During the month of September I have been helping my local scout troupe attain their Geocaching Scout Merit Badge. During my first lesson, I only had three boys show up. Then the word started spreading and by the last week we had all six boys in attendance.
I enjoy geocaching, but I have to admit, I am a novice at it. Sister Pat Nell, who is also in our ward has more experience with geocaching. She also has several handheld GPS units that she uses with her family whenever the go geo-hunting. So, when I agreed to help, I enlisted her assistance.
On Saturday, September 30th the boys who had attended all of the preparatory lessons, where ready for this last requirement. We took them out to the Jordan River Parkway geocaching. We all had a blast. Later that same weekend, our family went to the same spot for family pictures and our kids were able to find some of the same caches with me.


Together, we can create the Circle of Support your employees need to help ensure a full recovery.
Their circle can celebrate their integrity as they complete a simple, daily online check-in.
Hayver has been built on sound research. Programs that include a daily check-in, verified by random urine screens, show results that people improve their chances of staying in recovery with monitoring.
Uniquely, the Hayver system is the only one that is 1) online, 2) involves a Circle of Support that is informed about your Daily Check-In status, and 3) provides state-of-the-art urine testing that detects alcohol or drug use in the previous 5 days.
Based on the Hayver plan that is chosen, the minimum number of urine tests is two per month, up to 8 per month. Each test registers alcohol or drugs taken in the last 5 days.
So, rather than having to ask you, anyone in your Circle of Support can ask the system to have 'today' be the day you are required to do a urine test. However, you are not told whether today's test was ordered by the system, or ordered by someone in your Circle of Support.
Pricing is listed on the 'Getting Started' page of the website. However, the Hayver system starts as low as $29.95 per month, billed to a credit card for the Long-Term Recovery Plan. This includes the Daily Check In and one randomized alcohol and drug screen per month.
Many people start with the Quick Start Recovery Plan which includes the Daily Check In, three randomized alcohol and drug screens per month, along with three Spot Checks for the Circle of Support to implement. The price, with all of these extras, is just $74.95 per month. Our primary concern is that you are getting the program and support that will achieve the objective of you staying clean — forever.


Thyme - Kings County Nurseries, INC.
We have a 20,000 sq. ft. Nursery One of Brooklyn's Largest!
Description: Perennial Herb. The leaves have a marked, but agreeable, aroma and pungent flavor. They are used fresh or dried for seasoning clam chowder and other fish soups, stews, sauces and meat dishes. Thyme is noted for attracting bees.
© 2019 Kings County Nurseries, INC.


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The Tournament of the Golden Skull
By Julian | May 24, 2021
The Story Every five years a secretive council of elders hosts a grand competition in the Coliseum of Chrome to discover the greatest champion of the realm. This is the Tournament of the Golden Skull. This year combatants from across the land have trained hard and journeyed here to participate in deadly games, for glory and riches. The tournament consists of a variety of challenges in the form of a Battle Royale, where the contestants fight for their lives to win the Golden Skull, a glorious trophy which is given to the greatest champion of the realm. Each round, the arena's layout changes, and new hazards, traps, and challenges await each time the fighters enter the mystical battleground.
Style of Play Custom rules of my own design, inspired mostly by D&D and Pathfinder, as well as other roleplaying games. The rules are easy-to-learn and meant to make gameplay exciting and fast-paced! You will each build your own heroes from a set of skills, spells, abilities, and equipment. Every player will have a roster of 3 different heroes, and each round they will choose which hero to use. If your hero is knocked out during the fight, they've lost that round. If your hero is killed, you must choose from one of your remaining heroes to continue playing through the competition in future rounds.
After each round, the surviving heroes will get points / gold with which to upgrade their abilities and equipment. The winner of each round not only wins the most points / gold, but also gets a special bonus item that will help them in the next round. Spend your upgrades wisely! It could mean the difference between victory and utter defeat!
THIS IS A PVP (player vs. player) GAME — you will each be competing against your fellow players. Even though your heroes will be fighting for their lives, the players should focus on good sportsmanship and keeping the game friendly between you and the other players. Because this is a Battle Royale competition (a free-for-all fight until one hero remains) each player will be on their own, with no alliances or secret team-ups allowed (basically, no ganging up on anyone unfairly).
GM: Marc Berg
suggested Grades: 5th - 7th
limit: 5 players
6 Sessions, June 1, 2, 3, 4, 7 & 8
4-6pm CST
Custom Rules Inspired By D&D
Hosted on Roll20
Preparation A MICROPHONE IS REQUIRED. We will be using voice chat to communicate, and it is crucial that we can all hear each other. Headphones are encouraged, because they help cut down on feedback and echoing. We will spend the first 30-60 minutes of the first session learning how to play and choosing characters. You may receive some background information before the first session, but don't get ahead of yourself — all final decisions about our characters will be made on the first day. You'll be choosing from a series of pre-made characters, each with their own strengths and weaknesses. You will all need to register a free account on roll20.net, if you don't already have one. Everyone will receive an invitation link to the game in their email on the first day, before we play. PLEASE NOTE: it is NOT recommended that you use a tablet to play this game. A laptop or desktop computer works the best. But if a tablet is your only means of playing the game, you can still use one, just know that there may be some unintended issues or glitches that might arise because tablets are not "officially" supported by Roll20. Please let me know ahead of time if you plan on using a tablet, and I will send you some helpful tips that can help make the process as smooth as possible.
About the GM Marc Berg is a seasoned Game Master with over a decade of experience running tabletop games such as Pathfinder and D&D. He likes to run games with an equal focus on roleplaying, combat, and exploration; rewarding cooperation, creativity, and curiosity.
You can contact me at [email protected] with any questions you have about the game.
Posted in Finished Online RPG

redpajama_set_name: RedPajamaCommonCrawl


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FunderMax, one of Europe's leading providers of wood materials and decorative laminates for furniture, interior design and facades, is expanding worldwide using SAP software.
As a leader in innovation, the Austrian company will soon launch the use of iOS apps based on the SAP Cloud Platform and the SAP Cloud Platform SDK for iOS.
FunderMax offers materials that combine beauty with tangible quality – boasting an extensive product range which enables new design freedom, and leaves no limits to the creativity of architects, designers and for the furniture industry as a whole.
For indoor applications such as furniture design, shop interiors, wall cladding and partition walls, it's important to have good intuition for refined decors and sophisticated surfaces, as well as good taste and stylistic confidence in colours and shapes. When choosing materials, particularly for exterior applications, such as a facade or a balcony covering, design quality is just as important as product quality. FunderMax's comprehensive range of products creates upholds this, offering the perfect balance between interior and exterior.
For an innovative company such as FunderMax, the issue of digitisation is of great importance. "We have been working on our digital transformation for quite some time. We have invested a lot in training and education for our employees," explains Hartwig Schwarzlmüller, FunderMax's vice president of Organisation & Information Management/IT Management.
In order to drive the digital transformation, FunderMax uses powerful software tools: Two years ago the SAP HANA database was introduced. For one and a half years, FunderMax has been working on SAP Fiori, the UX-based user interface. Management is able to use a range of applications, enabling them to carry out everyday tasks quickly and easily on the move.
If an expense claim is submitted, the line manager receives a push notification and can react immediately by using an app on his or her iPhone. The next step on the path to digital transformation is the development of native iOS apps which run on the SAP Cloud Platform for FunderMax.
Digital Sales Support iOS App Under Development
150 sales employees are employed by FunderMax. Together with their sales partners, they are in contact with about 45,000 architects, processors and traders. To illustrate different application areas for the products, sales representatives refer to projects which have already been implemented. For the exterior product sector, an iOS app will soon help visualise these reference projects: Buildings can be sorted according to decors, surfaces or specific location and thereby presented to customers virtually.
Additional information, such as contact details of the architect or awards associated with a particular project, can also be retrieved – they can also be added to favourites and rated. In addition to the current 'real-life' sample collection, the entire decor collection can also be viewed digitally via the iPad app and displayed to customers on site.
Information can be filtered effortlessly according to desired criteria and subsequently suitable decor combinations can be presented to the client, much more easily and conveniently.
In a further version of the app, customers should be able to experience a three-dimensional reference object with desired decors and individual surroundings – for example in a winter landscape or at sunset. In doing so, it should not only be possible to take a 360° view, but also to view a building from different angles, for example, from the top of a hill.
FunderMax is currently working with its implementation partner Atos on the fast implementation of the Digital Sales Support app with the SAP Cloud Platform SDK for iOS (software development kit). The requirements are already documented and first results are expected at the end of April 2017. A key business area of ATOS's corporate strategy focuses on innovation in connection with SAP technologies and products. As a strong SAP Partner in the field of SAP HANA and SAP Cloud Platform, the largest European IT service provider assists companies in the definition and implementation of their digital roadmap. FunderMax is already progressing its digital roadmap; the sales app is just the beginning on the path towards a digital future.
Schwarzlmüller's vision of the future can be described in the following scenario: Customers upload a 3D model of their object and design the facade individually. Using Augmented Reality customers could see a building on-site and change its decors with simple gestures or hand movements. The app conveys a comprehensive impression, which allows the customer to experience the future result on a whole new, emotional level. As soon as the customer has decided, he or she can get a quotation directly from the app. When the order is placed with FunderMax, production can begin immediately.
Intuitive Customer Experience Thanks to SAP and Apple Integration
The intuitive state-of-the-art Fiori for iOS design not only improves the service of our sales force, but also strengthens our response to customers' needs and intensifies the customer experience: "With the app, we want our customers to experience a world that truly inspires them and helps them to make their ideas a reality" says Schwarzlmüller.
For Schwarzlmüller, the possibilities of native iOS apps are endless. For example, Sales Representatives could dictate visit reports straight into Siri, Apples speech robot, rather than painstakingly typing them up.
It is clear to see that FunderMax, a leader in innovation, is pushing ahead with its strategy of digital transformation. The company is truly focusing on the needs of its customers and employees with the implementation of an optimal mobile workplace, through the use of native iOS apps.
Top imave via FunderMax/Christof Lackner
via SAP News Center http://ift.tt/2lk8roI


I am a technical illustrator working in pen & ink, Illustrator and Photoshop, specializing in cutaway illustrations, step by step graphics, exploded views, info-graphics, medical art, conceptual artwork, charts and maps.
A product of the School of Visual Arts in New York City, I like to merge traditional techniques with digital art to get an organic look with the versatility of a digital file – the best of both worlds. I now find inspiration for my technical drawings on a hilltop in upstate Connecticut.
I meet project deadlines with attention to detail and deliver digital art on-target.


text:
Core Subjects
Subjects Home
You are here: Information > Virtual Open Evening > Creative Arts
The Snaith School Creative Arts
The Art and Design department is made up of three Art teachers, Mrs Nichols (Head of Art), Miss Speedy and Miss Bentley. The department also has a valued classroom assistant, Miss Barker. The department has two specialist teaching rooms with 20 computers contained within. In addition the department has a darkroom, kiln and facilities for print making.
Art is a popular and successful subject in both KS3 and KS4 with a large number of KS4 pupils progressing to further study at a number of colleges both near and further afield.
The Art department now offers Photography at KS4 as a GCSE option.
At Key Stage 3 pupils receive one lesson of Music every week. They study the various topics below, incorporating performing, composing, listening and appraising. Activities vary from whole class work (such as a class performance) and smaller group work to paired and individual work.
At The Snaith School we understand both the importance and relevance of Drama and recognize the wide range of ways Drama can influence and develop pupils in today's society. Drama is an exciting and creative subject that allows all pupils, of all backgrounds and abilities, to learn in a different and imaginative way. Pupils develop their confidence and performance skills while working in groups
and individually.
Subject Leader - Mrs Sampson
Teacher of Drama & Music – Miss Baines
Creative Arts Technical Subjects Humanities Home


Knowing what to invest and when to invest it, good financial advisors make the most of business resources. Infrastructure and Project Finance —We guide clients such as infrastructure equity funds, lenders, municipalities and other financing authorities, and private and public entities, in the acquisition, development, finance, and operation of a variety of infrastructure assets including tunnels, offsite airport parking lots, hospitals, power plants and transmission and distribution grids.
Perusahaan yang tidak menerapkan Good Corporate Governance malah dinilai lebih maju, karena prinsip keterbukaan perusahaan bagi sebagian pihak dianggap negative namun disisi lain, banyak juga perusahaan – perusahaan yang sudah merasakan ilai tambah dari aplikasi Good Corporate Governance, seperti lebih mudahnya akses pasar modal internasional serta banyaknya investor yang bersedia membayar premi yang lebih tinggi bagi saham perusahaan yang menerapakan Good Corporate Governance.
Provide overdraft agreements for the temporary advancement of the bank's own money to meet monthly spending commitments of a customer in their current account. Some customers may not want to visit a branch yet the knowledge that a branch exists instills a level of confidence amongst customers who might have relationships with banks through their deposits, loans, mortgages and so on. The recent financial downturn has made customers exercise more caution while dealing with finances and financial institutions.
In any commercial or industrial organization, whether big or small, accounting is always of great consequence towards effective administration. A few states allow a number of years of public accounting experience to substitute for a college degree. Along with a traditional undergraduate accounting major, SLU also offers an integrated undergraduate and graduate accounting program. The Work Environment tab includes the number of jobs held in the occupation and describes the workplace, the level of physical activity expected, and typical hours worked. Zahir Accounting Standard Versi 5.1 memberikan kemudahan dalam mengelola proyek, menghitung penyusutan fixed asset, sesuai untuk bidang usaha kontraktor, realestate, pabrik kecil, ritail, dll. In the career profiles below you'll learn how accounting careers differ from one another, along with what it takes to truly succeed in your career of choice. That is why cloud accounting systems are highly recommended for startups and SMEs.
Feel free to contact us: [email protected] , I have the Book Resources for the above textbook. The link between these decisions and firm value can be made by recognizing that the value of a firm is the present value of its expected cash flows, discounted back at a rate that reflects both the riskiness of the projects of the firm and the financing mix used to finance them.
To help the board fulfill its oversight function, it is important for the Executive Director and the Finance Committee to present the information in as clear and concise a manner as possible. If you have your heart set on corporate finance and analysis, do a knockout job during that particular rotation and develop a good relationship with your manager. That means more corporate spending, more mergers and acquisitions, and so on-and more work for corporate finance types.
Well, there are many reasons and one of the most prominent among them is that offshore outsourcing is carried out mainly from developing countries having a vast talent pool of young and ambitious professionals, for whom it is a dream come true to work for large financial companies listed on Wall Street. This is an all too common situation which can prove disastrous to many companies – especially smaller businesses and start-ups who are usually the least able to effectively deal with these types of situations due to generally low levels of liquid assets in the first place.


(MFWA/IFEX) - Armed operatives of the Bureau of National Investigation (BNI), Ghana's intelligence agency, on 12 January 2012 violently assaulted Gifty Lawson, a photojournalist of the "Daily Guide", a privately-owned newspaper on the premises of the Human Rights Court in Accra.
Two of the newspaper's security personnel, Michael Awampaga and Anthony Kwame Antwi, who were guarding her, suffered a similar fate. After their ordeal, Lawson and Awampaga were bundled into a vehicle and detained at the BNI headquarters. Lawson was released after an hour. Awampaga, however, is being held on a trumped up charge of "slapping" one of the BNI operatives.
The "Daily Guide" team had gone to the court to cover the story of bail being granted to a senior police officer, a prime suspect in a missing cocaine scandal that hit the country. The BNI had detained the police officer beyond the 48 hours that the constitution allows.
Confirming the attack to Media Foundation for West Africa (MFWA), Lawson said the armed officers of the BNI violently attacked her and the two security guards after she took photographs of the accused person. She stated that one official pulled her by the trousers exposing parts of her body in the process and bundled her into their vehicle to the national headquarters of the BNI.
On 11 January, Lawson and Richard Sky, another reporter of the Accra-based Citi FM were attacked by persons suspected to be relatives of the accused. Lawson, according to the privately-owned newspaper "The Enquirer", was beaten up and her camera destroyed beyond repair. The attacks were in the presence of policemen who took no action to stop the attack.
There has been widespread condemnation of the attack on the journalist. MFWA has called on the National Security Advisor and the Council of State to immediately bring to book officials of the BNI who violated the rights of these three persons.
"The MFWA fears there is a concerted attempt by some security operatives to obstruct journalists from performing their duties, especially in the coverage of suspected drug criminals. The violent attack on the "Daily Guide" staff is one of many acts perpetrated by operatives of the BNI and the police officers that violate rights of journalists to do their legitimate work in recent years. Indeed, it should be noted that in the Fourth Republic, the BNI and the police have been the major and only source of attacks on the rights of journalists in Ghana," stated MFWA.


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High mobility group box-1 contributes to anti-myeloperoxidase antibody-induced glomerular endothelial cell injury through a moesin-dependent route
Hui Deng1,
Chen Wang1,
Dong-Yuan Chang1,
Nan Hu1,
Min Chen1 &
Ming-Hui Zhao1
Arthritis Research & Therapy volume 19, Article number: 125 (2017) Cite this article
Our previous study found that circulating and urinary levels of high mobility group box-1 (HMGB1) were closely associated with disease activity in patients with antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Moreover, HMGB1 participates in ANCA-induced neutrophil activation. Cross-reactivity between moesin and anti-myeloperoxidase (MPO) antibody has been reported in both human and mouse. The current study investigated whether HMGB1 participated in MPO-ANCA-induced glomerular endothelial cell (GEnC) injury, which is one of the most important aspects in the pathogenesis of AAV.
The effects of HMGB1 on expression of moesin on GEnCs and anti-MPO antibody binding to GEnCs were measured. MPO expression on GEnCs was explored. The effects of HMGB1 in MPO-ANCA induced GEnC injury were measured, during which the role of moesin was explored. Antagonists for various relevant receptors were employed.
Sera from AAV patients at the active stage could mediate GEnC injury, while this effect could be attenuated by preblocking HMGB1. HMGB1 could increase the expression of moesin on GEnCs and the binding of anti-MPO antibody to moesin. The colocalization of moesin expression and anti-MPO antibody binding can be detected. Little, if any, MPO was expressed in GEnCs. HMGB1 increased GEnC activation and injury in the presence of patient-derived MPO-ANCA-positive IgGs through moesin. The effects of HMGB1 on expression of moesin on GEnCs, anti-MPO antibody binding to GEnCs, GEnC activation and injury were mainly toll like receptor 4 (TLR4) dependent.
HMGB1 can increase the expression of moesin but not MPO on GEnCs, and can further participate in MPO-ANCA-induced GEnC activation and injury by cross-reactivity between moesin and anti-MPO antibody.
Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) consists of granulomatosis with polyangiitis (GPA, previously named Wegener's granulomatosis), microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA) [1]. The serological markers for the aforementioned primary small vessel vasculitis are ANCAs, which recognize a variety of target antigens, in particular proteinase 3 (PR3) and myeloperoxidase (MPO). It is worth noting that Chinese patients with AAV are predominantly MPO-ANCA-positive, as demonstrated by our previous studies [2, 3]. One of the hallmarks of AAV is massive endothelial injury, especially glomerular endothelial cell (GEnC) injury, resulting in necrotizing vasculitis. ANCAs are proved to be involved in inducing and amplifying neutrophil-mediated endothelial injury in AAV [4, 5]. Nevertheless, there is also evidence supporting the direct ability of MPO-ANCA to produce vessel damage [6,7,8].
High mobility group box-1 (HMGB1) exists ubiquitously within the nucleus, playing its role of stabilizing the structure of nucleosomes and inducing DNA bending [9]. Upon certain stimulations, HMGB1 is released from various cells and becomes a proinflammatory mediator [10]. The signal pathways of HMGB1 involve a number of signaling molecules and receptors, including receptor for advanced glycation end products (RAGE) and Toll-like receptors TLR2 and TLR4 [11,12,13]. In our recent studies, we found that circulating and urinary levels of HMGB1 are associated with disease activity and renal damage in AAV patients [14, 15]. Moreover, HMGB1 participates in ANCA-induced neutrophil activation, indicating a pathogenic role of HMGB1 in AAV [16, 17].
Recently, Lee et al. [18] demonstrated that the HMGB1–RAGE–moesin axis could elicit severe inflammatory responses on human umbilical vein endothelial cells (HUVEC), during which HMGB1 exhibited an increase in phosphorylation of moesin and further secretion of moesin. Moesin, with the full name of membrane-organizing extension spike protein, is previously described as a cytoskeletal protein that belongs to the ezrin–radixin–moesin (ERM) family, which could function as links between the plasma membrane and the actin cytoskeleton [19]. More interestingly, Nagao et al. recently reported a direct activation of mouse GEnCs by anti-moesin activity of anti-MPO antibody [8]. In their study, the authors identified a cross-reactive molecule, which could be recognized by anti-MPO antibody, existing on mouse GEnCs. Later, the molecule was confirmed as moesin by mass spectrometry [8]. Given the potential effect of HMGB1 on upgrading moesin and the anti-moesin activity of anti-MPO antibody on GEnCs, we hypothesized that there is a moesin-dependent way through which HMGB1 can contribute to MPO-ANCA-induced GEnC injury.
Recombinant HMGB1 protein was purchased from R&D Systems (C23-C45 disulfide C106 thiol form; Abingdon, UK). The endotoxin level of HMGB1 was below the detection limit (0.125 EU/ml) of the Limulus assay (Sigma, St Louis, MO, USA). Anti-HMGB1 IgY was purchased from Shino-TEST (Sagamihara, Japan). Tumor necrosis factor alpha (TNF-α), lipopolysaccharides (LPS) and polymyxin B were purchased from Sigma. Monoclonal antibodies (mAbs) recognizing human moesin and MPO were purchased from Abcam (Cambridge, UK), with irrelevant IgG control antibodies. Polyclonal antibodies to MPO were purchased from Millipore (CA, USA). For inhibition assay, antibodies blocking moesin were purchased from BD Biosciences (San Jose, CA, USA). Normal human IgG and rabbit IgG were purchased from Sigma. Antibodies blocking the TLR2 and TLR4 were purchased from eBioscience (San Diego, CA, USA). RAGE-Fc was purchased from R&D Systems (Minneapolis, MN, USA). For immunofluorescence assay, AF488-labeled donkey anti-rabbit IgG and Cy3-labeled donkey anti-mouse IgG were purchased from Jackson ImmunoResearch Laboratories (Westgrove, PA, USA). For enzyme-linked immunosorbent assay (ELISA) experiments, human moesin protein was purchased from Sino Biological Inc. (Beijing, China). For flow cytometry analysis, phycoerythrin (PE)-conjugated goat anti-rabbit antibody and allophycocyanin (APC)-conjugated goat anti-mouse antibody were purchased from Abcam. For immunoprecipitation assay, rabbit anti-MPO antibody was purchased from BD Pharmingen (San Jose, CA, USA), while the control rabbit IgG was purchased from Santa Cruz (CA, USA). Peroxidase conjugated donkey anti-mouse IgG was purchased from Santa Cruz.
Blood samples of 10 active AAV patients with positive MPO-ANCA at initial onset and before commencing immunosuppressive therapy, diagnosed at Peking University First Hospital from August 2013 to June 2014, were collected in this study. All of the patients met the Chapel Hill Consensus Conference (CHCC) definition of AAV [1]. Patients with secondary vasculitis or with comorbid renal diseases, such as anti-glomerular basement membrane (GBM) nephritis, IgA nephropathy, diabetic nephropathy, lupus nephritis or membranous nephropathy, were excluded. Blood samples of five age and sex-matched healthy controls were collected. Venous blood was collected into red cap vacuum blood collection tubes. Collections of the whole blood sample were centrifuged at 3000 rpm for 10 min at 4 °C. Sera were then obtained and kept at –80 °C until use. The general information for these patients and healthy controls is presented in Table 1.
Table 1 General data for AAV patients and controls
Preparation of IgG
MPO-ANCA-positive IgGs were prepared from plasma exchange liquid of patients with active MPO-ANCA-positive primary small vessel vasculitis, using a High-Trap-protein G column on an AKTA-FPLC system (GE Biosciences, South San Francisco, CA, USA). Preparation of IgG was performed according to the methods described previously [20, 21]. In brief, plasma exchange liquid was filtered through a 0.2-mm syringe filter (Schleicher & Schuell, Duesseldorf, Germany) and applied to a High-Trap-protein G column on an AKTA-FPLC system (GE Biosciences). The column was treated with equal volume of 20 mmol/L Tris–HCl buffer, pH 7.2 (binding buffer), and IgG was eluted with 0.1 mol/L glycine–HCl buffer, pH 2.7 (elution buffer). After the antibodies emerged from the column, the pH was immediately adjusted to pH 7.0 using 2 mol/L Tris–HCl (pH 9.0). The protein concentration of the antibodies was measured using the Nandrop-1000 (Pierce, Rockford, IL, USA), and the level of anti-MPO IgG was measured by the ELISA kit (EUROIMMUN, Lubeck, Germany). We obtained written informed consent from the participants involved in our study. The research was in compliance of the Declaration of Helsinki and approved by the clinical research ethics committee of the Peking University First Hospital.
Primary GEnCs (ScienCell, San Diego, CA, USA) were cultured in endothelial cell basal medium (ECM) (ScienCell) with additional 10% fetal bovine serum (FBS), 1% penicillin/streptomycin and 1% endothelial cell growth factor in the formation of a confluent endothelial cell monolayer. The flasks for cell subculture were biocoated with human plasma fibronectin (Millipore, Billerica, MA, USA) beforehand according to the manufacturer's recommendation. For synchronization of the cell cycle, GEnC monolayers were starved in basal medium without FBS and endothelial cell growth supplement for 12 h without biocoating. All experiments were performed using GEnCs at passages 3–5. All cultures were incubated at 37 °C in 5% CO2. In order to investigate the effect of HMGB1 in the sera on GEnC injury, GEnC monolayers were incubated with ECM with additional 10% sera from either AAV patients or healthy controls for 4 h at 37 °C. For HMGB1 inhibition, GEnC monolayers were preincubated with 10 μg/ml anti-HMGB1 IgY for 1 h, which is the commercial anti-HMGB1 blocking antibodies isolated and purified from the egg yolk of HMGB1-immunized hens, followed by other treatments.
Measurement of moesin expression and the binding of anti-MPO mAb on GEnCs
The GEnC monolayers were incubated for 4 h with HMGB1 at a concentration of 10 ng/ml, which was comparable with the circulating HMGB1 level in active AAV patients [15], TNF-α, LPS, polymyxin B or buffer control. The time was set according to the result of time-dependent curve and cell conditions. In order to further investigate the role of candidate receptors of HMGB1 on GEnCs, the cells were first incubated with blocking antibodies and inhibitors (anti-TLR2 at 5 μg/ml; anti-TLR4 at 5 μg/ml; RAGE-Fc at 5 μM) or buffer control for 2 h. Next, cells were digested using trypsin to maintain in suspension. After washing, suspended cells were incubated with Trustain FcX™ Fc receptor blocking solution (Biolegend, CA, USA) for 10 min, and then stained with a saturating dose of rabbit antibody against human moesin or with an irrelevant IgG control antibody for 30 min, followed by incubation with PE-conjugated goat anti-rabbit antibody for another 30 min. For double staining of moesin and MPO on GEnCs, the suspended cells were stained with saturating doses of rabbit antibody against human moesin and mouse antibody against human MPO, or with irrelevant IgG control antibodies for 30 min, followed by incubated with both PE-conjugated goat anti-rabbit antibody and APC-conjugated goat anti-mouse antibody for 30 min consistently. After washing with phosphate buffer saline (PBS), prepared cells were analyzed using a FACSCalibur flow cytometer (Influx™). Cells were gated in forward/sideward scatter (FSC/SSC) and data were collected from 10,000 cells per sample.
For fluorescent microscopy, GEnCs were grown for 48–72 h in slide culture chambers (Nunc, Roskilde, Denmark) until confluent. The procedures were conducted as described previously [22]. After treatment, the GEnC monolayers were fixed in cold 4% paraformaldehyde (PFA) for 4 h at 4 °C. After multiple washing with PBS, coverslips with the fixed cells were removed from the plates. Nonspecific binding sites were blocked in PBS containing 5% donkey serum. We incubated the specimens with rabbit mAb against moesin (1:100 dilution) combined with mouse antibody against MPO (1:100 dilution) in blocking buffer for 1 h at 37 °C. After washing with PBS, AF488-labeled donkey anti-rabbit IgG or Cy3-labeled donkey anti-mouse IgG (both 1:500 dilution) was applied for 1 h. The samples were washed with PBS and distilled water, stained with DAPI and mounted with Mowiol. All images were collected and analyzed with a fluorescent microscope (Nikon 80I). The percentage of anti-MPO antibody binding with moesin was analyzed with Image-Pro Plus v6.0.
GEnCs were cultured on a culture plate until confluence. After washing twice with PBS, the cells were lysed and the sample was separated by SDS-PAGE. After identification by coomassie blue staining, the bands on gel were divided into 10 parts according to protein abundance and molecular weights. Each part of the sample was applied to mass spectrometry to detect the level of MPO expression in GEnCs, in order to explore the possibility of anti-MPO antibody binding with intrinsic MPO in GEnCs.
After treatment, the GEnC monolayers were lysed in ice-cold RIPA buffer at 4 °C for 30 min. The cell lysates were then mixed with polyclonal antibodies to MPO, or rabbit normal IgG as control, and incubated overnight at 4 °C on a rotating device. After mixing with Protein A/G PLUS-Agarose (Santa Cruz) for another 3 h at 4 °C, the immunoprecipitates were washed in RIPA buffer five times, and then the boiled samples were separated by SDS-PAGE and detected by anti-moesin mAb. Finally, the strips were incubated with peroxidase-conjugated affinity-purified donkey anti-mouse IgG (1:5000 dilution) for 1 h at room temperature with gentle agitation and then were revealed on autoradiographic film using the ECL Plus Western Blotting Detection System (GE Healthcare, Piscataway, NJ, USA).
The binding of moesin and anti-MPO antibody was further detected by ELISA with human moesin at 2 μg/ml as the solid-phase antigen, bovine serum albumin (BSA) at the same concentration as control and anti-MPO polyclonal antibody (1:2000 dilution) as the detection antibody. Anti-MPO antibody was added to the antigen-coated microtiter plate and incubated for 1 h at 37 °C. Anti-rabbit IgG alkaline phosphatase antibody produced in goat was then added to the wells. After adding to the stop solution, the absorption measurement was obtained at 405 nm using a microtiter plate reader (Bio-Rad iMark™ Microplate Reader).
Measurements of GEnC activation and injury in vitro
Measurement of soluble ICAM-1 and soluble VCAM-1
Levels of soluble intercellular cell adhesion molecule-1 (sICAM-1) and soluble vascular cell adhesion molecule-1 (sVCAM-1) were considered biomarkers of endothelial cell activation and injury [23]. After treatment, the cell culture supernatant was collected for the ICAM-1 and VCAM-1 assays. Samples were tested using the human ICAM-1/CD54 and human VCAM-1/CD106 ELISA kit (R&D, Abingdon, UK). The assay was conducted according to the manufacturer's instructions. In brief, samples were added to the microtiter plate coated with capture antibody and incubated for 2 h at room temperature, followed by detection antibody incubation for another 2 h. Horseradish peroxidase-conjugated streptavidin was then added. After 20 min of incubation avoiding direction light, the plates were washed and substrate solution was added to the wells. After adding to the stop solution, the absorption measurements were obtained at 450 nm (with a correction of 570 nm to eliminate optical imperfections in the plate) using a microtiter plate reader (Bio-Rad iMark™ Microplate Reader). All samples and standards were performed in duplicate.
The cytotoxic effect of HMGB1 or/and MPO-ANCA-positive IgGs was determined by measuring the secretion of lactate dehydrogenase (LDH) using the Cytotoxicity Detection Kit (Roche Diagnostics, Mannheim, Germany) according to manufacturer's recommendation. To demonstrate the effects of moesin in this process, the cells were preincubated with blocking antibodies to moesin with a saturated dosage before treated with HMGB1 and/or MPO-ANCA-positive IgGs.
Permeability assay
GEnC monolayer permeability was determined with fluorescein isothiocyanate (FITC)-labeled BSA (Sigma), as described previously [24]. Cells were grown on Costar Transwell 0.4-μm porous filters (Coming, Acton, USA) until confluent. Also, in order to demonstrate the effect of moesin in this process, the cells were preincubated with blocking antibody to moesin before treatment with anti-MPO antibody, MPO-ANCA-positive IgGs and/or HMGB1. After treatment, the tracer protein FITC-albumin was added to the luminal chamber for 45 min, and samples were taken from both the luminal and abluminal chambers for fluorometry analysis. Fluorescence signals were measured in a microplate fluorescence reader (Tristar™ LB941) with filter settings of 485 nm (excitation) and 538 nm (emission). These readings were then used to determine the permeability coefficient of albumin which could stand for the vascular barrier disruption. All data are shown as the ratio of control at the corresponding test.
Quantitative data were expressed as mean ± SD (for data that were normally distributed) or median and quartiles (for data that were not normally distributed) as appropriate. Differences of quantitative parameters between groups were assessed using one-way ANOVA (for data that were normally distributed) or Mann–Whitney U test (for data that were not normally distributed) as appropriate. Differences were considered significant when p < 0.05. Analysis was performed with SPSS statistical software package (version 13.0; Chicago, IL, USA).
HMGB1 participated in GEnC injury mediated by sera from AAV patients
Compared with GEnCs incubated with ECM with additional 10% sera from healthy controls, the levels of LDH release increased significantly in GEnCs incubated with ECM with additional 10% sera from AAV patients (1.44 ± 0.16 vs 1.18 ± 0.11, p < 0.01). By preincubating anti-HMGB1 IgY, the levels of LDH release decreased significantly in GEnCs incubated with ECM with additional 10% sera from AAV patients (1.44 ± 0.16 vs 1.30 ± 0.21, p = 0.02) (Fig. 1).
HMGB1 participated in the GEnC injury mediated by sera from AAV patients. Levels of LDH release increased significantly in GEnCs incubated with ECM containing sera from AAV patients and decreased significantly by preincubating anti-HMGB1 IgY. Bars represent mean ± SD of repeated measurements from three independent experiments. AAV ANCA-associated vasculitis, ANCA antineutrophil cytoplasmic antibody, HMGB1 high mobility group box-1, LDH lactate dehydrogenase, OD optical density
HMGB1 increased the expression of moesin on GEnCs
Expression of moesin on GEnCs in different experimental groups in vitro was analyzed using flow cytometry. Compared with untreated cells, the level of moesin expression was significantly higher on GEnCs treated with HMGB1 at concentration of 10 ng/ml, TNF-α at 15 ng/ml or LPS at 90 ng/ml (343 ± 55 vs 207 ± 33, p < 0.01; 293 ± 66 vs 207 ± 33, p = 0.03; or 303 ± 41 vs 207 ± 33, p = 0.02, respectively). No obvious moesin expression increase was observed in cells treated with polymyxin B at 20 μg/ml (224 ± 47 vs 207 ± 33, p = 0.65) (Fig. 2).
HMGB1 increased the expression of moesin on GEnCs. Expression of moesin on GEnCs was significantly increased with the treatment of HMGB1, LPS and TNF-α. Bars represent mean ± SD of repeated measurements from three independent experiments. HMGB1 high mobility group box-1, LPS lipopolysaccharides, MFI mean fluorescence intensity, TNF-α, Tumor necrosis factor alpha
Binding of anti-MPO mAb to GEnCs treated with HMGB1 through moesin increased significantly
Given the cross-reaction to moesin of anti-MPO antibody [8], we studied whether the binding of anti-MPO mAb to GEnCs increased with the treatment of HMGB1.
Indirect immunofluorescent assay was performed to study the expression of moesin and the binding of anti-MPO mAb to GEnCs. Compared with untreated cells, moesin expression and the binding of anti-MPO mAb significantly increased on HMGB1-treated GEnCs. Moreover, colocalization of moesin expression and anti-MPO mAb binding were more obvious in HMGB1-treated cells than in untreated cells (Fig. 3a, b). The percentage of colocalization for moesin and anti-MPO mAb was about 6% after HMGB1 stimulation.
HMGB1 increased the binding of anti-MPO mAb to GEnCs. Compared with untreated cells, moesin expression and the binding of anti-MPO monoclonal antibody (mAb) were upregulated on HMGB1-treated GEnCs (a, b). Binding of anti-MPO mAb significantly increased on HMGB1-treated GEnCs compared with untreated cells (c). Bars represent mean ± SD of repeated measurements from three independent experiments. Representative histogram of effects of HMGB1 on the binding of anti-MPO monoclonal antibody to GEnCs (d). Proportion of GEnCs with both upregulated moesin expression and increased anti-MPO monoclonal antibody binding elevated in HMGB1-treated cells (e, f). Moesin on GEnCs could be captured by polyclonal antibodies to MPO by immunoprecipitation (g). HMGB1 high mobility group box-1, MPO myeloperoxidase, MFI mean fluorescence intensity
Mass spectrometry showed that little, if any, MPO was expressed in GEnCs, suggesting that the binding of anti-MPO antibody with moesin detected by immunofluorescent assay was not via MPO (see Additional file 1: Table S1 and Additional file 2: Table S2).
Flow cytometry assay was further performed to study the change of proportions of GEnCs with both upregulated moesin expression and increased anti-MPO mAb binding. Compared with untreated cells, the binding of anti-MPO mAb increased significantly (2418 ± 248 vs 1497 ± 139, p < 0.01), and the proportion of GEnCs with both upregulated moesin expression and increased anti-MPO mAb binding elevated in HMGB1-treated cells also increased (Fig. 3c–f).
The binding of anti-MPO IgG to moesin was confirmed by immunoprecipitation. It was found that moesin in GEnCs was recognized by anti-MPO antibody (Fig. 3g). The binding was further detected by ELISA, showing that the level of anti-MPO antibody binding to coated moesin was significantly higher than that of anti-MPO antibody binding to BSA control (expressed by OD values: 0.03 ± 0.0040 vs –0.016 ± 0.011, p < 0.01).
HMGB1 increased GEnC activation and injury in the presence of patient-derived MPO-ANCA-positive IgGs, while HMGB1-induced GEnC injury was attenuated by blocking moesin
We next studied the effect of HMGB1 on activation and subsequent injury of GEnCs.
sICAM-1 and sVCAM-1 were considered markers of endothelial cell activation as already mentioned. Compared with untreated cells, the levels of both sICAM-1 and sVCAM-1 increased significantly in the supernatants of GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs (expressed as percentages of the control: 180 ± 19% vs 100%, p < 0.01; and 484 ± 29% vs 100%, p < 0.01, respectively). Levels of these markers also increased in the supernatants of GEnCs treated with patient-derived MPO-ANCA-positive IgGs alone (160 ± 32% vs 100%, p < 0.01; and 453 ± 16% vs 100%, p < 0.01, respectively). However, compared with untreated cells, sICAM-1 and sVCAM-1 levels still increased significantly in the supernatants of GEnCs treated with HMGB1 plus anti-MPO polyclonal antibody (166 ± 27% vs 100%, p < 0.01; and 289 ± 10% vs 100%, p < 0.01, respectively). No obvious GEnC activation was observed in cells treated with normal human IgG, rabbit IgG or HMGB1 alone (Fig. 4a, b).
HMGB1 increased GEnC activation and injury in the presence of patient-derived MPO-ANCA-positive IgGs through moesin. Levels of both sICAM-1 and sVCAM-1 increased significantly in the supernatants of GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs compared with untreated cells, cells treated with HMGB1 or patient-derived MPO-ANCA-positive IgGs alone (a, b). Levels of LDH release increased significantly in the supernatants of GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs compared with untreated cells, cells treated with HMGB1 or patient-derived MPO-ANCA-positive IgGs alone (c). Levels of vascular barrier disruption increased significantly in GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs compared with untreated cells, cells treated with HMGB1 or patient-derived MPO-ANCA-positive IgGs alone (d). By preincubating with anti-moesin antibodies, levels of cell activation, LDH release and levels of vascular barrier disruption decreased significantly (a–d). Bars represent mean ± SD of repeated measurements from five independent experiments. Ab antibody, ANCA antineutrophil cytoplasmic antibody, GEnC glomerular endothelial cell, HMGB1 high mobility group box-1, LDH lactate dehydrogenase, MPO myeloperoxidase, OD optical density, sICAM-1, soluble intercellular cell adhesion molecule-1, sVCAM-1, soluble vascular cell adhesion molecule-1
LDH assay was performed to study the viability of GEnCs in different experimental groups. Compared with untreated cells, cells treated with HMGB1 or patient-derived MPO-ANCA-positive IgGs alone, the levels of LDH release increased significantly in the supernatants of GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs (expressed by OD values: 1.03 ± 0.03 vs 0.41 ± 0.10, p < 0.01; 1.03 ± 0.03 vs 0.56 ± 0.20, p < 0.01; and 1.03 ± 0.03 vs 0.79 ± 0.06, p < 0.01, respectively). Similarly, compared with cells treated with HMGB1 or anti-MPO polyclonal antibody alone, the levels of LDH release increased significantly in cells treated with HMGB1 plus anti-MPO polyclonal antibody (expressed by OD values: 0.85 ± 0.04 vs 0.56 ± 0.19, p < 0.05; and 0.85 ± 0.04 vs 0.61 ± 0.02, p < 0.01, respectively). No obvious LDH release was observed in cells treated with normal human IgG, rabbit IgG or HMGB1 alone (Fig. 4c).
Endothelial cell permeability assay was performed to study the vascular barrier disruption in different experimental groups. Compared with untreated cells, cells treated with HMGB1 or patient-derived MPO-ANCA-positive IgGs alone, the levels of vascular barrier disruption increased significantly in GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs (expressed as percentages of the control: 148 ± 8.2% vs 100%, p < 0.01; 148 ± 8.2% vs 96 ± 14%, p < 0.01; and 148 ± 8.2% vs 134 ± 8.9%, p < 0.01, respectively). Similarly, significant increase of vascular barrier disruption was also detected in GEnCs treated with HMGB1 plus anti-MPO polyclonal antibody, as compared with cells treated with anti-MPO polyclonal antibody alone (173 ± 6.5% vs 129 ± 15%, p < 0.01) (Fig. 4d). By preincubating with anti-moesin antibodies, the levels of sICAM-1 and sVCAM-1, LDH release and the levels of vascular barrier disruption in cells treated with MPO-ANCA-positive IgGs plus HMGB1 decreased significantly (163 ± 28% vs 181 ± 19%, p < 0.05; 448 ± 26% vs 484 ± 29%, p < 0.05; 0.58 ± 0.12 vs 1.03 ± 0.03, p < 0.01; and 128 ± 22% vs 148 ± 8.2%, p = 0.02, respectively) (Fig. 4a–d).
Collectively, these data suggested that HMGB1 increased GEnC activation and injury in the presence of patient-derived MPO-ANCA-positive IgGs via a moesin-dependent route.
Effects of HMGB1 on expression of moesin on GEnCs, anti-MPO mAb binding to GEnCs, GEnC activation and injury were mainly TLR4 dependent
To further investigate the role of candidate receptors through which HMGB1 exerts its effect, GEnCs were preincubated with anti-TLR2 at 10 μg/ml, anti-TLR4 at 10 μg/ml or RAGE-Fc at 10 μM to block corresponsive receptors.
Using flow cytometry, parallel experiments blocking TLR4 and RAGE resulted in a significant decrease in HMGB1-induced expression of moesin. Moesin expression decreased from 2258 ± 618 in HMGB1-treated GEnCs, to 991 ± 79 by preincubating with anti-TLR4 antibody (p < 0.01) or to 1641 ± 179 by preincubating with RAGE antagonist (p = 0.02), while there was no significant decrease in cells preincubated with anti-TLR2 antibody (2258 ± 618 vs 2102 ± 86, p = 0.53) (Fig. 5a, b). Subsequently, anti-MPO mAb binding decreased on preincubation with anti-TLR4 antibody and RAGE antagonists, while no significant decrease occurred in GEnCs preincubated with anti-TLR2 antibody (Fig. 5c–g). Although blocking RAGE resulted in a decrease in HMGB1-induced expression of moesin and increased binding of anti-MPO mAb, the change seemed to be mild.
Effects of HMGB1 on expression of moesin on GEnCs, anti-MPO mAb binding to GEnCs, GEnC activation and injury were mainly TLR4 dependent. Representative histogram for expression of moesin on GEnCs (a). Blockage of TLR4 and RAGE rather than TLR2 decreased expression of moesin on HMGB1-treated GEnCs (b). Among them, TLR4 had a dominate effect. Bars represent mean ± SD of repeated measurements on neutrophils for five independent experiments. Blockage of TLR4 rather than RAGE or TLR2 decreased the binding of anti-MPO mAb to GEnCs (c–g). Blockage of TLR4 rather than RAGE or TLR2 decreased the levels of LDH release (h). Blockage of TLR4 and RAGE rather than TLR2 decreased the levels of vascular barrier disruption decreased significantly (i). Bars represent mean ± SD of repeated measurements on neutrophils from five independent experiments. mAb monoclonal antibody, ANCA antineutrophil cytoplasmic antibody, GEnC glomerular endothelial cell, HMGB1 high mobility group box-1, LDH lactate dehydrogenase, MPO myeloperoxidase, MFI mean fluorescence intensity, OD optical density, RAGE receptor for advanced glycation end product, TLR Toll-like receptor
Consistently, the injury induced by HMGB1 and patient-derived MPO-ANCA-positive IgGs also decreased in GEnCs preincubated with anti-TLR4 antibody or RAGE antagonist. In LDH assay, the levels of LDH release decreased significantly in the supernatants of GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs by preincubating with anti-TLR4 antibody (expressed by OD values: 0.74 ± 0.06 vs 1.04 ± 0.22, p < 0.01) (Fig. 5h). In endothelial cell permeability assay, the levels of vascular barrier disruption decreased significantly in GEnCs treated with HMGB1 plus patient-derived MPO-ANCA-positive IgGs by preincubating with anti-TLR4 antibody and preincubating with RAGE antagonist (expressed as percentages of the control: 144 ± 16% vs 171 ± 7%, p < 0.01; and 152 ± 4% vs 171 ± 7%, p = 0.04, respectively) (Fig. 5i). The effects caused by preincubating with RAGE antagonist seemed to be mild. Collectively, the effects of HMGB1 on expression of moesin on GEnCs, anti-MPO mAb binding to GEnCs, GEnC activation and injury were mainly TLR4 dependent.
In the current study, we observed that sera from AAV patients at the active stage could mediate GEnC injury, while the effect could be attenuated by preblocking HMGB1, indicating that HMGB1 participated in the GEnC injury in AAV. Then we further found that HMGB1 could increase the expression of moesin on GEnCs and the binding of anti-MPO mAb to GEnCs due to the cross-reaction of anti-MPO antibody to moesin. In such circumstances, GEnCs could be activated and sequentially be damaged.
As reported by Pendergraft et al. [25], MPO is not expressed in endothelial cells. Nagao et al. identified a cross-reactive molecule on mouse GEnCs, which could be recognized by anti-MPO antibody, termed moesin [8]. They assumed that moesin shares certain similar sequences with those on the N-terminal region of the MPO heavy chain. Furthermore, the same group also demonstrated that MPO-ANCA from patients with active AAV had high reactivity to the aforementioned sequences in human [26, 27]. In our study, we detected the binding of anti-MPO antibody to moesin by immunofluorescence. Also, MPO was scarcely expressed in GEnCs, as measured by protein mass spectrometry, suggesting that the binding of anti-MPO antibody to the GEnC surface was not via MPO. In addition, binding of anti-MPO antibody to moesin was further confirmed by immunoprecipitation and ELISA. However, human MPO has much lower homology with human moesin. Other than molecular mimicry with similar sequences [28], the cross-reaction observed in our study might also be attributed to other mechanisms such as spatial distribution of charge or certain intermediators [29,30,31], which need further investigations to confirm.
In the current study, sICAM-1 and sVCAM-1 were employed as markers of endothelial cell activation. Generally, biomarkers of endothelial cell activation can be divided into soluble markers, among which sICAM-1, sVCAM-1 and VEGF are typical, and cell component markers, including circulating endothelial cells (CECs) and endothelial microparticles (EMPs) [23, 32]. In AAV, the activated endothelial cells would be further damaged mainly in two ways. First, upregulated cell adhesion molecules make endothelial cells more vulnerable to the attack of neutrophils [4, 33]. Second, the activated cells would then detach and undergo necrosis and apoptosis, thus increasing vascular permeability and causing vascular barrier disruption in specific stimulating conditions [34].
Among the various receptors which HMGB1 can recognize on the cell surface of GEnCs, we found TLR4 is the dominant receptor through which HMGB1 exerts the aforementioned moesin-dependent effects. Lee et al.'s study [18] found that the HMGB1–RAGE–moesin axis could elicit severe inflammatory responses on HUVEC. Different types of endothelial cells and dosages of HMGB1 employed might contribute to such discrepancy of receptors. To be specific, different types of endothelial cells might have different responses to inflammatory conditions or mediators [35, 36], which is also indicates necessity to start with the main affected endothelial cell types involved in the pathogenesis of AAV. We applied HMGB1 at a concentration of 10 ng/ml, which was comparable with the circulating HMGB1 level in active AAV patients [15], in order to simulate the pathophysiologic conditions in AAV.
There are some limitations in this study. First, patient-derived MPO-ANCA-positive IgG rather than affinity-purity MPO-ANCA was used in most assays. The reason for this was that we were not able to acquire sufficient purified MPO-ANCA to accomplish the total experiments. However, commercial anti-MPO antibody was used; similar levels of cell activation and LDH release were observed, as compared with patient-derived MPO-ANCA-positive IgG. Second, we detected that the HMGB1 inhibition did not totally abrogate GEnC injury mediated by serum from AAV patients. The possible reason for this is that, besides HMGB1, some other proinflammatory factors in serum of AAV patients might also upregulate moesin expression. However, GEnC injury mediated by sera from AAV patients can be significantly attenuated by blocking HMGB1, suggesting that HMGB1 was important for GEnC injury.
HMGB1 participates in MPO-ANCA-induced GEnC activation and injury through a moesin-dependent route, as schematized in Fig. 6. The current finding could explain, at least to some extent, the direct ability of MPO-ANCA to produce vessel damage, which provides us with more clues to determine the exact pathogenic role of ANCA in AAV.
Proposed working model for the role of HMGB1 in anti-MPO antibody-induced GEnC injury. HMGB1 could increase the expression of moesin on GEnCs and further the binding of anti-MPO antibody to GEnCs due to the cross-reaction of anti-MPO antibody to moesin. In such circumstances, GEnCs could be activated and sequentially be damaged. HMGB1 high mobility group box-1, LDH lactate dehydrogenase, MPO myeloperoxidase, RAGE receptor for advanced glycation end product, sICAM-1 soluble intercellular cell adhesion molecule-1, sVCAM-1 soluble vascular cell adhesion molecule-1, TLR Toll-like receptor
AAV:
ANCA-associated vasculitis
ANCA:
Antineutrophil cytoplasmic antibody
BVAS:
Birmingham Vasculitis Activity Score
ECM:
Endothelial cell basal medium
EGPA:
Eosinophilic granulomatosis with polyangiitis
ERM:
Ezrin–radixin–moesin
GEnC:
Glomerular endothelial cell
HMGB1:
High mobility group box-1
HUVEC:
Human umbilical vein endothelial cells
LDH:
Lactate dehydrogenase
LPS:
Lipopolysaccharides
mAb:
MPA:
Microscopic polyangiitis
PBS:
Phosphate buffer saline
PR3:
Proteinase 3
RAGE:
Receptor for advanced glycation end product
sICAM-1:
Soluble intercellular cell adhesion molecule-1
Soluble vascular cell adhesion molecule-1
Toll-like receptor
TNF-α:
Tumor necrosis factor alpha
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This study is supported by three grants of the National Natural Science Fund (No. 81425008, No. 81501392 and No. 81621092), by a grant from the National Key Research and Development Program (No. 2016YFC0906102), and by a grant from the University of Michigan Health System and Peking University Health Sciences Center Joint Institute for Translational and Clinical Research.
The datasets used or analyzed during the current study are available from the corresponding author on reasonable request.
HD collected samples and clinical information, carried out cell culture, IgG preparation, measurement of GEnC activation and injury, ELISA and data analysis, and was a major contributor in writing the manuscript. CW participated in immunofluorescence and immunoprecipitation, and helped to draft the article. D-YC and NH participated in sample collection and data analysis. MC and M-HZ participated in study design and approved the final manuscript. All authors read and approved the final manuscript.
The research was approved by the clinical research ethics committee of the Peking University First Hospital. All patients provided written informed consent including consent to publish and report individual patient data.
Renal Division, Department of Medicine, Peking University First Hospital, Institute of Nephrology, Peking University; Key Laboratory of Renal Disease, Ministry of Health of China, Beijing, 100034, China
Hui Deng, Chen Wang, Dong-Yuan Chang, Nan Hu, Min Chen & Ming-Hui Zhao
Hui Deng
Dong-Yuan Chang
Nan Hu
Min Chen
Ming-Hui Zhao
Correspondence to Min Chen.
Presenting a summary of the mass spectrometry results. (XLS 23 kb)
Presenting information for proteins detected in GEnCs. (XLS 632 kb)
Deng, H., Wang, C., Chang, DY. et al. High mobility group box-1 contributes to anti-myeloperoxidase antibody-induced glomerular endothelial cell injury through a moesin-dependent route. Arthritis Res Ther 19, 125 (2017). https://doi.org/10.1186/s13075-017-1339-4


text:
package org.elasticsearch.search.aggregations.matrix;

import org.elasticsearch.plugins.Plugin;
import org.elasticsearch.search.SearchModule;
import org.elasticsearch.search.aggregations.matrix.stats.InternalMatrixStats;
import org.elasticsearch.search.aggregations.matrix.stats.MatrixStatsAggregationBuilder;
import org.elasticsearch.search.aggregations.matrix.stats.MatrixStatsParser;

import java.io.IOException;

public class MatrixAggregationPlugin extends Plugin {

    public void onModule(SearchModule searchModule) {
        InternalMatrixStats.registerStreams();
        searchModule.registerAggregation(MatrixStatsAggregationBuilder::new, new MatrixStatsParser(),
            MatrixStatsAggregationBuilder.AGGREGATION_NAME_FIELD);
    }
}

redpajama_set_name: RedPajamaGithub


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Inyathelo, the South African Institute for Advancement, has announced the appointment of Jessica Rees-Jones as its new executive director from 1 April.
Ms Rees-Jones takes over from acting executive director Nomfundo Walaza, renowned for her human rights-related work both in South Africa and internationally.
Ms Rees-Jones's experience includes working as a specialist consultant to both the Graca Machel Trust and power producer Cennergi; and CEO of the Royal Bafokeng Nation Foundation. She was foundation director and director of advancement for the Diocesan College in Cape Town (Bishops) and development director for Vista Nova School for children with special needs. She has qualifications in non-profit leadership and strategy from Stellenbosch University.
Zenariah Barends, chairperson of the Inyathelo Board of Trustees, said: "Inyathelo is at the helm of providing organisational development and support services to an extensive array of civil society and related organisations.


This sweet crochet baby bib is adapted from a vintage pattern and perfect for a toddler. Almost too pretty to use as a bib but adorable to dress up an outfit. Using a pattern from my husband's, Aunt Marie, I made a crochet baby bib for little Chloe.
Our sweet baby girl is just getting the hang of walking.
Aunt Marie liked to crochet too. She made these two little cotton bibs back in 1978 for our oldest daughter, Erin.
Last month, we took Chloe's, older sisters, Keri and Cambrie with us to visit Aunt Marie who will be celebrating her 96th birthday in June. I brought my work-in-progress along to show her.
These photos show the progress of this larger, slightly adapted baby bib.
As you can see, my bib is much larger than the one Aunt Marie made. I used TLC Cotton Plus (color: light rose) and F crochet hook, so the finished size of my bib is much larger than the original pattern bib (below).
*Knit-Cro-Sheen is a registered trademark of the Coats & Clark Co.
Starting at Neck with White, ch 56.
Row 1: Sc in 2nd ch from hook and in each ch across. ch 3, turn.
Row 2: Skip first sc, dc in next 9 sc, *ch 1, skip next 2 sc, in next sc make 2 dc, ch 2 and 2 dc (shell made); ch 1, skip next 2 sc, dc in next 10 sc. Repeat from * across. Ch 3, turn.
Row 3-6 : Skip first dc, *dc in each dc across to next ch 1 sp, dc in sp, ch 1, shell in sp of next shell (shell made over shell), ch 1, dc in ch 1 sp. Repeat from * across. Dc in top of turning chain. Break off at end of 6th row. Turn.
Row 7: Attach White to first dc following first shell on previous row, ch 3, dc in each dc across to next ch 1 sp, dc in sp, ch 1, shell over shell, ch 1, dc in next ch 1 sp, dc in each dc across to next ch 1 sp. Ch 3, turn. Work in pattern until 19 dc rows in all are completed. Break off.
Row 1: Attach White to top of turning chain at beginning of 6th row, sc in same place, (ch 2, skip next 2 dc, shell in next dc, ch 2, skip next 2 dc, sc in next sc) twice; ch 2, shell over shell, ch 2, sc in base of first st on 7th row, (ch 2, skip next row, shell in end st of next row, ch 2, skip next row, sc in end st of next row) 3 times; ch 2, skip next row, in end st of next row make (2 dc, ch 2) twice and 2 dc; (ch 2, skip next 2 dc, sc in next dc, ch 2, skip next 2 dc, shell in next dc) 5 times; ch 2, sc in first dc of next shell, ch 2, shell over same shell, ch 2, skip next dc of same shell, sc in next dc, ch 2, shell in next dc, ch 2, skip next 2 dc, sc in next dc. Complete row as for other side. Break off. Attach Blue to first sc on previous row, sc closely around entire Bib. Break off.
Starting at center with White, ch 44.
Row 1: Dc in 4th ch from hook and in each ch across, ch 6 for buttonhole, working along opposite side of starting chain, make dc in each ch across. Break off.
Row 2: Attach Blue to first st of previous row, sc in same place, sc in each dc across, 7 sc in ch 6 sp, sc in each remaining dc across to end of row. Break off.
Sew strap in place. Sew button to other side to correspond. Cut 2 pieces of ribbon 4 inches long and lace through sps at each side of bib. Sew ends in place. Lace remaining ribbon through spaces at center and tie into a bow at top.
Here is the completed baby bib.
Reminded me of great grandma. Thank you for sharing the pattern. I made it and love it; can't wait to made a couple more.
I looked this pattern up online in hopes of finding something "close" to the one I have made for many babies throughout my lifetime including my grandaughter, Chloe. My original pattern came from my dear mother, whose limited funds made it necessary to create crocheted, sewn and embroidered clothing for her nine children as well as countless others. The years of use by my mother and myself have left the original pattern books rather tattered and fragile. I want to thank you for sharing the pattern that is identical to my moms and for version that you created for your beautiful Chloe. I plan to follow your wonderful advice and make some for new babies coming into my family this year! Thank you and bless you and your family!
Beautiful bib, dress and girl and what a lovely idea to crochet a vintage pattern to celebrate a ripe old age!
The bob came out truly beautiful and Chloe well, shes just beyond precious!
Such a lovely Bib and post. The bib is adorable. What a treasure.
I LOVE this bib! It reminds me of a sweet, little bib my dear mother-in-law crocheted for my sweetheart. I still have that bib and you've inspired me to follow your pattern to make one for my youngest granddaughter. Thank you for the inspiration and the pattern.
That is the most prettiest bib I've ever seen. Wonderful work! New Follower. Would love for you to share at Simple & Sweet Fridays.
That is adorable. I miss babies here. I just want to pick up that sweetness and give her a kiss.
Awesome work! dear Lorraine. How so talented you are….!
Love little Chloe's precious baby face and oh, those pierced ears! Tooo sweet! The bib is just beautiful and Chloe makes it even more so.
I could NOT do that! It's beautiful but oh my, the work. 🙂 My favorite grandmother used to crochet some incredibly beautiful items…she rarely even looked at the needle. Thanks for the wonderful memory!
Chloe is a beautiful baby! The last photo with her holding the flower is definitely frame worthy! 🙂 Can you even stand for her to go home?
what a great idea! it's very beautiful!
Hi there, Firstly, Chloe is adorable!!!! I love this bib-so sweet and delicate. My daughter knits and I'm sending her a photo so she can try to make one for a family's little one. I love the color-it is just perfect. Seeing your aunt was very special-how blessed you are to still have her.
Oh, my goodness, that is so beautiful. She should just wear it with her clothing as an accessory. What talent!
So totally sweet! And the pattern too! Love how it's continued through the generations. It looks like you had a wonderful mother's day with one on one time!
I clearly remember my grandma having a bib like this, although she would have been thrilled to have such a pretty one like you did. I like anything vintage, but I must admit, this is one of the most beautiful bibs I have ever seen. Thanks so much for sharing the time and skill you put into this for sweet little Chloe.
Precious Chloe is darling in this bib. God bless Aunt Marie. Her crochet for Erin is gorgeous, too. I know she was thrilled to see your work, as well. Every photograph you take looks like it belongs in an expensive magazine. You are very talented.
That is the prettiest bib I've seen. I love the color and it looks so sweet on your little granddaughter. You really do beautiful work. I bet Aunt Marie was so proud.
I love the bib. Your grandaughter looks so sweet in it. I have a new granddaughter arriving in Sept. 🙂 I may see if I can try to make one for her. Thanks for the pattern.


text:
Arctic sea ice continues its downward spiral, reaching a record low extent for the month of May
ImaGeoBy Tom YulsmanJune 9, 2016 6:27 AM
If the trend continues, sea ice coverage will decline to an historic minimum by summer's end in September
A May 21, 2016 satellite view of the Beaufort Sea off the coast of Alaska and Canada shows highly fragmented sea ice. (Source: LANCE System/NASA/GSFC via National Snow and Ice Data Center) Back in mid-May, unusually warm conditions, and shrinking sea ice coverage, prompted Ted Scambos of the National Snow and Ice Data Center to predict that "the Arctic is going to go through hell this year." Almost a month later, that prediction seems right on track. Each and every day during May, the extent of Arctic sea ice shrunk by an area roughly equal to the state of West Virginia. Sea ice does decline naturally in May, as the weather warms up. But this rate of loss was more rapid than during any other May in the 38-year satellite record. So it should come as no surprise that overall, May saw the lowest average extent on record for the entire month, according to provisional data from NSIDC. This follows record lows set in January, February and April. (In March, Arctic sea ice extent was the second lowest in the 38-year satellite record.)
SEE ALSO: Second dramatic spike in thawing on the Greenland Ice Sheet heralds an early start to the melt season.
Source: NSIDC The monthly average extent for May 2016 was more than 386,000 square miles below that observed in May 2012. That's an area roughly equivalent to California, Nevada and Arizona combined. I mention this because 2012 went on to experience the lowest minimum Arctic sea ice extent on record, a mark set in September of that year at the end of the warm season. What we are seeing now puts 2016 well ahead of the declines seen in 2012. The ice has been taking big hits from ocean heat flowing in from the Atlantic, along with warm southerly winds. Unusual warmth has also contributed to what the NSIDC describes as "exceptionally low" snow coverage in the Northern Hemisphere. This past spring — March, April and May — saw the lowest snow cover in a record extending back 50 years.
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