Impermeable graphenic encasement of bacteria.

Transmission electron microscopy (TEM) of hygroscopic, permeable, and electron-absorbing biological cells has been an important challenge due to the volumetric shrinkage, electrostatic charging, and structural degradation of cells under high vacuum and fixed electron beam.(1-3) Here we show that bacterial cells can be encased within a graphenic chamber to preserve their dimensional and topological characteristics under high vacuum (10(-5) Torr) and beam current (150 A/cm(2)). The strongly repelling π clouds in the interstitial sites of graphene's lattice(4) reduces the graphene-encased-cell's permeability(5) from 7.6-20 nm/s to 0 nm/s. The C-C bond flexibility(5,6) enables conformal encasement of cells. Additionally, graphene's high Young's modulus(6,7) retains cell's structural integrity under TEM conditions, while its high electrical(8) and thermal conductivity(9) significantly abates electrostatic charging. We envision that the graphenic encasement approach will facilitate real-time TEM imaging of fluidic samples and potentially biochemical activity.

Writing an effective National Institutes of Health (NIH) budget: How to get the money for your science.

In the scheme of developing an application for funding from any federal or foundation source, it is reasonable to place significant attention on the science. However, it is also imperative to remember that your budget is what will provide the resources to make sure you can complete your proposed investigations and, as such, deserves appropriate consideration. In the competitive arena of extramural funding, funding agencies are incentivized to ensure that the funds committed to research will yield maximum impact. A well-thought-out budget demonstrates to the funding agency 2 key factors: (1) that you understand the needs of the project and (2) you have a realistic expectation of the project costs. When these 2 things are communicated to the funding agency, in addition to the significance of your science, it is more likely that you will receive the budget you request. Herein, we put forth the fundamentals for preparing your budget and the nuances that may help you not only be in compliance but also improve your chances of success. This article will discuss issues to consider when designing a budget for large research grants, using the NIH R&R Budget as a prototype.

Mentored career development awards for the development of surgeon-scientists.

Career development awards are important sources of support for surgeons who aim to become independent investigators. However, many challenges and opportunities need to be considered when deciding whether an individual is an appropriate career development award candidate. A quintessential example of the mentor-based career development award to support scientific training is the National Institutes of Health K award. In this article, we focus on issues that face surgeons interested in applying for these K series National Institutes of Health-mentored career development awards. We discuss the different types of K awards and the challenges they may pose for surgeons and provide recommendations for how to determine if a career development award is an appropriate approach given one's career track and institutional environment. Lastly, we discuss how to effectively manage K awards and how to increase the odds of achieving a K to R award transition. The career development award can be a highly effective mechanism to help develop the careers of the next generation of surgeon-scientists, but successfully obtaining these awards requires an assessment of whether the career development award is the appropriate mechanism for the applicant and how to optimize the probability for success.

A practical guide to writing a competitive K award application.

Career Development Awards, including K-series grants from the National Institutes of Health, are often the first external award that developing surgeon-scientists will receive, and can lead to higher success rates in obtaining later independent funding. However, just like learning a new surgical technique, learning to create a competitive Career Development Award application requires good instruction and dedicated practice. This article is geared to deliver practical instruction for how to approach an initial Career Development Award application, so that aspiring surgeon-scientists will be equipped to tackle this daunting task in practice. Based on insights gleaned from published sources and the authors' own experiences as K awardees, the discussion will cover preapplication considerations, including when to apply and how to get started, as well as specific advice for crafting well-developed components of the Career Development Award application. The objective of this article is to provide potential applicants with information and strategies to produce the highest quality, cohesive Career Development Award application possible. In sum, the authors hope that this article provides helpful insights to guide applicants toward successfully securing Career Development Award funding and establishing a solid foundation for their academic research careers.

Interfacial Coating Method for Amine-Rich Surfaces using Poly(ethylene glycol) Diacrylate Applied to Bioprosthetic Valve Tissue Models.

Bioprosthetic heart valve implants are beset by calcification and failure due to the interactions between the body and the transplant. Hydrogels can be used as biological blank slates that may help to shield implants from these interactions; however, traditional light-based hydrogel polymerization is impeded by tissue opacity and topography. Therefore, new methods must be created to bind hydrogel to implant tissues. To address these complications, a two-step surface-coating method for bioprosthetic valves was developed. A previously developed bioprosthetic valve model (VM) was used to investigate and optimize the coating method. Generally, this coating is achieved by first reacting surface amine groups with an NHS-PEG-acrylate while also allowing glucose to absorb into the bulk. Then, glucose oxidase, poly(ethylene glycol) diacrylate (PEGDA), and iron ions are added to the system to initiate free-radical polymerization that bonds the PEGDA hydrogel to the acrylates sites on the surface. Results showed a thin (∼8 µm), continuous coating on VM samples that is capable of repelling protein adhesion (2% surface fouling versus 20% on uncoated samples) and does not significantly affect the surface mechanical properties. Based on this success, the coating method was translated to glutaraldehyde-fixed valve tissue samples. Results showed noncontinuous but evident coating on the surface, which was further improved by adjusting the coating solution. These results demonstrate the feasibility of the proposed two-step surface coating method for modifying the surface of bioprosthetic valve replacements.

Cancer-Associated Fibroblasts Induce a Collagen Cross-link Switch in Tumor Stroma.

UNLABELLED: Intratumoral collagen cross-links heighten stromal stiffness and stimulate tumor cell invasion, but it is unclear how collagen cross-linking is regulated in epithelial tumors. To address this question, we used Kras(LA1) mice, which develop lung adenocarcinomas from somatic activation of a Kras(G12D) allele. The lung tumors in Kras(LA1) mice were highly fibrotic and contained cancer-associated fibroblasts (CAF) that produced collagen and generated stiffness in collagen gels. In xenograft tumors generated by injection of wild-type mice with lung adenocarcinoma cells alone or in combination with CAFs, the total concentration of collagen cross-links was the same in tumors generated with or without CAFs, but coinjected tumors had higher hydroxylysine aldehyde-derived collagen cross-links (HLCC) and lower lysine-aldehyde-derived collagen cross-links (LCCs). Therefore, we postulated that an LCC-to-HLCC switch induced by CAFs promotes the migratory and invasive properties of lung adenocarcinoma cells. To test this hypothesis, we created coculture models in which CAFs are positioned interstitially or peripherally in tumor cell aggregates, mimicking distinct spatial orientations of CAFs in human lung cancer. In both contexts, CAFs enhanced the invasive properties of tumor cells in three-dimensional (3D) collagen gels. Tumor cell aggregates that attached to CAF networks on a Matrigel surface dissociated and migrated on the networks. Lysyl hydroxylase 2 (PLOD2/LH2), which drives HLCC formation, was expressed in CAFs, and LH2 depletion abrogated the ability of CAFs to promote tumor cell invasion and migration. IMPLICATIONS: CAFs induce a collagen cross-link switch in tumor stroma to influence the invasive properties of tumor cells.

Development of a heart valve model surface for optimization of surface modifications.

Current bioprosthetic valve replacements (BPVs) are susceptible to myriad complications, including calcification and thrombosis; however, recent research has explored surface modifications to encourage re-endothelialization of the tissue, preventing unwanted blood-tissue interactions. A bioprosthetic valve surface model (BVSM) was developed to facilitate rapid in vitro optimization of surface modification techniques for BPVs. The BVSM was manufactured by photopolymerization of PEGDA and collagen type I and subsequent addition of amine-rich peptide to provide reactive sites for surface modification. This BVSM mimics surface mechanical properties of bioprosthetic valve tissue, as measured by micropipette aspiration. The BVSM successfully mimics the latent toxic effects of glutaraldehyde fixation, as shown through MTT assay results. Amine content, assessed by XPS, was shown to be significantly lower in the BVSM than unfixed tissue. However, incubation of the surface with amine-reactive NHS-PEG-Cy5 revealed even coverage of the BVSM surface, suggesting that there exists sufficient surface reactive groups to anchor surface modifications, and that translation of the modification process to tissue will yield more complete modification of the BPV surface. These results indicate successful construction of a BVSM that mimics essential properties of bioprosthetic valve tissue and its usefulness for rapid in vitro optimization of surface modification methods for endothelialization. STATEMENT OF SIGNIFICANCE: Current bioprosthetic valve replacements are susceptible to many complications, including calcification and thrombosis; however, recent research has explored surface modifications to encourage the integration of the replacement with the native tissue, which would prevent unwanted blood-tissue interactions. However, methods to analyze and optimize such modifications are limited by the complex surface topography, individual variability, and opacity of native tissue. Thus, we have developed a novel bioprosthetic valve tissue model (BVM) which mimics the important features of the bioprosthetic valve tissue and serves as a platform for rapid optimization and testing of surface modification strategies for tissue valves. Thus, the BVM will provide a needed platform to support rapid improvement of clinically available cardiovascular implants.

Cellular and Extracellular Matrix Basis for Heterogeneity in Mitral Annular Contraction.

PURPOSE: Regional heterogeneity in mitral annular contraction, which is generally ascribed to the fibrous vs. muscular annular composition, ensures proper leaflet motion and timing of coaptation. It is unknown whether the fibroblast-like cells in the annulus modulate this heterogeneity, even though valvular interstitial cells (VICs) can be mechanically "activated." METHODS: Fourteen sheep underwent implantation of radiopaque markers around the mitral annulus defining four segments: septal (SEPT), lateral (LAT), and anterior (ANT-C) and posterior (POST-C) commissures. Segmental annular contraction was calculated using biplane videofluoroscopy. Immunohistochemistry of annular cross sections assessed regional matrix content, matrix turnover, and cell phenotype. Micropipette aspiration measured the Young's modulus of the leaflets adjacent to the myocardial border. RESULTS: Whereas SEPT contained more collagen I and III, LAT demonstrated more collagen and elastin turnover as shown by greater decorin, lysyl oxidase, and matrix metalloprotease (MMP)-13 and smooth muscle alpha-actin (SMaA). This greater matrix turnover paralleled greater annular contraction in LAT vs. SEPT (22.5% vs. 4.1%). Similarly, POST-C had more SMaA and MMP13 than ANT-C, consistent with greater annular contraction in POST-C (18.8% vs. 11.1%). Interestingly, POST-C had the greatest effective modulus, significantly higher than LAT. CONCLUSIONS: These data suggest that matrix turnover by activated VICs relates to annular motion heterogeneity, maintains steady-state mechanical properties in the annulus, and could be a therapeutic target when annular motion is impaired. Conversely, alterations in this heterogeneous annular contraction, whether through disease or secondary to ring annuloplasty, could disrupt this normal pattern of cell-mediated matrix remodeling and further adversely impact mitral valve function.

Heterogeneity of Mitral Leaflet Matrix Composition and Turnover Correlates with Regional Leaflet Strain.

To determine how extracellular matrix and contractile valvular cells contribute to the heterogeneous motion and strain across the mitral valve (MV) during the cardiac cycle, regional MV material properties, matrix composition, matrix turnover, and cell phenotype were related to regional leaflet strain. Radiopaque markers were implanted into 14 sheep to delineate the septal (SEPT), lateral (LAT), and anterior and posterior commissural leaflets (ANT-C, POST-C). Videofluoroscopy imaging was used to calculate radial and circumferential strains. Mechanical properties were assessed using uniaxial tensile testing and micropipette aspiration. Matrix composition and cell phenotypes were immunohistochemically evaluated within each leaflet region [basal leaflet (BL), mid-leaflet (ML), and free edge]. SEPT-BL segments were stiffer and stronger than other valve tissues, while LAT segments demonstrated more extensibility and strain. Collagens I and III in SEPT were greater than in LAT, although LAT showed greater collagen turnover [matrix metalloprotease (MMP)-13, lysyl oxidase] and cell activation [smooth muscle alpha-actin (SMaA), and non-muscle myosin (NMM)]. MMP13, NMM, and SMaA were strongly correlated with each other, as well as with radial and circumferential strains in both SEPT and LAT. SMaA and MMP13 in POST-C ML was greater than ANT-C, corresponding to greater radial strains in POST-C. This work directly relates leaflet strain, material properties, and matrix turnover, and suggests a role for myofibroblasts in the heterogeneity of leaflet composition and strain. New approaches to MV repair techniques and ring design should preserve this normal coupling between leaflet composition and motion.

Osteogenic and angiogenic potentials of monocultured and co-cultured human-bone-marrow-derived mesenchymal stem cells and human-umbilical-vein endothelial cells on three-dimensional porous beta-tricalcium phosphate scaffold.

The use of biodegradable beta-tricalcium phosphate (ß-TCP) scaffolds holds great promise for bone tissue engineering. However, the effects of ß-TCP on bone and endothelial cells are not fully understood. This study aimed to investigate cell proliferation and differentiation of mono- or co-cultured human-bone-marrow-derived mesenchymal stem cells (hBMSCs) and human-umbilical-vein endothelial cells (HUVECs) on a three-dimensional porous, biodegradable ß-TCP scaffold. In co-culture studies, the ratios of hBMSCs:HUVECs were 5:1, 1:1 and 1:5. Cellular morphologies of HUVECs, hBMSCs and co-cultured HUVECs/hBMSCs on the ß-TCP scaffolds were monitored using confocal and scanning electron microscopy. Cell proliferation was monitored by measuring the amount of double-stranded DNA (dsDNA) whereas hBMSC and HUVEC differentiation was assessed using the osteogenic and angiogenic markers, alkaline phosphatase (ALP) and PECAM-1 (CD31), respectively. Results show that HUVECs, hBMSCs and hBMSCs/HUVECs adhered to and proliferated well on the ß-TCP scaffolds. In monoculture, hBMSCs grew faster than HUVECs on the ß-TCP scaffolds after 7 days, but HUVECs reached similar levels of proliferation after 14 days. In monoculture, ß-TCP scaffolds promoted ALP activities of both hBMSCs and HUVECs when compared to those grown on tissue culture well plates. ALP activity of cells in co-culture was higher than that of hBMSCs in monoculture. Real-time polymerase chain reaction results indicate that runx2 and alp gene expression in monocultured hBMSCs remained unchanged at days 7 and 14, but alp gene expression was significantly increased in hBMSC co-cultures when the contribution of individual cell types was not distinguished.