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Elevated Kallikrein-binding protein in diabetes impairs wound healing through inducing macrophage M1 polarization
Cell Communication and Signaling volume 17, Article number: 60 (2019)
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The accumulation of M1-polarized macrophages and excessive inflammation are important in the pathogenesis of diabetic foot ulcer (DFU). However, the underlying mechanism of DFU pathogenesis and the crucial regulators of DFU are less well known. Our previous study reported that kallikrein-binding protein (KBP), an angiogenesis inhibitor, was significantly upregulated in diabetic patients compared to its levels in controls. The effects of KBP on monocyte chemotaxis and macrophage M1 polarization were elucidated in this study.
Plasma KBP levels and monocyte counts were assessed by ELISA and flow cytometry. Wound closure rates in different groups were monitored daily. The phenotype and recruitment of macrophages were measured by real-time PCR, western blot and immunofluorescence assays. The expression of Notch and NF-κB signalling pathway members was determined by the methods mentioned above. ChIP and dual-luciferase reporter gene assays were employed to explore the binding and transcriptional regulation of Hes1 and iNOS.
We found that plasma KBP levels and circulating monocytes were elevated in diabetic patients compared to those in nondiabetic controls, and both were higher in diabetic patients with DFU than in diabetic patients without DFU. KBP delayed wound healing in normal mice; correspondingly, KBP-neutralizing antibody ameliorated delayed wound healing in diabetic mice. Circulating monocytes and macrophage infiltration in the wound were upregulated in KBP-TG mice compared to those in control mice. KBP promoted the recruitment and M1 polarization of macrophages. Mechanistically, KBP upregulated iNOS by activating the Notch1/RBP-Jκ/Hes1 signalling pathway. Hes1 downregulated CYLD, a negative regulator of NF-κB signalling, and then activated the IKK/IκBα/NF-κB signalling pathway.
Our findings demonstrate that KBP is the key regulator of excessive inflammation in DFUs and provide a novel target for DFU therapy.
Diabetic foot ulcer (DFU) is one of the most intractable complications of diabetes mellitus and leads to nontraumatic amputation in more than 70,000 patients worldwide [1, 2]. The pathological impairment of wound healing is the foremost reason for DFU. Wound healing consists of the following overlapping dynamic phases: inflammation, re-epithelization and neovascularization, and tissue remodelling [3, 4]. The local inflammatory response initiated during wound healing includes the migration and proliferation of diverse cells in addition to the regulation of inflammatory factors and cytokines [3].
Macrophages, which derive from monocytes and upstream progenitor cells, are involved in all the phases of wound healing [5]. Macrophage colony-stimulating factor (M-CSF) and monocyte chemoattractant protein-1 (MCP-1) are vital cytokines for macrophage survival, differentiation and mobilization [5,6,7]. Furthermore, the recruitment of monocyte-macrophages to wounds depends on MCP-1 secreted by diverse skin cells and the expression of its receptor chemokine receptor 2 (CCR2) on monocyte-macrophage surfaces [5, 8]. Macrophages 2assume a spectrum of activation states ranging from pro-inflammatory M1 macrophages that induce an inflammatory response with the secretion of inflammatory factor [9] to anti-inflammatory M2 macrophages that promote the absorption of inflammation and wound healing [10,11,12]. M1 macrophages are characterized by the production of inflammatory mediators, such as inducible nitric oxide synthase (iNOS), IL-6, IL-12, and TNF-α, in response to IFN-γ and LPS [13]. M2 macrophages express anti-inflammatory mediators; promote angiogenesis mediators, such as arginase-1 (ARG1), IL-10, TGF-β1, and VEGF; and play a pivotal role in tissue repair, reconstruction and tumours [14, 15]. Delayed diabetic healing is characterized by excessive inflammation with the prolonged accumulation of M1 macrophages and elevated pro-inflammatory cytokines. In addition, anti-inflammatory factors and growth factors secreted by M2-polarized macrophages are also downregulated [16]. However, the reason for this abnormal phenotypic transformation in M1/M2 macrophages in diabetic patients is not well defined.
Kallikrein-binding protein (KBP), also named SERPINA3K, was originally identified as a member of the serine proteinase inhibitor (serpin) family [17]. KBP is a plasma protein mainly synthesized and secreted by the liver that has a wide-ranging spectrum of activities, including the relaxation of blood vessels and the inhibition of angiogenesis and antioxidative stress [18, 19]. Our previous studies have shown that circulating KBP levels are increased in diabetic patients with microvascular complications compared to those in diabetic patients without microvascular complications; furthermore, KBP delays diabetic wound healing through inhibiting angiogenesis [20, 21]. Although the effect of KBP on angiogenesis in diabetic wound healing has been reported [21], the effects of KBP on macrophage polarization and the excessive inflammatory reaction in diabetic wound healing have not been documented.
The Notch family is a family of evolutionarily conserved proteins that regulate cell differentiation, proliferation, survival and development [22]. Notch ligands bind with their receptors, resulting in intramembranous cleavage by γ-secretase to release Notch intracellular domain (NICD). NICD translocates into the nucleus and binds to the DNA-binding protein RBP-Jκ to activate Notch target genes, such as Hes1 and Deltex [22]. Notch signalling plays a pivotal role in regulating the development and differentiation of monocyte-macrophages [23, 24]. Nevertheless, the role of KBP in regulating monocyte-macrophages through Notch signalling during wound healing has not been verified.
The NF-κB signalling pathway is a classic pathway that promotes the M1 polarization of macrophages [13]. The activation of inhibitor of κBα (IκBα) kinase (IKK) promotes the phosphorylation of IκBα, which is the inhibitory form of IκBα, following which NF-κB p65 is activated and translocated into the nucleus to activate the expression of target genes [25, 26]. The activation of the Notch signalling pathway can promote the activation of the NF-κB signalling pathway [27,28,29,30]. It remains to be explored whether KBP promotes the M1 polarization of macrophages via activating the Notch and NF-κB signalling pathways.
In this study, we elucidated the role of KBP in the excessive inflammatory response during diabetic wound healing. We additionally tested the hypothesis that KBP regulates the numbers and polarization of monocyte-macrophages by activating the Notch and NF-κB signalling pathways, consequently delaying wound healing.
Human subjects
The collection of human samples adhered to the Declaration of Helsinki and was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital. All patients provided their informed consent. All diabetic patients with or without DFU were diagnosed by a medical doctor.
Animal experiments
All the animal experiments were carried out with the approval of the Animal Care and Use Committee of Sun Yat-sen University (approval ID: SCXK 2011–0029). Wild-type C57BL/6 mice were purchased from the Laboratory Animal Center of Sun Yat-sen University. A human KBP transgenic C57BL/6 mouse strain (KBP-TG) generated as previously described was provided as a gift from Dr. Jianxing Ma (University of Oklahoma Health Sciences Center) [21]. Six-week-old male mice were fed a high-fat diet (60% of calories, D12492, Research Diets, Inc.) for one month and then intraperitoneally injected with streptozotocin (STZ; 40 mg/kg/day) daily for 7 days to induce type 2 diabetes [4, 31, 32]. The type 2 diabetic mice were randomly divided into two groups: an IgG group and a KBP antibody (0.4 mg/kg/day) group. IgG (Sigma-Aldrich, St. Louis, MO, USA) or KBP-neutralizing antibody (Genscript, China) was intraperitoneally administered to the diabetic mice every day beginning three days before the establishment of a wound model for 15 days. BSA or KBP (20 mg/kg/day) was intraperitoneally administered to the WT mice every day beginning three days before the establishment of a wound model for 15 days. Male db/db mice, which is also a type 2 diabetes mouse model, were purchased from Nanjing Model Animal Center. Wound healing rates were observed, the wounds were photographed every other day, and wound tissues from different mouse models were collected.
Cell culture
Bone marrow-derived macrophages (BMDM) were generated as previously described [33]. BMDMs and mouse RAW264.7 macrophages were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin. THP-1 cells were cultured in RPMI-1640 with 10% FBS and 1% penicillin/streptomycin. THP-1 cells were differentiated with phorbol 12-myristate 13-acetate (PMA) (20 ng/mL, Sigma) for 72 h.
ELISA to detect KBP, GM-CSF/M-CSF, TNFα, IL-6 and MCP-1
The plasma level of KBP was detected using a human KBP ELISA kit (R&D Systems, Minneapolis, MN, USA, #DY1669) according to the manufacturer's instructions. The levels of GM-CSF, M-CSF, MCP-1, TNFα and IL-6 in mouse plasma or cellular supernatants were measured with a mouse GM-CSF ELISA kit (R&D Systems, #MGM00), mouse M-CSF ELISA kit (RayBiotech, RayBiotech, Norcross, GA, USA, #ELM-MCSF-1), mouse MCP-1 ELISA kit (RayBiotech, #ELM-MCP-1), mouse TNFα ELISA kit (R&D Systems, #DY410–05) and mouse IL-6 ELISA kit (R&D Systems, #DY406–05).
Wound healing assays
The dorsa of anaesthetized mice were clipped to remove hair, and then standardized circular wounds were made with a full-thickness 6-mm biopsy punch (Acuderm, Fort Lauderdale, FL). Wound closure rates were monitored by tracing the wound area daily through photographs that were quantified with ImageJ software. Frozen wound tissue slides were stained with F4/80 antibody (1:200, Abcam, Cambridge, MA, USA, #ab6640), iNOS antibody (1:200, Abcam, #ab3523) or ARG1 antibody (1200, Santa Cruz, CA, USA, sc-20,150).
RNA extraction, reverse transcription of cDNA, and real-time quantitative PCR
RNA extraction, reverse transcription and real-time PCR were performed as described previously [34]. The primers used for real-time PCR are listed in Additional file 1: Table S2.
Western blotting
Western blot analysis was performed as described previously [4, 35]. The proteins were transferred to a PVDF membrane (Millipore, Billerica, MA, USA) and probed with primary antibodies specific for iNOS (1:1000, Abcam, #ab3523), ARG1 (1:200, Santa Cruz, sc-20,150), Notch1 (1:1000, CST, Danvers, MA, USA, #4380S), Hes1 (1:1000, CST, #11988) and β-actin (1:10000, Sigma-Aldrich, #A5441) overnight at 4 °C. The following secondary antibodies were used: goat anti-rabbit IgG/HRP (1:1000, Vector Laboratories, Burlingame, CA, USA, #PI1000) and goat anti-mouse IgG/HRP (1:5000, Vector Laboratories, #PI2000). Chemiluminescence was developed using ECL Western blotting substrate.
Immunofluorescence staining and immunohistochemistry
For immunofluorescence staining, wound sections were fixed in 4% paraformaldehyde and permeabilized with 0.01% Triton X-100 in PBS. The samples were incubated with F4/80 (1:200, Abcam, #ab6640), iNOS (1:200, Abcam, #ab3523) or ARG-1 (1:200, Santa Cruz, #sc-20,150) antibodies overnight at 4 °C and then incubated with Alexa Fluor 488-donkey anti-rat IgG (H + L) (1:200, Life Technologies, Gaithersburg, MD, USA, #A21208) and Alexa Fluor 594-donkey anti-rabbit IgG (1:200, Life Technologies, #R37119) for 1 h. The slices were digitally photographed with a confocal microscope. For immunohistochemistry, tissue slices were prepared as described before [36]. The sections were incubated with F4/80 antibody overnight at 4 °C and then incubated with a biotin-conjugated secondary antibody for 30 min, followed by incubation with DAB for 10 s and haematoxylin staining for 30 s. The IHC signal for F4/80 was analysed using ImageJ.
Transwell migration assay
Chemotaxis experiments were performed using 24-well Boyden chambers (Corning, NY) as described previously [4, 37]. Briefly, DMEM containing 10% FBS was placed in the lower chamber. A total of 1 × 105 RAW264.7 cells in 200 μL medium were seeded into the upper chamber. Macrophages were preincubated with 640 nM KBP for 48 h prior to seeding. The chamber was then incubated for 12 h. The number of macrophages that migrated to the lower surface of the membrane was counted in 10 random high-power fields under a light microscope (Nikon Eclipse, USA). Each assay was performed in triplicate wells.
ChIP (chromatin immunoprecipitation) assay
RAW264.7 cells were grown in a 10-cm dish (90–95% confluence), and histones were crosslinked to DNA with 1% formaldehyde for 15 min at 37 °C. The cells were washed three times with ice-cold PBS and scraped into a tube for nuclear protein extraction using the NE-PERTM reagent kit (Pierce). Subsequent steps were performed as described previously [38]. PCR was performed to amplify fragments of the iNOS promoter using 2 μL of the extracted DNA (with or without antibody) as a template. The primers used to amplify the iNOS promoter were 5′-TGTACATGCAAGGCAAGCAC-3′ and 5′-TGGCCTCAATAGTTGGGAGAAC-3′.
siRNA transfection
Hes1 siRNA, RBP-Jκ siRNA and control siRNA were purchased from RiboBio. Transfections were performed at approximately 60% confluency using Lipofectamine® 3000 transfection reagent (Invitrogen) according to the manufacturer's instructions.
Flow cytometry
To quantify the circulating monocytes by FACS analysis, whole mouse blood cells were collected in anticoagulant tubes, and PE-labelled CD115 antibody was added (1:100, BD); the cells were then incubated at 37 °C in the dark for 1 h. Ten millilitres of red cell lysate was added to the cells for 5 min at room temperature, following which the cells were centrifuged at 2000 rpm for 3 min, the supernatant was removed, and the cells were washed twice with PBS and resuspended in 300 μl PBS for flow cytometry (Beckman Coulter, CytoFLEX). The data were analysed via CytExpert2.0 software and at least 10,000 gated events were acquired from each sample.
Data analysis
All the data are expressed as the mean ± standard deviation. Student's t-test was applied for comparisons between two groups, and one-way ANOVA followed by LSD t-test was used to compare differences between more than two different groups (GraphPad Prism software). A P value less than 0.05 indicated statistical significance.
Elevated KBP and monocyte counts in diabetic patients with diabetic foot ulcer
The level of KBP in plasma samples from 61 nondiabetic controls (NDM), 44 diabetic patients without diabetic foot ulcer (DM w/o DFU) and 25 diabetic patients with diabetic foot ulcer (DM w/DFU) was examined by ELISA. The clinical data, such as age, monocyte counts and some biochemical indicators, were analysed (Additional file 1: Table S1). We found that the plasma level of KBP in the DM group (17.9 ± 13.8 μg/mL) was higher than that in the NDM group (6.4 ± 2.0 μg/mL), and the level in the DM w/DFU group (25.9 ± 14 μg/mL) was higher than that in the DM w/o DFU group (12.3 ± 10.6 μg/mL) (Fig. 1a). Furthermore, there were more monocytes in the peripheral blood of the DM group (5.0 ± 2.1 × 109/L) than in the peripheral blood of the NDM group (3.8 ± 1.1 × 109/L), and the number of monocytes was much higher in the DM w/DFU group (6.2 ± 2.1 × 109/L) than in the DM w/o DFU (4.1 ± 1.6 × 109/L) and NDM groups (Fig. 1b). The collected data refer to the clinical data detected by a blood cell analyser. Correlation analysis indicated that circulating KBP levels were positively associated with the number of circulating monocytes in the patients in all groups (Fig. 1c, R = 0.48, P < 0.01). Collectively, circulating KBP levels were elevated in the DM group, especially in the DM w/DFU group, which was associated with elevated numbers of circulating monocytes.
KBP delays wound healing, and the administration of KBP-neutralizing antibody improves wound healing in diabetic mice
Wound healing in KBP-TG mice was delayed compared with that observed in wild type (WT) littermates (Fig. 2a, b). Consistently, wound healing in the recombinant KBP-treated group was slower than that in the control group treated with BSA (Fig. 2c, d). Furthermore, the administration of KBP-neutralizing antibody accelerated wound healing in diabetic mice (Fig. 2e, f) whose KBP level was elevated (Additional file 2: Figure S2). Taken together, our results suggested that KBP administration alone impaired wound healing, while wound healing in diabetic mice was accelerated via blocking KBP.
KBP increases the number of circulating monocytes and macrophage infiltration in wounds
Compared with that in the WT mice, there was more F4/80+ (a macrophage marker) macrophage infiltration in the wounds of KBP-TG mice (Fig. 3a, b), and the mRNA expression of F4/80 was correspondingly increased in the wounds of KBP-TG mice at different time points (Fig. 3e). The administration of KBP antibody decreased the infiltration of macrophages (Fig. 3c, d) as well as the mRNA expression of F4/80 (Fig. 3f). The statistical analysis of the data, which was indicated by greyscale values, is shown in Fig. 3b and Fig. 3d. In addition, the percentage of circulating monocytes was increased in KBP-TG mice (Fig. 3g) as well as db/db diabetic mice (Additional file 3: Figure S3) compared to that in control mice. Therefore, our results indicated that KBP increased the number of circulating monocytes and macrophage infiltration in wounds.
KBP promotes the M1 polarization of macrophages
We further examined the expression of an M1 marker (iNOS) and an M2 marker (ARG1) in vivo. There were more iNOS and F4/80 double-positive cells (Fig. 4a, c) and fewer ARG1 and F4/80 double-positive cells (Fig. 4b, d) in the wounds of DM and KBP-TG mice than those in the wounds of control mice. In contrast, the KBP antibody reduced the number of M1 macrophages and increased M2 macrophage infiltration in the wounds of diabetic mice (Fig. 4e, f). In the late stage of wound healing (D10), the mRNA expression of iNOS was increased (Fig. 4g), while the expression of ARG1 was decreased in KBP-TG mice (Fig. 4h). Similarly, treatment with KBP-neutralizing antibody reversed this phenomenon, as the expression of iNOS was downregulated (Fig. 4i), while the expression of ARG1 was upregulated in diabetic mouse wounds (Fig. 4j). Consequently, KBP increased the number of pro-inflammatory M1 macrophages and reduced M2 macrophage infiltration in diabetic wounds, especially in the late stage of wound healing, resulting in a persistent inflammatory state in the diabetic wounds.
To further explore the role of KBP in macrophage polarization, we measured different M1/M2 markers/cytokines in diverse monocyte-macrophages (Additional file 4: Figure S4A-C). The M1/M2 ratios in THP-1 cells (Fig. 5a), RAW264.7 cells (Fig. 5b) and BMDMs (Fig. 5c) stimulated with KBP were significantly increased compared to those in control cells. The expression of the inflammatory cytokines TNFα and IL-6 was also increased in the supernatant of RAW264.7 cells stimulated with KBP compared to that in unstimulated cells (Fig. 5d). The results of western blotting indicated the increased protein level of iNOS, while the expression of ARG1 was decreased or not significantly changed in the abovementioned monocyte-macrophage cell lines (Fig. 5e-g). Collectively, these results indicate that KBP stimulated the M1 polarization of macrophages both in vivo and in vitro.
KBP promotes M1 polarization via activating the notch Signalling pathway
Further, we explored the effect of KBP on macrophage M1 polarization. The expression of Notch1 was upregulated in the wound bed of KBP-TG mice compared with that in WT mice (Fig. 6a). In RAW264.7 cells, the mRNA expression of Notch1 and its downstream transcription factor or target genes (such as RBP-Jκ, Hes1, Hes5 and Socs3) were increased after KBP treatment (Fig. 6b). DAPT, a Notch signalling inhibitor, repressed the upregulation of Notch1, Hes1, Hes5 and SOCS3 following treatment with KBP (Fig. 6c). The results of immunofluorescent staining of Notch1 were consistent with those above (Additional file 5: Figure S5A), and the expression of delta-like 4 (DLL4, a Notch receptor) was also increased after KBP treatment in RAW264.7 macrophages (Additional file 5: Figure S5B). In addition, DAPT inhibited the expression of iNOS and upregulated the expression of ARG1 via inhibiting the Notch signalling pathway following treatment with recombinant KBP (Fig. 6d). Therefore, inhibition of the Notch signalling pathway reversed the effect of KBP on macrophage polarization. To further explore the molecular mechanism of this effect, we employed three pairs of siRNA sequences that knocked down RBP-Jκ or Hes1, reduced the expression of iNOS and increased ARG1 compared with that in the control in RAW264.7 macrophages (Fig. 6e, g). In addition, macrophages were treated with KBP and siRBP-Jκ 03 or siHes1 03, which was most effective, and the effect of KBP on macrophage polarization was reversed following the interference of RBP-Jκ and Hes1 (Fig. 6f, h). Our observations indicated that KBP promoted the M1 polarization of macrophages through activating the Notch signalling pathway.
Hes1, a downstream target gene of the notch Signalling pathway, does not directly activate iNOS expression
Furthermore, we used a bioinformatics method to predict the possible transcription factors that bind to the promoter region of iNOS via the PROMO website. Bioinformatics prediction indicated that Hes1, a downstream target gene of Notch signalling, could bind to the promoter region of the iNOS gene (Fig. 7a). The mRNA expression of iNOS was decreased after the interference of Hes1 (Additional file 6: Figure S6A). A ChIP assay showed that Hes1 bound to the promoter region of the iNOS gene (Additional file 6: Figure S6B), while a dual-luciferase reporter gene assay indicated that the transcription of iNOS was not activated by Hes1 in 293 T cells (Additional file 6: Figure S6C). Our observation indicated that Hes1 bound the promoter region of the iNOS gene but did not activate the expression of iNOS directly.
KBP promotes the M1 polarization of macrophages via activation of the notch Signalling pathway and the cross-activation of the NF-κB inflammatory Signalling pathway
NF-κB is a classical inflammatory signalling pathway that promotes the M1 polarization of macrophages, and NF-κB is also a possible transcription factor that binds to the promoter region of iNOS (Fig. 7a). The activation of Notch signalling activated the NF-κB signalling pathway in breast cancer cells, and NF-κB activated the transcription of iNOS directly [28]. Our results indicated that KBP promoted the phosphorylation and activation of NF-κB p65 (Fig. 7b, e) as well as the translocation of p65 into the nucleus (Fig. 7c, d). Furthermore, KBP promoted the phosphorylation of inhibitor of κB (IκBα) kinase (IKK) to activate the phosphorylation of downstream IκBα, which inhibits the NF-κB transcription factor and is inactive in its phosphorylated form (Fig. 7e). In addition, KBP downregulated the expression of cylindromatosis tumour-suppressor protein (CYLD) (Fig. 7e), which is a deubiquitinase and negative regulator of NF-κB signalling [39]. Treatment with the NF-κB signalling inhibitor JSH23 and the Notch signalling inhibitor DAPT inhibited the effect of KBP on the activation of the NF-κB signalling pathway (Fig. 7b-e), while the overexpression of Hes1 activated the NF-κB signalling pathway (Fig. 7f). The above results indicated that KBP promoted the M1 polarization of macrophages via activation of Notch signalling and cross-activation of the NF-κB signalling pathway.
KBP upregulates the expression of M-CSF and MCP-1
M-CSF was upregulated in the plasma of KBP-TG mice (Fig. 8a) as well as the supernatant of RAW264.7 macrophages treated with KBP (Fig. 8b). KBP increased M-CSF mRNA expression in RAW264.7 cells (Fig. 8c). In a Transwell migration assay, RAW264.7 cells treated with KBP exhibited increased migration compared to that of the control group (Fig. 8d), which may be associated with the upregulation of MCP-1. Furthermore, the increased expression of MCP-1 was detected in the wounds of KBP-TG mice, especially in the late stage of wound healing, while KBP antibody reversed this phenomenon (Fig. 8e, f). MCP-1 was upregulated in the plasma of KBP-TG mice (Fig. 8g) and the supernatant of the RAW264.7 cells (Fig. 8h), and the mRNA expression of MCP-1 was correspondingly increased (Fig. 8i). Consequently, our results indicated that KBP promoted the differentiation and chemotactic migration of monocytes-macrophages via the upregulation of M-CSF and MCP-1.
An excessive inflammatory reaction delays diabetic healing, which is a conjoint cause of amputation in diabetic patients [16, 40]. The molecular basis underlying the pathogenesis of excessive inflammatory reactions in diabetes-induced wound healing deficiency has not been completely illuminated. The present study demonstrated an association between elevated levels of circulating KBP and increased numbers of monocyte-macrophages in DFU for the first time. Furthermore, circulating monocyte-macrophages and macrophage infiltration were upregulated in KBP-TG mice compared to those in control mice. We have demonstrated that 1) high levels of KBP contributed to a delay in wound healing in diabetic mice through regulating monocyte-macrophages that induced an excessive inflammatory reaction and that 2) KBP promoted the M1 polarization of macrophages, resulting in the accumulation of pro-inflammatory M1 macrophages and a prolonged inflammatory state. Our studies suggest for the first time that KBP may promote M1 polarization through activating the Notch and NF-κB signalling pathways and that Hes1 may activate the NF-κB signalling pathway via inhibiting CYLD. These observations established for the first time an association between elevated KBP levels and an excessive inflammatory reaction with delayed diabetic healing and DFU, which may provide a new theoretical basis for and targets to intervene in DFU.
Our previous studies demonstrated that circulating KBP levels were increased in diabetic patients associated with microvascular complications [20, 21]. Here, we revealed that circulating KBP levels were elevated in DM patients, especially DM w/DFU patients, compared to those in nondiabetic individuals, which was associated with elevated monocyte counts (Fig. 1). Furthermore, our results revealed delayed wound healing in KBP-TG mice and recombinant KBP-treated mice compared with that in WT littermates (Fig. 2a-d). This is authoritative evidence suggesting that KBP may be a factor in the regulation of wound healing. To further establish the role of KBP in wound healing, the administration of a KBP-neutralizing antibody was used to block KBP activity, which accelerated wound healing in diabetic mice (Fig. 2e, f). Taken together, these results all suggest that elevated levels of circulating KBP indeed contribute to a delay in wound healing in diabetes.
Diabetic patients have delayed healing characterized by persistent inflammatory responses accompanied by the prolonged accumulation of M1 macrophages, which can eventually necessitate lower limb amputation [16]. Elevated KBP was associated with an increased number of circulating monocytes in diabetic patients with DFU compared with those in diabetic patients without DFU. Therefore, we hypothesized that a high level of KBP might influence persistent inflammatory responses in diabetes through regulating the recruitment and polarization of macrophages. Consistent with this prediction, our study demonstrated that the number of circulating monocytes and macrophage infiltration in the wound were increased in KBP-TG mice compared to those in control mice (Fig. 3a, b, e, g). KBP administration promoted the recruitment of macrophages and M1 polarization in an animal model and various monocyte-macrophage cell lines (Figs. 4 and 5), which suggesting KBP induced the persistent inflammatory responses in diabetic wound tissue. However, previous studies have suggested that KBP has potent anti-inflammatory activities: such as Liu's study demonstrated KBP decreased inflammatory cell infiltration and TNFα express in the corneal, which represent a superficial angiogenesis and acute inflammation model [41]. While chronic inflammation is a hallmark of impaired diabetic wound healing [42]. These results suggested that KBP may play the diverse roles in different patterns of inflammation. Our results confirmed that the polarization and recruitment of macrophages are crucial in the inflammatory response during wound healing [10,11,12]. Nevertheless, the underlying molecular mechanism is not well understood.
Notch signalling plays a pivotal role in regulating the development and differentiation of monocyte-macrophages [23, 24]. Increased M1 macrophage infiltration was correlated with the activation of Notch signalling in the wounds of KBP-TG mice (Fig. 6A). To further confirm that Notch signalling contributes to macrophage polarization, DAPT, an inhibitor of the Notch pathway, and knockdown of the transcription factor RBP-Jκ and Hes1 by siRNA were employed to explore the effects of KBP on Notch signalling. DAPT downregulated the expression of iNOS and upregulated the expression of ARG1 via inhibiting the Notch signalling pathway under KBP treatment (Fig. 6b-h). Taken together, our observations indicate for the first time that KBP promoted the M1 polarization of macrophages through activating the Notch signalling pathway.
Bioinformatics prediction and a ChIP assay showed that Hes1 could bind to the promoter of iNOS, while a dual-luciferase reporter gene assay showed that Hes1 could not activate the expression of iNOS directly (Additional file 6: Figure S6). Since Hes1 could not activate iNOS expression directly, we wondered whether KBP activated iNOS expression via an indirect pathway. The NF-κB signalling pathway, which is a classic pathway that promotes the M1 polarization of macrophages, is closely related to the inflammatory response [13, 43]. Hes1, which is downstream of Notch signalling, can inhibit the transcription of the deubiquitinase CYLD, which negatively regulates IKK [44]. CYLD inhibits the ubiquitination of TNFα receptor-associated factor (TRAF6), while TRAF6 conjugated to a Lys-63 (K63)-linked polyubiquitin chain is required for the activation of IKK and downstream signalling events [45,46,47]. KBP activated the Notch signalling pathway to upregulate Hes1, which inhibited the expression of CYLD to activate the phosphorylation of IKK in macrophages. After the activation of IKK, NF-κB signalling was activated, and the subsequent nuclear translocation of p65 further promotes iNOS expression, which keeps macrophages in an M1 polarization state (Fig. 7e, f). We first discovered the effect of KBP in promoting the M1 polarization of macrophages by cross-activating the Notch pathway and NF-κB signalling pathways (Additional file 7: Figure S1). A similar mechanism was also found in breast cancer cells [28].
Macrophages come from monocytes and upstream progenitor cells that are regulated by M-CSF [5]. In wounded tissue, macrophage recruitment depended on the ischaemia-induced upregulation of MCP-1 and the increased expression of CCR2 on the cell surface. We also explored the possible mechanism by which KBP regulates monocyte-macrophage numbers. Since the differentiation, mobilization and recruitment of macrophages are regulated by M-CSF and MCP-1, we found the increased expression of M-CSF and MCP-1 following treatment with recombinant KBP in KBP-TG mice (Fig. 8). The detailed mechanism by which KBP regulates M-CSF and MCP-1 remains to be clarified in the future.
As shown by these results, KBP aggravated the inflammatory response in wound tissue by targeting macrophages. We first demonstrated that a high level of KBP in DFUs activated Notch signalling and the NF-κB signalling pathway, leading to M1 polarization, increased numbers of macrophages in the wound, and, consequently, excessive inflammatory reactions during wound healing. These activities contribute to a delay in wound healing in diabetic patients. Hence, the blockade of KBP may benefit DFU treatment and prevent amputation.
Availability of data and materials
All data generated or analysed during this study are included in this published article and its supplementary information files.
Chemokine receptor 2
Diabetic foot ulcer
- DM w/DFU:
Diabetic patients with diabetic foot ulcer
- DM w/o DFU:
Diabetic patients without diabetic foot ulcer
Inhibitor of κB kinase
- iNOS:
Nitric oxide synthase
- IκBα:
Inhibitor of κB
Kallikrein-binding protein
Monocyte chemoattractant protein-1
Macrophage colony-stimulating factor
Nondiabetic control
Notch intracellular domain
- 1.
Boulton AJ, Vileikyte L, Ragnarson-Tennvall G, Apelqvist J. The global burden of diabetic foot disease. Lancet. 2005;366:1719–24.
- 2.
White R, McIntosh C. A review of the literature on topical therapies for diabetic foot ulcers. Part 2: advanced treatments. J Wound Care. 2009;18:335–41.
- 3.
Singer AJ, Clark RA. Cutaneous wound healing. N Engl J Med. 1999;341:738–46.
- 4.
Qi W, Yang C, Dai Z, Che D, Feng J, Mao Y, Cheng R, Wang Z, He X, Zhou T, et al. High levels of pigment epithelium-derived factor in diabetes impair wound healing through suppression of Wnt signaling. Diabetes. 2015;64:1407–19.
- 5.
Bennett CL, Djulbegovic B, Norris LB, Armitage JO. Colony-stimulating factors for febrile neutropenia during cancer therapy. N Engl J Med. 2013;368:1131–9.
- 6.
Ushach I, Zlotnik A. Biological role of granulocyte macrophage colony-stimulating factor (GM-CSF) and macrophage colony-stimulating factor (M-CSF) on cells of the myeloid lineage. J Leukoc Biol. 2016;100:481–9.
- 7.
Lee HW, Choi HJ, Ha SJ, Lee KT, Kwon YG. Recruitment of monocytes/macrophages in different tumor microenvironments. Biochim Biophys Acta. 2013;1835:170–9.
- 8.
Kolattukudy PE, Niu J. Inflammation, endoplasmic reticulum stress, autophagy, and the monocyte chemoattractant protein-1/CCR2 pathway. Circ Res. 2012;110:174–89.
- 9.
Daley JM, Brancato SK, Thomay AA, Reichner JS, Albina JE. The phenotype of murine wound macrophages. J Leukoc Biol. 2010;87:59–67.
- 10.
Porcheray F, Viaud S, Rimaniol AC, Leone C, Samah B, Dereuddre-Bosquet N, Dormont D, Gras G. Macrophage activation switching: an asset for the resolution of inflammation. Clin Exp Immunol. 2005;142:481–9.
- 11.
Gordon S. Alternative activation of macrophages. Nat Rev Immunol. 2003;3:23–35.
- 12.
Mosser DM, Edwards JP. Exploring the full spectrum of macrophage activation. Nat Rev Immunol. 2008;8:958–69.
- 13.
Sica A, Mantovani A. Macrophage plasticity and polarization: in vivo veritas. J Clin Invest. 2012;122:787–95.
- 14.
Brancato SK, Albina JE. Wound macrophages as key regulators of repair Origin, Phenotype, and Function. Am J Pathol. 2011;178:19–25.
- 15.
Goren I, Allmann N, Yogev N, Schurmann C, Linke A, Holdener M, Waisman A, Pfeilschifter J, Frank S. A transgenic mouse model of inducible macrophage depletion: effects of diphtheria toxin-driven lysozyme M-specific cell lineage ablation on wound inflammatory, angiogenic, and contractive processes. Am J Pathol. 2009;175:132–47.
- 16.
Loots MA, Lamme EN, Zeegelaar J, Mekkes JR, Bos JD, Middelkoop E. Differences in cellular infiltrate and extracellular matrix of chronic diabetic and venous ulcers versus acute wounds. J Invest Dermatol. 1998;111:850–7.
- 17.
Chai KX, Ma JX, Murray SR, Chao J, Chao L. Molecular cloning and analysis of the rat kallikrein-binding protein gene. J Biol Chem. 1991;266:16029–36.
- 18.
Miao RQ, Agata J, Chao L, Chao J. Kallistatin is a new inhibitor of angiogenesis and tumor growth. Blood. 2002;100:3245–52.
- 19.
Shen B, Hagiwara M, Yao YY, Chao L, Chao J. Salutary effect of kallistatin in salt-induced renal injury, inflammation, and fibrosis via antioxidative stress. Hypertension. 2008;51:1358–65.
- 20.
Jenkins AJ, McBride JD, Januszewski AS, Karschimkus CS, Zhang B, O'Neal DN, Nelson CL, Chung JS, Harper CA, Lyons TJ, Ma JX. Increased serum kallistatin levels in type 1 diabetes patients with vascular complications. J Angiogenes Res. 2010;2:19.
- 21.
McBride JD, Jenkins AJ, Liu XC, Zhang B, Lee K, Berry WL, Janknecht R, Griffin CT, Aston CE, Lyons TJ, et al. Elevated circulation levels of an antiangiogenic SERPIN in patients with diabetic microvascular complications impair wound healing through suppression of Wnt signaling. J Investig Dermatol. 2014;134:1725–34.
- 22.
Kopan R, Ilagan MX. The canonical notch signaling pathway: unfolding the activation mechanism. Cell. 2009;137:216–33.
- 23.
Xu H, Zhu J, Smith S, Foldi J, Zhao B, Chung AY, Outtz H, Kitajewski J, Shi C, Weber S, et al. Notch-RBP-J signaling regulates the transcription factor IRF8 to promote inflammatory macrophage polarization. Nat Immunol. 2012;13:642–50.
- 24.
Wang YC, He F, Feng F, Liu XW, Dong GY, Qin HY, Hu XB, Zheng MH, Liang L, Feng L, et al. Notch signaling determines the M1 versus M2 polarization of macrophages in antitumor immune responses. Cancer Res. 2010;70:4840–9.
- 25.
Wan F, Lenardo MJ. The nuclear signaling of NF-kappaB: current knowledge, new insights, and future perspectives. Cell Res. 2010;20:24–33.
- 26.
Hacker H, Karin M. Regulation and function of IKK and IKK-related kinases. Sci STKE. 2006;2006:re13.
- 27.
Gonzalez DM, Medici D. Signaling mechanisms of the epithelial-mesenchymal transition. Sci Signal. 2014;7:re8.
- 28.
Li L, Zhang J, Xiong N, Li S, Chen Y, Yang H, Wu C, Zeng H, Liu Y. Notch-1 signaling activates NF-kappaB in human breast carcinoma MDA-MB-231 cells via PP2A-dependent AKT pathway. Med Oncol. 2016;33:33.
- 29.
Maniati E, Bossard M, Cook N, Candido JB, Emami-Shahri N, Nedospasov SA, Balkwill FR, Tuveson DA, Hagemann T. Crosstalk between the canonical NF-kappaB and notch signaling pathways inhibits Ppargamma expression and promotes pancreatic cancer progression in mice. J Clin Invest. 2011;121:4685–99.
- 30.
Fukuda D, Aikawa E, Swirski FK, Novobrantseva TI, Kotelianski V, Gorgun CZ, Chudnovskiy A, Yamazaki H, Croce K, Weissleder R, et al. Notch ligand delta-like 4 blockade attenuates atherosclerosis and metabolic disorders. Proc Natl Acad Sci U S A. 2012;109:E1868–77.
- 31.
Furman BL. Streptozotocin-induced diabetic models in mice and rats. Curr Protoc Pharmacol. 2015;70(5):47 41–20.
- 32.
Yu T, Sungelo MJ, Goldberg IJ, Wang H, Eckel RH. Streptozotocin-treated high fat fed mice: a new type 2 diabetes model used to study Canagliflozin-induced alterations in lipids and lipoproteins. Horm Metab Res. 2017;49:400–6.
- 33.
Yang Z, Grinchuk V, Urban JF Jr, Bohl J, Sun R, Notari L, Yan S, Ramalingam T, Keegan AD, Wynn TA, et al. Macrophages as IL-25/IL-33-responsive cells play an important role in the induction of type 2 immunity. PLoS One. 2013;8:e59441.
- 34.
Dai Z, Qi W, Li C, Lu J, Mao Y, Yao Y, Li L, Zhang T, Hong H, Li S, et al. Dual regulation of adipose triglyceride lipase by pigment epithelium-derived factor: a novel mechanistic insight into progressive obesity. Mol Cell Endocrinol. 2013;377:123–34.
- 35.
Zhang B, Zhou KK, Ma JX. Inhibition of connective tissue growth factor overexpression in diabetic retinopathy by SERPINA3K via blocking the WNT/beta-catenin pathway. Diabetes. 2010;59:1809–16.
- 36.
Li S, Mao Y, Zhou T, Luo C, Xie J, Qi W, Yang Z, Ma J, Gao G, Yang X. Manganese superoxide dismutase mediates anoikis resistance and tumor metastasis in nasopharyngeal carcinoma. Oncotarget. 2016;7:32408–20.
- 37.
Hong H, Zhou T, Fang S, Jia M, Xu Z, Dai Z, Li C, Li S, Li L, Zhang T, et al. Pigment epithelium-derived factor (PEDF) inhibits breast cancer metastasis by down-regulating fibronectin. Breast Cancer Res Treat. 2014;148:61–72.
- 38.
Kadariya Y, Nakatani K, Nishioka J, Fujikawa T, Kruger WD, Nobori T. Regulation of human methylthioadenosine phosphorylase gene by the CBF (CCAAT binding factor)/NF-Y (nuclear factor-Y). Biochem J. 2005;387:175–83.
- 39.
Hahn M, Burckert JP, Luttenberger CA, Klebow S, Hess M, Al-Maarri M, Vogt M, Reissig S, Hallek M, Wienecke-Baldacchino A, et al. Aberrant splicing of the tumor suppressor CYLD promotes the development of chronic lymphocytic leukemia via sustained NF-kappaB signaling. Leukemia. 2017.
- 40.
Brem H, Tomic-Canic M. Cellular and molecular basis of wound healing in diabetes. J Clin Invest. 2007;117:1219–22.
- 41.
Liu XC, Lin ZR, Zhou T, Zong RR, He H, Liu Z, Ma JX, Liu ZG, Zhou YP. Anti-Angiogenic and anti-inflammatory effects of SERPINA3K on corneal injury. PLoS One. 2011;6.
- 42.
Torbica T, Wicks K, Umehara T, Gungordu L, Alrdahe S, Wemyss K, Grainger JR, Mace KA. Chronic inflammation in response to injury: retention of myeloid cells in injured tissue is driven by myeloid cell-intrinsic factors. J Invest Dermatol. 2019.
- 43.
Simon PS, Sharman SK, Lu C, Yang D, Paschall AV, Tulachan SS, Liu K. The NF-kappaB p65 and p50 homodimer cooperate with IRF8 to activate iNOS transcription. BMC Cancer. 2015;15:770.
- 44.
Espinosa L, Cathelin S, D'Altri T, Trimarchi T, Statnikov A, Guiu J, Rodilla V, Ingles-Esteve J, Nomdedeu J, Bellosillo B, et al. The notch/Hes1 pathway sustains NF-kappaB activation through CYLD repression in T cell leukemia. Cancer Cell. 2010;18:268–81.
- 45.
Jin W, Chang M, Paul EM, Babu G, Lee AJ, Reiley W, Wright A, Zhang M, You J, Sun SC. Deubiquitinating enzyme CYLD negatively regulates RANK signaling and osteoclastogenesis in mice. J Clin Invest. 2008;118:1858–66.
- 46.
Deng L, Wang C, Spencer E, Yang L, Braun A, You J, Slaughter C, Pickart C, Chen ZJ. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell. 2000;103:351–61.
- 47.
Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature. 2001;412:346–51.
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This study was supported by the National Nature Science Foundation of China, grant numbers: 81600641, 81471033, 81572342, 81770808, 81370945, 81570871, 81570764, 81572409, and 81872165; the National Key SciTech Special Project of China, grant numbers: 2013ZX09102–053 and 2015GKS-355; the Key Project of Nature Science Foundation of Guangdong Province, China, grant numbers: 2015A030311043 and 2016A030311035; the Guangdong Natural Science Fund, grant numbers: 2014A020212023, 2014A030313073, 2015A030313029, 2015A030313103, and 2015A030313010; the Guangdong Science Technology Project, grant numbers: 2017A020215075 and 2015B090903063; Initiate Research Funds for the Central Universities of China (Youth Program), grant numbers: 14ykpy05, and 16ykpy24; the Key Sci-tech Research Project of Guangzhou Municipality, China, grant numbers: 201508020033, 201707010084, 201803010017, and 201807010069; the Pearl River Nova Program of Guangzhou Municipality, China, grant number: 201610010186; and the 2017, and 2019 Milstein Medical Asian American Partnership Foundation Research Project Award in Translational Medicine.
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Additional files
Additional file 1:
Table S1. Clinical characteristics in non-diabetic (NDM) control subjects, patients with Type 2 diabetes (DM) and diabetic patients with diabetic foot ulcer (DM + DFU). Table S2. Primer sequences for real-time quantitative PCR. (DOC 65 kb)
Additional file 2:
Figure S2. The expression of KBP in the liver tissue of WT and diabetic mice (n = 3). (DOCX 214 kb) (TIFF 1695 kb)
Additional file 3:
Figure S3. Representative FACS results and quantification of CD115+ monocytes in peripheral blood of WT and db/db male mice. All data were presented as the mean ± SD. n = 3; * p < 0.05. (TIFF 2174 kb)
Additional file 4:
Figure S4. A. The mRNA expression of M1 and M2 markers/cytokines in THP-1 cells. B. The mRNA expression of M1 and M2 markers/cytokines in RAW264.7 cells. C. The mRNA expression of M1 and M2 markers/cytokines in BMDM. Data are presented as the mean ± SD. n = 3; * p < 0.05. (TIFF 22 kb)
Additional file 5:
Figure S5. A. The immunofluorescence staining results of notch1 in different groups of RAW264.7 macrophages. Scale bar = 5 μm. B. The expression of Jagged1 and DLL4 in RAW264.7 cells. (TIFF 4456 kb)
Additional file 6:
Figure S6. A. The mRNA expression of iNOS in RAW264.7 cells after using sihes1. Data were presented as the mean ± SD. n = 3; * p < 0.05 versus sicon group. B. The ChIP assay of transcription factor hes1 and iNOS promoter region. C. The dual-luciferase report gene assay for hes1 and iNOS promoter in 293 T cells. (TIFF 1669 kb)
Additional file 7:
Figure S1. The schematic overview of KBP in promoting the M1 polarization of macrophages by cross-activating the notch signaling pathway and NF-κB signaling pathway. KBP upregulates DLL4 and Notch1 to activate notch signaling, promoting NICD to bind to the CSL/RBPJ-κ, and then increases the expression of hes1. Hes1 suppresses the expression of CYLD, which could inhibit the ubiquitination of TRAF6. Because of the reduction of CYLD, the weakened deubiquitination of TRAF6 could activate NF-κB p65 via phosphorylation of IκBα. NF-κB p65 translocases into the nucleus to activate the expression of target gene-iNOS, to promote the M1 polarization of macrophages. (TIFF 2700 kb)
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Feng, J., Dong, C., Long, Y. et al. Elevated Kallikrein-binding protein in diabetes impairs wound healing through inducing macrophage M1 polarization. Cell Commun Signal 17, 60 (2019). https://doi.org/10.1186/s12964-019-0376-9
- Diabetic wound healing
- Kallikrein-binding protein
- Monocyte-macrophages
- Notch/NF-κB signalling


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The cost of healthcare, not only in the United States but in the rest of the world as well, has proven to be a hot-button issue these days. With arguing about who should pay and how much should be paid going on throughout most every segment of society, one thing is clear: there's a lot of money going around in healthcare and it needs to come from somewhere. But there's one move that's offering some substantial potential for savings, and that's the electronic healthcare record (EHR).
A report from Juniper Research, titled "Digital Health, Remote Monitoring & EHR Cost Savings 2014-2019," took a look at the matter and suggested that, between 2014 and 2019, EHRs would save the global healthcare industry a healthy—so to speak—$78 billion. With that kind of money on the table, it became clear to most observers that EHRs will represent a major part of the future of healthcare, as more hospitals and the like brought said systems into play and took advantage of substantial savings.
It's not just about savings, either, but also about outright revenue generation. Increasingly, healthcare organizations are reportedly encountering Accountable Care Organization (ACO) initiatives that offer incentives according to the measured "wellness" of the patients in its charge, which in turn is changing the way healthcare organizations look at providing that service. Digital healthcare, meanwhile, is offering a much greater potential for monitoring patients and thus being able to track long-term trends, providing a more effective overall measure of "wellness."
Juniper Research's Anthony Cox offered up a bit of commentary around the results of his study, noting that, as EHRs advanced in nature, such things would ultimately become the "glue" in unifying the somewhat disparate picture of devices and records that is seen today. With more healthcare workers finding digital healthcare a smart idea overall, adding EHRs in increasing numbers is likely to help drive that phenomenon forth to a greater degree.
But even Cox offers a note of caution: with the rise of EHRs needs to come an accompanying rise in controlled mobile health (mHealth) trials, and the differences among various portions of the global health industry mean that each EHR solution is going to have to be specifically tailored to account for those differences. That also means a lot of buy-in from the various parts; unless there's reason enough for businesses to enter a sector—a sufficiently large market—there won't be offerings in that market. However, with market offerings already in place like Samsung's Samsung Architecture for Multimodal Interactions (SAMI) and Apple's HealthKit—not to mention increasing support from regulators—it's clear that this market will have at least some presence in the field for some time to come.
Any business—and healthcare here is truly no exception—is looking to do a combination of two things: increase revenue and decrease expenses. If expenses must increase, then so too must revenue increase to offset and surpass the rise in expenses. It's a basic universal survival recipe for business of all types. With EHRs, healthcare workers can not only access records faster and thus prove more efficient in the field, but can also use these records as part of a wellness cycle.
While that not only means incentive programs can kick in from regulators, it also has the potential to reduce expenses down the road. Only time will tell just how well it works, but it's got some great potential to do well for those putting EHRs to work on all sides of the healthcare spectrum.
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Lenten Meditations from the Pastoral Concerns Dept – Day 33 "FOURTH WORD ON THE CROSS: MY GOD, MY GOD, WHY HAVE YOU FORSAKEN ME"
[The Pastoral Concerns Department of the Church of South India brings out devotions for the 40 Lenten Days in 2019 beginning from the Ash Wednesday. A group of CSI Presbyters from the five states of South India prepared these devotions and published on this official website of the CSI Synod, Official Facebook group https://www.facebook.com/groups/csisynodcommunication/ and the official WhatsApp Broadcast from the number +91 9840577404. You can read/download the English version of the devotion here. The writer of the devotion presents the same in a video on the day. Watch here Rev. C. Christopher David, Presbyter, CSI Vellore Diocese gives a meditation "FOURTH WORD ON THE CROSS: My God, My God, why have you Forsaken Me"
Lenten Meditations- Day 33 (for 12th April 2019)
Selected Texts: Mathew 27: 45-49 | Mark 15: 33- 36 | Exodus 10: 21-23 | I Peter 2:9
Eli, Eli, lema sabachthani?" That is, "My God, my God, why have you forsaken me?" Mathew 27: 46
The world where we live in is filled with bittersweet experiences. Everyone faces desperate situations and separations from the beloved ones whom they think, are close to their heart. It is uncommon and unusual as we see Jesus' cry and anguish in the passage (Mathew 27: 45-49/Mark15: 33-36). He experienced segregation and deep sense of separation from God and the people who are close to his life. Every Human being encounters such situation of bitterness and exclusion as Jesus experienced on the cross. The words that Jesus uttered from the cross are close to our minds; He spoke in his vernacular language to express the deep sense of loss, disconnection and alienation from God. The people who were around the cross misunderstood that Jesus is calling Elijah to vindicate him from this situation, some of them even suggested to wait and see that Elijah would come and rescue Him from this bitter experience. Most of the time we express our feelings in our mother tongue rather in foreign language. We get different descriptions and interpretations of our problems as Jesus received from the people. Jesus' authentic humanity is clearly exemplified in the fourth word saying that from the Cross. This passage clearly raises the fundamental question of the suffering of the innocent and just. As we read in Psalm 22, the righteous can face sufferings but they can be relieved from their circumstances.
As we perceive in Jesus' condition on the brutality of the cross the emancipation is not accessible as per Psalmist's imagination. The Gospel writers portrayed Jesus' loud cry and groaning to get rid of the unbearable agony. Another important aspect we get it from this passage is, Jesus' question to God. Jesus accepted the form of incarnation; His connection with God was intense, but, here Jesus experiences the loneliness and discomfort for not having the connection as He had with God in the past. Darkness is a powerful symbol in the Bible. The element of darkness preceded creation (Gen. 1:2-3), represented God's judgment on Egypt (Exo. 10: 21-23) and concealed God's glory from the people (Exo. 20: 21). Darkness is also used as a metaphor for people's ignorance of God (Mt. 4: 16) and their hopelessness without God (I Pet. 2:9) picks up all these meanings, but above all it conveys a sense of doom and hopelessness as God seems absent. Lamenting is another powerful symbol which exposes the agony of sufferer. The lamentation is a powerful tool of dissent in the history of Israelites (Lam1:11-12). Even Jesus cried out in distress at being separated, though temporarily from His Father: 'My God, my God, why have you forsaken me?'
The lament of Jesus, clearly projects the brokenness and abandonment of God. Such occasions of pain and agony give different ideas and perspectives on our lives. At the same time it kindles us to suspect and question: Can God leave us alone? Or Can God allow suffering? Having had the sense of doubt and question it is very important to face the challenges and tribulations of the world. Jesus' experience teaches us to overcome the trials and tribulations through our faith experience.
As we meditate this verse, there are heart burning issues that common folk faced in everyday life. One of the incidents was the cyclone effects of Gaja, especially to the farmers. If we approach this incident from farmer's perspective, we come across, exploitation, desperation, separation and deep sense of loss. Though they did not expect such an unexpected catastrophe it has distorted the normal course of the common people. Here the same question arises, "Why God?"
Another important context is the plight of the farmers in India. Farmers are the backbone of the economy of India and the supporters of life of our nation. But, the farmers are not recognized by the government. For the sake of development huge amount of money is spent on the technological and scientific sectors, at the same time Government is not ready to spend money on agriculture which is the vital source of Life. Farmers' voices are not recognized by the authority. In the name of developmental projects most of the farmers' lands are taken and they are in desperate situation, their cries are sloganeering. At present our farmers are facing desperate sense of loss, pain and segregation by our own government. The suicide rates among farmers are increasing day by day. Their pleas are not heard and they experience the deep sense of separation as Jesus experienced on the Cross. Why God?
It is very important for us to re-think our commitment to God. When we face desperate situations, we fail to overcome but we raise the question "why should this happen to me". Jesus had overcome the problem by questioning God. He had overcome by asking questions and finding remedy for them, he realized that the Cross is the resolution for separation. Another important aspect is, we see the intimacy between God and Jesus. The intimate relation made Him to accomplish the task that is given by God. It's a personal experience of every individual's life. Most of the time we never find the alternative way to get things done. Sometimes we have to face the tribulations but at the same time it can be taken out with our alternative way of thinking and approach with focused mind and commitment. Jesus had taken the alternate way that dying on the Cross alone can emancipate him, so He takes the risk. We never take challenges and come out of from safe zones. We want salvation for sale. As a Christian community let us unite and make our commitment alive and be focused to win the battle as Jesus had overcome trials and tribulations on the Cross. As Martin Luther King Jr says, "Darkness cannot drive out darkness; only light can do that. Hate cannot drive out hate; only love can do that" so, love is the transforming force which can change an enemy into a friend. As a Christian community let us be united in the love of Jesus Christ and march towards to win the race by witnessing Christ in our deeds and action.
1. O sacred Head, now wounded,
with grief and shame weighed down,
now scornfully surrounded
with thorns, Thine only crown.
O sacred Head, what glory,
what bliss till now was Thine!
Yet, though despised and gory,
I joy to call Thee mine.
2. What language shall I borrow
to thank Thee, dearest Friend,
for this, Thy dying sorrow,
Thy pity without end?
O make me Thine forever!
And should I fainting be,
Lord, let me never, never,
outlive my love for Thee.
-Bernard of Clairvaux
Merciful God, we seek your mercy to move forward to witness for you in our actions and deeds. God of hope, we believe that you are our power to overcome trials, tribulations and desperation that we face in our everyday life. Grant us knowledge and wisdom to win the battle and be the testimony of Jesus Christ till we depart from this world. God of justice, we seek your presence for the people who face exploitation, separation and segregation all over the world, let your divine presence be upon them to overcome the circumstances that they face. Lord, help us to be constantly in contact with you even if we go through trials and temptations. Amen
Rev. C. Christopher David
CSI Vellore Diocese


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Blood is thicker than water, but it isn't bondage— an unyielding tie to our relations— it's more like a bond— a tie that connects us to one another in an unbreakable and inexplicable manner. Under the skin, we're all family, tracing our roots back to the most basic of human origins, and playwright Michael Hollinger articulates that in his touching familial drama Under the Skin. Directed by Vincent M. Lancisi, this poignant and evocative drama performs explorative theatrical surgery on the construct of human relationships when it comes to the boundaries of the body and the limits of love.
A hospital room is hardly warming or welcoming but there is something in the nature of Set Designer Brandon McNeel's fabrication that encompasses the sterility of what that sort of room ought to be while still bringing a sense of comfortable homeliness into the atmosphere. The drop-tile ceiling with harsh fluorescent lighting (compliments of Lighting Designer Jay A. Herzog) invites the antiseptic verve of hospital spaces in great detail, from the ventilation ducts to the overhead speakers aligned in an exacting pattern into each of the tiles. McNeel uses rather impressive rotation panels in the white walling of the overall set to ease transitions in and out of each scene, but it's Herzog's lighting that provides the ultimate shift. A single square of light over a well-placed piece of furniture takes the broad stage into a narrow office. This style of lighting trick, as well as shifting focus-lights, achieve moments of flashback and inner-outer meta-occurrence throughout the performance.
Sound Designer Stowe Nelson adds a fifth character element to the production with his subtle soundscape. There is a particular beat structure that playwright Michael Hollinger has written into the play, which Nelson augments and calls attention to frequently with bell-tones, many of which sound somewhere serene and yet simultaneously unearthly. Nelson's overall aural approach to the show creates a pulse of energy, particularly in the transitional music, which adds to the underlying current that moves the show forward.
While Hollinger captures the essence of conversation, conflict, and dramatic twists exceptionally well throughout his script there appears to be a slight imbalance in the way in which he includes meta-components in the work as a whole. There are moments when the characters actualize that they are characters in a play that feel out of place but this only happens in select moments— sometimes feeling ill-placed and other times feeling superfluous. This occurs most in their semi-direct addresses to the audiences and only when Hollinger attempts to use these moments to illustrate humor between them. Otherwise the subject matter is brilliantly articulated, from factual accuracy to emotional intention, and the play moves smoothly along.
Director Vincent M. Lancisi, working with Dialect Coach Steven J. Satta takes two of the four performers in the show and transforms them into multiple characters, distinguished through the use of their foreign accents. Satta applies the sound of a native from Santo Domingo and one from Africa when the situation calls for it. These informed applications authenticates the character existence as they are experienced in Hector and Dr. Badu respectively. Lancisi navigates the peculiar blend of active in-play dramatic action and meta-external moments of characters through the fourth wall with practiced ease. The show moves swiftly, keeps the audience engaged, and is a deeply relevant emotional and familial experience.
The play's great success is accomplished by the authentic conversations and relationship dynamics portrayed with ample consistency throughout the course of the show. Alice M. Gatling comes to the fray with multiple engaging characters, whether it's her very formal and aloof portrayal of Dr. Badu or her snappy and sassy attitude-driven barista character that engages in heated passive-aggressive arguments with the Raina character every time they find themselves in the coffee shop scene. Gatling delivers a wholesome and level-headed character portrayal of Marlene, not without her own hints of humor or wise words of wisdom to unearth as the twists in the plot unfold. Grounded, and calm of mind, her Marlene portrayal is fully present and engaging in the moments where it is most needed
Keith L. Royal Smith, whose primary character is the son of Gatling's Marlene, is equally tranquil for the majority of his on-stage existence. There is a gentle peace that resonates in his character's presence, except for moments of heated debate and slightly superfluous rivalry of a very specific nature that occurs near the end of the performance. Smith's primary interactions occur with Raina (Megan Anderson) and its their discourse that drives a great deal of the show's rising action, particularly in the way that Smith's tranquility balances the high-strung nature of Anderson's character.
Anderson perpetuates an edgy and jittery character, fully embodying the awkward and frustrated tensions of a character that is plagued with anxiety, discomfort, and internalized emotions that have been bottled up for far too long. Her frigid interactions with Lou (Mitchell Hèbert) are the exacting epitome of an estranged relationship between father and daughter. There is a casualness with which Hèbert approaches his character that further fuels Anderson's fire, particularly in moments of heated discussion. The conversations and overall interactions between this character pairing feel particularly authentic and exist solely without artifice. Strikingly real and raw performances across the board keep the show compelling, as well as allowing the audience to focus on the bigger picture, ultimately making the drama a truly relatable one.
Running Time: 2 hours with one intermission
Under the Skin plays through February 21, 2016 at Everyman Theatre— 315 W. Fayette Street in the West End Entertainment District of Baltimore, MD. For tickets call the box office at (410) 752-2208 or purchase them online.


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From Brooklyn Center Minnesota's Surly Brewing Co comes their "Coffee Bender" an American Brown Ale flavoured with coffee. Coffee used in the beer is Guatemalan Finca Vista Hermosa Full City. The beer pours a deep brown colour with a tan head. The aroma consists of coffee, toasty malts, smoke and bitterness. The flavour is of burnt coffee, roasted malts, smoke and hops bitterness. The alcohol content comes in at 5.5% with an IBU of 45. This is a great coffee beer and tastes much like an iced coffee.
Commercial Description: Coffee Bender refreshes like an iced-coffee, is aromatic as a bag of whole beans as satisfies like your favorite beer. The Surly brew team has developed a cold extraction process that results in intense coffee aromatics and flavor bringing together two of our favorite beverages. Your only dilemma will be whether to finish your day or start it with a Coffee Bender.


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Website Saturation And Popularity
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It's not just about writing content, it's about writing content that's unique. There are many websites out there than deem themselves as unique content suppliers, but find themselves regurgitating copy that's already out in the wild wild web. WeSell.com can supply you with fresh content that you won't find anywhere else. Our talented team of copy creators are known to come up with content on the fly. We'll become familiar with the type of writing that you need, and adjust our styles and writing voices to fit your website. This is an essential part of the SEO process, and it's a timely process that will have an overall positive impact on every page of your site.
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blink again
a raindrop
a tear drop
all the same
a call to consciousness
whispering a name
do you hear what they are saying ?
soul come to me
to thy chamber
where your memories
have been kept
where the virgins have wept
for love's embrace
can you taste the sweetness
or the salt
i have exalted thee
above the Angels
and you still yet
to learn to fly
now it is my turn
to ask why
have i flawed
for the day soon comes
when you child
shall realize
but a small portion
of your sum
and you shall live
out your earthly days in a wonder
of your magnificence
a raindrop
a teardrop
for they are all the same
blink again
© 8 November 2012 : william s. peters, sr.
my Newest Book . . .
"The Vine Keeper . . . messages in Poetry and Prose"
is available at


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News in brief:
- Emrill has opened a new training base in in Al Quoz, Dubai
- The centre is known as 'Emrill Centre of Excellence' and it provides both BICSc-certified and NICEIC-accredited training
-The centre will give students a hands-on experience in facilities management
-The centre also contains common spaces and rooms for collaboration, workshops, seminars and open learning sessions
Emrill has opened a brand new training base as part of its plans to drive staff development and quality-driven growth.
Investment in the new regional training centre, located in Al Quoz, Dubai, reinforces Emrill's commitment to bringing the highest internationally-benchmarked standards, the FM Company said in a press statement.
The news follows Emrill's decision earlier this year to open satellite BICSc-certified training rooms at selected client sites, including Dubai International Airport, to fully embed training standards into working environments.
Known as the 'Emrill Centre of Excellence' the training centre contains both BICSc-certified and NICEIC-accredited training and assessment rooms and will give students a place for hands-on experience in facilities FM alongside a range of classroom-based and digital training.
The centre also contains common spaces and rooms for collaboration, workshops, seminars and open learning sessions.
A notable aspect of the new training facility is its large lecture theatre, known as the Knowledge Theatre, with tiered seating and a raised stage area that resembles a movie theatre set-up.
Alex Davies, managing director, Emrill said, "Our investment in this facility further reinforces Emrill's commitment to talent development and upskilling our people by offering team members the most advanced training experience in the market.
"In the highly competitive FM industry, a key differentiator for Emrill is the quality of our people combined with the high level of training customisation we offer, which sets us apart in the industry. Last year alone, we delivered 134,667 training man-hours, an increase of 4.87%, which led to an increase of 17% in internal staff promotions."
Sarah Rodgers, who recently took over as head of soft services at Emrill, and is responsible for the learning and development department, added, "Our objective is to create a world class working environment where continuous learning and development can take place; where employees gain more fulfilment from their roles, and develop intrinsic motivation to contribute to the organisation's objectives.
The new facility is significantly larger and more technologically-advanced than Emrill's previous training centre.


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Company wants power meters installed in Magiagi village
Samoa's Electric Power Corporation is in a stand off with the Magiagi village, a suburb of Apia, over the government's proposed cash power meters installation project.
Most families are rejecting the proposal.
One hundred households of Magiagi were given free electricity as part of an agreement from the 1980s, since the village water resources were used to generate power.
This is now to change.
The government has reportedly offered to pay some compensation for the village resources such as land if they accept to pay their power bills and install cash power meters in their homes.
However the Minister of Works, Tuisugaletaua Sofara Aveau, denied any mention of the 28 year old free power supply agreement during a meeting with villagers.
The minister says the village council should come u with a decision in another meeting next month.


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Understanding wildfire season
With California's wildfire season bringing yearly evacuations, damage to communities and lasting health effects, Stanford faculty have been exploring ways of preventing fires and managing health risks.
As fires force evacuations in both Northern and Southern California, Gov. Gavin Newsom declared a statewide emergency. Roughly 200,000 people have so far been evacuated from their homes and more than a million are without power in the Bay Area.
And this scenario is becoming a yearly event following devastating fires the past two years.
Stanford faculty have been studying the health effects of breathing smoke and particulates in communities surrounding California's wildfires as well as investigating policies and technologies for preventing fires. Other experts are thinking about the lasting financial effects of wildfires on the state – particularly for the state's largest electric utility, which was found responsible for three of the fires in 2018.


text: Rolling Game Station is the Ultimate Party FUN in Chester, Montgomery, Delaware Berks and Lancaster County! We bring the party games, you bring your guests and they get their game on in a Luxury Mobile Video Game Theater with 2- 46″ and 3 -50″ Huge HDTVs! When you book with Rolling Game Station you get 5+ years of experience in bringing Video Game Parties to the customer. We are not new at this and know just the right way to bring you the BEST PARTY ON WHEELS!


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North American Network Operators Group
Date Prev | Date Next | Date Index | Thread Index | Author Index | Historical
Re: flap flap: AS 10916
At Friday 04:59 PM 8/11/00, John Todd wrote: >701 > \ > \____________ > | > | link 2 > 1239---10916--------6138 > / \________________/ > / link 1 >13789 > > >JT > > Thank you, Todd, this was the scenario I had in mind. Nice to see someone still excells in the field of ASCII art, too :) The POC of AS10916 has emailed me back since, and is looking into this issue. I was also intrigued by: > (b) Link that both BGP and traffic pass through is insufficient for continued keepalives once traffic moves in that direction (line becomes preferred by a large amount of traffic, traffic floods line, BGP keepalives fail, BGP session fails, traffic moves away, wash, rinse, repeat - see RED discussion archives some months ago for more detailed discussions on traffic flow dampening with similar patterns.) What can be done to prevent flapping in this situation, other than putting QoS mechanisms into place to prefer the BGP traffic over everything else? Is there a good and automated (Cisco-leaning, sorry) way to keep BGP sessions down if they have flapped too often?


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One of the components of the Worldview course I teach is a study of comparative religions. It's important to know what other people believe, especially in this shrinking world of ours. Growing up I often heard people quote I Peter 3:15 "But in your hearts revere Christ as Lord. Always be prepared to give an answer to everyone who asks you to give the reason for the hope that you have…" Rarely did anyone complete the verse. Here's how it ends: "But do this with gentleness and respect." Gentleness and respect is important. It should never, ever, be left out. Learning what other people believe is essential to gentleness and respect.
Usually, teaching this part of the course seems more like teaching social studies than a Bible class. We talk about culture and history and sometimes even geography as we examine other faiths. This year, though, God has been teaching me very definite and important spiritual lessons as we move through this part of the class. This week we looked at Judaism.
One of the questions my students answered from our text concerned the history of Judaism. They were supposed to pick an ancient Jewish leader and explain why that person stood out to them, what they admired about their life and story. Listening to my students' answers was a tremendous blessing. I have found these last few years that young people know less and less about literature in general. Unless the book has been made into a recent movie, students rarely get literary analogies and, even in a Christian school, I often find my students ignorant of the details of basic Bible stories. Sunday School curriculum and attendance are not what they used to be. However, this time I was very pleasantly surprised. My students had lots to say and they had great reasons for their answers.
Students talked about King David, the man after God's own heart, and what a great example he is of faithfulness and repentance. Moses was another favorite–the way that God used a man who really would have preferred to hang out in the desert with his father-in-law's sheep. I chose Joseph, as did several of my students. The story of Joseph has so many fascinating twists and turns as God changes him from a proud, self-righteous goody-goody into a faithful and forgiving leader used by God to save a nation and his own family from starvation. But the overwhelming favorite of the day was Abraham. Listening to my students talk about Abraham was exactly what I needed this week.
I'm in a tough place right now. On Friday I was smiling and taking attendance and moving through my day as usual, but inside my heart was breaking. This world is a hard place. People are cruel and truth is often purposely distorted. I think God might be asking me to face some big changes in my life. I am not a fan of change. Looming change makes me insecure and anxious. There are dreams lying, shattered, at my feet. It's hard to let go of dreams, but sometimes God asks us to do it. He certainly required that of Abraham. One of my students spoke about the faith of Abraham and how, as he walked up that mountain to sacrifice Isaac in obedience to God, he had confidence that God would provide. As she said it, I knew in my heart that God was speaking directly to me. God will provide. I needed to hear that. Another student spoke of how Isaac was the result of years and years of patient waiting and belief in God's promise, so it must have been so hard to think about offering him up, of starting all over again, with nothing. I needed that, too. Abraham believed that God would provide somehow, that He would raise Isaac up from the dead if necessary, but most of all that God, being a GOOD and LOVING God, would do what was best. Abraham believed that God could be trusted. I needed the reminder that he still can. It was difficult to control my emotions as student after student, class after class, was used by the Lord to remind me of the truth. God knows. He always knows. And underneath are the everlasting arms.
There's an old Rich Mullins song that often comes on my playlist as I run. It's about the boyhood years of Jesus and it poses some interesting questions. My favorite lines ask, "Did they tell you stories about the saints of old, stories about their faith? They say stories like that make a boy grow bold, stories like that make a man walk straight." It works for women, too. Thank you, Jesus!


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You can think of lebkuchen as gingerbread's German cousin. Both cookies rely heavily on cinnamon and spices, taste best glazed, and are traditionally made around the holidays.
Photos via CakeSpy
However, lebkuchen and gingerbread have a few important differences.
Unlike gingerbread, which is made with molasses, lebkuchen is often (though not always) made with honey. Many believe that this is because honey was one of the only widely available sweeteners during the 13th century, when lebkuchen was first made.
Another difference is that lebkuchen recipes frequently include candied citrus and nuts, which give lebkuchen a distinctly different taste than gingerbread.
But despite their differences, both gingerbread and lebkuchen taste great with a cup of hot tea on a cold winter day. Make sure to include this lebkuchen recipe in your holiday cookie baking plans this year!
Lebkuchen recipe
Makes about 4 dozen small cookies
For the cookies:
- 2¼ cups all-purpose flour
- ¼ teaspoon baking soda
- 1 teaspoon ground ginger
- 1 teaspoon ground nutmeg
- 1 teaspoon ground cloves
- 2 teaspoons ground cinnamon
- 1/8 teaspoon salt
- 3/4 cup honey
- ½ cup light brown sugar, packed
- 1 large egg, at room temperature
- ½ teaspoon lemon extract
- ½ cup finely chopped blanched almonds
- 2 teaspoons finely chopped orange peel, lemon peel, or a combination
For the glaze:
- 1 cup confectioners' sugar
- 4 tablespoons milk
- 1 teaspoon brandy or lemon extract
Step 1:
In a medium bowl, sift together the flour, baking soda, spices and salt. Set aside.
Step 2:
In a medium saucepan, combine the honey and brown sugar. Heat over medium-low heat, stirring frequently, until the sugar has dissolved and the mixture begins to bubble. Remove from heat, and let cool to room temperature. Transfer to a large bowl, or the bowl of a stand mixer.
Step 3:
Add the egg, lemon extract, almonds and citrus peel. Stir to combine.
Step 4:
Add the flour mixture, and stir until combined. The dough will be somewhat like drop cookie dough in consistency: firm, but sticky. Cover the dough in the bowl, and refrigerate for at least 4 hours, or overnight (overnight is even better).
Step 5:
Preheat the oven to 350 F. Lightly flour a work surface. Roll the dough into a rectangle measuring approximately 9" x 13". Trim the edges to make them neat (your rectangle is now probably more like 8" x 12").
Step 6:
Cut into small rectangles, approximately 1" x 2" each (though you can alter this based on your preference). Place them on baking sheets lined with parchment paper with a little space around each portion.
Step 7:
Bake for 16-20 minutes, or until puffy, golden on top and set in the centers. Remove from the oven and let cool on the tray for a few minutes before transferring to a wire rack to cool completely.
Step 8:
While the cookies cool, make the glaze. Whisk together all of the ingredients until smooth and combined. Drizzle each cookie with the glaze, and garnish with a few extra almond pieces if desired. If garnishing with almond pieces, scatter them on top of the cookies directly after applying glaze, as they won't stick once the glaze has set.
Store in an airtight container for up to 1 week. These cookies freeze beautifully for up to 1 month.
Note: These cookies may be lighter or darker in color depending on the type of honey you use.
More Holiday Favorite — Free
We rounded up our favorite holiday dessert recipes from folks around the office. And in the spirit of giving (and hosting a fabulous spread) we're sharing them with you.Get the Guide


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Jennifer HigginsRoom number: 108 (Children's House)Subject(s): Reading, Grammar, Spelling, Math, HandwritingFor homework or additional information, please visitFor more class information, please read our class brochure: Student BrochureConferences available by appointment. Please call me at 748-9307.
Let me introduce myself by saying I love learning. Since I love learning, I love to teach students how to enjoy learning as well. I am a firm believer that the student is "our student", the family's and mine. We share in teaching our student and encouraging them to be the best they can be.
My room has a relaxed and family atmosphere. Please feel free to call or email me at any time with questions or concerns. My first priority is our student being the best they can be.


text:
Johnny Dryburgh is an Incorporated Member of the Chartered Institute of Building. He has over 30 years experience working in the property sector in the UK.
Rob James is a Chartered Construction Manager and Builder (MCIOB) with over 25 years' experience in the construction and property sector.
Richard is a member of the Royal Institution of Chartered Surveyors and has 32 years' experience in the property development market including 14 years within Primary Care.
Stuart has been with the Apollo business since its formation in 2001. During his career with Apollo, he has been involved in various aspects of the business, including the management of Apollo's own investment portfolio.
Campbell is a Chartered Architect with experience of working in Europe and the UK. He has predominantly worked in the private sector, with an intervention of four-and-a-half years within local government.
Originally from London, Jo is an Association of Chartered Certified Accountants finalist. Prior to joining Apollo in 2010 Jo held a number of key finance posts, building up a wealth of experience within both property and public-sector fields.
22nd June 2020
15th April 2020
Tel: 01473 659912
Email: email@example.com
Copyright Apollo Capital Projects 2017


text:
Journal of Open, Flexible, and Distance Learning
Dec 23, 2014 Volume 18, Number 2
Alison J. Fields
Table of Contents
Number of articles: 4
Establishing a Sense of Connectedness Amongst Theology Students in Distance Education
Nigel Smith, Charles Erlam, Naomi Quirke & Grace Sylvester, Laidlaw College
Student engagement is required for effective distance learning. Amongst other things, it is built by fostering connectedness amongst tutors, academic advisors, and students, and by providing high... More
pp. 11-28
Student Engagement in Distance-based Vocational Education
Anne Yates & Wendy Brindley-Richards, Victoria University of Wellington; Tony Thistoll, The Open Polytechnic of New Zealand
Students enrolled in distance education courses tend to have lower course completion rates than those who attend face-to-face classes (Simpson, 2013). This article reports on a collective case... More
pp. 29-43
Take AIM and Keep Your Students Engaged
Catherine Nash, Open Polytechnic of New Zealand
This paper outlines the benefits to distance education teachers of formatting a weekly online newsletter in accordance with motivational learning theory. It reflects on the delivery of weekly AIM... More
pp. 45-57
Empowering Teachers? An Exploratory Study of Personnel Practices in Virtual Charter Schools in the United States
Dennis Beck & Robert Maranto, University of Arkansas
Virtual charter schools have the potential to transform teacher personnel management. However, there is currently little evidence that they are doing so. This study examined how 89 teachers at two ... More
pp. 59-81


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She lives!
Aug 17, 2009 update on Carrera30's 1983 Porsche 944
Finally had a chance to work on the car and get new speed and reference sensors installed. After a few coughs, she started! Now to give her a good once over - fluid changes, new belts, new clutch master and slave.
All 1983 Porsche 944 updates
She lives!


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We can complain because rose bushes have thorns, or rejoice because thorn bushes have roses.
– Abraham Lincoln
When a professional evaluates your child and offers a diagnosis, it might provide some relief. Finally, your concerns and suspicions are validated — and you know what to call it. But are labels powerful or problematic?
Over the years, I've talked with many parents about what they wish they had known about diagnostic labels. Here are the top 3 tips:
- Psychiatric Labels Are Not Like a Throat Culture
Ever wonder how diagnostic labels were created – or how they are determined? Sixty years ago, the American Psychiatric Association developed a system to help clinicians across mental health fields make consistent diagnoses. The system provides diagnostic criteria for mental disorders by organizing clusters of symptoms that tend to occur together into categories. All this is presented in a kind of psychiatric bible called the Diagnostic and Statistical Manual of Mental Disorders or "DSM" (the current version is "DSM 5", the fifth edition).
Sounds official, right? But this pathology-based system can be deceiving, because labels don't correspond with known, measurable biological processes. Unlike diabetes, for example, which can be diagnosed by measuring insulin levels, mental "disorders" are determined by what amounts to a symptom checklist. Although the DSM provides standardized guidelines, there is still room for subjective interpretation. A child may have symptoms from more than one category, but a clinician will determine which category (or categories) fits best.
Since diagnosing is as much an art as it is a science, it's crucial to find a qualified practitioner whom you trust.
- Use with Care: Labels Give Us Shorthand, But They Don't Describe an Individual
Labels come with their own baggage, stigma, and misunderstanding. Diagnoses are a shorthand way of characterizing a mental condition, giving parents, teachers, and clinicians some idea of what to expect. But labels should be used with care. Keep in mind, everyone has different interpretations and experiences related to these terms. There's a saying that "if you've met one child with autism, you've met one child with autism." This holds true for any diagnosis. Two children might have the same label, but seem to have little in common. Using a label to describe a child can leave more misunderstanding than not. As you have no doubt figured out already, every child is different.
Sometimes, labels can cause people to lose sight of the whole person. By looking at individual strengths, talents, and interests – in addition to offering ways around the difficulties – we can separate the child from the diagnosis. Many (but not all) parents follow the rule of "person first". For example: There is a big difference between saying he or she is a/n "(diagnostic label) kid", and saying he or she is a "kid who is affected by / living with (diagnostic label)".
Diagnostic labels can create a set of limiting expectations, not only for the adults who are trying to help, but for the child as well. When we lead with a label, we're telling our kids "you have a disability, a disorder, a deficit", which sends messages that can be internalized as "you are broken…you have less potential than others…you will be trouble…here's your excuse for not trying." None of these messages are constructive.
Dr. Ned Hallowell, who is well known in the realm of learning and attentional issues, eloquently highlights the damaging effects this labeling system can have. (Note: I've crossed out his references to specific issues, so you can superimpose your own kind of complicated.) Hallowell says:
"Using our pathology-based model, when we diagnose and treat these kids we often, inadvertently, instill the most dangerous learning disabilities. By far, the most dangerous learning disabilities, what truly holds people back in life, are not ADD or dyslexia. The dangerous disabilities are fear, shame, loss of hope, broken confidence, shattered dreams, and a feeling of being less-than. Those are the disablers. Without meaning to, teachers, parents, doctors, and other professionals instill these disablers every day–in the name of helping children."
The good news is that you don't have to rely on labels to help professionals, friends, or family members provide effective support. How about saying "This is a kid who enjoys doing ____, is good at ____, and is really interested in ___, but has some challenges related to (difficulty) that makes it harder for him / her when it comes to ___." BIG difference!
Why bother with diagnostic labels then, if they can be so damaging? Two reasons: special education and insurance. A diagnosis is required for a child to qualify for certain special education services, and a diagnostic code is required for insurance coverage. What's important to remember is that the system may be a necessary evil, but you don't need to allow a diagnostic label to become the paradigm that defines your child and his or her place in the world.
- Labels Can Be Fluid
As children grow and evolve, sometimes they shed or collect new labels. Over time, it could be that a different diagnosis is more descriptive. Or, it could be that the practitioner was looking for what they know best. Sometimes specialists tend to see what they're looking for. Whatever the case, it's not uncommon for a child's label to change. It can be frustrating when you go down a rabbit hole, trying to learn everything you can about "x", and then you learn that actually "y" is what you need to know about. At least now you know that labels aren't necessarily set in stone.
Kids who are complicated may be facing a whole range of issues, some scarier than others, but let's face it — everyone has something. (As one friend said "The DSM is a good read. You'll find so many aspects of yourself in it!") A label doesn't have to define your life, or your child's self-concept. It's not a sentence. Don't underestimate the ability of the human mind to adapt and overcome. Getting an accurate diagnosis is a useful step towards understanding your child's mind. It can help you and your child gain insight into strengths that can be tapped and vulnerabilities to be supported. Sure, there are thorns, but there are also rosebuds to cultivate.


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Has the tide of public opinion turned to public outrage? Are the liberal sheeple of Mass-a-Two-Shits finally waking up – as their rights and their sovereignty are being squandered by 21st Century One-World No-Borders Socialists? Deval Patrick and Obama were both elected – then re-elected – on the basis of built-up white guilt, – and […]
Posts tagged 'MS13'
Mass Citizens – In Deval Patrick's FACE!
July 27th, 2014


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China: Truck crash leaves ten dead in Huashi (Hunan province) September 22
Truck crash leaves ten dead and 16 others injured in Huashi (Hunan province) September 22; brake failure investigated as cause of crash
A truck crashed into a crowded market in Huashi (Hunan province) around 08:50 (local time) on Sunday, September 22, leaving at least ten people dead and 16 others injured. An initial investigation indicated the vehicle's brakes had failed before the deadly crash.
A heightened security presence and localized traffic disruptions are possible in the incident area over the coming hours.
Individuals in Huashi are advised to avoid the incident area, avoid all related demonstrations as a precaution, and adhere to all instructions issued by the local authorities.
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This property meets all the requirements as an investment or owner occupier . Only 2 units on the block no body corp.
As you enter the property you will be impressed by the size of the kitchen , living area . Kitchen has ample cupboard space , bench space , electric hotplate & dishwasher. From dining and living there is access to decking area.
Master bedroom has walk in robe & full ensuite
Second bedroom has built in robe
Third bedroom or study
Spacious family bathroom including bath tub , shower and toilet
Laundry has wall to wall built in cabinets and access to decking
Single lockable garage plus carport
Timber flooring throughout , ducting heating & cooling , vertical blinds
Currently rented , periodic lease
|Premium Listing (Priority Ranking)
|$99 a month
|Standard Listing (Date Added)
We were told by other agents in the town that because we had one of the larger, more expensive houses in Dandenong that we would never get the price we wanted. But you proved them wrong! $180,000 more then we wonted!!!! Your marketing program sold it within six weeks! So thank you for getting us the price we never dreamed of and breaking the price record in Dandenong. Would we recommend you? We don't know. What do you think???
When we put our business for sale with Steadfast Real Estate, we were anxious about how thing would go, we soon become relaxed and reassured by your sales team in particular with Dane's professional handling of the sale process. Only qualified buyers and no tyre kickers are brought through our business. Sales did not suffer as the result of your marketing campaign and most importantly - you got us the money we wanted. Thank you all and YES, we would recommend you to all.
Thank you Dane for selling our 37 apartments development within 16 day of commencement of your marketing campaign. As you said to me: There is no need for 1000's to be spent to achieve successful sale. We certainly believe you now.