K30, H150, and H168 Are Essential Residues for Coordinating Pyridoxal 5′-Phosphate of O-Acetylserine Sulfhydrylase from Acidithiobacillus ferrooxidans

O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV–vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5′-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization

Effects of Impurity Content on the Sintering Characteristics of Plasma-Sprayed Zirconia

Yttria-stabilized zirconia powders, containing different levels of SiO2 and Al2O3, have been plasma sprayed onto metallic substrates. The coatings were detached from their substrates and a dilatometer was used to monitor the dimensional changes they exhibited during prolonged heat treatments. It was found that specimens containing higher levels of silica and alumina exhibited higher rates of linear contraction, in both in-plane and through-thickness directions. The in-plane stiffness and the through-thickness thermal conductivity were also measured after different heat treatments and these were found to increase at a greater rate for specimens with higher impurity (silica and alumina) levels. Changes in the pore architecture during heat treatments were studied using Mercury Intrusion Porosimetry (MIP). Fine scale porosity (<_50 nm) was found to be sharply reduced even by relatively short heat treatments. This is correlated with improvements in inter-splat bonding and partial healing of intra-splat microcracks, which are responsible for the observed changes in stiffness and conductivity, as well as the dimensional changes

Color-scalable flow cytometry with Raman tags

Flow cytometry is an indispensable tool in biology and medicine for counting and analyzing cells in large heterogeneous populations. It identifies multiple characteristics of every single cell, typically via fluorescent probes that specifically bind to target molecules on the cell surface or within the cell. However, flow cytometry has a critical limitation: the color barrier. The number of chemical traits that can be simultaneously resolved is typically limited to several due to the spectral overlap between fluorescence signals from different fluorescent probes. Here, we present color-scalable flow cytometry based on coherent Raman flow cytometry with Raman tags to break the color barrier. This is made possible by combining a broadband Fourier-transform coherent anti-Stokes Raman scattering (FT-CARS) flow cytometer, resonance-enhanced cyanine-based Raman tags, and Raman-active dots (Rdots). Specifically, we synthesized 20 cyanine-based Raman tags whose Raman spectra are linearly independent in the fingerprint region (400 to 1,600 cm-1). For highly sensitive detection, we produced Rdots composed of 12 different Raman tags in polymer nanoparticles whose detection limit was as low as 12 nM for a short FT-CARS signal integration time of 420 µs. We performed multiplex flow cytometry of MCF-7 breast cancer cells stained by 12 different Rdots with a high classification accuracy of 98%. Moreover, we demonstrated a large-scale time-course analysis of endocytosis via the multiplex Raman flow cytometer. Our method can theoretically achieve flow cytometry of live cells with >140 colors based on a single excitation laser and a single detector without increasing instrument size, cost, or complexity

Bioenergetics Failure and Oxidative Stress in Brain Stem Mediates Cardiovascular Collapse Associated with Fatal Methamphetamine Intoxication

Background: Whereas sudden death, most often associated with cardiovascular collapse, occurs in abusers of the psychostimulant methamphetamine (METH), the underlying mechanism is much less understood. The demonstration that successful resuscitation of an arrested heart depends on maintained functionality of the rostral ventrolateral medulla (RVLM), which is responsible for the maintenance of stable blood pressure, suggests that failure of brain stem cardiovascular regulation, rather than the heart, holds the key to cardiovascular collapse. We tested the hypothesis that cessation of brain stem cardiovascular regulation because of a loss of functionality in RVLM mediated by bioenergetics failure and oxidative stress underlies the cardiovascular collapse elicited by lethal doses of METH. Methodology/Principal Findings: Survival rate, cardiovascular responses and biochemical or morphological changes in RVLM induced by intravenous administration of METH in Sprague-Dawley rats were investigated. High doses of METH induced significant mortality within 20 min that paralleled concomitant the collapse of arterial pressure or heart rate and loss of functionality in RVLM. There were concurrent increases in the concentration of METH in serum and ventrolateral medulla, along with tissue anoxia, cessation of microvascular perfusion and necrotic cell death in RVLM. Furthermore, mitochondrial respiratory chain enzyme activity or electron transport capacity and ATP production in RVLM were reduced, and mitochondria-derived superoxide anion level was augmented. All those detrimental physiological and biochemica