Mechanical properties and cellular response of novel electrospun nanofibers for ligament tissue engineering: Effects of orientation and geometry

Electrospun nanofibers are a promising material for ligamentous tissue engineering, however weak mechanical properties of fibers to date have limited their clinical usage. The goal of this work was to modify electrospun nanofibers to create a robust structure that mimics the complex hierarchy of native tendons and ligaments. The scaffolds that were fabricated in this study consisted of either random or aligned nanofibers in flat sheets or rolled nanofiber bundles that mimic the size scale of fascicle units in primarily tensile load bearing soft musculoskeletal tissues. Altering nanofiber orientation and geometry significantly affected mechanical properties; most notably aligned nanofiber sheets had the greatest modulus; 125% higher than that of random nanofiber sheets; and 45% higher than aligned nanofiber bundles. Modifying aligned nanofiber sheets to form aligned nanofiber bundles also resulted in approximately 107% higher yield stresses and 140% higher yield strains. The mechanical properties of aligned nanofiber bundles were in the range of the mechanical properties of the native ACL: modulus=158±32MPa, yield stress=57±23MPa and yield strain=0.38±0.08. Adipose derived stem cells cultured on all surfaces remained viable and proliferated extensively over a 7 day culture period and cells elongated on nanofiber bundles. The results of the study suggest that aligned nanofiber bundles may be useful for ligament and tendon tissue engineering based on their mechanical properties and ability to support cell adhesion, proliferation, and elongation.

The cellular and mechanistic study of nisin inhibition of bacillus anthracis spore outgrowth, nisin alteration of infection, and native producer immunity

A critical step during Bacillus anthracis infection is the outgrowth of germinated spores into vegetative bacilli that proliferate and disseminate rapidly within the host. An important challenge exists for developing chemotherapeutic agents that act upon and kill B. anthracis immediately after germination initiation when antibiotic resistance is lost, but prior to the outgrowth into vegetative bacilli, which is accompanied by toxin production. Chemical agents must also function in a manner refractive to the development of antimicrobial resistance. In this thesis we have identified the lantibiotics as a class of chemotherapeutics that are predicted to satisfy these two criteria. The objective of this thesis was to evaluate the efficacy of nisin, a prototypical lantibiotic, in prevention of outgrowth of germinated B. anthracis spores. Like all lantibiotics, nisin is a ribosomally translated peptide that undergoes post-translational modification to form (methyl)lanthionine rings that are critical for antimicrobial activity. Our studies indicate that nisin rapidly inhibits the in vitro outgrowth of germinated B. anthracis Sterne 7702 spores. Although germination initiation was shown to be essential for nisin-dependent antimicrobial activity, nisin did not inhibit or promote germination initiation. Nisin irreversibly killed germinated spores by blocking the establishment of a membrane potential and oxidative metabolism, while not affecting the dissolution of the outer spore structures. The membrane permeability of the spore was increased by nisin, but germinated spores did not undergo full lysis. Nisin was demonstrated to localize to lipid II, which is the penultimate precursor for cell wall biogenesis. This localization suggests two possible independent mechanisms of action, membrane pore formation and inhibition of peptidoglycan synthesis. Structure-activity studies with a truncated form of nisin lacking the two C-terminal (methyl)lanthionine rings and with non-pore forming mutants indicated that membrane disruption is essential for nisin-dependent inhibition of spore outgrowth to prevent membrane potential establishment. Finally, utilizing an in vitro infection model, it was shown that nisin reduced the viability of B. anthracis spores within an infection resulting in increased survival of immune cells while reducing infection-mediated cytokine expression. Fluorescence microscopy indicated that nisin localizes with spores within phagosomes of peritioneal macrophages in germinating conditions. These data demonstrate the effectiveness of nisin, as a model lantibiotic, for preventing spore outgrowth. It is speculated that nisin targeting of lipid II, resulting in membrane perturbations, may be effective at inhibiting the outgrowth of spores prepared from bacteria across a number of species.

Matrix Metalloproteinases And Left Ventricular Function And Structure In Spinal Cord Injured Subjects.

Subjects with spinal cord injury (SCI) exhibit impaired left ventricular (LV) diastolic function, which has been reported to be attenuated by regular physical activity. This study investigated the relationship between circulating matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) and echocardiographic parameters in SCI subjects and the role of physical activity in this regard. Forty-two men with SCI [19 sedentary (S-SCI) and 23 physically-active (PA-SCI)] were evaluated by clinical, anthropometric, laboratory, and echocardiographic analysis. Plasmatic pro-MMP-2, MMP-2, MMP-8, pro-MMP-9, MMP-9, TIMP-1 and TIMP-2 levels were determined by enzyme-linked immunosorbent assay and zymography. PA-SCI subjects presented lower pro-MMP-2 and pro-MMP-2/TIMP-2 levels and improved markers of LV diastolic function (lower E/Em and higher Em and E/A values) than S-SCI ones. Bivariate analysis showed that pro-MMP-2 correlated inversely with Em and directly with E/Em, while MMP-9 correlated directly with LV mass index and LV end-diastolic diameter in the whole sample. Following multiple regression analysis, pro-MMP-2, but not physical activity, remained associated with Em, while MMP-9 was associated with LV mass index in the whole sample. These findings suggest differing roles for MMPs in LV structure and function regulation and an interaction among pro-MMP-2, diastolic function and physical activity in SCI subjects.

Growth Factor Stimulation Improves The Structure And Properties Of Scaffold-free Engineered Auricular Cartilage Constructs.

The reconstruction of the external ear to correct congenital deformities or repair following trauma remains a significant challenge in reconstructive surgery. Previously, we have developed a novel approach to create scaffold-free, tissue engineering elastic cartilage constructs directly from a small population of donor cells. Although the developed constructs appeared to adopt the structural appearance of native auricular cartilage, the constructs displayed limited expression and poor localization of elastin. In the present study, the effect of growth factor supplementation (insulin, IGF-1, or TGF-β1) was investigated to stimulate elastogenesis as well as to improve overall tissue formation. Using rabbit auricular chondrocytes, bioreactor-cultivated constructs supplemented with either insulin or IGF-1 displayed increased deposition of cartilaginous ECM, improved mechanical properties, and thicknesses comparable to native auricular cartilage after 4 weeks of growth. Similarly, growth factor supplementation resulted in increased expression and improved localization of elastin, primarily restricted within the cartilaginous region of the tissue construct. Additional studies were conducted to determine whether scaffold-free engineered auricular cartilage constructs could be developed in the 3D shape of the external ear. Isolated auricular chondrocytes were grown in rapid-prototyped tissue culture molds with additional insulin or IGF-1 supplementation during bioreactor cultivation. Using this approach, the developed tissue constructs were flexible and had a 3D shape in very good agreement to the culture mold (average error <400 µm). While scaffold-free, engineered auricular cartilage constructs can be created with both the appropriate tissue structure and 3D shape of the external ear, future studies will be aimed assessing potential changes in construct shape and properties after subcutaneous implantation.

Ssr-based Genetic Diversity And Structure Of Garlic Accessions From Brazil.

Garlic is a spice and a medicinal plant; hence, there is an increasing interest in 'developing' new varieties with different culinary properties or with high content of nutraceutical compounds. Phenotypic traits and dominant molecular markers are predominantly used to evaluate the genetic diversity of garlic clones. However, 24 SSR markers (codominant) specific for garlic are available in the literature, fostering germplasm researches. In this study, we genotyped 130 garlic accessions from Brazil and abroad using 17 polymorphic SSR markers to assess the genetic diversity and structure. This is the first attempt to evaluate a large set of accessions maintained by Brazilian institutions. A high level of redundancy was detected in the collection (50 % of the accessions represented eight haplotypes). However, non-redundant accessions presented high genetic diversity. We detected on average five alleles per locus, Shannon index of 1.2, HO of 0.5, and HE of 0.6. A core collection was set with 17 accessions, covering 100 % of the alleles with minimum redundancy. Overall FST and D values indicate a strong genetic structure within accessions. Two major groups identified by both model-based (Bayesian approach) and hierarchical clustering (UPGMA dendrogram) techniques were coherent with the classification of accessions according to maturity time (growth cycle): early-late and midseason accessions. Assessing genetic diversity and structure of garlic collections is the first step towards an efficient management and conservation of accessions in genebanks, as well as to advance future genetic studies and improvement of garlic worldwide.

Ki-1/57 And Cgi-55 Ectopic Expression Impact Cellular Pathways Involved In Proliferation And Stress Response Regulation.

Ki-1/57 (HABP4) and CGI-55 (SERBP1) are regulatory proteins and paralogs with 40.7% amino acid sequence identity and 67.4% similarity. Functionally, they have been implicated in the regulation of gene expression on both the transcriptional and mRNA metabolism levels. A link with tumorigenesis is suggested, since both paralogs show altered expression levels in tumor cells and the Ki-1/57 gene is found in a region of chromosome 9q that represents a haplotype for familiar colon cancer. However, the target genes regulated by Ki-1/57 and CGI-55 are unknown. Here, we analyzed the alterations of the global transcriptome profile after Ki-1/57 or CGI-55 overexpression in HEK293T cells by DNA microchip technology. We were able to identify 363 or 190 down-regulated and 50 or 27 up-regulated genes for Ki-1/57 and CGI-55, respectively, of which 20 were shared between both proteins. Expression levels of selected genes were confirmed by qRT-PCR both after protein overexpression and siRNA knockdown. The majority of the genes with altered expression were associated to proliferation, apoptosis and cell cycle control processes, prompting us to further explore these contexts experimentally. We observed that overexpression of Ki-1/57 or CGI-55 results in reduced cell proliferation, mainly due to a G1 phase arrest, whereas siRNA knockdown of CGI-55 caused an increase in proliferation. In the case of Ki-1/57 overexpression, we found protection from apoptosis after treatment with the ER-stress inducer thapsigargin. Together, our data give important new insights that may help to explain these proteins putative involvement in tumorigenic events.

The Atomic Structure Of A Bare Buffer Layer On Sic(0001) Chemically Resolved.

A chemical-specific photoelectron diffraction structure determination of a carbon rich buffer layer on SiC is reported. In addition to the long-range ripple of this surface, a local buckling in the hexagonal sublattice, which breaks the local range order symmetry, was unraveled.

Crystal Structure Of (3e)-3-[(4-nitro-phen-oxy)-meth-yl]-4-phenyl-but-3-en-2-one.

In the title compound, C17H15NO4, the conformation about the C=C double bond [1.348 (2) Å] is E with the ketone group almost co-planar [C-C-C-C torsion angle = 7.2 (2)°] but the phenyl group twisted away [C-C-C-C = 160.93 (17)°]. The terminal aromatic rings are almost perpendicular to each other [dihedral angle = 81.61 (9)°] giving the mol-ecule an overall U-shape. The crystal packing feature benzene-C-H⋯O(ketone) contacts that lead to supra-molecular helical chains along the b axis. These are connected by π-π inter-actions between benzene and phenyl rings [inter-centroid distance = 3.6648 (14) Å], resulting in the formation of a supra-molecular layer in the bc plane.

Crystal Structure Of (3e)-3-(2,4-di-nitro-phen-oxy-meth-yl)-4-phenyl-but-3-en-2-one.

In the title compound, C17H14N2O6, the conformation about the C=C double bond [1.345 (2) Å] is E, with the ketone moiety almost coplanar [C-C-C-C torsion angle = 9.5 (2)°] along with the phenyl ring [C-C-C-C = 5.9 (2)°]. The aromatic rings are almost perpendicular to each other [dihedral angle = 86.66 (7)°]. The 4-nitro moiety is approximately coplanar with the benzene ring to which it is attached [O-N-C-C = 4.2 (2)°], whereas the one in the ortho position is twisted [O-N-C-C = 138.28 (13)°]. The mol-ecules associate via C-H⋯O inter-actions, involving both O atoms from the 2-nitro group, to form a helical supra-molecular chain along [010]. Nitro-nitro N⋯O inter-actions [2.8461 (19) Å] connect the chains into layers that stack along [001].

Human Herpesvirus-6 And Cytomegalovirus Dna In Liver Donor Biopsies And Their Correlation With Hla Matches And Acute Cellular Rejection.

Herpesvirus reactivation is common after liver transplantation. Analyze the presence of cytomegalovirus (HCMV) and human herpesvirus-6 (HHV-6) DNA in liver donor biopsies, seeking to better understand issues involving human donor leukocyte antigens (HLA)-A, B and DR, as well as correlations with acute cellular rejection. Fifty-nine liver transplantation patients were investigated for the presence of HCMV and HHV-6 DNA in liver donor biopsies, using the Nested-PCR technique. The clinical donor information and HLA matches were obtained from the São Paulo State Transplant System. The recipients' records regarding acute cellular rejection were studied. Seven (11.8%) biopsies were positive for HCMV DNA and 29 (49%) were positive for HHV-6 DNA. In 14 donors with HLA-DR 15 nine had HHV-6 DNA positive liver biopsy with a tendency for significant association (p=0.09), 22 recipients developed acute cellular rejection and 9/22 were positive for HLA-DR 15 (p=0.03; χ(2)=4.51), which was statistically significant in univariate analysis and showed a tendency after multivariate analysis (p=0.08). HHV-6 DNA was prevalent in liver donors studied as well as HLA-DR 15. These findings suggest that patients with HLA-DR 15 in liver donor biopsies develop more rejection after liver transplantation.

On The Consistency Of Monte Carlo Track Structure Dna Damage Simulations.

Monte Carlo track structures (MCTS) simulations have been recognized as useful tools for radiobiological modeling. However, the authors noticed several issues regarding the consistency of reported data. Therefore, in this work, they analyze the impact of various user defined parameters on simulated direct DNA damage yields. In addition, they draw attention to discrepancies in published literature in DNA strand break (SB) yields and selected methodologies. The MCTS code Geant4-DNA was used to compare radial dose profiles in a nanometer-scale region of interest (ROI) for photon sources of varying sizes and energies. Then, electron tracks of 0.28 keV-220 keV were superimposed on a geometric DNA model composed of 2.7 × 10(6) nucleosomes, and SBs were simulated according to four definitions based on energy deposits or energy transfers in DNA strand targets compared to a threshold energy ETH. The SB frequencies and complexities in nucleosomes as a function of incident electron energies were obtained. SBs were classified into higher order clusters such as single and double strand breaks (SSBs and DSBs) based on inter-SB distances and on the number of affected strands. Comparisons of different nonuniform dose distributions lacking charged particle equilibrium may lead to erroneous conclusions regarding the effect of energy on relative biological effectiveness. The energy transfer-based SB definitions give similar SB yields as the one based on energy deposit when ETH ≈ 10.79 eV, but deviate significantly for higher ETH values. Between 30 and 40 nucleosomes/Gy show at least one SB in the ROI. The number of nucleosomes that present a complex damage pattern of more than 2 SBs and the degree of complexity of the damage in these nucleosomes diminish as the incident electron energy increases. DNA damage classification into SSB and DSB is highly dependent on the definitions of these higher order structures and their implementations. The authors' show that, for the four studied models, different yields are expected by up to 54% for SSBs and by up to 32% for DSBs, as a function of the incident electrons energy and of the models being compared. MCTS simulations allow to compare direct DNA damage types and complexities induced by ionizing radiation. However, simulation results depend to a large degree on user-defined parameters, definitions, and algorithms such as: DNA model, dose distribution, SB definition, and the DNA damage clustering algorithm. These interdependencies should be well controlled during the simulations and explicitly reported when comparing results to experiments or calculations.

Graphene Oxide: A Carrier For Pharmaceuticals And A Scaffold For Cell Interactions.

During the last ten years, graphene oxide has been explored in many applications due to its remarkable electroconductivity, thermal properties and mobility of charge carriers, among other properties. As discussed in this review, the literature suggests that a total characterization of graphene oxide must be conducted because oxidation debris (synthesis impurities) present in the graphene oxides could act as a graphene oxide surfactant, stabilizing aqueous dispersions. It is also important to note that the structure models of graphene oxide need to be revisited because of significant implications for its chemical composition and its direct covalent functionalization. Another aspect that is discussed is the need to consider graphene oxide surface chemistry. The hemolysis assay is recommended as a reliable test for the preliminary assessment of graphene oxide toxicity, biocompatibility and cell membrane interaction. More recently, graphene oxide has been extensively explored for drug delivery applications. An important increase in research efforts in this emerging field is clearly represented by the hundreds of related publications per year, including some reviews. Many studies have been performed to explore the graphene oxide properties that enable it to deliver more than one activity simultaneously and to combine multidrug systems with photothermal therapy, indicating that graphene oxide is an attractive tool to overcome hurdles in cancer therapies. Some strategic aspects of the application of these materials in cancer treatment are also discussed. In vitro studies have indicated that graphene oxide can also promote stem cell adhesion, growth and differentiation, and this review discusses the recent and pertinent findings regarding graphene oxide as a valuable nanomaterial for stem cell research in medicine. The protein corona is a key concept in nanomedicine and nanotoxicology because it provides a biomolecular identity for nanomaterials in a biological environment. Understanding protein corona-nanomaterial interactions and their influence on cellular responses is a challenging task at the nanobiointerface. New aspects and developments in this area are discussed.

Analysis Of Cellular Adhesion On Superhydrophobic And Superhydrophilic Vertically Aligned Carbon Nanotube Scaffolds.

We analyzed GFP cells after 24h cultivated on superhydrophilic vertically aligned carbon nanotube scaffolds. We produced two different densities of VACNT scaffolds on Ti using Ni or Fe catalysts. A simple and fast oxygen plasma treatment promoted the superhydrophilicity of them. We used five different substrates, such as: as-grown VACNT produced using Ni as catalyst (Ni), as-grown VACNT produced using Fe as catalyst (Fe), VACNT-O produced using Ni as catalyst (NiO), VACNT-O produced using Fe as catalyst (FeO) and Ti (control). The 4',6-diamidino-2-phenylindole reagent nuclei stained the adherent cells cultivated on five different analyzed scaffolds. We used fluorescence microscopy for image collect, ImageJ® to count adhered cell and GraphPad Prism 5® for statistical analysis. We demonstrated in crescent order: Fe, Ni, NiO, FeO and Ti scaffolds that had an improved cellular adhesion. Oxygen treatment associated to high VACNT density (group FeO) presented significantly superior cell adhesion up to 24h. However, they do not show significant differences compared with Ti substrates (control). We demonstrated that all the analyzed substrates were nontoxic. Also, we proposed that the density and hydrophilicity influenced the cell adhesion behavior.

Human Mitochondrial Hsp70 (mortalin): Shedding Light On Atpase Activity, Interaction With Adenosine Nucleotides, Solution Structure And Domain Organization.

The human mitochondrial Hsp70, also called mortalin, is of considerable importance for mitochondria biogenesis and the correct functioning of the cell machinery. In the mitochondrial matrix, mortalin acts in the importing and folding process of nucleus-encoded proteins. The in vivo deregulation of mortalin expression and/or function has been correlated with age-related diseases and certain cancers due to its interaction with the p53 protein. In spite of its critical biological roles, structural and functional studies on mortalin are limited by its insoluble recombinant production. This study provides the first report of the production of folded and soluble recombinant mortalin when co-expressed with the human Hsp70-escort protein 1, but it is still likely prone to self-association. The monomeric fraction of mortalin presented a slightly elongated shape and basal ATPase activity that is higher than that of its cytoplasmic counterpart Hsp70-1A, suggesting that it was obtained in the functional state. Through small angle X-ray scattering, we assessed the low-resolution structural model of monomeric mortalin that is characterized by an elongated shape. This model adequately accommodated high resolution structures of Hsp70 domains indicating its quality. We also observed that mortalin interacts with adenosine nucleotides with high affinity. Thermally induced unfolding experiments indicated that mortalin is formed by at least two domains and that the transition is sensitive to the presence of adenosine nucleotides and that this process is dependent on the presence of Mg2+ ions. Interestingly, the thermal-induced unfolding assays of mortalin suggested the presence of an aggregation/association event, which was not observed for human Hsp70-1A, and this finding may explain its natural tendency for in vivo aggregation. Our study may contribute to the structural understanding of mortalin as well as to contribute for its recombinant production for antitumor compound screenings.

Nectar Robbery By A Hermit Hummingbird: Association To Floral Phenotype And Its Influence On Flowers And Network Structure.

Interactions between flowers and their visitors span the spectrum from mutualism to antagonism. The literature is rich in studies focusing on mutualism, but nectar robbery has mostly been investigated using phytocentric approaches focused on only a few plant species. To fill this gap, we studied the interactions between a nectar-robbing hermit hummingbird, Phaethornis ruber, and the array of flowers it visits. First, based on a literature review of the interactions involving  P. ruber, we characterized the association of floral larceny to floral phenotype. We then experimentally examined the effects of nectar robbing on nectar standing crop and number of visits of the pollinators to the flowers of Canna paniculata. Finally, we asked whether the incorporation of illegitimate interactions into the analysis affects plant-hummingbird network structure. We identified 97 plant species visited by P. ruber and found that P. ruber engaged in floral larceny in almost 30 % of these species. Nectar robbery was especially common in flowers with longer corolla. In terms of the effect on C. paniculata, the depletion of nectar due to robbery by P. ruber was associated with decreased visitation rates of legitimate pollinators. At the community level, the inclusion of the illegitimate visits of P. ruber resulted in modifications of how modules within the network were organized, notably giving rise to a new module consisting of P. ruber and mostly robbed flowers. However, although illegitimate visits constituted approximately 9 % of all interactions in the network, changes in nestedness, modularity, and network-level specialization were minor. Our results indicate that although a flower robber may have a strong effect on the pollination of a particular plant species, the inclusion of its illegitimate interactions has limited capacity to change overall network structure.