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Platelet Derived Extracellular Vesicles Promote in Vitro Gingival Wound Healing.
Miquel Antich-Rosselló, Marta Munar-Bestard, Maria Antònia Forteza-Genestra, and 4 more
posted 21 Sep, 2021
Purpose: Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates like platelet lysate (PL). In this article, the use of extracellular vesicles (EVs) derived from PL and their combination with hyaluronic acid biomaterials (HA) in wound healing was investigated.
Methods: EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro for biocompatibility (LDH activity and metabolic activity) and by a wound healing assay and gene expression analysis.
Results: EVs and EVs-HA treatments were biocompatible and showed an increase in wound healing compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed in gingival keratinocytes and gingival fibroblasts after the treatment with EVs.
Conclusion: EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet derived EVs emerge as promising candidates for oral regeneration with the possibility to combine them with biomaterials in order to enhance their application in clinical use.
Stem Cell & Developmental Cell Biology
Extracellular vesicles
gingival fibroblasts
gingival keratinocytes
Wound healing is a physiological process which aims at restoring the architecture and functionality of damaged tissue. The wound repair includes different phases that involve a complex cellular cascade: haemostasis, inflammation, proliferation and remodelling.1, 2 The regeneration process is particularly important in gingival tissue. Epithelial wound healing is essential to assure tooth and periodontal tissue protection against trauma or infection, especially after dental intervention.1, 3
Gingival epithelial regeneration involves different cell types such as gingival fibroblasts and gingival keratinocytes. On the one hand, gingival fibroblasts are the major constituents of periodontal connective tissue. They maintain gingival tissue integrity by producing and organizing different extracellular matrix components such as collagen and proteoglycans.4 On the other hand, gingival keratinocytes form the outer layer of gingival tissue and create a protective barrier against external agents.5 In wound healing processes, gingival keratinocytes rapidly move to the injured area in order to cover the exposed connective tissue.4
Current regenerative approaches pursue a rapid wound healing response and tissue regeneration.2 Among the different strategies under research, the use of platelet concentrates, such as Platelet Rich Plasma (PRP) or Platelet Lysate (PL), has emerged due to platelet major role during wound healing.2 Research has shown that platelet concentrates can ease the regeneration of dental or periodontal tissues.6 Moreover, some studies show that platelets induce a wound healing response in fibroblasts and keratinocytes.7–9 Although the wound healing response induced by platelets have traditionally been associated to the direct effects of secreted biomolecules such as growth factors, recently it has been suggested that Extracellular Vesicles (EVs) could also be involved in the wound healing response.10, 11
EVs are membranous particles, naturally released by cells, which play a key role in cell communication.12 They present a wide range of sizes, which extend from 30 nm up to 1000 nm. EVs have been classified according to their origin. Thus, exosomes derive from the endosomal system, while microvesicles are generated by plasmatic membrane budding.13, 14 EVs are enriched in protein, lipids and nucleic acids (DNA, mRNA, miRNA, tRNA), which allows the communication with the receptor cell.15 Even more, the lipid bilayer of EVs protects the biomolecules they contain, preventing their degradation once released to the extracellular space.12 Their specific cargo depends on the cellular origin and conditions, being immune cells, mesenchymal stem cells, body fluids and dental related cells the most studied for oral regenerative medicine.11 However, only few studies have explored the role of platelet-derived EVs on oral wound healing, although EVs have already been suggested as major effectors of platelet concentrate activity.16
Moreover, the combination of biomaterials with EVs may improve its tissue regeneration capability.17 Among the different biomaterials that exist, hyaluronic acid biomaterials have been widely studied in periodontal regeneration.18, 19 Hyaluronic acid is a component of the extracellular matrix able to modulate the regenerative process.20 Therefore, hyaluronic acid based biomaterials combined with platelet EVs could be good candidates for oral regenerative treatments.
Although platelet-derived EVs have been proposed as promoters of dermal fibroblast and keratinocyte migration and wound healing,21, 22 lack of evidence remains in the use of platelet EVs in gingival tissue. Thus, here we have evaluated the gingival regenerative effects of PL derived EVs on gingival keratinocytes and fibroblasts on its own as well as their combination with HA gels for periodontal applications.
Human PL preparation
Fresh buffy coats were obtained from the IdISBa Biobank and their use for the current project was approved by its Ethics Committee (IB 1995/12 BIO). Platelet concentrates were obtained as previously described.23 Briefly, six buffy coats were pulled and a platelet concentrate was obtained after a centrifugation at 650 × g for 10 min and leucocyte filtration, then, at least three freeze/thaw cycles at -80 ºC and 37 ºC were performed to lyse platelets. Cell debris were discarded by centrifugation at 5050 × g during 20 minutes at room temperature and supernatant was filtered by 40.0 µm pore size membrane (Sartorius, Goettingen, Germany). PL was stored at -20 ºC until use.
EVs isolation
PL was centrifuged at 1,500 × g for 15 min at 4 ºC. The supernatant was filtered through 0.8 µm porous membrane (Sartorius) for large cell debris elimination and then through 0.2 µm porous membrane (Sartorius). PL was centrifuged at 10,000 × g for 30 minutes at 4 ºC. The supernatant (5 ml) was loaded on a Sepharose CL-2B precast column (GE Healthcare, Pittsburg, PA, USA). AKTA purifier system coupled with a collector Frac 950 (GE Healthcare) was used to set a flow rate at 0.5 ml/min. EVs were eluted with PBS (Capricorn, Ebsdorfergrund, Germany) in 5 ml fractions, which were collected and characterized.
Platelet-derived EV were fixed in 2 % formaldehyde (Sigma-Aldrich) solution. Fixed EVs were set on copper Formvar-Carbon-coated grids (Ted Pella, Redding, California, USA) during 20 min and washed with PBS. Then, samples were incubated with 1 % glutaraldehyde (Sigma-Aldrich) for 5 minutes and washed with deionized water. The samples were stained for 1 minute with 2 % uranyl acetate (Electron Microscopy Sciences Hatfield, PA, USA) and washed with PBS. Images were taken using a TEM-H600 (Hitachi, Tokyo, Japan) at 50 kV
Protein quantification
Total protein amount of PL and EVs samples was quantified with BCA Protein Assay kit (Thermo Fisher, Waltham, MA, USA) following the manufacturer's instructions. Absorbance was read at λ = 562 nm (BioTek Instruments, Winooski, VT, USA).
PL and EVs samples were prepared with non-reducing Laemli loading buffer and denatured at 70 ºC. Samples were loaded in a 12 % gradient SDS-PAGE gel and proteins were separated by electrophoresis. The transfer was performed in humid conditions onto nitrocellulose membrane (GE Healthcare, Pittsburgh, PA, USA). A Ponceau S (Sigma-Aldrich) solution at 0.2 % v/v was used for total protein visualization.
After several washes, membranes were blocked and incubated with anti-human CD9 (Thermo Fisher) and anti-human CD63 (Abcam, Cambridge, UK) antibodies. Secondary antibody incubation was performed with HRP-coupled anti-mouse IgG (Thermo Fisher). Chemiluminescence was induced with Clarity Western ECL Substrate (Bio-Rad, Hercules, CA, USA) and visualized after exposure on autoradiographic films (GE Healthcare).
Nanoparticle Tracking Analysis (NTA)
Size distribution and particle concentration were analysed with Nanosight NS300 (Malvern Instruments, Malvern, UK). Samples were diluted (1:100) to a final volume of 1 ml and recorded with a laser at λ = 532 nm and an sCMOS camera. Data was analysed with NTA 3.2 Dev Build 3.2.16 Software. Once the number of particles was set, purity ratio was calculated by the formula described by Webber et al. (Eq. 1): 24
(Eq. 1)
Hydrogel preparations
Hydrogels based on hyaluronic acid (Bioibérica, F002103, Mw 800–1,200 kDa, Spain) were prepared at 2 % (w/v) by incubating overnight at 25°C. Thus, three different hydrogels were obtained: (1) Hyaluronic acid hydrogel (HA) for which PBS was used; (2) PL containing hyaluronic acid hydrogel (HA-PL) for which PBS containing PLwas used to obtain a final protein concentration of 0.167 µg/µl; (3) EVs containing hyaluronic acid hydrogel (HA-EVs), for which PBS containing EVs was used to obtain a final protein concentration of 0.167 µg/µl.
Equilibrium swelling ratio determination
The equilibrium swelling ratio (ESR) of the hydrogels was determined for HA, HA-PL and HA-EVs. 1 ml of each gel was incubated in PBS at 37°C for 3 h or 24 h. Then each gel was centrifuged at 16,000 × g for 15 min and the wet pellets were weighted (Ww); supernatants were stored at -80°C for release studies. Then, hydrogels pellets were frozen at -80°C and lyophilized during 72 h. Dried products were weighted again (Wd). The experiment was performed in triplicate. ESR values were determined using the following equation (Eq. 2):
EVs Release
The supernatants obtained after the 16,000 × g centrifugation step of the ESR determination were stored at -80°C. HA-EVs supernatants were analyzed by NTA to determine the number of particles released after 3 h or 24 h incubation during the swelling experiments.
Immortalized Human Gingival Keratinocytes (ihGK, Applied Biological Materials Inc., Richmond, BC, Canada) and Immortalized Human Gingival Fibroblasts-hTERT (ihGF, Applied Biological Materials Inc) were grown at 37 ℃ and 5 % CO2 atmosphere. The culture medium was renewed twice per week.
Keratinocytes were cultured on tissue culture flasks for sensitive adherent cells (Sarstedt, Germany) using Dulbecco's modified Eagle's medium (DMEM) without magnesium and calcium (Gibco, Grand Island, NY, US) and Ham's F12 (Biowest, Nuaille, France) in a 2:3 proportion, supplemented with 0.01 mg/ml insulin (Sigma-Aldrich), 0.4 ng/ml hydrocortisone (Sigma-Aldrich), 6.7 ng/ml selenium (Sigma-Aldrich), 0.01 µg/ml human epithelial growth factor (ThermoFisher Scientific, Waltham MA, USA), 1M HEPES-buffer (Biowest), 5.5 µg/ml transferrin (Sigma-Aldrich), 0.1 nM cholera toxin (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), 5 % (v/v) fetal bovine serum embryonic stem cells tested (FBS, Biowest) and 100 µg/ml penicillin and 100 µg/ml streptomycin (Biowest).
Fibroblasts were cultured in DMEM low glucose (Biowest) and Ham's F12 (Biowest) in a 2:1 proportion, supplemented with 10 % (v/v) FBS (Biowest) and 100 µg/ml penicillin, and 100 µg/ml streptomycin (Biowest).
Wound healing assay
Cells were seeded in 48-well plates at a density of 20,000 cells/well. When confluence was reached, cells were washed twice with PBS and the medium was replaced by medium without supplements and containing 1 % (v/v) EV's depleted FBS. EV's depletion was performed by ultracentrifugation at 120 000 × g for 18 h at 4 ℃. Three independent experiments were performed having triplicates in each (n = 9). All experiments were conducted in parallel and under the same conditions. Thus, PL and EVs in vitro studies, either alone or combined with HA, present the same control group to compare the different effects.
Wound was performed by scraping the cell monolayer with a 100 µl sterile pipette tip in a straight line to create a scratch, cell medium was renewed, and the treatments were applied according to the group. The different groups tested were Control (it had the medium renewed but no treatment), PL (5 µg of PL per well), EVs (5 µg of EVs per well), HA (30 µl of HA gel per well), HA-PL (30 µl of HA-PL gel per well, which contains 5 µg of PL) and HA-EVs (30 µl of HA-EVs gel per well, which contains 5 µg of EVs).
Images of the same areas were taken using a bright-field inverted microscope (Nikon Eclipse TS100) before treatment and 3 hours after healing in ihGK and 24 h after healing in ihGF. The images were analysed with ImajeJ software. The wound closure area (%) was defined as the difference of the scratch area before (Ai) and after the treatment (Af) and normalized by the initial scratch area (Eq. 3).
Cell Cytotoxicity
Cell media was collected after 3 h of treatment for ihGK and 24 h of treatment for ihGF. Lactate dehydrogenase (LDH) activity was measured with Cytotoxicity Detection kit (Roche Diagnostics, Manheim, Germany) following the manufacturer's instructions. For cytotoxicity calculation (Eq. 4), 0.1 % Triton-X100 treated wells were used as high control, 100 % cell death, while non-treated wells were used as low control, 0 % cell death.
Metabolic activity
Total metabolic activity was evaluated after 3 h of treatment for ihGK and 24 h of treatment for ihGF. Presto Blue reagent (Life Technologies, Carlsbad, CA) was used during 1 h of reagent incubation time following manufacturer's protocol. Non-treated cells were set as 100 %.
Gene Expression by Real-Time RT-PCR
RNA was isolated using RNAzol® RT (Molecular Research Center, Cincinnati, OH, USA). RNA concentration was quantified with NanoDrop spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA) and normalized for reverse transcription to cDNA using High Capacity RNA-to-cDNA kit (Applied Biosystems, Foster City, CA, USA).
Real-time PCR was performed for three reference genes and different target genes25 (Table 1) using the Lightcycler 480 thermocycler (Roche Diagnostics) and SYBR green detection. Each reaction well contained Lightcycler 480 SYBR Green I Master (Roche Diagnostics), 0.5 µM of each primer, the sense and the antisense, and 3 µl of the cDNA dilution in a final volume of 10 µl. The amplification program started with 5 min pre-incubation step for cDNA template denaturation at 95°C, followed by 45 cycles consisting of 10 s steps of denaturation at 95 ºC, annealing at 60°C and an extension at 72 ºC. Fluorescence was measured at 72°C after each cycle.
Genes and their sense (S) and antisense (A) primer sequences used in the real-time RT-PCR.
Primer sequence (5'-3')
Product size (bp)
Collagen I α1 (COL1A1)
S: CCTGACGCACGGCCAAGAGG
A: GGCAGGGCTCGGGTTTCCAC
NM_000088.3
Decorin (DCN)
S: ATCTCAGCTTTGAGGGCTCC
A: GCCTCTCTGTTGAAACGGTC
Matrix metalloproteinase- 1 (MMP-1)
S: TGTCAGGGGAGATCATCGGGAC
A: TGGCCGAGTTATGAGCTGCA
Tissue inhibitor of metalloproteinases 1 (TIMP-1)
S: TTCCGACCTCGTCATCAGGG
A: TAGACGAACCGGATGTCAGC
α-Smooth muscle actin 2 (ACTA2)
S: TAAGACGGGAATCCTGTGAAGC
A: TGTCCCATTCCCACCATCAC
NM_001141945.1
Transforming growth factor-β1 (TGF-B)
S: TGTCACCGGAGTTGTGCGGC
A: GGCCGGTAGTGAACCCGTTG
Endothelin-1 (EDN)
S: ACGGCGGGGAGAAACCCACT
A: ACGGAACAACGTGCTCGGGA
Fibronectin (FN1)
S: CGGAGAGACAGGAGGAAATAGCCCT
A: TTGCTGCTTGCGGGGCTGTC
Vimentin (VIM)
S: GGCCGCCTGCAGGATGAGATTC
A: CAGAGAAATCCTGCTCTCCTCGC
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
S: TGCACCACCAACTGCTTAGC
A: AAGGGACTTCCTGTAACAA
Beta-Actin (ACTBL2)
S: CTGGAACGGTGAAGGTGACA
18S ribosomal RNA (18S rRNA)
S: GTAACCCGTTGAACCCCATT
A: CCATCCAATCGGTAGTAGCG
NR_146156.1
Notes: Abbreviations: 18S rRNA, 18S ribosomal RNA; A, Sequence of antisense; ACTA2, α-Smooth muscle actin 2; ACTBL2, Beta-actin; bp, Base pairs; COL1A1, Collagen I α1; DCN, Decorin; END, Endothelin-1; ESR, Equilibrium swelling ratio; EVs, Extracellular vesicles; FBS, Fetal bovin serum embryonic stem cells tested; FN1, Fibronectin; GAPDH, Glyceraldehyde-3-phosphate dehydrogenase; HA, Hyaluronic acid; HA-EVs, Hyaluronic acid gel containing EVs; HA-PL, Hyaluronic acid gel containing PL; ihGF, Immortalized Human Gingival Fibroblasts-hTERT; ihGK, Immortalized Human Gingival Keratinocytes; LDH, Lactate dehydrogenase; MMP-1, Matrix metalloproteinase-1; NTA, Nanoparticle Tracking Analysis; PBS, Dulbecco's Phosphate Buffered Saline; PL, Platelet Lysate; PRP, Platelet Rich Plasma; S, Sequence of sense; TEM, Transmission electron microscopy; TGF-B1, Transforming growth factor-β1; TIMP-1, Tissue inhibitor of metalloproteinase-1; VIM, Vimentin.
For relative quantification, standard curves were constructed for all genes. The Second Derivative Maximum Method provided by the LightCycler480® analysis software version 1.5 (Roche Diagnostics) was used to calculate the amount of each gene from the crossing point data. Reference genes were used to normalize the target genes expression levels and changes were compared to Control group, which was set to 100 %.
For statistical analysis, SPSS program version 25.0 (SPSS Inc., Chicago, IL, USA) and GraphPad Prism (version 7, La Jolla, CA, USA) were used. All data are presented as mean values ± SEM. Normality was determined using Shapiro-Wilk test. For parametric distributed groups, one-way ANOVA was performed to identify differences between the groups while Levene's test for variance homogeneity. When homogeneities were comparable DMS post-hoc was used; if they were not, Games-Howell test was used. Differences between non-parametric groups were checked with Kruskal-Wallis test. Results were considered statistically significant at p < 0.05.
Platelet-derived EVs characterization
EV isolation and characterization were performed according to ISEV recommendations (Fig. 1).26 Wide-field TEM images were used to confirm the presence of vesicles bodies (Fig. 1A). Then, EVs were analysed by NTA showing a heterogeneous population with a median size ± standard error of 106.0 ± 2.5 nm (Fig. 1B). Moreover, through the particle content (3.8·1011 particles/ml) and the protein concentration (167 µg/ml) the purity ratio of 2.3·109 particles/µg was obtained.24 Specific EV markers (CD9 and CD63) were confirmed by western blot (Fig. 1C).
PL and EVs effect on ihGK and ihGF
To evaluate the effects of PL and the PL-derived EVs, an in vitro functional study was performed using ihGK and ihGF (Fig. 2). The wound closure area, metabolic activity and LDH activity were determined 3 h after the wound for ihGK (Fig. 2A, 2B, 2C, 2D) and 24 h after the wound for ihGF (Fig. 2E, 2F, 2G, 2H). In both cell lines, EVs presented a statistically significant increase of wound closure compared to the Control (Fig. 2A, 2B, 2E, 2F). Moreover, PL and EVs treated fibroblasts showed higher metabolic activity than the Control 24h after wound and treatment (Fig. 2G). No cytotoxicity was found for any of the groups (Fig. 2D and 2H).
Additionally, mRNA expression levels for different marker genes were evaluated for both cell lines (Fig. 3). No differences were observed in FN1 and VIM expression for ihGK (Fig. 3A). However, ihGF treated with EVs presented increased levels for all the genes but for DCN, reaching significance for COL1A1, MMP1 compared to control and PL and TGFβ compared to control (Fig. 3B, 3C).
HA gels characterization
The equilibrium swelling ratio was determined for the different HA gels (Fig. 4A). As expected, higher ESR were observed for for all gel formulations at 24 h than at 3 h. However, HA-PL and HA-EVs presented lower ratios than HA in both time points, reaching statistical significance the ESR for HA EVs at 3 h compared to control. Additionally, the EVs release over time for HA-EVs group was determined by NTA (Fig. 4B). HA-EVs presented a sustained release over the time period evaluated, with higher amount of particle released at 24 h than at 3 h (Fig. 4B).
In vitro effect of the different HA gels.
The different HA gels were evaluated in an in vitro functional study (Fig. 5). For ihGK, cell closure area was significantly higher for HA-PL and HA-EVs after 3 h of treatment compared to the Control (Fig. 5A, 5B). Moreover, an increase in metabolic activity was observed for both groups, HA-PL and HA-EVs (Fig. 5C). No cytotoxicity was induced by any of the treatment groups in ihGK cells (Fig. 5D). In addition, ihGF treated with HA-PL or HA-EVs also showed increased wound closure area compared to the control. Even more, HA-EVs treated cells presented a significantly more closed wound compared to the HA treated group (Fig. 5E, 5F). Higher metabolic activity in ihGF was observed for all treated groups compared to the Control one (Fig. 5G). Furthermore, any of the treatments induced any cytotoxicity as measured by LDH activity released to the cell culture media (Fig. 5H).
Gene expression was determined for cells treated with the different HA formulated gels (Fig. 6). ihGK treated with HA-EVs presented lower expression levels of VIM compared to the Control group and the HA treated group, while no differences were observed for FN1 expression levels (Fig. 6A). Furthermore, ihGF treated with HA-EVs showed higher expression levels of COL1A1 and MMP1 mRNA and lower levels of DCN mRNA compared to all other groups. HA and HA-PL presented a lower level of COL1A1 and DCN expression compared to the Control, while HA-PL also showed higher MMP1 expression levels compared to the Control (Fig. 6B). As for the gene expression levels of TGFβ, HA treated cells showed lower expression levels than the Control and the HA-EVs groups, while HA-PL showed lower TGFβ expression levels compared to the Control. Finally, HA-EVs showed decreased expression levels of ACTA than the Control and HA groups, while presented increased EDN levels compared to all the other groups (Fig. 6C).
Gingival epithelial wound healing and regeneration is essential for tooth and periodontal tissue protection. In the last decade, platelet concentrates have increasingly been used for dental and periodontal tissue regeneration.2,6 Here, we propose the use of platelet derived Extracellular Vesicles for gingival regeneration, proving its in vitro regenerative effect on gingival keratinocytes and fibroblasts, both, on its own and combined with hyaluronic acid gels.
In this study, we demonstrate that PL-derived EVs increase wound closure both in gingival keratinocytes and fibroblasts. Previous studies had already proven the regenerative effect of platelets, but these effects were mainly attributed to the released proteins, like growth factors.7–9 Our results, show higher effects for the EVs groups, suggesting higher healing effects for EVs than for the secreted proteins. Therefore, platelet-derived EVs could be an effective substitute for platelet concentrates in regenerative medicine. A highlight of this work is the high purity of the used EVs, since a purification by size exclusion chromatography was performed and only the fractions rich on EVs and poor on protein content were used. Isolated EVs were characterized according to the ISEV recommendations,26 presenting an heterogeneous population of small EVs positive in CD9 and CD63.
Additionally, in this study, PL and the EVs have been combined with HA gels. The HA gels formulation enables a topical or intradermal application which would allow a future clinical use and industrial development for oral interventions.27 HA is a highly porous and hydrophilic biomaterial which allows a proper interaction with the physiological environment.28 The ESR values obtained are relatively high, but they decrease when HA is combined with PL or EVs, probably due to their lipophilic nature. In vitro studies show that wound healing response was improved in both gingival cell types by HA treatment as expected from previous reports.27 What is more, this effect was enhanced by the combination of HA with PL or EVs, showing HA-EVs the better result in agreement with the effects observed for non-formulated EVs.
After a wound, a series of sequential responses are activated to reconstitute the damaged tissues. On one hand, diverse cell types must migrate, proliferate and differentiate; on the other hand, these cells must secrete and organize several molecules of the extracellular matrix, including collagens, fibronectin and proteoglycans.29 These process are governed by a complex array of signals and involve the regulation of the expression of numerous genes.30 For instance, fibronectin is a component of the extracellular matrix that induces wound healing and cell adhesion, migration and differentiation,31 while VIM is associated with the cytoskeleton reorganization and induce cell proliferation and migration during the regeneration process in keratinocytes.32 The expression of these two genes in ihGK 3 hours after wounding and treatment with PL or EVs, was evaluated finding no significant differences. However, HA combined treatments present a decreasing tendency in VIM and FN1 on ihGK, probably because hyaluronic acid is an extracellular matrix glycosaminoglycan involved in cell matrix formation,33 therefore neither VIM nor FN1 production are required when HA is supplied to the cells. Similar effects are observed towards COL1A1 and DCN expression in ihGF 24 hours after wounding and treatment. COL1A1 and DCN are components of the extracellular matrix,34 thus HA treated group presented a decrease in both, corroborating the direct functionality associated to the hyaluronic acid itself. In addition, the decrease of DCN can be associated to a higher cellular proliferation as higher levels of DCN expression are related to quiescence and growth inhibition.35
In addition, COL1A1 is a component of the extracellular matrix while MMP1 is an enzyme that degrade collagen proteins.36 When EVs were used as treatment for their own or combined with HA, an increase on COL1A1 and MMP1 mRNA levels was observed. The expression of both proteins simultaneously suggests a remodeling process of the extracellular matrix which promotes a scarless and not fibrotic repair.37 Moreover, EVs treated groups presented higher levels of TGF-β1 or END. These are proteins that promote COL1A1 production and enhance cell proliferation.36 However, TGF-β1 and EDN are also responsible for an increase in ACTA levels which can lead to myofibroblast differentiation.38 In our study, ACTA presents a non-significant increasing tendency in PL or EV treated cells, while it decreases for HA-PL and HA-EVs groups. Therefore, HA-EV treatment would reduce the myofibrotic regeneration risk while maintaining the regenerative properties of EVs. As regards to the biocompatibility, HA combined treatments presented low levels of LDH activity, under the 30 %, which is set as the maximum accepted cytotoxicity value for medical devices according to ISO-10993:5, and improved levels of metabolic activity, indicating both the safety of the treatment.
Overall, EVs achieve improved regenerative effects compared to the direct use of PL. These effects may be caused due to different properties of EVs. On the one hand, despite having normalized the protein amount, EVs may contain other functional biomolecules such as nucleic acids or active lipids.39 On the other hand, EVs protect the cargo from degradation and enhance their delivery to the target cells.40 Therefore, PL seems to present an increment of wound closure, probably caused mainly by proteins, while EVs have higher effects thanks to the encapsulation of these proteins and other active biomolecules and an enhanced delivery.41,42 In fact, different miRNAs contained in EVs derived from different cell types have been suggested to modulate wound healing.43,44 Nevertheless, further research should be performed to confirm the presence of these biomolecules in platelet derived EVs and their mechanism of action.
In conclusion, platelet derived EVs present a good biocompatibility in vitro and a regenerative effect on both gingival keratinocytes and fibroblasts. Moreover, EVs induce changes on expression levels of genes associated with the remodeling process during wound healing. Finally, EVs can be combined with HA gels to produce more applicable treatments while preserving their regenerative effects.
Ethics approval and consent to participate: Not applicable
Consent for publication: Not applicable
Availability of data and material: Data available on request due to restrictions. The data present in this study are available on request from the corresponding authors.
Funding: This research was funded by Instituto de Salud Carlos III, co-funded by the ESF European Social Fund and the ERDF European Regional Development Fund (contract to J.M.R; MS16/00124 and M.M.B; FI18/00104 and project PI17/01605) and the Direcció General d'Investigació, Conselleria d'Investigació, Govern Balear (contract to M.A.R.; FPI/2046/2017) and the Institut d'Investigació Sanitària de les Illes Balears (contract to M.A.F.G; ITS2018-002-TALENT PLUS JUNIOR PROGRAM, JUNIOR18/01).
Authors' contributions: M.A.R., M.M.B., M.M. and J.M.R. contributed to the conception and design. J.C. and A.G. provided hUC-MSCs and PL. M.A.F.G. and M.A.R. produced EVs samples. M.M.B and M.A.R. acquired and analysed data in the in vitro study. M.A.R., M.M.B, M.M. and J.M.R. contributed to interpretation of the results. M.A.R. wrote the main manuscript text and M.A.R., M.M.B, M.A.F.G., J.C., A.G., M.M. and J.M.R reviewed the manuscript.
Acknowledgments: The authors thank SCT-UIB for access to their facilities.
Competing Interests: Authors declare no conflict of interest
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Torreggiani, E., Perut, F., Roncuzzi, L., Zini, N., Baglìo, S.R., Baldini, N. Exosomes: novel effectors of human platelet lysate activity. European cells & materials. 28, 137–51; discussion 151, doi: 10.22203/ecm.v028a11 (2014).
Brennan, M., Layrolle, P., Mooney, D.J. Biomaterials Functionalized with MSC Secreted Extracellular Vesicles and Soluble Factors for Tissue Regeneration. Advanced Functional Materials. 30 (37), doi: 10.1002/adfm.201909125 (2020).
Fujioka-Kobayashi, M. et al. In vitro effects of hyaluronic acid on human periodontal ligament cells. BMC Oral Health. 17 (1), 1–12, doi: 10.1186/s12903-017-0341-1 (2017).
Babo, P.S. et al. Platelet Lysate-Loaded Photocrosslinkable Hyaluronic Acid Hydrogels for Periodontal Endogenous Regenerative Technology. ACS Biomaterials Science and Engineering. 3 (7), 1359–1369, doi: 10.1021/acsbiomaterials.6b00508 (2017).
Graça, M.F.P., Miguel, S.P., Cabral, C.S.D., Correia, I.J. Hyaluronic acid—Based wound dressings: A review. Carbohydrate Polymers. 241 (February), 116364, doi: 10.1016/j.carbpol.2020.116364 (2020).
Guo, S.-C., Tao, S.-C., Yin, W.-J., Qi, X., Yuan, T., Zhang, C.-Q. Exosomes derived from platelet-rich plasma promote the re-epithelization of chronic cutaneous wounds via activation of YAP in a diabetic rat model. Theranostics. 7 (1), 81–96, doi: 10.7150/thno.16803 (2017).
Lovisolo, F., Carton, F., Gino, S., Migliario, M., Renò, F. Platelet rich plasma-derived microvesicles increased in vitro wound healing. European Review for Medical and Pharmacological Sciences. 24 (18), 9658–9664, doi: 10.26355/eurrev_202009_23055 (2020).
Antich-Rosselló, M., Forteza-Genestra, M.A., Calvo, J., Gayà, A., Monjo, M., Ramis, J.M. Platelet-derived extracellular vesicles promote osteoinduction of mesenchymal stromal cells. Bone and Joint Research. 9 (10), 667–674, doi: 10.1302/2046-3758.910.BJR-2020-0111.R2 (2020).
Webber, J., Clayton, A. How pure are your vesicles? Journal of Extracellular Vesicles. 2 (1), 1–6, doi: 10.3402/jev.v2i0.19861 (2013).
Gómez-Florit, M. et al. Human gingival fibroblasts function is stimulated on machined hydrided titanium zirconium dental implants. Journal of Dentistry. 42 (1), 30–38, doi: 10.1016/j.jdent.2013.11.003 (2014).
Théry, C. et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. Journal of extracellular vesicles. 7 (1), 1535750, doi: 10.1080/20013078.2018.1535750 (2018).


text:
Avaleht > Adam Cullen
Adam Cullen (1986) is a poet and translator of Estonian prose, poetry, theater, and children's literature into English. He has been a member of the Estonian Writers' Union since 2014 and is on the editorial board of the Estonian Literary Magazine. Originally from Minnesota, he has lived in Estonia for over a decade.
Translating children's literature kindles a state of lightness. It is a dance in sudden spring drizzle; a gleeful dash through shifting shadows beneath a spring-green forest canopy. Children's literature invigorates and inspires me for my work on denser prose and poetry. Above all, I enjoy writing that is rich in lively dialogue, deep (yet buoyant) in meaning, and far-reaching but still honed in terms of subject matter. Naturally, a book's humorous and intricate illustrations can be just as important as the writing itself, and may serve as signs that point out pathways I might otherwise have missed if I'd concentrated on the language alone.
I've found that Estonian children's authors and illustrators rarely make the mistake of underestimating young readers – they challenge, nurture, and prompt children to imagine and explore every magical nook of this dazzling world while assuring them they can always rely on the support of others, no matter how unexpected those sources of care may be.
Eno Raud, The Gothamites. Elsewhere Editions 2019
Currently translating
Andrus Kivirähk, Oscar and the Things. Emma Press 2020
Piret Raud, Trööömmmpffff or Eli's Voice. Restless Books 2020
2016 Nominee for the Cultural Endowment of Estonia's Award for Translated Literature (Mihkel Mutt, The Inner Immigrant; Rein Raud, The Reconstruction)
2015 Nominee for the Cultural Endowment of Estonia's Award for Translated Literature (Mihkel Mutt, The Cavemen Chronicle)
2014 Nominee for the Cultural Endowment of Estonia's Award for Translated Literature (Tõnu Õnnepalu, Radio)
chakcha(at)gmail.com
Photo: Dmitri Kotjuh

redpajama_set_name: RedPajamaCommonCrawl


Hope you will be back in commission soon.
Oh dear – hope you are ok! We all have to disappear at times. Come back soon.
Hope you didn't get caught in an avalanche of paper!
Get better soon!! We miss you!!
Oh nooooo!! Better wear thick gloves next time!!
Aww, sorry to hear that. I know migraines can be horrific!
I would love a pic!
I hope you feel better soon. I wanted to let you know that I have progressed some on my paper… instead of just looking at the piles of paper… this morning I actually moved one stack on top of another stack! I feel that is major progress. Ha ha ha!
Hope you are feeling better. Pain of any sort should be banned – but I guess it does serve a purpose, except with headaches, why make our head hurt?!?


text:
Coalition Calls Federal Plan for National Inquiry too Vague
(Coast Salish Territory/Vancouver, B.C. – August 3, 2016) Today a Coalition on Missing and Murdered Indigenous Women and Girls responded to the Federal Government's announcement of the national Inquiry. The Federal Government has released the names of the Commissioners and the Terms of Reference (TOR) for the Inquiry.
The Coalition continues to demand justice for missing and murdered Indigenous women and girls. We have the following three main concerns with today's announcement:
1. The Terms of Reference do not include a robust and binding commitment from the provinces and territories to participating in the Inquiry or implementing any recommendations that may emerge. The participation of the provinces has been described as supportive but there is no legally binding mechanism to compel the production of documents or other evidence. Without proper disclosure, the national inquiry cannot do its work.
2. Despite repeated calls for the explicit inclusion of policing in the Inquiry's scope, policing is not mentioned in the terms of reference. The Coalition does not have confidence that the role of policing will be appropriately addressed.
3. The Terms of Reference fail to provide families with redress for inadequate police investigations
into their missing or murdered relatives. The TOR only provides the Commission with the authority to send families back to the local authorities whose very conduct is being questioned, providing no confidence that allegations of police misconduct will be meaningfully addressed. Decades of ceaseless work by women across the country calling for solutions grounded in Indigenous laws, has culminated in this Inquiry. The Coalition is pleased that the federal government has mandated the Inquiry to examine systemic causes of violence and has taken into account the needs of vulnerable communities and witnesses. However, given the importance and the promise of the Inquiry, we are deeply concerned about the gaps in the framework that stand to undermine the good intentions that have led to the formation of the Inquiry.
The Coalition calls upon the federal, provincial and territorial governments to address the critical gaps in the Terms of Reference and make a legally binding commitment to addressing violence against Indigenous women and girls. Without concrete commitments from the provincial and federal governments to systemic change, the Inquiry risks being more about words than action.
Amnesty International Canada
Atira Housing
Battered Women's Support Services
BC Assembly of First Nations
BC Civil Liberties Association
Butterflies in Spirit
Carrier Sekani Family Services
Carrier Sekani Tribal Council, Tribal Chief Terry Teegee
Downtown Eastside Women's Centre
February 14th Women's Memorial March Committee
First Nations Summit
First United Church
Lookout Emergency Aid Society
Michele Pineault, family member
Neskonlith Indian Band, Kukpi7
PHS Community Services Society
Pivot Legal Society
Poverty and Human Rights Centre
RainCity Housing
Spirit of the Children Society
Union of B.C. Indian Chiefs
Vancouver Council of Women
Vancouver Rape Relief and Women's Shelter
West Coast LEAF
WISH Drop-in Centre Society
Women Against Violence Against Women
The Coalition on Missing and Murdered Indigenous Women and Girls initially came together in response to the Missing Women Commission of Inquiry in British Columbia overseen by Commissioner Wally Oppal. The Coalition membership includes representation from families, survivors, Indigenous organizations, front-line service organizations, feminist and women's organizations, legal advocacy sector, faith-based groups, and provincial organizations. Our breadth of representation and our experience with the Oppal Commission of Inquiry make the Coalition well-positioned to respond to and inform the national inquiry.


OAKLAND — A marine consultant who bought a flag that draped a Korean War veteran's casket at a local flea market has located the veteran's family — ending a months-long search to return it to the rightful owners.
After a story about the search ran in Tuesday's Tribune, Derek Conant said his voicemail and e-mail account were filled with positive messages in reaction to the story.
One anonymous, female caller furnished a last name and a local phone number.
In December, Conant, who spends most of his time working on tugboats between Oakland and San Rafael, stumbled across the flag in a triangular box at a flea market at Laney College.
He recognized what it was and bought it for $12. Conant said he bought the flag just to get it off the ground and save it from the wet weather. He also wanted to honor the man behind the flag who served his country by locating his family or someone who knew him, he said.
The box had a bronze nameplate with Arnold's name, and his year of birth, 1933, and the year he died, 1999.
His weeks of unsuccessful Internet and telephone searches to find the veteran's family led him to call the Oakland Tribune to ask about an obituary. No obituary could be found, but the Tribune ran a story about the quest. The messages started coming.
Messages he received, including some from Hawaii, ranged anywhere from "You are a heck of a guy! We're behind you all the way" to thanking him for his patriotism, some offering to buy the flag box from him and others suggesting to donate it, Conant said.
But one anonymous message from a woman gave Conant a last name, Miranda, and a phone number, which later turned out to belong to Rose Arnold's brother, Tom Miranda, in San Leandro. He gave Conant his sister's number.
When Arnold heard the whole story, she was shocked.
"I had no idea (the flag and its box) was not in my storage," Arnold said.
Arnold was more surprised when she learned about Conant's efforts to reunite the memento with family members.
Here's what she thinks happened to the flag: When her husband died, Arnold moved from Oakland to Las Vegas with her son.
In 2005, when her son moved back to Oakland, Arnold said he must have accidentally loaded the flag-box in his U-Haul truck.
He apparently kept his items, including the flag, in a storage unit. But he couldn't keep up the storage fees and contents of the unit were auctioned off, she said.
Neither Arnold, nor her son, knew the flag was in the pile.
"My instincts were right on the mark," Conant said.
When Conant first saw the flag-box and the bronze name plate, he said he was convinced it did not belong at the flea market.
Arnold had been handed the folded flag at the San Joaquin Valley National Cemetery, where her husband was buried, she said.
Rose and James Arnold did not have children together. The war veteran has five children from a previous marriage living scattered around the nation, including one in Oakland, and Rose Arnold has two, she said.
Arnold intends to come to California next week on a visit and plans are being made to return the flag to her.
Friends and family members in the Bay Area have offered to receive the box on her behalf, but she wanted Conant to directly hand it to her, she said.
"If I had gone through my belongings and if I had not found my flag, I would have been upset," she said.
According to her e-mail message, Arnold hopes to find the anonymous person who provided her brother's number, so she can thank her too.
Conant is also looking forward to meeting with Arnold.
"It is wonderful I got closure to this," he said.


Dwight R. Messimer is a U.S. Army veteran and former lecturer in history at California State University San Jose. His most recent book is The Baltimore Sabotage Cell: German Agents, American Traitors and the U-boat Deutschland during World War I. He resides in Northern California.


text:
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redpajama_set_name: RedPajamaGithub


Just a quick one today concerning the baby pig dish. Twice every week the kitchen receives a suckling pig. Somewhere between 20-30 lbs. They hang it whole in their walk-in for two days - this dries and slightly tenderizes the proteins. The open air drying process also lends a hand to the final flavor of the pork.
Each and every piece of the pig is used. The head is simmered gently, meat is picked and pressed into a terrine. Later, it is sliced, breaded, and deep fried. The loins, hams, shoulders, and rack are all deboned, brined, and circulated for 90 minutes. A simple sear and baste and it hits the plate with a beautiful medium pink hue. The belly is cured and circulated for 16 hours. Any trim is processed into a sausage that is seared just before the plate up. Lastly, the bones go into a stock that is later reduced and fortified to create the final sauce.


There are surprisingly few good spring gardens around. So often, a 'spring garden' consists of drifts of daffodils let loose in grass. Very nice, but so easy to do, it hardly counts as gardening once its done, just mowing at the right time. Then there are that small number of gardens where there are thousands of tulips, which, given that tulips are generally one-year wonders, means that the cost, not only of tulips but of putting them in, limits this form of horticultural expression to the very wealthy. And in times of austerity, such flaunting of wealth is perhaps a little rude (I remember Prince Charles planting tulips in meadow grass once, I don't suppose even his budget allows a repeat of that gesture; if it is, I look forward to the appropriate peasants' revolt throwing down the gates to Highgrove).
So, I was very glad to finally get round to visiting Olive Mason's garden in Worcestershire a few weeks ago, a garden well and truly full of the joys of spring. She opens for her snowdrops in Feb (for the NGS) and hopefully one day might do later too. A medium-sized and very intensely planted garden clearly relies on clipped box and perennials for its impact most of the year, but amongst these are a great number of bulbs and spring-flowering perennials. For many years, Olive has looked after a severely disabled son at home, and making and maintaining a garden has been something which she could do, whilst caring for him. Clearly, she spends a lot of time gardening, but at the same time it is an example of what I call intelligent management – a great deal of self-seeding and spreading is allowed or even encouraged, and so weeding and other plant control is very much like sub-editing – dealing with the detail.
Olive's plant management encourages what is key to making a good spring garden which then turns into a good summer garden – integration. Most spring-performing plants have a different annual cycle to summer-performing; they grow at lower temperatures, and by the time they are dying back, the summer species are coming into growth. Their growth cycles are complementary, so it is possible to shoehorn both into almost the same space. Summer perennials, and bulbs, and spring-flowering but summer-dormant perennials can be cheek by jowl. Those which are not summer dormant, such as many primulas, cannot be quite so close, but will still tolerate summer shading by taller perennials. Given this neat complementarity, it is possible to think of having two, completely different, planting schemes: spring and summer/autumn. A summer planting scheme, as it uses larger plants with a longer period of growth, can be the 'main' one, and then the spring planting can be thought of as another layer, dropped on top. The only real points of interaction will be perennial clumps emerging amongst the bulbs and spring perennials; for the most part these will just be blobs of background green, but a few with dark foliage (peonies and Anthriscus sylvestris 'Ravenswing') do look very nice alongside the predominantly blues and yellows of spring.
Primula wanda types seed attractively, low enough to fit in a variety of places.
...even seeding beneath a box hedge.
Visiting Dial Park made me realise how much harder I must work at this. We have plenty of Barnhaven primulas and Primula elatior which is simply the toughest primula out, some daffodils (not as many as I'd like, as so many have rotted off in our wet ground) and a few scatterings of scillas and muscari. Primula wanda will be on the seed shopping list this summer, nicely coloured very short and very spready primulas for seeding around the front of borders and amongst perennials. And I must try harder with corydalis – C. solida clearly seeds well.
Thinking of a garden as a two layer project: spring and summer, seems a good thing to keep in mind, one of those ways of looking, which can be very useful.
Good integration between bulbs and summer perennials in this and following pictures.
Council contractor planting team April 2006. Good people. They deserve better.
Margaret Thatcher had an impact on everything that happened in Britain, and in a way still does. She was not known to have any particular interest in gardening or landscape, but her effect on Britain's public urban landscapes was enormous, and arguably, very indirectly, on a particular era of private garden design too (but more of that another time). Here I'd like to reflect on what she set in motion has affected our public parks and other planted greenspace. It has important lessons.
Those who are not British, were too young at the time or indeed who benefited from her rule (1979-1990) have no idea of the sheer loathing, the deep and visceral hatred she inspired in those at the sharp end of her policies. Change is always difficult (and an awful lot in Britain in 1979, when she was elected, was in desperate need of change), but it was her seeming contempt for the whole idea of community which caused so much suffering and anger. For everyone who admired her leadership and policies there was at least another who found her arrogant to the point of being dictatorial, viciously divisive and intolerant (of opposition and of minorities). After a decade or so of privatising state assets (sometimes to our benefit, but more usually to the benefit of those who bought the underpriced shares) she turned her attention to our public parks – which are a community resource if they are anything, and it is an irony that the privatisation of their management occurred in the very last year of her rule.
In 1990, Compulsory Competitive Tendering was introduced, so that the management of parks and greenspace had to be put put to companies who bid for the services – the most competitive (i.e. the cheapest) winning the contract. The impact was "horrendous" in the words of Ivor Stokes, who had a career in Swansea parks and ended up as Curator of Swansea Botanical Complex. "A lot of young people used to get apprenticeships with the parks... that all went" he recalls. Park services, like the railways, and to some extent the other state-owned industries had something which cannot be expressed in monetary terms – a culture, which passes on knowledge from one generation of the workforce to another, but also shares that knowledge within the profession. Park services used to be run "very hierarchically" says Ivor, but everyone would have horticultural training and people could work their way up. The new system involved contractor companies providing services, with managers who knew nothing about horticulture and unskilled staff, who only knew how to mow and cut, "you had time and motion people assessing jobs, and staff decision making replaced by computer-driven programmes instead... its week ten, so we can to cut all the shrubs now, just the other day I saw some forsythia which isn't going to flower because it had been cut back by someone on schedule". Horticulture cannot be managed with this kind of dogmatic inflexibility.
Not surprisingly, standards plummeted, young people lost the opportunity to move into a healthy and interesting (if badly paid) profession where there was a real opportunity for progression, and many public spaces simply got duller, less well managed and often more dangerous. I remember in Bristol staff employed by one company (contracted to manage parks) would throw rubbish into the playgrounds (managed by another company), who would throw it back again. It summed up the shambles of the whole exercise. My conversations with parks staff always revealed deep frustration at the lack of opportunity to use and develop their skills, at the way management failed to respect them, and how everything was dominated by the need to cut costs.
Although quality plummeted, the very best parks were rescued, thanks to Baroness Trumpington who introduced a bill in the House of Lords to keep parks of botanical and historical interest in public ownership – hence Ivor's job at Swansea. It was a rare victory in a decade of government assets (which we all own and have some political control over) being sold off, and workforces being made redundant or de-skilled.
The decline in quality in public greenspace, the loss of jobs, the loss of skills and accumulated knowledge was symptomatic of what happened in the Thatcher years. The well-off generally did not notice, but for people who lived in working class communities, who saw factories and mines shut, public services reduced, public housing sold off and not replaced – what they saw was a heartless contemptuous vandalism. Contracting out in the parks was like so much else done in these years, not done to improve quality but done for reasons of political ideology and to save money.
Not that pre-1990 parks and greenspace was a golden age. Not at all. (Small 'c') conservatism, funding cuts and a rather dreary lack of imagination meant that there had been little in the way of innovation for years. I came across a rather interesting survival of this old mentality a few weeks ago. Shouldn't say where yet, until I do more research, but it is a council-run botanical garden that was in danger of sliding into being yet another mediocre park. The council wanted shot of it, sold it to a Community Interest Company who then engaged a business consultant who was passionate about the place. The chap in question has now invested a considerable sum of his own money into the place in order to turn it around. The whole thing was like one of those TV programmes where the business whizz kid goes into to rescue the failing country house or whatever. The entrepreneur described to me the great difficulty of finding out what went on, who did what, when they did it, or to get staff to change age-old working practices, such as working at weekends (when most of the public visit) etc. etc. It was a good example of 'the bad old days' that has given local government in Britain a bad name, and of course helped open the door to Thatcherism. The garden in question will be very interesting to watch. First signs are very hopeful - and again, more later.
There has to be a better way – to modernise the way public space is managed yet maintain flexibility. Germany, Sweden, Holland, France, all have much better parks and greenspace, although all very different. There are different emphases on public and private ownership, but what actually counts is a political commitment to continually improving quality, openness to innovation and a career path for staff.


text:
Q: Query to get all primary keys in a database Is this the best way to - Get a List of all Primary Keys in a Database - or is there something better?
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redpajama_set_name: RedPajamaStackExchange


text:
Home / Law / United States / Trump Impeachment Process
Trump's China Policy Pages: 4 (1256 words)
Trump Versus Hawaii Pages: 3 (835 words)
Has Trump been a Beneficial President? Pages: 4 (1255 words)
Effects of Immigration under Trump Administration Pages: 3 (868 words)
Donald Trump in the United States Pages: 5 (1350 words)
American Grand Strategy under Trump Pages: 4 (1271 words)
Trump Impeachment Process
Check out more papers on Donald Trump Government Justice
President Donald Trump has been in office for a year and 10 months, which for some people its been enough. It is time for Congress to introduce an impeachment inspection on President Trump for obstruction of justice. Impeachment was designed to remove any executive branch officials that have over used their power to commit a misdemeanor or a high crime, which they no longer can be trusted to continue in office. Donald Trump has influenced a mood of anger towards journalist or citizens, he's obstructed justice, Breaking the Emoluments clause of the United States Constitution, supporting violence and unconstitutionally prisoning families. Trump has been over using his power ever since he has stepped in foot as President and needs to be impeached before things gets way out of hands.
According to Google, a press officer is part of an organization like the New York Times or Daily Mail, that is responsible of organizing press releases, media coverage, and interviews for its firm. President Trump has been accusing the press of "fake news" or referring them as " the enemy of the American people." He has fired threats to sue news companies or to get their license revoked due to the fact that he doesn't like the way they run their new business. Also, he has suggested in changing the U.S. liberal laws in order to make it unchallenging to punish news firms. According to the Columbia Journalism Review, President Trump has used his twitter account to tweet over a thousand of tweets criticizing and disparaging the press. He has also tweeted words to press like "garbage", "sad" or "failing" and has tried to fire certain journalists or boycott the media organizations. Out of all, the worst thing our president has done is insult or attack a New York Times reporter, by the name of Serge Kovaleski, for having joint problems called arthrogryposis, a congenital condition that impacts the movements of the joints. Our president that is supposed to care for our people is actually insulting people or reporters by using their personal problems to shut them down, which isn't right at all. He is also black mailing reporters in order to silence them, which he is violating their freedom of speech, first amendment, and free society.
Before stepping in as office, a president is to oath, as stated, "I do solemnly swear that i will faithfully execute the Office of President of the United States, and will to the best of my ability, preserve, protect, and defend the constitution of the United States. Which would mean that president trump has already violated the constitution by obstructing justice, right before he took office. He persuade or asked the FBI Director, James Comey to desert the inspection of the Russian intrusion in the election of 2016, but James refused to terminate the investigation, which lead him to getting fire by the president. Our president is supposed to follow the constitution not break them and believe they can get away with it due to the amount of power they have. According to The Guardian, Donald admitted of firing James ,on a televised interview, over the "Russian thing", because he believe that it was a story that was made up by the democrats in excuse of losing the election, which could've or could've not been true. But why would Trump prevent James from continuing his investigation if it was just a made up story? Or was he hiding something? Either one, Trump was breaking the law when he obstructed justice. What kind of president would feel okay with breaking the law and believing that he or she should not get punished for because I wouldn't. Should us, citizens, be bale to break the law and not pay any consequences for our actions? Cuz our president thinks so.
On April 6, 2018, President Donald Trump passed a zero tolerance policy that prosecutes all individuals who cross the borders illegally to the United States, separating children from their parents when they attempt to cross the border together. Some people believe that the policy is a " cruel new child separation policy," according to Politifact. Just with in 2 weeks of passing the policy, about 700 children were divided from their parents in May, according to Politifact. Before Trump passed the zero tolerance policy, families who were detained together were sent back immediately or paroled into the country, stated by Peter Margulies, a professor at Roger Williams University in immigration laws. With the new policy being passed, the zero tolerance, families are now being separated and put into cages or camps like animals, in which the conditions are in humane, heartless or cruel. President Trump has zero plans of reuniting families with their children, even if the child is being deported back to. If the parents of the child is being prosecuted, the child is then handed to the Health and Human Services Department of the United States. The agencies duty is to find a sponsor for the child until he or she find outs about its status will be. Donald Trump believes that harsh punishment would prevent more and any immigrants from from wanting to cross the border. Immigrants, especially children, should not be treated as caged animals just because they come from another country. They deserve to be held in custody with their families in a appropriate detention center.
The emolument clause of the constitution is stated in Britannica, "generally prohibits federal office holders from receiving any gift, payment, or other thing value from a foreign state or its rulers, officers, or representatives, explain that Trump is not allowed to receive any gifts or money from anyone in general. Which every time any foreign President or official stays at any of Trumps hotels, he or she is supporting any new organization projects or trade marks that Trump has planned, which means that President Trump is breaking the Constitution of the United States. Just like when the Chinese President, XI Jinping, approved 40 trade marks of Trump and in June approved another 9 of Donalds Trademarks. Also, anytime that Trump takes any foreign office holders to his gold course or they just go voluntarily without Trump presents, he or she is sending taxpayer money into his business, which is violating the Domestic Emoluments Clause.
According to Google, equal protection of the laws is in the United States fourth amendment saying "states guarantee the same rights, privileges, and protection to all citizens." On August 12, 2017, there was a neo-Nazi Riot in Charlottesville that ended with people being killing and injuring. President Trump decided to protect the new-Nazi that decided to kill a protestor, which Trump was violating duty to shield the citizen against violence. Trump also gave specific instructions to the police offices to be rough with the protestors who were under arrest which he was violating his duty to devote executions of the laws. Trump has shown a pattern of ignoring the simple responsibilities that are in the constitution that a president must follow.
In my opinion, President Trump should be impeached for over using his power, neglecting immigrants, receiving cash and breaking laws. A proper President should look out for his people, not insult them. He should treat everyone fairly no matter what skin color or place you come from. A president should not receive any gifts from any foreign presidents. A President should show his people that you need to be kind, loving and understanding, not like Trump who is showing his people that if you commit a crime you won't be prosecuted. Trump needs to change his way in order for him to continue as the President of the United States.
In conclusion, An impeachment was created to abolish any executive branch officials who have mistreated their power, committing misdemeanors or crimes in which they can't be trusted to advance in office. President Trump has over used his powers in many situation from attacking the press to obstructing justice. How much longer are we going to left President Trump get away with these misdemeanors or crimes till we are able to impeach him from office. We have him confessing on live television on the accusations, what more do you need? It's time to end Donald Trump from his Presidency.
Trump Impeachment Process. (2021, Mar 24). Retrieved January 27, 2023 , from
https://studydriver.com/trump-impeachment-process/
Conflict of Interest between President Trump and Businessman Trump
The Impeachment of Andrew Jackson
Donald Trump is Using Kanye West for Civil War
Comparing and Contrasting George Washington and Donald Trump
Has Donald Trump Kept his Word?
Is Donald Trump Becoming a Great Presidnet?
Trump's Election has Changed the United States
About Donald Trump


While you're busy caring for your precious new baby, it's important to take care of yourself, too.
Eat at least three healthy meals a day.
Return to a healthy weight gradually. Aim to lose about 1 pound per week.
If you had gestational diabetes when you were pregnant, you and your child are at a higher risk for getting diabetes later in life. Take action now to prevent diabetes.
Learn more about how to breastfeed and the health benefits for both you and your baby.
Try to get enough sleep — sit down and rest when your baby is resting.
Take a walk — get some exercise.
Stay smoke-free and avoid secondhand smoke, including marijuana.
Learn more about the risks of drinking alcohol if you are breastfeeding.
Depression is the most common complication of pregnancy — get help handling difficult postpartum emotions. Learn about pregnancy-related depression.
Talk to a supportive person and express your feelings — both positive and negative.
Learn what to expect developmentally from your child and how you can help your child learn at each stage.
Touch, hold and cuddle your baby.
Visit www.earlieriseasier.org for ideas and tips on how to read, sing, talk, play, write and laugh with your child every day! Activities are arranged by age and are available in English and Spanish.
Learn how your baby's brain is built and what you can do to help your baby!
As surprising as it may seem, you can get pregnant again just two weeks after giving birth. For your health and for the health of your next baby, it's never too early to start talking about family planning.


text:
Q: Future for an alternative plan + I'm afraid
Are you coming with us tomorrow to the beach?

Intended answer is: "No. I prefer going to the mountain."

*

*I'm afraid I'll be going to the mountain.

*I'm afraid I will go to the mountain.

*I'm afraid I'm going to the mountain

I would use 1. Right? What about 2. and 3.?

A: I would find (3) the most natural - or, more explicitly

I'm afraid I've arranged to go to the mountains with [person/group].

(We use mountain to refer to a particular one, but mountains to mean an area of mountain scenery.)
I'm afraid I will [verb] sounds as though you are literally afraid that you will end up doing something unpleasant.


I have been in a purging mode lately… you know the kind, when you start to look around your home and wonder why you have so much stuff? I am pretty good at giving away the stuff that I am obviously not using, but there have been some items I have been holding onto for reasons I wasn't quite sure about anymore.
Three particular items really stood out to me, each representing a past life of mine. I am pretty sure I was only holding onto them for sentimental reasons.
I finally made the decision to give them away to Value Village, so that someone else can truly appreciate them. Past Wardrobe: Since having four kids, I have given myself a wardrobe makeover, opting for pieces that are still stylish but also practical. I bought these black Michael Kors pumps years ago when I found them for a crazy price. Unfortunately, they never quite fit my wide feet right and sat on my dresser as a useless trophy. Goodbye, shoes!
Past Baby Stage: I think this one was the hardest item to giveaway: my coveted Petunia Pickle Bottom diaper bag that I got when we had our first baby (I have a twin sized one now). I had truly been holding on to this prized possession as a memento. But I know another mom could get a lot more use out of it. Goodbye, diaper bag!
Past Career: I used to get dressed up every morning in very fancy clothes and sport some pretty trendy jewelry to head off to my glamorous interior design job, including this over-sized Swarovski cocktail ring. Is it practical for my lifestyle today as a work-from-home blogger and mommy of four? Nope. Goodbye, ring!
Previous Previous post: What Advice Would You Go Back And Give Yourself As A New Mom?
Next Next post: How Going Through A Twin Pregnancy Woke Me The ____ Up!


Oscar de la Renta gown in eyelet floral-lace.
High-low, asymmetric hem with train.
Brandon Maxwell evening gown in satin pineapple-jacquard. Deep V neckline. Long, flared sleeves. Fitted waist. A-line skirt. Center front slit. Hidden back zip. Floor-sweeping hem. Viscose/cotton. Made in USA of Italian materials.
Carolina Herrera evening gown with floral-print. Off-the-shoulder neckline. Button front. Short flounce sleeves. Fitted bodice. Pleated A-line skirt. Floor-sweeping hem. Cotton/spandex. Made in USA of Italian materials.
Exclusively ours. La Petite Robe di Chiara Boni "Dilia" evening gown in stretch-jersey. Sculpted ruffle down left side. Bateau neckline. Long sleeves. Trumpet silhouette. Step-in style. Floor sweeping hem. Nylon/spandex. Made in Italy.
La Petite Robe di Chiara Boni "Etheline" evening gown with beaded-bodice. Bateau neckline. Cap sleeves. Asymmetric peplum waist. Trumpet silhouette. Hidden back zip. Floor-length. Nylon/spandex. Dry clean. Made in Italy.


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text:
Women's Basketball Host Notre Dame Wednesday on Senior Night
Notre Dame (Ohio)
Notre Dame (Ohio) (13-12) 26 40 66
Lake Erie (4-21) 23 28 51
Sarah Wojciechowski
Pts: MACER, Lauren - 17
Reb: ENTINGHE, Erin - 10
Ast: 5 Players (#01, #22, #23, #24, #33) - 2
Pts: Sarah Wojciechowski - 14
Reb: Chrissy Delaney - 9
Ast: Chrissy Delaney - 2
PAINESVILLE, OHIO -- Following a weekend split in conference play last weekend, the Lake Erie College women's basketball team plays its final nonconference game of the season Thursday (Feb. 27) against traditional rival Notre Dame College on Senior Night. The Storm (4-20) defeated Tiffin University on Thursday, 50-43, before falling to Ohio Dominican University on Saturday, 68-58. Prior to tonight's game, the penultimate of the season, Lake Erie College will recognize senior guards Sarah Wojciechowski and Kiarra Jones for their contributions to the women's basketball program.
LIGHTNING QUICK RECAP: Despite a career day from Chloe Irish, the Storm were prevented from recording back-to-back wins by visiting Ohio Dominican. Irish set career bests with 25 points and 12 rebounds for her first career double-double, but Lake Erie was ultimately done in by a 15-0 Panthers run to close the first half.
FALCONS FLASH: After a 2-11 start, Notre Dame (12-12) went on a 10-game winning streak that was finally snapped by Cedarville University on Saturday, 82-76. The Falcons have been paced this year by senior guard Lauren Macer, averaging 18.7 points per game this season with 10 games of 20 or more.
SERIES HISTORY: Notre Dame leads the all-time series 29-9 and has won seven of the last nine meetings. However, the Storm won last year in South Euclid, 96-72, behind double-doubles from both Alyssa Wagers (23 points, 11 rebounds) and Stephanie Rogers (21, 10).
UP NEXT: The Storm hosts Ashland University in the season finale Saturday, March 2.


text:
using Aurora.Settings;
using Newtonsoft.Json;
using System;
using System.IO;
using System.Text;
using System.Windows.Controls;
using System.Windows.Media;
using System.Windows.Media.Imaging;

namespace Aurora.Profiles.Evolve
{
    public class EvolveProfileManager : ProfileManager
    {
        public EvolveProfileManager()
            : base("Evolve Stage 2", "Evolve", "evolve.exe", new EvolveSettings(), new GameEvent_Evolve())
        {
        }

        public override UserControl GetUserControl()
        {
            if (Control == null)
                Control = new Control_Evolve();

            return Control;
        }

        public override ImageSource GetIcon()
        {
            if (Icon == null)
                Icon = new BitmapImage(new Uri(@"Resources/evolve_48x48.png", UriKind.Relative));

            return Icon;
        }

        internal override ProfileSettings LoadProfile(string path)
        {
            try
            {
                if (File.Exists(path))
                {
                    string profile_content = File.ReadAllText(path, Encoding.UTF8);

                    if (!String.IsNullOrWhiteSpace(profile_content))
                        return JsonConvert.DeserializeObject<EvolveSettings>(profile_content, new JsonSerializerSettings { ObjectCreationHandling = ObjectCreationHandling.Replace });
                }
            }
            catch (Exception exc)
            {
                Global.logger.LogLine(string.Format("Exception Loading Profile: {0}, Exception: {1}", path, exc), Logging_Level.Error);
            }

            return null;
        }
    }
}


text:
Апия Интернешънъл Сидни 2012 е 120-ото издание на Апия Интернешънъл Сидни. Турнирът е част от категория "Висши" на WTA Тур 2012. Провежда се в Сидни, Австралия от 8 до 14 януари. На Ли защитава титлата си и отново достига финала, но този път губи от поставената под номер 3 Виктория Азаренка с 1:2 сета. За беларуската това е девета титла в кариерата ѝ. На двойки победителки от Медибанк Интернешънъл Сидни 2011 са Ивета Бенешова и Барбора Захлавова-Стрицова, които обаче са елиминирани на старта от Пешке / Среботник. Водачките в схемата впоследствие печелят турнира след победа с 6 – 1, 4 – 6, [13 – 11] над Лизел Хубер и Лиса Реймънд.

Поставени тенисистки
  Каролине Возняцки (четвъртфинал)
  Петра Квитова (полуфинал)
  Виктория Азаренка (шампионка)
  На Ли (финал)
  Саманта Стосър (1 кръг)
  Вера Звонарьова (1 кръг)
  Агнешка Радванска (полуфинал)
  Марион Бартоли (четвъртфинал)

Схема на сингъл

Финална фаза

Горна половина

Долна половина

Поставени двойки
  Квета Пешке /  Катарина Среботник (шампионки)
  Лизел Хубер /  Лиса Реймънд (финал)
  Жисела Дулко /  Флавия Пенета (осминафинал)
  Мария Кириленко /  Надя Петрова (полуфинал)

Схема на двойки

Външни препратки
 Схема на сингъл
 Схема на двойки

WTA Тур 2012
Апия Интернешънъл Сидни

redpajama_set_name: RedPajamaWikipedia


text:
Updated 3 August, 2013 - 06:38 aprilholloway
1,300-Year-Old Feathered Shield Found in Peru Temple
Archaeologists have found an ancient feathered sh ield in a sealed portion of a temple at the site of Pañamarca in Peru, which was made by the Moche people.
The Moche was a mysterious civilization who ruled the northern coast of Peru approximately two thousand years ago. They built huge pyramids made of millions of mud bricks and were pioneers of metal working techniques like gilding and soldering, which enabled them to created extraordinarily intricate jewellery and artefacts.
Little was known about the Moche civilization because they left no written texts to help explain their beliefs and customs. However, the discovery of detailed paintings and murals on pottery work and on temple walls has helped to provide insights into their culture and beliefs.
The shield measures about 25 centimetres in diameter and was found face down near two ancient murals, one of which depicts a supernatural monster. It has a base made of carefully woven basketry with a handle and has a surface covered with red and brown textiles along with about a dozen yellow feathers sewn on. It may have originally held more than 100 feathers arranged in concentric circles.
The shield would have served a ritualistic rather than a practical use, either in ceremonial performances or in ritualized battles similar to gladiatorial combat, and the placement of the shield on the bench or altar appears to have been the last act carried out before the temple was sealed and a new temple built on top of it.
By April Holloway
feathered
April Holloway is a Co-Owner, Editor and Writer of Ancient Origins. For privacy reasons, she has previously written on Ancient Origins under the pen name April Holloway, but is now choosing to use her real name, Joanna Gillan.
Joanna... Read More
18 January, 2022 - 22:00 Cecilia Bogaard
2,000-Year-Old Peruvian Underwent Successful Skull Surgery
A skull housed at the Museum of Osteology in Oklahoma is causing quite a stir. The ancient elongated skull, dating back 2,000 years, once belonged to a Peruvian warrior. After being injured during...
Read more about 2,000-Year-Old Peruvian Underwent Successful Skull Surgery
12 January, 2022 - 00:00 Sahir
Wari Culture Used Alcohol and Drugs to Maintain Political Control
An altered state of consciousness to rule an ancient empire? Not a plot of a television series or a fantasy novella, but a page out of the fascinating book that is human history. A new study...
Read more about Wari Culture Used Alcohol and Drugs to Maintain Political Control
10 January, 2022 - 18:00 Nathan Falde
Fiberglass Moche Phallic Statue Damaged by Vandals in Peru
Just a few short days after its controversial installation, vandals have damaged a unique Moche phallic statue that was erected outside the city of Trujillo in northern Peru. The 9-foot (2.7-meter)...
Read more about Fiberglass Moche Phallic Statue Damaged by Vandals in Peru
26 November, 2021 - 00:57 ashley cowie
Adobe Brick Building Used In Peru As Early As 5,000 Years Ago
A recent study in pre-Hispanic northern Peru shows previously unacknowledged use of adobe brick building as early as 5,000 years ago. What's more, these adobe bricks were created by annual El Niño...
Read more about Adobe Brick Building Used In Peru As Early As 5,000 Years Ago
29 October, 2021 - 18:50 Nathan Falde
Ancient Peruvian Gold Mask Was Painted with Human Blood!
Archaeologists with the Sicán Archaeological Project , a decades-long study of a lost pre-Incan culture, unearthed a tomb and its contents along the northern coast of Peru in the early 1990s...
Read more about Ancient Peruvian Gold Mask Was Painted with Human Blood!
29 October, 2021 - 15:00 Bipin Dimri
Andean Agriculture: What Were The Inca Building At Moray?
In the South American nation of Peru can be found the ruins of the last Pre-Columbian civilization, the glorious Inca Empire. What was once a thriving, rich civilization is today's tourist attraction...
Read more about Andean Agriculture: What Were The Inca Building At Moray?


Ooh, Tim Curry would be great for those. Thanks!
We've listened to The Twits and several other Roald Dahl books in the car. They have super fun expressive readers.
Hi Paula! We've listened to Charlie and the Chocolate Factory, but no other Roald Dahl so far. Thanks for the reminder!
We just started listening to the How To Train Your Dragon books by Cressida Cowell. They're read by David Tennant and are marvellous!
Ooh, thanks, I'll have to look for those!


If there's one thing I greatly enjoy, it is beautiful stationery. I loved connecting with one of the owners of The Enchanted Envelope and seeing all the beautiful pieces they could create.
This is a creative, Father/Daughter ran business. Mandy's father, Sam learned all he could the various printing methods, as well as all the differences between paper.
He got the business started, and when his daughter, Mandy, and her husband had their own daughter, she helped him bring the business to Etsy!
They work wonderfully together as a team, and love that The Enchanted Envelope is a family focused business.
They love working to create fun pieces for their customers, and specialize in personalized pieces. The quality of their products are very high, you can see and feel their hard work in each piece.
I am in love with the sets they created for me, and can't decide which printed card is my favorite, the Leopard Print Shoe or the Floral Print Flat Card!
I really like the formal look of the Gold Foil personalized card, as well as the Embossed Panel card. Both of these pieces would work perfectly for any type of client driven business. What a professional look to send a thank you, or just a "keeping in touch" note on.
The Enchanted Envelope also specializes in a variety of other gorgeous home and office products! This would be the perfect Etsy shop to get wedding invitations, baby announcements, thank you cards, and really anything you can think of.
Their Instagram account showcases the variety of products with stunning, clear pictures, and they are quick to respond with any questions. I hope that you will check them out for your next home/office needs!
I'm also a fan of the enchanted envelope and have been thrilled with the quality and service.


My favorite scene in "Planes, Trains and Automobiles" is when Steve Martin finally blows his stack at John Candy's irritating character.
The thing that finally sends Martin over the edge isn't when he finds he's washed his face with Candy's dirty underwear. It isn't even when he wakes up to discover that he and Candy are snuggling together in the hotel room's only bed.
I often think of that line when I'm writing and editing. "Here's an idea: Have a point!" Every piece you write needs a point. It needs a tight story angle. It needs focus.
Why is selection, or finding your focus, the best way to condense your copy? Because the best pieces are an inch wide and a mile deep: They cover a small topic, but they do so fully and colorfully.
That's why Jon Ziomek, associate professor at the Medill School of Journalism, keeps a one-inch picture frame at his computer. It's a reminder to tighten his story angle.
I was reminded of the importance of starting with a tight story angle recently when the editor of an airline magazine asked me to write a piece about Kansas City.
Well, I know Kansas City. I lived there for 30 years, and I covered it from my desk as a magazine editor for nearly five. But I've never toiled so hard on a simple piece. And I've never been so disappointed in the results.
Might as well ask me to write 900 words on traveling to the United States.
My problem, of course, was that my copy lacked focus. And a lack of focus makes it difficult for you — and for your reader — to get through your story.
1. Focus on a single idea. You can communicate one idea well, a handful poorly or several not at all.
Think of your piece as a tree. Your story angle is the trunk. The tree can branch out in several directions. But when you find a sapling, yank it out. Don't tie a rope from the sapling to your tree and call it a branch.
Your summary sentence will keep you from getting scattered and from including information that isn't pertinent to the copy.
4. Test for focus. Finally, make sure every paragraph, every sentence and every word in your piece work together to support your theme.
Focus has more to do with what you eliminate from your piece than with what you leave in it. So if a section, paragraph or sentence doesn't pass the test, take it out.
Tighten your angle after you write.
Tightening your story angle upfront should help you write short. But if you write a piece that's too long, you may need to narrow your story again after you write.
When New Yorker writer Cynthia Gorney wrote a feature about knees, for instance, her notes filled 13 notebooks, and her first draft was 8,000 words long.
"A piece about knees is not a story," she realized. Her real topic was baby boomer jock knees. And out went 4,000 words.
"It's easier to whack an entire paragraph than a single word," writes Chip Scanlan, The Poynter Institute's writing workshop director.
So cut sections, not syllables.