text:
The Warhammer franchise is pretty popular in gaming these days. We have strategy games, Coop shooters and turn-based fighting games, even sports games. Warhammer 40k Inquisitor Martyr wants to be the Diablo-competitor. You pick your hero and dive into random generated maps fighting dozens of enemies and collecting more and more epic loot. That’s the overview, but does it actually work?
You start simply by selecting the class you want to play as. Currently you can choose between two classes with three sub-categories, so you have up to six heroes to start with. There is no customization at the start though, your individual hero gets individual by the loot he collects during the game. As a fan of custom created stuff I hope that the devs are working on that for the final version.
When you have selected your hero and press start you’ll find yourself on a ship/spacestation and have no idea what to do. There is no real tutorial to tell you what to do or what all these different things and locations on your screen are. Well, with a little use of your brain you can find out the most important and basic stuff, like selecting missions and managing your inventory. But right now it’s more of a “find out yourself” thing but I’m sure there are people who want the game to explain everything which is not the case.
Luckily it is pretty obvious how to select missions. You go to the star map and navigate to the system and then the mission you want to play. There are different types and difficulties of missions. So you select a beginner mission and let’s go!
You start right away. After the loading has finished you spawn and get your mission objective marked on the map. And then you go fighting, mostly by shooting sometimes by sword fighting. The focus of the game is clearly on shooting, you can take cover and better keep your distance between the enemies. The maps are random so each mission should feel different, that’s what the devs are promoting. But sadly each mission was very repetitive. The “random” maps were mostly a mix of the same corridors which lead to the same square halls. In these halls you would find an almost identical combination of enemies compared to the rooms before. Even in the first mission I felt like I was doing the same stuff over and over again and that is an absolute no-go. After a couple hours you can get the feeling of repetitive grinding and even that is bad for a game. But the very first mission already got me waiting for the end. Too few types of enemies, map diversity, skills, weapons, overall possibilities. Plus, there are not many options to fight and try different tactis because your skills depend on the weapons you choose. That means you always have six attacks, the basic attacks included. For example you have, basic shot, sniper shot, triple shot and just spray your magazine empty. Then a key for a grenade/mine and a special skill, like two clones fighting for you, invisible etc. That’s not much and changing weapons doesn’t do the trick either. It’s cool that your skills depend on the weapons you have equipped, but first it’s not new and second the weapons almost have identical abilities. If I do a sniper shot with a rifle or a machine gun, the only thing that changes is the distance from where I can shoot.
Skills to unlock are hardly existent, your skill tree consists of slight improvements like “Increase Stun duration by 5%”, so basically minor changes that have an impact, but you barely notice it. There goes the motivation to level up …
Right at the beginning you have seen most of the weapons. Of course after each mission you get your rewards through the basic system: Mostly common, some rares and epics/legendaries, … basic concept. Beside weapons you can change your armor and small improvements for your weapons (only stats, nothing visual). Maps, enemies, items, mission types as well … you see everything pretty fast. The basic concept is there, but the content is missing.
I have played multiple missions with multiple heroes, but it gets repetitive and therefore boring way too fast. I know this is Early Access (which isn’t an excuse forever), so I really hope that the devs have lots of plans for new content and mission design, because right now it feels like a Demo you play once to get an idea of the game.
But it’s not a Demo, it’s a full-priced AAA-game, and I cannot recommend spending that much money on this game at this certain stage of development.


text:
Kalbadevi fire: Age-old cables, BEST staff crunch keep nine buildings in the dark
Officials from the undertaking say that it will take at least a week to give a permanent power connection to the nine buildings that have been in the dark since Saturday’s fire in Gokul Niwas
Two days after a blaze gutted the 50-year-old, four-storeyed Gokul Niwas in Kalbadevi on Saturday, the Brihanmumbai Electricity Supply and Transport (BEST) workers who went to restore the electricity connection were seen cursing the age-old cables, which are at least as old as the building itself.
Two firemen had lost their lives in the blaze, which was reportedly triggered by a short-circuit. Pic/Sayyed Sameer Abedi
One of the workers said a network of old power cables is spread across Kalbadevi, Bhuleshwar and the neighbouring areas. He said the cables are very tough to replace and the narrow lanes just make the job harder.
Commenting on the restoration of power supply to the nine buildings surrounding Gokul Niwas that were plunged into darkness after the mishap, a BEST official said, “We will be providing the residents with a temporary connection by the night of May 11. These lines will be connected to the nearby junction boxes, from where they will draw electricity.”
Though the BEST officials want to restore the power supply at their earliest, the civic authorities have asked them to wait until the debris is removed from the site. It was learnt that a JCB that was pressed into action to clear the rubble had uprooted an important junction box and cables supplying electricity.
“Lanes here are extremely narrow. Executing trenching work and laying new cables is an uphill task. It will take at least a week to provide the residents with a permanent connection,” said a BEST official. A few officials also stressed on the need for immediate redevelopment of the area, as it is congested.
Sources in the BEST said that congested locations like Kalbadevi have several connections, which increase the load tremendously. Officials claim that the load for the entire Kalbadevi area, which also has offices and markets, could be around 25 megawatt a day.
Manpower shortage at the BEST administration has hampered the power supply restoration work. Sources claimed that the position of chief engineers in-charge of maintenance and additional general manager for electric supply are lying vacant. Besides, there are over 200 vacancies across the supply division.

domain: news


text:
About the Organization
A vital strength at Rush Health Systems has been the highly-dedicated physicians who provide a wealth of medical expertise and knowledge to the community we serve. For almost 100 years, our physician network has delivered the highest quality health care services combining the latest in technology and treatments. Our medical staff represents all major medical and surgical specialities. With more than 150 physicians and advance practice providers affiliated with 7 hospitals and over 25 clinics, our network is one of the largest in the state of Mississippi. As an employed physician, you would receive numerous benefits such as an attractive base salary plus productivity incentive, coverage of professional malpractice insurance, health and disabililty insurance, attractive pension plan options, CME allowance, PTO, coverage of relocation expense and a very competitive recruitment package.

domain: medical


text:
This article is only available in the PDF format. Download the PDF to view the article, as well as its associated figures and tables.
The growing concern which has developed recently relative to the relation of cholesterol intake and metabolism to atherosclerosis has created an unusual need for a reliable source of information about dietary sources of cholesterol and the practical management of low cholesterol diets. This need has been admirably filled by the volume "The Low Fat, Low Cholesterol Diet" by Dobbin, Lyon, and Gofman.
This book is written for both the physician and the layman by a group of capable dietitians and one physician. It is filled with useful information concerning the fat and cholesterol content of various foods and with dozens of carefully prepared menus which are applicable to conditions of slight, moderate, or extreme dietary restriction of fat or cholesterol, or both. The exact manner of use of special purpose foods, the sources of such foods, and the actual recipes for their use in the diet are all included. Indeed,
The Low Fat, Low Cholesterol Diet: What to Eat and How to Prepare It.. JAMA. 1951;146(10):975. doi:10.1001/jama.1951.03670100095042

domain: literature


text:
In the realm of drilling operations, technology plays a vital role in improving efficiency and productivity. One technology that has revolutionized drilling processes is the decanter centrifuge. This sophisticated piece of equipment has seen significant advancements over the years, enhancing its capabilities and making it an indispensable asset in the industry. This article takes a deep dive into the evolution of decanter centrifuges, exploring the innovations that have shaped its future.
Enhancing Efficiency with Improved Design
Decanter centrifuges have come a long way from their early prototypes. Initially, their design was simple, consisting of a rotating drum and a conveyor system to separate solids from liquids. However, as the demand for more efficient drilling operations grew, so did the need for improved decanter centrifuge designs. Manufacturers began incorporating advanced technologies and refining the design to enhance separation efficiency, reduce maintenance requirements, and increase operational flexibility.
The Advent of Variable Speed Control
One of the significant breakthroughs in decanter centrifuge technology was the introduction of variable speed control. Traditionally, decanter centrifuges operated at a fixed speed, limiting their adaptability in handling different drilling fluids and solids. The incorporation of variable speed control allowed operators to adjust the centrifuge rotation speed to optimize separation efficiency based on the specific drilling conditions. This innovation not only improved overall performance but also minimized downtime, as operators no longer needed to replace fixed-speed centrifuges for varied drilling applications.
Automation and Remote Monitoring
Automation has become a buzzword across various industries, and drilling operations are no exception. Decanter centrifuges have seen substantial advancements in automation, making them more efficient and user-friendly. Modern decanter centrifuges are equipped with advanced control systems that enable automated adjustment of operating parameters based on real-time data. This ensures the optimal separation efficiency is maintained continuously, minimizing human intervention and maximizing productivity.
Furthermore, remote monitoring capabilities have been integrated into decanter centrifuges, allowing operators to access real-time performance data from anywhere. This remote monitoring feature enables quick troubleshooting, performance analysis, and preventive maintenance, saving time and reducing costs associated with manual supervision.
Improved Solids Discharge Systems
In drilling operations, the efficient discharge of solids is crucial to avoid disruptions and maintain drilling fluid quality. Decanter centrifuges have witnessed significant advancements in the design of their solids discharge systems. Early designs often faced challenges such as blockages and uneven solids distribution, causing operational inefficiencies.
To overcome these issues, modern decanter centrifuges now feature improved solids discharge systems. These systems employ innovative mechanisms, such as scroll conveyors and adjustable pool depths, to ensure a consistent and controlled discharge of solids. These enhancements not only improve separation efficiency but also reduce maintenance requirements and prolong the lifespan of the centrifuge.
Enhanced Material Durability
In drilling operations, the harsh environments encountered can take a toll on equipment durability. Traditional decanter centrifuges were often not designed to withstand the wear and tear of long-duration drilling activities. However, advancements in materials and manufacturing techniques have resulted in the development of more robust decanter centrifuges.
Modern decanter centrifuges are constructed using high-grade materials that offer superior resistance to corrosion, erosion, and abrasion. These materials, coupled with improved manufacturing processes, have significantly enhanced the lifespan and durability of decanter centrifuges. This not only reduces downtime due to maintenance and repairs but also leads to substantial cost savings for drilling operations.
The evolution of decanter centrifuges in drilling operations highlights the industry's commitment to innovation and continuous improvement. From basic designs to sophisticated machines, decanter centrifuges have undergone significant advancements to cater to the dynamic needs of drilling operations. The incorporation of variable speed control, automation and remote monitoring, improved solids discharge systems, and enhanced material durability has boosted their efficiency, reliability, and performance.
As we look into the future, the evolution of decanter centrifuges is expected to continue. Research and development efforts are focused on further improving separation efficiency, reducing energy consumption, and enhancing environmental sustainability. With these ongoing advancements, decanter centrifuges will remain an invaluable asset in drilling operations, driving the industry towards greater efficiency and productivity..

domain: design


text:
Rated 10 out of 10 by retribution55 AWESOME!!!!!!!
first of all i have been a fan of gundam since i was 4 or 5 so i have been looking forward to this game for a long time. This game is really cool there are 2 different modes. one of them mixes 3 or more gundam series for an awesome experience. the other follows the storyline of the gundam series the character is from, and from that characters point of view. The graphics are cool i cant really describe them because i cant find the word im looking for but they are really good. this game is pretty long to complete each characters story takes about two hour and there are over 10 people to play as and the voices sound a lot like the origin voices from the tv shows. the guys you have to fight arent tough at all they are usually in groups of about 30-150 and the groups can get really big. and when you beat enough of the guys a guard or stronger mobile suit will appear and they are tough. i would recommend this to all gundam fans, people who are looking for a game that will take a while to beat, andthose who are looking for an awesome thrill.
Rated 8 out of 10 by RPGamer Please Read BEFORE Purchasing
As a fan of the Gundam franchise I have been following the onslaught of video games that have been released. Lets just say so far.. I'm not so impressed. Then I see this game sitting on the shelf at my local Gamestop, hand twiched and I gave. Bought the game headed home, put it in and after an hour... I was satisfied. Like all the other Dynasty Warrior games the point is to Hack & Slash your way through hordes of enemies, Gundam has shooting and flying to add to the mix. The game has little to no story but if your a Gundam fan and just want to blow everything to hell this is the game for you. There are playable mobile suits spanning across the entire Gundam Timeline, I have yet to unlock them all. All in all this one is for the die hards ONLY. Basicly if you own more then one Gundam game and enjoy those games then get this. If you are a casual fan or not a fan at all either forget about it or wait a while for the price to drop.
Rated 0 out of 10 by A Customer Marriage made in heaven,at least for me
I never cared for what the game mags say about Dynasty Warrior and Gundam games.After class,work,and other tedious activity,maybe what i want is kill 500 pathetic troops in a matter of mintues or destroying a whole city block in a gigantic robot to find a another gigantic robot that when it blows up,another city block goes up in smoke.I could careless of the mediocre cpu,average graphics,1st grader controls,and loser voice actors,these games are the only of their kind,and putting them together makes it even better for me cause i dont even have to change games.This game is more than likely going to be rated poorly,and described as dumb and useless by the game mags,but tell me where else are you going to find a game where in a matter of minutes,you and your robotic self go out and destroy 500 others just like you,tearin up a city like applesauce in a old folks home in the process?Nowhere,thus,i cant wait for this game!
Rated 10 out of 10 by Plasmawarrior Dynasty Warriors fans: Must have
Please make sure you read 'Dynasty Warriors Fans' though, since Gundam fans may not like the idea. But the fact is, this game is made for both. It's orignal story is quite well done, and I love how the art of each battle has it's own style. Space fighting is the same as ground fighting, so it's not going to thow you off like a bad re-make of Star Wars. This is one of the best Dynasty Warriors games out there. In fact, it's the best Gundam game out there... which isn't much to prase to say the least, but consitering how this game would beat the next closest Gundam Game by about 8 notches, it's still very well done. If this is your first Gundam or Dynasty Warriors game, you've got one of the best. It appeals to both sides very well, the fans of the Gundam Series, and the fans of the Dynasty Warriors Series. You can't go wrong, not at all. New commers, hardcore fans, you'll be talking about this game above the rest.
Rated 0 out of 10 by peg-leg omega demo was good
for a Gundam Game, this one looks not bad! for a DW game, it's a good direction, but while playing you realize something: this is just a mod! a really good looking mod, though. more entertaining. and i only have a few complains about the voice acting. 1) one of the girl enemies is really annoying and could've been left out. 2) when master asia says "without a strategy, your strength is useless. first, start bashing the enemy.", that's STUPID. it's a hack 'n slash, with almost only 1 direction to go. finally, with the gameplay: when everyone's got guns, laser swords, and axes and cannons and whatnot, why don't they shoot or fight back unless they're a boss or if you arn't doing anything? i killed over a thousand enemies the first time i beat the demo (it keeps track like previous DW games). not many other gundams who weren't bosses or guards hit me. it's not like they do alot of damage! make it harder!!!
Rated 10 out of 10 by MvC2_Roch This game is good. Not great.
I finished 1k achievements with 50hours of playing time. All in all it was a good game... I've played nearly all the DW/SW/WO series from DW2 going forward. And although I'm a fan of DW and Gundam.. I'd give it a 7.0 overall.. the storyline could've really been better (include more cutscenes and more variety).. afterall hack n slash for however many titles can only get you so far (additional content outside of that would help gameplay tremendously)... there are 16 different pilots in the original mode.. but some of the pilots use the same Mobile Suits.. (which gets kinda gay and repetitive because the playstyle is the EXACT same).. It just seems like they were just throwing different pilots on the same suit to fill up the slots.... they could've atleast thrown in additional mobile suits with diff pilots instead of new pilots with the same mobile suits... like throw in some Gundam Wing suits (my fave).. Part 2 also has the addition of Seed Destiny(another fave) suits which is AWESOME. My take -- Buy the game, enjoy it.. and play part 2.. you won't regret it either way XD
Rated 10 out of 10 by Herman Melville and the Destroyers Gundam? Damn Near Killed 'Em!
Woah. So I randomly hopped on XBOX Live and found a demo for this and decided i'd give it a shot: blew me away. I ran out and got it right away. If you enjoy any hack and slash and/or any game in the Dynasty Warriors series then this is so far up your alley it's essentially rape. With the addition of the ability to shoot guns as apposed to arrows and the amazingly helpful ability to fly and "super-strafe" it's definitely one of the top picks of all DW games and absolutely in the top tier of Gundam games. The story isn't anything fantastic, but does that really bother you in a hack and slash? No? Good. Additionally, any fan of the Gundam series of shows or games will be enthralled at the amount of characters you can play as (which, of course, the Dynasty Warriors series is notorious for). So all in all its nothing esoteric, but for playability and robotic sword swinging fun, this is the game for all.
Rated 0 out of 10 by Dynasty Warrriors Fan awww come on!
Okay I love the dynasty warriors series and especially dynasty warriors 3. after playing the demo of this game, i was shocked at how horrible the graphics were compared to other ps3 games. the graphics on the demo of this game can be compared to the best graphics game on the ps2, but not the ps3. the gameplay is alrite. it is just the plain old slash and kill slash and kill. it gets boring sometimes. the a.i. is extremely horrible. i thought they'll be smarter but from the demo, the a.I. is the same compared to other dynasty series on the ps3. from what the demo looks like, I dont think this game is a must-have. Your just buying another dynasty warriors game except with gundams this time. KOei has definitely dissappointed me with this game.
Rated 0 out of 10 by 4th Kingdom One Question
Why is it called 'Dynasty Warriors: Gundam'? it has nothing to do with the 3 kingdoms story of dynasty warriors, its not set in the same time period, and it has none of the same characters/maps/etc. It looks like a good game but the title doesnt make sense. KOEI should have just called it 'Gundam Warriors' or something. I probably wont buy it because I'm not a big gundam fan and I'm saving my money for Warriors Orochi but it does look good like the rest of the 'warriors' games in terms of gameplay, etc. Like any game, especially new series games, it probably has some stuff they could change and a lot more they could add I'm sure. Content-wise it'll probably give you about as much gameplay time as any other KOEI game and thats quite a bit.
Rated 0 out of 10 by JCeaser08 Alright, Alright
okay, I've been a fan of dynasty warriors since DW3 and yet while it grew a little boring with the fifth and empires versions, I've still been loyal to the franchise. I'm also a great fan of Gundam owning all episodes of both Gundam Wing and the original Gundam on my computer er, DVD. This game however, is a splendid mix of both. The ONE level alone that is offered gives a very "Wing" series feel yet has the Warriors elements down pact. I am curious to know how Pilot level and Gundam level will affect the overall game, as they grew kind of pointless in empires but I must say, I along with several friends, have already played the demo many times through. We can not wait for this game!

domain: game


text:
How to Play “Give It To Me Baby” on Guitar
The classic hit song “Give It To Me Baby” by Rick James is sure to get your toes tapping and have you singing along! Playing this fun track on guitar is fairly simple and can easily be mastered by players of any skill level. Here’s a quick guide on how to get started:
Step 1: Learn the Chords
To play the song, you’ll need to know the following chords: A, E, D, F#, and G. You’ll want to practice switching between the chords with a steady rhythm.
Step 2: Learn the Strumming Pattern
Once you’re comfortable with the chords, you’ll need to learn the strumming pattern. The basic pattern is down-down-down-up-down-up. Practice this pattern by itself a few times before moving on to the rest of the song.
Step 3: The Intro
At the beginning of the song, you’ll be playing the A chord up and down twice. Then, switch to the E chord and pick the first three strings (E, B, and G). This is then repeated twice.
Step 4: The Verse
For the verse, you’ll need to switch between the A, E, and D chords. This should be done using the strumming pattern you learned in step two. Then, finish the verse by playing the E chord on the 3rd string twice.
Step 5: The Chorus
For the chorus, you’ll play a slightly different pattern. Instead of switching between A and E chords, you’ll switch between F# and G chords. This should be done while strumming the same pattern you used in step 2.
Step 6: Play It Together
Once you’ve practiced the chords, strumming, and verse and chorus patterns, you’ll be ready to put it all together and play the full song!
Tips and Tricks:
- Start slow to make sure you’re playing all of the chords correctly.
- Listen to the song and sing along when you play it.
- Practice with a metronome to get the timing right.
With a bit of practice, you’ll soon be playing “Give It To Me Baby” on guitar like a pro! Good luck and have fun!

domain: artistic


With hybrid technology, DuoWIN combines heating with wood and pellets in one unit. This biomass boiler system allows users to either use logs or wood pellets and its intelligent fuel switching is designed to detect when the wood gassifier is in burnout and switches to pellet operation, either automatically or at a desired time.
Available in four models from 33 kW – 56 kW output, the DuoWIN has a filling volume of 145 litres for 50cm split logs and 164 litres of wood pellets, the feed for which can be either manual or fully automatic.
At Ecobuild, Windhager will also be promoting its new scheme for installers – WIN, the Windhager Installer Network. Installers undertaking training at Windhager under this scheme will benefit from being able to offer extended seven year warranties on all Windhager biomass boilers.


text:
Culture and Agriculture
Volunteered - Oral Presentation Session
Navneet Gill (Panjab University)
Abhik Ghosh (Panjab University)
Climate change significantly poses a threat to agrarian societies. Even mild climate variation affects the crop adversely resulting in a challenge for farmers in coping with the changing climatic conditions. Punjab, being an agrarian economy, was facing a crisis and was rapidly turning into a farmers' graveyard. Neo-liberal policies by central and state government deepened the agrarian crisis. In view of the prevailing agrarian crisis in Punjab, farmers’ voices were used to understand the local farming systems, farmer's experiences and perceptions regarding farming practices from an anthropological perspective. In this study, the researcher also comprehends the local knowledge of farmers towards climate change and their adaptation strategies to cope with it through an ethnographic account of Sarawan village of Faridkot District, Punjab. In-depth interviews were conducted from 250 respondents which included small, marginal and large farmers, cooperative societies, arhtiyas, bank officials and procurement field officials. Inferences drawn from the data reveals that modernized farming practices had led to the deterioration of local cultural farming knowledge, loss of soil fertility, the decline in the water table, instability in crop yield, among other issues. Narratives from fieldwork highlighted farmers who opposed the policies of the government. With the changing climate, farmers were not able to cope with this situation due to a lack of knowledge regarding new varieties of seeds, the high cost of agricultural machinery and poor agricultural extension services. Farming systems had developed in ways reflecting the activities and choices of farmers influenced by environmental, socio-cultural, economic and political conditions.

domain: agronomy


text:
Hi there Libby, just to let you and your readers know, I have been low carb for over a year now, ( sometimes I fall down and have processed carbs, mostly when I am travelling away from home) and the good news is I was diabetic, and now I am pre-diabetic, which is huge :O) I have also lost a small amount of weight in the process ( still more to go :O) but this eating plan makes me feel normal again, not always hungry, and I am a lot healthier than I was :O) now all I need to do is make exercise a habit :O) So thank you so much for all the time and energy you put in to this site, and all of these wonderful recipes :O)
There are vegetables that are high in carbs and others low in carbs. The keto diet recommends sticking to the ones low on carbs but encourages you to eat a lot of them. Best vegetables are all green ones to make it easy. And vegetables that grow above the ground (e.g. lettuce) are always better than the ones that grow below the ground (e.g. potatoes)
Meal plan Cheese lovers, your time has finally come! It’s basic knowledge (among cheese lovers, at least) that cheese makes everything taste better, so we’re dedicating this week to this wonderful ingredient. You’ll enjoy various types of cheese such as mozzarella, cheddar, feta, goat cheese… and we won’t skimp on the cream cheese either. Not a member yet? Try it out 1 month for free.
Keto recipes that include nachos?! Oh yes. You’ll begin by making the fat head tortilla chips first. Did I mention you’ll use two types of cheese for this step? Delicious. Next, you’ll load them up with a meaty sauce and finish them off with your favorite toppings, like guac, salsa or sour cream. While these make a delicious keto snack, they’re frankly filling enough to share as a meal.
Keep up electrolytes. The major electrolytes in our bodies are sodium, potassium and magnesium. Because a low carb diet (especially a keto diet!) reduces the amount of water you store, this can flush out electrolytes and make you feel sick (called “keto flu”). This is temporary, but you can avoid or eliminate it by salting your food liberally, drinking broth (especially bone broth), and eating pickled vegetables. Some people also choose to take supplements for electrolytes, but it’s best to first consult a doctor that understands and supports keto/low carb lifestyles.

domain: food


text:
In his collection of Essays and Aphorisms, Arthur Schopenhauer tells about what he considers a criteria of a “good novel”.
A novel will be the higher and nobler the more inner and less outer life depicts… The art lies in setting the inner life into the most violent motion with the smallest possible expenditure of outer life: for it is the inner life which is the real object of our interest. – The task of the novelist is not to narrate great events but to make small ones interesting.
Schopenhauer highlights four novels that are the “crown of the genre.”
Laurence Stern – The Life and Opinions of Tristram Shandy, Gentleman (1759)
Laurence Sterne’s great masterpiece of bawdy humour and rich satire defies any attempt to categorize it, with a rich metafictional narrative that might classify it as the first ‘postmodern’ novel. Part novel, part digression, its gloriously disordered narrative interweaves the birth and life of the unfortunate ‘hero’ Tristram Shandy, the eccentric philosophy of his father Walter, the amours and military obsessions of Uncle Toby, and a host of other characters, including Dr Slop, Corporal Trim and the parson Yorick. A joyful celebration of the endless possibilities of the art of fiction, Tristram Shandy is also a wry demonstration of its limitations.
Johann Wolfgang Von Goethe – Wilhelm Meister’s Apprenticeship (1796)
Wilhelm Meister’s Apprenticeship, a novel of self-realization greatly admired by the Romantics, has been called the first Bildungsroman and has had a tremendous influence on the history of the German novel. The story centers on Wilhelm, a young man living in the mid-1700s who strives to break free from the restrictive world of economics and seeks fulfillment as an actor and playwright.
Jean-Jacques Rousseau – Julie, or the New Eloise: Letters of Two Lovers, Inhabitants of a Small Town at the Foot of the Alps (1761)
In Julie, or the New Heloise, Rousseau reconceptualized the relationship of the individual to the collective and articulated a new moral paradigm. The story follows the fates and smoldering passions of Julie d’Etange and St. Preux, a one-time lover who re-enters Julie’s life at the invitation of her unsuspecting husband, M. de Wolmar. The complex tones of this work made it a commercial success and a continental sensation when it first appeared in 1761, and its embodiment of Rousseau’s system of thought, in which feelings and intellect are intertwined, redefined the function and form of fiction for decades. As the characters negotiate a complex maze of passion and virtue, their purity of soul and honest morality reveal, as Rousseau writes in his preface,”the subtleties of heart of which this work is full.” A comprehensive introduction and careful annotations make this novel accessible to contemporary readers, both as an embodiment of Rousseau’s philosophy and as a portrayal of the tension and power inherent in domestic life.
Miguel De Cervantes Saavedra – Don Quixote (1605)
Don Quixote has become so entranced reading tales of chivalry that he decides to turn knight errant himself. In the company of his faithful squire, Sancho Panza, these exploits blossom in all sorts of wonderful ways. While Quixote’s fancy often leads him astray—he tilts at windmills, imagining them to be giants—Sancho acquires cunning and a certain sagacity. Sane madman and wise fool, they roam the world together-and together they have haunted readers’ imaginations for nearly four hundred years.

domain: library


text:
Mr Rakhmon, a former cotton farm boss, was elected chairman of the Supreme Council of Tajikistan in 1992 after the country's first post-Soviet leader, Rahmon Nabiyev, was forced to resign.
He was elected president in 1994 and re-elected in 1999 when his term was extended to seven years.
In 2006 he won a third term in office in an election which international observers said was neither free nor fair. Opposition parties boycotted the vote, dismissing it as a Soviet-style staged attempt at democracy.
In 2013 he gained a further seven-year term in elections. The only serious opposition candidate was prevented from running by the electoral commission which said she hadn't collected enough signatures to become a candidate.
Mr Rakhmon was instrumental in the pro-Communist effort to remove Islamist rebels from Dushanbe in the early 1990s. He led troops from southern Kulob District and supported the intervention of forces from other former Soviet republics. After years of civil war and violence, some stability returned to Tajikistan.
The president has a firm grip on power. His People's Democratic Party holds virtually all seats in parliament. Western observers said legislative elections in 2005 and 2010 failed to meet international standards.
Mr Rakhmon does retain substantial public support. Tajikistan is still very poor, but many people are thankful they no longer have to face the civil war of the 1990s which killed tens of thousands and caused more than 10% of the population to flee the country.
Mr Rakhmon was born in 1952. His surname was Rakhmonov until 2007 when he ordered his countrymen to drop Russian-style surnames, in a break with the nation's Soviet past. He removed the Russian suffix "-ov" from his surname.
- Administrative divisions: This entry generally gives the numbers, designatory terms, and first-order administrative divisions as approved by the US Board on Geographic Names (BGN). Changes that have been reported but not yet acted on by BGN are noted.
- Capital city > Geographic coordinates: This entry gives the name of the seat of government, its geographic coordinates, the time difference relative to Coordinated Universal Time (UTC) and the time observed in Washington, DC, and, if applicable, information on daylight saving time (DST). Where appropriate, a special note has been added to highlight those countries that have multiple time zones.
- Capital city > Name: This entry gives the name of the seat of government, its geographic coordinates, the time difference relative to Coordinated Universal Time (UTC) and the time observed in Washington, DC, and, if applicable, information on daylight saving time (DST). Where appropriate, a special note has been added to highlight those countries that have multiple time zones.
- Constitution: The dates of adoption, revisions, and major amendments to a nation's constitution
- Diplomatic representation from the US > Mailing address: This entry includes the chief of mission, embassy address, mailing address, telephone number, FAX number, branch office locations, consulate general locations, and consulate locations.
- Executive branch > Cabinet: Cabinet includes the official name for any body of high-ranking advisers roughly comparable to a U.S. Cabinet. Also notes the method for selection of members.
- Executive branch > Chief of state: The name and title of any person or role roughly equivalent to a U.S. Chief of State. This means the titular leader of the country who represents the state at official and ceremonial functions but may not be involved with the day-to-day activities of the government
- Executive branch > Head of government: Head of government includes the name and title of the top administrative leader who is designated to manage the day-to-day activities of the government. For example, in the UK, the monarch is the chief of state, and the prime minister is the head of government. In the US, the president is both the chief of state and the head of government.
- Government type: A description of the basic form of government (e.g., republic, constitutional monarchy, federal republic, parliamentary democracy, military dictatorship).
- Judicial branch: The name(s) of the highest court(s) and a brief description of the selection process for members.
- Legal system: A brief description of the legal system's historical roots, role in government, and acceptance of International Court of Justice (ICJ) jurisdiction.
- Legislative branch: This entry contains information on the structure (unicameral, bicameral, tricameral), formal name, number of seats, and term of office. Elections includes the nature of election process or accession to power, date of the last election, and date of the next election. Election results includes the percent of vote and/or number of seats held by each party in the last election.
- Political parties and leaders: Significant political organizations and their leaders.
- Political pressure groups and leaders: Organizations with leaders involved in politics, but not standing for legislative election.
- Suffrage: The age at enfranchisement and whether the right to vote is universal or restricted
"Tajikistan Government Stats", NationMaster. Retrieved from http://www.nationmaster.com/country-info/profiles/Tajikistan/Government
"Tajikistan Government Stats, NationMaster." 1990-2014. <http://www.nationmaster.com/country-info/profiles/Tajikistan/Government>.
'Tajikistan Government Stats, NationMaster', <http://www.nationmaster.com/country-info/profiles/Tajikistan/Government> [assessed 1990-2014]
"Tajikistan Government Stats", NationMaster [Internet]. 1990-2014. Avaliable from: <http://www.nationmaster.com/country-info/profiles/Tajikistan/Government>.
"Tajikistan Government Stats", NationMaster. Avaliable at: nationmaster.com. Assessed 1990-2014.
"Tajikistan Government Stats, NationMaster," http://www.nationmaster.com/country-info/profiles/Tajikistan/Government (assessed 1990-2014)
"Tajikistan Government Stats", NationMaster, http://www.nationmaster.com/country-info/profiles/Tajikistan/Government (last visited 1990-2014)
"Tajikistan Government Stats", NationMaster, http://www.nationmaster.com/country-info/profiles/Tajikistan/Government (as of 1990-2014)

domain: geography


text:
Should I Pay Off Debt or Invest?
This calculator will help determine whether you should invest funds or pay down debt.
You are viewing this website at a small screen resolution which doesn't support calculators. Click here to view this calculator.
Coaches have helped you your whole life, in ways big and small. We’d like to be one of them.
Money Draining Food Myths
These food myths will really put a drain on your wallet.
Interested in a Fuel Efficient Car?
Estimate how many months it may take to recover the out-of-pocket costs when buying a more efficient vehicle.

domain: economics


text:
Saturday, November 21, 2015
40 Great Nail Art Ideas- Grey and Color
So, here's this week's 40GNAI prompt. The color prompt was grey and color, grey being fitting because it is very grey this morning. My nail art prompt was zig zags. Here is what I cam up with.....
Base color is Zoya Tris, which looks more green than grey in my pic, but I promise you it is grey. The zig zags are from Vivid Lacquer 010, stamped in MdU fiji.
Grey & Color Inlinkz-


text:
Here we introduce a series of thoroughly tested and well standardized research protocols adapted for use in remote marine environments. The sampling protocols include the assessment of resources available to the microbial community (dissolved organic carbon, particulate organic matter, inorganic nutrients), and a comprehensive description of the viral and bacterial communities (via direct viral and microbial counts, enumeration of autofluorescent microbes, and construction of viral and microbial metagenomes). We use a combination of methods, which represent a dispersed field of scientific disciplines comprising already established protocols and some of the most recent techniques developed. Especially metagenomic sequencing techniques used for viral and bacterial community characterization, have been established only in recent years, and are thus still subjected to constant improvement. This has led to a variety of sampling and sample processing procedures currently in use. The set of methods presented here provides an up to date approach to collect and process environmental samples. Parameters addressed with these protocols yield the minimum on information essential to characterize and understand the underlying mechanisms of viral and microbial community dynamics. It gives easy to follow guidelines to conduct comprehensive surveys and discusses critical steps and potential caveats pertinent to each technique.
25 Related JoVE Articles!
Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease
Institutions: SRI International, University of California-Santa Cruz.
Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer's disease, but its effects in Parkinson's disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone. To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e.
5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder. Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD. The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates. This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.
Medicine, Issue 83, MPTP, dopamine, Iba1, TH, GFAP, lipoxygenase, transgenic, gene-environment interactions, mouse, Parkinson's disease, neurodegeneration, neuroinflammation
2D and 3D Chromosome Painting in Malaria Mosquitoes
Institutions: Virginia Tech.
Fluorescent in situ
hybridization (FISH) of whole arm chromosome probes is a robust technique for mapping genomic regions of interest, detecting chromosomal rearrangements, and studying three-dimensional (3D) organization of chromosomes in the cell nucleus. The advent of laser capture microdissection (LCM) and whole genome amplification (WGA) allows obtaining large quantities of DNA from single cells. The increased sensitivity of WGA kits prompted us to develop chromosome paints and to use them for exploring chromosome organization and evolution in non-model organisms. Here, we present a simple method for isolating and amplifying the euchromatic segments of single polytene chromosome arms from ovarian nurse cells of the African malaria mosquito Anopheles gambiae
. This procedure provides an efficient platform for obtaining chromosome paints, while reducing the overall risk of introducing foreign DNA to the sample. The use of WGA allows for several rounds of re-amplification, resulting in high quantities of DNA that can be utilized for multiple experiments, including 2D and 3D FISH. We demonstrated that the developed chromosome paints can be successfully used to establish the correspondence between euchromatic portions of polytene and mitotic chromosome arms in An. gambiae
. Overall, the union of LCM and single-chromosome WGA provides an efficient tool for creating significant amounts of target DNA for future cytogenetic and genomic studies.
Immunology, Issue 83, Microdissection, whole genome amplification, malaria mosquito, polytene chromosome, mitotic chromosomes, fluorescence in situ hybridization, chromosome painting
Barnes Maze Testing Strategies with Small and Large Rodent Models
Institutions: University of Missouri, Food and Drug Administration.
Spatial learning and memory of laboratory rodents is often assessed via navigational ability in mazes, most popular of which are the water and dry-land (Barnes) mazes. Improved performance over sessions or trials is thought to reflect learning and memory of the escape cage/platform location. Considered less stressful than water mazes, the Barnes maze is a relatively simple design of a circular platform top with several holes equally spaced around the perimeter edge. All but one of the holes are false-bottomed or blind-ending, while one leads to an escape cage. Mildly aversive stimuli (e.g.
bright overhead lights) provide motivation to locate the escape cage. Latency to locate the escape cage can be measured during the session; however, additional endpoints typically require video recording. From those video recordings, use of automated tracking software can generate a variety of endpoints that are similar to those produced in water mazes (e.g.
distance traveled, velocity/speed, time spent in the correct quadrant, time spent moving/resting, and confirmation of latency). Type of search strategy (i.e.
random, serial, or direct) can be categorized as well. Barnes maze construction and testing methodologies can differ for small rodents, such as mice, and large rodents, such as rats. For example, while extra-maze cues are effective for rats, smaller wild rodents may require intra-maze cues with a visual barrier around the maze. Appropriate stimuli must be identified which motivate the rodent to locate the escape cage. Both Barnes and water mazes can be time consuming as 4-7 test trials are typically required to detect improved learning and memory performance (e.g.
shorter latencies or path lengths to locate the escape platform or cage) and/or differences between experimental groups. Even so, the Barnes maze is a widely employed behavioral assessment measuring spatial navigational abilities and their potential disruption by genetic, neurobehavioral manipulations, or drug/ toxicant exposure.
Behavior, Issue 84, spatial navigation, rats, Peromyscus, mice, intra- and extra-maze cues, learning, memory, latency, search strategy, escape motivation
The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin
Institutions: European Institute of Oncology.
Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics
), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP
in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.
Biochemistry, Issue 86, chromatin, histone post-translational modifications (hPTMs), epigenetics, mass spectrometry, proteomics, SILAC, chromatin immunoprecipitation , histone variants, chromatome, hPTMs cross-talks
Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities
Institutions: Université de Strasbourg.
Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols.
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.
Neuroscience, Issue 89, mice, nociception, tail immersion test, tail pressure test, morphine, analgesia, opioid-induced hyperalgesia, tolerance
Analysis of Oxidative Stress in Zebrafish Embryos
Institutions: University of Torino, Vesalius Research Center, VIB.
High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo
system to perform such studies and present a protocol to measure in vivo
oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo
: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.
Developmental Biology, Issue 89, Danio rerio, zebrafish embryos, endothelial cells, redox state analysis, oxidative stress detection, in vivo ROS measurements, FACS (fluorescence activated cell sorter), molecular probes
Preparation of Primary Myogenic Precursor Cell/Myoblast Cultures from Basal Vertebrate Lineages
Institutions: University of Alabama at Birmingham, INRA UR1067, INRA UR1037.
Due to the inherent difficulty and time involved with studying the myogenic program in vivo
, primary culture systems derived from the resident adult stem cells of skeletal muscle, the myogenic precursor cells (MPCs), have proven indispensible to our understanding of mammalian skeletal muscle development and growth. Particularly among the basal taxa of Vertebrata,
however, data are limited describing the molecular mechanisms controlling the self-renewal, proliferation, and differentiation of MPCs. Of particular interest are potential mechanisms that underlie the ability of basal vertebrates to undergo considerable postlarval skeletal myofiber hyperplasia (i.e.
teleost fish) and full regeneration following appendage loss (i.e.
urodele amphibians). Additionally, the use of cultured myoblasts could aid in the understanding of regeneration and the recapitulation of the myogenic program and the differences between them. To this end, we describe in detail a robust and efficient protocol (and variations therein) for isolating and maintaining MPCs and their progeny, myoblasts and immature myotubes, in cell culture as a platform for understanding the evolution of the myogenic program, beginning with the more basal vertebrates. Capitalizing on the model organism status of the zebrafish (Danio rerio
), we report on the application of this protocol to small fishes of the cyprinid clade Danioninae
. In tandem, this protocol can be utilized to realize a broader comparative approach by isolating MPCs from the Mexican axolotl (Ambystomamexicanum
) and even laboratory rodents. This protocol is now widely used in studying myogenesis in several fish species, including rainbow trout, salmon, and sea bream1-4
Basic Protocol, Issue 86, myogenesis, zebrafish, myoblast, cell culture, giant danio, moustached danio, myotubes, proliferation, differentiation, Danioninae, axolotl
A Strategy for Sensitive, Large Scale Quantitative Metabolomics
Institutions: Cornell University, Cornell University.
Metabolite profiling has been a valuable asset in the study of metabolism in health and disease. However, current platforms have different limiting factors, such as labor intensive sample preparations, low detection limits, slow scan speeds, intensive method optimization for each metabolite, and the inability to measure both positively and negatively charged ions in single experiments. Therefore, a novel metabolomics protocol could advance metabolomics studies. Amide-based hydrophilic chromatography enables polar metabolite analysis without any chemical derivatization. High resolution MS using the Q-Exactive (QE-MS) has improved ion optics, increased scan speeds (256 msec at resolution 70,000), and has the capability of carrying out positive/negative switching. Using a cold methanol extraction strategy, and coupling an amide column with QE-MS enables robust detection of 168 targeted polar metabolites and thousands of additional features simultaneously. Data processing is carried out with commercially available software in a highly efficient way, and unknown features extracted from the mass spectra can be queried in databases.
Chemistry, Issue 87, high-resolution mass spectrometry, metabolomics, positive/negative switching, low mass calibration, Orbitrap
Who is Who? Non-invasive Methods to Individually Sex and Mark Altricial Chicks
Institutions: Freie Universität Berlin.
Many experiments require early determination of offspring's sex as well as early marking of newborns for individual recognition. According to animal welfare guidelines, non-invasive techniques should be preferred whenever applicable. In our group, we work on different species of song birds in the lab and in the field, and we successfully apply non-invasive methods to sex and individually mark chicks. This paper presents a comprehensive non-invasive tool-box. Sexing birds prior to the expression of secondary sexual traits requires the collection of DNA-bearing material for PCR. We established a quick and easy method to sex birds of any age (post hatching) by extracting DNA from buccal swabs. Results can be obtained within 3 hours. For individual marking chick's down feathers are trimmed in specific patterns allowing fast identification within the hatching order. This set of methods is easily applicable in a standard equipped lab and especially suitable for working in the field as no special equipment is required for sampling and storage. Handling of chicks is minimized and marking and sexing techniques are non-invasive thereby supporting the RRR-principle of animal welfare guidelines.
Developmental Biology, Issue 87, songbird, molecular sexing, PCR, individual marking, down feather, DNA extraction, sample storage, zebra finch, buccal swabs, saliva, gender
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)
DNA-affinity-purified Chip (DAP-chip) Method to Determine Gene Targets for Bacterial Two component Regulatory Systems
Institutions: Lawrence Berkeley National Laboratory.
methods such as ChIP-chip are well-established techniques used to determine global gene targets for transcription factors. However, they are of limited use in exploring bacterial two component regulatory systems with uncharacterized activation conditions. Such systems regulate transcription only when activated in the presence of unique signals. Since these signals are often unknown, the in vitro
microarray based method described in this video article can be used to determine gene targets and binding sites for response regulators. This DNA-affinity-purified-chip method may be used for any purified regulator in any organism with a sequenced genome. The protocol involves allowing the purified tagged protein to bind to sheared genomic DNA and then affinity purifying the protein-bound DNA, followed by fluorescent labeling of the DNA and hybridization to a custom tiling array. Preceding steps that may be used to optimize the assay for specific regulators are also described. The peaks generated by the array data analysis are used to predict binding site motifs, which are then experimentally validated. The motif predictions can be further used to determine gene targets of orthologous response regulators in closely related species. We demonstrate the applicability of this method by determining the gene targets and binding site motifs and thus predicting the function for a sigma54-dependent response regulator DVU3023 in the environmental bacterium Desulfovibrio vulgaris
Genetics, Issue 89, DNA-Affinity-Purified-chip, response regulator, transcription factor binding site, two component system, signal transduction, Desulfovibrio, lactate utilization regulator, ChIP-chip
Slide Preparation Method to Preserve Three-dimensional Chromatin Architecture of Testicular Germ Cells
Institutions: Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine.
During testicular germ cell differentiation, the structure of nuclear chromatin dynamically changes. The following describes a method designed to preserve the three-dimensional chromatin arrangement of testicular germ cells found in mice; this method has been termed as the three-dimensional (3D) slide method. In this method, testicular tubules are directly treated with a permeabilization step that removes cytoplasmic material, followed by a fixation step that fixes nuclear materials. Tubules are then dissociated, the cell suspension is cytospun, and cells adhere to slides. This method improves sensitivity towards detection of subnuclear structures and is applicable for immunofluorescence, DNA, and RNA fluorescence in situ
hybridization (FISH) and the combination of these detection methods. As an example of a possible application of the 3D slide method, a Cot-1 RNA FISH is shown to detect nascent RNAs. The 3D slide method will facilitate the detailed examination of spatial relationships between chromatin structure, DNA, and RNA during testicular germ cell differentiation.
Basic Protocol, Issue 83, Chromatin, Germ cells, Sex chromosomes, Testis, Meiotic sex chromosome inactivation, Postmeiotic sex chromatin
Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways
Institutions: University of Colorado School of Medicine, Oregon Health & Science University, University of Colorado School of Medicine.
Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.
Neuroscience, Issue 82, male, female, sex, neuronal culture, ischemia, cell death, neuroprotection
Making Gynogenetic Diploid Zebrafish by Early Pressure
Institutions: University of Oregon, Fred Hutchinson Cancer Research Center - FHCRC.
Heterozygosity in diploid eukaryotes often makes genetic studies cumbersome. Methods that produce viable homozygous diploid offspring directly from heterozygous females allow F1 mutagenized females to be screened directly for deleterious mutations in an accelerated forward genetic screen. Streisinger et al.1,2
described methods for making gynogenetic (homozygous) diploid zebrafish by activating zebrafish eggs with ultraviolet light-inactivated sperm and preventing either the second meiotic or the first zygotic cell division using physical treatments (heat or pressure) that deploymerize microtubules. The "early pressure" (EP) method blocks the meiosis II, which occurs shortly after fertilization. The EP method produces a high percentage of viable embryos that can develop to fertile adults of either sex. The method generates embryos that are homozygous at all loci except those that were separated from their centromere by recombination during meiosis I. Homozygous mutations are detected in EP clutches at between 50% for centromeric loci and less than 1% for telomeric loci. This method is reproduced verbatim from the Zebrafish Book3
Developmental Biology, Issue 28, Zebrafish, Early Pressure, Homozygous Diploid, Haploid, Gynogenesis
FISH for Pre-implantation Genetic Diagnosis
Institutions: Guy’s & St Thomas’ Centre for Preimplantation Genetic Diagnosis.
Pre-implantation genetic diagnosis (PGD) is an established alternative to pre-natal diagnosis, and involves selecting pre-implantation embryos from a cohort generated by assisted reproduction technology (ART). This selection may be required because of familial monogenic disease (e.g. cystic fibrosis), or because one partner carries a chromosome rearrangement (e.g. a two-way reciprocal translocation). PGD is available for couples who have had previous affected children, and/or in the case of chromosome rearrangements, recurrent miscarriages, or infertility. Oocytes aspirated following ovarian stimulation are fertilized by in vitro
immersion in semen (IVF) or by intracytoplasmic injection of an individual spermatozoon (ICSI). Pre-implantation cleavage-stage embryos are biopsied, usually by the removal of a single cell on day 3 post-fertilization, and the biopsied cell is tested to establish the genetic status of the embryo. Fluorescence in situ
hybridization (FISH) on the fixed nuclei of biopsied cells with target-specific DNA probes is the technique of choice to detect chromosome imbalance associated with chromosome rearrangements, and to select female embryos in families with X-linked disease for which there is no mutation-specific test. FISH has also been used to screen embryos for spontaneous chromosome aneuploidy (also known as PGS or PGD-AS) in order to try and improve the efficiency of assisted reproduction; however, the predictive value of this test using the spreading and FISH technique described here is likely to be unacceptably low in most people's hands and it is not recommended for routine clinical use. We describe the selection of suitable probes for single-cell FISH, spreading techniques for blastomere nuclei, and in situ
hybridization and signal scoring, applied to PGD in a clinical setting.
Medicine, Issue 48, Fluorescence in situ hybridization, Pre-implantation genetic diagnosis, PGD, Sex determination, Translocations, Chromosome aneuploidy
Swimming Performance Assessment in Fishes
Institutions: University of Alberta.
Swimming performance tests of fish have been integral to studies of muscle energetics, swimming mechanics, gas exchange, cardiac physiology, disease, pollution, hypoxia and temperature. This paper describes a flexible protocol to assess fish swimming performance using equipment in which water velocity can be controlled. The protocol involves one to several stepped increases in flow speed that are intended to cause fish to fatigue. Step speeds and their duration can be set to capture swimming abilities of different physiological and ecological relevance. Most frequently step size is set to determine critical swimming velocity (Ucrit
), which is intended to capture maximum sustained swimming ability. Traditionally this test has consisted of approximately ten steps each of 20 min duration. However, steps of shorter duration (e.g. 1 min) are increasingly being utilized to capture acceleration ability or burst swimming performance. Regardless of step size, swimming tests can be repeated over time to gauge individual variation and recovery ability. Endpoints related to swimming such as measures of metabolic rate, fin use, ventilation rate, and of behavior, such as the distance between schooling fish, are often included before, during and after swimming tests. Given the diversity of fish species, the number of unexplored research questions, and the importance of many species to global ecology and economic health, studies of fish swimming performance will remain popular and invaluable for the foreseeable future.
Physiology, Issue 51, fish, swimming, Ucrit, burst, sustained, prolonged, schooling performance
Experimental Manipulation of Body Size to Estimate Morphological Scaling Relationships in Drosophila
Institutions: University of Houston, Michigan State University.
The scaling of body parts is a central feature of animal morphology1-7
. Within species, morphological traits need to be correctly proportioned to the body for the organism to function; larger individuals typically have larger body parts and smaller individuals generally have smaller body parts, such that overall body shape is maintained across a range of adult body sizes. The requirement for correct proportions means that individuals within species usually exhibit low variation in relative trait size. In contrast, relative trait size can vary dramatically among species and is a primary mechanism by which morphological diversity is produced. Over a century of comparative work has established these intra- and interspecific patterns3,4
Perhaps the most widely used approach to describe this variation is to calculate the scaling relationship between the size of two morphological traits using the allometric equation y=bxα, where x and y are the size of the two traits, such as organ and body size8,9
. This equation describes the within-group (e.g., species, population) scaling relationship between two traits as both vary in size. Log-transformation of this equation produces a simple linear equation, log(y) = log(b) + αlog(x) and log-log plots of the size of different traits among individuals of the same species typically reveal linear scaling with an intercept of log(b) and a slope of α, called the 'allometric coefficient'9,10
. Morphological variation among groups is described by differences in scaling relationship intercepts or slopes for a given trait pair. Consequently, variation in the parameters of the allometric equation (b and α) elegantly describes the shape variation captured in the relationship between organ and body size within and among biological groups (see 11,12
Not all traits scale linearly with each other or with body size (e.g., 13,14
) Hence, morphological scaling relationships are most informative when the data are taken from the full range of trait sizes. Here we describe how simple experimental manipulation of diet can be used to produce the full range of body size in insects. This permits an estimation of the full scaling relationship for any given pair of traits, allowing a complete description of how shape covaries with size and a robust comparison of scaling relationship parameters among biological groups. Although we focus on Drosophila
, our methodology should be applicable to nearly any fully metamorphic insect.
Developmental Biology, Issue 56, Drosophila, allometry, morphology, body size, scaling, insect
Mapping Bacterial Functional Networks and Pathways in Escherichia Coli using Synthetic Genetic Arrays
Institutions: University of Toronto, University of Toronto, University of Regina.
Phenotypes are determined by a complex series of physical (e.g.
protein-protein) and functional (e.g.
gene-gene or genetic) interactions (GI)1
. While physical interactions can indicate which bacterial proteins are associated as complexes, they do not necessarily reveal pathway-level functional relationships1. GI screens, in which the growth of double mutants bearing two deleted or inactivated genes is measured and compared to the corresponding single mutants, can illuminate epistatic dependencies between loci and hence provide a means to query and discover novel functional relationships2
. Large-scale GI maps have been reported for eukaryotic organisms like yeast3-7
, but GI information remains sparse for prokaryotes8
, which hinders the functional annotation of bacterial genomes. To this end, we and others have developed high-throughput quantitative bacterial GI screening methods9, 10
Here, we present the key steps required to perform quantitative E. coli
Synthetic Genetic Array (eSGA) screening procedure on a genome-scale9
, using natural bacterial conjugation and homologous recombination to systemically generate and measure the fitness of large numbers of double mutants in a colony array format.
Briefly, a robot is used to transfer, through conjugation, chloramphenicol (Cm) - marked mutant alleles from engineered Hfr (High frequency of recombination) 'donor strains' into an ordered array of kanamycin (Kan) - marked F- recipient strains. Typically, we use loss-of-function single mutants bearing non-essential gene deletions (e.g.
the 'Keio' collection11
) and essential gene hypomorphic mutations (i.e.
alleles conferring reduced protein expression, stability, or activity9, 12, 13
) to query the functional associations of non-essential and essential genes, respectively. After conjugation and ensuing genetic exchange mediated by homologous recombination, the resulting double mutants are selected on solid medium containing both antibiotics. After outgrowth, the plates are digitally imaged and colony sizes are quantitatively scored using an in-house automated image processing system14
. GIs are revealed when the growth rate of a double mutant is either significantly better or worse than expected9
. Aggravating (or negative) GIs often result between loss-of-function mutations in pairs of genes from compensatory pathways that impinge on the same essential process2
. Here, the loss of a single gene is buffered, such that either single mutant is viable. However, the loss of both pathways is deleterious and results in synthetic lethality or sickness (i.e.
slow growth). Conversely, alleviating (or positive) interactions can occur between genes in the same pathway or protein complex2
as the deletion of either gene alone is often sufficient to perturb the normal function of the pathway or complex such that additional perturbations do not reduce activity, and hence growth, further. Overall, systematically identifying and analyzing GI networks can provide unbiased, global maps of the functional relationships between large numbers of genes, from which pathway-level information missed by other approaches can be inferred9
Genetics, Issue 69, Molecular Biology, Medicine, Biochemistry, Microbiology, Aggravating, alleviating, conjugation, double mutant, Escherichia coli, genetic interaction, Gram-negative bacteria, homologous recombination, network, synthetic lethality or sickness, suppression
Recording Electrical Activity from Identified Neurons in the Intact Brain of Transgenic Fish
Institutions: University of California, Los Angeles .
Understanding the cell physiology of neural circuits that regulate complex behaviors is greatly enhanced by using model systems in which this work can be performed in an intact brain preparation where the neural circuitry of the CNS remains intact. We use transgenic fish in which gonadotropin-releasing hormone (GnRH) neurons are genetically tagged with green fluorescent protein for identification in the intact brain. Fish have multiple populations of GnRH neurons, and their functions are dependent on their location in the brain and the GnRH gene that they express1
. We have focused our demonstration on GnRH3 neurons located in the terminal nerves (TN) associated with the olfactory bulbs using the intact brain of transgenic medaka fish (Figure 1B
). Studies suggest that medaka TN-GnRH3 neurons are neuromodulatory, acting as a transmitter of information from the external environment to the central nervous system; they do not play a direct role in regulating pituitary-gonadal functions, as do the well-known hypothalamic GnRH1 neurons2, 3
.The tonic pattern of spontaneous action potential firing of TN-GnRH3 neurons is an intrinsic property4-6
, the frequency of which is modulated by visual cues from conspecifics2
and the neuropeptide kisspeptin 15
. In this video, we use a stable line of transgenic medaka in which TN-GnRH3 neurons express a transgene containing the promoter region of Gnrh3
linked to enhanced green fluorescent protein7
to show you how to identify neurons and monitor their electrical activity in the whole brain preparation6
Neuroscience, Issue 74, Neurobiology, Cellular Biology, Molecular Biology, Anatomy, Physiology, Neuroendocrinology, Neurophysiology, Electrophysiology, Comparative, action potential, gonadotropin-releasing hormone, neuron, brain, teleost, animal model
A Noninvasive Method For In situ Determination of Mating Success in Female American Lobsters (Homarus americanus)
Institutions: University of New Hampshire, Massachusetts Division of Marine Fisheries, Boston University, Middle College.
Despite being one of the most productive fisheries in the Northwest Atlantic, much remains unknown about the natural reproductive dynamics of American lobsters. Recent work in exploited crustacean populations (crabs and lobsters) suggests that there are circumstances where mature females are unable to achieve their full reproductive potential due to sperm limitation. To examine this possibility in different regions of the American lobster fishery, a reliable and noninvasive method was developed for sampling large numbers of female lobsters at sea. This method involves inserting a blunt-tipped needle into the female's seminal receptacle to determine the presence or absence of a sperm plug and to withdraw a sample that can be examined for the presence of sperm. A series of control studies were conducted at the dock and in the laboratory to test the reliability of this technique. These efforts entailed sampling 294 female lobsters to confirm that the presence of a sperm plug was a reliable indicator of sperm within the receptacle and thus, mating. This paper details the methodology and the results obtained from a subset of the total females sampled. Of the 230 female lobsters sampled from George's Bank and Cape Ann, MA (size range = 71-145 mm in carapace length), 90.3% were positive for sperm. Potential explanations for the absence of sperm in some females include: immaturity (lack of physiological maturity), breakdown of the sperm plug after being used to fertilize a clutch of eggs, and lack of mating activity. The surveys indicate that this technique for examining the mating success of female lobsters is a reliable proxy that can be used in the field to document reproductive activity in natural populations.
Environmental Sciences, Issue 84, sperm limitation, spermatophore, lobster fishery, sex ratios, sperm receptacle, mating, American lobster, Homarus americanus
Genetic Manipulation in Δku80 Strains for Functional Genomic Analysis of Toxoplasma gondii
Institutions: The Geisel School of Medicine at Dartmouth.
Targeted genetic manipulation using homologous recombination is the method of choice for functional genomic analysis to obtain a detailed view of gene function and phenotype(s). The development of mutant strains with targeted gene deletions, targeted mutations, complemented gene function, and/or tagged genes provides powerful strategies to address gene function, particularly if these genetic manipulations can be efficiently targeted to the gene locus of interest using integration mediated by double cross over homologous recombination.
Due to very high rates of nonhomologous recombination, functional genomic analysis of Toxoplasma gondii
has been previously limited by the absence of efficient methods for targeting gene deletions and gene replacements to specific genetic loci. Recently, we abolished the major pathway of nonhomologous recombination in type I and type II strains of T. gondii
by deleting the gene encoding the KU80 protein1,2
. The Δku80
strains behave normally during tachyzoite (acute) and bradyzoite (chronic) stages in vitro
and in vivo
and exhibit essentially a 100% frequency of homologous recombination. The Δku80
strains make functional genomic studies feasible on the single gene as well as on the genome scale1-4
Here, we report methods for using type I and type II Δku80Δhxgprt
strains to advance gene targeting approaches in T. gondii
. We outline efficient methods for generating gene deletions, gene replacements, and tagged genes by targeted insertion or deletion of the hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT
) selectable marker. The described gene targeting protocol can be used in a variety of ways in Δku80
strains to advance functional analysis of the parasite genome and to develop single strains that carry multiple targeted genetic manipulations. The application of this genetic method and subsequent phenotypic assays will reveal fundamental and unique aspects of the biology of T. gondii
and related significant human pathogens that cause malaria (Plasmodium
sp.) and cryptosporidiosis (Cryptosporidium
Infectious Diseases, Issue 77, Genetics, Microbiology, Infection, Medicine, Immunology, Molecular Biology, Cellular Biology, Biomedical Engineering, Bioengineering, Genomics, Parasitology, Pathology, Apicomplexa, Coccidia, Toxoplasma, Genetic Techniques, Gene Targeting, Eukaryota, Toxoplasma gondii, genetic manipulation, gene targeting, gene deletion, gene replacement, gene tagging, homologous recombination, DNA, sequencing
Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish
Like many aquatic animals, zebrafish (Danio rerio
) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The presented automatic video tracking system accomplishes this by using a mirror system and a calibration procedure that corrects for the considerable error introduced by the transition of light from water to air. With this system it is possible to record both single and groups of adult zebrafish. Before use, the system has to be calibrated. The system consists of three modules: Recording, Path Reconstruction, and Data Processing. The step-by-step protocols for calibration and using the three modules are presented. Depending on the experimental setup, the system can be used for testing neophobia, white aversion, social cohesion, motor impairments, novel object exploration etc
. It is especially promising as a first-step tool to study the effects of drugs or mutations on basic behavioral patterns. The system provides information about vertical and horizontal distribution of the zebrafish, about the xyz-components of kinematic parameters (such as locomotion, velocity, acceleration, and turning angle) and it provides the data necessary to calculate parameters for social cohesions when testing shoals.
Behavior, Issue 82, neuroscience, Zebrafish, Danio rerio, anxiety, Shoaling, Pharmacology, 3D-tracking, MK801
Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer
Institutions: Joint Unit Hospices de Lyon-bioMérieux, BioMérieux, Hospices Civils de Lyon, Lyon 1 University, BioMérieux, Hospices Civils de Lyon, Hospices Civils de Lyon.
The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1
. ‘How to use PSA' remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2
or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4
. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g.
PCA3 in prostate cancer5,6
and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10
. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1
Medicine, Issue 81, Cancer Biology, Genetics, Molecular Biology, Prostate, Retroviridae, Biomarkers, Pharmacological, Tumor Markers, Biological, Prostatectomy, Microarray Analysis, Gene Expression, Diagnosis, Human Endogenous Retroviruses, HERV, microarray, Transcriptome, prostate cancer, Affymetrix
Quantitative Comparison of cis-Regulatory Element (CRE) Activities in Transgenic Drosophila melanogaster
Institutions: University of Dayton, University of Dayton.
Gene expression patterns are specified by cis
-regulatory element (CRE) sequences, which are also called enhancers or cis-regulatory modules. A typical CRE possesses an arrangement of binding sites for several transcription factor proteins that confer a regulatory logic specifying when, where, and at what level the regulated gene(s) is expressed. The full set of CREs within an animal genome encodes the organism′s program for development1
, and empirical as well as theoretical studies indicate that mutations in CREs played a prominent role in morphological evolution2-4
. Moreover, human genome wide association studies indicate that genetic variation in CREs contribute substantially to phenotypic variation5,6
. Thus, understanding regulatory logic and how mutations affect such logic is a central goal of genetics.
Reporter transgenes provide a powerful method to study the in vivo
function of CREs. Here a known or suspected CRE sequence is coupled to heterologous promoter and coding sequences for a reporter gene encoding an easily observable protein product. When a reporter transgene is inserted into a host organism, the CRE′s activity becomes visible in the form of the encoded reporter protein. P-element mediated transgenesis in the fruit fly species Drosophila (D.) melanogaster7
has been used for decades to introduce reporter transgenes into this model organism, though the genomic placement of transgenes is random. Hence, reporter gene activity is strongly influenced by the local chromatin and gene environment, limiting CRE comparisons to being qualitative. In recent years, the phiC31 based integration system was adapted for use in D. melanogaster
to insert transgenes into specific genome landing sites8-10
. This capability has made the quantitative measurement of gene and, relevant here, CRE activity11-13
feasible. The production of transgenic fruit flies can be outsourced, including phiC31-based integration, eliminating the need to purchase expensive equipment and/or have proficiency at specialized transgene injection protocols.
Here, we present a general protocol to quantitatively evaluate a CRE′s activity, and show how this approach can be used to measure the effects of an introduced mutation on a CRE′s activity and to compare the activities of orthologous CREs. Although the examples given are for a CRE active during fruit fly metamorphosis, the approach can be applied to other developmental stages, fruit fly species, or model organisms. Ultimately, a more widespread use of this approach to study CREs should advance an understanding of regulatory logic and how logic can vary and evolve.
Developmental Biology, Issue 58, Cis-regulatory element, CRE, cis-regulatory module, enhancer, site-specific integration, reporter transgenes, confocal microscopy, regulatory logic, transcription factors, binding sites, Drosophila melanogaster, Drosophila
Transplantation of Whole Kidney Marrow in Adult Zebrafish
Institutions: Harvard Medical School.
Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (<3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.
Developmental Biology, Issue 2, zebrafish, HSC, stem cells, transplant

domain: biology


text:
Cursed to never speak again, Kaede Oshiro hasn’t uttered a word in decades. And then a curvy witch storms into his life and makes him moan her name.
Banished to the Frostlands by his jealous cousin and his cousin’s wife, Kaede Oshiro has spent years fighting to stay alive on a frozen tundra that’s filled with creatures straight out of nightmares. Battling day after day, killing night after night has turned him into a cold-hearted shifter. His hope of returning to the mortal realm and reclaiming his title as pack alpha seems impossible. Then one day, an opportunity for him to regain his freedom arises. All he has to do is kidnap a witch and bring her to The Deciders. He thought finding the witch, Sinclair Rose, and turning her over to The Deciders would be a simple task. She proved him wrong. The moment he touched her, he experienced pain unlike anything he’d felt before.
When he finally came to, he realized six things:
1. The captor had become the captive.
2. The cottage he was being held captive in was filled with Christmas decorations.
3. The witch holding him hostage was the most beautiful creature he’d ever seen.
4. When her hands were on him, he was able to speak.
5. Her presence was awaking the dormant dragon inside him.
6. He must protect this woman at all costs.
However, if he chooses protecting Sinclair over completing the task assigned to him by The Deciders, he’s putting both their lives in danger. Obey The Deciders or protect a voluptuous witch who makes his dragon roar? There’s only one answer both he and his inner animal can agree on.
Protect the witch. And death to any who try to harm her.
*A peek into the book.*
“I’m ready,” she whispered.
He nodded, but didn’t move.
“I’m not a small dragon.” He rubbed one finger between her wet folds. “Two of my fingers barely fit. What if I don’t fit?”
She chuckled. He looked to her face.
“I was made for you,” she told him. “You’ll fit.”
He swallowed. “I don’t ever want to hurt you.”
“Sometimes a little pain isn’t a bad thing. I want to feel you inside me, Kaede. Don’t make me wait any longer.”
He didn’t want to wait any longer. He lowered himself to her body.
“You’re not heavy,” she said, before he could ask.
It was like she was reading his mind.
“I’m a thick girl,” she told him. “I can handle it.
BK 1: https://amzn.to/2CPWTrp
BK 2: https://amzn.to/2OCYjbD
BK 3 https://amzn.to/2DwtBit
Book 4 coming 07/31/2021.


text:
London-based housewares retailer Elmageta opened a third outlet last week.
The 3,500sq ft store is in Whetstone, North London, and joins other branches in Swiss Cottage and East Finchley.
“We do virtually everything in the way of homewares and kitchenwares,” consultant Jeffrey Goodman told HousewaresLive.net. “We’re very comprehensive.”
He added that the new branch would be developed as a destination store.
Key suppliers include Brabantia, Meyer, Kitchen Craft, Premier Housewares, Grunwerg, Rayware, Le Creuset, JWP, Kuhn Rikon, Arc International and EFG Housewares. “And we do a lot of buying through BHF and Home Hardware,” said Goodman.
The manager of the Whetstone store is Anita Mazzotta, who for many years was manager of The Scullery in Muswell Hill.
Goodman said that there were no plans yet to grow the chain further: “We’re happy with three, we’re not racing away.”
But he added: “This is the best time to open a new shop, when it hopefully only gets better!”


text:
I might heard so many complaints about this art piece and others like it that I used to be completely skeptical about buying it. Nonetheless, I am glad I took the chance. Any sort of image or any subject may be transformed into a unique canvas print. Too ceaselessly in life ‘professional’ means ‘extortionately priced’, but putting your photographs on canvas does not necessarily need to interrupt the financial institution. Listed here are some simple suggestions and decor ideas to create that nice beach and tropical really feel in your house.
Store framed and canvas wall art prints or body photographs and your favorite family pictures. Secure Online Ordering: With our online system, it is very secure and easy to upload your pictures then select all the options you need in your canvas. This is one of the best ways to keep the prices low; nonetheless you will additionally get all you want from an artwork supply store.
This is the so-known as Gallery Wrap” (in style in Europe) by which a portion of the picture is definitely printed over the edges of the canvas to create a more 3-d appearance. Simply look around a bit and you’ll absolutely find just a few first rate printers in your vicinity providing you canvas printing providers.
Inks are not simply inks – The Canvas Art Manufacturing facility makes use of HP latex inks – fade resistant for as much as eighty years out of direct daylight and completely scratch resistant not to mention non-toxic and bio-degradable! Unleash the potential locked away in your pictures for cheap images on canvas with a distinction.
You too can select canvas prints on paper that you may also frame to make them look extra presentable. Picture printing on canvas has change into more and more standard as a result of it’s inexpensive and in the same means straightforward to pick out a picture that you just wish to convert to a canvas picture.