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The mediator protein Claspin is critical for the activation of the checkpoint kinase Chk1 during checkpoint responses to stalled replication forks. This function involves the Chk1-activating domain (CKAD) of Claspin, which undergoes phosphorylation on multiple conserved sites. These phosphorylations promote binding of Chk1 to Claspin and ensuing activation of Chk1 by ATR. However, despite the importance of this regulatory process, the kinase responsible for these phosphorylations has remained unknown. By using a multifaceted approach, we have found that casein kinase 1 gamma 1 (CK1γ1) carries out this function. CK1γ1 phosphorylates the CKAD of Claspin efficiently in vitro, and depletion of CK1γ1 from human cells by small interfering RNA (siRNA) results in dramatically diminished phosphorylation of Claspin. Consequently, the siRNA-treated cells display impaired activation of Chk1 and resultant checkpoint defects. These results indicate that CK1γ1 is a novel component of checkpoint responses that controls the interaction of a key checkpoint effector kinase with its cognate mediator protein.
In eukaryotic cells, duplication of the genome and cell division must be coordinated faithfully. Otherwise, genomic defects might be propagated to progeny cells. In vertebrates, the checkpoint kinase Chk1 plays a critical role in preventing the continuation of the cell cycle if the genome has undergone any perturbation (Cimprich and Cortez, 2008 ). Genomic disruptions trigger the prompt activation of Chk1, which in turn phosphorylates key cell cycle control enzymes such as Cdc25 and Wee1. These reactions dampen the activation of cyclin-dependent kinases (Cdks) until the cell can rectify lesions in the DNA. The activation of Chk1 involves phosphorylation of its C-terminal regulatory domain on multiple sites by a master checkpoint regulatory kinase called ATM- and Rad3-related kinase (ATR). ATR exists in a tight complex with a partner called ATR-interacting protein (ATRIP; Cortez et al., 2001 ; Zou and Elledge, 2003 ). Upon occurrence of replication blockages in the nucleus, for example, the ATR–ATRIP complex undergoes activation in a manner that depends upon an intricate upstream regulatory pathway. In vertebrates, the culmination of this pathway results in the physical association of ATR–ATRIP with a specific activating protein known as topoisomerase IIβ-binding protein 1 (TopBP1; Kumagai et al., 2006 ; Mordes et al., 2008 ).
Significantly, the activated ATR–ATRIP complex also requires the assistance of a mediator or adaptor protein to effectively phosphorylate Chk1. In vertebrates, this protein is known as Claspin (Kumagai and Dunphy, 2000 ; Chini and Chen, 2003 ; Lee et al., 2003 ; Lin et al., 2004 ). ATR has a higher propensity to recognize the Claspin–Chk1 complex than the isolated Chk1 protein (Kumagai et al., 2004 ; Lindsey-Boltz et al., 2009 ). Significantly, Claspin also interacts in a highly specific manner with replication forks, which suggests this protein helps the cell to monitor the progress of DNA replication (Lee et al., 2005 ; Petermann et al., 2008 ; Scorah and McGowan, 2009 ). The association of Chk1 with Claspin depends upon phosphorylation of Claspin on multiple conserved residues within its Chk1-activating domain (CKAD; Kumagai and Dunphy, 2003 ; Clarke and Clarke, 2005 ; Lee et al., 2005 ; Chini and Chen, 2006 ; Bennett et al., 2008 ). These sites reside in highly conserved repeats of about ten amino acids (there are two such repeats in Xenopus Claspin and three in its human homologue). During a checkpoint response, serine and threonine residues (e.g., Ser864 and Ser895 in Xenopus and Thr916, Ser945, and Ser982 in humans) within each repeat undergo phosphorylation. These phosphorylations create phosphopeptide sequences that dock with the catalytic domain of Chk1, thereby promoting an increase in its kinase activity (Jeong et al., 2003 ). Mutagenesis studies have indicated that both phosphorylations of Xenopus Claspin and at least two out of three phosphorylations of the human protein are critical for the ultimate activation of Chk1 (Kumagai and Dunphy, 2003 ; Bennett et al., 2008 ).
Despite the importance of these processes, the kinase responsible for these modifications has not yet been identified. These phosphorylations are dependent upon ATR, but ATR does not phosphorylate these sites directly (Kumagai and Dunphy, 2003 ). Although it has been reported that Chk1 can phosphorylate Thr916 of human Claspin in vitro (Chini and Chen, 2006 ), more recent evidence has established that Chk1 is not responsible for the phosphorylation of the CKAD in vivo (Bennett et al., 2008 ). Thus, the identity of the Claspin-activating kinase has remained an open question. In this study, we have adopted a multifaceted approach to identify kinase(s) responsible for phosphorylation of the CKAD of Claspin. We first performed a whole-kinome RNAi screen in Drosophila S2R+ cells to identify kinases that influence the activation of Chk1 (Bettencourt-Dias et al., 2004 ). We followed through with these studies by carrying out various functional tests in both Drosophila S2R+ and human cells. Our results have indicated that one or more forms of casein kinase 1 gamma 1 (CK1γ1) can phosphorylate Claspin very effectively and regulate its ability to activate Chk1. Therefore, we have identified a novel player in the regulatory mechanisms through which metazoan cells safeguard genomic integrity.
To identify the kinase(s) responsible for phosphorylation of the CKAD of Claspin, we carried out a kinome-wide RNA interference (RNAi) screen in Drosophila S2R+ tissue culture cells. Initially, we screened for kinases whose knockdown would diminish phosphorylation of Chk1. We reasoned that this approach might identify both the Claspin-regulatory kinase and other kinases that could affect the Chk1-activating pathway. Owing to technical limitations in detecting phosphorylation of the endogenous Drosophila Chk1 (Grapes), we decided to express an inducible version of Xenopus Chk1 in the S2R+ cells (Figure 1A). There are various high-quality antibodies available that detect the whole Xenopus Chk1 protein and phosphorylation of two residues involved in its activation (Thr314 and Ser344). As shown in Figure 1B, treatment of cells with either aphidicolin (APH) or hydroxyurea (HU) resulted in robust phosphorylation of the exogenously expressed Chk1. The kinetics of this activation closely mirrored that of the endogenous Chk1 in human cells (Figure 1C). For example, in both Drosophila and human cells, strong phosphorylation of Chk1 occurred within 5 min after addition of APH. To assess whether this phosphorylation involves the same regulators as in other organisms, we treated the cells with interfering dsRNAs directed against the Drosophila versions of ATR (Mei-41) and Claspin (CG32251/CG1326). We found that both dsRNAs dramatically reduced the phosphorylation of Chk1 in response to APH and HU (Figure 1, D and E). Taken together, these tests indicate that this Chk1 reporter system recapitulates a faithful and specific checkpoint response.
After transfecting individual dsRNAs targeting each of 228 Drosophila kinases into the reporter cells, we monitored the effect on the APH-induced phosphorylation of Chk1 by immunoblotting with antiphosphopeptide antibodies. After multiple rounds of screening, we found approximately 16 dsRNAs that elicited a moderate to strong reduction in the phosphorylation of Chk1 (unpublished data). The results of the entire screen will be reported elsewhere. One positive dsRNA was directed against the casein kinase Gish. Moreover, dsRNAs against the casein kinases CG7094 and CG12147 caused some reduction in the phosphorylation of Chk1 in the initial round of screening, although this effect was not apparent in a secondary round of screening. In general, casein kinases have a preference for acidic substrates (Cheong and Virshup, 2011 ). Interestingly, the sequences around the critical phosphorylation sites in the CKAD of Claspin are also quite acidic. For example, the Xenopus CKAD repeat 1 sequence is MDELLDLCSGQF.
On the basis of these considerations, we decided to coexpress Gish as well as other Drosophila casein kinases in S2R+ cells along with a fragment containing the CKAD from Xenopus Claspin. We monitored phosphorylation of this fragment by using antibodies that detect the Ser864-phosphorylated form of Claspin (Kumagai and Dunphy, 2003 ). In these experiments, we observed that cells overexpressing Gish exhibited strong phosphorylation of Claspin, but there was no phosphorylation in cells expressing CG7094 and CG12147 (Figure 2A). There was also significant phosphorylation of Claspin in cells expressing the CG9962 kinase. However, CG9962 was negative in our dsRNA screen. To test if these kinases could phosphorylate Claspin directly, we expressed FLAG-tagged versions of these proteins in S2R+ cells. We isolated the kinases with anti-FLAG antibody beads, and then performed in vitro phosphorylation assays using the purified glutathione S-transferase (GST)-CKAD fragment as substrate. We observed that only Gish could phosphorylate the GST-CKAD fragment efficiently, whereas the CG9962 kinase was considerably less effective (Figure 2, B and C). Taken together, these results indicate that Gish can phosphorylate Claspin both in vivo and in vitro.
To investigate further the functional significance of these findings, we extended our studies to human cells. Among human proteins, Drosophila Gish is most similar to the CK1 gamma family of kinases (CK1γ), which includes three subfamilies (CK1γ1, CK1γ2, and CK1γ3; Zhai et al., 1995 ; Kusuda et al., 2000 ; Knippschild et al., 2005 ). Although all three kinases exhibit a high degree of homology within the kinase domain, there are significant differences in their N- and C-terminal extensions, which suggests these proteins are functionally distinct. To test whether these kinases can directly phosphorylate Claspin, we prepared recombinant versions of CK1γ1, CK1γ2, and CK1γ3 in Sf9 insect cells and then performed in vitro kinase assays (Figure 3A). In these experiments, we observed that all three kinases could phosphorylate the GST-CKAD fragment well (Figure 3B). In control experiments, we observed that recombinant Chk1 could not phosphorylate this fragment significantly (Figure 3B). Moreover, we demonstrated that the CK1γ kinases could not phosphorylate a mutant form of the CKAD (2AG) in which the two critical phosphorylation sites (Ser864 and Ser895) had been changed to alanine (Figure 3C). Finally, we also produced wild-type and kinase-dead (N169A mutant) forms of GST-tagged CK1γ1 in bacteria. We observed that the wild-type GST-CK1γ1 could phosphorylate Ser864 of Claspin efficiently, whereas the kinase-dead mutant was inactive (Figure 3D).
We pursued these findings by comparing the abilities of these kinases to phosphorylate Claspin in human cells. We proceeded to express recombinant forms of CK1γ1, CK1γ2, and CK1γ3 at similar levels in human U2OS cells. Concomitantly, we also introduced the GST-CKAD fragment into the cells. We observed that only cell lines expressing CK1γ1 could phosphorylate the fragment of Claspin above the background level found in cells containing no recombinant kinase (Figure 3E, lanes 2, 4, 6, and 8). Moreover, cells expressing exogenous CK1γ1 also displayed a modest increase in phosphorylation of the CKAD in response to APH (Figure 3E, lanes 3, 5, 7, and 9).
To further evaluate the role of CK1γ1 in the phosphorylation of Claspin, we utilized small interfering RNAs (siRNAs) to knock down the expression of the various CK1γ kinases in human cells. We monitored the efficiency of siRNA knockdown at both the mRNA and protein level for CK1γ1 and CK1γ2 (Figure 4A and Supplemental Figure S1, A, B, and D–F). Due to the fact that a suitable antibody for detection of CK1γ3 is not available, we were able to monitor only the mRNA level for this kinase (Figure S1C). As an initial test for the effect of the CK1γ1 knockdown, we examined phosphorylation of the transiently expressed form of the Xenopus CKAD in human U2OS cells. For this purpose, we generated a line of U2OS cells in which expression of this fragment is under control of a doxycycline-inducible promoter. We observed a large increase in the phosphorylation of the CKAD fragment within 5 min after addition of APH, which correlated well with the activation of Chk1 (Figure S1G). We found that this phosphorylation could be abolished almost completely if the cells had been transfected with CK1γ1 siRNA (Figure 4B). In contrast, when the cells were transfected with siRNAs against CK1γ2 or CK1γ3, there was no such reduction (Figure 4, C and D). We could also observe reduced phosphorylation of Claspin with multiple CK1γ1 siRNAs, which suggests the results are not due to off-target effects (Figure 4E). Finally, depletion of CK1γ1 (as well as that of CK1γ2 or CK1γ3) did not affect the cellular levels of other regulators of Chk1, such as ATR, Claspin, TopBP1, and Rad17 (Figure S1, H and I). Thus, removal of CK1γ1 does not generally affect components of the ATR-mediated regulatory pathway.
Since phosphorylation of Claspin is necessary for binding to Chk1, we also examined whether knockdown of CK1γ1 would affect the Claspin–Chk1 interaction. For this purpose, we performed pulldown experiments with the GST-CKAD fragment, which is known to interact well with Chk1. As expected, there was increased binding of Chk1 to the GST-CKAD in cells treated with APH (Figure 4F). By contrast, this increase was abrogated in cells transfected with CK1γ1 siRNA. Altogether, our data indicate CK1γ1, but not CK1γ2 or CK1γ3, is a specific kinase responsible for the checkpoint-induced phosphorylation of Claspin in human cells.
To pursue these findings further, we examined the effect of ablation of CK1γ1 on the ultimate activation of Chk1. We observed that removal of CK1γ1 by siRNA treatment from U2OS cells substantially diminished the phosphorylation of Chk1 (Figure 5, A and B). The reduction was similar to that observed in cells treated with siRNA directed against TopBP1, the upstream activator of ATR–ATRIP. By contrast, depletion of CK1γ2 or CK1γ3 in the same experiments had no effect on phosphorylation of Chk1. We observed a reduction in the phosphorylation of Chk1 following treatment with three different siRNAs against CK1γ1 (Figure 5B). Moreover, we obtained very similar results in a different cell line, namely, HeLa cells (Figure S2A–C). Finally, as a complementary means to confirm the effect of the siRNAs, we also generated a series of endoribonuclease-prepared siRNAs (esiRNAs), which are a mixture of siRNA oligonucleotides generated in vitro by cleavage of long double-stranded RNA with RNase III. As shown in Figure S2D, esiRNAs directed against CK1γ1 strongly inhibited phosphorylation of Chk1 but comparably generated esiRNAs against CK1γ2 and CK1γ3 had no effect.
Among the various substrates of ATR–ATRIP, Chk1 appears to be unique in its need for assistance by Claspin (Liu et al., 2006 ; Smits et al., 2010 ). Accordingly, we asked whether ablation of CK1γ1 might affect other substrates of ATR–ATRIP. For this purpose, we examined the ATR-dependent phosphorylation of Rad17 on Ser645 (Bao et al., 2001 ; Post et al., 2001 ). As anticipated, depletion of TopBP1 diminished phosphorylation of Rad17 in response to APH (Figure S2E). On the other hand, removal of CK1γ1 (or CK1γ2 and CK1γ3) had no effect on this phosphorylation. This result lends further support to the hypothesis that CK1γ1 acts specifically at the level of the Claspin–Chk1 interaction. This finding also indicates depletion of CK1γ1 does not generally perturb the ATR-mediated signaling pathway.
Once we established that CK1γ1 is responsible for the phosphorylation of Claspin and activation of Chk1, we sought to extend our studies by examining the roles of CK1γ1 in various aspects of cell physiology, including checkpoint responses. We noticed depletion of CK1γ1 eventually led to a decrease in cellular proliferation (Figures 6A and S3A). The effect was relatively mild within 48 h after siRNA transfection, but became more noticeable by 72 h. To analyze this effect in more detail, we examined cell cycle progression in the siRNA-treated cells. By performing flow cytometry, we observed that the percentage of cells in G2/M remained about the same upon depletion of CK1γ1, but there appeared to be a significant drop in the percentage of S phase cells as well as an increase in G1 cells (Figure 6B). As another means to examine the effect on S phase, we labeled cells with 5-ethynyl-2′-deoxyuridine (EdU) and detected the EdU-positive cells by flow cytometry in order to assess DNA replication. In these experiments, we observed an approximately 50–60% reduction of incorporated EdU in CK1γ1-depleted cells compared with control cells (Figure 6C).
To examine potential causes for the decrease in DNA replication, we asked whether depletion of CK1γ1 might lead to the occurrence of DNA damage. For this purpose, we stained cells with antibodies directed against Ser139-phosphorylated H2AX, known as γ-H2AX, which is a well-known marker for the presence of damaged DNA (Paull et al., 2000 ). We observed the highly elevated presence of nuclear foci containing γ-H2AX in CK1γ1-depleted cells compared with control cells (Figures 6D and S3B). Thus, lack of CK1γ1 apparently leads to the accumulation of damage without exposure to any exogenous damaging agent. As expected from published results, depletion of Chk1 also resulted in elevated staining for γ-H2AX (Paulsen et al., 2009 ). On the other hand, removal of CK1γ2 did not have this effect. It is well known that the presence of damaged DNA suppresses DNA replication. Therefore, chronic DNA damage in CK1γ1-depleted cells may at least partially explain the reduced levels of DNA synthesis. However, we do not have direct evidence for this possibility.
We next examined various physiological responses to genotoxic stress following ablation of CK1γ1. Toward this end, we examined cell survival upon treatment with replication inhibitors. In one type of assay, we treated cells with HU for 24 h, and then examined the ability of the cells to form colonies upon removal of the drug. We also monitored survival of cells incubated continuously with various doses of APH. In both types of assays, we observed depletion of CK1γ1 significantly compromised the ability of cells to withstand replication stress (Figure 6, E and F). In another line of experiments, we monitored recovery of replication forks after stalling. For this purpose, we used the experimental design shown in Figure 6G. We first challenged cells with a transient (10 min) treatment with APH to block DNA replication. Subsequently, we removed the drug, allowed the cells to recover for 60 min, and finally performed pulse-labeling with EdU to assess the replicative capacity of the cells. We observed control cells exhibited nearly complete recovery from the challenge to DNA replication, whereas the CK1γ1-depleted cells were not able to recover effectively (Figure 6, G and H). This result further supports a role for CK1γ1 in mediating a proper DNA damage response in the S phase of the cell cycle.
Finally, we examined whether CK1γ1 also plays a role in mediating the G2/M checkpoint. To test this possibility, we irradiated control or CK1γ1-depleted cells with ultraviolet (UV) light, labeled the cells with antibodies against the M-phase marker phospho-histone H3, and quantitated the staining by flow cytometry. In these experiments, we observed the expected large reduction in the mitotic marker in the irradiated control cells, but this response was substantially defective in the CK1γ1-depleted cells (Figure 6, I and J). As part of these experiments, we also verified that depletion of CK1γ1 also compromised activation of Chk1 in response to UV (Figure S3C). Together, our results indicate that CK1γ1 plays an important role in regulating DNA damage checkpoint responses in both the S and G2/M phases.
As part of these studies, we sought to rescue the effects of depletion of CK1γ1 by introducing an siRNA-resistant form of CK1γ1 into human cells. However, as shown in Figure S4A, we were able to obtain only a partial rescue of these phenotypes in stable lines of U2OS cells harboring an inducible, siRNA-resistant version of the published form of CK1γ1 (Zhai et al., 1995 ; Kusuda et al., 2000 ). An important consideration for these experiments is that there appear to be multiple isoforms of CK1γ1 in human cells. After performing a systematic search of the human Expressed Sequence Tags database, we could readily identify numerous potentially distinct forms of CK1γ1. After aligning the sequences, we noticed that all of them contained the same sequence at their 5′ end (which encodes the kinase domain), but that their 3′ ends were substantially different. After carefully analyzing possible polyadenylation signal sites and stop codons and performing comparisons with genomic DNA data, we identified at least four potential new isoforms of CK1γ1. On the basis of these sequences, we designed primers specific to each isoform and were able to amplify three out of the four isoforms from human U2OS cells by reverse transcription PCR (RT-PCR; see sequence alignments in Figure S4B). For clarity of presentation, we have designated the published version of CK1γ1 as isoform A, and the remaining three new forms that we were able to isolate as B, C, and D.
To evaluate the differences between these forms, we expressed these proteins as FLAG-tagged fusions in human cells and examined their intracellular localization by indirect immunofluorescence (Figure 7A). Interestingly, their localizations appeared to be quite distinct. We observed that immunofluorescence staining for isoform A covered the entire cell with a prominent staining of the plasma membrane. This distribution is consistent with the fact that isoform A contains a lipid attachment sequence near the C-terminal end of the protein that would confer membrane localization. By contrast, isoforms B and C exhibited both nuclear and cytoplasmic staining, whereas isoform D appeared to reside only in the cytoplasm. None of the isoforms B, C, and D appeared to exhibit membrane localization, which is consistent with the fact that all three proteins lack the lipid attachment sequence.
We then compared the activities of these different proteins in phosphorylating the GST-CKAD fragment of Claspin upon coexpression in human cells (Figure 7B). In this in vivo assay, phosphorylation of Claspin by isoform A was comparatively weak. Significantly, however, both isoforms B and C were very effective in phosphorylating S864 of Claspin. We could not observe good phosphorylation with isoform D, but it was difficult to express this protein at high levels. As another assessment of specificity, we also coexpressed the GST-CKAD with other members of the casein kinase family (namely, CK1α , two different forms of CK1δ, and CK1ε). As shown in Figure S4C, none of these kinases could phosphorylate S864 of Claspin significantly in a side-by-side comparison with CK1γ1 (isoform C).
In an attempt to dissect the relative contributions of these isoforms to phosphorylation of Claspin, we designed isoform-specific siRNAs against one of the nuclear isoforms (isoform C). Unfortunately, the unique sequence of isoform B is not long enough for design of multiple nonoverlapping siRNAs. We observed that depletion of isoform C by two distinct siRNAs resulted in an approximately 50% inhibition of the checkpoint-dependent phosphorylation of Claspin and Chk1 (Figure 7, C–E). These findings suggest isoform C is partially responsible for the phosphorylation of Claspin, and imply that one or more of the other isoforms would carry out the remainder of the phosphorylation.
Finally, we sought to rescue the decreased phosphorylation of Claspin and Chk1 in CK1γ1-depleted cells by introducing siRNA-resistant versions of one or more isoforms of CK1γ1 into the cells. For this purpose, we generated lentiviruses encoding siRNA-resistant versions of isoforms A, B, and C. As shown in Figure 7, F and G, coinfection of siRNA-treated cells with three lentiviruses encoding all three isoforms resulted in good rescue of both the phosphorylation of Claspin (monitored with the exogenously expressed GST-CKAD) and the phosphorylation of Chk1 in response to treatment with APH. In various experiments, we obtained the best rescue with this combination of viruses. We obtained a similar rescue of the phosphorylation of Chk1 in cells lacking the exogenously expressed GST-CKAD (unpublished data). Furthermore, these rescued cells also exhibited a significantly restored cell cycle profile and G2/M checkpoint (Figure S5, A and B). Taken together, these results suggest that multiple isoforms of CK1γ1 are necessary for the normal phosphorylation of Claspin.
In this study, we sought to resolve the molecular basis of a key step in the checkpoint-dependent activation of Chk1 in response to genomic stress. The activation of Chk1 involves phosphorylation-dependent docking of Chk1 with its cognate mediator protein (Claspin) and recognition of the resulting Claspin–Chk1 complex by ATR–ATRIP. In particular, phosphorylation of Claspin on multiple residues in its CKAD mediates the binding of Chk1. Direct biochemical evidence has indicated the presence of Claspin in this complex enhances the ability of ATR–ATRIP to phosphorylate Chk1 on key residues necessary for the ultimate activation of Chk1 (Kumagai et al., 2004 ; Lindsey-Boltz et al., 2009 ). Thus, the kinase that phosphorylates Claspin on the CKAD plays a key role in this pathway.
To address the identification of this kinase(s) systematically, we initially performed a kinome-wide RNAi screen in Drosophila S2 cells. Various RNAi screens in these cells have been used to identify numerous regulators in diverse cellular pathways (Bettencourt-Dias et al., 2004 ; Bjorklund et al., 2006 ; Boutros and Ahringer, 2008 ). For our purposes, the fact that Drosophila contains approximately one-third as many kinases as humans was a considerable advantage. S2 cells appear to possess checkpoint-signaling pathways generally similar to those in other metazoan cells (de Vries et al., 2005 ; Yi et al., 2009 ). In particular, the Drosophila Chk1 homologue Grapes exhibits a conserved checkpoint function in S2 cells. It functions downstream of Mei-41 (the Drosophila homologue of ATR) and is critical for a proper checkpoint response to genotoxic stress.
Nonetheless, the reagents available for the study of checkpoint responses in Drosophila S2 cells are still relatively limited. To circumvent this problem, we introduced the Xenopus version of Chk1 into the Drosophila cells as a more readily traceable marker to monitor the checkpoint response. We were able to establish a system in which phosphorylation of this reporter could be induced following treatment with a variety of replication inhibitors. Moreover, this response seems to have molecular features similar to those present in vertebrate cells. For example, RNAi-mediated knockdown of Drosophila homologues of ATR and Claspin abolished checkpoint-dependent phosphorylation of the exogenously introduced Chk1 reporter molecule.
The results of our screen indicated that knockdown of several casein kinases led to the reduced phosphorylation of Chk1. Following this lead, we next employed a candidate-based cDNA overexpression approach to further pinpoint a specific kinase that can directly phosphorylate the CKAD of Claspin. From these tests, we eventually found that Drosophila Gish, a homologue of CK1γ, could phosphorylate the CKAD of Claspin quite effectively both in vitro and in the Drosophila tissue culture cells. At this juncture, we chose to extend our studies to human cells. Interestingly, humans possess three different versions of CK1γ, namely, CK1γ1, CK1γ2, and CK1γ3. All three proteins have very similar central kinase domains but are significantly different in their N- and C-terminal extensions. We observed that all three kinases could phosphorylate the CKAD relatively well in vitro. However, our further characterizations established that CK1γ1 appears to be the form primarily responsible for the phosphorylation of CKAD in human cells. Upon expression in human cells, only CK1γ1 could phosphorylate the CKAD of Claspin in vivo. More importantly, siRNA-mediated knockdown of CK1γ1 resulted in greatly reduced phosphorylation of Claspin, whereas knockdowns of CK1γ2 and CK1γ3 did not have a significant effect. As expected from the known function of the phosphorylation of Claspin, cells with diminished levels of CK1γ1 were greatly compromised in their ability to carry out the activation of Chk1 in response to a variety of genotoxic agents, including APH, HU, and UV. Furthermore, these cells had the physiological defects characteristic of cells with impairment of the Chk1-mediated signaling pathway. In particular, these cells showed reduced survival following treatment with genotoxic agents, impaired recovery of stalled replication forks, a defective G2/M checkpoint response, and spontaneous DNA damage in the absence of exogenous stress. Taken together, our results indicated CK1γ1 is an important regulator in Chk1-mediated cellular checkpoint responses.
The casein kinase 1 family of serine/threonine kinases is highly conserved and ubiquitously expressed. The functions of CK1 encompass a wide variety of processes, including cell proliferation, cell division, apoptosis, circadian rhythms, and others (Gross and Anderson, 1998 ; Knippschild et al., 2005 ; Cheong and Virshup, 2011 ). In mammals, this family contains at least seven members (α, β, δ, ε, γ1, γ2, and γ3) with multiple splicing variants. All of the CK1 proteins share significant homology in the central kinase domain (53–98% identical), but differ significantly in the flanking N- and C-terminal sequences, which most likely confer unique properties to the various kinases. In the case of CK1γ1, we identified and isolated multiple splicing variants of this kinase from human U2OS cells. These isoforms contain distinct C-terminal sequences (ranging from 3 to 106 amino acids), display discrete subcellular localizations, and exhibit differences in their abilities to phosphorylate the CKAD. A similar phenomenon has also been observed in various organisms in the case of CK1α, which contains four types of isoforms with distinct localizations and kinase activities (Zhang et al., 1996 ; Green and Bennett, 1998 ; Burzio et al., 2002 ). These findings strongly suggest various isoforms of CK1 may participate in distinct cellular activities due to differences in access to and/or affinity for substrates.
Interestingly, we initially found it difficult to achieve good rescue of CK1γ1-depleted cells with vectors encoding an siRNA-resistant version of the major published form of CK1γ1. However, as described in the last part of Results, there are multiple forms of CK1γ1 in human cells. We were able to clone and express three additional versions of CK1γ1, which we designated as isoforms B, C, and D to distinguish them from the published form (isoform A). In cellular localization studies, we found that isoform A had the expected prominent localization to cell membranes (Davidson et al., 2005 ). However, isoforms B and C resided in both the nucleus and cytoplasm, whereas isoform D was mainly cytoplasmic. In coexpression studies, we found that isoforms A, B, and C (but not D) could phosphorylate the CKAD of Claspin, although isoforms B and C were more effective than isoform A in this in vivo assay. Ultimately, we were able to rescue depletion of CK1γ1 by introducing siRNA-resistant forms of isoforms A, B, and C into the cells. It is straightforward to understand how the nuclear isoforms could regulate Claspin, but it is intriguing that a membrane-bound enzyme is also partially responsible. Conceivably, some fraction of this isoform could be absent from the membrane. It is also possible that Claspin could shuttle between the nucleus and cytoplasm and thus be subject to regulation by enzymes in both locations. In this regard, a recent study has shown that perturbed cell-surface signaling through the Sonic Hedgehog (Shh) pathway inhibits ATR-mediated signaling by disrupting the interaction between Claspin and Chk1 (Leonard et al., 2008 ).
It will be important to understand the mechanisms that control the phosphorylation of Claspin by CK1γ1 (see Figure 7H). It is known that phosphorylation of the CKAD in Xenopus egg extracts is dependent upon ATR. However, ATR itself is unable to phosphorylate the critical sites in the CKAD directly. One possibility would be that ATR might somehow regulate the activity of CK1γ1. However, we have been able to detect only a subtle increase in the activity of CK1γ1 in response to genomic stress. Moreover, when we mutated the only apparent potential target site for ATR (Ser361) to Ala in CK1γ1, we could not observe a change in its kinase activity toward the CKAD upon coexpression in U2OS cells (unpublished data). Another possibility is that ATR might regulate the accessibility of Claspin to CK1γ1. Further studies will be required to understand the dynamics of this phosphorylation.
In summary, we identified a conserved casein kinase, Gish/CK1γ1, from Drosophila and humans as a specific enzyme that controls phosphorylation of the CKAD of Claspin. Functional studies have revealed that this kinase is critical for mediating activation of Chk1 and ensuring a proper checkpoint response under conditions of genotoxic stress. Further studies of its regulation and function should help us gain more insight into the molecular basis of checkpoint responses.
Drosophila S2R+ cells were purchased from the Drosophila Genomics Resource Center (Bloomington, IN) and cultured at room temperature (23°C) in Schneider's Drosophila culture medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and 100 U/ml penicillin and 100 μg/ml streptomycin. Stable S2R+ cell lines expressing the pMT-Chk1-NLS-GTS reporter were generated by cotransfection of the reporter plasmid with the pCoHygro plasmid (19:1) and selection with 300 μg/ml hygromycin for ~21 d. Expression of the reporter gene was induced with CuSO4 (500 μM) for 16 h. DNA replication blocks were induced by treating cells with APH (10 μg/ml) for 10 min or HU (2 mM) for 16 h.
Plasmid DNA transfections were performed using FuGENE 6 reagent (Roche, Indianapolis, IN) according to the manufacturer's instructions. Briefly, S2R+ cells (0.6 × 106) were seeded in 12-well plates and grown overnight. On the following day, a mixture of plasmid DNA (0.6 μg) and FuGENE 6 reagent (5 μl) was diluted in 100 μl H2O and incubated with cells in serum-free medium for 4 h. Next, medium containing FBS was added, and cells were incubated for a total of 48 h.
dsRNA-mediated RNA interference in S2R+ cells was performed essentially as described previously (Clemens et al., 2000 ). Briefly, dsRNAs (~500 base pairs long) were produced by in vitro transcription using DNA templates containing T7 promoters at both ends. The quality of dsRNAs was monitored by gel electrophoresis to ensure that only single bands were present. dsRNAs (15 μg) were added directly to 0.5 ml of serum-free medium and incubated with cells for 60 min. Subsequently, 1 ml complete medium with 15% FBS was added, and cells were incubated for another 96 h to allow for turnover of the proteins of interest.
Whole-cell lysates were prepared in TEB150 buffer (50 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 2 mM MgCl2, 5 mM ethylene glycol tetraacetic acid [EGTA], 0.5% Triton X-100, and 10% glycerol) containing freshly added 1 mM dithiothreitol (DTT), 1 mM NaF, 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 μg/ml each of pepstatin, chymostatin, and leupeptin. Lysates were briefly sonicated, diluted in 2× protein sample buffer, and resolved by SDS–PAGE.
HeLa cells, U2OS cells, and U2OS T-REx cells (a kind gift from Thomas C. Spelsberg, Mayo Clinic, Rochester, MN) were cultured in DMEM containing 10% FBS, penicillin, and streptomycin. Plasmid DNA transfections were performed using the FuGENE 6 reagent, and siRNA (50 nM) transfections were carried out with Lipofectamine RNAiMAX (Invitrogen), according to the manufacturer's instructions. For cotransfection experiments with the U2OS T-Rex cells, a 2:1 ratio of GST-CKAD reporter plasmid to the kinase expression plasmid was used. Transfections were carried out for a total of 48 h, and kinase expression was induced by treatment with doxycycline (100 ng/ml) for 16 h.
The pMT-Chk1-NLS-GST reporter plasmid was generated by a two-step cloning method. First, the NLS-GST fragment was generated by PCR and inserted into the Drosophila expression vector pMT/V5-His B plasmid (Invitrogen). Next, the sequence encoding the full-length Xenopus Chk1 was ligated in-frame to the 5′ end of the NLS-GST sequence. The pUAST Drosophila casein kinase expression plasmids were kind gifts from Jin Jiang (University of Texas Southwestern Medical School, Dallas, TX). The full-length cDNA clones of human CK1γ1, CK1γ2, and CK1γ3 were purchased from Open Biosystems (Thermo Scientific, Lafayette, CO). We also obtained cDNAs encoding CK1α, a short and long form of CK1δ (409 and 415 amino acids in length, respectively), and CK1ε from the same source. For expression in human cells, these cDNAs were cloned into the tetracycline-inducible pcDNA5/TO (Invitrogen) vector, which encodes an N-terminal FLAG tag. siRNA-resistant versions of CK1γ1 were created through introduction of silent mutations into three nucleotides in the middle of siRNA-targeting regions by site-directed mutagenesis.
Stealth siRNA duplexes specific for CK1 gamma kinases and a control siRNA (low GC) were purchased from Invitrogen. The sense strand sequences of these siRNAs are listed in Supplemental Table S1. These CK1γ1 siRNAs are directed against common regions present in all isoforms. esiRNAs were produced in vitro essentially by following the protocol kindly provided by Eric Lau and Wei Jiang (Burnham Institute, La Jolla, CA; Lau et al., 2006 ).
Immunoblotting and enhanced chemiluminescence (ECL) were performed by standard methods. For detection of phosphorylation signals, 2 mM Na3VO4 and 20 mM NaF were included in all immunoblotting solutions to block potential phosphatase activities. For quantification of immunoblotting signals, membranes were stripped and blotted with IRDye-800- or IRDye-680-conjugated secondary antibodies and scanned with the Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE).
For generation of anti-CK1γ1 antibodies, the sequence encoding the full-length human CK1γ1 isoform A was cloned into the pET30a vector and expressed in bacterial cells. Recombinant protein was solubilized in 6 M urea and purified with nickel agarose beads. This protein was used to immunize rabbits, and the generated antibody was affinity purified. Because the expression level of CK1γ1 in U2OS cells is low, we carried out immunoprecipitation and then immunoblotting on a relatively large scale for detection of endogenous CK1γ1. Whole-cell lysates were prepared in TEB150 buffer with phosphatase and protease inhibitors as described above. Next, whole-cell lysates were incubated with anti-CK1γ1 antibodies coupled to the protein-A Dynabeads (Invitrogen) for 90 min at 4°C. Finally, the beads were washed two times with Buffer A (10 mM HEPES-KOH, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40, 2.5 mM EGTA, and 20 mM β-glycerolphosphate) and two times with HEPES-buffered saline (HBS; 10 mM HEPES-KOH, pH 7.5, and 150 mM NaCl). Finally, samples were boiled in protein sample buffer and subjected to SDS–PAGE.
Antibodies against the following targets were obtained from the indicated sources: Chk1 P-Ser345 and Chk1 P-Ser317 (Cell Signaling Technology, Beverly, MA); Chk1 (G-4), CK1γ2 (N-20), GST (Z-5), and Rad17 (H-3; Santa Cruz Biotechnology, Santa Cruz, CA); Claspin P-Ser864 (Kumagai and Dunphy, 2003 ); γ-H2AX (Millipore, Billerica, MA); ATR (Affinity BioReagents, Golden, CO); TopBP1 (Bethyl Laboratories, Montgomery, TX), Rad17 P-Ser645 (a kind gift from Eva Y. Lee, University of California, Irvine, CA); FLAG epitope (M2, Stratagene, Agilent, Santa Clara, CA); and α-tubulin (Calbiochem, San Diego, CA).
Recombinant CK1 gamma kinases with FLAG, hemagglutinin (HA), and His6 tags were produced in Sf9 insect cells and purified by following a previously described protocol (Kumagai and Dunphy, 2000 ). Briefly, the full-length cDNAs of CK1 gamma kinases were cloned into the pFastBac-HA-HTa vector. Recombinant baculoviruses were generated with the Bac-to-Bac system (Invitrogen), and recombinant proteins were purified using nickel agarose beads. For production of recombinant wild-type or kinase-dead (N169A) versions of GST-tagged CK1γ1, the appropriate cDNAs were cloned into the pGEX-4T-3 vector. Recombinant proteins were produced in Escherichia coli cells and purified using the Glutathione Sepharose 4 Fast Flow beads (GE Healthcare, Waukesha, WI), according to the manufacturer's instructions.
Total RNA was isolated using the TRIzol RNA isolation reagent (Invitrogen). Reverse transcription PCR (RT-PCR) reactions were carried out using the SuperScript III One-Step RT-PCR System (Invitrogen) according to the manufacturer's instructions. Specific primers were designed to span exon–intron boundaries of respective genes, ensuring the results were not affected by genomic DNA contamination. The linear ranges of amplification cycle for each gene were predetermined by electrophoresis and quantification of the PCR products. The sequences of RT-PCR primers are listed in Table S2.
FLAG-tagged Drosophila casein kinases were expressed in S2R+ cells for 48 h. Whole-cell lysates were prepared and incubated with anti-FLAG antibody-conjugated Dynabeads at 4°C for 60 min. Beads were washed two times with buffer A and three times with HBS. The bead-bound kinases were incubated with the GST-CKAD fragment (500 ng) in kinase buffer (10 mM Tris-HCl, pH 7.5, 10 mM MgCl2, and 1 mM DTT) containing 10 mM ATP at room temperature for 30 min. Finally, the reactions were stopped by addition of protein sample buffer and boiling. In the case of in vitro kinase assays using recombinant proteins, 250 ng of purified CK1γ1, CK1γ2, CK1γ3, or Xenopus Chk1-GST-His6 (Kumagai and Dunphy, 2000 ) protein, instead of the immunoprecipitated kinases, were added to the reactions.
U2OS cells expressing GST-CKAD were lysed in TEB150 buffer as described above. Whole-cell lysates were incubated with Glutathione Sepharose 4 Fast Flow beads (GE Healthcare) at 4°C for 1 h. Beads were washed two times with buffer A and three times with HBS. Finally, the beads were boiled in an equal volume of 2× protein sample buffer and subjected to SDS–PAGE.
For immunofluorescence staining, cells grown on coverslips were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) and permeabilized with 0.5% Triton X-100 in PBS. After undergoing blocking with 5% goat serum in PBS for 30 min, cells were incubated with primary antibodies at room temperature for 60 min. Following three washes with PBS, cells were incubated with Alexa Fluor 594-conjugated secondary antibodies for 60 min. Finally, cells were washed, counterstained with 5 μg/ml Hoechst 33258, and mounted in VECTASHIELD mounting medium. For detection of EdU incorporation to assess DNA replication, the Click-iT system (Invitrogen) was used as described previously (Kumagai et al., 2010 ). For immunofluorescence staining of the FLAG-tagged CK1γ1 isoforms, U2OS cells were first transfected with pcDNA5-FLAG-CK1γ1 plasmids for 48 h. Cells were then fixed and stained with anti-FLAG (M2) antibody and Alexa Fluor 488–conjugated anti–mouse secondary antibodies.
For detection of replicating cells in S phase, cells were labeled with 10 μM EdU for 45 min and stained with the Click-iT system. Briefly, cells were fixed with 70% ethanol and incubated overnight. After undergoing washing with PBS containing 1% bovine serum albumin (BSA), cells were stained with freshly prepared Click-iT reaction cocktail containing Alexa Fluor 488 azide for 30 min at room temperature. Finally, cells were washed again, treated with RNase A (50 μg/ml), and counterstained with 7-amino-actinomycin D (7-AAD). Flow-cytometry analyses were performed with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). For G2/M checkpoint assays, cells were treated with UV (20 J/m2) and incubated for 1 h. Cells were then fixed and stained with anti–phospho-histone H3 (Ser10) antibody conjugated to Alexa Fluor 488.
U2OS cells transfected with control or CK1γ1 siRNAs were seeded in 6-well plates at low density (~200 cells/well). Cells were treated with HU (0, 0.1, 0.5, 1.0, and 2.0 mM) for 24 h. After removal of HU and addition of fresh medium, cells were continuously incubated for an additional 8 d and stained with crystal violet. Surviving colonies were counted by scanning plates with the Odyssey Imaging System.
Rescue experiments with lentiviruses were essentially performed as described previously (Qin et al., 2003 ). Briefly, lentiviruses expressing siRNA-resistant versions of CK1γ1 isoforms were produced by cotransfecting 293T cells with four lentivector plasmids (pFUW, pRSV-Rev, pMDLg/pRRE, and pVSV) in a 2:1:1:1 ratio using the FuGENE 6 reagent. Viral supernatants were harvested 48 h later and filtered. To rescue the effect of CK1γ1 siRNA, U2OS cells were infected with viruses in the presence of 10 μg/ml of Polybrene (Millipore) for 8 h. After an additional incubation of 16 h, cells were transfected with control or CK1γ1 siRNA. Two days after siRNA transfection, cells were treated with aphidicolin for 10 min, and whole-cell lysates were then immediately prepared, resolved on SDS–PAGE, and processed for immunoblotting.
We are grateful to laboratory members for helpful advice and comments on the manuscript. We also thank Rochelle Diamond for assistance with flow cytometry and J. Jiang (University of Texas Southwestern Medical School, Dallas, TX) for Drosophila casein kinase expression vectors. This work was supported by National Institutes of Health grants GM-043974 and GM-070891 and an Ellison Senior Scholar in Aging award to W.G.D. Work in the laboratory of D.M.G. was supported by Cancer Research UK and the Biotechnology and Biological Sciences Research Council (United Kingdom).
This article was published online ahead of print in MBoC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E11-01-0048) on June 16, 2011.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Dear Attorneys.The thing is me and my husband get married May 2 2011, it'll be 2 years next month.We had applied for 2 years green card and got our interview at February 6, 2013. Im.officer sad, that she need couple more documents .We get it RFE in a couple weeks, and a dead line to send it back - is May 6(90 days from interview)Is it any chance that if officer will receive our request after 2 years anniversary (after may 2)than she will gave us a 10 years green card instead of 2 ?
Immigration Attorney
If the approval comes after 2 years of marriage, then a 10 year permanent card will be granted.
Immigration Attorney
The determining date is the date you are awarded permanent residency. The LPR is conditional if it is based on a marriage that was less than 2 years old "on the day [the alien is] given permanent residence". http://www.uscis.gov/portal/site/uscis/menuitem.5af9bb95919f35e66f614176543f6d1a/?vgnextoid=745218a1f8b73210VgnVCM100000082ca60aRCRD
Legal disclaimer: The statement above is provided by CC Abbott is based on general assistance and not intended to be a legal opinion because not all the facts are provided. The person requesting information and all others reading the answer should retain an attorney who is permitted by the state bar within the jurisdiction who can examine the complete facts and provide a legal opinion on your case. All information provided in the above answer and other information provided by CC Abbott does not create an attorney/client relationship within any state of Federal law.
Immigration Attorney
Whether you get a conditional card or a permanent card depends on how long you have been married at the time the status is granted.
J Charles Ferrari Eng & Nishimura 213.622.2255 The statement above is general in nature and does not constitute legal advice, as not all the facts are known. You should retain an attorney to review all the facts specific to your case in order to receive advise specific to your case. The statement above does not create an attorney/client relationship. Answers on Avvo can only be general ones, as specific answers would require knowledge of all the facts. As such, they may or may not apply to the question.
Immigration Attorney
Samuel Ouya Maina, Esq. 415.391.6612 firstname.lastname@example.com Law Offices of S. Ouya Maina, PC 332 Pine Street, Suite 707 San Francisco, CA 94104
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subset: commoncrawl


text:
AP names Roger Schneider as Michigan news editor
DETROIT (AP) – Roger Schneider, a veteran editor in Wisconsin who has led The Associated Press' news operations there for the past seven years, is moving across Lake Michigan to become the cooperative's news editor in Michigan.
The appointment was announced Friday by David Scott, the AP's regional editor for the central U.S.
In Wisconsin, Schneider has directed the AP's coverage of news as varied as Gov. Scott Walker's push to curb the union rights of state workers to this past summer's deadly shooting at a Sikh temple in suburban Milwaukee.
"Roger is an editor who has a history of working with reporters to deliver both scoops and insightful enterprise reporting based off those scoops," Scott said. "Getting the news to AP's customers before all others is a hallmark of our Wisconsin report under his leadership, and we're excited to see the same in Michigan once he begins working with our staff in Detroit, Lansing and Traverse City."
Schneider is a 1979 graduate of the University of Wisconsin-Milwaukee, where he earned a bachelor of science degree in journalism and political science. He worked as an editor at Beloit Daily News and the Green Bay Press-Gazette in Wisconsin, and the Asbury Park Press in New Jersey, before joining the AP as Wisconsin News Editor in 2005.
The addition of Schneider to the Michigan staff is the second in the past month for the AP, which recently hired reporter David Eggert to cover state government in Lansing. Both moves are signs of AP's strong commitment to providing members of the cooperative with the best in state news, said AP's Regional Director-East Eva Parziale.
Schneider, 55, will be based in Detroit and begin work as Michigan News Editor on Feb. 1.
(Copyright 2013 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.)
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text:
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All About Jazz needs your help and we have a deal. Contribute $20 (or more) and we'll hide those six pesky Google ads that appear on every page for a full year! You'll also fund website expansion.
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text:
For some reason wordpress displays an entire post within paragraph tags
ie. < p > YOUR ENTIRE POST < / p>
So instead of having proper paragraphs the post's paragraphs are done using the < BR / > tag.
How can I get wordpress to use the paragraph tag correctly for posts.


text:
The Connecticut Citizens Defense League is a non-partisan, grassroots organization devoted to advocating rights affirmed by the Constitutions of the United States of America and the State of Connecticut.
For the upcoming 2016 election, the CCDL Executive Committee has considered the past voting record and views that each candidate holds with respect to our constitutional rights.
This year, our job of vetting candidates has come with some regret. Some legislators we endorsed in the past have voted for a gun control bill (HB5054) this past session. Interestingly enough, some legislators who voted for gun control in 2013 reversed course and did not support gun control efforts this year. While we applaud this turn around, at this time we simply cannot endorse candidates who supported SB1160 in 2013.
One important point: This year, more legislators from both parties voted for anti-gun bills, than voted for them during the emotionally charged 2013 session. This trend should concern all gun owners, and be a cause for them to actively support the following candidates:
CCDL Endorsed Candidate List updated 7/4/16
CT House:
District Candidate Name Towns Represented
8 Timothy J. 'Tim' Ackert – Columbia, Coventry, Tolland, Vernon
13 Mark Tweedie – Glastonbury, Manchester
23 Devin R Carney – Lyme, Old Lyme, Saybrook, Westbrook
24 Jim Sanders – New Britain, Newington
30 Chris Morelli – Berlin, Southington
33 Linda J Szynkowicz – Middletown
34 Melissa H. Ziobron – Colchester, East Haddam, East Hampton
35 Jesse D MacLachlan – Clinton, Killingworth, Westbrook
36 Bob Siegrist – Chester, Deep River, Essex, Haddam
37 Holly H Cheeseman – East Lyme, Salem
38 Lauren Shaw – Montville, Waterford
40 John F Scott – Groton, Ledyard
41 Aundre P Bumgardner – Groton, New London
42 Mike France – Ledyard, Montville, Preston
44 Anne Dauphinais – Killingly, Plainfield
47 Doug Dubitsky – Norwich, Sprague, Franklin, Lebanon, Lisbon, Hampton, Chaplin, Canterbury, Scotland
49 Tony Fantoli – Windham
52 Kurt Vail – Somers, Stafford
53 Sam Belsito – Ashford, Tolland, Willington
57 Christopher C Davis – East Windsor, Ellington
61 Tami Zawistowski – Suffield, East Granby, Windsor
62 Bill Simanski – Barkhamsted, East Granby, Granby, New Hartford
63 Jay M. Case – Barkhamsted, Canaan, Colebrook, Hartland, Norfolk, North Canaan, Winchester
64 Brian Ohler – Canaan, Cornwall, Goshen, Kent, Norfolk, North Canaan, Salisbury, Sharon,Torrington
68 Eric C. Berthel – Oakville, Watertown, Woodbury
70 Rosa Rebimbas – Naugatuck
73 Steve Giacomi – Waterbury
76 John E Piscopo – Burlington, Harwinton, Litchfield, Thomaston
77 Cara C Pavalock – Bristol
78 Whit Betts – Bristol, Plymouth, Terryville
80 Robert C. 'Rob' Sampson – Wolcott, Southington
85 Serge G Mihaly – Wallingford
86 Vincent Candelora – Durham, Guilford, North Branford, Wallingford
89 Lezlye Zupkus – Bethany, Cheshire, Prospect
90 Craig Fishbein – Cheshire, Wallingford
103 Andy Falvey – Cheshire, Southington, Wallingford
113 Jason Perillo – Shelton
119 Pam Staneski – Orange, Milford
122 Ben McGorty – Shelton, Stratford, Trumbull
131 David K. Labriola – Naugatuck, Oxford, Southbury
CT Senate:
District Candidate Name Towns Represented
2 Theresa Tillett – Hartford, Bloomfield, Windsor
7 John A. Kissel – East Granby, Enfield, Granby, Somers, Suffield, Windsor, Windsor Locks
8 Kevin D Witkos – Avon, Barkhamsted, Canton, Colebrook, Granby, Hartland, Harwinton, New Hartford, Norfolk, Simsbury, Torrington
13 Len Suzio – Cheshire, Meriden, Middletown, Middlefield
16 Joe Markley – Cheshire, Prospect, Southington, Wolcott, Waterbury
18 Heather Somers – Griswold, Groton, North Stonington, Plainfield, Preston, Sterling, Stonington, Voluntown
19 Barbara Crouch – Columbia, Franklin, Hebron, Lebanon, Ledyard, Lisbon, Marlborough, Montville, Norwich, Sprague
19 Catherine A. 'Cathy' Osten – Columbia, Franklin, Hebron, Lebanon, Ledyard, Lisbon, Marlborough, Montville, Norwich, Sprague
20 Paul M Formica – Bozrah, East Lyme, Montville, New London, Old Lyme, Old Saybrook, Salem, Waterford
29 John French – Brooklyn, Canterbury, Killingly, Mansfield, Putnam, Scotland, Thompson, Windham
30 Craig A Miner – Brookfield, Canaan, Cromwell, Goshen, Kent, Litchfield, Morris, New Milford, North Canaan, Salisbury, Sharon, Torrington, Warren, Winchester
31 Henri R Martin – Bristol, Harwinton, Plainville, Plymouth, Thomaston
32 Robert J. 'Rob' Kane – Bethlehem, Bridgewater, Middlebury, Oxford, Seymour, Southbury, Roxbury, Washington, Watertown, Woodbury
33 Art Linares – Chester, Clinton, Colchester, Deep River, East Haddam, East Hampton, Essex, Haddam, Lyme, Old Saybrook, Portland, Westbrook
35 Tony Guglielmo – Ashford, Chaplin, Coventry, Eastford, Ellington, Hampton, Pomfret, Stafford, Tolland, Union, Vernon, Willington, Woodstock
US House:
District Candidate Name
1 Matthew Corey
2 Daria Novak
3 Angel Cadena
5 Clay Cope
US Senate:
Candidate Name
Dan Carter
*CCDL reserves the right to add or remove candidates as they are vetted by the Executive Committee. This page may be updated to reflect that. 7/4/2016 – Added: 6/29/2016 – Added:
CT House 68 Eric Berthel
CT House 86 Vin Candelora
CT House 78 Whit Betts
US House 1 Matthew Corey
7/4/2016 – Added:
6/29/2016 – Added:


text:
Chiefs depart Ballast Point
Head brewer Yuseff Cherney joins exodus of executives
Ian Anderson 6 p.m., July 29
Peter (B-boy) Alan and Donna.
Image posted by Peter Trevino
December 20, 2013
Ian Anderson 6 p.m., July 29
Garrett Harris 4 p.m., July 29
Ken Harrison 2:30 p.m., July 29
Andrew Hamlin 1 p.m., July 29
Kate Peterson 12:30 p.m., July 29
Matthew Lickona noon, July 29
Ian Anderson 11 a.m., July 29
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text:
Future of Lipid Oxidation, Antioxidants, Nutrition, Genomics and Metabolomic Aspects
Saturday, April 28
Organizers: Edwin N, Frankel, Adjunct Professor, Department of Food Science and Technology, University of California–Davis, USA
Designed to help chemists, food scientists, researchers, and industrial managers understand the major factors that affect the stability of oil-bearing foods, how antioxidants can be used to minimize the effects of lipid oxidation in foods and biological systems, and how antioxidants increase their oxidative stability as a basis for development of healthful products.
|Free Radical Lipid Oxidation: Mechanisms.
Edwin N. Frankel, University of California, Davis, USA.
|Control of Oxidation, Oxidative Stability, and Antioxidants.
Edwin N. Frankel, University of California, Davis, USA.
|Lipid Oxidation in Biology—A Systems Approach.
J. Bruce German, University of California, Davis, USA.
|Personalizing Oxidation and Protection: Genomics to Metabolomics.
J. Bruce German, University of California, Davis, USA.
|Fish Oils and Algal Oils.
Neil MacFarlane, Consultant, UK.
|Lipid Oxidation Volatiles and Flavors.
Neil MacFarlane, Consultant, UK.
|Improving Oxidative Stability of Vegetable Oils without Hydrogenation.
Kathleen Warner, Fats & Oils Consulting, USA.
|Questions & Answers / Closing Comments.
Edwin Frankel, University of California, Davis, USA.


text:
JEGS Dragster Cover
Manufactured from strong, long wearing and durable black 600 Denier fabric to offer the best protection from harmful elements. This cover is nearly bulletproof! Designed for 215" - 240" wheelbase dragsters without a rear wing This cover features sewn in straps and buckles for easy installation and maximum retention. Includes a handy storage bag.
JEGS Dragster Cover
Dragster Cover
Ships Monday
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text:
|Publication number
|Publication type
|Application number
|Publication date
|Jun 22, 2004
|Filing date
|Dec 9, 2002
|Priority date
|Dec 10, 2001
|Fee status
|Publication number
|Boris Altshuler, James F. Reynolds
|Original Assignee
|Boris Altshuler, James F. Reynolds
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (7), Referenced by (27), Classifications (14), Legal Events (4)
|External Links: USPTO, USPTO Assignment, Espacenet
This application claims the benefit of provisional application Serial No. 60/339,839, filed Dec. 10, 2001.
The present invention relates generally to anti-terrorist attack measures available to civilians and pertains, more specifically, to a gas mask available to civilians as an emergency measure to counter biological, germ and gas contaminants in the air.
Recent events have prompted the need for the emergency protection of civilians from biological, germ and poisonous gas pollutants in the air, generally designated herein as hazardous air pollutants (HAPs). The most effective methods for controlling HAPs utilize adsorption to remove HAPs from the ambient air. The most common adsorbent employed in such methods is activated carbon. Activated particles in a bed of activated carbon are very porous and have large surface-to-volume ratios. Gases penetrate pore spaces in the bed and contact the large surface areas provided in the bed, where the HAPs adhere so as to be eliminated from the ambient air.
The HAP is held on the activated carbon surface either by physical attractive forces or, in certain cases, depending upon the chemical nature of the molecule and the surface, by chemical forces (chemisorption). In any particular system, both types of adsorption can occur, as well as some intermediate types of adsorption. The adsorption capacity of activated carbon for any given HAP may be represented by an adsorption isotherm that relates the amount of HAP adsorbed (adsorbate) to the equilibrium pressure (concentration) at a constant temperature. Typically the adsorption capacity of activated carbon increases as molecular weight of the HAP increases. Unsaturated compounds are more completely adsorbed than saturated compounds, and cyclical compounds are more easily adsorbed than linearly structured materials.
Adsorption is not unlimited. As the exposed surfaces become increasingly covered with molecules of adsorbate, the rate of adsorption diminishes, reaching zero when saturation of the surface is complete. Thus, the useful life of an activated carbon bed depends upon the concentration of HAP in the air, the amount of gas which passes through the carbon bed, and the total amount of carbon in the bed. A good grade of activated carbon in a bed reaches saturation under high concentrations of HAP when the amount of HAP adsorbed reaches about twenty percent of the weight of carbon in the bed. Lesser grades adsorb down to about five to eight percent of the weight of carbon in the bed.
Adsorption in an activated carbon bed also effectively removes very low concentrations of organics and such beds frequently are specified for air deodorization where the concentration of pollutants often is below five parts-per-million (ppm). Bed depth in such current commercial carbon bed cleaners utilizing granular activated carbon generally is in the range of about 0.5 inch to 3.0 inches, with nominal bed residence times of about 0.025 second to 0.1 second. Carbon systems which are combined with air conditioning filters have a suitable design value of air flow rate of 0.1 ft/second for a bed depth of 0.5 inch.
The properties of different activated carbons can vary widely, and all activated carbons are selective to a certain degree. Activated carbon can be utilized in the form of particles or granules, as well as in the form of activated carbon fibers and carbon-coated fibers. Activated carbon remains as the only reliable physical adsorbing agent for protection against anticipated poison gas attacks. Accordingly, activated carbon is one of the ingredients of civilian and military gas masks and is installed in air conditioning equipment used in connection with underground shelters.
The present invention utilizes the above attributes of activated carbon in a civilian gas mask which can protect personnel against chemical and biological terrorist attacks. As a rule, such attacks take place under indoor conditions, within buildings, shopping malls, subways and the like, where the highest concentration of chemical and biological agents can serve as weapons utilizing a minimum amount of toxic compounds. In such situations, a civilian gas mask need be used for only the relatively short period of time during which personnel can be evacuated to a safe environment. Typically, that duration will be less than one hour.
The present invention provides several objects and advantages, some of which are summarized as follows: Provides a small and compact gas mask which can be stored conveniently in a drawer, or readily carried in a briefcase or coat pocket for emergency use; is available for ready use during a period sufficient to allow a user to escape a contaminated area; is simple in construction and effective in combating biological, germ and poisonous gas contamination; allows full visual effectiveness with enough roominess to avoid claustrophobic reactions, as well as other stressful and emotional effects; enables continuous circulation of fresh, breathable air for promoting well-being; precludes condensation and concomitant impairment of vision by supplying continuously circulated fresh air; provides a simplified construction which is economical to manufacture and easy to use; adapts a universally accepted cap construction for effective and reliable performance readily made available for widespread use.
The above objects and advantages, as well as further objects and advantages, are attained by the present invention which may be described briefly as a civilian anti-terrorist attack gas mask for use by a person as an emergency measure to remove hazardous air pollutants present in air drawn from an ambient atmosphere having an ambient air pressure, and provide the person with suitably breathable air, the gas mask being configured for fitting over the person's head, above the person's neck and adjacent the person's face, the gas mask comprising: a support structure configured for fitting to the person's head; a brim carried by the support structure for projecting outwardly from the person's head upon fitting the support structure to the person's head, the brim having an outside and an inside; a hood carried by the brim and extending from the brim for establishing a chamber having at least a portion contiguous with the person's face, the chamber being bounded by the hood and the inside of the brim; a passage in the brim, the passage extending from the inside of the brim to the outside of the brim so as to communicate with the chamber and with the ambient atmosphere; an adsorption element in the passage for communicating with the chamber and with the ambient atmosphere; and an impeller assembly for drawing air from the ambient atmosphere into the passage and passing the air through the adsorption element and into the chamber to remove hazardous air pollutants from the air drawn into the passage and deliver an essentially continuous supply of fresh breathable air to the chamber while maintaining an air pressure in the chamber raised above the ambient air pressure.
The invention will be understood more fully, while still further objects and advantages will become apparent, in the following detailed description of a preferred embodiment of the invention illustrated in the accompanying drawing, in which the single FIGURE is a longitudinal cross-sectional view showing a civilian gas mask constructed in accordance with the present invention.
Referring now to the single FIGURE of the drawing, a civilian gas mask constructed in accordance with the present invention is illustrated at 10 and is seen to include a support structure having an overall configuration resembling a common baseball cap 12 including a crown 14 and a brim in the form of a peak 16. The crown 14 incorporates an adjustable flexible band 18 which enables the crown 14 to be fitted appropriately to the head 20 of a person 22, as shown.
Peak 16 extends forward from the crown 14 to a forward end 24, and a hood 26 is carried by the cap 12 and depends from the cap 12 to drape downwardly over the head 20 of the person 22, as shown. Hood 26 is constructed of a pliable material and extends downwardly to a lowermost terminal end 28 where the hood 26 is gathered around the neck 30 of the person 22, as by a gathering arrangement which includes a drawstring 32, for fitting loosely about neck 30 for purposes described below. At least a forward portion 34 of the hood 26 is transparent and falls in front of the face 36 of the person 22 to provide a transparent window through which the person 22 can view surroundings. To that end, hood 26 preferably is constructed of a synthetic polymeric material, such as PVC, which provides the hood 26 with a requisite pliability and flexibility as well as the desired transparency at portion 34.
Peak 16 includes an activated carbon adsorption element 40 placed within a passage 41 extending between an upper support screen 42, located at the outside of the peak 16, and a lower support screen 44, located at the inside of the peak 16. A fine particle filter element 46 is placed within the passage 41, beneath the carbon element 40. Carbon element 40 may be in the form of a bed of carbon particles or granules, or activated carbon fibers or carbon-coated fibers. An impeller assembly in the form of a fan 50 includes a motor 52 secured at peak 16 and an impeller 54 housed within a safety cage 56 which includes a tubular shroud 58 depending from the inside of the peak 16 and extending circumferentially around impeller 54. A power supply shown in the form of a battery pack 60 is secured to the cap 12 at the band 18 and is connected selectively to the fan 50 through a power switch 62.
With the cap 12 in place on the person 22, as shown, the switch 62 is operated to actuate the fan 50. Ambient air is drawn through the passage 41 and into the hood 26 through the activated carbon adsorption element 40 and the filter element 46 so that any toxic compounds in the ambient air are adsorbed, any fine particulate materials, including biological agents, are filtered from the air, and the air supplied to the person 22 is suitable for breathing, free of toxic compounds and other harmful particulate materials. At the same time air pressure within the hood 26 is raised above ambient air pressure so as to preclude the leakage of contaminated ambient air into the hood 26, especially at the loosely fitted terminal end 28. An essentially continuous supply of fresh breathable air is assured by the continued operation of fan 50, with the exhaust of air from beneath the hood 26 taking place largely at the loosely fitting terminal end 28 of the hood 26. Unwanted condensation of water on the transparent portion 34 is prevented by the continuous circulation of fresh air within the hood 26. In addition, the continuous circulation of fresh air, as accomplished by the fan 50, enables easy breathing, especially for older people and people with breathing problems and, combined with the roominess provided under hood 26, promotes a certain amount of well-being in that any claustrophobic effects are reduced, with a concomitant reduction of stress and nervous emotional effects. Further, the roominess provided under the hood 26 enables effective use of the gas mask 10 by persons having facial hair and persons wearing glasses.
It will be seen that the present invention attains the several objects and advantages summarized above, namely: Provides a small and compact gas mask which can be stored conveniently in a drawer, or readily carried in a briefcase or coat pocket for emergency use; is available for ready use during a period sufficient to allow a user to escape a contaminated area; is simple in construction and effective in combating biological, germ and poisonous gas contamination; allows full visual effectiveness with enough roominess to avoid claustrophobic reactions, as well as other stressful and emotional effects; enables continuous circulation of fresh, breathable air for promoting well-being; precludes condensation and concomitant impairment of vision by supplying continuously circulated fresh air; provides a simplified construction which is economical to manufacture and easy to use; adapts a universally accepted cap construction for effective and reliable performance readily made available for widespread use.
It is to be understood that the above detailed description of a preferred embodiment of the invention is provided by way of example only. Various details of design and construction may be modified without departing from the true spirit and scope of the invention, as set forth in the appended claims.
|Cited Patent
|Filing date
|Publication date
|Jul 25, 1983
|Oct 29, 1985
|Chien Chao Huei
|Multiple-effect respirator
|Feb 22, 1988
|Aug 1, 1989
|Coal Industry (Patents) Limited
|Sep 22, 1987
|Jan 16, 1990
|Waters William A
|Air conditioned headwear having convertible power module
|Sep 4, 1992
|Jun 20, 1995
|Stroud; Kevin J.
|Hat-mounted fan
|Jul 30, 1993
|Nov 26, 1996
|Mine Safety Appliances Company
|Helmet respirator apparatus
|Mar 24, 1998
|Dec 8, 1998
|Lu; Yao
|Safety helmet with alarm means for calling for help
|Jul 2, 2002
|Sep 23, 2003
|Stryker Instruments
|Air filtration system including a helmet assembly
|Citing Patent
|Filing date
|Publication date
|Apr 7, 2003
|May 2, 2006
|Joseph Manne
|Air curtain device
|Dec 20, 2006
|Nov 10, 2009
|The United States Of America As Represented By The Secretary Of The Army
|Quantitative fit test system and method for assessing respirator biological fit factors
|Aug 27, 2004
|May 11, 2010
|Campus Housing Company LLC
|Inflatable protective enclosure
|May 21, 2003
|Feb 1, 2011
|3M Innovative Properties Company
|Heat management system for industrial safety equipment
|Sep 20, 2011
|Microtek Medical, Inc.
|Helmets and methods of making and using the same
|Mar 7, 2007
|Nov 15, 2011
|Pericles Deavila
|Mobile safety compliance apparatus
|Nov 6, 2009
|Apr 10, 2012
|The United States Of America As Represented By The Secretary Of The Army
|Quantitative fit test system and method for assessing respirator biological fit factors
|May 4, 2004
|Jul 9, 2013
|The United States Of America As Represented By The Secretary Of The Army
|Enhanced chemical/biological respiratory protection system
|Jan 8, 2010
|Aug 27, 2013
|Hancock Medical, Inc.
|Self-contained, intermittent positive airway pressure systems and methods for treating sleep apnea, snoring, and other respiratory disorders
|Nov 25, 2013
|Dec 30, 2014
|Hancock Medical, Inc.
|Positive airway pressure system with head position control
|Nov 2, 2012
|Jan 6, 2015
|Hancock Medical, Inc.
|Positive airway pressure system with head position control
|Dec 15, 2014
|Nov 10, 2015
|Hancock Medical, Inc.
|Positive airway pressure system with head position control
|Jul 22, 2010
|Dec 29, 2015
|Vent-A-Hood, Ltd.
|Duct-free cooking air filtration systems and methods
|Apr 7, 2003
|Oct 9, 2003
|Joseph Manne
|Air curtain device
|May 21, 2003
|Jul 29, 2004
|Alvey Jeffrey A.
|Heat management system for industrial safety equipment
|May 4, 2004
|Nov 10, 2005
|Grove Corey M
|Enhanced chemical/biological respiratory protection system
|Aug 27, 2004
|Mar 2, 2006
|University Service Corporation Llc
|Inflatable protective enclosure
|Apr 23, 2007
|Nov 1, 2007
|Tiara Medical Systems, Inc.
|Self-contained respiratory therapy apparatus for enhanced patient compliance and therapeutic efficacy
|Feb 26, 2007
|Aug 28, 2008
|Microtek Medical, Inc.
|Helmets and methods of making and using the same
|Jul 31, 2008
|Feb 5, 2009
|Robert Ochoa
|Cap having illuminating and pivotably movable fan
|Oct 8, 2007
|Apr 9, 2009
|Sector 10 Holdings, Inc.
|Distributed safety apparatus
|Nov 15, 2007
|May 21, 2009
|Robert Ochoa
|Cap having an illuminating fan and heating device
|Apr 15, 2008
|Oct 15, 2009
|Robert Ochoa
|Safety helmet having pivotably movable fan
|Dec 24, 2009
|E.D. Bullard Company
|Powered air purifying respirator
|Apr 30, 2008
|Jan 7, 2010
|Wood Jess W
|Hat with a Fan and Filter
|Jul 8, 2010
|Bowditch Nathaniel L
|Self-contained, intermittent positive airway pressure systems and methods for treating sleep apnea, snoring, and other respiratory disorders
|Jan 28, 2015
|May 21, 2015
|Resmed Motor Technologies Inc
|U.S. Classification
|128/201.25, 128/205.12, 128/205.27, 2/209.12, 2/195.1, 128/204.18, 2/171.3, 2/175.1
|International Classification
|Cooperative Classification
|European Classification
|Dec 21, 2007
|Fee payment
Year of fee payment: 4
|Feb 6, 2012
|Maintenance fee reminder mailed
|Jun 22, 2012
|Lapse for failure to pay maintenance fees
|Aug 14, 2012
|Expired due to failure to pay maintenance fee
Effective date: 20120622


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EVENT China and Latin America: sustainability and development
As part of the series on New and Old Silk Roads in...
The University of Texas at Austin Department of Asian Studies Graduate Conference (i.e. the 2013 Texas Asia Conference) will be held this year on Fri and Sat, November 1st & 2nd, 2013 in Austin, Texas (USA). The Texas Asia Conference (TAC) is a wonderful opportunity for graduate students to present their original research on all aspects of Asia (ancient and modern), become familiar with other graduate students' work, and meet burgeoning scholars within the broad and interdisciplinary field of Asian Studies.
Our keynote speakers will be:
The Texas Asia Conference (TAC) is a biennial and international graduate student conference organized by the graduate students of the Department of Asian Studies at the University of Texas, Austin. This year's conference will take place on Friday and Saturday, November 1st & 2nd 2013. Our theme is Tradition and Transition. We welcome papers that explore various definitions of and approaches to the term "tradition" in the study of Asia as well as the ways in which change has been enacted and negotiated within and across traditions. Traditions encompass a broad scope of overlapping modes and contexts including socio-cultural, literary, religious, economic, artistic, and performative, as well as traditions in academic research investigating these areas.
We are accepting individual paper abstracts as well as proposals for group panels. The deadline for both is August 10th, 2013. Individual abstracts must include a title and topic. For panel proposals, the organizer of the panel must email the panel abstract, proposed title, topic, and names of presenters. Each panel can include 3 or 4 presenters who are also required to submit individual abstracts. All abstracts should be limited to 300 words. For abstract submissions and additional information or questions, please contact TAC conference co-chair Vibha Shetiya at firstname.lastname@example.com
The Texas Asia Conference provides graduate students with a platform to present their research and to benefit from a scholarly exchange of ideas. We welcome applications from graduate students in all academic disciplines engaged in research on South, Southeast, Central, and East Asia. These disciplines include, but are not limited to, anthropology, art history, communication, ethnomusicology, history, literature, philology, political science, religion, sociology and women's studies. Our previous conference, held in 2011, drew over 100 participants and 30 academic panels, from both the United States and abroad.


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CHARLOTTE, N.C. (AP) — Free agent center Kwame Brown has agreed to terms on a one-year, $7 million contract with the Golden State Warriors.
SEE ALSO: Occupy Protesters Shut Down Ports
Agent Mark Bartelstein says the offer in Golden State was "too good to pass up."
The loss of Brown leaves the Bobcats with a hole at center. Gana Diop, who is coming off a torn Achilles tendon, is the only center on the Bobcats roster.
Bartelstein says Brown is "extremely grateful" for the opportunity the Bobcats gave him, adding that "it was a huge step to come back and play for Michael."
Also On News One:
55 Photos That Will Make You Miss The Obama Family Already
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cPro+ Craigslist Mobile Client
Escargot Studios, LLC
- July 15, 2016
Offers in-app purchases
( 343,879)
cPro, an application for accessing Craigslist from your Android device, iPhone or iPad.
Craigslist Mobile Search • Posting • Account Management • Notifications • Photo Wall • Thumbnail Photo Previews • Map Browser
The New York Times: "Craigslist users have a secret weapon in cPro, which lets you search Craigslist much more easily than the Web site, and it offers a handful of other great features."
cPro features an intuitive interface and essential set of features to browse and post to Craigslist. It also includes Notifiers that run your searches in the background and notify you of new matches.
• Multi-City search
• Map browser
• Photo gallery
• 20+ enhanced search features
• Thumbnail photos and listing previews
• Neighborhood support
• etc, etc.
• post an ad in under 1 minute
• over 1 million successfully posted ads from cPro!
• attach pictures from your Camera or Photo Albums
• full-fledged Account Management (including reposting, editing, and renewing)
• full support of multiple accounts
• Multi-City notifiers with rapid Alerts
• notifiers keep checking Craigslist until a new listing matching your search is posted
• Favorite listings
• add Notes to your favorite listings
• Saved Searches
• Maps and Directions
• Email, Call, or Text posters directly from cPro
• Email or Text listings to your friends
• Flag inappropriate and/or best-of-craigslist ads
• Photo Wall
• Already-Seen indicators
• Sorting by date, best match, low or high price
• PIN code lock protection
The craigslist and associated trademarks are owned by craigslist, Inc. and are being used under licensed permission from craigslist, Inc. for a limited purpose. www.craigslist.org
AppAdvice: "cPro isn't just a website wrapper; it takes searching, posting, and managing your Craigslist account to a whole new level"
Forbes: "All of the features of Craigslist, but with a search agent option that notifies you of new results that match your terms."
Don't buy the premium version Great concept, but using the notification feature of the premium version causes craigslist to block my IP address after about 8 hours. Craigslist won't respond to my queries regarding the reason. I haven't been posting any ads, so flagging isn't the reason. This app also uses a lot of data, probably because the notification feature frequently sends messages from the phone to craigslist. This could be tuned by the user to save data. Or better yet, the notification requests could be handled by a cPRO server.
i bought this app years ago. it then changed to premium-free tiers, and in phone transition i lost my paid benefits. recently, they added a support menu item that specified "recover old account" or something to that tune. i messaged them, and they _immediately_ restored my account. im talking <60s after sending my message, i reopened the app and my premium permissions were back. wow! 5* for great customer service, -1* for dropping my perms to begin with. 100% quality delivery is hard. would buy from escargot studios again.
Liked the app, but, Turning on notifications for new search items got my IP address blocked by CL twice. Had to delete notifications, now I hope I can get my IP address unblocked.. sorting ads by date (newest first) doesn't work correctly.
Usually works fine Use Craigslist all the time. Just today everytime I go to contact a seller it does this annoying thing where it asks if im a robot and makes me select these different pictures before I contact the seller. Its probably to avoid scams but its super annoying have to go through that each time I want to contact someone.
Works for basics I bought the paid version to be able to get multiple notifiers when items become available; sometimes it works, sometimes it doesn't. For example, I might get a notification that an item has become available to purchase in my area and when I click on it I see that item plus items that were listed days before that I did not receive a notification for. This happens about 80% of the time; it's very frustrating if you're really looking to purchase an item.
Disappointing I purchase the pro version to get more notifications, turns out the notifications don't really work at all. I contacted customer service and never got a reply. Don't waste your time or money on this app.
What's New
Bug fixes and performance improvements.


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Norwich — Hanging out before Friday's game, Phil Orbe looked nothing like the beaten down man that appeared on the verge of walking off into the sunset last August.
Orbe enthusiastically talked about the Mystic Schooners' fast start from the Dodd Stadium dugout prior to their New England Collegiate Baseball League game against Newport.
It is the same Newport that ended Mystic's season last summer in the Eastern Division Championship series.
Orbe took the loss hard and expressed doubts about returning for his third season as Mystic manager. But he never seriously considered stepping down.
"Physically and emotionally, I was drained from just the ups and downs of the season," Orbe said. "I always think that's a good thing because you're emotionally invested into something. The kids gave us everything they had and I felt as if we let them down by not being able to get to the next round because we played well enough to win.
"At that point, it's like a boxer who just went through 15 rounds of fighting and was beat to heck. I'm not sure he's thinking about his next fight. But it's a wonderful organization. Our management team that we have is second to none. So it makes it very easy to decide to spend a summer doing this."
Mystic's management team includes dedicated owner Tom Marra and general manager/pitching coach Dennis Long, two people who share Orbe's passion for the game. Marra celebrated his birthday Friday by throwing out the first pitch.
During a dinner meeting last fall, Marra reassured Orbe that he remained fully committed to pushing the Schooners, who had the best season in Mystic franchise history last summer, in the right direction.
"It made me feel good," Orbe said.
Orbe couldn't survive the daily grind of summer baseball without help from Long. Orbe also received the backing of the MVP in his life - his wife, Paula, who plays a vital behind the scenes role.
There's certainly no doubt that Orbe has baseball in his blood. Take what happened just a few weeks ago.
All in a matter of hours, Orbe went from coaching his Montville High School team in a crushing 11-inning loss in the Class M quarterfinals to driving to Wakefield, R.I. for Mystic's season opener against Ocean State. He arrived in the fourth inning.
The next morning, Orbe went back to the high school field with Paula to close up for the season.
"It was a good thing my wife was with me," Orbe said, "because those moments of solitude when I'm cleaning the field at the end of the season are not always my shiniest moments. It was good but I'm still not over the high school season."
Orbe has shifted his focus to the Schooners, who saw a six-game winning streak end with a 9-5 loss on Friday.
Mystic (7-2) is tied for first place in the Southern Division.
Mystic has its usual cast of good characters. The Schooners also can point to superb pitching and timely hitting as reasons for a successful start. Their defense still needs work, though.
It should be another enjoyable summer season for Orbe, who doesn't appear to be slowing down.
He says he'll know where it's time to quit.
"The one day I wake up and I don't want to go to a practice and I don't want to go to a game, I'll start thinking about it," Orbe said. "But it's not all of me. I'd like to think I'm a lot more than just baseball. For Christmas and Father's Day, believe it or not, I get some books that have nothing to do with baseball. I have some interests other than that.
"It's certainly a big part of who I've become, but I don't think it defines me as a whole."
Mad About Madeline Facebook page helped family cope with daughter's fatal leukemia
For newest police recruits, helping the community comes first
Waterford fabricator, national barbecue champion team up to design and manufacture pits
Casey and The Sports Doctor with Jim Buonocore on schools of choice
Casey & The Sports Doctor: Caroline Taber, rules changes, Matt Harvey
Blue Lives Matter rally postponed after backlash in New London
Trump accepts GOP nomination, promises security, prosperity, a return to 'law and order'


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Lammo welcomes Comedian Jeremy Hardy, TV and Radio Presenter Matthew Wright and Badly Drawn Boy AKA Damon Gough into the studio to chat about some interesting new releases. You too can comment on all the tracks by entering the Listener's Roundtable. You can find it via the BBC 6 Music website and enter your opinion with a collection of other 6Music listeners.
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Asperger's Syndrome: A Guide for Parents and Professionals 1st Edition
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I had the good fortune of hearing Dr Attwood speak on television one day, addressing a national press club. I realised within 15 minutes that I had Asperger's Syndrome, and suddenly a lot of problems that I had experienced, mainly social in nature, were explained.
In his book, Dr Attwood attempts to explain AS to the neuro-typical (NT) person, mainly parents and.or associated carers. As an AS sufferer, I found Dt Attwood had a deep understanding on the syndrome and the way it affects people. The book has a lot of practical value, and his insights and associated advice are extremely well thought out.
One of the overriding themes of the book is Dr Attwood's positive outlook for AS sufferers, and he emphasises that it can be overcome with time and perseverence. I found this approach refreshing from the outlook presented in other tomes for AS, which suggest that AS sufferers must accept the inherent limitations imposed on them by the AS. I feel that the majority, if not all, of AS sufferers can overcome the condition. All people go through learning curves in life, and must 'ride uphill' to get there. For AS sufferers, the hill is merely longer and steeper. Dr Attwood makes this point very well, and I'd like to quote a passage from his book (p183):
'The author has met many adults with Asperger's Syndrome who have described how in their late twenties or thirties they eventually managed to intellectually grasp the mechanisms of social skills. From then on the only people who knew of their condition were their family and those who knew them intimately.Read more ›
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Asperger's is a newer term for high functioning autism. Neither term is very well understood.
The author compares Asperger's to a thousand piece puzzle without a defining... Read more
I am really disappointed with this book. I guess I thought I was getting more a self-help book to learn how to handle and cope with things in regards to my Aspie. Read morePublished 8 months ago by Momof5
Product as described. On time delivery. Good book for learning about Autism.Published 8 months ago by Dennis J. Bird


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Britannica Web sites
Articles from Britannica encyclopedias for elementary and high school students.
- Charlotte Cushman - Student Encyclopedia (Ages 11 and up)
(1816-76). The tragedian Charlotte Cushman was the first native-born star of the American stage. While lacking subtlety and a talent for comedy, she became enormously popular in both the United States and Europe playing powerfully emotional roles, such as Lady Macbeth.


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American English: /ˈɛkjəməˌnɪzəm/ , /ɪˈkjumənɪzəm/ British English: /ɪˈkjuːmənɪz(ə)m/
Translation of ecumenism in Spanish:
- Example sentences
- Among the subjects he considers are the diaconate, the priestly office, the office of the bishop, the place of canon law in the life of the church, and ecumenism.
- As irrevocably committed as the Catholic Church is to ecumenism, I cannot imagine the Magisterium ever endorsing the demotion of defined dogmas to the status of venerable opinion.
- The 'Evangelical' world (forgetting for a moment, churches explicitly committed to ecumenism and multifaith) is in a mess.
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