Highly parallel magnetic tweezers by targeted DNA tethering

Single-molecule force-spectroscopy methods such as magnetic and optical tweezers have emerged as powerful tools for the detailed study of biomechanical aspects of DNA-enzyme interactions. As typically only a single molecule of DNA is addressed in an individual experiment, these methods suffer from a low data throughput. Here, we report a novel method for targeted, nonrandom immobilization of DNA-tethered magnetic beads in regular arrays through microcontact printing of DNA end-binding labels. We show that the increase in density due to the arrangement of DNA-bead tethers in regular arrays can give rise to a one-order-of-magnitude improvement in data-throughput in magnetic tweezers experiments. We demonstrate the applicability of this technique in tweezers experiments where up to 450 beads are simultaneously tracked in parallel, yielding statistical data on the mechanics of DNA for 357 molecules from a single experimental run. Our technique paves the way for kilo-molecule force spectroscopy experiments, enabling the study of rare events in DNA-protein interactions and the acquisition of large statistical data sets from individual experimental runs.     

Covalent tethering of protruding arms for addressable DNA nanostructures

Functionalization of self-assembled DNA nanostructures is of fundamental importance for the realization of their application in nanotechnology and biosensing. Approaches reported so far suffer from lack of general applicability and usually require careful system design to avoid poor yields in the assembly of target structures. A novel approach well suited for fabrication of addressable DNA superstructures is reported here to generate DNA tile motifs. The method is based on the covalent linkage of a single-stranded protruding arm (covPA) to one of the oligomers forming the tile. Subsequent to assembly of tile motifs and superlattices, the covPA can be addressed by hybridization with complementary oligonucleotides or DNA-protein conjugates. The covPA can be located at arbitrary positions in a given tile motif without changing the general design and without compromising the structural integrity of the tile. The covPA strategy can also be readily extended to different PA sequences and multiple covPA arms can be linked to a tile. Superlattices obtained by self-assembly of covPA tiles reveal partial folding into double layers which possess an intrinsic order at the ultrastructural level. This phenomenon is likely associated with the increased flexibility of the covPA and might open up novel ways for DNA-based functionalization of solid surfaces and other applications of structural DNA nanotechnology.     

Regulation of cell fate by near infrared-responsive telomerase activity

Here we develop a strategy using near infrared (NIR) modulation of telomerase activity based on gold nanocage@smart polymer system. Using this biocompatible design, we can regulate cellular behavior. This system has been used in vivo by taking advantages of NIR. This is the first example for optical modulation of telomerase activity in living cells and tissues.

     

Mantle shear-wave tomography and the fate of subducted slabs

A new seismic model of the three-dimensional variation in shear velocity throughout the Earth's mantle is presented. The model is derived entirely from shear bodywave travel times. Multibounce shear waves, core-reflected waves and SKS and SKKS waves that travel through the core are used in the analysis. A unique aspect of the dataset used in this study is the use of bodywaves that turn at shallow depths in the mantle, some of which are triplicated. The new model is compared with other global shear models. Although competing models show significant variations, several large-scale structures are common to most of the models. The high-velocity anomalies are mostly associated with subduction zones. In some regions the anomalies only extend into the shallow lower mantle, whereas in other regions tabular high-velocity structures seem to extend to the deepest mantle. The base of the mantle shows long-wavelength high-velocity zones also associated with subduction zones. The heterogeneity seen in global tomography models is difficult to interpret in terms of mantle flow due to variations in structure from one subduction zone to another. The simplest interpretation of the seismic images is that slabs in general penetrate to the deepest mantle, although the flow is likely to be sporadic. The interruption in slab sinking is likely to be associated with the 660 km discontinuity.     

Systems biology approaches to understanding stem cell fate choice

Stem cells have the capability to self-renew and maintain their undifferentiated state or to differentiate into one or more specialised cell types. Stem cell expansion and manipulation ex vivo is a promising approach for engineering cell replacement therapies, and endogenous stem cells represent potential drugable targets for tissue repair. Before we can harness stem cells' therapeutic potential, we must first understand the intracellular mechanisms controlling their fate choices. These mechanisms involve complex signal transduction and gene regulation networks that feature, for example, intricate feed-forward loops, feedback loops and cross-talk between multiple signalling pathways. Systems biology applies computational and experimental approaches to investigate the emergent behaviour of collections of molecules and strives to explain how these numerous components interact to regulate molecular, cellular and organismal behaviour. Here we review systems biology, and in particular computational, efforts to understand the intracellular mechanisms of stem cell fate choice. We first discuss deterministic and stochastic models that synthesise molecular knowledge into mathematical formalism, enable simulation of important system behaviours and stimulate further experimentation. In addition, statistical analyses such as Bayesian networks and principal components analysis (PCA)/partial least squares (PLS) regression can distill large datasets into more readily managed networks and principal components that provide insights into the critical aspects and components of regulatory networks. Collectively, integrating modelling with experimentation has strong potential for enabling a deeper understanding of stem cell fate choice and thereby aiding the development of therapies to harness stem cells' therapeutic potential.     

Aerobic fate and impact of canola oil in aquatic media

The fate of canola oil in water under aerobic conditions was studied in respirometry tests. First-order biodegradation rates were estimated for a refined oil (commercial canola oil, CCO), 0.0037 h⁻¹, and for two synthetic oils, one prepared with mono-acid triacylglycerols (TAO) 0.0048 h⁻¹, and the other with free fatty acids and glycerol (FAO) 0.0038 h⁻¹. Two abiotic factors limited substrate bioavailability: the autoxidation of the oleoyl chains and the insolubility of fully saturated triacylglycerols. In addition, the ecotoxic impact of the oils was assessed by Microtox^? toxicity. For the acylglycerol oils, luminescent bacteria inhibition was not detected in the water phase, whereas solid-phase EC₅₀ values below 1.8% sample volume were measured. Toxicity was observed in the aqueous phase (EC₅₀ < 16%) during the biodegradation of FAO. In this case, however, solid-phase toxicity was extremely high at the beginning of the test (0.02%) but the toxicity dissipated 480 h into the experiment.     

Biomaterials control of pluripotent stem cell fate for regenerative therapy

Pluripotent stem cells (PSCs) derived from either the embryo or reprogramming processes have the capacity to self-renew and differentiate into various cells in the body, thereby offering a valuable cell source for regenerative therapy of intractable disease and serious tissue damage. Traditionally, methods to expand and differentiate PSCs have been confined to 2D culture through the use of biochemical signals; the use of biomaterials beyond the commercially available culture dish has not been widespread. Nevertheless, biomaterials with tailored physical, chemical, and geometrical cues can mimic the native stem cell niche to tune the microenvironmental conditions for PSCs to preserve their self-renewal capacity or to switch their phenotype, a status ultimately needed to gain regenerative functions ex vivo and in vivo. Recently efforts to explore biomaterials to regulate PSC behavior have accelerated. The biomaterials properties investigated include surface chemistry, immobilized ligand, nano-/micro-topography, matrix stiffness, geometrical complexity, 3D configuration, and combinations thereof. This review aims to cover the current advances of biomaterials-based control over PSCs, particularly for the preservation of self-renewal capacity as well as for their differentiation into target cells. Furthermore, it aims to suggest future research directions that would facilitate the eventual translation of these advances.     

Nanosize and vitality: TiO2 nanotube diameter directs cell fate

We generated, on titanium surfaces, self-assembled layers of vertically oriented TiO2 nanotubes with defined diameters between 15 and 100 nm and show that adhesion, spreading, growth, and differentiation of mesenchymal stem cells are critically dependent on the tube diameter. A spacing less than 30 nm with a maximum at 15 nm provided an effective length scale for accelerated integrin clustering/focal contact formation and strongly enhances cellular activities compared to smooth TiO2 surfaces. Cell adhesion and spreading were severely impaired on nanotube layers with a tube diameter larger than 50 nm, resulting in dramatically reduced cellular activity and a high extent of programmed cell death. Thus, on a TiO2 nanotube surface, a lateral spacing geometry with openings of 30-50 nm represents a critical borderline for cell fate.     

Zirconium oxide regulates RNA interfering of osteoblast-like cells

Zirconium oxide (ZO) has outstanding mechanical properties, high biocompatibility and high resistance to scratching. Since dental implants are made with ZO and the genetic effects of ZO on osteoblasts are incompletely understood, we used microRNA microarray techniques to investigate the translation process in osteoblasts exposed to ZO. By using miRNA microarrays containing 329 probes designed from Human miRNA sequences, we identified in osteoblast-like cells line (MG-63) cultured on ZO disks several miRNA whose expression was significantly modified. The most notable regulated genes acting on osteoblasts are: NOG, SHOX, IGF1, BMP1 and FGFR1. The data reported below represent the first study on translation regulation in osteoblasts exposed to zirconium and one in which the effect of ZO on bone formation has been detected.     

Logic programming to predict cell fate patterns and retrodict genotypes in organogenesis

Caenorhabditis elegans vulval development is a paradigm system for understanding cell differentiation in the process of organogenesis. Through temporal and spatial controls, the fate pattern of six cells is determined by the competition of the LET-23 and the Notch signalling pathways. Modelling cell fate determination in vulval development using state-based models, coupled with formal analysis techniques, has been established as a powerful approach in predicting the outcome of combinations of mutations. However, computing the outcomes of complex and highly concurrent models can become prohibitive. Here, we show how logic programs derived from state machines describing the differentiation of C. elegans vulval precursor cells can increase the speed of prediction by four orders of magnitude relative to previous approaches. Moreover, this increase in speed allows us to infer, or ‘retrodict’, compatible genomes from cell fate patterns. We exploit this technique to predict highly variable cell fate patterns resulting from dig-1 reduced-function mutations and let-23 mosaics. In addition to the new insights offered, we propose our technique as a platform for aiding the design and analysis of experimental data.     

Influence of calcium hydroxide on the fate of perfluorooctanesulfonate under thermal conditions

To explore the potential fate and transport of perfluorochemicals in the thermal treatment of sludge, perfluorooctanesulfonate (PFOS), a perfluorochemical species commonly dominant in wastewater sludge, was mixed with hydrated lime (Ca(OH)₂) to quantitatively observe their interaction under different temperatures. The phase compositions of the mixtures after the reactions were qualitatively identified and quantitatively determined using X-ray diffraction technique. The results of the thermogravimetry and differential scanning calorimetry analyses indicate that PFOS gasified directly during the thermal treatment process when the temperature was increased to around 425 °C. However, the formation of CaF₂ at 350 °C suggests that the presence of Ca(OH)₂ in the mixture can lead to the decomposition of PFOS at 350 °C, which is lower than the decomposition temperature of PFOS alone (425 °C). The increase of temperature promoted a solid state reaction between PFOS and Ca(OH)₂, and also enhanced the interaction between the gaseous products of PFOS and CaO (or Ca(OH)₂). The preferred Ca/F molar ratio to achieve fluorine stabilization by Ca(OH)₂ was above 1:1 in the experiment involving 400 °C and 600 °C treatment. It also showed that equilibrium efficiency is achieved within 5 min at 400 °C and within 1 min above 600 °C.     

The fate and management of high mercury-containing lamps from high technology industry

This study investigated the fate and management of high mercury-contained lamps, such as cold cathode fluorescent lamps (CCFLs), ultraviolet lamps (UV lamps), and super high pressure mercury lamps (SHPs), from high technology industries in Taiwan, using material flow analysis (MFA) method. Several organizations, such as Taiwan Environmental Protection Administration, Taiwan External Trade Development Council, the light sources manufactories, mercury-containing lamps importer, high technology industrial user, and waste mercury-containing lamps treatment facilities were interviewed in this study. According to this survey, the total mercury contained in CCFLs, UV lamps, and SHPs produced in Taiwan or imported from other countries was 886kg in year 2004. Among the various lamps containing mercury, 57kg mercury was exported as primary CCFLs, 7kg mercury was wasted as defective CCFLs, and 820kg mercury was used in the high technology industries, including 463kg mercury contained in exported industrial products using CCFLs as components. On the contrary, only 59kg of mercury was exported, including 57kg in CCFLs and 2kg in UV lamps. It reveals that 364kg mercury was consumed in Taiwan during year 2004. In addition, 140kg of the 364kg mercury contained in lamps used by high technology industry was well treated through industrial waste treatment system. Among the waste mercury from high technology industry, 80kg (57%), 53kg (38%), and 7kg (5%) of mercury were through domestic treatment, offshore treatment, and emission in air, respectively. Unfortunately, 224kg waste mercury was not suitable treated, including 199kg mercury contained in CCFL, which is a component of monitor for personal computer and liquid crystal display television, and 25kg non-treated mercury. Thus, how to recover the mercury from the waste monitors is an important challenge of zero wastage policy in Taiwan.     

High throughput screening for discovery of materials that control stem cell fate

Insights into the complex stem cell niche have identified the cell–material interface to be a potent regulator of stem cell fate via material properties such as chemistry, topography and stiffness. In light of this, materials scientists have the opportunity to develop bioactive materials for stem cell culture that elicit specific cellular responses. To accelerate materials discovery, high throughput screening platforms have been designed which can rapidly evaluate combinatorial material libraries in two and three-dimensional environments. In this review, we present screening platforms for the discovery of material properties that influence stem cell behavior.     

Mechanics regulates ATP-stimulated collective calcium response in fibroblast cells

Cells constantly sense their chemical and mechanical environments. We study the effect of mechanics on the ATP-induced collective calcium response of fibroblast cells in experiments that mimic various tissue environments. We find that closely packed two-dimensional cell cultures on a soft polyacrylamide gel (Young''s modulus E = 690 Pa) contain more cells exhibiting calcium oscillations than cultures on a rigid substrate (E = 36 000 Pa). Calcium responses of cells on soft substrates show a slower decay of calcium level relative to those on rigid substrates. Actin enhancement and disruption experiments for the cell cultures allow us to conclude that actin filaments determine the collective Ca²⁺ oscillatory behaviour in the culture. Inhibition of gap junctions results in a decrease of the oscillation period and reduced correlation of calcium responses, which suggests additional complexity of signalling upon cell–cell contact. Moreover, the frequency of calcium oscillations is independent of the rigidity of the substrate but depends on ATP concentration. We compare our results with those from similar experiments on individual cells. Overall, our observations show that collective chemical signalling in cell cultures via calcium depends critically on the mechanical environment.     

An LNG release, transport, and fate model system for marine spills

LNGMAP, a fully integrated, geographic information based modular system, has been developed to predict the fate and transport of marine spills of LNG. The model is organized as a discrete set of linked algorithms that represent the processes (time dependent release rate, spreading, transport on the water surface, evaporation from the water surface, transport and dispersion in the atmosphere, and, if ignited, burning and associated radiated heat fields) affecting LNG once it is released into the environment. A particle-based approach is employed in which discrete masses of LNG released from the source are modeled as individual masses of LNG or spillets. The model is designed to predict the gas mass balance as a function of time and to display the spatial and temporal evolution of the gas (and radiated energy field).

LNGMAP has been validated by comparisons to predictions of models developed by ABS Consulting and Sandia for time dependent point releases from a draining tank, with and without burning. Simulations were in excellent agreement with those performed by ABS Consulting and consistent with Sandia's steady state results.

To illustrate the model predictive capability for realistic emergency scenarios, simulations were performed for a tanker entering Block Island Sound. Three hypothetical cases were studied: the first assumes the vessel continues on course after the spill starts, the second that the vessel stops as soon as practical after the release begins (3 min), and the third that the vessel grounds at the closest site practical. The model shows that the areas of the surface pool and the incident thermal radiation field (with burning) are minimized and dispersed vapor cloud area (without burning) maximized if the vessel continues on course. For this case the surface pool area, with burning, is substantially smaller than for the without burning case because of the higher mass loss rate from the surface pool due to burning. Since the vessel speed substantially exceeds the spill spreading rate, both the thermal radiation fields and surface pool trail the vessel. The relative directions and speeds of the wind and vessel movement govern the orientation of the dispersed plume.

If the vessel stops, the areas of the surface pool and incident radiation field (with burning) are maximized and the dispersed cloud area (without burning) minimized. The longer the delay in stopping the vessel, the smaller the peak values are for the pool area and the size of the thermal radiation field. Once the vessel stops, the spill pool is adjacent to the vessel and moving down current. The thermal radiation field is oriented similarly. These results may be particularly useful in contingency planning for underway vessels.