Protein dynamics in iron-starved Mycobacterium tuberculosis revealed by turnover and abundance measurement using hybrid-linear ion trap-Fourier transform mass spectrometry

To study the proteome response of Mycobacterium tuberculosis H37Rv to a change in iron level, iron-starved late-log-phase cells were diluted in fresh low- and high-iron media containing [ (15)N]-labeled asparagine as the sole nitrogen source for labeling the proteins synthesized upon dilution. We determined the relative protein abundance and protein turnover in M. tuberculosis H37Rv under these two conditions. For measurements, we used a high-resolution hybrid-linear ion trap-Fourier transform mass spectrometer coupled with nanoliquid chromatography separation. While relative protein abundance analysis shows that only 5 proteins were upregulated by high iron, 24 proteins had elevated protein turnover for the cells in the high-iron medium. This suggests that protein turnover is a sensitive parameter to assess the proteome dynamics. Cluster analysis was used to explore the interconnection of protein abundance and turnover, revealing coordination of the cellular processes of protein synthesis, degradation, and secretion that determine the abundance and allocation of a protein in the cytosol and the extracellular matrix of the cells. Further potential utility of the approach is discussed.     

Direct voltammetry and catalysis with Mycobacterium tuberculosis catalase-peroxidase, peroxidases, and catalase in lipid films.

Stable films of dimyristoylphosphatidylcholine and M. tuberculosis catalase-peroxidase (KatG), several peroxidases, myoglobin, and catalase showed reversible FeIII/FeII voltammetry on pyrolytic graphite electrodes and catalytic current for hydrogen peroxide and oxygen. Amperometric responses for these films to H2O2 at 0 V are likely to contain significant contributions from catalytic reduction of oxygen produced during the catalytic cycles. Relative apparent turnover rates at pH 6 based on steady-state currents at 0 V versus SCE in the presence of H2O2 were in the order horseradish peroxidase > cytochrome c peroxidase (CcP) > soybean peroxidase > myoglobin > KatG > catalase. Lower currents for the very efficient peroxide scavengers KatG and catalase may be related to the instability of their compounds I in the presence of H2O2. KatG catalyzed the electrochemical reduction of oxygen more efficiently than catalase and CcP but less efficiently than the other peroxidases. DMPC films incorporating glucose oxidase and peroxidases gave good analytical responses to glucose, demonstrating the feasibility of dual enzyme-lipid films for biosensor fabrication.     

Changes in Gene Expression of E. coli under Conditions of Modeled Reduced Gravity

Relatively few studies have examined bacterial responses to the reduced gravity conditions that are experienced by bacteria grown in space. In this study, whole genome expression of Escherichia coli K12 under clinorotation (which models some of the conditions found under reduced gravity) was analyzed. We hypothesized that phenotypic differences at cellular and population levels under clinorotation (hereafter referred to as modeled reduced gravity) are directly coupled to changes in gene expression. Further, we hypothesized that these responses may be due to indirect effects of these environmental conditions on nutrient accessibility for bacteria. Overall, 430 genes were identified as significantly different between modeled reduced gravity conditions and controls. Up-regulated genes included those involved in the starvation response (csiD, cspD, ygaF, gabDTP, ygiG, fliY, cysK) and redirecting metabolism under starvation (ddpX, acs, actP, gdhA); responses to multiple stresses, such as acid stress (asr, yhiW), osmotic stress (yehZYW), oxidative stress (katE, btuDE); biofilm formation (lldR, lamB, yneA, fadB, ydeY); curli biosynthesis (csgDEF), and lipid biosynthesis (yfbEFG). Our results support the previously proposed hypothesis that under conditions of modeled reduced gravity, zones of nutrient depletion develop around bacteria eliciting responses similar to entrance into stationary phase which is generally characterized by expression of starvation inducible genes and genes associated with multiple stress responses.     

Effect of Hypergravity on the Level of Heat Shock Proteins 70 and 90 in Pea Seedlings

Exposure to hypergravity induces significant changes in gene expression of plants which are indicative of stress conditions. A substantial part of the general stress response is up-regulation of heat shock proteins (Hsp) which function as molecular chaperones. The objective of this research was to test the possible changes in the Hsp70 and Hsp90 level in response to short-term hypergravity exposure. In this study 5-day-old etiolated pea seedlings were exposed to centrifuge-induced hypergravity (3–14 g) for 15 min and 1 h and a part of the seedlings was sampled at 1.5 and 24 h after the exposures. Western blot analysis showed time-dependent changes in Hsp70 and Hsp90 levels: an increase under hypergravity and a tendency towards recovery of the normal content during re-adaptation. The quantity and time of their expression was correlated with the g-force level. These data suggest that short-term hypergravity acts as a stress which could increase the risk of protein denaturation and aggregation. Molecular chaperons induced during the stress may have an essential role in counteracting this risk.     

Thermal shock cracking of lithium niobate single crystal

The quantitative estimation of failure stress of a lithium niobate (LN) single crystal due to thermal shock was investigated. Cylindrical test specimens were used in the thermal shock tests. The thermal stress of an LN test specimen under conditions of thermal shock cracking was calculated from a computer program which takes account of the crystal anisotropy, using the surface temperature measured in the thermal shock test. Four-point bending tests were also carried out to examine the relationship between the thermal shock cracking and the failure of a small test specimen due to mechanical load. LN single crystals fractured at the cleavage planes {0 1 1 2} in the thermal shock test and the four-point bending test. Although the failure stress data obtained from both tests obey the Weibull distribution, the Weibull distribution depends not only on specimen size but also on loading type. According to the Weibull distribution of thermal shock test data, if the normal stress σ_n acting on the cleavage planes {0 1 1 2} is lower than 10 MPa, the probability of thermal shock cracking becomes very small — less than 2%. This revised version was published online in July 2006 with corrections to the Cover Date.     

Proteome Analysis of Thyroid Cancer Cells After Long-Term Exposure to a Random Positioning Machine

Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1?g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11?days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1?g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71?kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.     

Shock compaction of silicon carbide powder

A series of shock compaction experiments on SiC powder were carried out over a wide range of shock pressures and shock temperatures up to 30 GPa and 3400 K. Large changes in some physical properties and a variety of unique microstructures were observed in the shock-treated samples with changes in impact conditions. For an iron plate impactor, the optimum impact condition, which depends on the initial density, is 2.5 km sec^–1 for 70% initial density and 2.0 km sec^–1 for 50%. The best-sintered compact of SiC with 97% density and micro-Vicker's hardness of 2700 kg mm^–2 was obtained under optimum conditions. Good compacts with high relative density and high strength exhibit the disruptive effects of the shock wave, which are indicated by microstrain increase and crystallite size reduction. The skin model is presented here in order to estimate the heterogeneous shock state which is realized under and after shock loading of the initial powder aggregates.     

Dynamic behaviour of dry and water-saturated sand under planar shock conditions

Plane shock-wave experiments were performed on dry and partially water-saturated sand, using three water contents, in order to validate predictive models of material behaviour at stress levels between 1 and 10 GPa. Gas and powder guns were used to load the sample under uni-axial strain conditions at low and high stress levels, respectively. Wave motions were detected by piezoelectric pins in the samples and a VISAR (Velocity Interferometer System for Any Reflector) recorded the free-surface velocity on the back target. This study presents both experimental and simulated results. Experimental data are used to determine shock Hugoniot states. Significant differences are observed in the dynamic response of the materials under various water-saturated conditions, and are reproduced with good agreement by numerical simulations using the ARMORS (A Rheological MOdel of Rocks Saturated) model.     

Dynamics of iron release from transferrin N-lobe studied by electrospray ionization mass spectrometry

Transferrins constitute a class of metalloproteins that are involved in circulatory iron transport in a variety of species. The metal ion-binding properties of these proteins have been the focus of extensive research efforts in the past decade due to their extreme importance in a variety of biological and healthcare-related fields. The large size of these proteins, as well as the presence of high-spin metal ions (e.g., Fe3+), limits the use of NMR. In this work, we report on the use of electrospray ionization mass spectrometry (ESI MS) to study dynamics of the transferrin system in vitro under conditions that are designed to mimic the endosomal environment. ESI MS is shown to provide valuable insights into the mechanistic aspects of metal ion-binding/release by transferrins and is complementary to other spectroscopic techniques. Conformational stability of the complex is evaluated based on the appearance of the charge-state distribution of protein ions, while the composition of the protein-ligand complex is determined based on the mass of the protein ions. In the absence of iron chelators, a stepwise dissociation of the ternary complex (protein-metal ion-synergistic anion) is observed as the solution pH is gradually decreased. Although the release of synergistic anion from the complex is initiated at typical endosomal pH levels (i.e., 5.5), metal ion remains largely bound to the protein until the pH is lowered to a level of approximately 4.5. Under these conditions, a significant fraction of the protein populates unfolded conformations. In stark contrast to this behavior, addition of an iron chelating agent (citrate) to the protein solution results in facile iron release at typical endosomal pH levels without any detectable unfolding of the protein. The mass spectral data lends further credibility to the notion that the holoprotein samples conformations that are specific to the apo form (e.g., "open conformation"), from which iron dissociation most likely occurs. The results of the present study demonstrate that ESI MS can be used to model metal ion release from transferrin under conditions that are designed to mimic the physiological environment.     

Magnetically Guided Rat Erythrocytes Bearing Isoniazid: Preparation,Characterization, and Evaluation

Abstract

Rat erythrocytes were loaded with isoniazid and magnetite by the preswell technique. Various parameters such as drug concentration, magnetite concentration, and volume of aqueous solution were optimized to study the maximum loading of drug into erythrocytes (67.2 ± 1.6%). The loaded cells were characterized for drug and magnetite content, hemoglobin content, percent cell recovery, morphology, osmotic fragility, turbulence shock, in-vivo drug and hemoglobin efflux, and magnetic responsiveness. No appreciable detrimental effect on cell morphology, osmotic fragility, and turbulence shock in comparison to normal cells was noted. However, drug and magnetite showed little detrimental effect on cells. Drug release from these systems followed approximately zero-order kinetics. Re drug- and magnetite-loaded cells effectively responded to an external magnetic field of 8.0 ± 1.0 K.Oe. The in-vivo studies showed that an erythrocyte-based delivery system has potential to increase drug concentration many fold at the target site under influence of an external magnetic field. The drug-loaded erythrocytes appeared to be promising carriers of isoniazid to infected organ/tissue.     

Magnetically Guided Rat Erythrocytes Bearing Isoniazid: Preparation, Characterization, and Evaluation

Rat erythrocytes were loaded with isoniazid and magnetite by the preswell technique. Various parameters such as drug concentration, magnetite concentration, and volume of aqueous solution were optimized to study the maximum loading of drug into erythrocytes (67.2 ± 1.6%). The loaded cells were characterized for drug and magnetite content, hemoglobin content, percent cell recovery, morphology, osmotic fragility, turbulence shock, in-vivo drug and hemoglobin efflux, and magnetic responsiveness. No appreciable detrimental effect on cell morphology, osmotic fragility, and turbulence shock in comparison to normal cells was noted. However, drug and magnetite showed little detrimental effect on cells. Drug release from these systems followed approximately zero-order kinetics. Re drug- and magnetite-loaded cells effectively responded to an external magnetic field of 8.0 ± 1.0 K.Oe. The in-vivo studies showed that an erythrocyte-based delivery system has potential to increase drug concentration many fold at the target site under influence of an external magnetic field. The drug-loaded erythrocytes appeared to be promising carriers of isoniazid to infected organ/tissue.     

Antimycobacterial efficacy of silver nanoparticles as deposited on porous membrane filters

Environmental mycobacteria pose a significant health burden. Non-tuberculous mycobacteria infections have been traced to water treatment networks, where mycobacterial biofilms are ubiquitous. Filters that remove potential pathogens have significant medical applications. The purpose of this study is to demonstrate that an antibacterial silver nanoparticle (AgNP) coating can prevent colonization and growth of a mycobacterial biofilm on a filter material. The antibacterial efficacy of commercially available AgNPs was measured against Mycobacterium avium, Mycobacterium smegmatis, and Mycobacterium marinum after 48 h in liquid culture. Nanoparticles were deposited on micro-porous track etched polycarbonate membranes. The growth of biofilms on the membranes was observed by microscopy and counting colony forming units. M. smegmatis was most susceptible to AgNPs, with a 98.7% reduction at 100 μM AgNP concentration. M. avium was reduced by 97.3% at 539 μM AgNP after 48 h. Deposited nanoparticles inhibited colonization and growth for both M. smegmatis and M. avium on the membrane surface. Similar to the liquid culture, M. avium (84.2% survival) was more resistant than M. smegmatis (0.03% survival).     

Behavior of decomposition of rifampicin in the presence of isoniazid in the pH range 1-3

The extent of decomposition of rifampicin in the presence of isoniazid was determined in the pH range 1-3 at 37°C in 50 min, the mean stomach residence time. With increase in pH, the degradation initially increased from pH 1 to 2 and then decreased, resulting in a bell-shaped pH-decomposition profile. This showed that rifampicin degraded in the presence of isoniazid to a higher extent at pH 2, the maximum pH in the fasting condition, under which antituberculosis fixed-dose combination (FDC) products are administered. At this pH and in 50 min, rifampicin decomposed by ~ 34%, while the fall of isoniazid was 10%. The extent of decomposition for the two drugs was also determined in marketed formulations, and the values ranged between 13-35% and 4-11%, respectively. The extents of decomposition at stomach residence times of 15 min and 3 h were 11.94% and 62.57%, respectively, for rifampicin and 4.78% and 11.12%, respectively, for isoniazid. The results show that quite an extensive loss of rifampicin and isoniazid can occur as a result of interaction between them in fasting pH conditions. This emphasizes that antituberculosis FDC formulations, which contain both drugs, should be designed in a manner that the interaction of the two drugs is prevented when the formulations are administered on an empty stomach.     

基于非傅里叶热传导对热冲击下带裂纹厚壁圆筒的分析

随着科学技术、工业水平的发展,传统的傅里叶导热在极端条件下不再适用。基于双曲型单相延迟非傅里叶热传导方程,推导了热冲击下有限元方程,编写了有限元算法程序,研究了在热冲击载荷下含裂纹厚壁圆筒结构的热力学响应,计算出厚壁圆筒在非经典传热条件下的温度场、位移场和裂纹尖端应力强度因子的数值解,分析不同热冲击载荷、不同裂纹长度、不同相位延迟下非傅里叶热传导的波动性效应以及温度应力强度因子的变化,得到相应的结论。为非经典工程条件下,带裂纹厚壁圆筒构件的可靠性以及构件的优化设计提供了数值上的参考。     

Behavior of Decomposition of Rifampicin in the Presence of Isoniazid in the pH Range 1–3

Abstract

The extent of decomposition of rifampicin in the presence of isoniazid was determined in the pH range 1–3 at 37°C in 50 min, the mean stomach residence time. With increase in pH, the degradation initially increased from pH 1 to 2 and then decreased, resulting in a bell-shaped pH-decomposition profile. This showed that rifampicin degraded in the presence of isoniazid to a higher extent at pH 2, the maximum pH in the fasting condition, under which antituberculosis fixed-dose combination (FDC) products are administered. At this pH and in 50 min, rifampicin decomposed by ? 34%, while the fall of isoniazid was 10%. The extent of decomposition for the two drugs was also determined in marketed formulations, and the values ranged between 13–35% and 4–11%, respectively. The extents of decomposition at stomach residence times of 15 min and 3 h were 11.94% and 62.57%, respectively, for rifampicin and 4.78% and 11.12%, respectively, for isoniazid. The results show that quite an extensive loss of rifampicin and isoniazid can occur as a result of interaction between them in fasting pH conditions. This emphasizes that antituberculosis FDC formulations, which contain both drugs, should be designed in a manner that the interaction of the two drugs is prevented when the formulations are administered on an empty stomach.     

The effect of perfusion culture on proliferation and differentiation of human mesenchymal stem cells on biocorrodible bone replacement material

Biocorrodible iron foams were coated with different calcium phosphate phases (CPP) to obtain a bioactive surface and controlled degradation. Further adhesion, proliferation and differentiation of SaOs-2 and human mesenchymal stem cells were investigated under both static and dynamic culture conditions. Hydroxyapatite (HA; [Ca₁₀(PO₄)₆OH₂]) coated foams released 500 μg/g iron per day for Dulbecco's modified eagle medium (DMEM) and 250 μg/g iron per day for McCoys, the unmodified reference 1000 μg/g iron per day for DMEM and 500 μg/g iron per day for McCoys, while no corrosion could be detected on brushite (CaHPO₄) coated foams. Using a perfusion culture system with conditions closer to the in vivo situation, cells proliferated and differentiated on iron foams coated with either brushite or HA while in static cell culture cells could proliferate only on Fe-brushite. We conclude that the degradation behaviour of biocorrodible iron foams can be varied by different calcium phosphate coatings, offering opportunities for design of novel bone implants. Further studies will focus on the influence of different modifications of iron foams on the expression of oxidative stress enzymes. Additional information about in vivo reactions and remodelling behaviour are expected from testing in implantation studies.     

Surface-protein interactions on different stainless steel grades: effects of protein adsorption, surface changes and metal release

Implantation using stainless steels (SS) is an example where an understanding of protein-induced metal release from SS is important when assessing potential toxicological risks. Here, the protein-induced metal release was investigated for austenitic (AISI 304, 310, and 316L), ferritic (AISI 430), and duplex (AISI 2205) grades in a phosphate buffered saline (PBS, pH 7.4) solution containing either bovine serum albumin (BSA) or lysozyme (LSZ). The results show that both BSA and LSZ induce a significant enrichment of chromium in the surface oxide of all stainless steel grades. Both proteins induced an enhanced extent of released iron, chromium, nickel and manganese, very significant in the case of BSA (up to 40-fold increase), whereas both proteins reduced the corrosion resistance of SS, with the reverse situation for iron metal (reduced corrosion rates and reduced metal release in the presence of proteins). A full monolayer coverage is necessary to induce the effects observed.     

塑料瓦楞板组合式周转箱设计及跌落分析

目的设计一款可替代塑料周转箱和瓦楞纸箱的可拼接式塑料瓦楞周转箱,并通过仿真寻找所设计塑料瓦楞周转箱在跌落时的易损部位,以便对所设计结构进行更合理的设计及优化。方法基于HyperMesh LS-DYNA及LS-Pre Post对模型进行日常运输时跌落的模拟仿真,箱体总质量为60 kg,高度为1 m,分别采用斜15°和水平(箱底面相较于跌落地面的角度)这2种工况下进行跌落仿真。结果所设计周转箱在2种工况下底部包边边框刚碰撞时,边框局部等效应力大于200 MPa,超过铝合金的屈服强度,会发生塑性应变,但边框有效塑性应变均未达到铝合金的断面伸长率(16%),因此未发生断裂失效。结论所设计的周转箱抗跌落性能良好,在改进时需着重对4个底角进行加强防护设计。