Improving Breast Cancer Treatment Specificity Using Aptamers Obtained by 3D Cell-SELEX

Three-dimensional spheroids of non-malignant MCF10A and malignant SKBR3 breast cells were used for subsequent 3D Cell-SELEX to generate aptamers for specific binding and treatment of breast cancer cells. Using 3D Cell-SELEX combined with Next-Generation Sequencing and bioinformatics, ten abundant aptamer families with specific structures were identified that selectively bind to SKBR3, and not to MCF10A cells. Multivalent aptamer polymers were synthesized by co-polymerization and analyzed for binding performance as well as therapeutic efficacy. Binding performance was determined by confocal fluorescence imaging and revealed specific binding and efficient internalization of aptamer polymers into SKBR3 spheroids. For therapeutic purposes, DNA sequences that intercalate the cytotoxic drug doxorubicin were co-polymerized into the aptamer polymers. Viability tests show that the drug-loaded polymers are specific and effective in killing SKBR3 breast cancer cells. Thus, the 3D-selected aptamers enhanced the specificity of doxorubicin against malignant over non-malignant breast cells. The innovative modular DNA aptamer platform based on 3D Cell SELEX and polymer multivalency holds great promise for diagnostics and treatment of breast cancer

Determinants of Beat-to-Beat Variability of Repolarization Duration in the Canine Ventricular Myocyte: A Computational Analysis

Beat-to-beat variability of repolarization duration (BVR) is an intrinsic characteristic of cardiac function and a better marker of proarrhythmia than repolarization prolongation alone. The ionic mechanisms underlying baseline BVR in physiological conditions, its rate dependence, and the factors contributing to increased BVR in pathologies remain incompletely understood. Here, we employed computer modeling to provide novel insights into the subcellular mechanisms of BVR under physiological conditions and during simulated drug-induced repolarization prolongation, mimicking long-QT syndromes type 1, 2, and 3. We developed stochastic implementations of 13 major ionic currents and fluxes in a model of canine ventricular-myocyte electrophysiology. Combined stochastic gating of these components resulted in short- and long-term variability, consistent with experimental data from isolated canine ventricular myocytes. The model indicated that the magnitude of stochastic fluctuations is rate dependent due to the rate dependence of action-potential (AP) duration (APD). This process (the “active” component) and the intrinsic nonlinear relationship between membrane current and APD (“intrinsic component”) contribute to the rate dependence of BVR. We identified a major role in physiological BVR for stochastic gating of the persistent Na+ current (INa) and rapidly activating delayed-rectifier K+ current (IKr). Inhibition of IKr or augmentation of INa significantly increased BVR, whereas subsequent β-adrenergic receptor stimulation reduced it, similar to experimental findings in isolated myocytes. In contrast, β-adrenergic stimulation increased BVR in simulated long-QT syndrome type 1. In addition to stochastic channel gating, AP morphology, APD, and beat-to-beat variations in Ca2+ were found to modulate single-cell BVR. Cell-to-cell coupling decreased BVR and this was more pronounced when a model cell with increased BVR was coupled to a model cell with normal BVR. In conclusion, our results provide new insights into the ionic mechanisms underlying BVR and suggest that BVR reflects multiple potentially proarrhythmic parameters, including increased ion-channel stochasticity, prolonged APD, and abnormal Ca2+ handling

Early changes in the extracellular matrix of the degenerating intervertebral disc, assessed by Fourier transform infrared imaging.

Mechanical overloading induces a degenerative cell response in the intervertebral disc. However, early changes in the extracellular matrix (ECM) are challenging to assess with conventional techniques. Fourier Transform Infrared (FTIR) imaging allows visualization and quantification of the ECM. We aim to identify markers for disc degeneration and apply these to investigate early degenerative changes due to overloading and katabolic cell activity. Three experiments were conducted; Exp 1.: In vivo, lumbar spines of seven goats were operated: one disc was injected with chondroitinase ABC (mild degeneration) and compared to the adjacent disc (control) after 24 weeks. Exp 2a: Ex vivo, caprine discs received physiological loading (n=10) or overloading (n=10) in a bioreactor. Exp 2b: Cell activity was diminished prior to testing by freeze-thaw cycles, 18 discs were then tested as in Exp 2a. In all experiments, FTIR images (spectral region: 1000-1300 cm ) of mid-sagittal slices were analyzed using multivariate curve resolution. In vivo, FTIR was more sensitive than biochemical and histological analysis in identifying reduced proteoglycan content (p=0.046) and increased collagen content in degenerated discs (p<0.01). Notably, FTIR analysis additionally showed disorganization of the ECM, indicated by increased collagen entropy (p=0.011). Ex vivo, the proteoglycan/collagen ratio decreased due to overloading (p=0.047) and collagen entropy increased (p=0.047). Cell activity affected collagen content only (p=0.044). FTIR imaging allows a more detailed investigation of early disc degeneration than traditional measures. Changes due to mild overloading could be assessed and quantified. Matrix remodeling is the first detectable step towards intervertebral disc degeneration. [Abstract copyright: Copyright © 2018. Published by Elsevier Ltd.

Multifactorial intervention to reduce falls in older people at high risk of recurrent falls a randomized controlled trial

Background: Falls occur frequently in older people and strongly affect quality of life. Guidelines recommend multifactorial, targeted fall prevention. We evaluated the effectiveness of a multifactorial intervention in older persons with a high risk of recurrent falls. Methods: A randomized controlled trial was conducted from April 3, 2005, to July 21, 2008, at the geriatric outpatient clinic of a university hospital and regional general practices in the Netherlands. Of 2015 persons identified, 217 persons aged 65 years or older were selected to participate. They had a high risk of recurrent falls and no cognitive impairment and had visited the emergency department or their family physician after a fall. The geriatric assessment and intervention were aimed at reduction of fall risk factors. Primary outcome measures were time to first and second falls after randomization. Secondary outcome measures were fractures, activities of daily living, quality of life, and physical performance. Results: Within 1 year, 55 (51.9%) of the 106 intervention participants and 62 (55.9%) of the 111 usual care (control) participants fell at least once. No significant treatment effect was demonstrated for the time to first fall (hazard ratio, 0.96; 95% confidence interval, 0.67-1.37) or the time to second fall (1.13; 0.71-1.80). Similar results were obtained for secondary outcome measures and for perprotocol analysis. One intervention participant died vs 7 in the control group (hazard ratio, 0.15; 95% confidence interval, 0.02-1.21). Conclusion: This multifactorial fall-prevention program does not reduce falls in high-risk, cognitively intact older persons. Trial Registration: isrctn.org Identifier: ISRCTN11546541

Differences in gene expression and cytokine production by crystalline vs. amorphous silica in human lung epithelial cells

<p>Abstract</p> <p>Background</p> <p>Exposure to respirable crystalline silica particles, as opposed to amorphous silica, is associated with lung inflammation, pulmonary fibrosis (silicosis), and potentially with lung cancer. We used Affymetrix/GeneSifter microarray analysis to determine whether gene expression profiles differed in a human bronchial epithelial cell line (BEAS 2B) exposed to cristobalite vs. amorphous silica particles at non-toxic and equal surface areas (75 and 150 × 10⁶μm²/cm²). Bio-Plex analysis was also used to determine profiles of secreted cytokines and chemokines in response to both particles. Finally, primary human bronchial epithelial cells (NHBE) were used to comparatively assess silica particle-induced alterations in gene expression.</p> <p>Results</p> <p>Microarray analysis at 24 hours in BEAS 2B revealed 333 and 631 significant alterations in gene expression induced by cristobalite at low (75) and high (150 × 10⁶μm²/cm²) amounts, respectively (p < 0.05/cut off ≥ 2.0-fold change). Exposure to amorphous silica micro-particles at high amounts (150 × 10⁶μm²/cm²) induced 108 significant gene changes. Bio-Plex analysis of 27 human cytokines and chemokines revealed 9 secreted mediators (p < 0.05) induced by crystalline silica, but none were induced by amorphous silica. QRT-PCR revealed that cristobalite selectively up-regulated stress-related genes and cytokines (<it>FOS, ATF3, IL6 </it>and <it>IL8</it>) early and over time (2, 4, 8, and 24 h). Patterns of gene expression in NHBE cells were similar overall to BEAS 2B cells. At 75 × 10⁶μm²/cm², there were 339 significant alterations in gene expression induced by cristobalite and 42 by amorphous silica. Comparison of genes in response to cristobalite (75 × 10⁶μm²/cm²) revealed 60 common, significant gene alterations in NHBE and BEAS 2B cells.</p> <p>Conclusions</p> <p>Cristobalite silica, as compared to synthetic amorphous silica particles at equal surface area concentrations, had comparable effects on the viability of human bronchial epithelial cells. However, effects on gene expression, as well as secretion of cytokines and chemokines, drastically differed, as the crystalline silica induced more intense responses. Our studies indicate that toxicological testing of particulates by surveying viability and/or metabolic activity is insufficient to predict their pathogenicity. Moreover, they show that acute responses of the lung epithelium, including up-regulation of genes linked to inflammation, oxidative stress, and proliferation, as well as secretion of inflammatory and proliferative mediators, can be indicative of pathologic potential using either immortalized lines (BEAS 2B) or primary cells (NHBE). Assessment of the degree and magnitude of these responses <it>in vitro </it>are suggested as predictive in determining the pathogenicity of potentially harmful particulates.</p

How to overcome the detrimental effects of noise in social interaction: the benefits of generosity

Interpersonal misunderstanding is often rooted in noise, or discrepancies between intended and actual outcomes for an interaction partner due to unintended errors (e.g., not being able to respond to an E-mail because of a local network breakdown). How can one effectively cope with noise in social dilemmas, situations in which self-interest and collective interests are conflicting? Consistent with hypotheses, the present research revealed that incidents of noise exert a detrimental effect on level of cooperation when a partner follows strict reciprocity (i.e., tit for tat) but that this effect can be overcome if a partner behaves somewhat more cooperatively than the actor did in the previous interaction (i.e., tit for tat plus 1). Also, when noise was present, tit for tat plus 1 elicited greater levels of cooperation than did tit for tat, thereby underscoring the benefits of adding generosity to reciprocity in coping with noise in social dilemmas. The Discussion outlines implications of the present work for theories focusing on self-presentation and attribution, communication, and trust and prorelationship behavior