Cells use force as a mechanical signal to sense and respond to their microenvironment. Understanding how mechanical forces affect living cells requires the development of tool sets that can apply ...nanoscale forces and also measure cellular traction forces. However, there has been a lack of techniques that integrate actuation and sensing components to study force as a mechanical signal. Here, we describe a system that uses an array of elastomeric microposts to apply external forces to cells through cobalt nanowires embedded inside the microposts. We first biochemically treat the posts' surfaces to restrict cell adhesion to the posts' tips. Then by applying a uniform magnetic field (B<0.3 T), we induce magnetic torque on the nanowires that is transmitted to a cell's adhesion site as an external force. We have achieved external forces of up to 45 nN, which is in the upper range of current nanoscale force-probing techniques. Nonmagnetic microposts, similarly prepared but without nanowires, surround the magnetic microposts and are used to measure the traction forces and changes in cell mechanics. We record the magnitude and direction of the external force and the traction forces by optically measuring the deflection of the microposts, which linearly deflect as cantilever springs. With this approach, we can measure traction forces before and after force stimulation in order to monitor cellular response to forces. We present the fabrication methods, magnetic force characterization, and image analysis techniques used to achieve the measurements.
Spatial regulation of angiogenesis is important for the generation of functional engineered vasculature in regenerative medicine. The Notch ligands Jag1 and Dll4 show distinct expression patterns in ...endothelial cells and, respectively, promote and inhibit endothelial sprouting. Therefore, patterns of Notch ligands may be utilized to spatially control sprouting, but their potential and the underlying mechanisms of action are unclear. Here, we coupled in vitro and in silico models to analyze the ability of micropatterned Jag1 and Dll4 ligands to spatially control endothelial sprouting. Dll4 patterns, but not Jag1 patterns, elicited spatial control. Computational simulations of the underlying signaling dynamics suggest that different timing of Notch activation by Jag1 and Dll4 underlie their distinct ability to spatially control sprouting. Hence, Dll4 patterns efficiently direct the sprouts, whereas longer exposure to Jag1 patterns is required to achieve spatial control. These insights in sprouting regulation offer therapeutic handles for spatial regulation of angiogenesis.
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•Dll4-functionalized patterns guide direction and location of angiogenic sprouts•Jag1-functionalized patterns do not have relevant effects on angiogenic sprouts•Modeling predicts that differences are due to distinct signaling temporal dynamics
Bioengineering; Biological sciences; Developmental biology; Tissue engineering
Abstract We present simulations of cell-cell adhesion as reported in a recent study Liu et al., 2010 , PNAS, 107(22), 9944-9 for two cells seeded on an array of micro-posts. The micro-post array ...allows for the measurement of forces exerted by the cell and these show that the cell-cell tugging stress is a constant and independent of the cell-cell junction area. In the current study, we demonstrate that a material model which includes the underlying cellular processes of stress fibre contractility and adhesion formation can capture these results. The simulations explain the experimentally observed phenomena whereby the cell-cell junction forces increase with junction size but the tractions exerted by the cell on the micro-post array are independent of the junction size. Further simulations on different types of micro-post arrays and cell phenotypes are presented as a guide to future experiments.
Summary Background Previous studies have suggested that any benefits of folic acid-based therapy to lower serum homocysteine in prevention of cardiovascular events might be offset by concomitant use ...of antiplatelet therapy. We aimed to establish whether there is an interaction between antiplatelet therapy and the effects of folic acid-based homocysteine-lowering therapy on major vascular events in patients with stroke or transient ischaemic attack enrolled in the vitamins to prevent stroke (VITATOPS) trial. Methods In the VITATOPS trial, 8164 patients with recent stroke or transient ischaemic attack were randomly allocated to double-blind treatment with one tablet daily of placebo or B vitamins (2 mg folic acid, 25 mg vitamin B6 , and 500 μg vitamin B12 ) and followed up for a median 3·4 years (IQR 2·0–5·5) for the primary composite outcome of stroke, myocardial infarction, or death from vascular causes. In our post-hoc analysis of the interaction between antiplatelet therapy and the effects of treatment with B vitamins on the primary outcome, we used Cox proportional hazards regression before and after adjusting for imbalances in baseline prognostic factors in participants who were and were not taking antiplatelet drugs at baseline and in participants assigned to receive B vitamins or placebo. We also assessed the interaction in different subgroups of patients and different secondary outcomes. The VITATOPS trial is registered with ClinicalTrials.gov , number NCT00097669 , and Current Controlled Trials, number ISRCTN74743444. Findings At baseline, 6609 patients were taking antiplatelet therapy and 1463 were not. Patients not receiving antiplatelet therapy were more likely to be younger, east Asian, and disabled, to have a haemorrhagic stroke or cardioembolic ischaemic stroke, and to have a history of hypertension or atrial fibrillation. They were less likely to be smokers and to have a history of peripheral artery disease, hypercholesterolaemia, diabetes, ischaemic heart disease, and a revascularisation procedure. Of the participants taking antiplatelet drugs at baseline, B vitamins had no significant effect on the primary outcome (488 patients in the B-vitamins group 15% vs 519 in the placebo group 16%; hazard ratio HR 0·94, 95% CI 0·83–1·07). By contrast, of the participants not taking antiplatelet drugs at baseline, B vitamins had a significant effect on the primary outcome (123 in the B-vitamins group 17% vs 153 in the placebo group 21%; HR 0·76, 0·60–0·96). The interaction between antiplatelet therapy and the effect of B vitamins on the primary outcome was significant after adjusting for imbalance in the baseline variables (adjusted p for interaction=0·0204). Interpretation Our findings support the hypothesis that antiplatelet therapy modifies the potential benefits of lowering homocysteine with B-vitamin supplementation in the secondary prevention of major vascular events. If validated, B vitamins might have a role in the prevention of ischaemic events in high-risk individuals with an allergy, intolerance, or lack of indication for antiplatelet therapy. Funding Australia National Health and Medical Research Council, UK Medical Research Council, Singapore Biomedical Research Council, and Singapore National Medical Research Council.
Adenovirus (Ad) early region 4 (E4) mutants activate cellular DNA damage responses (DDRs) that include non-homologous end joining (NHEJ) pathways mediated by the DNA repair kinase DNA-PK and its ...associated factors Ku70/Ku86. NHEJ results in concatenation of the viral linear double-stranded DNA genome and inhibits a productive infection. E4 proteins normally prevent activation of cellular DDRs in wild-type Ad type 5 (Ad5) infections, thereby promoting efficient viral growth. The purpose of this study was to evaluate the factors that govern DNA-PK activation during adenovirus infection. Our data indicate that viral DNA replication promotes DNA-PK activation, which is required for genome concatenation by NHEJ. Although the Mre11/Rad50/Nbs1 (MRN) DDR sensor complex is not required for DNA-PK activation, Mre11 is important for recruitment of the NHEJ factor Ku86 to viral replication centers. Our study addresses the interplay between the DNA-PK and MRN complexes during viral genome concatenation by NHEJ.
•Viral DNA replication promotes DNA-PK activation and genome concatenation during E4-infection.•The MRN complex is not essential for DNA-PK autophosphorylation at Ser2056.•Ku86 is transiently localized to viral replication centers, which is promoted by the MRN complex.
The distribution of viruses and gene therapy vectors is difficult to assess in a living organism. For instance, trafficking in murine models can usually only be assessed after sacrificing the animal ...for tissue sectioning or extraction. These assays are laborious requiring whole animal sectioning to ascertain tissue localization. They also obviate the ability to perform longitudinal or kinetic studies in one animal. To track viruses after systemic infection, we have labeled adenoviruses with a near-infrared (NIR) fluorophore and imaged these after intravenous injection in mice. Imaging was able to track and quantitate virus particles entering the jugular vein simultaneous with injection, appearing in the heart within 500 milliseconds, distributing in the bloodstream and throughout the animal within 7 seconds, and that the bulk of virus distribution was essentially complete within 3 minutes. These data provide the first in vivo real-time tracking of the rapid initial events of systemic virus infection.
We present a novel microfabricated device to simultaneously and actively trap thousands of single mammalian cells in alignment with a planar microelectrode array. Thousands of 3
μm
diameter trapping ...electrodes were fabricated within the bottom of a parallel-plate flow chamber. Cells were trapped on the electrodes and held against destabilizing fluid flows by dielectrophoretic forces generated in the device. In general, each electrode trapped only one cell. Adhesive regions were patterned onto the surface in alignment with the traps such that cells adhered to the array surface and remained in alignment with the electrodes. By driving the device with different voltages, we showed that trapped cells could be killed by stronger electric fields. However, with weaker fields, cells were not damaged during trapping, as indicated by the similar morphologies and proliferation rates of trapped cells versus controls. As a test of the device, we patterned ∼20,000 cells onto a 1
cm
2 grid of rectangular adhesive regions, with two electrodes and thus two cells per rectangle. Our method obtained 70±1% fidelity versus 17±1% when using an existing cell-registration technique. By allowing the placement of desired numbers of cells at specified locations, this approach addresses many needs to manipulate and register cells to the surfaces of biosensors and other devices with high precision and fidelity.
Rising temperatures in most agricultural regions of the world are associated with a higher incidence of extreme weather events such as heat waves. We performed an experiment to mitigate the impact of ...heat waves and exposure of berries in grapevine (
Vitis vinifera
cv. “Cabernet Sauvignon”) with untreated vines (Exposed) or with fruit-zone partial shading (Shaded) under 40 and 80% replacement of crop evapotranspiration (ET
c
) with sustained deficit irrigation in a factorially arranged experiment. The trial was performed in a vineyard with vertically shoot positioned trellis with a row orientation that concentrated solar radiation exposure on the southwest aspect of the fruit zone. Leaf stomatal conductance (
g
s
) and net carbon assimilation (
A
N
) were significantly lower in shaded leaves under partial fruit-zone shading that resulted in lower pruning mass for Shaded treatments. Stem water potential (Ψ
stem
) responded to a large extent to increased irrigation. However, grapevines with partial fruit-zone shading had transiently better water status under 40% ET
c
. Cluster maximum temperatures were 3.9°C greater in Exposed grapevines. Exposed clusters had transiently lower acidity and higher pH. However, Exposed clusters on 40% ET
c
had higher total soluble solids (TSS). The experimental vineyard suffered a 4-day heat wave 21 days before harvest, resulting in 25% of the clusters being damaged in Exposed treatment, regardless of irrigation amount. Furthermore, berries in Exposed treatments suffered a great loss of anthocyanins and flavonols even if they were not damaged by direct solar exposure. The pre-planting decision of using a vertically shoot positioned trellis that concentrated solar radiation on the Southwest aspect offered mild protection in a hot climate region with a sunny growing season with extreme heat events during the execution of study. The extreme conditions under which this study was conducted are not unusual, and have become more expected. Our work provided evidence of the vulnerability of grape berry to heat waves and exposure during heat wave events and possible protection methods to mitigate these effects
in situ
in context of climate change.
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