One-Dimensional Radial Diffusion of Small Molecules (376 Da) in Bone Tissue

The flow of nutrients through any biological tissue is important to maintain homeostasis. If the transport process is understood, medical research teams can better design medications, prosthetic implants, and tissue scaffolds. Additionally, transport rates help physicians to better understand disease states and wound healing, including minor injuries such as breaks and sprains, which will aid in better diagnoses. We developed a novel method that measures the rate of diffusion in vitro, of fluorescein sodium salt. Samples were incubated at 37°C in a 5 CO2 atmosphere for various periods of time. Samples were sliced and analyzed using Image-Pro Plus and MATLAB to obtain concentration profiles. The diffusivity was estimated from the data using the model equation for one-dimensional transport in a finite medium. We found that radial diffusivity in canine bone in 1-dimension was 1.27 x 10-7±177 1.96 x 10-8 cm2/s. As a point of reference, the diffusivity of fluorescein sodium salt in PBS is 2.7 x 10-6 cm2/s. Given the average distance between a Haversian canal and an osteon radius is 250 um, our data shows it would take approximately 20 minutes for a nutrient of a weight of 376 Da to travel between the two locations. This indicates that the diffusion time of key nutrients, such as vitamin D, with molecular weight of 384 Da, would be about 20 minute

One-Dimensional Radial Diffusion of Small Molecules (376 Da) in Bone Tissue

The flow of nutrients through any biological tissue is important to maintain homeostasis. If the transport process is understood, medical research teams can better design medications, prosthetic implants, and tissue scaffolds. Additionally, transport rates help physicians to better understand disease states and wound healing, including minor injuries such as breaks and sprains, which will aid in better diagnoses. We developed a novel method that measures the rate of diffusion in vitro, of fluorescein sodium salt. Samples were incubated at 37°C in a 5 CO2 atmosphere for various periods of time. Samples were sliced and analyzed using Image-Pro Plus and MATLAB to obtain concentration profiles. The diffusivity was estimated from the data using the model equation for one-dimensional transport in a finite medium. We found that radial diffusivity in canine bone in 1-dimension was 1.27 x 10-7±177 1.96 x 10-8 cm2/s. As a point of reference, the diffusivity of fluorescein sodium salt in PBS is 2.7 x 10-6 cm2/s. Given the average distance between a Haversian canal and an osteon radius is 250 um, our data shows it would take approximately 20 minutes for a nutrient of a weight of 376 Da to travel between the two locations. This indicates that the diffusion time of key nutrients, such as vitamin D, with molecular weight of 384 Da, would be about 20 minute

Sensitivity of Neural Stem Cell Survival, Differentiation and Neurite Outgrowth Within 3D Hydrogels to Environmental Heavy Metals

© 2015 Elsevier Ireland Ltd. We investigated the sensitivity of embryonic murine neural stem cells exposed to 10 pM-10. μM concentrations of three heavy metals (Cd, Hg, Pb), continuously for 14 days within 3D collagen hydrogels. Critical endpoints for neurogenesis such as survival, differentiation and neurite outgrowth were assessed. Results suggest significant compromise in cell viability within the first four days at concentrations ≥10. nM, while lower concentrations induced a more delayed effect. Mercury and lead suppressed neural differentiation at as low as 10 pM concentration within 7 days, while all three metals inhibited neural and glial differentiation by day 14. Neurite outgrowth remained unaffected at lower cadmium or mercury concentrations (≤100. pM), but was completely repressed beyond day 1 at higher concentrations. Higher metal concentrations (≥100. pM) suppressed NSC differentiation to motor or dopaminergic neurons. Cytokines and chemokines released by NSCs, and the sub-cellular mechanisms by which metals induce damage to NSCs have been quantified and correlated to phenotypic data. The observed degree of toxicity in NSC cultures is in the order: lead. \u3e. mercury. \u3e. cadmium. Results point to the use of biomimetic 3D culture models to screen the toxic effects of heavy metals during developmental stages, and investigate their underlying mechanistic pathways

Postmortem changes in brain cell structure: a review

Brain cell structure is a key determinant of neural function that is frequently altered in neurobiological disorders. Following the global loss of blood flow to the brain that initiates the postmortem interval (PMI), cells rapidly become depleted of energy and begin to decompose. To ensure that our methods for studying the brain using autopsy tissue are robust and reproducible, there is a critical need to delineate the expected changes in brain cell morphometry during the PMI. We searched multiple databases to identify studies measuring the effects of PMI on the morphometry (i.e. external dimensions) of brain cells. We screened 2119 abstracts, 361 full texts, and included 172 studies. Mechanistically, fluid shifts causing cell volume alterations and vacuolization are an early event in the PMI, while the loss of the ability to visualize cell membranes altogether is a later event. Decomposition rates are highly heterogenous and depend on the methods for visualization, the structural feature of interest, and modifying variables such as the storage temperature or the species. Geometrically, deformations of cell membranes are common early events that initiate within minutes. On the other hand, topological relationships between cellular features appear to remain intact for more extended periods. Taken together, there is an uncertain period of time, usually ranging from several hours to several days, over which cell membrane structure is progressively lost. This review may be helpful for investigators studying human postmortem brain tissue, wherein the PMI is an unavoidable aspect of the research

Substrate Concentration Influences Effective Radial Diffusion Coefficient in Canine Cortical Bone

Transport of nutrients and waste across osseous tissue is dependent on the dynamic micro and macrostructure of the tissue; however little quantitative data exists examining how this transport occurs across the entire tissue. Here we investigate in vitro radial diffusion across a section of canine tissue, at dimensions of several hundred microns to millimeters, specifically between several osteons connected through a porous microstructure of Volkmann\u27s canals and canaliculi. The effective diffusion coefficient is measured by a sample immersion technique presented here, in which the tissue sample was immersed in solution for 18-30 h, image analysis software was used to quantify the solute concentration profile in the tissue, and the data were fit to a mathematical model of diffusion in the tissue. Measurements of the effective diffusivity of sodium fluorescein using this technique were confirmed using a standard two-chamber diffusion system. As the solute concentration increased, the effective diffusivity decreased, ranging from 1.6 x 10(-7) +/- A 3.2 x 10(-8) cm(2)/s at 0.3 mu M to 1.4 x 10(-8) +/- A 1.9 x 10(-9) cm(2)/s at 300 mu M. The results show that there is no significant difference in mean diffusivity obtained using the two measurement techniques on the same sample, 3.3 x 10(-8) +/- A 3.3 x 10(-9) cm(2)/s (sample immersion), compared to 4.4 x 10(-8) +/- A 1.1 x 10(-8) cm(2)/s (diffusion chamber)

Alterations in phenotype and gene expression of adult human aneurysmal smooth muscle cells by exogenous nitric oxide

International audienceAbdominal aortic aneurysms (AAA) are characterized by matrix remodeling, elastin degradation, absence of nitric oxide (NO) signaling, and inflammation, influencing smooth muscle cell (SMC) phenotype and gene expression. Little is known about the biomolecular release and intrinsic biomechanics of human AAA-SMCs. NO delivery could be an attractive therapeutic strategy to restore lost functionality of AAA-SMCs by inhibiting inflammation and cell stiffening. We aim to establish the differences in phenotype and gene expression of adult human AAA-SMCs from healthy SMCs. Based on our previous study which showed benefits of optimal NO dosage delivered via S-Nitrosoglutathione (GSNO) to healthy aortic SMCs, we tested whether such benefits would occur in AAA-SMCs. The mRNA expression of three genes involved in matrix degradation (ACE, ADAMTS5 and ADAMTS8) was significantly downregulated in AAA-SMCs. Total protein and glycosaminoglycans synthesis were higher in AAA-SMCs than healthy-SMCs (p < 0.05 for AAA-vs. healthy-SMC cultures) and was enhanced by GSNO and 3D cultures (p < 0.05 for 3D vs. 2D cultures; p < 0.05 for GSNO vs. non-GSNO cases). Elastin gene expression, synthesis and deposition, desmosine crosslinker levels, and lysyl oxidase (LOX) functional activity were lower, while cell proliferation, iNOS, LOX and fibrillin-1 gene expressions were higher in AAA-SMCs (p < 0.05 between respective cases), with differential benefits from GSNO exposure. GSNO and 3D cultures reduced MMPs −2, −9, and increased TIMP-1 release in AAA-SMC cultures (p < 0.05 for GSNO vs. non-GSNO cultures). AAA-SMCs were inherently stiffer and had smoother surface than healthy SMCs (p < 0.01 in both cases), but GSNO reduced stiffness (~25%; p < 0.01) and increased roughness (p < 0.05) of both cell types. In conclusion, exogenously-delivered NO offers an attractive strategy by providing therapeutic benefits to AAA-SMCs

High Angular Resolution Imaging of Solar Radio Bursts from the Lunar Surface

Locating low frequency radio observatories on the lunar surface has a number of advantages, including positional stability and a very low ionospheric radio cutoff. Here, we describe the Radio Observatory on the lunar Surface for Solar studies (ROLSS), a concept for a low frequency, radio imaging interferometric array designed to study particle acceleration in the corona and inner heliosphere. ROLSS would be deployed during an early lunar sortie or by a robotic rover as part of an unmanned landing. The preferred site is on the lunar near side to simplify the data downlink to Earth. The prime science mission is to image type II and type III solar radio bursts with the aim of determining the sites at and mechanisms by which the radiating particles are accelerated. Secondary science goals include constraining the density of the lunar ionosphere by measuring the low radio frequency cutoff of the solar radio emissions or background galactic radio emission, measuring the flux, particle mass, and arrival direction of interplanetary and interstellar dust, and constraining the low energy electron population in astrophysical sources. Furthermore, ROLSS serves a pathfinder function for larger lunar radio arrays. Key design requirements on ROLSS include the operational frequency and angular resolution. The electron densities in the solar corona and inner heliosphere are such that the relevant emission occurs below 10 M Hz, essentially unobservable from Earth's surface due to the terrestrial ionospheric cutoff. Resolving the potential sites of particle acceleration requires an instrument with an angular resolution of at least 2 deg at 10 MHz, equivalent to a linear array size of approximately one kilometer. The major components of the ROLSS array are 3 antenna arms, each of 500 m length, arranged in a Y formation, with a central electronics package (CEP) at their intersection. Each antenna arm is a linear strip of polyimide film (e.g., Kapton(TradeMark)) on which 16 single polarization dipole antennas are located by depositing a conductor (e.g., silver). The arms also contain transmission lines for carrying the radio signals from the science antennas to the CEP. Operations would consist of data acquisition during the lunar day, with data downlinks to Earth one or more times every 24 hours

Epithelial to mesenchymal transition is associated with rapamycin resistance

Rapamycin analogues have antitumor efficacy in several tumor types, however few patients demonstrate tumor regression. Thus, there is a pressing need for markers of intrinsic response/resistance and rational combination therapies. We hypothesized that epithelial-to-mesenchymal transition (EMT) confers rapamycin resistance. We found that the epithelial marker E-cadherin protein is higher in rapamycin sensitive (RS) cells and mesenchymal breast cancer cell lines selected by transcriptional EMT signatures are less sensitive to rapamycin. MCF7 cells, transfected with constitutively active mutant Snail, had increased rapamycin resistance (RR) compared to cells transfected with wild-type Snail. Conversely, we transfected two RR mesenchymal cell lines—ACHN and MDA-MB-231—with miR-200b/c or ZEB1 siRNA to promote mesenchymal-to-epithelial transition. This induced E-cadherin expression in both cell lines, and ACHN demonstrated a significant increase in RS. Treatment of ACHN and MDA-MB-231 with trametinib modulated EMT in ACHN cells in vitro. Treatment of MDA-MB-231 and ACHN xenografts with trametinib in combination with rapamycin resulted in significant growth inhibition in both but without an apparent effect on EMT. Future studies are needed to determine whether EMT status is predictive of sensitivity to rapalogs and to determine whether combination therapy with EMT modulating agents can enhance antitumor effects of PI3K/mTOR inhibitors

Trefoil factor 2 rapidly induces interleukin 33 to promote type 2 immunity during allergic asthma and hookworm infection

The molecular mechanisms that drive mucosal T helper type 2 (T[subscript H]2) responses against parasitic helminths and allergens remain unclear. In this study, we demonstrate in mice that TFF2 (trefoil factor 2), an epithelial cell–derived repair molecule, is needed for the control of lung injury caused by the hookworm parasite Nippostrongylus brasiliensis and for type 2 immunity after infection. TFF2 is also necessary for the rapid production of IL-33, a T[subscript H]2-promoting cytokine, by lung epithelia, alveolar macrophages, and inflammatory dendritic cells in infected mice. TFF2 also increases the severity of allergic lung disease caused by house dust mite antigens or IL-13. Moreover, TFF2 messenger RNA expression is significantly increased in nasal mucosal brushings during asthma exacerbations in children. These experiments extend the biological functions of TFF2 from tissue repair to the initiation and maintenance of mucosal T[subscript H]2 responses

Poxvirus Protein N1L Targets the I-κB Kinase Complex, Inhibits Signaling to NF-κB by the Tumor Necrosis Factor Superfamily of Receptors, and Inhibits NF-κB and IRF3 Signaling by Toll-like Receptors

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or interferon regulatory factor 3 (IRF3) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-alpha and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF3 signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF3 signaling pathways