Top Reasons to Go With Managed WordPress Hosting 2011: The Year the Check-in DiedOnce Again, Twitter Drops Ruby for JavaFacebook is Great, But Does It Make Businesses Any Money?UX Evolutions: The Future of the CameraApple Hiring a Team to Build “the Future of Cloud Services”More coverage and analysis from ReadWriteWebGet Ready For The ReadWriteWeb 2WAY Summit: June 13-14 in New York City Join ReadWriteWeb for one of the top tech events of the summer. Day one of the summit will feature talks from some of the smartest folks in technology and media, including Fred Wilson, Gawker CEO Nick Denton, Jason Calacanis, danah boyd, Flipboard CEO Mike McCue, Chris Dixon, NPR’s Andy Carvin and more. Day two is all about learning and interaction, with breakout discussions and enriching workshops that cater to all levels of Web knowledge.Visit the event site now for more information and to buy your ticket.LocationWhat to Expect from Where 2.0 in 2011: Context, Crowdsourcing & ProximityCheck-ins Are Dead? Location App Life360 Adds 1 Million Users in 10 WeeksMore Location coverageMobile Tags:#web#Weekly Wrap-ups Using Windows Phone 7: One Week with an HD7HTC Dominating Online Chatter, Says Market Research FirmMore Mobile coverageInternet of ThingsMicrosoft Announces Kinect SDK: Why This is the Future of WindowsThe State of the Internet of Things – Is There Enough Commercial Activity?More Internet of Things coverageCheck Out The ReadWriteWeb iPhone App As well as enabling you to read ReadWriteWeb while on the go or lying on the couch, we’ve made it easy to share ReadWriteWeb posts directly from your iPhone, on Twitter and Facebook using the official ReadWriteWeb iPhone app. You can also follow the RWW team on Twitter, directly from the app. We invite you to download it now from iTunes.ReadWriteEnterpriseReadWriteEnterprise is devoted to enterprise 2.0 and using social software inside organizations. 5 Top Marketing Blogs by Entrepreneurs5 Tools to Improve Your Idea Before You Write a Line of Code4 Tips for Taking Your Startup InternationalReadWriteCloudReadWriteCloud is dedicated to virtualization and cloud computing. Why Tech Companies Need Simpler Terms of Servic… Facebook is Great, But Does It Make Businesses Any Money?How to Manage Your Business’s Presence on BingiContact Offers Free Email Marketing Service for Small BusinessesReadWriteHackReadWriteHack is a resource and guide for developers. Once Again, Twitter Drops Ruby for JavaApple Hiring a Team to Build “the Future of Cloud Services”The Cloud Stratosphere [Infographic]ReadWriteBizReadWriteBiz is a resource and guide for small to medium businesses. All About “Mango:” New Version of Windows Phone Arriving this FalliPad to Dominate Tablet Market Until 2015Nokia Reveals Symbian Update, New Phones & Ovi Store NumbersEnjoy your weekend everyone!Subscribe to the Weekly Wrap-upYou can subscribe to the Weekly Wrap-up by RSS or by email below.RWW Weekly Wrap-up Email Subscription form: 8 Best WordPress Hosting Solutions on the Market ReadWrite Sponsors Related Posts Live Blog: Microsoft MIX 2011 Day 1 – Internet Explorer 10 Preview, and MoreMicrosoft MIXed Messages on Silverlight, Internet Explorer 10Live Blog: Microsoft MIX 2011 Day 2 – New Windows Phone 7 Features, Silverlight 5, Kinect SDKReadWriteMobileReadWriteMobile is dedicated to helping its community understand the strategic business and technical implications of developing mobile applications. Government Agrees With Microsoft: Google Wasn’t Certified [Update]Mixed Reviews for Blackberry’s Tablet, But Will It Be a Good Enterprise Device?IT Poll: Which NoSQL Company Will Be Acquired by a Major Player First?ReadWriteStartReadWriteStart is a resource for startups and entrepreneurs. One of our top posts this week was Richard MacManus’ look at the future of the camera. We all know how smartphones integrated cameras. “Could we be about to see the inverse – cameras integrating smartphone technology?” he asked. The story is part of our ongoing series looking at what it means to consume and produce media in post-PC-centric world.After the jump you’ll find more of this week’s top news stories on some of the key trends that are shaping the Web – mobile, location, Internet of Things – plus highlights from our six channels. Read on for more.Top Stories of the Week A Web Developer’s New Best Friend is the AI Wai…
Get the Free eBook! Learn how to sell without a sales manager. Download my free eBook! You need to make sales. You need help now. We’ve got you covered. This eBook will help you Seize Your Sales Destiny, with or without a manager. Download Now If you want to move from transactional to consultative, here is where you start.Spend More Time in Discovery: Spend more time in discovery understanding your dream client’s real needs. You want to understand the needs that they know how to express, and you want to understand the real needs that they have not yet identified. You want to deal with the root cause, not the presenting problem. To create the most compelling, value creating opportunity, you need to work to understand what needs to change, why it needs to change, what that change needs to look like, and how you can help your dream client with their transformation. Most of the value you create as a salesperson happens early in the process.Go Deep and Wide: You can’t do real discovery work with a single contact in a complex sale. You need to meet with stakeholders throughout your dream client’s company. You need to work your way up to leadership to understand their strategic needs, and you need to work down to end users to capture their needs—and a lot of insight around the real obstacles you are likely to encounter. The consensus you need later is built on your having captured the organization’s needs—not just the CEO of the Problem.Be Bigger and Bolder: You need to present a differentiated, compelling level of value. You can’t sell what you believe is easy to sell (that makes you something less than a trusted advisor). You can’t sell what you believe is easy to sell, or what you believe you can sell fast. Both are transactional behaviors. You have to sell the greatest outcome you are capable of producing, even if it is a more difficult sale, and even if it takes more time.Don’t Sell Product: If you want to focus on value creation, then you can’t lead with product. The value you create cannot be in the product or service. It has to be greater than that. It has to be outcomes.Don’t Sell Price: If you lead with price, you aren’t selling the value you create. Price is the absence of value. Leading with price means you aren’t competing on the real value you create.Don’t Sell to Your Coach/Sponsor/Authority Alone: Selling greater value requires that you sell to the whole organization. Selling to only one person is transactional.The first three behaviors here allow you to create and develop compelling value. They move you from transactional to consultative. The last three behaviors subtract value and move you in the wrong direction.QuestionsHow do you create value early in the sales process?What do you do to create value in the discovery phase of the buying cycle?Where do you find your dream client’s real needs and real obstacles?Do you always present the best solution possible, even when it may cost you time?
OTTAWA – A report by the Canadian Centre for Policy Alternatives recommends that payments to shareholders such as dividends and share buybacks by companies should be limited if their pension plans are underfunded.The report says pension regulations must expand to consider broader financial decisions within companies.It says that in many instances, firms are complying with the minimum required payments under the rules, but they are not making up the shortfalls in the pension plans as fast as they could.Companies with defined-benefit pension plans have been hurt by the financial crisis and low interest rates, which have increased the amount of money they are required to have in their pension plans to pay future benefits.When a pension plan is not fully funded, members face the possibility of seeing their pensions reduced if the plan is forced to wind up.The report noted that the pension plan at Sears Canada has a $267-million shortfall, but the retailer which is in the process of liquidating has paid $1.5 billion in shareholders in dividends and share buybacks since 2010.
Entering its game against Minnesota, the Ohio State women’s soccer team had high hopes for its season-opening run. Those high hopes were shattered on one serious play during the seventh minute, when redshirt senior goalkeeper Jillian McVicker tried to make a save.McVicker collided with a Minnesota player, and sustained fractured ribs, a punctured lung and a lacerated kidney. She was quickly removed from the field and taken to a Minnesota hospital,where she remained in intensive care for two days before being downgraded to standard care.“Ohio State senior goalkeeper Jillian McVicker is being held at a Minneapolis hospital through the end of the week for further treatment and testing after suffering an injury Sunday at Minnesota,” the athletic department said in a statement from an OSU spokesperson on Friday.McVicker has appeared in over 50 matches for the Buckeyes since joining the team in 2012. McVicker is a former assistant sports director for Lantern TV and also reported for The Lantern during the 2016 Spring Semester.After being released from the hospital on Saturday afternoon, she and her family drove back to Columbus, stopping in Chicago along the way before arriving back at OSU on Monday.Although not the first time the Metuchen, New Jersey, native has sustained an injury, this time, was different from the rest.OSU senior goaltender Jillian McVicker flexes as she recovers in her hospital bed in Minnesota. Credit: Courtesy of Jillian McVicker“I knew that it definitely wasn’t my muscle or anything. I just came out, I knew there was going to be a collision but I just had to get the ball,” McVicker said. “When I came out, I thought I had the wind knocked out of me at first, and then I couldn’t breathe for like a minute.”According to McVicker, her doctor said this type of injury is most common after being involved in a car crash, not with a sport like soccer.The injury has effectively ended the OSU career of McVicker, with a little over a month left in the regular season. Although her time as a Buckeye athlete has been cut short, she has still maintained a positive attitude and high spirits. “All the adversity I have faced, whether it be from injury or typical adversity that every athlete goes through, you just have to keep your head up and keep your mind on the process,” McVicker said. “Take it day-by-day and step-by-step. Which is actually very ironic because that’s exactly what I’m doing in my rehab right now.”As part of the healing process, McVicker can only lift things under 10 pounds, while limiting the amount of overall movement she has throughout the day.On social media, McVicker has received an outpouring of support from family, friends, teammates, players from other teams and, most notably, former United States Women’s National Team midfielder Julie Foudy. McVicker, a four-time OSU scholar-athlete and three-time Academic All-Big Ten selection, has appeared in over 60 matches during her career with the Buckeyes. She is a double major in strategic communications and journalism, and will be graduating this December.After graduation and her injuries have fully healed, McVicker has intentions to continue her playing career outside of OSU.“I definitely want to graduate and want to look at playing overseas in Germany professionally or in America,” McVicker said. “I’m going to probably try to get an agent and figure that out once I graduate. It’s definitely not the last time I’m going to put on my keeper gloves and my jersey again.”Although she will no longer be lacing up her cleats for the Scarlet and Gray, McVicker said she will be embracing her new role of cheering her team on. Without the redshirt senior in the net, the Buckeyes will now be looking to the sideline when when she returns. From here on out, the focus of McVicker is to rally the troops to continue the team’s success. “Although this injury happened to me, my main thing for this season is for my team to be successful,” McVicker said. “Yes, this is horrible and I wouldn’t wish this upon anyone. But at the same time, all of my energy and everything when I get back is to prepare my the team the best I can in my new role.”
Ernesto Valverde believes his team is extra motivated to win the Champions League, as they bid to end their barren run since 2015.The Spanish Champions were dumped out of the competition in the Quarter-final stage by Roma but Valverde thinks they have the right mindset to enjoy European success once again.“We would like to win this competition, and surely this feeling is being felt by other teams too,” he told a pre-match press conference.”Teams like Atletico Madrid, Valencia, Manchester City and Real Madrid all have the same desire, yet I like that there is an extra motivation to win the Champions League this year.”Many pundits have tipped Barca to win the tie PSV, however, Valverde is wary of their attacking talents.Quiz: How much do you know about David Villa? Boro Tanchev – September 14, 2019 Time to test your knowledge about Spanish legendary forward David Villa.“They are the Dutch champions and are used to taking the initiative,” he said.”I don’t see them changing that. Most teams want to maintain their style. They will want to attack and we are going to have to defend well against them.”We try to play the same way every game and we will try to do the same again tomorrow.”They have some very talented wingers, especially on the left. They have some very good full-backs as well, and we have to deal with that regardless of who plays for us.”
His advice is not an uncommon cry and for the Dominican Community which is said to have at least 4,000 registered in country, Hernandez said this is a huge loss for them, but not just for them but for the Turks and Caicos. He added, “The TCI is a high class tourist destination, but if things continue going in the current direction, we will lose everything…” The Assistant Consul for the Dominican Community, Edwin Hernandez called the surge in killings, scary. “if the relevant authorities don’t put a handle on this type of crimes, it will come to a point where it will be totally out of hand and will be impossible to control crime.” Juan Carlos Morla was shot reportedly at point blank range in an armed robbery shortly after 3:30pm on Friday October 7, 2016 in an area officially known as Times Square… steps away from two very popular family eateries and steps away from the Public Library. Morla worked in construction in the TCI. Facebook Twitter Google+LinkedInPinterestWhatsApp Hernandez pleaded with authorities, and said as a citizen he believes it is high time the authorities respond also with better, stronger cases that they can win in the courts. I quote him again, “..please give the general public an answer, but not just saying who they believe did it, but bringing those guilty before the courts with sufficient evidence to secure a conviction.” Facebook Twitter Google+LinkedInPinterestWhatsAppProvidenciales, TCI, October 13, 2016 – Four Dominicans are dead this year at the hands of cold-hearted criminals and that community is saddened by losses. Two women, whose suspect is behind bars awaiting a sufficiency hearing set for next month so that the double homicide trial can begin and two men; both gunned down in separate instances of armed robberies. Police confirm that the 27 year old killed in down town Providenciales last week was Juan Carlos Morla. Related Items:27 year old Dominican male gunned down in broad daylight, danny depaula killed in tci, fourth dominican killed this year in provo
Lightning put on a show over Clark County late Thursday and early Friday, but appeared to spare the region of any damage, fires or headaches, local officials said.About 40 lightning strikes lit up the sky in Clark County, said Andy Bryant, a hydrologist with the National Weather Service in Portland. Most of those flashed between 8 and 10 p.m. Thursday, he said, largely in east county near Camas and Washougal.Despite the potent storm, fire officials in the two cities reported no major calls associated with the weather.“Frankly, I’m surprised, as prolific as the lightning was,” said Scott Koehler, fire chief at East County Fire & Rescue.Lightning lasted until around 11 p.m. Thursday, and then started again around 5 or 6 a.m. Friday, Koehler said.Though no injuries or property damage were reported Thursday night or Friday morning, Koehler advised residents to remain vigilant. Lightning strikes can cause roots underground to smolder for days before a fire ignites, he said.Camas-Washougal Fire Chief Nick Swinhart was out of town Thursday night, but noted he did not receive any calls notifying him of any major weather-related events. People should stay inside, avoid standing near tall metal structures and remain on the bottom floor, if they’re in a multifloor building, when lightning storms occur, he said.
Bob Daemmrich for The Texas TribuneU.S. Sen. Ted Cruz meets with Austin-area home schoolers on August 4, 2018.As Ted Cruz took questions at a Republican women’s event here Saturday evening, Bastrop retiree Ronnie Ann Burt wanted to know: Should she really trust the growing barrage of chatter online that the senator’s re-election bid is in peril?Cruz’s response: Believe it.“It’s clear we have a real and contested race where the margin is far too close for comfort,” said Cruz, who’s facing a vigorous, massively funded challenge from U.S. Rep. Beto O’Rourke, D-El Paso.Cruz’s stop in this small Central Texas town was part of a return to the campaign trail Saturday in which the incumbent cranked up his long-building warnings that Democratic enthusiasm in the era of President Donald Trump should not be discounted, even in a state as red as Texas.The timing couldn’t have been more fitting: A trio of polls came out this week showing Cruz’s race tightening and a national political forecaster shifted the contest in O’Rourke’s favor. Meanwhile, Cruz launched his first TV ads Friday, including three targeting O’Rourke, and the challenger moved quickly to turn them into a fundraising boon for him.Appearing Saturday afternoon at the conservative Resurgent Gathering in Austin, Cruz delivered a nearly 10-minute assessment of the uncertain political landscape he faces in November.“The biggest challenge I have in this race … is complacency,” Cruz said. “People say all the time, ‘Oh, come on, it’s a Texas re-elect. How could you possibly lose?’ Well, in an ordinary cycle, that might be true. But this is not an ordinary cycle. The far left is filled with anger and rage and we underestimate that anger at our peril.”Cruz added that there is reason to be skeptical of the polls — his campaign has criticized their methodologies — but the trendline “ought to be a cause for concern for everyone.”After the Resurgent conference, Cruz headed to a meeting with supporters and home school families in east Austin, where he continued to press the argument that the GOP base cannot take November for granted. Cruz said Travis County was the “base of the support” for O’Rourke but other “bright red” counties like Denton, Tarrant and Collin need to turn out hard as a counterweight.“There are a lot of good, strong conservatives [in Travis County] too — you’re outnumbered, but it does make you sturdier when you’re withstanding criticism and abuse,” Cruz said. “What [Democrats] are doing is to find every liberal in the state of Texas and get them energized and get them to show up.”Cruz’s remarks at events Saturday came a day after Gov. Greg Abbott offered a more reassuring forecast for November while addressing the Resurgentconference. He dismissed the idea of a “blue wave” in November as media hype that “sells papers” and reminded the audience that he ended up defeating his much-ballyhooed Democratic opponent, Wendy Davis, by over 20 points in 2014.“Texas is going to stay red,” said Abbott, whose Democratic opponent, Lupe Valdez, has not caught traction in the way O’Rourke has against Cruz.Cruz did not sound as sure as Abbott on Saturday — and his supporters appeared to get the message.“I think what Sen. Cruz said is true: The Democrats are unhappy that they lost [the 2016 presidential election] because they never anticipated it, and so they’re coming out in force, and I see it in my own county,” said Jeanne Raley, vice president of the Lost Pines Republican Women group that hosted Cruz in Smithville. “That just means we have to work harder.”“Complacency will kill any of us,” she added.O’Rourke spent Saturday in the border city of Del Rio, the latest stop on his 34-day tour of the state during the August congressional recess. Holding an evening town hall there, O’Rourke geared up supporters for a final three months of the race with momentum on their side.“They say there are two points that separate us, the campaign we’re running and Ted Cruz — two points is all we’re down right now,” O’Rourke said. “There are 94 days to go in this election. We can totally win this, but it is 100 percent on us.”O’Rourke’s campaign continued to show momentum Saturday afternoon, when it said it had raised more than $500,000 over the last 24 hours in response to Cruz’s commercials. The campaign has set a goal of topping $1 million by the end of the weekend.Cruz got a taste of the opposition several minutes into his appearance at the Resurgent Gathering, when a protester interrupted with a sign reading, “Russian Bootlicker,” called Cruz a coward and used an expletive to denounce the crowd before breaking out in chants of “Beto!” Speaking afterward, Cruz wasted little time turning the incident into a rallying point for the fall.“That anger, by the way, is dangerous,” Cruz said. “Every one of us needs to be taking this November election deadly serious.”Sydney Greene contributed to this report. Share
As it turns out someone with some serious skills also wanted that. Cesar Herada, a researcher who has formerly been associated with Ushahidi and MIT’s Senseable City Lab, has created the Protei oil-spill cleaning drone. The Protei oil-spill cleaning drone is designed to be a semi-autonomously device that can sail into the sea and scoop up the oil in a spill, leaving the oil in the container and the water in the ocean. How is this accomplished? With the help of powerful oil-sucking booms that are built into the device. The oil-sucking boom is detachable, and each one is able to hold up to two tons of crude oil per trip. The advantage to using one of these devices is that no humans have to be exposed to toxic substances in order to clean up the mess. © 2010 PhysOrg.com (PhysOrg.com) — Oil spills represent a significant danger to the oceans of the world. Many of us watched the DeepWater Horizon Oil Spill in the Gulf of Mexico and wished that there was a simple way to clean it up. Citation: Cesar Herada designs oil sucking drones to help clean the seas after a spill (2011, April 12) retrieved 18 August 2019 from https://phys.org/news/2011-04-cesar-herada-oil-drones-seas.html Explore further More information: sites.google.com/a/opensailing.net/protei/ The Protei drones are also able to be modified for other types of disasters. In the future modified versions of the Protei drones may possibly be sent in to detect the levels of radiation in water supplies, or to collect samples of other potentially polluted waters. The designers have also mentioned that there may be some commercial uses for the Protei drones as well, but they did not give any specifics on this point. Clean-up tools may help protect wetlands from Gulf of Mexico oil spill The best part is that Protei is an Open Source Hardware project. This means that its design will be available to the public, so it can be built by anyone. The remote controlled Protei is relatively inexpensive to produce and inflatable. This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only.
Re-engineering the cell membrane for improved biofunction is an emerging, powerful tool in cell biology to develop next-generation cell therapies. The process can allow users to supplement cells with added therapeutic functionalities. Additional functionalities can include cell homing, surface adhesion or resistance to hypoxia for enhanced cellular capabilities. However, the number of such examples on re-engineered plasma-membranes to activate membrane-bound enzymes that promote the assembly of extracellular matrix (ECM) proteins to promote cell functionalities are limited. 3D projection of fibrin gel containing fibrinogen stained with Alexa 594 (red) fibrinogen and hMSCs incorporating sc_thrombin [ox890] stained with Hoeschst 33342, imaged after 60 min of cell associated fibrin formation in cell culture. Credit: Nature Communications, doi: 10.1038/s41467-019-09763-0. They synthesized the artificial membrane binding thrombin complex using a two-step process to generate an active supercationic thrombin construct (sc_thrombin). Deller et al. also generated a polymer surfactant corona or green halo using electrostatic coupling of glycolic acid ethoxylate 4-nonylphenyl ether (ox890) to sc_thrombin to engineer a third variant sc_thrombin [ox890]. The scientists controlled the reaction conditions (pH, temperature and chemical composition) carefully and monitored the reaction progress using zeta potentiometry across a period of two hours. They observed the activity of thrombin by monitoring/characterizing the increasing turbidity of the fibrinogen solution. They then obtained MALDI-TOF spectra (matrix-assisted laser desorption ionization time of flight mass spectroscopy) of the native and modified thrombin to show full cationization of the construct. When Deller et al. conducted compression testing of the resultant self-supporting structures, the Young’s moduli were similar to soft fibrin hydrogels, indicating consistency. To investigate thrombin adhesion to cell membranes, the scientists chose a monolayer of bone marrow derived hMSCs (with well-characterized adipogenic, chondrogenic and osteogenic pathways). First, they incubated a monolayer of hMSCs with fluorescently-labelled analogues of thrombin; thereafter, they labeled the hMSCs with a plasma membrane-specific dye and imaged immediately to confirm the thrombin-plasma membrane binding. Using time-lapse confocal fluorescence microscopy, they showed the nucleation and fibrin growth from the cells thereafter. Welding with stem cells for next-generation surgical glues Advanced cell therapies are currently approaching clinical translation in response to an increasing demand for newly modified, cell-specific matrices (scaffolds) for biocompatible therapeutic performance. However, the rational design of matrices is extremely challenging since the cell phenotype and cell fate can be intertwined to a wide-range of scaffold-dependent factors; including cell adhesion, surrounding chemical composition, cell receptor stimulation, surface micro-/nano-morphology and mechanical stiffness. These factors immediately impact cells during in vitro tissue engineering, typically when cells are seeded to adhere on biocompatible and biodegradable scaffolds in the lab, where the scaffolds act as a surrogate extracellular matrix (ECM). Eventually, when the cells grow and differentiate, they can produce natural ECM of their own, to gradually replace the biomimetic scaffold material and form a structurally self-supporting biological entity. Deller et al. also completed cell growth assays to determine relative metabolic activities of labelled hMSCs-thrombin to show the modified cells were without observable cytotoxicity in varying concentrations of thrombin (1 µm to 25 mM). Using confocal microscopy again, the scientists showed the arrangement of fibrin structures emanating from the plasma membrane of the hMSC monolayer, in contrast to hMSCs without thrombin. The work protocol thereby generated a 3-D fibrin hydrogel construct with dense cellular aggregates surrounded by a 3-D fibrin matrix. The scientists also investigated the ability of the fibrin hydrogel system to sustain 3-D cultures for long-term growth; a requisite for tissue engineering, to show hMSC differentiation via adipogenic and osteogenic pathways. To verify the results, the scientists conducted extensive biomechanical tests on the cell types and tested for the upregulation of specific genes SOX9 and RUNX2 involved in chondrogenesis and osteogenesis respectively, using RT-PCR (reverse transcription polymerase chain reaction), to substantiate the fibrin hydrogel system sustained long-term hMSC proliferation in vitro.After confirming the membrane re-engineering approach for in lab tissue engineering applications, Deller et al. investigated the ability to produce thrombin coated cells in lab for their injection at a site of injury to initiate a healing response for tissue-engineering applications in vivo. For this, the scientists used an in vivo Zebrafish model system to perform preliminary cell transplant studies. Zebrafish is a model organism, established for fluorescently labelled live cell imaging and thrombolytic and hemostatic processes; suited for the present work. The scientists isolated, labelled and delivered fluorescently labelled primary Zebrafish fibroblasts, labelled with sc_thrombin[ox890] conjugate via microinjection to show cell survival after 3 days at a site of incisional injury. The synthesis and characterisation of the supercationic thrombin-polymer surfactant conjugate. a Schematic showing the electrostatic surface potential of native and supercationic thrombin (sc_thrombin) (PDB; 1UVS) at pH 7, highlighting the anionic (blue) and cationic (red) charged regions. Generation of the polymer surfactant corona (green halo) via electrostatic coupling of glycolic acid ethoxylate 4-nonylphenyl ether (ox890) to sc_thrombin gives [sc_thrombin][ox890]. b Zeta potential (ca. pH7; n = 3) of thrombin as a function of cationization times (0–120 min). Data reported as means ± standard deviation (s.d.). c Rate of fibrinogen solution (3.125 mg mL−1) gelation as measured by changes in turbidity (600 nm) catalysed by sc_thrombin subjected to various cationization times (0–120 min). Data shown as one-phase association curves of raw data. d MALDI-TOF MS spectra (m/z = 3) of native and sc_thrombin (60 min). Credit: Nature Communications, doi: 10.1038/s41467-019-09763-0 Schematic diagram showing in situ fibrin hydrogel formation from the membranes of bone-marrow derived human mesenchymal stem cells (hMSCs). Artificial membrane binding thrombin constructs comprising supercationic thrombin molecules (white) surrounded by a polymer surfactant corona (yellow) that associates with surface exposed cationic (red) residues spontaneously insert into bilayer regions of hMSC plasma membranes. In the presence of fibrinogen, the membrane-immobilised thrombin catalyses fibrin formation (blue fibres) within the interstitial spaces between the cells giving rise to a self-supporting hydrogel monolith. Credit: Nature Communications, doi: 10.1038/s41467-019-09763-0 More information: Robert C. Deller et al. Artificial cell membrane binding thrombin constructs drive in situ fibrin hydrogel formation, Nature Communications (2019). DOI: 10.1038/s41467-019-09763-0 D. E. Discher. Tissue Cells Feel and Respond to the Stiffness of Their Substrate, Science (2005). DOI: 10.1126/science.1116995 Tamer A.E. Ahmed et al. Fibrin: A Versatile Scaffold for Tissue Engineering Applications, Tissue Engineering Part B: Reviews (2008). DOI: 10.1089/ten.teb.2007.0435 © 2019 Science X Network In a recent study, Robert C. Deller and co-workers at the interdisciplinary departments of Cellular and Molecular Medicine, Engineering, Functional Nanomaterials and Pharmacology, and Neuroscience in the UK, bioengineered a self-contained cell matrix-forming system. In the experiments, they modified the plasma membrane of human mesenchymal stem cells (hMSCs) to integrate a new thrombin construct, which gave rise to spontaneous fibrin hydrogel nucleation and growth when supplemented with human plasma concentration levels of fibrinogen in cell culture media. The scientists bioengineered the cell membrane by synthesizing a membrane-binding supercationic thrombin-polymer surfactant complex. Thereafter, they observed cell differentiation in the resulting robust, stem cell-containing fibrin hydrogel constructs to form osteogenic and adipogenic cell lineages. The differentiated cells could eventually secrete fibrin to form self-supported bioengineered cellular monoliths that exhibited Young’s moduli as expected of the native extracellular matrix. The results are now published in Nature Communications. A range of natural biocompatible polymers have produced such transient hydrogel scaffolds for tissue engineering; including chitosan, gelatin and fibrin. Fibrin hydrogels are the most popular among them, since they can be produced readily at room temperature using proteolytic cleavage. Biological fibrin formation occurs in response to injury, culminating from a biochemical cascade of proteolytic cleavage, which converts prothrombin to thrombin and forms a fibrin-hydrogel clot. Fibrin hydrogels can therefore mediate cellular biomolecular functions and regulate the osteogenic and chondrogenic differentiation of human stem cells such as hMSCs. They can also be conveniently delivered using syringes, albeit with complications related to reduced cell viability.In the present work, Deller and co-workers first described a method to synthesize supercationic thrombin-polymer surfactant complexes that spontaneously bound to the plasma membrane of hMSCs to drive in situ fibrin hydrogel nucleation and growth. The resulting self-supporting hydrogel construct allowed high levels of metabolic activity as an artificial matrix for effective differentiation of stem cells to form adipogenic or osteogenic cell lineages. The scientists then showed the feasibility of the method of cell functionalization by injecting thrombin-labelled GFP-expressing fibroblasts (GFP: Green fluorescence protein) into a zebrafish (Danio rerio) skin wound model to demonstrate their in vivo biocompatibility for hemostatic applications. Explore further Journal information: Nature Communications In vivo zebrafish injury and [sc_thrombin][ox890] labelled GFP + fibroblast addition. Schematic representation of the in vivo adult zebrafish injury model. a Wildtype (non-transgenic) recipient zebrafish were anaesthetized and a 4 mm incisional injury made on the ventral upper thorax. A lateral view is shown. b Unlabelled or [sc_thrombin][ox890] labelled, FACS sorted GFP+ fibroblasts were injected at six sites around the edge of the incisional injury. At the desired time-point, fish were sacrificed and the tissue surrounding the incision was fixed, imaged and embedded for sectioning. A ventral view is shown. Ventral view of the area of tissue surrounding the incision at 3 dpi following transfer of c unlabelled or d [sc_thrombin][ox890] labelled GFP+ fibroblasts. Similar numbers of cells were retained at all wounds. The red line depicts the approximate position of the incisional injury which is fully re-epithelialized at this stage. Sections through the injury region at 3 dpi following transfer of e unlabelled or f [sc_thrombin][ox890] labelled GFP+ fibroblasts. No obvious differences were observed between wounds containing labelled or unlabelled cells. Arrows indicate the position of the incision. Credit: Nature Communications, doi: 10.1038/s41467-019-09763-0. Citation: Engineering artificial cell membranes to drive in situ fibrin hydrogel formation (2019, May 6) retrieved 18 August 2019 from https://phys.org/news/2019-05-artificial-cell-membranes-situ-fibrin.html Using macroscopic observations and histological assays, the scientists further revealed that there were no adverse effects between fish injected with engineered or native fibroblasts. However, Deller et al. expect to complete further investigations to understand the precise effects on the specific wound healing response using bioengineered cells in the future.In this way, Deller et al. synthesized and characterized a new membrane active thrombin construct and demonstrated its application to drive in situ fibrin formation in the plasma membranes of stem cells. The scientists showed that thrombin-based fibrin hydrogel constructs prepared using the new protocol supported high levels of cell growth and viability to eventually produce a self-supporting tissue engineered construct. The stem cells were also able to differentiate along the well-defined adipogenic and osteogenic pathways while demonstrating Young’s moduli similar to the native cells to indicate high levels of integration of the modifications. Deller et al. propose to optimize the protocols for further experiments in vitro prior to in vivo translation, to gain further insight to the enzymatic activity of cell membrane bound bioengineered proteins to develop biocompatible, hemostatic products. Evaluating rhodamine (rh) labelled rh_thrombin, rh_sc_thrombin and [rh_sc_thrombin][ox890] on hMSC plasma membrane affinity. Cells labelled with CellMask (green) and rhodamine labelled thrombin (magenta) visualized with confocal microscopy. Video shows the rh_sc_thrombin [ox890] labelled hMSCs supplemented with fibrin gel conjugating with fibrinogen (green) to highlight fibrin formation emanating from the bioengineered cell surface. Credit: Nature Communications, doi: 10.1038/s41467-019-09763-0 , Science This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only.