Studying patterns in bacterial organization

credit to Gerard Wong, of the California NanoSystems Institute

Bacterial biofilms, at first glance, may seem to be spontaneous, random phenomena from which we have no power to protect our environment or ourselves.

They’re potentially useful as an aid to wastewater treatment, but they also cause infections that account for $6 billion a year in health care costs. Biofilms are also more resistant to antibiotic drugs, making them difficult to eradicate.

Dr. Kun Zhao, of the California NanoSystems Institute at UCLA, refuses to see biofilms as arbitrary: he emphasizes the fact that biofilms are communities of bacteria in self-produced polymeric matrices of polysaccharides, and using a biophysical approach, he studies the pattern behind their organization.

Central questions in Zhao’s research include how bacterial colonies transition from reversible to irreversible attachment, how they migrate, and how they ultimately disperse. Specifically, Zhao examines the polysaccharide Psl, which poses a positive feedback loop because it is both secreted by moving bacteria and serves as a chemo-attractant for future bacteria movement. The positive feedback creates an inherent pattern, as bacteria are more likely to visit a location they have been to before.

Zhao and colleagues have also discovered that bacterial mutants that cannot produce Psl exhibit more random and uniform movement.

To better quantify bacterial movement,Zhao has created a computer algorithm that shows the full movement history of each individual bacterium on a dish, and that provides a “search engine” allowing researchers to find every bacterium performing specific life cycle activities, like division.

Zhao has postulated a “rich get richer” mechanism for biofilms. He compares bacterial organization to Wall Street because concentrated movement ensures that some cells become extremely enriched. In the future, he hopes to model colloidal structures for biological problems, like the growth of the bacterial cell wall. Zhao currently uses colloids, which in physics are used as models for atomic systems, to observe how shapes affect self-assembly. He also would like to look at cell-substrate interactions, which are implicated in bacterial territoriality and social interactions.

by Olivia Zhu

Source: Duke University

EGFR-Mediated Beclin 1 Phosphorylation in Autophagy Suppression, Tumor Progression, and Tumor Chemoresistance

EGFR negatively regulates autophagy by binding to Beclin 1.
Active EGFR phosphorylates Beclin 1 and alters its interactome.
EGFR suppression of Beclin 1 may contribute to tumor progression in lung cancer.

Lung cancer responses to EGFR inhibitors may involve activation of Beclin 1. Image Credit: Cell Press

Summary
Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.

Introduction
Epidermal growth factor receptor (EGFR), an oncogenic receptor tyrosine kinase, links extracellular signals to cellular homeostasis. In normal cells, EGFR signaling is triggered by the binding of growth factors, such as epidermal growth factor (EGF), leading to homodimerization or heterodimerization with other EGFR family members (such as HER2/neu) and autophosphorylation of the intracellular domain (Lemmon and Schlessinger, 2010). The phosphotyrosines formed serve as a docking site for adaptor molecules, which results in the activation of signaling pathways including the Ras/MAPK pathway, the PI3K/Akt pathway, and STAT signaling pathways. In tumor cells, the tyrosine kinase activity of EGFR may be dysregulated by EGFR gene mutation, increased EGFR gene copy number, or EGFR protein overexpression, leading to aberrant EGFR signaling and increased tumor cell survival, proliferation, invasion, and metastasis ( Ciardiello and Tortora, 2008). EGFR signaling is deregulated in many human cancers, including those of the lung, head and neck, colon, pancreas, and brain.

The deregulation of EGFR in human cancers has led to the development of anticancer agents that target EGFR, including: (1) anti-EGFR antibodies that inhibit ligand binding and (2) small-molecule receptor tyrosine kinase inhibitors (TKIs), erlotinib and gefitinib, that block EGFR intracellular tyrosine kinase activity. Although the EGFR TKIs have shown limited clinical benefit in the majority of solid tumors, they are effective in non-small-cell lung carcinomas (NSCLCs) that harbor specific mutations in the tyrosine kinase domain of EGFR (most commonly, in-frame deletion in exon 19 around codons 746–750 or single-base substitution, L858R, in exon 21) (Ciardiello and Tortora, 2008, Lynch et al., 2004 and Pao and Chmielecki, 2010). Most patients with NSCLCs with EGFR mutations initially respond favorably to erlotinib or gefitinib, suggesting these mutations drive tumorigenesis. However, among tumors that initially respond to EGFR TKIs, most eventually acquire resistance, often due to the emergence of a secondary mutation, T790M, in the kinase domain of EGFR (Pao and Chmielecki, 2010).

Several studies have shown that EGFR signaling regulates autophagy, a lysosomal degradation pathway that functions in cellular homeostasis and protection against a variety of diseases, including cancer (Levine and Kroemer, 2008). The downstream targets of EGFR—PI3K, Akt, and mammalian target of rapamycin (mTOR)—are well-established negative regulators of autophagy (Botti et al., 2006). Moreover, EGFR inhibitors induce autophagy in NSCLCs (Gorzalczany et al., 2011 and Han et al., 2011) and other cancer cells (Fung et al., 2012). However, the links between EGFR signaling and autophagy remain poorly understood, particularly (1) the molecular mechanisms by which EGFR signaling suppresses autophagy, (2) the role of EGFR suppression of autophagy in lung cancer pathogenesis, and (3) the role of autophagy induction in the response to TKI therapy. EGFR inhibitor-induced autophagy in lung cancer cells has been postulated to exert either cytoprotective (Han et al., 2011) or cytotoxic (Gorzalczany et al., 2011) effects.

Conflicting results regarding the role of autophagy in the response or resistance to EGFR TKI treatment reflects broader uncertainties in the role of autophagy in cancer therapy (Rubinsztein et al., 2012). It is not understood in what contexts autophagy induction contributes to tumor progression or suppression and to tumor chemoresistance or chemosensitivity. There is a general consensus that autophagy prevents tumor initiation, as loss-of-function mutations of several different autophagy genes results in spontaneous tumorigenesis (beclin 1, Atg5, and Atg7) and/or increased chemical-induced tumorigenesis (Atg4C) in mice ( Rubinsztein et al., 2012). Despite this inhibitory role in tumor initiation, it has been proposed that autophagy may promote the growth of established tumors and contribute to chemoresistance, principally through its actions to prolong the survival of metabolically stressed neoplastic cells ( Rubinsztein et al., 2012).

To understand the relationship between oncogenic signaling, autophagy, and distinct stages of tumorigenesis, it is important to define the molecular mechanisms by which oncogenic signaling regulates autophagy. We recently showed that the oncogene Akt inhibits autophagy independently of mTOR signaling via serine phosphorylation of the essential autophagy protein, Beclin 1 (Wang et al., 2012), a haploinsufficient tumor suppressor protein frequently monoallelically deleted in human breast and ovarian cancer (Levine and Kroemer, 2008). Moreover, Akt-mediated phosphorylation of Beclin 1 contributes to Akt-dependent fibroblast transformation, supporting the concept that inactivation of Beclin 1-dependent autophagy plays a role in tumor initiation. However, it is not known whether oncogenic inactivation of Beclin 1 (or other autophagy proteins) influences progression of established tumors and/or their response to therapy.


Here, we identify the molecular basis by which EGFR tyrosine kinase activity regulates autophagy. We show that active EGFR binds to Beclin 1, leading to its tyrosine phosphorylation, alteration of its interactome, and inhibition of its autophagy function. A mutant of Beclin 1 containing phosphomimetic mutations in the EGFR-dependent tyrosine phosphorylation sites enhances autophagy suppression in EGFR-mutated NSCLC cells, resulting in enhanced tumor progression, altered tumor cell differentiation, and partial tumor resistance to EGFR TKI therapy. These findings demonstrate a heretofore unknown link between oncogenic receptor tyrosine kinases and the autophagy machinery, which may contribute to tumor progression and resistance to targeted therapy.

Source: Full Artical At - CELL PRESS

Drug combo supresses growth of late-stage prostate cancer turmors

By Natalie van Hoose

Low doses of metformin, a widely used diabetes medication, and a gene inhibitor known as BI2536 can successfully halt the growth of late-stage prostate cancer tumors, a Purdue University study finds.

Prostate cancer causes the second-highest number of cancer-related deaths in men in the U.S., and methods of treating advanced prostate cancer are limited.

Xiaoqi Liu (pronounced zhow-CHEE' LEE'-oo), associate professor of biochemistry and cancer research, and fellow researchers found that the drugs metformin and BI2536 can work together to suppress the spread of prostate cancer that resists all other available treatments, potentially prolonging patients' lives.

"We've found a promising way to treat late-stage prostate cancer," Liu said. "By combining low levels of two well-tolerated drugs, the progression of this disease could be significantly delayed. Completely curing the cancer at the advanced stage is pretty much impossible, but this treatment might manage it for a while - that's exciting."

A number of treatments exist for the earlier stages of prostate cancer, which grows slowly compared with many other cancers. Because prostate cancer cells need the male sex hormone androgen to develop, one way to treat the disease is to suppress androgen - a process known as castration. If the cancer continues to spread, the patient often undergoes chemotherapy. As a last resort, drugs that block the synthesis of androgen by prostate cancer cells can be used, but these medications only extend a patient's lifespan for several months.

New approaches to treating the most persistent forms of prostate cancer are "urgently needed," Liu said.

Adding to the challenge is the fact that castration treatment can inadvertently encourage the cancer to get tougher. It can heighten oxidative stress on the prostate gland, which increases the expression of Plk1, a gene that has been linked to many cancers. Over-expression of Plk1 can also trigger the synthesis of androgen.

"The goal of castration is to block androgen synthesis," Liu said. "But cancer cells eventually become 'smart' enough to make androgen anyhow, which is why the cancer continues to grow."

Additionally, castration can disrupt the body's metabolism and lead to insulin resistance, which also can stimulate the production of androgen. The cancer will spread until both of these side effects are stopped, Liu said.

Previous studies showed that metformin - an inexpensive, antidiabetic drug that has been commonly used for more than 40 years - is particularly potent to prostate cancer tumors.

Working with fellow researchers from Purdue, the University of Wisconsin-Madison and the Indiana University School of Medicine, Liu found that a combination of low levels of metformin and BI2536, a drug that stifles the activity of Plk1, could work in tandem to slow the growth of prostate tumors too advanced for current treatments by promoting the self-destruction of cancer cells and preventing androgen synthesis.

The drugs did not impact healthy prostate cells, a "key finding," Liu said. "Ideally, cancer therapy will have minimal effects on normal cells."

Because metformin helps regulate metabolism, it may reverse some of the metabolic damage caused by castration, he said.

The researchers tested the drugs in a classical cell culture assay of prostate cancer cells and in advanced prostate tumors in mice. Low concentrations of the drugs significantly slowed the development of cancer in both trials. The mice tumors were grown from the tumor cells of a late-stage prostate cancer patient, suggesting that the treatment would prove effective in humans.

"Those results were amazing," Liu said. "These are the first data we've generated from a real patient, so I was almost jumping in the air when I saw that it worked."

Liu said that the next step in the research is to test the combination of drugs in clinical trials. Further research is also needed to understand the underlying mechanism of metformin and why it is effective at suppressing prostate cancer

Source: Purdue Univesity

Stanford bioengineers develop tool for reprogramming genetic code

Stanford bioengineers have developed a new tool that allows them to preferentially activate or deactivate genes in living cells. VITSTUDIO/SHUTTERSTOCK

Biology relies upon the precise activation of specific genes to work properly. If that sequence gets out of whack, or one gene turns on only partially, the outcome can often lead to a disease.

Now, bioengineers at Stanford and other universities have developed a sort of programmable genetic code that allows them to preferentially activate or deactivate genes in living cells. The work is published in the current issue of Cell, and could help usher in a new generation of gene therapies.

The technique is an adaptation of CRISPR, itself a relatively new genetic tool that makes use of a natural defense mechanism that bacteria evolved over millions of years to slice up infectious virus DNA.

Standard CRISPR consists of two components: a short RNA that matches a particular spot in the genome, and a protein called Cas9 that snips the DNA in that location. For the purposes of gene editing, scientists can control where the protein snips the genome, insert a new gene into the cut and patch it back together.

Inserting new genetic code, however, is just one way to influence how the genome is expressed. Another involves telling the cell how much or how little to activate a particular gene, thus controlling how much protein a cell produces from that gene and altering its behavior.

It's this action that Lei Stanley Qi, an assistant professor of bioengineering and of chemical and systems biology at Stanford, and his colleagues aim to manipulate.

Influencing the genome
In the new work, the researchers describe how they have designed the CRISPR molecule to include a second piece of information on the RNA, instructing the molecule to either increase (upregulate) or decrease (downregulate) a target gene's activity, or turn it on/off entirely.

Additionally, they designed it so that it could affect two different genes at once. In a cell, the order or degree in which multiple genes are activated can produce different metabolic products.

"It's like driving a car. You control the wheel to control direction, and the engine to control the speed, and how you balance the two determines how the car moves," Qi said. "We can do the same thing in the cell by up- or downregulating genes, and produce different outcomes."

As a proof of principle, the scientists used the technique to take control of a yeast metabolic pathway, turning genes on and off in various orders to produce four different end products. They then tested it on two mammalian genes that are important in cell mobility, and were able to control the cell's direction and how fast it moved.

Future therapies
The ability to control genes is an attractive approach in designing genetic therapies for complex diseases that involve multiple genes, Qi said, and the new system may overcome several of the challenges of existing experimental therapies.

"Our technique allows us to directly control multiple specific genes and pathways in the genome without expressing new transgenes or uncontrolled behaviors, such as producing too much of a protein, or doing so in the wrong cells," Qi said. "We could eventually synthesize tens of thousands of RNA molecules to control the genome over a whole organism."

Next, Qi plans to test the technique in mice and refine the delivery method. Currently the scientists use a virus to insert the molecule into a cell, but he would eventually like to simply inject the molecules into an organism's blood.

"That is what is so exciting about working at Stanford, because the School of Medicine's immunology group is just around the corner, and working with them will help us address how to do this without triggering an immune response," said Qi, who is a member of the interdisciplinary Stanford ChEM-H institute. "I'm optimistic because everything about this system comes naturally from cells, and should be compatible with any organism."

Source: Stanford university

Structure of Neuron-Connecting Synaptic Adhesion Molecules Discovered

Figure 1: Overview of the PTPd Ig1–3/Slitrk1 LRR1 complex. Copyright : Korea Advanced Institute of Science and Technology

A research team has found the three-dimensional structure of synaptic adhesion molecules, which orchestrate synaptogenesis. The research findings also propose the mechanism of synapses in its initial formation.

Some brain diseases such as obsessive compulsive disorder (OCD) or bipolar disorders arise from a malfunction of synapses. The team expects the findings to be applied in investigating pathogenesis and developing medicines for such diseases.

The research was conducted by a Master’s candidate Kee Hun Kim, Professor Ji Won Um from Yonsei University, and Professor Beom Seok Park from Eulji University under the guidance of Professor Homin Kim from the Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology (KAIST), and Professor Jaewon Ko from Yonsei University. Sponsored by the Ministry of Science, ICT and Future Planning and the National Research Foundation of Korea, the research findings were published online in the November 14th issue of Nature Communications.

Figure 2: Representative negative-stained electron microscopy images of Slitrk1 Full ectodomain (yellow arrows indicate the horseshoe-shaped LRR domains). The typical horseshoe-shaped structures and the randomness of the relative positions of each LRR domain can be observed from the two-dimensional class averages displayed in the orange box. Copyright : Korea Advanced Institute of Science and Technology

A protein that exists in the neuronal transmembrane, Slitrk, interacts with the presynaptic leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) and forms a protein complex. It is involved in the development of synapses in the initial stage, and balances excitatory and inhibitory signals of neurons.

It is known that a disorder in those two proteins cause a malfunction of synapses, resulting in neuropsychosis such as autism, epilepsy, OCD, and bipolar disorders. However, because the structure as well as synaptogenic function of these proteins were not understood, the development of cures could not progress.

The research team discovered the three-dimensional structure of two synaptic adhesion molecules like Slitrk and LAR-RPTPs and identified the regions of interaction through protein crystallography and transmission electron microscopy (TEM). Furthermore, they found that the formation of the synapse is induced after the combination of two synaptic adhesion molecules develops a cluster.

Figure 3: Model of the two-step presynaptic differentiation process mediated by the biding of Slitrks to LAR-RPTPs and subsequent lateral assembly of trans-synaptic LAR-RPTPs/Slitrik complexes. Copyright : Korea Advanced Institute of Science and Technology

Professor Kim said, “The research findings will serve as a basis of understanding the pathogenesis of brain diseases which arises from a malfunction of synaptic adhesion molecules. In particular, this is a good example in which collaboration between structural biology and neurobiology has led to a fruitful result.” Professor Ko commented that “this will give new directions to synaptic formation-related-researches by revealing the molecular mechanism of synaptic adhesion molecules.”

Source: KAIST

In mice, vaccine stops urinary tract infections linked to catheters

To adhere to catheters and start urinary tract infections, bacteria extend microscopic fibers with sticky proteins at their ends. Scientists have developed a vaccine that blocks the EbpA protein, visible as a white bulge above, and stops infections in mice. Credit: John Heuser

The most common type of hospital-associated infection may be preventable with a vaccine, new research in mice suggests.

The experimental vaccine, developed by researchers at Washington University School of Medicine in St. Louis, prevented urinary tract infections associated with catheters, the tubes used in hospitals and other care facilities to drain urine from a patient's bladder.

Each day a catheter is present in the urethra and the bladder, the risk of urinary tract infection increases. Nearly every patient who has a catheter for more than 30 days acquires a urinary tract infection. The infections make urination painful and can damage the bladder. If untreated, bacteria can cross into the bloodstream and cause sepsis, a potentially life-threatening complication.

"Catheter-associated urinary tract infections are very common," said first author Ana Lidia Flores-Mireles, PhD, a postdoctoral research associate at the School of Medicine. "Antibiotic resistance is increasing rapidly in the bacteria that cause these infections, so developing new treatments is a priority."

The study is available online Sept. 17 in Science Translational Medicine.

Manufacturers typically coat catheters with antibiotics to reduce the risk of infection. But Flores-Mireles and her colleagues in the laboratory of Scott Hultgren, PhD, showed that inserting catheters into the bladder provokes an inflammatory response that results in the catheter being covered with fibrinogen, a blood-clotting protein.

Fibrinogen shields bacteria from the antibiotics and provides bacteria with a landing pad to adhere to and food to consume as they establish an infection, the research revealed.

"The bacteria use long, thin hairs known as pili to anchor themselves to the fibrinogen, and then they can start to form biofilms, which are slimy coatings on the surface of the catheter composed of many bacteria," said co-author Michael Caparon Jr., PhD, professor of molecular microbiology. "The biofilms protect the bacteria from antibiotics and immune cells, further prevent them from being washed from the body by the flow of urine, and make it possible for bacteria to seed the lining of the bladder with infections."

The urethra and bladder of a mouse are too small to insert a full catheter into, but the scientists showed that surgically implanting a small segment of catheter into the bladder via the urethra increased vulnerability to infection in a similar fashion.

Working with Enterococcus faecalis, a common cause of catheter-associated urinary tract infections, Flores-Mireles showed that a protein on the end of the pili, EbpA, binds to fibrinogen and makes it possible for the bacteria to begin forming biofilms.

When Flores-Mireles prevented the bacteria from making EbpA, they couldn't start infections.

"This protein is like the anchor of a boat," she said. "Without the anchor, the infection is at the mercy of the waves and gets washed away."

Next, the researchers injected the mice with a vaccine containing EbpA. The vaccine caused the animal's immune systems to produce antibodies that blocked EbpA and stopped the infectious process.

The scientists are testing to see if the vaccine helps mice clear established infections of E. faecalis. They also are working to develop a monoclonal antibody that blocks EbpA to prevent catheter-associated infections in the urinary tract and elsewhere in the body.

"We took a closer look at this protein and found that one-half of it is essential for binding to fibrinogen to induce infections," Flores-Mireles said. "The segment of genetic code that makes this part of the protein is also found in the genes of many other bacteria that cause urinary tract infections, so a vaccine, antibody or drug that blocks this part of the protein may help prevent other infections linked to catheters in the urinary tract and in other parts of the body."

Source: Washington University in St. Louis

Source of most cases of invasive bladder cancer identified

Philip Beachy and his team found a single type of cell in mice that gives rise to invasive bladder cancers. Credit: Steve Fisch

A single type of cell in the lining of the bladder is responsible for most cases of invasive bladder cancer, according to researchers at the Stanford University School of Medicine.

Their study, conducted in mice, is the first to pinpoint the normal cell type that can give rise to invasive bladder cancers. It's also the first to show that most bladder cancers and their associated precancerous lesions arise from just one cell, and explains why many human bladder cancers recur after therapy.

"We've learned that, at an intermediate stage during cancer progression, a single cancer stem cell and its progeny can quickly and completely replace the entire bladder lining," said Philip Beachy, PhD, professor of biochemistry and of developmental biology. "All of these cells have already taken several steps along the path to becoming an aggressive tumor. Thus, even when invasive carcinomas are successfully removed through surgery, this corrupted lining remains in place and has a high probability of progression."

Although the cancer stem cells, and the precancerous lesions they form in the bladder lining, universally express an important signaling protein called sonic hedgehog, the cells of subsequent invasive cancers invariably do not -- a critical switch that appears vital for invasion and metastasis. This switch may explain certain confusing aspects of previous studies on the cellular origins of bladder cancer in humans. It also pinpoints a possible weak link in cancer progression that could be targeted by therapies.

"This could be a game changer in terms of therapeutic and diagnostic approaches," said Michael Hsieh, MD, PhD, assistant professor of urology and a co-author of the study. "Until now, it's not been clear whether bladder cancers arise as the result of cancerous mutations in many cells in the bladder lining as the result of ongoing exposure to toxins excreted in the urine, or if it's due instead to a defect in one cell or cell type. If we can better understand how bladder cancers begin and progress, we may be able to target the cancer stem cell, or to find molecular markers to enable earlier diagnosis and disease monitoring."

Beachy is the senior author of the study, which will be published online April 20 in Nature Cell Biology. He is the Ernest and Amelia Gallo Professor in the School of Medicine and a member of the Stanford Cancer Institute and the Stanford Institute for Stem Cell Biology and Regenerative Medicine. He is also a Howard Hughes Medical Institute investigator. Kunyoo Shin, PhD, an instructor at the institute, is the lead author.

Bladder cancer is the fourth most common cancer in men and the ninth most common in women. Smoking is a significant risk factor. There are two main types of the disease: one that invades the muscle around the bladder and metastasizes to other organs, and another that remains confined to the bladder lining. Unlike the more-treatable, noninvasive cancer -- which comprises about 70 percent of bladder cancers -- the invasive form is largely incurable. It is expensive and difficult to treat, and the high likelihood of recurrence requires ongoing monitoring after treatment.

In 2011, Shin and Beachy and their colleagues identified a cell type in the bladder that is capable of completely replacing the lining of the organ after infection or damage. The fact that it could give rise to multiple cell types (even forming small, multilayered, bladder-like spheres when cultured in vitro), and also self-renew, showed that it was a bladder stem cell. 

They found that the cell, which came from the basal layer of the bladder epithelium, used a protein called sonic hedgehog to "talk" to other cells in the bladder and stimulate proliferation and specialization into other cell types. (Beachy identified the first hedgehog protein in fruit flies in 1992; the hedgehog signaling pathway has since been shown to play a vital role in embryonic development and in many types of cancers.)

Many animal models of cancer rely on prior knowledge or hunches as to what genes or cell types are involved. Researchers may genetically alter an animal, or a certain cell type, to induce the overexpression of a gene known to be involved in tumorigenesis, for example, or block the expression of a gene that inhibits cancer development.

Although prior work suggested that basal cells may play a role in bladder cancer, the researchers chose an unbiased approach when developing their mouse model that more closely mimicked what happens in humans: They put a chemical compound called N-butyl-N-4-hydroxybutyl nitrosamine, or BBN, in the mice's drinking water and watched the animals over a period of months.

Nitrosamines are carcinogens found in cigarette smoke; BBN is a form of the chemical that is specifically activated in the bladder. After four months, many of the animals had developed precancerous lesions, or carcinomas in situ, in their bladders that very closely resemble those seen in human patients. By six months, all of the animals had developed invasive bladder cancers.

With their model in place, the researchers then conducted two main experiments in the mice: In the first experiment, they looked to see what would happen in animals exposed to BBN when the sonic-hedgehog-expressing cells were marked with a distinctive fluorescent color. In the second, they used genetic techniques to selectively kill those same cells in animals prior to exposure with BBN.

In the first case, they saw something startling: After just a few months of BBN exposure, nearly the entire lining of the bladder was labeled with the fluorescent green marker that indicated the cells had arisen from the sonic-hedgehog-expressing basal stem cells. When transplanted into other mice, those labeled cells were able to give rise to bladder cancers, but cells not expressing sonic hedgehog did not.

In the second case, no tumors grew in the animals in which the stem cells had been selectively killed -- although the bladder architecture became severely compromised in the absence of stem cells to regenerate cells lost during the normal course of bladder function.

"So now we have two lines of evidence indicating that the bladder stem cells are solely responsible for tumorigenesis," Shin said. "When we mark the bladder stem cells, the tumors are also marked. When we remove, or ablate, the stem cells, no tumors arise after BBN treatment."

Next the researchers tackled the question of whether bladder cancers arise as the result of genetic changes to one or more of these bladder stem cells. To do so, they used a genetically engineered mouse with cells that fluoresce green, but which can be triggered to randomly fluoresce one of three other colors: red, blue or yellow. Known as a "rainbow mouse," the animal allows researchers to more precisely determine the origin of groups of cells. If all cells in a tumor are red, for example, it is much more likely that they originated from a single cell.

"After four months of BBN treatment," Beachy said, "we'd most often see just one color dominating the entire epithelium. This clearly indicates that a single cell has taken over the lining of the entire bladder, elbowing out its neighbors in a way that's not been seen in other organs."

Further studies showed that, surprisingly, none of the cells in the most advanced, invasive carcinomas in the BNN-treated animals expressed sonic hedgehog -- despite the fact that only sonic-hedgehog-expressing cells are able to give rise to the earlier stages of bladder cancer. One obvious implication of the lack of sonic hedgehog expression in these cells is that the hedgehog pathway somehow inhibits steps required for tissue invasion or metastasis.

"We know that the hedgehog pathway is widely used throughout the animal kingdom to tightly regulate cellular and tissue differentiation," Hsieh said. "So its loss could make sense in this context because cancer is essentially a loss of normal regulation."

"One really important lesson from this study," Beachy said, "is the idea that, by the time you get to a full-blown tumor, the properties of the cells in that tumor may have changed quite significantly from the cell type that gives rise to the tumors. This can complicate understanding how human tumors arise, because even if you identify the tumor-propagating cells within a mature tumor, conclusions about the origins of a cancer based on properties of these cells may be inaccurate."

Source: Stanford University Medical Center

Breathing in diesel exhaust leads to changes 'deep under the hood'

A student participates in the study while seated in a booth.
Credit: Image courtesy of University of British Columbia

Just two hours of exposure to diesel exhaust fumes can lead to fundamental health-related changes in biology by switching some genes on, while switching others off, according to researchers at the University of British Columbia and Vancouver Coastal Health.

The study involved putting volunteers in a polycarbonate-enclosed booth -- about the size of a standard bathroom -- while breathing in diluted and aged exhaust fumes that are about equal to the air quality along a Beijing highway, or a busy port in British Columbia.

The researchers examined how such exposure affected the chemical "coating" that attaches to many parts of a person's DNA. That carbon-hydrogen coating, called methylation, can silence or dampen a gene, preventing it from producing a protein -- sometimes to a person's benefit, sometimes not. Methylation is one of several mechanisms for controlling gene expression, which is the focus of a rapidly growing field of study called epigenetics.

The study, published this month in Particle and Fibre Toxicology, found that diesel exhaust caused changes in methylation at about 2,800 different points on people's DNA, affecting about 400 genes. In some places it led to more methylation; in more cases, it decreased methylation.

How these changes in gene expression translate to health is the next step for researchers. But this study shows how vulnerable our genetic machinery can be to air pollution, and that changes are taking place even if there are no obvious symptoms.

"Usually when we look at the effects of air pollution, we measure things that are clinically obvious -- air flow, blood pressure, heart rhythm," said senior author Dr. Chris Carlsten, an associate professor in the Division of Respiratory Medicine. "But asthma, higher blood pressure or arrhythmia might just be the gradual accumulation of epigenetic changes. So we've revealed a window into how these long-term problems arise. We're looking at changes 'deep under the hood.'"

The fact that DNA methylation was affected after only two hours of exposure has positive implications, said Carlsten, the AstraZeneca Chair in Occupational and Environmental Lung Disease.

"Any time you can show something happens that quickly, it means you can probably reverse it -- either through a therapy, a change in environment, or even diet," he said.

Carlsten's team, having catalogued the changes along the entire human genome, is now sharing its data with scientists who are further exploring the function of specific genes.

Source: University of British Columbia

Special delivery: Hitchhiking microparticles deliver drugs directly

Disc-shaped microparticles use monocytes to get to their destination. Credit: Peter Allen illustration

Inflammation is a normal and often beneficial response to injury or infection. The swelling, heat and even pain are the body's attempts to protect its soft tissue, remove offending objects, substances or microbes and initiate healing. However, persistent inflammation is often indicative of more serious conditions and can lead to problems of its own, including impaired healing, loss of function or even tissue death.

"Many diseases result in inflammation," said Samir Mitragotri, professor of chemical engineering at UC Santa Barbara and director of the campus's Center for Bioengineering. Whether inflammation is a byproduct of the disease or the inflammation is the disease, it is a common indicator of a problem with the system. "If we could target the common denominator, whether the inflammation is coming from cancer or arthritis, we could deliver the drug there," said Mitragotri, who specializes in targeted drug delivery.

By taking advantage of natural body processes, researchers at UC Santa Barbara and MIT have developed a method of targeting inflamed tissues, creating a way to treat both the inflammation and its underlying cause.

"It's a cell-mediated approach to targeted drug delivery," said UCSB grad student researcher Aaron Anselmo, lead author of a study in the current issue of the Journal of Controlled Release.

Key to this technology is the utilization of monocytes, the type of white blood cell known for its ability to penetrate into deep sections of tissue. Under normal circumstances, the job of these monocytes is to circulate in the blood and respond to biochemical signals that indicate inflammation -- a sign of injury or infection. Once at the site, these monocytes transform into macrophages, cells that reside in the affected tissues to engulf and digest foreign material.

Working with the expertise of chemical engineering and materials science researchers at MIT, including graduate researcher Jonathan Gilbert and professors Robert Cohen and Michael Rubner, the UCSB researchers developed an approach based on "cellular backpacks" -- flat, disc-shaped polymeric particles that could, in the near future, hold therapeutic agents that can be released at the site of the inflammation. These polymeric discs are coated on one side with a single layer of an antibody that can bind to receptors on the monocyte's surface.

To prevent the cellular backpack from being engulfed and devoured by the very cell that is transporting it, the researchers chose a flexible particle that is nonspherical in shape, which, according to the study, has proved to be more durable and resistant to phagocytosis than a rigid spherical particle. The shape and flexibility gives the backpack the ability to bind strongly while resisting phagocytosis to hitchhike onto monocytes and reach the inflamed tissue.

In-vitro and in-vivo tests have proved that cellular backpacks are successful in attaching to and being transported by monocytes to target areas without impairing the monocytes' natural functions, said Anselmo. Further studies will include research into how much drug can be loaded into the cellular backpacks. Ideally, Anselmo said, the cellular backpacks loaded with drugs would be injected into the bloodstream, whereupon they would attach to these traveling monocytes and hitchhike to the target region. At the inflamed site, the particles would simultaneously degrade and release their drugs.

The development of effective cellular backpacks has broad potential, say the researchers.
"Basically the main benefit is that you can deliver the drug in a more effective dose," Mitragotri said. Take for example the case of chemotherapy, which often has a narrow therapeutic range: Too little and the treatment is not effective, too much and it can be lethal. 
Because chemo travels through the bloodstream and affects all the tissues it comes in contact with, dosages are restricted at least in part based on the deleterious effect it has on other, unafflicted organs and their functions. Not only can targeted therapy ensure other body systems remain unaffected, Mitragotri explained, but it could allow for higher doses of drug to the site, which could decrease treatment time.

Source: University of California - Santa Barbara

Technique for cardiovascular diagnostics shows promise

Researcher Elira Maksuti is involved in developing and testing a promising method for diagnosing atherosclerosis, or hardening of the arteries. Credit: Staffan Larsson

A new technique developed at Sweden's KTH Royal Institute of Technology shows promise for early diagnosis and treatment of cardiovascular disease.

Hardening of the arteries, or atherosclerosis, is a common disorder that occurs when fat, cholesterol, and other substances build up in the walls of arteries and form hard structures called plaques. The condition can lead to heart attacks and strokes.

To diagnose atherosclerosis, doctors today rely on ultrasonic grayscale images to visually assess vascular function and how very large arteries move. The less mobility, the more developed the case of atherosclerosis.

But it is an indirect measurement. Better diagnoses can be made when the stiffness of the blood vessels can be analysed, explains Elira Maksuti, a researcher at the Department of Medical Imaging Technology at KTH.

"You need a doctor who is an expert and has extensive experience in order to get a good diagnosis," she says.

But by combining the technologies of shear wave elastography and ultrasound, Maksuti and researcher Erik Widmanh, have developed an inexpensive and non-invasive method not only for checking the stiffness of blood vessels, but for analysing the type of plaque present in the artery.

Maksuit says their method not only offers a potentially more effective way to diagnose atherosclerosis, but the ultrasound technology that it relies on is less expensive -- and safer -- than other imaging alternatives, such as magnetic resonance imaging (MRI) or computed tomography (CT).

The technique was tested on artificial blood vessels, or "phantom" vessels, which allowed the researchers to experiment with not only vascular stiffness, but also pressure and flow.

Maksuti says that with the success of tests on these phantoms, the next step is testing the technique with blood vessels from pigs. "These tests also look very promising," she says.

"We see two major future applications before us," she says. "The first is to determine when a patient's blood vessels are becoming rigid, that is, when the atherosclerosis process begins.

"The second application is to be able to diagnose the type of calcification -- or plaque -- present in the blood vessel." Not all plaque is the same: it ranges from hard to soft. If the plaque is soft and has a thin, hard shell, it is more likely to come loose inside the blood vessel.

It's a difficult distinction to determine. But the information is critical in deciding whether to open the artery surgically. "Today it is rather a matter of guessing. A doctor cannot know," she says. And to complicate matters, such operations can also generate strokes.

Source: KTH The Royal Institute of Technology

High-intensity sound waves may aid regenerative medicine

This is a cross section through a histotripsy lesion created in bovine liver tissue with the liquified cellular contents washed out revealing the remaining extracellular matrix. The scale bar represents 5mm. Credit: T.Khoklova/UW

Researchers at the University of Washington have developed a way to use sound to create cellular scaffolding for tissue engineering, a unique approach that could help overcome one of regenerative medicine's significant obstacles. The researchers will present their technique at the 168th meeting of the Acoustical Society of America (ASA), held October 27-31, 2014, at the Indianapolis Marriott Downtown Hotel.

The development of the new technique started with somewhat of a serendipitous discovery. The University of Washington team had been studying boiling histotripsy -- a technique that uses millisecond-long bursts of high-intensity ultrasound waves to break apart tissue -- as a method to eliminate cancerous tumors by liquefying them with ultrasound waves. After the sound waves destroy the tumors, the body should eliminate them as cellular waste. When the researchers examined these 'decellularized' tissues, however, they were surprised by what the boiling left intact.

"In some of our experiments, we discovered that some of the stromal tissue and vasculature was being left behind," said Yak-Nam Wang, a senior engineer at the University of Washington's Applied Physics Laboratory. "So we had the idea about using this to decellularize tissues for tissue engineering and regenerative medicine."

The structure that remains after decellularizing tissues is known as the extracellular matrix, a fibrous network that provides a scaffold for cells to grow upon. Most other methods for decellularizing tissues and organs involve chemical and enzymatic treatments that can cause damage to the tissues and fibers and takes multiple days. Histrostipsy, on the other hand, offers the possibility of fast decellularization of tissue with minimal damage to the matrix.

"In tissue engineering, one of the holy grails is to develop biomimetic structures so that you can replace tissues with native tissue," Wang said. Stripping away cells from already developed tissue could provide a good candidate for these structures, since the extracellular matrix already acts as the cellular framework for tissue systems, Wang said.

Due to its bare composition, the matrix also induces only a relatively weak immune response from the host. The matrix could then theoretically be fed with stem cells or cells from the same person to effectively re-grow an organ.

"The other thought is that maybe you could just implant the extracellular matrix and then the body itself would self-seed the tissues, if it's just a small patch of tissue that you're replacing," Wang said. "You won't have any immune issues, and because you have this biomimetic scaffold that's closer to the native tissue, healing would be better, and the body would recognize it as normal tissue."

Wang is currently investigating decellularization of kidney and liver tissue from large animals. Future work involves increasing the size of the decellularized tissues and assessing their in-vivo regenerative efficacy.

Source: Acoustical Society of America (ASA)

WHACK! Study measures head blows in girls' lacrosse

Trey Crisco invited lacrosse-playing girls to the lab to measure the impact of their blows as they whacked the head of a laboratory dummy — and to evaluate the performance of protective headgear. Credit: Mike Cohea/Brown University

Lacrosse players swing hard, which is why errant stick blows are the leading cause of concussion in girls' and women's lacrosse. In a new study, researchers measured how much the worst blows accelerate the head and how much different kinds of headgear could reduce those accelerations.

Girls' and women's lacrosse is a different game from the version played by males, said Joseph Crisco, the Henry Frederick Lippitt Professor of Orthopaedic Research in the Alpert Medical School of Brown University and a researcher at Rhode Island Hospital. Females wear far less protective equipment than males do, and injuries -- especially severe head injuries -- are comparatively rare. But recently the debate about whether female players should wear headgear has gained prominence.

Coming to blows

The girls delivered peak performance averaging 60 times the acceleration of Earth's gravity (60g) when they struck the headforms with their lacrosse sticks.

"The goal of our study was to answer the question of what types of head accelerations would you see if you were hit in the head with a stick," said Crisco, who used to coach his daughters in girls lacrosse and also sits on the Sports Science and Safety Committee of US Lacrosse, the national governing body of lacrosse.

To conduct the study, published online in the Journal of Applied Biomechanics, Crisco's team asked seven female lacrosse players aged 12 to 14 to deliver at least 36 whacks each, as hard as they possibly could, to various places on two dummy headforms in the lab.

"The kinds of hits recorded were basically aggressive street fights," Crisco said. "They were really whacking at it, every shaft was broken by the end of the study, which would never happen in a game. The goal was just to give US Lacrosse and the manufacturers some baseline information on the types of accelerations they could expect to see in a worst-case scenario."

They used six different sticks, each outfitted with motion capture markers. The headforms had embedded accelerometers. In a second set of experiments the headforms donned one of four different kinds of protective headgear.

On average across 508 successful blows in the first experiment, the girls swung their sticks about 18 miles an hour, enough to complete two revolutions in less than a second. (One of Crisco's prior studies showed, perhaps not surprisingly, that high school and college players swung their sticks even faster). The peak acceleration the girls delivered to the headforms when they struck them with the shafts of their sticks averaged 60 times the acceleration of Earth's gravity (60g).

That's about three times more force than, say, football players with the kind of celebratory head butt that teammates exchange after a big play, Crisco said.

Headgear dampens blows

The second set of experiments examined what effect headgear might have on the girls' harder whacks (those with speeds around 23 miles an hour). Crisco's team measured the accelerations delivered by 20 whacks from the shaft of each volunteer's stick on both the back and the side of each headform. The headforms wore either nothing, a hard-sided men's lacrosse helmet, a rugby scrum cap, mixed martial arts headgear, or soft headgear designed for girls' and women's field hockey and lacrosse.

The average peak accelerations measured on bare headgear were 81.6g for blows to the side and 150.7g for blows to the back. The men's lacrosse helmet brought the average peak acceleration all the way down to 28.2g on the side and 23.1g on the back. The martial arts and girls lacrosse/field hockey headgear each reduced the accelerations significantly as well, but not nearly as much as the men's helmet. The rugby cap failed to reduce acceleration for blows to the side but dampened blows to the back a little better than the martial arts or lacrosse/field hockey gear.

Headgear, therefore, significantly reduced head accelerations. But Crisco cautioned against a run on headgear at the sporting goods store based on the study.

Generally research has shown that helmets do not protect against concussion -- only against skull fractures and traumatic brain injury. Indeed very little data connects accelerations to concussion risk, and individual susceptibility varies widely. Though some research hints at a figure around 100g, only the hard-sided men's helmet brought accelerations for blows to the back significantly below that figure. And in many game situations, given how little other protective equipment female players wear, Crisco said, a hard-sided helmet could easily cause more injuries that it prevents.

"It could actually make the game more aggressive," Crisco said.

Source: Brown University

Charged graphene gives DNA a stage to perform molecular gymnastics

DNA interacts with charged graphene and contorts into sequence-specific shapes when the charge is changed. Credit: Photo courtesy Alek Aksimentiev

When Illinois researchers set out to investigate a method to control how DNA moves through a tiny sequencing device, they did not know they were about to witness a display of molecular gymnastics.

Fast, accurate and affordable DNA sequencing is the first step toward personalized medicine. Threading a DNA molecule through a tiny hole, called a nanopore, in a sheet of graphene allows researchers to read the DNA sequence; however, they have limited control over how fast the DNA moves through the pore. In a new study published in the journal Nature Communications, University of Illinois physics professor Aleksei Aksimentiev and graduate student Manish Shankla applied an electric charge to the graphene sheet, hoping that the DNA would react to the charge in a way that would let them control its movement down to each individual link, or nucleotide, in the DNA chain.

"Ideally, you would want to step the DNA through the nanopore one nucleotide at a time," said Aksimentiev. "Take a measurement and then have another nucleotide in the sensing hole. That's the goal, and it hasn't been realized yet. We show that, to some degree, we can control the process by charging the graphene."

The researchers found that a positive charge in the graphene speeds up DNA movement through the nanopore, while a negative charge stops the DNA in its tracks. However, as they watched, the DNA seemed to dance across the graphene surface, pirouetting into shapes they had never seen, specific to the sequence of the DNA nucleotides.

"It reminds me of Swan Lake," Aksimentiev said. "It's very acrobatic. We were very surprised by the variety of DNA conformations that we can observe at the surface of graphene when we charge it. There is one sequence that starts out laying down on the surface, and when we change the charge, they all tilt on the side like they are doing a one-armed push-up. Then we also have nucleotides that would lay back, or go up like a ballerina en pointe."

Aksimentiev hypothesizes that the conformations are so different and so specific to the sequence because each nucleotide has a slightly different distribution of electrons, the negatively charged parts of the atoms. There is even a visible difference when a nucleotide is methylated, a tiny chemical change that can turn a gene on or off.

By switching the charge in the graphene, the researchers can control not only the DNA's motion through the pore, but also the shape the DNA contorts into.

"Because it's reversible, we can force it to adopt one conformation and then force it to go back. That's why we call it gymnastics," Aksimentiev said.

The researchers extensively used the Blue Waters supercomputer at the National Center for Supercomputing Applications, housed at the University of Illinois. They mapped each individual atom in the complex DNA molecule and ran numerous simulations of many different DNA sequences. Supercomputing power was essential to carrying out the work, Aksimentiev said.

"This is a really computationally intensive project," he said. "Having access to Blue Waters was essential because with the sheer number of simulations, we would not have been able to finish them. It would have taken too long."

The next step is to combine a charged nanopore setup with a sensor to build a DNA sequencing device that would incorporate both motion control and nucleotide recognition. The researchers also hope to explore the unexpected conformational changes for insights into epigenetics, the field that studies how genes are expressed and moderated.

"DNA is much more complicated than just a double helix. It's a complex molecule that has many properties, and we are still uncovering them," Aksimentiev said.

Video animation of DNA dancing as the graphene charge changes:


Source: University of Illinois at Urbana-Champaign

Mode of action of protein channelrhodopsin-2 decoded: Findings facilitate manufacture of optogenetic tools

The pore of the ion channel is opened by removing the amino acid E90. Water molecules enter and tilt Helix H2, thus opening the continuous channel. Credit: © RUB, graphics: Eisenhauer

Researchers have shed light upon the mode of action of the light-controlled channelrhodopsin-2 with high spatiotemporal resolution. This biomolecule is used in optogenetic applications, which is deployed to control the activity of living cells with light.

"The model we developed makes it possible to create customised optogenetic tools for individual applications," says Prof Dr Klaus Gerwert from the Department of Biophysics at the Ruhr-Universität Bochum. Together with colleagues at the Humboldt Universität zu Berlin from the team headed by Prof Dr Peter Hegemann, the Bochum researchers report about their finding in the magazine "Angewandte Chemie."

Channelrhodopsin-2 has revolutionised optogenetics

Discovered by Peter Hegemann in green algae, channelrhodopsin-2 is the central light-activated channel protein in optogenetics. If this ion channel is applied to nerve cells, the channels can be opened by light, thus activating the cell. "The application of channelrhodopsin-2 in optogenetics has revolutionised neurobiology in the recent years," says Klaus Gerwert. The magazine "Nature Methods" awarded this process as "Method of the Year" in 2010. "However, scientists had not been aware of what is actually happening inside a protein and thus ultimately triggers its activation," continues the Bochum researcher. But it is the understanding of processes on the atomic level that is essential for optimising the protein specifically for its applications.

"EHT" model describes the mode of action of channelrhodopsin-2

With time-resolved vibrational spectroscopy and bio-molecular simulations, the Bochum-Berlin team has now closed that gap. The EHT (E90-Helix2-tilt) model describes the mode of action of channelrhodopsin-2 as follows: the light-sensitive group of the protein, i.e. the retinal, is twistedunder incidence of light. This twist then continues in the protein and opens a pore ultra fast, which is closed by amino acid E90 in the dark. E90 marks the narrowest place in the pore and opens it through a downward move , similar to the motion of a swing door, so that water can enter an empty vestibule above the narrowest place in the pore. The entering water then tilts the protein helix H2, which eventually triggers a protein-traversing open ion channel. When forming this model, the Bochum researchers benefitted from their comprehensive experience that they had gained resolving the mechanism of light-driven proton pump bacteriorhodopsin in detail.

"Protein engineering": pioneering novel optogenetic tools

"With this structural model, the next step, i.e. protein engineering, will become possible," explains Klaus Gerwert. Through mutation of the amino acid E90, the protein's properties can be controlled in a targeted manner. The conductivity or the selectivity for certain ions could be customised for specific applications, and the protein could be specifically activated with different wavelengths.

Source: Ruhr-Universitaet-Bochum