text:
cricketworldcup
India power to World Cup triumph
India beat Sri Lanka by six wickets in a pulsating final to deliver World Cup glory to their cricket-mad population for the first time since 1983.
Sri Lanka smashed 91 from their last 10 overs to post 274-6 in Mumbai, with Mahela Jayawardene making a superb 103
Categories Cricket, Other Headlines Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhoni, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, India, india vs sri lanka, india world champs, india-sri lanka final, kumar sangakkara, ms dhoni, mumbai wankhede stadium, sachin, sachin tendulkar, sangakkara, sri lanka, tendulkar, wankhede stadium, wc, wc final, wcfinal, world cup, world cup 2011, world cup final, worldcup, worldcup2011, worldcupfinal
Battle for glory
India-Sri Lanka WC final today
The curtain will fall on cricket's biggest event today.
Besides some fortunate ones who have got the precious tickets to watch the match from the stands, billion of cricket-mad people around the world will stay glued to their TV screens when India lock horns with Sri Lanka in the all-Asian final of the ICC Cricket World Cup at the Wankhede Stadium in
Categories Other Headlines Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhoni, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, India, india vs sri lanka, india-sri lanka final, kumar sangakkara, ms dhoni, mumbai wankhede stadium, sachin, sachin tendulkar, sangakkara, sri lanka, tendulkar, wankhede stadium, wc, wc final, wcfinal, world cup, world cup 2011, world cup final, worldcup, worldcup2011, worldcupfinal
Muralitharan's mom-in-law will cheer both teams
Mumbai: When Sri Lanka's Muttiah Muralitharan played his last Test match (vs. India at Galle, in July last year), he had invited his Indian mother-in-law, Dr. Nithiya Rammurthy, to witness his 800th wicket. She will be in Mumbai on Saturday for the World Cup final between India and Sri Lanka. "My daughter, Madhi, will obviously support her husband's team. and as per Indian tradition, I have
Categories Cricket Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, India, india vs sri lanka, india-sri lanka final, mumbai, muralitharan, muttiah muralitharan, sri lanka, wankhede stadium, wc, wc final, wcfinal, world cup, world cup 2011, world cup final, worldcup, worldcup2011, worldcupfinal
It's India vs SL in the final
Hosts edge out arch-rivals Pakistan in tense battle
An atypical Sachin Tendulkar, a menacing Munaf Patel and a wily spell of spin bowling from Harbhajan Singh propelled India into the final of the 2011 World Cup, with a hard fought 29 win over arch-rivals Pakistan at Mohali yesterday.
India will now face Sri Lanka in a battle for sub-continental and world supremacy in the final
Categories Other Headlines Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhoni, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, India, india vs pakistan, india-pakistan match, india-pakistan semifinal, ms dhoni, pakistan, sachin, sachin tendulkar, semifinal, shahid afridi, tendulkar, wc, world cup, world cup 2011, worldcup, worldcup2011
SL set all-Asia final
The Colombo sky was ablaze with fireworks last night as Sri Lanka fought their way over murky waters and into the World Cup final, giving their legend Muttiah Muralidaran a fitting farewell from international cricket. The five-wicket win was not an easy ride, thanks to a late rally by New Zealand at the flashbulb-laden R Premadasa Stadium, but it was closer than imagined after Sri Lanka's dominance for the
Categories Other Headlines Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, new zealand, semifinal, sri lanka, sri lanka vs new zealand, sri lanka-new zealand, wc, world cup, world cup 2011, world cup final, worldcup, worldcup2011, worldcupfinal
More than a semi
The World Cup 2011 will reach its zenith as the two fiercest rivals of world cricket, India and Pakistan, square off against each other in a semifinal match in Mohali today. The excitement has already reached fever pitch, with Indian police leaving no stone unturned to ensure peaceful progress of a match where both the prime ministers of the two countries, Manmohan Singh of India and Yousuf Raza Gilani of
Categories Cricket Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhoni, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, India, india vs pakistan, india-pakistan match, india-pakistan semifinal, ms dhoni, pakistan, semifinal, shahid afridi, wc, world cup, world cup 2011, worldcup, worldcup2011
Kiwis clinch semi-final place
New Zealand sealed their place in the March 29 semi-finals in Colombo, Sri Lanka, after stunning South Africa by 49 runs in the third quarter-final match at Sher-e-Bangla National Stadium in Mirpur, Dhaka on Friday.
The protease were bowled out for 172 runs in 43.2 overs as a gritty Jacob Oram captured four wickets followed by NL McCullum three and Luke Woodcock and Tim Southee took one wicket
Categories Cricket Tags BCB, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhaka, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, mirpur, new zealand, quarterfinal, s africa vs new zealand, s africa-new zealand, South Africa, wc, world cup, world cup 2011, worldcup, worldcup2011
Champs gone
India set up semis with Pakistan
Yuvraj Singh smashed an unbeaten half-century under pressure as India dispatched defending champions Australia from the World Cup with a five-wicket win at Ahmedabad yesterday. Their victory set up a dream semifinal against archrivals Pakistan at Mohali on March 30.
It had looked to be slipping away from the hosts when captain MS Dhoni perished to a
Categories Cricket, Other Headlines Tags Australia, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, India, india vs australia, india-pakistan semifinal, ms dhoni, quarterfinal, ricky ponting, wc, world cup, world cup 2011, worldcup, worldcup2011, yuvraj singh
S Africa-New Zealand quarter-final today
The curtain on the six-week long World Cup fiesta will fall in Bangladesh with South Africa facing New Zealand in a quarter-final match at the Sher-e-Bangla National Stadium today.
The carnival began here on February 12 with the Bangladesh-Canada warm-up game in Chittagong and it has gone through a lot of highs and lows before it finally comes to an
Categories Cricket, Other Headlines Tags BCB, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhaka, icc cricket world cup, icc world cup, icccricketworldcup, iccworldcup, mirpur, new zealand, quarterfinal, s africa vs new zealand, s africa-new zealand, South Africa, wc, world cup, world cup 2011, worldcup, worldcup2011
Pakistan outplay Windies to storm into semis
Dhaka: Pakistan thrashed West Indies by ten wickets to move into the World Cup semi-finals here at the Sher-e-Bangla National Stadium, Mirpur on Wednesday.
Chasing an easy target of 113 runs, openers Kamran Akmal and Mohammad Hafeez completed the Pakistan victory without being separated, in just 20.5 overs
Categories Cricket Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, darren sammy, dhaka, first quarterfinal, icc cricket world cup, icc world cup, iccworldcup, mirpur, pak, pakistan, pakistan vs west indies, sammy, shahid afridi, wc, west indies, windies, world cup, world cup 2011, worldcup, worldcup2011
Afridi leaves Imran behind
It is still too early to think that Shahid Afridi can emulate Imran Khan by leading Pakistan to their second World Cup trophy, but he did at least one thing better than his legendary countryman on Wednesday.
With his 4-30 against the West Indies in the first quarter-final, Afridi overtook Imran to become Pakistan's most successful bowler in a single World Cup as his tally in the ongoing tournament
WC set for knock-out stage
Pakistan take on Windies today
The much-anticipated knock-out phase of the World Cup is set for a cracking start as unpredictable Pakistan take on a youthful West Indian side in the first quarter-final in Dhaka today.
More than a semi-final place will be at stake for Pakistan, who missed out on a second-round place in the last World Cup and has since been plagued by many controversies including a
Categories Other Headlines Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, darren sammy, dhaka, first quarterfinal, icc cricket world cup, icc world cup, iccworldcup, mirpur, pak, pakistan, pakistan vs west indies, sammy, shahid afridi, wc, west indies, windies, world cup, world cup 2011, worldcup, worldcup2011
Only good-looking cops for World Cup final
Only good-looking police officers will be allowed to work on security for the cricket World Cup final in Mumbai, the city's police commissioner has reportedly said.
Arup Patnaik told senior officers during a briefing that he wanted only "tall, well-built and slim policemen" to be working on April 2 when the last match of the competition is played at
Categories Cricket Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, final match, good-looking cops, good-looking police, icc cricket world cup, icc world cup, iccworldcup, mumbai, wankhede stadium, wc, world cup, world cup 2011, world cup final, worldcup, worldcup2011
Pak, Windies in city to play first quarterfinals
Dhaka: Pakistan and West Indies teams arrived in Dhaka on Monday evening to take part in the first quarter-final match of the ICC Cricket World Cup to be held at the Sher-e-Bangla National Cricket Stadium on March 23. Pakistan team led by the mercurial all-rounder Shahid Afridi arrived in the capital directly from Sri Lanka. They will practice at the Mirpur stadium from 2pm to 5pm on
West Indies team to get 'state-level' security
The security personnel have spread a safety blanket ranking to 'state-level' for the West Indies team in an attempt to ensure a hassle free quarter-final match of the Cricket World Cup 2011 tomorrow, officials confirmed.
To avoid repetition of the embarrassing March 4 like incident, the security experts are asked to
Categories Cricket Tags cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, darren sammy, dhaka, first quarterfinal, icc cricket world cup, icc world cup, iccworldcup, mirpur, pak, pakistan, pakistan vs west indies, sammy, state-level security, wc, west indies, west indies team, windies, world cup, world cup 2011, worldcup, worldcup2011
Struggling Aussies face knock-out clash with India
Australia will fly from Sri Lanka to India facing arguably the most daunting challenge of the cricket World Cup: beating India in a knock-out quarter-final on its own turf.
India's stuttering victory over the West Indies overnight gave it a second-placed finish in group B, ensuring it would be drawn against a fellow tournament heavyweight in the quarter-finals at
Categories Cricket Tags Australia, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, icc world cup, iccworldcup, India, knock-out clash, ponting, ricky ponting, struggling aussies face india, wc, world cup, world cup 2011, worldcup, worldcup2011
Defeat spoils Tamim's birthday party
Mumbai: Tamim Iqbal, who turned 22 on Sunday, accepted the congratulatory message, saying, "Thank you", to this scribe. Had Bangladesh won its crucial World Cup match against South Africa on Saturday, the team could have had a grand party and his birthday celebration as well. "After leaving the team hotel, he preferred to celebrate the day in Dhaka (Tamim has a house in the same building in Dhaka, where his
Categories Cricket Tags bangladesh, bangladesh cricket team, bangladesh team, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhaka, icc world cup, iccworldcup, mirpur, Tamim, tamim Iqbal, tamim's birthday, tamim's birthday party, tigers, wc, world cup, world cup 2011, worldcup, worldcup2011
Gloomy face all over after sad exit
Dhaka: The euphoria which gripped the country for the last one month is over now. Bangladesh are now a mere spectator of the cricket's showcase event in their own den as they will hold two quarterfinal matches on March 23 and 25. Surroundings of Sher-e-Bangla National Cricket Stadium (SBNCS) are beautifully lit up on the eve of the World Cup which is a sight to behold. Over the one month, for people it
Categories Cricket Tags bangladesh, bangladesh cricket team, bangladesh team, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhaka, gloomy face all over, icc world cup, iccworldcup, mirpur, south africa team, tigers, wc, world cup, world cup 2011, worldcup, worldcup2011
Tigers' WC dream shattered
The Proteas proved far too strong for the Tigers who failed firstly to check South Africa's run making and later to withstand their bowling attack, folding for only 78 and thus ending their Cricket World Cup dream at Sher-e-Bangla National Stadium in Mirpur on Saturday.
South Africa secured its place at the top on the point table and among top eight moving into
Categories Other Headlines Tags bangladesh, bangladesh captain, bangladesh cricket team, bangladesh team, bangladesh v south africa, bangladesh-south africa match, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhaka, graeme smith, icc world cup, iccworldcup, mirpur, shakib, shakib al hasan, smith, South Africa, south africa captain, south africa team, tigers, tigers' wc dream end, wc, world cup, world cup 2011, worldcup, worldcup2011
Aiming for the quarterfinals
Tigers face SA today
Prior to today's crunch Bangladesh-South Africa match, the question on everybody's lips was what awaited the millions of cricket crazy fans in the country? Will it be heartbreak or will today usher in an era of hope?
Shakib Al Hasan's men can write a new chapter for Bangladesh cricket when they face off against an inform South Africa in a make or break battle at the Sher-e-Bangla National Stadium in
Categories Cricket Tags bangladesh, bangladesh captain, bangladesh cricket team, bangladesh team, bangladesh v south africa, bangladesh-south africa match, cricket, cricket world cup, cricket-craz, cricketer, cricketworldcup, dhaka, graeme smith, icc world cup, iccworldcup, mirpur, shakib, shakib al hasan, smith, South Africa, south africa captain, south africa team, tigers, wc, world cup, world cup 2011, worldcup, worldcup2011


In a boost to those working to turn Odenton into a vibrant town center, County Executive Janet S. Owens has approved a nearly $201,000 contract for walkways geared to make train and bus service more accessible to pedestrians.
Almost $150,000 in additional county funds will go to landscaping along Morgan and Odenton roads, and to a Department of Public Works project to extend public sewer service to Epiphany Episcopal Church and 11 other properties.
"I'm elated to find that Odenton has received money for some of the projects that are well-needed in the community," said Katharina E. DeHaas, chairman of the Odenton Small Area Planning Committee.
"The pedestrian access will allow more people to commute within the area."
Cossentino Contracting will install the new sidewalks beginning in mid-August.
Ultimately, the Odenton area will have an interconnecting pedestrian system, a feature deemed essential for a town center.
"We're trying to enable people to get out of their cars," said Planning and Zoning spokesman John A. Morris. "We want them to be able to use the alternative transportation."
Odenton, at the center of multiple transportation networks, was planned as a town center in the 1960s, when the county started examining growth. County lawmakers adopted a formal Odenton plan in 1995, Morris said.
Since then, community representatives have come up with 30 projects they say are needed in the area, including improved septic and water systems, a library, an adult learning facility and more pedestrian walkways.
"The forecast is that the county needs to put money in the projects before developers will come," DeHaas said.
"They have to see the county has a real interest in getting the town center."
In the past two years, the county has acquired the potential site for a regional library, widened Piney Orchard Parkway east of the train station and laid the foundation for Town Center Boulevard, according to a county news release.


Hello there! Today is April 16th, 2018. In this episode I talk a little bit about Overwatch's newest update, Retribution. Feel free to hit me up on Twitter @Perfectnomad or call me here on Anchor with your questions and comments!
Blizzard games are still fun, it's okay to be loud about your favorite things, and what happens when a tweet goes viral?
My Week in Blizzard gaming! TechieTaco and I are making something new, Frazley is a cool guy, Apex Legends is really fun! Blizzard talk weekly.
It's 2019! I missed podcasting. Got inspired to raid again, and I'm active in Overwatch again!
*MAJOR SPOILERS AHEAD* Battle for Azeroth is finally out! Follow me on my journey through Zuldazar.
Nomad talks about the War of Thorns! Huge shout out to Rho from Realm Maintenance for the interview! Future expansion predictions.
Hello everyone! This bonus episode is all about the time Blizzard came to Medieval Times, and we changed the show for them!
Disclaimer: I do not own any audio used in this episode. All audio belongs to Blizzard Ent.
Freezing Nomad Episode 10: Gaming with OPSEAT!
A Blizzard centered podcast with the occasional hidden gem! All about what I've been playing. Game talks weekly!
Need a new gaming chair? Try OPSEAT! BEST. CHAIR. EVER!
Episode 9: Nomad is evolving!
My experience as a Medieval Times Knight walking out the LAValiant for Overwatch League.
Hello there! Today is April 2nd, 2018. In this episode I talk about a game I really enjoy, Dead By Daylight. If you enjoyed this episode, feel free to hit me up on Twitter @PerfectNomad, or call in with your questions!
I'm back on that Overwatch grind!
Hello there! It's 3/28/18 and in this episode I talk about my climb back to Plat, share some tips that helped me, with a sprinkle of mechanical tips!
Toxicity and how we can stop it.


text:
Small extracellular vesicles from human adipose-derived mesenchymal stromal cells: a potential promoter of fat graft survival
Aizhen Chen1,2,3 na1,
Shijie Tang1,2,3 na1,
Jiawei He1,2,
Xiangyu Li1,3,
Guohao Peng1,2,3,
Haoruo Zhang1,2,3,
Jinghua Chen4,
Liangwan Chen5 &
Xiaosong Chen ORCID: orcid.org/0000-0002-3780-55101,2
Small extracellular vesicles (sEVs) with genetic information secreted by cells play a crucial role in the cellular microenvironment. In this study, our purpose is to explore the characteristics of the small extracellular vesicles of human adipose-derived mesenchymal stromal cells (hADMSC-sEVs) and studied the role of hADMSC-sEVs in improving the survival rate of grafted fat.
In the present study, we used the transmission electron microscopy, nano-tracking analysis, nanoflow surface protein analysis, and zeta potential value to identify sEVs. SEVs' trajectory was traced dynamically to verify whether hADMSC-sEVs can be internalized into human umbilical vein endothelial cells (HUVECs) in vitro at different times. The angiogenic property of hADMSC-sEVs was observed by measuring the volume, weight, and histological analysis of the grafted fats in nude mouse models.
Our research showed that the hADMSC-sEVs were sEVs with double-layer membrane structure and the diameter of which is within 30–150 nm. hADMSC-sEVs exert biological influence mainly through internalization into cells. Compared with the control group, the hADMSC-sEVs group had a significantly higher survival rate of grafted fat, morphological integrity, and a lower degree of inflammation and fibrosis. And immunohistochemistry showed that hADMSC-sEVs significantly increased the neovascularisation and the expression of CD34, VEGFR2, and Ki-67 in the graft tissue.
As a potential nanomaterial, hADMSC-sEVs have been explored in the field of cell-free application of stem cell technology. hADMSC-sEVs promoted the survival of grafted fats by promoting the formation of new blood vessels, which is another promising progress in the field of regenerative medicine. We believe that hADMSC-sEVs will have a broad application prospect in the field of regenerative medicine in the future.
Small extracellular vesicles (sEVs) are 30–150 nm membranous vesicles actively released by cells [1], containing various kinds of molecules—proteins, mRNA, miRNA, lipids—that are actively being studied as potential biomarkers and play essential roles in intercellular communication [2]. In particular, sEVs derived from adipose-derived mesenchymal stromal cells (hADMSC) have strong therapeutic potential and may offer a new therapeutic strategy [3]. Upon the literature review, we found that sEVs can promote angiogenesis [4, 5] and account for the beneficial paracrine effects of hADMSCs therapy [6]. Previous studies have confirmed that hADMSC-sEVs can effectively attenuate myocardial ischemia/reperfusion injury [7] and accelerate cutaneous wound healing [8].
In clinical, autologous fat grafting is playing an increasingly important role in the esthetic and reconstructive field for its extensive utility for soft tissue rejuvenation, volume augmentation, and body contouring. In contrast, the unpredictable fat resorption and low survival rates limit its further application and development [9, 10]. Mounting data have demonstrated that adequate neovascularization is vital for the survival and maintenance of grafted fat [11]. Therefore, many approaches have been developed to promote angiogenesis and improve fat graft retention. Among these, co-transplantation of autologous adipose tissue with hADMSCs or stromal vascular fraction (SVF), known as cell-assisted lipotransfer (CAL) technique [12], can enhance the survival rates by stimulating angiogenesis through a paracrine effect [13]. Adipose tissue seems to be the most advantageous tissue from which to isolate mesenchymal stromal cells because of its abundancy, subcutaneous location, and the need for less invasive techniques [14]. Furthermore, the primary dilemma of stem cell therapy lies in its difficultly to extend clinical applications for its safety concerns [15, 16]. Given that, we turned to explore a kind of biomaterial, which were equipped with the function of cells but no cells' framework and maybe promising in clinical application. Based on the access to previous researches, we hypothesized that hADMSC-sEVs could be a kind of biomaterial applying in promoting vessel reconstruction after transplantation, with satisfactory biocompatibility and retention.
In this context, we extracted sEVs from the fourth passage of hADMSCs and identified the characteristics of hADMSCs and hADMSC-sEVs. Then, we explore the mechanism of hADMSC-sEVs' exerting influence in this biological progress. We chose a nude mice fat grafting model to identify whether hADMSC-sEVs could potentially promote angiogenesis after fat grafting and studied the underlying mechanism of hADMSC-sEVs' effect in improving the retention of fat graft.
Animal maintenance
All animal protocols were implemented under the Animal Ethical Committee of Fujian Medical University's supervision and approval. Eighteen male nude mice (6 weeks of age) were raised in the Experimental Animal Center of Fujian Medical University. Animals were kept in cages individually after grafting and maintained under ambient temperature.
We obtained lipoaspirates from three healthy female patients who underwent liposuction of the thigh at the Department of Plastic Surgery, Fujian Medical University Union Hospital. The donors ranged in age between 25 and 45 years. All participants signed the informed consent. This study was approved by the Ethics Committee of Union hospital of Fujian Medicine University and performed following the principles described in the Declaration of Helsinki. hADMSCs were isolated as previously described [15]. Briefly, the lipoaspirate was washed with phosphate-buffered saline (PBS) and digested with 0.25% collagenase I (Sigma-Aldrich, St. Louis, MO, USA). After filtration and centrifugation, the cell pellet was resuspended in Dulbecco's modified Eagle's medium (DMEM) (Hyclone, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Carlsbad, CA, USA) and then cultured in an incubator with 37 °C and 5% CO2. The seeding density of cells was 5 × 105/25 cm2. The fourth passage of hADMSCs, cultivated until around 80% confluency, was used in future experiments. hADMSCs from different donors were cultivated or kept separately. The collected hADMSCs were observed by inverted microscope (CNOPTEC, Chongqing, P. R. China) and characterized by osteogenic and adipogenic induction and flow cytometry (BD Biosciences, San Jose, CA, USA).
Identification of hADMSCs
We chose the fourth-passage hADMSCs, which were in the state of logarithmic growth. When the cells grow to more than 80% confluence, we added adipogenesis induction media (a mixture of 1 μmol/L dexamethasone, 10 μmol/L insulin, 200 μmol/L indomethacin, 0.5 mmol/L isobutylmethylxanthine, and completely medium) and changed the medium every 3 days. Oil Red Assay kit (KeyGEN BioTECH, Jiangsu, China) was for lipid droplets staining according to the manufacturer's specifications, and the results were observed under a microscope after 2 weeks.
Similarly, we used the fourth-passage hADMSCs which proliferated in logarithmically and added osteogenic induction media (a mixture of 10 mmol/L β-glycerol sodium phosphate, 0.1 μmol/L dexamethasone, 50 μmol/L vitamin C, and complete medium) after the cells grew to more than 80% confluence. Accordingly, we changed the medium every 3 days and used an alkaline phosphatase calcium cobalt staining kit (KeyGEN BioTECH, Jiangsu, China) for staining cells after 3 weeks according to the manufacturer's instructions, and the results were observed under the microscope.
The fourth passage of hADMSCs was selected for flow cytometry analysis for phenotypic identification of mesenchymal stromal cells. CD29, CD90, CD31, and CD45 along with related isotype controls (Abcam, Cambridge, UK) were used for hADMSCs' immunofluorescence staining. Flow cytometry was performed by using the BD Accuri C6 System (BD Biosciences, San Jose, CA, USA).
Acquisition of hADMSC-sEVs
hADMSC-sEVs were collected and purified according to the following processes. We selected the fourth-passage hADMSCs to extract sEVs. After cells' confluency reaching 70–80%, hADMSCs' culture medium was replaced with serum-free low glucose DMEM for 48 h to collect cells' supernatant. To isolate and remove cell particles, dead cells, and cell debris of the obtained supernatant, we performed a series of differential centrifugal precipitation (300×g for 10 min, 2000×g for 10 min, and 10,000×g for 30 min). The supernatant removed the sediment was then filtered through a 0.22-μm filter (Millipore, USA) to remove the large extracellular vesicles further and ultracentrifuged at 100,000×g for 70 min by using the High-Speed Refrigerated Centrifuge (Beckman Coulter, USA). The supernatant was discarded, and the precipitation was resuspended with PBS. Finally, the suspension was ultracentrifuged at 100,000×g for 70 min again, and sEVs were obtained after precipitation collection. The obtained sEVs concentration was measured with bicinchoninic acid (BCA) protein detection kit (Beyotime, Shanghai, China) and stored at − 80 °C for further use. All centrifugations are operated at 4 °C.
Nanoparticle tracking analysis
Particle size, particle size distribution, and concentration of sEVs were identified by nanoparticle tracking analysis (NTA) (NanoFCM, China). Compared with polystyrene beads (RI = 1.59), in the Nano-FCM system, monodisperse silica nanoparticles (RI = 1.46) are employed as the reference to calibrate the size of EVs. In the nanoFCM system, the detection efficiency is 100%. Particle concentration can be determined via single-particle enumeration, which defines the particle concentration of the number of particles collected in a given period. Finally, the size, distribution, and total concentration of EVs were calculated by NTA software.
Zeta potential assay of sEVs
Zeta potentials of sEVs were measured three times using a Nano laser particle size analyzer (Litesizer 500, Anton Paar, Austria). Data were collected and analyzed using Anton Paar Kalliope software.
EVs were imaged by transmission electron microscopy (TEM) to verify their morphology. The sample with a volume of 5 μl (366 μg/ml) was prepared and dropped on the sealing film. Covered with a copper mesh and stood for 20 min so that the copper mesh fully absorbed sEVs. The copper mesh with sEVs adsorbed was transferred to 4% paraformaldehyde for fixation for 5 min. Then using 50 μl of 2% uranyl acetate to stain negatively with copper mesh for 5 min, and then copper mesh was dried at room temperature for 30 min. Finally, using FEI transmission electron microscopy (FEI Tecnai G2, USA) for imaging at 100 kV.
Take 20 μl sEVs samples and freeze-dry them in a freeze dryer for 16 h. Then, the lyophilized sEVs powder was evenly dispersed on the conductive tape of the sample holder, and the sample holder was placed in the gold evaporation chamber for ion sputtering gold plating. Finally, the shape and quantity of sEVs were observed under a high-low vacuum scanning electron microscope (SEM) (FEI QUANTA 450, USA), and the images were taken and recorded.
Nanoflow analysis
Taking 30 μL of sEVs diluent and adding FITC Mouse Anti-Human CD9, CD81 (BD, Franklin Lake, New Jersey, USA) and FITC Mouse IgG (BioLegend, San Diego, USA) to mix, then incubating at 37 °C for 30 min in the dark, adding 1 ml of pre-cooled PBS, 110,000×g ultracentrifuging for 70 min. Carefully remove the supernatant, resuspend it in 50 μL of pre-cooled PBS, and detect the protein index results with a NanoFCM instrument (NanoFCM, China).
Internalization of hADMSC-sEVs into human umbilical vein endothelial cells (HUVECs)
The hADMSC-sEVs were labeled with a PKH67 dye (Sigma Aldrich, USA) for 4 min. Then, Bovine Serum Albumin (BSA) was used to terminate staining. The excess dye after labeling was removed by ultracentrifugation at 100,000g for 1 h at 4°. HUVECs (Cellcook, Guangzhou, China) and hADMSC-sEVs labeled with PKH67 were incubated in 37 °C with serum-free medium for 6 h and 12 h. Meanwhile, HUVECs and PKH67 dye were incubated in 37 °C for 6 h and 12 h as control. After fixation with 4% paraformaldehyde (PFA) for 30 min and staining with 4, 6-diamino-2-phenylindoles (DAPI, CST, USA), the samples were observed under a fluorescence microscope (Nikon, Japan).
Histological analysis and immunostaining
The grafted fat paraffin-embedded sections were stained with hematoxylin, eosin, and immunohistochemistry. The sections were primarily incubated with rabbit anti-human and mouse CD34 (Abcam, USA), VEGFR2 (Abcam, USA), and Ki-67 (Abcam, USA) separately, followed by incubation with horseradish peroxidase-conjugated secondary antibody. Finally, the staining color was developed using the DAB Detection Kit (Maixin, Fuzhou, China). Histologic parameters were examined under a light microscope. HE staining was observed in 5 low magnification field analysis methods for the assessment of fat graft integrity, as evidenced by the presence of intact and nucleated adipocytes and the presence of cysts and vacuoles. Each parameter was graded by two observers independently on a semiquantitative scale ranging from 0 to 5 (0 = absence; 1 = minimal presence; 2 = minimal to moderate presence; 3 = moderate presence; 4 = moderate to extensive presence; and 5 = extensive presence).
Six-week-old, male nude mice were obtained from the Laboratory Animal Center of Fujian medicine University (Fuzhou, P. R. China). The experimental protocol was approved by the Animal Ethical Committee of Fujian Medical University (Permit Number: FJMU IACUC 2019-0131). The mice were randomly assigned to hADMSC-sEVs or control groups (six mice in each group). A mixture of 0.4 ml of fat particles and 0.1 ml (1010 particles/ml) hADMSC-sEVs solution (hADMSC-sEVs group) or 0.1 ml PBS (control group) was injected subcutaneously into the back of nude mice (the fat particles was from the other patient who had liposuction of thighs). The mice were sacrificed at 1, 2, and 3 months after fat grafting. Grafted fat samples were harvested and measured by the weight, volume, hematoxylin-eosin (HE), and immunochemistry staining. The weights of fat grafts were determined by a balance and the volumes were measured by the liquid overflow method. The analysis of HE and IHC staning has been discussed above.
Statistical analysis was performed using GraphPad Prism 8.0.1 software (GraphPad Software, Inc., La Jolla, CA, USA). Results were presented as the mean ± standard deviation (SD). The two-tailed Student's t test was used to evaluate differences between groups. P < 0.05 was considered as statistically significant.
Characterizations of hADMSCs
Under light microscope, a characteristic morphology of slender spindle-like cells of fourth-passage hADMSCs was observed (Fig. 1a). Mature adipocytes and mineralized nodules can be formed from fourth-passage hADMSCs induced by adipogenic and osteoblastic induction media, respectively (Fig. 1b, right; c, right). The results of oil red O staining showed that there were red fat droplets with different sizes in the cells. And the alkaline phosphatase calcium cobalt method staining showed positive osteoblastic nodules. To determine the mesenchymal phenotype of hADMSCs, we investigated the purified hADMSCs by using flow cytometry analysis. Approximately 98% of hADMSCs were positive for CD29 and CD90, but negative for CD31 and CD45 (Fig. 1d). These suggested that the cultured cells were adipose-derived mesenchymal stromal cells [17].
Characterizations of human adipose-derived mesenchymal stromal cells (hADMSCs). a Morphological features of the fourth-passage hADMSCs under light microscopy (× 100 magnification, scale bar =75 μm). b Representative images of Oil Red O staining (left: non-induced control group; right: induced group; × 200 magnification, scale bar = 75 μm). c Representative images of the alkaline phosphatase calcium cobalt staining (left: non-induced control group; right: induced group; × 200 magnification, scale bar =75 μm). d Flow cytometry analysis of cell surface marker CD29, CD90, CD31, and CD45 was showed in hADMSCs
Characterizations of hADMSC-sEVs
The classic ultracentrifugation method was used to obtain hADMSC-sEVs. We obtained the sEVs pellet of Fig. 2b from the cell supernatant of Fig. 2a after a series of differential centrifugation. According to the results of NTA data analysis, most of the sEVs'particle size ranged between 50 and 120 nm (Fig. 2c). We estimated the ratio of the number of sEVs particles (× 108) to 1 × 106 cells and the ratio of protein amount (μg) to 1 × 106 cells through the measurement of NTA and previous BCA (Fig. 2d, e). It can be seen from the ratio of Fig. 2d and e that a large amount of cell-derived supernatant was needed to extract a sufficient amount of sEVs precipitate. Zeta potential measurement of sEVs showed that its average potential was − 16.68 mV (Fig. 2f). The morphology of sEVs was observed under transmission electron microscopy, which was a saucer-like membrane structure with a diameter of 50 ~ 120 nm (Fig. 2g). Under the scanning electron microscopy, the sEVs can be seen in a more objective and accurate spherical three-dimensional shape, with particle size similar to that of the transmission electron microscope (Fig. 2h). Specific markers CD9 and CD81 of EVs were confirmed to be expressed in hADMSC-sEVs [18], and IgG was used as a negative control (Fig. 2i).
Characterizations of hADMSC-sEVs. a Gross observation: the number of T75 flasks for culturing cells at one time. b The gross visible sEVs precipitate was obtained from a. c NTA measured the size distribution of sEVs. d The ratio of the number of sEVs particles(× 108) to 1 × 106 cells. e The ratio of protein amount (μg) to 1 × 106 cells. f The potential of hADMSC-sEVs was measured by Zeta potential. g Representative transmission electron microscopy image of ADMSC-sEVs with double circular vesicle structures was shown by directional arrow (× 100,000 magnification, scale bar = 200 nm). h Morphological characterization of sEVs with three-dimensional shapes under scanning electron microscopy (× 10,000magnification; scale bar = 10 μm). i Nanoflow analysis of the hADMSC-sEVs' surface protein marker (CD9 and CD81), and IgG as positive controls
hADMSC-sEVs can be transferred to HUVECs
As shown in Fig. 3, the PKH67-labeled hADMSC-sEVs and PKH67 dye were co-incubated with HUVECs for 6 h and 12 h, seperately. We can find that the PKH67-labeled hADMSC-sEVs were increasingly scattered around the nucleus of HUVECs as time went on. However, the control group of pure PKH67 dye did not find any green fluorescence accumulation. That was, only the substances with biological activity can enter the cells and the pure dye cannot through the cell membrane. The large stained bodies that we saw were the aggregation of a large number of sEVs labeled with pKH67 fluorescence, rather than individual tiny sEV, which means hADMSC-sEVs play their role mainly through internalization into cells. This result was mutually corroborated with related articles [19,20,21].
hADMSC-sEVs internalization to HUVECs. hADMSC-sEVs (labeled with PKH67 dye, green) and HUVECs (nuclei stained with DAPI) were co-incubated for 6 h and 12 h, respectively. In the control group, PKH67 dye was co-incubated with HUVECs for 6 h and 12 h, respectively. Representative fluorescence images were shown above(× 1000 magnification; scale bar = 25 μm)
hADMSC-sEVs improved the fat graft survival rate in the nude mouse model
To assess the beneficial effects of hADMSC-sEVs, we adopted a nude mouse model of fat grafting. The grafts were harvested 1, 2, and 3 months after fat grafting. Gross observation of the graft specimens demonstrated that the hADMSC-sEVs group had larger graft sizes compared to that in the control group (Fig. 4a). As shown in Fig. 4b, c, a significantly higher graft survival rate was observed in the hADMSC-sEVs group when compared with the control group from 1 to 3 months after fat grafting (P < 0.05), which was confirmed by the better quantitative results of weight and volume of grafted fat were better in the hADMSC-sEVs group compared to the control group, indicating a protective effect of hADMSC-sEVs on grafted fat survival. For micro-evaluation, we analyzed the HE staining, which revealed that the grafted fat in the hADMSC-sEVs groups exhibited better survival and morphologic integrity compared to the control group, as shown in Fig. 5a. We observed extensive cystic changes and fibrous septa in the control group. There were significant differences between the hADMSC-sEVs groups and control group in the histological evaluation of integrity, cysts/vacuoles, fibrosis, and inflammation (P < 0.05) (Fig. 5b, c).
hADMSC-sEVs improved the fat graft survival rate in the nude mouse model. a Gross observation of the grafted fat demonstrated that the hADMSC-sEVs group (the left side of each picture) had larger graft sizes and heavier weight compared to that in the control group (the right side of each picture). b, c 1, 2, and 3 months after fat grafting, the weight (measured by the balance) and volume (measured by the liqiud outflow method) of grafted fat were significantly better in the hADMSC-sEVs group compared to the control group (*p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001)
Histological changes in adipose tissue over time. a HE staining revealed that the grafted fat in the hADMSC-sEVs groups exhibited better survival and morphologic integrity (evidenced by the presence of intact and nucleated adipocytes and the presence of cysts and vacuoles) compared to the control group. b, c Semiquantitative scale of integrity and cysts/vacuoles of HE staining (***p < 0.001,****p < 0.0001)
hADMSC-sEVs promoted neovascularization in the nude mouse model of fat grafting
We used immunohistologic staining for histological evaluation. Studies have shown that vascularisation is crucial for fat survival and repassage [22]. So we measured capillary density within the grafts via immunohistochemical (IHC) staining of anti-CD34 antibody in tissue sections to evaluate the effect of hADMSC-sEVs on neoangiogenesis of grafted fat. Consistent with the HE staining and histologic examination, IHC staining showed a significant increase in CD34-positive (a specific marker of capillary [23]) in the hADMSC-sEVs group with the control groups (Fig. 6; See supplemental materials for the enlarged subsections of the images), indicating increased capillary density in the hADMSC-sEVs group. Together, these results showed that hADMSC-sEVs could effectively improve the vascularisation of the grafted fat.
hADMSC-sEVs promoted neovascularization in the nude mice fat grafting model (rows means different staining and lines represents different groups and months). CD34 was selectively expressed in the small vessel endothelium membrane. The VEGF-kinase ligand/receptor signaling system plays a key role in vascular development and regulation of vascular permeability. The VEGFR2 was mainly localized in membrane. Signs of proliferation (nuclei stained by Ki-67) in the vessel endothelium (blue line at the bottom left of each figure represents scale bar; scale bar = 50 μm)
Mechanism of hADMSC-sEVs-mediated angiogenesis in vivo
The previous study has revealed that VEGF secreted by stromal cells stimulates the proliferation and survival of endothelial cells leading to the formation of new blood vessels [7]. We further evaluated the potential mechanism of hADMSC-sEVs in promoting survival of grafted fat by IHC analysis of anti-VEGFR2. The results demonstrated that the expression of VEGFR2 was increased in the hADMSC-sEVs group compared to the control group in 1, 2 and 3 months after transplantation (Fig. 6), suggesting that VEGF/VEGFR signaling pathway was involved in hADMSC-sEVs-mediated angiogenesis. We also semi-quantified endothelium cell proliferation by analyzing Ki-67 staining [24], and we found increased immunostaining of Ki-67 in the hADMSC-sEVs group compared with the control group (Fig. 6), suggesting that the proliferation of vascular endothelial cells were enhanced after hADMSC-sEVs treatment.
The study of EVs spans decades. In recent years, it has been brought to the fore, which is mainly due to the remarkable advancement of the identification of EVs, and related mechanisms have been continuously explored [18]. In particular, hADMSC-sEVs have shown great potential in a variety of disease models [25,26,27,28].
In this study, we used a classic ultracentrifugation method to separate the target sEVs, which obtained sEVs with higher purity. After acquiring sEVs, we further analyzed the morphology, surface markers, and physical properties of hADMSC-sEVs in combination with various methods as follows. The results of two-dimensional and three-dimensional images showed that the sEVs we obtained were spherical double-layer membrane structures with a particle size range of about 50–150 nm, which was similar to other related literature [29]. Most of those vesicles (98%) were 50–120 nm based on NTA analysis, and the mean size was 77 nm, which was consistent with the acknowledged size range (30–150 nm) [30]. The above showed that the classic ultracentrifugation method could be used to enrich nano-level EVs at a higher purity. Among membrane proteins, we used CD9 and CD81 as positive markers and IgG as the negative control. CD9 and CD81 are usually associated with EVs and are often regarded as surface protein markers for EVs [31]. Through nanoflow analysis, we successfully detected the sEVs marker proteins CD9 (3.0%), CD81 (10.1%), and IgG (0.2%). In terms of physical properties, hADMSC-sEVs displayed negative zeta potential values. All these data indicated that the extracellular vesicles of hADMSCs we extracted are small EVs, which is the operational term referred to EVs' size, actually [24].
In our animal model of fat grafting, we found an interesting phenomenon that the weight of the grafts was much less at 3 months of implantation both in the control and the hADSC-sVEs group compared to the other months studied. That can be explained according to the rationale of the progression of grafted fat. Which mainly includes cysts, nodules, calcification, necrosis, fibrosis, and survival, and the progress basically takes about 3 months to reach stability [15]. The reason why the weight did not drop significantly at 2 months was mainly because part of the fat necrosis formed cysts or nodules, which had not yet or had just begun to be absorbed. By 3 months, these inflammatory responses, calcification absorption, etc., tend to stabilize. The surviving fat body weight and volume also tend to stabilize. So we found that there was a significant decrease in body weight in the 3 months. We generally think that the body weight after the third month can represent the live fat cells that survived the grafted fat.
The relationship between sEVs and vascular repassage is the focus of research in the field of regenerative medicine. Studies have shown that sEVs can change the angiogenesis steps including proliferation, migration, and endothelial cell structure, as well as increase the expression of angiogenesis-related genes and the secretion of related proteins, including VEGFA, CXCL8, IL-6, FGF2, and miRNA-23a [32,33,34,35]. To test whether sEVs can improve the survival of grafted fat by promoting angiogenesis, we adopted the nude mice fat grafting model. Consistent with previous studies [32, 36], we demonstrated that fat grafts in the hADMSC-sEVs groups exhibited better survival and morphologic integrity compared to the control group.
Several factors are contributing to fat graft survival [9]. Since the grafted fat show lower tolerance for ischemia caused by devascularisation, it is quickly absorbed and replaced by fibrous tissues and oil sacs [37, 38]. The development of a neovascular supply, or angiogenesis, serves crucial homeostatic roles since blood vessels carry nutrients to tissues and organs and remove catabolic products [39]. Therefore, timely and adequate neoangiogenesis is essential for the survival of grafted fat [40, 41]. Substantial evidence demonstrates CD34 is expressed not only by MSC but by a multitude of other nonhematopoietic cell types, including vascular endothelial progenitors [42]. Therefore, we adopted CD34 as a marker of neovascularisation analysis. Our results showed a significant increase in CD34-positive rate in the hADMSC-sEVs group with the control groups (Fig. 6), indicating increased capillary density in the hADMSC-sEVs group. We can observe the improvement of angiogenesis in the hADMSC-sEVs group, and then we began to focus on the underlying mechanism of those improvements. Studies have revealed that VEGF is identified as a principal pro-angiogenic factor that enhances the production of new blood vessels from the existing vascular network [43]. While research suggested that VEGF undergoes alternative exon splicing that leads to multiple isoforms, which has different functions and affinities to the same targets [44]. So there is some concern of low positive rate or false negative rate of anti-VEGFA. That is the reason why we chose VEGFR but not secreted VEGF, because we think the up-regulation of receptors may reflect the real condition about enhancements. In addition, it is well established that VEGF/VEGFR2 signaling pathway plays a vital role in regulating the process of neoangiogenesis. And it was demonstrated that the lower-affinity, highly homologous VEGFR2 was the primary signaling receptor for VEGF [45]. We think the VEGF family has complicated and extensive participation on the VEGF signaling pathway. So we prefer to study VEGFR2. Theoretically, an increase in ligands can lead to upregulation of receptors. Therefore, if the expression of VEGFR2 increases, on the one hand, the upstream stimulus signal is enhanced, and on the other hand, the secreted VEGF (A, C, or D) is also in a state of high expression [39]. So we chose VEGFR2 as our research target. Our IHC analysis demonstrated elevated VEGFR2 expression in grafted tissue of the hADMSC-sEVs group. Therefore, the high expression of VEGFR2 in the hADMSC-sEVs group may account for its pro-angiogenic effects. For further verification and exploration on the mechanism about that if there were the circumstance on proliferation of endothelia, so we analyzed the Ki-67 IHC staining and found increased immunostaining of Ki-67 in the hADMSC-sEVs group compared with the control group, suggesting that hADMSC-sEVs treatment could enhance the proliferation of vascular endothelial cells.
As a potential type of nanomaterial, sEVs have attracted growing attention from researchers in different fields. Interdisciplinary integration and the use of nanotechnology continuously promote the development of sEVs. However, in the process of extraction, identification, and subsequent mechanism research of sEVs, we found that a large amount of supernatant was needed to extract only a little bit of sEVs (Fig. 2a and b). Therefore, how to obtain a large number of sEVs has become a critical preclinical and clinical research direction. Only by breaking the bottleneck of sEVs production can the clinical transformation and application of sEVs have infinite development possibilities. We think that there are two main ways to increase the output of sEVs. One is to increase the number of sEVs secreted by cells from the source, which is, microcarrier-based three-dimensional (3D) cell culture technology. The other is to reduce the loss of sEVs, which means in the process of extracting EVs, it is necessary not only to ensure its purity but also to minimize the loss of sEVs.
3D cell culture technology is a common strategy for large-scale adherent cell culture, which adopts a kind of device equipped with three-dimensional culture system based on a hollow fiber bioreactor, and a large amount of conditioned medium (CM) can be obtained by using this device. Studies have shown that compared with the traditional 2D culture, the total amount of sEVs in the 3D culture system has increased 19.4 times [46, 47]. Moreover, compared with 2D-sEVs, 3D-sEVs have no significant differences in surface markers, size, and shape. In particular, 3D-sEVs can significantly improve the symptoms of related diseases in animal models and are more effective than 2D-sEVs [48]. In conclusion, sEVs obtained by 3D cell culture are more in line with the needs of the body's biological functions, which is an important measure for the clinical development of sEVs.
In terms of sEVs extraction, the mainstream sEVs extraction methods mainly include ultracentrifugation (UC), density gradient centrifugation (DGC), exclusion chromatography (SEC), ultrafiltration (UF), Immune capture (IC) and polymer precipitation (Precip) [49]. Among them, the most classic method is UC [50], but its main limitations are time-consuming and low yield. So people began the exploration of how to increase the output of sEVs further and reduce the cost while ensuring its purity. In recent years, preclinical studies have found that the extraction method of UF combined with SEC is superior to the ultracentrifugation method under the comprehensive conditions of purity, efficiency and cost [51,52,53,54]. In addition, in industrial production, mass production of high-quality sEVs is the most critical factor in its therapeutic applications. Among various separation methods, tangential flow filtration (TFF) is considered as an ideal method for industrial-scale production of sEVs [55,56,57]. TFF can provide GMP level sEVs from a large amount of CM [58]. Some studies have even shown that sEVs separated by TFF have higher yield and activity than those separated by ultracentrifugation [45, 59]. And the analysis of multiple batches of isolated MSC-sEVs showed that the TFF method could generate stable sEVs in a large volume of media. Therefore, TFF is suitable for large-scale production of high-quality sEVs that meet GMP requirements [48].
The present study had several limitations. First, the downstream molecules in the VEGF/VEGFR2 signaling pathway remain to be defined in our further investigation. Second, the theory of graft retention or endogenous adipose repassage is still being defined. Consequently, further studies are warranted to address this issue.
Small extracellular vesicles, as a novel kind of nanoparticle without nuclear structure, do not show apparent side effects, such as immunogenicity or tumorigenicity when applied in animal models. Studies have found that EVs can replicate the function of the cells which they are derived. Our research has proved that hADMSC-sEVs play a considerable role in fat grafting nude mouse model. hADMSC-sEVs can promote neovascularization and increase the retention of grafted fat, whose mechanism may be explained by VEGF/VEGFR2 signal transduction. These findings indicate that hADMSC-sEVs can be regarded as a potential treatment option for fat transplantation. As a new type of nanomaterial, we need further and more in-depth studies to promote hADMSC-sEVs to apply in a broader range of diseases.
ADMSCs:
Adipose-derived mesenchymal stromal cells
DMEM:
Dulbecco's modified Eagle's medium
PBS:
Phosphate-buffered saline
sEVs:
Small extracellular vesicles
hADMSC-sEVs:
The small extracellular vesicles of human adipose-derived mesenchymal stromal cells
HUVECs:
Human umbilical vein endothelial cells
Mesenchymal stem/stromal cells
SVF:
Stromal vascular fraction
CAL:
Cell-assisted lipotransfer
FBS:
Fetal bovine serum
BCA:
Bicinchoninic acid
TEM:
Scanning electron microscope
NTA:
PFA:
Oaraformaldehyde
DAPI:
4, 6-diamino-2-phenylindoles
HE:
Hematoxylin-eosin
SD:
Conditioned medium
UC:
Ultracentrifugation
DGC:
Density gradient centrifugation
Exclusion chromatography
UF:
Immune capture
Precip:
Polymer precipitation
TFF:
Vidal M. Exosomes and GPI-anchored proteins: judicious pairs for investigating biomarkers from body fluids. Adv Drug Deliv Rev. 2020;161-162:110–23. https://doi.org/10.1016/j.addr.2020.08.006.
Borrelli DA, Yankson K, Shukla N, Vilanilam G, Ticer T, Wolfram J. Extracellular vesicle therapeutics for liver disease. J Control Release. 2018;273:86–98. https://doi.org/10.1016/j.jconrel.2018.01.022.
van Balkom BWM, Gremmels H, Giebel B, Lim SK. Proteomic signature of Mesenchymal stromal cell-derived small extracellular vesicles. Proteomics. 2019;19(1–2):e1800163. https://doi.org/10.1002/pmic.201800163.
Cui X, He Z, Liang Z, Chen Z, Wang H, Zhang J. Exosomes from adipose-derived Mesenchymal stem cells protect the myocardium against ischemia/reperfusion injury through Wnt/β-catenin signaling pathway. J Cardiovasc Pharmacol. 2017;70(4):225–31. https://doi.org/10.1097/FJC.0000000000000507.
Hu L, Wang J, Zhou X, Xiong Z, Zhao J, Yu R, Huang F, Zhang H, Chen L. Exosomes derived from human adipose mensenchymal stem cells accelerates cutaneous wound healing via optimizing the characteristics of fibroblasts. Sci Rep. 2016;6(1):32993. https://doi.org/10.1038/srep32993.
Merino CG, Zuniga FA, Escudero C, Ormazabal V, Reyes C, Nova EL, et al. Mesenchymal stem cell-derived extracellular vesicles promote angiogenesis: potencial clinical application. Front Physiol. 2016;7:24.
Liu X, Li Q, Niu X, Hu B, Chen S, Song W, Ding J, Zhang C, Wang Y. Exosomes secreted from human-induced pluripotent stem cell-derived mesenchymal stem cells prevent osteonecrosis of the femoral head by promoting angiogenesis. Int J Biol Sci. 2017;13(2):232–44. https://doi.org/10.7150/ijbs.16951.
Basu J, Ludlow JW. Exosomes for repair, repassage and rejuvenation. Expert Opin Biol Ther. 2016;16(4):489–506. https://doi.org/10.1517/14712598.2016.1131976.
Yu Q, Cai Y, Huang H, Wang Z, Xu P, Wang X, Zhang L, Zhang W, Li W. Co-transplantation of Nanofat enhances neovascularization and fat graft survival in nude mice. Aesthet Surg J. 2018;38(6):667–75. https://doi.org/10.1093/asj/sjx211.
Bellini E, Grieco MP, Raposio E. The science behind autologous fat grafting. Ann Med Surg (Lond). 2017;24:65–73.
Konczalik W, Siemionow M. Experimental and clinical methods used for fat volume maintenance after autologous fat grafting. Ann Plast Surg. 2014;72(4):475–83. https://doi.org/10.1097/SAP.0000000000000117.
Yoshimura K, Sato K, Aoi N, Kurita M, Hirohi T, Harii K. Cell-assisted Lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells. Aesthet Plast Surg. 2020;44(4):1258–65.
Suga H, Glotzbach JP, Sorkin M, Longaker MT, Gurtner GC. Paracrine mechanism of angiogenesis in adipose-derived stem cell transplantation. Ann Plast Surg. 2014;72(2):234–41. https://doi.org/10.1097/SAP.0b013e318264fd6a.
Bacakova L, Zarubova J, Travnickova M, Musilkova J, Pajorova J, Slepicka P, Kasalkova NS, Svorcik V, Kolska Z, Motarjemi H, Molitor M. Stem cells: their source, potency and use in regenerative therapies with focus on adipose-derived stem cells - a review. Biotechnol Adv. 2018;36(4):1111–26. https://doi.org/10.1016/j.biotechadv.2018.03.011.
Laloze J, Varin A, Gilhodes J, Bertheuil N, Grolleau JL, Brie J, Usseglio J, Sensebe L, Filleron T, Chaput B. Cell-assisted lipotransfer: friend or foe in fat grafting? Systematic review and meta-analysis. J Tissue Eng Regen Med. 2018;12(2):e1237–e50. https://doi.org/10.1002/term.2524.
Eun SC. Stem cell and research in plastic surgery. J Korean Med Sci. 2014;29(Suppl 3):S167–9. https://doi.org/10.3346/jkms.2014.29.S3.S167.
Wang D, Liu N, Xie Y, Song B, Kong S, Sun X. Different culture method changing CD105 expression in amniotic fluid MSCs without affecting differentiation ability or immune function. J Cell Mol Med. 2020;24(7):4212–22. https://doi.org/10.1111/jcmm.15081.
Thery C, Witwer KW, Aikawa E, Alcaraz MJ, Anderson JD, Andriantsitohaina R, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 2018;7(1):1535750. https://doi.org/10.1080/20013078.2018.1535750.
Woo CH, Kim HK, Jung GY, Jung YJ, Lee KS, Yun YE, Han J, Lee J, Kim WS, Choi JS, Yang S, Park JH, Jo DG, Cho YW. Small extracellular vesicles from human adipose-derived stem cells attenuate cartilage degeneration. J Extracell Vesicles. 2020;9(1):1735249. https://doi.org/10.1080/20013078.2020.1735249.
Wang K, Li F, Yuan Y, Shan L, Cui Y, Qu J, Lian F. Synovial mesenchymal stem cell-derived EV-packaged miR-31 downregulates histone demethylase KDM2A to prevent knee osteoarthritis. Mol Ther Nucleic Acids. 2020;22:1078–91. https://doi.org/10.1016/j.omtn.2020.09.014.
Bian X, Li B, Yang J, Ma K, Sun M, Zhang C, Fu X. Regenerative and protective effects of dMSC-sEVs on high-glucose-induced senescent fibroblasts by suppressing RAGE pathway and activating Smad pathway. Stem Cell Res Ther. 2020;11(1):166. https://doi.org/10.1186/s13287-020-01681-z.
Zheng H, Yu Z, Deng M, Cai Y, Wang X, Xu Y, Zhang L, Zhang W, Li W. Fat extract improves fat graft survival via pro-angiogenic, anti-apoptotic and pro-proliferative activities. Stem Cell Res Ther. 2019;10(1):174. https://doi.org/10.1186/s13287-019-1290-1.
Xue Y, Lim S, Brakenhielm E, Cao Y. Adipose angiogenesis: quantitative methods to study microvessel growth, regression and remodeling in vivo. Nat Protoc. 2010;5(5):912–20. https://doi.org/10.1038/nprot.2010.46.
Rotti PG, Xie W, Poudel A, Yi Y, Sun X, Tyler SR, Uc A, Norris AW, Hara M, Engelhardt JF, Gibson-Corley KN. Pancreatic and islet remodeling in cystic fibrosis transmembrane conductance regulator (CFTR) knockout ferrets. Am J Pathol. 2018;188(4):876–90. https://doi.org/10.1016/j.ajpath.2017.12.015.
Kholia S, Ranghino A, Garnieri P, Lopatina T, Deregibus MC, Rispoli P, Brizzi MF, Camussi G. Extracellular vesicles as new players in angiogenesis. Vasc Pharmacol. 2016;86:64–70. https://doi.org/10.1016/j.vph.2016.03.005.
Ribeiro-Rodrigues TM, Laundos TL, Pereira-Carvalho R, Batista-Almeida D, Pereira R, Coelho-Santos V, Silva AP, Fernandes R, Zuzarte M, Enguita FJ, Costa MC, Pinto-do-Ó P, Pinto MT, Gouveia P, Ferreira L, Mason JC, Pereira P, Kwak BR, Nascimento DS, Girão H. Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis. Cardiovasc Res. 2017;113(11):1338–50. https://doi.org/10.1093/cvr/cvx118.
Zhang B, Wu X, Zhang X, Sun Y, Yan Y, Shi H, Zhu Y, Wu L, Pan Z, Zhu W, Qian H, Xu W. Human umbilical cord mesenchymal stem cell exosomes enhance angiogenesis through the Wnt4/beta-catenin pathway. Stem Cells Transl Med. 2015;4(5):513–22. https://doi.org/10.5966/sctm.2014-0267.
Wong DE, Banyard DA, Santos PJF, Sayadi LR, Evans GRD, Widgerow AD. Adipose-derived stem cell extracellular vesicles: a systematic review. J Plast Reconstr Aesthet Surg. 2019;72(7):1207–18. https://doi.org/10.1016/j.bjps.2019.03.008.
Kondratov KA, Petrova TA, Mikhailovskii VY, Ivanova AN, Kostareva AA, Fedorov AV. Extracellular vesicles from blood plasma studied by low voltage scanning electron microscopy. Tsitologiia. 2017;59(3):169–77.
Boukouris S, Mathivanan S. Exosomes in bodily fluids are a highly stable resource of disease biomarkers. Proteomics Clin Appl. 2015;9(3–4):358–67. https://doi.org/10.1002/prca.201400114.
Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–83. https://doi.org/10.1083/jcb.201211138.
Han Y, Ren J, Bai Y, Pei X, Han Y. Exosomes from hypoxia-treated human adipose-derived mesenchymal stem cells enhance angiogenesis through VEGF/VEGF-R. Int J Biochem Cell Biol. 2019;109:59–68. https://doi.org/10.1016/j.biocel.2019.01.017.
Montemurro T, Vigano M, Ragni E, Barilani M, Parazzi V, Boldrin V, et al. Angiogenic and anti-inflammatory properties of mesenchymal stem cells from cord blood: soluble factors and extracellular vesicles for cell repassage. Eur J Cell Biol. 2016;95(6–7):228–38. https://doi.org/10.1016/j.ejcb.2016.04.003.
McBride JD, Rodriguez-Menocal L, Guzman W, Candanedo A, Garcia-Contreras M, Badiavas EV. Bone marrow Mesenchymal stem cell-derived CD63 Exosomes transport Wnt3a exteriorly and enhance dermal fibroblast proliferation, migration, and angiogenesis in vitro. Stem Cells Dev. 2017;26(19):1384–98. https://doi.org/10.1089/scd.2017.0087.
Mathiyalagan P, Liang Y, Kim D, Misener S, Thorne T, Kamide CE, Klyachko E, Losordo DW, Hajjar RJ, Sahoo S. Angiogenic mechanisms of human CD34 stem cell Exosomes in the repair of ischemic hindlimb. Circ Res. 2017;120(9):1466–76. https://doi.org/10.1161/CIRCRESAHA.116.310557.
Cui X, He Z, Liang Z, Chen Z, Wang H, Zhang J. Exosomes from adipose-derived mesenchymal stem cells protect the myocardium against ischemia/reperfusion injury through Wnt/b-catenin signaling pathway. J Cardiovasc Pharmacol. 2017;70(4):225–31. https://doi.org/10.1097/FJC.0000000000000507.
Dong Z, Peng Z, Chang Q, Zhan W, Zeng Z, Zhang S, et al. The angiogenic and adipogenic modes of adipose tissue after free fat grafting. Plast Reconstr Surg. 2015;135(3):556e–567e.
Mizoguchi T, Kijima Y, Hirata M, Kaneko K, Arima H, Nakajo A, Higashi M, Tabata K, Koriyama C, Arigami T, Uenosono Y, Okumura H, Maemura K, Ishigami S, Yoshinaka H, Shinden Y, Ueno S, Natsugoe S. Histological findings of an autologous dermal fat graft implanted onto the pectoralis major muscle of a rat model. Breast Cancer. 2015;22(6):578–85. https://doi.org/10.1007/s12282-014-0523-5.
Apte RS, Chen DS, Ferrara N. VEGF in signaling and disease: beyond discovery and development. Cell. 2019;176(6):1248–64. https://doi.org/10.1016/j.cell.2019.01.021.
Locke MB, de Chalain TMB. Current practice in autologous fat transplantation: suggested clinical guidelines based on a review of recent literature. Ann Plast Surg. 2008;60(1):98–102.
Yamaguchi MMF, Bujo H, et al. Revascularisation determines volume retention and gene expression by fat grafts in mice. Exp Biol Med (Maywood). 2005;230(10):742–8. https://doi.org/10.1177/153537020523001007.
Sidney LE, Branch MJ, Dunphy SE, Dua HS, Hopkinson A. Concise review: evidence for CD34 as a common marker for diverse progenitors. Stem Cells. 2014;32(6):1380–9. https://doi.org/10.1002/stem.1661.
Alkharsah KR. VEGF upregulation in viral infections and its possible therapeutic implications. Int J Mol Sci. 2018;19(6):1642.
McFee RM, Rozell TG, Cupp AS. The balance of proangiogenic and antiangiogenic VEGFA isoforms regulate follicle development. Cell Tissue Res. 2012;349(3):635–47. https://doi.org/10.1007/s00441-012-1330-y.
Abhinand CS, Raju R, Soumya SJ, Arya PS, Sudhakaran PR. VEGF-A/VEGFR2 signaling network in endothelial cells relevant to angiogenesis. J Cell Commun Signal. 2016;10(4):347–54. https://doi.org/10.1007/s12079-016-0352-8.
Haraszti RA, Miller R, Stoppato M, Sere YY, Coles A, Didiot MC, Wollacott R, Sapp E, Dubuke ML, Li X, Shaffer SA, DiFiglia M, Wang Y, Aronin N, Khvorova A. Exosomes produced from 3D cultures of MSCs by tangential flow filtration show higher yield and improved activity. Mol Ther. 2018;26(12):2838–47. https://doi.org/10.1016/j.ymthe.2018.09.015.
Cao J, Wang B, Tang T, Lv L, Ding Z, Li Z, Hu R, Wei Q, Shen A, Fu Y, Liu B. Three-dimensional culture of MSCs produces exosomes with improved yield and enhanced therapeutic efficacy for cisplatin-induced acute kidney injury. Stem Cell Res Ther. 2020;11(1):206. https://doi.org/10.1186/s13287-020-01719-2.
Yan L, Wu X. Exosomes produced from 3D cultures of umbilical cord mesenchymal stem cells in a hollow-fiber bioreactor show improved osteochondral repassage activity. Cell Biol Toxicol. 2020;36(2):165–78. https://doi.org/10.1007/s10565-019-09504-5.
Coumans FAW, Brisson AR, Buzas EI, Dignat-George F, Drees EEE, El-Andaloussi S, et al. Methodological guidelines to study extracellular vesicles. Circ Res. 2017;120(10):1632–48. https://doi.org/10.1161/CIRCRESAHA.117.309417.
Park SK, Bandeira E, Shelke GV, Lässer C, Lötvall J. Enhancement of therapeutic potential of mesenchymal stem cell-derived extracellular vesicles. Stem Cell Res Ther. 2019;10(1):288. https://doi.org/10.1186/s13287-019-1398-3.
Takov K, Yellon DM, Davidson SM. Comparison of small extracellular vesicles isolated from plasma by ultracentrifugation or size-exclusion chromatography: yield, purity and functional potential. J Extracell Vesicles. 2019;8(1):1560809. https://doi.org/10.1080/20013078.2018.1560809.
Corso G, Mäger I, Lee Y, Görgens A, Bultema J, Giebel B, Wood MJA, Nordin JZ, Andaloussi SEL. Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography. Sci Rep. 2017;7(1):11561. https://doi.org/10.1038/s41598-017-10646-x.
Buschmann D, Kirchner B, Hermann S, Märte M, Wurmser C, Brandes F, Kotschote S, Bonin M, Steinlein OK, Pfaffl MW, Schelling G, Reithmair M. Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next-passage sequencing. J Extracell Vesicles. 2018;7(1):1481321. https://doi.org/10.1080/20013078.2018.1481321.
Mol EA, Goumans MJ, Doevendans PA, Sluijter JPG, Vader P. Higher functionality of extracellular vesicles isolated using size-exclusion chromatography compared to ultracentrifugation. Nanomedicine. 2017;13(6):2061–5. https://doi.org/10.1016/j.nano.2017.03.011.
Reiner AT, Witwer KW, van Balkom BWM, de Beer J, Brodie C, Corteling RL, et al. Concise Review: Developing Best-Practice Models for the Therapeutic Use of Extracellular Vesicles. Stem Cells Transl Med. 2017;6(8):1730–9.
Ha DH, Kim H-K, Lee J, Kwon HH, Park G-H, Yang SH, Jung JY, Choi H, Lee JH, Sung S, Yi YW, Cho BS. Mesenchymal stem/stromal cell-derived exosomes for immunomodulatory therapeutics and skin repassage. Cells. 2020;9(5):1157. https://doi.org/10.3390/cells9051157.
Dizon-Maspat J, Bourret J, D'Agostini A, Li F. Single pass tangential flow filtration to debottleneck downstream processing for therapeutic antibody production. Biotechnol Bioeng. 2012;109(4):962–70. https://doi.org/10.1002/bit.24377.
Lee JH, Ha DH, Go HK, Youn J, Kim HK, Jin RC, et al. Reproducible large-scale isolation of exosomes from adipose tissue-derived mesenchymal stem/stromal cells and their application in acute kidney injury. Int J Mol Sci. 2020;21(13):4774.
Busatto S, Vilanilam G, Ticer T, Lin LW, Dickson DW, Shapiro S, et al. Tangential flow filtration for highly efficient concentration of extracellular vesicles from large volumes of fluid. Cells. 2018;7(12):273. https://doi.org/10.3390/cells7120273.
We thank the Public Technology Service Center of Fujian Medical University (PR China) and Central Laboratory of Fujian Medical University Union Hospital (PR China) for providing technical support as well as experimental platforms. We appreciate Xuejing Zeng, Deye Zeng's (Fujian Medical University Union Hospital, PR China) assistance for the interpretation, and preparation of the histological section.
This study was financed by National Natural Science Foundation of China (grant No.81971855, 81671930), Joint Funding Project of Science and Technology Innovation in Fujian Province (grant No. 2017Y9101), Special Financial Funds of Fujian Province (grant No. 2018B054).
Aizhen Chen and Shijie Tang contributed equally to this work.
Department of Plastic Surgery, Fujian Medical University Union Hospital, Fuzhou, China
Aizhen Chen, Shijie Tang, Jiawei He, Xiangyu Li, Guohao Peng, Haoruo Zhang & Xiaosong Chen
Department of Plastic Surgery and Regenerative Medicine Institute, Fujian Medical University, Fuzhou, China
Aizhen Chen, Shijie Tang, Jiawei He, Guohao Peng, Haoruo Zhang & Xiaosong Chen
Department of Stem Cell Research Institute, Fujian Medical University, Fuzhou, China
Aizhen Chen, Shijie Tang, Xiangyu Li, Guohao Peng & Haoruo Zhang
Department of Pharmaceutical Analysis, the School of Pharmacy, Fujian Medical University, Fuzhou, China
Jinghua Chen
Department of Cardiac Surgery, Fujian Medical University Union Hospital, Fuzhou, China
Liangwan Chen
Aizhen Chen
Shijie Tang
Jiawei He
Xiangyu Li
Guohao Peng
Haoruo Zhang
Xiaosong Chen
Conceptualization: AZC and SJT designed, performed experiments, analyzed data, and wrote the manuscript. JWH and XYL performed experiments. JHC and GHP edited manuscript. LWC and HRZ conceived, designed experiments, and reviewed/edited the manuscript. XSC is the guarantor of this work and, as such, had full access to all the data in this study and takes responsibility for the integrity of the data and the accuracy of the data analysis. All authors have read and agreed to the published version of the manuscript.
Correspondence to Liangwan Chen or Xiaosong Chen.
All animal protocols were implemented under the Animal Ethical Committee of Fujian Medical University's (PR China) supervision and approval, which conforms to the guide for the National Institutes of Health to use laboratory animals.
The authors have declared that no competing interest exists.
Supplemntal material (enlarged figures) hADMSC-sEVs promoted neovascularization in the nude mice fat grafting model (rows meaned different staining and lines represents different groups and months).
Chen, A., Tang, S., He, J. et al. Small extracellular vesicles from human adipose-derived mesenchymal stromal cells: a potential promoter of fat graft survival. Stem Cell Res Ther 12, 263 (2021). https://doi.org/10.1186/s13287-021-02319-4
Accepted: 31 March 2021
Human adipose-derived mesenchymal stromal cells

redpajama_set_name: RedPajamaCommonCrawl


Korean haddock soup is one of my favorite soups. I learned to make it from my mom. When I moved to America, I missed my mom's soup badly. I didn't know what kind of fish to buy for this soup. One day, I found some frozen haddock at Walmart and tried this soup with it. It was almost the same taste as the fish used in my mom's soup, so I was very happy to find it. Since that time, I often buy haddock and make this soup. I'm glad to share this recipe with you. Thanks!
To make the broth, add the broth ingredients in a medium sized pan: 5 cups of water, 6 pieces of kelp, and 6-7 dried anchovies. (I used a dried anchovy pack instead.) Boil it for 10 minutes on medium-high.
While the broth is boiling, slice half an onion and ½ cup of Korean radish thinly. Cut 2 green onions into half inch pieces. Cut ½ a hot pepper. If you don't like spicy food, just add 1-2 pieces.
Cut 12 oz of haddock into 1-inch cubes.
After 10 minutes, remove the anchovy pack and kelp from the broth.
Add the haddock, the radish, and the onion into the broth.
When the soup starts to boil again, add ½ Tbsp of minced garlic and 1 tsp of salt. Cook for another 10 minutes on high.
Occasionally remove the foam that forms on the top of the soup. This will make the broth clear, and it will help to remove the bad fish taste.
Add the hot pepper and green onions. Then cook for 2 more minutes.
Serve this soup with rice and other Korean side dishes. Enjoy!
Did you defrost the haddock before adding it to the soup, or can you use frozen fish?
Only reason I defrost it a little is ..because I had to cut it.. 🙂 frozen fish can be added to it too.
can you substitute korean radish for regular red radishes?
Sorry I didn't try this soup with regular red radishes.. so I can't tell .. just my guessing is.. for the taste and texture it will not work that well.. you can still try though.
Aeri can this be made with black cod? I am craving a spicy black cod soup my mom used to make.
When Aeri first mentioned that she would make fish soup, I about puked my guts out. Luckily, I maintained my presence of mind and said, "That sounds like a great idea!" Wow was I surprised! This tasted nothing at all like I imagined. It is great! The broth is very clear and light tasting. It complements the fish very well. The soup does not have any strong fish taste. I highly recommend it to anyone that likes white fish.


Many of us, after desensitization and having learned a few techniques, often wonder: What next? Where do I go and practice my new communication skills? Many pws keep waiting for the "right opportunity". Some, smart ones, create these opportunities by inventing a social role for themselves.
Pankaj, a young man from Almora, has done Diploma in Mechanical Engineering and is now preparing for competitions. I met him a year ago- then again, he visited me at SMTA sometime back. We discussed a few basic ideas about stepping out of comfort zone. He was also active in MOOC 3 then.
Then, as it happens frequently, we lost touch; we were busy in our own mad worlds. Yesterday, I received a phone call from him, followed up by some pictures on whatsapp. He had followed ONE idea to its fruition.
Above all, he felt that he was connected to an idea bigger than himself. He was feeling happy and fulfilled. No therapy, could have given him this feeling and this sense of achievement and Self-confidence- he was convinced.
How did he do this? He identified a social need, he created a role for himself and stepped into it, without worrying about - "Mujhe kya milega?" (What will I get out of this?).
If we look around ourselves, Life is offering us many such opportunities to help and serve others every day. Why wait? Look carefully at your neighborhood, your office, your city. Surely someone needs your skills, your help. Jump into it with joy. This is your McMera Program - 100 percent free and 100 percent effective.


text:
Truck driver blown off bridge; dies after helicopter rescue
VIRGINIA BEACH, Va. (AP) — Authorities say a Navy helicopter plucked a driver from the roof of a tractor-trailer after it was swept off the Chesapeake Bay Bridge-Tunnel by high winds and began floating in the water. The driver later died.
Bridge and tunnel deputy director Thomas Anderson says the unidentified driver died after his rescue Thursday.
U.S. Coast Guard Petty Officer Corinne Zilnicki says the tractor-trailer was blown off the bridge shortly before 12:30 p.m. and the driver was rescued a little after 1.
Winds on the bridge-tunnel were about 40 mph Thursday, although Anderson says they had been much higher earlier in the day.


text:
COVID-19 AV library
Home / NEWS & INSIGHTS / News / McCullough Robertson cements its IPO status with Wagners listing
News 11 December 2017
McCullough Robertson cements its IPO status with Wagners listing
Leading independent Australian law firm McCullough Robertson has built on its strong initial public offering (IPO) credentials topping off the year with the third largest ASX listing of 2017 for Toowoomba-based construction group, Wagners.
Wagners (ASX:WGN) shares soared 23 percent immediately after opening at 1.30pm on Friday (8 December 2017). The stock, which listed at $2.71, rose 63¢ to $3.34 in the first 10 minutes of trading.
Lead legal Partner on the IPO Reece Walker said this 137th listing of the year and the third largest of 2017 was significant not only for the Wagner family and the history of the company but for Queensland as a whole.
"As Queensland's largest law firm, we were delighted that Wagners put its trust in us to manage what is an iconic moment for the company and for the Australian construction industry," Mr Walker said.
"Wagners exports its products to a variety of countries, including New Zealand, across Asia and the USA but the heart of the business has always been its Toowoomba foundations and base. For us, as a law firm that grew up in regional Queensland before expanding nationally, Friday's listing was a proud day, unlocking success for our client.
Mr Walker and his team, including lead Senior Associate Ben Wood and Senior Associate Naomi Omundson, advised on the IPO, as well as the restructuring of the group ahead of the IPO and demerger of the Wagners family shareholding (to occur following completion of the IPO).
The Wagner family raised $197 million from the IPO after selling 45 percent of the company, with more than 72 million shares purchased by new investors. The family will retain 55 percent of the stock, keeping a controlling stake.
"The fact that the stock rose immediately is an indication of the confidence the market has in the Wagners IPO. We look forward to seeing the business continue to grow in the coming years, including with the company's new generation materials," Mr Walker said
Wagners was established in 1989 by Henry, John, Denis, Neill, and Joe Wagner. It delivers high quality construction products and services both domestically and internationally through its Concrete, Quarries and Transport divisions.
In 2014, the Wagner family completed construction of their own airport, Wellcamp, in Toowoomba (not part of the listed company) with more than 80 weekly passenger flights to various destinations, including Sydney, Melbourne, Townsville and Cairns as well as an international freighter service.
Morgans and Credit Suisse acted as joint lead managers to the IPO and PwC also advised on the transaction.
In addition to publishing regular newsletters and focus articles for our clients and contacts, our lawyers are frequently asked by mainstream and specialist media to comment on a range of topical issues and trends affecting the Australian legal industry and Australian business.
Please contact Chris Saxby on +61 2 8241 5655 or by email for further information.
New Industrial Relations Laws – What it means for you
22 December 2020Insight
Payment Times Reporting Scheme
Australian Government proposes new broadcaster's licence and forced investment quotas in Media Reform Green Paper
Verification of identity – does it always need to be in person?
6 December 2020Insight
VIEW ALL NEWS & INSIGHTS
Level 11, 66 Eagle Street
Brisbane enquirySydney enquiryCanberra enquiryMelbourne enquiryNewcastle enquiry
We handle your personal information in accordance with our privacy policy.
Level 32, MLC Centre
Sydney enquiryBrisbane enquiryMelbourne enquiryNewcastle enquiryCanberra enquiry
Melbourne enquiryBrisbane enquiryCanberra enquirySydney enquiryNewcastle enquiry
Level 2, 16 Telford Street
Newcastle NSW 2300
Newcastle enquiryBrisbane enquiryCanberra enquiryMelbourne enquirySydney enquiry
Level 9, 2 Phillip Law Street
Canberra enquirySydney enquiryBrisbane enquiryMelbourne enquiryNewcastle enquiry
© 2017 McCullough Robertson. Site map Disclaimer Privacy Policy Credit Reporting Policy


text:
Q: IIS windows authentication (NTLM) We are using IIS 7.5 with only windows authentication enabled. providers are ntlm and negogiate ( since we want it to be accessible via internet).
The client is silverlight calling wcf services. even though we have session established the client sends the negotiate and server return 401 with some authentication token. this happends intermettinetly , with many sucessful calls ( intermingled with failed calls)
Many calls work fine and just send sessionid and everthing works fine.
In fiddler we see below
Client sends
Authorization: Negotiate TlRMTVNTUAABAAAAl4II4gAAAAAAAAAAAAAAAAAAAAAGAbEdAAAADw==
 ASP.NET_SessionId=0ix0fqf02j1imrpfc4awit3w
Server sends
WWW-Authenticate: Negotiate TlRMTVNTUAACAAAACgAKADgAAAAVgonitqRU/FVLp9EAAAAAAAAAAI4AjgBCAAAABgGxHQAAAA9BAEQATABBAEIAAgAKAEEARABMAEEAQgABAA4AQwBJAFIAVAAtAEQAMwAEABYAYQBkAGwAYQBiAC4AbABvAGMAYQBsAAMAJgBDAEkAUgBUAC0ARAAzAC4AYQBkAGwAYQBiAC4AbABvAGMAYQBsAAUAFgBhAGQAbABhAGIALgBsAG8AYwBhAGwABwAIAMsTFgtFNc4BAAAAAA

A: I'm not positive I understand your question but I'm guessing that you want to resolve the intermittent NTLM failures?
The headers you describe are part of the challenge response protocol of NTLM. Here's a more complete example:
Client: Get / HTTP/1.1

Server: HTTP/1.1 401 Unauthorized
        WWW-Authenticate: Negotiate
        WWW-Authenticate: NTLM

Client: Get / HTTP/1.1
        Authorization: Negotiate ###################### (encrypted Negotiate)

Server: HTTP/1.1 401 Unauthorized
        WWW-Authenticate: Negotiate ###################### (encrypted Challenge)

Client: Get / HTTP/1.1
        WWW-Authenticate: Negotiate ###################### (encrypted Response)

Server: HTTP/1.1 200 OK

It's probably a bad idea to use this Authentication method across the public internet; it's intended to be used within private intranets where both client and server can access a common Windows Active Directory Domain.
I suspect that your issue may be addressed in the "You are intermittently prompted for credentials or experience time-outs when you connect to Authenticated Services" KB article.
Here are some other related references:

*

*NTLM Bottlenecks and the RPC runtime

*NTLM and MaxConcurrentApi Concerns

*Performance tuning for NTLM authentication

redpajama_set_name: RedPajamaStackExchange


text:
« NOW BUY THIS BOOK!
THE REAL DIVERSITY: THAT OF BELIEF »
MORE FROM PAUL HARVEY #21
Tags: briefcase, City Hall, George Downs, Paul Harvey's For What It's Worth, Speedy Morris, Worth Department
Here are two more timeless items from Paul Harvey's For What It's Worth:
Our For What It's Worth Department has learned that Duluth, Minnesota, is where a city council-man . . .
George Downs . . .
In City Hall . . .
Put his briefcase down while he put his coat on . . .
Put his briefcase down behind a statue in the lobby . . .
And forgot it . . .
Left it there . . .
When he went across the street for dinner.
He returned to City Hall in time to hear there'd been a "bomb threat".
But the bomb squad had taken care of it.
They had opened a mysterious briefcase–with a blast of high-pressure water–and the whole lobby was wallpapered with George's soggy, shredded papers.
Our For What It's Worth Department hears that Speedy Morris–basketball coach for La Salle University–was shaving when his wife called out to tell him he was wanted on the phone by Sports Illustrated.
Speedy Morris was so excited by the prospect of national recognition that he nicked himself with his razor and ran–with a mixture of blood and lather on his face–and fell down the steps.
But he got to the phone.
And the voice on the other end said:
"For just seventy-five cents an issue you can get a one-year trial subscription . . ."
0 Responses to "MORE FROM PAUL HARVEY #21"


Looking for Dr Kincaid Chair at Runions' Furniture in Orrville, OH?
Runions' Furniture in Orrville, OH is an authorized dealer of Dr Kincaid Chair Products. It is DR Kincaid's promise to offer chairs of integrity and enduring quality. Each unique piece is crafted of the finest hardwoods and hand-finished to a patina characteristic of fine furniture. Upholstery is meticulously applied for lasting pleasure. They are extremely proud to be able to offer pieces that are produced at their plant in the foothills of North Carolina by their team of professional craftsmen.
So if you are looking for Dr Kincaid Chair products in Orrville, Wooster, Wadsworth, Massillon, Barberton, Norton, Green, Portage Lake, North Canton, Akron and Medina, or if you have any questions about Dr Kincaid Chair products, please feel free to call us at 330-682-4926 or simply stop by Runions' Furniture at any time and we would be glad to help you.


A simple space invader game, but this is just the start of a bigger project.
Vector Zero was an idea I had many years ago which I created a proto-type using XNA and C# in 2011. Nothing came of this due to me being busy with University. However I have always kept the concept in the back of my mind and added things to it for many years but have never worked on it since.
In the beginning of December I realised I had not released anything in 2018 because of another project that is in the works. So before the end I set out to create a simple version of Vector Zero for Android and then built upon it later.
The game took a total of about 2 weeks, the project was started at the beginning of December but due to christmas and all that, time was a little scarce. The Unity project was done with relative ease, except for the damn aspect ratio for different devices, but I had to implement the letter box effect.


Originally Published: June 1, 2013 1:03 p.m.
My wife and I recently spent a week on vacation in the Verde Valley and had a truly wonderful time.
We toured wineries, historic landmarks, went hiking, rented a jeep for a day and spent a good deal of time in and around Cottonwood.
We especially liked Old Town with its great restaurants and interesting shops. Yes, we toured Sedona too, but liked the "vibe" of your area much better.
I also attended a meeting of the Rotary Club of Verde Valley and met some great people doing great things in your community.
I am employed as a Parks & Recreation Director for a small city near Tacoma, Wash.
So, naturally, when I found out about the Cottonwood Recreation Center, I just had to see it for myself. My wife and I stopped by the facility one afternoon and were thoroughly impressed by this terrific community asset.
I spoke to the friendly staff at the front desk and was introduced to one of the Recreation Supervisors who gave us the VIP tour. This place has it all.
How proud you should be to have supported the construction of a first-class recreation center in a community the size of Cottonwood.
Your citizens and those in the surrounding area will reap the benefits for decades to come.
I also happened to read a letter to patrons regarding the recent budget cuts at the Recreation Center. I am all too familiar with this situation. It has occurred in my community also.
Loyal patron or not, I urge all of you to continue to support the operation of the Recreation Center.
While it may not be apparent at this point, you will look back a few years from now and be very thankful that you possess this community treasure that improves the quality of life for all of your citizens.


MAKE YOUR FAVORITE ZOMBIE ROAR WITH LAUGHTER! From the creator of the Bag of Unicorn Farts.
DO YOU NEED A UNIQUE BIRTHDAY GIFT IDEA? How about a fun Halloween gift item for that special someone?
DO YOU LIKE TO MAKE PEOPLE LAUGH? Bag of Zombie Farts will brighten any kid's day.
Here comes the most unique zombie gift of 2018.
Kids and adults boys and girls will laugh over the hilarious birthday gift idea of receiving farts for a present. Looking for a unique stocking stuffer or white elephant gift? We've got you covered! Each bag contains a packed 3oz size sealed bag of packed fruit punch cotton candy. The cotton candy is Made in the USA. Zombie farts are the perfect Halloween gift when you want to make someone laugh.
The Bags are cute, the cotton candy is awesome. - MAGICAL!


Liriodendron Tulipifera Fastigiatum is a medium sized, deciduous, pollution tolerant, compact form of tulip tree with a columnar habit and considered to be highly ornamental making it suitable for public parks. Due to its columnar shape, this makes it suitable for roadside planting. One of the features of the tree is that the leaves are missing a toe, which is an easier way of saying the apex is absent from the tip of the leaf and actually points inwards. The leaves have four lobes or toes and unfold like a book in the Spring.
It seems the more spectacular a flower, the longer you have to wait for it. Using that reasoning, these must be amazing as they can take from 7 years to make an appearance (must be female) although some estimates state 20 years. This age is taken from day one, our tulip trees are supplied at around the three year point or more. The flowers are tulip shaped and a green/yellow colour with bands of red and orange on the outside petals and come out in early Summer.
Apparently the Liriodendron Tulipifera Fastigiatum is a little bit of a non-conformist in the Autumn. The leaves turn yellow before turning brown.
At the 10 year point the tree will be 10 years old.....ok, just checking you are awake. On its tenth birthday, it will be around 6 metres tall but could go on to be much taller than that dependant upon local weather and soil conditions. Heights of 20 metres are common with a 5 metre spread.
We know how much you green fingered lot like to go out and tend to your garden children. To touch them is to love them, to touch them more is to love them more or other such philosophies however this tree will not thank you for it. Basically plant it and forget it in pruning terms. Only take out dead, diseased or crossing over branches.
A well draining site will give you much better results with acidic soil although the tree is chalk and clay tolerant. Full sun preferred with a sheltered site if possible. Try for a deep, moist loam especially for the early years but as it becomes established it will be drought tolerant.
Ideal conditions will give you a moderate to fast growth rate. Average growth is around 30cm a year but juvenile growth (the first 10 years) can be much more. Considered to be fully UK frost hardy.
This tree is supplied at a height of between 1.8 and 2.20 metres in a 10-20 litre container for year round planting and better results than from either bare-root or rootballed stock.
This section used to be Brigadier Tulip Information but since being upgraded, we now call it General Tuplip Tree Information!
Tulip trees are long lived (up to 300 years), very ornamental and will tolerate urban pollution well making them very suitable for parks and urban planting. Tulip trees with an upright shape are suitable for roadside planting. Usually a medium to large tree meaning they will grow to around 6m at 10 years. They could go on to grow much more but this is dependant upon local weather and soil conditions. Heights of over 50m have been recorded.
Most Tulip trees prefer a well draining site, will tolerate chalk but prefer acidic soil.
For those really into Tulip trees, you maybe interested to know that they are part of the Magnolia family. We are not sure if it was adopted or if there was some sordid affair between a Tulip and Magnolia many years ago. The flowers are a rich source of nectar and will attract bees to your garden. Any fruits produced will last long into the winter.
A hardwood tree with many uses because of its height and long length of clear trunk. The wood is light, straight grained, resists splitting and was the "tree of choice" for Native Americans to make canoes.
Useful as a shade tree because of its size and dense canopy. Younger Tulip trees can be fast growing reaching 30-100cm growth a year.


TOP English clubs including Arsenal, Manchester City and Chelsea have been tipped off that Edinson Cavani is open to a move away from Napoli - by his mum!
Express Online reported earlier this week that the trio were interested in signing the Uruguay hitman - and now Cavani's mum Berta Gomez has waded in.
She told a Uruguayan radio station: "He told me he wants to go elsewhere and to leave Napoli.
"Edi doesn't know what will happen yet. Naples is a city that lives for football. The affection of the people is marvellous and I have always told my son: 'you can go anywhere - but nobody will love you like they do at Napoli'."
The 26-year-old is widely expected to leave Naples this summer and admitted as much in a recent interview, despite being cautious of ruining his good relationship with the fans.
City are understood to be favourites to sign the 26-year-old although Real Madrid are also interested in his sevices.


For tea lovers, this combines the floral quality of jasmine tea with the clean vegetal aspects of matcha.
This simple matcha cappuccino is a favorite drink of mine. Matcha provides clean, vegetal notes with a good kick of caffeine. while the milk balances with sweet creaminess.


text:
Q: link error lnk2019 so im getting this error
Error   1   error LNK2019: unresolved external symbol "public: enum LL_ERR __thiscall linkedList<int>::addData(int)" (?addData@?$linkedList@H@@QAE?AW4LL_ERR@@H@Z) referenced in function "public: enum S_ERROR __thiscall stack<int>::push(int const &)" (?push@?$stack@H@@QAE?AW4S_ERROR@@ABH@Z)  C:\Users\Spyder\Documents\Visual Studio 2010\Projects\stack_ll\stack_ll\stack__ll.obj   stack_ll

plus the same errors for each function I call that is in the linked list
I have 4 files.  a linked_list.h, linked_list.cpp, stack_ll.h, stack_ll.cpp
Linked_list.h has the class definition.  linked_list.cpp has all the functions of said class.  stack_ll.h has another class for a stack that stores everything using the linked list.  and finally stack_ll.cpp that has the functions for the stack plus main().  For some reason the linker is saying that none of the functions of the linked list class have been defined.  linked_list.cpp is included in the project.  Any help would be greatly appreciated.

A: You haven't shown the code, but the problem is almost certainly that you've defined some class-template member functions in your .cpp file, rather than your .h file.
See the C++ FAQ: How can I avoid linker errors with my template classes?.


Tube Recycling Hero:Christopher Czambel is the Operations Manager for Bike and Roll Chicago, a bicycle and Segway rental and tour company. Christopher started out his 2012 season as a member of the distribution team, which required him to move equipment and resources between all rental locations. With a fleet of over 300 bicycles, they go through a lot of tubes.
Why Chris Recycles: Christopher began to notice the amount of broken rims, bicycle parts, tires, and tubes that where simply being thrown out. He began to investigate into company's who re-purpose these materials and found Alchemy.
After presenting companies to the head mechanic, he was told that if Bike and Roll had to pay for shipping, it wasn't worth their time to spend the money. Determined to reuse these tubes, Christopher stored used tubes at his house and donated them to other bike shops around Chicago who where already sending to Alchemy. It was through these shops, he learned that being an authorized Trek dealer, which Bike and Roll is, SHIPPING IS FREE!
Christopher started the 2013 season, making tube recycling a number one priority. He installed a collection bin for members as well as a bin at each mechanic location. Using boxes from QBP orders, from April until the end of the Alchemy tube drive last week, Bike and Roll collected over 800 tubes. He also made friends with the local Bike Police Patrol Unit mechanics and got them to donate their used tubes to the cause.
But Christopher won't stop there: his mission is to get bike tube recycling stations installed throughout Chicago, and he's partnering with The Active Transportation Alliance to make it happen.
Why they joined the Tube Collection Challenge: We joined the tube campaign because we couldn't stand throwing away our tubes and knowing they were just going to a landfill. We jumped at the chance to help turn bike waste into something useful and help the earth out, just a little bit! It was a win-win!
Why They Recycle: All of us at the Two Wheeler Dealer love to recycle and tubes are only a small part of our belief in recycling. We are happy to participate and find it fun to see how many tubes we can collect.
Staff Bios: Our staff is diverse and very active in numerous sports/activities such as surfing, stand-up paddling, racing bikes, long distance touring, photography, volunteering, commuting, fishing, school, raising families, art, and the list goes on…! The picture above is from a shop bike ride one morning.