University of Delaware researchers have identified a protein, hiding in plain sight, that acts like a bodyguard to help protect and stabilize another key protein, that when unstable, is involved in Crohn’s disease. The fundamental research points to a possible pathway for developing an effective therapy for the inflammatory bowel disease.

The research was conducted by Catherine Leimkuhler Grimes, assistant professor of chemistry and biochemistry at UD, and Vishnu Mohanan, doctoral student in biological sciences,

As the scientists point out, our immune system provides the first line of defence against invading pathogens, a task even more challenging in the human gut, where over a trillion commensal bacteria live — resident microorganisms that help convert food into protein, vitamins and minerals.

To distinguish “bad” versus “good” bacteria, our bodies rely on a complex network of receptors that can sense patterns that are unique to bacteria, such as small fragments of bacterial cell wall. The receptors recognize and bind to these fragments, triggering an immune response to take out the “bad guys” or control the growth of the “good guys.”

However, when one of these receptors breaks down, or mutates, an abnormal immune response can occur, causing the body to mount an immune response against the “good” bacteria. Chronic inflammatory disorders, such as Crohn’s disease, are hypothesized to arise as a result.

The UD team focused on a protein called NOD2 — nucleotide-binding oligomerisation domain containing protein 2. More than 58 mutations in the NOD2 gene have been linked with various diseases, and 80 percent of these mutations are connected specifically to Crohn’s disease, according to Grimes.

In experiments to unveil NOD2’s signalling mechanisms and where they break down, “we stumbled on this chaperone molecule,” says Mohanan, who was the lead author of the scientific article.

The chaperone molecule was HSP70, which stands for “heat shock protein 70.” It assists with the folding of proteins into their correct three-dimensional shapes, even when cells are under stress from elevated body temperatures, such as a fever.

Grimes said she was a little sceptical at first about pursuing studies with HSP70 because it is a commonly known protein, but she found Mohanan’s initial data intriguing.

“Vishnu found that if we increased the expression level of HSP70, the NOD2 Crohn’s mutants were able to respond to bacterial cell wall fragments. A hallmark of the NOD2 mutations is inability to respond to these fragments. Essentially, Vishnu found a fix for NOD2, and we wanted to determine how we were fixing it.”

In further experiments, Mohanan created a tagged-wild-type NOD2 cell line in which NOD2 levels nearly matched the levels found in nature (versus “super” levels that might stimulate an artificial response) and found that NOD2 became more stabilized and degraded more slowly when treated with HSP70. In fact, HSP70 increased the half-life of NOD2 by more than four hours.

“Basically, HSP70 keeps the protein around — it kind of watches over and protects NOD2, and keeps it from going in the cellular trash can,” Grimes explains.

The researchers tested three human cell lines in their study: kidney cells, colon cells and white blood cells. In the next phase of the study, patient tissue will be examined through a collaboration with Nemours/A.I. duPont Hospital for Children to determine if NOD2 levels can be controlled via HSP70 expression.

“We want to figure out why the mutation in NOD2 results in an increase in inflammation,” says Mohanan. “Right now, we have limited knowledge. Once the signalling mechanism is figured out, we will have the keystone.” University of Delaware

The fungal infection invasive aspergillosis (IA) can be life threatening, especially in patients whose immune systems are weakened by chemotherapy or immunosuppressive drugs. Despite the critical need for early detection, IA remains difficult to diagnose. A study compared three diagnostic tests and found that the combination of nucleic acid sequence-based amplification (NASBA) and real-time quantitative PCR (qPCR) detects aspergillosis with 100% accuracy.

IA is caused by the fungus Aspergillus fumigatus, which is considered by many pathologists to be the world’s most harmful mold. ‘Traditional diagnostic methods, such as culture and histopathology of infected tissues, often fail to detect Aspergillus,’ comments lead investigator Yun Xia, PhD, of the First Affiliated Hospital of Chongqing Medical University, Chongqing, China.

In this retrospective study, scientists evaluated the diagnostic performance of two nucleic acid amplification assays (qPCR and NASBA) and one antigen detection method (galactomannan enzyme-linked immunosorbent assay [GM-ELISA]) using blood samples collected from 80 patients at high risk of IA. Of the 80 patients, 42.5% had proven or probable IA. The patients came from intensive care, haematology, neurology, nephrology, geriatrics, and other hospital departments.

The tests were evaluated singly and in combination. Individually, NASBA had the highest sensitivity (76.47%) whereas qPCR offered the highest specificity (89.13%). NASBA also was the test that best indicated that a patient did not have the infection (negative predictive value). NASBA and qPCR each had a high Youden index, a measure of the effectiveness of a diagnostic marker.

Combining the tests improved the outcomes. The combination of NASBA and qPCR led to 100% specificity and 100% positive predictive value (the probability that subjects truly have the infection).

‘Because each test has advantages and disadvantages, a combination of different tests may be able to provide better diagnostic value than is provided by a single test,’ says Dr. Xia. The combination of NASBA and qPCR should be useful in excluding IA in suspect cases, thus reducing both suffering and expense for immunocompromised patients. On the other hand, the combination of NASBA and qPCR could be more suitable for screening patients suspected of infection, because this assay had the highest sensitivity.’

The authors note that NASBA offers the advantages of rapid amplification (90 minutes) and simple operation with low instrument cost compared with qPCR and GM-ELISA. They caution that although GM-ELISA is widely and routinely used for aspergillosis diagnosis, this study indicates that it had low sensitivity (52.94%) with reasonable specificity (80.43%), making it ‘inferior to both NASBA and qPCR.’  EurekAlert

Dartmouth Institute for Quantitative Biomedical Sciences (iQBS) researchers developed a new gene expression analysis approach for identifying cancer genes. The study results challenge the current paradigm of microarray data analysis and suggest that the new method may improve identification of cancer-associated genes.

Typical microarray-based gene expression analyses compare gene expression in adjacent normal and cancerous tissues. In these analyses, genes with strong statistical differences in expression are identified. However, many genes are aberrantly expressed in tumours as a byproduct of tumorigenesis. These ‘passenger’ genes are differentially expressed between normal and tumour tissues, but they are not ‘drivers’ of tumorigenesis. Therefore, better analytical approaches that enrich the list of candidate genes with authentic cancer-associated ‘driver’ genes are needed.

Lead authors of the study, Ivan P. Gorlov, PhD, Associate Professor of Community and Family Medicine and Christopher Amos, PhD, Professor of Community and Family Medicine and Director of the Center for Genomic Medicine described a new method to analyse microarray data. The research team demonstrated that ranking genes based on inter-tumour variation in gene expression outperforms traditional analytical approaches. The results were consistent across four major cancer types: breast, colorectal, lung, and prostate cancer.

The team used text-mining to identify genes known to be associated with breast, colorectal, lung, and prostate cancers. Then, they estimated enrichment factors by determining how frequently those known cancer-associated genes occurred among the top gene candidates identified by different analysis methods. The enrichment factor described how frequently cancer associated genes were identified compared to the frequency of identification that one could expect by pure chance. Across all four cancer types, the new method of selecting candidate genes based on inter-tumour variation in gene expression outperformed the other methods, including the standard method of comparing mean expression in adjacent normal and tumour tissues. Dr. Gorlov and colleagues also used this approach to identify novel cancer-associated genes.

The authors cite tumour heterogeneity as the most likely reason for the success of their variance-based approach. The method is based on the knowledge that different tumours can be driven by different subsets of cancer genes. By identifying genes with high variation in expression between tumours, the method preferentially identifies genes specifically associated with cancer. This same feature, tumour heterogeneity, may reduce the ability to identify critical gene expression changes when comparing mean gene expression in adjacent tumor and normal tissues, as tumors of the same type may have different sets of genes differentially expressed.

The results of the study challenge the model that comparing mean gene expression in adjacent normal and cancer tissues is the best approach to identifying cancer-associated genes. Indeed, the team identified high variation in adjacent ‘normal’ tissue samples, which are typically used as control samples for comparison in analyses based on mean gene expression. The study suggests that methods based on variance may help get the most from existing and future global gene expression studies. Dartmouth Institute for Quantitative Biomedical Sciences

Understanding eye diseases is tricky enough. Knowing what causes them at the molecular level is even more confounding.

Now, University of Iowa researchers have created the most detailed map to date of a region of the human eye long associated with blinding diseases, such as age-related macular degeneration. The high-resolution molecular map catalogues thousands of proteins in the choroid, which supplies blood and oxygen to the outer retina, itself critical in vision. By seeing differences in the abundance of proteins in different areas of the choroid, the researchers can begin to figure out which proteins may be the critical actors in vision loss and eye disease.

“This molecular map now gives us clues why certain areas of the choroid are more sensitive to certain diseases, as well as where to target therapies and why,” says Vinit Mahajan, assistant professor in ophthalmology at the UI and corresponding author on the paper. “Before this, we just didn’t know what was where.”

What vision specialists know is many eye diseases, including age-related macular degeneration (AMD), are caused by inflammation that damages the choroid and the accompanying cellular network known as the retinal pigment epithelium (RPE). Yet they’ve been vexed by the anatomy: Why does it seem that some areas of the choroid-RPE are more susceptible to disease than others, and what is happening at the molecular level? The researchers set about to answer that question with non-diseased eye tissue donated by three deceased older individuals through the Iowa Lions Eye Bank. From there, Mahajan and Jessica Skeie, a post-doctoral researcher in ophthalmology at the UI, created a map that catalogues more than 4,000 unique proteins in each of the three areas of the choroid-RPE: the fovea, macula, and the periphery.

Why that’s important is now the researchers can see which proteins are more abundant in certain areas, and why. One such example is a protein known as CFH, which helps prevent a molecular cascade that can lead to AMD, much like a levee can keep flooding waters at bay. The UI researchers learned, though the map, that CFH is most abundant in the fovea. That helps, because now they know to monitor CFH abundance there; fewer numbers of the protein could mean increased risk for AMD, for instance.

“Now you can see all those differences that you couldn’t see before,” explains Mahajan, whose primary appointment is in the Carver College of Medicine. University of Iowa

A study by researchers at the University of Kansas shows a new role for the protein adenomatous polyposis coli (APC) in suppressing colorectal cancer — the second-leading cause of cancer-related deaths in the U.S.

Lead author Kristi Neufeld, associate professor in the Department of Molecular Biosciences and co-leader of the Cancer Biology program at the KU Cancer Center, has spent the better part of her career trying to understand the various activities of APC, a protein whose functional loss is thought to initiate roughly 80 percent of all colon polyps, a precursor to colon cancer. Neufeld, along with her postdoctoral fellow Maged Zeineldin, undergraduate student Mathew Miller and veterinary pathologist Ruth Sullivan, now reports that APC found in a particular subcellular compartment, the nucleus, protects from inflammation as well as tumour development associated with chronic colitis.

Whether APC reaches the nucleus may well affect the ability of intestinal stem cells to produce differentiated cells with specialized functions, Neufeld said.

“It’s not widely appreciated, but there is still plenty of cell growth going on in adults, with the colon being a good example,” she said. “On average, we shed and replace about 70 pounds of intestinal tissue annually, so you can imagine that this process requires exquisite control to prevent tumour formation.”

Regular renewal of the colon lining occurs through stem cells that are capable of constantly dividing. These cells produce descendants that take up specific roles: By secreting mucin, for instance, goblet cells generate a mucus layer that serves as the colon’s physical barrier against its many microbial tenants. But if APC can’t find its way to the nucleus, Neufeld and her team have noted far fewer goblet cells as one outcome.

“We introduced a specific APC mutation into mice that took away the nuclear zip code, so to speak, leaving APC stuck in the cytoplasm,” Neufeld said. The researchers studied this mouse model under conditions that induce ulcerative colitis, a form of inflammatory bowel disease that can be a prelude to colon cancer.

Observing significantly more colon tumours in these mice compared to those with normal APC in the same disease setting, they hypothesized that functional nuclear APC might somehow guard against inflammation and its downstream effects, including tumour development. Now, Neufeld thinks she and her team may have a clue as to how this happens.

“The drop in goblet cell numbers we observed was striking,” she said. “We then examined one of the proteins found in mucus, called Muc2, and found that its RNA levels were greatly decreased. If there are fewer goblet cells as a result of APC being unable to reach the nucleus, there will also be less mucus, which could increase the colon’s sensitivity to bacteria and other microorganisms in the gut that are capable of promoting inflammation.”

Neufeld said while there are still no quick fixes for mutant genes, perhaps tools could be developed to synthetically replace this less-than-ideally thick mucus layer in affected people.

One known function of APC is that it halts cell proliferation: by muzzling the canonical arm of the Wnt signaling pathway, which otherwise instructs cells to go forth and multiply. Neufeld and her group have already shown, using the same mouse model, that APC stationed in the nucleus is necessary to suppress Wnt and its signaling partners — particularly β-catenin, a key target of normal APC. With a role for nuclear APC in controlling goblet cell differentiation now supported, the researchers are probing possible mechanisms to learn if and how Wnt pathway members might be involved. Kansas University