Immunogold FIB‐SEM: Combining Volumetric Ultrastructure Visualization with 3D Biomolecular Analysis to Dissect Cell–Environment Interactions

Volumetric imaging techniques capable of correlating structural and functional information with nanoscale resolution are necessary to broaden the insight into cellular processes within complex biological systems. The recent emergence of focused ion beam scanning electron microscopy (FIB‐SEM) has provided unparalleled insight through the volumetric investigation of ultrastructure; however, it does not provide biomolecular information at equivalent resolution. Here, immunogold FIB‐SEM, which combines antigen labeling with in situ FIB‐SEM imaging, is developed in order to spatially map ultrastructural and biomolecular information simultaneously. This method is applied to investigate two different cell–material systems: the localization of histone epigenetic modifications in neural stem cells cultured on microstructured substrates and the distribution of nuclear pore complexes in myoblasts differentiated on a soft hydrogel surface. Immunogold FIB‐SEM offers the potential for broad applicability to correlate structure and function with nanoscale resolution when addressing questions across cell biology, biomaterials, and regenerative medicine.     

Utilizing yield‐stress fluids to suppress chaining during magnetic alignment of microdisks via rotating fields

Inkjet printing can be used to deposit photopolymerizable magnetic inks for the creation of functionalized composites. Anisotropic properties in the composites can be achieved when inkjet printing is combined with external magnetic fields to align magnetically orientable particles (MOPs). When a MOP is induced by an external magnetic field, it will create its own magnetic field that can attract neighboring particles. The coarsening of particles into higher‐order structures like chains and sheets is unwanted in certain areas, such as high‐frequency applications. We show that this particle–particle attraction and subsequent particle migration can be inhibited with the introduction of a yield stress into the suspending medium, while still allowing the alignment of the particle to proceed. For magnetically induced rotational and translational motions for oblate spheroids in a rotating magnetic field, theoretical scalings are presented for the characteristic timescales in a linear fluid and for the characteristic stresses in a yield‐stress fluid. © 2018 American Institute of Chemical Engineers AIChE J, 64: 3215–3226, 2018     

Bi-level Programming Formulation and Heuristic Solution Approach for Dynamic Traffic Signal Optimization

Abstract:   Although dynamic traffic control and traffic assignment are intimately connected in the framework of Intelligent Transportation Systems (ITS), they have been developed independent of one another by most existing research. Conventional methods of signal timing optimization assume given traffic flow pattern, whereas traffic assignment is performed with the assumption of fixed signal timing. This study develops a bi-level programming formulation and heuristic solution approach (HSA) for dynamic traffic signal optimization in networks with time-dependent demand and stochastic route choice. In the bi-level programming model, the upper level problem represents the decision-making behavior (signal control) of the system manager, while the user travel behavior is represented at the lower level. The HSA consists of a Genetic Algorithm (GA) and a Cell Transmission Simulation (CTS) based Incremental Logit Assignment (ILA) procedure. GA is used to seek the upper level signal control variables. ILA is developed to find user optimal flow pattern at the lower level, and CTS is implemented to propagate traffic and collect real-time traffic information. The performance of the HSA is investigated in numerical applications in a sample network. These applications compare the efficiency and quality of the global optima achieved by Elitist GA and Micro GA. Furthermore, the impact of different frequencies of updating information and different population sizes of GA on system performance is analyzed.     

Novel transmit protection scheme for ultrasound systems

The problem of protecting or isolating extremely sensitive receive circuitry from high-voltage transmit circuitry is commonly addressed through the use of diode bridges, transformers, or high-voltage switches, which prove to be prohibitively expensive, bulky, and power consuming for use in portable, low-cost, battery-powered systems. These approaches also compound the interconnect difficulties associated with two-dimensional (2-D) transducer arrays. In this paper we present a novel transmit protection scheme that allows compact MOSFET shunting devices to be brought on-chip within each receive channel implemented in a standard CMOS integrated circuit process. During transmit, the high voltage transmit pulse is driven onto the common connection of the transducer array and the on-chip MOSFET devices shunt the current to ground. During receive, these devices are turned off, the common connection of the transducer array is shunted to ground, and the received echo can be detected as usual. The transmit protection scheme was experimentally shown to shunt a 16 mA peak current resulting from the equivalent of a 100-V, 25-ns-risetime transmit pulse through a 4 pF transducer element. The scheme was also incorporated into a prototype 1024-channel, low-cost, ultrasound system successfully used to form pulse echo images.     

Profiling of organic acids during fermentation by ultraperformance liquid chromatography-tandem mass spectrometry

A method has been developed for rapid quantification of organic acids using ultraperformance liquid chromatography/electrospray-tandem mass spectrometry (UPLC/ESI-MS-MS) to monitor the metabolism of 10 organic acids during microbial fermentation. Because comprehensive chromatographic separation is not required, analysis time is less than traditional ion chromatography assays, with complete organic acid analyses by UPLC/ESI-MS-MS being achieved in less than 3 min. Quantification is accomplished using nine isotopically labeled organic acids as internal standards. Intrasample precisions for organic acid measurements in fermentation supernatants using this method average 8.9% (RSD). Calibration curves are linear over the range of 0.06-100 microg/mL, and detection limits are estimated at 0.06-1 microg/mL. This method has the potential to demonstrate correlation of organic acid consumption and production by microorganisms with observed growth profiles, novel media formulations, and cellular growth events. Data visualization software has been used to profile organic acid levels during fermentation and correlate these profiles to nutrient supplementation protocols employed during microbial production. The potential use of this capability in computational modeling and simulation of microbial metabolism to accelerate the bioprocess development cycle is recognized.     

Manure-borne estrogens as potential environmental contaminants: a review

Livestock wastes are potential sources of endocrine disrupting compounds to the environment. Steroidal estrogen hormones such as estradiol, estrone, and estriol are a particular concern because there is evidence that low nanogram per liter concentrations of estrogens in water can adversely affect the reproductive biology of fish and other aquatic vertebrate species. We performed a literature review to assess the current state of science regarding estrogen physicochemical properties, livestock excretion, and the fate of manure-borne estrogens in the environment. Unconjugated steroidal estrogens have low solubility in water (0.8-13.3 mg L(-1)) and are moderately hydrophobic (log Kow 2.6-4.0). Cattle excrete mostly 17alpha-estradiol, 17beta-estradiol, estrone, and respective sulfated and glucuronidated counterparts, whereas swine and poultry excrete mostly 17beta-estradiol, estrone, estriol, and respective sulfated and glucuronidated counterparts. The environmental fate of estrogens is not clearly known. Laboratory-based studies have found that the biological activity of these compounds is greatly reduced or eliminated within several hours to days due to degradation and sorption. On the other hand, field studies have demonstrated that estrogens are sufficiently mobile and persistent to impact surface and groundwater quality. Future research should use standardized methods for the analysis of manure, soil, and water. More information is needed about the types and amounts of estrogens that exist in livestock wastes and the fate of manure-borne estrogens applied to agricultural lands. Field and laboratory studies should work toward revealing the mechanisms of estrogen degradation, sorption, and transport so that the risk of estrogen contamination of waterways can be minimized.     

Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae

Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for beta-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated beta-tubulin gene into a S. cerevisiae strain with diminished transporter activity and developed the first paclitaxel-sensitive budding yeast strain. In the presence of paclitaxel, cytoplasmic microtubules were stable to cold depolymerization. Paclitaxel-treated cells showed evidence of a mitotic block, with an increase in large-budded cells and cells with a 2N DNA content and DNA fragmentation, identified by FACS analysis and the TUNEL assay. In the presence of paclitaxel, the number of dead cells in cultures increased three-fold and cells containing reactive oxygen species were present. We conclude that paclitaxel blocks mitosis in this strain, leading to an apoptotic-like cell death. This strain will also be useful in further studies of the effect of microtubule dynamics on various cellular processes in S. cerevisiae.     

Chemical characterization and sorption capacity measurements of degraded newsprint from a landfill

Newsprint samples collected from 12-16 ft (top layer (TNP)), 20-24 ft (middle layer (MNP)), and 32-36 ft (bottom layer (BNP)) below the surface of the Norman Landfill (NLF) were characterized by infrared (IR) spectroscopy, cross-polarization, magic-angle spinning 13C nuclear magnetic resonance (CP-MAS 13C NMR) spectroscopy, and tetramethylammonium hydroxide (TMAH) thermochemolysis gas chromatography/mass spectrometry (GC/MS). The extent of NLF newsprint degradation was evaluated by comparing the chemical composition of NLF newsprint to that of fresh newsprint (FNP) and newsprint degraded in the laboratory under methanogenic conditions (DNP). The O-alkyl/alkyl, cellulose/lignin, and lignin/resin acid ratios showed that BNP was the most degraded, and that all three NLF newsprint samples were more degraded than DNP. 13C NMR and TMAH thermochemolysis data demonstrated selective enrichment of lignin over cellulose, and TMAH thermochemolysis further exhibited selective enrichment of resin acids over lignin. In addition, the crystallinity of cellulose in NLF newsprint samples was significantly lower relative to that of FNP and DNP as shown by 13C NMR spectra. The yield of lignin monomers from TMAH thermochemolysis suggested that hydroxyl groups were removed from the propyl side chain of lignin during the anaerobic decomposition of newsprint in the NLF. Moreover, the vanillyl acid/aldehyde ratio, which successfully describes aerobic lignin degradation, was not a good indicator of the anaerobic degradation of lignin on the basis of the TMAH data. The toluene sorption capacity increased as the degree of newsprint degradation increased or as the O-alkyl/alkyl ratio of newsprint decreased. The results of this study further verified that the sorbent O-alkyl/ alkyl ratio is useful for predicting sorption capacities of natural organic materials for hydrophobic organic contaminants.     

Creating three-dimensional lattice patterns using programmable Dammann gratings

We demonstrate the creation of a three dimensional (3D) lattice of focus spots using a 3D Dammann grating structure. Such a 3D lattice of focus spots can be used for probing 3D structures or for creating 3D photonic crystal structures in optically sensitive media. Experimental results are included where the patterns are encoded onto a programmable liquid crystal display. We demonstrate the generation of five planar arrays each having 6×6 points surrounding another set of four planar arrays each having 5×5 points with a single pattern.     

Interactions of Tc(IV) with humic substances

To understand the key processes affecting 99Tc mobility in the subsurface and help with the remediation of contaminated sites, the binding constants of several humic substances (humic and fulvic acids) with Tc(IV) were determined, using a solvent extraction technique. The novelty of this paper lies in the determination of the binding constants of the complexes formed with the individual species TcO(OH)+ and TcO(OH)2(0). Binding constants were found to be 6.8 and between 3.9 and 4.3, for logβ1,-1,1 and logβ1,-2,1, respectively; these values were little modified by a change of ionic strength, in most cases, between 0.1 and 1.0 M, nor were they by the nature and origin of the humic substances. Modeling calculations based on these show TcO(OH)-HA to be the predominant complex in a system containing 20 ppm HA and in the 4-6 pH range, whereas TcO(OH)2(0) and TcO(OH)2-HA are the major species, in the pH 6-8 range.