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Sirt6 mRNA-incorporated endothelial microparticles (EMPs) attenuates DM patient-derived EMP-induced endothelial dysfunction
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Tong Jing1,*, Kuang Ya-Shu1,*, Wang Xue-Jun1, Hou Han-Jing1, Lai Yan1, Yao Yi-An1, Chen Fei1,** and Liu Xue-Bo1,**
1Department of Cardiology, Shanghai Tongji Hospital, Tongji University, Shanghai, China
*These authors have contributed equally to this work
Chen Fei, email: [email protected]
Liu Xue-Bo, email: [email protected]
Keywords: EMPs; sirtuin 6; endothelial dysfunction; diabetes; vascular complication
Received: June 15, 2017 Accepted: December 03, 2017 Published: December 15, 2017
Background: Endothelial microparticles (EMPs) are small vesicles released by endothelial cells (ECs); they are considered biomarkers for endothelial dysfunction and therapeutic targets in diabetes-related vascular disease. Sirtuins have also been shown to play important roles in diabetes by regulating endothelial dysfunction. However, the effect of sirtuin-incorporated EMPs on their parental ECs remains unknown.
Aim: The present study aims to investigate the diagnostic value of EMPs in diabetes and detect the protective effects of sirtuin 6 (Sirt6) mRNA -incorporated EMPs on endothelial dysfunction.
Methods: EMPs were prepared from cultured HUVECs and venous blood from patients with diabetes (n=10) and from healthy volunteers (n=6) after sequential centrifugation. Adv-Sirt6 or Sirt6 siRNA was used to alter Sirt6 expression. EC angiogenesis, inflammatory phenotypes, nitric oxide (NO) formation and eNOS phosphorylation were used to evaluate endothelial dysfunction.
Results: The levels of EMPs in diabetic patients and high glucose-cultured HUVECs are high, whereas Sirt6 expression in plasma and EMPs is low. EMPs generated from diabetic patients or high glucose-cultured HUVECs increase inflammatory chemokine release and blunt EC angiogenesis. Furthermore, EMPs enriched with Sirt6 mRNA induces EC angiogenesis, increases eNOS phosphorylation and impedes inflammatory chemokine release. Inhibition of Sirt6 mRNA expression in EMPs by siRNA hinders angiogenesis and eNOS phosphorylation but increases cellular inflammation.
Conclusion: The Sirt6 mRNA-carrying EMPs may ameliorate endothelial dysfunction in diabetic patients.
The incidence of diabetes mellitus (DM) is prevalent in both developed and developing countries. As of 2035, an estimated 592 million individuals worldwide will be diagnosed with DM, which is increased from 387 million in 2014 . DM-induced macrovascular and microvascular complications lead to reduced quality of life, added health care costs and even early death . It is noteworthy that vascular endothelial dysfunction is the main factor in the pathogenesis of diabetic vascular complications via increased inflammation, dysregulated blood flow, cellular trafficking, decreased nitric oxide (NO) production and the induction of endothelial cell death, thus contributing to increased mortality rates [3, 4]. Consequently, the comprehensive analysis and amelioration of endothelial impairment may inhibit the progression of DM-related vascular complications.
Endothelial dysfunction is associated with inordinate intercellular information exchange between endothelial cells (ECs) and immune cells, smooth muscle cells, fibroblasts or even parent ECs . In addition, intercellular information exchange may involve the release of extracellular vesicles, which can function as intercellular carriers of ligands, enzymes, RNA and miRNA . However, the precise mechanism of intercellular information exchange between ECs in DM remains largely unknown.
Recently, documents have verified that endothelial microparticles (EMPs) are novel, complex membrane-shed vesicles that facilitate endothelial dysfunction in DM-related vascular complications . EMPs range from 0.1-1 μm in size and are generated from ECs during apoptosis, activation or injury . Importantly, previous data reveal that the biological content (protein, lipid, miRNA and mRNA) and functional effect of EMPs depend on the condition of the ECs from which they are released . Hyperglycemic conditions significantly increase EMP expression, which is low under normal conditions, and subsequently change their biological effect on target cells . Under DM conditions, microparticles (MPs) have shown noxious effects on ECs in a previous study [6, 7, 10]. in vivo studies have shown that endothelial cells or platelet derived-MPs impair angiogenesis, reduce cerebral microvascular density and accelerate the progression and severity of retinopathy in DM; meanwhile, MPs, especially CD31+ MPs, mediated EC dysfunction in ex vivo studies [10-12]. In addition, EMPs also act as vectors during intercellular information exchange though coupling with membrane-associated receptors, releasing signaling molecules, such as active proteins and miRNA, and finally, mediating adaptive responses , but the effects of EMPs on their parent ECs are still unclear. Therefore, the present study aims to understand the release levels of EMPs and they cause endothelial dysfunction in DM and high glucose mediated-HUVECs.
Sirtuins (Sirts), a family of evolutionarily conserved nicotinamide adenine dinucleotide-dependent histone deacetylases, contains 7 Sirt genes, which have been tested to participate in multiple pathophysiological processes, such as DNA damage repair, apoptosis, senescence and neurodegeneration . Recently, mounting evidence suggests that Sirts exerts effects in multiple cardiovascular disease models, including atherosclerosis, cardiac hypertrophy, chronic inflammation and vascular aging . Additionally, previous studies have revealed that Sirt6 expression is downregulated in response to DM. Moreover, Sirt6 deficiency exacerbates the DM-induced impairment of wound healing and endothelial senescence [16, 17]. However, Sirt6 expression in EMPs remains unknown. This study aims to investigate the possible roles of Sirt6-containing EMPs in endothelial dysfunction in DM.
Increased plasma EMPs levels and reduced Sirt6 expression in DM patients
When compared with the healthy group, plasma CD31+/CD42- EMPs levels were significantly increased in the DM group (Figure 1A). Meanwhile, CD62+/CD42- EMP levels were augmented in DM patients (Figure 1B). However, the levels of CD42+ MPs were not significantly different between the two groups (Figure 1C). This indicated the increased release of EMPs into plasma in DM patients.
Figure 1: EMP levels and Sirt6 expression in DM patients. (A) Increased apoptosis-derived EMP (CD31+) levels in DM patients compared to healthy controls. (B) Increased activated EMP (CD62+) levels in DM patients compared to healthy controls. (C) There is no difference in platelet-derived (CD42+) MPs between healthy controls and DM patients. (D) Decreased serum Sirt6 levels in DM patients. (E) There is no Sirt6 protein in EMPs. (F) Decreased incorporation of Sirt6 mRNA in DM patient-derived EMPs compared to EMPs from healthy controls. (G) Boosted CD31+ EMP levels related with decreased serum Sirt6 levels and increased 2-hour post-meal blood glucose levels. Healthy control, n=6; and DM, n=10. EMPs, endothelial microparticles; MPs, microparticles; HCT, healthy control; DM, diabetes mellitus; FBG, fast blood glucose; 2h PBG, 2-hour post-meal blood glucose; p(n), patient numble. * p<0.05. GAPDH mRNA serves as an internal control.
Decreased expression of Sirt6 is a widely accepted risk factor of DM and atherosclerosis. Therefore, we speculated that EMP-mediated injury may be caused by Sirt6 deficiency. In this study, we first detected the expression of plasma Sirt6 using ELISA. The results revealed that Sirt6 expression in plasma from DM patients was significantly reduced compared with plasma from healthy volunteers (Figure 1D and Supplementary Figure 1A). Then, we measured Sirt6 mRNA or protein expression in EMPs. In contrast to Sirt6 mRNA, immunoblot experiments revealed nearly no expression of Sirt6 protein in different EMPs (Figure 1E), suggesting that Sirt6 mRNA is selectively packaged in EMPs. Additionally, we noted that Sirt6 mRNA-containing EMPs were obviously decreased in DM patients (Figure 1F). These data indicated that Sirt6-containing EMPs may play important roles in DM patients.
To estimate the EMPs and plasma Sirt6 levels whether related with blood glucose levels, we examined the blood glucose levels in DM patients and found that there was significant difference in 2-hour post-meal blood glucose but not in fasting blood-glucose. Additionally, the increased EMP levels and decreased Sirt6 levels were observed in patients with high blood-glucose levels accordingly (Figure 1G).
EMPs from DM patients induced EC dysfunction ex vivo
As previous reported, the level of EMP at 1x105 EMPs/ml is sufficient to generate effects for target cells , therefore we applied EMPs at 1x105 EMPs/ml to cultured HUVECs. As shown in Figure 2A, the plasma EMPs could effectively be engulfed by cultured HUVECs. Then, we measured IL-1b, TNF-a and IL-10 mRNA levels after EMP stimulation. We found that DM patient-derived EMPs significantly induced IL-1b and TNF-a mRNA expression, whereas they inhibited IL-10 mRNA expression compared to healthy volunteer-derived EMPs (Figure 2B), which indicated that DM patient-derived EMPs increased inflammatory release in cultured ECs. Additionally, we also measured NO expression. As shown in Figure 2C, DM patient-derived EMPs decreased NO expression in ECs. Combining these data, we deduced that DM patient-derived EMPs induced EC dysfunction ex vivo.
Figure 2: DM patient-derived EMPs induce HUVEC dysfunction. (A) HUVECs effectively engulf EMPs using confocal. Red, PE-labelled EMPs (600x). (B) DM patient-derived EMPs, but not healthy control-derived EMPs, increase the expression of TNF-a and IL-1b mRNA, whereas they decrease the expression of IL-10 mRNA. (C) DM patient-derived EMPs, but not healthy control-derived EMPs, decrease NO expression in HUVECs. Healthy control, n=6; and DM, n=10. HC EMPs, healthy control-derived endothelial microparticles; and DM patient-derived EMPs, diabetes mellitus patient-derived endothelial microparticles. * p<0.05. GAPDH mRNA serves as an internal control.
High glucose levels promoted EMP release ex vivo
As we shown in previous document , 33 mM glucose had sufficient effect to induce HUVECs dysfunction, therefore, we directly applied 33 mM glucose to cultured HUVECs to verify the relationship between DM and EMP release. As expected, high glucose levels increased CD31+ EMP production (Figure 3A). Additionally, we detected the expression of Sirt6 mRNA and protein in EMPs and HUVECs. As with DM patients, high glucose levels inhibited Sirt6 mRNA and protein expression in EMPs and ECs (Figure 3B, 3C and Supplementary Figure 1B).
Figure 3: EMP levels and Sirt6 expression in high glucose-cultured HUVECs. (A) High glucose induces the formation of apoptotic (CD31+) EMPs but not activated (CD62+) EMPs. (B) High glucose decreases Sirt6 mRNA expression in HUVECs and EMPs. (C) High glucose decreases Sirt6 protein expression in HUVECs but not EMPs. n=3. EMPs, endothelial microparticles; CT, control; and HG, high glucose. * p<0.05. GAPDH mRNA serves as an internal control.
Sirt6 mRNA-containing EMPs control EMP-mediated HUVEC dysfunction ex vivo
As Sirt6 mRNA expression decreased in DM patient- or high glucose-derived EMPs, we suspected that Sirt6 deficiency may be related to EMP-mediated HUVEC dysfunction. First, we applied Sirt6 siRNA or Adv-Sirt6 to generate Sirt6-deficient or Sirt6-enriched EMPs (Figure 4A, 4B). Then, we added these EMPs to cultured-HUVECs. Interestingly, Sirt6-deficient EMPs effectively mimicked high glucose- derived EMPs and mediated EC dysfunction, including increased inflammatory cytokine production and decreased NO production, whereas Sirt6-enriched EMPs reversed the high glucose-derived EMP-mediated EC dysfunction (Figure 4C-4E). Angiogenesis is a very important sign of ECs favorable function, therefore we added EMPs to HUVECs. As shown in Figure 4I and Supplementary Figure 1D, high glucose and Sirt6-mRNA-deficient EMPs impeded HUVECs tube formation, whereas Adv-Sirt6-treated EMPs induced tube formation in high glucose-cultured HUVECs.
Figure 4: Sirt6 mRNA plays important roles in high glucose-derived EMP-induced HUVEC dysfunction. (A) Sirt6 siRNA or Adv-Sirt6 alters Sirt6 mRNA levels in HUVECs. (B) Sirt6 siRNA or Adv-Sirt6 alters Sirt6 mRNA levels in EMPs. (C) Sirt6 siRNA-treated EMPs mimic high glucose-induced IL-10 decreases and TNF-a and IL-1b increases. (D) Adv-Sirt6-treated EMPs protect against high glucose-induced IL-10 decreases and TNF-a and IL-1b increases. (E) Sirt6 siRNA-treated EMPs mimic high glucose-induced NO decreases, whereas Adv-Sirt6-treated EMPs protect against high glucose-induced NO decreases. (F) PCR of Sirt6 mRNA incorporated in EMPs and HUVEC. (G) Adv-Sirt6 EMPs rescues Sirt6 protein expression in Sirt6 KD HUVECs. si-Sirt6, Sirt6 siRNA. (H) Sirt6 protein in EMPs is undetectable using ELISA. (I) High glucose- and Sirt6 siRNA-treated EMPs impair HUVEC tube formation, whereas Adv-Sirt6-treated EMPs induce tube formation in high glucose-cultured HUVECs. n=3. CT, control; HG, high glucose; EMPs, endothelial microparticles; siRNA EMP, Sirt6 siRNA treated-EMP; Adv Sirt6 EMP, adv-Sirt6 treated-EMP. * p<0.05 vs CT; # p<0.05 vs HG. GAPDH mRNA serves as an internal control. “CT EMPs” means that EMPs sourced HUVECs did not undergo siRNA- or adv- contamination.
To prove that mRNAs incorporated in the EMPs are mature and complete Sirt6 mRNA, we then measured the EMPs Sirt6 mRNA sequence using RNAseq. As shown in Figure 4F and Supplementary Figure 2, the Sirt6 mRNAs incorporated in the EMPs are mature. To further confer the Sirt6 mRNA incorporated in the EMPs are functional, we added Sirt6 mRNA-enriched EMPs to Sirt6 knock down (KD) HUVECs, which induced by Sirt6 siRNA. As shown in Figure 4G and Supplementary 1C, Sirt6 mRNA-enriched EMPs partially rescued Sirt6 protein expression in Sirt6 KD HUVECs. Additionally, to exclude whether these effects were caused by free Sirt6 protein, which is centrifuged together with EMPs, or Sirt6 proteins which is contained in EMPs, we lysed the EMPs precipitate directly and then measured using ELISA. Interestingly, the Sirt6 levels were undetectable (Figure 4H).
Undisputedly, the primary nucleotides species found and the most popular research focused in EMPs are microRNAs (miRs) , therefore we first using Drosha siRNA in cultured-HUVECs to impede miRs package, then added Sirt6 siRNA or Adv-Sirt6 into these cells to generate EMPs. After careful collection, these miRs-deficient EMPs were added to cultured-HUVECs. Interestingly, Sirt6 mRNA, which is incorporated in the EMPs, rescued the ECs dysfunction caused by high glucose, including decreased inflammatory cytokine production and increased NO production, still preserved (Figure 5A, 5B). Additionally, we also packaged Sirt6 mRNA into EMPs using mRNA electroporation method. As shown in Figure 5C-5D, Sirt6 mRNA can effectively transferred into EMPs and then conveyed to target HUVECs. We also observed the inflammatory factors release and NO secretion. As shown in Figure 5E, EMPs packaged Sirt6 mRNA inhibited IL-1b and TNF-a release but induced IL-10 release. Also the NO expression is increase following Sirt6-enriched EMPs treatment (Figure 5F).
Figure 5: Sirt6 mRNA incorporated in EMPs is a key factor for HUVEC function. Drosha siRNA was used to generate miRs-deficient EMPs. (A) Sirt6 siRNA-treated EMPs mimic high glucose-induced IL-10 decreases and TNF-a and IL-1b increases whether Drosha siRNA presence or absence in EMP formation. Whereas Adv-Sirt6-treated EMPs protect against high glucose-induced IL-10 decreases and TNF-a and IL-1b increases whether Drosha siRNA presence or absence in EMP formation. (B) Sirt6 siRNA-treated EMPs mimic high glucose-induced NO decreases, whereas Adv-Sirt6-treated EMPs protect against high glucose-induced NO decreases whether Drosha siRNA presence or absence in EMP formation. (C) Electroporation transfers Sirt6 mRNA into EMPs. (D) Eletroporation-EMP effectively transfers Sirt6 mRNA into target HUVECs. (E) Electroporation-EMPs protect against high glucose-induced IL-10 decreases and TNF-a and IL-1b increases. (F) Electroporation-EMPs protect against high glucose-induced NO decreases. n=3. EMPs, endothelial microparticles; CT, control; and HG, high glucose; Electro, electroporation. * p<0.05 vs CT; # p<0.05 vs HG. GAPDH mRNA serves as an internal control.
eNOS expression differed in HUVECs following exposure to EMPs
To verify that Sirt6 mRNA did not degrade after transfection, we evaluated Sirt6 protein expression in HUVECs following EMP administration in spite of miRs elimination (using Drosha siRNA) or mRNA electroporation (Figure 6A-6B and Supplementary Figure 1E-1F). Then, we evaluated the activation of eNOS, which is a key mediator in the Sirt6 pathway. Immunoblotting analysis revealed decreased phosphorylation of eNOS when HUVECs were exposed to DM patient-derived EMPs compared with exposure to healthy volunteer-derived EMPs, suggesting that eNOS is inactive following stimulation by DM patient-derived EMPs (Figure 6C and Supplementary Figure 1G). The effect of Sirt6 mRNA-containing EMPs on eNOS was further confirmed. The relative amount of p-eNOS following Sirt6 mRNA-enriched EMP exposure was significantly increased, whereas Sirt6 mRNA-deficient EMPs mediated a significant downregulation of eNOS phosphorylation (Figure 6D-6F and Supplementary Figure 1H-1J). These data indicated that eNOS phosphorylation may participate in EMP-mediated EC dysfunction in DM.
Figure 6: Sirt6 protein and phosphorylation of eNOS is different in HUVECs following exposure to EMPs. (A) Sirt6 mRNA-incorporated EMPs, in spite of miRs-enrichment (Drosha -) or miRs-deficient (Drosha +), alter Sirt6 protein expression in HUVECs. (B) Electroporation-EMPs alter Sirt6 protein expression in HUVEC. (C) DM patient-derived EMPs, but not healthy control-derived EMPs, cause a decrease in p-eNOS in HUVECs. (D) High glucose- and Sirt6-siRNA-treated EMPs decease p-eNOS levels, whereas Adv-Sirt6-treated EMPs increase p-eNOS levels in HUVECs following high glucose administration. (E) Sirt6 mRNA-incorporated EMPs, which are miRs-deficient, induce p-eNOS increase in HUVECs. (F) Sirt6 mRNA electroporation-EMPs increases p-eNOS levels in HUVECs following high glucose administration. n=3. CT, control; HCT, healthy control; HG, high glucose; DM, diabetes mellitus; EMPs, endothelial microparticles; Electro, electroporation.
Sirt6 mRNA-containing EMPs improved ECs function is independent of Sirt1-dependent manner
Previous documents proven that Sirt1-dependent mechanism was wide agreement in DM-associated endothelial dysfunction , therefore we added Sirt6 mRNA- containing EMPs into Sirt1 deficient HUVECs (Figure 7A, 7B). We found that the IL-1b and TNFa mRNAs were decrease, whereas IL-10 mRNA was increase (Figure 7C). Additionally, NO production was induced following Sirt6 mRNA-enriched EMPs treatment (Figure 7D). These data indicated that Sirt6 mRNA-containing EMPs improved ECs function is independent of, at least partially, Sirt1-dependent manner.
Figure 7: Sirt6 mRNA-mediated protective effects are independent of Sirt1. (A) Sirt1 siRNA abrogates Sirt1 mRNA expression in HUVECs. (B) Adv-Sirt6-treated EMPs increase Sirt6 mRNA levels in Sirt1 KD HUVECs. (C) Adv-Sirt6-treated EMPs protect against high glucose-induced IL-10 decreases and TNF-a and IL-1b increases in Sirt1 KD HUVECs. (D) Adv-Sirt6-treated EMPs protect against high glucose-induced NO decreases in Sirt1 KD HUVECs. CT, control; HG, high glucose; EMPs, endothelial microparticles. * p<0.05 vs CT; # p<0.05 vs HG. GAPDH mRNA serves as an internal control.
The present study demonstrated that DM-derived EMPs activate inflammatory cytokines and decrease the production of NO in cultured HUVECs, thereby facilitating EC damage. Notably, EMPs carrying Sirt6 mRNA are critically involved in the process, and regulation of EMPs containing Sirt6 could effectively affect EMP-mediated inflammatory cytokine activation, NO decrease and impaired tube formation. In addition, the phosphorylation of eNOS was revealed to be associated with the stimulation of ECs by EMPs.
The potential role of endothelial dysfunction in cardiovascular complications has recently been investigated using cultured ECs and experimental models of DM. Indeed, endothelial dysfunction manifests as endothelial erosion, apoptosis, inflammation and premature senescence with pronounced EMP release [10, 18]. Therefore, the possibility that dysfunctional endothelial cells are a major source of deleterious EMPs in DM patients was evaluated. The present findings further confirmed these previous hypotheses, showing that both EMPs released from activated endothelial cells and apoptotic endothelial cells are upregulated in DM patients, whereas the appearance of a pronounced CD31+/CD42-, apoptotic EMP profile, but not a CD62+/CD42-, activated EMP profile, is observed in high glucose-exposed HUVECs. Notably, the prevalence of CD31+/CD42- EMPs is much higher in DM patients than CD62+/CD42- EMPs. In addition to representing a surrogate diagnostic and therapeutic marker of endothelial dysfunction, EMPs might also contribute to aggravating the function of circulating ECs, as EMPs from patients with DM exerted prothrombotic, proinflammatory, disseminated intravascular coagulopathic, microvascular injury and immunosuppressive effects on circulating ECs . They also blunted endothelium-dependent relaxation in arterial rings [21, 22] and promoted premature endothelial senescence and impaired tube formation in cultured ECs . At present, the uptake of EMPs from DM patients or high-glucose incubated HUVECs increases proinflammatory cytokine release, reduces NO release and impairs tube formation, whereas a similar concentration of EMPs from healthy volunteers are inactive on ECs. Altogether, these findings support the concept that DM patient-derived EMPs carry a pro-damage message to neighboring ECs.
In addition to classical short-distance intercellular communication via signaling molecules and long-distance communication via hormones, extracellular vesicles, such as microparticles and exosomes, have been proposed to be essential for information exchange that affects the physiology of neighboring and remote recipient cells [23, 24]. Mostly evidence suggests that the information transferred and then elicited pathophysiological effects on the target cells by microparticles and exosomes are mediated by microRNA or protein-dependent mechanisms [25, 26]. Recently, researchers found that mRNA incorporated into MPs also played key roles in tumor biology and predicted prognostic risk stratification . However, there is no doubt that the cargo that is incorporated into the EMPs is dependent on the stimulus received by the parent ECs . miR-126, membrane-associated proteins and glycoproteins that are incorporated into the EMPs are accepted as the major mechanism by which information is transferred to neighboring and remote recipient cells [22, 29]. Sirt6 is a sirtuin family member that participates in the control of a wide range of pathophysiological processes, including glucose homeostasis, cardiovascular diseases, cancer and longevity . The relationship between Sirt6 and diabetes is complex, although Sociali G. reported that pharmacological Sirt6 inhibition improved glucose tolerance as well as reduced triglycerides and cholesterol levels in a T2DM mouse model ; most models have confirmed the hypothesis that Sirt6 serves as a surrogate diagnostic and therapeutic marker of DM with or without cardiovascular complications. Sirt6 expression decreases in diabetic atherosclerotic plaque and endothelium , whereas overexpression of Sirt6 protects against endothelial senescence , inhibits macrophage-derived foam cell formation , represses aging , delays pancreatic β-cell dysfunction and apoptosis , and enhances insulin sensitivity . Downregulation of Sirt6 accelerates ROS- or high glucose-induced endothelial senescence [17, 37] and leads to obesity and insulin resistance . At present, we also determined that serum Sirt6 levels are much lower in diabetic patients than in healthy controls. However, there is limited information available regarding Sirt6 expression in EMPs from DM patients. Interestingly, the expression of Sirt6 mRNA, but not protein, in the EMPs from DM patients and high glucose-incubated HUVECs is consistently decreased; additionally, Sirt6 mRNA and protein deficiency has also been confirmed in HUVECs following high glucose treatment in our study, suggesting that Sirt6 might have significant effects on the pathogenesis of diabetic complications. Additionally, altering Sirt6 mRNA concentrations in EMPs using Adv-Sirt6, Sirt6 siRNA or direct mRNA-electroporation can consistently direct EMP-mediated effects on target HUVECs, including an inflammatory phenotype, NO production and tube formation. These effects also exist using miRs-deficient EMPs. Combining these data, we may deduce that Sirt6 mRNA-incorporated EMPs may mediate an important mechanism in the transfer of information to neighboring and remote ECs in diabetes.
It is well known that eNOS-derived NO formation plays a very important role in maintaining endothelial functional integrity. Indeed, both senescent and apoptotic ECs are observed with a pronounced downregulation of eNOS expression and NO formation [18, 39]. Recently, researchers have observed that EMPs from CAD or CHF patients inhibit angiogenesis and induce inflammation in ECs via disturbed eNOS phosphorylation and NO formation [40, 41]. The present findings further extend these previous reports by showing that blunted eNOS phosphorylation and NO formation are observed in HUVECs following exposure to EMPs from DM patients or high glucose-incubated ECs, suggesting that the eNOS pathway is very important for the transfer of information via EMPs in DM. Whether the EMP-mediated decrease of eNOS phosphorylation and NO formation is related to Sirt6 is still unclear. Although Liu R reported that the downregulation of Sirt6 decreased eNOS phosphorylation and played key roles in oxidative stress-induced EC senescence , the results contrasted with those from Freitas M who showed that Sirt3 and Sirt7, but not Sirt6, had a close relationship with eNOS phosphorylation and NO formation in aged patients . At present, our findings further extend these previous reports by showing that blunted eNOS phosphorylation and NO formation can be observed in target ECs following Sirt6-deficient EMP exposure, whereas Sirt6 mRNA-overexpressing EMPs cause significant eNOS phosphorylation and NO formation. Thus, it may be possible that Sirt6 deficiency in DM patient-derived EMPs induces endothelial dysfunction via the eNOS pathway.
There is a limitation in this study that should be noted. Although we have shown that Sirt6 mRNA-incorporated EMPs are very important for the maintenance of neighboring and remote EC function, the mechanism underlying the effects of Sirt6 on the eNOS pathway has not been completely elucidated. Additionally, direct eNOS regulation in target HUVECs was not shown in this study. Therefore, additional in vivo and ex vivo studies are needed to verify the abovementioned mechanisms.
In conclusion, diabetes leads to increased EMP release and decreased serum Sirt6 levels. Sirt6 mRNA-incorporated EMPs are decreased in DM patients and high glucose-incubated HUVECs. EMPs containing Sirt6 mRNA inhibits inflammatory cytokine release and induces tube formation, whereas deficiency of Sirt6 mRNA in EMPs amplifies inflammation and impairs tube formation. Moreover, eNOS phosphorylation and NO formation may be involved in this progress. Many questions must be answered regarding the molecular network underlying these responses. However, the possibility that the Sirt6 mRNA-incorporated EMPs have an essential effect on neighboring and remote ECs in DM is very exciting.
MATERIALS AND METHODS
Glucose and TRIzol were purchased from Sigma (St Louis, Missouri, USA). Monoclonal antibodies against CD62-PE, CD31-PE, CD42-FITC and IgG-PE were obtained from BD (Shanghai, China), and the monoclonal rabbit antibodies against Sirt6, p-eNOS, eNOS and β-actin were obtained from Cell Signaling (Denver, Colorado, USA). The fetal bovine serum (FBS), DMEM culture medium with or without glucose, Lipofectamine® 2000 Transfection Reagent and ECMatirmTM solution were purchased from Invitrogen (Carlsbad, CA, USA). The cDNA Synthesis Kit and Premix Ex Taq SYBR Green PCR Kit were purchased from Takara (Shiga, Japan). The adv-Sirt6, Sirt6 siRNA, CMV-Sirt6 shRNA, Sirt1 siRNA and Drosha siRNA were obtained from HanBio. (Shanghai, China). Other unmentioned reagents were purchased from Shenggong Bio. (Shanghai, China).
Patients and blood collection
Informed consent was obtained from all participants in accordance with the guidelines of the Human Subjects Committee of the Medical Ethics Commission of the Tongji Hospital Affiliated with Tongji University (Shanghai, China). Between August 2014 to March 2015, 10 diabetes patients without hypertension, smoking, chronic kidney disease, peripheral atherosclerosis, inflammation, malignant tumors, severe hepatic dysfunction or coronary atherosclerosis who were diagnosed by coronary angiogram at our inpatient department were screened for inclusion in the study. Six age-matched healthy volunteers were also included in our present study. All DM patients were treated with either oral antidiabetic drugs or subcutaneous insulin administration.
Venous blood was obtained from the cubital vein under sterile conditions and then buffered with sodium citrate (for EMP analysis) or with anticoagulant sodium (for Sirt6 quantification). Blood was centrifuged at 1,500 g for 10 min, and then, the supernatant was centrifuged at 13,000 g for 20 min to obtain MPs, which were subsequently resuspended in PBS. The blood was coagulated for 45 min, and then, the serum was collected to measure Sirt6 levels.
The human umbilical vein endothelial cell (HUVEC) line and endothelial cell media (ECM) were obtained from ScienCell (Carlsbad, California, USA). The HUVECs were grown in ECM (glucose: 5.5 mM), starved by culturing them without FBS for 12 h and then exposed to glucose at concentrations of 0 mM for 24 h or 33 mM for 72 h to generate EMPs. Additionally, Adv-Sirt6 or Sirt6 siRNA was used to alter EMP components.
Adv-control or Adv-Sirt6 was directly added to cultured cells and incubated for 24 h.
Cells were transfected with either scramble siRNA, Sirt6 siRNA, Sirt1 siRNA or Drosha siRNA using Lipofectamine® 2000 Transfection Reagent following the manufacturer’s procedure. The siRNA sequence is shown on Table 1.
Table 1: Oligonucleotide sequences of the primers and siRNAs
stage1: 95°C, 30 sec
stage2: 95°C, 5 sec
55°C, 30 sec
72°C, 35 sec
EMP collection from HUVECs
After incubation with Adv-Sirt6, Sirt6 siRNA or Drosha siRNA for 24 h, the supernatant was removed, and the cells were carefully washed with PBS 3 times for 5 min. Then, the cells were exposed to glucose at concentrations of 0 mM for 24 h or 33 mM for 72 h. The medium was collected and centrifuged at 1000 rpm for 5 min, and then, the supernatant was centrifuged at 13000 g for 20 min to obtain MP, which were resuspended in PBS.
Quantitative determination of the plasma EMP levels or cultured HUVEC EMP levels
As previous description, CD31 and CD62 were biomarkers of endothelial cells, whereas CD42 was biomarker of platelets, therefore, CD62+/CD42- or CD31+/CD42- were considered to be EMPs in plasma. Usually, documents reported that CD62+ EMPs released from activated endothelial cells, whereas CD31+ released from apoptotic endothelial cells . CD31+/CD42- or CD62+/CD42- MPs levels were measured freshly with flow cytometry by using CD42-FITC and CD31-PE/CD62-PE.
Real-time reverse transcription PCR (RT-PCR)
Total RNA from HUVECs or MPs (obtained using the TRIzol procedure) was converted to cDNA using Takara reverse transcriptase. RT-PCR was performed using the samples and SYBR Premix regent on an ABI 7500. The primers (Shenggong Bio., China) are displayed on Table 1.
Total RNA from HUVECs or MPs was tested for quality and treated with DNase and RiboZero Kits (Illumina, Singapore) to remove DNA and rRNA. RNA-seq was performed by Shenggong Bio., China.
After purified of mRNA using Oligotex mRNA Mini Kit (Qiagen, Germany), the Sirt6 mRNA was executed of electroporation into EMPs was performed using GenePulser XcellTM electroporation system (BioRad, USA) as previously described . Briefly, 1x105 EMPs and 4 ug mRNA were mixed in 200 ul of electroporation buffer and electroporated at 350 V and 150 uF in a 10 msec. After incubated at 37°C for 30 min, the EMPs were transferred to cultured cells.
Equal amounts of total protein from HUVEC or MPs (obtained using cell total protein lysis buffer, Beyotime, Haimen, China) were subjected to 8-10% SDS-PAGE, transferred to PVDF membranes and then blocked using 5% non-fat milk. After incubation with primary antibodies at 4 °C overnight, HRP-conjugated secondary antibodies were applied at room temperature for 1 h. Immunoblots were visualized using enhanced chemiluminescence reagents (Bio-Max, Israel).
Enzyme-linked immunosorbent assay of Sirt6 production in blood and EMPs
Sirt6 production was detected in blood or EMPs using human ELISA kits according to the manufacturer’s instructions.
HUVEC tube formation assay in ECMatirmTM solution
To assess the vasculogenic effects of EMPs on endothelial cells ex vivo, an ECMatirmTM solution tube formation assay was performed according to the manufacturer’s instructions.
NO levels in culture supernatants were measured using the nitrite method (Jiancheng, Nanjing, China). Briefly, the reaction substrate was mixed with supernatants, vortexed, incubated and then centrifuged at 4,500 rpm for 15 min. The supernatant was then incubated with the chromogenic reagent for 15 min. Finally, the output was detected at an optical absorbance of 540 nm.
Significant differences were determined by one-way ANOVA followed by Tukey’s HSD test using SPSS14.0 software, and all data are presented as the mean ± SEM. A value of p<0.05 was considered statistically significant.
DM: diabetes mellitus; EMPs: endothelial microparticles; ECs: endothelial cells; Sirt6: sirtuin 6; Sirt1: sirtuin 1; MPs: microparticles; HUVEC: the human umbilical vein endothelial cells; CAD: coronary artery disease; CHF: chronic heart failure.
CF, LXB, TJ, KYS conceived and designed the study. TJ, KYS, WXJ, HHJ performed the experiments. CF, TJ and KYS wrote the paper. LXB, LY and YYA reviewed and edited the manuscript. All authors have reviewed and approved the manuscript.
We thank Prof. Fei Zheng for helping us carry out some of the western-blotting tests.
CONFLICTS OF INTEREST
This work was supported by the China National Natural Science Foundation, Nos: 81370390, 81500381 and 81670403.
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4. Wells BJ, Roth R, Nowacki AS, Arrigain S, Yu C, Rosenkrans WA Jr, Kattan MW. Prediction of morbidity and mortality in patients with type 2 diabetes. Peer J. 2013; 1:e87.
5. Liebner S, Cavallaro U, Dejana E. The multiple languages of endothelial cell-to-cell communication. Aeterioscler Thromb Vasc Bio. 2006; 26:1431–1438.
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10. Bernard S, Loffroy R, Sérusclat A, Boussel L, Bonnefoy E, Thévenon C, Rabilloud M, Revel D, Moulin P, Douek P. Increased levels of endothelial microparticles CD144 (VE-Cadherin) positives in type 2 diabetic patients with coronary noncalcified plaqued evaluated by multidetector computed tomography (MDCT). Atherosclerosis. 2009; 203:429–435.
11. Chen J, Chen S, Chen Y, Zhang C, Wang J, Zhang W, Liu G, Zhao B, Chen Y. Circulating endothelial progenitor cells and cellular membrane microparticles in db/db diabetic mouse: possible implications in cerebral ischemic damage. Am J Physiol Endocrinol Metab.2011; 301:e62–71.
12. Tsimerman G, Roguin A, Bachar A, Melamed E, Brenner B, Aharon A. Involvement of microparticles in diabetic vascular complications. Thromb Haemost. 2011; 106:310–321.
13. Chironi GN, Boulanger CM, Simon A, Dignat-George F, Freyssinet JM, Tedgui A. Endothelial microparticles in diseases. Cell Tissue Res. 2009; 335:143–151.
14. Tian K, Liu Z, Wang J, Xu S, You T, Liu P. Sirtuin-6 inhibits cardiac fibroblasts differentiation into myofibroblasts via inactivation of nuclear factor kB signaling. Transl Res. 2015; 165:374–386.
15. Conti V, Forte M, Corbi G, Russomanno G, Formisano L, Landolfi A, Izzo V, Filippelli A, Vecchione C, Carrizzo A. Sirtuins: possible clinical implications in cardio and cerebrovascular diseases. Curr Drug Targets. 2017; 18:473–484.
16. Thandavarayan RA, Garikipati VN, Joladarashi D, Suresh Babu S, Jeyabal P, Verma SK, Mackie AR, Khan M, Arumugam S, Watanabe K, Kishore R, Krishnamurthy P. Sirtuin-6 deficiency exacerbates diabetes-induced impairment of wound healing. Exp Dermatol. 2015; 24:773–778.
17. D’Onofrio N, Servillo L, Giovane A, Casale R, Vitiello M, Marfella R, Paolisso G, Balestrieri ML. Ergothioneine oxidation in the protection against high-glucose induced endothelial senescence: involvement of SIRT1 and SIRT6. Free Radic Biol Med. 2016; 96:211–222.
18. Chen F, Chen B, Xiao FQ, Wu YT, Wang RH, Sun ZW, Fu GS, Mou Y, Tao W, Hu XS, Hu SJ. Autophagy protects against senescence and apoptosis via the RAS-mitochondria in high-glucose-induced endothelial cells. Cell Physiol Biochem. 2014; 33:1058–1074.
19. Kumar S, Kim YR, Vikram A, Naqvi A, Li Q, Kassan M, Bachschmid MM, Jacobs JS, Kumar A, Irani K. Siruin-1 regulated lysine acetylation of p66Shc governs diabetes-induced vascular oxidative stress and endothelial dysfunction. PNAS. 2017; 114:1714–1719.
20. Souza AC, Yuen PS, Star RA. Microparticles: markers and mediators of sepsis-induced microvascular dysfunction, immunosuppression, and AKI. Kidney Int. 2015; 87:1100–1108.
21. Abbas M, Jesel L, Auger C, Amoura L, Messas N, Manin G, Rumig C, León-González AJ, Ribeiro TP, Silva GC, Abou-Merhi R, Hamage E, Hecker M, et al. Endothelial microparticles from acute coronary syndrome patients induce premature coronary artery endothelial cell aging and thrombogenicity: role of the Ang II/AT1 receptor/NADPH oxidase-mediated activation of MAPKs and PI3-kinase pathways. Circulation. 2017; 135:280–296.
22. Jansen F, Stumpf T, Proebsting S, Franklin BS, Wenzel D, Pferfer P, Flender A, Schmitz T, Yang X, Fleischmann BK, Nickenig G, Werner N. Intercellular transfer of miR-126-3p by endothelial microparticles reduces vascular smooth muscle cell proliferation and limits neointima formation by inhibiting LRP6. J Mol Cell Cardiol. 2107; 104:43–52.
23. Yellon DM, Davidson SM. Exosomes: nanoparticles involved in cardioprotection? Circ Res. 2014; 114:325–332.
24. Hulsmans M, Holvoet P. MicroRNA-containing microvesicles regulating inflammation in association with atherosclerotic disease. Cardiovasc Res. 2013; 100:7–18.
25. Nie DM, Wu QL, Zheng P, Chen P, Zhang R, Li BB, Fang J, Xia LH, Hong M. Endothelial microparticles carrying hedgehog-interacting protein induce continuous endothelial damage in the pathogenesis of acute graft-versus-host disease. Am J Physiol Cell Physiol. 2016; 310:C821–835.
26. Martinez MC, Larbret F, Zobairi F, Coulombe J, Debili N, Vainchenker W, Ruat M, Freyssinet JM. Transfer of differentiation signal by membrane microvesicles harboring hedgehog morphogens. Blood. 2006; 108:3012–3020.
27. Xu JF, Wang YP, Zhang SJ, Chen Y, Gu HF, Dou XF, Xia B, Bi Q, Fan SW. Exosomes containing differential expression of microRNA and mRNA in osteosarcoma that can predict response to chemotherapy. Oncotarget. 2017; 8:75968–75978. https://doi.org/10.18632/oncotarget.18373.
28. Peterson DB, Sander T, Kaul S, Wakim BT, Halligan B, Twigger S, Pritchard KA Jr, Oldham KT, Ou JS. Comparative proteomic analysis of PAI-1 and TNF-alpha-derived endothelial microparticles. Proteomics. 2008; 8:2430–2446.
29. Burnier L, Fontana P, Kwak BR, Angelillo-Scherrer A. Cell-derived microparticles in haemostasis and vascular medicine. Thromb Haemost. 2009; 101:439–451.
30. Vitiello M, Zullo A, Servillo L, Mancini FP, Borriello A, Giovane A, Della Ragione F, D’Onofrio N, Balestrieri ML. Multiple pathways of SIRT6 at the crossroads in the control of longevity, cancer, and cardiovascular diseases. Ageing Res Rev. 2017; 35:301–311.
31. Sociali G, Magnone M, Ravera S, Damonte P, Vigliarolo T, Von Holtey M, Vellone VG, Millo E, Caffa I, Cea M, Parenti MD, Del Rio A, Murone M, et al. Pharmacological Sirt6 inhibition improves glucose tolerance in a type 2 diabetes mouse model. FASEB J. 2017; Epub.
32. Freitas M, Rodrigues AR, Tomada N, Fonseca J, Magalhães A, Gouveia AM, Neves D. Effects of aging and cardiovascular disease risk factors on the expression of sirtuins in the human corpus cavernosum. J Sex Med. 2015; 12:2141–2152. https://doi.org/10.18632/aging.100975.
33. Breitenstein A, Stein S, Holy EW, Camici GG, Lohmann C, Akhmedov A, Spescha R, Elliott PJ, Westphal CH, Matter CM, Luscher TF, Tanner FC. Sirt1 inhibition promotes in vivo arterial thrombosis and tissue factor expression in stimulated cells. Cardiovasc Res. 2011; 89:464–472.
34. Xu S, Yin M, Koroleva M, Mastrangelo MA, Zhang W, Bai P, Little PJ, Jin ZG. SIRT6 protects against endothelial dysfunction and atherosclerosis in mice. Aging (Albany NY). 2016; 8:1064–1082. https://doi.org/10.18632/aging.100975.
35. Xiong X, Sun X, Wang Q, Qian X, Zhang Y, Pan X, Dong XC. SIRT6 protects against palmitate-induced pancreatic β-cell dysfunction and apoptosis. J Endocrinol. 2016; 231:159–165.
36. Anderson JG, Ramadori G, Ioris RM, Galiè M, Berglund ED, Coate KC, Fujikawa T, Pucciarelli S, Moreschini B, Amici A, Andreani C, Coppari R. Enhanced insulin sensitivity in skeletal muscle and liver by physiological overexpression of SIRT6. Mol Metab. 2015; 4:846–856.
37. Liu R, Liu H, Ha Y, Tilton RG, Zhang W. Oxidative stress induces endothelial cell senescence via downregulation of Sirt6. Biomed Res Int. 2014; 2014:902842.
38. Kuang J, Zhang Y, Liu Q, Shen J, Pu S, Cheng S, Chen L, Li H, Wu T, Li R, Li Y, Zou M, Zhang Z, et al. Fat-specific Sirt6 ablation sensitizes mice to high-fat diet-induced obesity and insulin resistance by inhibiting lipolysis. Diabetes. 2017; 66:1159–1171.
39. Donato AJ, Morgan RG, Walker AE, Lesniewski LA. Cellular and molecular biology of aging endothelial cells. J Mol Cell Cardiol. 2015; 89:122–135.
40. Ou ZJ, Chang FJ, Luo D, Liao XL, Wang ZP, Zhang X, Xu YQ, Ou JS. Endothelium-derived microparticles inhibit angiogenesis in the heart and enhance the inhibitory effects of hypercholesterolemia on angiogenesis. Am J Physiol Endocrinol Metab. 2011; 300:E661–668.
41. Lin ZB, Ci HB, Li Y, Cheng TP, Liu DH, Wang YS, Xu J, Yuan HX, Li HM, Chen J, Zhou L, Wang ZP, Zhang X, et al. Endothelial microparticles are increased in congenital heart diseases and contribute to endothelial dysfunction. J Transl Med. 2017; 15:4.
42. Qu L, Ding J, Chen C, Wu ZJ, Liu B, Gao Y, Chen W, Liu F, Sun W, Li XF, Wang X, Wang Y, Xu ZY, et al. Exosome-transmitted lncASAR promotes Sunitinib resistance in renal cancer by acting as a competing endogenous RNA. Cancer Cell. 2016; 29:653–668.
All site content, except where otherwise noted, is licensed under a Creative Commons Attribution 3.0 License.

domain: medical


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Ideal during cancer treatments thanks to its immediate calming action - 30ml
The organic cicabaume is ideal during cancer treatments thanks to its immediate calming action. It restores the epidermis, easing any irritations and leaving your skin soothed.
Its repairing and regenerating action is perfectly suited for damaged, fragile or irritated skin, including the lips. This balm can also used in the case of hand-foot syndrome caused by chemotherapy treatments.
Apply several times per day, if necessary.
You can also use it for your hands and feet in case of cracking or hand-foot syndrome.
Extract from organic Calendula flowers (50 %): antiseptic
Organic musk rose oil (27 %): regenerating
Natural vitamin E
Essential oils of lavender, carrot, geranium: stimulating
Calendula Officinalis, Helianthus Annuus Seed Oil, Rosa Moschata Seed Oil, Cera Alba, Tocopheryl Acetate, Lavandula Angustifolia Oil, Daucus Carota, Tocopherol, Pelargonium Graveolens Flower Oil, Usnea Barbata Extract, Geraniol, Linalool, Citronellol.

domain: health


According to a prominent think-tank, people should not expect a higher wage increase this year than inflation, as wages stagnate until the end of 2018.
The resolution foundation found that wage pressures could get worse before things get better, with wage packages no meaningful recovery until the end of next year.
Real wage growth for the whole of 2018 is expected to be zero, meaning that the stress of living standards will continue.
Inflation, which began to exceed wage gains in early 2017, marked the return of pay squeeze after the financial crisis and finally began to slow in 2015.
The resolution foundation said there were reasons to be optimistic about the next year, as there were signs that wage pressures could begin to rise by the end of 2018.
It also highlights the lowest income workers a minimum wage rise, their hourly rate rose 4.3% in April, 7.83 pounds, to three months in October, productivity to pioneer this is likely to be 1.2% pay rise.
2017 is a “difficult year for living standards because of falling wages,” said the resolution foundation director, thorspell.
“The good news is that things will get better next year,” he said. “The bad news is that we may have to go back to rest, and the promise of meaningful revenue recovery is still invisible.
“While the public has been grumbling about the recent economic downturn, the public expectation does seem to be in line with the experts’ gloomy forecasts. More than half predicted no pay rises next year, and families could expect their finances to deteriorate further next year. This pessimism is strongest among low-income people and, unsurprisingly, there will be substantial cuts in benefits. ”
It also found that public expectations for a pay rise were low, with more than half expecting a pay rise next year, according to analysis of bank of England data.
More than a quarter of working-age families -27 per cent – expect their financial situation to deteriorate over the next 12 months, roughly the same proportion as they think they will improve – up to 28 per cent.
More than a third of the poorest households thought their situation would worsen – 35 percent – and the richest families only had a sixth to 17 percent.
Another Lloyds survey shows that men expect higher wages next year than women.
On average, men expect their annual income to rise by 770.50 pounds by the end of 2018, according to the bank’s spending power report, while women can only expect their salaries to rise by 429.70 pounds.
A survey of more than 2,000 people in November in the UK showed that 63 per cent were dissatisfied with the current level of inflation, up 14 percentage points since November 2016.
Two-thirds of the people feel negative for the UK economy, compared with the same period last year rose five percent, and 65% of them feel about the housing market is bad or not so good, six percent higher than last year.


Ten students from the the London Academy of Excellence Tottenham will visit the festival next month
The Cliveden Literary Festival has announced an educational partnership with the London Academy of Excellence Tottenham.
Students from the free sixth form school, which opened as part of the redevelopment of White Hart Lane in September 2017, are invited to submit papers on why they would like to attend the festival. From the submissions, the head teacher will select 10 students to attend the festival, which takes place on 29 and 30 September. As well as meeting speakers, they will each receive a copy of The Mistresses of Cliveden by festival chair Natalie Livingstone, as well as a choice of book from John Sandoe Books, the festival bookshop.
Livingstone said: "History and education go hand-in-hand and nothing is more important than involving the next generation of historians and writers in the literary festival. We are therefore delighted to work with the London Academy of Excellence Tottenham and look forward to welcoming their students to the festival in September and to continuing the collaboration in the years to come."
Jan Balon, headteacher, said: "Our students at London Academy of Excellence Tottenham are academically motivated and ambitious and the opportunity to visit the Cliveden Literary Festival will expose them to a rich world of culture to which they have not previously had access. It will broaden their academic horizons significantly and the exposure provided to such a wide range of incredible speakers in an historic location will help them to develop their intellectual curiosity beyond measure."
Stars at the festival include Naomi Wolf, Armando Iannucci, Simon Sebag Montefiore, Antony Beevor, Alan Mallinson, Tina Brown, and Tristram Hunt. Programme here.


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Home Asia Pacific South East Asia Myanmar Myanmar Protest News
Burma admits holding 468 protesters
DPA, Oct 16, 2007
Rangoon, Burma -- The Burmese junta has confirmed that it continues to imprison at least 468 of the 2,927 people it admits to having arrested for participating in last month's peaceful protests.
State-run media said Wednesday, in a short announcement on Burmese TV at 10 pm Tuesday, that the regime had rounded up 2,284 people in Rangoon for their role in last month's anti-military protests, while another 643 were arrested in the provinces.
Of those arrested in Rangoon, the epicentre of the recent protests, some 190 remain under detention in Rangoon and 278 in the provinces.
The news was repeated in state-run newspapers Wednesday.
The announcement was deemed odd because of its briefness and lack of typical anti-western propaganda, leading some to speculate that it may have been made in response to mounting international pressure on the Burmese junta to release all political prisoners including those who were detained in a brutal crackdown on September 26 and 27 on a peaceful monk-led rebellion.
The junta, however, has continued to arrest people weeks after the demonstrations in defiance of world condemnation.
On October 13 it arrested three leaders of the 88 Generation Students group and another activist named Ko Ko who had been in hiding for weeks after participating in marches against fuel price hikes announced on August 15.
The sudden doubling of fuel prices, at a time when Burmese are already suffering from double-digit inflation, sparked the largest demonstrations the country has witnessed since 1988.
After Buddhist monks took the lead in the protest movement, the demonstrations peaked at 100,000 participants on September 24 and 25 before authorities brought in riot police and troops to crush the movement.
The crackdown has drawn Burma, a country that has been under a military dictatorship since 1962, back into the world's spotlight.
The United Nations Security Council on October 11 issued a statement "deploring" the crackdown on monks and laymen and has dispatched special envoy Ibrahim Gambari to pressure the regime to start a dialogue with opposition members while freeing political prisoners including Nobel peace prize laureate Aung San Suu Kyi and thousands of others.
Those still in danger of getting arrested and tortured consider the UN's actions insufficient.
"Thousands of protests, including monks and students, continue to suffer ill-treatment and severe torture in detention centres and some have passed away in custody," said a letter from three members of the 88 Generation Students group to UN Secretary General Ban Ki-moon.
The message to Ban, signed by Tun Myint Aung, Nilar Thein and Soe Htun, noted that "this may be the last letter we send to you before our own arrest and torture."
The group urged Ban to station Gambari permanently in Burma for "as long as is necessary to facilitate a political dialogue," and to personally telephone the junta's chief Senior General Than Shwe to demand an end to the torture of those being detained.
"As long as effective action from the international community is delayed, Burma continues to be a lawless society and the regime will continue to kill peaceful demonstrators systematically and quietly in torture chambers," it concluded.

domain: politics


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A few weeks ago, we made the trip from Brooklyn to Saratoga Springs to visit my sister and her boyfriend Alex. It was a relaxing weekend, seeped in autumn colors, amazing food and beautiful scenery.
About thirty minutes outside of Albany, Saratoga Springs makes a beautiful weekend retreat. Home of the famed Saratoga Races (July-September) and summer home of the New York City Ballet, Saratoga offers world class entertainment with a charming and village-like feel. Saratoga's downtown is walkable and full of top notch restaurants, boutiques, gallery spaces and bars.
A few paces from the main drag, you'll find block upon block of immaculately restored Victorian homes. Colorful gables and shutters decorate the streets. One almost expects to see parasols, top hats and stagecoaches.
Saratoga is splendid, but it is more than just a pretty setting. Saratoga is seeped in history and tradition.
Saratoga is home to the famed Saratoga springs, a series of naturally carbonated mineral water springs lauded for their healing and curative powers.
The Iroquois and Mowhawk Indians celebrated the medicinal properties of the springs bathing in and drinking the waters. Later, George Washington was said to drink and bathe in the springs, he even tried unsuccessfully to purchase the springs which were considered during the Colonial times to heal ailments from rheumatism, diabetes, heartburn, liver and kidney problems, cancer, malaria and hangovers.
By the 19th century, wealthy and famous New Yorker's would retreat to Saratoga Springs, New York for luxurious spa getaways in the springs.
Today, you can sample the water from about 17 different springs. Each spring has a slightly different taste and apparently depending upon the time of the year, the water tastes different. An ambitious visitor to Saratoga can take a walking tour and sample each spring. I myself, made it to two of the spring fountains before deciding that the water was not for me. I couldn't get past the strong odor of sulfur.
An easy weekend trip to take from many points in the Northeast (New York, Philadelphia, Boston, Albany, Rochester, Syracuse, Buffalo, to name a few) Saratoga is well worth exploring.
During the summer there are races and cultural events like the New York City ballet. In the autumn, Saratoga is bright with fall leaves and apple picking. Saratoga transforms into a winter wonderland during the holiday season and is home to manicured gardens during the spring.

domain: geography


text:
Welcome to Picket Fence Blogs! Our site provides you with the top lifestyle blogs
on the web. Feel free to just look around, or
add your own blog so
others can find you and benefit from what you have to say. Plus, it's free to join!
Crafts, games, activities, and more to keep our kids happy, active, and artistic! Now featuring Friday Food Day and Mandi's Menu, as well as more ideas for toddler friendly crafts! 2011 Parents Best Craft For Kids Blog Nominee.
#education# children#art and crafts
This is a go-to site for busy moms looking for recipes, learning activities, menu planners, tips, printables, validation, community, and encouragement. None of us are perfect, we're just chasing "Supermom."
A place to collect & borrow teaching tools, share & receive inspiration, and explore & review professional resources. For all who love to learn, create, explore & teach!
Our goal is to cooperate with parents and teachers to create a valuable resource.
"One Cramazing Life" is about the life of a mom, a dad, 2 kids, and a life that is crazy and amazing. Topics include: Family life, prematurity, product reviews, and just plain fun stuff!
#family fun#family #boys#family activities
Just some snippets of my life as a Christian wife, homeschool teacher, scrapbooker, easy party planner, and mom to three manifestations of my mother's curse to have "one just like me" - not necessarily in that order...
From the moment babies are born, they are constantly taking in information. Everyone has their own ideas on how children learn best, but whatever they may be, there is no doubt they are learning in some way, shape, or form.
Hi!, I'm a Mexican artisan, here I share pictures of my items, their crafting process and what I'm thinking while making them, as well as other things happening in my life. Sometimes with my favourite music to read along to :) I also sing in a choir.
This blog offers detailed videos and step by step instructions on lessons that integrate drama. The lessons can be used as is or changed to fit your needs. The videos can be used as part of your lesson or you can try to make your own.

domain: hobby


Using GIS software ARCVIEW, 7 landscape indices (Diversity, Evenness, Dominance, Fractal dimension, FN, FS and Isolation ) were chosen to study the spatial patterns of the land use and soil erosion in Bishan County.
Using GIS software ARCVIEW3.3 landscape indices (Diversity Evenness DominanceFractal dimension and Isolation et.) were chosen to study the spatial patterns of the land useand soil erosion in Meihekou City.
The information Tupu of space pattern and evolutionary process were analyzed in detail taking time interval from 1987 to2001 as an example.
Choosing Pinus sglvestris Var. mongolica forest distributing area in Honghuaerji of east Inner Mongolia as research object, using GIS software such as Arcview3.2 / ArcGis8.12 and landscape pattern analysis software Fragstats3.3. This article, that get going from vegetation distributing influence issue of earth environment and human being activity which is hotspot of plant ecology and plant geography, has studied landscape space pattern and change of Pinus sglvestris Var. mongolica forest in Honghuaerji.
By using three-period aerial photo as basic information resource and withthe support of ARC/INFO , a space information storehouse is built up. Through selecting patches size , fractal dimension, dominance, diversity index, evenness index, etc,the dynamic development of land sight space pattern of Nihegou Valley in the loessplateau gully area is analysized.
The result from 1987～1995 shows that Nihegou's landsight space pattern changes significantly with the diversity and evenness index being onthe rise. The pattern shows a varied development tendency of agriculutre, fruit,forestry and animal husbandry, i. e. the obvious results obtained from integrated control .
However soil moisture in grassland after rainfall could be expressed by exponential and spherical model, and the spatial correlation coefficient, range and fractal dimension were 0.62 and 0.98, 181.80 m and 4.55 m, 1.91 and 1.99 at 0～20 cm and 20～40 cm soil layers, respectively, having the larger difference of spatial variability between two layers. Kriging maps expressed the spatial structure characters in detail.
It is widely acknowledged that it is a basic subject in remote sensing applications. Image information with different scale displays distinct spatial structure, so image analysis with single scale could not meet the heterogeneity and dynamic of pattern and process.
tabulaeformis forests with a clumped spatial pattern but rarely appeared in other forest types, indicating that P.
Based on the sampling data, a correct approach to the boundary effect on aggregation index was put forward to the spatial pattern analysis of the broad-leaved Korean pine forest in its different stages of development.
The spatial pattern of the broad-leaved Korean pine forest changing from clumped to random distribution is influenced by biological characteristic, the adaptive strategy of tree species and the natural disturbance in its entire development.
Spatial pattern analysis on individual trees in different age-classes of Larix principis-rupprechtii was made in this paper.
The spatial pattern analysis of population and community is important to understand community structure and has become one key topic in modern plant ecology.
Here, we suggest a method for adjusting boundary effect in the analysis of spatial patterns at different stages of development of the broad-leaved Korean pine forest.
In order to explain the spatial patterns of interception, we adopted grid-sampling method to install rain-gauges to measure throughfall.
Many studies have demonstrated the changes in the spatial patterns of plant and animal communities with respect to habitat fragmentation.
The DRT was found to be an effective approach to the anisotropy analysis of spatial patterns of plant populations.
Comparison of the genetic variation surveyed by starch gel electrophoresis to allozyme polymorphism indicated that morphological diversity is a good metric for quantifying spatial patterns of biodiversity.


The most successful facility projects follow distinct delivery strategies. In particular, those strategies that align the core project team - owner, designers, primary building and key specialty trades - are more effective in meeting or exceeding their cost, schedule and quality goals. Best leveraged in a workshop setting, this guide will illustrate the process for selecting a delivery strategy that meets the need of your project. Learn more >
Sponsored by the Charles Pankow Foundation and the Construction Industry Institute (2013 - 2016)
Interactive Workspaces are team meeting rooms that have been augmented with technology to enable visualization, interaction with digital information, and team collaboration for enhanced decision-making. This guide assists a capital facility project team in the identification and selection of workspace requirements for their integrative team meetings.
The Virtual Construction Simulator (VCS) is a simulation game that teaches students the dynamic nature of the construction process and frequent changes to construction schedules. The VCS provides the user with an advanced 3D graphical user interface (GUI) for the development of the planned schedule, together with a 3D simulation environment. The purpose of the game is to allow the user to experiment with the planning and managing of construction schedules, while learning the difference between the as-planned and the as-built schedule.
Sponsored by the National Science Foundation (2012 - 2016)
OpenStudio Enhancements to Support Energy Retrofit Projects
The enhancements will further the development of OpenStudio and Radiance to support modeling of physically based lighting layouts and systems including daylight integrated photocontrol. These additional enhancements to OpenStudio, as well as Radiance, will improve energy efficient design for small and medium sized retrofit projects, while achieving high levels of lighting quality. These capabilities will continue to be supported by developing increasing levels of interoperability with BIM applications via BIMserver integration with a focus on extending beyond the core geometric information for energy modeling to enable more analyses related to lighting.
A well documented BIM Project Execution guide will help ensure all parties are clearly aware of opportunities and responsibilities associated with the incorporation of BIM into a project work flow. This guide provides a structured procedure for creating and implementing a BIM Project Execution Plan. The goal for developing this structured procedure is to ensure planning and elaborate communication among the project participants during the early phases of a project.
During the research and creation of the BIM Project Execution Planning Guide, the group determined there was a need to develop a guide for facility owners and operators that includes a procedure to develop a strategy for integrating BIM throughout their organization. This guide, developed primarily for facility owners, will focus on the decisions required to define their organization's standard BIM processes and practices; design information integration strategies; and identify appropriate BIM contracting strategies.
Sponsored by The Charles Pankow Foundation, Penn State OPP, Kaiser Permanente, US Department of Defense Military Health System, U.S. Department of Veterans Affairs and the The Partnership for Achieving Construction Excellence
Integrated Building Life Cycle Process Model for Advanced Energy Retrofit Project
Advanced energy retrofit projects are critical to contributing to the reduction of energy consumption within the United States and abroad. Successful retrofit projects require detailed coordination throughout the design, construction, and operations process. As a part of the consortium for building energy innovation, we developed an integrated building lifecycle process model for performing advanced energy retrofit projects.
This process model identifies the interaction between detailed system level processes, and tasks which must be performed by integrated teams to effectively leverage the knowledge and expertise of the project participants. The process map includes approximately 1000 activities within the design and construction process for advanced energy retrofit projects. This map can guide the development of a project specific plan for implementing a retrofit project, and has also been developed to guide the future creation of data standards to support information flow between different activities throughout the lifecycle process. The model is created using business process mapping notation, and divides the various activities into integrated activities, managerial activities, and activities that are more specifically focus on system-level details. The model was tested using various detailed case study projects of energy retrofit projects. The links below will take you to the final process maps for the planning and design phases of a project as well as the construction phases of a project. Additional details are included in project reports as well as the thesis from Sreelatha Chunduri and Ruo ‘Tony’ Gao.
Sponsored by the U.S. Department of Energy Energy Efficient Buildings Hub (currently the Consortium for Building Energy Innovation) (2012 - 2015)
Fostering Innovation and Learning through Immersive Virtual Facility Prototyping
The design and construction of facilities requires the conceptualization of complex systems and processes. While many industries have embraced rapid prototyping processes and technologies, the use of prototypes in the building industry remains in its infancy. Expensive scale models and mock-ups of building components are used to analyze design and construction. However, they represent only a single snapshot or configuration of the construction process. 3D and 4D computer aided design tools allow virtual models to be constructed, however they are typically challenging to manipulate and limited by small scale display for individual users.
Learn more >
Sponsored by the National Science Foundation (2004 - 2009)
The Pennsylvania State University 104 Engineering Unit A University Park, PA 16802


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KACEY FIFIELD, “Confused”
Kacey Fifield is a 16 year old singer/songwriter from L.A. whose burgeoning talent is popping in 2021. “Confused” is her second release for Tipsy Records, following up last year’s uncharacteristic and unique duet with Robbie Rosen called “Last Breath”. “Confused” is a thoughtful electronic ballad that should capture the ears of many listeners within her age range. Its relatable words pinpoint the feelings of many teens and young adults in these unusual times who are trying their best to cope with the demands of relationships, social media, puberty, school, work, and more. Kacey sings right from the heart, there is no embellishing, and she is fortunate to have ongoing guidance with her music from friend, occasional co-writer and producer Robbie. Kacey is well-established in social media circles and her fans should flock to the authenticity of “Confused”.

domain: artistic


Spending on trees 25 times lower in England than in Scotland
Grant spending on trees & woodlands per person is 25 times lower in England than in Scotland
Danny Gross, trees campaigner at Friends of the Earth, said:
“Westminster’s strategy for creating more woodland has a huge imbalance. It’s failing to do nearly enough in England, while leaving Scotland to do the heavy lifting.
“Growing more trees is a key part of fighting the climate crisis, so the government really needs to step up. At the moment it stands to miss even its own meagre manifesto commitment on trees. The upcoming English Tree Strategy is a big opportunity to turn this around so must focus on boosting spending on trees in England. We know that England has the land to double tree cover, it’s just the political will that’s lacking.” Notes:
- In 2018-2019, £20.5 million was spent on woodland grants in England, which is the second lowest amount in the past 10 years, according to the government’s latest forestry statistics (p135 of Forestry Statistics 2019). In contrast, £50.2 million was spent on woodland grants in Scotland.
- In 2018-2019, grant spending on trees & woodlands per person was 25 times higher in Scotland than England.
- In Scotland, spending was £9.23 per person.
- In England, spending was £0.37 per person.
- The Budget promised that 30,000 hectares of trees would be planted in England over the next 5 years, which is 6,000 hectares per year (full Budget document here). This is only 20% of the government’s manifesto commitment (see p43) to “reach an additional 75,000 acres [30,000 hectares] of trees a year by the end of the next Parliament”. This would leave Scotland, Northern Ireland and Wales to grow 80% of our new trees.
- In 2018-19, Scotland created 8 times more woodland than England, despite England being a lot larger than Scotland (p27 of Forestry Statistics 2019).
- Scotland created 11,210 hectares of new woodland.
- England created 1,420 hectares of new woodland.
- 19% of Scotland is already covered in Woodlands, compared to just 10% in England (p3 of Forestry Statistics 2019).


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Description: Columbia River M1601KS M16-10KZ Folder AUS-4 Spear Point Blade Black
The M16-10 features a dual hollow grind tanto-style blade with combined razor-sharp and triple-point serrated edges similar to the "Big Dog" M16 models. The result is maximum blade strength and ability to saw through cord, webbing, nets or vegetation quickly. The M16-10KZ model has a black EDP blade, black frame and hardware for a non flective tactical look.
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In August 2020, the Nursing and Midwifery Services Department (NMSD) of the Ministry of Heath, Community Development, Gender, Elderly, and Children (MoHCDEC) approved the inclusion of adolescent and youth sexual and reproductive health (AYSRH) content in the midwifery curriculum for all diploma programs by 2022. The three-year midwifery curriculum currently has eleven modules, none of which address reproductive health or AYSRH. The approval will increase the number of nurse midwives who graduate with the skills and confidence to provide quality, youth-friendly information, counseling, and services. An estimated 7,300 nurse midwives graduate every year and go on to provide services at all levels of public and private health facilities, suggesting the curriculum update could have far-reaching impact (1).
Teenage pregnancy is a major health and development concern in Tanzania. Twenty-seven percent of girls between the ages of 15 and 19 are either pregnant or have begun childbearing, and an estimated 8,000 girls in Tanzania drop out of school every year due to a pregnancy (2-4). Contraceptive use among young people 15 to 19 years old is 13% for modern methods, suggesting that access to AYSRH services is an issue for this group (5).
In 2017 Tanzania made a Family Planning 2020 commitment to increase the number of health facilities providing youth-friendly services from 30% (2016) to 80% by 2020. In 2019, an estimated 63% of health facilities were providing youth-friendly services (6); however, only 30% of health service delivery points met national standards for AYSRH (5). At the same time, the pace of training youth-friendly service providers in Tanzania has been slow. Only 57% of the 2,400 providers that were scheduled to receive the current youth-friendly services training in 2019 received training (7).
Advance Family Planning local partner Tanzania Men as Equal Partners in Development (TMEPiD) held advocacy meetings with officials from the NMSD to discuss ongoing AYSRH services challenges. TMEPiD analyzed the midwifery curriculum and identified gaps affecting the delivery of youth-friendly services, particularly the skills and attitudes of nurse midwives. They packaged this evidence into a policy brief and presentation to make the case for the inclusion of AYSRH and youth-friendly services training in the midwifery curriculum. TMEPiD then presented their ask during a meeting with the department team, where it was approved to be included in the 2022 curriculum review and revision.
In the short term, the NMSD Director, Ms. Ziada Sellah, instructed the department to form a task force to develop an addendum to the current curriculum, allowing for earlier access to AYSRH training for current midwifery students. The multi-department task force includes the NMSD assistant director, nursing officers, technical training officers, and reproductive and child health section officers. Ms. Sellah said the ministry’s decision will complement their ongoing efforts to strengthen access to AYSRH services through trained youth-friendly service providers in Tanzania.
Millions of adolescents stand to benefit from improved AYSRH service provision. The increase in AYSRH-trained nurse midwives will contribute towards attaining strategic priority four of the National Family Planning Costed Implementation Plan (NFPCIP II 2019-2023). Priority four focuses on expanding access to age-appropriate sexual and reproductive health information and contraceptives for young people (8). It also supports the achievement of priorities one and two of the National Accelerated Action and Integrated Agenda for Adolescent Health and Wellbeing to expand adolescent access to contraceptive information, services, and supplies (5).
Although in-person meetings and engagements have been delayed due to the COVID-19 pandemic, advocates are optimistic that the curriculum revision will continue despite these setbacks. TMEPiD and Tanzania Youth and Adolescent Reproductive Health Coalition (TAYARH), another Advance Family Planning local partner, will continue to collaborate with the ministry’s task force to develop the addendum in 2021 and support the curriculum revision in 2022. TAYARH is also supporting the ministry to include youth-friendly services training in all pre-service health training curricula by August 2021.
- United Republic of Tanzania. 2018. Budget Speech for the Financial Year 2018-19 delivered by the Minister of Health, Community Development, Gender, Elderly and Children, Honourable Ummy A. Mwalimu (MP).
- Ministry of Health, Community Development, Gender, Elderly and Children [Tanzania Mainland], Ministry of Health [Zanzibar], National Bureau of Statistics, Office of the Chief Government Statistician, and ICF. 2016. Tanzania Demographic and Health Survey and Malaria Indicator Survey (TDHS-MIS) 2015-16. Dar es Salaam, Tanzania, and Rockville, Maryland, USA: MoHCDGEC, MoH, NBS, OCGS, and ICF. Retrieved from https://dhsprogram.com/pubs/pdf/FR321/FR321.pdf
- Ministry of Health, Community Development, Gender, Elderly and Children (MoHCDGEC). 2013. Service Availability and Readiness Assessment. Retrieved from http://apps.who.int/healthinfo/systems/datacatalog/index.php/ddibrowser/58/download/179
- United Nations Children’s Fund (UNICEF). 2011. Adolescence in Tanzania. Retrieved from https://www.unicef.org/infobycountry/files/TANZANIA_ADOLESCENT_REPORT_Final.pdf
- United Republic of Tanzania, Ministry of Health, Community Development, Gender, Elderly and Children. National Accelerated Investment Agenda for Adolescent Health and Wellbeing (2019-2022).
- United Nations Children’s Fund (UNICEF). 2019. HIV and AIDS fact sheet. Retrieved from https://www.unicef.org/tanzania/media/2436/file/HIV%20Programme%20Fact%20Sheet.pdf
- Human Rights Watch. 2017. Tanzania: 1.5 Million Adolescents Not in School. Retrieved from https://www.hrw.org/news/2017/02/14/tanzania-15-million-adolescents-not-school
- United Republic of Tanzania, Ministry of Health, Community Development, Gender, Elderly and Children. 2019. Tanzania National Family Planning Costed Implementation Plan (2019-2023) Progress Review. Retrieved from https://tciurbanhealth.org/wp-content/uploads/2019/07/National-Family-Planning-Costed-Implementation-Plan-2019-2023-INSIDE-%E2%80%A2-F....pdf


We all know by now how fake social media accounts influenced the outcome of the 2016 U.S. Presidential election.
With their proven ability to rapidly sway political opinion, a more concerning issue centers on the spread of fake medical news , which I wrote about in the run-up to the election in 2016.
From cancer cures to fake medical treatments for any number of diseases, the power of such fake news can adversely impact a patient’s decision regarding treatment and outcome of their condition.
Recent research from the Keck School of Medicine of USC reveals just how such automated social media accounts on Facebook and Twitter can disseminate fake medical news and ultimately put your health at risk.
With the rise of artificial intelligence (AI), and armed with powerful social bots--automated accounts that use AI to promote ideas or products and influence discussions—consumers must be vigilant to avoid their powerful grip.
For their study, the researchers from USC decided to focus on public messaging regarding the dangers of e-cigarettes. They specifically chose to focus on how such bots were able to convince consumers that using e-cigarettes can help promote smoking cessation, something which has not been supported conclusively by scientific research to date.
Based on data from the CDC, tobacco use is the number one cause of preventable disease, disability and death in the US. And on the heels of this data, we now find that e-cigarettes are the most commonly used tobacco product among children and teens.


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The disgraced Disney starlet appeared to forget all about the blow-up over her Salvia-smoking, bong-sucking antics last week and for good reason, it turns out her people have known for weeks and allegedly been buying people off!
At a New Orleans Saints game Dec. 12, noted Salvia enthusiast Miley Cyrus ignored the hits she took after her bong brouhaha last week and took in another game involving hot air. It turns out however there wasn’t much for her to ignore as she had known about this video for weeks, and her team has allegedly been trying to buy people off to keep it a secret.
TMZ spoke to sources who said, “a college student who came into possession of the video contacted one of Miley’s reps shortly after Thanksgiving, asking if the rep wanted his copy. We’re told money was discussed, in exchange for turning over the video.”
A man in his forties was then sent to the campus and the student, “gave the man his MacBook Pro which contained the ‘bong’ video … in return the man gave the student a new MacBook Pro. We’re told no money changed hands, though we don’t know why.”
This happened twice, but clearly not enough times, as the video and stills eventually leaked.
For her part Miley has been seemingly nonchalant about the whole thing, stopping by a football game as mentioned above and working on the set of her new film, So Undercover, alongside former addict Kelly Osbourne.

domain: news


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Since 2014 Diamond Field has released four "maxi-singles" – 'Neon Summer' (with Nina Yasmineh aka NINA LUNA), 'This City' (with Matthew Ruys), 'Closer' (with Rat Rios) and 'Won't Compromise' (with Bob Haro). All four singles feature 12" extended versions, instrumental version and remixes from the likes of Sunglasses Kid, Phantom Ride, Who Ha, Dream Fiend, Vincenzo Salvia, Von Hertzog, Stereospread, Furniteur, Saint-Samuel, Morgan Willis, VHS Dreams, MIDWAVE, Circuit Work, Le Flex and Dashcam. Diamond Field has also contributed to compilations such as Vehlinggo Presents "5 Years" (with Dana Jean Phoenix), Retro Promenade's "Next Peak" Twin Peaks tribute series and annual Christmas synth albums. Remixes for other artists include Roxi Drive, Retroglyphs, Junica, Dream Shore, Phantom Ride, Von Hertzog, Delete Delete, David Bridie, Little Bark, Stereospread, Go Stop Go, Nina Yasmineh, Milaro, Bunny X, Matthew J. Ruys and MIDWAVE.
A full-length Diamond Field album is due for release in 2020.


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A pastry shop is an industrial establishment that makes flour-based baked goods and sells them to individuals. Some retail pastry shops additionally serve coffee as well as tea. These businesses lie in retail stores. The term bakeshop has various meanings. A bakery is one of the most preferred types of services in the USA. However, some retail bakeshops are actually cafés that offer baked goods.
A bakeshop’s revenues are biggest throughout the summertime as well as the end of the year when events prevail. The holiday season is also a busy time for pastry shops. Bakers also need to consider seasonal optimals and other elements to estimate their manufacturing capability. By doing this, they can approximate their day-to-day capability, costs, as well as possible profit. Using this details, they can much better prepare their procedures and also boost productivity. Nevertheless, they have to bear in mind to keep an eye on their budget plan.
The main ingredients used in baking are flour, water, and leavening agents. Other ingredients, such as eggs, milk, as well as salt, are utilized to customize the top quality of the end item. Different minor ingredients are utilized also. A few of these ingredients also influence the final taste as well as color. These components are used in the production of pastry shop products, such as breads, pastries, and pizza crusts.
Bread manufacturing is a sector that has experienced development as well as adjustment. A range of modern technology has actually resulted in improvements in the sector. Boosted active ingredients with boosted useful qualities are currently readily available. Using equipment that is much easier to use decreases the need for skilled workers. Batch handling and also fermentation are now automated. In the mid-20th century, an additional technology in cooking was the enhancement of minerals and vitamins to bread. This is a major milestone in the field of baking.
Some pastry shops are run by relative. Some bakers pick to run out of their houses, while others pick to rent commercial kitchen space. In both situations, there are benefits and drawbacks to both choices. If you are a home baker, you’ll be able to utilize the kitchen area area, yet you will certainly need an official space for cooking and inviting consumers.
Cooking has actually been around for countless years, yet its advancement became much more prevalent in the Roman Empire. Roman residents enjoyed baked goods and would certainly require them on special occasions. By 300 BC, baking was a recognized career. Roman bakers were experienced at making bread and using mills to grind grain right into flour. In 168 BC, a baker’s guild was established in Rome. The appeal of baked items spread out throughout Europe and also eastern Asia.
The secret to success in a pastry shop is to discover just how to handle your pastry shop’s service version. This entails determining just how much you can pay for to invest in marketing, technology, and also sales. An effective bakeshop can be rewarding within three years of procedure. The earnings of a bakery relies on the variety of sales, food expenses, and labor prices.
In general, bakery items are composed of flour, sugar, and chocolate. They are typically sold at coffee bar, tea houses, and dining establishments. Regardless of the name, the ingredients and items marketed in a bakery will certainly vary significantly. These foods are typically high in carbohydrates as well as sugar, and also are usually used as part of parties.
A pastry shop can be a retail company or a wholesale business. Many pastry shops operate both. The baker has to be certified to sell their products as well as have to have bakers’ profession papers. When she or he has actually gotten certification, she or he can focus on retail sales. Otherwise, they can concentrate on wholesale sales.
Cooking powder is just one of the most frequently made use of active ingredients in business bakeshops. It is a mix of acids and soda, with diluents added to make the powder extra secure. During the baking process, the cooking powder reacts with the batter and launches safe salts and carbon dioxide. The majority of business baking powders are double-acting, implying that they give off a percentage of carbon dioxide while the batter is being blended. When the batter reaches the right temperature, the carbon dioxide will be completely inert.
The process of cooking is most likely the earliest technique of cooking food as well as it is utilized to make pastry shop products. Many pastry shop products are made with flour and dish, which are derived from grains. These items are very important to human nutrition because they supply a selection of nutrients.
A pastry shop is a company that creates flour-based food and also markets it to customers. Some retail bakeshops are also coffee shops, offering coffee and tea. Bakeries create as well as sell a wide range of food. The most popular items are breads, pastries, and cakes. On top of that, some pastry shops likewise offer food solutions such as gelato and coffee.
A pastry shop’s earnings depends upon the period. Summer months are generally one of the most lucrative for a bakeshop. Holidays likewise produce active durations for pastry shops. The end of the year is additionally a superb time to open a bakery. A bakery’s sales will certainly also depend upon exactly how swiftly it can prepare the food needed to fulfill demand.
Most bakeshop products include flour, water, as well as leavening agents. Other typical active ingredients include eggs, butter, sugar, and also salt. Other active ingredients are used to alter the taste and structure of the ended up item. Some bakeshops are additionally regulated by federal governments, so it is important to adhere to the legislations of your house nation prior to starting a company.
A bakeshop can be a retail or wholesale procedure. Numerous pastry shops additionally integrate retail as well as wholesale operations. Picking the ideal room is important to the success of a bakery. See to it you have enough room for the kitchen area and the general public area. You should also create a contingency fund to cover unforeseen costs. This fund can cover any type of added costs, such as the cost of labor. Informative post
The background of cooking dates back to ancient Egypt. The Egyptians were the very first to develop the stove. The earliest stoves were round vessels made from baked Nile clay. These ovens had a tapered top and also a horizontal shelf-like dividers that divided the top as well as bottom parts. The upper area of the stove was the baking chamber, and also items of dough were placed inside through an opening on top.

domain: food