Control of Mammalian Cell and Bacteria Adhesion on Substrates Micropatterned with Poly(ethylene glycol) Hydrogels

A simple method for controlling the spatial positioning of mammalian cells and bacteria on substrates using patterned poly(ethylene glycol) (PEG) hydrogel microstructures is described. These microstructures were fabricated using photolithography on silicon, glass or poly (dimethylsiloxane) (PDMS) surfaces modified with a 3-(trichlorosilyl) propyl methacrylate (TPM) monolayer. During the photogelation reaction, the resulting hydrogel microstructures were covalently bound to the substrate via the TPM monolayer and did not detached from the substrate upon hydration. For mammalian cell patterning, microwell arrays of different dimensions were fabricated. These microwells were composed of hydrophilic PEG hydrogel walls surrounding hydrophobic TPM floors inside the microwells. Murine 3T3 fibroblasts and transformed hepatocytes were shown to selectively adhere to the TPM monolayer inside the microwells, maintaining their viability, while adherent cells were not present on the hydrogel walls. The number of cells inside one microwell could be controled by changing the lateral dimension of the microwells, thus allowing only a single cell per microwell if desired. In the case of 30×30 m microwells, as many as 400 microwells were fabricated in 1 mm². In addition, PEG hydrogel microstructures were also shown to effectively resist the adhesion of bacteria such as Escherichia coli.     

Hardening of Additive Manufactured 316L Stainless Steel by Using Bimodal Powder Containing Nanoscale Fraction

The particle size distribution significantly affects the material properties of the additively manufactured parts. In this work, the influence of bimodal powder containing nano- and micro-scale particles on microstructure and materials properties is studied. Moreover, to study the effect of the protective atmosphere, the test samples were additively manufactured from 316L stainless steel powder in argon and nitrogen. The samples fabricated from the bimodal powder demonstrate a finer subgrain structure, regardless of protective atmospheres and an increase in the Vickers microhardness, which is in accordance with the Hall-Petch relation. The porosity analysis revealed the deterioration in the quality of as-built parts due to the poor powder flowability. The surface roughness of fabricated samples was the same regardless of the powder feedstock materials used and protective atmospheres. The results suggest that the improvement of mechanical properties is achieved by adding a nano-dispersed fraction, which dramatically increases the total surface area, thereby contributing to the nitrogen absorption by the material.     

Bleomycin in octaarginine-modified fusogenic liposomes results in improved tumor growth inhibition

Bleomycin (BLM) is an example of an anticancer drug that should be delivered into cytosol for its efficient therapeutic action. With this in mind, we developed octaarginine (R8)-modified fusogenic DOPE-liposomes (R8-DOPE-BLM). R8-modification dramatically increased (up to 50-fold) the cell-liposome interaction. R8-DOPE-liposomes were internalized via macropinocytosis and did not end up in the lysosomes. R8-DOPE-BLM led to a significantly stronger cell death and DNA damage in vitro relative to all controls. R8-DOPE-BLM demonstrated a prominent anticancer effect in the BALB/c mice bearing 4T1 tumors. Thus, R8-DOPE-BLM provided efficient intracellular delivery of BLM leading to strong tumor growth inhibition in vivo.     

Dynamic force spectroscopy of parallel individual Mucin1-antibody bonds

We used atomic force microscopy to measure the binding forces between Mucin1 (MUC1) peptide and a single-chain variable fragment (scFv) antibody selected from a scFv library screened against MUC1. This binding interaction is central to the design of molecules used for targeted delivery of radioimmunotherapeutic agents for prostate and breast cancer treatment. Our experiments separated the specific binding interaction from nonspecific interactions by tethering the antibody and MUC1 molecules to the atomic force microscope tip and sample surface with flexible polymer spacers. Rupture force magnitude and elastic characteristics of the spacers allowed identification of the rupture events corresponding to different numbers of interacting proteins. We used dynamic force spectroscopy to estimate the intermolecular potential widths and equivalent thermodynamic off rates for monovalent, bivalent, and trivalent interactions. Measured interaction potential parameters agree with the results of molecular docking simulation. Our results demonstrate that an increase of the interaction valency leads to a precipitous decline in the dissociation rate. Binding forces measured for monovalent and multivalent interactions match the predictions of a Markovian model for the strength of multiple uncorrelated bonds in a parallel configuration. Our approach is promising for comparison of the specific effects of molecular modifications as well as for determination of the best configuration of antibody-based multivalent targeting agents.     

Bioavailability of beryllium oxide particles: an in vitro study in the murine J774A.1 macrophage cell line model

Beryllium metal and its oxide and alloys are materials of industrial significance with recognized adverse effects on worker health. Currently, the degree of risk associated with exposure to these materials in the workplace is assessed through measurement of beryllium aerosol mass concentration. Compliance with the current mass-based occupational exposure limit has proven ineffective at eliminating the occurrence of chronic beryllium disease (CBD). The rationale for this research was to examine the mechanism of beryllium bioavailability, which may be pertinent to risk. The authors tested the hypothesis in vitro that dissolution of particles engulfed by macrophages is greater than dissolution in cellular medium alone. Physicochemical changes were evaluated in vitro for well-characterized high-purity beryllium oxide (BeO) particles in cell-free media alone and engulfed by and retained within murine J774A.1 monocyte-macrophage cells. The BeO particles were from a commercially available powder and consisted of diffuse clusters (aerodynamic diameter range 1.5 to 2.5 microm) of 200-nm diameter primary particles. Following incubation for 124 to 144 hours, particles were recovered and recharacterized. Recovered particles were similar in morphology, chemical composition, and size relative to the original material, confirming the relatively insoluble nature of the BeO particles. Measurable levels of dissolved beryllium, representing 0.3% to 4.8% of the estimated total beryllium mass added, were measured in the recovered intracellular fluid. Dissolved beryllium was not detected in the extracellular media. The BeO chemical dissolution rate constant in the J774A. 1 cells was 2.1 +/- 1.7 x 10(-8)g/(cm2 . day). In contrast, the BeO chemical dissolution rate constant in cell-free media was < 8.1 x 10(-9)g/(cm2 . day). In vivo, beryllium dissolved by macrophages may be released in the pulmonary alveolar environment, in the lymphatic system after transport of beryllium by macrophages, or in the alveolar interstitium after migration and dissolution of beryllium particles in tissue. These findings demonstrate a mechanism of bioavailability for beryllium, are consistent with previously observed results in canine alveolar macrophages, and provide insights into additional research needs to understand and prevent beryllium sensitization and CBD.     

Absence of late gadolinium enhancement on cardiac magnetic resonance imaging in ventricular fibrillation and nonischemic cardiomyopathy

Introduction

Cardiac magnetic resonance (CMR)‐identified late gadolinium enhancement (LGE), representing regional fibrosis, is often used to predict ventricular arrhythmia risk in nonischemic cardiomyopathy (NICM). However, LGE is more closely correlated with sustained monomorphic ventricular tachycardia (SMVT) than ventricular fibrillation (VF). We characterized CMR findings of ventricular LGE in VF survivors.

Methods

We examined consecutively resuscitated VF survivors undergoing contrast‐enhanced 1.5T CMR between 9/2007 and 7/2016. We excluded coronary artery disease, hypertrophic cardiomyopathy, amyloid, sarcoid, arrhythmogenic right ventricular cardiomyopathy, and channelopathy. Preexisting implantable cardioverter‐defibrillator (ICD) was a CMR contraindication. VF patients were divided into three groups: (1) NICM, (2) left ventricular (LV) dilatation with normal LV ejection fraction (LVEF), and (3) normal LV size and LVEF. Two groups of NICM patients with and without SMVT were examined for comparison.

Results

We analyzed 87 VF patients, and found that LGE was seen in 8/22 (36%) with NICM (LVEF 38 ± 11%, LV end‐diastolic volume index [LVEDVI] 134 ± 68 mL/BSA), 11/40 (28%) with LV dilatation and normal LVEF (LVEDVI 103 ± 17 mL/BSA), 4/25 (16%) with normal LV size and LVEF. Incidence of LGE in NICM patients without prior ventricular tachycardia/VF (LVEF 36 ± 12%, LVEDVI 141 ± 46 mL/body surface area [BSA]) was 117/277 and was not lower than those with VF and NICM (42% vs 36%; P = 0.59). By contrast, 22/37 NICM patients with SMVT (LVEF 42 ± 11%, LVEDVI 123 ± 48 mL/BSA) were LGE‐positive (59% NICM‐SMVT vs 36% NICM‐VF; P = 0.04).

Conclusion

Most VF survivors with a diagnosis of NICM did not have LGE on CMR and would not have met primary prevention ICD criteria based on LVEF. Absence of LGE may not portend a benign prognosis in NICM. Novel strategies for determining SCD risk in this cohort are required.