DNA double-strand breaks (DSBs) are among the most deleterious cellular lesions since they threaten genomic integrity and cell viability. To counteract cell degeneration and to preserve genomic integrity, a complex network of DSB repair and signaling processes has evolved –.
Two main DSB repair pathways exist, canonical non-homologous end-joining (c-NHEJ) and homologous recombination (HR) , . In mammalian cells, c-NHEJ represents the major repair pathway for ionizing radiation (IR)-induced DSBs . C-NHEJ repairs unresected break ends without the need for sequence homologies and can function throughout the cell cycle . The key factors in c-NHEJ involve the KU70/80 heterodimer, which binds to the DSB end, and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which, together with KU70/80, constitutes the DNA-PK holoenzyme. The repair process is completed by a complex of DNA ligase IV, XRCC4, and XLF/Cernunnos . In contrast to c-NHEJ, HR is restricted to the S and G2 phases of the cell cycle where break ends undergo extensive resection and homologous DNA sequences on the sister chromatid serve as a template for repair. In addition to the repair of DSBs, HR functions during the S phase to restart stalled or collapsed replication forks . HR is initiated by CtIP-dependent resection to create 3′-overhangs at the DSB ends , . Following extended resection by EXO1 or BLM/DNA2, loading of RAD51 onto single-stranded DNA (ssDNA) is facilitated by BRCA2, XRCC2, and XRCC3. RAD54-mediated homology search then promotes strand exchange and Holliday junction formation . HR is completed after repair synthesis by Holliday junction resolution and DNA end ligation. In the absence of c-NHEJ factors, DSB repair can also occur by an alternative end-joining mechanism, termed alt-NHEJ , . In contrast to c-NHEJ but similar to HR, alt-NHEJ involves CtIP-dependent resection. The resected break ends are subsequently rejoined by a process involving micro-homologies and various repair factors such as poly (ADP-ribose) polymerase (PARP), DNA ligase I or III, and XRCC1 –. Although alt-NHEJ can efficiently operate in cells devoid of c-NHEJ factors, little is known about its ability to compensate for HR defects.
It has become clear over the last years that higher order chromatin structure impacts on the response to DSBs . Thus, IR-induced DSBs in densely compacted heterochromatin (HC) are more difficult to repair than euchromatic (EC) DSBs and they require additional structural changes in the surrounding chromatin , . One example are ATM-mediated chromatin changes due to KAP-1 phosphorylation . In undamaged cells, KAP-1 forms HC by recruiting HP1, CHD3 and other remodeling factors , . DSB-induced KAP-1 phosphorylation leads to release of CHD3 which locally relaxes HC and facilitates repair . Other studies involving HP-1 mobilization have observed either a release from  or a recruitment to damaged chromatin –. These apparently conflicting findings have led to the suggestion that a transient release might be followed by an accumulation of HP1 at sites of DNA damage , . However, it is often unclear how the various processes of chromatin modification impact on DSB repair and if different repair pathways are differentially affected.
Repair kinetics for IR-induced DSBs are biphasic, exhibiting a fast and a slow component . The slow component accounts for the repair of a subset (15–20%) of IR-induced DSBs that are localized to HC DNA regions, whereas DSBs induced in EC regions are typically repaired with fast kinetics. In G1 phase, the fast and the slow component of DSB repair comprise a c-NHEJ mechanism . ATM-dependent phosphorylation of KAP-1 on serine 824 (S824) is specifically required for the slow component , . In G2 phase, in contrast, c-NHEJ accounts only for the fast DSB repair process, while the slow ATM-dependent HC component represents HR . Thus, in G2, defined DSB populations, EC vs. HC breaks, are repaired by either c-NHEJ or HR, respectively. Despite the existence of two repair pathways in G2, a mutation in one of them leads to elevated unrepaired DSBs. Thus, c-NHEJ and HR cannot compensate for each other which might be attributed to the fact that c-NHEJ is unable to repair DSBs which have undergone extensive resection. Consistent with this notion, c-NHEJ can compensate for HR if resection is prevented by CtIP depletion . What remains unclear is why alt-NHEJ, which in principal is able to rejoin resected break ends, cannot compensate for a loss of down-stream HR factors such as BRCA2 or RAD51.
In the present study, we analyzed the process of HR at HC DSBs in G2 phase. We show that the intensity of phosphorylated ATM at DSBs decreases during the process of resection, suggesting that ATM initially binds to but is then released from DSBs which undergo repair by HR. Consistent with this notion, chemical inhibition of ATM prior to but not after resection causes a repair defect. Thus, ATM has an early role during HR but is dispensable for later stages. This contradicts the situation in G1 where continuous ATM activity is required for HC DSB repair by c-NHEJ . In G1, ATM functions to phosphorylate KAP-1, leading to its inactivation and local relaxation of the HC structure . Moreover, depletion of KAP-1 by siRNA overcomes the requirement for ATM in G1 but leads to reduced HR usage in G2. Finally, following KAP-1 siRNA or expression of a phospho-mimic form of KAP-1, both of which cause HC relaxation, resected DSBs can be repaired by a PARP-dependent alt-NHEJ process. Together, these data show that the HC structure represents a barrier for repair by c-NHEJ and alt-NHEJ but facilitates usage of HR. ATM, which initially binds to DSBs, is released from break ends during the process of resection. This prevents usage of c-NHEJ and alt-NHEJ and commits resected DSBs to repair by HR.
We have previously demonstrated that BRCA2-deficient cells exhibit elevated γH2AX foci levels at 8 h post irradiation in G2 , . These unrepaired DSBs have undergone efficient end-resection as evidenced by RPA loading (Figure 1A) which might explain why they cannot be repaired by NHEJ. We sought to further characterize these breaks and observed that the pATM focal intensity in G2- but not in G1-phase cells is greatly diminished at 8 h compared with 30 min time points (Figure 1A and Figure S1A). In contrast, the γH2AX focal signal is equally intensive at 30 min and 8 h in G1 and G2 (Figure S1B). We also measured the pATM focal intensity at 2 h post IR, a time point when resected and unresected DSBs are present in G2-phase cells. Of note, the pATM focal intensity of RAD51-foci-positive resected breaks is reduced compared with RAD51-foci-negative unresected breaks. In contrast, the γH2AX focal intensity is similar or even slightly increased at resected versus unresected DSBs (Figure 1B). These findings suggest that the pATM focal intensity decreases during resection in G2. pATM contributes, together with DNA-PKcs and ATR, to the phosphorylation of H2AX , . To test if the loss of pATM intensity at the break site leads to reduced ATM activity, we measured the γH2AX focal intensity in cells with strongly diminished levels of ATR, a kinase which is activated by ssDNA regions . Significantly, although ATR-deficient cells show γH2AX focal intensities at unresected DSBs similar to wildtype (wt) cells, they exhibit greatly diminished intensities at resected breaks (Figure 1C). Consistent with the notion that ATM is active at unresected but not at resected DSBs, chemical inhibition of ATM only affects γH2AX foci intensities at unresected but not at resected DSBs (Figure 1D and Figure S1C).
Fig. 1. phosphoATM focal intensity decreases at DSBs undergoing resection.
(A) A549 tumor cells treated with BRCA2 siRNA were irradiated with 1 Gy (0.5 h) or 2 Gy (8 h) and immunostained with the indicated antibodies. Using EdU and cell cycle markers to distinguish G1- from G2-phase cells , focal intensities of pATM were measured using ImageJ software (see Figure S1A). BRCA2 siRNA was used in this analysis to accumulate resected DSBs. (B) 2BN hTert (XLF-deficient) human fibroblasts were analyzed 2 h post IR with 1 Gy. Cells were stained against γH2AX and RAD51 or pATM and RAD51, and γH2AX or pATM focal intensities were measured at RAD51-foci-positive or RAD51-foci-negative foci. XLF-deficient cells were used in this analysis to prevent repair of EC DSBs during the time needed for resection of HC DSBs. (C) 82-6 hTert (wt) and F02-98 hTert (ATR-deficient) human fibroblasts were stained against γH2AX and RAD51 at 2 h post 1 Gy, and γH2AX focal intensities were measured as in (B). (D) 2BN hTert (XLF-deficient) human fibroblasts were stained against γH2AX and RAD51 at 2 h post 1 Gy, and γH2AX focal intensities were measured as in (B). Since both DNA-PK and ATM can phosphorylate γH2AX, cells were treated with DNA-PK inhibitor under all conditions. Inhibitors were added 1 h post IR, a time sufficient to allow for ATM-dependent resection and RAD51 loading (see Figure S1C). In (A–D), at least 300 foci from 3 independent experiments were analyzed for each point. Box plots were used with a maximum whisker-length of 1.5-fold the inter-quartile range; the lower and upper "x" indicates the 1% or 99% margin of the data range, respectively.
We next sought to confirm the immunofluorescence (IF) measurements by Western blotting. We used A549 tumor cells which can be efficiently synchronized in G1 by serum starvation and moderately enriched in G2 by double thymidine blocking (Figure S2A). The level of chromatin-bound pATM decreases with time after IR due to ongoing repair in G1 and in G2 but, importantly, at later times the pATM level per γH2AX level is smaller in G2-enriched than in G1-synchronized cells (Figure 2A). We also measured pKAP-1 (S824) levels as a specific read-out for ATM activity  and obtained similar results (Figure 2A). We next wished to measure pATM bound to DSBs and employed immunoprecipitation (IP) experiments. For this, we used HeLa tumor cells which can be efficiently synchronized in G2 (Figure S2B). Strikingly, pATM bound to γH2AX is readily detected at 30 min but nearly absent at 8 h post IR in G2 (Figure 2B). To directly show that the diminished pATM activity in G2 is a result of resection, we inhibited resection by depleting CtIP or BLM  and measured pKAP-1 levels. G2-synchronized HeLa tumor cells show a strongly reduced pKAP-1 level at 4 h post IR compared with unsynchronized cells which is fully or partly restored after CtIP or BLM depletion (Figure 2C and Figure S2C). To provide evidence for the restoration of chromatin condensation at resected DSBs, we performed IP experiments as in Figure 2B. We observed that the level of KAP-1 bound to γH2AX continuously increases with repair time (Figure 2D), possibly due to an enrichment of HC DSBs at longer times and the recruitment of KAP-1 to damaged sites as previously reported . Importantly, γH2AX-bound KAP-1 is substantially phosphorylated at early times post IR but largely unphosphorylated at later times (Figure 2D). Together, these biochemical approaches confirm the IF data above and provide strong evidence that ATM accumulation and activity is strongly reduced at DSBs which undergo resection. This leads to KAP-1 dephosphorylation and possibly the restoration of HC. The observed diminished ATM activity at resected DSBs is consistent with studies using a human cell extract-based assay in which ATM is activated by blunt DSB ends and ends with short ss overhangs but not by extended ssDNA regions which arise during the process of resection .
Fig. 2. phosphoATM activity is diminished at resected DSBs.
(A) G1-synchronized and G2-enriched A549 tumor cells were irradiated with 10 Gy and harvested at indicated time points. Fractionated chromatin (chromatin) was immunoblotted (left panel), and pATM, pKAP-1 and γH2AX levels of the chromatin fraction from the same blot were quantified using ImageJ. The right panels show the ratio of pATM or pKAP-1 relative to γH2AX for G1 and G2 cells at various time points. The data for G2 was normalized to G1 which was set to 100% (mean ± SEM from ≥2 experiments). (B) G2-synchronized HeLa tumor cells were irradiated with 30 Gy, harvested at the indicated time points, immunoprecipitated (IP) with γH2AX antibody and analyzed by immunoblotting. In G2 cells, pATM is co-immuno-precipitated with γH2AX at 30 min but not at 8 h post IR. The depicted FACs distributions in panels (A) and (B) represent the cell populations at the time of irradiation. How these populations change during repair incubation is shown in Figure S2. (C) HeLa tumor cells were treated with siRNA, synchronized in G2, and whole cell extracts were analyzed by immunoblotting 4 h post 10 Gy. pKAP-1 is detected in unsynchronized but not in G2-synchronized cells unless either CtIP or BLM is depleted. Depletion of CtIP or BLM did not affect the cell cycle distribution (see Figure S2C). (D) G2-synchronized HeLa tumor cells were irradiated with 30 Gy, harvested at the indicated time points, immunoprecipitated (IP) with γH2AX antibody and analyzed by immunoblotting. The level of KAP-1 co-immuno-precipitated with γH2AX increases with increasing repair time post IR. KAP-1 is substantially phosphorylated at early but not at later times.
ATM has been implicated in early steps of HR , , . A prediction of our findings above is that ATM is no longer required for HR after resection has occurred. To test this, we inactivated ATM either before or at 2 h post IR, a time point when resection has occurred (Figure S1C), and investigated the efficiency of DSB repair. γH2AX foci numbers at 8 h post IR were substantially elevated both in G1- and G2-phase cells treated with ATM inhibitor (ATMi) before IR but only in G1-phase and not in G2-phase cells if ATMi was added 2 h post IR (Figure 3A). We also analyzed mitotic chromatid breakage in G2-irradiated cells and observed substantially elevated break levels if ATMi is administered before irradiation but not if it is added 2 h post IR (Figure 3B). HR in G2 leads to sister chromatid exchanges (SCEs)  which are diminished if ATM is inhibited before but not at 2 h after IR (Figure 3C). Together, these data show that ATM is dispensable for HR stages that occur after resection has taken place.
Fig. 3. ATM is not required for later stages of HR.
(A) γH2AX foci were analyzed in G1- and G2-irradiated A549 tumor cells as previously described . Cells were treated with ATMi 0.5 h prior to or 2 h post IR to investigate the impact of ATM inactivation at various stages during repair. Background foci numbers were subtracted. At least 40 cells were analyzed per data point and experiment (mean ± SEM from ≥3 experiments). (B–C) Chromatid breaks (B) and SCEs (C) were analyzed in mitotic HeLa tumor cells at 8 h post 2 Gy. Cells were treated with caffeine and colcemid at 5 h post IR to abolish the G2 checkpoint and collect cells in mitosis. The addition of caffeine does not affect homologous recombination levels as assessed by SCE formation . Cells were labeled with EdU, and only EdU-negative cells (i.e. cells in G2 at the time of irradiation) were included in the analysis. Cells were treated with ATMi as in (A). At least 40 metaphases were analyzed per data point and experiment (mean ± SEM from ≥3 experiments). Example of a DAPI-stained metaphase spread with an enlarged SCE (left panel) and chromatid break (right panel). P values were obtained by t-test and represent a comparison of all cells analyzed in the indicated cell populations (***: p<0.001).
It was previously shown that ATM operates in G1 by continuously phosphorylating KAP-1 at heterochromatic DSBs and that KAP-1 depletion overcomes the requirement for this ATM function . Since ATM accumulation and activity is reduced at resected DSBs, we next asked if KAP-1 depletion might affect DSB repair in G2. KAP-1 siRNA did not alter γH2AX foci numbers in wt cells but strikingly rescued the repair defect in BRCA2 mutants and cells treated with BRCA2 siRNA (Figure 4A and Figure S3A). The same effect was observed in CHO cells deficient for the HR factor XRCC3 as well as in RAD51-depleted CHO cells (Figures S3B and S3C). Moreover, KAP-1 siRNA reduced the elevated level of chromatid breaks in BRCA2-deficient cells to that of wt cells (Figure 4A). We also measured the formation of SCEs and did not observe any IR-induced SCE formation in BRCA2/KAP-1-depleted cells (Figure S3D). Finally, we investigated cells containing an integrated HR reporter with two differentially mutated GFP genes . Expression of the endonuclease I-SceI generates a DSB in one of the two genes which can be repaired by HR (gene conversion) with the second gene copy as a template, resulting in a cell with functional GFP. HR frequencies assessed by the fraction of GFP-positive cells are significantly decreased after BRCA2 depletion and dual depletion of BRCA2 and KAP-1, confirming that the repair events occurring in BRCA2/KAP-1-depleted cells do not represent HR (Figure S3E). A pathway switch from HR to c-NHEJ has recently been demonstrated for heterochromatic DSBs after the inhibition of resection by CtIP siRNA, consistent with the idea that resection determines DSB repair pathway choice . Therefore, we asked if RPA foci formation, as a read-out for resection, is affected by KAP-1 depletion. Significantly, wt and BRCA2-depleted cells show the same initial level of RPA foci at 2 h post IR which is unaffected by KAP-1 siRNA. These RPA foci persist in BRCA2-depleted cells up to 8 h post IR consistent with their elevated γH2AX foci level. In contrast, RPA foci numbers decrease with time due to ongoing repair in wt and BRCA2-depleted cells treated with KAP-1 siRNA (Figure 4B and Figure S3F). We also investigated RAD51 loading at resected DSBs and observed normal RAD51 foci numbers after KAP-1 siRNA in wt but not in BRCA2-depleted cells (Figure 4B).
Fig. 4. KAP-1 depletion allows HC DSB repair in the absence of BRCA2.
(A) γH2AX foci and PCC analysis in G2-irradiated 82-6 hTert (wt) and HSC62 hTert (BRCA2-deficient) human fibroblasts. (B) RPA and RAD51 foci analysis in G2-irradiated A549 tumor cells. (C) Endogenous KAP-1 and BRCA2 was depleted in HeLa tumor cells by siRNA, and cells were transfected with GFP-tagged and siRNA-resistant empty (GFP), wt or mutated (phospho-mutant S824A or phospho-mimic S824D) KAP-1 plasmids. γH2AX foci were analyzed in GFP-positive G2-irradiated cells. EdU and cell cycle markers were used to distinguish G2- from S- and G1-phase cells . In (A), (B) and (C), foci numbers or PCC breaks from unirradiated cells were subtracted. At least 40 cells or PCC spreads were analyzed per data point and experiment (mean ± SEM from ≥3 experiments). KAP-1 and BRCA2 depletion in this and subsequent experiments was highly efficient (>90% as assessed by Western blotting). P values were obtained by t-test and represent a comparison of all cells analyzed in the indicated cell populations (***: p<0.001).
The finding that a BRCA2-independent process repairs resected DSBs after combined BRCA2 and KAP-1 siRNA suggests that the commitment for HR results from the loss of pATM at resected DSBs which is overcome by KAP-1 depletion. To consolidate this finding, we investigated DSB repair in cells treated with KAP-1 siRNA and complemented with siRNA-resistant KAP-1 constructs which were mutated at the ATM-dependent phosphorylation site on S824 . The BRCA2 repair defect, which is rescued after KAP-1 siRNA, is restored after complementation with wt KAP-1 or with KAP-1 rendered unphosphorylatable by mutating serine at position 824 to alanine (S824A). Significantly, however, KAP-1 mutated to a phospho-mimic aspartate at position 824 (S824D) fails to restore the BRCA2 repair defect (Figure 4C). Thus, KAP-1 phosphorylation at the established ATM site 824 overcomes the commitment for HR and DSB repair in the absence of BRCA2 can proceed by an HR-independent process.
Next, we wanted to investigate the process which is employed in BRCA2-deficient cells for the repair of resected DSBs. For this, we depleted BRCA2 and/or KAP-1 in cells deficient in the c-NHEJ factor XLF. XLF-defective cells show greatly elevated γH2AX foci and chromatid breaks consistent with the notion that c-NHEJ represents the predominant repair pathway in G2 . Interestingly, depletion of BRCA2 leads to a similar increase in γH2AX foci/chromatid break numbers in wt cells and XLF mutants, demonstrating additivity of the two major repair pathways in G2, c-NHEJ and HR (Figure 5A). But most importantly in the present context, dual depletion of BRCA2 and KAP-1 did not affect γH2AX foci/chromatid break numbers in XLF mutants, demonstrating that the HR defect is rescued by KAP-1 depletion even in the absence of the c-NHEJ factor XLF (Figure 5A). The same effect was observed in CHO cells deficient in the c-NHEJ factor KU80 (Figure S4A).
Fig. 5. Alt-NHEJ can function as a back-up pathway for HR.
(A) γH2AX foci and PCC analysis in G2-irradiated 82-6 hTert (wt) and 2BN hTert (XLF-deficient) human fibroblasts. (B) γH2AX foci and PCC analysis in G2-irradiated 82-6 hTert (wt) human fibroblasts treated with PARPi 0.5 h prior to IR. (C) PCC analysis from G2-irradiated HSC62 hTert (BRCA2-deficient) human fibroblasts treated with PARPi as in (B). (D) γH2AX foci analysis in G2-irradiated 82-6 hTert (wt) and HSC62 hTert (BRCA2-deficient) human fibroblasts. (E–G) Chromatid fusions and breaks in G2-irradiated mitotic HeLa tumor cells at 8 h post 2 Gy. Cells were treated with caffeine and colcemid at 5 h post IR to abolish the G2 checkpoint and collected in mitosis. Chromosomes were stained with Giemsa (panel E) or analyzed by FISH with probes specific for chromosomes 1 (red), 2 (green) and 4 (yellow) (panel F) or to chromosomes 18 and 19 (panel G). Foci numbers, chromatid breaks and fusions from unirradiated cells were subtracted. For panels A–E, at least 40 cells or 40 PCC/mitotic spreads were analyzed per data point and experiment (mean ± SEM from ≥3 experiments). For panel G, 50 mitotic spreads were analyzed per data point and experiment (mean ± SEM from ≥2 experiments). N.d. indicates that no chromatid fusions were observed under these conditions. P values were obtained by t-test and represent a comparison of all cells analyzed in the indicated cell populations (***: p<0.001).
We then tested if an alt-NHEJ pathway repairs DSBs in BRCA2/KAP-1-depleted cells and employed chemical inhibition of PARP (PARPi), a factor which has been implicated in alt-NHEJ , . γH2AX foci and chromatid breaks were not significantly affected in wt cells treated with PARPi, demonstrating that alt-NHEJ processes do not contribute substantially to IR-induced DSB repair in normal cells. However, the elevated level of γH2AX foci/chromatid breaks in BRCA2-deficient cells, which is rescued after KAP-1 siRNA, is restored by PARPi (Figures 5B and 5C). Thus, PARPi precluded the repair events which arose in BRCA2-deficient cells after KAP-1 siRNA, demonstrating that a PARP-dependent process can function as a back-up pathway for HR. We also investigated other factors which have been described to function in alt-NHEJ. In CHO mutants deficient in XRCC1 as well as in cells deficient for DNA ligase I and III, KAP-1 failed to rescue the elevated γH2AX foci level which is conferred by a deficiency in BRCA2 or RAD51 (Figure 5D and Figure S4B). Consistent with the notion that alt-NHEJ can function as a back-up pathway for HR, we observed greatly increased levels of chromatid fusions in BRCA2/KAP-1-depleted cells (Figure 5E). To characterize the nature of these chromatid fusion events, we employed fluorescence-in-situ-hybridization (FISH) analysis with chromosome-specific probes. In one set of experiments, we used probes for chromosomes 1, 2 and 4 and observed that all fusion events (∼40 fusions from the analysis of ∼800 cells) occurred between heterologous chromosomes, that is, between a stained and an unstained chromosome or between two differently stained chromosomes (Figure 5F). Further, we employed probes for chromosome 19 which is exceptionally rich in KAP-1 binding sites and for the similar-sized chromosome 18 which is largely devoid of these sites . Following BRCA2 depletion, we observed significantly higher breakage levels in chromosome 19 compared with chromosome 18, confirming that HR in G2 occurs mainly in KAP-1-dependent HC (Figure 5G). Importantly, following dual depletion of BRCA2 and KAP-1, chromosome fusions occur more often in chromosome 19 than in chromosome 18 confirming the notion that they arise from the misrejoining of chromatid breaks in KAP-1-dependent HC (Figure 5G).
The data above show that KAP-1 depletion allows heterochromatic DSBs to be repaired by an alt-NHEJ pathway in the absence of BRCA2, XRCC3 or RAD51. It is, however, unclear how the efficiency of HR in wt cells is affected by KAP-1-mediated chromatin changes. As shown above, γH2AX foci and chromatid breaks are repaired with similar kinetics with and without KAP-1 siRNA (see Figure 4A) but it is not known if repair after KAP-1 siRNA involves HR or, as in the case of HR mutants, an alt-NHEJ pathway. To address this question, we investigated the formation of SCEs in mitotic cells and observed greatly diminished SCE levels after KAP-1 siRNA in wt cells (Figure 6A). We also employed the HR reporter assay described above (Figure S3E) and observed strongly reduced HR levels following KAP-1 depletion (Figure 6B). Thus, KAP-1-depleted cells do not employ HR although repair occurs efficiently. We also analyzed chromatid fusion events as a read-out for incorrect end-joining. Strikingly, KAP-1-depleted cells show elevated chromosomal fusions, suggesting that the DSBs are repaired by an error-prone alt-NHEJ pathway (Figure 6C). This notion is consolidated by the observation that PARPi increases γH2AX foci and chromatid break numbers in cells depleted for KAP-1 or complemented with phospho-mimic KAP-1 (S824D) (Figures 6D and 6E). Further, cells deficient in DNA ligase I and III or in XRCC1 show elevated γH2AX foci levels following KAP-1 depletion (Figures 6F and 6G). Taken together, this data shows that HR is efficiently used in cells with unphosphorylatable KAP-1 and cannot occur if KAP-1 is depleted.
Fig. 6. HR requires KAP-1-dependent heterochromatin.
(A) SCEs in G2-irradiated mitotic HeLa tumor cells at 8 h post 2 Gy. Cells were treated with caffeine and colcemid at 5 h post IR to abolish the G2 checkpoint and collected in mitosis. (B) HR frequencies (gene conversion) after I-SceI expression in HeLa pGC cells carrying an integrated GFP reporter system. (C) Chromatid fusions analyzed from cells in panel A. (D) γH2AX foci and PCC analysis in G2-irradiated 82-6 hTert (wt) human fibroblasts. Cells were treated with PARPi 0.5 h prior to IR. (E) Endogenous KAP-1 was depleted in HeLa tumor cells by siRNA, and cells were transfected with GFP-tagged and siRNA-resistant phospho-mimic (S824D) KAP-1 plasmid. γH2AX foci were analyzed in GFP-positive G2-irradiated HeLa tumor cells treated with PARPi 0.5 h prior to IR. (F) γH2AX foci analysis in G2-irradiated 82-6 hTert (wt) human fibroblasts. (G) γH2AX foci analysis in G2-irradiated CHO9 (wt) and EMC11 (XRCC1-deficient) hamster cells. In (C–G), foci numbers, PCC breaks or chromatid fusions from unirradiated cells were subtracted. At least 40 cells or 40 PCC/mitotic spreads were analyzed per data point and experiment (mean ± SEM from ≥3 experiments). (H) Model of heterochromatic IR-induced DSB repair. In wt cells, ATM activates CtIP to initiate resection and phosphorylates KAP-1 to facilitate chromatin decondensation. Following extended resection, ATM is released from chromatin and KAP-1 is dephosphorylated, which likely results in restoration of condensed chromatin and a commitment to HR (left). In the case of decondensed chromatin due to KAP-1 depletion or expression of phospho-mimic KAP-1 (KAP-1 S824D), repair of resected DSBs occurs by alt-NHEJ, which is suppressed by chromatin condensation (right). P values were obtained by t-test and represent a comparison of all cells analyzed in the indicated cell populations (***: p<0.001).
HR involves resection of DSB ends. Here, we investigated the process of HR at HC DSBs in G2 and showed that pATM, which initially binds to DSB ends, is released from the break sites during the process of resection. This leads to diminished KAP-1 phosphorylation at HC breaks and a commitment to repair such resected DSBs by HR. If the loss of KAP-1 phosphorylation is overcome by KAP-1 depletion or expression of phospho-mimic KAP-1, both of which are known to cause local HC relaxation, this commitment to HR is abolished and resected DSBs are repaired by an alt-NHEJ process. Thus, KAP-1-dependent HC facilitates later stages of HR whereas c-NHEJ and alt-NHEJ both require continuous HC relaxation due to ATM-dependent KAP-1 phosphorylation (see Figure 6H).
ATM binding and activation at DSB ends occurs within minutes after damage induction and is important for the initiation of various signaling processes . Concomitant with the induction of signaling pathways, a variety of chromatin remodeling processes are initiated. This involves modifications which either relax or condense the chromatin structure in the surrounding of DSBs. However, it is currently unclear how these changes are chronologically orchestrated and how they differentially affect different DSB repair pathways in different chromatin compartments. Therefore, we focused our investigation on chromatin modifications which occur in HC regions due to the process of resection in order to specifically investigate how such chromatin changes impact on later stages of HR. We did not examine chromatin remodeling processes at early times which affect the decision to initiate resection.
We have previously shown that ATM is dispensable for the majority of DSB repair in G1 but that HC breaks strictly require ATM . ATM's function during HC DSB repair in G1 involves continuous KAP-1 phosphorylation which leads to local HC relaxation . Our finding that ATM is released from resected DSBs in G2 was therefore unexpected. However, there is precedence in the literature that ATM changes binding properties upon resection of DSBs. First, ATM's binding affinity to break ends has been reported to be attenuated with the progressive presence of ssDNA at resected DSBs . This ATM attenuation is accompanied by increasing ATR activity , consistent with our result that H2AX phosphorylation at RAD51-foci-positive DSBs requires ATR. Second, 53BP1, a damage response factor which localizes to and facilitates pATM accumulation at DSB sites , has been reported to show reduced occupancy at resected DSBs in G2 . Although the reported reduction of ATM accumulation and activity at resected breaks is consistent with published data, the functional consequence of this finding was hitherto unclear.
In G2 phase, DSB repair can be performed by NHEJ and HR. It is therefore remarkable that cells with mutations in BRCA2, XRCC3 or RAD51 exhibit unrejoined DSBs, which obviously are refractory to repair by NHEJ. Thus, it has been suggested that the process of resection commits DSB repair to HR and prevents usage of NHEJ . Here, we provide mechanistic insight into the processes determining pathway usage upon resection. Since ATM is released from resected DSBs we reasoned that the concomitant reduction in KAP-1 phosphorylation prevents repair of resected breaks by NHEJ. Indeed, if loss of ATM-dependent KAP-1 phosphorylation is overcome by KAP-1 depletion or expression of phospho-mimic KAP-1, BRCA2-, XRCC3- or RAD51-deficient cells exhibit normal repair kinetics. Thus, it is not the resection per se but the loss of ATM activity at resected breaks which commits repair to HR.
HC DSBs which remain unrepaired in BRCA2-, XRCC3- or RAD51-deficient cells can be repaired if HC relaxation is provided by KAP-1 depletion or expression of phospho-mimic KAP-1. Interestingly, these DSBs undergo resection as evidenced by normal RPA foci formation. Thus, HC repair occurring in the absence of BRCA2, XRCC3 or RAD51 must involve a pathway which is capable of dealing with resected breaks. Consistent with the notion that alt-NHEJ can repair resected DSBs, we showed that the HC repair events occurring in the absence of BRCA2, XRCC3 or RAD51 require PARP, XRCC1 and DNA ligase I/III. We also observed that HC repair in the absence of BRCA2 has a significant propensity to lead to chromatid exchanges in G2-irradiated cells. Because alt-NHEJ has been implicated in the formation of genomic exchanges –, this finding supports our contention that HC repair in the absence of BRCA2, XRCC3 or RAD51 involves alt-NHEJ.
Perhaps surprisingly, we observed that the process of HR is nearly abolished in cells with depleted KAP-1, even in the presence of functional HR factors. This suggests that DSB repair pathway usage is significantly affected by chromatin modifications, favoring HR in condensed genomic regions. This notion is further supported by the observation that PARP inhibition or the loss of XRCC1 or DNA ligase I and III leads to elevated unrepaired breaks in KAP-1-depleted cells, which not only demonstrates that cells use alt-NHEJ but also, that they cannot employ HR in the absence of KAP-1-dependent HC. In summary, these findings establish that KAP-1-dependent HC is not only a barrier to repair by c-NHEJ or alt-NHEJ but, unexpectedly, also facilitates the process of HR.
Consistent with our results, depletion of HP1α or KAP-1 strongly reduces gene conversion frequencies in a I-SceI-based HR assay . Furthermore, HP1α and KAP-1 is recruited to chromatin damaged by laser- or X-irradiation ,  and depletion of HP1α diminishes SCE formation after treatment with camptothecin . One explanation of how HC might promote HR is that a reduced spatial distance between sister chromatids in HC regions facilitates homology search . In support of this idea, we have recently obtained preliminary evidence that the average distance between sister chromatids, measured by FISH analysis with locus-specific probes, is substantially larger in EC versus HC regions (Geuting et al., unpublished data). A similar mechanism has been suggested for cohesin proteins which might promote HR by providing the required proximity of sister chromatids in G2 phase . Another explanation of how HC might facilitate HR is by suppressing alt-NHEJ processes. Although it is well established that the presence of KU70/80 at DSB ends prevents repair by alt-NHEJ, KU70/80 is likely released from resected DSB ends. Chromatin condensation occurring due to ATM release at resected DSBs might represent an alternative mechanism to keep error-prone alt-NHEJ processes in check.
In conclusion, our study provides mechanistic insight into sequential events determining DSB repair pathway usage. First, we demonstrate that ATM activity is diminished at DSBs which undergo resection during the process of HR. Second, the concomitant loss of pKAP-1 at resected DSBs leads to local reconstitution of the HC superstructure and prevents repair of resected DSBs by alt-NHEJ. Thus, our study links two seemingly unrelated findings by showing how modifications at DSBs undergoing resection affect chromatin remodeling processes and DSB repair pathway usage.
Immortalized and transformed cell lines were 82-6 hTert (wt), HSC62 hTert (BRCA2-deficient, kindly provided by Dr. M. Digweed), 2BN hTert (XLF-deficient, kindly provided by Dr. P. Jeggo) and F02-98 hTert (ATR-deficient, kindly provided by Dr. P. Jeggo) human fibroblasts, HeLa-S3, HeLa pGC (kindly provided by Dr. J. Dahm-Daphi) and A549 human tumor cells, and CHO-AA8 (wt), IRS1SF (XRCC3-deficient; kindly provided by Dr. L. Thompson), CHO-K1 (wt), XRS6 (KU80-deficient, kindly provided by Dr. P. Jeggo), CHO-9 (wt) and EMC11 (XRCC1-deficient, kindly provided by Dr. B. Kaina) hamster cells. HeLa-S3 and A549 tumor cells were cultured in DMEM with 10% FCS and 1% NEAA; HeLa pGC cells additionally in 0.3 µg/ml puromycin. Human fibroblasts and CHO cells were cultured in MEM with 20% FCS, 1% NEAA. All cells were maintained at 37°C in a 5% CO2 incubator.
SiRNA transfection was carried out with HiPerFect Transfection Reagent (Qiagen) following the manufacturer's instructions. siRNAs used in the experiments were: BLM (50 nM), Control (10 nM), CtIP (20 nM), KAP-1 (25 nM), RAD51 (20 nM), Lig I (20 nM), Lig III (20 nM) (Qiagen), and BRCA2 (25 nM) (SmartPool, Dharmacon). SiRNA sequences were: BLM (AAG CUA GGA GUC UGC GUG CGA), BRCA2 (GAA ACG GAC UUG CUA UUU A; GUA AAG AAA UGC AGA AUU C; GGU AUC AGA UGC UUC AUU A; GAA GAA UGC AGG UUU AAU A), Control (AAU UCU CCG AAC GUG UCA CGU), CtIP (UCC ACA ACA UAA UCC UAA UUU), KAP-1_A (CAG UGC UGC ACU AGC UGU GAG), KAP-1_B (CAU GAA CCC CUU GUG CUG UUU), RAD51 (AAG GGA AUU AGU GAA GCC AAA), Lig I (AAG GCA UGA UCC UGA AGC AGA), Lig III (AAC CAC AAA AAA AAU CGA GGA). Experiments were performed 48 h following siRNA transfection. For GFP-tagged siRNA-resistant KAP-1 plasmid transfection, HeLa tumor cells were incubated with KAP-1_B or KAP-1_B and BRCA2 siRNA and, 8 h later, transfected with 1 µg plasmid DNA using Lipofectamine LTX Transfection Reagent (Life Technologies). Cells were irradiated with 2 Gy, fixed and stained for γH2AX, EdU and GFP. Only GFP-positive cells were analyzed.
A549 tumor cells were used for G1 synchronization and G2 enrichment. HeLa tumor cells were only used for G2 enrichment. G1 synchronization was carried out by 48 h serum starvation in DMEM without FCS and NEAA. 0.5 h before irradiation, medium was replaced by DMEM with FCS and NEAA. For G2 enrichment, a double thymidine blocking was used. Cells were blocked 16 h with 2 mM thymidine (Sigma), released in fresh medium for 9 h, blocked again with 2 mM thymidine for 16 h and released in fresh medium for 7–8 h. Synchronization was controlled by FACs analysis as described previously . X-irradiation was performed at 90 kV and 19 mA with an aluminum filter (dose rate: 2 Gy/min). Chemical inhibitors were added 0.5 h prior to IR and maintained during repair incubation. The ATM inhibitor (Tocris KU 60019), the DNA-PK inhibitor Nu7441 (Tocris NU7026) and the PARP inhibitor PJ34 (Calbiochem PARP inhibitor VIII PJ34) were used at concentrations of 5 µM, 10 µM and 20 µM, respectively. Repair incubation was limited to time periods which provided that the majority of G2-irradiated cells remained in G2 (controlled by FACs analysis).
Cells were grown on glass coverslips. EdU (10 µM) was added 0.5 h prior to IR to discriminate between S- and G2-phase cells. In experiments analyzing G1-phase cells, nocodazol (100 ng/ml) was added 0.5 h prior to IR to prevent G2-phase cells progressing into G1 during repair incubation . Cells were fixed and stained as described  and additionally stained with Click-it EdU (Life technologies). Antibodies used were: mouse-α-γH2AX at 1∶2000 (Millipore); rabbit-α-γH2AX at 1∶2000 (Abcam), mouse-α-pATM at 1∶1000 (Biomol), rabbit-α-RAD51 at 1∶15000 (Abcam), mouse-α-RPA at 1∶2000 (Neomarkers) and mouse-α-GFP at 1∶200 (Roche). Cells were analyzed with a Zeiss microscope and Metafer software (Metasystems). Samples were evaluated in a blinded manner. Foci intensities were analyzed using ImageJ software (see Figure S1A).
HeLa pGC cells were incubated with siRNA and, 24 h later, transfected with 3 µg pBL464-pCBASce plasmid DNA using MaTra transfection (IBA). After 24 h, cells were again siRNA treated and, 48 h later, fixed and stained. 10000 cells per sample were analyzed with a Zeiss microscope and Metafer software (Metasystems).
Whole cell extracts were prepared as described . For chromatin fractionation, cells were resuspended two times in NP-40 buffer (10 mM Tris/HCl pH 7.5, 10 mM NaCl, 3 mM MgCl2, 30 mM sucrose, 0.5% NP-40, 0.2 mM sodiumvanadate, 0.5 mM PMSF) and centrifuged for 10 min at 1500× g. Cell pellet was resuspended in Glycerol buffer (20 mM Tris/HCl pH 7.9, 100 mM KCl, 0.2 mM EDTA, 20% glycerol, 0.2 mM sodiumvanadate, 0.5 mM PMSF) and incubated 10 min on ice. After centrifugation (10 min, 1500× g) chromatin fraction was lysed and sonicated in RIPA buffer (50 mM Tris/HCl pH 8, 150 mM NaCl, 0.5 Na-deoxycholate, 1% Triton, 0.1% SDS). For immunoprecipitation, cells were fixed with 3% paraformaldehyd containing 2% sucrose for 5 min at 4°C, immediately washed with PBS, scraped in medium and centrifuged for 10 min at 400× g. Cells were resuspended two times in NP-40 buffer containing 15 mM caffeine and centrifuged for 10 min at 1500× g. Cell pellet was resuspended in equal volume Nuclease buffer (10 mM HEPES pH 7.5, 10 mM KCl, 1 mM CaCl2, 1.5 mM MgCl2, 0.34 M sucrose, 10% glycerol, 0.1% Triton-X-100, 0.2 mM sodiumvanadate, 0.5 mM PMSF, 15 mM caffeine), micrococcal nuclease (500 U/ml) was added and suspension was incubated for 45 min at 37°C. Equal volume of Solubilization buffer (2% NP-40, 2% Triton-X-100, 600 mM NaCl in Nuclease buffer) was added before mixing, brief sonication and clearifing for 10 min at 8000× g. Dynabead Protein G (Invitrogen) were blocked 1 h with 100 µg/ml salmon sperm DNA in 0.1% BSA/PBS and antibodies (4 µg) were linked to the beads, washed two times in 0.1% BSA/PBS and then incubated with the cell extract at 4°C over night. Beads were washed three times in Wash buffer (equal volume of Nuclease buffer and Solubilization buffer) and boiled in 2× Laemmli buffer for 5 min at 95°C.
Western blotting was carried out at 300 mA for 1 h or at 80 mA over night. Nitrocellulose membrane (Roth) was blocked for 1 h in 5% low fat milk or 5% BSA in TBS/0.1% Tween20. Antibody incubation was carried out in TBS/0.1% Tween20/1% low fat milk or 5% BSA over night at 4°C, followed by HRP-conjugated secondary antibody incubation in PBS/0.1% Tween20/1% low fat milk or 5% BSA for 1 h. Immunoblots were developed using ECL (Roche). Signal detection was carried out with a chemi-smart-system (Vilber Lourmat). Primary antibodies used were: rabbit-α-pATM at 1∶1000 (Epitomics); rabbit-α-pKAP-1 (S824) at 1∶10000 (Epitomics); rabbit-α-KAP-1 at 1∶1000 (abcam); mouse-α-BRCA2 at 1∶1000 (Cell signaling); rabbit-α-GAPDH at 1∶1000 (Santa Cruz); mouse-α-γH2AX at 1∶1000 (Millipore); mouse-α-H3 at 1∶1000 (abcam); mouse-α-RPA2 at 1∶1000 (Calbiochem); rabbit-α-pRPA2 (S4/8) at 1∶10000 (Bethyl).
EdU (10 µM) was added 0.5 h prior to IR and maintained to discriminate between S- and G2-phase cells. PCCs were harvested at 8 h, mitotic cells for SCE or FISH analysis between 5–8 h after IR as described . Microscope slides were stained with DAPI (0.2 µg/ml) and Click-it EdU. For FISH analysis, whole chromosome probes for chromosomes 1, 2, and 4 or for chromosomes 18 and 19 were used (Metasystems). Chromosome spreads were recorded by Metafer software (Metasystems). Only EdU-negative chromosome spreads were analyzed.
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text:
Njazi Kuqi (ur. 25 marca 1983 w Vučitrn) – fiński piłkarz występujący na pozycji napastnika. Jego starszym bratem jest Shefki Kuqi.

Kariera klubowa
Kuqi zawodową karierę rozpoczynał w sezonie 2002 w FC Lahti. W debiutanckim sezonie był tam podstawowym graczem. W barwach Lahti rozegrał wówczas 24 ligowe spotkania i zdobył 5 bramek. Od następnego sezonu zaczął pełnić rolę rezerwowego. W Lahti spędził trzy sezony. Łącznie zagrał tam w 53 ligowych meczach i strzelił 16 goli.

1 stycznia 2005 roku za 500 tysięcy euro został sprzedany do angielskiego Birmingham City. 23 stycznia 2006 został wypożyczony na miesiąc do Blackpool. W marcu 2006 wypożyczono go do końca sezonu do Peterborough United. W kwietniu 2006 powrócił do Birmingham. Grał tam do końca sezonu 2005/2006. W barwach Birmingham przez półtora roku nie zagrał ani razu.

W lipcu 2006 odszedł do holenderskiego FC Groningen. Spędził tam pół roku, jednak nie wystąpił tam w żadnym spotkaniu. W styczniu 2007 przeszedł do niemieckiego FC Carl Zeiss Jena. Zadebiutował tam 19 stycznia 2007 w przegranym 2:3 meczu z Kickers Offenbach rozegranym w ramach rozgrywek 2. Bundesligi. Było to jednak jedyne spotkanie rozegrane przez niego w barwach Carl Zeiss. W sierpniu 2007 rozwiązano z nim kontrakt.

W styczniu 2008 podpisał kontrakt z drugoligowym TuS Koblenz. Pierwszy ligowy występ zanotował tam 3 lutego 2008 w zremisowanym 1:1 meczu z 1. FSV Mainz 05.

W sierpniu 2010 zagrał w 1 meczu Stevenage F.C., w którym grał 7 minut We wrześniu 2010 podpisał roczny kontrakt z Dundee F.C., jednakże po miesiącu opuścił klub. W marcu 2011 podpisał kontrakt na jeden sezon z Turun Palloseura. W sierpniu 2011 podpisał dwuletni kontrakt z Panioniosem Ateny. W czerwcu 2012 podpisał dwuletni kontrakt z Atromitosem Ateny. W grudniu 2013 przeszedł do US Pro Vercelli Calcio. W sierpniu 2014 trafił do Olympiakosu Wolos. W styczniu 2015 podpisał kontrakt do końca sezonu z SG Sonnenhof Großaspach. W maju 2015 opuścił ten klub. W lutym 2016 podpisał roczny kontrakt z PK-35 Vantaa. W sierpniu 2016 podpisał kontrakt do końca sezonu z Interem Turku, natomiast w listopadzie tegoż roku przedłużył ją o rok z możliwością przedłużenia o kolejny rok.

Kariera reprezentacyjna
W reprezentacji Finlandii zadebiutował 12 marca 2005 w wygranym 1:0 towarzyskim meczu z Kuwejtem, w którym strzelił gola. W kadrze rozegrał łącznie 12 spotkań i strzelił 5 goli.

Przypisy

Bibliografia
 Profil na stronie btm.touch-line.com
 Profil na stronie national-football-teams.com

Linki zewnętrzne

Reprezentanci Finlandii w piłce nożnej
Kosowscy piłkarze
Piłkarze FC Lahti
Piłkarze Birmingham City F.C.
Piłkarze Blackpool F.C.
Piłkarze Peterborough United F.C.
Piłkarze FC Groningen
Piłkarze FC Carl Zeiss Jena
Piłkarze TuS Koblenz
Piłkarze Stevenage F.C.
Piłkarze Dundee F.C.
Piłkarze Turun Palloseura
Piłkarze Panioniosu GSS
Piłkarze PAE Atromitos
Piłkarze Olympiakosu Wolos
Piłkarze Pro Vercelli 1892
Piłkarze Interu Turku
Urodzeni w 1983

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A seat that's all about the mold--we're talking form here, not fungus. The GUBI 3D collection is innovative and unique--the key to contemporary style--and is the first of its kind based on the technique of molding three-dimensional veneer and plastic into a comfortable seat with a design that keeps all edges pointing away from you. Perfect for around the dining table, bellied up to the bar, or in the conference room, 3D holds up in any environment, 3D or otherwise. Choose your favorite seat, upholstery, base, and finish, and sit back to relax with the results.
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text:
Home » Music » Linda Lusardi signs up to The Real Full Monty to face her fears and save lives
Linda Lusardi signs up to The Real Full Monty to face her fears and save lives
Linda Lusardi says she is facing her fears in a bid to save lives.
The Page 3 legend, 62, has signed up to strip off on the latest series of The Real Full Monty.
It aims to highlight how important it is for women to check their breasts for cancer symptoms.
Linda said: "It's a wonderful cause and a really important message to get out there. If just one person checks themselves because of this programme, then it's all been worth it.
"Early detection is the key in fighting breast cancer. At the moment people aren't going to the doctors with their worries and they really should.
"I've watched the programme in the past and I have always been very moved by it and it has made me want to check myself for lumps and bumps. I just think it's so important."
The new series has a twist — the celebs will do a striptease while performing an ice skating routine.
Linda has previously had experience on skates as a contestant on Dancing on Ice.
But she admits she is more nervous about the skating than she is about baring all on TV.
She said: "Obviously it was a concern having to expose myself, but, I put that aside and said yes. Then I found out it was on ice, so that was a bit of a shock, as I've always feared getting back on the ice.
Page 3's Linda Lusardi wows fans with photo of lookalike model daughter Lucy
"I broke a bone in my foot during training for Dancing on Ice, before I even got to the live shows. So I do have a fear of hurting myself on the ice.
"I think the skating is more worrying than the reveal at the moment, I just hope I remember the routine properly and don't mess up. I don't want to fall on the ice or expose myself at the wrong point during the dance."
Other celebs taking part include Loose Woman Coleen Nolan and former Corrie star Hayley Tamaddon.
The Real Full Monty on Ice starts tomorrow at 9pm on ITV.
Tagged Cancer, ITV
Bill Rieflin, Drummer for King Crimson, R.E.M., Ministry, Dead at 59
Chance the Rapper Reveals He Can Call Kanye West Whenever He Wants: 'I've Got a Direct Line'
Elvis Presley's Hair Color Was Faked Using Shoe Polish
Strictly finalists 2020: Who are the Strictly Come Dancing finalists?
LadBaby releases Christmas single as he recalls living off £20 a week


text:
Get Lost in Los Angeles
Check out LA photos of the Griffith Observatory, Hollywood Walk of Fame, Rodeo Drive and more.
Visit the Griffith Observatory to gaze at the stars and get great views of downtown LA and the Pacific Coast.
Enjoy the beauty of the LA skyline at night.
A Charlie Chaplin impersonator stops for a photograph on the Hollywood Walk of Fame.
Universal Studios in LA is an exciting, fun-filled vacation spot for families with children.
Go shopping on Rodeo Drive in Beverly Hills at high-end stores, including Fendi, Chanel, De Beers and Christian Dior.
Explore the J. Paul Getty Museum to see European and American visual art, including paintings, sculptures and photographs.
Acres of shopping, dining, strolling at The Grove.
Explore the playgrounds and thrill rides located along the Santa Monica Pier.
Visit Hollywood's Forever Cemetery, the final resting place for famous celebrities, including rock star Johnny Ramone.
Linda Vista Haunted History
Royal Spotting 41 Photos
10 Budget-Friendly Things to Do With Kids in LA 10 Photos


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With many mobile platform options available to chose, it is important to align your App strategy to the mobile hardware platform, App market, and pricing so that it can deliver better business results today and support more than one platform and app-markets in future. We can help you to think through these available choices and make qualified decisions.
We can help you to implement your mobile App ideas using onshore and offshore model to deliver the cost efficiencies and speed. We will be your technology partner in designing, implementing and rollout of the Application.
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The Lonben team is comprised of experienced technical developers and managers who have for years have worked with leading outsourcing and technology firms.


Each episode of the series is a story of saved infant planted in the so-called "baby box", a special spot arranged in a maternity or a children's hospital for those who wish to anonymously get rid of the baby. The film is set in a children's hospital of a perinatal center, with the doctors and the medical staff put forward as characters. They give the foundling a start in life, providing treatment and care. Then come the parents who reached a conscious decision to adopt another one's child. Still, the baby remains the central character as the events are unfolding around the little boy or girl.The plot of each film episode is full of unexpected twists and turns.
"The Baby Box" is a melodrama series about salvation and kindness, human vices and a huge will to live; these are stories about making a choice and becoming a whole world for someone else.


text:
NextVolunteering increases skills for employment
Pottery is 'a way of life' for local artist
IWC Team	on May 1, 2012
Making pottery is not an occupation that many people would consider pursuing, but for Lamyaa AlQadhi, the art-form is a way of life. AlQadhi, an Iraq native, has worked as a professional potter since 1995, earning herself an excellent reputation in her country's artistic community. Even before beginning to work independently, she was very involved in pottery.
"I started in 1986," she explained," and worked under the supervision of a famous Iraqi, Ablaa Alzawi, for five years. After that she said to me 'you can start on your own.'"
Given permission to work alone, AlQadhi quickly became involved in her chosen field, opening her own workshop and presenting exhibitions. As involved as she was in her own career, though, she still found time to enrich her community.
"I was an instructor in a summer school, and a children's counsellor in a hospital. Mostly [I worked] with children who [were] ill and [had] to stay in the hospital… [I] started doing just simple pottery and art [with them] to keep them busy and active."
"AlQadhi quickly became involved in her chosen field, opening her own workshop and presenting exhibitions."
AlQadhi enjoyed several years of prosperity and success in Iraq, but with the escalation of the war, and the increased danger it presented, her situation rapidly deteriorated.
Forced to leave Iraq in what she described as "a hurry," AlQadhi was unable to take any of her work with her to Egypt, where she initially fled. Now, living in Canada, AlQadhi is still attempting to rebuild her life.
"Back home," she explained, "I didn't have any barriers, but you know, since I came here, the [biggest] barrier for me is the financial things. I don't know the labour market very well."
While she intends to continue her career as an artist, AlQadhi concedes that it is difficult to survive in such a field without financial support, and considers it necessary to have a separate occupation which can provide additional revenue.
"My first profession was an administrative assistant," she stated, adding that she has a diploma in office management.
Despite the difficulty of her situation, AlQadhi has persevered. Upon arriving in Canada, she participated in several programs at the Immigrant Women's Centre, including Job Search Workshops, and Leading and Learning. Now, she volunteers as a peer support worker at the Centre, and has even managed to find employment with the Immigrant Culture Art Association of Hamilton. She currently works there as an advanced pottery instructor, and hopes to eventually attain the same popularity and success she experienced in Iraq.
"My long-term goal," she explained, "is to open my ceramic studio again, here in Canada."
AlQadhi, thankful for her position, expressed gratitude to the ICAA for providing her with the opportunity. She feels that the importance of art in a community is unparalleled, and was quick to recommend that any recent immigrants with an interest in art become involved with the organization.
– Article and photo by Evelyna Kay, Westdale Secondary School
Tagged in: artists, hamilton, immigrant, newcomer, pottery
Volunteering increases skills for employment
Breaking Barriers, Building Pathways


Thank you for the valuable work that you do. I don't understand how the analysis of CREE went from "CREE capped at $29" to a much higher $62.63 less than a year latter. I appreciate your nimbleness in changing your position but drastic changes make me lose confidence in the stability and usefulness of the analysis. But I do want to be a believer so help me.


Yoginfinity Founder Stefania Brunori on why TECOM Group's in5 is a perfect platform to launch a startup business in Dubai.
Researchers suggest that "the autonomy enjoyed by 'being one's own boss' more than compensates entrepreneurs for the hardships otherwise associated with self-employment."
Taka Solutions founder and Managing Director Charles Blaschke on his startup's mission to reducing energy consumption, entrepreneurship in UAE, and representing the GCC region on the global competition The Venture.
Founded by 36-year-old Leslie Mallart, Dubai Confidential is a lifestyle website geared towards helping expat women "discover and explore hidden gems around the UAE."
Ali Abdel-Hafiz talks about his passion for renewable energy, education and entrepreneurship.
From Back to the Future to augmented reality, three co-founders launched pixelbug, a tech company developing advanced augmented reality applications.
If you're interested in using Instagram for your business, this event would be beneficial for you.


text:
Q: Create and edit object using modal in rails I've been trying to create and edit an object using a modal view in the index html in Rails. I'm using twitter bootstrap in the project. I m so far successful in creating the object using the modal.
For things to work, I had to create an object called @post = Post.new in my index action.
Since before the edit modal is displayed, the edit object must be ready as @post = Post.find(params[:id]), but it happens in the edit action.
Is their a way using which I can initialize @post for my edit modal view before it is displayed?
Here's my code:
index.html.erb

Posts

<% @posts do |post| %>

    <%= post.name %>
    <%= post.description %>

    <%= link_to 'Edit', edit_post_path(post), :class => 'btn btn-mini btn-min-standard-width',  :data => {:toggle => "modal", :target => "#editItemModal"} , :remote => true %>
        <%= link_to 'Destroy', post, confirm: 'Are you sure?', method: :delete, :class => 'btn btn-mini btn-danger btn-min-standard-width'  %>

<% end %>

<%= link_to 'New Item', new_post_path,
        :class => 'btn btn-primary btn-standard-width' ,  :data => {:toggle => "modal", :target => "#newItemModal"} , :remote => true %>

    <button type="button" class="close" data-dismiss="modal">×</button>

New item

    <%= render :partial => 'form' %>

    <button type="button" class="close" data-dismiss="modal">×</button>

Edit item

    <%= render :partial => 'form' %>

_form.html.erb
<%if @post%>

    <%= form_for(@post, :html => { :class => 'form-horizontal', :remote => true } ) do |f| %>

        <%= f.label :name, :class => 'control-label' %>

           <%= f.text_field :name %>

        <%= f.label :description, :class => 'control-label' %>

            <%= f.text_area :description %>

       <%= f.submit nil, :class => 'btn btn-primary' %>

    <% end %>
<% end %>

PostController
class PostsController < ApplicationController

 def index
    @post = Post.new
    @posts = Post.all

    respond_to do |format|
      format.html # index.html.erb
      format.json { render json: @posts }
    end
  end

 def show
    @post = Post.find(params[:id])

    respond_to do |format|
       format.html # show.html.erb
       format.json { render json: @post }
     end
  end

 def new
    @post = Post.new
 end

  def edit
    @post = Post.find(params[:id])
    respond_to do |format|
      format.html
     format.json { render json: @post }
    end
  end

............

A: As long as I understand you want to use instance variable @post in the edit modal view.
Without knowing your code base, what can I say:

*

*Find the corresponding action in the controller that renders the edit modal view, create one if there is no

*Initialize @post with the model Post found by id, just simply copy and paste that into the proper action

*Call it within the view scope

Hope that answers your question

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We drove across the state last week to attend the opening of Into The Forest. There was so much to look at! And the opening was packed. I'm glad I was able to go back to the gallery the next day and get another look. I was constantly seeing things I hadn't noticed the night before. What a treat! But I'm afraid I have run out of words about now so here are some pictures from Into The Forest.
You can buy the exhibition catalog which contains a picture of every contribution along with the name and locale of the artists. For more information, press here. But the pictures and the catalog are not a substitute for walking into the forest yourself. The exhibit runs until December 3 and is worth a trip to Pittsburgh.


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Swinging Sixties - Harrogate musician Stuart Colman in 1960s pop band Pinkertons Colors and an early single on Decca Records.
Shakin Stevens has paid tribute to a Harrogate-born music industry legend who died yesterday after a long battle with cancer.
The 80s hitmaker paid tribute to his friend and former producer Stuart Colman on Twitter saying: "In memory of our close colleague and long time friend, who still had a lot more to give.
"Rest in peace, Stuart, and carry on listening to the music you loved so much. Much love to Annie, who was the rock he needed. "
Musician, producer and broadcaster supreme - The late, great Stuart Colman fom Harrogate playing his beloved bass.
But who was Stuart Colman and why was he so famous in the music industry in both the UK and USA?
Put simply, he was the single most important music industry figure to emerge from Harrogate - or most other places - since the war.
One of a kind, he made his mark everywhere he travelled in an illustrious career, and he travelled far.
Born Ian Stuart Colman in Harrogate on December 19, 1944 to a well-known musical family, Colman was a musician, record producer and broadcaster who excelled at the very top of the musical tree.
Colman sold a million records in America at the height of the 1960s, became an influential champion of 50s rock n roll and rockabilly on his own show on BBC Radio 1 in the 1970s, before moving onto BBC Radio London and Capital Radio.
He also wrote a regular column for music paper Melody Maker, produced successful records for a host of top country acts over several decades and worked with everyone from Cliff Richard to Kate Bush.
He was best known, perhaps, for turning Shakin' Stevens into a number one hit-making machine in the 1980s.
But his music career began young as a teenager in Harrogate in the pre-Beatles era of the very early 1960s when he joined rock band The Denvers at Harrogate Grammar School with fellow music-mad schoolboys including Bob Mason.
After he left Harrogate to move to Rugby, Stuart went on to play bass with Swinging Sixties pop band Pinkerton's Colors, who scored a sole top ten single in the UK 1965 before a change of line-up and name to Flying Machine in 1969.
As part of the new outfit, Stuart enjoyed a top five hit in the USA with Smile a Little Smile for Me, which sold a million copies.
After the band split up in the early 1970s, Stuart became a session musician, music producer and radio broadcaster - all at the same time.
After he was brought in the early 1980s by Epic Records to help with the Welsh rock n roller's solo career, Colman found a hit formula with Stevens almost instantaneously, producing early hits like Hot Dog and Marie, Marie followed by a series number one singles including This Old House, Green Door and Oh Julie.
Colman enjoyed another number one hit in 1986 when he produced that year's Comic Relief record, Livin Doll with Cliff Richard and the cast of BBC comedy show The Young Ones.
By the mid-1990s he had moved with his family to Nashville, Tennessee, partly in order to devote his talents to country acts.
A health scare in 2002 saw him diagnosed with cancer of the oesophagus and he had to endure chemotherapy, radiation and surgery.
Colman then returned to Britain in 2014 but continued to run music projects from his new home in the Cotswolds despite his cancer.
Despite all the many things he achieved in his music career, Stuart never lost his love for the bass, holding his six-stringed 1962 Fender Bass VI and, in particular, his Fender Telecaster which he described as "my Special One" in high regard.
A close family friend issued an initial statement yesterday on the behalf of Stuart's family to mark his sad passing.
It said: "Stuart Coleman, aged 73, has died after a long battle with cancer in a Sue Ryder hospice outside Cheltenham. He lived in the Cotswolds and was active in the music industry until the end. He leaves a wife, son and two daughters."


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Jersey County
Senior Living in Jersey County, IL
To help you with your search, browse the 5 reviews below for senior living in Jersey County. On average, consumers rate senior living in Jersey County 4.5 out of 5 stars.
Caring.com has helped thousands of families find high-quality senior care. To speak with one of our Family Advisors about senior living options and costs in Jersey County, call (855) 863-8283.
Browse Senior Living by city: Senior Living in Jerseyville
4 Senior Living Communities in Jersey County, IL
Villas of Holly Brook Assisted & Refl...
1318 State Highway 109, Jerseyville, IL 62052
"My uncle moved into The Villas of Holly Brook & Reflections Jerseyville, IL. We go there all the time. That's only about 15 miles from where he had his home. It's very nice. The people are very..." More
Jerseyville Manor
1251 NORTH STATE STREET, Jerseyville, IL 62052
"I placed my wife in a skilled nursing facility called Jerseyville Manor. Everything was excellent; it's very clean, nice and modern. The staff has been very helpful, nice, and friendly. I haven't..." More
Find Local Costs for Jersey County, IL
Jerseyville Estates
1210 E. Fairgrounds, Jerseyville, IL 62052
"All the assistance you need, based on your likes and wishes. That's supportive living at Jerseyville Estates. When it comes to Supportive Living, we're not what you'd expect. No boring days, dull..." More
Jerseyville Nsg & Rehab Center
1001 SOUTH STATE STREET, Jerseyville, IL 62052
Willow Rose Rehab & Health
Provides: Nursing Homes and Memory Care
410 Fletcher St, Jerseyville, IL 62052
Willow Rose Rehab & Health Care
"Our main objective is to provide excellent nursing care. We have a very dedicated and caring nursing staff to meet your needs. We also offer an exciting activity and social service department to..." More
Other Options in Jersey County, IL
Senior Living near Jersey County, IL
Macoupin County
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Recent Reviews of Senior Living in Jersey County
Review of Villas of Holly Brook Assisted & Reflections Jerseyville, IL
My aunt is at Villas of Holly Brook in Jerseyville. It's like a 4-star hotel, it's a beautiful place. She knew several of the nurses and the aides that were there because they came from a place whe... Read more
My uncle moved into The Villas of Holly Brook & Reflections Jerseyville, IL. We go there all the time. That's only about 15 miles from where he had his home. It's very nice. The people are very... Read more
Review of Jerseyville Manor
Do not send family members here. My dad was sent for Rehab. Due to Covid we were not able to visit and the communication with this place was horrible. They did not bother to tell us they moved h... Read more
RUN AWAY QUICKLY
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Chapman's Greenhouse offers a large variety of plants to the Vancouver Area. From Annuals and Perennials to Vegetables and Herbs, we have selection that is sure to have you leaving with your complete planting space filled! We continue to grow all of our plants on site from seed or unrooted cuttings, so new varieties become available daily! While you are here talk with our experienced gardeners for helpful tips on where and when to plant your selection. We'll make sure that you are taking home plants that compliment your style and space, making your buying experience an enjoyable one.
While you are here make sure to pick up a bag of Black Gold Potting Soil or Ed Hume seed packets to help with your planting project.
As the season grows on make sure to check back to see updated pictures of this years growing season in the Photo Gallery section of our website!
Living only one growing season, these are plants that you must replant every year. Great for adding color to spaces where there used to be none!
These flora have a life cycle lasting more than two years. They do not need replanting each year in contrast to annuals.
We have over 15 types of Vegetables to choose from to get your garden growing! Make sure to take a peak at our 50+ varieties of Tomatoes and 40+ varieties of Peppers!
Whether you use them for their taste, scent, or medicinal purposes we have a variety for you to choose from all season long.
Choose from 10" or 12" hanging or patio pots, mixed or single flora. All are designed in house by our experienced gardeners!


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Grove City- Ron Casto
Ron Casto
Congratulations to our June Hometown Hero, Ron Casto of Grove City. Ron began his career as a city of Columbus Firefighter in 1972 and retired as a House Captain from 14's. Ron is also a veteran of the Marine Corps and served in the Combined Action Program in the Vietnam War from 1964-1965.
Ron is not only a courageous veteran, but also spends his free time volunteering at the USO reception area of John Glenn International Airport and with the Honor Flight. He took on the responsibility of running the Firefighters VFW Post 3426, making it known throughout the state for its outstanding charity work.
Congratulations Ron and thank you for your service and heroism!
© 2021 Local Hometown Hero | Privacy Policy


Stick out your tongue and take a good look in the mirror. A healthy tongue should be pink and covered in small nodules, also known as papillae. If you notice any discolouration that doesn't go away within a week or two, there may be cause for concern. Even though there's a variety of common tongue symptoms and conditions, many of these problems are not serious and can be resolved quickly with good oral care habits.
White tongue: This can be caused by a fungal infection, also known as 'thrush,' that occurs in the mouth or throat due to an overgrowth of yeast. Symptoms include white spots inside the mouth or on the tongue, sore throat and difficulty swallowing. Dehydration, poor dental hygiene or Leukoplakia, excessive growth of cells in your mouth and tongue, might also be the reason.
Red (or strawberry) tongue: When your tongue turns bright red it may signify a deficiency in folic acid and vitamin B-12, a bacterial infection (often paired with a high fever) or Kawasaki disease. This is more common in children and causes a victim's blood vessels to become inflamed, which explains the redness of the tongue.
Black tongue: A tongue with black patches, mainly along the centre could indicate trapped bacteria and yeast. Diet and smoking habits can blacken the surface area, as well as certain medications, dehydration and bad oral hygiene. Often times the bacteria build up inhibits the natural shedding process of the papillae and they start to grow, creating a hair-like appearance.
Yellow tongue: This usually occurs as a result of a harmless buildup of dead skin cells on the surface of your tongue. It's often associated with acid reflux or mouth breathing and can be an early sign of black tongue. If your skin or the whites of your eyes are also yellow this may suggest jaundice and you should seek medical care.
Every time you brush and floss your teeth, finish your oral care routine with proper tongue cleaning.
Clean your tongue on a regular basis to prevent the growth of bacteria. Using your toothbrush works fine but you can also try a tongue scraper.
While scraping, don't apply too much pressure or you may end up hurting your tongue. Scrape it in a downward motion at least two or three times to remove excess plaque.
You can also coat your tongue with an all-natural toothpaste before scraping. The toothpaste works to neutralize the bacteria.
Rinse your mouth with water or a gentle, alcohol-free mouthwash thoroughly after scraping to help get rid of bad odour and moisturize the mouth.
You should visit your dentist for diagnosis and treatment if your tongue problem is severe, unexplained, or persists for several weeks with no signs of improvement.


What a great collection piece for the ultimate Marshall Faulk collector. Bobblehead is in great shape and worth every penny! The item "CURTIS MARTIN New York Jets BOBBLEHEAD with Box EUC Bobble Dobbles SUPER RARE" is in sale since Friday, August 05, 2016.
This item is in the category "Sports Mem, Cards & Fan Shop\Fan Apparel & Souvenirs\Football-NFL". The seller is "jerry49412" and is located in Twin Lake, Michigan. This item can be shipped to United States, to Canada, to United Kingdom, DK, RO, SK, BG, CZ, FI, HU, LV, LT, MT, EE, to Australia, GR, PT, CY, SI, to Japan, to China, SE, KR, ID, to Taiwan, ZA, TH, to Belgium, to France, to Hong Kong, to Ireland, to Netherlands, PL, to Spain, to Italy, to Germany, to Austria, RU, IL, to Mexico, to New Zealand, PH, SG, to Switzerland, NO, SA, UA, AE, QA, KW, BH, HR, MY, BR, CL, CO, CR, PA, TT, GT, HN, JM.