0.2, 0.6, 2.0, 6.0, and 20 μmol/L). The concentration of DMSO in the medium was adjusted to 0.1%. After 72 h of incubation, viable cell numbers were measured by using 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS; absorption of formazan at 490 nm; CellTiter96, Promega) according to the manufacturer's protocol. Each assay consisted of six replicate wells of the same drug concentration. The IC50 was determined from the dose-response curve.
Apoptosis analysis. Parental NIH-H1975 cells and CL-387,785–resistant H1975 clones were plated at a density of 3 × 105 per well in six-well plates. Twenty-four hours after plating, cell culture medium was replaced by fresh medium containing 10% FBS with or without 1 or 2 μmol/L of CL-387,785 or EGFR/ErbB2/ErbB4 inhibitor, and incubated for another 48 h. Apoptosis was assessed with the Annexin V-FLUOS staining kit (Roche) according to the instructions of the manufacturer.
Cell cycle analysis. Cells were incubated with Cdk4 inhibitor (0–6 μmol/L) in six-well plates for 24 and 72 h before collection. Cells were pulse labeled for 45 min with 10 μmol/L bromodeoxyuridine (BrdUrd; BD PharMingen, BD Biosciences), collected by trypsinization, washed, fixed, and stained with anti-bromodeoxyuridine-FITC conjugates (BD Biosciences), then counterstained with 7-amino-actinomycin D and analyzed by flow cytometry (FACSort; BD Biosciences). Data were analyzed using WINMDI software.
Structural analysis. The crystal structures of the kinase domain were downloaded from the Protein Data Bank (accession codes 1M14, 1M17, 1XKK, 2GS2, 2GS6, and 2GS7; refs. 22– 24) and the structures analyzed using the program O ( 25).
Random mutagenesis strategy for the identification of EGFR mutations conferring resistance to CL-387,785. To generate a library of point mutants of the EGFR tyrosine kinase binding domain, we randomly mutagenized a pCDNA 3.1(−)-EGFR-L858R-T790M-HA plasmid construct using PCR conditions of reduced Taq polymerase fidelity ( Fig. 1 ). Following digestion with BsmBI and ClaI, we religated a 1,308-bp region spanning the entire tyrosine kinase domain of the mutated plasmid pool back into the original plasmid backbone. Parental H1975 cells were then transfected with this plasmid pool and subsequently selected for colony growth in the presence of both 500 μg/mL G418 and 1 μmol/L CL-387,785 to identify transformation-competent and drug-resistant variants. H1975 cells carry double-mutant EGFR-L858R-T790M ( 10) and were previously shown to be resistant to gefitinib or erlotinib (IC50 >10 μmol/L) but sensitive to CL-387,785 (IC50, 0.36 μmol/L; ref. 12). Because growth curves and MTS assays showed that at 1 μmol/L CL-387,785 there is complete and sustained proliferation arrest of H1975 cells (Supplementary Fig. S1), this concentration was used for these studies. Thirty days after transfection, well-separated individual colonies were plucked with the use of cloning rings and 31 clones were successfully expanded. Limiting dilution was used as a second selection strategy and three colonies identified through this selection were further expanded.
Strategy for the random mutagenesis of EGFR and steps followed to identify the mutations that confer resistance to CL-387,785.
From the expanded colonies, genomic DNA was isolated and the entire mutated kinase domain region of the EGFR gene was amplified by PCR. The resultant PCR products were bidirectionally sequenced to identify mutations. Sequence analysis revealed several previously undescribed point mutations. From 34 drug-resistant colonies (31 from the first and 3 from the second selection strategy), we identified 29 distinct amino acid substitutions affecting 14 residues ( Table 1 ). Substitutions at six positions were identified more than once: E931G (8 cases), L658P (4 cases), L655H (3 cases), H773L (2 cases), Q684R (2 cases), and E545G (2 cases). From the first selection strategy, 12 colonies contained only wild-type EGFR sequences (one of these had a silent basepair change at K713), 13 clones contained a single EGFR mutation, and 6 clones contained double mutations (E545G/M793T, R533W/L658P, E931G/H773L, G649R/K929E, and E931G/Q684R twice). All of the three resistant clones identified through our limiting dilution strategy contained sequence alterations: one clone contained two mutations, T629A and T725S, and a silent basepair change at position I891, whereas the other two contained a single mutation each (clone 2, L655H; clone 3, L656S). Interestingly, the L655H mutation was identified in both selection strategies. The fact that several mutations were repeatedly identified strongly suggested a functional role for the identified mutations.
Point mutants recovered in 1 μmol/L CL-387,785
Characterization of the identified EGFR mutants. To preclude that drug resistance could be mediated by altered expression of EGFR, we examined cell-surface EGFR expression by flow cytometry in multiple identified colonies including five with detected mutations (L658P, E931G, L655H, H773L, and L656S) as well as four without detectable mutations. There was no difference in EGFR expression between parental H1975 cells and mutant H1975 colonies, whereas significantly higher expression of EGFR was noted in all nonmutant, drug-resistant colonies examined ( Fig. 2A ). This suggests that in these nonmutant colonies, resistance to CL-387,785 might be mediated by higher overall expression of EGFR.
Biochemical and cellular assays of mutant sensitivity. A, EGFR expression in H1975, four nonmutant clones, and five mutant clones (L658P, E931G, L655H, H773L, and L656S) detected by flow cytometry through staining with EGFR-phycoerythrin (PE) antibody. The overlay histogram shows representative histograms of control H1975, L658P, and a nonmutant clone. B, dose-dependent growth inhibition of H1975 cells expressing different mutant and nonmutant clones as detected by the MTS assay. C, Western blotting shows equal expression of transfected EGFR as detected by expression of hemagglutinin (HA) tag by the use of an anti-hemagglutinin antibody. β-Actin serves as control. Dose response of CL-387,785 on phosphorylation of EGFR, AKT, and mitogen-activated protein kinase in parental H1975 and EGFR-mutant cells. Cells were treated with CL-387,785 at indicated concentrations for 3 h before treatment with 100 ng/mL EGF. Western blots are shown for phospho- and total EGFR, AKT, and ERK. β-Actin serves as control. D, CL-387,785 induces apoptosis in H1975 but not in mutant H1975 cells. Top, representative Annexin V/propidium iodide flow cytometry histograms; the numbers represent percent cells in the appropriate quadrant. Left bottom quadrant, viable cells; right bottom quadrant, early apoptotic cells; right top quadrant, late apoptotic cells. Bottom, columns, fold changes in apoptosis; bars, SD. H1975 and stably mutant H1975 were grown in the absence or presence of 1 or 2 μmol/L of CL-387,785 for 48 h.
To verify the functional relevance of the isolated EGFR variants, we studied H1975 clones stably expressing four of the frequently encountered mutations (E931G, H773L, L658P, and L655H). We also generated stably transfected H1975 cell lines expressing pCDNA3.1(−)-EGFR-L858R-T790M-C797S-HA plasmid constructs to model resistance as a result of this hypothesized resistance mutation that abrogates covalent binding of irreversible EGFR inhibitors such as CL-387,785 ( 18, 21). We confirmed equivalent expression of the transfected EGFR protein by detecting uniform expression of the hemagglutinin tag in these stable cell lines ( Fig. 2C). We assessed the level of resistance that each of the identified mutations conferred to CL-387,785 by generating dose-response curves of each cell line by MTS assays. Cells were cultured in the presence of increasing concentrations of CL-387,785 ranging from 0 to 20 μmol/L. Parental H1975 cells were potently inhibited by CL-387,785 (IC50, 0.36 μmol/L) as previously reported ( 12). H1975 cells expressing mutant EGFR forms were less sensitive to CL-387,785 with IC50s ranging from 1.12 to 6.70 μmol/L. Two of the nonmutant clones were also examined and were similarly found to have increased IC50s of 1.21 and 4.29 μmol/L, respectively ( Fig. 2B).
Immunoblot analyses on protein lysates of H1975 cells incubated with increasing doses of CL-387,785 were also done to examine the effect of the drug on the phosphorylation of EGFR and its main downstream signaling effectors, AKT and ERK. In parental H1975 cells, 1 μmol/L CL-387,785 completely inhibited phosphorylation of EGFR and all of its examined downstream signaling effectors ( Fig. 2C). In contrast, all the variants showed persistent EGFR phosphorylation in the presence of CL-387,785 at concentrations ranging from 1 to 3 μmol/L, confirming an ∼3- to 10-fold shift in drug sensitivity corresponding to the findings of the MTS assays. Similarly, both ERK and AKT phosphorylation persisted at higher drug concentrations than in parental H1975 cells whereas total levels of EGFR, ERK, and AKT remained unchanged.
Next, all four mutations (E931G, H773L, L658P, and L655H) were regenerated by site-directed mutagenesis and used for transient transfection studies in Cos-7 cells. Expression of the mutant EGFRs was confirmed by detection of the hemagglutinin-tagged EGFR. Immunoblotting studies to detect phospho- and total EGFR, AKT, and ERK expression levels were then done with varying concentrations of CL-387,785 in the presence of EGF stimulation. These transfection studies confirmed resistance to CL-387,785 similar to the findings in H1975 stable transfectants (Supplementary Fig. S2). We further analyzed the functional consequences of these mutations by assessing cellular apoptosis induced by CL-387,785 as defined by Annexin V/propidium iodide apoptosis assays. Treatment of NIH-H1975 cells with 1 and 2 μmol/L of CL-387,785 resulted in a concentration-dependent prominent increase in the percentage of apoptotic cells, whereas the same concentrations had essentially no effect on apoptosis in any of the five mutant H1975 cell clones examined ( Fig. 2D).
Next, we regenerated two of these mutant forms, E931G and L658P, in a EGFR-L858R-HA background to test whether these mutations confer resistance to reversible inhibitors such as erlotinib. Transient transfection studies of Cos-7 cells with these constructs showed an ∼3-fold shift of the IC50 of these two mutant forms versus the parental plasmid, suggestive of functional resistance against reversible EGFR tyrosine kinase inhibitors such as erlotinib (Supplementary Fig. S3).
Structural analysis of the identified EGFR mutations. There are now multiple published EGFR kinase domain crystal structures ( 22– 24, 26, 27). We mapped the three-dimensional locations of the residues affected by the point mutations onto these solved crystal structures of the EGFR tyrosine kinase domain ( Fig. 3 ). Of the four mutations that were further tested in this study, E931G, L658P, L655H, and H773L, two occur in the kinase domain. Mutation E931G occurs in a residue proximal to the proposed dimerization activation interface ( 24). In the EGFR crystal structures, E931 can form an intermolecular salt bridge with K708; however, E931 is not completely buried in the dimerization interface. Residue H773 is located in the loop between helix αC and strand β4. This residue forms a hydrogen bond to the backbone carbonyl oxygen of V851 and is above 10 Å distal from the kinase catalytic cleft. Both the L655H and L658P mutations occur outside of the solved EGFR kinase domain crystal structures; however, sequence analysis suggests that these residues are located in a conserved stretch of leucines that is part of the receptor single-pass transmembrane helix. For the mutations that were not further tested in this study, four occur in domain IV of the extracellular region (R533W, E545G, Y626H, and T629A), two occur in the transmembrane helix (G649R and L656S), and one occurs in the juxtamembrane region (Q684R). The remaining mutations are located in the kinase domain, two in close proximity to the adenosine pocket of the catalytic cleft (T725S and M793T) and one adjacent to the frequently seen E931G mutation (K929E).
Structural analysis of the EGFR kinase domain mutations. The EGFR kinase domain crystal structure (PDB accession code 2GS6) is shown in complex with an ATP analogue-peptide conjugate. Two kinase domains are depicted here to illustrate the Cdk/cyclin-like activating asymmetrical dimerization interface elegantly proposed by Zhang et al. ( 24). The two EGFR kinase domain molecules are shown in cartoon format and colored blue and brown with their activation loops colored orange. The kinase domain mutations identified in this study are indicated with space-filling spheres and are colored with oxygen atoms in red and nitrogen atoms in blue. The locations of activating mutation L858R and the resistance mutation T790M are indicated with red spheres. The ATP analogue-peptide conjugate solved in this crystal structure is indicated with the ATP moiety shown in stick format and colored according to atom type. The top exploded view shows the location of residues E931 and K929 in the intermolecular interface packed against the surface (in gray) of the partner kinase domain. The bottom exploded view shows the location of the mutations found in the vicinity of the ATP-binding pocket. The figure was prepared using the program PYMOL (http://www.pymol.org).
An alternative EGFR inhibitor can overcome resistance. Next, we tested the above examined five CL-387,785–resistant H1975 clones against a panel of EGFR inhibitors including AG1478 (reversible EGFR inhibitor), GW583340 (reversible EGFR/ErbB2 inhibitor), and an irreversible EGFR/ErbB2/ErbB4 inhibitor (Calbiochem). We carried out standard MTS assays following 72 h of drug treatment using a range of concentrations from 0 to 20 μmol/L. Whereas all mutant clones showed high-level resistance to AG1478 and GW583340 (data not shown), the EGFR/ErbB2/ErbB4 inhibitor (chemical structure shown in Supplementary Fig. S4) showed varying degrees of maintained efficacy against all the mutant clones tested ( Fig. 4A ). As compared with parental H1975 cells, this inhibitor was as effective against L655H and only slightly less effective against C797S and H773L mutant cells. Whereas a higher level of resistance was noted against L658P and E931G cells, the drug concentrations required to overcome resistance still suggest potential clinical relevance. Signaling studies also showed that treatment with the EGFR/ErbB2/ErbB4 inhibitor can potently inhibit EGFR Y-1068 phosphorylation as well as downstream signaling correlates such as phospho-AKT and ERK ( Fig. 4B). Annexin/propidium iodide studies further showed the induction of apoptosis by this compound in both parental H1975 cells and all five clones (E931G, L665H, L658P, H773L, and C797S) examined ( Fig. 4C and D).
A, cellular proliferation of H1975 and mutant H1975 clones at different concentrations of the EGFR/ErbB2/ErbB4 inhibitor and CL-387,785 as determined by the MTS assay. The percentage of cell viability is shown relative to untreated controls. IC50 shown in the table was determined from these dose-response curves. B, signaling inhibition by EGFR/ErbB2/ErbB4 inhibitor. Cells were incubated in the presence of EGFR/ErbB2/ErbB4 inhibitor at the indicated concentrations for 3 h before stimulation with 100 ng/mL EGF for 15 min. Cell lysates were subjected to SDS-PAGE. Blots were probed for phospho- and total EGFR, AKT, and ERK. β-Actin served as control. C, EGFR/ErbB2/ErbB4 inhibitor induces apoptosis in parental and mutant H1975 cells. Representative Annexin V/propidium iodide flow cytometry histograms; the numbers represent percent cells in the appropriate quadrant. D, quantification of apoptosis.
Downstream signaling inhibition identifies the use of Cdk4 inhibitors as a potential treatment strategy. We further sought to determine whether inhibition of downstream signaling pathways might also be a potentially effective treatment strategy. We were particularly interested in studying the effect of inhibition of the cyclin D/Cdk4 axis because our prior studies showed the functional significance of this pathway in oncogenic EGFR signaling ( 28). Therefore, we focused our studies on the potential efficacy of Cdk4 inhibition. The Cdk4 inhibitor used in these studies showed uniform activity against both parental H1975 cells and all five mutant clones tested in BrdUrd proliferation studies. Consistent with the effects on retinoblastoma (Rb) dephosphorylation, the Cdk4 inhibitor drug blocked cell cycle progression, resulting in a sharp decrease in the number of cells in the S phase of the cell cycle at a concentration of 2 μmol/L ( Fig. 5A and B ). An increased number of cells in the sub-G1 fraction at 72 h of drug treatment was also observed in all clones examined, consistent with the induction of apoptosis ( Fig. 5A and B). Immunoblotting showed that Cdk4 inhibitor treatment resulted in dephosphorylation of Rb protein at Ser780, Ser795, and Ser807/811 in all mutants, consistent with inhibition of cyclin D/Cdk4 ( Fig. 5C). Dephosphorylation occurred at drug concentrations (0.6–2 μmol/L) where the induction of growth arrest was also observed. These results suggest that blockade of the cyclin D/Cdk4 axis could be a potential alternative target in EGFR-mutant cells.
A, cells were treated with 2 μmol/L of Cdk4 inhibitor for 24 h. Untreated and treated cells were pulse labeled for 45 min with BrdUrd. Cells were stained with BrdUrd-FITC, counterstained with 7-amino-actinomycin D (7-AAD), and analyzed by flow cytometry. R1, cells in G1; R2, cells in G2-M; R3, cells in S phase; R4, sub-G1 cells. B, percentage of cells in S and sub-G1 phases of the cell cycle after treatment with Cdk4 inhibitor for 24 and 72 h, respectively. C, Cdk4 inhibitor treatment leads to hypophosphorylation of Rb. Cells were incubated with Cdk4 inhibitor for 24 h. Protein extracts were analyzed by immunoblotting with phospho-Rb and total Rb specific antibodies. β-Actin was used as control.
In our current study, we show the functional relevance of secondary EGFR mutations as potential resistance mechanisms against irreversible EGFR inhibitors, modeled by CL-387,785, in EGFR-mutant non–small cell lung cancer. Our cell-based random mutagenesis approach identified numerous secondary mutations of EGFR. Several of these mutations were repeatedly identified, strongly suggestive of their functional relevance. Four of these mutants were chosen for further studies, and all four were confirmed to lead to functional resistance to CL-387,785. The IC50 of these mutants ranged from 1.1 to 6.7 μmol/L (3–19-fold resistance over parental cells). Because many imatinib-resistant mutations of BCR-ABL as well as the recently identified D761Y mutation of EGFR lead to an IC50 shift in this range, we predict that these mutations will be clinically relevant ( 15, 29, 30).
The mutations described in our study broadly fall into four categories: kinase domain mutations close to the activating interface, mutations in the kinase domain close to the adenosine binding region of the catalytic cleft, mutations within the transmembrane helix, and extracellular mutations. Approximately half of the mutations that we discovered were in the kinase domain of EGFR and were dominated by E931G. This mutation was recovered in eight independent clones and the nearby mutation K929E occurred in one further clone. Both E931 and K929 are conserved residues located on the periphery of the proposed kinase-activating asymmetrical dimer interface in the kinase domain crystal structures. Indeed, in the study of Zhang et al. ( 24), both K929 and E931 were predicted to be important in the activation of the cyclin/Cdk-like asymmetrical EGFR dimer, suggesting that these mutations could potentially affect the dimerization and activation potential of EGFR. It is interesting to note that the double mutation E931G + Q684R arose twice in this study; the mutation Q684R is in the EGFR juxtamembrane region. This may suggest a role in regulation and resistance for an interaction between the juxtamembrane region and the C-lobe of the EGFR kinase domain. Our structural analysis also suggested that many of the mutations may have an effect on the kinase catalytic cleft. Mutation H773L occurs at a residue position dominated by histidine and asparagine residues in human kinases. These residues are able to form hydrogen bonds; in the EGFR kinase domain crystal structures, H773 bonds to the backbone carbonyl oxygen of V851. Mutation of histidine to leucine here may disrupt the local conformation of this loop and influence the catalytic cleft. The other mutations that occur proximal to the kinase catalytic cleft, M793T and T725S, are also close to the adenosine-binding region, with M793T occurring in the kinase hinge segment and T725S in the glycine-rich nucleotide-binding loop. The recent study of Yun et al. ( 26) illustrates the effects of EGFR point mutations on inhibitor and ATP binding; it is possible that, similar to the Yun study, alterations in the relative kinetics for ATP and CL-387,785 could also play a role in resistance. The transmembrane helix mutations G649R, L655H, L656S, and L658P are intriguing. Previous work has shown that the transmembrane region of EGFR self-associates in cell membranes ( 31) and that disulfide-induced helix rotation can induce kinase activity ( 32). Helix rotation was also seen on binding of ATP-competitive inhibitors to the cytoplasmic kinase domain of the protein ( 32). The discovery of these resistance mutations supports the role of the transmembrane helix in EGFR activation and raises the possibility that a distortion in the interface can interfere with kinase inhibition by certain ATP-competitive inhibitors. The mechanism by which this occurs will need to be further investigated. Interestingly, the originally described oncogenic rat ErbB2 mutation V664E ( 33) seems to be similar to these transmembrane helix mutations.
It is not clear how the four extracellular domain IV mutations cause resistance to CL-387,785, although expression levels may play a role. Of note, several of these were found as part of double mutant clones, potentially suggesting that some of these might be "carrier" mutations without functional relevance. The EGFR expression level of two of the mutants (Y626H and E545G) was examined and indeed was as high as in nonmutant, high-EGFR colonies (data not shown), suggesting that these mutations might be "silent" and altered expression levels might explain resistance. Whether these mutations affect EGFR expression per se is not yet clear. Irreversible inhibitors covalently bind to C797 of the protein through their Michael acceptor functional group ( 18, 19, 21). In our studies, this mutation leads to relatively low-level resistance, potentially explaining why this mutant was not recovered. In non–small cell lung cancer cells, EGFR mutations are often linked with amplification of the EGFR gene. It currently remains unknown whether EGFR amplification leads to resistance against EGFR inhibitors; nonetheless, this mechanism is suggested by our finding that CL-387,785–resistant, transfected H1975 cells without a detectable mutation consistently express EGFR at a higher level as compared with their mutant counterparts or parental H1975 cells. This suggests that similar resistance mechanisms also exist in vivo in EGFR-mutant non–small cell lung cancer. In the study of Balak et al. ( 29), one patient was indeed identified with a significant increase in EGFR gene copy number after EGFR tyrosine kinase inhibitor therapy. Whether the mutations identified in our study lead to functional resistance against other irreversible EGFR inhibitors, such as HKI-272 and EKB-569, will need to be defined individually. At the same time, we believe our model should provide a fair sample of potential resistance mechanisms to be encountered and should also be readily applicable for future studies of EGFR inhibitors. Of note is that our model system focuses on reactivation of EGFR signaling as the mechanism of resistance and therefore is not expected to identify alternative mechanisms, such as the recently described MET amplification ( 34).
Our studies suggest that tertiary resistance against such inhibitors can be overcome by alternative EGFR inhibitors. Here we used an irreversible pyrido(3,4)-pyrimidine pan-ErbB inhibitor that has nanomolar potency against EGFR, ErbB2, and ErbB4. This compound has previously showed great selectivity for EGFR family members over other receptors (e.g., platelet-derived growth factor, fibroblast growth factor, and insulin receptors; ref. 35). This drug showed high-level efficacy against L655H and was only slightly less effective against the C797S and H773L mutants. L658P and E931G cells were more resistant to this compound but the concentrations required to overcome resistance still suggest clinical usefulness. Whether its differential effect is due its higher potency overall or is dependent on its slight structural difference from CL-387,785 will need to be further investigated. This inhibitor, similar to CL-387,785 and other irreversible ErbB inhibitors, is thought to covalently bind to C797 of EGFR. However, the mechanism by which this inhibitor is able to sustain efficacy against the C797S mutation is unclear and may suggest that kinase inhibition does not depend on covalent binding. Clearly, further studies and crystallographic analysis are required and may provide insights such as the Cl-D855 halogen bond seen for gefitinib bound to L858R mutant EGFR ( 26). The EGFR inhibitor used in our study is multitargeted and blocks the activity of both ErbB2 and ErbB4. Our interpretation of EGFR blockade as its main mechanism of inhibition is supported by our use of the dual EGFR/ErbB2 inhibitor GW583340 that showed no efficacy and signaling studies showing blockade of EGFR activation by this compound on EGF stimulation.
We previously showed that cyclin D1 and downstream regulation of E2F activation are a critical downstream pathway in oncogenic EGFR signaling, suggestive of the therapeutic potential of the cyclin D/Cdk axis ( 28). In our current studies, we used a selective ATP-competitive Cdk inhibitor ( 36). This compound in our studies led to S-phase depletion and inhibition of phosphorylation at Cdk phosphorylation sites of Rb in both wild-type H1975 cells and all EGFR-resistant mutant cells tested, consistent with Cdk inhibition. Our current studies further corroborate the importance of the cyclin D/Cdk axis and suggest potential therapeutic benefit in patients with EGFR-mutant non–small cell lung cancer. It will be important to identify whether more potent inhibitors, such as the EGFR inhibitor used in our current studies or combinations of inhibitors (e.g., combined EGFR plus Cdk4 inhibitors), might synergize and thereby prevent or delay the emergence of resistance. Our in vitro model system should allow the testing of these concepts.
Grant support: Case Comprehensive Cancer Center (B. Halmos), a Young Clinical Scientist Award from the Flight Attendant Medical Research Institute (B. Halmos), NIH grant 1 K99 CA126026-01A1 (S. Kobayashi), NIH/National Cancer Institute Specialized Program of Research Excellence in Human Lung Cancer grant P50 CA90578-04 (B. Halmos and D.G. Tenen), and a scholarship from the American Society of Hematology Junior Faculty (T.J. Boggon).
Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).
Received April 4, 2007.
Revision received July 12, 2007.
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Rosell R, Taron M, Sanchez JJ, Paz-Ares L. Setting the benchmark for tailoring treatment with EGFR tyrosine kinase inhibitors. Future Oncol 2007; 3: 277–83.
Kobayashi S, Boggon TJ, Dayaram T, et al. EGFR mutation and resistance of non-small-cell lung cancer to gefitinib. N Engl J Med 2005; 352: 786–92.
Pao W, Miller VA, Politi KA, et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med 2005; 2: e73.
Kwak EL, Sordella R, Bell DW, et al. Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib. Proc Natl Acad Sci U S A 2005; 102: 7665–70.
Kobayashi S, Ji H, Yuza Y, et al. An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor. Cancer Res 2005; 65: 7096–101.
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Rabindran SK, Discafani CM, Rosfjord EC, et al. Antitumor activity of HKI-272, an orally active, irreversible inhibitor of the HER-2 tyrosine kinase. Cancer Res 2004; 64: 3958–65.
Discafani CM, Carroll ML, Floyd MB, Jr., et al. Irreversible inhibition of epidermal growth factor receptor tyrosine kinase with in vivo activity by N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide (CL-387,785). Biochem Pharmacol 1999; 57: 917–25.
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Kobayashi S, Shimamura T, Monti S, et al. Transcriptional profiling identifies cyclin D1 as a critical downstream effector of mutant epidermal growth factor receptor signaling. Cancer Res 2006; 66: 11389–98.
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November 1 Cancer Research Highlights
You are going to email the following Resistance to an Irreversible Epidermal Growth Factor Receptor (EGFR) Inhibitor in EGFR-Mutant Lung Cancer Reveals Novel Treatment Strategies
Cancer Res November 1 2007 (67) (21) 10417-10427; DOI: 10.1158/0008-5472.CAN-07-1248
Trastuzumab and Pertuzumab: Enhanced Antitumor Activity
Imatinib Sensitizes Bcr-Abl+ Cells to Cisplatin
Loss of S1P Lyase Upregulates Bcl-2
Show more Experimental Therapeutics, Molecular Targets, and Chemical Biology


text:
OFF BEAT: Guitarist's family on 'Rhoad' to court over book
A documentary on the life of rock 'n' roller Randy Rhoads might sound appealing to the family of the late Ozzy Osbourne and Quiet Riot guitarist, but a coffee table book making use of materials provided for the film strikes a sour note.
The musician's family and estate filed suit last month in Los Angeles County Superior Court against the authors and publishers of the "Randy Rhoads" book, according to reports. The lawsuit claims that the book makes unauthorized use of photos and interview excerpts and other information that was provided exclusively for use in a planned documentary about the guitarist.
The suit names the book's authors, Andrew Klein and Steven Rosen, its editor, Peter Margolis, and its publisher, Velocity Publishing Group Inc. It claims fraud, breach of contract, invasion of privacy and misappropriation of rights of publicity.
Mr. Rhoads' family entered into an agreement with Mr. Margolis in 2007 to produce a documentary about the guitarist, according to reports. With the documentary never completed, however, materials provided exclusively for the film were subsequently used to produce the book, the suit claims, without the family's authorization or cooperation.
Mr. Rhoads died at age 25 in a 1982 airplane crash while on tour with Mr. Osbourne.

redpajama_set_name: RedPajamaCommonCrawl


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White Ports come in a range of styles from very sweet to very dry. We believe that Cockburn's Fine White has the best of both worlds. It has the natural sugars of ripe grapes as well as the dryness of a slightly longer fermentation. The result: a slightly drier, easy-going, easy to drink White Port perfect for a summer's afternoon, so sit back relax and if it's not summer yet, you can certainly imagine it is.
We like to drink our Fine White Port as an aperitif before lunch or dinner. And on special occasions here in Portugal, we serve chilled White Port to welcome our guests. Serve chilled, straight from the ice bucket or the fridge.
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Although religious and moral beliefs are widely held, doubts about them are also very common and skepticism about or even outright rejection of religious and moral beliefs is increasingly a live option for people. But genuine doubt or skepticism about our perceptual beliefs in the existence of an external world is almost unheard of outside of philosophy departments, and even among people there, it is rarely taken seriously in their everyday lives. This project on "Knowing in Religion and Morality" will critically examine to what degree, if any, this difference in attitude toward perceptual beliefs, on the one hand, and religious and moral beliefs, on the other, is justified.
The aim of this project is twofold: (i) to develop and evaluate defenses of the view that moral and religious beliefs are relevantly similar to perceptual beliefs, both in terms of how they are produced and in terms of their rationality; and (ii) to critically examine objections to moral and religious belief—in particular, objections based on the prevalence of disagreement about their subject matter and on evolutionary accounts of their origins (both of which are objections that don't seem to apply to perceptual belief).
A monograph by Bergmann, provisionally entitled Perceptual, Moral, and Religious Skepticism.
A 2011 summer seminar led by Bergmann on the topics of the monograph.
A 2012 interdisciplinary conference co-organized by Bergmann and Kain and focused on challenges to religious and moral belief.
A volume called "Challenges to Moral and Religious Belief" (Oxford, 2014) edited by Bergmann and Kain that collects together the papers from the 2012 conference.
Generous support for this project has been provided by the John Templeton Foundation and Purdue University. For more information, see this news release from the Purdue Newsroom.
Copyright © 2011 Knowing in Religion and Morality | Template.


text:
Das ISOE – Institut für sozial-ökologische Forschung GmbH ist eine national und international tätige, gemeinnützige Forschungseinrichtung mit Sitz in Frankfurt am Main. Das ISOE entwickelt als unabhängiges Forschungsinstitut sozial-ökologische Konzepte für eine nachhaltige Entwicklung. Als innovativer Think Tank arbeitet das Forschungsinstitut transdisziplinär und entwickelt gemeinsam mit Partnern aus Wissenschaft und Praxis fundierte Entscheidungsgrundlagen für Zivilgesellschaft, Politik und Wirtschaft.  Dabei werden sozial- und naturwissenschaftliche Aspekte systematisch bereits von Anfang an aufeinander bezogen und integriert.  Das ISOE ist Mitbegründer des nationalen Forschungsnetzwerks Ecological Research Network (Ecornet) und des Senckenberg Biodiversität und Klima Forschungszentrum (SBiK-F).

Fokus
Im Fokus der Forschungsarbeit stehen komplexe gesellschaftliche Probleme nachhaltiger Entwicklung, wie beispielsweise nicht-nachhaltiges Konsum- oder Mobilitätsverhalten, lokale und globale Wasserprobleme oder der Verlust von Biodiversität. Bei ihrer Untersuchung werden Perspektiven der Bevölkerungsentwicklung und der Geschlechtergerechtigkeit mitbehandelt. Ziel der Institutsarbeit ist es, zukunftsfähige Lösungskonzepte zu entwickeln, um praxisrelevante Beiträge zur nachhaltigen Entwicklung und Nachhaltigkeitsforschung zu liefern.

Geschichte
Das Institut wurde 1989 als interdisziplinäre unabhängige Forschungseinrichtung gegründet. Die Basis für die Institutsgründung hat 1986 und 1987 die Forschungsgruppe Soziale Ökologie erarbeitet, die im Auftrag der Hessischen Landesregierung ein Gutachten zur sozial-ökologischen Forschung in Hessen erstellt hatte. Der Wissenschaftsrat hat bereits 1994 in seiner Stellungnahme zur Umweltforschung in Deutschland die "bedeutsame und förderungswürdige sozial- und gesellschaftswissenschaftliche Grundlagenforschung im Spannungsfeld von Ökonomie und Ökologie" des ISOE hervorgehoben. Im Jahr 2000 wurde das ISOE für das Projekt "Nachhaltigkeit - ein fachübergreifendes Konzept für sozial-ökologische Transformationen" mit dem zum ersten Mal vergebenen Schweizer Wissenschaftspreis für hervorragende transdisziplinäre Forschungsprojekte ausgezeichnet. Das ISOE war maßgeblich an der Etablierung des BMBF-Förderschwerpunkts "Sozial-ökologische Forschung" beteiligt. Der Evaluierung durch den Wissenschaftsrat 2016 zufolge wird das vom ISOE mitgeprägte Konzept der Transdisziplinarität in seiner "Forschungspraxis überzeugend umgesetzt". Seit April 2021 ist Flurina Schneider neue wissenschaftliche Geschäftsführerin und Sprecherin der Institutsleitung. Sie übernimmt gleichzeitig eine Professur für Soziale Ökologie am Fachbereich Biowissenschaften der Goethe-Universität Frankfurt am Main. Hier ist das ISOE bereits seit 2008/09 mit dem Fachbereich Gesellschaftswissenschaften im Masterstudiengang Umweltwissenschaften verantwortlich für das Schwerpunktfach "Soziale Ökologie".

Struktur
Derzeit arbeiten rund 50 Mitarbeitende in einer Vielzahl unterschiedlicher disziplinübergreifender Forschungsprojekte. Unterstützt wird das Institut durch einen Wissenschaftlichen Beirat.

Das wissenschaftliche Team arbeitet in folgenden Forschungsschwerpunkten:
 Wasserressourcen und Landnutzung
 Wasserinfrastruktur und Risikoanalysen
 Energie und Klimaschutz im Alltag
 Mobilität und Urbane Räume
 Biodiversität und Bevölkerung
 Transdisziplinäre Methoden und Konzepte

Finanzierung

Als unabhängige und außeruniversitäre Forschungseinrichtung finanziert sich das Institut im Wesentlichen aus öffentlichen Projektmitteln und Aufträgen und durch private Auftraggeber und Verbände. Darüber hinaus erhält es eine institutionelle Förderung durch das Land Hessen sowie eine Unterstützung durch die Stadt Frankfurt am Main.

Forschungsprogramm
Ausgangspunkt der Forschungsarbeit des ISOE ist die Erkenntnis, dass sich in ökologischen Problemlagen gesellschaftliche Handlungsmuster und natürliche Wirkungszusammenhänge wechselseitig durchdringen und eine sozial-ökologische Krisendynamik auslösen. Aus dieser Erkenntnis folgt ein spezifischer sozial-ökologischer Forschungszugang: Natur und Gesellschaft werden gleichzeitig in den Blick genommen und deren wechselseitige Beziehungen untersucht. Im Begriff der gesellschaftlichen Naturverhältnisse wird diese Verflechtung deutlich. Soziale Ökologie als Wissenschaft von den gesellschaftlichen Naturverhältnissen bewegt sich damitzwischen den beiden Polen Lösungen für lebenspraktische Probleme als praxisbezogene, transdisziplinäre Forschung zu erarbeiten und methodisch erzeugtes neues Wissen als theorieorientierte Wissenschaft begrifflich zu ordnen.

Literatur
 Egon Becker, Thomas Jahn (Hrsg.): Soziale Ökologie. Grundzüge einer Wissenschaft von den gesellschaftlichen Naturverhältnissen. Frankfurt a. M., New York: Campus 2006
 Matthias Bergmann, Engelbert Schramm (Hrsg.): Transdisziplinäre Forschung – Integrative Forschungsprozesse verstehen und bewerten Frankfurt a. M., New York: Campus 2008
 Diana Hummel (ed.): Population Dynamics and Supply Systems – A Transdisciplinary Approach Frankfurt a. M., New York: Campus 2008
 Matthias Bergmann, Thomas Jahn, Tobias Knobloch, Wolfgang Krohn, Christian Pohl, Engelbert Schramm (Hrsg.): Methoden transdisziplinärer Forschung – Ein Überblick mit Anwendungsbeispielen Frankfurt a. M., New York: Campus 2010
 Thomas Jahn (2013): Wissenschaft für eine Nachhaltige Entwicklung braucht eine kritische Orientierung. GAIA 1, 29–33
 Thomas Jahn, Diana Hummel, Engelbert Schramm (2015): Nachhaltige Wissenschaft im Anthropozän. GAIA 24 (2), 92–95
 Johanna Kramm, Melanie Pichler, Anke Schaffartzik, Martin Zimmermann (Hrsg.): Social Ecology: State of the Art and Future Prospects. Printed Edition of the Special Issue Published in Sustainability. MDPI, Basel usw. 2017
 Thomas Jahn, Diana Hummel, Lukas Drees, Stefan Liehr, Alexandra Lux, Marion Mehring, Immanuel Stieß, Carolin Völker, Martina Winker, Martin Zimmermann (2020): Sozial-ökologische Gestaltung im Anthropozän. GAIA 29 (2), 93–97

Weblinks
 Offizielle Website

Fußnoten

Sozialokologische Forschung
Interdisziplinäres Forschungsinstitut
Organisation (Ökologie)
Organisation (Nachhaltigkeit)
Soziologische Forschungsinstitution
Denkfabrik in Deutschland
Gegründet 1989
Unternehmen (Frankfurt am Main)
Forschungseinrichtung in Frankfurt am Main

redpajama_set_name: RedPajamaWikipedia


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These disorders are intrinsic to the individual, presumed to be due to central nervous system dysfunction ( or may occur across the life span).
Prevention is usually described as primary, secondary, tertiary depending on when why preventive action is taken.
Dyslexia - Difficulty processing Language.
Dyscalculia - Difficulty with math.
Dyspraxia (SIT)- Difficulty with fine motor skills.
ADP ( Auditory Processing Disorder)- Difficulty hearing differences between sound.
VDP ( Visual Processing Disorder)- Difficulty interpreting visual information.


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Capstar for cats are orally administered tablets for short-term flea treatment. The tablets contain nitenpyram, which is neurotoxin that blocks flow of neural messages in fleas thus resulting in the death of the parasites. By attacking the central nervous system, it instantly kills the parasites. By killing fleas immediately, it prevents them from laying eggs and stops multiplication of these parasites.
Cats start scratching more in the first few hours as fleas react to nitenpyram. Within a few hours, it kills fleas putting an end to itching and irritation. Since it is a short-term treatment, do follow up with long-term treatment for long-lasting effect.
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For now, we have looked at personal beliefs about money as such. Today, we move into another level of beliefs – those about being rich. Because that's what most of us dream of, right? We want financial independence, want to have enough money for a nice home, a garden, vacations, and maybe, just maybe work much less.
It's such a dream: Sit at the beach or under palm trees, with a book or your smartphone in hand and just enjoy life. Sip a cocktail every now and then, feel the gentle, warm breeze on your skin, maybe chat with a friend… bliss.
Read it out aloud to yourself and watch your belief system react. It can be fascinating. We all have lots and lots of inherited beliefs about rich people.
In my family, the underlying thought when talking about rich people was that they must be crooks, or they wouldn't be rich. They must be lying about their taxes, have exploited others, be involved in shady dealings or otherwise have accumulated their wealth in a shady manner.
Because they were rich. That was enough to condemn them.
Rich people must be bad people.
I mean, just remember that pharma guy, who raised the price of a much needed medication by 5000% after buying the company that made it. Putting pressure on chronically ill people for personal gain. The picture of his self-satisfied smirk in front of Congress went around the world.
He's the poster child for the ugly rich person.
Now, this ties in with the belief that money will make me a bad person, but it's also even deeper: Rich people are crooks by definition. Bad people. Or they wouldn't be rich.
This belief is very, very old, btw. Remember this?
Nobody wants to be that kind of a bad person.
And you instinctively avoid doing things that might promote your business, you shy away from success, and possibly sales. You avoid coaching and learning new things. You don't go to meetings or networking events. You may not write that cracking description that will make your product sell instantly – because all of this might take you closer to being one of those bad, rich people.
And there goes that beach, the cocktail and the gentle breeze as perfect spot to relax.
Unless we eliminate that kind of belief.
It'll be much easier to earn money – and even really good money – without that one holding you back.
And that's where tapping is so useful.
Even though I learned that rich people must be crooks by definition, and I really avoid getting rich because I don't want to be a crook, I'm totally okay the way I am, and I'm now ready to recognize that this is simply a belief.
Even though it's even written in the bible that rich people are bad people, and I really want to avoid becoming a bad person, I'm totally okay the way I am, and I'm open to the possibility that this is simply a belief – and that I can be rich and a good person.
Even though there is so much hatred around rich people, including the belief that they must all be crooks, and I bought into that belief, I'm still totally okay, and I'm giving myself permission to let go of that belief, and to become financially independent now.
Can you imagine being really, really rich?
PS: My newsletter contains a full tapping round to go with my blog posts, so you can start getting results. Sign up through the form on the upper right hand corner, and receive that tapping round plus occasional special offers. If you're on a mobile and can't see the sidebar, you can sign up through this link: Newsletter Sign-up.
This entry was posted in EFT and tagged abundance, belief, filthy rich, limiting beliefs, Money Mindset, negative belief, rich. Bookmark the permalink.


text:
Risbury Hill round barrows
Bell barrow and bowl barrow 1025m west of Lyburn Farm.
Bell barrows, the most visually impressive form of round barrow, are funerary monuments dating to the Early and Middle Bronze Age, with most examples belonging to the period 1500-1100 BC. They occur either in isolation or in round barrow cemeteries and were constructed as single or multiple mounds covering burials, often in pits, and surrounded by an enclosure ditch. The burials are frequently accompanied by weapons, personal ornaments and pottery and appear to be those of aristocratic individuals, usually men. Bell barrows (particularly multiple barrows) are rare nationally, with less than 250 known examples, most of which are in Wessex. Their richness in terms of grave goods provides evidence for chronological and cultural links amongst early prehistoric communities over most of southern and eastern England as well as providing an insight into their beliefs and social organisation. As a particularly rare form of round barrow, all identified bell barrows are important. Bowl barrows are the most numerous form of round barrow, dating from the Late Neolithic period to the Late Bronze Age, with most examples belonging to the period 2400-1500 BC. They were constructed as earthen or rubble mounds, sometimes ditched, which covered single or multiple	burials. They occur either in isolation or grouped as cemeteries and often acted as a focus for burials in later periods. Often superficially similar, although differing widely in size, they exhibit regional variations in form and a diversity of burial practices. Often occupying prominent locations, they are a major historic element in the modern landscape and their considerable variation of form and longevity as a monument type provide important information on the diversity of beliefs and social organisations amongst early prehistoric communities. They are particularly representative of their period. Despite tree growth and some later interference the bell barrow and bowl barrow 1025m west of Lyburn Farm survive well and will contain archaeological and environmental evidence relating to their construction, relative chronologies, territorial significance, social organisation, ritual and funerary practices and overall landscape context.
This record was the subject of a minor enhancement on 17 September 2015. This record has been generated from an "old county number" (OCN) scheduling record. These are monuments that were not reviewed under the Monuments Protection Programme and are some of our oldest designation records.
This monument, which falls into two areas, includes a bell barrow and a bowl barrow situated on the summit and upper north east ridge of the prominent Risbury Hill on Hamptworth Common overlooking three tributaries to the River Blackwater. The bell barrow to the south west survives as a circular mound of 23m in diameter and 2m high, with a 5m wide and 0.8m high berm surrounded by a 3m wide and 0.8m deep quarry ditch. The surface has been cut by practice trenches made during the Second World War. To the north east is a bowl barrow which survives as a circular mound of up to 18m in diameter and 0.6m high surrounded by a buried quarry ditch from which the construction material was derived.
PastScape 222660 and 222657, Wiltshire HER SU21NW608 and SU21 NW607


text:
Barto-Prize
Tzimon Barto – triumphant finale with the SWR
In the coming weeks, Tzimon Barto will end his residency with the SWR Symphony Orchestra on a high note with performances of Beethoven's Fourth Piano Concerto and Dvorak's Piano Quintet No. 2. In mid-March, Tzimon Barto will delight audiences at concerts in Stuttgart, Ulm and Mannheim under the principle conductor of the Bamberg Symphony, Jakub Hrusa, who has risen to international acclaim at a young age. The concerts by the internationally renowned American and award-winning native of the Czech Republic promise a unique musical experience.
As a triumphant finale to end his residency, Tzimon Barto will join the musicians of the SWR Symphony Orchestra and, with his characteristic charm and technical excellence, delight audiences in Freiburg, Baden-Baden and Stuttgart.
© 2023 - Tzimon Barto


text:
Q: BMO2 2011 Question 3 - How to Find the Pattern in this Function? The function $f$ is defined on the positive integers as follows:
$$f(1) = 1$$
$$f(2n) = f(n), \text{if $n$ is even}$$
$$f(2n) = 2f(n), \text{if $n$ is odd}$$
$$f(2n + 1) = 2f(n) + 1, \text{if $n$ is even}$$
$$f(2n + 1) = f(n), \text{if $n$ is odd}$$
Find the number of positive integers n which are less than $2011$ and
have the property that $f(n) = f(2011)$.
Calculating the values of $f(n)$ up to $20$ didn't yield any pattern, so I am not sure where to go from here. Usually I try to figure out whether the function is increasing, decreasing or doing something predictable, but here I do not see it.

A: The pattern is more obvious if you write in base $2$:
$$\begin{align}f(10011_2)&=101_2\\
f(10100_2)&=1010_2\\
f(10101_2)&=10101_2\\
f(10110_2)&=1010_2\\
f(10111_2)&=101_2\\
...
\end{align}$$
Show, in general, the value of $f(n)$ is the binary digits of $n$, removing repeated digits.
So, since $2011= 11111011011_2$, you get $f(n)=10101_2$, and you need to find all $n<2011$ which can be written by taking $10101$ and expanding any digit by repeating digits.
These can be written as $2^{a}-2^{b}+2^{c}-2^d+2^e-1$ where $a>b>c>d>e> 0$.
For example, $2011=2^{11}-2^{6}+2^{5}-2^{3}+2^{2}-1$.
There are $\binom{10}{5}=252$ cases $a\leq 10$. The cases where $a=11$ is harder - you need $b\geq 6$, and then have to continue counting cases.
The full count is, I think:
$$\binom{11}{5}-\binom{6}{4}+\binom{5}{3}-\binom{3}{2}+\binom{2}{1}-1$$
Essentially, $\binom{11}{5}-\binom{6}{4}$ counts the cases where $a\leq 11$, but removing the cases where $a=11$ and $b\leq 6$. Then $\binom{5}{3}-\binom{3}{2}$ count the cases where $a=11,b=6$, excepting the cases where $c=5,d\leq 3$. And $\binom{2}{1}-1$ counts the cases where $a=11,b=6,c=5,d=3.$ and the resulting number is not $n$.
Note that the top parts of the binomials are $11,6,5,3,2$, which are the exponents of $2011=2^{11}-2^6+2^5-2^3+2^2-1$. That might give you an idea for the general formula, given an $m$ for the count of $n< m$ with $f(n)=f(m)$.
Every natural number $m\neq 0$ can be written uniquely as $m=\sum_{i=0}^{2k-1} (-1)^{i+1}2^{a_i}$, where $k\geq 1$ and $0\leq a_0<a_1<\dots<a_{2k-1}$. If $m$ is odd, then $a_0=0$, and the number of $n<m$ such that $f(n)=f(m)$ is:
$$\sum_{i=0}^{2k-1}(-1)^{i+1}\binom{a_i}{i}$$
So, for example, if $m=17=2^{5}-2^{4}+2^1-2^0$, you get $$\binom{5}{3}-\binom{4}{2}+\binom{1}{1}-\binom{0}{0}=10-6+1-1=4.$$
And the $n<m$ with $f(n)=f(m)$ are $101_2,1001_2,1011_2,1101_2$.
If $m$ is even, then $a_0>0$ and you get:
$$\sum_{i=0}^{2k-1}(-1)^{i+1}\binom{a_i}{i+1}$$
For example, if $m=26=11010_2=2^5-2^3+2^2-2^1$ then:
$$\binom{5}{4}-\binom{3}{3}+\binom{2}{2}-\binom{1}{1}=4$$
And there are $1010_2,10100_2,10110_2,10110_2$.

A: (Hint) Another way of viewing the function is by looking at the input upon divisibility by 4. For example, if the input is of the form $2n$ where $n$ is odd, then the input is of the form $2(2k+1) = 4k+2$. Thus we can envision the functions as
$$
f(n) = \begin{cases}
f(\lfloor n/2\rfloor)& n \equiv 0,3 \mod 4 \\
2f(\lfloor n/2\rfloor)+1& n \equiv 1\mod 4 \\
2f(\lfloor n/2)& n\equiv 2\mod 4
\end{cases}
$$
Moreover, using J.G.'s suggestion, we can work in binary where $\lfloor n/2\rfloor$ is equivalent to deleting the rightmost bit. In binary, $2011 = 11111011011$ so
\begin{align}
f(11111011011) &= f(1111101101) \\
&= 2f(111110110)+1 \\
&= 4f(11111011)+1 \\
&= 4f(1111101)+1 \\
&= 8f(111110)+5 \\
&= 16f(11111)+5 \\
&= 16f(1)+5 = 21
\end{align}
Notice immediately that long strings of 1s or long strings of 0s are invariant under this function. This will allow you to construct binary strings whose values don't change. For example, $f(1110110)=f(2011)$.

A: This is a partial answer:
Note that any binary string can be divided into alternating blocks of $0$'s and $1$'s, for example, $1111001101001110=B_1B_2B_3B_4B_5B_6B_7B_8$, where $B_1=1111,\ B_2=00,\ B_3=11,\ B_4=0,\ B_5=1,\ B_6=00,\ B_7=111,\ B_8=0$.  Now note that $f(n)$ will be unchanged if the trailing block is replaced by a single $1$ or a single $0$ (depending on whether the block consisted of $0$'s or $1$'s). Now, note that a trailing $0$ implies $f(n)=2f(n/2)$, and trailing $1$ implies. $f(n)=2f((n-1)/2)+1$, which means $f(n)$ has the binary expansion of where the trailing bit is $0$ or $1$ dependeing on whether the trailing block of   binary expansion of $n$ were a block of $0$ or $1$.
Repeating this procedure, it is easy to argue that the binary expansion of $f(n)$ is alternating binary number $1010\cdots$ with number of bits equal to the number of alternating blocks in the binary expansion of $n$. For example $f(13)=f(1101_b)=101_b=3$.
To find the number $g(n)$ of $m\le n$ such that $f(m)=f(n)$, following is an idea:
Let the number of bits in the expansion of $n$ is $k$, and let the expansion of $n$ has $l$ alternating blocks. Then, any number $m<n$ such that $m$ also has $l$ blocks is a candidate to count. Now, if we count all numbers $m$ such that $m\le 2^{k}$, (call that number $h(k)$) and $m$ has $l$ alternating blocks, then that number will overestimate $g(n)$ by some amount, which we denote $e(n,k)$. Then, $g(n)=h(k)-e(n,k)$.
Let $z(n),u(n)$ be the number of blocks of $0$ and $1$, respec., in the binary expansion of $f(n)$, and let it has $c_1,\cdots,\ c_{z(n)}$ and $d_1,\cdots,d_{u(n)}$ elements in those blocks, respec.
Define the following set of $l$-tuples of positive integers: $$S_k=\{m_1,\cdots,\ m_l: 1\le m_j,\ j=1,2,\cdots,\ l,\ \sum_{j=1}^l m_j\le k\}$$
After some observation, we can conclude that $h(k)$ is the cardinality of the set $S_k$.
Then it follows that $h(k)$ is the sum of coefficients of $x^{l+j}$ for $j=0,\cdots, k-l$ in the expnasion of $x^l(1-x)^{-l}$, which results in $$h(k)=1+\sum_{j=1}^{k-l}\binom{l+j-1}{j}=\binom{k}{l}$$
However, finding $e(k,n)$ seem to be quite difficult. The difficulty lies in finding the pattern in which the number of $0$'s and $1$'s in the blocks can vary for numbers larger than $n$ and having $k$ bits. I can understand that the number of $1$'s in the leading block will always be nondecreasing but how does the number of $0$'s and $1$'s in the latter blocks changes, might require some careful analysis.
So, for example, for the given problem, $f(n)=10101,\ l=5,\ z(n)=2,\ u(n)=3,\ $, $c_1=c_2=1,\ d_1=5,\ d_2=2,\ d_3=2,\ k=11$.
Then, $$h(k)=1+\sum_{j=1}^{6}\binom{4+j}{j}=462$$

redpajama_set_name: RedPajamaStackExchange


text:
Q: Meaning of the phrase "without breaking down"
Computers are very reliable; they can work for a long time without
  breaking down.

What does the phrase "without breaking down" mean in this sentence?

A: It means to become inoperative or ineffective. It is an intransitive verb, and you can see it in the Merriam Webster Dictionary under definition 1 B.

A: Without encountering a fault that renders them non-workable.


text:
That Every Mensch Be Armed
I could see it in my website traffic: Someone in the public eye had said something stupid about Jews and guns. People were trying to fact-check the comment, and they were heading straight for my site. A piece I wrote in January 2013 ridiculing a familiar gun rights meme (the one that goes something like "€œif the German Jews had owned guns, there"€™d"€™a been no Holocaust"€) began racking up thousands of new hits last week, with search terms like "€œJews,"€ "€œguns,"€ "€œ2nd Amendment,"€ and "€œHolocaust"€ leading the charge.
A quick check of my morning news feed revealed the culprit"€"GOP presidential contender Dr. Ben Carson, who seems to think Holocaust historiography is so simple even a brain surgeon can do it. Carson was joined in his repetition of the shopworn gun rights cliché by his buddy and fund-raiser, Fox News personality Dr. Keith Ablow, a psychiatrist known for his wacky and unverified on-air claims.
Every few years, the "€œgun ownership would have stopped the Holocaust"€ argument pops up in the aftermath of a tragedy like a mass shooting, or during a worse tragedy like a U.S. presidential election. It's important to understand why the "€œsheeny get your gun"€ meme is, from a Holocaust-history point of view, utter rubbish, but it's even more important to understand why the meme is symbolic of how the gun rights lobby often loses in the court of public opinion.
"€œBeing armed might be practical in many situations, but it isn"€™t a panacea."€
Let's start with the historical stuff. The notion that if the German Jewish population had been well armed the Holocaust would have been avoided is asinine. Jews represented less than 1% of the prewar German population (500,000 people in a nation of 67 million). Even if every single Jew had a rifle, the Nazis had a massive police force and an army with the most modern weaponry available at the time. During the first eight years of Nazi rule, Jews were not the primary target of the police state. Rather, the Gestapo busied itself with real or perceived political enemies, many of them communists, some Russian-backed. Those people did have weapons and access to weapons, but that made little difference. The Nazis were a superior force. That the Nazis successfully rounded up and imprisoned trained, armed partisans is not theory, it's fact.
FNC's Ablow claims that if only the German Jews had physically resisted, if only they"€™d attempted to "€œcrush the skull or scratch out the eyes of any Nazi who turned his back for one moment,"€ it would have led "€œthe people of that country and the world to be moved to action by their bold refusal to be enslaved."€ That's rich. From 1933 through 1941, the legal rights of German Jews were indeed curtailed, but not to an extent worse than the restrictions imposed upon blacks in America or nonwhites in England's vast colonial empire. The ruling powers in the West would not likely have stood up and applauded the idea that an oppressed people should skull-crush and eye-gouge anyone who deprives them of the right to marry who they please or own property where they choose. To say that rampant eye-poking and skull-cracking in the face of government oppression (officially known as the Stooge Strategy) would have been a reward in its own right, as many of my anarcho-libertarian friends would argue, is one thing; but to claim that it would have prevented the Holocaust is another. Such an assertion is pure pseudohistorical fantasy.
Every time resistance did occur, it merely worsened the Jews"€™ precarious situation. In November 1938, Herschel Grynszpan, a 17-year-old German-born Jew living illegally in France, walked into a gun shop and purchased a revolver and bullets. France had no waiting period, no background check, no restrictions on him being a Jew, an illegal alien, or 17 years old. The seller even helped him choose the right model, and he gave the kid a quick tutorial on shooting. Armed with his new purchase, Grynszpan walked to the German embassy and assassinated a German diplomat as revenge for the fact that his parents had been deported from Germany back to Poland (yes, he was upset that his parents were forced to leave Germany, but that's a whole "€™nother story). The assassination led to the orgy of anti-Jewish destruction known as Kristallnacht. Kristallnacht was condemned by many in the U.S. and England, but the fact that Kristallnacht "€œmoved the world"€ did nothing to prevent the Holocaust.
As the Grynszpan case illustrates, Jews in France had easy access to guns, and a fat lot of good that did in preventing the Nazi invasion of that nation and the deportation of many Jews from French territory. This rather handily blows Carson and Ablow's theory out of the water. Plus, the majority of Jews killed during the Holocaust were not German Jews. German gun laws didn"€™t apply to preinvasion Poland and Russia. The Polish and Russian Jews killed during the Holocaust were citizens of nations that did indeed fight back against the Nazis. They fought, and they lost to a superior military force.
And here we see the problem with what I call "€œutopian"€ pro-gun arguments, the kinds that claim "€œthis wouldn"€™t have happened to a well-armed populace."€ It's bunk. Armed people are defeated by other armed people every day of the week, whether on a grand scale (one army triumphing over another) or a small one (an armed hood shot dead by an armed cop). Being armed might be practical in many situations, but it isn"€™t a panacea. Unfortunately, like anyone with a good idea, gun rights people often become evangelists, making promises that are simply unrealistic.
The Stupid Party
Dr. Kizzmekia Corbett Presents "Wacky Vaxxers!"
The GOP's Big Black Suicide Tent
Something Worse Than Voter Fraud
40 Acres and a Mall


text:
ABOUT DOMINIC
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Irwell Hill Residences
Launched with great success 2021, Irwell Hill Residences is the epitome of luxurious city living, perched right at the crossroads of exclusivity and connectivity.
Register now for priority access. Get the floorplans, price list, latest news and access to the showflat ahead of the pack.
Launching with previews starting 27 March 2021
Launching soon in 2021, Irwell Hill Residences is the epitome of luxurious city living, perched right at the crossroads of exclusivity and connectivity.
Concept Architect: MVRDV
Principal Architect: ADDP
Awarded with BCI Asia Top 10 architectural firm in Singapore from 2005-2007.
Renowned Netherlands architect
District 09 is known as the shopping haven in Singapore. It also serves as a prime residential area, with affluent housing estates that include top-notch luxury condominiums.
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CDL is a leading global real estate company in Singapore with a network spanning 106 locations in 29 countries and regions.
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This new luxury condominium sits on approximately 138,000 sqft with 2 towers of 36 storey each for a total of 540 units which range from studio, 1-bedroom to 4-bedroom and penthouses. Views from the apartments are curated to provide great city vistas.
CDL is a leading global real estate company with a network spanning 106 locations in 29 countries and regions.
One Sail @ Marina Bay • One Shenton Way • Sentosa Cove
This new luxury condominium sits on approximately 138, 000 sqft with 2 towers of 36 storey each for a total of 540 units which range from studio, 1-bedroom to 4-bedroom and penthouses. Views of every unit are maximised by staggering the blocks.
View Siteplan
District 09 is known as the shopping haven in Singapore. It also serves as a prime residential area, with affluent housing estates that include top-notch condominiums
Inspired And Progressive
Founded in 1993 by Winy Maas, Jacob van Rijs and Nathalie de Vries in the Netherlands, MVRDV provides solutions to contemporary architectural and urban issues globally and delivers future-facing works with highly collaborative and research-based design methods. With iconic works like Depot Boijmans Van Beuningen, The Markthal, and upcoming projects like RED7 and Valley in over 45 countries, MVRDV is a thought leader in the industry and is now making its mark with Irwell Hill Residences in Singapore.
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Widely respected for award-winning build quality, CDL commissioned the world-renowned architecture practice MVRDV from the Netherlands and ADDP from Singapore to create Irwell Hill Residences. Configured to optimise the living environment, the architecture by MVRDV & ADDP seamlessly integrates the conserved heritage raintrees into the landscape while placing attention to the details within the grounds. Incorporating the efficiency of a sustainable construction method, the architecture also connects the botanical landscape with the outdoor spaces of the balconies to create a green and enjoyable dwelling experience. Envisioned by MVRDV, the champagne gold pixel-patterned façade of the two towers is a deliberate departure from the predictable, while injecting a bold breath of design to Singapore's skyline.
About Concept Architect
Widely respected for award-winning build quality, CDL commissioned the world-renowned architecture practice MVRDV from the Netherlands and ADDP from Singapore to create Irwell Hill Residences. Configured to optimise the living environment, the architecture by MVRDV & ADDP seamlessly integrates the conserved heritage raintrees into the landscape while placing attention to the details within the grounds. Incorporating the efficiency of a sustainable construction method, the architecture also connects the botanical landscape with the outdoor spaces of the balconies to create a green and enjoyable dwelling experience.
Great World MRT Station (TE15)
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City Developments Limited (CDL) is a leading global real estate company with a network spanning 106 locations in 29 countries and regions. Listed on the Singapore Exchange, the Group is one of the largest companies by market capitalisation. Its income-stable and geographically-diverse portfolio comprises residences, offices, hotels, serviced apartments, shopping malls and integrated developments.
With a proven track record of over 55 years in real estate development, investment and management, the Group has developed over 46,000 homes and owns over 24 million square feet of gross floor area in residential, commercial and hospitality assets globally. Its diversified global land bank offers 3.4 million square feet of land area.
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text:
using System;
using System.Collections.Generic;
using System.Diagnostics;
using System.IO;

namespace Zio.FileSystems
{
    /// <summary>
    /// Provides an abstract base <see cref="IFileSystem"/> for composing a filesystem with another FileSystem.
    /// This implementation delegates by default its implementation to the filesystem passed to the constructor.
    /// </summary>
    public abstract class ComposeFileSystem : FileSystem
    {
        protected bool Owned { get; }

        /// <summary>
        /// Initializes a new instance of the <see cref="ComposeFileSystem"/> class.
        /// </summary>
        /// <param name="fileSystem">The delegated file system (can be null).</param>
        /// <param name="owned">True if <paramref name="fileSystem"/> should be disposed when this instance is disposed.</param>
        protected ComposeFileSystem(IFileSystem? fileSystem, bool owned = true)
        {
            Fallback = fileSystem;
            Owned = owned;
        }

        protected override void Dispose(bool disposing)
        {
            if (disposing && Owned)
            {
                Fallback?.Dispose();
            }
        }

        /// <summary>
        /// Gets the next delegated file system (may be null).
        /// </summary>
        protected IFileSystem? Fallback { get; }

        /// <summary>
        /// Gets the next delegated file system or throws an error if it is null.
        /// </summary>
        [DebuggerBrowsable(DebuggerBrowsableState.Never)]
        protected IFileSystem FallbackSafe
        {
            get
            {
                if (Fallback is null)
                {
                    throw new InvalidOperationException("The delegate filesystem for this instance is null");
                }
                return Fallback;
            }
        }

        protected override string DebuggerDisplay()
        {
            return $"{base.DebuggerDisplay()} (Fallback: {(Fallback is FileSystem fs ? fs.DebuggerKindName() : Fallback?.GetType().Name.Replace("FileSystem", "fs").ToLowerInvariant())})";
        }

        // ----------------------------------------------
        // Directory API
        // ----------------------------------------------

        /// <inheritdoc />
        protected override void CreateDirectoryImpl(UPath path)
        {
            FallbackSafe.CreateDirectory(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override bool DirectoryExistsImpl(UPath path)
        {
            return FallbackSafe.DirectoryExists(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override void MoveDirectoryImpl(UPath srcPath, UPath destPath)
        {
            FallbackSafe.MoveDirectory(ConvertPathToDelegate(srcPath), ConvertPathToDelegate(destPath));
        }

        /// <inheritdoc />
        protected override void DeleteDirectoryImpl(UPath path, bool isRecursive)
        {
            FallbackSafe.DeleteDirectory(ConvertPathToDelegate(path), isRecursive);
        }

        // ----------------------------------------------
        // File API
        // ----------------------------------------------

        /// <inheritdoc />
        protected override void CopyFileImpl(UPath srcPath, UPath destPath, bool overwrite)
        {
            FallbackSafe.CopyFile(ConvertPathToDelegate(srcPath), ConvertPathToDelegate(destPath), overwrite);
        }

        /// <inheritdoc />
        protected override void ReplaceFileImpl(UPath srcPath, UPath destPath, UPath destBackupPath,
            bool ignoreMetadataErrors)
        {
            FallbackSafe.ReplaceFile(ConvertPathToDelegate(srcPath), ConvertPathToDelegate(destPath), destBackupPath.IsNull ? destBackupPath : ConvertPathToDelegate(destBackupPath), ignoreMetadataErrors);
        }

        /// <inheritdoc />
        protected override long GetFileLengthImpl(UPath path)
        {
            return FallbackSafe.GetFileLength(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override bool FileExistsImpl(UPath path)
        {
            return FallbackSafe.FileExists(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override void MoveFileImpl(UPath srcPath, UPath destPath)
        {
            FallbackSafe.MoveFile(ConvertPathToDelegate(srcPath), ConvertPathToDelegate(destPath));
        }

        /// <inheritdoc />
        protected override void DeleteFileImpl(UPath path)
        {
            FallbackSafe.DeleteFile(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override Stream OpenFileImpl(UPath path, FileMode mode, FileAccess access, FileShare share = FileShare.None)
        {
            return FallbackSafe.OpenFile(ConvertPathToDelegate(path), mode, access, share);
        }

        // ----------------------------------------------
        // Metadata API
        // ----------------------------------------------

        /// <inheritdoc />
        protected override FileAttributes GetAttributesImpl(UPath path)
        {
            return FallbackSafe.GetAttributes(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override void SetAttributesImpl(UPath path, FileAttributes attributes)
        {
            FallbackSafe.SetAttributes(ConvertPathToDelegate(path), attributes);
        }

        /// <inheritdoc />
        protected override DateTime GetCreationTimeImpl(UPath path)
        {
            return FallbackSafe.GetCreationTime(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override void SetCreationTimeImpl(UPath path, DateTime time)
        {
            FallbackSafe.SetCreationTime(ConvertPathToDelegate(path), time);
        }

        /// <inheritdoc />
        protected override DateTime GetLastAccessTimeImpl(UPath path)
        {
            return FallbackSafe.GetLastAccessTime(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override void SetLastAccessTimeImpl(UPath path, DateTime time)
        {
            FallbackSafe.SetLastAccessTime(ConvertPathToDelegate(path), time);
        }

        /// <inheritdoc />
        protected override DateTime GetLastWriteTimeImpl(UPath path)
        {
            return FallbackSafe.GetLastWriteTime(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override void SetLastWriteTimeImpl(UPath path, DateTime time)
        {
            FallbackSafe.SetLastWriteTime(ConvertPathToDelegate(path), time);
        }

        // ----------------------------------------------
        // Search API
        // ----------------------------------------------

        /// <inheritdoc />
        protected override IEnumerable<UPath> EnumeratePathsImpl(UPath path, string searchPattern, SearchOption searchOption, SearchTarget searchTarget)
        {
            foreach (var subPath in FallbackSafe.EnumeratePaths(ConvertPathToDelegate(path), searchPattern, searchOption, searchTarget))
            {
                yield return ConvertPathFromDelegate(subPath);
            }
        }

        /// <inheritdoc />
        protected override IEnumerable<FileSystemItem> EnumerateItemsImpl(UPath path, SearchOption searchOption, SearchPredicate? searchPredicate)
        {
            foreach (var subItem in FallbackSafe.EnumerateItems(ConvertPathToDelegate(path), searchOption, searchPredicate))
            {
                var localItem = subItem;
                localItem.Path = ConvertPathFromDelegate(localItem.Path);
                yield return localItem;
            }
        }

        // ----------------------------------------------
        // Watch API
        // ----------------------------------------------

        /// <inheritdoc />
        protected override bool CanWatchImpl(UPath path)
        {
            return FallbackSafe.CanWatch(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override IFileSystemWatcher WatchImpl(UPath path)
        {
            return FallbackSafe.Watch(ConvertPathToDelegate(path));
        }

        // ----------------------------------------------
        // Path API
        // ----------------------------------------------

        /// <inheritdoc />
        protected override string ConvertPathToInternalImpl(UPath path)
        {
            return FallbackSafe.ConvertPathToInternal(ConvertPathToDelegate(path));
        }

        /// <inheritdoc />
        protected override UPath ConvertPathFromInternalImpl(string innerPath)
        {
            return ConvertPathFromDelegate(FallbackSafe.ConvertPathFromInternal(innerPath));
        }

        /// <summary>
        /// Converts the specified path to the path supported by the underlying <see cref="Fallback"/>
        /// </summary>
        /// <param name="path">The path exposed by this filesystem</param>
        /// <returns>A new path translated to the delegate path</returns>
        protected abstract UPath ConvertPathToDelegate(UPath path);

        /// <summary>
        /// Converts the specified delegate path to the path exposed by this filesystem.
        /// </summary>
        /// <param name="path">The path used by the underlying <see cref="Fallback"/></param>
        /// <returns>A new path translated to this filesystem</returns>
        protected abstract UPath ConvertPathFromDelegate(UPath path);
    }
}

redpajama_set_name: RedPajamaGithub


Lie on the floor on your back with your knees bent. Bring the soles of your feet together, so that your knees angle out.
Perform the crunch: slowly raise your head and shoulders in a forward-curling motion off the floor.
Hold for two seconds and lower yourself back to the starting position.


text:
Food Justice Film Series at O Cinema
Mandy Baca
Mandy Baca | September 13, 2011 | 1:29pm
O Cinema in Wynwood has been a hit since its opening in February. It even rocked the food world earlier in the summer with two dinner-and-a-movie specials with local favorite Michael's Genuine Food & Drink.
The cinema is about to rock the socks off the food world again with the premiere of a food-conscious film series à la Slow Food on Film -- a first around town. All films will focus on the importance of food justice and responsible eating.
Hosted by Youth L.E.A.D. and sponsored by Slow Food Miami and Raw One, the three-month series will kicks off this Thursday with the acclaimed film Forks Over Knives. Created by Erin Healy, Youth L.E.A.D. aims to raise awareness of the city's main food issues and provide everyone with access to healthful food.
Here's the full schedule:
Thursday, September 14, at 7 p.m.
Tasting and wine reception sponsored by Raw One, 6 to 7 p.m.
Saturday, October 22, at 1: p.m.
What's on Your Plate?
The feature coincides with National Food Day, a national convergence for food justice. Like TEDxManhattan's Changing the Way We Eat, various cities throughout the country will simultaneously work toward better food. To celebrate, Urban Oasis Projects' Farmer's Market and local food truck Mac N Cheese will be on-site.
Tuesday, November 8, at 7 p.m.
Wine reception, 6 to 7 p.m.
Tickets are still available. For more information, visit o-cinema.org.