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Nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) is a constitutively active fusion tyrosine kinase involved in lymphomagenesis of human anaplastic large cell lymphomas (ALCL), the maturation and activity of which depend on the association with the heat shock protein (hsp) 90 protein chaperone. Targeting hsp90 by the ansamycins geldanamycin and 17-allyl-amino-demethoxygeldanamycin (17-AAG) promotes degradation of several proteins through the ubiquitin-proteasome pathway, including oncogenic Raf, v-Src, erbB2, and BCR-ABL. We have previously shown that 17-AAG prevents hsp90/NPM-ALK complex formation and fosters NPM-ALK turnover, perhaps through its association with the hsp70 chaperone. Here, we show that inhibition of the proteasome activity by the potent and specific compound pyrazylcarbonyl-Phe-Leu-boronate (PS-341) blocks 17-AAG-induced down-regulation of NPM-ALK, which becomes detergent-insoluble and relocates into ubiquitin-rich perinuclear vesicles that represent aggregated polyubiquitinated forms of the protein. Kinase activity was not mandatory for proteasomal degradation of NPM-ALK, because kinase-defective NPM-ALK was even more rapidly degraded upon 17-AAG treatment. Prolonged exposure to the proteasome inhibitor was shown to trigger caspase-3-mediated apoptosis in proliferating ALCL cells at nanomolar concentrations. However, we verified that the accumulation of detergent-insoluble NPM-ALK in ALCL cells was not a spurious consequence of PS341-committed apoptosis, because caspase inhibitors prevented poly(ADP-ribose) polymerase cleavage whereas they did not affect par-titioning of aggregated NPM-ALK. In line with these observations, the carboxyl hsp70-interacting ubiquitin ligase (CHIP), was shown to increase basal ubiquitination and turnover of NPM-ALK kinase, supporting a mechanism whereby NPM-ALK proceeds rapidly toward hsp70-assisted ubiquitin-dependent proteasomal degradation, when chaperoning activity of hsp90 is prohibited by 17-AAG.
Anaplastic large cell lymphomas (ALCLs) show recurring chromosomal translocations between chromosome 2 and chromosomes 1, 3, or 5 and in a few cases, chromosomes 17 and 19 (1) . The majority of the anaplastic lymphoma kinase (ALK)-positive ALCLs carries a t(2;5)(p23;q35) reciprocal translocation that originates a Mr 80,000 constitutively active tyrosine kinase protein, termed nucleophosmin (NPM)-ALK or p80, where the oligomerization motif within the NH2-terminal portion of the NPM protein is juxtaposed to the catalytic domain of the insulin-like ALK tyrosine kinase receptor (2) . The oncogenic potential of NPM-ALK has been demonstrated both in vivo and in vitro, because mice that receive injections of NPM-ALK-transformed murine BaF3 or Fr3T3 cells develop hematological malignancies, and ectopic expression of NPM-ALK accelerates cell cycle entry of rat fibroblasts, which also acquire the capability to grow on semi-solid support (3) . Moreover, transgenic mice in which the full-length cDNA of the npm-alk gene is under control of the murine CD4 promoter spontaneously develop T-cell lymphomas or plasma cell neoplasms (4) and lethally irradiated interleukin 9 transgenic mice reconstituted with NPM-ALK-transduced mouse bone marrow progenitors develop either B-cell lymphomas or T-cell lymphoblastic lymphomas (5) . Cytoplasm-localized NPM-ALK homodimers phosphorylate canonical receptor tyrosine kinase adapter proteins such as Shc, IRS-1, Grb2, or PLCγ1, leading to activation of signaling pathways involved in both cell proliferation and survival (6 , 7) . Indeed, whereas NPM-ALK association with and phosphorylation of p85 regulatory subunit of phosphatidylinositol 3′-kinase activates Akt and reduces Bad-dependent apoptosis (8 , 9) , phosphorylation of STAT3 transcription factor results in the up-regulation of antiapoptotic protein Bcl-xL, as well as in transcription of cell-cycle-related genes (10 , 11) . We have recently shown that expression of mature NPM-ALK kinase requires the interaction with the molecular chaperone heat shock protein (hsp) 90, and inhibition of the chaperoning activity by the benzoquinone 17-allyl-amino-demethoxygeldanamycin (17-AAG) reduces NPM-ALK content and negatively regulates its signaling pathway in ALCL cells (12) . The exact mechanism of NPM-ALK degradation is still unknown, but our previous findings that destabilization of NPM-ALK kinase is preceded by binding hsp70 (a chaperone involved both in folding and proteasomal degradation of certain hsp90-client proteins) led us to speculate in favor of the 26S proteasome as degradation pathway of misfolded NPM-ALK. Several other hsp90 chaperone substrates, such as Akt (13) , Raf-1 (14) , BCR-ABL (15) , and CFTR (16) , have been reported to bind hsp70, before degradation, in cells treated with hsp90 inhibitors. The ability of hsp70 to drive protein degradation, rather than folding, depends on the interaction with the E3 ubiquitin ligase carboxyl terminus hsp70-interacting protein (CHIP). CHIP binds to hsp70 through NH2-terminal tetratricopeptide (TPR) motifs (17) while mediating ubiquitination of chaperone-bound polypeptides through a COOH-terminal U-box domain (18) . Here, we demonstrate that the inhibition of proteasome activity prevents 17-AAG-dependent degradation of NPM-ALK, resulting in the accumulation of NPM-ALK kinase in the detergent-insoluble fraction of treated cells. Furthermore, we provide evidence that CHIP ubiquitin ligase associates with NPM-ALK and negatively regulates NPM-ALK steady state in transfected cells by promoting its ubiquitination.
Cell Culture.
The human ALK-positive ALCL cell lines Karpas299 and SR786 and the ALK-negative FE-PD (19) were maintained in RPMI 1640 containing 15% heat-inactivated FCS, 2 mmol/liter glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin under standard tissue-culture conditions. COS-7 monkey renal epithelial cells were cultivated in Dulbecco's modified medium, supplemented with 10% heat-inactivated FCS, glutamine, penicillin, and streptomycin, as described above.
Reagents and Antibodies.
17-AAG and pyrazylcarbonyl-Phe-Leu-boronate (PS-341) were obtained from L. Neckers and Edward Mimnaugh (National Cancer Institute, Bethesda, MD). Anti-poly(ADP-ribose) polymerase (PARP) and anti-CHIP antibodies were purchased from Calbiochem-Novabiochem Corp. (San Diego, CA). Monoclonal antiactin IgM ascites and horseradish peroxidase goat antimouse IgM were bought from Oncogene Research Products (Boston, MA). Rabbit polyclonal antihistone deacetylase 1 and monoclonal antiubiquitin (P4D1) antibodies and antiphosphotyrosine (PY99) and anti-hsp70 monoclonal antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ALK (ALKc) and anti-NPM (NPM-376) monoclonal antibodies were a generous gift from Prof. B. Falini (University of Perugia, Perugia, Italy). Caspase inhibitors Z-VAD-FMK, Z-AEVD-FMK, DEVD-CHO, and Z-LEHD-FMK and protease inhibitors N-acetyl-l-leucyl-l-leucyl-norleucinal (ALLnL), N-acetyl-Leu-Leu-Met-CHO (LLM), pepstatin, [4-(2-aminoethyl) benzenesulfonylfluoride, HCl] (AEBSF), calpeptin, and chathepsin inhibitor I were purchased from Calbiochem. Phenylmethylsulfonyl fluoride was purchased from Sigma-Aldrich Co. (St. Louis, MO). Leupeptin, aprotinin, and rabbit antimouse IgG1 were obtained from CAPPEL (ICN Biomedicals Inc., Costa Mesa, CA). 4′,6-Diamidino-2-phenylindole nucleic acid stain, fluorophore-conjugated goat antimouse (Alexa546), and goat antirabbit (Alexa488) antibodies were bought from Molecular Probes (Eugene, OR). Horseradish peroxidase-conjugated sheep antimouse and donkey antirabbit antibodies were purchased from Amersham Biosciences (Uppsala, Sweden), as well as protein A-Sepharose beads and protein G-Sepharose Fast-Flow beads. BCA protein assay and Western blot chemiluminescence reagents were all from Pierce Chemical Co. (Rockford, IL). Protran nitrocellulose membranes were from Schleicher & Schuell. All of the other chemicals used in this study were purchased from Sigma-Aldrich.
Immunoprecipitation and Immunoblotting.
ALCL and COS-7 cells were lysed as described previously (12) . In brief, cells were washed twice in ice-cold 1× PBS and incubated on ice for 20 min in 1% Triton X-100 lysis buffer. After high-speed clarification at 4°C, supernatants were recovered, and pellets were resuspended in the same volume of lysis buffer and sonicated on ice. Thirty to 60 μg of lysates from both fractions were loaded onto 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. Alternatively, when immunoprecipitation was performed, 500 μg of lysates were incubated for 2–12 h at 4°C with anti-ALK, anti-CHIP, or anti-hsp70 antibody, where indicated. Immunocomplexes were adsorbed to 30 μl of protein G-Sepharose beads or to 150 μl of protein A-Sepharose beads previously conjugated to rabbit antimouse IgG1 antibody and incubated 2 h at 4°C. The immunoadsorbed pellets were washed in 1% Triton X-100 lysis buffer and heated at 95°C in 1× reducing Laemmli loading buffer, before loading onto SDS-PAGE. To assess NPM-ALK ubiquitination, cell lysates were prepared in radioimmunoprecipitation assay buffer (Triton lysis buffer supplemented with 0.1% SDS, 0.5% sodium deoxycholate, 1 mm phenylmethylsulfonyl fluoride (PMSF), 1 mm sodium orthovanadate, and 10 mm sodium fluoride), and SDS-PAGE resolved samples were transferred onto polyvinylidene difluoride membranes. Proteins were visualized by chemiluminescence using a commercial kit (Pierce Chemical Co.). Films (Hyperfilm; Amersham Biosciences) were scanned, and results were analyzed by using image analysis software (NIH Image).
Preparation of Cytoplasmic and Nuclear Fractions.
To obtain purified cytoplasmic and nuclear fractions, cells were processed according to the protocol described by Rosette and Karin (20) . After washing in PBS, cells were frozen in dry ice and immediately thawed on ice in pH 7.9 hypotonic buffer [10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 mm KCl, 0.2 mm EDTA, 0.1 mm EGTA, 1 mm DTT, 1 mm phenylmethylsulfonyl fluoride, 20 μg/ml leupeptin, and 20 μg/ml aprotinin]. NP40 (2%) was then added, and cells were further lysed by passing through a 26-gauge needle. Soluble cytoplasmic proteins were recovered by clarification at 10,000 × g, whereas intact nuclei were resuspended in pH 7.9 hypertonic buffer [20 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 0.4 m NaCl, 1 mm EDTA, and 1 mm DTT] containing protease inhibitors. Nuclear extracts were recovered from pestle-broken nuclei (Kimble/Kontes Scientific Glassware Instruments, Vineland, NJ), rocked on ice for 30 min, and clarified by high-speed centrifugation (10,000 × g). Supernatant represents soluble nuclear extracts.
Preparation of Expression Constructs and Transient Transfection.
The whole npm-alk coding region, originally cloned into the pSRαMSVtkneo retroviral vector (3) , was subcloned into the pcDNA3 vector (Invitrogen). Point mutant NPM-ALKK210A (K210A) was created with QuikChange Site-Directed Mutagenesis Kit system (Stratagene, La Jolla, CA). Wild-type CHIP and the two point mutants K30A and H260Q, cloned into the pcDNA3.1 vector, are kind gifts from L. Neckers. To transiently express the constructs described above, COS-7 cells (0.2 × 106) were plated in 100-mm dishes; after 72 h, log-phase growing cells were transfected with 35 μg each of NPM-ALK or NPM-ALKK210 or with 20 μg when either construct was cotransfected with equal amounts of β-galactosidase (β-gal) or CHIP expression vectors, by calcium-phosphate DNA coprecipitation. Thirty-six h after transfection, COS-7 cells were lysed in ice-cold lysis buffer or additionally treated with 17-AAG and/or PS-341, where indicated. Lysates were clarified, immunoprecipitated, and resolved by SDS-PAGE as described above.
Fluorescence Microscopy.
Karpas299 cells (3 × 103) were plated onto 8 well/chamber slides (Becton Dickinson, Franklin Lakes, NJ) and grown for 72 h. Cells were then incubated for 24 h with 17-AAG, PS-341, or both compounds, as reported in Fig. 3 ⇓ . Exponentially growing cells were fixed in 3.7% formaldehyde, permeabilized in 0.2% Triton X-100, and blocked in 10% FCS-PBS, followed by incubation in 100 mm glycin. After permeabilization, cells were incubated 1 h at 37°C with ALK-11 rabbit anti-ALK antibody (a kind gift of S. W. Morris, St. Jude Children's Research Hospital, Memphis, TN) and with a mouse antiubiquitin antibody. After extensive washing in PBS, cells were incubated with fluorophore-conjugated secondary antibodies goat antirabbit (Alexa488) and goat antimouse (Alexa546) at a 1:500 dilution of 2 mg/ml stock. Cells were then washed in PBS and mounted onto slides with 1:1 PBS/glycerol, with addition of 4′,6-diamidino-2-phenylindole nucleic acid stain at a 1:1000 dilution. Cells were observed at ×63/0.75 numerical aperature in a Leica DMBL microscope. Images were obtained with a Leica DC 300F digital camera and prepared for reproduction with Leica IM1000 imaging software (Leica Microsystems Ltd., Wetzlar, Germany).
NPM-ALK Accumulates in the Detergent-Insoluble Cell Fraction after Proteasome Inhibition.
Degradation of receptor tyrosine kinases still remains an intriguing issue, because immature receptor proteins are usually degraded through the 26S proteasome, whereas proteolysis of mature receptors upon ligand-dependent endocytosis and ubiquitination may be both lysosome and proteasome dependent (21, 22, 23, 24, 25, 26) . Because we have previously shown that the ALK receptor protein moiety is responsible for the accelerated degradation of NPM-ALK kinase when hsp90-folding activity is inhibited by 17-AAG (12) , we screened a panel of selective protease inhibitors. We included inhibitors of both proteasomal and lysosomal function to characterize the degradation pathway of NPM-ALK in 17-AAG-treated ALCL cells Karpas299 and SR786 (Fig. 1, A and B ⇓ , respectively). Exponentially growing cells were treated for 8 h with inhibitors of lysosomal (leupeptin and pepstatin), calpain (cathepsin inhibitor I and calpeptin), proteasomal (PS-341, ALLnL and LLM), and serine/threonine (AEBSF, PMSF) proteases in the presence of 10 μm 17-AAG. Surprisingly, none of the inhibitors used had an effect on NPM-ALK expression in the clarified lysates of both cell lines. However, in ALCL cells incubated with proteasome inhibitors PS-341, ALLnL, and LLM, degradation of NPM-ALK caused by 17-AAG was somehow prevented, because NPM-ALK accumulated consistently in detergent-insoluble pellet fractions (Fig. 1, A and B ⇓ , graph). Given that boronic acid dipeptides (PS-341) and peptide aldehydes (ALLnL and LLM) are promiscuous inhibitors, because they also target certain serine and cysteine proteases besides the proteasome (27) , we analyzed more specific inhibitors of such enzymes AEBSF, cathepsin inhibitor I, and calpeptin. Yet, because no similar accumulation of NPM-ALK protein occurred with such more specific compounds, it is likely that NPM-ALK is subjected to proteasomal degradation when its hsp90-assisted maturation is impeded by 17-AAG.
To investigate the kinetics of NPM-ALK degradation, Karpas299 cells were exposed to 0.5 μm 17-AAG for 24 h, and proteasome activity was inhibited for 16 or 24 h (Fig. 1C ⇓ , Lanes 1–7). Unlike the Mr 38,000 protein NPM (Fig. 1C ⇓ , Lanes 8–14), NPM-ALK protein level was reduced both in clarified lysates and detergent-insoluble pellet fractions after treatment with 17-AAG (Fig. 1C ⇓ , Lane 3). When added to 17-AAG, PS-341 inhibitor did not prevent the down-regulation of detergent-soluble NPM-ALK but caused time-dependent partitioning of NPM-ALK in the insoluble pool (Fig. 1C ⇓ , Lanes 6 and 7). When Karpas299 cells were grown in the presence of both 17-AAG and PS-341 for increasing time intervals, time-dependent down-regulation of soluble NPM-ALK caused by 17-AAG paralleled exactly the up-regulation of insoluble NPM-ALK mediated by PS-341 (Fig. 1D) ⇓ , in accordance with what was shown previously for other hsp90-client proteins (28, 29, 30) .
PS-341-Induced Apoptosis Does Not Affect Proteasomal Degradation of NPM-ALK.
PS-341 was the first proteasome inhibitor to enter Phase I clinical trial for its antitumor activity, because tumor cells are more sensitive than normal cells to PS-341-dependent impairment of cell growth and induction apoptosis (31 , 32) , as recently shown in multiple myeloma cells both in vivo and in vitro (33 , 34) . Pro-apoptotic activity of PS-341 was assessed both in t(2;5)-positive (Karpas299, SUDHL1, and SR786) and t(2;5)-negative (FE-PD) ALCL cell lines, by exposing cells for 48 h to nanomolar concentrations of inhibitor. As shown in Fig. 2A ⇓ , in vitro cell proliferation was inhibited by more than 90% at 10 nm in all of the cell lines and regardless of NPM-ALK expression (Fig. 2A ⇓ , graph). Indeed, exposure to PS-341 caused time-dependent cleavage of the Mr 116,000 apoptotic hallmark PARP (Fig. 2A ⇓ , panels) into its caspase-cleaved fragment of Mr 89,000 (Fig. 2A ⇓ , arrowheads) in both ALK-positive (SUDHL1 and SR786) and ALK-negative (FE-PD) cells.
Thus, to rule out that the accumulation of insoluble NPM-ALK might be an artifact of PS-341-driven apoptosis rather than inhibition of its proteasomal degradation, Karpas299 and SR786 cells (Fig. 2B ⇓ , Lanes 1–6 and 7–12, respectively) were treated with two pan-caspase inhibitors before being exposed for as long as 8 h to higher doses of 17-AAG and PS-341. Consistently with PARP cleavage shown in Fig. 2A ⇓ , the two cell lines responded differently to PS-341 treatment, because down-regulation of mature PARP protein was strongly induced in SR786 but not in Karpas299. In SR786 cells pretreated with Z-VAD-FMK and Z-AEVD-FMK inhibitors, caspase-dependent PARP cleavage was completely blocked (Fig. 2B ⇓ , graph), and PARP protein level rose even higher than in untreated cells, which might possess a constitutive caspase activity, as appears from PARP immunoblots of Fig. 2A ⇓ . In contrast, after incubation with both 17-AAG and PS-341, aggregated NPM-ALK was found in the pellet fraction both in the absence of apoptosis (Fig. 2B ⇓ , Lanes 4–6) and when PS-341-induced apoptosis (SR786) was prevented by the two caspase inhibitors (Fig. 2B ⇓ , Lanes 10–12). When Karpas299 cells were treated longer (24 h) with PS-341, extensive PARP cleavage occurred (Fig. 2C ⇓ , graph), and it was totally prevented by the two specific caspase-3 and caspase-9 inhibitors, DEVD-CHO and Z-LEHD-FMK, respectively. However, redistribution of NPM-ALK upon proteasome inhibition observed in the detergent-insoluble fraction of 17-AAG-treated cells (Fig. 2C ⇓ , Lane 4) was not prevented when both caspase-3 and caspase-9 enzymes were inhibited (Fig. 2C ⇓ , Lanes 5 and 6), hence proving that increase of insoluble NPM-ALK and apoptosis are two independent phenomena in PS-341-treated ALCL cells.
NPM-ALK Relocates in Ubiquitin-Rich Perinuclear Structures after Proteasome Inhibition.
Covalent attachment of multiple molecules of ubiquitin to specific lysine residues of target proteins is a strict prerequisite for recognition and subsequent degradation by the 26S proteasome, and inhibition of the proteasome activity normally leads to accumulation of polyubiquitinated proteins into cells (35) . Thus, we performed fluorescent microscopy analysis of ALCL cells stained for NPM-ALK and ubiquitin (Fig. 3A ⇓ , panels A–D and E–H, respectively), to verify whether detergent-insoluble NPM-ALK corresponds to deposition of ubiquitinated forms of the protein in cells treated with both 17-AAG and PS-341. As shown in Fig. 3A ⇓ , exposing Karpas299 to PS-341 alone caused accumulation of ubiquitinated proteins into the cytoplasm (Fig. 3A ⇓ , panel G), which had no correspondence with the wide distribution of NPM-ALK (Fig. 3A ⇓ , panel C). On the other hand, when the cells were exposed to both 17-AAG and PS-341, NPM-ALK fluorescence showed a perinuclear distribution that perfectly merged with the ubiquitin staining (Fig. 3A ⇓ , panels D, H, and N, arrowheads). Besides, when part of the cells were lysed and cytosolic and nuclear soluble fractions were purified and analyzed together with sonicated detergent-insoluble pellets, accumulation of NPM-ALK was found only in the pellet fraction of cells exposed to both 17-AAG and PS-341 (Fig. 3 ⇓ B, Lane 12). This indicates that juxtanuclear-localized NPM-ALK observed in fixed cells represents insoluble ubiquitinated NPM-ALK. Furthermore, we observed that chaperones hsp70 and hsp40, but not hsp90, were increased consistently in the detergent-insoluble fraction of NPM-ALK-transfected cells treated with 17-AAG and PS-341 (data not shown). Because this is consistent with the evidence that, upon prolonged inhibition of proteasome activity, molecular chaperones relocate in large perinuclear structures with polyubiquitinated misfolded client proteins (36 , 37) , it is likely that the deposition of detergent-insoluble NPM-ALK may result from the impairment of its chaperone-assisted proteasomal degradation.
17-AAG-Induced Degradation of NPM-ALK Is Independent of Its Tyrosine Kinase Activity.
Ubiquitin-dependent proteasomal degradation of several hsp90-client proteins has been shown to rely upon interaction with the hsp70 chaperone, independently from their biological functions (38, 39, 40) . To test whether kinase activity was mandatory for NPM-ALK proteasomal degradation, we generated a kinase-defective mutant of NPM-ALK by replacing lysine 210 of the nucleotide-binding domain with alanine (K210A). Wild-type NPM-ALK and kinase-defective K210A mutant, when transiently expressed in COS-7 cells, displayed comparable steady-state levels, although the K210A mutant failed to react with the antiphosphotyrosine antibody that marked NPM-ALK immunoprecipitates (Fig. 4A) ⇓ . As expected, 17-AAG inhibited hsp90-driven maturation of both wild-type NPM-ALK and NPM-ALK K210A, causing their rapid depletion in COS-7 cells (Fig. 4B ⇓ , Lane 2). Nevertheless, when added to 17-AAG, PS-341 prevented the loss of the two misfolded proteins and enhanced the extent of their aggregation in the detergent-insoluble pellet fraction (Fig. 4B ⇓ , Lane 8). Interestingly, the K210A mutant showed enhanced sensitivity to hsp90 inhibition (Fig. 4B ⇓ , Lane 2), similarly to what was found recently for kinase-dead EGFR (41) . When the kinetics of the down-regulation of the two proteins were compared, 10 μm 17-AAG led to almost complete depletion of the K210A mutant after just 2 h of treatment, whereas wild-type NPM-ALK was still detectable even after 8 h (Fig. 4C ⇓ , arrowhead).
The Ubiquitin Ligase CHIP Down-Regulates NPM-ALK Kinase Expression.
Chaperone-assisted folding requires the cooperation of several scaffold proteins and cofactors of the hsp70 and hsp90 multimolecular chaperone complexes (42) . The TPR-containing protein CHIP has been shown to suppress the folding and catalyze the ubiquitination of certain hsp90-client proteins when bound to hsp70, by virtue of a COOH-terminal U-box domain (43 , 44) . Point mutations at lysine 30 within the TPR domain or at histidine 260 of the U-box domain abrogate respectively CHIP ability to complex with hsp70 and its ubiquitin ligase activity, both mandatory for ubiquitin-dependent proteasomal degradation of chaperone-bound substrates (45) . Because we have recently shown that after 17-AAG treatment, NPM-ALK/hsp70 association increases and precedes NPM-ALK down-regulation in ALCL cells, we wondered whether CHIP could regulate NPM-ALK turnover, perhaps throughout hsp70. Therefore, we examined NPM-ALK steady state in COS-7 cells expressing wild-type CHIP or each of the two point mutants described above (CHIP K30A and CHIP H260Q) or β-gal protein. In accordance with our hypothesis, wild-type CHIP protein markedly diminished NPM-ALK expression when compared with β-gal or the two point mutants (Fig. 5A ⇓ , Lanes 1–4), and it stimulated kinase-defective NPM-ALK K210A degradation (Fig. 5A ⇓ , Lanes 5–9). When an anti-CHIP antibody was used for immunoprecipitation, NPM-ALK precipitated from clarified lysates of CHIP-transfected and CHIP H260Q-transfected cells (Fig. 5B ⇓ , Lanes 4 and 6) but not from β-gal or K30A cell lysates (Fig. 5B ⇓ , Lanes 2 and 5). Control immunoprecipitation, performed by immunoadsorbing NPM-ALK with ALK-C antibody, ruled out any spurious effect by detecting NPM-ALK in all of the above described cell lysates, except in lysates of untransfected COS-7 (Fig. 5B ⇓ , Lane 8). In addition, we carried out reverse co-immunoprecipitation and confirmed that, unlike CHIP K30A, wild-type CHIP and CHIP H260Q associated with hsp70 and NPM-ALK (Fig. 5B ⇓ , Lanes 13 and 14 and 19 and 20, respectively) as well as with kinase-defective NPM-ALK K210A (data not shown). Taken together, these data indicate that recruitment of CHIP and subsequent degradation of NPM-ALK kinase depends on NPM-ALK binding to hsp70 rather than on its kinase activity.
Additionally, we investigated the combinatorial effect of CHIP expression and 17-AAG treatment, because we noticed a similar inhibitory effect on NPM-ALK steady state. Indeed, CHIP and 17-AAG seemed to cooperate in this activity, because 17-AAG-induced depletion of soluble NPM-ALK further increased in the presence of CHIP (Fig. 5C) ⇓ . In contrast, CHIP H260Q mutant, which lacks enzymatic activity, neither affected soluble NPM-ALK level in untreated cells nor prevented its down-regulation in the presence of 17-AAG. Yet, it is worth pointing out that CHIP may cause apparent down-regulation of hsp90 substrates independently of its ubiquitin ligase activity, by partitioning misfolded hsp90 substrates in the insoluble pool (46) . In fact, cotransfection of NPM-ALK with both wild-type CHIP and CHIP H260Q resulted in the redistribution of NPM-ALK from the soluble to the insoluble pool even in the absence of 17-AAG (Fig. 5C) ⇓ , whereas exposure to 17-AAG treatment was additive to such an effect.
CHIP Enhances NPM-ALK Ubiquitination in COS-7 Cells.
To assess whether CHIP ubiquitin ligase activity stimulates NPM-ALK ubiquitination, COS-7 cells untransfected or transfected with plasmids expressing NPM-ALK, β-gal, or CHIP were incubated with 10 μm 17-AAG, 3 μm PS-341, and the combination thereof for 8 h. As shown in Fig. 6A ⇓ , treating β-gal/NPM-ALK-expressing cells with 17-AAG was enough to elicit NPM-ALK ubiquitination, thus reinforcing the concept that NPM-ALK is efficiently ubiquitinated before proteasome degradation when hsp90-folding activity is inhibited. Blockade of proteasome activity by PS-341 had only a slight effect on NPM-ALK ubiquitination, whereas it enhanced the level stabilized by 17-AAG (Fig. 6A ⇓ , Lanes 1–4). In the presence of CHIP, however, polyubiquitinated NPM-ALK was precipitated even from the clarified lysates of untreated cells, and the extent of NPM-ALK ubiquitination was augmented dramatically after treatment with 17-AAG and PS-341 (Fig. 6A ⇓ , Lanes 9–12). As expected, immunoadsorbed lysates from untransfected (Fig. 6A ⇓ , Lanes 5–8) or β-gal/CHIP-transfected cells (Fig. 6A ⇓ , Lanes 13 and 14) did not contain any higher molecular-weight form of ubiquitinated NPM-ALK, which indicates the relative specificity of CHIP-induced NPM-ALK ubiquitination. Remarkably, we found that the association between NPM-ALK and CHIP was stabilized by the proteasome inhibitor PS-341 in the presence of 17-AAG (Fig. 6A ⇓ , bottom), although CHIP steady state remained unaltered (Fig. 6B) ⇓ . Thus, we can conclude that CHIP supports the ubiquitination of NPM-ALK per se and in the presence of 17-AAG, and blockade of the proteasome catalytic activity results in the accumulation of CHIP-bound polyubiquitinated NPM-ALK.
NPM-ALK is a Mr 80,000 fusion tyrosine kinase originating from a reciprocal translocation between chromosomes 2 and 5, which regulates cell-cycle progression of ALK-positive ALCL lymphomas (47) and antiapoptotic behavior of transformed cells (3) upon self-association and phosphorylation. Ansamycin drugs such as herbimycin, geldanamycin, and its derivative 17-AAG have been found to diminish NPM-ALK expression and tyrosine phosphorylation in ALCL cells, leading to G1-phase arrest and apoptosis (48 , 49) . Despite their initial use as tyrosine kinase inhibitors, ansamycins are a class of small molecules with no inhibitory kinase activity in vitro (50) but are capable of binding to and competing with ATP for the ATP-binding site of the molecular chaperone hsp90, which regulates late-stage folding and maturation of several signaling proteins. Drug-induced impairment of hsp90-chaperoning activity has been shown to promote ubiquitin-dependent proteasomal degradation of non-native substrates (51 , 52) , and we have previously shown that inhibition of hsp90 activity by 17-AAG prevents NPM-ALK/hsp90 complex formation and accelerates NPM-ALK turnover (12) .
Covalent attachment of multiple ubiquitin molecules to selected lysine residues targets for ATP-dependent proteolysis by the 26S proteasome both normal native and unfolded proteins, as well as damaged polypeptides (35) . However, ubiquitin-dependent degradation of growth factor receptors is still an intriguing process, because poly-ubiquitination of ligand-activated receptors at the plasma membrane has been shown to accelerate endocytosis (22 , 53, 54, 55) , whereas subsequent proteolysis of endosome-sequestered receptors may be hindered either by proteasomal or lysosomal inhibitors (24 , 25) . To investigate the degradation pathway of NPM-ALK, we screened a panel of lysosomal, endosomal, and proteasomal inhibitors, because the COOH-terminal portion of NPM-ALK corresponds to the entire cytoplasmic domain of the transmembrane receptor ALK, possibly including sequences relevant for both proteasomal and/or lysosomal proteolysis. When the ALCL cell lines Karpas299 and SR786 were maintained in the presence of both 17-AAG and the different protease inhibitors described in Fig. 1 ⇓ , 17-AAG-induced degradation was prevented and NPM-ALK accumulated in the detergent-insoluble fraction only in the presence of proteasome inhibitors. The observation that aggregated NPM-ALK did not accumulate when selective inhibitors of serine, cysteine, and lysosomal proteases were used suggests that the proteasome may represent the preferred degradation pathway of NPM-ALK in 17-AAG-treated cells.
Abrogation of proteasomal activity by PS-341 inhibitor has been found to block cell cycle progression of tumor cells (31 , 32) and to induce apoptosis through nuclear factor κB (NFκB) antiapoptotic activity overcome (56) , cytochrome c release (57) , Bcl-2 cleavage (58) , and c-Jun NH2-terminal kinase-dependent caspase-3 and caspase-8 activation (59) . The effect, which increases tumor cell sensitivity to DNA-damaging chemotherapy both in vitro and in vivo (60 , 61) , can be prevented by pan-caspase or caspase-3 inhibitors (58) . In line with these observations, we found that PS-341 did block in vitro growth of all of the ALCL cell lines tested, regardless of NPM-ALK kinase expression, and caused apoptosis in a time- and dose-dependent manner, as assessed by caspase-dependent cleavage of PARP protein (Fig. 2A) ⇓ . However, even though PS-341-induced fragmentation of PARP was prevented by inactivation of both caspase-3 and caspase-9 (Fig. 2C) ⇓ or by using pan-caspase inhibitors (Fig. 2B) ⇓ , aggregated NPM-ALK was still found in the detergent-insoluble pellet fraction of 17-AAG/PS-341-treated cells, proving that proteasomal degradation of NPM-ALK and apoptosis occur independently of each other.
Down-regulation of detergent-soluble NPM-ALK and increase of its detergent-insoluble pool after exposing 17-AAG-treated cells to PS-341 occurred simultaneously in a time-dependent manner (Fig. 1, C and D) ⇓ , similarly to what was described recently for the hsp90 client protein Akt (28) . The loss of solubility seems to be featuring many other misfolded chaperone substrates when their binding to hsp90 is prevented and proteasome activity is sequentially inhibited (15 , 29 , 30 , 62 , 63) , and this correlates to intracellular deposition of polyubiquitinated protein aggregates with a preferred juxtanuclear distribution (64 , 65) into vimentin-surrounded inclusions known as aggresomes (37 , 65) . Indeed, PS-341 caused accumulation of ubiquitinated proteins in Karpas299 cells, and the extent further increased in the presence of 17-AAG (Fig. 3A) ⇓ . PS-341 treatment did not significantly alter steady-state and cellular localization of NPM-ALK fusion kinase in the absence of 17-AAG, but it caused NPM-ALK to colocalize with ubiquitin, when combined to the hsp90 inhibitor, into perinuclear vesicles resembling the polyubiquitinated protein aggregates described (Fig. 3) ⇓ . The data presented above support a mechanism whereby 17-AAG-induced misfolding and degradation of NPM-ALK occurs rapidly and efficiently unless proteasome activity is perturbed, likely indicating a relationship between chaperone-assisted folding of NPM-ALK and the ubiquitin/proteasome pathway.
As recently shown, the balance between protein folding and destruction is reached through the activity of chaperone-bound cofactors, such as CHIP and BAG-1, that diverge misfolded or damaged chaperone-client proteins to proteasomal degradation rather than allowing their folding. CHIP, in particular, acts as an E3 ubiquitin ligase and promotes ubiquitination of hsp90-client proteins by virtue of a COOH-terminal U-box domain (43) that recruits the E2-conjugating enzyme UBCH5a (18) . Through multiple TPR repeats, CHIP binds to hsp70 and hsp90 in a mutually exclusive manner, preventing hsp70-folding activity and active hsp90/p23 complex formation, respectively (17) . Point mutation analysis has shown that both the TPR repeats and U-box domain are mandatory for CHIP ubiquitin ligase activity, because rapid degradation of misfolded proteins in the presence of wild-type CHIP cannot be elicited by either TPR (CHIP K30A) or U-box (CHIP H260Q) point mutants (44 , 45 , 64 , 66) . Consistent with this mechanism, we found that CHIP associated with and increased protein turnover of NPM-ALK when expressed in COS-7 cells (Fig. 5) ⇓ . In contrast, in the presence of CHIP H260Q, which binds stronger to hsp70 and NPM-ALK but shows impaired ubiquitin-ligase activity, or CHIP K30A, which does not bind hsp70, NPM-ALK steady state was unaffected, hence proving that ubiquitin-dependent degradation of NPM-ALK takes place when the mutual interaction between CHIP and hsp70 exists. Interestingly, CHIP and 17-AAG exert a similar effect on NPM-ALK stability, which indicates that although some redundancy is likely to occur, a cooperation between the two mechanisms, as previously found for erbB2 (45 , 67) , may be feasible. Indeed, when COS-7 cells were exposed to 17-AAG, drug-induced degradation of exogenous NPM-ALK was enhanced in the presence of wild-type CHIP, whereas U-box mutant CHIP H260Q did not counteract 17-AAG-induced depletion of soluble NPM-ALK but rather stabilized its insoluble pool (Fig. 5C) ⇓ . By acting as an hsp70-folding inhibitor, CHIP may indeed decrease soluble protein levels without stimulating the ubiquitin-dependent degradation pathway but by causing aggregation of misfolded proteins (46) . Accordingly, the partition of aggregated NPM-ALK in the detergent-insoluble pellet fraction was observed in cells expressing wild-type or U-box mutant CHIP (Fig. 5C) ⇓ but not in those cells transfected with CHIP K30A (data not shown). However, when the ubiquitination status was assessed, we verified that CHIP dramatically raised the content of ubiquitinated NPM-ALK when proteasome activity was inhibited in the presence of 17-AAG (Fig. 6) ⇓ , thus indicating that CHIP represents the E3 ubiquitin ligase responsible for the ubiquitination of misfolded NPM-ALK in 17-AAG-treated cells.
Ubiquitin-dependent down-regulation of activated receptor tyrosine kinases at the plasma membrane has been shown to occur upon recruitment of another E3 ubiquitin ligase, Cbl, although that drives endocytosis and lysosomal degradation of monoubiquitinated receptor proteins (68) . Unlike CHIP, Cbl needs to be tyrosine phosphorylated before activation and catalytically inactive tyrosine kinases are not affected by Cbl (24 , 69 , 70) . In contrast, in transiently transfected COS-7 cells, kinase-dead NPM-ALK K210A and CHIP associated (data not shown) and CHIP dictated the down-regulation of kinase-dead NPM-ALK (Fig. 5A) ⇓ . Besides, kinase-defective NPM-ALK K210A appeared to be more dependent on hsp90-chaperoning activity than wild-type NPM-ALK, as confirmed by the higher responsiveness to and the enhanced decay rate of K210A mutant in the presence of 17-AAG (Fig. 4) ⇓ . In agreement with our data, kinase-dead EGFR was recently shown to bind to hsp90 with higher affinity than wild-type EGFR and to be more rapidly degraded by the hsp90 inhibitor geldanamycin (41) . Similarly, folding and stabilization of newly synthesized kinase-inactive heme-regulated eukaryotic initiation factor kinase has been found to depend on hsp90 and to be sensitive to the inhibitory effect of geldanamycin until its kinase domain is activated (71) .
In conclusion, the present study strongly supports the hypothesis that 17-AAG-induced down-regulation of NPM-ALK kinase occurs through the ubiquitin/proteasome degradation pathway, because inhibition of proteasome activity by PS-341 prevents the depletion of both endogenous and exogenous NPM-ALK induced by 17-AAG. We have hereby shown that combined inhibition of hsp90 and proteasome activity causes NPM-ALK to accumulate insoluble and to relocate in ubiquitin-rich perinuclear vesicles that, resembling aggresome formations, couple NPM-ALK dislocation to degradation. Furthermore, we found a correlation between chaperone-assisted folding and proteasomal degradation of NPM-ALK by showing that CHIP ubiquitin ligase, when bound to hsp70, associates with and enhances the turnover of ubiquitinated NPM-ALK. Finally, what we find particularly interesting from a pharmacological viewpoint, is that inactivating mutations of kinase activity were found to increase NPM-ALK responsiveness toward inhibitors of the hsp90-chaperoning machinery, which makes the development of NPM-ALK kinase inhibitors a promising anticancer strategy when used in combination with hsp90 inhibitors.
We thank Dr. B. Falini for anti-ALK (ALK-C) and anti-NPM (NPM-376) monoclonal antibodies; Dr. S. W. Morris (St. Jude Children's Research Hospital) for ALK-11 polyclonal antibody; Dr. Martina Boscaro (Education and Communication Office, Istituto Oncologico Veneto) for help in editing the manuscript; and E. Tosato for technical support.
Grant support: Consiglio Nazionale delle Ricerche-Ministero Istruzione Università e Ricerca, Settore Oncologia, Legge 449/97, and Fondazione Città della Speranza. P. Bonvini was a recipient of a fellowship from Istituto Oncologico Veneto.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Paolo Bonvini, Clinica di Oncoematologia Pediatrica, Azienda Ospedaliera-Università di Padova, via Giustiniani 3, 35128 Padova, Italy. Phone: 39-049-8215678; Fax: 39-049-8215679; E-mail: paolobonvini{at}hotmail.com
- Received November 11, 2003.
- Revision received February 10, 2004.
- Accepted February 26, 2004.
- ©2004 American Association for Cancer Research.


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|He's happy because he invests in the lives of others!
Click on one of the gigs below to sign up to volunteer:
Free Market : Monday - Friday, 10:00 am - 5:00 pm
This is our daily resource of food and other basic supplies for anyone in need who walks through our doors. Volunteers are needed to staff the market, which includes stocking shelves and tidying up, taking information from our guests, and weighing the outgoing food. This is a great way to put in hours volunteering in a low-key environment that allows time to chat with neighbors and share stories.
The Good Sam: We serve a meal on Tuesday nights at a local hostel for their residents and visitors. We go as a team of 4-8 people to bring and serve the food and to spend time eating and talking with the people.
The Banquet: We serve a meal at the Underground on Thursday nights and do it up restaurant-style. A team of 8-10 people give our best to our neighbors and also open up the Free Market afterwards. This is our chance to really show the kind of hospitality industry care that people on the streets rarely receive.
The Conscious Party: A lot of ingredients and people come together to make the Conscious Party each month. Please contact firstname.lastname@example.com if you would like to help in any of these areas.
- Provide food
- Photography or videography
- Invite people on the streets
- Invite artists to perform
Don't let that dolphin show you up. Jump in!


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$149.99 $227
Our BRIANA shoulder bag has a sculpted silhouette with an interior, roomy enough to carry all your essentials from day-to-night. Constructed in genuine pebbled leather, its geometric shape is offset with polished gunmetal micro-beading and hardware. Internal duo-slip pockets and a zipped security pocket ensure contents are easily accessed and organized. Carry it as a hand-held bag or attach the adjustable strap and wear it as a shoulder bag. Either way, this style's versatility and modern feel will enhance any look in your wardrobe.
- Colour: GREY
- Dimensions: H 9.75'' X W 10.5'' X D 5''
- Hardware: Polished Gunmetal
- Lining: Cotton/Polyester Twill
- Genuine Leather
- Leather Care Products Recommended
- Imported


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Brazil says 19 migrants feared drowned off Bahamas
At least 19 Brazilian migrants who went missing last month are feared drowned off the Bahamas as they tried to cross illegally to the United States, officials in Brasilia said Monday.
"The fear is that the boat the Brazilians were traveling in went down in the Caribbean as it left the Bahamas," according to Brazilian authorities on the official government information website, Portal Brasil.
The migrants' families have not heard from them since November 6, the Brazilian foreign ministry said.
Brazilian media reports said there were also dozens of other migrants aboard the boat, which officials suspect went down while attempting the roughly 50-mile (80-kilometer) crossing between the Bahamas and the Florida coast.
"The Brazilian embassy in Nassau, Bahamas and the Brazilian consulate in Miami, Florida are in contact with family members and Caribbean and US authorities to try to locate the missing persons," said Portal Brasil.
The Brazilian foreign ministry said it was not ruling out the possibility that the migrants could be in prison or missing for other reasons.
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Pervacio Inc. operates a Web-based mobile device automation platform that offers a suite of individual applications, features, services, and agents for managing mobile phones and handheld devices. Its Revolution mobile device automation platform delivers SaaS based products.
Looking for a Project Manager for the PMO team in Pervacio. In this role you will be responsible for scoping and delivering large projects end-to-end. Responsibilities include collection of business and systems requirements from internal and external stakeholders, driving project schedules from design to release, and managing till the final release. Coordinate design/implementation efforts between internal teams to develop optimal solutions.
- 10+ years of technical project management experience in Java, Springs & Hibernate Frameworks
- Deep understanding of all aspects of software design with a demonstrated track record of increasing responsibility
- Should have experience in dealing with high level architecture
- Strong coding experience; Ability to get into the code with team members and drive to solutions
- Able to work with Product Managers to prioritize features for ongoing sprints and managing a list of technical requirements based on customer trends, new technologies, known defects, and issues
- Plan, execute and finalize according to strict deadlines and within budget
- Define project success criteria and disseminate them to involved parties throughout project/product life cycle
- Develop best practices and tools for project execution and management
- Estimate the resources and participants needed to achieve project goals
Basic Qualifications
- Excellent problem-solving skills
- Experience developing and delivering on project milestones
- 7+ years of relevant work experience in Technical program and/or technical project management in a software applications environment
- A bachelor's degree in computer science, engineering or a related technical discipline
- Ability to fully engage with technical/operations teams and business teams and effectively manage customer expectations and resolve conflicts that balance client and company needs
- Good understanding in JAVA / J2EE technologies
Preferred Qualifications
- Experience communicating with technical and non-technical stakeholders across multiple units
- Experience with agile or other program management methodologies
- Master's degree in business or engineering or a related technical discipline
- Relevant experience in mobile / mobility domain will be appreciated


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I really cannot emphasize enough how absolutely screwed this is: Every Concealed Carry Permit in eastern NC Invalid Due to Hurricane Irene.
The idea of a state of emergency declared by the governor as we knew it a few years ago used to be a legal formality to be eligible for federal aid money to recover from anything deemed an emergency. At this point, the idea of declaring a state of emergency is totally perfunctory.
And so politicians are able to pass laws that restrict things like the law of supply and demand and otherwise lawful concealed or open carry under the guise of only doing it during "emergencies".
But the result is that the Governor Perdue, in her rush to invoke a state of emergency in order to ostensibly help people, has deprived lawful concealed carry holders of the ability to keep and bear arms in a potential disaster area.
There's a saying in the self-defense community, "When seconds count, the police are only minutes away." I wonder how that adage goes when the power and phone lines are knocked out by a category three hurricane, and the major thoroughfares are washed out.
Edit: I'm not sure what to make of this. Reading the text (Section 7) of the executive order declaring the state of emergency itself says "This order . . . does not trigger the limitations on weapons in G.S. 14-288.7 [the above-linked restrictions on guns] or impose any limitations on the consumption, transportation, sale or purchase of alcoholic beverages."
It's not clear why the law was passed if it is going to be disregarded in a state of emergency, except to change the default to being disallowed to carry. I guess that means we should be grateful when we're allowed to carry during a declared state of emergency.


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It may start with a tingle on the inside of your lip. Or maybe a bit of a burning sensation on your inner cheek. You might wonder, did you poke yourself with a pointy end of a potato chip again? Or worse, who have you been kissing?
There's more than one type of sore that can develop inside your mouth. Some appear as flat lesions or shallow abrasions, while others may be raised bumps. They may be red, white, yellow or gray.
Aphthous ulcers (or aphthae), commonly known as canker sores, are small sores that develop in the soft tissues inside the mouth: the inside of the lips, the inner cheeks and on the tongue. These are the most common type of mouth ulcer.
There are three types of aphthous ulcers: minor ulcers, major ulcers and herpetiform ulcers. Chances are good that if you find yourself with a mouth ulcer it will be a minor aphthous type -- minor ulcers account for at least 80 percent of all aphthae, while major and herpetiform ulcers make up the remaining 20 percent [source: Cleveland Clinic].
Minor sores are usually less than 5 millimeters in diameter (although they can be anywhere from 1 to 10 millimeters and still be considered minor). Often, people develop their first minor ulceration as a child, teen or young adult. Major ulcerations, which affect adults, are similar to minor sores but are larger than one-half inch (1.3 centimeters) in diameter; and the rarest form, herpetiform ulcerations, are clusters of very small ulcers that usually affect only older adults. While some people will have just one or two ulcers in a lifetime, others -- at least one out of five people -- have chronic ulcerations, a condition called recurrent aphthous ulcers (RAU).
While canker sores are not contagious, another type of mouth sore is: the cold sore. Cold sores are sores that appear on the outside of the mouth. They're caused by a virus -- the herpes simplex virus -- and are so contagious that during ancient Roman times an outbreak of cold sores led Emperor Tiberius to ban kissing during public ceremonies.
But it's not the herpes simplex virus that causes ulcers inside the mouth. Let's find out what does, and if you're at risk.


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Mobile Apps notifying users when something new and interesting is nearby are now enjoying a big influx of excitement. Why is the technology so, trending right now? This is not a new innovation. User Experience, bandwidth availability Timing, smartphone ubiquity and also the explosion of Android devices – more than 70% of people have smartphones that can really leverage the tech. Or is it the acceptable level that changed drastically?
The retail industry is going wireless by adapting new technology of proximity device, Smartphone, Bluetooth promotion – Bluetooth proximity sensor, Beacon and much more. The in-stores are thus taking the privileges of opportunities and earning high revenue on investment.
Shopping Apps: Shopping is an in-store feature. A large number of stores offer in-store apps that enable the buyer to use digital coupons, find out whether the store carries the particular product, and enable the store to manage their products in the store. It also comes with easy to swipe and share options for the shoppers.
Some of the retailers also offer reward points, which can cut down the expense while making payment. In addition to time and energy saving, the shopping apps maximize the retailer's checkout range, thus enabling a customer to buy more products with fewer employees. With the delivering coupons of the customers, the retailers can get the consumer data and analyze that for any future use. mega-stores like Walmart launched several features that allow customer profile creation, browsing weekly in-store ads, the creation of shopping lists, in-store pick up, etc. Members of the app can also earn additional gift points that they can redeem later at the store outlets. This encourages shoppers in coming back again and again into the store.
Personalization and Affinity Marketing:
With so many buyers moving toward online purchases, in-store retailers must put an extra effort to put relevant products (that can be predicted by predictive analysis) in front of their users to escalate the store sales.
Affinity marketing creates customers tailored to brands. Suppose you have a Company selling "Running Shoes" and your target is the sports fans for a good sale. But that is not something suggested by Marketing Strategies. You need to target avid marathon athletes instead to enjoy a good sale.
With tracking process and personalization, customers can track, analyze and understand the buying behavior of the customers studying their past purchase record. By curated system, a retailer can also recommend merchandise that was related or compatible with the ones already purchased by the customer to enhance sales and build customer satisfaction.
Predictive Analytics and Retail Insights:
Retailers are utilizing predictive analysis tools to understand retail insight and thus personalize the shopping experience of the users and thereby improve in-store sales. Customer data is pretty essential to influence the business decision of any in-store that includes staffing, inventory management, plan reward program and redesign their in-store.
In-Store Wi-Fi:
According to the latest report, more than 28% of participating in-store of all types and all sizes, experienced increased customer loyalty after launching in-store consumer Wi-Fi, which came along with more than 2% increase in overall sales. Nearly 21% of retailers also reinforced the fact that their shoppers spent more time in the brick-and-mortar, and so… Big Thanks to the In-Store Wi-Fi.
In-store Wi-Fi empowers both customers and in-store sales representatives to gain more information about stocks while in the store.
Online Retailing is growing by the day with the breadth of utility and customer appreciation. If brick-and-mortar wants to stay competitive, adoption of these tech newbies can be the key to sustaining a cutting edge in the era.


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Posts Tagged 'Global Ocean Trust'
GLORES Partner Spotlight: Global Ocean Trust
We are thrilled to shine this week's Global Ocean Refuge System (GLORES) Partner Spotlight on Global Ocean Trust! Global Ocean Trust (GOT) focuses on finding innovative finance mechanisms for better ocean governance and promotes science-based solutions to ocean issues. GOT works through international partnerships to establish marine protected areas representing diverse ecosystems around the globe. GOT helps…Read More


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Our Present & Future
Green Sheep Group (GSG) has been established since 2006 and specialises in the design and manufacture of nursery baby products. Two brands operate under the company umbrella of GSG; The Little Green Sheep and Snüz
In 2017, Green Sheep Group was named in the Sunday Times Fast Track Top 10 Ones to Watch, a prestigious shortlist of some of the UK's most exciting and fast-growing businesses.
Green Sheep Group has a Global retail support network. GSG works directly with leading national retailers and an extensive network of independant stores.
Management Team
Roger Allen
Chief Executive
Roger joined the Company in 2013 as Chairman of the board of directors. He is married with two children and lives locally in Warwickshire. As a professional strategist, his responsibilities are the oversight and support of the directors along with the long term planning of the Company's corporate direction.
He has led a number of UK companies and also owns a commercial property portfolio in South Warwickshire. Roger has led growth strategy over recent years; alongside a recent property search, selection and transition planning. He was appointed Chief Executive in April 2017.
His best 3 moments so far*… Skydiving 2½ miles down into Warwickshire, watching Coventry City win the FA Cup and walking into the Oval Office, West Wing of The White House in Washington DC.
Rosie Pritchard
Commercial Director
Rosie has been with the Company since her first role as an executive and has steadily progressed through the ranks in a number of senior management positions. She became the first director to be promoted internally in 2015 and has overseen the diversification of the customer base, which includes major nationals (Mamas & Papas, Mothercare plc, Amazon, Boots etc) and a very good spread of independent outlets. The business also generates £1Mpa of e-commerce business.
She employs a growing sales and customer service division, which is now supporting clients in continental Europe and the Middle East. Rosie lives locally in Stratford upon Avon.
Her best 3 moments so far*…Becoming Commercial Director at 25, visiting Angkor Wat - a wonder of the world and celebrating New Year in Hong Kong.
Paul Maurice
Product Director
An academic background in Product Design with commercial awareness gained through roles covering a wide variety of industries sees Paul head up the product development of the business. Paul has a focus on product development and logistics, covering product compliance and safety, R&D, product range mapping and separately, the storage and logistics processes within the business.
With an eye for detail on product creation combined with his forward thinking commercial awareness, ensures the products delivered are safe and sought after he product range now boasts multiple award-winning products, the UK's best-selling bedside crib and has attracted a customer base in over 30 countries.
His best 3 moments so far*… Driving a Ferrari around the Monaco Grand Prix circuit, seeing a product I'd designed in a high street store and starting what became the biggest student society in Birmingham.
Mark Nicholls
Marketing Director
Mark has a degree in Marketing and a career history including roles at Johnson & Johnson and Kerry Foods. He is the creator and ongoing guardian of the two brands the business operates; Snüz and The Little Green Sheep.
Mark's passion and flair lie in creating and managing brands that bring something different to existing markets, whilst ensuring they're consistent and relevant to their customer base. Between the two brands the business operates, it can attract and retain customers from across the broad spectrum of parents, from style-aware and feature led customers, to organic followers who want the purest environment for their baby.
His best 3 moments so far*…Watching Newcastle United score a last minute winner in the Champions League to progress to the knockout stages, Graduating in Marketing BSc from Aston University, Birmingham and Seeing Coldplay perform at the Royal Albert Hall and Wembley.
Lianne Bishop
Company Secretary
Lianne is Company Secretary and an experienced CIMA Management Accountant. She has worked closely in the leadership and the running of a number of successful UK businesses.
Her background is in office and financial management following an earlier career change as a professional model with Premier Model Management working for Giorgio Armani, Vivienne Westwood and others..
Her best 3 moments so far*… Spending six weeks living and working in New York, starting the final level of CIMA and modelling for Giorgio Armani in London and Milan..
Jacquie Habershon
Finance Manager
Jacquie has led the finance departments of a number of West Midlands businesses since completing her accountancy training at KPMG Birmingham. She has enjoyed contributing to the growth of these companies, overseeing the introduction of new systems and controls whilst always looking out for the staff - arguably a firm's greatest asset.
After a break to indulge her passion for property renovations, Jacquie is delighted to back at the heart of a dedicated and professional team.
Her best 3 moments so far*... flying with 3 friends to a deserted Caribbean island and camping till the ice ran out, being appointed Finance Director of an aluminium foundry (the only female director in the group), fighting to the front at Wembley for the Nelson Mandela concert to celebrate his release.


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Welcome to the (Un)Masked Blog Tour with Anyta Sunday, Andy Gallo & A Novel Take PR. Check out the re-release of (Un)Masked, an intriguing paranormal romance and see below for excerpts, review, and more. Also, scroll down to see how to enter in the giveaway for a chance to win 3 e-copies of any backlist book by either Anyta Sunday or Andy Gallo.
"What made the book for me? One word: Gristle.
Gristle, the straight boy, Jay's best friend, his "bro," is one of the best characters I've had the joy to read this year. His devotion to Jay is breathtaking, his snark so very snappy […]. Read this book if nothing for the character of Gristle. […] Spectacular."
–T.J. Klune, Author of For Real, Bear, Otter, and the Kid, and Lightning-Struck Heart
Title: (Un)Masked
Publisher: Anyta Sunday & Andy Gallo (Self-Published)
Release Date (Print & Ebook): October 30, 2017
Subgenre: MM gay paranormal romance, magical realism, new adult
Book blurb:
Walker has two wishes: to perform the play of his dreams alongside his best friend at Wellington's Tory Street Theatre, and to meet that special someone. Someone he'd go to the ends of the earth for. Someone who might only exist in fairy tales.
When Jay meets accordion busker Lethe Cross, it's like living a dream come true. Lethe's music captivates Jay, and he resolves to meet the man who plays so beautifully. But then he discovers Lethe's life is more like a nightmare. The phrase "down on his luck" can't begin to cover it. Determined to help, Jay does some snooping for answers—and winds up on the wrong end of a centuries-old curse. The good news is there's a way to break it. The bad news is it might cost Jay his life.
This book was previously published by Dreamspinner Press. This updated 2nd edition has some rewritten parts and has been line edited again.
Buy Links: http://www.anytasunday.com/#unmasked
Excerpt #1:
His music sailed over me, beautiful, seductive; the call of a siren—and I went willingly.
He stood on a concrete wall dividing the beach and pavement, cutting notes into a thousand beautiful fragments that the air caught and whipped around me.
His hips twisted and his fingers slid over the accordion keyboard as he commanded the bellows. Like magic, the disappointment that had clung to me all morning dulled, and I sank against a pylon across and watched him. Watched, and wished the lip of the hoodie he wore would lift so I could see his face.
He swayed with the music, and I glimpsed blond hair and a scar splitting one brow.
Nearer and nearer, I inched, until I stood before him.
His song ended abruptly, and he stared down at me with haunted, tired eyes.
I opened my mouth to ask him to play again, to tell him he was truly talented, but he jumped onto the beach and fled.
My soul prickled, and I knew I would never be the same again.
One never was, when death lurked nearby.
Author Bios:
Heart-stopping slow burn.
Anyta Sunday is a big, BIG fan of slow-burn romances. She loves to read and write stories with characters who slowly fall in love.
Some of her favorite tropes to read and write are: Enemies to Lovers, Friends to Lovers, Clueless Guys, Bisexual, Pansexual, Demisexual, Oblivious MCs, Everyone (Else) Can See It, Slow Burn, Love Has No Boundaries.
Anyta writes a variety of stories, Contemporary MM Romances with a good dollop of angst, Contemporary lighthearted MM Romances, and even a splash of fantasy. Her books have been translated into German, Italian and French.
Member of Romance Writers of America.
Connect with Anyta: Website | Twitter | Facebook | To receive a free e-book, sign up for Anyta's newsletter here!
Andy Gallo's stories capture how he wished he'd spent his formative years, instead of how it really happened. Unfortunately for his characters, they find themselves infused with some of Andy's less noble qualities.
A hopeless romantic, Andy writes seated next to a hundred year old Smith Brothers typewriter he inherited from his grandfather. He also dreams of superheroes and wizards and sees no reason why two men with superpowers can't fall in love just like everyone else. Although not all of his stories have a paranormal bent, a touch of the supernatural never derailed a good read in his mind.
Married and living his happy ever after, Andy helps others find their happy endings in the pages of his stories. He and his husband of more than twenty years spend their days rubbing elbows with other parents as they raise their daughter. Embracing his status as the gay dad, Andy sometimes has to remind others that one does want a hint of color even when chasing after their child.
Connect with Andy: Website | Facebook
Giveaway: 3 e-copies of any backlist book by either Anyta Sunday or Andy Gallo:
a Rafflecopter giveaway
Direct link: http://www.rafflecopter.com/rafl/display/88d45f0332/
Review by Lost in a Book
4.25 Stars
I get it. I totally get why there are mixed reviews on this book. Especially if you're comparing them to some of the Anyta books we know and love. You know, those light hearted, quirky, with witty banter, slow burned to the brink of death, and a lightness that you just feel like you can float away on? Yep, those. If you're an established Anyta fan girl/boy like I am, I had a hard time not comparing the feels in this to the ones I've read in the past. And when I really thought about it, not all of her previous writes had that light hearted feeling. One of my favorites is her novella Bird Meets Cage that isn't at all like her other books. Or Rock. Once I checked my preconceived notions at the door, I enjoyed the hell out of this one with all of its sad, heartbreaking heaviness; Because on the outskirts of all that melancholy is hope and light and self-sacrifice
"Do you believe there are things in this world that can't be explained?"
In (Un)Masked it's possible. Lethe is the last living member of a cursed bloodline and the curse makes it almost impossible for him to let his guard down to live and love, until he meets Jay. Jay sees Lethe for who he is. Their journey is difficult and many times seems impossible. This book is completely unique and very little can be revealed without ruining the heartbreakingly beautiful story that Anyta Sunday and Andy Gallo created. It has a mysterious feel and I was rooting desperately for them to beat all the odds stacked against them.
The love between Lethe and Jay was perfectly imperfect. They got their HEA that almost bordered on the HFN end of the spectrum. They had chemistry but there was very little sexy times and those that were on page weren't explicit. Normally I'm all gimme the sex, but the quantity and heat level fit the bill for the obstacles the MCs faced.
Gristle. Oh Gristle. I loved you so hard. I was planning little Gristle/Jay babies. But alas, they were just
friends soulmates. Gristle and Jay's love ran so deep that I hurt for them. My face leaked at the sadness surrounding Gristle from the very beginning all the way through. He added so much life to this story that I considered him a third main character.
This book was really well written and I couldn't put it down. I had to know what was going to happen which is my favorite type of read. Definitely recommended.
Buddy read with Vir. 🙂
Copy provided for honest review.


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Among the very best coverings planned for Eiderdown is generally Swiss Batiste, the lightest of all down-proof cambric cottons integrated with the largest thread matter, sateen finish in addition to a smooth soft touch, woven specifically for Eiderdown, the softest down on the earth. Thread matter is the amount of threads which are found in a single square inch of material made use of relating to the cover which is where the down is included. Lighter and softer material will have a higher thread matter. Thread counts above 300 tend to be most desirable and are going to continue to be down-proof for 10-15 years.
But how can you select the ideal duvet for you? Many homeowner are not even aware of what differentiates a high-quality duvet from a typical one. Fortunately, so long as you examine a couple of essential characteristics of the duvets that you consider, you should be able to find bedding for your space that is both appealing and useful.
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Silk Luxury Baby Bedding sets were as soon as reserved just for the resting quarters of nobility and the extremely rich. Nowadays, however, everybody can luxurious themselves in the extravagant feel of the product. There are lots of advantages of having silk sheets on your bed. Being lightweight, hypoallergenic, easy to clean, and long lasting are just a few of the reasons you have to purchase silk bedding.
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But, you can not stay clear of washing down comforters at some point even if they are protected. Right here is a great way think of it and keep in mind the formula. Down are feathers and are usually the feathers of geese. Exactly how do you expect a goose keeps its feather clean? Cold water is the right response to that. And, think of this. Exactly how would a goose perform in an agitating washing device? It's an unsightly thought but a fantastic picture of exactly what not to do. So, exactly what does this mean? It implies you must clean you down comforter in a front packing device like they have in laundry mats not leading packing agitating device like you discover in many houses. The agitating device can damage the comforter completely. The secret is a gentle cycle of cold water.
When selecting your bedding collection, you should pay attention to four necessary elements, including style, color, quality and fabric of the material. You should balance these four elements so about create your bedroom design which can most mirror your personal style. The very first important thing for you to consider is exactly what you need them for. You should also keep in mind that where you would want to utilize the bedding sets and your specific bed size.
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These sheets have elastic edges that make use of to fit over the mattress which is likewise called as fitted sheet. The coverlets are made-up of decorative great material coverings that do not touch the floor. These coverlets do not cover the pillows. The woven coverlets and quilts are among the most popular coverlets. Bedspreads are more like the coverlets with the tin decorative coverings but these cover the entire bed and touch the floor. The blankets or throws are the little coverings that make use of to position at the foot of the bed and cover the body for heat. These are important decorative accents forbedrooms.
These sets give a fantastic impression without a huge investment. You can often purchase a set of around six pieces consisting of pillow cases and sheets as well as the cover. The majority of high street shops have a large range of sets at various rates and this is one of the major advantages of choosing this product kind.
Things Dangerous Zombies Can Teach You About Bedding
Not everyone can make the most of quality down filled Luxury Baby Bedding. You might dislike it. This is where alternative down is available in. It is the best selection for allergy patients. This bedding will be basic to keep clean. You can easily care for it. It will keep you warm and look great in all kinds of bedroom decoration. It might be a little heaver than a down comforter.
A natural goose down duvet is highly helpful over many of its equivalents due to the fact it is exceptionally resilient and breathable. Assisting to manage your body temperature level far better than anything else on the market, a natural duvet will assist to keep you warm in the winter whilst guaranteeing you are cool in the summer months. It is this flexibility that has actually rapidly developed the use of down within duvets as the leading, the majority of kind after item offered.
Eiderdown includes a characteristic referred to as "hooks.". These hooks allow the clusters to stick on to one someone else, for that reason capturing teeny air pockets. All these air pockets form barriers. Eiderdown, while used in bed comforters or duvets plus bedroom pillows, checks out at around 700 fill power. Nevertheless, because of the "hooks" within the eiderdown cluster it brings an unequaled density as well as adhesiveness and warms at a wonderful 850 to 900 fill power.
Not everybody can pay for to have luxury houses. However you can make luxury and deluxe rooms including the right furniture, making use of the right wall colors, making use of wall hangings, making use of stunning window drapes and door drapes, the right mattresses and pillows and naturally making use of mesmerizing bedspreads and pillow covers. It is bedspreads that are clean, that are either sober or colorful, that which exhibition detailed designs or patterns that define the décor visual appeals of rooms. The effort of relaxing on your bed or taking pleasure in a blissful sleep gets rewarded; getting rejuvenated after a long day's job then appears reasonable.
There are all kinds of fabrics and materials that are used to create comfy sheets and pillow cases, however just the finest are used to create luxury bed linens. From Egyptian cotton to pure silk, these are the materials that provide us the most luxurious nights sleep. Product is not the only thing that makes luxury bed linens so fantastic though; there are other factors included also.
If You Want To Be A Winner, Change Your Bedding Now!
It's all well and good to choose that you want to make sleeping even more enjoyable by buying some luxury bedding but, if you dont even understand what luxury bedding sets are expected to include, you're sure to wind up disappointed. To further complex matters, the terms surrounding luxury bed linens appear to be among those things everybody in the understand expects everybody else to currently understand. Well, let's punch a hole in the member's only club. Here's a quick rundown of the various terms in luxury bedding.
Because this is a distinct event, you should choose bedding and devices which are gorgeous and pleasing to the eyes. If you have the ability to afford it, you will use laced sheets with elaborate floral styles. Such sheets will give your bed an elegant, clean and fresh look. If you are on a budget, simply choose bedding with stunning colors and romantic designs like trendy roses and hearts in deep red or burgundy.
What Everyone Ought To Know About Luxury Baby Bedding
Duvet covers are usage to position over the duvets and can be utilized for the comforters. These covers have openings with buttons for inserting the duvet or comforter. These are ornamental coverings that make your bedrooms indoor more enticing and sophisticated. Some of the duvet covers featured sheet of material sewn into one side and do not need top sheet. It streamlines the bed making. Euro or Continental pillows are the big square size pillows that sit back against the headboard of the bed. These look very ornamental. The sleeping pillows are available in different sizes such as the requirement, Queen, King and so on. These are usage to lay the head on while sleeping.
Wonderful Suggestions To Improve The Look And Feel Of Bedding
The majority of moms and dads generally get the kid's baby room ready even prior to the kid can show up. Oftentimes such moms and dads overlook the gender of the kid, hence leading to embellishing the whole space in the most inappropriate color possible. That is when you see a baby boy in a pink space and a baby woman in a blue space with toys and clothing that match that specific color.
Whilst they clearly still have a vested financial interest in the bedding sets that they promote, they all take the field of developing really seriously. This indicates that you can be sure that they have actually done their best to produce quality designs that that they are delighted to put their name to, in contrast to simply searching for the fast payday.
All your bed room needs is charm. With comforters and duvets, which hare versatile and functional elements to always provide your be that appealing appearance and style day in day out. You can be ensured of adequate design and warmth for your bedroom. Their qualities of inexpensiveness, most painless and also the quickest response to the bedroom desires for charm grants you every opportunity to enjoy your bedroom.
Many of those alternate alternatives are made of synthetic products that imitate the appear and feel of down. Primaloft Comforters, for instance, are produced from a hypoallergenic synthetic product that has actually verified being a close to match to along in warmth and convenience. Primaloft Comforters are readily offered On the Company Shop, Eddie Bauer and Property's Stop.
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The other element you will check out is the gentleness of the material. Therefore, you will search for a cotton material that is highly threaded. Numerous companies focus on highly threaded material. Study on them to obtain the one that provide Luxury Baby Bedding with the very best quality. This will enable you to not just get the very best deign however also the very best quality.


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Check out this intelligent, stimulating, and thoughtful website where a wide, wide range of leaders and thinkers, including Jim Lehrer, Moby, Richard Branson, and Anna Deavere Smith, talk about what's important to them and to the world. This is just the blog, the site will launch in January, but this amuse-bouche has got us hungry for a whole bigthink dinner.
by Winsome


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Are Your Customers Getting Your Message?
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Sales & Product Launches
If you are looking for a way to tell customers about a sale or new product, SMS marketing is a great way to do it. Linking it to your website or app would guide customers right into your "store", increasing the chances of a purchase. Domino's Pizza has taken advantage of SMS marketing tools, offering customers the chance to opt into the program.
As a company, you want customers to have an easy access point into your company. When there is less hassle for your customer's, their chances of purchasing are greater. So when the lunch cravings hit, their customers don't have to go to a website and fill out long forms, they just text for a pizza!
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Benefits of SMS and Text Marketing
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3.1 Rapid Response to an Unexpected Child Death
Show amendments
A Guide to Investigating Child Deaths (College of Policing, 2014)
At all stages of these procedures, practitioners should consider:
- Whether a referral for child protection under Section 47 is required;
- Whether the criteria for a Serious Case Review (or other review) might apply in which case, the MKSCB office should be contacted, the Serious Case Review Procedure should be followed, and a MKSCB SCR referral form should be completed and submitted to the MKSCB office;
- Sensitivity to the needs of parents and siblings including bereavement support;
- Does the child death meet the criteria for reporting by the local authority to Ofsted as a notifiable incident (see Working Together to Safeguard Children 2015).
This chapter was revised in October 2015 to take account of Working Together to Safeguard Children 2015, changes in Coronial procedures and revised interagency working processes following the implementation of Milton Keynes Multi-Agency Safeguarding Hub (MASH)
- 1. Introduction
- 2. Notes on Definitions
- 3. Agency Roles and Responsibilities
- 4. Initial Case Review Discussion
- 5. Coroner's Investigation and Inquest
- 6. After the Inquest
- 7. When a Child Dies Out of Area
- 8. Abbreviations and Acronyms
1. Introduction
|This procedure was first produced in response to 'Working Together to Safeguard Children, 2006'. It has since been updated in response to local and national developments, including the revised Working Together 2015. It forms the basis of the inter-agency response to an unexpected death of a child in Milton Keynes.
2. Notes on Definitions
|"Childhood" is defined as the time from live birth (irrespective of gestation) until the attainment of the age of 18 years. By far the largest group of child deaths occurs in the perinatal period (0 - 7 days). For "rapid response" procedures these infants will not be routinely included unless involved professionals express concerns about the circumstances of the pregnancy or the death.
|"Responsible consultant" has been used to identify the senior involved clinician, this is most likely to be a paediatrician but when the death is that of a young person of over 16 years the clinician will be an adult specialist and some interpretation of the protocol depending on age and mode of death may be required.
|"Unexpected death" is defined in Working Together to Safeguard Children 2015 (Chapter 5, paragraph 12) as the death of an infant or child (less than 18 years old) which was not anticipated as a significant possibility for example 24 hours before the death; or where there was an unexpected collapse or incident leading to or precipitating the events which led to the death.
"Death" means the time that death is formally certified. It is clearly not appropriate to instigate this process whilst the child is alive and receiving medical care. However, in cases where the medical team have any concerns that there are issues of abuse or neglect when a child has presented in a critical condition, especially where there are other children in the household, then early discussion with Police and Children's Social Care (CSC) should take place. This will allow scrutiny of the CSC and Police databases as a minimum precaution and further inter-agency involvement as considered necessary. These preliminary enquiries can be made without informing the parents if it is considered that this will add to their distress.Rapid Response procedures may need to be considered in a young person over 18 years old in the exceptional circumstance where the unexpected collapse or incident leading to or precipitating the events which led to death occurred when the young person was under 18 years old.
3. Agency Roles and Responsibilities
|Once a child has been declared dead, the Coroner has jurisdiction over the body and all that pertains to it.
|The majority of unexpected deaths in childhood are natural tragedies, but a minority are a consequence of ignorance, neglect, abuse or homicide. The investigation should keep an appropriate balance between medical and forensic requirements and the needs of the family in coping with the tragedy. Account should be taken of possible risks to other children in the household.
|Professionals should approach the investigation with an open mind and families should be treated with sensitivity, discretion and respect. Professionals must be aware that as the number of child deaths due to natural causes decreases, the proportion of such deaths which could be attributed to neglect or abuse is likely to increase.
|There should be a multi-agency approach involving collaboration among: emergency department (ED) staff, ambulance staff, named and designated doctors and nurses in child protection, Coroners, coroners' officers, general practitioners (GP's), health visitors, midwives, paediatricians, pathologists, police, social workers and education.
|In each agency a senior person with suitable training and experience should be identified as having responsibility for implementation of the protocol, including continuing training for all relevant staff.
|The Designated Paediatrician for Unexpected Deaths in Childhood will take the strategic lead for rapid response within Health, but the input at senior level in individual cases will be from the consultant involved in the initial event as at present (responsible consultant).
|The Duty Detective Inspector for the Local Police area will lead for the Police on initial rapid response to Child Death. Subsequent enquiries will be led by the Local Police area Detective Inspector for the Child Abuse Investigation Unit (CAIU) and the Team Manager (Multi Agency Safeguarding Hub) for the local authority.
|Children found dead at home should usually be taken into the emergency department, not to the mortuary, and resuscitation should be initiated unless clearly inappropriate. Ambulance crew must contact the police with full incident details as soon as it is apparent that a child is in cardiac/ respiratory arrest. There are situations where it is obvious that a body is beyond resuscitation and needs either to remain at the death scene for forensic purposes or to be moved to a mortuary. This will be a decision for the police in consultation with the Coroner as outlined in the Murder Investigation Manual or in the case of road deaths, the Road Death Investigation Manual.
|On arrival in the emergency department the parents/carers should be allocated a member of staff to care for them and should normally be given the opportunity to hold and spend time with their child at a later point while in the department. They should also be offered mementoes e.g. a lock of hair or a photo.
|As soon as possible after arrival, the child should be examined by the responsible consultant and a careful history should be taken from the parents.
|A standard set of investigative samples should be taken. This list is available in the emergency department and on the MKHFT safeguarding children intranet pages.
|When the child is pronounced dead, the responsible consultant should inform the parents/carers, and explain the Coroner, Police and Children's Social Care involvement and the need for a post-mortem examination. The opportunity to donate tissues or organs would only occur in the situation where life support on an Intensive Care Unit following collapse was being withdrawn and there was an opportunity to seek the Coroner's permission.
|As soon as death has been confirmed, notification should be given to the Coroner (via the Coroner's Officer), the Police, Children's Social Care and the primary care team.
|Further notification of the child's death, should be completed, as per the "Procedure for sudden unexpected death of a child' within the Safeguarding Children policy for Milton Keynes University Hospital NHS Foundation Trust", this includes completion of the checklist which is then forwarded to the MKSCB CDOP Co-ordinator.
|When a child goes directly to the mortuary the on-call clinical team (paediatric or adult) should be informed so that urgent specimens can be obtained as necessary and support for the bereaved can be organised.
4. Initial Case Review Discussion
The duty Social Worker is contacted by the responsible consultant or Duty Detective and a Multi Agency Referral Form (MARF) is completed by them and forwarded to the Milton Keynes Multi Agency Safeguarding Hub (MASH). An initial Case Review Discussion should be held between the lead professionals (the involved clinician, the senior investigating officer in consultation with the CAIU DI and the MASH at Children's Social Care). This is usually done by telephone by the involved clinician but should be initiated by the agency that has the initial contact with the child if not Health.
The circumstances of the child's death and initial discussions involving the clinicians in consultation with the Police Senior Investigating Officer (SIO) needs to be incorporated into the MARF.
In the event of a child dying out of hours, the out of hour's duty team will be involved in the case review discussion and refer into MASH for the next working day.
The purpose of the Case Review Discussion is for agencies:
In most cases a multi agency discussion will take place within the Milton Keynes MASH. Depending on the outcome of this discussion a Strategy Meeting under Section 47 Children Act may be convened by Children's Social Care. There will be situations where the circumstances of the death are straightforward and this is not required.
The outcome from the MASH case review discussion or any Section 47 Strategy Meeting should be sent to the Coroner's Office by a practitioner in the MASH. Depending on the discussions at the meeting, reports for the Coroner may be requested from:
|If the death is suspicious or significant concerns are raised at any stage about the possibility of abuse or neglect, a decision will be taken for the police to become the lead agency, and take primacy in the investigation. In these circumstances the Duty DI will inform a Senior Investigating Officer (SIO) from Major Crime. A decision will then be made about who will take responsibility for investigating the child's death. In these circumstances consultation must take place with the police, to ensure no compromise of information to the parents/carers or those close to them, who may be responsible for or contributed to the cause of death. In all other cases, Health remains the lead agency.
|If it is thought at any time that the criteria for a Serious Case Review might apply, the Chair of the MKSCB should be contacted and the Serious Case Review Proceduresshould be followed.
|When a baby or older child dies unexpectedly, the police are responsible for investigating the circumstances of the death, and may have to secure the death scene. There will be a visit to the scene of the death (preferably within 24 hours). This home visit is by the Police as part of the forensic investigative process; additional clinical expertise will be accessed as part of this process where necessary. Following an initial review of the scene by the Police, if expert clinical opinion is required the material gathered by the police will be reviewed and assessed jointly. The Police are responsible for providing the Coroner with a report from the scene visit and subsequent investigation into the death. In addition to notifying the Coroner, the police will notify the MASH of the outcome of the home visit to enable re-evaluation of the information and reconsideration of whether a s47 strategy meeting is required. The CDOP co-ordinator should also be notified in order to track and coordination of the Rapid Response process.
|A copy of the medical notes will be made available to the Coroner and the pathologist. The details of the involved clinician MUST be clearly recorded on the medical records. It must be clear on the medical records who the lead clinician is, to facilitate discussion between the pathologist and the lead clinician. This is particularly important in cases where a forensic pathologist is required.
|The Coroner's Office will complete a Form A which will indicate whether or not a post-mortem examination will be carried out. Completed A forms should be sent to the MKSCB CDOP Co-ordinator The coroner will order a post-mortem examination to be carried out as soon as possible, preferably within 48 hours, by the most appropriate pathologist. In most cases, this will be a paediatric pathologist, following a recommended protocol, but if significant concerns have been raised about the possibility of homicide, abuse or neglect, a Home Office approved paediatric pathologist should take the lead. If the post-mortem examination reveals no sufficient identifiable cause of death, whether or not any concerns have been raised during the post-mortem examination or previously about the possibility of abuse or neglect, the pathologist should categorise the death as "unexplained pending further investigations". The coroner should hold an investigation into every case which may lead to an inquest, if death is not due to natural causes. This process would apply when the final post-mortem report is available.
|Following the initial Post-mortem results, an MDT meeting/discussion occurs with all professionals involved. The Coroner/Police/Paediatrician will discuss the results of the initial post-mortem (either directly or by email). This discussion, the decisions made and any actions taken should be to be copied to the CDOP Co-ordinator. Confirmation of the discussion, and the post-mortem report should also be sent to the MKSCB CDOP Co-ordinator.
If the post mortem indicates death from abuse or neglect:
The autopsy report will be forwarded to the responsible consultant and to the Lead Paediatrician for Child Death. Either (or both together) should discuss the results of the autopsy with the parents/carers at the earliest opportunity:
|The parents/carers will receive written information advising that the child's death will be subject to a review by the Child Death Overview Panel (CDOP) in order to learn any lessons that may help to prevent future deaths of children (see Child Death Overview Panel Procedure).
5. Coroner's Investigation and Inquest
|HM Coroner in Milton Keynes holds an investigation into all sudden unexpected deaths in childhood and an inquest will be held if a cause of death is not ascertained. The information shared as in the above paragraphs will contribute to this process.
The purpose of an Inquest is to determine
6. After the Inquest
|If the verdict of the inquest is that abuse and neglect caused or contributed to the death the Police and Children's Social Care will act accordingly and the need for a Serious Case Review will be considered (if these have not previously occurred).
|If the child's family have any unanswered questions concerning the death the responsible consultant or Designated Paediatrician for unexpected deaths in Childhood (or both) will meet them to answer their questions. If the family do not wish contact this may be done by letter.
|The Designated Paediatrician for unexpected deaths in childhood will consider whether a professionals meeting is required to consider the needs of other children in the family or future children and will convene and chair this meeting. It is expected that SUDI cases are discussed in this way.
|In the case of SUDI it should be noted that if there are subsequent pregnancies in the mother will be offered a referral to the Care of the Next Infant (CONI) programme at booking.
|All information and the standard data set will be forwarded to the Child Death Overview Panel
|Where it is anticipated that this may become a high-profile case the MKSCB will co-ordinate a media response/communication strategy between MKSCB partners.
7. When a Child Dies Out of Area
Where a child (usually resident in Milton Keynes) dies out of area, communication should go through the MKSCB single point of contact: 01908 254373 who and we will invoke these procedures.
8. Abbreviations and Acronyms
|Child Abuse Investigation Unit
|Care of Next Infant
|Children's Social Care
|Detective Inspector
|General Practitioner
|Local Policing Area
|Sudden Infant Death
|Senior Investigating Officer (Police)
|Sudden Unexpected Death of an Infant