Four examiners have lost their jobs with a Cambridge exam board after some students were graded incorrectly.
The OCR Board says mistakes were made in the transcribing and totalling of marks. The board says it's apologised and corrected mistakes.
78 markers have been told to improve and the exam board says the errors were unacceptable.
It's reassuring students who've just taken their exams that marking systems have now significantly improved.
Mark Dawe, the Chief Executive of the OCR exam board tells ITV Anglia's Education Correspondent Elodie Harper how he's dealing with a series of errors in the marking of last year's GCSE and A-Level papers.
Mark Dawe, the Chief Executive of the OCR exam board, apologises for errors in the marking of last year's GCSE and A-Level papers.
A level students in Cambridge say they have lost trust in examiners, following one exam board's marking mistakes.


text:
Pierre Duchesne (Le Mesnil-Saint-Georges, 1637- Saint-François-de-l'Île-d'Orléans, ) est l'un des 96 Picards qui vinrent au Canada, au .

Biographie
Pierre Duchesne (dit Lapierre) affirmait à son contrat de mariage être natif de Le Mesnil-Saint-Georges, minuscule commune située en bordure de la Somme, qui se divise en quatre arrondissements : Amiens, Abbeville, Péronne et Montdidier. Le Mesnil-Saint-Georges est l'une des 34 communes qui encerclent Montdidier. Comme ses coreligionnaires, il dépendait au point de vue religieux de l'évêque d'Amiens.

Pierre mit le pied en Nouvelle-France avant 1664. La preuve en est facile à établir. Le , à Château-Richer, il se présente comme parrain de Catherine Lereau dit L'Heureux, fille de Simon et de Suzanne Jaroussel, de Sainte-Famille, Île d'Orléans. Il accompagne comme marraine Catherine Colin, femme de Claude Guyon, également de l'Île d'Orléans. Pierre semble avantageusement connu par les insulaires. Le Gouverneur de Courcelles, le marquis de Tracy et l'intendant Jean Talon signent au contrat de mariage.

Pierre Duchesne dit Lapierre consent à devenir parrain d'une autre petite fille Marie, dont les parents répondent aux noms de Pierre Lat ou Lelat et de Françoise Crépeau, famille vivant non loin des Lereau. L'épouse d'Abel Turcot, Marie Giraud, est marraine de l'enfant.

Le , Pierre Duchesne obtenait officiellement une concession de terre de veuve d'Aillebout, Barbe de Boulogne, la protectrice de son épouse à son arrivée de France au Canada.

Pierre épouse Catherine Rivet le  à Notre-Dame de Québec.

Pierre Duchesne décède le  à Saint-François-de-l'Île-d'Orléans. Il est inhumé au cimetière de Sainte-Famille de l'Île d'Orléans.

Il est l'ancêtre de l'Honorable Pierre Duchesne, lieutenant-gouverneur du Québec (2007- ).

Pionnier en Nouvelle-France
Personnalité de la Nouvelle-France
Naissance en 1637
Décès en mars 1697

redpajama_set_name: RedPajamaWikipedia


text:
Q: Cannot be used as type parameter 'TElement' in the generic type or method I am using Entity Model, C#, .NET Framework 3.5.
I have generated entity classes from POCO, but it had issues on ObjectSet and CreateObjectSet which I fixed by replacing them with ObjectQuery and CreateQuery.
But I still have one issue in following code in EntityGenerator.Context.cs,
The code is
public ObjectResult<lead> GetLeads()
        {
            **return base.ExecuteFunction<lead>("GetLeads");**
        }

And the error is

Proj.EntityModel.employee cannot be used as type parameter 'TElement'
  in the generic type or method
  'System.Data.Objects.ObjectContext.ExecuteFunction(string,
  params System.Data.Objects.ObjectParameter[])'. There is no implicit
  reference conversion from 'Proj.EntityModel.employee' to
  'System.Data.Objects.DataClasses.IEntityWithChangeTracker'.

What's this error? What's solution?

A: It sounds like you just need to make employee implement IEntityWithChangeTracker.
(I suspect you've cut and paste the wrong section of code though, given that the error talks about employee and your code talks about lead. I'd also encourage you to change your names so that they follow .NET naming conventions.)

redpajama_set_name: RedPajamaStackExchange


Birch Design Associates is an interior design firm based in Ann Arbor, Michigan.
and would love the opportunity to discuss your next project.
"We have collaborated with Tiffany on many important projects. Her firm is the gold standard for refined interior design".


Arianna Alexander has worked hard over the last four years in a high school and it's safe to say her hard work paid off. Alexander, a senior at Kenwood Academy High School, graduated at the top of her class with her high school diploma in one hand and 26 acceptance letters and over $3 million in scholarships in the other.
The valedictorian earned a 5.1 grade point average on a 4.0 scale proving that she's no armature to hard work. "It was a lot to take in. I received emails, letters. It was just like, 'Come here, come here!' They were bombarding me with all this information," Arianna expressed.
She was encouraged to accomplish such a major goal when a previous student earned over $1 million in scholarships and if they could do it, so could she. And she did. Her dad's encouraging words, "Work hard and pray on it and don't give and no matter what happens, you did your best," kept her focus during the entire process.
With the help of teachers and mentors, Alexander has decided to continue her academic journey at The University of Pennsylvania to study business at the Wharton School of Business. She has hopes of becoming an entrepreneur and pursuing a career in the restaurant industry.


I think people already know if their trash can is filthy dirty or not. Probably most people just think of it as a mess that lives outside so it's of little importance. But for others, the idea that the bacteria growing hot mess of a can could attract rodents, racoons, roaches and other crawly insects, is worth some attention.
Apparently the outdoor trash can is a breeding ground for E. Coli, Salmonella, Baylisascaris & Listeria Leptospirosis. That's not good, right?
Historically the options for cleaning out that trash can was to drag it out in the yard, untangle the hose, spray it out, and dump the nasty water on the lawn or in the street where it washes down to the sewer and who knows where.
Cleaning your own trash cans is a nice effort but almost hardly worth the energy to do. While spraying out your disgusting trash can with cold water might make you feel better, it's nowhere as good as washing it out with near boiling 200 degree fahrenheit scalding water.
Local Wake Forest resident Joel Odegaard, who should probably change his name to Odorguard, has launched an almost first in the nation business that will leave you with fresh and environmentally friendly clean trash cans. Apparently this type of service is more well known and popular in the UK and Australia.
Look, I was just as skeptical as you are so I asked Joel to come give my dirty disgusting trash bins a clean to see how the service worked. Bottom line: impressive.
Joel arrives in his high pressure bacteria killing vehicle. He also does pressure washing of stuff like driveways, homes, and pollen encrusted porches. Ask him to show you the driveway cleaning thing on his truck. Pretty cool and it melts the grime away.
On the back of the truck is that big green thing which contains all the nasty stuff that gets cleaned out of the trash cans.
The can first gets sprayed with some cleaning stuff that smells nice to give you that new can smell.
Up it goes and it will emerge with all the nasty stuff gone.
After several minutes of high pressure hot water washing with some fancy multi-nozzle thing the can steams and all the junk that washes out is captured on Joel's truck. Not a drop hit my driveway. That nasty wash water gets disposed of properly at a local facility.
Joel gives the can some final extra attention and it emerged like I've never seen it before. Washed away were those old remnants of that crab party we had years ago that always reintroduced themselves on a hot summer day.
To make this service even easier Joel will tiptoe by your house on trash day and clean the cans after the trash vanishes. You can also schedule him to come by every couple of months or do it just once. Prices are very affordable.
If you hate germs, bacteria, attracting rodents, or you are just an OCD neat freak – this might be the service for you. Especially if you are an environmentally conscious person.
You can find out more over at BlueGreenBinClean.com or give Joel a call at 503-730-4983.


In present day Japan, the life of a school girl is never dull. The easily-bored Konata never finds time to study because of her otaku habits, which frustrates hard-working Kagami to no end. On the other hand, laid-back Tsukasa always manages to go with the flow, while Miyuki is concerned with keeping her status as resident know-it-all. Join these four girls as they muse and meander their way through everyday events such as eating chocolate cones, doing homework, gaming, and trips to the beach galore.


Zyad Limam is a Franco-Tunisian publisher and journalist. He heads up AM International, an independent media company based in Paris. AM International publishes AM – Afrique Magazine and AMB – Afrique Méditerranée Business, along with their websites www.afriquemagazine.com. Next December, AM will celebrate 35 years of existence and uninterrupted publication. AMB is positioned as the high-end magazine for the world of business and development. Zyad Limam is a regular guest on a number of French television channels (France 24, TV5, Arte, etc.).


text:
Chronic inflammation-induced senescence impairs immunomodulatory properties of synovial fluid mesenchymal stem cells in rheumatoid arthritis
Hyeon-Jeong Lee1 na1,
Won-Jae Lee2 na1,
Sun-Chul Hwang3,
Yongho Choe1,
Saetbyul Kim1,
Eunyeong Bok1,
Sangyeob Lee1,
Seung-Joon Kim2,
Hyun-Ok Kim4,
Sun-A Ock5,
Hae-Sook Noh4,
Gyu-Jin Rho1,6,
Sang-Il Lee4 &
Sung-Lim Lee ORCID: orcid.org/0000-0002-1055-80971,6
Stem Cell Research & Therapy volume 12, Article number: 502 (2021) Cite this article
Although the immunomodulatory properties of mesenchymal stem cells (MSCs) have been highlighted as a new therapy for autoimmune diseases, including rheumatoid arthritis (RA), the disease-specific characteristics of MSCs derived from elderly RA patients are not well understood.
We established MSCs derived from synovial fluid (SF) from age-matched early (average duration of the disease: 1.7 years) and long-standing (average duration of the disease: 13.8 years) RA patients (E-/L-SF-MSCs) and then analyzed the MSC characteristics such as stemness, proliferation, cellular senescence, in vitro differentiation, and in vivo immunomodulatory properties.
The presence of MSC populations in the SF from RA patients was identified. We found that L-SF-MSCs exhibited impaired proliferation, intensified cellular senescence, reduced immunomodulatory properties, and attenuated anti-arthritic capacity in an RA animal model. In particular, E-SF-MSCs demonstrated cellular senescence progression and attenuated immunomodulatory properties similar to those of L-SF-MSC in an RA joint-mimetic milieu due to hypoxia and pro-inflammatory cytokine exposure. Due to a long-term exposure to the chronic inflammatory milieu, cellular senescence, attenuated immunomodulatory properties, and the loss of anti-arthritic potentials were more often identified in SF-MSCs in a long-term RA than early RA.
We conclude that a chronic RA inflammatory milieu affects the MSC potential. Therefore, this work addresses the importance of understanding MSC characteristics during disease states prior to their application in patients.
The onset of rheumatoid arthritis (RA) is associated with loss of systemic immunological self-tolerance, which results in the activation of autoreactive immune cells that target collagen-rich joint regions and present symptoms characterized as chronic, destructive joint inflammation [1, 2]. As an autoimmune disease, there are limited curative options for RA that provide immunomodulation and articular cartilage or subchondral bone regeneration in the damaged joints. Mesenchymal stem cells (MSCs) can inhibit the immune response and are a potential candidate to treat various inflammatory autoimmune diseases, including RA [3]. Once MSCs are exposed to pro-inflammatory cytokines in an inflammatory milieu, they are activated and inhibit immune cell proliferation and activation by secreting anti-inflammatory cytokines [4, 5].
The utilization of MSC immunomodulation properties in RA patients is supported by the anti-arthritic potentials of MSCs observed in an RA animal model [3, 6]. However, there are some gaps in the in vivo studies, particularly regarding limited therapeutic effects, worsening of symptoms, and highly variable efficacy after MSC administration [6,7,8]. In addition, most of these studies were conducted using MSCs derived from mouse or non-patient specimens. Thus, investigating how RA affects MSCs derived from disease regions or MSCs transplanted into disease regions is required for clinical evaluation of potential therapeutic applications. Synovial fluid-derived MSCs (SF-MSCs) from RA patients are important for understanding disease pathogenesis and are easily obtained during the diagnosis or treatment of RA. Importantly, SF-MSCs have enhanced immunomodulation properties compared to bone marrow-derived MSCs (BM-MSCs) in collagen-induced arthritis (CIA) mouse models [3, 9]. However, the distinctive characteristics of SF-MSCs in an arthritis milieu are not well understood. Further, synovial fluid in an inflamed pathologic environment may result in alterations to the cells around inflammatory regions (genotoxic stress) and reduced ability to recover injuries [3, 10, 11].
Comparative studies of the immunomodulatory properties of activated MSCs from RA patients is a prerequisite for understanding the RA disease-affected cellular mechanisms of MSCs and how MSCs can be prepared for clinical applications in RA therapy. Given that SF-MSCs from RA patients (RA-SF-MSCs) have been exposed to the inflammatory milieu, we hypothesized that the progression of cellular senescence and the potential immunomodulatory properties are affected by pathological events and cellular environmental factors during RA. Therefore, we aimed to uncover alterations in duration-dependent immunomodulatory properties caused by inflammation-induced senescence in RA-SF-MSCs in an RA disease model.
Chemicals used in experiments and ethical approval
All chemicals and media were purchased from ThermoFisher (Waltham, MA, USA) or Sigma-Aldrich Chemical Company (St. Louis, MO, USA), unless otherwise specified. Collection of synovial fluid (SF) specimens and PBMCs was performed after obtaining informed consent from patients and volunteers (Approval number GNUH 2012-05-009). The protocol for animal experiments in CIA mice was approved by the Animal Center for Biomedical Experimentation at Gyeongsang National University (GNU-131209-M068).
Collection of SF and establishment of SF-MSCs from RA patients
Control SF was obtained from donors without evidence of inflammatory joint disease. SF from the RA groups was obtained from the joints of RA patients who were divided into early (E-RA, disease duration < 2 years) or long-standing (L-RA, disease duration > 10 years) groups. Then, the SF-MSCs were divided into three groups: CTL-SF-MSCs (n = 10), E-SF-MSCs (n = 9) and L-SF-MSCs (n = 12). The clinical histories of the RA patients are presented in Table 1. Cells were isolated from the aspirated and cultured SF and were processed as previously described [3]. The SF specimens were filtered through a 40-μm nylon cell strainer (BD Falcon, NJ, USA) to remove debris and centrifuged at 400×g for 10 min. The supernatants were stored at −80°C until the inflammatory cytokine analysis, while the cell pellet was resuspended and explanted onto 35 mm dishes (Nunc, Roskilde, Denmark). The cells were allowed to adhere for 2 days in culture medium before non-adherent cells were discarded. The adherent cells were cultured with advanced Dulbecco's modified Eagle's medium (ADMEM) supplemented with 10% fetal bovine serum (FBS), 1% GlutaMax, 10 ng/mL bFGF, and 1% penicillin and streptomycin (10,000 IU and 10,000 μg/mL) at 36.5°C in a humidified incubator with 5% CO2. The expanded cells were passaged four times before use for further analysis.
Table 1 Demographic and disease characteristics of donors
Characterization of SF-MSCs
Expression of MSC-specific cell surface molecules in SF-MSCs were validated in triplicate by flow cytometry using a BD FACS Calibur instrument (BD Biosciences, NJ, USA). A total of 1 × 104 cells were harvested and fixed with 4% paraformaldehyde at 4°C. All antibodies were diluted (1:200) with 1% bovine serum albumin (Table S1). Fluorescein isothiocyanate (FITC)-conjugated primary antibodies were incubated with the harvested cells for 1 h, with mouse IgG1-FITC used as an isotype control. Approximately ~80% of confluent SF-MSCs differentiated into adipocytes and osteoblasts after 3 weeks. Adipogenesis was induced with Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS, 100 mM indomethacin, 10 mM insulin, and 1 mM dexamethasone. Then, adipogenesis was confirmed by intracellular lipid vacuole staining with 0.5% Oil red O solution and by gene expression (FABP4 and PPARγ). Osteogenesis was induced with DMEM supplemented with 10% FBS, 200 mM ascorbic acid, 10 mM β-glycerophosphate, and 0.1 mM dexamethasone. Then, osteogenesis was determined by the accumulation of calcium deposits visualized with Alizarin-red S solution and by gene expression (ON and OCN). For chondrogenesis, 1 x 106 SF-MSCs were cultured for 3 weeks in 15 mL tubes containing STEMPRO Osteocyte/Chondrocyte basal medium supplemented with 10% chondrogenesis supplement. Cell pellets were embedded in paraffin, cut into 5 mm sections, and stained with 1% Alcian blue and 0.1% nuclear fast red counterstain to confirm proteoglycan synthesis. Chondrogenesis was also verified by gene expression analysis (COL2 and COL10A1). The protocol for the gene expression analysis is described below.
Gene expression by quantitative PCR (qPCR)
qPCR was used for gene expression studies to determine pluripotency (Oct3/4, Sox2, and Nanog), apoptosis (Bax, Bak, p53, Bcl2, and Birc), differentiation (FABP4, PPARγ, ON, OCN, COL2, and COL10A1), and the expression of hypoxia-related genes (GLUT1, LDHA, LOX, and PGK1). Three replicates of each sample were analyzed by qPCR. Relevant primer information is displayed in Table S2. The total RNA was extracted using an RNeasy Minikit (Qiagen, CA, USA) and quantified using an OPTIZEN 3220 UV BIO spectrophotometer (Mecasys, Sungnam, Korea). Next, cDNA synthesis was performed from 1 μg total RNA using an Omniscript Reverse Transcription Kit (Qiagen) with a oligo dT primers at 60°C for 1 h. qRT-PCR was performed using a Rotor Gene Q qRT-PCR instrument (Qiagen) with Rotor-Gene 2× SYBR Green mix (Qiagen), 2 μL cDNA per reaction, and 0.5 mM forward and reverse primers. The qPCR program settings included of pre-denaturation (95°C for 10 min), 45 PCR cycles (95°C for 10 s, 60°C for 6s, and 72°C for 4 s), melting curve analysis (60°C to 95°C ramp, 1°C per seceond ramp rate) and cooling (40°C for 30 s). Transcript levels of all target genes were normalized against those of TBP, which is a stable reference gene in human MSCs [12].
Proliferation and cell cycling in SF-MSCs
Vybrant MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] Cell Proliferation Assays (Molecular Probes, Eugene, OR, USA) were used to evaluate SF-MSC proliferation. Absorbance at 540 nm was measured using a microplate reader (Molecular Devices). To analyze cell cycle changes, SF-MSCs were fixed with 70% ethanol, stained with 10 μg/ml propidium iodide (PI) solution, and evaluated using flow cytometry.
Senescence-associated β-galactosidase activity staining
Cellular senescence was evaluated using Senescence β-Galactosidase Staining Kits (Cell Signaling Technology, Danvers, MA, USA). SF-MSCs were fixed for 15 min in fixation solution at room temperature, stained with β-Galactosidase staining solution, and incubated at 37°C overnight. To measure β-galactosidase activity, Mammalian β-Galactosidase Assay Kits (ThermoFisher, Rockford, IL, USA) were used. SF-MSCs were harvested, incubated with M-PER reagent for 10 min, and centrifuged for 10 min at 27,000xg. The supernatant was transferred into 96-well plates and treated with β-galactosidase reagent for 30 min at 37°C. The optical density was determined at 405 nm using a microplate reader (Molecular Devices).
Evaluation of telomere length and telomerase activity
Telomere lengths in SF-MSCs were investigated using a nonradioactive chemiluminescent TeloTAGGG telomere restriction fragment (TRF) length assay kit (Roche, Indianapolis, IN, USA).
Suppression of PBMC proliferation by SF-MSCs
Human PBMCs were isolated from healthy donors (n = 6) by density gradient centrifugation using Ficoll-Paque PLUS (GE Healthcare, Uppsala, Sweden). PBMCs were resuspended in RPMI 1640 complete medium supplemented with 10% FBS, 1% penicillin, and 1% streptomycin (10,000 IU and 10,000 μg/mL). Then, the cultures were stimulated with 1 μg/mL PHAL to activate T-cell proliferation. The PHAL-activated PBMCs (6.25 × 103 cells/well) were seeded in a 96-well plates. After 12 h, 10 μg/mL Mitomycin-C (Sigma-Aldrich, USA) was added 2 h to inhibit cell proliferation. The PHAL-activated PBMCs were co-cultured for 5 days in a 96-well plate with pre-seeded SF-MSCs at PBMC to MSC ratios of 1:4, 1:2, and 1:1 before the addition of 5-bromo-2-deoxyuridine (BrdU). PBMC proliferation was evaluated using a Cell Proliferation ELISA, BrdU (colorimetric) Kit (Roche Diagnostics, Mannheim, Germany).
Analysis of cytokine levels in SFs and SF-MSCs
The frozen SF supernatant samples were thawed and used to evaluate inflammatory cytokine levels. The levels of tumor necrosis factor (TNF)-α and interleukin (IL)-1β in the SF were determined with Quantikine ELISA kits (R&D Systems, Minneapolis, MN, USA). Briefly, standards and samples were incubated in wells pre-coated with the respective human primary antibody. The resulting antigen-antibody complexes were detected using human TNF-α or IL-1β conjugated to horseradish peroxidase, and the conjugate was quantified by a colorimetric reaction with 3,3′,5,5′-tetramethylbenzidine substrate. The resultant color intensity was read at 450 nm using a microplate reader (Molecular Devices). For SF-MSCs, 6.25 × 103 cells/well were cultured in 96 well plates in serum-starvation medium (1% FBS in ADMEM), followed by supplementation with human recombinant TNF-α (50 ng/mL; R&D Systems) for 2 days to activate inflammatory cytokine production. After collecting the supernatant, the levels of matrix metalloproteinases (MMPs; MMP-1, MMP-3, and MMP-13) and other cytokines [IL-6 and indoleamine-pyrrole 2,3-dioxygenase (IDO)] were analyzed in the same manner as SF samples. All samples were assayed in duplicate and the concentration of target proteins in each sample was determined by interpolation from a standard curve.
SF-MSC administration to CIA mice
Injection of MSCs into CIA mice was conducted as previously described [3]. Briefly, pathogen-free male DBA/1 mice (7–9 weeks old; Orient Bio, Seoul, Korea) were immunized with 100 μg bovine type II collagen (Chondrex, Redmond, WA, USA) emulsified in complete Freund's adjuvant (CFA, Chondrex) by injection into the intradermal region of the tail on day 0. Mice received a booster immunization of an equal volume of bovine type II collagen and incomplete Freund's adjuvant (IFA, Chondrex) on day 21. The experiment included 4 groups (n = 8 per group): a PBS injection control, and CTL-SF-MSC-, E-SF-MSC-, or L-SF-MSC-injected groups. SF-MSCs were intraperitoneally injected on day 21 and for five consecutive days with 200 μL PBS or SF-MSCs (5 × 106 cells in 200 μL PBS). Clinical arthritis scores (0–4 scale) were evaluated for each limb in accordance with a well-defined standard. The total possible score was 16. To measure hind paw thickness, a caliper was placed across the ankle joint at the widest point. On day 48, CIA mice were sacrificed by cervical dislocation. The hind paws were scanned with a SkyScan 1076 micro-CT apparatus (Bruker, Kontich, Belgium) and reconstructed into a three-dimensional structure with a voxel size of 18 μm using NRecon and CT Analyzer software (Bruker). Joint tissue specimens from CIA mice were fixed with 10% formalin, decalcified for 3–4 weeks in 10% EDTA, and embedded in a paraffin block. Joint sections (5 μm) were stained with hematoxylin and eosin (H&E), Safranin O, or tartrate-resistant acid phosphatase (TRAP) to evaluate articular inflammation, cartilage damage, and TRAP-positive multinucleated cells (osteoclasts), respectively. The total number of TRAP-positive multinucleated cells containing three or more nuclei was counted in 10 areas of each CIA mouse ankle [3, 13].
Induction of the RA-like inflammatory milieu
Because both low partial oxygen pressure (hypoxia) and inflammation are relevant features in the synovial joints of RA patients [14], an in vitro RA-like inflammation milieu was induced in E-SF-MSCs to explore whether immunomodulatory properties and senescence were altered in inflammation-exposed SF-MSCs. E-SF-MSCs were cultured in normal culture conditions for 3 days with in various gas compositions: 21% O2, 5% CO2, and 74% N2; or 3% O2, 5% CO2, and 92% N2 in a 95% humidified atmosphere. The cells were maintained in multi-gas incubators (ASTEC, Fukuoka, Japan) to reflect normoxia or hypoxia. In addition, the media for hypoxic E-SF-MSCs was supplemented with 20 ng/mL TNF-α and 20 ng/mL IL-1β (R&D Systems) as representative inflammatory cytokines. Both E-SF-MSCs and L-SF-MSCs cultured in normoxic conditions were used as controls.
Western blot analysis
The induction of hypoxia was validated by upregulated hypoxia-inducible factor 1-α (HIF1α) expression. Cell extracts of E-SF-MSCs with or without hypoxia were prepared with RIPA buffer supplemented with Halt Protease Inhibitor Cocktail (Pierce Biotechnology, Rockford, IL, USA). The total protein concentration in the cell extracts was quantified using Bicinchoninic Acid Protein Assay Reagent Kits (Pierce Biotechnology). A 25-μg aliquot from each sample was fractionated by 10% SDS-PAGE and was transferred onto a polyvinylidene difluoride membrane (Millipore, Darmstadt, Germany). The membranes were blocked with 0.1% bovine serum albumin (BSA), incubated with anti-HIF-1α or anti-GAPDH primary antibodies (1:100 dilution with BSA) at 4°C overnight, incubated with horseradish peroxidase-conjugated secondary antibodies (1:3,000 dilution with BSA) at RT for 1 h, and detected using a chemiluminescence assay (Amersham Biosciences Corp, Piscataway, NJ, USA) with X-ray film for visualization.
Apoptosis assays
The proportion of apoptosis in E-SF-MSCs with or without the induction of an RA-like inflammatory milieu was determined using an Annexin V-FITC Apoptosis Detection Kit (Invitrogen, Eugene, OR, USA). For these assays, 1 × 104 cells were harvested, washed twice in PBS, and resuspended in 200 μL binding buffer. Then, the cells were treated with 10 μL Annexin V stock solution, incubated at 4°C for 30 min, counterstained with PI, and analyzed by flow cytometry (BD FACS Calibur).
IDO activity measurements
IDO activity was measured as previously described [15]. Cultures were harvested and 2.5 × 104 E-SF-MSCs with or without induction of an RA-like inflammatory milieu were cultured for 4 days. Then, the cells were supplemented with 100 μM L-tryptophan (Sigma) for 4 h. The supernatant was harvested and mixed with 30% trichloroacetic acid (Sigma) before an additional incubation at 50°C for 30 min. This solution was diluted in Ehrlich reagent (1:1) (Sigma, USA), and the optical density was measured at 492 nm using a microplate reader (Molecular Devices). Serially-diluted l-kynurenine (Sigma) made with fresh culture medium was used as the standard.
Statistical significance was analyzed using paired T tests, one-way analysis of variance (ANOVA), and Tukey's multiple comparison tests followed by Games-Howell post hoc analysis using SPSS 21.0 (IBM, Armonk, NY. USA). All data are presented as mean ± standard deviation (SD). P < 0.05 was considered significantly different.
RA-SF-MSCs exhibit a normal phenotype and differentiation regardless of disease status
RA patients were classified as having early (E-RA) or long-standing (L-RA) RA according to disease duration and the degree of joint destruction. Thus, L-RA patients had a longer disease duration and more severe joint destruction (modified sharp score) than E-RA patients (Table 1). Three types of SF-MSCs were derived from healthy controls (CTL-SF-MSCs, n = 10), E-RA patients (E-SF-MSCs, n = 9), and L-RA patients (L-SF-MSCs, n = 12). All SF-MSCs presented normal MSC phenotypes, such as plastic-adherent populations with fibroblastic morphology and positive MSC-specific or -negative hematopoietic-specific cell surface molecule expression (Fig. 1a, b). In specific cytochemical staining, adipogenic- and osteogenic-differentiated SF-MSCs showed strong positive stains, as confirmed by the cytoplasmic accumulation of lipid vacuoles and the deposition of calcified extracellular matrix by Oil red O and Alizarin-red S staining, respectively. When induced toward chondrogenic differentiation, all SF-MSCs were successfully aggregated were positively stained with Alcian blue. The staining intensity did not differ among the differentiated SF-MSCs upon gross observation (Fig. 1c). Adipogenesis-, osteogenesis-, and chondrogenesis-related genes in differentiated SF-MSCs were analyzed before and after the induction of differentiation. Adipocyte-related genes (FABP4 and PPARγ) were significantly increased (P < 0.05) after differentiation into adipocytes compared to the undifferentiated state, but the levels of FABP4 in E- and L-RA-SF-MSCs (246.1±1.1 and 294.5±1.1) were significantly (P < 0.05) lower than in C-SF-MSCs (868.5±0.9). After differentiation, the levels of osteocyte (ON and OCN)- and chondrocyte (COL2 and COL10A1)-related genes in SF-MSCs were significantly increased (P < 0.05) compared to undifferentiated cells (Fig. 1d). Though there were slight differences in degrees, all SF-MSCs exhibited the ability to differentiate into multiple mesenchymal lineages such adipocytes, osteoblasts, and chondrocytes, with significantly increased (P < 0.05) lineage-specific gene expression. Therefore, the differentiation ability of E- and L-SF-MSCs did not decrease compared to C-SF-MSCs (Fig. 1c, d). Overall, we conclude that the phenotype and differentiation ability of SF-MSCs were not reduced by the RA disease status.
Characteristics and differentiation abilities of RA patient-derived SF-MSCs depend on disease duration. a A plastic-adherent population with fibroblastic morphology. b Flow cytometry indicates the positive expression of MSC-specific molecules (CD44, CD90, and CD105) and the negative expression of a hematopoietic cell surface molecule (CD45) within this SF-MSC population. c Cytochemical staining of differentiated SF-MSCs showing adipocytes (lipid droplets), osteoblasts (calcium deposits), and chondrocytes (proteoglycan synthesis) when grown in vitro and d changes in lineage-specific gene expression. The asterisks indicate significant differences. The data represent percentage mean values ± SD. 40× magnification. 0W, undifferentiated SF-MSC at the start of the culture; 4W, differentiated SF-MSCs for 4 W
Impaired proliferation and increased senescence in L-SF-MSCs
RA-SF-MSCs had significantly lower (P < 0.05) proliferation ability with a slower cell cycle than CTL-SF-MSCs (Fig. 2a, b). Telomere shortening, inactivation of telomerase, and increased β-galactosidase activity (β-gal) were significantly increased (P < 0.05) in L-SF-MSCs, indicating altered cellular senescence patterns (Fig. 2c, d). Moreover, inflammatory cytokine-treated L-SF-MSCs had a significantly higher (P < 0.05) senescence-associated secretory phenotype (SASP) than CTL-SF-MSCs and E-SF-MSCs (Fig. 2e). In E- and L-SF-MSCs, the expression level of Oct3/4 and Sox2 was not differ but Nanog (0.55 ± 0.05 and 0.51 ± 0.07), a pluripotent factor in stem cells was significantly lower (P < 0.05) than CTL-SF-MSCs (1.0 ± 0.11) (Fig. 6). The apoptosis-related factors, the expression levels of Bax and Bcl-2 in E-and L-SF-MSCs (1.21 ± 0.05 and 0.76 ± 0.04) was significantly (P < 0.05) higher and lower than in CTL-SF-MSCs (1.17 ± 0.09 and 0.78 ± 0.05). The expression levels of Bak and Birc in L-SF-MSCs (1.01 ± 0.07 and 0.7 ± 0.08) was significantly (P < 0.05) higher and lower than in E- (0.75 ± 0.03 and 2.55 ± 0.5) and CTL-SF-MSCs (0.64 ± 0.03 and 2.40 ± 0.3), respectively. However, expression level of p53 did not differ between all groups. Collectively, these results suggest that L-SF-MSCs are more senescent than both CTL-SF-MSCs and E-SF-MSCs.
Proliferative ability and cellular senescence of RA patient-derived SF-MSCs. a Comparison of SF-MSC proliferation by MTT assay. b Cell cycle status of SF-MSCs by flow cytometry. c The range of telomere restriction fragment (TRF) lengths (kb) in SF-MSCs by non-radioactive chemiluminescent assay. d Evaluation of the relative telomerase activity (RTA) in SF-MSCs by qRT-PCR. e Comparison of β-galactosidase-positive cell populations in SF-MSCs. 100× magnification. The asterisks indicate significant differences. The data represent mean values ± SD. N/C, negative control; P/C, positive control; SA β-gal, senescence-associated β-galactosidase; SASP, senescence associated secretory phenotype; MMPs, matrix metalloproteinases
Reduced in vitro immunomodulatory properties of L-SF-MSCs
Although all SF-MSC types suppressed PBMC proliferation (Fig. 3a), the intensity of this suppression varied depending on the group (Fig. 3b). Compared to the control group (PBMC + PHAL), the suppression of PBMC proliferation (PBMC to MSC = 1:1) of CTL-SF-MSCs and L-SF-MSCs was significantly (P < 0.05) higher and lower, respectively. In addition, IDO secretion in L-SF-MSCs post-TNF-α treatment (40 ± 15) did not reach that of CTL-SF-MSCs (175 ± 50) (Fig. 3c). Furthermore, IDO activity in L-SF-MSC after 50 ng and 100 ng IFN-γ treatment (36.72 ± 1.08 and 37.91 ± 0.71) significantly decreased (P < 0.05) more than that of CTL-SF-MSC (58.41 ± 0.77 and 69.77 ± 0.62) (Fig. 3d). Overall, the in vitro immunomodulatory properties of L-SF-MSCs were attenuated more than in other SF-MSCs.
In vitro immunomodulatory properties of RA patient-derived SF-MSCs. a Evaluation of inhibited PBMC proliferation. The ratios 1:4, 1:2, and 1:1 indicate the cell population of pre-seeded SF-MSCs and PBMCs. PBMC and PBMC+PHAL indicate PBMC cultures without or with PHAL, respectively. b Comparison of inhibited PBMC proliferation in the highest PBMC to MSC ratio (1:1). c Quantification of IDO secretion after treatment with inflammatory cytokines by ELISA. Con or T indicate cells that were untreated or treated with inflammatory cytokines, respectively. d Evaluation of IDO activity after treatment with IFN-γ by measurement of L-kynurenine. The asterisks indicate significant differences. The data represent mean values ± SD. PBMC, peripheral blood mononuclear cells; PHAL, phytohemagglutinin-L; IDO, indoleamine 2,3-dioxygenase; IFN-γ, interferon-γ
Attenuation of the anti-arthritic properties of L-SF-MSCs in CIA mice
A significantly lower (P < 0.05) mean arthritis score and ankle thickness of CIA mice injected with CTL- and E-SF-MSCs indicated the therapeutic effects of SF-MSCs. However, L-SF-MSCs demonstrated no therapeutic potential and had worse symptoms 44 d post-MSCs application (Fig. 4a). Bone destruction did not progress in CTL- and E-SF-MSC-injected CIA mice, but was detected in the joints of L-SF-MSC-injected mice (Fig. 4b). Histopathological observation of the joints of CIA mice post-CTL- and E-SF-MSC application demonstrated arthritis prevention, mice injected with L-SF-MSCs presented arthritis-related changes such as synovial inflammation, cartilage damage, and osteoclast activity (Fig. 4c). Based on these in vitro and in vivo results, we conclude that the preventative effects of SF-MSCs are varied and depend on disease duration. Furthermore, L-SF-MSCs lost most of their immunomodulatory properties, possibly due to prolonged exposure to the RA-associated inflammatory milieu.
In vivo anti-arthritic potential of RA patient-derived SF-MSCs. a Evaluation of the clinical scores and measurements of hind paw thickness in CIA mice after intraperitoneal administration of SF-MSCs or PBS. b Images of micro-CT scanning and bone surface/bone volume (BS/BV) analysis. c and d Histological examination of therapeutic effects of SF-MSCs. In the HE staining, Ti represents tibia, Ta represents talus, and Ca represents calcaneus as the anatomical locations. 40× or 200× magnification. Graphs summarize the pathological scores in terms of inflammation, cartilage damage, and TRAP + cell (osteoclast) populations in joints from CIA mice. The asterisks indicate significant differences. The data represent the mean values ± SD. PBS, phosphate-buffered saline; H&E, hematoxylin and eosin; TRAP, tartrate resistant acid phosphatase. In the HE staining (panel c), Ti represents tibia, Ta represents talus, and Ca represents calcaneus as the anatomical locations
Cellular alteration of E-SF-MSCs into L-SF-MSCs in the RA-mimetic milieu
The synovial tissues of RA patients are exposed to an inflammatory milieu that includes inflammatory cytokines and hypoxia [14]. In particular, L-SF-MSCs derived from the SF of L-RA patients were exposed to prominent pro-inflammatory cytokines for a significantly longer (P < 0.05) time than E-SF-MSCs (Figure S1). Therefore, we asked whether an RA joint-mimetic milieu generated by treatment with inflammatory cytokines and hypoxia (h+/i+) could provoke cellular and immunomodulatory alterations in E-SF-MSCs when compared with non-treated L-SF-MSCs (h-/i- L-SF-MSCs). This experiment was performed to clarify the effect of a chronic inflammatory milieu on RA-SF-MSCs. Hypoxia in E-SF-MSCs was validated by significantly increased (P < 0.05) HIF-1α levels and the activation of downstream signaling cascades (Fig. 5a). Increased apoptosis and cellular senescence were observed in h+/i+ E-SF-MSCs in the RA joint-mimetic milieu, but not in non-treated E- or hypoxic E-SF-MSCs (h-/i- and h+/i- E-SF-MSCs) (Fig. 5b, c). Although h+/i+ E-SF-MSCs maintained IDO activity and the ability to significantly suppress (P < 0.05) PBMC proliferation, the intensities of these activities were weaker than in h+/i- E-SF-MSCs (Fig. 5d). Interestingly, these changes in h+/i+ E-SF-MSCs were similar to those measured in h-/i- L-SF-MSCs, suggesting that the chronic inflammatory milieu of the joints of L-RA patients influences the MSC cellular senescence and immunomodulatory status.
Alteration of SF-MSC senescence by a long-term RA-mimetic milieu. a Confirmation of hypoxic culture conditions in E-SF-MSCs by Western blotting and qRT-PCR. 21% O2 or 3% O2 indicate normoxic or hypoxic culture conditions in SF-MSCs, respectively. b Measurement of apoptosis in SF-MSCs with or without a RA joint-mimetic milieu using flow cytometry with Annexin V/PI staining. Cells with Annexin V-/PI-, Annexin V-/PI+ or Annexin V+/PI-, or Annexin V+/PI+ staining were alive, necrotic, or apoptotic, respectively. c The range of TRF lengths (kb) in SF-MSCs with or without induced RA joint-mimetic milieu by Southern blotting. d Comparison of positive β-gal cell populations in SF-MSCs with or without induction of the RA joint-mimetic milieu. The asterisks indicate significant differences. The data represent the mean ± SD. h- or h+ indicate normoxic or hypoxic conditions, respectively; i- or i+ indicate the absence or presence of pro-inflammatory cytokines, respectively
MSCs display a remarkable potential for immunomodulatory properties as a cell-based regimen for inflammatory disease. However, accurate understanding of the therapeutic mechanism of MSCs and their characteristics during disease are particularly important to the development of safe and effective therapeutic strategies. The present study demonstrates the presence of SF-MSC populations in RA patients regardless of disease duration and reveals that immunomodulatory properties of RA-SF-MSCs are attenuated due to inflammation-induced senescence in the inflammatory milieu of RA joints. The immunomodulatory properties of RA-SF-MSCs are especially altered in an RA disease duration-dependent manner. Therefore, our findings suggest that the therapeutic effects of disease-affected MSCs on autoimmune diseases are highly associated with pathology and cellular environmental factors.
The significant populations of MSCs collected from the SF of arthritic patients have been clearly addressed in previous research. In a study of mesenchymal progenitor cells (MPCs) obtained from RA patients, there were no significant differences between the number of MPCs in the SF from E-RA and L-RA patients, nor in characteristics such as differentiation ability and phenotype [16]. Consistently, the presence of MSCs in SF from RA patients was identified through characterization of the minimal criteria for MSCs, as defined by the International Society of Cellular Therapy [17]. However, our results provide clear evidence for the progression of cellular senescence in RA-SF-MSCs in an RA disease duration-dependent manner, as shown by elevated β-galactosidase-positive cell populations, shortened telomere length, attenuated telomerase activity, apoptosis-related gene expression, and enhanced SASP secretions in L-SF-MSCs exposed to inflammatory cytokines for a longer time compared to CTL- and E-SF-MSCs. As in other autoimmune diseases, MSCs from patients with SLE exhibited increased frequencies of apoptosis, as evidenced by the downregulation of Bcl-2 and upregulation of Bax, and higher intracellular ROS levels than those in normal MSCs [18]. Bcl-2 is the best-characterized gene of the anti-apoptotic Bcl-2 family that has been associated with the prevention of cell death initiation [19], and Birc is involved in inhibition of apoptosis and cell cycle regulation [20]. By contrast, the pro-apoptotic Bcl-2 family members Bax and Bak play apoptosis regulatory functions during development and in tissue homeostasis [21]. The SF-MSCs from RA patients also shared similar alterations, consistent with the results of the present study. Both E-SF-MSCs and L-SF-MSCs showed diminished proliferation ability, with upregulation of pro-apoptotic genes (Bax and Bak), and downregulation of anti-apoptotic genes (Bcl-2 and Birc), than those of C-SF-MSCs (Fig. 6).
Analysis of gene expression levels in SF-MSCs. Quantitative RT-PCR was used to evaluate a pluripotency, b pro-apoptosis, and c anti-apoptosis gene expression in SF-MSCs. The asterisks indicate significant differences. The data present mean values ± SD
Cellular senescence is activated during intrinsic stress such as extensive cell replication and/or extrinsic stress like ultraviolet radiation, oxidative damage, activated oncogenes, and chronic inflammation. In particular, during the chronic inflammation that occurs with liver cirrhosis, hematopoietic stem cell-associated disorders, chronic human immunodeficiency virus (HIV) infection, myelodysplastic syndromes, ulcerative colitis, and L-RA, immune cells produce strong oxidizing genotoxic substances that induce cellular senescence [22,23,24,25]. In the inflammatory milieu, pro-inflammatory cytokines are produced from lymphocytes stimulated by antigens or pathogens. These cytokines activate immune cells, inhibit proliferation of transformed cells, and intensify cellular anti-viral/-tumor effects. Moreover, they induce cellular senescence in various cell types including melanocytes, endothelial cells, and MSCs [10, 25,26,27,28,29]. Cellular senescence is defined as irreversible changes that inhibit cellular division, growth, and function. Senescent MSCs from experimentally induced models, RA patients, and systemic lupus erythematosus (SLE) patients exhibit degradation of distinct cellular features, including differentiation potential, immunomodulatory properties, dysregulated pro-inflammatory cytokine production, and reduced migratory ability [2, 5, 10, 11, 18, 23, 25, 27,28,29,30,31]. Therefore, these studies show that the chronic inflammatory milieu of L-RA is closely associated with cellular senescence in L-SF-MSCs.
Because MSCs modulate responses by both the adaptive and innate immune systems, there is an interest in using MSCs to develop new cell-based approaches for the treatment of various inflammatory diseases [5, 32]. The immunomodulatory properties of MSCs are not spontaneously acquired, but are initiated when MSCs are primed through exposure to pro-inflammatory cytokines and the secretion of anti-inflammatory factors that have inhibitory effects on immune cells [26, 33, 34]. Therefore, administration of MSCs improves the clinical signs associated with autoimmune encephalomyelitis, autoimmune diabetes, multiple sclerosis, polymyositis, atopic dermatitis, and RA [35,36,37]. Indeed, pre-clinical research of MSCs applications for RA treatment have been actively conducted to clarify the pathogenesis of arthritis and the therapeutic mechanisms of MSCs using CIA and antigen-induced arthritis (AIA) mouse models. MSC-injected CIA or AIA mice display reduced inflammation, ameliorated cartilage destruction, integration of injected MSCs into the synovium, decreased inflammation-induced systemic bone erosion, reduced osteoclast precursors, inhibited receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis, elevated anti-inflammatory cytokines, and pro-inflammatory cytokine suppression [3, 38, 39]. However, in the present study, we verified that SF-MSCs do not have the same immunomodulatory properties (Figs. 3 and 4). While CTL-SF-MSCs demonstrated intensive suppression of PBMC proliferation and significant anti-arthritic effects in mice, the immunomodulatory properties of RA-SF-MSCs were attenuated depending on the RA disease status. Moreover, several studies suggest that MSCs are incapable of therapeutically modulating arthritis in CIA mice [6,7,8]. When considering these and our current results, environmental parameters such as the MSC administration route, the degree of the inflammatory milieu, and/or the condition of the MSCs could influence their immunomodulatory properties. Because the L-SF-MSCs used in the present study were directly exposed in the inflammatory milieu for 13.8 years (Figure S1), we further investigated whether the genotoxic stress caused by chronic inflammation in L-RA patients could alter the characteristics and senescence status of RA-SF-MSCs.
The synovial tissues, including SF, of RA patients are characterized by both chronic inflammation and hypoxic regions compared to synovial tissues in osteoarthritis and healthy patients. The synovial oxygen tension in RA patients was hypoxic from 2 to 4% compared to 9 to 12% at normal [40], and thus, 3% oxygen was exposed as RA hypoxic culture condition (Fig. 5). These symptoms are due to increased oxygen consumption by metabolically active tissue because of the formation of a pannus mass and the recruitment of inflammatory cells. Additionally, newly formed and immature vessels (angiogenesis) cannot supply enough oxygen to the tissue [14, 41,42,43,44]. Further, hypoxia can alter mitochondrial activity and increase ROS production and oxidative damage to the inflamed tissues in RA, potentially inducing cell senescence in response to genotoxic substances [42]. Likewise, both hypoxia-conditioned E-SF-MSCs (h+/i- E-SF-MSCs) and non-treated E-SF-MSCs (h-/i- E-SF-MSCs) demonstrated the maintenance of immunomodulatory properties and a non-senescent status. In contrast, E-SF-MSCs in an RA joint-mimetic milieu with hypoxia and pro-inflammatory cytokines (h+/i+ E-SF-MSCs) exhibited senescence-related cellular effects, including increased apoptosis, telomere shortening, elevated β-galactosidase activity, attenuation of IDO activity, and inhibited PBMC proliferation, which were similar to observations made in non-treated L-SF-MSCs (h-/i- L-SF-MSC). Therefore, it is likely that the chronic inflammatory milieu that contains hypoxia and pro-inflammatory cytokines in inflamed RA joints causes genotoxic stress that increases over time, indicating that RA-SF-MSCs have limited immunomodulatory properties at the inflamed site due to inflammation-induced senescence.
In conclusion, we characterized and investigated how the immunomodulatory properties of RA-SF-MSCs change in response to disease duration. To our knowledge, the present study is the first effort to comparatively evaluate cellular senescence and immunomodulatory characteristics of SF-MSCs under different inflammatory milieus. The three types of SF-MSCs derived from RA patients with different disease prognoses were verified by the differentiation ability and expression of MSC-specific cell surface molecules. Although both E- and L-SF-MSCs were exposed to the inflammatory milieu of RA, the immunomodulatory properties in CIA mice were attenuated depending on disease progression. Cellular senescence was induced by long-term exposure to chronic inflammation and was one reason that L-SF-MSCs had decreased immunomodulatory properties. Therefore, since the fate and potential of MSCs depends on their exposure to the inflammatory environment, the duration of inflammation must be considered prior to the initiation of autologous stem cell applications for inflammatory disease treatment or the clinical application of inflammatory stem cells.
Main data have been listed in the primary figures and supplemental data. All the original data are available from the corresponding author on reasonable request.
MSCs:
SF-MSC:
Synovial fluid-derived MSC
RA-SF-MSC:
SF-MSCs from RA patients
E-SF-MSC:
MSC-derived from synovial fluid of early RA patients
L-SF-MSC:
MSC-derived from synovial fluid of long-standing RA patients
CTL-SF-MSC:
MSC-derived from synovial fluid of healthy controls
CIA:
Collagen-induced arthritis
BM-MSC:
Bone marrow-derived MSC
ADMEM:
Advanced Dulbecco's modified Eagle's medium
FBS:
Fetal bovine serum
FITC:
Fluorescein isothiocyanate
DMEM:
Dulbecco's modified Eagle's medium
Propidium iodide
TRF:
Telomere restriction fragment
PHAL:
Phytohemagglutinin-L
BrdU:
5-Bromo-2-deoxyuridine
H&E:
Hematoxylin and eosin
TRAP:
Tartrate-resistant acid phosphatase
β-gal:
β-galactosidase activity
SASP:
Senescence-associated secretory phenotype
PBMC:
Peripheral blood mononuclear cell
IDO:
Indoleamine 2,3-dioxygenase
MPCs:
Mesenchymal progenitor cells
HIV:
SLE:
AIA:
Antigen-induced arthritis
RANKL:
Receptor activator of NF-κB ligand
RTA:
Relative telomerase activity
MMPs:
Matrix metalloproteinases family
BS/BV:
Bone surface/bone volume
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This study was supported by a grant from the Korea Health Technology R&D project through the Korea Health Industry Development Institute (KHIDI) funded by the Korean Ministry of Health and Welfare (grant number: HI14C1277) and the National Research Foundation (NRF) of Korea, funded by the government of the Republic of Korea (grant no. NRF-2018R1D1A3B07048602 and NRF-2021R1A2C1007054).
Hyeon-Jeong Lee and Won-Jae Lee contributed equally to this work.
College of Veterinary Medicine, Gyeongsang National University, Jinju, 52828, Republic of Korea
Hyeon-Jeong Lee, Yongho Choe, Saetbyul Kim, Eunyeong Bok, Sangyeob Lee, Gyu-Jin Rho & Sung-Lim Lee
College of Veterinary Medicine, Kyungpook National University, Daegu, 41566, Republic of Korea
Won-Jae Lee & Seung-Joon Kim
Department of Orthopaedic Surgery, Gyeongsang National University School of Medicine and Hospital, Jinju, 52727, Republic of Korea
Sun-Chul Hwang
Department of Internal Medicine and Institute of Health Sciences, Gyeongsang National University School of Medicine and Hospital, Jinju, 52727, Republic of Korea
Hyun-Ok Kim, Hae-Sook Noh & Sang-Il Lee
Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, 1500 Kongjwipatjwi-ro, Isero-myeon, Wanju-gun, Jeollabuk-do, 565-851, Republic of Korea
Sun-A Ock
Research Institute of Life Sciences, Gyeongsang National University, Jinju, 52828, Republic of Korea
Gyu-Jin Rho & Sung-Lim Lee
Hyeon-Jeong Lee
Won-Jae Lee
Yongho Choe
Saetbyul Kim
Eunyeong Bok
Sangyeob Lee
Seung-Joon Kim
Hyun-Ok Kim
Hae-Sook Noh
Gyu-Jin Rho
Sang-Il Lee
Sung-Lim Lee
H.-J.L.: conception and design, collection and assembly of data, data analysis and interpretation, manuscript writing; W. and J.L: conception and design, collection and assembly of data, data analysis and interpretation, and manuscript writing; S.-C.H.: administrative support and provision of study material or patients; Y.C., S.K., E.B., and S.L.: collection and assembly of data, data analysis, and interpretation; S.-J.K.: administrative support; H.-O.K.: administrative support and provision of study material or patients; S.-A.O.: administrative support; H.-S.N.: collection and assembly of data, data analysis, and interpretation; G.-J.R.: financial support, conception, and design; S.-I.L.: financial support, conception and design, provision of study material or patients, and manuscript writing; S-L.L.: financial support, conception and design, and final approval of manuscript. The authors read and approved the final manuscript.
Corresponding authors
Correspondence to Sang-Il Lee or Sung-Lim Lee.
All work was performed within the guidelines of the Gyeongsang National University School of Medicine and Hospital, and collections of SF specimens and PBMCs were authorized by "GNUH 2012-05-009" after obtaining informed consent from patients and volunteers. The protocol for animal experiments in CIA mice was approved by the Animal Center for Biomedical Experimentation at Gyeongsang National University (GNU-131209-M068).
Analysis of cytokine levels in SF. Significant differences between SF-MSCs are indicated by asterisks and the associated P values are given above the graphs. The data represent the mean values ± SD.
Additional file 2: Table S1.
FACS antibodies used in the evaluation of cell surface markers in SF-MSCs.
Primer sequences for qRT-PCR.
Lee, HJ., Lee, WJ., Hwang, SC. et al. Chronic inflammation-induced senescence impairs immunomodulatory properties of synovial fluid mesenchymal stem cells in rheumatoid arthritis. Stem Cell Res Ther 12, 502 (2021). https://doi.org/10.1186/s13287-021-02453-z
Mesenchymal stem cell-derived from the patient
Duration of inflammatory disease
Immunomodulation
Cellular senescence

redpajama_set_name: RedPajamaCommonCrawl


text:
package com.github.blackanthrax.iounbot.persistence.domain;

import javax.persistence.Entity;
import javax.persistence.Id;
import javax.persistence.IdClass;
import javax.persistence.ManyToOne;
import javax.persistence.PrimaryKeyJoinColumn;

@Entity
@IdClass(MembershipId.class)
public class Membership {

    private Person person;
    private Organization organization;
    private Role role;

    @Id
    @ManyToOne
    @PrimaryKeyJoinColumn(name = "personId", referencedColumnName = "id")
    public Person getPerson() {
        return person;
    }

    public void setPerson(Person person) {
        this.person = person;
    }

    @Id
    @ManyToOne
    @PrimaryKeyJoinColumn(name = "OrganizationId", referencedColumnName = "id")
    public Organization getOrganization() {
        return organization;
    }

    public void setOrganization(Organization organization) {
        this.organization = organization;
    }

    @Id
    @ManyToOne
    @PrimaryKeyJoinColumn(name = "roleId", referencedColumnName = "id")
    public Role getRole() {
        return role;
    }

    public void setRole(Role role) {
        this.role = role;
    }

}

redpajama_set_name: RedPajamaGithub


text:
10 AppleScripts to make you love your Mac (even more)
Hands on: Geolocation using HTML5
Data manipulation tricks: Even better in R
HTML5 client-side storage tutorial: Beyond cookies
Introduction to Windows PowerShell
By Brian Knittel
Computerworld |
Previous 1 2 3 4 5 6 7 8 9 10 11 12 Page 5 Next
There are some other function keys that you might want to know about. These shortcuts actually work in any Powershell or Command Prompt window, and with any console application:
Does the following...
F2 followed by a single character copies text from the previously entered command line into the current command line, up to but not including the first occurrence of the character you type. For example, if the previous command was
get-childitem c:\temp | get-member
then pressing F2 | copies get-childitem c:\temp into the current input line.
Types in whatever was in the previous command line from the cursor position to the end of the line. For example, if you mistype a single character in a command line:
fet-childitem c:\temp
and don't realize it until you've pressed Enter, just type g and then press F3 to recall the rest of the line. You'll end up with
get-children c:\temp.
F4 followed by a single character deletes text from the cursor up to but not including the first occurrence of that character.
Pops up a list of recently typed commands. You can scroll through it using the arrow keys and press Enter to reissue the command.
Lets you type in the number of a previous command. The numbers are the ones shown in the F7 pop-up. (This one would have been useful if the numbers ran backward, so that 3 meant the third command back, but they don't, so it's not.)
To be honest, you can work through an entire IT career without ever using these F-key shortcuts and not suffer for it, but if you can manage to remember them, they could come in handy some day. I keep meaning to remember F7, but I never do.
Copying and Pasting
Copying and pasting in the PowerShell environment works exactly as it does in any Command Prompt or console program window, again, because PowerShell is a normal console program, just like cmd.exe.
You can't cut text out of the window; you can only copy text from a rectangular region to the clipboard. To copy from the PowerShell screen with the mouse, click the upper-left corner of the PowerShell window and select Edit, Mark. Point to the upper-left corner of the text you want to copy, press the mouse button, and drag to the lower-right corner of the text you want. Then, right-click the screen, or press Enter, to copy the text.
Note - If all the text you want isn't visible, remember that you can scroll the window up and down during the process.
There are also keyboard shortcuts for these steps. Press Alt+Space E K to begin. You can then use the arrow keys to move the cursor to the upper-left corner of the desired text; then, hold down Shift and while holding it, use the arrow keys to move to the lower corner. Press Enter to complete the operation.
Personally, I find it easiest to type Alt+Space E K to start, switch to the mouse to mark the rectangle, and then go back to the keyboard to press Enter.
To paste text from the clipboard into the PowerShell window, click the upper-left corner of the window and select Edit, Paste. You might find it quicker to use to the keyboard shortcut: Alt+Space E P.


text:
It Smile Direct Club
In June, the thin, transparent plastic trays that fit over her leading and bottom teeth was available in the mail. McKinney is working through the series slowly, swapping the very first set for the next, and the next, as her teeth move. It'll take about nine months in all. But after 3 months of wearing these trays almost every day and night, McKinney is delighted about flaunting her new smile (It Smile Direct Club).
Danny Menendez/ BuzzFeed News Inside one of SmileDirectClub's New York City locations. SmileDirectClub's business design seems working. Align Innovation– the company that makes Invisalign, SmileDirect's main competitor– invested $46.7 million to become SmileDirect's unique provider in 2015, and this year invested another $12.8 million, to own an overall of 19% of the company.
Meanwhile, lookalikes have introduced with comparable services: Orthly, launched by 2 University of Pennsylvania trainees, now uses services to customers in Philadelphia, and Honest Co. released in New York this summer season. A SmileDirect consumer can be matched with among 225 associated dental experts or orthodontists in all 50 states. These dental specialists from another location review patient cases and then design the aligners that SmileDirect sends out in the mail.
The company declined to name any of the physician within its network, and would not provide details about its financial relationships with them. "Essentially they are gathering payments and paying us marketing costs," Fenkell said."I think that claim is rather fake," AAO's basic counsel Dillard said. "The customers do not even see a dental expert.
(SmileDirectClub has a two-star rating on Yelp.) And lots of professional dentists are alarmed that customers never ever see a certified medical professional prior to or during their treatment."It blew my mind," Casi Stubbs, a Florida orthodontist and member of the American Association of Orthodontists, informed BuzzFeed News. Like normal orthodontists, SmileDirect's dental professionals base their prescriptions on computer simulations of mouth scans.
"The teeth don't move biologically like they do on the computer system," Stubbs stated. Among her patients, 60-year-old Charlene Burnham, is a SmileDirect client whose too-tight aligners cut her gums, exposing part of her tooth near the root. Burnham informed BuzzFeed News she spent a week trying– and stopping working– to talk to a SmileDirect dental professional on the phone, then went to Stubbs for help."They were bleeding, they were cut to the root," Burnham said.
"I was appalled."A SmileDirect agent informed BuzzFeed News by email that company-affiliated dental practitioners "track the client's progress throughout treatment at regular intervals."The business "helps with the communications" of customers and dental suppliers, the agent stated. "Dental suppliers communicate straight with patients through the customer portal and by means of SMS or phone when appropriate or shown."In a YouTube video that's been viewed over 21,000 times given that June, Baltimore orthodontist Jeffrey Miller warned people away from the service, stating that no orthodontist would ever recommend aligners without first taking the X-rays that SmileDirect avoids.
(The video was removed after this story published.)Grant Olson, a dental professional in Springfield, Missouri, told BuzzFeed News that the company's item marketing, which compares itself with orthodontist-fitted Invisalign, is misleading. Unlike Invisalign trays, which orthodontists can enhance by sticking unique attachments on a tooth, SmileDirect's item is limited to very minor corrections: The aligners can correct a tilt, but can't move a tooth's orientation or position together with its root.
He described that SmileDirect-affiliated orthodontists just authorize cases that are suitable for a remote assessment and don't require follow-up dental visits (It Smile Direct Club). Laws for dental professionals vary throughout states, but there are some common themes in the 36 grievances sent by the AAO: Some states think about making impressions and providing retainers to be the role of a certified doctor, however SmileDirectClub normally supplies those services.
AAO basic counsel Dillard also informed BuzzFeed News that the company's business model incentivizes dental practitioners associated with SmileDirect to prescribe the trays even if they aren't essential. Danny Menendez/ BuzzFeed News Although the company hasn't drew in the attention of regulators yet, client complaints are plentiful online. TL; DR: Prepare for a bumpy flight.
The vast bulk of consumers in these groups resign themselves to dealing with the snags due to the fact that there are simply no other choices (It Smile Direct Club)."If you can be client, want an inexpensive strategy, SDC IS properly to go! Having a favorable mindset and being patient goes a LONG way," Melanie Masso composed to BuzzFeed News.
For lots of other clients, the flight is a bust. Seattle resident Melissa Gulick informed BuzzFeed News that at the end of nearly a year of treatment, the aligners have actually destroyed her bite: Her front teeth touch prior to her molars can make contact (It Smile Direct Club). "I'm quite frustrated– food is difficult to chew now and they will not fix it!"Gulick stated that after problems to the company, SmileDirectClub accepted continue treatment for totally free.
When inquired about complaints that the trays ruined clients' bites, a SmileDirect representative told BuzzFeed News: "Over 95% of our consumers complete treatment and approve their ended up brand-new smile. It is necessary to keep in mind that with any orthodontic treatment, bites will continue to shift until the treatment is ended up."Another customer, 24-year-old Drexel University trainee Kacie Durham, informed BuzzFeed News that SmileDirectClub sent her a simulated treatment plan that made her teeth look worse."It in fact made my teeth appear more crooked at the bottom," she stated.
Durham returned her trays and used for a refund. Later on, an orthodontist not connected with the business told her that her teeth would need to be submitted and shaped prior to they could be lined up. Durham felt deceived: SmileDirect had authorized her for a treatment that would never have worked."If a possible client is unhappy with the 3d sneak peek of his/her smile, there is no obligation to purchase aligners," the SmileDirectClub representative said (It Smile Direct Club).
Minelle Tendler, a Boca Raton orthodontist, joined one of the biggest Facebook groups, the 11,800-member SmileDirectClub Support, after 2 of her patients asked about the item. In perusing the posts that appear in her Facebook feed, Tendler said that she's not seen anybody say they've had the ability to speak with a dental professional at the company.
"You're asking somebody who has no oral background, 'Is this going to work?'"More than once, Tendler has actually stopped short of responding to threads on the online forum. She rests on her hands because the patients are not hers. "I'm looking at the pictures and thinking, Oh my god, you need to get teeth secured, don't do it."Primarily, she is stressed that people who are charmed by SmileDirectClub's low cost aren't completely conscious of the drawbacks of skipping an oral check out.
A previous version of this post misidentified him as an orthodontist. Align Technology owns 19% of SmileDirectClub. A previous version of this story mentioned it was 17%. Likewise, SmileDirectClub started running in North Carolina in early October. A previous variation of this story, based on an August interview with cofounder Alex Fenkell, stated that the business was not operating in North Carolina.
Mail-in aligners might appear like a great option for a slight repair. However bear in mind you're not the professional on how minimal a repair your teeth require. "It's challenging to go from something that's 90% excellent to make it 100% perfect; in my experience, I discover that these are the harder cases," Dr.
The results of using unnoticeable aligners can be very unforeseeable, due to the fact that some individuals react well to them and others won't. "It's really hard to monitor this on your own, because– like whatever in medication– not everyone responds the exact same," he states. The real difficulty comes with figuring out how to fix your smile if you're not responding correctly to the retainers, he says.


text:
List of Country music Bands
A reed tool created during the early nineteenth century European countries, the accordion is worn like a vest and is made from right and left-hand keyboards that are connected by a bellows. Notes are manufactured by the bellows pushing environment through valves that are managed by the keyboard.
The accordion is used mainly in conjunto, tejano and cowboy musics.
The late accordionist Clifton Chenier put the conventional for modern Cajun people like Zachary Richard. Basil Duhon, whom works closely with Grand Ole Opry celebrity Jimmy C. Newman, provides a cajun-style method of the tool. Flaco Jimenez is one of popular accordionist playing conjunto these days.
Have a listen:
An associate associated with the zither household, the autoharp is played by strumming its strings with one hand although the contrary settings a bar which damps those strings not into the chord.
Very first delivered to prominence in country music by Ernest "Pop" Stoneman when you look at the 1920s, it absolutely was later made popular by Sara Carter for the Carter Family. Folk musician Bryan Bowers is one of today's leading professionals associated with the autoharp.
Have actually a listen:
Banjos are plucked or strummed stringed devices whoever unique shades stem from strings becoming supported by a bridge that rests on a firmly stretched skin membrane. Historically, American banjos are descendants of a broadly associated category of lutes developed in western Africa from earlier in the day center Eastern models. The slave-trade brought banjo prototypes towards the "" new world "", in which these types of effective transforming forces as nineteenth-century minstrelsy and mass manufacture changed the banjo and its own associated playing types many times over.
The family of banjos these days includes four-string tenors (just like the standard banjo however with a reduced throat and no 5th sequence), plectrums (so called since they are used a plectrum and, in form, identical to the typical banjo but with no fifth string) and six-string guitar-banjos. Typical now's the five-string banjo, where the "fifth sequence" is a brief sequence usually tuned to function as a top drone or "chanterelle." Five-string banjos are present open-back folk or old-time kinds making use of instinct or steel strings also in resonator-backed variants, almost always steel-strung. Banjos might be fretted or fretless, acoustic or electric, mass-manufactured or separately handcrafted.
The banjo is visually and aurally very familiar instruments related to nation songs. To early tracks and broadcasts of country songs, the banjo brought not merely its unique frailing or finger-picked noises, but in addition its African and minstrel connotations. Both in the nineteenth-century minstrel program plus the early twentieth-century nation music tv show, banjo players usually played comedy roles and were frequently musically marginal, while they were considerable symbolically as well as their tonal contributions within an ensemble.
Beginning in the 1940s, Pete Seeger's revival for the five-string banjo to come with folk music began to introduce the tool to brand new northern and urban audiences. At the same time, the emergent sound of bluegrass music, built-in big component round the stylistic breakthrough into a smooth three-finger selecting style of Earl Scruggs, began to stimulate still another renaissance with this ancient instrument. Today the banjo is enormously preferred throughout the world, particularly the five-string kind played in bluegrass alongside kinds of folk and nation songs.
- Thomas A. Adler
- adjusted from the Country musical Hall of Fame® and Museum's Encyclopedia of nation musical, posted by Oxford University Press.
The bass is a stringed tool which provides a rhythmic "bottom" for the melody line.
Basses are offered in all sizes and shapes including the one-string washtub bass present in jug bands, the four-string acoustic standup (or "bull fiddle") favored by bluegrass and rockabilly bands, and also the electric four and five strings utilized in most contemporary nation.
a precursor towards the steel electric guitar, the Dobro ended up being developed by the Dopyera Brothers in the 1920s and modeled after the Hawaiian "slack" or resonator electric guitar. A twangy relative on slip guitar, the Dobro is played face up with some finger selections and a metal club which is used to fret strings.
Grand Ole Opry member "Bashful Brother Oswald" first popularized the Dobro as one of Roy Acuff's Smoky hill Boys before Flatt and Scruggs sideman Josh Graves made it a mainstay in bluegrass songs. Jerry Douglas and Mike Auldridge (Seldom Scene, Chesapeake) are today's acknowledged masters associated with the Dobro, both making use of pyrotechnics and an enlarged music palette to present the tool to a wider audience.
The drums weren't a part of the original tool setup utilized in nation songs. a modern drum or "pitfall" set frequently is made from a bass or "kick" drum, a snare, tom-toms, cymbals and a couple of "sticks" with which hitting the drum "heads."
Drums had been scorned by early nation performers to be "too loud" and "maybe not pure" - so much so, actually, one-story has Bob Wills' drummer having to relax and play behind a curtain at Grand Ole Opry in December 1944. With all the rise of rock 'n roll inside 1950s, Nashville purists had been much more adamant than ever about keeping drums out of their songs. By the early sixties, but ended up being unusual that a country band don't have a drummer.
Initially delivered to America from British Isles, the fiddle is without question one of the major instruments in nation songs. Recorded nation music began whenever Tx fiddler A. C. "Eck" Robertson recorded for Victor in ny in 1922. From 1923, sales of tracks by another fiddler, John Carson of Atlanta, resulted in the active exploitation of white southern rural music because of the phonograph business. Fiddlers eg Clark Kessinger from western Virginia, Clayton McMichen from Georgia, Charlie Bowman and Arthur Smith from Tennessee, and Doc Roberts from Kentucky recorded thoroughly during nation songs's first two decades and influenced numerous fiddlers in succeeding generations.
The fiddle lies at the heart of numerous country songs styles. Bob Wills and Milton Brown built the western move noise across the fiddle. Although fiddlers had been rare in mainstream jazz, many youthful fiddlers in Texas and Oklahoma into the 1930s and 1940s excitedly heard and took musical tips from jazz violinists Joe Venuti, Stuff Smith, and Stephane Grappelli. Cecil Brower, J. R. Chatwell, Hugh Farr (Sons for the Pioneers), Cliff Bruner, Joe Holley, Johnny Gimble, and others discovered making standard fiddle tunes swing, and just take hot choruses on new songs such as for example "remain all-night, Stay somewhat Longer, " "just take myself back once again to Tulsa, " "Fat child Rag, " and "San Antonio Rose."
Source: countrymusichalloffame.org
The Band Perry - DONE - 2013 Academy of Country Music ...
The Band Perry《If I Die Young (Academy Of Country Music ...
Act of Congress Country music band
Pussycat was a Dutch country and pop music group from Brunssum, driven by the three Kowalczyk sisters: Tonny, Betty and Marianne. Other members of the band were Lou Willé (Tonny's husband), Theo Wetzels, Theo Coumans and John Theunissen.
Prior to forming the band...
Marching Band Music video
40s Big Band music
What is Big Band music?
Band of Brothers theme sheet music


One of the first ways people interact with brands is via their website. It doesn't matter what you provide, you will always be searched. We work to provide a brand focused and distinct interactive tools for customers to experience your brand through website design. Using brand guidelines, values and overall ideals, we look to induce brand experience through a digital platform. Your brand should be experienced the same way everywhere, and online is no different.
A successful brand understands its target market. In order to understand that we need to empathize with both the brand and the customer. How can we create a connection between both?
In order to portray anything, there needs to be a culture and identity behind each brand. In developing this culture and identity, you are able to replicate the brand values and voice across any platform, including your website.
Online presence should replicate brand presence, whether it is in-store, value-based, culture-based, etc. Your website should tell your story and influence your target demographic.
Website interface should reflect and be reflected through all brand output. Whether customers see an ad or a post, it should always maintain the same design. At the same time, that design should be aesthetically practical.


Your dentist in South Philadelphia can help by providing professional treatments like dental sealants that cover the chewing surfaces to teeth to prevent bacteria and plaque from collecting in the natural pits and grooves of molars. Professional fluoride treatments may also be useful, especially if your children do not get enough fluoride from water consumption. Regular dental visits are the best way to ensure that tooth decay is addressed early. If you keep your children's dental appointments now, there will be fewer in the future.


Energy! Base Metals! Agriculture! and more!
Please give yourself an additional 10 minutes to sign in with security downstairs, and you will need photo ID.
There's no good story about parking where we are in midtown Manhattan.