Identifying a minimal rheological configuration: a tool for effective and efficient constitutive modeling of soft tissues

We describe a modeling methodology intended as a preliminary step in the identification of appropriate constitutive frameworks for the time-dependent response of biological tissues. The modeling approach comprises a customizable rheological network of viscous and elastic elements governed by user-defined 1D constitutive relationships. The model parameters are identified by iterative nonlinear optimization, minimizing the error between experimental and model-predicted structural (load-displacement) tissue response under a specific mode of deformation. We demonstrate the use of this methodology by determining the minimal rheological arrangement, constitutive relationships, and model parameters for the structural response of various soft tissues, including ex vivo perfused porcine liver in indentation, ex vivo porcine brain cortical tissue in indentation, and ex vivo human cervical tissue in unconfined compression. Our results indicate that the identified rheological configurations provide good agreement with experimental data, including multiple constant strain rate load/unload tests and stress relaxation tests. Our experience suggests that the described modeling framework is an efficient tool for exploring a wide array of constitutive relationships and rheological arrangements, which can subsequently serve as a basis for 3D constitutive model development and finite-element implementations. The proposed approach can also be employed as a self-contained tool to obtain simplified 1D phenomenological models of the structural response of biological tissue to single-axis manipulations for applications in haptic technologies.     

Solubilization and assay of a colony-stimulating factor receptor from murine macrophages

The colony-stimulating factor, CSF-1, selectively stimulates the survival, proliferation, and differentiation of mononuclear phagocytes. The solubilization, assay, and characteristics of the CSF-1 receptor from the J774.2 murine macrophage cell line are described. The recovery of cell-surface receptor in the postnuclear supernatant membrane fraction of hypotonically disrupted cells was 76%. Recovery of the ligand binding activity of the receptor after solubilization of this fraction with 1% Triton X-100 was approximately 150%. The binding of 125I-CSF-1 to intact cells and membrane preparations was consistent with the existence of a single class of high-affinity receptor sites. In contrast, the equilibrium binding of 125I-CSF-1 to the solubilized postnuclear fraction indicated the existence of two distinct classes of binding site (apparent Kds 0.15 nM and 10 nM). A rapid assay was developed for the high-affinity sites, which were shown to be associated with the CSF-1 receptor. The function of the low-affinity sites, which have not been demonstrated on intact cells or cell membranes and which are 13 times more abundant than the high-affinity sites, is unknown. The solubilized high-affinity receptor-CSF-1 complex was stable on storage at 0 degrees C and -70 degrees C but dissociated at 37 degrees C. Dissociation also occurred at 0 degrees C in buffers of low pH (4.0) or high ionic strength (0.7 M NaCl).     

The BMP-binding protein Crossveinless 2 is a short-range, concentration-dependent, biphasic modulator of BMP signaling in Drosophila

In Drosophila, the secreted BMP-binding protein Short gastrulation (Sog) inhibits signaling by sequestering BMPs from receptors, but enhances signaling by transporting BMPs through tissues. We show that Crossveinless 2 (Cv-2) is also a secreted BMP-binding protein that enhances or inhibits BMP signaling. Unlike Sog, however, Cv-2 does not promote signaling by transporting BMPs. Rather, Cv-2 binds cell surfaces and heparan sulfate proteoglygans and acts over a short range. Cv-2 binds the type I BMP receptor Thickveins (Tkv), and we demonstrate how the exchange of BMPs between Cv-2 and receptor can produce the observed biphasic response to Cv-2 concentration, where low levels promote and high levels inhibit signaling. Importantly, we show also how the concentration or type of BMP present can determine whether Cv-2 promotes or inhibits signaling. We also find that Cv-2 expression is controlled by BMP signaling, and these combined properties enable Cv-2 to exquisitely tune BMP signaling.     

Ancestral polymorphism and linkage disequilibrium at the het-6 region in pseudohomothallic Neurospora tetrasperma

In species of Neurospora, non-self recognition is mediated by at least 11 heterokaryon (het) incompatibility loci. Previously, we identified ancient allelic variation at het-c in pseudohomothallic N. tetrasperma, which confirmed outcrossing in this species. Here, we report distinct ancestral alleles at het-6 and un-24, two closely linked genes with het incompatibility function in N. crassa. The pattern of variation at het-6 and un-24 in N. tetrasperma is similar to that observed for N. crassa, where two ancestral allele specificities exist for each locus, Oak Ridge (het-6(OR), un-24(OR)) and Panama (het-6(PA), un-24(PA)). Only het-6(OR)/un-24(OR) and het-6(PA)/un-24(PA) allele combinations have been observed. The absence of recombinant haplotypes (e.g., het-6(OR)/un-24(PA)) appears to derive from an ancestral chromosomal rearrangement that limits recombination. Allelic variation at het-6 and un-24 in N. tetrasperma provides further evidence of outcrossing in this predominantly selfing species and indicates that selection maintains ancient allelic diversity at het loci.     

Association of FCGR2A and FCGR2A-FCGR3A haplotypes with susceptibility to giant cell arteritis

The Fc gamma receptors have been shown to play important roles in the initiation and regulation of many immunological and inflammatory processes and to amplify and refine the immune response to an infection. We have investigated the hypothesis that polymorphism within the FCGR genetic locus is associated with giant cell arteritis (GCA). Biallelic polymorphisms in FCGR2A, FCGR3A, FCGR3B and FCGR2B were examined for association with biopsy-proven GCA (n = 85) and healthy ethnically matched controls (n = 132) in a well-characterised cohort from Lugo, Spain. Haplotype frequencies and linkage disequilibrium (D') were estimated across the FCGR locus and a model-free analysis performed to determine association with GCA. There was a significant association between FCGR2A-131RR homozygosity (odds ratio (OR) 2.10, 95% confidence interval (CI) 1.12 to 3.77, P = 0.02, compared with all others) and carriage of FCGR3A-158F (OR 3.09, 95% CI 1.10 to 8.64, P = 0.03, compared with non-carriers) with susceptibility to GCA. FCGR haplotypes were examined to refine the extent of the association. The haplotype showing the strongest association with GCA susceptibility was the FCGR2A-FCGR3A 131R-158F haplotype (OR 2.84, P = 0.01 for homozygotes compared with all others). There was evidence of a multiplicative joint effect between homozygosity for FCGR2A-131R and HLA-DRB1*04 positivity, consistent with both of these two genetic factors contributing to the risk of disease. The risk of GCA in HLA-DRB1*04 positive individuals homozygous for the FCGR2A-131R allele is increased almost six-fold compared with those with other FCGR2A genotypes who are HLA-DRB1*04 negative. We have demonstrated that FCGR2A may contribute to the 'susceptibility' of GCA in this Spanish population. The increased association observed with a FCGR2A-FCGR3A haplotype suggests the presence of additional genetic polymorphisms in linkage disequilibrium with this haplotype that may contribute to disease susceptibility. These findings may ultimately provide new insights into disease pathogenesis.     

The passionvine mealybug,Planococcus minor (Maskell) (Hemiptera: Pseudococcidae), and its natural enemies in the cocoa agroecosystem in Trinidad

Planococcus minor (Maskell) is native to South Asia, but it is also present in several Neotropical locations including the island of Trinidad in the southern Caribbean. The mealybug poses a serious threat to uninfested countries in this region as well as the mainland U.S. As part of an effort to gather much needed information on P. minor, 33 cocoa (Theobroma cacao L.) field sites on the island were surveyed in 2006 with a view to assess the occurrence and pest status of the mealybug. P. minor was identified from 20 field sites, indicating that it was well distributed across the island on this crop, which appeared to be a reliable indicator host plant. Infestation levels were generally low and populations were sparsely distributed across the field sites categorized into three habitat types. The following year, nine field sites were surveyed for natural enemies of P. minor using laboratory-infested potatoes in sentinel traps. Species from four insect orders and six families were collected and identified. The major predators belonged to the families Cecidomyiidae and Coccinellidae. Two primary parasitoids, Leptomastix dactylopii Howard (Encyrtidae) and Coccidoxenoides perminutus (Girault) (=Pauridia peregrina Timberlake, =Coccidoxenoides peregrinus (Timberlake)) (Encyrtidae), were reared from different mealybug stages, along with several hyperparasitoids. The primary parasitoids were probably introduced fortuitously. These diverse natural enemies were recovered throughout the sampling period from the different habitat types. The identification of key natural enemies associated with P. minor has important implications for the implementation of biological control in newly infested areas.     

TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.     

MSC Therapy Attenuates Obliterative Bronchiolitis after Murine Bone Marrow Transplant

Rationale

Obliterative bronchiolitis (OB) is a significant cause of morbidity and mortality after lung transplant and hematopoietic cell transplant. Mesenchymal stromal cells (MSCs) have been shown to possess immunomodulatory properties in chronic inflammatory disease.

Objective

Administration of MSCs was evaluated for the ability to ameliorate OB in mice using our established allogeneic bone marrow transplant (BMT) model.

Methods

Mice were lethally conditioned and received allogeneic bone marrow without (BM) or with spleen cells (BMS), as a source of OB-causing T-cells. Cell therapy was started at 2 weeks post-transplant, or delayed to 4 weeks when mice developed airway injury, defined as increased airway resistance measured by pulmonary function test (PFT). BM-derived MSC or control cells [mouse pulmonary vein endothelial cells (PVECs) or lung fibroblasts (LFs)] were administered. Route of administration [intratracheally (IT) and IV] and frequency (every 1, 2 or 3 weeks) were compared. Mice were evaluated at 3 months post-BMT.

Measurements and Main Results

No ectopic tissue formation was identified in any mice. When compared to BMS mice receiving control cells or no cells, those receiving MSCs showed improved resistance, compliance and inspiratory capacity. Interim PFT analysis showed no difference in route of administration. Improvements in PFTs were found regardless of dose frequency; but once per week worked best even when administration began late. Mice given MSC also had decreased peribronchiolar inflammation, lower levels of hydroxyproline (collagen) and higher frequencies of macrophages staining for the alternatively activated macrophage (AAM) marker CD206.

Conclusions

These results warrant study of MSCs as a potential management option for OB in lung transplant and BMT recipients.     

β-Catenin Inactivation Is a Pre-Requisite for Chick Retina Regeneration

In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration.