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DNA DYNAMICS AND CHROMOSOME STRUCTURE
The Absence of the DNA-Dependent Protein Kinase Catalytic Subunit in Mice Results in Anaphase Bridges and in Increased Telomeric Fusions with Normal Telomere Length and G-Strand Overhang
Fermı́n A. Goytisolo, Enrique Samper, Scott Edmonson, Guillermo E. Taccioli, Marı́a A. Blasco
Fermı́n A. Goytisolo
Department of Immunology and Oncology, Centro Nacional de Biotecnologı́a, Madrid E-28049, Spain, 1 and
Enrique Samper
Scott Edmonson
Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118-2526 2
Guillermo E. Taccioli
Marı́a A. Blasco
DOI: 10.1128/MCB.21.11.3642-3651.2001
The major pathway in mammalian cells for repairing DNA double-strand breaks (DSB) is via nonhomologous end joining. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK). Mammalian Ku proteins bind to DSB and recruit DNA-PKcs to the break. Interestingly, besides their role in DSB repair, Ku proteins bind to chromosome ends, or telomeres, protecting them from end-to-end fusions. Here we show that DNA-PKcs−/− cells display an increased frequency of spontaneous telomeric fusions and anaphase bridges. However, DNA-PKcs deficiency does not result in significant changes in telomere length or in deregulation of the G-strand overhang at the telomeres. Although less severe, this phenotype is reminiscent of the one recently described for Ku86-defective cells. Here we show that, besides DNA repair, a role for DNA-PKcs is to protect telomeres, which in turn are essential for chromosomal stability.
One of the most lethal lesions that can occur in a cell after ionizing irradiation is a double-strand break (DSB), because it disrupts the integrity of the DNA molecule. The importance of this lesion is evident by the existence of evolutionarily conserved DNA repair systems that act on DSBs. Moreover, DSBs are also generated under physiological conditions, such as during transposition, meiosis, and recombination. It is important that these endogenous breaks be resolved in order for cells to function properly. Cells have evolved two fundamentally different pathways for repairing DSBs: homologous recombination (HR), which requires extensive regions of homology, and DNA nonhomologous end joining (NHEJ). In contrast to what has been described for Saccharomyces cerevisiae, the major pathway in mammalian cells is NHEJ. Five components function in this pathway, of which three (Ku70, Ku80, and the DNA-dependent protein kinase catalytic subunit [DNA-PKcs]) constitute a complex termed DNA-dependent protein kinase (DNA-PK) (48). Xrcc4 and DNA ligase IV are the two additional proteins known to function in the NHEJ pathway (19, 22, 37). The essential role of the DNA-PK complex in NHEJ has been well documented in mice generated by HR and carrying null mutations for any of the three subunits. The phenotypes shared by these animals are defective DSB repair (dsbr), and thus hypersensitivity to ionizing radiation, and an impaired V(D)J recombination process (18, 25, 43, 44, 49, 52). However, these animals present differential phenotypes, which provides unequivocal evidence for additional roles for each component of the DNA-PK complex separate from their function in NHEJ. In this context, Ku-deficient mice grow poorly and senesce early (25, 36,43, 51), a differential feature not observed in DNA-PKcs-deficient mice (18, 49).
Telomeres are unique structures present at the end of eukaryotic chromosomes and are composed of tandem DNA repeats and specific proteins, conferring properties that keep the ends from being detected as DSBs by the cell (7). The fundamental difference between telomeres and DSBs is that telomeres are protected from end-to-end fusions, recombination, and unregulated nucleolytic degradation, whereas DSBs are subjected to such processing events in order to promote repair of the break. In fact, when normal telomere function is affected, either by loss of telomeric sequences or by mutation of a protective telomere binding protein, i.e., TRF2, end-to-end fusions occur (9, 14, 50). Telomeric sequences are lost during in vitro culture of primary cells and with increasing age in some adult tissues. Besides, an impairment of telomere function due to a loss of telomeric sequences has been shown to limit the proliferative capacity of cultured cells and to affect the life span of the organism (4, 10).
Studies of yeast, where the major DNA repair pathway is HR instead of NHEJ, show that Ku has an important role at the telomere. In particular, yeast defective in either Ku subunit show a 60% loss of telomeric repeats, loss of telomere clustering, loss of telomeric silencing, and deregulation of the G-strand overhang (12, 13, 21,35, 42). Furthermore, yeast Ku moves from the telomeres to the DSB upon induction of damage, suggesting a link between DNA repair and telomeres (38, 40). In mammals, Ku proteins have been reported to bind to telomeric sequences (6, 31) and to prevent end-to-end fusions (5, 32, 47); but in contrast to what has been reported for yeast, Ku86 deficiency in mice does not result in telomere shortening or in deregulation of the G-strand overhang (47). It is likely that Ku protects mammalian telomeres from fusions through its interaction with another telomeric protein, TRF1 (32). DNA-PKcs is a member of the phosphatidylinositol 3-kinase superfamily (27, 45), which includes, among others, the yeast Tel1 protein and human ATM, which have been implicated in telomere metabolism (23, 39, 41). Preliminary evidence suggesting a role for DNA-PKcs at the telomeres came from studies conducted with severe combined immunodeficient (SCID) mice carrying a leaky mutation in the DNA-PKcs locus. These studies showed that SCID mouse telomeres were elongated compared to those of wild-type mice and that SCID mouse cells had an increased frequency of end-to-end fusions (2, 5,11, 15, 26, 33). In addition, the Mre11-Rad50-NsbI dsbr complex is also present at the mammalian telomere and interacts with telomeric protein TRF2 (53). Collectively, the recruitment of DSB DNA repair proteins to the telomeres suggests that chromosome ends are binding sites for dsbr proteins in mammals.
In the present study, the use of mice with a null mutation in the DNA-PKcs gene (49) allowed us to demonstrate that the absence of DNA-PKcs in mammals results in an increased frequency of telomeric fusions and anaphase bridges, suggesting a protective role of DNA-PKcs at the telomere independent to that reported for Ku proteins (32, 47). Interestingly, DNA-PKcs deficiency, similar to Ku86 deficiency, does not result in an alteration of telomere length or the integrity of the G-strand overhang. In this context, the contrasting results found for SCID mice that show elongated telomeres compared to those of wild-type controls (26) (see below) might serve as a means to discover important differences between these two mouse models.
Mice and cells.DNA-PKcs null mice were described elsewhere (49). Wild-type and DNA-PKcs−/−mice or cells were derived from heterozygous crosses and, for all work, littermate mice or cells were used. SCID mice used were BALB/cJHanHsd-Scid (Harlam, Barcelona, Spain), and the corresponding wild-type mice in the same genetic background were BALB/cOlaHsd (Harlam). Mice used for quantitative fluorescent in situ hybridization (Q-FISH) and flow cytometry FISH (Flow-FISH) studies were between 8 and 12 weeks old. Mouse embryonic fibroblasts (MEFs) were prepared from day 13.5 embryos derived from heterozygous crosses as described previously (9). First-passage MEFs used in the different experiments corresponded to approximately two population doublings (PDL 2). Mice or cells from the same litter are indicated with the same letter.
Scoring of chromosomal abnormalities.Between 80 and 100 (each) wild-type, DNA-PKcs+/−, and DNA-PKcs−/− metaphases were scored for telomere fusions, chromatid breaks, and chromosome fragments by superimposing the telomere image on the DAPI (4′,6′-diamidino-2-phenylindole) chromosome image in the TFL-telo program. The following criteria were applied: telomeric fusions, chromosomes fused by their telomeres showing at least two overlapping telomeric signals; Robertson-like fusions, chromosomes fused by their p arms (they may or may not show telomeres at the fusion point; all the Robertson-like fusions found in this study showed telomeres at the fusion point and were included in the group of telomeric fusions); telomere associations, chromosomes with four distinct telomere signals but aligned less than one-half chromatid apart; breaks, gaps in a chromatid whose corresponding chromosome was identified; chromosome fragments, chromosome pieces (with two telomeres or less) whose corresponding chromosome was not easily identified.
To score for anaphase bridges, primary MEF cultures were seeded on microscope slides and stained with DAPI to visualize the DNA. At least 50 anaphases were scored for wild-type and DNA-PKcs−/− cultures, and the anaphase bridges were counted.
Statistical analysis.Statistical calculation was done using Microsoft Excel. For statistical significance, Student's ttest values were calculated.
Telomere length analysis. (i) Q-FISH.First-passage MEFs were prepared for Q-FISH as described previously (29). Q-FISH was carried out as described previously (29, 34,47). To correct for lamp intensity and alignment, images from fluorescent beads (Molecular Probes) were analyzed using the TFL-Telo program. Telomere fluorescence values were extrapolated from the telomere fluorescence of LY-R and LY-S lymphoma cell lines (1) of known lengths of 80 and 10 kb (E. Samper et al., unpublished results). There was a linear correlation (r 2 = 0.999) between the fluorescence intensities of the LY-R and -S telomeres with a slope of 38.6. The calibration-corrected telomere fluorescence intensity was calculated as described previously (29).
Images were recorded using a COHU charge-coupled device camera on a Leica Leitz DMRB fluorescence microscope. A Philips CS 100W-2 mercury vapor lamp was used as the source. Images were captured using Leica Q-FISH software at a 400-ms integration time in a linear-acquisition mode to prevent oversaturation of fluorescence intensity.
TFL-Telo software (gift from P. M. Lansdorp, Vancouver, British Columbia, Canada) was used to quantify the fluorescence intensity of telomeres from at least 15 metaphases or fusions of each data point. The images from littermate wild-type and DNA-PKcs−/− metaphases were captured on the same day, in parallel, and blindly. All the images from the MEFs were captured in a 3-day period after the hybridization.
(ii) Flow-FISH.Fresh bone marrow (BM) cells, splenocytes, and primary MEFs from littermate wild-type, DNA-PKcs+/−, and DNA-PKcs−/− mice, as well as splenocytes and BM cells from SCID and control wild-type animals, were prepared as described previously (9, 29, 30). Flow-FISH was performed as described previously(46). To normalize Flow-FISH data two mouse leukemia cell lines (LY-R and LY-S; described above) were used as internal controls in each experiment. The telomere fluorescence of at least 2,000 cells gated at the G1-G0 cell cycle stage was measured using a Coulter flow EPICS XL cytometer with SYSTEM 2 software.
(iii) TRF analysis.Fresh BM cells from wild-type, DNA-PKcs+/−, and DNA-PKcs−/− littermate mice, as well as from SCID mice and their corresponding wild types, were isolated as described above, and telomere restriction fragment (TRF) analysis was done as described by Blasco et al. (9).
G-strand overhang assay.The G-strand assay was performed as described previously (28) with minor modifications. Fresh BM cells and MEFs (106) from several pairs of wild-type mice and DNA-PKcs−/− littermates were included in restriction analysis grade agarose plugs in accordance with instructions provided by the manufacturer (Bio-Rad). After overnight digestion in LDS buffer (1% lithium dodecyl sulfate, 100 mM EDTA [pH 8.0], 10 mM Tris [pH 8.0]), the plugs were digested with either 0, 40, or 100 U of mung bean nuclease (MBN) for 15 min. Then the plugs were digested with MboI overnight and subjected to pulsed-field gel electrophoresis as described previously (9). The sequential in-gel hybridizations in native and denaturing conditions to visualize G-strand overhangs and telomeres, respectively, were carried out as described before (28). Quantification of the G-strand overhang radioactive signals was carried out using a STORM 860 PhosphorImager (Molecular Dynamics), using the software provided by the manufacturer. These values were corrected by the TRF signal in denaturing gel conditions.
Telomerase assay.S-100 extracts were prepared from wild-type, DNA-PKcs+/−, and DNA-PKcs−/− primary MEF cultures, and a modified version of the TRAP assay was used to measure telomerase activity (8). An internal control for PCR efficiency was included (TRAPeze kit; Oncor).
DNA-PKcs is necessary to prevent telomere fusions and the formation of anaphase bridges during mitosis.Telomeres protect chromosome ends from degradation, end-to-end fusions, and recombination activities. To study the impact of DNA-PKcs deficiency on telomere function, we analyzed the involvement of telomeres in the chromosomal aberrations spontaneously arising in primary (passage 1) wild-type and DNA-PKcs−/− MEFs derived from heterozygous crosses. For this, we performed Q-FISH of metaphasic nuclei with a fluorescent telomeric peptide nucleic acid probe (47, 54) (see Materials and Methods for a description of different aberrations) and then scored spontaneously arising chromosome aberrations for 80 to 100 metaphases of each primary MEF culture. As displayed in Table 1, no statistically significant differences between the frequency of chromosome breaks and fragments detected in MEFs isolated from DNA-PKcs−/− cultures and that detected in wild-type controls were found (0.026 and 0.032 breaks plus fragments per metaphase, respectively; Student's t test,P = 0.67475). Similarly, no significant difference in the frequency of telomeric associations (chromosomes with four distinct telomere signals that are aligned less than one-half chromatid apart; Fig. 1A) between genotypes was found (0.015 and 0.012 telomeric associations per metaphase for DNA-PKcs−/− and wild-type MEFs, respectively) (Table 1). Interestingly, DNA-PKcs−/− MEFs showed an elevated frequency of telomeric fusions (two chromosomes which are fused by at least one telomere; Fig. 1A) compared to wild-type controls; the average frequencies of fusions per metaphase were 0.065 and 0.017 for DNA-PKcs−/− and wild-type MEFs, respectively (Fig. 1; Table 1). All Robersonian-like fusions found (chromosomes fused by their p arms) showed telomeres at the fusion point and were included in the group of telomeric fusions. It is worth noting that the difference in telomeric fusions between wild-type and DNA-PKcs−/− cells is highly significant (Student's t test, P = 0.00037174). The frequency of telomeric fusions found in the DNA-PKcs−/− primary MEFs was lower than that previously reported for SCID mouse cells (0.165 telomeric fusions per metaphase) which carry a leaky mutation in the DNA-PKcs gene (5). We cannot rule out the possibility that the higher chromosomal instability described for SCID cells is due to the higher passage number of the SCID MEFs used by Bailey et al. (5) compared with the passage 1 DNA-PKcs−/− MEFs used in this study.
Q-FISH and cytogenetic analysis of wild-type and DNA-PKcs−/− MEFs
Chromosomal instability in wild-type (WT) and DNA-PKcs−/− MEFs. (A) Cytogenetic alterations detected in DNA-PKcs−/− metaphases from primary MEFs after hybridization with DAPI and a fluorescent Cy-3-labeled telomeric peptide nucleic acid probe. For quantifications see Table 1. Blue, chromosome DNA stained with DAPI; yellow and white dots, TTAGGG repeats; yellow arrows, chromosomal abnormalities. For definition of the different aberrations see Materials and Methods. (B) Representative images of anaphase bridges in DNA-PKcs−/− cells. Blue, chromosome DNA stained with DAPI. Notice the presence of DNA bridges in DNA-PKcs-deficient cells even in late-anaphase and telophase stages.
Importantly, all chromosomal abnormalities present in DNA-PKcs−/− cells were also detected in primary Ku86−/− MEFs but at a significantly higher frequency (47), suggesting additional roles for the Ku86 protein at the telomeres independent of the activity of the DNA-PK complex.
It is important to note that Q-FISH analysis allowed us to determine that all telomeric fusions present in DNA-PKcs−/− cells contained intact telomeres at the fusion point with an average length of 64.4 ± 9.39 kb, suggesting that these fusions did not originate from a loss of telomeric sequences. A similar phenotype has been previously described for Ku86 deficiency (47).
Finally, loss of telomere function has been proposed to trigger breakage-fusion-bridge cycles (3, 16) through the formation of telomeric fusions and the occurrence of anaphase bridges during mitosis. Both types of chromosomal aberrations occur in tumors and are thought to be important for their clonal evolution and progression (20). In agreement with a role for DNA-PKcs in telomere function and chromosomal stability, we found that DNA-PKcs−/− MEFs showed a significantly higher frequency of anaphase bridges than control cells (0.16 and 0.70 anaphase bridges per anaphase scored for wild-type and DNA-PKcs−/− MEFs, respectively; 50 anaphases of each cell type were scored) (Fig. 1B shows examples). This difference is highly significant, as indicated by the Student t test value (P = 0.0014). Some anaphase bridges were still present at telophase in the DNA-PKcs−/− cells (Fig. 1B shows examples). Altogether, these results strongly suggest a role for DNA-PKcs in protecting chromosome ends and thus a role in genomic stability.
Normal length of TTAGGG repeats in DNA-PKcs-deficient cells.To determine if the protective role of DNA-PKcs at the telomere is mediated by the length of TTAGGG repeats, quantification of telomere length was carried out for littermate wild-type and DNA-PKcs−/− mice or embryos derived from heterozygous crosses. It is essential to compare littermate mice since mouse telomeres show individual variability (54). Q-FISH analysis of MEFs from wild-type and DNA-PKcs−/− littermate mice revealed that DNA-PKcs−/− cells had a telomere length similar to that of the wild type (Table 1). The average telomere lengths were 33.95 ± 0.2 and 35.0 ± 0.2 kb for MEFs from DNA-PKcs−/− (average of B6, C2, and C9) and wild-type (average of B5, C5, and C3) littermate mice, respectively (Fig. 2). The Q-FISH data on MEFs were confirmed by using a different technique to measure telomere fluorescence based on flow cytometry (Flow-FISH; described in Materials and Methods) (Table 2). In this case, average telomere fluorescence levels expressed in arbitrary units were 2.58 ± 0.1, 2.72 ± 0.2, and 2.71 ± 0.1 for DNA-PKcs−/− (average of A1, B4, B6, C2, and C9), DNA-PKcs−/+ (average of A2 and B3), and wild-type (average of B5, C3, and C5) MEFs, respectively. Histograms showing the frequency of a given telomere fluorescence in MEFs from littermate wild-type (B5, C5, and C3) and DNA-PKcs−/− (B6, C2, and C9) mice are presented in Fig. 2. These histograms confirmed that the mean telomere fluorescence levels in DNA-PKcs−/− and wild-type MEFs are similar and furthermore showed that the levels of heterogeneity of telomeric lengths in both genotypes are similar (Fig.2).
Telomere fluorescence distribution in wild-type (wt), DNA-PKcs+/−, and DNA-PKcs−/− MEFs. Shown is the telomere length distribution of at least 7,100 telomeres in primary MEFs from three different littermate wt (B5, C5, and C3) and DNA-PKcs−/− (B6, C2, and C9) mice. The histograms depict similar telomeres in DNA-PKcs−/− and wt cells. One telomere fluorescence unit corresponds to 1 kb of TTAGGG repeats (see Tables 1 and 2 for telomere length values).
Measurement by Flow-FISH of telomere length in MEFs obtained from DNA-PKcs+/− heterozygous crosses
Flow-FISH studies on fresh splenocytes and BM cells derived from wild-type, DNA-PKcs+/−, and DNA-PKcs−/− littermate mice (8 to 12 weeks old) also indicated that DNA-PKcs−/− telomeres were similar in length to those of wild-type littermates (Table3), in agreement with the Q-FISH and Flow-FISH data for MEFs. These results were reproducible in at least six independent litters (Table 3).
Measurement by Flow-FISH of telomere length in splenocytes and BM cells obtained from DNA-PKcs+/+, DNA-PKcs+/−, and DNA-PKcs−/− mice
Finally, telomere length was also evaluated by Southern blotting, as an alternative technique to measure telomere length not based on fluorescence. Primary BM cells from three wild-type, three DNA-PKcs+/−, and four DNA-PKcs−/− mice were subjected to TRF analysis as described in Materials and Methods. As shown in Fig.3, TRF analysis also showed similar telomere lengths in littermate mouse wild-type and DNA-PKcs−/− cells.
TRF analysis of wild-type, DNA-PKcs+/−, and DNA-PKcs−/− primary MEFs. Wild-type (+/+; DA45, DA46, and DA60), DNA-PKcs+/− (+/−; DA50, DA51, and DA59), and DNA-PKcs−/− (−/−; DA47, DA48, DA52, and DA58) littermate mice were studied. Notice that TRF signals are similar in DNA-PKcs−/−, DNA-PKcs+/−, and wild-type BM cells (see also Table 3 and Fig. 5 for other analyses of the same BM cells). Three litters were analyzed.
Altogether, these data demonstrate that a DNA-PKcs−/− deficiency in mammals does not result in significant telomere length alterations.
A spontaneous mouse mutation that also affects the DNA-PKcs locus but that produces a leaky phenotype is the mutation resulting in SCID mice (2, 11, 15, 33). Curiously, a previous report showed elongated telomeres in SCID mice compared to those in wild-type controls (26), which is in contrast to the normal-telomere-length phenotype displayed by DNA-PKcs−/− animals in this study. To rule out the possibility that possible technical differences between laboratories were the reason for this discrepancy, we measured telomere length for wild-type and SCID mice using the same methods used to measure telomere length in DNA-PKcs-deficient cells. First, we performed Flow-FISH of fresh splenocytes and BM cells isolated from five SCID and five wild-type mice in the same genetic background (see Materials and Methods). In agreement with results previously reported by Hande et al. (26), we found that SCID mouse telomeres were elongated compared with those of wild-type mice (Table4; Fig.4A). Average levels of telomere fluorescence for freshly isolated splenocytes from five different wild-type and SCID mice were 3.13 ± 0.33 and 5.27 ± 0.53, respectively. This difference was highly significant (Student'st test, P = 1.2 × 10−5). Similarly, average levels of telomere fluorescence for fresh BM cells from five different wild-type and SCID mice were 3.24 ± 0.72 and 4.64 ± 1.24, respectively. This difference was also significant (Student's t test,P = 0.0170). Furthermore, TRF analysis of five different wild-type and SCID mice also showed dramatically elongated telomeres in the SCID mice compared with the wild types (Fig. 4B). The elongated-telomere phenotype of SCID mice could also explain the difference in the frequency of telomeric fusions between SCID and DNA-PKcs−/− mice (see above). These results indicate fundamental differences between these two mouse models, at least in terms of telomere function. These differences can be explained if SCID mice carry other mutations besides the DNA-PKcs leaky mutation. Alternatively, the fact that SCID mice are not completely kinase null for the DNA-PKcs gene, and indeed express a different truncated form of the DNA-PKcs gene, could differentially affect telomere length and/or function.
Measurement by Flow-FISH of telomere length in splenocytes and BM cells obtained from SCID and wild-type mice
Telomere length analysis of fresh BM cells and splenocytes from SCID and wild-type (wt) mice. (A) Flow-FISH. Telomere fluorescence (in arbitrary units [a.u.]) of splenocytes and BM cells as measured by flow cytometry for five different SCID and wt mice is shown. The average telomere lengths and standard deviations are indicated. The statistical significance of the difference in telomere fluorescence between SCID and wt mice is indicated by the Student'st test numbers (P). (B) TRF analysis. BM from wt and SCID mice in the same genetic background (see Materials and Methods) was used for TRF analysis. Notice that TRFs were dramatically elongated in SCID mice compared with wt controls.
Normal-length telomeric G-strand overhangs in DNA-PKcs−/− cells.G-strand overhangs are regions of G-rich single-stranded telomeric DNA that protrude in the 3′direction from the double-stranded telomere. These single-stranded G-rich regions have been recently involved in the formation of a special structure at the chromosome end named the T loop, which has been proposed to protect the ends from recombination and DNA repair activities (24). Hence, examination of telomeric G-strand overhangs in DNA-PKcs null cells was crucial to gain more insight into the abnormal frequency of telomeric fusions and anaphase bridges detected in these cells. To study the telomeric G-strand overhangs, we carried out TRF analysis with a (CCCTAA)4probe as described previously (28) using nondenaturing pulsed-field agarose gels (see Materials and Methods). Detection of a signal with the (CCCTAA)4 probe hybridized to native DNA samples indicates the presence of the G-strand overhang. Freshly isolated BM cells, as well as primary MEFs from different littermate wild-type, DNA-PKcs+/−, and DNA-PKcs−/− mice showed G-strand-specific signals that were similar in size and intensity in all genotypes (Fig.5). Table5 shows the quantification of the G-strand signals; the wild-type values were normalized to 100 in each litter. The average G-strand signal for DNA-PKcs−/− MEFs or BM cells was 81.8 ± 21.7% that for the wild types; this difference is not statistically significant (Student's t test, P = 0.135). To show that the probe specifically recognized the single-stranded telomeric tail, treatment with MBN, which specifically degrades single-stranded DNA and RNA overhangs, was performed. As expected, the G-strand signal decreased in all genotypes upon treatment, as shown in Fig. 5 ("native gel"). As a control, the same gel was denatured and rehybridized with the (CCCTAA)4 probe, which highlighted the TRFs (Fig. 5; "denaturing gel"), again showing no difference in TRF lengths between wild-type, DNA-PKcs+/−, and DNA-PKcs−/− phenotypes.
Normal G-strand overhang in Ku86−/−-deficient primary cells. G-strand overhangs in fresh BM cells or primary MEFs, as indicated, from littermate wild-type and DNA-PKcs−/− mice were visualized in native gel after hybridization with a (CCCTAA)4 probe (see Materials and Methods). Notice that upon treatment with two different doses of MBN (40 and 100 U) the G-strand-specific signal decreases. As a control, the same gel was denatured and reprobed with the (CCCTAA)4 probe to visualize telomeres. DA40 and DA39 are littermate wild-type and DNA-PKcs−/− mice, respectively. B3, B4, B5, and B6 are primary MEF cultures from littermates, as are C2, C3, C5, and C9. See Table 5 for quantification of G-strand-specific signals. HMW, high molecular weight.
Quantification of G-strand overhang signals
Collectively, these results show that DNA-PKcs deficiency in mammals does not result in the loss or shortening of the G-strand overhang at the telomeres. A similar result has been reported for the Ku86 deficiency, another member of the NHEJ pathway and component of the DNA-PK complex (47).
DNA-PKcs activity does not regulate telomerase activity.To investigate if the protein kinase activity of the DNA-PKcs subunit could have a regulatory role in telomerase activity (i.e., by phosphorylating the catalytic subunit of telomerase Tert), we quantified telomerase activity in three wild-type, one DNA-PKcs+/−, and four DNA-PKcs−/− primary MEF cultures (passage 1) (see Materials and Methods). No significant difference in telomerase activity between wild-type and DNA-PKcs−/−littermates was detected (Fig. 6). This result indicates that lack of DNA-PK activity does not impact telomerase activity in mice and in turn is in agreement with the normal telomere length detected in DNA-PKcs-deficient cells.
Telomerase activity in wild-type and DNA-PKcs−/− MEFs. S-100 extracts were prepared from wild-type (C3, C5, and B5), DNA-PKcs+/− (B3), and DNA-PKcs−/− (C2, C9, B4, and B6) MEFs and assayed for telomerase activity. Extracts were pretreated (+) or not (−) with RNase. The protein concentrations used are indicated. Arrow, internal control (IC) for PCR efficiency. The same letter indicates littermate embryos.
The essential role of the DNA-PK complex in dsbr and V(D)J recombination in vertebrates is universally recognized. Biochemical analyses established that DNA-PK is a Ser/Thr kinase complex that must be bound to DNA in order to be activated. DNA-PK is composed of three subunits, a DNA end-binding component, which is a dimer of Ku70 and Ku86, and a large catalytic subunit of 460 kDa referred to as DNA-PKcs. Here we show that DNA-PKcs deficiency in mice results in increased end-to-end telomeric fusions and in a higher frequency of anaphase bridges in cells that otherwise show normal-length TTAGGG repeats at the telomeres and at the G-strand overhang. These results suggest a protective role for DNA-PKcs at the mammalian telomere that, in addition, is independent from the one conferred by telomeric repeats per se or by the length of the G-strand overhang. Telomeric fusions and anaphase bridges lead to breakage-fusion-bridge events, which in turn are important features found in human cancers (20).
An increase in the frequency of telomeric fusions has been recently reported for Ku86-deficient mice (47), suggesting that both Ku and DNA-PKcs have a role at the telomere. However, in contrast to DNA-PKcs-deficient cells, Ku86−/−cells show a slightly elongated telomere phenotype (47). These results suggest an additional role for the Ku86 protein at the telomere independent of that for the DNA-PK catalytic activity per se. This differential outcome between DNA-PKcs and Ku86 is not surprising if we consider that a mutation in Ku86 not only inactivates Ku86 per se but also impairs DNA-PK activity. This is also in agreement with the complex phenotype observed in Ku86−/− mice compared to DNA-PKcs−/− animals (18, 25,43, 49, 51). Although Ku86 and DNA-PKcs gene products belong to the same enzymatic complex, several studies have demonstrated the differential role of these proteins in DNA repair and V(D)J recombination (18, 25, 43, 44, 49).
Our finding of increased telomeric fusions in DNA-PKcs null cells has been confirmed in recent experiments using SCID mice carrying a spontaneous rodent DNA-PKcs gene mutation (5). In contrast to what we found for DNA-PKcs deficiency, however, Hande et al. (26) reported an elongated-telomere phenotype in the SCID mice compared to wild-type controls. Using Flow-FISH and TRF techniques, we have been able to confirm that SCID but not DNA-PKcs−/− mice show elongated telomeres compared with the corresponding wild-type controls. These results indicate fundamental differences between DNA-PKcs−/− and SCID mice, at least in terms of telomere function. These differences could be attributed (i) to the occurrence of still-to-be-defined additional mutations in the SCID strain which may affect telomere length, (ii) to the fact that SCID does not represent a null mutation for the DNA-PKcs gene and to potential residual kinase activity, and (iii) to the different types of truncation in the DNA-PKcs gene product in SCID and DNA-PKcs−/− cells that might impact the recruitment of other components necessary for telomere length regulation (2, 11, 15).
Collectively, these results indicate that the DNA-PK complex has a role in protecting telomeres from fusions. Interestingly, the DNA-PK complex is involved in dsbr in mammals by NHEJ; however, in combination with telomeric proteins, it might be involved in masking chromosome ends to prevent them from being recognized as DSBs. In fact, the telomere fusions detected in DNA-PKcs-deficient cells might represent NHEJ events. In this regard, mutations in DNA repair proteins do not abolish the capability of the cells to carry out NHEJ on broken ends (16,36).
The end-to-end fusion phenotype has been previously recreated in cells that lack telomerase activity and that undergo progressive telomere shortening with increasing cell divisions (9). Likewise, cells expressing a dominant-negative mutation of the TRF2 gene had an end-to-end fusion phenotype with normal telomere length but with loss of G-strand overhang (24, 50). Since the protective functions of DNA-PKcs and Ku86 at the telomere are independent of the length of TTAGGG repeats and of the integrity of the telomeric G-strand overhang, this suggests that the DNA-PK complex acts at the telomere in a fundamentally different way than telomerase (8) or TRF2 (24, 50). However, it is still possible that DNA-PKcs and Ku act similarly to TRF2 but that their functions are more redundant, giving rise to a less severe phenotype.
There are a number of ways, not necessarily mutually exclusive, that DNA-PK might function in telomere protection. One is that the protein kinase activity of DNA-PKcs is employed to regulate the binding activities of other telomeric components. Alternatively, in light of the large size of DNA-PKcs, it is tempting to speculate that it might have a role as a scaffolding protein, recruiting several other factors to the telomere. These results warrant future studies involving the evaluation of a putative synergistic effect between DNA-PK components and telomere components as means to shed more light on the biology of telomeres.
We thank R. Serrano and E. Santos for mouse care and genotyping, respectively.
E.S. and F.G. were supported by the Government of Madrid (CAM). G.E.T. is a scholar of the Leukemia and Lymphoma Society. The G.E.T. laboratory was supported by National Institutes of Health CA76409, American Cancer Society IN97T, and the Aids for Cancer Research Foundation. The M.A.B. laboratory was funded by the SWISS BRIDGE award, 2000, by the Ministry of Science and Technology (PM97-0133), Spain, by CAM 08.1/0030/98, by the European Union (EURATOM/991/0201, FIGH-CT-1999-00002, FIS5-1999-00055), and by the Department of Immunology and Oncology (DIO). The DIO is funded by the Spanish Council for Scientific Research and by Pharmacia Corporation.
F. A. Goytisolo and E. Samper contributed equally to this work.
Received 2 November 2000.
Returned for modification 11 December 2000.
Accepted 23 February 2001.
Copyright © 2001 American Society for Microbiology
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Molecular and Cellular Biology Jun 2001, 21 (11) 3642-3651; DOI: 10.1128/MCB.21.11.3642-3651.2001
You are going to email the following The Absence of the DNA-Dependent Protein Kinase Catalytic Subunit in Mice Results in Anaphase Bridges and in Increased Telomeric Fusions with Normal Telomere Length and G-Strand Overhang


text:
Ḩeşār kan syfta på följande platser:

Iran

 Ḩeşār (ort i Östazarbaijan),
 Ḩeşār-e Shālpūsh, ort, Teheran,
 Ḩeşār Qareh Bāghī, ort, Hamadan,
 Ḩeşār-e Gowjeh Bāghī, ort, Hamadan,
 Ḩeşār (ort i Nordkhorasan, lat 37,15, long 57,30),
 Ḩeşār (ort i Nordkhorasan, lat 37,52, long 57,48),
 Ḩeşār (ort i Nordkhorasan, lat 37,30, long 58,03),
 Ḩeşār Āndaf, ort, Nordkhorasan,
 Ḩeşārī-ye Gāzerānī, ort, Nordkhorasan,

Robotskapade Iranförgreningar

redpajama_set_name: RedPajamaWikipedia


This picture doesn't do it justice, but a lot of our drives look something like this.
The feed bunk in one of our heifer lots is so muddy that the animals can barely make it to the bunk anymore, so we borrowed a feed wagon from a neighbor to feed them else where in the pasture. I stole this picture from Theo as he leads them to the new feed location with a grain bucket.
Sold our old choppers, here is one of them on a truck getting hauled to Indiana. New one is supposed to be here in the next week or so.
Dave also upgraded his sprayer a little bit this winter. Should be able to cover some acres with this thing! Also going to use it to split our nitrogen application on our corn.
Here's my dog Sophie riding with me in the tractor on the way to pull the feed mixer tractor through the mud at the heifer lot mentioned above. She isn't a big fan of jumping up into the noisy tractor, but doesn't like being left behind! One in, she usually just sits on the buddy seat next to me.
Hopefully soon I can post about getting into the fields, but as I type this, it is snowing again… Bah humbug.
This entry was posted in Agronomy, Animals, General Information, Machinery and tagged agco, agriculture, agronomy, Animals, baby animals, corn, cows, dairy cows, dairy farm, dairy farming, dairy farming today, factory farming, family farm, family farming, farm, farm animals, farmer, farmers, farming, food production, loveable mutt, Modern Agricuture, modern farming, organic farming, pets, production agriculture, sustainable agriculture, sustainable agricuture, tractors, Wisconsin dairy farming. Bookmark the permalink.


text:
Q: dual stack IPv6 in6addr_loopback vs in6addr_any I have a dual stack set on my Linux host and trying to connect to a TCP/IPv6 server that can listen to in6addr_loopback or in6addr_any, here is a very minimal example of the server :
#include <stdio.h>
#include <sys/types.h>
#include <sys/socket.h>
#include <netinet/in.h>
#include <string.h>
#include <stdlib.h>
#include <arpa/inet.h>
#include <unistd.h>

int main(int argc, char *argv[])
{
    if (argc != 2)
        exit(1);

    struct sockaddr_in6 serv6;
    memset(&serv6, 0, sizeof(struct sockaddr_in6));

    int fd6 = socket(AF_INET6, SOCK_STREAM, 0);

    int loopback = atoi(argv[1]);

    if (loopback)
        serv6.sin6_addr = in6addr_loopback;
    else
        serv6.sin6_addr= in6addr_any;

    serv6.sin6_port = htons(3000);
    serv6.sin6_family = AF_INET6;

    int opt = 1;
    setsockopt(fd6, SOL_SOCKET, SO_REUSEADDR, &opt, sizeof(int));

    if (bind(fd6, (struct sockaddr *)&serv6, sizeof(struct sockaddr_in6)) < 0) {

        perror("bind");
        exit(1);
    }

    listen(fd6, 5);

    int cli = accept(fd6, NULL, NULL);

    char buf[10] = {0};
    int n = read(cli, buf, 9);

    if (n > 0)
        fprintf(stderr, "received %d bytes : %s from ipv6 stack\n", n, buf);
    else
        perror("read");

    exit(0);
}

I am using ncat on the same host to connect to the server in the following way :
server is listening to in6addr_any :
ncat 127.0.0.1 3000 // OK
ncat ::1 3000       // OK
ncat ::ffff:127.0.0.1 3000 // OK

server is listening to in6addr_loopback:
ncat 127.0.0.1 3000 // Ncat: Connection refused.
ncat ::1 3000       // OK
ncat ::ffff:127.0.0.1 3000 // Ncat: Connection refused.

Why does it work only with an IPv6 address for the loopback interface ?
Thank you.

A: To accept both IPv4 and IPv6 clients on a dual-stack listening socket, you should explicitly disable the IPV6_V6ONLY option via setsockopt() before bind()'ing the socket.
But, more importantly, a dual-stack socket bound to in6addr_any (::0) will bind to all local IPv4 and IPv6 interfaces, whereas a dual-stack socket bound to in6addr_loopback (::1) will only be able to bind to an IPv6 loopback interface but not to the IPv4 loopback interface (ie, ::1 != 127.0.0.1).

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Swivel chair for the home and for public facilities.
MAARTEN is available in a wide variety of base and seat finishes to fit with all kind of environments.
Swivel or 4 legs calibrated steel structure powder coated in thermoreinforced polyester in black, white, fluorescent orange and altea blue.
The swivel version is not stackable, but up to 4 units of the 4 leg version can be stacked.


USEMCO above ground petroleum storage tanks are available as horizontal cylindrical,vertical cylindrical or rectangular welded carbon steel. A variety of support saddle, skids and legs are offered. All types are available with secondary containment and multiple compartments. A urethane coating system is our standard, but many others are available.
USEMCO underground petroleum storage tanks are available as horizontal cylinder, welded carbon steel. they are available with secondary containment and multiple compartments. Anchor straps (when required) are provided along with detailed installation instructions. 10-year and 30-year limited warranties available.


text:
(791) Ani est un astéroïde de la ceinture principale découvert par Grigori Néouïmine à Simeiz le .

Il est nommé d'après Ani, la capitale médiévale arménienne du royaume Bagratouni.

Voir aussi

Liens internes
 Liste des principaux astéroïdes classés par taille
 Liste des planètes mineures (1-1000)

Liens externes

Astéroïde de type C
Famille de Mélibée
Astéroïde de la ceinture principale
Planète mineure découverte en 1914
Ani
Objet céleste découvert par Grigori Néouïmine


text:
Q: Excluding "Business View" panoramas when requesting Google Maps API Street Views I'd like to know if anyone knows how to exclude interior views when requesting the nearest street view panorama? I'm noticing more and more interior, "Business", views being returned for locations in well developed areas. I've searched the Javascript API reference for the google.maps.StreetViewPanorama and examined the data returned from the google.maps.StreetViewService class, but I can't see any telltale sign that would allow me to programmatically determine which I'm getting back for a particular location.
I have seen this question: How to know if street view panorama is indoors or outdoors and do not consider it an answer to mine.

A: For those coming from google and looking for an answer to this question the Google Maps Javascript API V3 has a way of requesting only outdoor street views. The getPanorama function takes a source parameter which can be set to outdoor (see this link: https://developers.google.com/maps/documentation/javascript/reference#StreetViewSource)
sv = new google.maps.StreetViewService();
sv.getPanorama({location: fenway, radius: 50, source: google.maps.StreetViewSource.OUTDOOR}, processSVData);


Lisbon, Portugal. Street Art. | The Incidental Tourist.
One of the things that I love to do when travelling to a new city, especially in Europe, is to learn more about the voice of the people from the street art that features in the various neighbourhoods. An expression of present self, of political and social-economic issues, of imagination and fantasy, of beauty and pride of place. Street Art as an artistic expression often gives more, if not equal, insight into the people. This is certainly true of Lisbon Street Art.
Beyond the picture-perfect capital full of medieval infrastructure, impressively preserved 19th-century and contemporary buildings, the many steep hills with their vantage point of the Tagus River, each side street and corner have different treasures to discover. One, possibly unexpected, example is Lisbon's street art and graffiti themed to cover the country's history, pop culture, and imaginative abstracts. I include a collection of my favourites, some as seen at the LxFactory, others under bridges, on tall walls and in intricate detail that caught my eye as I crossed a road. Stickers, stencils, murals and the power of words.
Me dreaming of being a tuk-tuk driver in my next life.
Take a look at my Lisbon, Portugal In Pics, read My First Lisbon Riverview Airbnb Apartment for more info and details of how and where I booked my flights and accommodation, and be inspired to learn more from my Lisbon, Portugal. Street Art post. Can't wait to get back there.


text:
Home » World News » France Distances itself from Trump's Golan Heights Move
France Distances itself from Trump's Golan Heights Move
Geneva (National Times) – French authorities have rebuffed US President Trump as they accuse the US of violating international law in its recognition of Israel's sovereignty over the Golan Heights.
The French deputy ambassador to the UN, Anne Guegen, told UN Security Council on Wednesday evening in New York that "the European Union had affirmed earlier in the day that its Member States did not recognize Israel's sovereignty, and that any efforts to override Council resolutions over the land, were 'doomed to failure'".
US President Trump took the unusual move of recognizing Israel's sovereignty over the Golan heights which Israel seized from Syria in 1967 and annexed in 1981. Most countries and the international community do not recognize Israel's sovereignty over the territory. America will be the first country to recognize Israel's rights over the Golan heights.
The UN Security Council believes Israel's annexation of the Golan Heights was illegal. Several Arab States including US allies in the Middle East have denounced Trump's recognition of Israel annexation of the Golan Heights. Most of these states see the move as inciting instability in the Middle East region.
The UN's head of Political and Peacebuilding Affairs, Rosemary DiCarlo, reiterated the view of the Secretary-General António Guterres, expressed earlier this week, that "the status of the Golan has not changed" in the light of the US decision.
"We hope that the recent developments will not be used as an excuse by anyone to pursue actions that could underimine the relative stability of the situation on Golan and beyond", said Ms. DiCarlo. "We will continue to closely monitor developments on the ground and will keep the Council informed, as appropriate."
With Contribution from UN News
Published in World News
More from World NewsMore posts in World News »
In the US, Justice is not Blind, It Knows the Colour of Your Skin
Simone Leigh: the Woman Using Arts inspired from Agriculture in Northwest Cameroon to Change how we think about Feminism
Militia in southern US state planned to assassinate Obama – FBI
Hundreds Killed as blasts hit Sri Lanka churches, hotels

redpajama_set_name: RedPajamaCommonCrawl


READ ALL ABOUT IT! The Catalyst Schools host Alderman Chandler and Alderman Graham.
One of the goals established by the Board of Directors of the Catalyst Schools is expansion of the Catalyst Network. In 2006 the first school opened in North Lawndale at 1616 South Spaulding. The second school openend in 2007 in Austin at 5608 West Washington Blvd. Both schools have seen steady progress over the years.
The new Catalyst School, to open in the fall of 2012 with grades K - 5 and grade 9, will be a K - 12 seamless system of values based education for families who live on the south side of Chicago.
More than 60 General Electric employees volunteered at the Catalyst Schools -- Howland and Circle Rock -- to help with necessary facilities projects on May 6, 2011. They worked on skylights, tiled floors, painted, cleaned empty lots, and a great many other things during the course of their day at the schools. THANK YOU GE!
The Catalyst Circle Rock Children's Choir joins students who are in Chicago Children's Choir school program for the May 16th Paint the Town Red celebration. This event will be part of the inauguration of Chicago's new Mayor-Elect, Rahm Emanuel. Governor Quinn will declare this day as Arts Education Day for the State. Join us for this exciting experience at the Pritzker Pavillion in Millennium Park. This will be a day that our children will remember for a long time.
The Graduate Support team brought Catalyst-Circle Rock Charter graduates back to the school for a reunion and to encourage them to drive toward success and their ultimate goal which is graduation from college. Graduates enjoyed renewing friendships, a great lunch, and a program that offered both encouragement and guidance. Congratulations to Catalyst graduates who are pursuing their dream.


text:
The Ripley Wind Power Project is a 76 megawatt (MW) wind power project near Ripley, Ontario.

The wind farm includes 38 Enercon E-82 wind turbines, each with two megawatt rated power, 82m rotor diameter and 79m hub height as well as a transmission line and two electrical sub-stations. The wind farm is located on the shores of Lake Huron in Huron-Kinloss Township, approximately 220 kilometres west of Toronto and 140 kilometres north of London.

The project is expected to have the capacity to generate enough zero-emission electricity to power 24,000 homes and offset approximately 66,000 tonnes of carbon dioxide annually. Project cost is estimated at $176 million Canadian dollars.

See also

List of wind farms in Canada
List of offshore wind farms

References

External links
 Canadian Wind Energy Association website
 Wind Energy Programs of Natural Resources Canada
 ENERCON homepage
  System Impact Assessment, Aug 2006
 Independent Electricity System Operator (IESO) website
Power Being Generated in Ontario by Site
"Where is my Electricity Coming From at this Hour? (if I lived in Ontario)" (Canadian Nuclear Society, with data from IESO)

Wind farms in Ontario
Buildings and structures in Bruce County


This one is going the usual way – the Police and DA get "experts" to declare that there was absolutely nothing wrong with the actions of the Police in the Tamir Rice murder. The Family hires actual experts in the specific area of "use of force" who find there are some very seriously wrong things with the actions of the Police. The experts hired by the family think that the officers are guilty, for the same reason I believe Officer Darren Wilson is guilty of killing MIchael Brown.
The reviews stand in direct contrast to three expert reports commissioned and released by Cuyahoga County Prosecutor Timothy J. McGinty, who Tamir's family, activists and religious leaders have repeatedly called to remove himself from the case.
Cleveland attorney Subodh Chandra and the New York law firm of Emery, Celli, Brinckerhoff & Abady have called McGinty's expert reports "utterly biased and deeply flawed." The attorneys represent Tamir's mother in a pending civil lawsuit filed against the city, the two officers involved in the shooting and the Cleveland police department.
At the legal team's request, police procedures consultant Roger Clark and former deputy police chief of the Irvine Police Department Jeffrey J. Noble, both California-based nationally renowned experts in police use-of-force issues, pored over investigative material and determined the shooting was not justified.
Clark and Noble, in a combined 31 pages of documents, reasoned that officers Timothy Loehmann and Frank Garmback placed themselves in harm's way by driving within feet of Tamir and shooting him Nov. 22, 2014 outside the Cudell Recreation Center on Cleveland's West Side.
The experts also partially blamed the shooting on a culture of corruption in the Cleveland police department that tolerates misconduct. They condemned the department for hiring Timothy Loehmann, the officer who shot Tamir, without examining his file from a former job that described him as an inept officer.
The officers' poor tactical decision-making and systemic failures within the department resulted in a death of a child that was "completely avoidable…and should never have occurred," Clark wrote.
The attorneys sent a letter to McGinty Saturday asking him to present their findings to the grand jury. The prosecutor invited the attorneys in June to offer input and evidence while a case for the grand jury is prepared.
McGinty after receiving the letter told cleveland.com that he would include the reports in the grand jury presentation.
Clark's and Noble's analysis examined the officers' tactics from the moment they were dispatched to the park.
The partners failed to follow police procedure that requires officers to develop a plan and call for backup before approaching a person who may be armed, the experts wrote.
"Reasonable police officers responding to a man-with-a-gun call would have stopped their vehicle prior to entering the park to visually survey the area to avoid driving upon a subject who may be armed," Noble wrote.
Clark also noted that the officers couldn't have known for sure whether Tamir was the subject being described by the 911 caller. The caller said the person was on the swings, but Tamir was seated at a table in the gazebo when police arrived.
"In my opinion there was nothing in the dispatch information that would positively identify Tamir as the certain target of the call to the responding officers," Clark wrote.
Further, if the officers determined that Tamir was the suspect in question, the park surveillance video makes it clear that Tamir was not a threat, Clark wrote. The man who called 911 told a dispatcher that the person was pointing a gun at people and scaring them.
But the video shows that Tamir didn't appear threatening as Loehmann and Garmback arrived, the experts said, and despite this, Garmback pulled the cruiser next to the gazebo.
The position of the car placed Loehmann, who was in the passenger's seat, in a difficult position, Noble wrote. Loehmann was forced to make a split-second decision about whether to use deadly force because he had no cover.
The experts bolstered this opinion with a statement from Judge Ronald Adrine of the Cleveland Municipal Court, who in June announced that he found probable cause to charge Loehmann with murder and other counts.
But the fault was not just Garmback's, the experts wrote. Loehmann also acted too quickly when he drew his gun and fired twice without warning.
While Loehmann has not provided an official statement in the investigation, a report from the Cuyahoga County Sheriff's Department shows that Loehmann says that he yelled commands to Tamir before he shot him.
But the fact that Loehmann shot the boy within 1.7 seconds of exiting the car proves that he did not give any verbal commands to Tamir, much less give him time to act on them, Noble wrote. The video makes it clear that there was no time for a "meaningful exchange" between the two, he said.


text:
package com.sensorberg.sdk.testUtils;

import com.sensorberg.sdk.internal.interfaces.BeaconHistoryUploadIntervalListener;
import com.sensorberg.sdk.internal.interfaces.BeaconResponseHandler;
import com.sensorberg.sdk.internal.transport.interfaces.Transport;
import com.sensorberg.sdk.internal.transport.interfaces.TransportHistoryCallback;
import com.sensorberg.sdk.internal.transport.interfaces.TransportSettingsCallback;
import com.sensorberg.sdk.model.persistence.ActionConversion;
import com.sensorberg.sdk.model.persistence.BeaconAction;
import com.sensorberg.sdk.model.persistence.BeaconScan;
import com.sensorberg.sdk.scanner.ScanEvent;

import java.util.List;
import java.util.SortedMap;

public class DumbSucessTransport implements Transport {

    @Override
    public void updateBeaconLayout(SortedMap<String, String> attributes) {

    }

    @Override
    public void setBeaconHistoryUploadIntervalListener(BeaconHistoryUploadIntervalListener listener) {

    }

    @Override
    public void setLoggingEnabled(boolean enabled) {

    }

    @Override
    public void setProximityUUIDUpdateHandler(ProximityUUIDUpdateHandler proximityUUIDUpdateHandler) {

    }

    @Override
    public void getBeacon(ScanEvent scanEvent, SortedMap<String, String> attributes, BeaconResponseHandler beaconResponseHandler) {
        beaconResponseHandler.onFailure(new IllegalArgumentException("this transport is dumb"));
    }

    @Override
    public boolean setApiToken(String apiToken) {
        return false;
    }

    @Override
    public void loadSettings(TransportSettingsCallback transportSettingsCallback) {

    }

    @Override
    public void publishHistory(List<BeaconScan> scans, List<BeaconAction> actions, List<ActionConversion> conversions, TransportHistoryCallback callback) {
        callback.onSuccess(scans, actions, conversions);
    }
}

redpajama_set_name: RedPajamaGithub


Homes, condos, lofts and commercial properties for sale in Ansnorveldt. This area is part of King. You can view listings in King as well. Last updated on April 23, 2019. Average listed price on site for Ansnorveldt is $1,684,720.
Salesperson, Century 21 Your Number One Realty Inc.
Accredited Buyer Representative, Homelife Galaxy Real Estate Ltd.


Definition: Input device for fine or precise control of cursor or brush when used in graphics, painting or image manipulation program. * Consists of the tablet, an electrostatically charged flat board connected to the computer, and the pen, which interacts with board to give precise information on the location of the pen tip. Most systems are also sensitive to the pressure applied to the tip. Movements of the pen cause corresponding movements of the cursor on screen. * The best systems use cordless pens.


Combatting botnets is a key challenge in cybersecurity. The classic crime-fighting approach of prosecuting perpetrators and confiscating crime tools fails here: botnets cannot be simply 'confiscated', and law-enforcement's reactive focus on prosecuting offenders is ill-suited to deal effectively with botnet threats. A wider set of anti-botnet strategies, including pro-active strategies and public-private co-operation, is needed to detect and dismantle botnets. Public-private anti-botnet operations, however, raise significant legal questions: can data about (possibly) infected computers be shared among private parties and public authorities? How far can private and public actors go in anti-botnet activities? And how legitimate are public-private partnerships in which private actors partly take up the intrinsically public task of crime-fighting? These questions will be addressed in the BotLeg project on the legal boundaries of public-private actions against botnets.
The BotLeg project is a consortium between TiU (TILT), SURFNet, SIDN, Abuse Information Exchange, and NHTCU. While the main focus of the research is the Netherlands, the project will develop a comparative analysis to include other EU countries. The project is financed via NWO and will last for 48 months. Among the expected legal research results, the BotLeg project will deliver sectorial guidelines and codes of conduct on anti-botnet operations.


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