N-WASP Is Required for Structural Integrity of the Blood-Testis Barrier

During spermatogenesis, the blood-testis barrier (BTB) segregates the adluminal (apical) and basal compartments in the seminiferous epithelium, thereby creating a privileged adluminal environment that allows post-meiotic spermatid development to proceed without interference of the host immune system. A key feature of the BTB is its continuous remodeling within the Sertoli cells, the major somatic component of the seminiferous epithelium. This remodeling is necessary to allow the transport of germ cells towards the seminiferous tubule interior, while maintaining intact barrier properties. Here we demonstrate that the actin nucleation promoting factor Neuronal Wiskott-Aldrich Syndrome Protein (N-WASP) provides an essential function necessary for BTB restructuring, and for maintaining spermatogenesis. Our data suggests that the N-WASP-Arp2/3 actin polymerization machinery generates branched-actin arrays at an advanced stage of BTB remodeling. These arrays are proposed to mediate the restructuring process through endocytic recycling of BTB components. Disruption of N-WASP in Sertoli cells results in major structural abnormalities to the BTB, including mis-localization of critical junctional and cytoskeletal elements, and leads to disruption of barrier function. These impairments result in a complete arrest of spermatogenesis, underscoring the critical involvement of the somatic compartment of the seminiferous tubules in germ cell maturation

Cold atmospheric plasma induces ATP-dependent endocytosis of nanoparticles and synergistic U373MG cancer cell death

Gold nanoparticles (AuNP) have potential as both diagnostic and therapeutic vehicles. However, selective targeting and uptake in cancer cells remains challenging. Cold atmospheric plasma (CAP) can be combined with AuNP to achieve synergistic anti-cancer cytotoxicity. To explore synergistic mechanisms, we demonstrate both rate of AuNP uptake and total amount accumulated in U373MG Glioblastoma multiforme (GBM) cells are significantly increased when exposed to 75 kV CAP generated by dielectric barrier discharge. No significant changes in the physical parameters of AuNP were caused by CAP but active transport mechanisms were stimulated in cells. Unlike many other biological effects of CAP, long-lived reactive species were not involved, and plasma-activated liquids did not replicate the effect. Chemical effects induced by direct and indirect exposure to CAP appears the dominant mediator of enhanced uptake. Transient physical alterations of membrane integrity played a minor role. 3D-reconstruction of deconvoluted confocal images confirmed AuNP accumulation in lysosomes and other acidic vesicles, which will be useful for future drug delivery and diagnostic strategies. Toxicity of AuNP significantly increased by 25-fold when combined with CAP. Our data indicate that direct exposure to CAP activates AuNP-dependent cytotoxicity by increasing AuNP endocytosis and trafficking to lysosomes in U373MG cells

Fluorescent D-amino-acids reveal bi-cellular cell wall modifications important for Bdellovibrio bacteriovorous predation

Modification of essential bacterial peptidoglycan (PG) containing cell walls can lead to antibiotic resistance, for example β-lactam resistance by L,D-transpeptidase activities. Predatory Bdellovibrio bacteriovorus are naturally antibacterial and combat infections by traversing, modifying and finally destroying walls of Gram-negative prey bacteria, modifying their own PG as they grow inside prey. Historically, these multi-enzymatic processes on two similar PG walls have proved challenging to elucidate. Here, with a PG labelling approach utilizing timed pulses of multiple fluorescent D-amino acids (FDAAs), we illuminate dynamic changes that predator and prey walls go through during the different phases of bacteria:bacteria invasion. We show formation of a reinforced circular port-hole in the prey wall; L,D-transpeptidaseBd mediated D-amino acid modifications strengthening prey PG during Bdellovibrio invasion and a zonal mode of predator-elongation. This process is followed by unconventional, multi-point and synchronous septation of the intracellular Bdellovibrio, accommodating odd- and even-numbered progeny formation by non-binary division

An Eye to a Kill: Using Predatory Bacteria to Control Gram-Negative Pathogens Associated with Ocular Infections

Ocular infections are a leading cause of vision loss. It has been previously suggested that predatory prokaryotes might be used as live antibiotics to control infections. In this study, Pseudomonas aeruginosa and Serratia marcescens ocular isolates were exposed to the predatory bacteria Micavibrio aeruginosavorus and Bdellovibrio bacteriovorus. All tested S. marcescens isolates were susceptible to predation by B. bacteriovorus strains 109J and HD100. Seven of the 10 P. aeruginosa isolates were susceptible to predation by B. bacteriovorus 109J with 80% being attacked by M. aeruginosavorus. All of the 19 tested isolates were found to be sensitive to at least one predator. To further investigate the effect of the predators on eukaryotic cells, human corneal-limbal epithelial (HCLE) cells were exposed to high concentrations of the predators. Cytotoxicity assays demonstrated that predatory bacteria do not damage ocular surface cells in vitro whereas the P. aeruginosa used as a positive control was highly toxic. Furthermore, no increase in the production of the proinflammatory cytokines IL-8 and TNF-alpha was measured in HCLE cells after exposure to the predators. Finally, injection of high concentration of predatory bacteria into the hemocoel of Galleria mellonella, an established model system used to study microbial pathogenesis, did not result in any measurable negative effect to the host. Our results suggest that predatory bacteria could be considered in the near future as a safe topical bio-control agent to treat ocular infections. © 2013 Shanks et al

Activity of Bdellovibrio Hit Locus Proteins, Bd0108 and Bd0109, Links Type IVa Pilus Extrusion/Retraction Status to Prey-Independent Growth Signalling

Bdellovibrio bacteriovorus are facultatively predatory bacteria that grow within gram-negative prey, using pili to invade their periplasmic niche. They also grow prey-independently on organic nutrients after undergoing a reversible switch. The nature of the growth switching mechanism has been elusive, but several independent reports suggested mutations in the hit (host-interaction) locus on the Bdellovibrio genome were associated with the transition to preyindependent growth. Pili are essential for prey entry by Bdellovibrio and sequence analysis of the hit locus predicted that it was part of a cluster of Type IVb pilus-associated genes, containing bd0108 and bd0109. In this study we have deleted the whole bd0108 gene, which is unique to Bdellovibrio, and compared its phenotype to strains containing spontaneous mutations in bd0108 and the common natural 42 bp deletion variant of bd0108. We find that deletion of the whole bd0108 gene greatly reduced the extrusion of pili, whereas the 42 bp deletion caused greater pilus extrusion than wild-type. The pili isolated from these strains were comprised of the Type IVa pilin protein; PilA. Attempts to similarly delete gene bd0109, which like bd0108 encodes a periplasmic/secreted protein, were not successful, suggesting that it is likely to be essential for Bdellovibrio viability in any growth mode. Bd0109 has a sugar binding YD- repeat motif and an N-terminus with a putative pilin-like fold and was found to interact directly with Bd0108. These results lead us to propose that the Bd0109/Bd0108 interaction regulates pilus production in Bdellovibrio (possibly by interaction with the pilus fibre at the cell wall), and that the presence (and possibly retraction state) of the pilus feeds back to alter the growth state of the Bdellovibrio cell. We further identify a novel small RNA encoded by the hit locus, the transcription of which is altered in different bd0108 mutation background

Частные стратегии маркетинга бизнес–отеля (на примере гостиницы «Беларусь» («ТЭУП «Бресттурист»))

В статье разобрана общая маркетинговая стратегия предприятия и частные стратегии по сегментам продаж. Установлены сегменты продаж гостиницы «Беларусь» (г.Брест). Общая стратегия маркетинга гостиницы распространяется на все сегменты спроса, причем для каждого существуют свои особенности. В ходе исследования были предложены рекомендации по развитию частных маркетинговых стратегий гостиницы «Беларусь». This article explores the overall marketing strategy of the enterprise and private strategies by segment sales. Sales segment of the hotel «Belarus» has been installed. The overall marketing strategy of the hotel extends to all segments of demand, and each has its own peculiarities. In the course of the study, recommendations were proposed for the development of the private marketing strategies of the hotel «Belarus»

dOCRL maintains immune cell quiescence in Drosophila by regulating endosomal traffic

Lowe Syndrome is a developmental disorder characterized by eye, kidney, and neurological pathologies, and is caused by mutations in the phosphatidylinositol-5-phosphatase OCRL. OCRL plays diverse roles in endocytic and endolysosomal trafficking, cytokinesis, and ciliogenesis, but it is unclear which of these cellular functions underlie specific patient symptoms. Here, we show that mutation of Drosophila OCRL causes cell-autonomous activation of hemocytes, which are macrophage-like cells of the innate immune system. Among many cell biological defects that we identified in docrl mutant hemocytes, we pinpointed the cause of innate immune cell activation to reduced Rab11-dependent recycling traffic and concomitantly increased Rab7-dependent late endosome traffic. Loss of docrl amplifies multiple immune-relevant signals, including Toll, Jun kinase, and STAT, and leads to Rab11-sensitive mis-sorting and excessive secretion of the Toll ligand Spåtzle. Thus, docrl regulation of endosomal traffic maintains hemocytes in a poised, but quiescent state, suggesting mechanisms by which endosomal misregulation of signaling may contribute to symptoms of Lowe syndrome

Micromechanical Properties of Injection-Molded Starch–Wood Particle Composites

The micromechanical properties of injection molded starch–wood particle composites were investigated as a function of particle content and humidity conditions. The composite materials were characterized by scanning electron microscopy and X-ray diffraction methods. The microhardness of the composites was shown to increase notably with the concentration of the wood particles. In addition,creep behavior under the indenter and temperature dependence were evaluated in terms of the independent contribution of the starch matrix and the wood microparticles to the hardness value. The influence of drying time on the density and weight uptake of the injection-molded composites was highlighted. The results revealed the role of the mechanism of water evaporation, showing that the dependence of water uptake and temperature was greater for the starch–wood composites than for the pure starch sample. Experiments performed during the drying process at 70°C indicated that the wood in the starch composites did not prevent water loss from the samples.Peer reviewe

ДОСЛІДЖЕННЯ ДИНАМІЧНИХ РЕЖИМІВ ЕЛЕКТРОДВИГУНА ПОСЛІДОВНОГО ЗБУДЖЕННЯ З ІМПУЛЬСНИМИ СХЕМАМИ РЕГУЛЮВАННЯ ЕЛЕКТРОПРИВОДУ

Purpose: The purpose of the work is to research the dynamics of DC motor of series excitation with pulse speed control and determine the possibility of the drive circuit simplification.Research methods: With the help of simulation and analytical methods of calculation techniques developed a simulation model, identified the advantages of using a modernized impulse control scheme.The obtained results: The modernized scheme of series excitation DC current motor pulse control is designed. For the proposed and existing pulse regulation schemes the models are developed; they help to investigate the electromechanical processes of electric drive in these schemes and to perform the comparative analysis. The results of the modernized DC motor speed impulse control scheme investigation in the acceleration mode, overrun and electro-dynamic braking are given.Scientific novelty: Developed advanced pulse regulating sequential excitation DC motor scheme. For existing and proposed schemes impulse regulation developed models that allow study of electromechanical processes in electric drive for existing and proposed impulse control schemes.The practical significance: It is shown that the addition of a shunt field winding diode to pulsed regulator scheme reducing the rate of current decay in the winding. It is determined that the proposed upgrade allows to use electro-dynamic braking mode at the motor self-excitation. The proposed scheme design avoids the use of additional independent power sources and simplify the technical implementation and reduce costs.Цель: Целью работы является исследование динамики двигателя постоянного тока последовательного возбуждения с импульсным регулированием частоты вращения и определениее возможности упрощения схемы электропривода.Методика: Методы имитационного моделирования и аналитические методы расчета и исследований электромеханических процессов в сериесном двигателе постоянного тока при различных схемах импульсного управления.Результаты: Разработана усовершенствованная схема импульсного регулирования двигателя постоянного тока последовательного возбуждения. Для предложенной и существующей схем импульсного регулирования разработаны модели, с помощью которых исследованы электромеханические процессы тягового электропривода в этих схемах и выполнен их сравнительный анализ. Приведены результаты исследования модернизированной импульсной схемы регулирования частоты вращения двигателя постоянного тока в режимах разгона, выбега и электродинамического торможения. Проведен анализ величины пульсаций тока якоря и влияние на нее частоты коммутации при использовании модернизированной схемы импульсного регулирования.Научная новизна: Разработаны новые имитационные модели, которые позволяют проводить исследования электромеханических процессов тягового электропривода в различных схемах импульсного регулирования.Практическая значимость Разработана усовершенствованная схема импульсного регулирования двигателя постоянного тока последовательного возбуждения. Проведено усовершенствование схемы импульсного регулирования двигателя постоянного тока последовательного возбуждения за счет введения в схему импульсного регулятора диода, шунтирующего обмотку возбуждения, что позволяет снизить скорость затухания тока в обмотке возбуждения и использовать режим электродинамического торможения при самовозбуждении электродвигателя. Предложенное схемное решение позволяет избежать применения дополнительных независимых источников тока, упростить техническую реализацию и уменьшить расходы.Мета: Метою роботи є дослідження динаміки двигуна постійного струму послідовного збудження з імпульсним регулюванням частоти обертання і визначення можливості спрощення схеми електропривода.Методика: Методи імітаційного моделювання й аналітичні методи розрахунку та дослідження електромеханічних процесів в серієсному двигуні постійного струму при різних схемах імпульсного керування.Результати: Розроблено вдосконалену схему імпульсного регулювання двигуна постійного струму послідовного збудження. Для запропонованої і існуючої схем імпульсного регулювання розроблені моделі, за допомогою яких досліджені електромеханічні процеси тягового електроприводу в цих схемах і виконано їх порівняльний аналіз. Наведено результати дослідження модернізованої імпульсної схеми регулювання частоти обертання двигуна постійного струму в режимах розгону, вибігу і електродинамічного гальмування. Проведено аналіз величини пульсацій струму якоря та вплив на неї частоти комутації при використанні модернізованої схеми імпульсного регулювання.Наукова новизна: Розроблено нові імітаційні моделі, які дозволяють виконувати дослідження електромеханічних процесів тягового електроприводу при різних схемах імпульсного регулювання.Практична значимість: Розроблено вдосконалену схему імпульсного регулювання двигуна постійного струму послідовного збудження. Вдосконалено схему імпульсного регулювання двигуна постійного струму послідовного збудження за рахунок введення в схему імпульсного регулятора діода, який шунтує обмотку збудження, що дозволяє знизити швидкість загасання струму в обмотці збудження та використовувати режим електродинамічного гальмування при самозбудженні електродвигуна. Запропоноване схемне рішення дозволяє уникнути застосування додаткових незалежних джерел струму та спростити технічну реалізацію і зменшити витрати