Center for Biomedical Research Members Hard at Work

Members of the Center for Biomedical Research have been hard at work this spring publishing articles that advance our understanding of important biomedical problems. Here is a sampler of what these researchers have been doing. 

CBR member Ken Mitton, of the Eye Research Institute, collaborated with a team from Beaumont Health System to develop new methods to study blocked arteries. The abstract of their paper Evaluation of Optimal RNA Extraction Method from Human Carotid Atherosclerotic Plaque (Cardiovascular Pathology, Volume 24, Pages 187-190, 2015) is given below. This research was funded in part by the Research Excellence Fund administered by the CBR. 


Investigating molecular mechanisms involved in the formation of carotid atherosclerotic plaques has been challenging. Isolating high-quality RNA from plaque tissue can be difficult because of acellularity, calcification, and degradation. It is essential that the mRNA isolated from this tissue preserves and reflects the actual relative gene expression. Two common methods for RNA preservation, snap-freezing and stabilizing reagent, were compared using surgically resected human carotid atherosclerotic tissue. In addition, isolation methods were compared for integrity and quantity: column-based extraction, phenol-based extraction, and a combination of the two. We found that using a stabilizing reagent with column filtration resulted in the lowest yield and quality. Phenol-based extraction resulted in higher yields but also increased fragmentation. Snap-frozen tissue coupled with column-based extraction yielded the highest quality. The higher quality and quantity RNA obtained when processing snap-frozen tissue with column-based extraction make it possible to use difficult sample types for molecular downstream applications. 


CBR member Libin Rong, of the Department of Mathematics and Statistics, analyzed a mathematical model of HIV infection and AIDS in his paper Global Stability of an Infection-Age Structured HIV-1 Model Linking Within-Host and Between-Host Dynamics (Mathematical Biosciences, Volume 263, Pages 37-50, 2015). Rong’s research is supported by grants from the National Science Foundation. 


Although much evidence shows the inseparable interaction between the within-host progression of HIV-1 infection and the transmission of the disease at the population level, few models coupling the within-host and between-host dynamics have been developed. In this paper, we adopt the nested approach, viewing the transmission rate at each stage (primary, chronic, and AIDS stage) of HIV-1 infection as a saturated function of the viral load, to formulate an infection-age structured epidemic model. We explicitly link the individual and the host population scale, and derive the basic reproduction number R-0 for the coupled system. To analyze the model and perform a detailed global dynamics analysis, two Lyapunov functionals are constructed to prove the global asymptotical stability of the disease-free and endemic equilibria. Theoretical results indicate that R-0 provides a threshold value determining whether or not the disease dies out. Numerical simulations are presented to quantitatively investigate the influence of the within-host viral dynamics on between-host transmission dynamics. The results suggest that increasing the effectiveness of inhibitors can decrease the basic reproduction number, but can also increase the overall infected population because of a lower disease-induced mortality rate and a longer lifespan of HIV infected individuals. 


CBR member Yang Xia, of the Department of Physics, studies the properties of articular cartilage and osteoarthritis. His graduate student David Kahn was lead author on an article about the Effects of Cryopreservation on the Depth-Dependent Elastic Modulus in Articular Cartilage and Implications for Osteochondral Grafting (Journal of Biomechanical Engineering, Volume 137, Article Number 054502, 2015). Xia’s laboratory is supported by a grant from the National Institutes of Health. 


Cryopreservation of articular cartilage is often used in storage of experimental samples and osteochondral grafts, but the depth-dependence and concentration of glycosaminoglycan (GAG) are significantly altered when cryogenically stored without a cryoprotectant, which will reduce cartilage stiffness and affect osteochondral graft function and long-term viability. This study investigates our ability to detect changes due to cryopreservation in the depth-dependent elastic modulus of osteochondral samples. Using a direct-visualization method requiring minimal histological alterations, unconfined stepwise stress relaxation tests were performed on four fresh (never frozen) and three cryopreserved (-20 degrees C) canine humeral head osteochondral slices 125 +/- 5 mu m thick. Applied force was measured and tissue images were taken at the end of each relaxation phase using a 4x objective. Intratissue displacements were calculated by tracking chondrocytes through consecutive images for various intratissue depths. The depth-dependent elastic modulus was compared between fresh and cryopreserved tissue for same-depth ranges using analysis of variance (ANOVA) with Tukey post-test with a 95% confidence interval. Cryopreservation was found to significantly alter the force-displacement profile and reduce the depth-dependent modulus of articular cartilage. Excessive collagen fiber folding occurred at 40-60% relative depth, producing a "black line" in cryopreserved tissue. Force-displacement curves exhibited elongated toe-region in cryopreserved tissue while fresh tissue had nonmeasurable toeregion. Statistical analysis showed significant reduction in the elastic modulus and GAG concentration throughout the tissue between same-depth ranges. This method of cryopreservation significantly reduces the depth-dependent modulus of canine humeral osteochondral samples. 

CBR member Xiangqun Zeng, of the Department of Chemistry, designs biosensors to detect bacteria. The abstract to her paper with Visiting Scholar Fen Ma, Antimicrobial Susceptibility Assays Based on the Quantification of Bacterial Lipopolysaccharides Via a Label Free Lectin Biosensor (Analytical Chemistry, Volume 87, Pages 4385-4393, 2015), is given below. Her work is funded in part by the Research Excellence Fund administered by the CBR. 


A label free lectin biosensor developed in our laboratory that can quantitatively measure the binding between the lectin immobilized at the carbohydrate sensor surface and the lipopolysaccharide (LPS) on Gram-negative bacteria was demonstrated for an antibiotic susceptibility assay. The biosensor utilizes a polythiophene interface containing fused quinone moieties glycosylated to form a carbohydrate platform for the immobilization of Concanavalin A (Con A) and is capable of LPS binding measurements via orthogonal quartz crystal microbalance and electrochemical readouts (EQCM). Such orthogonal transduction provides cross-validation, better sensor sensitivity, and a large dynamic range of the measurements. We have applied this label free lectin biosensor for a new antibiotic susceptibility assay by characterizing the antimicrobial activities of various antibiotics (i.e., ciprofloxacin, ceftriaxone, and tetracycline) against Escherichia coli W1485 as a model system. The label free biosensor allows both end point and real time measurements of antibiotic effects on the bacterial cell surface LPS, which is shown to correlate to their antibiotic effects. At the end point, after 18 h incubation of bacterial cells with these three antibiotics respectively, the bacterial LPS binding signal was reduced to 23%, 27%, and 38%, respectively, for the three antibiotics, indicating that ciprofloxacin is the most effective against this E. coli strain. Real time measurements at the 1 h time point showed a similar trend with a reduction of binding to 91%, 93%, and 95%, respectively. From the binding kinetic of these measurements, the relaxation time (tau) was obtained, where higher value means slow binding interactions between the lectin and the bacterial LPS. The obtained order of tau, (i.e., tau(ciprofloxacin) > tau(ceftriaxone) > tau(tetracycline)) again indicated that ciprofloxacin has more bactericidal activity than the other two antibiotics with the same concentrations. Thus, we are able to establish that the reduction in the binding of LPS with the lectin Con A sensor upon exposure to various antibiotics has a direct relation with the antibiotic dosages making this label free biosensor assay promising for therapeutic management of these drugs as well as for applications in antibiotic research and development.