Unsupported planar lipid membranes formed from mycolic acids of Mycobacterium tuberculosis

The cell wall of mycobacteria includes a thick, robust, and highly impermeable outer membrane made from long-chain mycolic acids. These outer membranes form a primary layer of protection for mycobacteria and directly contribute to the virulence of diseases such as tuberculosis and leprosy. We have formed in vitro planar membranes using pure mycolic acids on circular apertures 20 to 90 μm in diameter. We find these membranes to be long lived and highly resistant to irreversible electroporation, demonstrating their general strength. Insertion of the outer membrane channel MspA into the membranes was observed indicating that the artificial mycolic acid membranes are suitable for controlled studies of the mycobacterial outer membrane and can be used in nanopore DNA translocation experiments.     

Beta-1 integrins mediate substrate dependent effects of 1alpha,25(OH)2D3 on osteoblasts

Surface micron-scale and submicron scale features increase osteoblast differentiation and enhance responses of osteoblasts to 1,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)]. beta(1) integrin expression is increased in osteoblasts grown on Ti substrates with rough microarchitecture, and it is regulated by 1alpha,25(OH)(2)D(3) in a surface-dependent manner. To determine if beta(1) has a role in mediating osteoblast response, we silenced beta(1) expression in MG63 human osteoblast-like cells using small interfering RNA (siRNA). In addition, MG63 cells were treated with two different monoclonal antibodies to human beta(1) to block ligand binding. beta(1)-silenced MG63 cells grown on a tissue culture plastic had reduced alkaline phosphatase activity and levels of osteocalcin, transforming growth factor beta(1), prostaglandin E(2), and osteoprotegerin in comparison with control cells. Moreover, beta(1)-silencing inhibited the effects of surface roughness on these parameters and partially inhibited effects of 1alpha,25(OH)(2)D(3). Anti beta(1) antibodies decreased alkaline phosphatase but increase osteocalcin; effects of 1alpha,25(OH)(2)D(3) on cell number and alkaline phosphatase were reduced and effects on osteocalcin were increased. These findings indicate that beta(1) plays a major and complex role in osteoblastic differentiation modulated by either surface microarchitecture or 1alpha,25(OH)(2)D(3). The results also show that beta(1) mediates, in part, the synergistic effects of surface roughness and 1alpha,25(OH)(2)D(3).     

Spatial Compartmentalization Specializes the Function of Aurora A and Aurora B

Aurora kinase A and B share great similarity in sequences, structures, and phosphorylation motif, yet they show different localizations and play distinct crucial roles. The factors that determine such differences are largely unknown. Here we targeted Aurora A to the localization of Aurora B and found that Aurora A phosphorylates the substrate of Aurora B and substitutes its function in spindle checkpoint. In return, the centrosome targeting of Aurora B substitutes the function of Aurora A in the mitotic entry. Expressing the chimera proteins of the Auroras with exchanged N termini in cells indicates that the divergent N termini are also important for their spatiotemporal localizations and functions. Collectively, we demonstrate that functional divergence of Aurora kinases is determined by spatial compartmentalization, and their divergent N termini also contribute to their spatial and functional differentiation.     

The antibacterial toxin colicin N binds to the inner core of lipopolysaccharide and close to its translocator protein

Colicins are a diverse family of large antibacterial protein toxins, secreted by and active against Escherichia coli and must cross their target cell's outer membrane barrier to kill. To achieve this, most colicins require an abundant porin (e.g. OmpF) plus a low‐copy‐number, high‐affinity, outer membrane protein receptor (e.g. BtuB). Recently, genetic screens have suggested that colicin N (ColN), which has no high‐affinity receptor, targets highly abundant lipopolysaccharide (LPS) instead. Here we reveal the details of this interaction and demonstrate that the ColN receptor‐binding domain (ColN‐R) binds to a specific region of LPS close to the membrane surface. Data from in vitro studies using calorimetry and both liquid‐ and solid‐state NMR reveal the interactions behind the in vivo requirement for a defined oligosaccharide region of LPS. Delipidated LPS (LPS^ΔLIPID) shows weaker binding; and thus full affinity requires the lipid component. The site of LPS binding means that ColN will preferably bind at the interface and thus position itself close to the surface of its translocon component, OmpF. ColN is, currently, unique among colicins in requiring LPS and, combined with previous data, this implies that the ColN translocon is distinct from those of other known colicins.     

24,25-(OH)(2)D(3) regulates cartilage and bone via autocrine and endocrine mechanisms

The purpose of this paper is to summarize recent advances in our understanding of the physiological role of 24(R),25(OH)(2)D(3) in bone and cartilage and its mechanism of action. With the identification of a target cell, the growth plate resting zone (RC) chondrocyte, we have been able to use cell biology methodology to investigate specific functions of 24(R),25(OH)(2)D(3) and to determine how 24(R),25(OH)(2)D(3) elicits its effects. These studies indicate that there are specific membrane-associated signal transduction pathways that mediate both rapid, nongenomic and genomic responses of RC cells to 24(R),25(OH)(2)D(3). 24(R),25(OH)(2)D(3) binds RC chondrocyte membranes with high specificity, resulting in an increase in protein kinase C (PKC) activity. The effect is stereospecific; 24R,25(OH)(2)D(3), but not 24S,25-(OH)(2)D(3), causes the increase, indicating a receptor-mediated response. Phospholipase D-2 (PLD2) activity is increased, resulting in increased production of diacylglycerol (DAG), which in turn activates PKC. 24(R),25(OH)(2)D(3) does not cause translocation of PKC to the plasma membrane, but activates existing PKCalpha. There is a rapid decrease in Ca(2+) efflux, and influx is stimulated. 24(R),25(OH)(2)D(3) also reduces arachidonic acid release by decreasing phospholipase A(2) (PLA(2)) activity, thereby decreasing available substrate for prostaglandin production via the action of cyclooxygenase-1. PGE(2) that is produced acts on the EP1 and EP2 receptors expressed by RC cells to downregulate PKC via protein kinase A, but the reduction in PGE(2) decreases this negative feedback mechanism. Both pathways converge on MAP kinase, leading to new gene expression. One consequence of this is production of new matrix vesicles containing PKCalpha and PKCzeta and an increase in PKC activity. The chondrocytes also produce 24(R),25(OH)(2)D(3), and the secreted metabolite acts directly on the matrix vesicle membrane. Only PKCzeta is directly affected by 24(R),25(OH)(2)D(3) in the matrix vesicles, and activity of this isoform is inhibited. This effect may be involved in the control of matrix maturation and turnover. 24(R),25(OH)(2)D(3) causes RC cells to mature along the endochondral developmental pathway, where they become responsive to 1alpha,25(OH)(2)D(3) and lose responsiveness to 24(R),25(OH)(2)D(3), a characteristic of more mature growth zone (GC) chondrocytes. 1alpha,25(OH)(2)D(3) elicits its effects on GC through different signal transduction pathways than those used by 24(R),25(OH)(2)D(3). These studies indicate that 24(R),25(OH)(2)D(3) plays an important role in endochondral ossification by regulating less mature chondrocytes and promoting their maturation in the endochondral lineage.     

Neural and psychosocial contributions to sex differences in knee osteoarthritic pain

People with osteoarthritis (OA) can have significant pain that interferes with function and quality of life. Women with knee OA have greater pain and greater reductions in function and quality of life than men. In many cases, OA pain is directly related to sensitization and activation of nociceptors in the injured joint and correlates with the degree of joint effusion and synovial thickening. In some patients, however, the pain does not match the degree of injury and continues after removal of the nociceptors with a total joint replacement. Growth of new nociceptors, activation of nociceptors in the subchondral bone exposed after cartilage degradation, and nociceptors innervating synovium sensitized by inflammatory mediators could all augment the peripheral input to the central nervous system and result in pain. Enhanced central excitability and reduced central inhibition could lead to prolonged and enhanced pain that does not directly match the degree of injury. Psychosocial variables can influence pain and contribute to pain variability. This review explores the neural and psychosocial factors that contribute to knee OA pain with an emphasis on differences between the sexes and gaps in knowledge.     

Hepatitis C virus infection propagates through interactions between Syndecan‐1 and CD81 and impacts the hepatocyte glycocalyx

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan‐1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan‐1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan‐1 or Xylosyltransferase 2, a key enzyme of Syndecan‐1 biosynthesis. Simultaneous knockdown of Syndecan‐1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan‐1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan‐1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan‐1 and Xylosyltransferase 2 was altered within days post‐infection, and the remaining Syndecan‐1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.     

基于格网的伏牛山区土地利用变化对生态服务价值影响研究

以我国南水北调中线引水工程水源地所在的伏牛山区为例,测算、分析了1990—2015年土地利用变化和生态系统服务价值(ESV),在此基础上探究土地利用变化对ESV的影响。结果表明:(1)研究期间,伏牛山区建设用地大幅增加,耕地和草地大幅下降,耕地和建设用地相互均有较大规模转换。(2)林地是伏牛山区最主要的生态用地,调节服务和支持服务构成了伏牛山区ESV的主体;ESV高值区分布于伏牛山区中部高海拔林区,低值区分布于周围地势较平坦的耕地和建设用地区域;除林地、水域和未利用地ESV有小幅增加外,其他地类的ESV均快速减少,致使伏牛山区生态系统服务总价值减少。(3)ESV损失区域主要分布于各县的城镇边缘区以及栾川县的采矿区,可通过保护林地和水域、退耕还林、农居点整理、绿色矿山等土地调控政策提高生态服务价值。将土地利用变化图谱与ESV变化热点分析结合,可为从空间上研究土地利用变化对生态系统服务价值的影响提供新的研究框架,为伏牛山区土地利用精准调控提供政策依据。     

Carbonic anhydrase is required for statoconia homeostasis in organ cultures of statocysts from Aplysia californica

A novel organ culture system has been developed to study the regulation of statoconia production in the gravity sensing organ in Aplysia californica. Statocysts were cultured in Leibovitz (L15) medium supplemented with salts and Aplysia haemolymph for four days at 17°C. The viability of the system was evaluated by examining four parameters: statocyst morphology, the activity of the mechanosensory cilia in the statocyst, production of new statoconia during culture and change in statoconia volume after culture. There were no morphological differences in statocysts before and after culture when ciliary beating was maintained. There was a 29% increase in the number of statoconia after four days in culture. Mean statocyst, statolith and statoconia volumes were not affected by culture conditions. The presence of carbonic anhydrase in the statocysts was shown using immunohistochemistry. When statocysts were cultured in the presence of 4.0 × 10^–4 M acetazolamide to inhibit the enzyme activity, there was a decrease in statoconia production and statoconia volume, indicating a role for this enzyme in statoconia homeostasis, potentially via pH regulation. These studies are the first to report a novel system for the culture of statocysts and show that carbonic anhydrase is involved in the regulation of statoconia volume and production.     

Auditory neurones in the brain of the cricket Gryllus bimaculatus (De Geer): Ascending interneurones

Two types of auditory interneurone which ascend from the prothoracic ganglion to the brain in the cricket Gryllus bimaculatus (De Geer) are described. Intracellular recordings were made from the axons of the neurones in the brain under closed-field stimulus conditions and the recorded cells then stained with either cobalt or Lucifer Yellow. Both neurone types—the Plurisegmental ascending low frequency neurone 1 (PALF1), and the Plurisegmental ascending high frequency neurone 1 (PAHF1)—show response characteristics which would make them well suited to encoding the conspecific calling and courtship songs respectively. Further, the projection areas of both neurone types in the brain overlap those of previously identified intraganglionic interneurones, particularly in the anterior-ventral protocerebrum, and it is suggested that an auditory neuropile may exist in this region.