May 6, 2016 § Leave a comment
Niemann–Pick disease is a rare genetic disease with devastating effects. For one type of the disease, known as type C, defects in lysosomal storage within the cell lead to impaired neurological function. In infants, these symptoms can be especially difficult to recognize. They often include subtle changes in children’s behavior, such as failure to meet expected cognitive milestones or inability to control balance.
Until recently, the first-line diagnostic test for NPC disease has involved a skin biopsy and filipin staining, which is invasive, cumbersome and expensive. Patients with NPC typically often go up to five years without a diagnosis, drastically limiting the possibility of early interventions.
In a paper just published in the journal Science Translational Medicine, Daniel Ory of Washington University School of Medicine in St. Louis and colleagues lay the groundwork for a promising new diagnostic test for NPC. Importantly, the new noninvasive assay produces results within a day instead of months.
Here’s how it came to be.
The team used mass spectrometry to analyze dried blood spots, collected at various times after birth, from patients known to have NPC. They found three bile acids biomarkers that could clearly distinguish NPC patients from people without the disease.
The scientists then determined the structures of the bile acids. Ory and colleagues identified one bile acid as a trihydroxycholanic acid and another as its glycine conjugate.
As the second bile acid helped the team distinguish NPC patients from non-NPC patients more consistently, the researchers decided to use it to develop a new diagnostic test.
As a diagnostic test, Ory says, the assay already is being used at Washington University. He expects other centers to follow suit.
For its use in newborn screening, Ory said, researchers will need to put the assay to the test in the undiagnosed newborn population to ensure its usefulness in recognizing NPC in that age group, a process Ory believes will take several years.
Although the U.S. Food and Drug Administration hasn’t yet approved treatments for NPC, a promising drug called cyclodextrin is rapidly moving through clinical trials. The most effective treatment strategies will need to intervene early in the disease process, which is something the new test could accomplish. “We’re really trying to make an impact in this NPC community by being able to develop the therapies and being able to diagnose early,” says Ory. The approach “we’ve taken over the last 10 years, I feel like, it’s getting close to bearing fruit.”
March 17, 2016 § Leave a comment
When their lab mice unexpectedly packed on weight, Richard Huganir at the Johns Hopkins School of Medicine and his colleagues had to figure out why the mice suddenly turned obese. In a paper just out in the journal Science, the investigators describe their discovery of a protein-modification pathway in the brain that plays a surprising role in feeding control and satiety.
“This was a serendipitous discovery,” says Huganir, a neuroscientist. “We had to learn a whole new area of biology — feeding control, metabolism and obesity. Luckily, we had great collaborators at Hopkins who had the expertise to help us figure out what was going on. We eventually found out that the mice had impaired satiety and ate larger meals.”
The investigators originally were working on deciphering the role of an enzyme called O-GlcNac transferase, known as OGT, in regulating synaptic transmission and plasticity in the brain as well as its potential role in learning and memory. OGT catalyzes the attachment of a short sugar molecule to proteins; the sugar molecule then influences the function of the proteins.
As part of their project, Huganir and colleagues genetically modified the brains of mice so that the researchers could turn off the expression of OGT in the forebrain and hippocampus. These two regions of the brain are important for learning and memory.
“Much to our surprise, a couple of weeks after we knocked out OGT, the mice got very, very fat,” says Huganir. “We stopped studying learning and started studying feeding control.”
The parts of the brain the investigators had targeted in their mice are not usually associated with feeding control. But the hypothalamus is.
When the investigators looked at the hypothalamus, they discovered that they had inadvertently removed OGT in specific cells in a region of the hypothalamus called the paraventricular nucleus.
To make sure that OGT in the paraventricular nucleus cells was what was influencing the feeding and satiety of the mice, Huganir and colleagues created another set of genetically modified mice. These mice had OGT missing only in the paraventricular nucleus cells. “Knocking out OGT in only these cells inhibited their activity and produced the same overeating phenotype,” says Huganir.
The investigators now know that OGT plays an important role in the paraventricular nucleus cells in feeding control, but the molecular details are still unknown. For one, the investigators don’t know what substrates OGT acts on in the paraventricular nucleus cells to regulate their activity.
And, as with any work done on mice, the implications for humans have to be worked out. “This work in mice does suggest similar mechanisms are important in human satiety,” says Huganir. “However, much more work is needed to identify potential therapeutic targets to modify this pathway in humans to regulate food intake.”
March 14, 2016 § 1 Comment
Preeclampsia affects roughly three percent of pregnant women in the U.S., bringing on a host of complications that include premature births and even death. Unfortunately, there is no effective diagnostic test to predict the onset of disease.
In a recent paper published in the Journal of Lipid Research, Steven Graves of Brigham Young University and colleagues described a set of biomarkers that could help in the early detection of preeclampsia.
In spite of efforts to identify the mechanisms surrounding the disease, researchers haven’t been able to pinpoint a causative factor. When attempting to develop predictive assays for prenatal complications, scientists place the safety of the mothers’ and their unborn babies first. While a sampling of the placenta may provide critical information about preeclamptic processes, the placenta sampling procedure is risky. Scientists interested in developing prenatal diagnostic tests need to consider methods that are both informative and reasonable to use in a clinical setting.
Graves and colleagues decided to look at lipids although proteins tend to be the more conventional class of biomarker. Graves says the team focused on lipids in the blood because lipids tend to be more forgiving than their protein counterparts. Lipids, “are not particularly heat-sensitive compared to a protein or peptide and they’re not degraded rapidly by proteolytic enzymes which exist in the serum,” explains Graves. Additionally, serum samples can be collected in the clinic relatively easily with blood draws, keeping the risk to patients low.
The researchers took samples collected for another trial that was studying the early events of Down’s syndrome. Of the serum samples available, they used those collected at the earliest available time point which was 12–14 weeks into the pregnancy.
Using mass spectrometry data, the team compared the serum lipid profiles of women who went on to develop preeclampsia and those who did not. After an initial analysis and a second confirmatory run in another sample set, the team identified a set of 23 biomarkers in the form of mass spectral profiles that were able to predict those women who would go on to have a preeclamptic event.
Any biomarker on its own can’t provide sufficient predictive value, but combining the markers together into sets increased predictability. For their sample population, the investigators found that using six biomarkers helped with predicting preeclampsia; combining more than six markers failed to show an increase in predictive value. When the lipid test becomes publically available, Graves advises using all 23 biomarkers together to better account for individual patient factors.
Though the lipid biomarkers are intriguing, Graves is careful to point out these biomarkers aren’t ready for the clinic just yet. “What should happen now is one should establish a clear hypothesis that this set of markers would be useful and then carry out studies” focused on these markers. A lipid-based test will only be available after it passes through all the necessary studies and approval of a clinical test by the U.S. Food and Drug Administration.
Currently, the true advantage of this research isn’t in its immediate clinical value but the potential use of the biomarkers for streamlining the research process. Because the disease is so rare, one of the biggest issues with prospective studies for preeclampisa is the sheer number of women that need to be enrolled in order to have adequate numbers of preeclamptic cases. However, if researchers first can narrow down the population, using a set of predictive biomarkers such as the one proposed in the paper, fewer women would need to be enrolled. Graves proposes, “It could save time and allow for more things to be tested more efficiently.”
January 20, 2016 § 1 Comment
Many of us can attest to the rejuvenating effects of a manicure or a pedicure. The same applies to mice, as recently determined by Stanford University School of Medicine researchers. The investigators report that a simple pedicure can treat ulcerative dermatitis, a ubiquitous and often fatal condition among laboratory mice.
UD affects up to 21 percent of lab mice. Its specific cause remains unknown, although strong evidence suggests that it is behavior-related. Deep, itchy lesions often appear first on the neck. These lesions spread as the mouse scratches itself. UD is currently the most common cause of unplanned euthanasia among lab mice.
In a PLoS ONE paper, Sean Adams’ group described nail trimming as an alternative to the laborious and ineffective application of daily ointment to treat UD. This method was the first of several anecdotally-reported strategies that the researchers planned to explore. A pedicure, they found, reduces animal suffering, saves time and money, and boosts the integrity of mouse studies by reducing the need for medical intervention. Fewer euthanized mice means fewer mice needed per study.
The investigators then carried out a study. In one 14-day trial, 93.3 percent of mice were cured of UD with a pedicure compared with 25.4 percent of mice treated with daily ointment. The nail-trimmed mice received a one-time dose of topical treatment to prevent bacterial growth and soothe inflammation. These mice resisted scratching even after their nails grew back. “By intervening in the itch-scratch cycle, we are giving the animals the time that they need to recover, and also perhaps for that behavior to ramp down,” says Joseph Garner at Stanford University, a coauthor on the study.
The researchers later devised a plastic restraint, a modified conical tube, to keep the mice still while their nails are trimmed. With training, this process can take as little as 30 seconds. The researchers have been distributing these tubes when they present their findings at conferences, hoping to encourage more labs to adopt this humane and economical practice.
“There was a lot of serendipity and luck involved,” says Garner. “We never expected that we would find a solution that works in such a high percentage of mice, that it would be so simple, or that we would find it on the first try.”
Alexandra Taylor (firstname.lastname@example.org) is a staff science writer at ASBMB and a master’s candidate in science and medical writing at Johns Hopkins University.
January 11, 2016 § 1 Comment
Mammals, there’s a new enzyme in town. In a paper just published in the Proceedings of the National Academy of Sciences, scientists report the discovery of a new enzyme called glycerol-3-phosphate phosphatase. The enzyme plays a critical role in metabolism by overseeing the levels of different fuels in the body.
Metabolism has been a heavily trodden field of study ever since the dawn of molecular biology. These days, “it is extremely rare that a novel enzyme is discovered at the heart of intermediary metabolism in all mammalian tissues,” says S.R. Murthy Madiraju at the Montreal Diabetes Research Center, one of the corresponding authors on the paper.
Madiraju, along with Marc Prentki at the Montreal Diabetes Research Center and others, found the enzyme while grappling with a puzzle. They were studying pancreatic beta cells that produce insulin to control blood glucose levels. These cells get stressed when barraged with metabolic fuel from the diet, such as glucose and fatty acids. When that happens, the cells make and get rid of glycerol as a way of dealing with the excessive fuel supply.
Initially researchers thought the glycerol came from the breakdown of fats. But when Madiraju, Prentki and colleagues inhibited fat breakdown, the cells kept making glycerol. This suggested there was another glycerol source.
Microorganisms, plants and some fish have an enzyme that turns a molecule called glycerol-3-phosphate into glycerol. Mammals were thought not to have the enzyme.
But the investigators set out to check if mammals really and truly didn’t have a glycerol-3-phosphate phosphatase. It turns out they do. Madiraju, Prentki and colleagues confirmed the presence of the enzyme both in cell and animal models.
In discovering a mammalian glycerol-3-phosphate phosphatase, “we have to re-adjust our thinking that fat breakdown is not the only way by which mammalian cells generate glycerol, as has been believed so far!” says Prentki.
Glycerol-3-phosphate, the molecule on which the enzyme works, is made from glucose and gets incorporated into fats. It lies at the heart of glucose and lipid metabolism. With the discovery of an enzyme that can directly break down glycerol-3-phosphate, researchers now have a new player to contend with in understanding how the body maintains energy levels under normal circumstances and what goes wrong in different metabolic diseases.
The investigators say that they believe that, because of its critical metabolic role, glycerol-3-phosphate phosphatase will be a new target for treating chronic conditions such as obesity and type 2 diabetes as well as some cancers.
December 14, 2015 § Leave a comment
Much like Legos, proteins can come together in a number of ways to create complex structures. The various ways make it hard to organize protein complexes into categories.
But now, in a paper just out in Science, researchers describe an approach to classify protein complexes that creates a periodic table, like the periodic table that’s used in chemistry to organize elements. “We’re bringing a lot of order into the messy world of protein complexes,” explained Sebastian Ahnert at the University of Cambridge who is the first author on the paper in a press release.
Many proteins spend much of their time interacting with other proteins and assembling into complexes in order to carry out their functions. But the interactions and functions are specific, much like in the way different Lego bricks can latch onto each other only in certain ways. The underlying principles of protein interactions and assembly are not yet fully understood. But by organizing the different ways protein comes together into a table, Ahnert, along with Sarah Teichmann at the European Molecular Biology Laboratory–European Bioinformatics Institute, Joseph Marsh at the University of Edinburgh and others, wanted to see if some of the fundamental steps in protein complex evolution would become apparent.
They did. The investigators organized complexes based on simple rules so that they could find the most basic structures. “In the end, we discovered that three possible steps of interface evolution, combined in very specific ways, give rise to almost all known structures of protein complexes,” says Ahnert.
The investigators say that the fact that almost all known protein complexes could be arranged into a periodic table is revealing and will help understand how protein complexes come about. “Most heteromeric protein complexes—ones with more than one protein type—consist of identical repeated units of several protein types,” says Ahnert. “Because of this, heteromeric protein complexes can, in fact, be viewed as simpler, homomeric protein complexes—ones that only consist of a single type of protein—if we think of these repeated units as larger ‘single proteins.’”
(For an interactive version of the periodic table for proteins, go here)
October 22, 2015 § Leave a comment
Pollen, which consists of grains with the plant male gametes tucked inside, dries as it matures to increase its chances of survival. When carried over to neighboring plants, for example by insects or wind, pollen comes into contact with fluid in the female organ of a plant and then swells rapidly.
This swelling process a sensitive procedure; a pollen grain can die if it is not carefully rehydrated. Until now, the mechanism regulating this fluid uptake was unknown. Researchers writing in the journal Science today report that they have discovered an ion channel that helps pollen grains sense and respond to changes in internal water pressure.
Elizabeth Haswell at Washington University in St. Louis has been studying mechanical signals in plants for more than a decade. In the paper just published, she and her colleagues describe the discovery of ion channels on pollen membranes that monitor and respond to osmotic changes.
If the fluid content inside a membrane becomes too great, pores open to allow ions to leave. Water follows, relieving the pressure. The mechanosensitive ion channel, known as MSL8, senses pressure and makes adjustments as necessary. An incorrect amount of this protein deceases the pollen’s ability to fertilize.
By using RNA analysis, Haswell’s team determined that MSL8 transcripts are found in floral tissue but not in leaf or root tissue. They then fluorescently marked the proteins to show that the proteins were present on the plasma membranes of mature pollen grains.
After rehydrating, pollen grows a tube to carry its sperm cell to the eggs. Haswell and colleagues found that pollen without MSL8 germinated more effectively but generated so much pressure that the tube burst, impairing fertilization. Conversely, pollen that overexpressed MSL8 did not generate enough pressure for the pollen tube to break through the cell wall, rendering the pollen infertile.
This delicate osmotic balance demonstrates mechanical signals aiding in the developmental process. Researchers previously established that bacteria use stretch-activated channels to relieve internal pressure in response to environmental stress signals. The findings by Haswell and colleagues now indicate a previously unknown use for mechanically gated ion channels: reproduction. To cope with “the uncertain and potentially severe conditions of [the] pollen journey, pollen has developed some equally severe compensatory mechanisms, including this fascinating desiccation and rehydration process,” Haswell says. Other strategies include multiple nuclei and a tough cell wall.
While the function of MSL8 seems clear, the mechanism by which it operates — directly, by releasing osmolytes, or indirectly, through regulatory pathways — will be a target for further study. Haswell’s team also is interested in several related ion channels and in studying how membranes survive the dehydration/rehydration process.
This blog post was written by Alexandra Taylor who is a science writing intern at the American Society for Biochemistry and Molecular Biology.
September 29, 2015 § 1 Comment
Each year in the U.K., about 2 percent of horses die from grass sickness. No one knows what causes the disease, but it does occur almost exclusively in grass-fed animals, including ponies and donkeys. A similar disease is thought to afflict dogs, cats, rabbits, hares, llamas, and possibly sheep.
In an attempt to understand what happens at the molecular level of equine grass sickness, researchers recently reported in the journal Molecular & Cellular Proteomics their analysis of tissue samples taken from horses stricken with the disease. They found misfolded and dysregulated proteins in the tissues that resembled those found in human neurodegenerative conditions, such as Alzheimer disease, Parkinson disease and Huntington disease.
Animals with grass sickness usually suffer gut paralysis. The animals roll, sweat, drool and have trouble swallowing. Animals acutely afflicted with the disease usually have to be euthanized.
The disease is known to attack the neurons, but the causative agent is not known. To get a look at what goes on at the molecular level, Thomas Wishart at The Roslin Institute in Scotland, teamed up with Bruce McGorum at the University of Edinburgh’s veterinary school. The investigators applied proteomic techniques to samples taken from horses that came down with grass sickness.
“We do know which tissues are most consistently affected” by the disease, says Wishart. “We considered that a proteomic analysis would provide a snapshot of the molecular processes in play within those samples at that point in time.”
He points out that the work described in the MCP paper “is the first application of modern proteomic tools and in-silico analytical techniques to equine neuronal tissues and to an inherent neurodegenerative disease of large animals that is not a model of human disease.”
The investigators found that the expression levels of 506 proteins were changed in the ganglia taken from horses felled by grass sickness. Moreover, some of the proteins were misfolded, aggregated or in the wrong places. The proteins included amyloid precursor protein, the microtubule associated protein tau and several components of the ubiquitin proteasome system. These proteins have been implicated in human neurodegenerative disorders.
Finding this similarity between human and horse neurodegenerative diseases, says Wishart, suggests the aggregated or misregulated proteins are “more likely to be end-stage regulators or late consequences rather than initiators of the degenerative cascades.”
As equine grass sickness can be hard to diagnose in some horses, a next step for the investigators is to see if they can come up with a noninvasive diagnostic test.
July 29, 2015 § 1 Comment
Frustration has its perks. In a paper just out in Nature, researchers describe making an artificial ribosome because they couldn’t get normal ribosomes to do what they wanted. In creating this artificial ribosome, called Ribo-T, the investigators unwittingly turned conventional molecular biology wisdom on its head: Unlike regular ribosomes, Ribo-T doesn’t need to fall apart and come together again to support protein synthesis.
Alexander Mankin from the University of Illinois at Chicago says his group and that of Michael Jewett at the Northwestern University were trying to teach normal ribosomes new tricks, like getting it to translate “difficult-to-make” proteins or to take in unnatural amino acids to make special polymers. “We were frustrated with our inability to test or alter the functions of the ribosome,” says Mankin.
Trying to tweak the existing ribosomal RNA, which does much of the work of protein synthesis in the ribosome, didn’t go anywhere. Changes to it killed the cell.
So Mankin, Jewett and their teams considered making a portion of the ribosome that would be able to guide the ribosome into making the special polymers. But the problem is that the ribosome, made up of two subunits, falls apart and comes together in every cycle of protein synthesis. How would they stop the re-engineered portion of the ribosome from being swapped out by the normal subunit?
That’s when the idea of a tether came in. But “dissociation of ribosomal subunits was believed to be a prerequisite for efficient translation, and it was unclear whether ribosome with the tethered subunits would be functional,” says Mankin. Still, the investigators decided to give it a shot.
After many tries, one design worked: the Ribo-T. Mankin, Jewett and colleagues engineered a ribosomal RNA that combined sequences from the two subunits of the ribosome into a single unit. Short RNA linkers separated the two subunit RNAs in the contiguous stretch of nucleic acid.
And Ribo-T worked even better than anticipated. Not only did Ribo-T make proteins in a test tube, it also made proteins in bacterial cells that lacked naturally occurring ribosomes and keep the cells alive. Mankin still sounds surprised: “We have created probably the first-ever-on-Earth organism which lived with the ribosome where two subunits are combined into a single entity.”
He adds that Ribo-T could pave the way to exploring properties of the ribosome and to make a independent protein-synthesis system in cells that does not interfere with the ribosomes that take care of expression the rest of the cellular proteins. But, for now, the investigators are focusing on what sparked off the whole project in the first place: Getting Ribo-T to carry out the tasks that are difficult for normal ribosomes to do.
June 19, 2015 § 1 Comment
Sometimes the end doesn’t justify the means. In a recent paper in the Journal of Lipid Research, investigators describe how spinning high-density lipoproteins fast, a typical way to isolate them quickly, damages them. The finding suggests that the current understanding of the hydrodynamic properties and composition of HDL “is incorrect,” states William Munroe at the University of California, Los Angeles.
HDL, known as the “good cholesterol,” is an important lipoprotein in diagnosing cardiovascular disease. Its abundance in the bloodstream is considered to be a sign of good cardiovascular health because HDL carries away cholesterol.
Ever since the discovery in 1949 that lipoproteins can be separated and isolated in an ultracentrifuge, spinning lipoproteins like HDL at speeds 40,000 rpm or greater has been the norm. Samples often get spun at speeds of 65,000 to 120,000 rpm within 48 hours to hasten the isolation process.
But there have been whispers in the lipid community that the high speeds damage the molecules. So a trio of researchers at UCLA, led by Verne Schumaker, decided to see how speed affects HDL. “The phenomenon of HDL potentially exhibiting sensitivity to the ultracentrifuge speed is sometimes mentioned between lipoprotein researchers,” says Munroe, who is the first author on the paper. “However, there was little in the literature describing this phenomenon.”
In their JLR paper, Munroe, Schumaker and Martin Phillips showed that damage to HDL began as soon as the ultracentrifuge speed hit 30,000 rpm. Using mouse plasma samples, the investigators demonstrated that the damage got worse as the rotor went faster. Proteins, which are integral to the lipoproteins, got ripped out of the protein-lipid complexes, leaving few intact particles. “With enough gravitational force or time, this protein-deficient HDL undergoes further damage to lose lipid,” notes Munroe.
To try to circumvent the damage, the investigators tested out an alternative method for isolating HDL. They poured a potassium bromide density gradient over their sample. Next, they spun the gradient with the sample at a low speed of 15,000 rpm. Admittedly, the isolation took longer at 96 hours, but at least the amount of HDL that rose to the top of gradient was significantly higher than when using the conventional method.
Based on their findings, the investigators now want “to identify HDL-associated proteins that previous identification studies may have missed because certain proteins may have been completely lost from the recovered HDL particle during its isolation by ultracentrifugation,” says Munroe. “This may give insight into additional roles the HDL may participate in besides reverse cholesterol transport.”