New molecule shows promise in HIV vaccine design

Researchers at the University of Maryland and Duke University have designed a novel protein-sugar vaccine candidate that, in an animal model, stimulated an immune response against sugars that form a protective shield around HIV. The molecule could one day become part of a successful HIV vaccine.

“An obstacle to creating an effective HIV vaccine is the difficulty of getting the immune system to generate antibodies against the sugar shield of multiple HIV strains,” said Lai-Xi Wang, a professor of chemistry and biochemistry at UMD. “Our method addresses this problem by designing a vaccine component that mimics a protein-sugar part of this shield.”

Wang and collaborators designed a vaccine candidate using an HIV protein fragment linked to a sugar group. When injected into rabbits, the vaccine candidate stimulated antibody responses against the sugar shield in four different HIV strains. The results were published in the journal Cell Chemical Biology on October 26, 2017.

The protein fragment of the vaccine candidate comes from gp120, a protein that covers HIV like a protective envelope. A sugar shield covers the gp120 envelope, bolstering HIV’s defenses. The rare HIV-infected individuals who can keep the virus at bay without medication typically have antibodies that attack gp120.

Researchers have tried to create an HIV vaccine targeting gp120, but had little success for two reasons. First, the sugar shield on HIV resembles sugars found in the human body and therefore does not stimulate a strong immune response. Second, more than 60 strains of HIV exist and the virus mutates frequently. As a result, antibodies against gp120 from one HIV strain will not protect against other strains or a mutant strain.

To overcome these challenges, Wang and his collaborators focused on a small fragment of gp120 protein that is common among HIV strains. The researchers used a synthetic chemistry method they previously developed to combine the gp120 fragment with a sugar molecule, also shared among HIV strains, to mimic the sugar shield on the HIV envelope.

Next, the researchers injected the protein-sugar vaccine candidate into rabbits and found that the rabbits’ immune systems produced antibodies that physically bound to gp120 found in four dominant strains of HIV in circulation today. Injecting rabbits with a vaccine candidate that contained the protein fragment without the sugar group resulted in antibodies that primarily bound to gp120 from only one HIV strain.

“This result was significant because producing antibodies that directly target the defensive sugar shield is an important step in developing immunity against the target and therefore the first step in developing a truly effective vaccine,” Wang said.

Although the rabbits’ antibodies bound to gp120, they did not prevent live HIV from infecting cells. This result did not surprise Wang, who noted that it usually takes humans up to two years to build immunity against HIV and the animal study only lasted two months.

“We have not hit a home run yet,” Wang noted. “But the ability of the vaccine candidate to raise substantial antibodies against the sugar shield in only two months is encouraging; other studies took up to four years to achieve similar results. This means that our molecule is a relatively strong inducer of the immune response.”

The researchers’ next steps will be to conduct longer-term studies in combination with other vaccine candidates, hone in on what areas of gp120 the antibodies are binding to and determine how they can increase the antibodies’ effectiveness at neutralizing HIV.

Insights from a rare genetic disease may help treat multiple myeloma

A new class of drugs for blood cancers such as leukemia and multiple myeloma is showing promise. But it is hobbled by a problem that also plagues other cancer drugs: targeted cells can develop resistance. Now scientists, reporting in ACS Central Science, have found that insights into a rare genetic disease known as NGLY1 deficiency could help scientists understand how that resistance works — and potentially how drugs can outsmart it.

A class of compounds called proteasome inhibitors that include bortezomib and carfilzomib — both approved by the U.S. Food and Drug Administration — have been effective at treating certain types of blood cancers. The drugs work by jamming some of cancer cells’ machinery to induce cell death. But the drugs have been limited by cancer cells ability to development resistance, as well as the inhibitors inability to fight solid tumors effectively. Studies have suggested that resistance could be linked to a protein called Nrf1. When proteasome inhibitors go into action, Nrf1 is spurred into overdrive to restore the cells’ normal activities and keep them alive. If researchers could figure out how to block Nrf1, they might be able to address the resistance problem. Carolyn Bertozzi and colleagues, through studying NGLY1 deficiency, a seemingly unrelated condition, may have hit upon an approach to do this.

The researchers were investigating how lacking the enzyme NGLY1 causes a host of debilitating symptoms. They found that NGLY1 is responsible for activating Nrf1, the protein that is suspected of weakening proteasome inhibitors’ effectiveness against cancer. Further testing showed that dampening NGLY1 allowed a proteasome inhibitor to continue doing its work killing cancer cells without interference from Nrf1. This finding, the authors note, holds great promise for the development of combination therapeutics for blood cancers in the future.

MDI Biological Laboratory study finds immune system is critical to regeneration

The answer to regenerative medicine’s most compelling question — why some organisms can regenerate major body parts such as hearts and limbs while others, such as humans, cannot — may lie with the body’s innate immune system, according to a new study of heart regeneration in the axolotl, or Mexican salamander, an organism that takes the prize as nature’s champion of regeneration.

The study, which was conducted by James Godwin, Ph.D., of the MDI Biological Laboratory in Bar Harbor, Maine, found that the formation of new heart muscle tissue in the adult axolotl after an artificially induced heart attack is dependent on the presence of macrophages, a type of white blood cell. When macrophages were depleted, the salamanders formed permanent scar tissue that blocked regeneration.

The study has significant implications for human health. Since salamanders and humans have evolved from a common ancestor, it’s possible that the ability to regenerate is also built into our genetic code.

Godwin’s research demonstrates that scar formation plays a critical role in blocking the program for regeneration. “The scar shoots down the program for regeneration,” he said. “No macrophages means no cardiac regeneration.”

Godwin’s goal is to activate regeneration in humans through the use of drug therapies derived from macrophages that would promote scar-free healing directly, or those that would trigger the genetic programs controlling the formation of macrophages, which in turn could promote scar-free healing. His team is already looking at molecular targets for drug therapies to influence these genetic programs.

“If humans could get over the fibrosis hurdle in the same way that salamanders do, the system that blocks regeneration in humans could potentially be broken,” Godwin explained. “We don’t know yet if it’s only scarring that prevents regeneration or if other factors are involved. But if we’re really lucky, we might find that the suppression of scarring is sufficient in and of itself to unlock our endogenous ability to regenerate.”

The prevailing view in regenerative biology has been that the major obstacle to heart regeneration in mammals is insufficient proliferation of cardiomyocytes, or heart muscle cells. But Godwin found that cardiomyocyte proliferation is not the only driver of effective heart regeneration. His findings suggest that research efforts should pay more attention to the genetic signals controlling scarring.

The extraordinary incidence of disability and death from heart disease, which is the world’s biggest killer, is directly attributable to scarring. When a human experiences a heart attack, scar tissue forms at the site of the injury. While the scar limits further tissue damage in the short term, over time its stiffness interferes with the heart’s ability to pump, leading to disability and ultimately to terminal heart failure.

In addition to regenerating heart tissue following a heart attack, the ability to unlock dormant capabilities for regeneration through the suppression of scarring also has potential applications for the regeneration of tissues and organs lost to traumatic injury, surgery and other diseases, Godwin said.

Godwin’s findings are a validation of the MDI Biological Laboratory’s unique research approach, which is focused on studying regeneration in a diverse range of animal models with the goal of gaining insight into how to trigger dormant genetic pathways for regeneration in humans. In the past year and a half, laboratory scientists have discovered three drug candidates with the potential to activate regeneration in humans.

“Our focus on the study of animals with amazing capabilities for regenerating lost and damaged body parts has made us a global leader in the field of regenerative medicine,” said Kevin Strange, Ph.D., MDI Biological Laboratory president. “James Godwin’s discovery of the role of macrophages in heart regeneration demonstrates the value of this approach: we won’t be able to develop rational and effective therapies to enhance regeneration in humans until we first understand regeneration works in animals like salamanders.”

Godwin, who is an immunologist, originally chose to look at the function of the immune system in regeneration because its role as the equivalent of a first responder at the site of an injury means that it is responsible for preparing the ground for tissue repairs. The recent study was a follow-up to an earlier study which found that macrophages also play a critical role in limb regeneration.

The next step is to study the function of macrophages in salamanders and compare them with their human and mouse counterparts. Ultimately, Godwin would like to understand why macrophages produced by adult mice and humans don’t suppress scarring in the same way as in axolotls and then identify molecules and pathways that could be exploited for human therapies.

Godwin holds a dual appointment with The Jackson Laboratory, also located in Bar Harbor, which is focused on the mouse as a model animal. The dual appointment allows him to conduct experiments that compare genetic programming in the highly regenerative animals used as models at the MDI Biological Laboratory with genetic programming in neonatal and adult mice.

Stabilizing TREM2 — a potential strategy to combat Alzheimer’s disease

A gene called triggering receptor expressed on myeloid cells 2, or TREM2, has been associated with numerous neurodegenerative diseases, such as Alzheimer’s disease, Frontotemporal lobar degeneration, Parkinson’s disease, and Nasu-Hakola disease. Recently, a rare mutation in the gene has been shown to increase the risk for developing Alzheimer’s disease.

Independently from each other, two research groups have now revealed the molecular mechanism behind this mutation. Their research, published today in EMBO Molecular Medicine, sheds light on the role of TREM2 in normal brain function and suggests a new therapeutic target in Alzheimer’s disease treatment.

Alzheimer’s disease, just like other neurodegenerative diseases, is characterized by the accumulation of specific protein aggregates in the brain. Specialized brain immune cells called microglia strive to counter this process by engulfing the toxic buildup. But as the brain ages, microglia eventually lose out and fail to rid all the damaging material.

TREM2 is active on microglia and enables them to carry out their protective function. The protein spans the microglia cell membrane and uses its external region to detect dying cells or lipids associated with toxic protein aggregates. Subsequently, TREM2 is cut in two. The external part is shed from the protein and released, while the remaining part still present in the cell membrane is degraded. To better understand TREM2 function, the two research groups took a closer look at its cleavage. They were led by Christian Haass at the German Center for Neurodegenerative Diseases at the Ludwig-Maximilians-University in Munich, Germany, and Damian Crowther of AstraZeneca’s IMED Neuroscience group in Cambridge, UK together with colleagues at the Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto and the Cambridge Institute for Medical Research, University of Cambridge, UK.

Using different technological approaches, both groups first determined the exact site of protein shedding and found it to be at amino acid 157. Amino acid 157 was no unknown. Only recently, researchers from China had uncovered that a mutation at this exact position, referred to as p.H157Y, increased the risk of Alzheimer’s disease. Together, these observations indicate that protein cleavage is perturbed in the p.H157 mutant and that this alteration promotes disease development.

As a next step, Haass and Crowther’s groups investigated the biochemical properties of the p.H157Y mutant protein more closely. They found that the mutant was cleaved more rapidly than a healthy version of the protein. “Our results provide a detailed molecular mechanism for how this rare mutation alters the function of TREM2 and hence facilitates the progression of Alzheimer’s disease,” said Crowther.

While most TREM2 mutations affect protein production, the mechanism behind p.H157Y is somewhat different. The p.H157Y mutation allows the protein to be correctly manufactured and transported to the microglia cell surface, but then it is cleaved too quickly. “The end result is the same. In both cases, there is too little full-length TREM protein on microglia,” said Haass. “This suggests that stabilizing TREM2, by making it less susceptible to cleavage, may be a viable therapeutic strategy.”

Wistar scientists develop novel immunotherapy technology for prostate cancer

A study led by scientists at The Wistar Institute describes a novel immunotherapeutic strategy for the treatment of cancer based on the use of synthetic DNA to directly encode protective antibodies against a cancer specific protein. This is the first application of the new technology, called DNA-encoded monoclonal antibody (DMAb), for cancer immunotherapy. The study was published online in Cancer Immunology, Immunotherapy.

Prostate cancer is the second most common cancer in men worldwide. Traditional treatments are invasive and can impair the quality of life of patients, underscoring the need for alternative therapeutic strategies, including immunotherapy. One of the immunotherapeutic approaches that has been explored thus far relies on the use of monoclonal antibodies that specifically target a protein present on the surface of prostate cancer cells called prostate specific membrane antigen (PSMA) to elicit an anti-tumor immune response and control the cancer. Although promising, this strategy is limited by the production cost required to make these therapeutic antibodies. Additionally, multiple infusions are often required to achieve efficacy.

Wistar researchers devised a novel DNA-based approach in which an engineered DNA plasmid is constructed and used to deliver the instructions to make the desired anti-PSMA antibody so that the therapy can be generated in the patient’s body in a sustained manner. This research has important implications for the use of DNA-encoded monoclonal antibody technology as a platform for delivering the next generation of immunotherapies for cancer and many human diseases.

“This is an important demonstration of the possibilities opened up for immunotherapy by DMAb technology to direct in vivo production of antibodies of major relevance to human cancer,” said David B. Weiner, Ph.D., executive vice president of The Wistar Institute, director of The Wistar Institute Vaccine & Immunotherapy Center, W.W. Smith Charitable Trust Professor in Cancer Research, and senior author of the study. “There is a great need for such new approaches for prostate disease as well as many other cancers. As recent data suggest, PSMA is an important cancer antigen expressed on many human prostate, bladder, renal as well as ovarian cancers, so additional study of the possible benefits of this therapy are important.”

The new technology was tested in mice for the ability to generate antibodies in their blood stream that would target human PSMA as well as target PSMA-positive tumors. Results showed that antibodies were able to bind to the cancer cells and recruited specific immune cells called natural killer cells, resulting in shrinkage of the tumor, significantly improving survival.

“Our data provide proof of concept that DMAb engineered DNA plasmids can be successfully used to target important cancers,” said Kar Muthumani, M.Sc., Ph.D., assistant professor in the Translational Tumor Immunology Program at Wistar, member of the Vaccine & Immunotherapy Center and lead author of the study. “The unique features of our synthetic DNA-based system make it a promising novel approach for cancer therapy, alone or in combination with other treatments.”

Skewing the aim of targeted cancer therapies

Headlines, of late, have touted the successes of targeted gene-based cancer therapies, such as immunotherapies, but, unfortunately, also their failures.

Broad inadequacies in a widespread biological concept that affects cancer research could be significantly deflecting the aim of such targeted drugs, according to a new study. A team exploring genetic mechanisms in cancer at the Georgia Institute of Technology has found evidence that a prevailing concept about how cells produce protein molecules, particularly when applied to cancer, could be erroneous as much as two-thirds of the time.

Prior studies by other researchers have also critiqued this concept about the pathway leading from genetic code to proteins, but this new study, led by cancer researcher John McDonald, has employed rare analytical technology to explore it in unparalleled detail. The study also turned up novel evidence for regulating mechanisms that could account for the prevailing concept’s apparent shortcomings.

RNA concept incomplete

The concept stems from common knowledge about the assembly line inside cells that starts with code in DNA, is transcribed to messenger RNA, then translated into protein molecules, the cell’s building blocks.

That model seems to have left the impression that cellular protein production works analogously to an old-style factory production line: That the amount of a messenger RNA encoded by DNA on the front end translates directly into the amount of a corresponding protein produced on the back end. That idea is at the core of how gene-based cancer drug developers choose their targets.

To put that assumed congruence between RNA production and protein production to the test, the researchers examined — in ovarian cancer cells donated by a patient — 4,436 genes, their subsequently transcribed messenger RNA, and the resulting proteins. The assumption, that proverbial factory orders passed down the DNA-RNA line determine in a straightforward manner the amount of a protein being produced, proved incorrect 62 percent of the time.

RNA skews drug cues

“The messenger RNA-protein connection is important because proteins are usually the targets of gene-based cancer therapies,” McDonald said. “And drug developers typically measure messenger RNA levels thinking they will tell them what the protein levels are.” But the significant variations in ratios of messenger RNA to protein that the researchers found make the common method of targeting proteins via RNA seem much less than optimal.

McDonald, Mengnan Zhang and Ronghu Wu published their results on August 15, 2017 in the journal Scientific Reports. The work was funded by the Ovarian Cancer Institute, The Deborah Nash Endowment, Atlanta’s Northside Hospital and the National Science Foundation. The spectrophotometric technology needed to closely identify a high number of proteins is rare and costly but is available in Wu’s lab at Georgia Tech.

Whereas many studies look at normal tissue versus cancerous tissue, this new study focused on cancer progression, or metastasis, which is what usually makes cancer deadly. The researchers looked at primary tumor tissue and also metastatic tissue.

Hiding drug targets

“The idea that any change in RNA level in cancerous development flows all the way up to the protein level could be leading to drug targeting errors,” said McDonald, who heads Georgia Tech’s Integrated Cancer Research Center. Drug developers often look for oddly high messenger RNA levels in a cancer then go after what they believe must be the resulting oddly high levels of a corresponding protein.

Taking messenger RNA as a protein level indicator could actually work some of the time. In the McDonald team’s latest experiment, in 38 percent of the cases, the rise of RNA levels in cancerous cells did indeed reflect a comparable rise of protein levels. But in the rest of cases, they did not.

“So, there are going to be many instances where if you’re predicting what to give therapeutically to a patient based on RNA, your prescription could easily be incorrect,” McDonald said. “Drug developers could be aiming at targets that aren’t there and also not shooting for targets that are there.”

RNA muted or magnified

The analogy of a factory producing building materials can help illustrate what goes wrong in a cancerous cell, and also help describe the study’s new insights into protein production. To complete the metaphor: The materials produced are used in the construction of the factory’s own building, that is, the cell’s own structures.

In cancer cells, a mutation makes protein production go awry usually not by deforming proteins but by overproducing them. “A lot of mutations in cancer are mutations in production levels. The proteins are being overexpressed,” said McDonald, who is also a professor in Georgia Tech’s School of Biological Sciences.

A bad factory order can lead to the production of too much of a good material and then force it into the structures of the cell, distorting it. The question is: Where in the production line do bad factory orders appear?

According to the new study, the answer is less straightforward than perhaps previously thought.

Micro RNA managing

The orders don’t all appear on the front end of the assembly line with DNA over-transcribing messenger RNA. Additionally, some mutations that do over-transcribe messenger RNA on the front end are tamped down or canceled by regulating mechanisms further down the line, and may never end up boosting protein levels on the back end.

Regulating mechanisms also appear to be making other messenger RNA, transcribed in normal amounts, unexpectedly crank out inordinate levels of proteins.

At the heart of those regulating systems, another RNA called micro RNA may be micromanaging how much, or little, of a protein is actually produced in the end.

“We have evidence that micro RNAs may be responsible for the non-correlation between the proteins and the RNA, and that’s completely novel,” McDonald said. “It’s an emerging area of research.”

Micro RNA, or miRNA, is an extremely short strand of RNA.

No one at fault

McDonald would like to see tissues from more cancer patients undergo similar testing. “Right now, with just one patient, the data is limited, but I also really think it shows that the phenomenon is real,” McDonald said.

“Many past studies have looked at one particular protein and a particular gene, or a particular handful. We looked at more than 4,000,” McDonald said. “What that brings up is that the phenomenon is probably not isolated but instead genome-wide.”

The study’s authors would also like to see rarely accessible, advanced protein detecting technology become more widely available to biomolecular researchers, especially in the field of cancer drug development. “Targeted gene therapy is a good idea, but you need the full knowledge of whether it’s affecting the protein level,” McDonald said.

He pointed out that no one is at fault for the possible incompleteness of commonly held concepts about protein production.

As science progresses, it naturally illuminates new details, and formerly useful ideas need updating. With the existence of new technologies, it may be time to flesh out this particular concept for the sake of cancer research progress.

Cell mechanism discovery could lead to ‘fundamental’ change in leukaemia treatment

Researchers have identified a new cell mechanism that could lead to a fundamental change in the diagnosis and treatment of leukaemia.

A team in the University of Kent’s pharmacy school conducted a study that discovered that leukaemia cells release a protein, known as galctin-9, that prevents a patient’s own immune system from killing cancerous blood cells.

Acute Myeloid Leukaemia (AML) — a type of blood cancer that affects over 250,000 people every year worldwide — progresses rapidly because its cells are capable of avoiding the patient’s immune surveillance. It does this by inactivating the body’s immune cells, cytotoxic T lymphocytes and natural killer (NK) cells.

Existing treatment strategies consist of aggressive chemotherapy and stem cell transplantation, which often do not result in effective remission of the disease. This is because of a lack of understanding of the molecular mechanisms that allow malignant cells to escape attack by the body’s immune cells.

Now the researchers at the Medway School of Pharmacy, led by Dr Vadim Sumbayev, Dr Bernhard Gibbs and Professor Yuri Ushkaryov, have found that leukaemia cells — but not healthy blood cells — express a receptor called latrophilin 1 (LPHN1). Stimulation of this receptor causes these cancer cells to release galectin-9, which then prevents the patient’s immune system from fighting the cancer cells.

The discovery of this cell mechanism paves the way for new ‘biomarkers’ for AML diagnosis, as well as potential targets for AML immune therapy, say the researchers.

‘Targeting this pathway will crucially enhance patients own immune defences, helping them to eliminate leukaemia cells’, said Dr Sumbayev. He added that the discovery has the potential to also be beneficial in the treatment of other cancers.

Epilepsy drug therapies to be improved by new targeted approach

New research from the University of Liverpool, in collaboration with the Mario Negri Institute in Milan, published today in the Journal of Clinical Investigation, has identified a protein that could help patients with epilepsy respond more positively to drug therapies.

Epilepsy continues to be a serious health problem and is the most common serious neurological disease. Despite 30 years of drug development, approximately 30% of people with epilepsy do not become free of fits (also called seizures) with currently available drugs.

New, more effective drugs are therefore required for these individuals. We do not fully understand why some people develop seizures, why some go onto develop epilepsy (continuing seizures), and most importantly, why some patients cannot be controlled with current drugs.

Inflammation

There is now increasing body of evidence suggesting that local inflammation in the brain may be important in preventing control of seizures. Inflammation refers to the process by which the body reacts to insults such as having a fit. In most cases, the inflammation settles down, but in a small number of patients, the inflammation continues.

The aim of the research, undertaken by Dr Lauren Walker while she was a Medical Research Council (MRC) Clinical Training Fellow, was to address the important question of how can inflammation be detected by using blood samples, and whether this may provide us with new ways of treating patients in the future to reduce the inflammation and therefore improve seizure control.

The research focused on a protein called high mobility group box-1 (HMGB1), which exists in different forms in tissues and bloodstream (called isoforms), as it can provide a marker to gauge the level of inflammation present.

Predicting drug response

The results showed that there was a persistent increase in these isoforms in patients with newly-diagnosed epilepsy who had continuing seizure activity, despite anti-epileptic drug therapy, but not in those where the fits were controlled.

An accompanying drug study also found that HMGB1 isoforms may predict how an epilepsy patient’s seizures will respond to anti-inflammatory drugs.

Dr Lauren Walker, said: “Our data suggest that HMGB1 isoforms represent potential new drug targets, which could also identify which patients will respond to anti-inflammatory therapies. This will require evaluation in larger-scale prospective trials.”

Innovative scheme

Professor Sir Munir Pirmohamed, Director of the MRC Centre for Drug Safety Science and Programme lead for the MRC Clinical Pharmacology scheme, said: “The MRC Clinical Pharmacology scheme is a highly successful scheme to train “high flyers” who are likely to become future leaders in academia and industry.

“Dr Walker’s research is testament to this and shows how this innovative scheme, which was jointly funded by the MRC and Industry, can tackle areas of unmet clinical need, and identify new ways of treating patients with epilepsy using a personalised medicine approach”.

Why do aged muscles heal slowly?

As we age, the function and regenerative abilities of skeletal muscles deteriorate, which means it is difficult for the elderly to recover from injury or surgery. New work from Carnegie‘s Michelle Rozo, Liangji Li, and Chen-Ming Fan demonstrates that a protein called b1-integrin is crucial for muscle regeneration. Their findings, published by Nature Medicine, provide a promising target for therapeutic intervention to combat muscle aging or disease.

Muscle stem cells are the primary source of muscle regeneration after injury. These specialized adult stem cells lie dormant in the muscle tissue–off to the side of the individual muscle fibers, which is why they were originally dubbed satellite cells. When muscle fibers are damaged, they activate and proliferate. Most of the new cells go on to make new muscle fibers and restore muscle function. Some return to dormancy, which allows the muscle to keep repairing itself over and over again.

Rozo, the lead author, determined that the function of integrins (or, more specifically, the protein called β1-integrin) is absolutely crucial for maintaining the cycle of hibernation, activation, proliferation, and then return to hibernation, in muscle stem cells. Integrins are proteins that ‘integrate’ the outside to the inside of the cell, providing a connection to the immediate external environment, and without them, almost every stage of the regenerative process is disrupted.

The team theorized that defects in β1-integrin likely contribute to phenomena like aging, which is associated with reduced muscle stem cell function and decreased quantities of muscle stem cells. This means that healing after injury or surgery is very slow, which can cause a long period of immobility and an accompanying loss of muscle mass.

“Inefficient muscular healing in the elderly is a significant clinical problem and therapeutic approaches are much needed, especially given the aging population-and I am including myself in this population,” Fan explained. “Finding a way to target muscle stem cells could greatly improve muscle renewal in older individuals.”

Rozo and Li determined that the function of β1-integrin is diminished in aged muscle stem cells. Furthermore, when they artificially activated integrin in mice with aged muscles, their regenerative abilities were restored to youthful levels. Importantly, improvement in regeneration, strength, and function were also seen when this treatment was applied to animals with muscular dystrophy, underscoring its potential importance for the treatment of muscle disorders.

Muscle stem cells use b1-integrin to interact with many other proteins in the muscle external environment. Among these many proteins, they found a clue that one called fibronectin might be most relevant. To connect b1-integrin to fibronectin, they teamed up with another group led by scientists from the Nestlé Institute of Health Sciences, in addition to researchers from the EPFL EDBB Doctoral Program, the Leibniz Institute for Age Research, the Ottawa Hospital Research Institute, and the Max Planck Institute of Biochemistry.

They discovered that aged muscles contain substantially reduced levels of fibronectin compared to young muscles. Like b1-integrin, eliminating fibronectin from young muscles makes them appear as if they were old, and restoring fibronectin to aged muscle tissue restores muscle regeneration to youthful levels. Their joint efforts demonstrated a strong link between b1-integrin, fibronectin and muscle stem cell regeneration, which is the subject of a second paper, also published by Nature Medicine in the same issue.

“Taken together, our results show that aged muscle stem cells with compromised b1-integrin activity and aged muscles with insufficient amount of fibronectin both root causes of muscle aging. This makes b1-integrin and fibronectin very promising therapeutic targets,” Fan said.

UMN researchers find distinct differences in structure, features of retroviruses

In the most comprehensive study of its kind, researchers in the Institute for Molecular Virology and School of Dentistry at the University of Minnesota report that most types of retroviruses have distinct, non-identical virus structures.

Researchers analyzed seven different retroviruses including two types of HIV as well as HTLV-1, a virus that causes T-cell leukemia. They also examined retroviruses that infect birds, mice, chimpanzees and fish, that can cause cancer or immunodeficiency.

“Each kind of retrovirus has distinct structural features and each assembles virus particles differently,” said Louis Mansky, Ph.D., director of the Institute for Molecular Virology, who is also a member of the Masonic Cancer Center. “Most researchers assume that all retroviruses are just like HIV, but they’re not. We cannot take a one-size-fits-all approach when studying retroviruses and discovering new strategies for antiviral treatments or vaccines.”

Mansky’s team looked at the behavior of retrovirus Gag proteins, which drive retrovirus particle formation. Once the virus enters a cell, reverse transcriptase converts the viral RNA to DNA, which subsequently creates the Gag protein.

Understanding the nature of Gag protein interactions with one another and how the structures form will help scientists better understand how and why the virus works. It will also help identify ways to target the virus and prevent it from infecting a cell in the first place.

The study examined virus-like particle size, cellular distribution and basic morphological features through three distinct microscopy techniques.

The team noted that:

– HIV-1 and HIV-2 have Gag proteins that assemble retrovirus-like particles with distinct structures and sizes, which implies that differences exist in how the two HIV types form new virus particles. – HIV and HTLV-1 particles are quite distinct from one another in appearance, which also suggests fundamental differences in virus particle assembly.

“We found significant differences among the retroviruses,” said Jessica Martin, senior Ph.D. student in the Department of Pharmacology and lead author on the study. “A parallel comparative study evaluating retroviral Gag proteins and virus particle intermediates of this size and scope has never been done before.”

The team was surprised to find that one of the retroviruses, walleye dermal sarcoma virus (WDSV), did not readily produce virus particles.The disease can affect anything from 1-30 percent of walleye in a population, depending on the location. This research could help aquatic scientists better understand how to control the disease.

“Our study helps to highlight the importance of serendipity of basic science research,” Mansky said. “We set out to learn more about the differences among two important human retroviruses, namely HIV and HTLV, which we did, but our findings also shed light on important differences among all kinds of retroviruses that could inform not only the treatment of human viral diseases but could also impact aquatic health in fisheries.”

The study findings will help serve as a foundation for studying differences among retroviruses, including HIV.

“The scientific community can build off of our findings to develop new antiviral treatments, and hopefully determine how to stop these viruses from causing deadly diseases in humans such as cancer and AIDS,” Mansky said.

Get a clue: Biochemist studies fruit fly to understand Parkinson’s disease, muscle wasting

The fruit fly may help us be less clueless about human muscle development and Parkinson’s disease.

Erika Geisbrecht, Kansas State University associate professor of biochemistry and molecular biophysics, is studying the fruit fly, or Drosophila melanogaster, to understand a gene called clueless, or clu. Geisbrecht and her research team have found a connection between clu and genes that cause Parkinson’s disease.

Geisbrecht’s team is among the first to focus on the connection between clu and mitochondrial function in fruit fly muscle cells. The researchers recently published their work in the journal Human Molecular Genetics.

“We are trying to understand how muscles develop and how healthy muscles are maintained throughout the entire life of a fruit fly in the hopes of applying this knowledge to the human body,” Geisbrecht said.

Geisbrecht uses fruit fly muscles as a model for human muscles because of their similar structures — approximately 85 percent of the human disease genes have corresponding genes in the fruit fly. Fruit flies also have a short lifecycle of 10 days from when the egg is laid to when adults emerge, which allows for the rapid observation of muscle development and maintenance.

The connection between fruit fly and human muscles has made it possible to understand the role of the clu gene that — when mutated — causes defects in the localization and turnover of damaged mitochondria. A buildup of damaged mitochondria ultimately affects the ability of muscles and nerves to function properly. Geisbrecht and her team are just beginning to understand how clu interacts with a gene called parkin that — when mutated in humans — results in Parkinson’s disease.

People who are born with mutations in the parkin gene do not develop Parkinson’s disease until later in adult life. The same is true for fruit flies with defects in clu or parkin: These fruit flies proceed through the larval and pupal stage of insect development and emerge as adults, but quickly die because their muscles and neurons degenerate.

“If you think about a tissue in the body that uses more energy than anything else, of course it’s your muscle tissue,” Geisbrecht said. “Proper mitochondrial function is essential to having healthy, developed muscles. It’s an important connection.”

Geisbrecht’s team plans to continue studying fruit flies to better understand the connection between human disease genes and muscle function. Their work could lead to better treatment for Parkinson’s disease or other muscle diseases.

Aside from muscle or neurodegenerative diseases, maintaining healthy muscle tissue also is important in the general population, where common diseases can also lead to muscle problems. For example, muscle wasting, or muscle deterioration, can be a huge problem for people with diabetes or cancer.

“Muscle wasting for people with end-stage diabetes or cancer is often a bigger problem than the cancer or diabetes itself because it causes people to become immobile and lose the ability to make their muscles function well again,” Geisbrecht said. “We want to understand what happens at the cellular level — what these genes or proteins are doing.”

When it comes to developing stem cell treatments, seeing is half the battle

Stem cell therapies hold great promise in treating a variety of diseases, but in order to develop them researchers must first be able to monitor them inside the body. Enter the contrast agent.

“Often things go wrong right away when stem or therapeutic cells are injected into the body because some cells can migrate, while other dying cells are eaten by the body and expelled,” says Xiao-an Zhang, an assistant professor of chemistry at U of T Scarborough.

“It’s important to know if these cells are being injected in the right place, where they go once inside the body and whether they’re still alive. The only way to do that is with an effective contrast agent.”

Conventional contrast agents are directly injected into the body to make some tissues or diseases more clearly visible on MRI scans. The one developed by Zhang and his team, including Ph.D. student Inga Haedicke, U of T Assistant Professor Hai-Ling Margaret Cheng and two research groups (Dr. Timothy Scholl and Dr. Paula Foster) from Western University, can improve monitoring at the cellular level, which is a crucial element in measuring the effectiveness of stem and therapeutic cell treatments.

While still early in development, stem and therapeutic cells may one day offer effective treatments against diseases, particularly cancer. But one major hurdle in developing these treatments is an inability to effectively monitor them once inside the body.

“Current MRI methods are not great at looking at what’s going on at the cell or sub-cellular level. It’s just not powerful enough – the resolution and sensitivity needs to be higher and the contrast needs to be greater in order to distinguish one type of cell from another,” says Zhang.

“Traditional MRI can’t do quite what we need it to do right now, but there’s huge opportunity to improve the technology.”

Magnetic resonance imaging (MRI) uses a magnetic field and radio waves to produce a detailed image of tissues within the body. MRI is ideal for medical imaging because it’s milder than techniques like CT scans or X-rays since it’s not as invasive and does not rely on ionizing radiation.

There are quite a few technical challenges when developing an effective contrasting agent for cellular imaging. For one, it needs to be non-toxic or low in toxicity so it doesn’t damage cells. It also needs to be able to enter cells and accumulate in order to provide a clear enough contrast from the rest of objects in the image, notes Zhang.

The agent developed by Zhang and his team addresses these challenges by using manganese porphyrin. Manganese is a micronutrient that the body’s cells can handle and forms a very stable complex with the porphyrin, a type of pigment molecule that can help to achieve the necessary properties. Using a new porphyrin developed by Haedicke, the agent can be delivered to cells more efficiently in smaller concentrations and can accumulate more effectively than current alternatives.

“The real positive aspect of this design is that it’s cell permeable, and that the new mechanism allows it to stay inside the cell and accumulate,” says Haedicke. “This is extremely important for labelling which cells are being monitored.”

The research, which will be published in the journal Chemical Science, received funding from the Canada Foundation for Innovation (CFI) and the Natural Sciences and Engineering Research Council of Canada (NSERC).

The next steps for Zhang and his team is to focus mostly on using the agent on different types of stem cells and to test them in vivo.

No symptoms, but could there be cancer? Our chemosensor will detect it!

Many cancers could be successfully treated if the patient consulted the doctor sufficiently early. But how can a developing cancer be detected if it doesn’t give rise to any symptoms? In the near future, suitably early diagnosis could be provided by simple and cheap chemical sensors – thanks to special recognizing polymer films developed at the Institute of Physical Chemistry of the Polish Academy of Sciences in Warsaw.

These days, cancer is no longer a death sentence for the patient. However, the best chances of recovery are when the correct treatment is undertaken at an early stage of the disease. This is where the trouble starts, because many tumours develop over a long period without any symptoms. One solution to this problem could be diagnostic tests available to everyone that could be performed by people themselves and on a relatively regular basis. A step bringing us closer to this sort of personalized medical diagnosis and cancer prophylaxis is the chemical sensor devised and fabricated by Prof. Wlodzimierz Kutner’s group from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw using a grant from the National Science Centre, in collaboration with the team of Prof. Francis D’Souza of the University of North Texas in Denton TX, USA.

The most important element of the chemosensor devised at the IPC PAS is a thin film of the polymer that detects molecules of neopterin. Neopterin – in chemical terminology known as 2-amino-6-(1,2,3-trihydroxypropyl)-1H-pteridin-4-one) – is an aromatic compound present in human body fluids, such as serum, urine, and cerebrospinal fluid. Produced by the immune system, it is regarded as a universal marker in medical diagnosis. The concentration of this biomarker rises significantly particularly in the case of certain neoplastic diseases, e.g., malignant lymphoma, although elevated levels of neopterin are also seen in some viral and bacterial infections, as well as in diseases of parasitic aetiology. In turn, in transplant patients, increased levels of neopterin signal probable rejection.

“How can we detect the presence of neopterin? A reasonable approach is to use special recognizing materials for this purpose, prepared by molecular imprinting. This technique involves ‘stamping out’ molecules of the desired compound – their shape, but also at least some of the chemical characteristics – in a carefully designed polymer,” explains Dr. Piyush Sindhu Sharma (IPC PAS), the lead author of an article published in the Biosensors and Bioelectronics journal.

During the preparation of the polymer film, molecules of the substance being detected – in this case neopterin – are in a working solution in which their binding sites have to link with recognizing sites of so-called functional monomers. In turn, these monomers should be able to form connections with another monomer, a cross-linking agent which together, after polymerization, form a rigid support structure of the polymer. Next, the molecules of the compound used as a template are washed out from the structure. The result is a durable polymer with molecular cavities of a shape and chemical properties ensuring the capture of molecules of the desired compound from its surroundings.

The basic difficulty in molecular imprinting is the selection of the appropriate functional and cross-linking monomers as well as solvents, their proportions and reaction conditions. PhD student Agnieszka Wojnarowicz (IPC PAS) explains: “With the aid of quantum-chemical calculations, we first check whether there is bonding between our template molecule and selected functional monomers, and whether they will be stable in the solvent used. We also check whether the molecular cavities formed are sufficiently selective, i.e., whether they will primarily capture the molecules we are detecting, and not any that are similar to them. When the calculation results confirm our expectations, that is when we proceed to their experimental confirmation.”

At the IPC PAS a recognizing polymer film with molecular cavities from neopterin has been produced on the surface of an electrode. After immersion in artificial blood serum spiked with neopterin, the film on the electrode captured molecules of the latter, thus leading to a decrease in electrical potential in the connected measuring system. The tests showed that the molecular cavities of the polymer were almost entirely filled with molecules of neopterin despite the presence of molecules of similar structure and properties. This result means that the probability of false positive detection (detecting the presence of neopterin in body fluids not containing it) is negligibly small. The new chemical sensor therefore mainly reacts to what it should react to – and nothing else.

“At present, our chemosensor is a piece of laboratory equipment. However, the production of its key element, that is, the recognizing polymer film, does not pose major problems, and the electronics responsible for electrical measurements can easily be miniaturized. There is nothing standing in the way of building simple and reliable diagnostic equipment, based on our development, in just a few years’ time, which would be affordable not only for medical institutions and doctors’ surgeries, but also for the public in general,” predicts Prof. Kutner (IPC PAS).

Genetic Variations that Boost PKC Enzyme Contribute to Alzheimer’s Disease

In Alzheimer’s disease, plaques of amyloid beta protein accumulate in the brain, damaging connections between neurons. Now, researchers at University of California San Diego School of Medicine and Harvard Medical School have found that the enzyme Protein Kinase C (PKC) alpha is necessary for amyloid beta to damage neuronal connections. They also identified genetic variations that enhance PKC alpha activity in patients with Alzheimer’s disease.

The study, published May 10 in Science Signaling, may present a new therapeutic target for the disease.

“Until recently, it was thought that PKC helped cells survive, and that too much PKC activity led to cancer. Based on that assumption, many companies tested PKC inhibitors as drugs to treat cancer, but they didn’t work,” said co-senior author Alexandra Newton, PhD, professor of pharmacology at UC San Diego School of Medicine.

“Instead, we recently found that the opposite is true. PKC serves as the brakes to cell growth and survival, so cancer cells benefit when PKC is inactivated. Now, our latest study reveals that too much PKC activity is also bad, driving neurodegeneration. This means that drugs that failed in clinical trials for cancer may provide a new therapeutic opportunity for Alzheimer’s disease.”

The study was a three-way collaboration between experts in PKC (Newton), neuroscience (Roberto Malinow, MD, PhD, Distinguished Professor of Neurosciences and Neurobiology and holder of the Shiley-Marcos Endowed Chair in Alzheimer’s Disease in Honor of Dr. Leon Thal at UC San Diego School of Medicine) and genomics (Rudolph Tanzi, PhD, professor of neurology at Harvard Medical School).

Malinow’s team found that when mice are missing the PKC alpha gene, neurons functioned normally, even when amyloid beta was present. Then, when they restored PKC alpha, amyloid beta once again impaired neuronal function. In other words, amyloid beta doesn’t inhibit brain function unless PKC alpha is active.

Enter the Tanzi team, which has a database of genetic information for 1,345 people in 410 families with late-onset Alzheimer’s disease. Tanzi and team use this database to look for rare variants — genetic mutations found only in family members with the disease. Here, the team found three variants in one form of the PKC enzyme, PKC alpha that were associated with the disease in five families.

The researchers replicated these three PKC alpha gene variants in laboratory cell lines. In each instance, PKC alpha activity was increased.

While this study surfaced only five families with these rare mutations in the PKC alpha gene, there are many ways to influence PKC alpha’s activity, Newton said. She believes there could be many other inherited genetic variations that indirectly boost or inhibit PKC activity, and therefore also influence a person’s likelihood of developing Alzheimer’s disease.

“Next we want to identify more molecules participating in the pathophysiology,” said Malinow. “The more steps in the mechanism we can understand, the more therapeutic targets we’ll find for Alzheimer’s disease.”