Self-Irrigating Desert Plant Discovered



A desert plant has apparently figured out how to water itself.

Ecologists had been puzzling over the desert rhubarb for years: Instead of the tiny, spiky leaves found on most desert plants, this rare rhubarb boasts lush green leaves up to a meter wide.

Now scientists from the University of Haifa-Oranim in Israel have discovered that ridges in the plant’s giant leaves actually collect water and channel it down to the plant’s root system, harvesting up to 16 times more water than any other plant in the region.

“It is the first example of a self-irrigating plant,” said plant biologist Gidi Ne’eman, a co-author on the paper published in March in Naturwissenschaften, a German journal of ecology. “This is the only case we know, but in other places in the world there might be additional plants that use the same adaptions.”

The desert rhubarb grows in the mountainous deserts of Israel and Jordan, where there’s only about 75mm of rainfall each year. Even during the rainy season, the region’s light rainfalls often don’t penetrate the rocky soil of the desert. Plants with large leaves and a deep root system, like the desert rhubarb, typically can’t survive in such an arid climate.

But when the researchers measured the plant’s water absorption during a light rain, they discovered that water infiltrated the soil 10 times deeper around the desert rhubarb than in surrounding areas. Upon closer examination, scientists discovered deep grooves around the plant’s veins, which are coated in a waxy cuticle that helps channel water down to the root.

“Even in the slightest rains,” the researchers wrote, “the typical plant harvests more than 4,300 cubic centimeters of water per year and enjoys a water regime of about 427 millimeters per year, equivalent to the water supply in a Mediterranean climate.”

Some scientists say the desert rhubarb isn’t all that, however. “Many plants channel water to their base to be absorbed by the root,” Lindy Brigham, a plant ecologist from the University of Arizona, wrote in an email. “Just look at the way plant leaves are shaped and how they branch from the base in many cases.” The architecture of the desert rhubarb’s leaves is unusual, she said, but not necessarily the only example of this adaptation.

Square Watermelons! It's true.



It's not a fad. The technique actually has practical applications. "The reason they're doing this in Japan is because of lack of space," said Samantha Winters of the National Watermelon Promotion Board in Orlando, Florida.

A fat, round watermelon can take up a lot of room in a refrigerator, and the usually round fruit often sits awkwardly on refrigerator shelves. But clever Japanese farmers have solved this dilemma by forcing their watermelons to grow into a square shape.

Farmers insert the melons into square, tempered glass cases while the fruit is still growing on the vine.

The square boxes are the exact dimensions of Japanese refrigerators, allowing full-grown watermelons to fit conveniently and precisely onto refrigerator shelves.

But cubic fruit comes with a price: Each square watermelon costs 10,000 yen, the equivalent of about $82. Regular watermelons in Japan cost from $15 to $25 each.

Japanese farmers have perfected the art of growing square watermelons, but they aren’t about to reveal their secret process. When a square watermelon sells for $82 who can blame them.

Buddha shaped Pears

Gao Xianzhang has managed to create what some would call the holiest fruits ever, pears shaped like


Gao has been working on his pear-growing technique for six years and this season he managed to grow 10,000 Buddha-shaped baby pears. Each fruit is grown in an intricate Buddha mould and ends up looking like a juicy figurine. The ingenious farmer says the locals in his home village of Hexia, norther China, have been buying his Buddha pears as soon as he picks them from the trees. Most of them think they are cute and that they bring good luck.

Gao Xianzhang pears aren’t cheap, roughly $1.8 each, but their success in China convinced him to start exporting them into Europe.

Lifeline Hospitals & Loyola College joint PG Diplomo course on "Stem Cell Therapeutics"

Loyola College (LIVE), Chennai & Lifeline Institute of Regenerative Medicine (LIRM), Perungudi, Chennai jointly invites applications for the following course.


Course:
P.G. Diploma in “Stem cell therapeutics”.


Duration:
01 year program
03 hours of class per day on weekdays.


Eligibility:
BTech / Msc Life Sciences/ MBBS/ BDS/ BVSc and final year candidates also may apply.


Course Commencement:
17 August 2009


Application deadline:
10 August 2009


To apply:
Contact: 09884407195/ 09840940283

Email: stemcell.lirm@gmail.com / lirm@lifelinehospitals.com

Lifeline Institute of Regenerative Medicine also offers:

1. Special trainings and projects in stem cell research to all branches of life science students through out the year.
2. Special training on the advanced flowcytometric techniques (FACS Aria).

Loyola College (LIVE) also offers:
1) Summer training / project works on Bioinformatics and Medical Transcriptions to all branches of life science students through out the year.

TURTLES MAKE SENSE AFTER AL

Evolutionary development study describes a critical fold that sends the reptile off on its own By Susan Milius
Turtles may be weird, but according to new research, they’re not that weird. Their funny arrangement of shell and shoulder is just the same old land-dweller vertebrate stuff — with a little fold.

At first a turtle embryo grows much like a chicken or mouse. But then the developing body wall makes a critical fold, and the usual body plan starts to become an unusual turtle, Hiroshi Nagashima of Kobe University and his colleagues report in the July 10 Science.

Nothing else has a body plan like a turtle. Its ribs don’t grow inside its chest as a cage but instead fuse in the developing skin layer on its back to create one bony armored covering.

“It is not just that turtles 'grew a shell,'” says paleontologist Ben Kear of La Trobe University in Melbourne, Australia. In the evolution of that shell, bones and muscles had to shift around relative to other reptiles, birds and mammals, and turtle shoulders ended up inside the rib cage. “In essence this means that the turtle skeleton is inside-out,” he says.

Odd as they are, turtles clearly belong to the lineage of amniotes, which includes mammals, birds and reptiles. Turtles, which are at least 200 million years old, “have survived all kinds of stuff — we’re talking extinction of the dinosaurs and myriad climate changes,” Kear says. Yet there’s scant fossil evidence of turtles in the making to explain how their forms arose as they split off from birds and crocodiles.

Knowing how a basic amniote embryo ends up developing into something so radically different could shed light on turtle history, says paleontologist Michael Lee of the South Australian Museum in Adelaide. “Some intermediate stages in this process might resemble real intermediate — fossil — stages in evolution,” he says.

To sort out how turtles develop, Nagashima and his colleagues worked with eggs of Chinese soft-shelled turtles (Pelodiscus sinensis) bought from a farm. The researchers used tissue-specific stains as well as substances that detect activity of particular genes to figure out which bits of the tiny embryos were on their way to becoming the bones and muscles of the adult. At each stage in development, the researchers compared their embryos with developing chickens and mice at comparable stages.

Any features shared by all three embryos probably came from distant common ancestors of all amniotes, including people, the researchers note.

In turtles, chickens and mice, the earliest stages of development looked much the same, the researchers reported. Then the turtle embryos veered off on their own path. The developing muscle tissue that would lie along adult ribs in a standard amniote began to fold underneath itself in the turtle. This tissue tucked inward, bending up to lie below the developing ribs. On this kinked-under section, the shoulder blades, or scapulas, formed.

If this fold could be straightened out, the scapulas would lie outside the rib cage, as they do in chickens, mice and people. For turtles then, “the position of the scapula is not a novelty,” Nagashima says. Essentially, “turtles have the same body plan as other amniotes.”

That critical fold in the tissue maps out the line that becomes an important embryological feature of turtle embryos called the carapacial ridge. Earlier research has shown that this ridge drives the development of the bony back of the animal. The fold also allows developing muscles to form connections in ways that they don’t in the mouse and the chicken.

The researchers also noted that the turtle ribs stop short in comparison with mice and chickens. Turtle ribs grow out only along the sides of what will become the backbone instead of curving into the body wall to form the whole rib cage. Those short turtle ribs mingle with the skin tissue creating the fused bony shell on the turtle’s back.

“Very, very sophisticated work,” says reptile paleontologist Olivier Rieppel of the Field Museum in Chicago in describing the extensive detective work required to trace all the tissues and muscles.

He has studied the oldest known fossil of an ancestral turtle, and he says the new interpretation of turtle embryology may fit well with the fossil record. Last year he and colleagues described Odontochelys semitestacea from a fossil collected in 220-million-year-old marine sediments in southwestern China. The turtle had a standard armored underside but not a full shell on its back. Its ribs widened, but its shoulder blades still lay forward of instead of inside the ribs.

Nagashima speculates that the embryonic fold was evolving a bit at a time and maybe hadn’t reached as far around the body in this ancestral turtle as it does today. Clever suggestion, Rieppel says.

To understand turtle history, paleontologists really need more fossils, says Robert Reisz of the University of Toronto’s Mississauga campus in Canada. In the meantime, the new Japanese paper “clarifies a unique evolutionary event, one that gave rise to a really neat group of animals, our beloved turtles.”

Calorie-Counting Monkeys Live Longer


Rodents, yeast, and roundworms all have something in common: They live longer when they consume less. Now a primate has joined the calorie-restriction club. After 20 long years of waiting, scientists have concluded that rhesus monkeys that eat nearly a third less food than normal monkeys age more slowly. The results come as close as any can to proving that calorie restriction could significantly slow aging in humans--even if such a lean diet would not appeal to most of us.
Researchers first discovered the connection between lean diets and extended life spans in a 1935 study of calorie-restricted rats. In the past decade, studies in yeast and worms have pinpointed some genes that may be responsible. Scientists believe the genes somehow ramp up systems to protect an organism from environmental stress and may have evolved to help organisms survive in environments where food was scarce. In rodent studies, calorie restriction can extend life span by 20% to 80%. Whether calorie restriction also slows aging in primates wasn't known, however.

Two decades ago, three different research groups in the United States decided to fill this gap. The groups have previously published updates on their monkeys' health, but in tomorrow's issue of Science, one of them reports survival data from their colony of 76 rhesus monkeys. The team, led by gerontologist Richard Weindruch of the University of Wisconsin, Madison, began monitoring the animals when they hit 7 to 14 years old--monkey adulthood. Researchers allowed half of the monkeys to eat as much as they wanted during the day, while restricting the other half to a diet with 30% fewer calories. The scientists gave the restricted monkeys vitamin and mineral supplements to ensure they did not suffer malnutrition and treated any animals that fell sick, says Weindruch.

Studying aging in monkeys takes patience. Mice and rats only live for a couple of years, while these monkeys can live to 40, and the average life span is 27 years. Now that the surviving monkeys have reached their mid- to late 20s, the Wisconsin group could glean how calorie restriction was affecting their life span. Sixty-three percent of the calorie-restricted animals are still alive compared to only 45% of their free-feeding counterparts. For age-related deaths caused by illnesses such as cardiovascular disease and cancer, the voracious eaters died at three times the rate of restricted monkeys: 14 versus five monkeys, respectively. Another seven control and nine lean monkeys died from causes not related to aging such as complications from anesthesia or injuries. Leaner diets also reduced muscle and brain gray matter deterioration, two conditions associated with aging. (The team has not yet studied cognitive differences between the two groups.)

Researchers who study aging are split on how much stock to put in the study. Leonard Guarente, a molecular biologist at the Massachusetts Institute of Technology in Cambridge who has studied aging in yeast, believes that not enough monkeys have died yet to make definitive comparisons between the two groups. As of March, when Weindruch's group submitted the paper, about half of the colony was still alive. "The gap [in survival rates] may separate more, but it's still too early to tell," Guarente says. On the other hand, molecular biologist Matthew Kaeberlein of the University of Washington, Seattle, thinks the gap as it stands now is still compelling. He points to the difference in age-related deaths between the two groups as the more relevant statistic. "The fact that they see a significant effect at this point suggests there will be a robust effect when they finish the study," he says.

Weindruch and his collaborators plan to continue monitoring the remaining monkeys, which could stretch the study's length past 3 decades. "If we reach the 40-year-old life span, the study could continue for another 15 years," Weindruch said. "That would probably round out my career."

IISc to extract oil from Diatoms, algae


Driving will soon be a pollution-friendly activity if a small team of scientists from India and Canada have their way. Scientists at the Indian Institute of Science (IISc) have collaborated with their counterparts in Canada to ensure that global warming becomes a thing of the past.

According to the scientists, the answer to a clean and sustainable energy production lies in the microscopic algae — diatoms.

Some geologists believe that a majority of the world’s crude oil originated from diatoms. “Diatoms are the lowest in the order of the food chain, but are known to have oil glands that can yield an effective amount of oil. They also act as carbon sequesters trapping in carbon and releasing oxygen. We hope that this could work as a replacement for conventional energy or gasoline paving the way for a clean fuel that can effectively work as a solution to tackle global warming,” said Dr T.V. Ramachandra at IISc.

The research, that will soon be published in an international journal, indicates that a solution to the impending crude oil scarcity exists. It offers solutions for a cost-effective renewable source of alternative energy and also helps stop the emission of carbon dioxide into the atmosphere to an extent. Diatoms can trap and store carbon, sending out emissions free of any pollutants.

The team that comprises IISc professors Durga Madhab Mahapatra, Karthick B. and Dr Ramachandra and Richard Gordon from the University of Manitoba in Canada have also proposed a new approach to sustainable energy that uses solar panels by incorporating altered diatoms that secrete oil products.

IBAB- Job assured Biotechnology / Bioinformatics PG diplomo courses admissions- 2009

Institute of Bioinformatics and Applied Biotechnology (IBAB) (Government of Karnataka), Bangalore,invites applications for the following job oriented courses which almost has 100% placement record.


Courses:
Postgraduate Diploma in Bioinformatics
Postgraduate Diploma in Biotechniques
Postgraduate Diploma in Cheminformatics


Eligibility:
B.Sc /B.Tech /M.Sc /M.Tech and final year students awaiting final results.


Selection method:
Written test and Interview


Application deadline:
12 July 2009
Date of entrance test:
19 July 2009

Need Hydrogen Storage? Think Poultry

By Phil Berardelli
ScienceNOW Daily News
23 June 2009

Here's a case for which solving an energy problem could ease a challenging environmental problem as well. Researchers have discovered that carbonized chicken feathers could provide an inexpensive, environmentally friendly way to store hydrogen fuel for future motor vehicles. If the concept is proven--and perhaps a bigger if, accepted by the automobile industry--it could go a long way toward helping to dispose of the 2.7 billion kilograms of chicken feathers generated each year by commercial poultry operations.
Hydrogen is a leading alternative fuel for vehicles. The byproducts of its combustion are nonpolluting, and its source--water--is superabundant. One hitch is the amount of energy required to manufacture it, and another is storing enough of it onboard to give vehicles a cruising range that approaches that of gasoline or diesel fuel. Hydrogen has proven notoriously difficult to store in sufficient quantities without placing it under enormous pressure, something that greatly adds to the weight of a vehicle and adds a serious explosion hazard. The best idea so far has been carbon nanotubes--microscopic structures that can pack away large quantities of hydrogen at normal pressure within a relatively small space. But a storage tank made of the nanotubes would cost millions of dollars.

Now a team at the University of Delaware, Newark, says it has an unlikely candidate: chicken feathers. It turns out that the feathers, which are made of keratin--the same protein in fingernails and beaks--comprise strong, hollow tubes. The team, led by chemical engineer Richard Wool, had been investigating the feathers' potential for improving the performance of electronic microcircuits. The air inside the tubes helps to speed electrons along the printed wiring, but the feathers weren't stiff enough to hold the circuit boards together very well. So the team tried a heating technique to strengthen the bonds between the carbon atoms in the keratin.

As the team reported today at the 13th Annual Green Chemistry and Engineering Conference in College Park, Maryland, carbonizing the feathers gave them a strength approaching that of the nanotubes. They could also store up to 1.7% of their weight as hydrogen, about as much as carbon nanotubes could store. Moreover, the feathers cost virtually nothing to produce. "They're a nuisance commodity," says Wool.

The researchers estimate that a hydrogen-storage tank using the carbonized feathers would cost only about $200 when mass-produced. It's a major step forward, but the U.S. Department of Energy has set a target capacity for hydrogen-storage techniques of 6% of weight, so the carbonized feathers need improvement. Still, Wool is confident that the goal can be achieved. "There are all kinds of next steps," he says.

Even if hydrogen doesn't become the next primary transportation fuel, finding a safer and economical way to store the gas would still be of great value, says chemical engineer John Dorgan of the Colorado School of Mines in Golden. Hydrogen has several important nontransportation uses, he explains, such as a cooling medium in electricity generation. So the innovative storage technique developed by Wool and his team could be much less hazardous than pressurized tanks. In addition, he says, "it simply makes sense to use renewable materials to build the renewable energy infrastructure."

Soybeans Grow Where Nuclear Waste Glows


Soy crops are so tough they can flourish in the contaminated soil around Chernobyl and produce healthy offspring.

If scientists can understand how plants survive in ultra-hostile environments, it will help them engineer super hearty plants to withstand drought conditions or grow on marginal cropland.

“The fact that plants were able to adapt to the area of the world’s largest nuclear accident, is very encouraging,” says Martin Hajduch, a plant biotechnology expert at the Slovak Academy of Sciences and coauthor of the study in the Journal of Proteome Research. “So we were interested to know how plants can do such a job.”

Hajduch’s team built and harvested seeds from a garden near the village of Chistogalovka, which is roughly five kilometers from the ruined nuclear power plant. They analyzed the seeds with all sorts of modern proteomics tricks, going a step beyond the narrowly-focused studies that other scientists have done.

Biologists have been studying the effects of radiation on plants for decades, and they have identified a handful of proteins that seem to protect crops from genetic damage, but this is the first time that anyone has taken a snapshot of everything that’s going on inside of Chernobyl-grown vegetables.

The Slovak scientists started by freezing each seed with liquid nitrogen and crushing it to extract a mix of proteins. Then they sorted those molecules in an electrified block of gel, and identified each one with a mass spectrometer. As a reference, they did the same thing to seeds from a garden 100 kilometers from the disaster area.

Hajduch learned that the contaminated plants make a lot of changes to defend themselves, adjusting the levels of dozens of proteins that also guard against disease, heavy metals, and salt. All of that makes sense, but the biggest difference between plants from the wasteland and the controls was somewhat surprising. The levels of hundreds of proteins that are known for their ability to shuttle other proteins around — or lock them up in storage — had been lowered.

As a result of those adjustments, the levels of Cesium-137 in the beans was remarkably low. The plants are healthy and fertile, but definitely not safe to eat.

TEST MIGHT ASCERTAIN WHO NEEDS APPENDECTOMY

Biomarker in urine could minimize unnecessary surgery By Nathan Seppa

A compound identifiable in urine might help doctors distinguish appendicitis from other abdominal problems and avoid needless surgery, researchers report online June 23 in the Annals of Emergency Medicine.

Because signs of appendicitis are particularly difficult to assess in young children and elderly adults, surgeons unnecessarily remove a healthy appendix in 10 to 20 percent of appendectomies performed in the United States, says pediatrician Alex Kentsis of Harvard Medical School and Children’s Hospital Boston.

True appendicitis, on the other hand, often goes untreated because it may cause few symptoms until the appendix ruptures. At that point, a patient risks intestinal infection and severe complications, Kentsis says.

In an effort to find biomarkers that tip off appendicitis better, Kentsis teamed with biochemist Hanno Steen and physician Richard Bachur, both also at Children’s Hospital, to test for 57 compounds in the urine of 67 children being treated for suspected appendicitis. The children had an average age of 11.

Overall, 25 of these patients were found to have appendicitis and underwent surgery. The diagnoses resulted from physical examination, symptom assessment and tests such as CT scans, ultrasounds or other measures. Tissue analysis after surgery confirmed the original diagnoses.

In conducting the urine sample analysis, the researchers didn’t know which children were ultimately diagnosed with appendicitis and which had other diagnoses. These included ovarian cysts, constipation, abdominal pain or other problems that were ascertained by follow-up phone calls six to eight weeks later.

The compound that stood out among the children with appendicitis was leucine-rich alpha-2-glycoprotein, or LRG. Immune cells called neutrophils make LRG. “Release of LRG from neutrophils is a kind of specific feature of appendicitis,” Kentsis says.

LRG is not the only compound overproduced during an attack of appendicitis. But in this analysis, it was the most reliable biomarker to show up in the urine. High levels of LRG in the urine correctly identified a child who had appendicitis and low LRG levels suggested no appendicitis 97 percent of the time, the researchers found.

“They may have found a biomarker that’s really sensitive,” says Robert C. Barber, a geneticist at the University of Texas Southwestern Medical Center at Dallas. “This is a very interesting finding.” Nevertheless, he cautions, “appendicitis is unlikely to have a magic bullet biomarker.” More likely, researchers will eventually need more than one.

Kentsis agrees, noting that other teams have already found some promising biomarkers. “You could imagine using them in combination, if one isn’t sufficient,” he says.

The team will now concentrate on validating the new findings and creating a simpler, clinic-ready kit for testing urine, Kentsis says. Meanwhile, the researchers plan to look at whether the LRG test might also work in adults.

The use of CT scans and ultrasound has improved appendicitis diagnosis in recent years, but these tests still fail to catch some inflamed appendices and wrongly pinpoint healthy ones, the authors note. Also, in some regions of the world, Kentsis says, such high-tech diagnostics just aren’t readily available.

Gene Protects Alcoholism


In an interesting finding, a study revealed that a gene variant detected among a tribe in Orissa has been protecting them from harmful effects of alcohol.

The study conducted by the department of anthropology at Utkal University here has showed that the Bondas — one the most primitive tribes of Orissa- are immune to the side effects of alcoholism.

Alcohol is an agent of cirrhosis of liver, toxic psychosis, gastritis, pancreatitis, cardiac myopathy and so on. But surprisingly none of these diseases are seen among the Bonda highlanders, who are addicted to different kinds alcoholic beverages.

The reason: presence of a gene variant ALDH2.

Jayant Kumar Nayak, a research scholar of Anthropological Survey of India, in association of with the Utkal University has conducted a study on Bondas to know whether they are genetically protected from alcoholism. On a proportionate random sampling, out of 25 villages, he selected nine for the study covering 714 households of 2,700 population. Genomic DNA was extracted from 110 unrelated adult Bondas by the ASI following ethical guidelines after taking their consents. Both ADH and ADLH2 genes, considered protecting variants for alcohol, were detected.

Curry leaves Fights Tooth Decay


The curry leaf tree (Murraya Koenigii spreng – a green leafy vegetable) is grown all over India and other countries for its aromatic leaves which are used daily as an ingredient in Indian cuisine.

The fresh curry leaves contain 2.6% volatile essential oils (containing sesquiterpenes and monoterpenes) and the essential oils in the curry leaves are sufficiently soluble in water.

They contain 21000mug total carotene, 7100mug beta carotene, 93.9mug total folic acid, 0.21mg riboflavin, 0.93mg iron, 830mg calcium, 57mg phosphorus and 0.20mg zinc per 100g.

The cold extract of curry leaves (10g of cut fresh curry leaves in 200ml of distilled water) has a pH of 6.3 to 6.4. (unpublished personal observations). Chlorophyll has been proposed as an anticariogenic agent and it also helps to reduce halitosis8.

We have observed that holding curry leaves in the mouth for 5 to 7 minutes is helpful in reducing halitosis and that the terpenes have been found to reduce airborne chemicals and bacteria.

In addition to the presence of EO, the curry leaves contain chlorophyll, beta carotene and folic acid, riboflavin, calcium and zinc and all these can act on the oral tissues and help in keeping up good oral health. Chewing 2 to 4 fresh curry leaves with 10 to 15mls water in the mouth, swishing for 5 to 7 minutes and rinsing the mouth out with water can be of help in keeping good oral hygiene and as the curry leaf is a green leafy vegetable it will be safe and cheap to use as mouthwash. as an ingredient in Indian cuisine.

The fresh curry leaves contain 2.6% volatile essential oils (containing sesquiterpenes and monoterpenes) and the essential oils in the curry leaves are sufficiently soluble in water.

They contain 21000mug total carotene, 7100mug beta carotene, 93.9mug total folic acid, 0.21mg riboflavin, 0.93mg iron, 830mg calcium, 57mg phosphorus and 0.20mg zinc per 100g.

The cold extract of curry leaves (10g of cut fresh curry leaves in 200ml of distilled water) has a pH of 6.3 to 6.4. (unpublished personal observations). Chlorophyll has been proposed as an anticariogenic agent and it also helps to reduce halitosis8.

We have observed that holding curry leaves in the mouth for 5 to 7 minutes is helpful in reducing halitosis and that the terpenes have been found to reduce airborne chemicals and bacteria.

In addition to the presence of EO, the curry leaves contain chlorophyll, beta carotene and folic acid, riboflavin, calcium and zinc and all these can act on the oral tissues and help in keeping up good oral health. Chewing 2 to 4 fresh curry leaves with 10 to 15mls water in the mouth, swishing for 5 to 7 minutes and rinsing the mouth out with water can be of help in keeping good oral hygiene and as the curry leaf is a green leafy vegetable it will be safe and cheap to use as mouthwash.

How to Use a Hemacytometer

Classic Techniques in Cell Biology:
How to use a hemacytometer

by Heather Buschman

French physiologist Louis-Charles Malassez (1842-1909) studied a lot of things in his life. In dentistry, the residual cells of the epithelial root sheath in the periodontal ligament are now called the epithelial rests of Malassez. A genus of fungi is also named for him, which includes species that can cause dandruff and other skin infections.

Malassez also invented the hemacytometer, the thick glass microscope slide traditionally used to count the number of cells in a given volume of liquid. He made an indentation in a regular microscope slide to form a small space that could hold a few drops of cells in suspension. With etched lines and a known area and depth, the number of cells in that particular volume could be calculated. Since the invention was first used to count blood cells, it became known as the hemacytometer (hem = blood, cyto = cells), sometimes also spelled hemocytometer or haemocytometer.

Over the past few years, more than 4,000 people viewed a Scientist Solutions discussion on how to count cells with a hemacytometer. Even for people who learned cell cultures years ago, remembering the calculation can be tricky. Here’s a quick cheat sheet:

1. Transfer a small sample of cell suspension (~100 μl) to a microfuge tube.

2. Dilute 1:2 with Trypan blue or other cell stain.

3. Carefully transfer approximately 10 μl of cells to one of the semi-reflective panels on a hemacytometer covered with a cover slip. Allow the capillary action of the cover slip to gently draw in just enough liquid to fill the chamber liquid. Do not overload.

4. Under the microscope, you should see a grid of 9 squares. Focus the microscope on one of the 4 outer squares in the grid. The square should contain 16 smaller squares.

5. Count the number of live cells in at least 2 of the outer squares. (Dead cells turn dark blue with Trypan staining.)

6. Use the following equation to calculate the number of cells in the original volume:



Some tips for best results:
• Gently pipette the cell suspension up and down to mix well and reduce aggregation before removing a small sample.

• Remove any bubbles in the hemacytometer before counting.

• Count cells touching the top and right borders of each of the 4 squares, but not the cells touching the bottom and left borders.

• If there are less than 100 cells total in all 4 squares, count more squares or start again with a more concentrated cell suspension. If there are too many cells to count accurately, start again by diluting the sample 1:2 or 1:10 before adding Trypan blue.

REPLACING MICRORNA FOR CANCER TREATMENT



Inserting a missing molecule in mice may shrink liver tumors or limit their growth By Jenny Lauren Lee Web edition : Thursday, June 11th, 2009


It’s a simple idea: Treat cancer by finding out what’s absent in a cancer cell and replacing it.

Experiments in mice suggest that inserting one small missing molecule could fight cancer without harming normal tissue, researchers at Johns Hopkins University in Baltimore and the Nationwide Children’s Hospital in Columbus, Ohio, report in a study in the June 12 Cell.

The molecule in this case is a small RNA chain known as a microRNA. MicroRNAs (abbreviated as miRNAs) are involved in a wide range of body processes relating to cellular differentiation and tissue growth. They run about 22 units long, but their size belies their influence — they help regulate hundreds of thousands of genes.

In the past decade, certain flavors of miRNA have been shown to be missing from cancer cells. Scientists have previously explored the idea that replacing the missing miRNAs might reverse the cancer, but not in a way that could lead to viable treatments, said Joshua Mendell of Johns Hopkins, one of the study’s authors.

“One of the things that distinguishes this work is that we used a clinically relevant delivery vehicle to replace a microRNA that’s missing,” Mendell says. The team used a harmless virus that acts as a sort of mail carrier to deliver the miRNA to the cells.

The researchers worked with mice made to develop tumors similar to those in human liver cancer. Most of the untreated mice developed cancers that nearly consumed their livers. But eight out of 10 mice receiving the miRNA had small liver tumors or none at all.

“It’s door-opening research” that will encourage others to move into the field, says George Calin of the University of Texas M.D. Anderson Cancer Center in Houston.

Mendell and colleagues focused on a particular strain of miRNA called miR-26a, chosen because it was absent in liver cancer cells but abundant in normal liver cells. “We thought that the tumor cells might be sensitive to the miRNA” but that the normal parts of the liver would not be harmed, Mendell says. Restoring the miR-26a stopped the cancerous cells from creating two molecules called cyclins that are used in cell replication, effectively preventing the cells from multiplying.

“Although microRNAs have been shown to play an important role in cancer pathogenesis, not too many reports have shown that altering a single microRNA can suppress cancer development in vivo,” comments Baohong Zhang of East Carolina University in Greenville, N.C. “This result has opened a novel strategy for cancer therapy.”

The study has great practical benefit, Calin says, because it shows potential for treating a specific type of cancer — hepatocellular carcinoma, or HCC, which accounts for 80 to 90 percent of all liver cancers.

Calin also says that this research suggests it may be possible to use different miRNAs as therapies for many diseases, not just cancers. MiRNAs have a wide variety of functions, and some have been linked with autoimmune disorders, diabetes and heart disease, he says.

Despite the promising results, Mendell says, there are a lot of hurdles to jump before this type of treatment could be used for people.

For one thing, the liver cancer in the mice is much more aggressive than most human liver cancers. The mouse livers were infested with multiple tumors; humans with HCC typically have only one. And since much is still unknown about how miRNAs work, this type of therapy might have unwanted consequences.

“I think we’re at the earliest stages of understanding,” Mendell says. “There’s more we don’t know than we know.”

TUBERCULOSIS BACTERIUM SUBVERTS BASIC CELL FUNCTIONS

New findings reveal that the microbe achieves virulence by disrupting immune cells' internal processes By Nathan Seppa Web edition : Wednesday, June 10th, 2009

Tuberculosis microbes invading human immune cells carry a cargo that increases TB virulence by inducing the cells to act less like sentinels and more like bystanders, tests in mice show. In a report in the June 11 Nature, a team hypothesizes that this initial infection strategy lays the groundwork for TB’s uncanny ability to lie dormant in an infected person for years.

Even though TB has been studied for hundreds of years, it still guards many secrets — including precisely how it undercuts immune cells.

“Understanding the mechanisms by which the bacteria are having their way with our host cells will be very helpful in coming up with targets that we might hit,” says Kathleen McDonough, a microbiologist at the State University of New York at Albany and the Wadsworth Center of the New York State Department of Health, also in Albany.
Mycobacterium tuberculosis gets engulfed by cells called macrophages, the shock troops of the immune system. Scientists knew that the bacteria had evolved mechanisms to proliferate in these cells, but exactly how was unclear, says coauthor William Bishai, an infectious disease physician at Johns Hopkins University in Baltimore.

Bishai and his colleagues had noted that both TB bacteria and the macrophages they had entered had excess amounts of cyclic AMP, a compound that serves as a master regulator of many cell functions. So the researchers investigated an enzyme needed to make cyclic AMP. Curiously, M. tuberculosis has 17 genes that code for versions of this enzyme.

To test the effects of these 17 genes, the researchers infected mice with TB using aerosol sprays containing various combinations of the genes. Microbes harboring the enzyme encoded by a gene called Rv0386 out-competed the other microbes for survival in the mice and caused more severe lung disease. That suggests that the gene, along with the excess cyclic AMP production it induces, helps the microbe to sabotage the macrophage’s defensive abilities, thus making the TB more virulent.

In lab tests on human macrophages, the researchers showed that M. tuberculosis gins up cyclic AMP production upon entering a macrophage and unleashes this cargo once inside. That sets off a chain reaction that steers the cells to overproduce TNF alpha, a protein that causes inflammation, which contributes to tissue damage. Mice infected with real TB made 10 times as much TNF alpha in their lungs as did mice infected with TB lacking the Rv0386 gene.

This study is the first to pin down that M. tuberculosis is actively trafficking cyclic AMP into the cells and that “this really does affect the outcome of infections,” says McDonough.This reliance on cyclic AMP and the enzyme that makes it could prove useful to drug makers in the long run, she says.

Meanwhile, excess inflammation spurred by TNF alpha may be linked to the formation of lesions called granulomas, a hallmark of TB, Bishai says. TB granulomas have a central core of dead tissue surrounded by fatty debris and an outer rim of fibrous tissue and immune cells. Granuloma formation corrals the microbe but also enables it to lie latent in a person for decades. “We hypothesize that TB has a program that leads to granuloma formation, which gives the organism sanctuary,” Bishai says.

STRESSED-OUT DNA TURNS MOUSY BROWN HAIR GRAY

Stem cells responsible for hair color lose self-renewing abilities By Laura Sanders Web edition : Thursday, June 11th, 2009

Gray hair may be a mark of distinction in some circles, but it’s also a sign of a depleted stem cell population. DNA damage causes stem cells that produce hair-color cells in mice to lose their “stemness,” leaving brown hair gray, a report in the June 12 Cell shows. The results suggest a new way stem cell populations can be depleted as cells accumulate DNA damage over time.

The new study “opens up a new paradigm for how we’re going to study stem cell aging in many systems,” comments Kevin Mills of the Jackson Laboratory in Bar Harbor, Maine. The report “fills in what’s been a hole in our understanding of stem cell biology.”

Colorful locks depend on a group of special cells in hair follicles called melanocyte stem cells. Each of these cells divides into two cells: One that replaces itself and another that differentiates into a pigment-producing daughter cell called a melanocyte, which imbues hair with its browns, reds and blacks. Earlier research has suggested that the depletion of these stem cells was to blame for grayness. But how exactly these stem cells disappeared was mysterious. With no more stem cells around to produce melanocytes, hair turns gray.

Emi Nishimura at Tokyo Medical and Dental University in Japan and her colleagues tracked the fate of these stem cells and grayness in mice exposed to DNA-damaging radiation. The exposure level was fairly high, intended to magnify the effects of DNA damage that cells gradually accumulate with age.

Mice typically begin to go gray when they are 1 year to 1½ years old, after about 65 percent of their life, Nishimura says. But following exposure to high doses of radiation, hair on mice as young as 7 to 8 weeks grew in gray, while control mice remained brown, the team found. Other DNA-damaging agents, including hydrogen peroxide, had the same graying effect.

The team next looked at the stem cells in the hair follicles during this graying process. Researchers usually think of two ways stem cells stop working, say Paul Hasty, a geneticist at the University of Texas Health Science Center in San Antonio. The stem cells either die or stop dividing, he says. “But for these melanocyte stem cells, that’s not what happens.”

Instead, the DNA damage causes them to lose their “stemness,” the new report shows. Once the cells have racked up enough DNA damage, they become melanocytes and lose the ability to replace themselves or to replenish melanocyte cell populations. Once the melanocytes die, the hair is left with no pigment-producing cells.

“What’s really unexpected is that the cells differentiate in response to DNA damage,” instead of dying or halting division, Mills says. This irreversible pathway might be going on in stem cells in other tissues like the brain or blood, he notes. Figuring out the details of how these cells lose their “stemness” may ultimately lead to new ways to stave off stem cell depletion. “If you can modulate the stemness checkpoint, you can influence the activity of these stem cells,” Mills says.

ORIGINS OF THE SWINE FLU VIRUS

Researchers use evolutionary history to trace the early days of the pandemic By Laura Sanders Web edition : Thursday, June 11th, 2009

Closely related forms of the H1N1 strain of influenza virus circulated undetected in swine for years, a study published online June 11 in Nature reports. The virus, which has spread to multiple continents, has now been classified by the World Health Organization as a pandemic.

“Based on this report, we had a virus circulating in pigs for 10 years and nobody knew anything about it because we were not doing proper surveillance,” says Daniel Perez, an influenza expert at the University of Maryland in College Park.
Researchers traced the sordid past of the H1N1 virus by comparing mutations among different strains of the virus. Genetic sequences of 15 swine influenzas from Hong Kong and two human H1N1 viruses were compared with 796 sequences representing a large spectrum of related strains from humans, birds and pigs.

Analyzing numbers of mutations allowed an international team of researchers to estimate how long ago the strains first existed. Virus strains more than 90 percent identical to the current H1N1 strain were circulating in pigs between 9.2 and 17.2 years ago, the researchers found. The current strain “evidently spread without anyone noticing it for 10 years,” says Michael Worobey, an evolutionary biologist at the University of Arizona in Tucson and one of the study’s authors. “We need to spend more energy looking at what’s in pigs.”

The molecular clock method the team used assumes that genomes mutate at relatively constant rates, a tricky assumption for sporadically mutating influenza strains.

“Any estimate like this has a certain amount of uncertainty to it,” Worobey says. Although the numbers are not exact, he says, the data clearly show that a similar version of the virus was around long before anyone was aware of it.

The report also shows that each bit of the current virus’s DNA had been circulating on its own and primarily in pigs for years before combining to form the virus responsible for the current pandemic. Some genes have been in pigs for decades. “Across the genome, this is something that came from pigs,” Worobey says.

Some of these DNA segments came from a North American swine influenza virus, which itself is made of bits of avian, human and swine influenzas (called a triple-reassortant strain). Other segments came from Eurasian swine with avian virus components. The combination of the triple-reassortant strain from North America and the avianlike strain from Eurasia probably happened as live pigs were transported between North America and Eurasia, the authors say.

“We can do all the surveillance we want in humans, but if we really want to prevent pandemic influenza…, a fundamental change in efforts on the animal health side has to be made,” Perez says.

On the same day the new report appeared, the World Health Organization classified the H1N1 outbreak as a pandemic, defined as showing sustained person-to-person transmission in many parts of the world.

WHO Director-General Margaret Chan said that the organization is raising the alert level after determining that flu cases are now showing up in people who didn’t bring it from another region and weren’t in contact with such travelers. “Further spread is considered inevitable,” Chan said in a news conference.
Thomas Frieden, director of the Centers for Disease Control and Prevention, said in a June 11 press briefing that the new classification does not imply that the H1N1 virus has become more virulent.

“This does not mean that there is any difference in the level of severity of the flu,” he said. Rather, the pandemic label “is important because it does send the strong message that the virus is here, it’s in all likelihood here to stay, and it’s important that we continue our aggressive efforts to prepare and respond.”

So far no decision has been made to mobilize pharmaceutical companies to start mass-producing vaccines aimed specifically at the novel H1N1 virus. But preliminary steps to make that a seamless move have already been taken.

PLUMP YOUNGSTERS SHOW HEART-Y RISKS

By Janet Raloff Web edition : Friday, June 12th, 2009

Blood markers observed in obese children — some as young as 7 — indicate their bodies host chronic inflammation, a driver of heart disease, and elevations in chemicals that promote blood clots.

The findings, reported today at the Endocrine Society annual meeting, in Washington, D.C., indicate that school-age plumpness can prove more than a social stigma. It may signal that youngsters are on their way to developing cardiovascular disease — and years earlier than even a generation ago.

Nelly Mauras, chief of endocrinology at Nemours Children’s Clinic in Jacksonville, Fla., and her colleagues sampled the blood of 202 children aged 7 to 18. Half were lean and came from healthy, lean families. The rest were obese, but not due to disease. Moreover, for inclusion in the new study, heavy kids could not have metabolic syndrome, a condition whose constellation of symptoms — from high blood pressure and high cholesterol to elevated blood sugar — marks individuals at risk of heart disease, stroke and diabetes.

With childhood obesity reaching epidemic proportions, Mauras’ group wanted to see whether extreme pudginess in youngsters posed the same potential heart risks as it does in adults. And her disturbing data now indicate that it indeed appears to.

C-reactive protein is a blood marker of systemic inflammation. Concentrations of this CRP were, on average, eight times higher in fat adolescents (those who had at least entered puberty) than lean kids their age. And for kids who had not yet entered puberty, CRP values were 12 times higher among the very overweight kids, compared to lean children their age. Not surprisingly, the heavy kids also had elevated levels of interleukin-6, a signaling molecule that stimulates the liver to make CRP.

Adiponectin is a hormone secreted by fat cells that possesses anti-inflammatory properties. Its blood level was lower in the heavy kids than their lean counterparts, Mauras reports, while values of PAI-1, a compound that inhibits the breakdown of blood clots, were notably higher in overweight youngsters. Heavy kids also had significantly higher blood concentrations of fibrinogen, a material that fosters blood clotting.

At a briefing for reporters, Mauras concluded that “the unhealthy consequences of excess body fat start very early in childhood.”

“That’s kind of scary, isn’t it?” asked Daniel Bessesen, chief of endocrinology at the University of Colorado Denver School of Medicine, after hearing the new findings. “We know that cardiovascular disease develops not over a year or two but over or 20 and 30 years. And if kids have that kind of biochemical profile when they’re 7 to 9 years of age, you wonder how our country’s going to deal with that 30 or 40 years from now. “

Indeed, will we be in the throes of a middle-age heart-disease and stroke epidemic? And if health care costs are almost beyond our means to manage now, how much worse will they become?

Mauras didn’t go into what’s behind childhood obesity, but plenty of nutritionists point to one growing problem: Too little exercise. Elementary and secondary schools around the country have been eliminating physical education programs. Aggravating the problem, those computers and video games that well-meaning parents have been bringing into our homes have been seducing kids into becoming couch potatoes.

Kids may not know the long term implications of a sedentary lifestyle. But we parents do — and need to get the nation’s youngsters up and moving again.

Link between mammal, reptile, and bird

Check out this long beaked echidna! A very cool article on this wonderful animal just came out in the New York TImes. I had never heard of such a creature. It is a cousin to the platypus and has a fantastically large brain. (No wonder it avoids all human contact. It probably would have been hunted out of existence.) There is also a link to the very first field study on this animal in the Journal of Mammalogy.
Here are a couple of cool picks from the New York Times.


Chromosomes and Cytogenetics Topic Room

Cytogenetics is the study of chromosomes and their role in heredity. Thus, this topic room is all about chromosomes: chromosome structure and composition, the methods that scientists use to analyze chromosomes, chromosome abnormalities associated with disease, the roles that chromosomes play in sex determination, and changes in chromosomes during evolution.

The field of cytogenetics emerged in the early twentieth century, when scientists realized that chromosomes are the physical carriers of genes. As is always the case in science, researchers built on the observations of their fellow investigators to synthesize the chromosome theory of heredity. This groundbreaking theory had its foundations in the detailed observations that cytologists had made about chromosome movements during mitosis and meiosis, which suggested that chromosome behavior could explain Mendel's principles of inheritance.

In the early years of cytogenetics, scientists had a difficult time distinguishing individual chromosomes, but over the years, they continued to refine the conditions for preserving and staining chromosomes to the reproducible standard that is now expected in clinical cytogenetics. (Looking back, it seems incredible that the human chromosome number was not established until 1955.) In today's procedures, metaphase chromosomes are treated with stains that generate distinctive banding patterns, and chromosome pairs are then arranged into a standardized format known as a karyotype. Among the members of a species, karyotypes are remarkably uniform, which has made it possible for cytogeneticists to detect various deviations in chromosome number and structure that are associated with disease states and developmental defects.

A normal human karyotype contains 22 pairs of autosomes and one pair of sex chromosomes. Aneuploidies, or changes in chromosome number, are easily detected on karyotypes. In humans, most aneuploidies are lethal because of the ensuing imbalance in gene expression. A notable exception is trisomy 21, or Down syndrome, which is frequently detected during prenatal screening of older mothers. Sex chromosome aneuploidies are also tolerated in humans, most likely because X inactivation maintains near-normal expression levels for X-linked genes. In addition to changes in chromosome number, karyotypes can also reveal more subtle changes in chromosome structure. In effect, the normal banding pattern of a chromosome provides a "bar code" that can be translated into a map of the chromosome. Cytogeneticists can then use coordinates on these rough chromosome maps, or idiograms, to identify the positions of structural abnormalities, including deletions, duplications, and translocations, to within a few megabases of DNA.

Over the past few decades, versatile methods based on fluorescence in situ hybridization (FISH) have transformed cytogenetics into a molecular science and provided cytogeneticists with powerful new tools. In FISH procedures, labeled DNA or RNA probes are hybridized with their complementary target DNA sequences on chromosomes. FISH experiments often generate colorful results, because multiple probes, each of which is labeled with a spectrally distinct fluorescent dye, can be used in the same experiment. The target DNA sequences may consist of either a single gene or a collection of genes spread out along the length of a chromosome. FISH procedures are now routinely employed in clinical cytogenetics. Spectral karyotyping provides an overview of any gross rearrangements and changes in chromosome number that have occurred in a patient's cells. Using gene-specific probes, cytogeneticists can also positively identify the genes affected by chromosomal mutations. More recently, researchers have additionally begun to employ comparative genomic hybridization to analyze small quantitative differences between individuals' DNA, including copy number variations (CNVs).

Outside the clinic, FISH is one of many techniques biologists use to investigate the structure of chromosomes and their organization within the nucleus. Although chromosomes may appear to be static structures when viewed under a microscope, cytogeneticists know that chromosomes are actually dynamic assemblies made up of a DNA-protein complex called chromatin. Chromatin undergoes dramatic changes in packing during the cell cycle, and its structure also varies locally along the length of each chromosome. Transcriptionally active chromatin, or euchromatin, has a different composition than silent chromatin, or heterochromatin. (The inactive X chromosome in female mammals is a special case in which heterochromatin extends along the entire length of a chromosome.) Some chromatin specializations are essential for normal chromosome behavior. For example, centromeres contain a unique chromatin that is required for chromosome attachment to the mitotic spindle. Likewise, chromosome integrity depends on the assembly of a specialized chromatin found exclusively at the telomeres. Other less defined aspects of chromosome structure may also be important in positioning individual chromosomes with the nucleus. For instance, mounting evidence seems to indicate that chromosomes occupy discrete territories in the interphase nucleus; this marks a significant departure from the previously accepted idea that chromosomes are randomly organized during interphase.

In this era of comparative genomics, cytogenetics is also offering insights into evolution. Using cross-species FISH, scientists have identified groups of genes, called synteny groups, that maintain the same linkage relationships with each other across species boundaries. Synteny data reveal numerous chromosomal rearrangements that have occurred during the course of evolution. Taken together with DNA sequence information, synteny data are proving useful for detecting genome duplications and for constructing phylogenetic trees.

The collection of articles in this topic room is intended to provide students with an introduction to chromosome biology and an appreciation of the experimental evidence that has led to the current state of understanding. Cytogenetics is a broad and growing field of research, and many topics have not been discussed in detail. The editors hope that this collection will grow over time as new discoveries are made and gaps in the current collection are filled. To this end, teachers and researchers are encouraged to contribute new articles to the collection after consultation with the editors.

Gene Expression and Regulation

How does a gene, which consists of a string of DNA hidden in a cell's nucleus, know when it should express itself? How does this gene cause the production of a string of amino acids called a protein? How do different types of cells know which types of proteins they must manufacture? The answers to such questions lie in the study of gene expression. Thus, this topic room begins by showing how a quiet, well-guarded string of DNA is expressed to make RNA, and how the messenger RNA is translated from nucleic acid coding to protein coding to form a protein. Along the way, the room also examines the nature of the genetic code, how the elements of code were predicted, and how the actual codons were determined.

Next, this topic room turns to the regulation of genes. Genes can't control an organism on their own; rather, they must interact with and respond to the organism's environment. Some genes are constitutive, or always "on," regardless of environmental conditions. Such genes are among the most important elements of a cell's genome, and they control the ability of DNA to replicate, express itself, and repair itself. These genes also control protein synthesis and much of an organism's central metabolism. In contrast, regulated genes are needed only occasionally—but how do these genes get turned "on" and "off"? What specific molecules control when they are expressed?

It turns out that the regulation of such genes differs between prokaryotes and eukaryotes. For prokaryotes, most regulatory proteins are negative and therefore turn genes off. Here, the cells rely on protein–small molecule binding, in which a ligand or small molecule signals the state of the cell and whether gene expression is needed. The repressor or activator protein binds near its regulatory target: the gene. Some regulatory proteins must have a ligand attached to them to be able to bind, whereas others are unable to bind when attached to a ligand. In prokaryotes, most regulatory proteins are specific to one gene, although there are a few proteins that act more widely. For instance, some repressors bind near the start of mRNA production for an entire operon, or cluster of coregulated genes. Furthermore, some repressors have a fine-tuning system known as attenuation, which uses mRNA structure to stop both transcription and translation depending on the concentration of an operon's end-product enzymes. (In eukaryotes, there is no exact equivalent of attenuation, because transcription occurs in the nucleus and translation occurs in the cytoplasm, making this sort of coordinated effect impossible.) Yet another layer of prokaryotic regulation affects the structure of RNA polymerase, which turns on large groups of genes. Here, the sigma factor of RNA polymerase changes several times to produce heat- and desiccation-resistant spores. Within this topic room, the articles on prokaryotic regulation delve into each of these topics, leading to primary literature in many cases.

For eukaryotes, cell-cell differences are determined by expression of different sets of genes. For instance, an undifferentiated fertilized egg looks and acts quite different from a skin cell, a neuron, or a muscle cell because of differences in the genes each cell expresses. A cancer cell acts different from a normal cell for the same reason: It expresses different genes. (Using microarray analysis, scientists can use such differences to assist in diagnosis and selection of appropriate cancer treatment.) Interestingly, in eukaryotes, the default state of gene expression is "off" rather than "on," as in prokaryotes. Why is this the case? The secret lies in chromatin, or the complex of DNA and histone proteins found within the cellular nucleus. The histones are among the most evolutionarily conserved proteins known; they are vital for the well-being of eukaryotes and brook little change. When a specific gene is tightly bound with histone, that gene is "off." But how, then, do eukaryotic genes manage to escape this silencing? This is where the histone code comes into play. This code includes modifications of the histones' positively charged amino acids to create some domains in which DNA is more open and others in which it is very tightly bound up. DNA methylation is one mechanism that appears to be coordinated with histone modifications, particularly those that lead to silencing of gene expression. Small noncoding RNAs such as RNAi can also be involved in the regulatory processes that form "silent" chromatin. On the other hand, when the tails of histone molecules are acetylated at specific locations, these molecules have less interaction with DNA, thereby leaving it more open. The regulation of the opening of such domains is a hot topic in research. For instance, researchers now know that complexes of proteins called chromatin remodeling complexes use ATP to repackage DNA in more open configurations. Scientists have also determined that it is possible for cells to maintain the same histone code and DNA methylation patterns through many cell divisions. This persistence without reliance on base pairing is called epigenetics, and there is abundant evidence that epigenetic changes cause many human diseases.

In order for transcription to occur, the area around a prospective transcription zone needs to be unwound. This is a complex process requiring the coordination of histone modifications, transcription factor binding and other chromatin remodeling activities. Once the DNA is open, specific DNA sequences are then accessible for specific proteins to bind. Many of these proteins are activators, while others are repressors; in eukaryotes, all such proteins are often called transcription factors (TFs). Each TF has a specific DNA binding domain that recognizes a 6-10 base-pair motif in the DNA, as well as an effector domain. In the test tube, scientists can find a footprint of a TF if that protein binds to its matching motif in a piece of DNA. They can also see whether TF binding slows the migration of DNA in gel electrophoresis.

For an activating TF, the effector domain recruits RNA polymerase II, the eukaryotic mRNA-producing polymerase, to begin transcription of the corresponding gene. Some activating TFs even turn on multiple genes at once. All TFs bind at the promoters just upstream of eukaryotic genes, similar to bacterial regulatory proteins. However, they also bind at regions called enhancers, which can be oriented forward or backwards and located upstream or downstream or even in the introns of a gene, and still activate gene expression. Because many genes are coregulated, studying gene expression across the whole genome via microarrays or massively parallel sequencing allows investigators to see which groups of genes are coregulated during differentiation, cancer, and other states and processes.

Most eukaryotes also make use of small noncoding RNAs to regulate gene expression. For example, the enzyme Dicer finds double-stranded regions of RNA and cuts out short pieces that can serve in a regulatory role. Argonaute is another enzyme that is important in regulation of small noncoding RNA–dependent systems. This topic room contains an introductory article on these RNAs, but more content is needed; please contact the topic room editor if you are interested in contributing.

Imprinting is yet another process involved in eukaryotic gene regulation; this process involves the silencing of one of the two alleles of a gene for a cell's entire life span. Imprinting affects a minority of genes, but several important growth regulators are included. For some genes, the maternal copy is always silenced, while for different genes, the paternal copy is always silenced. The epigenetic marks placed on these genes during egg or sperm formation are faithfully copied into each subsequent cell, thereby affecting these genes throughout the life of the organism.

Still another mechanism that causes some genes to be silenced for an organism's entire lifetime is X inactivation. In female mammals, for instance, one of the two copies of the X chromosome is shut off and compacted greatly. This shutoff process requires transcription, the participation of two noncoding RNAs (one of which coats the inactive X chromosome), and the participation of a DNA-binding protein called CTCF. As the possible role of regulatory noncoding RNAs in this process is investigated, more information regarding X inactivation will continue to be discovered, and further additions to this topic room will therefore be warranted.

Protein biosynthesis

Protein biosynthesis (Synthesis) is the process in which cells build proteins.

Protein synthesis is the process in which cells build proteins. The term is sometimes used to refer only to protein translation but more often it refers to a multi-step process, beginning with amino acid synthesis and transcription of nuclear DNA into messenger RNA which is then used as input to translation.

The cistron DNA is transcribed into a variety of RNA intermediates. The last version is used as a template in synthesis of a polypeptide chain. Proteins can often be synthesized directly from genes by translating mRNA. When a protein is harmful and needs to be available on short notice or in large quantities, a protein precursor is produced. A proprotein is an inactive protein containing one or more inhibitory peptides that can be activated when the inhibitory sequence is removed by proteolysis during posttranslational modification. A preprotein is a form that contains a signal sequence (an N-terminal signal peptide) that specifies its insertion into or through membranes; i.e., targets them for secretion. The signal peptide is cleaved off in the endoplasmic reticulum. Preproproteins have both sequences (inhibitory and signal) still present.

For synthesis of protein, a succession of tRNA molecules charged with appropriate amino acids have to be brought together with an mRNA molecule and matched up by base-pairing through their anti-codons with each of its successive codons. The amino acids then have to be linked together to extend the growing protein chain, and the tRNAs, relieved of their burdens, have to be released. This whole complex of processes is carried out by a giant multimolecular machine, the ribosome, formed of two main chains of RNA, called ribosomal RNA (rRNA), and more than 50 different proteins. This molecular juggernaut latches onto the end of an mRNA molecule and then trundles along it, capturing loaded tRNA molecules and stitching together the amino acids they carry to form a new protein chain.

Protein biosynthesis, although very similar, is different for prokaryotes and eukaryotes.

Loss of the Alox5 gene impairs leukemia stem cells and prevents chronic myeloid leukemia

Targeting of cancer stem cells is believed to be essential for curative therapy of cancers, but supporting evidence is limited. Few selective target genes in cancer stem cells have been identified. Here we identify the arachidonate 5-lipoxygenase (5-LO) gene (Alox5) as a critical regulator for leukemia stem cells (LSCs) in BCR-ABL–induced chronic myeloid leukemia (CML). In the absence of Alox5, BCR-ABL failed to induce CML in mice. This Alox5 deficiency caused impairment of the function of LSCs but not normal hematopoietic stem cells (HSCs) through affecting differentiation, cell division and survival of long-term LSCs (LT-LSCs), consequently causing a depletion of LSCs and a failure of CML development. Treatment of CML mice with a 5-LO inhibitor also impaired the function of LSCs similarly by affecting LT-LSCs, and prolonged survival. These results demonstrate that a specific target gene can be found in cancer stem cells and its inhibition can completely inhibit the function of these stem cells.

Transfection of small RNAs globally perturbs gene regulation by endogenous microRNAs

Transfection of small RNAs (such as small interfering RNAs (siRNAs) and microRNAs (miRNAs)) into cells typically lowers expression of many genes. Unexpectedly, increased expression of genes also occurs. We investigated whether this upregulation results from a saturation effect—that is, competition among the transfected small RNAs and the endogenous pool of miRNAs for the intracellular machinery that processes small RNAs. To test this hypothesis, we analyzed genome-wide transcript responses from 151 published transfection experiments in seven different human cell types. We show that targets of endogenous miRNAs are expressed at significantly higher levels after transfection, consistent with impaired effectiveness of endogenous miRNA repression. This effect exhibited concentration and temporal dependence. Notably, the profile of endogenous miRNAs can be largely inferred by correlating miRNA sites with gene expression changes after transfections. The competition and saturation effects have practical implications for miRNA target prediction, the design of siRNA and short hairpin RNA (shRNA) genomic screens and siRNA therapeutics.

Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.

An essential tool for investigating the role of a gene during development is the ability to perform gene knockdown, overexpression, and misexpression studies. In zebrafish (Danio rerio), microinjection of RNA, DNA, proteins, antisense oligonucleotides and other small molecules into the developing embryo provides researchers a quick and robust assay for exploring gene function in vivo. In this video-article, we will demonstrate how to prepare and microinject in vitro synthesized EGFP mRNA and a translational-blocking morpholino oligo against pkd2, a gene associated with autosomal dominant polycystic kidney disease (ADPKD), into 1-cell stage zebrafish embryos. We will then analyze the success of the mRNA and morpholino microinjections by verifying GFP expression and phenotype analysis. Broad applications of this technique include generating transgenic animals and germ-line chimeras, cell-fate mapping and gene screening. Herein we describe a protocol for overexpression of EGFP and knockdown of pkd2 by mRNA and morpholino oligonucleotide injection.

The transformation of complex chromatographic raw data into quantitative and qualitative information

Using the new Chromatography Data System Geminyx III , an in-house software development from Goebel Instrumentelle Analytik, the detector raw data from complete sample sequences can be converted into meaningful quantitative and qualitative information with just a few mouse clicks:

The automatic integration ensures that complete sample sequences are automatically and correctly integrated without time-consuming parameter selection. This can be checked by simply paging through the individual samples.

The user can zoom in on details and, if required, use the graphical editor to move peak limits, reset the baseline or separate a peak shoulder using simply mouse clicks. Once all runs have been checked the sequence can be printed as a complete summary report.

All calibration functions usually used in the chromatography laboratory are supported in Geminyx III. Outliers in standard runs are automatically recognized using tightly defined limits, marked as outliers and are not then used for the calibration calculations.

The integrated report designer permits the professional design of the sample report. The method defines which results are shown in the integration report. Using a report template you define the positioning and formatting of the graphics and sample data and can also add the company logo to the template. The analysis results are thus printed using a professional layout and can be saved in all usual electronic formats or be sent as e-mail.

Alongside the quantitative calculations from the chromatograms Geminyx III also, in conjunction with a diode-array detector, supplies valuable qualitative information with respect to peak purity and component identity. The peak purity is displayed both numerically and graphically using comparisons made between high-resolution normalized spectra obtained across the peak. Using automatic solvent correction the system compensates for the changes in solvent spectrum during a gradient run.

The spectrum search, using self-created or commercially available spectral libraries, is a reliable indicator as to whether the detected component is in reality the substance expected at this retention time.

Naturally Geminyx III executes the quantitative and qualitative calculations defined in the method fully automatically so that checking only requires paging through the individual samples before the complete report of a sample sequence is generated with a single mouse click.

Goebel Instrumentelle Analytik offers complete HPLC solutions from a single source, alongside the configurable modules of the Celeno HPLC range we can also adapt our internally developed Geminyx Software to suit your needs. Further we also offer you reliable and cost-effective service support designed to preserve the value of your system and improve the quality of your analysis results!

Human Microbiome Project

Microbes occupy a number of niches on the surface of and inside human body. In fact, in an adult human being, microbe cells outnumber human cells 10 to 1. It makes us a supra-organism or a composite of species. The gut microbiome, for example, consists of at least many thousands of bacteria and archea species many of which can not be cultured. Arrival of fast and economic sequencing techniques e.g. 454 and SOLEXA has made the molecular (16S rRNA based) identification or microbes within the reach of labs. It appears that there is a lot of diversity in a specific niche across humans and other mammals.

NIH Human Microbiome Project is an intitiative to understand this composite of species. Specifically, the aim is to understand whether there is a core group of microbes that all humans share. The second one is to understand whether the composition of microbiota in a specific niche relates to health. For example there is strong evidence to suggest that gut microbiota of obese individuals is different from lean ones. Transfer of gut microbiota from a obese human to a germ free mouse (a humanized mouse) can induce adipocity which can again be transferred to other mice, yet again. Such a finding has far reaching consequences for a population dealing with calorie-rich diet and obesity.

Stem cells without virus infection

Recently there has been a lot of new studies showing how to reprogram adult cells into stem cells. Just a few months ago researchers showed that only four genes (c-Myc, Klf4, Oct4, and Sox2) were needed to obtain stem cells, and a few weeks ago Oct4 was shown to be the only gene needed to reprogram cells into stem cells.
All these studies required the use of viruses to reprogram cells into stem cells. The typical experiment was basically as follows: introduce your genes (Oct4, etc) into a retrovirus and then infect a cell with it. The retrovirus integrates into the cell's genome, expresses its genes (Oct4, etc) and the cell becomes a stem cell.
Now two studies, both published in the latest issue of Nature, have shown how to get around using a virus. One approach cloned the now called Yamanaka factors (c-Myc, Klf4, Oct4, and Sox2) into a vector and introduced it into cells. After the cells were reprogrammed the vector was digested using a restriction enzyme, only leaving a small trace of DNA. The other approached used a transposable element called piggyBac (from the cabbage looper moth). As with the vector, all four Yamanaka factors were cloned into piggyBac and then introduced into cells, which lead to reprograming into stem cells. The beauty of this new method is that this transposable element can be removed entirely with the use of a transposase (an enzyme that makes the transposable element completely excise itself from DNA, not leaving any trace of its existence). The transposase experiments remain to be performed, as well as a complete characterization of the pluripotent nature of these reprogrammed stem cells (there are a lot of experiments that need to be performed to fully characterize a cells as a stem cell).
These new techniques have great potential for genetic therapy. Up until now in order to introduce genetic modifications into cells to turn them into stem cell you had to infect them with a retrovirus (not the nicest thing and potentially dangerous). By getting rid of viruses altogether, these new techniques made genetic therapy and stem cell research significantly safer and easier.

IISER-Kolkatta recruits @ BT2020- JRF cum PhD position in Marine Microbiology/ Marine Molecular ecology

Dr. Punyasloke Bhadury, Assistant Professor, Indian Institute of Science Education and Research (IISER)- Kolkata,West Bengal would like to recruit suitable candidate for the following position.


Post:
JRF
(with PhD opportunity)


Area:
Marine Microbiology/Marine Molecular ecology


Eligibility:
M.Sc. Life Science / Microbiology / Marine Biology / Ecology (First Class);
CSIR-UGC NET qualifion or NET-LS qualification.
Participation in international oceanographic cruises will be required.


Duration:
03 years


Pay:
Rs 12,000 + HRA pm/-


Deadline:
12 June 2009


To apply:
Mail your resumes with the subject line "IISER" to
pbhadury@gmail.com,
aurobindobio@gmail.com

Macrophage Isolation: When things get cloudy

Macrophage Isolation: When things get cloudy
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Macrophage Isolation:
When things get a little cloudy

by Heather Buschman


Macrophages are dedicated phagocytes that love nothing more than seeking out infection or inflammation and gobbling up bacteria, dead cells, and other debris. Once activated, these innate immune defenders also release chemokines and cytokines that trigger an inflammatory cascade.

In the lab, macrophages are used to test all kinds of things, from phagocytosis to trans-endothelial migration. One of the most common ways to obtain macrophages is to elicit them from the mouse peritoneum. First, healthy mice are injected intraperitoneally (i.p.) with 3 mL of 3-4% thioglycollate. This causes inflammation, triggering monocyte differentiation and localization to the body cavity. Thus, this method results in a higher yield of macrophages than techniques that rely on collecting only resident macrophages. However, thioglycollate-derived macrophages are “activated”, and therefore are not appropriate for every experiment. (Alternatively, murine monocytes can be collected from the bone marrow and differentiated into macrophages, with high yields and little activation.)

After 3-5 days, the thioglycollate-injected mice are sacrificed, injected i.p. with 5-10 mL of sterile, ice cold phosphate buffered saline (PBS), and gently massaged. Then the skin is carefully snipped and peeled back to reveal the intact peritoneum, distended with fluid. At this point, a large gauge needle and syringe are used to draw back out as much of the injected PBS as possible, without disturbing the membrane or organs. This extraction should be cloudy with white cells, but mostly free of red blood cells. Keeping this solution cold, the cells are gently centrifuged and rinsed with fresh cold PBS several times before resuspending in tissue culture media.

As discussed in the mouse peritoneal macrophages isolation forum at Scientist Solutions®, the easiest method for separating the macrophages from the other white blood cells is by simple adherence. When plated in a tissue culture dish, the macrophages will stick overnight and the rest of the cells can be rinsed away the next day. To increase macrophage yields, several posterssuggest “aging” the thioglycollate. Nobody seems exactly sure why, but everyone agrees that autoclaved thioglycollate stored for a week to several months just works better. Some forum participants also report better results extracting with 16-30% sucrose rather than PBS.


ICMR Junior Research Fellowship -2009

Indian Council of Medical Research (ICMR), Delhi invites applications for its JRF-entrance exam -2009 .


No of fellowship:
120


Subject:
Biomedical sciences with emphasis on Life Sciences (like microbiology, physiology, molecular biology, genetics, human biology, bioinformatics, biotechnology, biochemistry, biophysics, immunology, Pharmacology, zoology, Environment Science, botany, veterinary sciences, bio-informatics etc.).


Fellowship:
Rs. 12000/- pm
To be revised


Qualification:
M.Sc. or equivalent degree with minimum 55% marks for General/OBC Candidates and 50% for the SC/ST & physically handicapped candidates .


Important dates:
Date of examination- 12 July 2009.
Deadline to apply - 29 April 2009


To download application form and more details,
ICMR- JRF exam details
PhD scholarship

USE OF THE LIGHT MICROSCOPE


  • Each time the microscope is to be used it should be set up correctly to give a good image. Most often users forget to adjust the iris diaphragm and focus the condenser to give its optimum performance, this could result in objects in the preparation being undetected.
  • There are many microscope set up procedures for specific functionality but the most versatile for general use is ‘Critical Illumination’ as detailed below.
  1. Examine the microscope and identify the parts as siting of some controls may vary and some have ‘Fixed’ or specialised condensers.
  2. Ensure the light intensity control is set to low, turn on the light and increase the brightness to give a white light output.
  3. Raise the substage condenser fully and open the iris diaphragm fully.
  4. Rotate the nosepiece to the lowest power objective (x4).
  5. Rack the stage down using the coarse adjustment control and place the specimen slide on the stage using the retaining mechanism to hold in place.
  6. While looking carefully rack the stage up close to the objective lens (do not rack the lens into the slide; this cannot normally occur when a low power objective is selected).
  7. Set the eye pieces to the correct width for your eyes so you can look down both together comfortably.
  8. Focus the Microscope.
  9. Look down the eyepieces with both eyes open and slowly lower the stage to bring the specimen into focus, (use the coarse adjustment control initially and then the fine). Slight movement of the specimen while focusing by using the mechanical stage controls may aid you to find the object. If the image is not sharp focus adjustment of the eyepieces may be necessary. If one eyepiece is fixed and one adjustable, close the eye over the adjustable one. Now focus using the fine control until a sharp image is obtained, (if you cannot achieve this ask for assistance as the problem may lie elsewhere). Now open the other eye (keep both open) and using the adjustment on the eyepiece itself rotate to obtain a sharp image.
  10. Focus the Condenser.
  11. The condenser should be fully raised to start with. Rotate the nose piece to select the x10 objective and check the focus using fine adjustment. Position the point of a pencil into the middle of the light source over the lamp housing. Using the condenser control, slowly rack it down until the outline image of the pencil becomes sharp and crisp, (this is the ‘Critical’ point for the condenser for optimum performance from where the technique derives it’s name). Note this must be done only after a specimen has been focused on the stage.
  12. Set the Iris Diaphragm.
  13. When the iris diaphragm is fully open the image is flooded with light and definition is lost due to ‘white-out’. As the diaphragm is closed controlling the amount of light passing through the condenser the image is darkened and outlines appear thickened and defined as more contrast is achieved. Remove one eyepiece and place safely to one side. Close the diaphragm down ,then open slowly until you achieve a dark ring blanking off about 1/5th of the original field of view to give a ‘bicycle tyre’ effect. The correct amount of light should thus be obtained.
  14. Examine the specimen, moving it by means of the mechanical stage controls. No further adjustment to the condenser or iris diaphragm is usually necessary to view other slides of the same type/density at low power. The Vernier scale readings may be used to identify and re-locate a point of interest on a slide provided it is a ‘mounted’ preparation and the slide is placed on the stage in the same orientation, i.e. well labeled.