The cryoEM structure of cytochrome bd from C. glutamicum provides novel insights into structural properties of actinobacterial terminal oxidases

Cytochromes bd are essential for microaerobic respiration of many prokaryotes including a number of human pathogens. These enzymes catalyze the reduction of molecular oxygen to water using quinols as electron donors. Their importance for prokaryotic survival and the absence of eukaryotic homologs make these enzyme ideal targets for antimicrobial drugs. Here, we determined the cryoEM structure of the menaquinol-oxidizing cytochrome bd-type oxygen reductase of the facultative anaerobic Actinobacterium Corynebacterium glutamicum at a resolution of 2.7 Å. The obtained structure adopts the signature pseudosymmetrical heterodimeric architecture of canonical cytochrome bd oxidases formed by the core subunits CydA and CydB. No accessory subunits were identified for this cytochrome bd homolog. The two b-type hemes and the oxygen binding heme d are organized in a triangular geometry with a protein environment around these redox cofactors similar to that of the closely related cytochrome bd from M. tuberculosis. We identified oxygen and a proton conducting channels emerging from the membrane space and the cytoplasm, respectively. Compared to the prototypical enzyme homolog from the E. coli, the most apparent difference is found in the location and size of the proton channel entry site. In canonical cytochrome bd oxidases quinol oxidation occurs at the highly flexible periplasmic Q-loop located in the loop region between TMHs six and seven. An alternative quinol-binding site near heme b595 was previously identified for cytochrome bd from M. tuberculosis. We discuss the relevance of the two quinol oxidation sites in actinobacterial bd-type oxidases and highlight important differences that may explain functional and electrochemical differences between C. glutamicum and M. tuberculosis. This study expands our current understanding of the structural diversity of actinobacterial and proteobacterial cytochrome bd oxygen reductases and provides deeper insights into the unique structural and functional properties of various cytochrome bd variants from different phylae

Induced shear stress measurement in overtopped floodwalls

The scour generated by water overtopping floodwalls results in tilting and sometimes failure of floodwalls. The water free fall induces shear stress, which is difficult to measure, at the levee surface and if it exceeds the critical shear stress, scouring initiates. It is perceived that once a pool of water is generated at the levee crest due to scour, the induced stresses on soil surface at the pool bottom will decrease. The scour progress is stopped once the induced shear stresses at the pool bottom are less than critical shear stress; however, due to the low values of critical shear stress, the scour most likely continues for a long time. A methodology was developed in this study to measure the induced shear stresses. Based on the tests conducted in this study, the generated pool will decrease the induced shear stress until constant erosion rate is achieved. This shear stress is defined as ‘equilibrium’ shear stress herein

Особенности использования статистического метода стабилизации частоты генераторов в распределенных информационно-измерительных системах

The article deals with the problems of frequency stabilization of generators in distributed information-measuring systems on the basis of the static frequency stabilization method. The examples of similar systems are given. The necessity to ensure high stability of signal frequency generated by subscribers to improve the efficiency of the stated systems is noted. Two main features of applying the static frequency stabilization method are stated. The first one is determined with a delay in signal input into the device for estimating the frequency-time signal parameters in relation to the signal phase measurement interval. The second feature involves the entry delay of the formed control signal of generator frequency correction. It is shown that the mentioned features lead to divergence of the current variation of generator frequency and the value under correction, obtained considering the delay. The description of information signals enabling to specify a mathematical model of the process of assessment forming of the information signal frequency variation, taking into account the mentioned factors, is offered. It is shown that the possibility of using a statistical method to stabilize the frequency of generators in distributed informationmeasuring systems is determined not only with the values of nominal frequencies and relative instabilities of generators included into the information-measuring system, but also by the autocorrelation function of a random process describing the change of information signal frequency. In case of small time delays between the evaluation of information signal frequency and frequency stabilization, the use of the method to be considered allows to reduce the relative instability of information signal frequency. When time delays are greater than the correlation interval, the use of the statistical method does not lead to the change of relative instability of information signal frequency. Within the correlation interval, the use of the statistical method of frequency stabilization can cause depending on the autocorrelation function values both decrease and increase of the relative information signal frequency instability.В статье рассмотрены вопросы стабилизации частоты генераторов в распределенных информационно-измерительных системах на основе использования метода статической стабилизации частоты. Приведены примеры таких систем и отмечена необходимость обеспечения высокой стабильности частоты формируемых абонентами сигналов для повышения эффективности функционирования указанных систем. Отмечены две основные особенности применения метода статистической стабилизации частоты. Первая определяется задержкой поступления сигнала в устройство оценивания частотно-временных параметров сигнала по отношению к интервалу измерения фазы сигнала. Вторая особенность связана с задержкой прихода сформированного управляющего сигнала коррекции частоты генератора. Показано, что отмеченные особенности приводят к несовпадению текущего отклонения частоты генератора и корректируемого значения, полученного с учетом задержки. Предложено описание информационных сигналов, позволяющих уточнить математическую модель процесса формирования оценки отклонения частоты информационного сигнала с учетом указанных факторов. Показано, что возможность использования статистического метода стабилизации частоты генераторов в распределенных информационно-измерительных системах определяется не только значениями номинальных частот и относительных нестабильностей генераторов, входящих в состав информационно-измерительной системы, но и автокорреляционной функцией случайного процесса, описывающего изменение частоты информационного сигнала. При малых временных задержках между оцениванием частоты информационного сигнала и стабилизацией частоты использование рассматриваемого метода позволяет уменьшить относительную нестабильность частоты информационного сигнала. При временных задержках больше интервала корреляции использование статистического метода не приводит к изменению относительной нестабильности частоты информационного сигнала. В пределах интервала корреляции использование статистического метода стабилизации частоты может приводить в зависимости от значений автокорреляционной функции как к уменьшению, так и увеличению относительной нестабильности частоты информационного сигнала

The correlation of RNase A enzymatic activity with the changes in the distance between Nepsilon2-His12 and N delta1-His119 upon addition of stabilizing and destabilizing salts.

The effect of stabilizing and destabilizing salts on the catalytic behavior of ribonuclease A (RNase A) was investigated at pH 7.5 and 25 degrees C, using spectrophotometric, viscometric and molecular dynamic methods. The changes in the distance between N(epsilon2) of His(12) and N(delta1) of His(119) at the catalytic center of RNase A upon the addition of sodium sulfate, sodium hydrogen sulfate and sodium thiocyanate were evaluated by molecular dynamic methods. The compactness and expansion in terms of Stokes radius of RNase A upon the addition of sulfate ions as kosmotropic salts, and thiocyanate ion as a chaotropic salt, were estimated by viscometric measurements. Enzyme activity was measured using cytidine 2', 3'-cyclic monophosphate as a substrate. The results from the measurements of distances between N(epsilon2) of His(12) and N(delta1) of His(119) and Stokes radius suggest (i) that the presence of sulfate ions decreases the distance between the catalytic His residues and increases the globular compactness, and (ii) that there is an expansion of the enzyme surface as well as elongation of the catalytic center in the presence of thiocyanate ion. These findings are in agreement with activity measurements

Sphingomimetic multiple sclerosis drug FTY720 activates vesicular synaptobrevin and augments neuroendocrine secretion

Neurotransmission and secretion of hormones involve a sequence of protein/lipid interactions with lipid turnover impacting on vesicle trafficking and ultimately fusion of secretory vesicles with the plasma membrane. We previously demonstrated that sphingosine, a sphingolipid metabolite, promotes formation of the SNARE complex required for membrane fusion and also increases the rate of exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and in hippocampal neurons. Recently a fungi-derived sphingosine homologue, FTY720, has been approved for treatment of multiple sclerosis. In its non-phosphorylated form FTY720 accumulates in the central nervous system, reaching high levels which could affect neuronal function. Considering close structural similarity of sphingosine and FTY720 we investigated whether FTY720 has an effect on regulated exocytosis. Our data demonstrate that FTY720 can activate vesicular synaptobrevin for SNARE complex formation and enhance exocytosis in neuroendocrine cells and neurons

Elite Suppressor–Derived HIV-1 Envelope Glycoproteins Exhibit Reduced Entry Efficiency and Kinetics

Elite suppressors (ES) are a rare subset of HIV-1–infected individuals who are able to maintain HIV-1 viral loads below the limit of detection by ultra-sensitive clinical assays in the absence of antiretroviral therapy. Mechanism(s) responsible for this elite control are poorly understood but likely involve both host and viral factors. This study assesses ES plasma-derived envelope glycoprotein (env) fitness as a function of entry efficiency as a possible contributor to viral suppression. Fitness of virus entry was first evaluated using a novel inducible cell line with controlled surface expression levels of CD4 (receptor) and CCR5 (co-receptor). In the context of physiologic CCR5 and CD4 surface densities, ES envs exhibited significantly decreased entry efficiency relative to chronically infected viremic progressors. ES envs also demonstrated slow entry kinetics indicating the presence of virus with reduced entry fitness. Overall, ES env clones were less efficient at mediating entry than chronic progressor envs. Interestingly, acute infection envs exhibited an intermediate phenotypic pattern not distinctly different from ES or chronic progressor envs. These results imply that lower env fitness may be established early and may directly contribute to viral suppression in ES individuals

Natural anti-CCR5 antibodies in HIV-infection and -exposure

Natural antibodies constitute a first-line of defence against pathogens; they may also play other roles in immune regulation and homeostasis, through their ability to bind host antigens, surface molecules and receptors. Natural anti-CCR5 antibodies can be decisive in preventing HIV infection in mucosal tissues and offer prompt and effective protection just at major sites of virus entry. Among natural anti-CCR5 antibodies, IgG and IgA to the ECL1 domain have been shown to block HIV effectively and durably without causing harm to the host. Their biological properties and their uncommon generation in subsets of HIV-infected and HIV-exposed individuals (so called ESN) will be introduced and discussed, with the aim at exploiting their potential in therapy and prevention

Cell-Cell Transmission Enables HIV-1 to Evade Inhibition by Potent CD4bs Directed Antibodies

HIV is known to spread efficiently both in a cell-free state and from cell to cell, however the relative importance of the cell-cell transmission mode in natural infection has not yet been resolved. Likewise to what extent cell-cell transmission is vulnerable to inhibition by neutralizing antibodies and entry inhibitors remains to be determined. Here we report on neutralizing antibody activity during cell-cell transmission using specifically tailored experimental strategies which enable unambiguous discrimination between the two transmission routes. We demonstrate that the activity of neutralizing monoclonal antibodies (mAbs) and entry inhibitors during cell-cell transmission varies depending on their mode of action. While gp41 directed agents remain active, CD4 binding site (CD4bs) directed inhibitors, including the potent neutralizing mAb VRC01, dramatically lose potency during cell-cell transmission. This implies that CD4bs mAbs act preferentially through blocking free virus transmission, while still allowing HIV to spread through cell-cell contacts. Thus providing a plausible explanation for how HIV maintains infectivity and rapidly escapes potent and broadly active CD4bs directed antibody responses in vivo

Horizontal pressures in cylindrical metal silos and comparison with different international standards

The focus of this research was to evaluate the horizontal pressures on a cylindrical metal silo of corrugated walls and flat bottom with 1.82m diameter and 5.4m high, and to compare the values with those obtained theoretically by the ISO 11697, EP 433 and AS 3774 standards. The silo was symmetrically filled and constant speed with wheat cv. soft red for two different height/diameter ratios (H/D) and was unloaded through three orifices with a diameter of 71.6mm, one concentric and two eccentrics. Horizontal pressures were measured on the walls of the silo at three positions using hydraulic type pressure cells. The results showed that shortly after the start of the unloading, there was a mass flow above the quota of H/D = 1.2, whereas below this quota funnel flow occurred. It can be said that the EP 433 standard was more appropriate to predict horizontal pressures in silos in H/D ratio = 1.0, with eccentric unloading. For the H/D ratio = 1.5, AS 3774 standard was the one that produced values closer to the experimental