Abstract Silks are appealing materials for numerous biomedical applications involving drug delivery, tissue engineering, or implantable devices, because of their tunable mechanical properties and ...wide range of physical structures. In addition to the functionalities needed for specific clinical applications, a key factor necessary for clinical success for any implanted material is appropriate interactions with the body in vivo . This review summarizes our current understanding of the in vivo biological responses to silks, including degradation, the immune and inflammatory response, and tissue remodeling with particular attention to vascularization. While we focus in this review on silkworm silk fibroin protein due to the large quantity of in vivo data thanks to its widespread use in medical materials and consumer products, spider silk information is also included if available. Silk proteins are degraded in the body on a time course that is dependent on the method of silk fabrication and can range from hours to years. Silk protein typically induces a mild inflammatory response that decreases within a few weeks of implantation. The response involves recruitment and activation of macrophages and may include activation of a mild foreign body response with the formation of multinuclear giant cells, depending on the material format and location of implantation. The number of immune cells present decreases with time and granulation tissue, if formed, is replaced by endogenous, not fibrous, tissue. Importantly, silk materials have not been demonstrated to induce mineralization, except when used in calcified tissues. Due to its ability to be degraded, silk can be remodeled in the body allowing for vascularization and tissue ingrowth with eventual complete replacement by native tissue. The degree of remodeling, tissue ingrowth, or other specific cell behaviors can be modulated with addition of growth or other signaling factors. Silk can also be combined with numerous other materials including proteins, synthetic polymers, and ceramics to enhance its characteristics for a particular function. Overall, the diverse array of silk materials shows excellent bioresponses in vivo with low immunogenicity and the ability to be remodeled and replaced by native tissue making it suitable for numerous clinical applications.
Single nucleotide polymorphisms (SNPs) within the SLC45A2/MATP, SLC24A5/NCKX5, and OCA2/P genes have been associated with natural variation of pigmentation traits in human populations. Here, we ...describe the characterization of human primary melanocytic cells genotyped for polymorphisms within the MATP, NCKX5, or OCA2 loci. On the basis of genotype, these cultured cells reflect the phenotypes observed by others in terms of both melanin content and tyrosinase (TYR) activity when comparing skin designated as either “White” or “Black”. We found a statistically significant association of MATP-374L (darker skin) with higher TYR protein abundance that was not observed for any NCKX5-111 or OCA2 rs12913832 allele. MATP-374L/L homozygous strains displayed significantly lower MATP transcript levels compared to MATP-374F/F homozygous cells, but this did not reach statistical significance based on NCKX5 or OCA2 genotype. Similarly, we observed significantly increased levels of OCA2 mRNA in rs12913832-T (brown eye) homozygotes compared to rs12913832-C (blue eye) homozygous strains, which was not observed for MATP or NCKX5 gene transcripts. In genotype–phenotype associations performed on a collection of 226 southern European individuals using these same SNPs, we were able to show strong correlations in MATP-L374F, OCA2, and melanocortin-1 receptor with skin, eye, and hair color variation, respectively.
Many potassium channel families are over-expressed in cancer, but their mechanistic role in disease progression is poorly understood. Potassium channels modulate membrane potential (Vmem) and thereby ...influence calcium ion dynamics and other voltage-sensitive signaling mechanisms, potentially acting as transcriptional regulators. This study investigated the differential response to over-expression and activation of a cancer-associated potassium channel, the intermediate conductance calcium-activated potassium channel (IK), on aggressive behaviors in mammary epithelial and breast cancer cell lines. IK was over-expressed in the highly metastatic breast cancer cell line MDA-MB-231 and the spontaneously immortalized breast epithelial cell line MCF-10A, and the effect on cancer-associated behaviors was assessed. IK over-expression increased primary tumor growth and metastasis of MDA-MB-231 in orthotopic xenografts, demonstrating for the first time in any cancer type that increased IK is sufficient to promote cancer aggression. The primary tumors had similar vascularization as determined by CD31 staining and similar histological characteristics. Interestingly, despite the increased in vivo growth and metastasis, neither IK over-expression nor activation with agonist had a significant effect on MDA-MB-231 proliferation, invasion, or migration in vitro. In contrast, IK decreased MCF-10A proliferation and invasion through Matrigel but had no effect on migration in a scratch-wound assay. We conclude that IK activity is sufficient to promote cell aggression in vivo. Our data provide novel evidence supporting IK and downstream signaling networks as potential targets for cancer therapies.
Millimeter-long axon tracts and individually addressable neuron populations in a circuit.
•A multi-compartment live neural circuit with 2mm-long axon tracts is generated.•Separate compartments can be ...simultaneously targeted with drugs and/or electrodes.•Electrical stimulation induced frequency-dependent intracellular calcium changes.•Axon connectivity was tracked within a local circuitry and in a 2mm-long tract.•Inhibitory connectivity of the axons was determined by GABA-induced hyperactivity.
The cortical circuitry in the brain consists of structurally and functionally distinct neuronal assemblies with reciprocal axon connections. To generate cell culture-based systems that emulate axon tract systems of an in vivo neural network, we developed a living neural circuit consisting of compartmentalized neuronal populations connected by arrays of two millimeter-long axon tracts that are integrated on a planar multi-electrode array (MEA). The millimeter-scale node-to-node separation allows for pharmacological and electrophysiological manipulations to simultaneously target multiple neuronal populations. The results show controlled selectivity of dye absorption by neurons in different compartments. MEA-transmitted electrical stimulation of targeted neurons shows ∼46% increase of intracellular calcium levels with 20Hz stimulation, but ∼22% decrease with 2k Hz stimulation. The unique feature of long distance axons promotes in vivo-like fasciculation. These axon tracts are determined to be inhibitory afferents by showing increased action potential firing of downstream node upon selective application of γ-aminobutyric acid (GABA) to the upstream node. Together, this model demonstrates integrated capabilities for assessing multiple endpoints including axon tract tracing, calcium influx, network architecture and activities. This system can be used as a multi-functional platform for studying axon tract-associated CNS disorders in vitro, such as diffuse axonal injury after brain trauma.
Computational analyses of a half-million circuit topologies provide a rationale for why certain fold-change detection topologies are more prevalent in nature.
Computational analyses of a half-million ...circuit topologies provide a rationale for why certain fold-change detection topologies are more prevalent in nature.
Polyamines such as spermine and spermidine are required for growth of Escherichia coli; they interact with nucleic acids, and they bind to ribosomes. Polyamines block porins and decrease membrane ...permeability, activities that may protect cells in acid. At high concentrations, however, polyamines impair growth. They impair growth more severely at high pH, probably due to their increased uptake as membrane-permeant weak bases. The role of pH is critical in understanding polyamine stress.
The effect of polyamines was tested on survival of Escherichia coli K-12 W3110 in extreme acid or base (pH conditions outside the growth range). At pH 2, 10 mM spermine increased survival by 2-fold, and putrescine increased survival by 30%. At pH 9.8, however, E. coli survival was decreased 100-fold by 10 mM spermine, putrescine, cadaverine, or spermidine. At pH 8.5, spermine decreased the growth rate substantially, whereas little effect was seen at pH 5.5. Spermidine required ten-fold higher concentrations to impair growth. On proteomic 2-D gels, spermine and spermidine caused differential expression of 31 different proteins. During log-phase growth at pH 7.0, 1 mM spermine induced eight proteins, including PykF, GlpK, SerS, DeaD, OmpC and OmpF. Proteins repressed included acetate-inducible enzymes (YfiD, Pta, Lpd) as well as RapA (HepA), and FabB. At pH 8.5, spermine induced additional proteins: TnaA, OmpA, YrdA and NanA (YhcJ) and also repressed 17 proteins. Four of the proteins that spermine induced (GlpK, OmpA, OmpF, TnaA) and five that were repressed (Lpd, Pta, SucB, TpiA, YfiD) show similar induction or repression, respectively, in base compared to acid. Most of these base stress proteins were also regulated by spermidine, but only at ten-fold higher concentration (10 mM) at high pH (pH 8.5).
Polyamines increase survival in extreme acid, but decrease E. coli survival in extreme base. Growth inhibition by spermine and spermidine requires neutral or higher pH. At or above pH 7, spermine and spermidine regulate specific proteins, many of which are known to be regulated by base stress. High pH amplifies polyamine stress; and naturally occurring polyamines may play an important role in base stress.
This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be ...found at http://www.elsevier.com/locate/withdrawalpolicy.
Peroxisome proliferator-activated receptor-gamma (PPARγ) is a member of the nuclear hormone receptor (NHR) superfamily of ligand-activated transcriptional regulators. Accumulating evidence suggests ...that PPARγ agonists such as the thiazolidinediones (TZDs) may prove to be useful anti-cancer agents exhibiting anti-proliferative and/or pro-apoptotic affects in a range of cancer cell types including melanoma, however, the mechanisms underlying this effect remain unclear. We have demonstrated the anti-proliferative effects of full and partial PPARγ modulators in human melanoma cell lines. Ablation of PPARγ expression in the MM96L melanoma cell line by siRNA mediated mechanisms attenuates the anti-proliferative effect of these agents suggesting this effect is directly mediated by PPARγ. The mechanisms underlying the anti-proliferative effects of PPARγ in melanoma cells involve the regulation of expression of a number of critical cell cycle genes and β-catenin. Moreover, our data indicate that PPARγ modulates Wnt/β-catenin mediated signalling in melanoma cells in an agonist dependent manner.
In many ways cancer is regeneration gone awry. Both processes involve many of the same behaviors and molecular pathways. However, regeneration occurs with impeccable coordination while in cancer many ...control mechanisms are lost leading to vastly different outcomes. Discovering the key factors responsible for the bifurcation between a path of regulated healing or unrestricted growth will provide valuable insight into new clinical strategies to both cure cancer and to improve healing after injury or disease. The determining factor is often thought to be mutations in DNA, however only a small percentage of cells carrying cancer causing mutations ever go on to form a tumor suggesting additional factors are at play. One mechanism with an early involvement in both regeneration and cancer is bioelectric signaling; changes in ion flows and the electric potential across a cell membrane. The purpose of this work was to explore this understudied signaling mechanism, specifically investigating whether changes in the electric membrane potential (Vmem) are sufficient to instruct regeneration and cancer-associated behaviors. Ostoegenic differentiation of human adult mesenchymal stem cells (hMSC) was used as a model of healthy regeneration. I utilized expression of specific ion channels, culture in different extracellular ion concentrations and inhibition or activation of ion channels with pharmacological agents to modulate Vmem of hMSC during differentiation. None of the conditions tested increased differentiation as determined by quantitative analysis of osteogenic markers. Differences in membrane potential have also been reported between healthy and cancerous cells. In order to test whether Vmem plays an instructive role in cancer, similar methods were used to modulate the Vmem of two breast epithelial cell lines. One of the cell lines was highly metastatic and thus modeled the highly diseased state and the other was a spontaneously immortalized non-tumorigenic cell line with a lower threshold of transformation than primary healthy cells, providing a model to investigate disease onset. While some of the treatments did lead to changes in cancer-related behaviors, overall the effects did not correlate with membrane potential suggesting they were enacted by Vmem -independent mechanisms. Together, these results are not supportive of an instructive role for Vmem, but instead suggest Vmem is a critical part of the cell machinery that is beneficial to most cell behaviors.
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