Thermal Memory in Self-Assembled Collagen Fibril Networks

Collagen fibrils form extracellular networks that regulate cell functions and provide mechanical strength to tissues. Collagen fibrillogenesis is an entropy-driven process promoted by warming and reversed by cooling. Here, we investigate the influence of noncovalent interactions mediated by the collagen triple helix on fibril stability. We measure the kinetics of cold-induced disassembly of fibrils formed from purified collagen I using turbimetry, probe the fibril morphology by atomic force microscopy, and measure the network connectivity by confocal microscopy and rheometry. We demonstrate that collagen fibrils disassemble by subunit release from their sides as well as their ends, with complex kinetics involving an initial fast release followed by a slow release. Surprisingly, the fibrils are gradually stabilized over time, leading to thermal memory. This dynamic stabilization may reflect structural plasticity of the collagen fibrils arising from their complex structure. In addition, we propose that the polymeric nature of collagen monomers may lead to slow kinetics of subunit desorption from the fibril surface. Dynamic stabilization of fibrils may be relevant in the initial stages of collagen assembly during embryogenesis, fibrosis, and wound healing. Moreover, our results are relevant for tissue repair and drug delivery applications, where it is crucial to control fibril stability.     

Increased PTH and 1.25(OH)(2)D levels associated with increased markers of bone turnover following bariatric surgery

The objective of this study was to characterize changes in metabolic bone parameters following bariatric surgery. Seventy-three obese adult patients who underwent either gastric banding (GB), Roux-en-Y gastric bypass (RYGB), or biliopancreatic diversion with duodenal switch (BPD/DS) were followed prospectively for 18 months postoperatively. Changes in the calcium-vitamin D axis (25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D (1,25(OH)(2)D), calcium, parathyroid hormone (PTH)), markers of bone formation (osteocalcin, bone-specific alkaline phosphatase) and resorption (urinary N-telopeptide (NTx)), as well as bone mineral density (BMD) were assessed at 3-month intervals during this time period. Bariatric surgery resulted in significant and progressive weight loss over 18 months. With supplementation, 25OHD levels increased 65.3% (P < 0.0001) by 3 months, but leveled off and decreased <30 ng/ml by 18 months. PTH initially decreased 21.4% (P = 0.01) at 3 months, but later approached presurgery levels. 1,25(OH)(2)D increased significantly starting at month 12 (50.3% increase from baseline, P = 0.008), and was positively associated with PTH (r = 0.82, P = 0.0001). When stratified by surgery type, median PTH and 1,25(OH)(2)D levels were higher following combined restrictive and malabsorptive operations (RYGB and BPD/DS) compared to GB. Bone formation/resorption markers were increased by 3 months (P < 0.05) and remained elevated through 18 months. Radial BMD decreased 3.5% by month 18, but this change was not significant (P = 0.23). Our findings show that after transient improvement, preoperative vitamin D insufficiency and secondary hyperparathyroidism persisted following surgery despite supplementation. Postoperative secondary hyperparathyroidism was associated with increased 1,25(OH)(2)D levels and increased bone turnover markers.     

An epistatic genetic basis for physical activity traits in mice

We recently identified several (4-8) quantitative trait loci (QTL) for 3 physical activity traits (daily distance, duration, and speed voluntarily run) in an F(2) population of mice derived from an original intercross of 2 strains that exhibited large differences in activity. These QTL cumulatively explained from 11% to 34% of the variation in these traits, but this was considerably less than their total genetic variability estimated from differences among inbred strains. We therefore decided to test whether epistatic interactions might account for additional genetic variation in these traits in this same population of mice. We conducted a full genome epistasis scan for all possible interactions of QTL between each pair of 20 chromosomes. The results of this scan revealed an abundance of epistasis, with QTL throughout the genome being involved in significant interactions. Overall, epistatic effects contributed an average of 26% of the total variation among the 3 activity traits. These results suggest that epistatic interactions of genes may play as important a role in the genetic architecture of physical activity traits as single-locus effects and need to be considered in future candidate gene identification studies.     

Genetic variation in the pleiotropic association between physical activity and body weight in mice

Background

A sedentary lifestyle is often assumed to lead to increases in body weight and potentially obesity and related diseases but in fact little is known about the genetic association between physical activity and body weight. We tested for such an association between body weight and the distance, duration, and speed voluntarily run by 310 mice from the F₂ generation produced from an intercross of two inbred lines that differed dramatically in their physical activity levels.

Methods

We used a conventional interval mapping approach with SNP markers to search for QTLs that affected both body weight and activity traits. We also conducted a genome scan to search for relationship QTLs (relQTLs), or chromosomal regions that affected an activity trait variably depending on the phenotypic value of body weight.

Results

We uncovered seven quantitative trait loci (QTLs) affecting body weight, but only one co-localized with another QTL previously found for activity traits. We discovered 19 relQTLs that provided evidence for a genetic (pleiotropic) association of physical activity and body weight. The three genotypes at each of these loci typically exhibited a combination of negative, zero, and positive regressions of the activity traits on body weight, the net effect of which was to produce overall independence of body weight from physical activity. We also demonstrated that the relQTLs produced these varying associations through differential epistatic interactions with a number of other epistatic QTLs throughout the genome.

Conclusion

It was concluded that individuals with specific combinations of genotypes at the relQTLs and epiQTLs might account for some of the variation typically seen in plots of the association of physical activity with body weight.     

Genotype × diet interactions in mice predisposed to mammary cancer. I. Body weight and fat

High dietary fat intake and obesity may increase susceptibility to certain forms of cancer. To study the interactions of dietary fat, obesity, and metastatic mammary cancer, we created a population of F₂ mice cosegregating obesity QTL and the MMTV-PyMT transgene. We fed the F₂ mice either a very-high-fat or a matched-control-fat diet and measured growth, body composition, age at mammary tumor onset, tumor number and severity, and formation of pulmonary metastases. SNP genotyping across the genome facilitated analyses of QTL and QTL × diet interaction effects. Here we describe development of the F₂ population (n = 615) which resulted from a cross between the polygenic obesity model M16i and FVB/NJ-TgN (MMTV-PyMT)^634Mul, effects of diet on growth and body composition, and QTL and QTL × diet and/or gender interaction effects for growth and obesity-related phenotypes. We identified 38 QTL for body composition traits that were significant at the genome-wide 0.05 level, likely representing nine distinct loci after accounting for pleiotropic effects. QTL × diet and/or gender interactions were present at 15 of these QTL, indicating that such interactions play a significant role in defining the genetic architecture of complex traits such as body weight and obesity.     

Patterns of cellular gene expression in cells infected with cytopathic or non-cytopathic bovine viral diarrhea virus

Bovine viral diarrhea virus (BVDV) infection in cattle is responsible for mucosal disease; an invariably fatal syndrome characterized by the recovery of two BVDV strains: cytopathic (cp) or noncytopathic (ncp). To understand the cellular responses to cp BVDV infection, we carried out differential display-polymerase chain reaction (DD-PCR) analysis of gene expression in infected cells. Altered expression of 14 genes involved in several functions was observed in cells infected with cp BVDV: (1) immune regulation, such as CD46, FKBP-12, and osteopontin (OPN); (2) apoptosis-related cysteine proteases like calpain; (3) signaling plasma membrane proteins such as integrin beta1, and prion protein; and (4) unknown function genes. Northern blot analysis of the expression of these genes in ncp BVDV infected cells revealed that while the expression of some genes was affected as in cp BVDV infected cells, others show a clearly contrary change. We postulate that a cause-effect relationship may exist between the differential gene expression alterations that characterize cp and ncp BVDV infections and the unique diseases associated with each BVDV biotype.     

Quantitative trait loci for directional but not fluctuating asymmetry of mandible characters in mice

Non-directional variation in right minus left differences in bilateral characters, referred to as fluctuating asymmetry (FA), often has been assumed to be largely or entirely environmental in origin. FA increasingly has been used as a measure of developmental stability, and its presumed environmental origin has facilitated the comparisons of populations believed to differ in their levels of stability. Directional asymmetry (DA), in which one side is consistently larger than the other, has been assumed to be at least partially heritable. Both these assumptions were tested with interval mapping techniques designed to detect any quantitative trait loci (QTLs) affecting FA or DA in 15 bilateral mandible characters in house mice resulting from a cross of the F1 between CAST/Ei (wild strain) and M16i (selected for rapid growth rate) back to M16i. For purposes of the analysis, all mandibles were triply measured and 92 microsatellite markers were scored in a total of 350 mice. No significant QTLs were found for FA, but three QTLs significantly affected DA in several characters, confirming both assumptions. The QTLs for DA were similar in location to those affecting the size of several of the mandible characters, although they accounted for an average of only 1% of the total phenotypic variation in DA.     

hMRAPα, but Not hMRAP2, Enhances hMC4R Constitutive Activity in HEK293 Cells and This Is Not Dependent on hMRAPα Induced Changes in hMC4R Complex N-linked Glycosylation

MRAP1 but not MRAP2, is essential for melanocortin receptor 2 functional expression. Human MRAP1 splice variant (hMRAPα) and human MRAP2 (hMRAP2) also interact with the other melanocortin receptor subtypes in vitro, although the physiological significance of these interactions is unknown. Previously we showed that HA-hMC4R co-expression with hMRAPα, but not hMRAP2, specifically alters HA-hMC4R complex N-linked glycosylation. hMRAPα-FLAG also enhances hMC4R constitutive activity in vitro. Here we directly compare hMRAPα and hMRAP2 effects on hMC4R constitutive activity in HEK293 cells. In contrast to hMRAPα, co-expression with hMRAP2 had no effect on HA-hMC4R or untagged hMC4R constitutive coupling to adenylyl cyclase. We used fixed and live cell imaging of HA-hMC4R and hMC4R-eGFP respectively, to further characterise effects of hMRAPα on hMC4R subcellular trafficking. hMRAPα-FLAG co-expression did not alter the partitioning of either HA-hMC4R or hMC4R-eGFP into either the ER or the Golgi apparatus, therefore the hMRAPα effect on hMC4R complex N-linked glycosylation is probably not due to hMC4R retention in the ER. We also observed that unlike HA-hMC4R, hMC4R-eGFP lacks complex glycosylation both in the presence and absence of hMRAPα, although both HA-hMC4R and hMC4R-eGFP exhibited increased constitutive coupling to adenylyl cyclase following co-expression with hMRAPα. We conclude that hMRAPα and not hMRAP2 modulates hMC4R constitutive activity. Furthermore, hMRAPα does not increase hMC4R constitutive activity by altering hMC4R complex N-linked glycosylation. Instead we hypothesise that hMRAPα alters hMC4R conformational states leading to increased hMC4R constitutive activity.