High-resolution spatial mapping of shear properties in cartilage

Structural properties of articular cartilage such as proteoglycan content, collagen content and collagen alignment are known to vary over length scales as small as a few microns (Bullough and Goodfellow, 1968; Bi et al., 2006). Characterizing the resulting variation in mechanical properties is critical for understanding how the inhomogeneous architecture of this tissue gives rise to its function. Previous studies have measured the depth-dependent shear modulus of articular cartilage using methods such as particle image velocimetry (PIV) that rely on cells and cell nuclei as fiducial markers to track tissue deformation (Buckley et al., 2008; Wong et al., 2008a). However, such techniques are limited by the density of trackable markers, which may be too low to take full advantage of optical microscopy. This limitation leads to noise in the acquired data, which is often exacerbated when the data is manipulated. In this study, we report on two techniques for increasing the accuracy of tissue deformation measurements. In the first technique, deformations were tracked in a grid that was photobleached on each tissue sample (Bruehlmann et al., 2004). In the second, a numerical technique was implemented that allowed for accurate differentiation of optical displacement measurements by minimizing the propagated experimental error while ensuring that truncation error associated with local averaging of the data remained small. To test their efficacy, we employed these techniques to compare the depth-dependent shear moduli of neonatal bovine and adult human articular cartilage. Using a photobleached grid and numerical optimization to gather and analyze data led to results consistent with those reported previously (Buckley et al., 2008; Wong et al., 2008a), but with increased spatial resolution and characteristic coefficients of variation that were reduced up to a factor of 3. This increased resolution allowed us to determine that the shear modulus of neonatal bovine and adult human tissue both exhibit a global minimum at a depth z of around 100 μm and plateau at large depths. The consistency of the depth dependence of |G*|(Z) for adult human and neonatal bovine tissue suggests a functional advantage resulting from this behavior.     

A comprehensive database of verified experimental data on protein folding kinetics

Insights into protein folding rely increasingly on the synergy between experimental and theoretical approaches. Developing successful computational models requires access to experimental data of sufficient quantity and high quality. We compiled folding rate constants for what initially appeared to be 184 proteins from 15 published collections/web databases. To generate the highest confidence in the dataset, we verified the reported lnk_f value and exact experimental construct and conditions from the original experimental report(s). The resulting comprehensive database of 126 verified entries, ACPro, will serve as a freely accessible resource ( https://www.ats.amherst.edu/protein/ ) for the protein folding community to enable confident testing of predictive models. In addition, we provide a streamlined submission form for researchers to add new folding kinetics results, requiring specification of all the relevant experimental information according to the standards proposed in 2005 by the protein folding consortium organized by Plaxco. As the number and diversity of proteins whose folding kinetics are studied expands, our curated database will enable efficient and confident incorporation of new experimental results into a standardized collection. This database will support a more robust symbiosis between experiment and theory, leading ultimately to more rapid and accurate insights into protein folding, stability, and dynamics.     

Pregnancy induced hypertension: evidence for increased cell membrane permeability to sodium.

In a cross sectional study of 137 women of childbearing age (16-40) the effects of normal pregnancy, hypertensive pregnancy, and oral contraceptives on red cell electrolyte content and sodium efflux rates were examined and the results compared with values in a control group of normotensive, non-pregnant women. Efflux rate constants were significantly increased in normotensive pregnancy and in women taking oral contraceptives. This was associated with a significant increase in sodium permeability in the contraceptive group. A much larger increase in sodium permeability and efflux rate constant was seen in the hypertensive group. The results permit a hypothesis that the hormonal changes induced by pregnancy and oral contraceptives increase membrane permeability to sodium and stimulate sodium efflux. The rise in blood pressure associated with use of oral contraceptives may have a similar aetiology to that occurring in pregnancy induced hypertension.     

Elastoviscous Transitions of Articular Cartilage Reveal a Mechanism of Synergy between Lubricin and Hyaluronic Acid

When lubricated by synovial fluid, articular cartilage provides some of the lowest friction coefficients found in nature. While it is known that macromolecular constituents of synovial fluid provide it with its lubricating ability, it is not fully understood how two of the main molecules, lubricin and hyaluronic acid, lubricate and interact with one another. Here, we develop a novel framework for cartilage lubrication based on the elastoviscous transition to show that lubricin and hyaluronic acid lubricate by distinct mechanisms. Such analysis revealed nonspecific interactions between these molecules in which lubricin acts to concentrate hyaluronic acid near the tissue surface and promotes a transition to a low friction regime consistent with the theory of viscous boundary lubrication. Understanding the mechanics of synovial fluid not only provides insight into the progression of diseases such as arthritis, but also may be applicable to the development of new biomimetic lubricants.     

Irisin and the Metabolic Phenotype of Adults with Prader-Willi Syndrome

Context

Hyperphagia, low resting energy expenditure, and abnormal body composition contribute to severe obesity in Prader Willi syndrome (PWS). Irisin, a circulating myokine, stimulates “browning” of white adipose tissue resulting in increased energy expenditure and improved insulin sensitivity. Irisin has not been previously studied in PWS.

Objectives

Compare plasma and salivary irisin in PWS adults and normal controls. Examine the relationship of irisin to insulin sensitivity and plasma lipids.

Design and Study Participants

A fasting blood sample for glucose, lipids, insulin, leptin, adinopectin, and irisin was obtained from 22 PWS adults and 54 healthy BMI-matched volunteers. Saliva was collected for irisin assay in PWS and controls.

Results

Fasting glucose (77±9 vs 83±7mg/dl, p = 0.004), insulin (4.1±2.0 vs 7.9±4.7μU/ml, p<0.001), and triglycerides (74±34 vs 109±71mg/dl, p = 0.007) were lower in PWS than in controls. Insulin resistance (HOMA-IR) was lower (0.79±0.041 vs 1.63±1.02, p<0.001) and insulin sensitivity (QUICKI) was higher (0.41±0.04 vs 0.36±0.03, p<0.001) in PWS. Plasma irisin was similar in both groups, but salivary irisin (64.5±52.0 vs 33.0±12.1ng/ml), plasma leptin (33.5±24.2 vs 19.7±19.3ng/ml) and plasma adinopectin (13.0±10.8 vs 7.6±4.5μg/ml) were significantly greater in PWS (p<0.001). In PWS, plasma irisin showed positive Pearson correlations with total cholesterol (r = 0.58, p = 0.005), LDL-cholesterol (r = 0.59, p = 0.004), and leptin (r = 0.43, p = 0.045). Salivary irisin correlated negatively with HDL-cholesterol (r = -0.50, p = 0.043) and positively with LDL-cholesterol (r = 0.51, p = 0.037) and triglycerides (r = 0.50, p = 0.041).

Conclusions

Salivary irisin was markedly elevated in PWS although plasma irisin was similar to levels in controls. Significant associations with plasma lipids suggest that irisin may contribute to the metabolic phenotype of PWS.     

Divergent effects of canonical and non‐canonical TGF‐β signalling on mixed contractile‐synthetic smooth muscle cell phenotype in human Marfan syndrome aortic root aneurysms

Aortic root aneurysm formation is a cardinal feature of Marfan syndrome (MFS) and likely TGF‐β driven via Smad (canonical) and ERK (non‐canonical) signalling. The current study assesses human MFS vascular smooth muscle cell (SMC) phenotype, focusing on individual contributions by Smad and ERK, with Notch3 signalling identified as a novel compensatory mechanism against TGF‐β‐driven pathology. Although significant ERK activation and mixed contractile gene expression patterns were observed by traditional analysis, this did not directly correlate with the anatomic site of the aneurysm. Smooth muscle cell phenotypic changes were TGF‐β‐dependent and opposed by ERK in vitro, implicating the canonical Smad pathway. Bulk SMC RNA sequencing after ERK inhibition showed that ERK modulates cell proliferation, apoptosis, inflammation, and Notch signalling via Notch3 in MFS. Reversing Notch3 overexpression with siRNA demonstrated that Notch3 promotes several protective remodelling pathways, including increased SMC proliferation, decreased apoptosis and reduced matrix metalloproteinase activity, in vitro. In conclusion, in human MFS aortic SMCs: (a) ERK activation is enhanced but not specific to the site of aneurysm formation; (b) ERK opposes TGF‐β‐dependent negative effects on SMC phenotype; (c) multiple distinct SMC subtypes contribute to a ‘mixed’ contractile‐synthetic phenotype in MFS aortic aneurysm; and (d) ERK drives Notch3 overexpression, a potential pathway for tissue remodelling in response to aneurysm formation.     

Rac-dependent doubling of HeLa cell area and impairment of cell migration and cell cycle by compounds from Iris germanica

Plants are an infinite source of bioactive compounds. We screened the Israeli flora for compounds that interfere with the organization of the actin cytoskeleton. We found an activity in lipidic extract from Iris germanica that was able to increase HeLa cell area and adhesion and augment the formation of actin stress fibers. This effect was not observed when Ref52 fibroblasts were tested and was not the result of disruption of microtubules. Further, the increase in cell area was Rac1-dependent, and the iris extract led to slight Rac activation. Inhibitor of RhoA kinase did not interfere with the ability of the iris extract to increase HeLa cell area. The increase in HeLa cell area in the presence of iris extract was accompanied by impairment of cell migration and arrest of the cell cycle at G1 although the involvement of Rac1 in these processes is not clear. Biochemical verification of the extract based on activity-mediated fractionation and nuclear magnetic resonance analysis revealed that the active compounds belong to the group of iridals, a known group of triterpenoid. Purified iripallidal was able to increase cell area of both HeLa and SW480 cells.