Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

A novel regulatory role for tissue transglutaminase in epithelial-mesenchymal transition in cystic fibrosis

Cystic fibrosis (CF) is a genetic disorder caused by mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) for which there is no overall effective treatment. Recent work indicates tissue transglutaminase (TG2) plays a pivotal intracellular role in proteostasis in CF epithelia and that the pan TG inhibitor cysteamine improves CFTR stability. Here we show TG2 has another role in CF pathology linked with TGFβ1 activation and signalling, induction of epithelial-mesenchymal transition (EMT), CFTR stability and induction of matrix deposition. We show that increased TG2 expression in normal and CF bronchial epithelial cells increases TGFβ1 levels, promoting EMT progression, and impairs tight junctions as measured by Transepithelial Electric Resistance (TEER) which can be reversed by selective inhibition of TG2 with an observed increase in CFTR stability. Our data indicate that selective inhibition of TG2 provides a potential therapeutic avenue for reducing fibrosis and increasing CFTR stability in CF.

Blood-brain barrier in vitro model: A tissue engineering approach and validation

This dissertation evaluated the feasibility of using commercially available immortalized cell lines in building a tissue engineered in vitro blood-brain barrier (BBB) co-culture model for preliminary drug development studies. Mouse endothelial cell line and rat astrocyte cell lines purchased from American Type Culture Collections (ATCC) were the building blocks of the co-culture model. An astrocyte derived acellular extracellular matrix (aECM) was introduced in the co-culture model to provide a novel in vitro biomimetic basement membrane for the endothelial cells to form endothelial tight junctions. Trans-endothelial electrical resistance (TEER) and solute mass transport studies were engaged to quantitatively evaluate the tight junction formation on the in-vitro BBB models. Immuno-fluorescence microscopy and Western Blot analysis were used to qualitatively verify the in vitro expression of occludin, one of the earliest discovered tight junction proteins. Experimental data from a total of 12 experiments conclusively showed that the novel BBB in vitro co-culture model with the astrocyte derived aECM (CO+aECM) was promising in terms of establishing tight junction formation represented by TEER values, transport profiles and tight junction protein expression when compared with traditional co-culture (CO) model setups and endothelial cells cultured alone. Experimental data were also found to be comparable with several existing in vitro BBB models built from various methods. In vitro colorimetric sulforhodamine B (SRB) assay revealed that the co-cultured samples with aECM resulted in less cell loss on the basal sides of the insert membranes than that from traditional co-culture samples. The novel tissue engineering approach using immortalized cell lines with the addition of aECM was proven to be a relevant alternative to the traditional BBB in vitro modeling.

Papel biológico del diadenosin tetrafosfato en el ojo: efecto sobre la composición lacrimal e implicación en la función de barrera corneal

El Ap4A es una molécula con un amplio papel biológico en el ojo. Se ha descrito su implicación en procesos de secreción lagrimal, cicatrización epitelial, regulación de la presión intraocular, y presenta también un papel neuroprotector sobre los terminales simpáticos que inervan el cuerpo ciliar. El objetivo de este trabajo ha sido analizar la participación del Ap4A en otras posibles funciones a nivel ocular. En concreto se ha estudiado la capacidad de este dinucléotido para estimular la liberación de proteínas lagrimales de acción antibacteriana tales como la lisozima y la lactoferrina. Por otra parte se ha investigado su efecto como modulador de la función de barrera de la córnea a través de la regulación de los niveles de expresión de proteínas constituyentes de las tight junctions (TJ). Dicho efecto sobre la barrera puede tener una importante repercusión en la entrada de fármacos y en la consiguiente eficacia terapéutica de los mismos. Por último, se ha estudiado la posible implicación del dinucleótido en el proceso de edematización descrito en modelos animales glaucomatosos tales como el ratón DBA/2J. Con el objetivo de averiguar si la activación de Ap4A inducía un efecto sobre la producción de dos proteínas antimicrobianas relevantes de la lágrima (lisozima y lactoferrina) se realizaron ensayos en la lágrima de conejos albinos de Nueva Zelanda, mediante las técnicas de agar-agar y ELISA. Los resultados obtenidos demostraron que Ap4A produce un aumento en la concentración de lisozima y de lactoferrina del 93% y 24%, respectivamente, frente a valores basales, y este efecto está mediado por receptores de tipo P2...

Characterization of in vitro effects of patulin on intestinal epithelial and immune cells

The intestinal mucosa is the first biological barrier encountered by natural toxins, and could possibly be exposed to high amounts of dietary mycotoxins. Patulin (PAT), a mycotoxin produced by Penicillium spp. during fruit spoilage, is one of the best known enteropathogenic mycotoxins able to alter functions of the intestine (Maresca et al., 2008). This study evaluated the effects of PAT on barrier function of the gut mucosa utilizing the intestinal epithelial cell model Caco-2, and scrutinized immunomodulatory effects using human peripheral blood mononuclear cells (PBMC) and human blood monocyte-derived dendritic cells (moDCs) as test systems. PAT exposure reduced Caco-2 cell viability at concentrations above 12 mM. As expected, the integrity of a polarized Caco-2 monolayer was affected by PAT exposure, as demonstrated by a decrease in TER values, becoming more pronounced at 50 mM. No effects were detected on the expression levels of the tight junction proteins occludin, claudin-1 and claudin-3 at 50 mM. However, the expression of zonula occludens-1 (ZO-1) and myosin light chain 2 (MLC2) declined. Also, levels of phospho-MLC2 (p-MLC2) increased after 24 h of exposure to 50 mM of PAT. T cell proliferation was highly sensitive to PAT with major effects for concentrations above 10 nM of PAT. The same conditions did not affect the maturation of moDC. PAT causes a reduction in Caco-2 barrier function mainly by perturbation of ZO-1 levels and the phosphorylation of MLC. Low doses of PAT strongly inhibited T cell proliferation induced by a polyclonal activator, but had no effect on the maturation of moDC. These results provide new information that strengthens the concept that the epithelium and immune cells of the intestinal mucosa are important targets for the toxic effects of food contaminants like mycotoxins

Role of phospholipase A(2) and tyrosine kinase in Clostridium difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion

Clostridium difficile-associated disease causes diarrhea to fulminant colitis and death. We investigated the role of phospholipase A(2) (PLA(2)) inhibitors, aristolochic acid (AA), bromophenacyl bromide BPB and quinacrine (QUIN) on the C. difficile toxin A-induced disruption of epithelial integrity, histologic inflammatory damage and intestinal secretion. Toxin A caused severe hemorrhagic and inflammatory fluid secretion at 6-8 h in rabbit ileal segments, an effect that was significantly inhibited by QUIN (71%, P < 0.01), AA (87%, P < 0.0001) or by BPB (51%, P < 0.01). The secretory effect of toxin A was also inhibited in segments adjacent to those with AA (89%, P < 0.01). Furthermore, QUIN or AA substantially reduced the histologic damage seen after 6-8 h in rabbit ileal segments. The cyclooxygenase inhibitor, indomethacin, also significantly inhibited (96%; n = 6) the secretory effects of toxin A in ligated rabbit intestinal segments. The destruction by toxin A of F-actin at the light junctions of T-84 cell monolayers was not inhibited by AA or BPB. AA or QUIN had no effect on the T-84 cell tissue resistance reduction over 8-24 h after toxin A exposure. All the inhibitors were shown to be effective in the doses administered direct in ileal loops to inhibit PLA(2) activity. The data suggest that PLA(2) is involved in the major pathway of toxin A-induced histologic inflammatory damage and hemorrhagic fluid secretion. Cop. right (C) 2008 John Wiley & Sons, Ltd.

Molecular and immunological analysis of food allergens.

 The study demonstrated that peanut allergens were able to adhere and cross a model of the intestinal epithelium. Peanut contact causes a decrease in barrier integrity and mislocalisation of tight junctions. Both allergens are endocytosed by multiple endocytotic mechanisms, which are useful targets for the development of future therapeutics.

Resolving Atomic Connectivity in Graphene Nanostructure Junctions

We report on the structural characterization of junctions between atomically well-defined graphene nanoribbons (GNRs) by means of low-temperature, noncontact scanning probe microscopy. We show that the combination of simultaneously acquired frequency shift and tunneling current maps with tight binding (TB) simulations allows a comprehensive characterization of the atomic connectivity in the GNR junctions. The proposed approach can be generally applied to the investigation of graphene nanomaterials and their interconnections and is thus expected to become an important tool in the development of graphene-based circuitry.

The challenges of abundance: epithelial junctions and small GTPase signalling

Small GTPases of the Ras superfamily play critical roles in epithelial biogenesis. Many key morphogenetic functions occur when small GTPases act at epithelial junctions, where they mediate an increasingly complex interplay between cell-cell adhesion molecules and fundamental cellular processes, such as cytoskeletal activity, polarity and trafficking. Important recent advances in this field include the role of additional members of the Ras superfamily in cell-cell contact stability and the capacity for polarity determinants to regulate small GTPase signalling. Interestingly, small GTPases may participate in the cross-talk between different adhesive receptors: in tissues classical cadherins can selectively regulate other junctions through cell signalling rather than through a global influence on cell-cell cohesion.

Optimal traffic control at railway junctions

Conflict occurs when two or more trains approach the same junction within a specified time. Such conflicts result in delays. Current practices to assign the right of way at junctions achieve orderly and safe passage of the trains, but do not attempt to reduce the delays. A traffic controller developed in the paper assigns right of way to impose minimum total weighted delay on the trains. The traffic flow model and the optimisation technique used in this controller are described. Simulation studies of the performance of the controller are given.

The conflict resolution in connected railway junctions

Purpose of study: Traffic conflicts occur when trains on different routes approach a converging junction in a railway network at the same time. To prevent collisions, a right-of-way assignment is needed to control the order in which the trains should pass the junction. Such control action inevitably requires the braking and/or stopping of trains, which lengthens their travelling times and leads to delays. Train delays cause a loss of punctuality and hence directly affect the quality of service. It is therefore important to minimise the delays by devising a suitable right-of-way assignment. One of the major difficulties in attaining the optimal right-of-way assignment is that the number of feasible assignments increases dramatically with the number of trains. Connected-junctions further complicate the problem. Exhaustive search for the optimal solution is time-consuming and infeasible for area control (multi-junction). Even with the more intelligent deterministic optimisation method revealed in [1], the computation demand is still considerable, which hinders real-time control. In practice, as suggested in [2], the optimality may be traded off by shorter computation time, and heuristic searches provide alternatives for this optimisation problem.

An event-based traffic flow model for traffic control at railway junctions

This paper introduces an event-based traffic model for railway systems adopting fixed-block signalling schemes. In this model, the events of trains' arrival at and departure from signalling blocks constitute the states of the traffic flow. A state transition is equivalent to the progress of the trains by one signalling block and it is realised by referring to past and present states, as well as a number of pre-calculated look-up tables of run-times in the signalling block under various signalling conditions. Simulation results are compared with those from a time-based multi-train simulator to study the improvement of processing time and accuracy.

Train control to reduce delays upon service disturbances at railway junctions

Train delay is one of the most important indexes to evaluate the service quality of the railway. Because of the interactions of movement among trains, a delayed train may conflict with trains scheduled on other lines at junction area. Train that loses conflict may be forced to stop or slow down because of restrictive signals, which consequently leads to the loss of run-time and probably enlarges more delays. This paper proposes a time-saving train control method to recover delays as soon as possible. In the proposed method, golden section search is adopted to identify the optimal train speed at the expected time of restrictive signal aspect upgrades, which enables the train to depart from the conflicting area as soon as possible. A heuristic method is then developed to attain the advisory train speed profile assisting drivers in train control. Simulation study indicates that the proposed method enables the train to recover delays as soon as possible in case of disturbances at railway junctions, in comparison with the traditional maximum traction strategy and the green wave strategy.

Managing older workers during a period of tight labour supply

This article reports on a recent survey of employer attitudes and policies towards older workers in Australia at a time of sustained economic growth and ongoing concerns about labour shortages. Findings from a survey of 590 employers with more than 50 employees in the State of Queensland point to an unusually strong orientation towards the recruitment of older workers among respondents, although the retraining of older workers is not prioritised by the majority. The issue of workforce ageing is viewed as being of medium-term importance by the majority of respondents, although for a substantial number the issue is of immediate concern. Both sector and organisation size are predictive of the application of a broad range of policies targeting older workers, with public-sector and larger organisations more likely to be active. Concerns about workforce ageing and labour supply are predictive of employer behaviours regarding older workers, suggesting that sustained policy making may be emerging in response to population ageing over and above more immediate concerns about labour shortages and that this broad thrust of organisational policy making may be immune to the point in the economic cycle. This study found no evidence that the flexible firm will not countenance an ageing workforce.