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MMH recommends everyone wear cloth masks
Everyone in the community should wear cloth masks in public to help prevent the spread of coronavirus by asymptomatic carriers, Midland Memorial Hospital officials said Monday. They estimated that 20 percent or more of the population could be asymptomatic carriers of the virus.
Hospital officials’ recommendation came after the CDC issued guidelines Friday that cloth face coverings should be worn in public places where social distancing is difficult to maintain, such as in grocery stores and pharmacies.
Midland Memorial CEO Russell Meyers said cloth masks will not prevent someone from being infected by the virus, because the aerosolized particles from an infected person are microscopic and can travel through openings in the masks. Cloth coverings can, however, prevent further spread of the virus.
“The purpose of the new CDC guidelines, which we will be implementing here in the hospital, is not so much to protect the person wearing the mask,” he said. “A cloth mask – no one is suggesting it will protect anyone from the aerosolized droplets that could come from a COVID patient’s cough or sneeze.”
Chief Medical Officer Dr. Larry Wilson agreed the masks are intended to protect the rest of the population from asymptomatic carriers.
“If I’m standing up here and I cough or sneeze, it’ll blend the distribution of my water droplets into the air around me,” he said. “So I am, by virtue of wearing a mask, protecting the people that I’m around when I’m in a public environment.”
Wilson said grocery stores and other retailers are the ideal places to wear these masks, as customers in those places are not practicing proper social distancing.
“I want to emphasize there are people with this virus in our community that don’t know they have it,” he said. “I promise you that’s true right now, and we are spreading it amongst ourselves if we’re not really, really careful with that.”
Additionally, he said MMH has expanded the criteria for testing patients at their drive-thru location.
He said they were originally testing only residents with a respiratory tract illness and fever who had traveled to a high-risk area or been on a cruise shop but are now testing more broadly.
“We’re looking more widely for the disease in the community, rather than just looking at people that have either been into a high-risk area or had known contact with somebody (positive for COVID-19),” he said.


text:
It has been reported about
It has been reported about the synthesis of benzyl bromide in our previous reports, as shown in , , The synthetic route of compounds – is depicted in , Briefly, the synthesis of pyrimidinedione derivatives was started from commercially available recently . After alkylation of material with 1-bromo-2-butyne or 2-cyanobenzo-5-fluorobenzyl bromide, the resulting precursor was transformed into through the -alkylation. Compound was obtained by a process of replacing the 6-chloro group with ()-3-(Boc-Amino) piperidine. Finally, removal of Boc with TFA produced the final compounds –. The inhibitory effects of compounds on DPP-4 activity were evaluated based on fluorescent probe. In this part, we found that three compounds , , showed potent inhibitory activity of DPP-4 at 100 nM with inhibitory rate of (100.12 ± 0.54)%, (95.59 ± 3.7)%, (98.84 ± 0.66)%, respectively (). As shown in , compounds , and exhibited potent DPP-4 inhibitory activity, with IC values of 64.47 nM, 188.7 nM and 65.36 nM. From the data of , we found that the degree of bromination of these compounds may have a close relationship with their DPP-4 inhibitory activity. For example, the number of bromine of compounds – gradually increases from 0 to 3. However, the DPP-4 inhibition rates of compounds – decrease from 100.12% to 0.66%. Similarly, compounds – were less potent than compound . It could be found that their inhibitory rates decreased with the increase of the degree of bromination on phenol moiety. Furthermore, the degree of bromination of these compounds may have a close relationship with their lipid/water partition coefficient (Log P). For example, the DPP-4 inhibition rates of compounds – decrease from 86.8% to -0.87% with the increase of Log P. In addition, the compounds (, ) with fluorocyanobenzyl substituent were more active than the compounds (, ) with butynyl group (). SAR studies showed that fluorocyanobenzyl was crucial since it formed a hydrogen bond with Arg125. Moreover, π-π interaction also explained its enhanced binding affinity (). To further study the hypoglycemic effect of compound , blood glucose levels were measured during 4 weeks treatment. In order to improve the solubility, the succinate salt of compound was prepared according to the method of Hu et al. The dynamic effects of compound on water intake and body weight were recorded during four weeks treatment in BKS mice. There was no difference in water intake and body weight at the baseline (A and B). As shown in A, there was a decrease trend in mean water intake from the second week in compound - or metformin-treated group. However, both compound and metformin had no effect on body weight of BKS mice (B). A lot of abdominal fat was developed in BKS mice at the fourth week (C). Compound or metformin was unable to block the increased ratio of abdominal fat (D). In comparison with BKS mice, diabetic BKS mice exhibited hyperglycaemia with blood levels of about 30 mM (). A trend towards decrease in blood glucose levels was revealed in compared with vehicle and compound /metformin-treated groups. The blood glucose levels were reduced from the second week in both compound and metformin treated group (). These results reveal that compound has anti-diabetic potency with an effective blood glucose-lowing effect. In summary, a series of pyrimidinedione derivatives were designed and evaluated for DPP-4 inhibitory activity and anti-hyperglycemic efficacy. The preliminary SARs analysis exhibits that: (a) hydrophobic property was important for the inhibitory activity of DPP-4. Lipid/water partition coefficient may affect their inhibitory activity of DPP-4; (b) the inhibitory rates increase with the decrease of the number of bromine atoms on phenol moiety; (c) the alkyl side chain could affect the DPP-4 inhibitory activity. (d) fluorocyanobenzyl was crucial for the inhibitory activity of DPP-4. Among them, the representative compounds , and showed strong inhibitory activity of DPP-4 with IC values of 64.47 nM, 188.7 nM and 65.36 nM, respectively. Further studies revealed that compound are potent hypoglycemic effect. Potentially, compound is a promising DPP-4 inhibitor and worth further development.

domain: biology


The long-awaited Vanity Fair article is now published (but is not freely available online, as far as I know). Relative to the Greek and Icelandic articles, this piece is more of a straightforward account of the Irish crisis (but maybe that impression is just because Irish readers would be quite familiar with many of the anecdotes in the article). But lots of interesting material, with a very good profile of Morgan Kelly and a striking quote from Colm McCarthy among the highlights.
A Q&A with Michael Lewis is here.
Update: Article available in PDF here.


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These components are known to have pharmacological effects. This is the substance responsible for the relief Renown CBD Gummies patients feel when using marijuana. Also called cannabis, the opportunity medicinal aspects of your plant lie in chemical substance cannabinoids within it, including THC, or even the most powerful active ingredient. During a busy Thanksgiving weekend Governor Chris Christie began to Tweet about the Garden State's still non-existent medical marijuana program.
Ellis is, they all said, better than sliced bread. The probable cause of death was morphine poisoning. It was hoped that all Renown CBD Gummies dispensaries and providers of cannabidiol would be a part of this organization, which would not only insure the safety of the medicine but provide a tool with which to regulate cultivation and distribution in the best interest of the patients. MADISON: Regular readers may recall two "Profiles in Cowardice" articles I wrote about cowardly state lawmakers who found ways to reject the heartfelt appeals from veterans for help getting their medicine - medical cannabis. At that point a friend brought her a joint and she continued to smoke at the hospital and also to eat marijuana in fudge and caramels.

domain: medical


text:
Haagen Dazs chocolate peanut butter used to be my drug of choice, and I shot up every chance I got. I was seriously a straight-up junkie. PB is my rehab.
_-J o u r n a l_--------- ---- ---- --- --- -- -- -
Turkey Hill's all natural line of ice cream deserves a mention too.Here's the nutrition facts and ingredient list for their vanilla,chocolate,and butter almond variety:
Serving Size 1/2 cup (69 g)
Servings Per Container 12
Amount Per Serving
Calories from Fat 90
% Daily Value
Total Fat 10 g
Saturated Fat 6 g
Trans Fat 0 g
Cholesterol 35 mg
Sodium 75 mg
Total Carbohydrates 17 g
Dietary Fiber 0 g
Sugars 16 g
Protein 4 g
Vitamin A 6% Vitamin C 0%
Iron 2% Calcium 10%
Caffeine: 5 mg to 10 mg
Ingredients: CREAM, NONFAT MILK, SUGAR, ALMONDS, COCOA, VANILLA, SALT, VANILLA BEAN.
Contains: MILK AND ALMONDS
Additional Info: Gluten-Free
As you can see it's pretty good by ice cream standards,it doesn't have any ingredients that the average person would need to google to get an idea of what it actually is.
Never did like ice cream that much, gourmet or otherwise, so that was a small loss going Primal.
I make my own, which iseasy to do IF you're at home anyway, but just labor intensive enough that you'll only make and eat it for special occasions. Also, it's rich enough--far richer than Haagan Daz, which I also adore--that you can't really eat more than one scoop. Made it for the in-laws once; the youngest, a boy, asked for a second helping of ice cream, and his parents stared at him in disbelief. He's a skinny wee thing who doesn't get sugar normally--I think he was actually wanting a second helping of the cream and egg!! No ice cream maker needed. David Lebovitz's
vanilla ice cream is a good one. Scratch the vanilla bean steeping if you want and double the vanilla extract. Add the extra egg yolks for more richness. Cut the sugar as low as you want to go. Sub heavy cream for the initial cup of milk if you want. I've tried it all ways, and it's always good.
If you don't have an ice cream maker, then when your custard is done, put it in a fairly wide container, if possible, and stick that container in your freezer. Take it out to stir it up good with a fork every hour or so, breaking up any ice crystals that have formed. Take it out to stir more often if you're so inclined. The ice cream should be done within four to five hours.
HD is my go to when I've beaten up my body and have to find a way to cram in an extra 1000 calories.

domain: food


text:
I took it too work, and within seconds it was fully approved. Three quarter of the cake were gone in only one afternoon. Fair enough, I saved the last quarter for my parents, to let them see what I transformed grandpa's cake into. I found it would be a perfect Christmas recipe, because of the booziness on one hand and the lovely Baileys taste on the other, which makes a great treat for the festive season.
For the cake you need:
100g good quality dark chocolate
200g brown sugar
45g cocoa powder
1 heaped tsp baking powder
For the ganache you need:
200g dark chocolate
120ml liquid cream
Preheat the oven to 175°
Chop the chocolate first, then take 100g for the cake. In a saucepan, heat the butter and chocolate over medium heat until completely melted. Add the sugar, cocoa powder as well as the Baileys and whisk together until you get a silky and lump-free mixture. Remove from heat and cool for a few minutes.
In a large bowl, whisk together the flour and baking powder. Add the chocolate mixture and mix until you get an even dough. Add the eggs and mix again. Now you can pour the dough into a buttered cake tin and bake for 40-45 minutes.
In a saucepan, bring the liquid cream and Baileys to a simmer. Add the remaining chocolate and stir regularly until it has completely molten.
Set the liquid ganache aside at room temperature. Stir every now and then, to avoid clumps, until thick enough to spread over the cake.


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Manitoba Music is a comprehensive platform dedicated to promoting and supporting the music industry in Manitoba. The organization provides a wide range of services and features that cater to every aspect of the music industry.
The organization offers an array of programs and initiatives designed to cater to the specific needs of its members. Some of the benefits of membership include a Member Discount Program, access to professional development resources, special workshops, and opportunities for grant writing. In terms of market access, there are several programs including the Export Market Development and Indigenous Music Development Program. The Black Professionals in Music and Programme de musique francophone are also significant initiatives towards promoting diversity and inclusivity.
Manitoba Music provides a platform for indigenous music as well as for new releases and member news. Music can be explored through various playlists provided by the organization, such as the Song Of The Week Manitoba Music Spotify Playlist, the Indigenous Music Spotify Playlist and others. These curated playlists offer an excellent way to discover new and emerging artists.
The organization also focuses on keeping its members well-informed about the latest industry trends and news through articles and updates. The 'Music Business Headlines' is a valuable source of information for anyone involved in the music industry.
In addition to these services, Manitoba Music also has a Mobile Stage Rental service, a unique offering that demonstrates their comprehensive support for live music. The platform provides invaluable opportunities for artists to get their music heard and to reach wider audiences.
Equity and inclusion are prioritized at Manitoba Music. The organization has a dedicated Equity and Accountability Policy, reflecting its commitment to fairness and accountability. There is also an emphasis on wellness and support for its members, with a Health/Wellness/Support section available on their website.
Overall, Manitoba Music does an impressive job of supporting all aspects of the music industry, from artists to industry professionals. It delivers a comprehensive suite of resources, tools, and programs that drive success in the music industry. It is a vital resource for anyone involved in music in Manitoba. Its commitment to diversity, inclusion, and member support underlines its role as a key player in the music industry.

domain: artistic


text:
Mar 27, 2013, 8:04 AM EDT
Throughout the season, NBCSN will feature the NHL’s fiercest rivalries on Wednesday nights. Tonight the Montreal Canadiens will head to TD Garden for a match against the Boston Bruins at 7:30 p.m. ET.
The Bruins and Canadiens have spent the better part of the season battling for the Northeast Division title and that battle is far from resolved. Going into tonight’s contest, the two teams are tied with 45 points, although Montreal has played in one more game than Boston.
Both of these teams have gotten this far thanks to solid defense, strong goaltending, and an offensive core that’s shown its depth this season.
The fact that Boston is in this race isn’t much of a surprise. There was some concern that goaltender Tuukka Rask might not be able to seamlessly assume Tim Thomas‘ starting duties, but after serving as a strong backup for years, Rask’s 2013 performance can’t be considered a shock.This video is no longer available. Click here to watch more NBC Sports videos!
Instead, the Canadiens are the Cinderella story. They’ve benefited from the emergence of rookies Alex Galchenyuk and Brendan Gallagher, but when the season started, their core wasn’t dramatically different than the one that finished in the basement of the Eastern Conference in 2011-12.
Perhaps the biggest difference has been behind the bench, where Michel Therrien is now running the show. Under him, the Canadiens have gone from one of the worst power-play teams in the NHL to one of the best. Of course, having a finally healthy Andrei Markov join P.K. Subban as the team’s top two blueliners with the man advantage helps too.
Not many teams in the league can afford to take penalties against the Canadiens, but if there’s one that can endure, it’s Boston. The Bruins have a league-leading 91.7 penalty killing percentage.
These two teams have played each other twice this season and both contests were decided by a goal. It wouldn’t be surprising if tonight’s game ended up being just as close.
Probable goaltenders: BOS – Rask (15-4-3, 1.90 GAA, .928 SV%) | MON – Peter Budaj (4-1-1, 2.47 GAA, .903 SV%)
Boston – Forwards: Chris Kelly (leg), Marc Savard (concussion) Defenseman: Adam McQuaid (shoulder), Johnny Boychuk (foot)
Montreal – Forwards: Brandon Prust (shoulder), Ryan White (lower body), Rene Bourque (concussion) Defenseman: Yannick Weber (knee), Raphael Diaz (concussion)
- Shots fired! ‘Canes owner Karmanos rips into Jim Rutherford (Updated) 30
- Here’s who’s left after Day 1 of free agency 23
- PHT’s 2015 free agent frenzy tracker 38
- Getting clutch? Capitals sign Justin Williams for two years, $6.5M 20
- Coyotes bring back Vermette with two-year deal 33
- Bruins sign Beleskey for five years, despite some warning signs 48
- Kessel trade shows Leafs are trying to ‘build this thing the right way’ 33
- Green signs with Red Wings — 3 years, $18 million 27
- ‘It’s hard to find players like Phil Kessel’ 46
- Kassian traded to Habs for Prust; Hodgson signs with Preds 17
- Blockbuster: Kessel traded to the Penguins (131)
- Trade: Flyers send Rinaldo to Bruins for 2017 third-rounder (105)
- Trade: Jackets land Saad; Anisimov and Dano headed to Chicago (93)
- Bypassing a buyout? Kings terminate Richards’ contract for ‘material breach’ (90)
- Trade: Flames land Dougie Hamilton for package of picks (75)

domain: news


University of Waterloo Develops Award-Winning Fuel Cell Technology Using Model-Based Design
- Ease of communication
- Substantial design time savings
- Innovative technology
Challenge X, a competition sponsored by General Motors and the U.S. Department of Energy, challenges 17 North American student teams to re-engineer a Chevrolet Equinox so as to reduce emissions and fuel consumption without sacrificing vehicle performance or safety.
The University of Waterloo Alternative Fuels Team (UWAFT) won first place overall in the first year of the three-year competition with their fuel-cell-powered vehicle design. UWAFT also won The MathWorks Crossover to Model-Based Design Award for outstanding achievement in creating, simulating, and analyzing models for vehicle design and subsystem control.
“We were the only team to use fuel cells in the powertrain,” says professor Roydon Fraser, UWAFT faculty advisor. “MathWorks software for Model-Based Design not only cut down on the time it took for our team to prototype and simulate our vehicle system designs, but also enabled us to establish the viability of the fuel cell technology.”
The first year of Challenge X focuses on vehicle design. Participants must use Model-Based Design to complete powertrain design and testing within 10 months, and must submit five major reports.
The University of Waterloo team sought design software that would enable them to use Model-Based Design throughout the project—from requirements capture to implementation. The software must accelerate control strategy development by facilitating model reuse. It must also be quick to learn and enable team members to share work easily.
“MathWorks tools enabled us to simulate various powertrains, develop accurate plant models, test control strategies, and validate the overall design,” says UWAFT captain, Matthew Stevens.
The MathWorks provided training in MATLAB®, Simulink®, Stateflow®, and PSAT, a modeling program based on Simulink. “Having a product with a quick learning curve or that students were already trained in was critical to the team’s success,” Stevens comments. “MathWorks tools could be used for multiple stages in the design process, minimizing the number of software programs that students needed to learn.”
UWAFT developed more than 400 PSAT simulations to compare fuels, technologies, and powertrain sizing. Optimization Toolbox™ and a sophisticated Design of Experiments enabled them to understand the relationship between component size and vehicle performance and then determine the optimal powertrain.
They used Simulink to develop a plant model of the fuel cell power system, which included the engine, the battery, the fuel cell, and a DC/DC converter.
MATLAB, Simulink, Stateflow, and Control System Toolbox™ enabled them to develop the hybrid control strategy (HCS), which determines the amount of power coming from the fuel cell. MATLAB helped them to find the optimal appropriation of power between the fuel cell and battery over a specific drive cycle.
The DC/DC converter boosts the fuel cell voltage and controls the power from the fuel cell. Simscape Electrical™ was used to model the circuit, which was controlled by a PI controller. The team investigated the frequency response and stability of the circuit using Bode plots and pole-zero maps in MATLAB. Simulation enabled them to verify correct operation, determine circuit efficiency, and calculate values and ratings of inductors and other components.
Because the fuel cell turns on and off, it creates a discontinuous function that is difficult to optimize using traditional methods. To find the optimal control benchmark, UWAFT therefore used Global Optimization Toolbox, which does not require the function to be continuous. They also used Deep Learning Toolbox™ to model the hydration of the membranes within the fuel cell stack.
The team used Embedded Coder® to target UWAFT satellite controllers throughout the vehicle for on-target rapid prototyping using an embedded controller.
They are currently testing powertrain components, refining the vehicle control strategy, integrating the advanced fuel cell into the vehicle, and researching weight-reduction possibilities.
“We are definitely interested in using other MathWorks products throughout the competition, and hopefully in our future careers in many other projects,” Stevens says.
Ease of communication. The MATLAB and Simulink environment increased efficiency by enabling team members to easily exchange shortcuts and tips and to e-mail their models and results to each other for further work.
Substantial design time savings. "Simulink in particular allowed UWAFT to use Model-Based Design to compare four different fuels and design a robust fuel cell sport utility vehicle and control strategy in less than 10 months, including training time—a truly incredible feat," notes Stevens. "Without Simulink, it probably would have taken us until the end of the three-year competition to complete the work required in the first year alone!"
Innovative technology. "We believe that our end product will be the first student-designed fuel cell vehicle to provide performance competitive with today’s vehicles while achieving the mileage and efficiency needs of the future,” Stevens says.


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Before I get started, I should mention that I never leave reviews. This is probably my first one on Yelp. These guys are just so great that I felt compelled to give them a shoutout. This is the shop I hoped to find when I began commuting two years ago. I'm a daily commuter and I love knowing that these guys will help me almost anytime that I need it. Andy, Jake and Brad listen and make sure to help me as best they can and are always speedy about it. Additionally, they encourage me to do my own work on my bikes when I have the time/when they know I'm capable and I really appreciate that encouragement. These guys will give you their best and provide some laughs as well. Couldn't recommend them more!
As you mentioned, it is surprising how often you can get multiple flat tires on while riding, particularly on longer sprints. The tip about bringing a patch kit in your bike repair kit can be incredibly helpful if you’ve already had one flat tire. I agree that all you listed seems like an awful lot to carry, but it’s always better to be safe than sorry, isn’t it?
Man, if you're starting out and you know you'll need several components for a bag... just get this. It will save you so much time picking everything out that even if you balk at the price (amazing value, by the way) I bet that if you did the math using your hourly wage and the time spent picking everything out and making sure it fits... you'd save a lot of money and a lot of irritation if you just got this. Just get this.
If you’re going to use the chain whip you just made, you’ll also need a way to get that cassette off. A lockring remover is a must-have; unless you use one of the shady removal methods I’ve seen on the internet—which I wouldn’t necessarily recommend unless you don’t care much about the condition of the wheel after you use them. I recommend Park Tool’s iterations here that cost about $6, but you’ll need to buy the specific one for your brand/type of cassette. (By the way, I’m assuming you already have an adjustable wrench laying around somewhere to use with this tool. If you don’t, well, you’ll need one of those too.)
All multi-tools are not created equal, but most with a plethora of features will get plenty of the jobs done. I personally like, and use often, the Lezyne RAP-21 LED tool, which will run you around $30. I like it because not only is it a high quality construction, but it has just about everything you’ll need, plus a sweet mini light. This will give you a chain breaker, spoke wrench, disc brake wedge, and all the most often used Allen keys and drivers. We’re talking a decent number of tools for 1/4 of what they would cost buying them separately.
Where to begin?! Long story made somewhat short. I bookmarked this place because I saw their dog Kaya and was sold. Lol. But they had 5 stars too so that helped. Amazing people. Very down-to-earth. Took the bikes down the path to Discovery Park. If you're in-shape and do mild cardio, then it should be an ok ride. I don't do cardio and I found myself trying to sell my soul like 15 times just to get to the top of some hills. Lol. Made it to the Ballard locks and actually got to see a boat pass through! This is the off-season so not much boat traffic. Steve, an engineer we met there, said peak time will start in May. Ate lunch at Ann's in Ballard. Delish shrimp sate! I busted my tire in a pothole and had to make a trip to REI. Which wasn't awful because we wanted to go there anyway. The city was tough to ride in at 5:00pm and we rode the sidewalk most of the way back due to the congestion. Some pedestrians are obvious to you and I almost ran over a few people.. By choice. Lol. Made it back right before they closed at 6:00pm. Easy peasy! Notes: Take some water. Take some pictures. The ride is beautiful so don't rush through. Get there before noon to give yourself some time to cruise.
I got some problems with my gears and I was wondering how to fix it, then got the idea on the internet about KELLY'S,I decided to go with them to fix the errors, that was my first time and was a great experience,they are all very accurate with their profession,very friendly,very polite and very honest as well,they just took half an hour to make me free.so,with the first experience I have become the fan of Kyle's.definitely suggested to everyone.
We focused on tools that would be useful to a commuter—someone trying to use a bike as a functional way to get around town, as opposed to riding recreationally (road biking and mountain biking). That said, it’s a wild and wooly bicycling world out there, and the streets are packed with so many different bikes, all shapes and sizes, new and old. Customizing your flat-fixing kit has advantages over buying a preassembled kit that always contains a tool (or two or three) that’s a piece of junk, or you don’t need. Or it’s missing something you do need. If you build your own kit, at least you know everything works. And you can add only what’s necessary for your specific bike without ending up with stuff you don’t need.
Do not take your Bike to Kyle's Bike Shop they handled my bike so poorly they can not work on Vintage Schwinn' and the attitude they had if it wasn't a high dollar bike don't expect good service. I had to find my own parts, had to take the bike back twice to get the wheel trued and they ruined my bike it is so scrapped up the Schwinn is gone on the side of the bike. If you care about your bike do not take it here.
Nice, helpful, friendly. Central location. Good bikes (I had two over two days.) Bikes had a water bottle cage (some places don't do that!) Provided helmets if needed (I didn't) and a one-sided pannier containing a decently strong but not super-heavy cable lock and a patch kit. Only quibbles: 1) The owner helping me with getting my bikes seemed to 'drop me' for too long when someone else walked in. He should have said to them, after asking what they were in for, that 'I'll be with you as soon as I'm finished with this person.' So I had to wait a little longer than desired, but not bad. 2) On my second day, the pannier bag fell off and got lost after I stopped for lunch, which required locking up the bike somewhere, so I took the bag into restaurant and re-attached it using the two velcro straps on the rear rack. I've used many panniers before, thought I did it correctly. But apparently I did not. So I paid $55 (cost) to replace everything in bag and bag. In hindsight, I wished I'd put a bungee around bag straps to really secure it to bike. PROBABLY MY FAULT. 3) Bring a second lock to lock quick-release front wheel, as decently strong but not super-heavy cable lock isn't long enough for both wheels and frame.I had a second cable lock.
I own the Lezyne CNC Chain Rod, which I’m a big fan of (yes, I know I’m partial to Lezyne here–they’re a local-to-me, San Luis Obispo company and they sponsored my collegiate team, so I got tons of gear) and it costs right around $30. But the truth is that even the cheapest of chain whips will likely do the job. In fact, I’ve found a dude who knows his stuff and shows you how to make this tool all by yourself. I would even go so far as to say that you probably have enough materials laying about your garage (if not, try your parents’ garage ? ) to put this together without spending anything. Check out the video below:
In spite of seat bags’ diminutive size, a lot of people really, really want them to accommodate more stuff. If that’s you, the medium and large seat bag (but not the small) have an extendable gill at the bottom, which you can see in the image up top. It zips open to create more room. With it unzipped, we could cram a wallet, keys, and phone inside the medium as well.
That’s a reality of all hand pumps—it’s really hard to get a tire up to pressure with these things. So another reason Lezyne pumps are so good is because you can actually keep pumping longer than any other brand we tested. With excruciating effort, we could get the Sport Drive HP to pump tires to 90 or 100 psi. It still wasn’t easy, but it wasn’t impossible the way it was with some other models.
It’s both surprising, and not, if you think about it. The way I’ve usually seen it happen is first the rear tire goes flat, then the front one. My theory is that both tires went through the same pile of glass/thorns/metal bits and they both picked up junk. the reason for the flat occurring on the back first is because there’s more weight on that tire, so the offending article works its way through the tread more quickly.
Professional mountain biker Rebecca Rusch has been on plenty of long rides and experienced enough mechanical failures to know what makes a fully-stocked repair kit. One of her recent journeys, a 1,200-mile journey through the jungles of Vietnam, Laos and Cambodia along the Ho Chi Minh Trail required the utmost degree of preparedness — “We brought with us a lot of extra bearings, o-rings, stuff that you would never be able to find in Laos and Cambodia. Before I go out on these big adventures, I replace every bearing in my bike, my fork got a total rebuild — everything is in pristine condition,” says Rusch.
The Pedro’s lever slipped only once throughout testing, though that may have been my fault because I was spaced out after changing 16 different tires. So they’re not foolproof, but they are far and away more reliable than the next closest competitor, from Park Tool, because the small, rounded tip on that one is hard to keep seated under the tire. Not impossible, but I wouldn’t choose it over Pedro’s.
3D Printers Baby Bath Tub Baby Strollers Bar Stool Bean Bag Chair best actor Bike cover Blackhead Remover Boys' Boxer Underwear Car Chargers Ceiling Fans Chair Cushions cleaner Dining Chair Diving Masks Elbow Braces Electric Air Fryer Electric Stove Fizik Saddles Folding Ladders gas cooktop Handheld CB Radio inflatable boat Kindle Paperwhite Makeup Brush Makeup Chairs Men's Cycling Shoes Paper Towel Holder Ping Pong Paddles pool cue rack Preqnancy body pillow Projector Stands RC Helicopter robot rocking horse Seagrass Rug Small Indoor Dog House sound bars Table Round toddler travel bed vacuum Vanity Makeup Mirrors Walmart Folding Table Women's Condoms Wood Table Lamps

domain: hobby


I have a Hyper V host server (Windows 2016) and I NIC teamed two network ports via power shell into a single virtual switch.
The host server now has a single DHCP IP address.
Should the host server have a static IP address instead?
Does a Hyper V host server supposed to have one static IP address AND another DCHP address or just one IP address?
I have used a few Hyper V host servers and they always had one static IP and a 2nd DCHP IP address.
When you team NICs together, you get a new NIC. When you attach a virtual switch to a NIC (the NIC can be teamed or stand alone), the NIC gets assigned to the switch and the host gets a new virtual NIC attached to the switch. Every time you get a new NIC, it will default to DHCP.
I would say that your host should have just 1 NIC and 1 IP address unless:
- You are using iSCSI for storage
- You are doing clustering, in which case new NICs will be created automatically for cluster operations
- You have a dedicated backup network
For a simple stand alone Hyper-V deployment I would team your NICs together. Attach the virtual switch to the NIC team and share with the management OS (host). Attach VMs to the same virtual switch.
I created a new virtual switch using the power script in the link above. Did this attach the virtual switch to my NIC team?
In the virtual switch manager in hyper v, the teamed virtual switch is the external network
When you NIC Team you are combining several physical ports into one virtual port. Think of it as bridging the physical adapters to make one virtual. You will have one ip associated with each mac address, if you are doing dhcp reservcations you need to reserve every mac address, with a static ip just set it on the windows host it will be easier.
It is better to assign a static IP address for Hyper-V host. I've recently checked SET teaming with my 4 x 40 GbE networking for management and it shows very bad results (around 40% of packet loss) on Windows 2016. So, I would rather suggest you using default LBFO teaming (can be configured within Server manager) with switch independent mode and then create vSwitch on top of it. To change the IP navigate to networking in control panel and change the IP for vSwitch NIC.


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The first season of CBS’ drama series Under The Dome will receive its UK premiere on Channel 5 on Monday August 19th at 10pm, it has been announced.
Based on the Stephen King novel of the same name, Under The Dome tells the story of a small town that is suddenly and inexplicably sealed off from the rest of the world by a massive transparent dome. The drama series, which has an order for 13 episodes, is produced by CBS Television Studios in association with Steven Spielberg’s Amblin Television and stars Mike Vogel, Rachelle Lefevre, Dean Norris, Natalie Martinez, Britt Robertson, Alexander Koch, Colin Ford, Nicholas Strong, Jolene Purdy and Aisha Hinds. The series, which pulled in nearly 20 million viewers for its premiere state-side on CBS, was recently renewed for a 13 episode second season.
TVWise’s UK Premiere Dates page has been updated with this new information.


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domain: design


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Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to “off-target” effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.
25 Related JoVE Articles!
Pseudomonas aeruginosa Induced Lung Injury Model
Institutions: University of Illinois at Chicago, Emory University, University of Illinois at Chicago.
In order to study human acute lung injury and pneumonia, it is important to develop animal models to mimic various pathological features of this disease. Here we have developed a mouse lung injury model by intra-tracheal injection of bacteria Pseudomonas aeruginosa
or PA). Using this model, we were able to show lung inflammation at the early phase of injury. In addition, alveolar epithelial barrier leakiness was observed by analyzing bronchoalveolar lavage (BAL); and alveolar cell death was observed by Tunel assay using tissue prepared from injured lungs. At a later phase following injury, we observed cell proliferation required for the repair process. The injury was resolved 7 days from the initiation of P. aeruginosa
injection. This model mimics the sequential course of lung inflammation, injury and repair during pneumonia. This clinically relevant animal model is suitable for studying pathology, mechanism of repair, following acute lung injury, and also can be used to test potential therapeutic agents for this disease.
Immunology, Issue 92, Lung, injury, pseudomonas, pneumonia, mouse model, alveoli
Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue
Institutions: UCLA, UC Irvine.
The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro
, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.
Cellular Biology, Issue 80, Myofibroblasts, Mesenchymal Stromal Cells, Gastrointestinal Tract, stroma, colon, primary cells
Microinjection of mRNA and Morpholino Antisense Oligonucleotides in Zebrafish Embryos.
Institutions: Yale University School of Medicine.
An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio
), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo
. In this video-article, we will demonstrate how to prepare and microinject in vitro
synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2
, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2
by mRNA and morpholino oligonucleotide injection.
Developmental Biology, Issue 27, Zebrafish, microinjection, morpholino antisense oligonucleotide, gene overexpression, gene knockdown
Long Term Chronic Pseudomonas aeruginosa Airway Infection in Mice
Institutions: San Raffaele Scientific Institute, Italian Cystic Fibrosis Research Foundation.
A mouse model of chronic airway infection is a key asset in cystic fibrosis (CF) research, although there are a number of concerns regarding the model itself. Early phases of inflammation and infection have been widely studied by using the Pseudomonas aeruginosa
agar-beads mouse model, while only few reports have focused on the long-term chronic infection in vivo
. The main challenge for long term chronic infection remains the low bacterial burden by P. aeruginosa
and the low percentage of infected mice weeks after challenge, indicating that bacterial cells are progressively cleared by the host.
This paper presents a method for obtaining efficient long-term chronic infection in mice. This method is based on the embedding of the P. aeruginosa
clinical strains in the agar-beads in vitro
, followed by intratracheal instillation in C57Bl/6NCrl mice. Bilateral lung infection is associated with several measurable read-outs including weight loss, mortality, chronic infection, and inflammatory response. The P. aeruginosa
RP73 clinical strain was preferred over the PAO1 reference laboratory strain since it resulted in a comparatively lower mortality, more severe lesions, and higher chronic infection. P. aeruginosa
colonization may persist in the lung for over three months. Murine lung pathology resembles that of CF patients with advanced chronic pulmonary disease.
This murine model most closely mimics the course of the human disease and can be used both for studies on the pathogenesis and for the evaluation of novel therapies.
Infection, Issue 85, Opportunistic Infections, Respiratory Tract Infections, Inflammation, Lung Diseases, Cystic Fibrosis, Pseudomonas aeruginosa
The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins
Institutions: Children's Hospital of Pittsburgh of UPMC, University of Pittsburgh School of Medicine.
Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e.
endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 ºC - a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 ºC. Next, the temperature is decreased again to 4 ºC to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 °C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 ºC as in the endocytic assay. Next, cells are incubated again at 37 °C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 ºC, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.
Basic Protocol, Issue 82, Endocytosis, recycling, plasma membrane, cell surface, EZLink, Sulfo-NHS-SS-Biotin, L-Glutathione, GSH, thiol group, disulfide bond, epithelial cells, cell polarization
Measuring Respiratory Function in Mice Using Unrestrained Whole-body Plethysmography
Institutions: Monash Institute of Medical Research, Monash Medical Centre, Animal Resource Centre, Perth, Australia, Wake Forest Institute for Regenerative Medicine.
Respiratory dysfunction is one of the leading causes of morbidity and mortality in the world and the rates of mortality continue to rise. Quantitative assessment of lung function in rodent models is an important tool in the development of future therapies. Commonly used techniques for assessing respiratory function including invasive plethysmography and forced oscillation. While these techniques provide valuable information, data collection can be fraught with artefacts and experimental variability due to the need for anesthesia and/or invasive instrumentation of the animal. In contrast, unrestrained whole-body plethysmography (UWBP) offers a precise, non-invasive, quantitative way by which to analyze respiratory parameters. This technique avoids the use of anesthesia and restraints, which is common to traditional plethysmography techniques. This video will demonstrate the UWBP procedure including the equipment set up, calibration and lung function recording. It will explain how to analyze the collected data, as well as identify experimental outliers and artefacts that results from animal movement. The respiratory parameters obtained using this technique include tidal volume, minute volume, inspiratory duty cycle, inspiratory flow rate and the ratio of inspiration time to expiration time. UWBP does not rely on specialized skills and is inexpensive to perform. A key feature of UWBP, and most appealing to potential users, is the ability to perform repeated measures of lung function on the same animal.
Physiology, Issue 90, Unrestrained Whole Body Plethysmography, Lung function, Respiratory Disease, Rodents
Induction and Analysis of Epithelial to Mesenchymal Transition
Institutions: R&D Systems, Inc., R&D Systems, Inc..
Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro
is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.
Molecular Biology, Issue 78, Cellular Biology, Biochemistry, Biomedical Engineering, Stem Cell Biology, Cancer Biology, Medicine, Bioengineering, Anatomy, Physiology, biology (general), Pathological Conditions, Signs and Symptoms, Wounds and Injuries, Neoplasms, Diagnosis, Therapeutics, Epithelial to mesenchymal transition, EMT, cancer, metastasis, cancer stem cell, cell, assay, immunohistochemistry
Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface
Institutions: The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill, The University of North Carolina at Chapel Hill.
models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens.
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc.
models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo
Cellular Biology, Issue 80, Epithelium, Cell culture models, ciliated, air pollution, co-culture models, nasal epithelium
Isolation of Pulmonary Artery Smooth Muscle Cells from Neonatal Mice
Institutions: Northwestern University Feinberg School of Medicine.
Pulmonary hypertension is a significant cause of morbidity and mortality in infants. Historically, there has been significant study of the signaling pathways involved in vascular smooth muscle contraction in PASMC from fetal sheep. While sheep make an excellent model of term pulmonary hypertension, they are very expensive and lack the advantage of genetic manipulation found in mice. Conversely, the inability to isolate PASMC from mice was a significant limitation of that system. Here we described the isolation of primary cultures of mouse PASMC from P7, P14, and P21 mice using a variation of the previously described technique of Marshall et al.26
that was previously used to isolate rat PASMC. These murine PASMC represent a novel tool for the study of signaling pathways in the neonatal period. Briefly, a slurry of 0.5% (w/v) agarose + 0.5% iron particles in M199 media is infused into the pulmonary vascular bed via the right ventricle (RV). The iron particles are 0.2 μM in diameter and cannot pass through the pulmonary capillary bed. Thus, the iron lodges in the small pulmonary arteries (PA). The lungs are inflated with agarose, removed and dissociated. The iron-containing vessels are pulled down with a magnet. After collagenase (80 U/ml) treatment and further dissociation, the vessels are put into a tissue culture dish in M199 media containing 20% fetal bovine serum (FBS), and antibiotics (M199 complete media) to allow cell migration onto the culture dish. This initial plate of cells is a 50-50 mixture of fibroblasts and PASMC. Thus, the pull down procedure is repeated multiple times to achieve a more pure PASMC population and remove any residual iron. Smooth muscle cell identity is confirmed by immunostaining for smooth muscle myosin and desmin.
Basic Protocol, Issue 80, Muscle, Smooth, Vascular, Cardiovascular Abnormalities, Hypertension, Pulmonary, vascular smooth muscle, pulmonary hypertension, development, phosphodiesterases, cGMP, immunostaining
Characterization of the Isolated, Ventilated, and Instrumented Mouse Lung Perfused with Pulsatile Flow
Institutions: University of Wisconsin – Madison.
The isolated, ventilated and instrumented mouse lung preparation allows steady and pulsatile pulmonary vascular pressure-flow relationships to be measured with independent control over pulmonary arterial flow rate, flow rate waveform, airway pressure and left atrial pressure. Pulmonary vascular resistance is calculated based on multi-point, steady pressure-flow curves; pulmonary vascular impedance is calculated from pulsatile pressure-flow curves obtained at a range of frequencies. As now recognized clinically, impedance is a superior measure of right ventricular afterload than resistance because it includes the effects of vascular compliance, which are not negligible, especially in the pulmonary circulation. Three important metrics of impedance - the zero hertz impedance Z0
, the characteristic impedance ZC
, and the index of wave reflection RW
- provide insight into distal arterial cross-sectional area available for flow, proximal arterial stiffness and the upstream-downstream impedance mismatch, respectively. All results obtained in isolated, ventilated and perfused lungs are independent of sympathetic nervous system tone, volume status and the effects of anesthesia. We have used this technique to quantify the impact of pulmonary emboli and chronic hypoxia on resistance and impedance, and to differentiate between sites of action (i.e., proximal vs. distal) of vasoactive agents and disease using the pressure dependency of ZC
. Furthermore, when these techniques are used with the lungs of genetically engineered strains of mice, the effects of molecular-level defects on pulmonary vascular structure and function can be determined.
Medicine, Issue 50, ex-vivo, mouse, lung, pulmonary vascular impedance, characteristic impedance
Analysis of Pulmonary Dendritic Cell Maturation and Migration during Allergic Airway Inflammation
Institutions: McMaster University, Hamilton, University of Toronto.
Dendritic cells (DCs) are the key players involved in initiation of adaptive immune response by activating antigen-specific T cells. DCs are present in peripheral tissues in steady state; however in response to antigen stimulation, DCs take up the antigen and rapidly migrate to the draining lymph nodes where they initiate T cell response against the antigen1,2
. Additionally, DCs also play a key role in initiating autoimmune as well as allergic immune response3
DCs play an essential role in both initiation of immune response and induction of tolerance in the setting of lung environment4
. Lung environment is largely tolerogenic, owing to the exposure to vast array of environmental antigens5
. However, in some individuals there is a break in tolerance, which leads to induction of allergy and asthma. In this study, we describe a strategy, which can be used to monitor airway DC maturation and migration in response to the antigen used for sensitization. The measurement of airway DC maturation and migration allows for assessment of the kinetics of immune response during airway allergic inflammation and also assists in understanding the magnitude of the subsequent immune response along with the underlying mechanisms.
Our strategy is based on the use of ovalbumin as a sensitizing agent. Ovalbumin-induced allergic asthma is a widely used model to reproduce the airway eosinophilia, pulmonary inflammation and elevated IgE levels found during asthma6,7
. After sensitization, mice are challenged by intranasal delivery of FITC labeled ovalbumin, which allows for specific labeling of airway DCs which uptake ovalbumin. Next, using several DC specific markers, we can assess the maturation of these DCs and can also assess their migration to the draining lymph nodes by employing flow cytometry.
Immunology, Issue 65, Medicine, Physiology, Dendritic Cells, allergic airway inflammation, ovalbumin, lymph nodes, lungs, dendritic cell maturation, dendritic cell migration, mediastinal lymph nodes
Using Eggs from Schistosoma mansoni as an In vivo Model of Helminth-induced Lung Inflammation
Institutions: University of Pennsylvania , University of Pennsylvania .
parasites are blood flukes that infect an estimated 200 million people worldwide 1
. In chronic infection with Schistosoma
, the severe pathology, including liver fibrosis and splenomegaly, is caused by the immune response to the parasite eggs rather than the parasite itself 2
. Parasite eggs induce a Th2 response characterized by the production of IL-4, IL-5 and IL-13, the alternative activation of macrophages and the recruitment of eosinophils. Here, we describe injection of Schistosoma mansoni
eggs as a model to examine parasite-specific Th2 cytokine responses in the lung and draining lymph nodes, the formation of pulmonary granulomas surrounding the egg, and airway inflammation.
Following intraperitoneal sensitization and intravenous challenge, S. mansoni
eggs are transported to the lung via the pulmonary arteries where they are trapped within the lung parenchyma by granulomas composed of lymphocytes, eosinophils and alternatively activated macrophages 3-6
. Associated with granuloma formation, inflammation in the broncho-alveolar spaces, expansion of the draining lymph nodes and CD4 T cell activation can be observed. Here we detail the protocol for isolating Schistosoma mansoni
eggs from infected livers (modified from 7
), sensitizing and challenging mice, and recovering the organs (broncho-alveolar lavage (BAL), lung and draining lymph nodes) for analysis. We also include representative histologic and immunologic data and suggestions for additional immunologic analysis.
Overall, this method provides an in vivo
model to investigate helminth-induced immunologic responses in the lung, which is broadly applicable to the study of Th2 inflammatory diseases including helminth infection, fibrotic diseases, allergic inflammation and asthma. Advantages of this model for the study of type 2 inflammation in the lung include the reproducibility of a potent Th2 inflammatory response in the lung and draining lymph nodes, the ease of assessment of inflammation by histologic examination of the granulomas surrounding the egg, and the potential for long-term storage of the parasite eggs.
Immunology, Issue 64, Infection, Microbiology, helminth, parasite, mouse, Th2, lung, inflammation, granuloma, alternative activation, macrophage
Flow Cytometric Isolation of Primary Murine Type II Alveolar Epithelial Cells for Functional and Molecular Studies
Institutions: Helmholtz Centre for Infection Research, Otto-von-Guericke University , Helmholtz Centre for Infection Research.
Throughout the last years, the contribution of alveolar type II epithelial cells (AECII) to various aspects of immune regulation in the lung has been increasingly recognized. AECII have been shown to participate in cytokine production in inflamed airways and to even act as antigen-presenting cells in both infection and T-cell mediated autoimmunity 1-8
. Therefore, they are especially interesting also in clinical contexts such as airway hyper-reactivity to foreign and self-antigens as well as infections that directly or indirectly target AECII. However, our understanding of the detailed immunologic functions served by alveolar type II epithelial cells in the healthy lung as well as in inflammation remains fragmentary. Many studies regarding AECII function are performed using mouse or human alveolar epithelial cell lines 9-12
. Working with cell lines certainly offers a range of benefits, such as the availability of large numbers of cells for extensive analyses. However, we believe the use of primary murine AECII allows a better understanding of the role of this cell type in complex processes like infection or autoimmune inflammation. Primary murine AECII can be isolated directly from animals suffering from such respiratory conditions, meaning they have been subject to all additional extrinsic factors playing a role in the analyzed setting. As an example, viable AECII can be isolated from mice intranasally infected with influenza A virus, which primarily targets these cells for replication 13
. Importantly, through ex vivo
infection of AECII isolated from healthy mice, studies of the cellular responses mounted upon infection can be further extended.
Our protocol for the isolation of primary murine AECII is based on enzymatic digestion of the mouse lung followed by labeling of the resulting cell suspension with antibodies specific for CD11c, CD11b, F4/80, CD19, CD45 and CD16/CD32. Granular AECII are then identified as the unlabeled and sideward scatter high (SSChigh
) cell population and are separated by fluorescence activated cell sorting 3
In comparison to alternative methods of isolating primary epithelial cells from mouse lungs, our protocol for flow cytometric isolation of AECII by negative selection yields untouched, highly viable and pure AECII in relatively short time. Additionally, and in contrast to conventional methods of isolation by panning and depletion of lymphocytes via binding of antibody-coupled magnetic beads 14, 15
, flow cytometric cell-sorting allows discrimination by means of cell size and granularity. Given that instrumentation for flow cytometric cell sorting is available, the described procedure can be applied at relatively low costs. Next to standard antibodies and enzymes for lung disintegration, no additional reagents such as magnetic beads are required. The isolated cells are suitable for a wide range of functional and molecular studies, which include in vitro
culture and T-cell stimulation assays as well as transcriptome, proteome or secretome analyses 3, 4
Immunology, Issue 70, Cellular Biology, Molecular Biology, Infection, Infectious Diseases, Microbiology, alveolar type II epithelial cells, mouse, respiratory tract, lung, cell sorting, flow cytometry, influenza, autoimmunity
In vitro Coculture Assay to Assess Pathogen Induced Neutrophil Trans-epithelial Migration
Institutions: Harvard Medical School, MGH for Children, Massachusetts General Hospital.
Mucosal surfaces serve as protective barriers against pathogenic organisms. Innate immune responses are activated upon sensing pathogen leading to the infiltration of tissues with migrating inflammatory cells, primarily neutrophils. This process has the potential to be destructive to tissues if excessive or held in an unresolved state. Cocultured in vitro
models can be utilized to study the unique molecular mechanisms involved in pathogen induced neutrophil trans-epithelial migration. This type of model provides versatility in experimental design with opportunity for controlled manipulation of the pathogen, epithelial barrier, or neutrophil. Pathogenic infection of the apical surface of polarized epithelial monolayers grown on permeable transwell filters instigates physiologically relevant basolateral to apical trans-epithelial migration of neutrophils applied to the basolateral surface. The in vitro
model described herein demonstrates the multiple steps necessary for demonstrating neutrophil migration across a polarized lung epithelial monolayer that has been infected with pathogenic P. aeruginosa
(PAO1). Seeding and culturing of permeable transwells with human derived lung epithelial cells is described, along with isolation of neutrophils from whole human blood and culturing of PAO1 and nonpathogenic K12 E. coli
(MC1000). The emigrational process and quantitative analysis of successfully migrated neutrophils that have been mobilized in response to pathogenic infection is shown with representative data, including positive and negative controls. This in vitro
model system can be manipulated and applied to other mucosal surfaces. Inflammatory responses that involve excessive neutrophil infiltration can be destructive to host tissues and can occur in the absence of pathogenic infections. A better understanding of the molecular mechanisms that promote neutrophil trans-epithelial migration through experimental manipulation of the in vitro
coculture assay system described herein has significant potential to identify novel therapeutic targets for a range of mucosal infectious as well as inflammatory diseases.
Infection, Issue 83, Cellular Biology, Epithelium, Neutrophils, Pseudomonas aeruginosa, Respiratory Tract Diseases, Neutrophils, epithelial barriers, pathogens, transmigration
The Bovine Lung in Biomedical Research: Visually Guided Bronchoscopy, Intrabronchial Inoculation and In Vivo Sampling Techniques
There is an ongoing search for alternative animal models in research of respiratory medicine. Depending on the goal of the research, large animals as models of pulmonary disease often resemble the situation of the human lung much better than mice do. Working with large animals also offers the opportunity to sample the same animal repeatedly over a certain course of time, which allows long-term studies without sacrificing the animals.
The aim was to establish in vivo
sampling methods for the use in a bovine model of a respiratory Chlamydia psittaci
infection. Sampling should be performed at various time points in each animal during the study, and the samples should be suitable to study the host response, as well as the pathogen under experimental conditions.
Bronchoscopy is a valuable diagnostic tool in human and veterinary medicine. It is a safe and minimally invasive procedure. This article describes the intrabronchial inoculation of calves as well as sampling methods for the lower respiratory tract. Videoendoscopic, intrabronchial inoculation leads to very consistent clinical and pathological findings in all inoculated animals and is, therefore, well-suited for use in models of infectious lung disease. The sampling methods described are bronchoalveolar lavage, bronchial brushing and transbronchial lung biopsy. All of these are valuable diagnostic tools in human medicine and could be adapted for experimental purposes to calves aged 6-8 weeks. The samples obtained were suitable for both pathogen detection and characterization of the severity of lung inflammation in the host.
Medicine, Issue 89, translational medicine, respiratory models, bovine lung, bronchoscopy, transbronchial lung biopsy, bronchoalveolar lavage, bronchial brushing, cytology brush
A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos
Institutions: University of Notre Dame.
Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ
hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.
Developmental Biology, Issue 93, zebrafish, chemical genetics, chemical screen, in vivo small molecule screen, drug discovery, whole mount in situ hybridization (WISH), high-throughput screening (HTS), high-content screening (HCS)
Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney
Institutions: University of Notre Dame.
The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.
Cellular Biology, Issue 90,
zebrafish; kidney; nephron; nephrology; renal; regeneration; proximal tubule; distal tubule; segment; mesonephros; physiology; acute kidney injury (AKI)
Videomorphometric Analysis of Hypoxic Pulmonary Vasoconstriction of Intra-pulmonary Arteries Using Murine Precision Cut Lung Slices
Acute alveolar hypoxia causes pulmonary vasoconstriction (HPV) - also known as von Euler-Liljestrand mechanism - which serves to match lung perfusion to ventilation. Up to now, the underlying mechanisms are not fully understood. The major vascular segment contributing to HPV is the intra-acinar artery. This vessel section is responsible for the blood supply of an individual acinus, which is defined as the portion of lung distal to a terminal bronchiole. Intra-acinar arteries are mostly located in that part of the lung that cannot be selectively reached by a number of commonly used techniques such as measurement of the pulmonary artery pressure in isolated perfused lungs or force recordings from dissected proximal pulmonary artery segments1,2
. The analysis of subpleural vessels by real-time confocal laser scanning luminescence microscopy is limited to vessels with up to 50 µm in diameter3
We provide a technique to study HPV of murine intra-pulmonary arteries in the range of 20-100 µm inner diameters. It is based on the videomorphometric analysis of cross-sectioned arteries in precision cut lung slices (PCLS). This method allows the quantitative measurement of vasoreactivity of small intra-acinar
arteries with inner diameter between 20-40 µm which are located at gussets of alveolar septa next to alveolar ducts and of larger pre-acinar
arteries with inner diameters between 40-100 µm which run adjacent to bronchi and bronchioles. In contrast to real-time imaging of subpleural vessels in anesthetized and ventilated mice, videomorphometric analysis of PCLS occurs under conditions free of shear stress. In our experimental model both arterial segments exhibit a monophasic HPV when exposed to medium gassed with 1% O2
and the response fades after 30-40 min at hypoxia.
Medicine, Issue 83, Hypoxic pulmonary vasoconstriction, murine lungs, precision cut lung slices, intra-pulmonary, pre- and intra-acinar arteries, videomorphometry
Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor
Institutions: Tulane University School of Medicine, Tulane National Primate Research Center, Tulane University School of Medicine, Tulane University School of Medicine.
There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ's natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo
. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.
Bioengineering, Issue 82, rhesus macaque, decellularization, recellularization, detergent, matrix, scaffold, large-organ bioreactor, mesenchymal stem cells
Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers
Institutions: University of Maryland, University of Maryland.
Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with 125
I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free 125
I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.
Bioengineering, Issue 80, Antigens, Enzymes, Biological Therapy, bioengineering (general), Pharmaceutical Preparations, Macromolecular Substances, Therapeutics, Digestive System and Oral Physiological Phenomena, Biological Phenomena, Cell Physiological Phenomena, drug delivery systems, targeted nanocarriers, transcellular transport, epithelial cells, tight junctions, transepithelial electrical resistance, endocytosis, transcytosis, radioisotope tracing, immunostaining
A Mouse Model for Pathogen-induced Chronic Inflammation at Local and Systemic Sites
Institutions: Boston University School of Medicine, Boston University School of Medicine.
Chronic inflammation is a major driver of pathological tissue damage and a unifying characteristic of many chronic diseases in humans including neoplastic, autoimmune, and chronic inflammatory diseases. Emerging evidence implicates pathogen-induced chronic inflammation in the development and progression of chronic diseases with a wide variety of clinical manifestations. Due to the complex and multifactorial etiology of chronic disease, designing experiments for proof of causality and the establishment of mechanistic links is nearly impossible in humans. An advantage of using animal models is that both genetic and environmental factors that may influence the course of a particular disease can be controlled. Thus, designing relevant animal models of infection represents a key step in identifying host and pathogen specific mechanisms that contribute to chronic inflammation.
Here we describe a mouse model of pathogen-induced chronic inflammation at local and systemic sites following infection with the oral pathogen Porphyromonas gingivalis
, a bacterium closely associated with human periodontal disease. Oral infection of specific-pathogen free mice induces a local inflammatory response resulting in destruction of tooth supporting alveolar bone, a hallmark of periodontal disease. In an established mouse model of atherosclerosis, infection with P. gingivalis
accelerates inflammatory plaque deposition within the aortic sinus and innominate artery, accompanied by activation of the vascular endothelium, an increased immune cell infiltrate, and elevated expression of inflammatory mediators within lesions. We detail methodologies for the assessment of inflammation at local and systemic sites. The use of transgenic mice and defined bacterial mutants makes this model particularly suitable for identifying both host and microbial factors involved in the initiation, progression, and outcome of disease. Additionally, the model can be used to screen for novel therapeutic strategies, including vaccination and pharmacological intervention.
Immunology, Issue 90,
Pathogen-Induced Chronic Inflammation; Porphyromonas gingivalis; Oral Bone Loss; Periodontal Disease; Atherosclerosis; Chronic Inflammation; Host-Pathogen Interaction; microCT; MRI
Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung
Institutions: National Institutes of Health.
The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.
Medicine, Issue 81, mouse, rodents, intratracheal intubation, delivery of exogenous substances, lung, study of airway pathology and physiology, pulmonary fibrosis
An Experimental System to Study Mechanotransduction in Fetal Lung Cells
Institutions: Alpert Medical School of Brown University.
Mechanical forces generated in utero
by repetitive breathing-like movements and by fluid distension are critical for normal lung development. A key component of lung development is the differentiation of alveolar type II epithelial cells, the major source of pulmonary surfactant. These cells also participate in fluid homeostasis in the alveolar lumen, host defense, and injury repair. In addition, distal lung parenchyma cells can be directly exposed to exaggerated stretch during mechanical ventilation after birth. However, the precise molecular and cellular mechanisms by which lung cells sense mechanical stimuli to influence lung development and to promote lung injury are not completely understood. Here, we provide a simple and high purity method to isolate type II cells and fibroblasts from rodent fetal lungs. Then, we describe an in vitro
system, The Flexcell Strain Unit, to provide mechanical stimulation to fetal cells, simulating mechanical forces in fetal lung development or lung injury. This experimental system provides an excellent tool to investigate molecular and cellular mechanisms in fetal lung cells exposed to stretch. Using this approach, our laboratory has identified several receptors and signaling proteins that participate in mechanotransduction in fetal lung development and lung injury.
Bioengineering, Issue 60, Mechanical stretch, differentiation, lung injury, isolation, fetal, type II epithelial cells, fibroblasts
Tracheotomy: A Method for Transplantation of Stem Cells to the Lung
Institutions: Harvard Medical School.
Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.
Cellular Biology, Issue 2, lung, stem cells, transplantation, trachea
Mouse Embryonic Lung Culture, A System to Evaluate the Molecular Mechanisms of Branching
Institutions: Childrens Hospital Los Angeles.
Lung primordial specification as well as branching morphogenesis, and the formation of various pulmonary cell lineages requires a specific interaction of the lung endoderm with its surrounding mesenchyme and mesothelium. Lung mesenchyme has been shown to be the source of inductive signals for lung branching morphogenesis. Epithelial-mesenchymal-mesothelial interactions are also critical to embryonic lung morphogenesis. Early embryonic lung organ culture is a very useful system to study epithelial-mesenchymal interactions. Both epithelial and mesenchymal morphogenesis proceeds under specific conditions that can be readily manipulated in this system (in the absence of maternal influence and blood flow). More importantly this technique can be readily done in a serumless, chemically defined culture media. Gain and loss of function can be achieved using expressed proteins, recombinant viral vectors and/or analysis of transgenic mouse strains, antisense RNA, as well as RNA interference gene knockdown.
Developmental Biology, Issue 40, lung, mice, culture

domain: health


Party night with a twist
The LA indie band Lady Danville has released their official video for ‘Better Side’ from the new EP “Operating”.
It’s an easy poppy song helped along by the carefree night out video with a twist. Bingo night at the seniors club meets Cocoon.
The band has a fresh sound with keyboards, lots of harmony and up-beat feel, or so they say, “With Michael Garner on keys, Matthew Frankel on percussion, and Dan Chang on the guitar LADY DANVILLE is most recognized for their well-crafted harmonies, thoughtful lyrics and refreshing dose of energy, wit and charm, evoking the sound of Simon and Garfunkel meets Death Cab for Cutie.”
They’ve been together since 2007 and have 2 EPs released. A full CD is in the works.
In 2007, they won the Star 98.7 Lounge Contest with “Tired Magician”, which is featured on the Star Lounge Compilation CD including John Mayer, Maroon 5, Pink and Nelly Furtado.
Lady Danville recently toured with the very hip Ben Folds, nice company.
I like the sound and the video. Fresh.
Available on iTunes and Amazon.com.