Asymmetrically decorated, doped porous graphene as an effective membrane for hydrogen isotope separation

We propose a new route to hydrogen isotope separation which exploits the quantum sieving effect in the context of transmission through asymmetrically decorated, doped porous graphenes. Selectivities of D2 over H2 as well as rate constants are calculated based on ab initio interaction potentials for passage through pure and nitrogen functionalized porous graphene. One-sided dressing of the membrane with metal provides the critical asymmetry needed for an energetically favorable pathway.

Desalination by a solar thermal membrane distillation process

A solar thermal membrane distillation pilot plant was operated for over 70 days in field conditions. The pilot plant incorporated a single spiral wound permeate gap membrane distillation style of module. All energy used to operate the unit was supplied by solar hot water collectors and photovoltaic panels. The process was able to produce a distillate stream of product water with a conductivity less than 10 µS/cm. Feed water concentration varied from 2,400 µS/cm to 106,000 µS/cm. The process is expected to find application in the production of drinking water for remote island and arid regions without the consumption of electrical energy.

Development of an ultra-thin fibroin membrane for RPE cell transplantation

Purpose: One of the challenges associated with cell-based therapies for repairing the retina is the development of suitable materials on which to grow and transplant retinal cells. Using the ARPE-19 cell line, we have previously demonstrated the feasibility of growing RPE-derived cells on membranes prepared from the silk protein fibroin. The present study was aimed at developing a porous, ultra-thin fibroin membrane that might better support development of apical-basal polarity in culture, and to extend this work to primary cultures of human RPE cells. Methods: Ultra-thin fibroin membranes were prepared using a highly polished casting table coated with Topas® (a cyclic olefin copolymer) and a 1:0.03 aqueous solution of fibroin and PEO (Mv 900 000 g/mol). Following drying, the membranes were water annealed to make them water-stable, washed in water to remove PEO, sterilised by treatment with 95% ethanol, and washed extensively in saline. Primary cultures containing human RPE cells were established from donor posterior eye cups and maintained in DMEM/F12 medium supplemented with 10% fetal bovine serum and antibiotics. First passage cultures were seeded onto fibroin membranes pre-coated with vitronectin and grown for 6 weeks in medium supplemented with 1% serum. Comparative cultures were established on porous 1.0 µm pore PET membrane (Millipore) and using ARPE-19 cells. Results: The fibroin membranes displayed an average thickness of 3 µm and contained numerous dimples/pore-like structures of up to 3-5 µm in diameter. The primary cultures predominantly contained pigmented epithelial cells, but mesenchymal cells (presumed fibroblasts) were also often present. Passaged cultures appeared to attach equally well to either fibroin or PET membranes. Over time cells on either material adopted a more cobblestoned morphology. Conclusions: Progress has been made towards developing a porous ultra-thin fibroin membrane that supports cultivation of RPE cells. Further studies are required to determine the degree of membrane permeability and RPE polarity.

An examination of MS candidate genes identified as differentially regulated in multiple sclerosis plaque tissue, using absolute and comparative real-time Q-PCR analysis

In our laboratory, we have developed methods in real-time detection and quantitative-polymerase chain reaction (Q-PCR) to analyse the relative levels of gene expression in post mortem brain tissues. We have then applied this method to examine differences in gene activity between normal white matter (NWM) and plaque tissue from multiple sclerosis (MS) patients. Genes were selected based on their association with pathology and through identification by previously conducted global gene expression analysis. Plaque tissue was obtained from secondary progressive (SP) patients displaying chronic active, as well as acute pathologies; while NWM from the same location was obtained from age- and sex-matched controls (normal patients). In this study, we used both SYBR Green I supplementation and commercially available mixes to assess both comparative and absolute levels of gene activity. The results of both methods compared favourably for four of the five genes examined (P < 0.05, Pearsons), while differences in gene expression between chronic active and acute pathologies were also identified. For example, a >50-fold increase in osteopontin (Spp1) and inositol 1-4-5 phosphate 3 kinase B (Itpkb) levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P < 0.05, unpaired t test). By contrast, there was no significant difference in the levels of the MS marker and calcium-dependent protease (Calpain, Capns1) in MS plaque tissue. In summary, Q-PCR analysis using SYBR Green I has allowed us to economically obtain what may be clinically significant information from small amounts of the CNS, providing an opportunity for further clinical investigations.

β-Actin—an unsuitable internal control for RT-PCR

Despite reports confirming cell-cycle dependent gene expression and a number of studies describing specific circumstances in which β-actin is also regulated, the mRNA for β-actin remains a widely used housekeeping gene internal control. Utilizing differential reverse transcriptase-polymerase chain reaction (RT-PCR), we report here the dose-dependent inhibition of β-actin by matrigel. This was detected by comparison to the very moderate inhibition of the target gene, membrane type-1 matrix metalloproteinase (MT1-MMP), with results independently confirmed by similar findings on MT1-MMP expression using competitive RT-PCR. Furthermore, RT-PCR of the housekeeping gene 18 Svedberg Units (S) rRNA demonstrated excellent consistency, reproducibility and non-regulation by a matrigel treatment. We conclude that β-actin is highly regulated by matrigel and therefore unsuitable as an internal control in this treatment. Hence, these findings suggest that researchers have a responsibility to ensure that the housekeeping gene of choice is not regulated in their specific application, as such regulation may dramatically affect the accuracy of their results. This study reinforces the necessity for minimally regulated housekeeping genes such as 18S rRNA, and the superiority of competitive templates as internal controls for quantitative applications of RT-PCR.

The role of immunoglobulins and their transporters in urogenital Chlamydial infections

Chlamydia trachomatis infections of the male and female reproductive tracts are the world's leading sexually transmitted bacterial disease, and can lead to damaging pathology, scarring and infertility. The resolution of chlamydial infection requires the development of adaptive immune responses to infection, and includes cell-mediated and humoral immunity. Whilst cluster of differentiation (CD)4+ T cells are known to be essential in clearance of infection [1], they are also associated with immune cell infiltration, autoimmunity and infertility in the testes [2-3]. Conversely, antibodies are less associated with inflammation, are readily transported into the reproductive tracts, and can offer lumenal neutralization of chlamydiae prior to infection. Antibodies, or immunoglobulins (Ig), play a supportive role in the resolution of chlamydial infections, and this thesis sought to define the function of IgA and IgG, against a variety of chlamydial antigens expressed during the intracellular and extracellular stages of the chlamydial developmental cycle. Transport of IgA and IgG into the mucosal lumen is facilitated by receptor-mediated transcytosis yet the expression profile (under normal conditions and during urogenital chlamydial infection) of the polymeric immunoglobulin receptor (pIgR) and the neonatal Fc receptor (FcRn) remains unknown. The expression profile of pIgR and FcRn in the murine male reproductive tract was found to be polarized to the lower and upper reproductive tract tissues respectively. This demonstrates that the two receptors have a tissue tropism, which must be considered when targeting pathogens that colonize different sites. In contrast, the expression of pIgR and FcRn in the female mouse was found to be distributed in both the upper and lower reproductive tracts. When urogenitally infected with Chlamydia muridarum, both male and female reproductive tracts up-regulated expression of pIgR and down-regulated expression of FcRn. Unsurprisingly, the up-regulation of pIgR increased the concentration of IgA in the lumen. However, down-regulation of FcRn, prevented IgG uptake and led to an increase or pooling of IgG in lumenal secretions. As previous studies have identified the importance of pIgR-mediated delivery of IgA, as well as the potential of IgA to bind and neutralize intracellular pathogens, IgA against a variety of chlamydial antigens was investigated. The protection afforded by IgA against the extracellular antigen major outer membrane protein (MOMP), was found to be dependent on pIgR expression in vitro and in vivo. It was also found that in the absence of pIgR, no protection was afforded to mice previously immunized with MOMP. The protection afforded from polyclonal IgA against the intracellular chlamydial antigens; inclusion membrane protein A (IncA), inclusion membrane proteins (IncMem) and secreted chlamydial protease-like activity factor (CPAF) were produced and investigated in vitro. Antigen-specific intracellular IgA was found to bind to the respective antigen within the infected cell, but did not significantly reduce inclusion formation (p > 0.05). This suggests that whilst IgA specific for the selected antigens was transported by pIgR to the chlamydial inclusion, it was unable to prevent growth. Similarly, immunization of male mice with intracellular chlamydial antigens (IncA or IncMem), followed by depletion CD4+ T cells, and subsequent urogenital C. muridarum challenge, provided minimal pIgR-mediated protection. Wild type male mice immunized with IncA showed a 57 % reduction (p < 0.05), and mice deficient in pIgR showed a 35 % reduction (p < 0.05) in reproductive tract chlamydial burden compared to control antigen, and in the absence of CD4+ T cells. This suggests that pIgR and secretory IgA (SIgA) were playing a protective role (21 % pIgR-mediated) in unison with another antigen-specific immune mechanism (36 %). Interestingly, IgA generated during a primary respiratory C. muridarum infection did not provide a significant amount of protection to secondary urogenital C. muridarum challenge. Together, these data suggest that IgA specific for an extracellular antigen (MOMP) can play a strong protective role in chlamydial infections, and that IgA targeting intracellular antigens is also effective but dependent on pIgR expression in tissues. However, whilst not investigated here, IgA targeting and blocking other intracellular chlamydial antigens, that are more essential for replication or type III secretion, may be more efficacious in subunit vaccines. Recently, studies have demonstrated that IgG can neutralize influenza virus by trafficking IgG-bound virus to lysosomes [4]. We sought to determine if this process could also traffic chlamydial antigens for degradation by lysosomes, despite Chlamydia spp. actively inhibiting fusion with the host endocytic pathway. As observed in pIgR-mediated delivery of anti-IncA IgA, FcRn similarly transported IgG specific for IncA which bound the inclusion membrane. Interestingly, FcRn-mediated delivery of anti-IncA IgG significantly decreased inclusion formation by 36 % (p < 0.01), and induced aberrant inclusion morphology. This suggests that unlike IgA, IgG can facilitate additional host cellular responses which affect the intracellular niche of chlamydial growth. Fluorescence microscopy revealed that IgG also bound the inclusion, but unlike influenza studies, did not induce the recruitment of lysosomes. Notably, anti-IncA IgG recruited sequestosomes to the inclusion membrane, markers of the ubiquitin/proteasome pathway and major histocompatibility complex (MHC) class I loading. To determine if the protection against C. muridarum infection afforded by IncA IgG in vitro translated in vivo, wild type mice and mice deficient in functional FcRn and MHC-I, were immunized, depleted of CD4+, and urogenitally infected with C. muridarum. Unlike in pIgR-deficient mice, the protection afforded from IncA immunization was completely abrogated in mice lacking functional FcRn and MHC-I/CD8+. Thus, both anti-IncA IgA and IgG can bind the inclusion in a pIgR and FcRn-mediated manner, respectively. However, only IgG mediates a higher reduction in chlamydial infection in vitro and in vivo suggesting more than steric blocking of IncA had occurred. Unlike anti-MOMP IgA, which reduced chlamydial infection of epithelial cells and male mouse tissues, IgG was found to enhance infectivity in vitro, and in vivo. Opsonization of EBs with MOMP-IgG enhanced inclusion formation of epithelial cells in a MOMP-IgG dose-dependent and FcRn-dependent manner. When MOMP-IgG opsonized EBs were inoculated into the vagina of female mice, a small but non-significant (p > 0.05) enhancement of cervicovaginal C. muridarum shedding was observed three days post infection in mice with functional FcRn. Interestingly, infection with opsonized EBs reduced the intensity of the peak of infection (day six) but protracted the duration of infection by 60 % in wild type mice only. Infection with EBs opsonized in IgG also significantly increased (p < 0.05) hydrosalpinx formation in the oviducts and induced lymphocyte infiltration uterine horns. As MOMP is an immunodominant antigen, and is widely used in vaccines, the ability of IgG specific to extracellular chlamydial antigens to enhance infection and induce pathology needs to be considered. Together, these data suggest that immunoglobulins play a dichotomous role in chlamydial infections, and are dependent on antigen specificity, FcRn and pIgR expression. FcRn was found to be highly expressed in upper male reproductive tract, whilst pIgR was dominantly expressed in the lower reproductive tract. Conversely, female mice expressed FcRn and pIgR in both the lower and upper reproductive tracts. In response to a normal chlamydial infection, pIgR is up-regulated increasing secretory IgA release, but FcRn is down-regulated preventing IgG uptake. Similarly to other studies [5-6], we demonstrate that IgA and IgG generated during primary chlamydial infections plays a minor role in recall immunity, and that antigen-specific subunit vaccines can offer more protection. We also show that both IgA and IgG can be used to target intracellular chlamydial antigens, but that IgG is more effective. Finally, IgA against the extracellular antigen MOMP can afford protection, whist IgG plays a deleterious role by increasing infectivity and inducing damaging immunopathology. Further investigations with additional antigens or combination subunit vaccines will enhance our understanding the protection afforded by antibodies against intracellular and extracellular pathogenic antigens, and help improve the development of an efficacious chlamydial vaccine.

Localization of Mineralocorticoid Receptors at Mammalian Synapses

In the brain, membrane associated nongenomic steroid receptors can induce fast-acting responses to ion conductance and second messenger systems of neurons. Emerging data suggest that membrane associated glucocorticoid and mineralocorticoid receptors may directly regulate synaptic excitability during times of stress when adrenal hormones are elevated. As the key neuron signaling interface, the synapse is involved in learning and memory, including traumatic memories during times of stress. The lateral amygdala is a key site for synaptic plasticity underlying conditioned fear, which can both trigger and be coincident with the stress response. A large body of electrophysiological data shows rapid regulation of neuronal excitability by steroid hormone receptors. Despite the importance of these receptors, to date, only the glucocorticoid receptor has been anatomically localized to the membrane. We investigated the subcellular sites of mineralocorticoid receptors in the lateral amygdala of the Sprague-Dawley rat. Immunoblot analysis revealed the presence of mineralocorticoid receptors in the amygdala. Using electron microscopy, we found mineralocorticoid receptors expressed at both nuclear including: glutamatergic and GABAergic neurons and extra nuclear sites including: presynaptic terminals, neuronal dendrites, and dendritic spines. Importantly we also observed mineralocorticoid receptors at postsynaptic membrane densities of excitatory synapses. These data provide direct anatomical evidence supporting the concept that, at some synapses, synaptic transmission is regulated by mineralocorticoid receptors. Thus part of the stress signaling response in the brain is a direct modulation of the synapse itself by adrenal steroids.

IL-20 is epigenetically regulated in NSCLC and down regulates the expression of VEGF

Background IL-20 is a pleiotrophic member of the IL-10 family and plays a role in skin biology and the development of haematopoietic cells. Recently, IL-20 has been demonstrated to have potential anti-angiogenic effects in non-small cell lung cancer (NSCLC) by down regulating COX-2. Methods The expression of IL-20 and its cognate receptors (IL-20RA/B and IL-22R1) was examined in a series of resected fresh frozen NSCLC tumours. Additionally, the expression and epigenetic regulation of this family was examined in normal bronchial epithelial and NSCLC cell lines. Furthermore, the effect of IL-20 on VEGF family members was examined. Results The expression of IL-20 and its receptors are frequently dysregulated in NSCLC. IL-20RB mRNA was significantly elevated in NSCLC tumours (p < 0.01). Protein levels of the receptors, IL-20RB and IL-22R1, were significantly increased (p < 0.01) in the tumours of NSCLC patients. IL-20 and its receptors were found to be epigenetically regulated through histone post-translational modifications and DNA CpG residue methylation. In addition, treatment with recombinant IL-20 resulted in decreased expression of the VEGF family members at the mRNA level. Conclusions This family of genes are dysregulated in NSCLC and are subject to epigenetic regulation. Whilst the anti-angiogenic properties of IL-20 require further clarification, targeting this family via epigenetic means may be a viable therapeutic option in lung cancer treatment. © 2011 Elsevier Ltd. All rights reserved.

IL-23 is pro-proliferative, epigenetically regulated and modulated by chemotherapy in non-small cell lung cancer

Background IL-23 is a member of the IL-6 super-family and plays key roles in cancer. Very little is currently known about the role of IL-23 in non-small cell lung cancer (NSCLC). Methods RT-PCR and chromatin immunopreciptiation (ChIP) were used to examine the levels, epigenetic regulation and effects of various drugs (DNA methyltransferase inhibitors, Histone Deacetylase inhibitors and Gemcitabine) on IL-23 expression in NSCLC cells and macrophages. The effects of recombinant IL-23 protein on cellular proliferation were examined by MTT assay. Statistical analysis consisted of Student's t-test or one way analysis of variance (ANOVA) where groups in the experiment were three or more. Results In a cohort of primary non-small cell lung cancer (NSCLC) tumours, IL-23A expression was significantly elevated in patient tumour samples (p<0.05). IL-23A expression is epigenetically regulated through histone post-translational modifications and DNA CpG methylation. Gemcitabine, a chemotherapy drug indicated for first-line treatment of NSCLC also induced IL-23A expression. Recombinant IL-23 significantly increased cellular proliferation in NSCLC cell lines. Conclusions These results may therefore have important implications for treating NSCLC patients with either epigenetic targeted therapies or Gemcitabine. © 2012 Elsevier Ireland Ltd.

Self-Regulated Learning : key strategies and their sources in a sample of adolescent males

This study identified the key self-regulated learning (SRL) strategies and their sources for nine school-aged adolescent males aged 15 to 17 years. The Self-Regulated Learning Interview Schedule (SRLIS) was used along with semi-structured interviews with the participants and their parents to elicit information on SRL strategies and contexts for the formation of self-regulatory habits. Early habit-forming experiences of the family home in relation to homework and study routines were found to form an important base for effective SRL. Teachers were identified as the most common source of SRL strategies with important formative experiences occurring during the first two years of high school.

The molecular basis for selective inhibition of unconventional mRNA splicing by an IRE1-binding small molecule

IRE1 couples endoplasmic reticulum unfolded protein load to RNA cleavage events that culminate in the sequence-specific splicing of the Xbp1 mRNA and in the regulated degradation of diverse membrane-bound mRNAs. We report on the identification of a small molecule inhibitor that attains its selectivity by forming an unusually stable Schiff base with lysine 907 in the IRE1 endonuclease domain, explained by solvent inaccessibility of the imine bond in the enzyme-inhibitor complex. The inhibitor (abbreviated 4μ8C) blocks substrate access to the active site of IRE1 and selectively inactivates both Xbp1 splicing and IRE1-mediated mRNA degradation. Surprisingly, inhibition of IRE1 endonuclease activity does not sensitize cells to the consequences of acute endoplasmic reticulum stress, but rather interferes with the expansion of secretory capacity. Thus, the chemical reactivity and sterics of a unique residue in the endonuclease active site of IRE1 can be exploited by selective inhibitors to interfere with protein secretion in pathological settings.

Quantitative proteomic approach to study subcellular localization of membrane proteins

As proteins within cells are spatially organized according to their role, knowledge about protein localization gives insight into protein function. Here, we describe the LOPIT technique (localization of organelle proteins by isotope tagging) developed for the simultaneous and confident determination of the steady-state distribution of hundreds of integral membrane proteins within organelles. The technique uses a partial membrane fractionation strategy in conjunction with quantitative proteomics. Localization of proteins is achieved by measuring their distribution pattern across the density gradient using amine-reactive isotope tagging and comparing these patterns with those of known organelle residents. LOPIT relies on the assumption that proteins belonging to the same organelle will co-fractionate. Multivariate statistical tools are then used to group proteins according to the similarities in their distributions, and hence localization without complete centrifugal separation is achieved. The protocol requires approximately 3 weeks to complete and can be applied in a high-throughput manner to material from many varied sources.

Sub-cellular localization of membrane proteins

In eukaryotes, numerous complex sub-cellular structures exist. The majority of these are delineated by membranes. Many proteins are trafficked to these in order to be able to carry out their correct physiological function. Assigning the sub-cellular location of a protein is of paramount importance to biologists in the elucidation of its role and in the refinement of knowledge of cellular processes by tracing certain activities to specific organelles. Membrane proteins are a key set of proteins as these form part of the boundary of the organelles and represent many important functions such as transporters, receptors, and trafficking. They are, however, some of the most challenging proteins to work with due to poor solubility, a wide concentration range within the cell and inaccessibility to many of the tools employed in proteomics studies. This review focuses on membrane proteins with particular emphasis on sub-cellular localization in terms of methodologies that can be used to determine the accurate location of membrane proteins to organelles. We also discuss what is known about the membrane protein cohorts of major organelles.

An extracellular signal-regulated kinase 2 survival pathway mediates resistance of human mesothelioma cells to asbestos-induced injury

We hypothesized that normal human mesothelial cells acquire resistance to asbestos-induced toxicity via induction of one or more epidermal growth factor receptor (EGFR) - linked survival pathways (phosphoinositol-3-kinase/AKT/ mammalian target of rapamycin and extracellular signal - regulated kinase [ERK] 1/2) during simian virus 40 (SV40) transformation and carcinogenesis. Both isolated HKNM-2 mesothelial cells and a telomerase-immortalized mesothelial line (LP9/TERT-1) were more sensitive to crocidolite asbestos toxicity than an SV40 Tag-immortalized mesothelial line (MET5A) and malignant mesothelioma cell lines (HMESO and PPM Mill). Whereas increases in phosphorylation of AKT (pAKT) were observed in MET5A cells in response to asbestos, LP9/TERT-1 cells exhibited dose-related decreases in pAKT levels. Pretreatment with an EGFR phosphorylation or mitogen-activated protein kinase kinase 1/2 inhibitor abrogated asbestos-induced phosphorylated ERK (pERK) 1/2 levels in both LP9/TERT-1 and MET5A cells as well as increases in pAKT levels in MET5A cells. Transient transfection of small interfering RNAs targeting ERK1, ERK2, or AKT revealed that ERK1/2 pathways were involved in cell death by asbestos in both cell lines. Asbestos-resistant HMESO or PPM Mill cells with high endogenous levels of ERKs or AKT did not show dose-responsive increases in pERK1/ERK1, pERK2/ERK2, or pAKT/AKT levels by asbestos. However, small hairpin ERK2 stable cell lines created from both malignant mesothelioma lines were more sensitive to asbestos toxicity than shERK1 and shControl lines, and exhibited unique, tumor-specific changes in endogenous cell death - related gene expression. Our results suggest that EGFR phosphorylation is causally linkedto pERK and pAKT activation by asbestos in normal and SV40 Tag - immortalized human mesothelial cells. They also indicate that ERK2 plays a role in modulating asbestos toxicity by regulating genes critical to cell injury and survival that are differentially expressed in human mesotheliomas.

Dying feet in ICU : why might extracorporeal membrane oxygenation machines cause necrotic feet?

Background Extracorporeal membrane oxygenation (ECMO) is used for severe lung and/or heart failure in intensive care units (ICU). The Prince Charles Hospital (TPCH) has one of the largest ECMO units in Australia. Its use rapidly increased during the H1N1 (“swine flu”) pandemic and an increase in pedal complications resulted. The relationship between ECMO and pedal complications has been described, particularly in children, though no strong data exists. This paper presents a case series of foot complications in patients having received ECMO treatment. Methods We present nine cases of severe foot complications resulting from patients receiving ECMO treatment at TPCH in 2009–2012. Results Case ages ranged from 16 - 58 years and three were male. Six cases had an unremarkable medical history prior to H1N1 or H1N2 infection, one had Cardiomyopathy, one had received a lung transplant, and one had multi-organ failure post-sepsis. Common medications prescribed included vasopressors, antibiotics, and sedatives. All cases showed signs of markedly impaired peripheral perfusion whilst on ECMO and seven developed increasing areas of foot necrosis. Outcomes include two bilateral below knee amputations, two multiple digital amputations, one Reflex Sympathetic Dystrophy Syndrome, three pressure injuries, and three deaths. Conclusion Necrosis of the feet appears to occur more readily in younger people requiring ECMO treatment than others in ICU. The authors are conducting further studies to investigate associations between particular infections, medical history, medications, or machine techniques and severe foot complications. Some of these early results will also be presented at this conference.