Blood–brain barrier structure and function and the challenges for CNS drug delivery

The neurons of the central nervous system (CNS) require precise control of their bathing microenvironment for optimal function, and an important element in this control is the blood–brain barrier (BBB). The BBB is formed by the endothelial cells lining the brain microvessels, under the inductive influence of neighbouring cell types within the ‘neurovascular unit’ (NVU) including astrocytes and pericytes. The endothelium forms the major interface between the blood and the CNS, and by a combination of low passive permeability and presence of specific transport systems, enzymes and receptors regulates molecular and cellular traffic across the barrier layer. A number of methods and models are available for examining BBB permeation in vivo and in vitro, and can give valuable information on the mechanisms by which therapeutic agents and constructs permeate, ways to optimize permeation, and implications for drug discovery, delivery and toxicity. For treating lysosomal storage diseases (LSDs), models can be included that mimic aspects of the disease, including genetically-modified animals, and in vitro models can be used to examine the effects of cells of the NVU on the BBB under pathological conditions. For testing CNS drug delivery, several in vitro models now provide reliable prediction of penetration of drugs including large molecules and artificial constructs with promising potential in treating LSDs. For many of these diseases it is still not clear how best to deliver appropriate drugs to the CNS, and a concerted approach using a variety of models and methods can give critical insights and indicate practical solutions.     

Overexpression of potassium channel ether à go-go in human osteosarcoma

Human ether à go-go (hEAG) potassium channels are primarily expressed in brain but also frequently overexpressed in solid tumors, which could indicate their potential value for cancer diagnosis and therapy. hEAG1, one member of the hEAG subfamily, has been shown to play a role in neoplastic process. Here we report the expression of hEAG1 in human osteosarcoma detected by a new polyclonal antibody. The full-length hEAG1 cDNA was cloned from human osteosarcoma cell line MG63 by RT-PCR and expressed in Escherichia coli as His tagged protein. The 6His-hEAG1F protein was purified by nickel agarose and used as the antigen to immunize rabbits following standard protocols. The obtained antiserum could detect hEAG1 exogenously expressed in HEK 293 cells. Furthermore, the polyclonal antibody was used to evaluate hEAG1 expression in 42 human osteosarcoma specimens and 19 osteochondromas specimens by immunohistochemistry. hEAG1 was expressed in 71.4% (30/42) osteosarcoma, and 15.8% (3/19) osteochondromas. Moreover, statistical analysis revealed that hEAG1 expression was not dependent on age, sex, site, histology, grade and type in the osteosarcoma specimens. Our data provide evidence that hEAG1 is overexpressed in human osteosarcoma and the hEAG1 polyclonal antibody offers a good tool for further characterization of the oncogenic function of hEAG1 in osteosarcoma.     

Is the central channel of nuclear pore complex really the exclusive gateway for macromolecular transport?

Examination of many nuclear pore complexes revealed in some of them very thin filaments presumably RNA with accompanying proteins, directing from inner nucleus to the edge of the complex. On one representative micrograph it is shown that this strand continues in the same direction through central part of the complex most probably through the peripheral channel. Next route of the strand is through the tunnel of a hollow rod - subunit of the cytoplasmic ring.     

Age-related changes in liver structure and function: Implications for disease ?

The geriatric populations of many countries are growing rapidly and they present major problems to healthcare infrastructures from both medical and economic perspectives. The elderly are predisposed to a variety of diseases, which contribute to a marked increase in morbidity in this subpopulation. The incidence of liver disease increases in the elderly, but the cellular and subcellular perturbations that underlie this suspected predisposition to pathology remain unresolved. Several age-related changes have been documented, including (a) a decline in liver volume, (b) an increase in the hepatic dense body compartment (lipofuscin), (c) moderate declines in the Phase I metabolism of certain drugs, (d) shifts in the expression of a variety of proteins and (e) diminished hepatobiliary functions. Other more subtle changes (e.g., muted responses to oxidative stress, reduced expression of growth regulatory genes, diminished rates of DNA repair, telomere shortening) may contribute to reduced hepatic regenerative capacity, shorter post-liver transplant survival and increased susceptibility to certain liver diseases in the elderly.     

Brain–machine interface for eye movements

A number of studies in tetraplegic humans and healthy nonhuman primates (NHPs) have shown that neuronal activity from reach-related cortical areas can be used to predict reach intentions using brain–machine interfaces (BMIs) and therefore assist tetraplegic patients by controlling external devices (e.g., robotic limbs and computer cursors). However, to our knowledge, there have been no studies that have applied BMIs to eye movement areas to decode intended eye movements. In this study, we recorded the activity from populations of neurons from the lateral intraparietal area (LIP), a cortical node in the NHP saccade system. Eye movement plans were predicted in real time using Bayesian inference from small ensembles of LIP neurons without the animal making an eye movement. Learning, defined as an increase in the prediction accuracy, occurred at the level of neuronal ensembles, particularly for difficult predictions. Population learning had two components: an update of the parameters of the BMI based on its history and a change in the responses of individual neurons. These results provide strong evidence that the responses of neuronal ensembles can be shaped with respect to a cost function, here the prediction accuracy of the BMI. Furthermore, eye movement plans could be decoded without the animals emitting any actual eye movements and could be used to control the position of a cursor on a computer screen. These findings show that BMIs for eye movements are promising aids for assisting paralyzed patients.Brain–machine interfaces (BMIs) have been successfully used to predict reaches and arm movements (1–7). However, little effort has been concentrated on building a BMI based on eye movements. This gap is surprising because the motor and neuronal mechanisms of eye movements are very well understood and arguably simpler than those of arm movements. Specifically, eye movements are rapid and ballistic. The lateral intraparietal cortex (LIP) is ideally suited to be the site for a BMI based on eye movements (8). LIP neurons are known to encode eye movement plans, among other signals such as eye position (9–16). We recently showed that eye movement plans can be accurately predicted from the responses of populations of LIP neurons using Bayesian inference (16). The aim of the present study was twofold. First, a BMI was used with small neuronal ensembles of LIP neurons to predict, in real time, eye movement plans without the animals actually making eye movements. Second, the BMI application induced learning-related changes in the saccade system. Learning can produce changes in reach areas, but how learning-related changes occur at the level of LIP neuronal ensembles is still unclear (17, 18).Here, we show that the intended eye movement activity can be used to accurately position a cursor on a computer screen. These results suggest that an eye movement BMI can be used as a prosthetic to assist locked-in patients who cannot produce eye movements. Moreover, such an eye movement BMI can also be used to assist tetraplegic persons to decode intended limb movements by providing an extra channel of target position information (19). Learning, defined as an increase in the prediction accuracy, occurred at the level of neuronal ensembles, particularly for difficult predictions. The population learning had two components: an update of the parameters of the BMI based on its history and a change in the responses of individual neurons. These results provide strong evidence that the responses of neuronal ensembles can be shaped with respect to a cost function, which here is the prediction accuracy of the BMI. Such learning adds additional support for the utility of an eye movement BMI based on LIP activity.     

Cog5–Cog7 crystal structure reveals interactions essential for the function of a multisubunit tethering complex

The conserved oligomeric Golgi (COG) complex is required, along with SNARE and Sec1/Munc18 (SM) proteins, for vesicle docking and fusion at the Golgi. COG, like other multisubunit tethering complexes (MTCs), is thought to function as a scaffold and/or chaperone to direct the assembly of productive SNARE complexes at the sites of membrane fusion. Reflecting this essential role, mutations in the COG complex can cause congenital disorders of glycosylation. A deeper understanding of COG function and dysfunction will likely depend on elucidating its molecular structure. Despite some progress toward this goal, including EM studies of COG lobe A (subunits 1–4) and higher-resolution structures of portions of Cog2 and Cog4, the structures of COG’s eight subunits and the principles governing their assembly are mostly unknown. Here, we report the crystal structure of a complex between two lobe B subunits, Cog5 and Cog7. The structure reveals that Cog5 is a member of the complexes associated with tethering containing helical rods (CATCHR) fold family, with homology to subunits of other MTCs including the Dsl1, exocyst, and Golgi-associated retrograde protein (GARP) complexes. The Cog5–Cog7 interaction is analyzed in relation to the Dsl1 complex, the only other CATCHR-family MTC for which subunit interactions have been characterized in detail. Biochemical and functional studies validate the physiological relevance of the observed Cog5–Cog7 interface, indicate that it is conserved from yeast to humans, and demonstrate that its disruption in human cells causes defects in trafficking and glycosylation.In eukaryotes, the transport of proteins and lipids among intracellular compartments is mediated by vesicular and tubular carriers under the direction of an elaborate protein machinery (1). Among the most complex and least well-characterized components of this machinery are the multisubunit tethering complexes (MTCs) (2). MTCs are thought to mediate the initial attachment (or tethering) between a trafficking vesicle and its target membrane through a constellation of interactions (3, 4). These may include binding of the MTC to activated Rab GTPases, coiled-coil proteins such as Golgins, vesicle coat proteins, SNAREs, Sec1/Munc18 (SM) proteins, and/or membrane lipids. Elucidating the 3D structures of MTCs represents an important step toward a better understanding of their molecular functions.Four of the known MTCs—termed complexes associated with tethering containing helical rods (CATCHR) or quatrefoil complexes (2, 5)—contain subunits whose shared 3D structure implies a single evolutionary progenitor (6–16). These CATCHR-family MTCs include the Dsl1, Golgi-associated retrograde protein (GARP), exocyst, and conserved oligomeric Golgi (COG) complexes, and they contain three, four, eight, and eight subunits, respectively. Although X-ray or NMR structures have been reported for 14 of these 23 subunits, only one of the structures contains the full-length polypeptide (14). Perhaps more critically, only two subunit interactions—both within the three-subunit Dsl1 complex—have been structurally characterized to date (11, 14). Defining the quaternary structure of the other CATCHR-family MTCs remains a major challenge.The COG complex is an MTC that is essential for vesicle transport within the Golgi apparatus and from endosomal compartments to the Golgi (3). Defects in individual COG subunits can lead to the aberrant distribution of glycosylation enzymes within the Golgi and thereby to severe genetic diseases known as congenital disorders of glycosylation (CDGs) (17, 18). The first CDG to be attributed to a COG complex defect was traced to a mutation in the COG7 gene, with infants homozygous for the mutation dying a few weeks after birth (19). Subsequent studies revealed that mutations in most other COG subunits can also give rise to congenital glycosylation disorders (17).Architecturally, the eight subunits that make up the COG complex can be divided into two subassemblies, lobe A (Cog1, Cog2, Cog3, and Cog4) and lobe B (Cog5, Cog6, Cog7, and Cog8) (20). Single-particle EM of lobe A revealed Y-shaped objects with three long, spindly legs (21). (Thus, the term “lobe”—defined as a roundish and flattish part of something—is a misnomer, at least with respect to Cog1–4.) Partial structures of Cog2 and Cog4 have been reported (6, 12), but the structure of the remainder of the complex, and the nature of the interactions among its subunits, are unknown.To initiate high-resolution studies of subunit interactions within the COG complex, we began with Cog5 and Cog7. This choice was guided by the observation that recombinant Saccharomyces cerevisiae Cog5 and Cog7 form an especially stable binary complex (22). Similarly, comprehensive in vitro cotranslation/coimmunoprecipitation experiments revealed that, among the eight human COG subunits, COG5 and COG7 were unusual in their ability to form a stable binary complex (23). We report here the X-ray structure of a complex containing Cog5 and Cog7 from the yeast Kluyveromyces lactis. Our structure reveals Cog5 as an example of the CATCHR fold and elucidates the nature of its interaction with Cog7. We find that the Cog5–Cog7 interface is conserved from yeast to humans and that its disruption causes glycosylation defects in human tissue culture cells and, probably, in a previously identified COG5-CDG patient (24, 25).     

Thyroid structure and function in bovine β-mannosidosis

Summary In bovine -mannosidosis, the thyroid in the affected newborn shows marked cytoplasmic vacuolation. There is an associated reduction in the serum concentrations of thyroxine and tri-iodothyronine.     

Heritability of insulin sensitivity and lipid profile depend on BMI: evidence for gene–obesity interaction

Aims/hypothesis  

Evidence from candidate gene studies suggests that obesity may modify genetic susceptibility to type 2 diabetes and dyslipidaemia. On an aggregate level, gene–obesity interactions are expected to result in different heritability estimates at different obesity levels. However, this hypothesis has never been tested.     

Structure–function analysis of myomaker domains required for myoblast fusion

During skeletal muscle development, myoblasts fuse to form multinucleated myofibers. Myomaker [Transmembrane protein 8c (TMEM8c)] is a muscle-specific protein that is essential for myoblast fusion and sufficient to promote fusion of fibroblasts with muscle cells; however, the structure and biochemical properties of this membrane protein have not been explored. Here, we used CRISPR/Cas9 mutagenesis to disrupt myomaker expression in the C2C12 muscle cell line, which resulted in complete blockade to fusion. To define the functional domains of myomaker required to direct fusion, we established a heterologous cell–cell fusion system, in which fibroblasts expressing mutant versions of myomaker were mixed with WT myoblasts. Our data indicate that the majority of myomaker is embedded in the plasma membrane with seven membrane-spanning regions and a required intracellular C-terminal tail. We show that myomaker function is conserved in other mammalian orthologs; however, related family members (TMEM8a and TMEM8b) do not exhibit fusogenic activity. These findings represent an important step toward deciphering the cellular components and mechanisms that control myoblast fusion and muscle formation.Plasma membrane fusion is a fundamental cellular process required for the conception, development, and physiology of multicellular organisms. Membrane fusion occurs during viral infection of a host cell, between intracellular membranes, and between two plasma membranes to form syncytial tissues (1). Cell–cell fusion is critical for a wide array of cellular processes, including sperm–egg fertilization, macrophage function, bone and placental development, and skeletal muscle formation. This form of fusion must be precisely controlled to prevent inappropriate cellular mixing.The fusion of myoblasts requires cell recognition, migration, adhesion, signaling, and finally, membrane coalescence (2). Much of our knowledge about myoblast fusion has originated from studies performed in Drosophila. In this system, intracellular signaling results in cytoskeletal alterations and actin polymerization, which drive the formation of cellular projections that invade neighboring cells to cause fusion (3). Recent evidence also indicates a critical function for branched actin polymerization during Drosophila indirect flight muscle fusion (4). The essential role of the cytoskeleton in fusion is conserved in mammals, in which the actin regulators Rac1, cdc42, and N-WASp are required for muscle development (5–8). In addition to actin dynamics, numerous proteins that regulate diverse cellular processes have been associated with myoblast fusion (9–13).We recently discovered a muscle-specific membrane protein named myomaker [annotated as Transmembrane protein 8c (TMEM8c)] that is absolutely required for skeletal myocyte fusion in the mouse (14, 15). To begin to decipher the mechanisms whereby myomaker controls cell–cell fusion, we designed a heterologous cell–cell fusion assay to monitor the ability of a series of myomaker mutants to direct the fusion of labeled fibroblasts with myoblasts in vitro. Here, we present a model of myomaker topology within the membrane, in which seven amphipathic α-helical transmembrane (TM) domains and an essential C-terminal cytoplasmic domain drive the fusion process. These findings begin to enlighten the mechanistic basis of myoblast fusion.     

Protective effect of morin on lipid peroxidation and lipid profile in ammonium chloride–induced hyperammonemic rats

ObjectiveTo evaluated the protective effects of morin (3, 5, 7, 2′, 4′-pentahydroxyflavone) on lipid peroxidation and lipid levels during ammonium chloride (AC) induced hyperammonemia in experimental rats.MethodsThirty two male albino Wistar rats, which are weighing between 180–200 g were used for the study. The hyperammonemia was induced by administration of 100 mg/kg body weight (i.p.) thrice in a week of AC for 8 weeks. Rats were treated with morin at dose (30 mg/kg body weight) via intragastric intubations together with AC. At the end of experimental duration, blood ammonia, plasma urea, lipid peroxidation indices [thiobarbituric acid reactive substances, hydroperoxides and lipid levels (cholesterol, triglycerides, free fatty acids and phospholipids)] in serum and tissues were analysed to evaluate the antiperoxidative and antilipidemic effects of morin.ResultsAmmonia, urea, lipid peroxidative indices and lipid levels were significantly increased in AC administered group. Morin treatment resulted in positive modulation of ammonia, urea, lipid peroxidative indices and lipid levels. Morin administration to normal rats did not exhibit any significant changes in any of the parameters studied.ConclusionsIt can be concluded that the beneficial effect of morin on ammonia, urea, lipid peroxidative indices and lipid levels could be due to its antioxidant property.     

Crystal structure of the octameric pore of staphylococcal γ-hemolysin reveals the β-barrel pore formation mechanism by two components

Staphylococcal γ-hemolysin is a bicomponent pore-forming toxin composed of LukF and Hlg2. These proteins are expressed as water-soluble monomers and then assemble into the oligomeric pore form on the target cell. Here, we report the crystal structure of the octameric pore form of γ-hemolysin at 2.5 Å resolution, which is the first high-resolution structure of a β-barrel transmembrane protein composed of two proteins reported to date. The octameric assembly consists of four molecules of LukF and Hlg2 located alternately in a circular pattern, which explains the biochemical data accumulated over the past two decades. The structure, in combination with the monomeric forms, demonstrates the elaborate molecular machinery involved in pore formation by two different molecules, in which interprotomer electrostatic interactions using loops connecting β2 and β3 (loop A: Asp43-Lys48 of LukF and Lys37-Lys43 of Hlg2) play pivotal roles as the structural determinants for assembly through unwinding of the N-terminal β-strands (amino-latch) of the adjacent protomer, releasing the transmembrane stem domain folded into a β-sheet in the monomer (prestem), and interaction with the adjacent protomer.Pathogenic bacteria secrete various virulence factors to attack host cells. The pore-forming toxins (PFTs) are among the most sophisticated virulence factors, and are expressed as water-soluble monomeric proteins that assemble on the membranes of the target cells to form bilayer-spanning pores (1). With the appearance of the pore on the membrane, the cells are killed through leakage. It is interesting to note that PFTs are expressed not only by bacteria but also by eukaryotes, such as the immune proteins perforin and complement C9, suggesting the universality of these molecules in a wide range of organisms (2). PFTs can be classified into two families according to the secondary structure of the transmembrane region in the pore structure; i.e., α-helical PFT (α-PFT) and β-barrel PFT (β-PFTs) (3, 4).Staphylococcus aureus, a ubiquitous and pernicious human pathogen, secretes several β-PFTs including αHL, γ-hemolysin (γHL), leukocidin (LUK), and Panton–Valentine leukocidin (PVL) (5). The αHL consists of a single polypeptide, whereas the others are bicomponent β-PFTs that require the synergistic association of a class F component and a class S component. The γHL, LUK, and PVL are composed of LukF and Hlg2, LukF and LukS, and LukF-PV and LukS-PV, as class F and S components, respectively. The components of bicomponent β-PFTs are similar to each other and to αHL in amino acid sequence: Within a class, S and F proteins are approximately 70% identical, whereas between classes the identity drops to approximately 30%. Class F proteins are more closely related to αHL (approximately 30%) than class S proteins (approximately 20%) (5, 6). Extensive experiments have been carried out for more than two decades, and have suggested that the pore formation mechanism of bicomponent toxins is as follows (7, 8). The soluble forms of F and S components bind sequentially to the target cells and form a heterodimer (9, 10). Each heterodimer assembles into an oligomer on the target cell to form a ring-shaped particle called a prepore, in which the β-barrel pore is not yet formed (11–14). After forming a stable prepore, the β-barrel pore is formed. Pore formation requires the binding of phosphatidylcholine (PC) head groups to a cleft in the LukF component surrounded by Trp177 and Arg198 (Trp176 and Arg197 of LukF-PV) (13, 15, 16). The crystal structures of the monomeric forms of bicomponent β-PFTs [i.e., LukF (15), LukF-PV (17), and LukS-PV (18)], have been determined. However, the structures of the pore forms have not been reported at atomic resolution, which has hindered detailed discussion of the complicated molecular mechanism of action of bicomponent pore-forming toxins. Although bicomponent PFTs are found in several species, such as the edible mushroom Pleurotus ostreatus (19), the structures of these pores have not been reported.One of the most important issues for staphylococcal bicomponent PFTs is the stoichiometry of the class F and S components. Electron microscopy and cross-linking experiments of purified γHL pores on human erythrocyte membranes demonstrated the existence of a heptamer with a 3∶4 or 4∶3 molar ratio of F to S components (20, 21), whereas biochemical analyses of pores of engineered covalent γHL heterodimers on erythrocytes and leukocytes suggested octameric stoichiometry (22). Several reports using LUK pores formed on rabbit erythrocytes also demonstrated the existence of an octamer consisting of 4-plus-4 subunits (23–25). However, hexamer was also proposed based on structure modeling using monomeric structures of PVL (17) and electron microscopy of LUK pores on leukocytes and erythrocytes (26). It is also important to determine the significance of using two components. Although a role was proposed for the F component in initiating the pore formation process (27), that of the S component remains unclear.In the present study, we determined the crystal structure of the pore form of bicomponent β-PFT, γHL. This is a unique report of the crystal structure of a heterocomponent β-barrel- type transmembrane protein. This is also a unique bicomponent β-PFT of which both monomer- and pore-form structures have been determined by X-ray crystallography, which allowed us to discuss the pore formation mechanism based on their real structures at atomic resolution. Based on the structural differences between pore and monomer forms in combination with biological data accumulated over the past two decades, we propose a mechanism of pore formation by β-PFTs along with the roles of each component. The electrochemical properties of the pore are also discussed from a structural viewpoint.     

Administration of insulin–like growth factor–1 (IGF–1) improves both structure and function of delta–sarcoglycan deficient cardiac muscle in the hamster

Dilated cardiomyopathies (DCM) are due to progressive dilatation of the cardiac cavities and thinning of the ventricular walls and lead unavoidably to heart failure. They represent a major cause for heart transplantation and, therefore, defining an efficient symptomatic treatment for DCM remains a challenge. We have taken advantage of the hamster strain CHF147 that displays progressive cardiomyopathy leading to heart failure to test whether stimulation of a hypertrophic pathway could delay the process of dilatation.Six month old CHF147 hamsters were treated with IGF–1 so that we could compare the efficacy of systemic administration of human recombinant IGF–1 protein (rh IGF–1) at low dose to that of direct myocardial injections of a plasmid DNA containing IGF–1 cDNA (pCMV–IGF1).IGF-1 treatment did not induce a significant variation of ventricle mass, but preserved left ventricular (LV) wall thickness and delayed dilatation of cardiac cavities when compared to non–treated hamsters. Together with this reduction of dilatation, we also noted a reduction in the amount of interstitial collagen. Furthermore, IGF–1 treatment induced beneficial effects on cardiac function since treated hamsters presented improved cardiac output and stroke volume, decreased end diastolic pressure when compared to nontreated hamsters and also showed a trend towards increased contractility (dP/dt_max).This study provides evidence that IGF–1 treatment induces beneficial structural and functional effects on DCM of CHF147 hamsters, hence making this molecule a promising candidate for future gene therapy of heart failure due to DCM.     

CRYPTOCHROME-mediated phototransduction by modulation of the potassium ion channel β-subunit redox sensor

Blue light activation of the photoreceptor CRYPTOCHROME (CRY) evokes rapid depolarization and increased action potential firing in a subset of circadian and arousal neurons in Drosophila melanogaster. Here we show that acute arousal behavioral responses to blue light significantly differ in mutants lacking CRY, as well as mutants with disrupted opsin-based phototransduction. Light-activated CRY couples to membrane depolarization via a well conserved redox sensor of the voltage-gated potassium (K⁺) channel β-subunit (Kvβ) Hyperkinetic (Hk). The neuronal light response is almost completely absent in hk^−/− mutants, but is functionally rescued by genetically targeted neuronal expression of WT Hk, but not by Hk point mutations that disable Hk redox sensor function. Multiple K⁺ channel α-subunits that coassemble with Hk, including Shaker, Ether-a-go-go, and Ether-a-go-go–related gene, are ion conducting channels for CRY/Hk-coupled light response. Light activation of CRY is transduced to membrane depolarization, increased firing rate, and acute behavioral responses by the Kvβ subunit redox sensor.CRYPTOCHROME (CRY) is a photoreceptor that mediates rapid membrane depolarization and increased spontaneous action potential firing rate in response to blue light in arousal and circadian neurons in Drosophila melanogaster (1, 2). CRY regulates circadian entrainment by targeting circadian clock proteins to proteasomal degradation in response to light (3–6). CRY is expressed in a small subset of central brain circadian, arousal, and photoreceptor neurons in D. melanogaster and other insects, including the large lateral ventral neuron (LNv; l-LNv) subset (1, 2, 7, 8). The l-LNvs are light-activated arousal neurons (1, 2, 9–11), whereas the small lateral ventral neurons (s-LNvs) are critical for circadian function (5, 12). Previous results suggest that light activated arousal is likely attenuated in cry-null mutants. In addition to modulating light-activated firing rate, membrane excitability in the LNv neurons helps maintain circadian rhythms (9, 13, 14), and LNv firing rate is circadian regulated (2, 16).Based on our previous work suggesting that l-LNv electrophysiological light response requires a flavin-specific redox reaction and modulation of membrane K⁺ channels, we investigated the molecular mechanism for CRY phototransduction to determine how light-activated CRY is coupled to rapid membrane electrical changes. Sequence and structural data suggest that the cytoplasmic Kvβs are redox sensors based on a highly conserved aldo-keto-reductase domain (AKR) (17–21). Although no functional role for redox sensing by Kvβ subunits has been established yet in vivo, studies with heterologously expressed WT and mutant Kvβ subunits show that they confer modulatory sensitivity to coexpressed K⁺ channels in response to oxidizing and reducing chemical agents (22–24). Mammals express six Kvβ genes, whereas Drosophila expresses a single Kvβ designated HYPERKINETIC (Hk) (18). We find that the light-activated redox reaction of the flavin adenine dinucleotide (FAD) chromophore in CRY has a distinct phototransduction mechanism that evokes membrane electrical responses via the Kvβ subunit Hk, which we show is a functional redox sensor in vivo.     

Product–process interface for manufacturing data management as a support for DFM and virtual manufacturing

In order to tackle a continuous improvement of virtual engineering, product modelling has to integrate more knowledge that refers to every decision taken during the product development process. Those decisions have to be related to the assessment of the whole product life cycle. This paper particularly addresses the domain of product’s industrialisation that aims at selecting the manufacturing processes. This selection must currently be done as soon as possible and has to be strongly linked with product definition and computer aided design (CAD) modelling. This work first presents some new results concerning a product–process interface to integrate manufacturing information in the product model and how it leads to the definition of the CAD model. Then this interface that also manages specific information coming from the manufacturing process (tolerances, stresses gradient...) is used to improve the whole manufacturing process plan simulation. This process plan has, indeed, to track every material transformation issued from each manufacturing operation.