A role for eisosomes in maintenance of plasma membrane phosphoinositide levels

The plasma membrane delineates the cell and mediates its communication and material exchange with the environment. Many processes of the plasma membrane occur through interactions of proteins with phosphatidylinositol(4,5)-bisphosphate (PI(4,5)P2), which is highly enriched in this membrane and is a key determinant of its identity. Eisosomes function in lateral organization of the plasma membrane, but the molecular function of their major protein subunits, the BAR domain–containing proteins Pil1 and Lsp1, is poorly understood. Here we show that eisosomes interact with the PI(4,5)P2 phosphatase Inp51/Sjl1, thereby recruiting it to the plasma membrane. Pil1 is essential for plasma membrane localization and function of Inp51 but not for the homologous phosphatidylinositol bisphosphate phosphatases Inp52/Sjl2 and Inp53/Sjl3. Consistent with this, absence of Pil1 increases total and available PI(4,5)P2 levels at the plasma membrane. On the basis of these findings, we propose a model in which the eisosomes function in maintaining PI(4,5)P2 levels by Inp51/Sjl1 recruitment

Regulation of Mammalian Autophagy by Class II and III PI 3-Kinases through PI3P Synthesis

Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis

Microencapsulation of TOMAC by suspension polymerisation: Process optimisation

The optimisation process for the synthesis of microcapsules containing trioctylmethylammonium chloride (TOMAC), a selective extractant agent for the removal of mercury from wastewaters, by the suspension copolymerisation of styrene (St) and divinylbenzene (DVB) was studied. The influence of the diluent, mass ratio of the suspending agents (arabic gum –AG– and polivinylalcohol –PVA–), TOMAC:diluent volume ratio and weight percentage of DVB respect to the monomers mixture (% DVB) on the encapsulation process and the physical properties of the resulting microcapsules were investigated. It was found that using heptane as diluent led to non-spherical microcapsules with poor reaction yield and conversion of the monomers. Nevertheless, when toluene was used these properties were improved. Furthermore, for a TOMAC:toluene ratio of 1:3 spherical beads were obtained. The combined use of the suspending agents was more appropriate than using them separately, due to the latex product and the low conversion of the monomers for AG and PVA, respectively. The increase of the % DVB from 18.8 to 50% enhanced the mechanical resistance of the polymeric shell, increasing the reaction yield up to an 84.7% and maintaining the TOMAC encapsulation (36.0%) and the sphericity of the microcapsules. The obtained particle size (40 μm) indicated that the main application of this material will be in fluidised beds or in perfectly mixed reactors. On the basis of the experimental results, a AG:PVA mass ratio of 1:1, a TOMAC:toluene volume ratio of 1:3 and 50% DVB were established as the best conditions to produce this kind of microcapsules, enabling its reproduction on a pilot scale plant. In addition, the high values of the distribution coefficient for mercury removal confirm the success of this optimisation process.Se estudió el proceso de optimización para la síntesis de microcápsulas que contienen cloruro de trioctilmetilamonio (TOMAC), un agente extractante selectivo para la eliminación de mercurio de aguas residuales, mediante la copolimerización en suspensión de estireno (St) y divinilbenceno (DVB). La influencia del diluyente, relación másica de los agentes de suspensión (goma arábiga –AG– y alcohol polivinílico –PVA–), relación volumétrica TOMAC:diluyente y porcentaje en peso de DVB respecto a la mezcla de monómeros (% DVB) sobre el proceso de encapsulación y el Se investigaron las propiedades físicas de las microcápsulas resultantes. Se encontró que el uso de heptano como diluyente conducía a microcápsulas no esféricas con un bajo rendimiento de reacción y conversión de los monómeros. Sin embargo, cuando se utilizó tolueno se mejoraron estas propiedades. Además, para una relación TOMAC:tolueno de 1:3 se obtuvieron perlas esféricas. El uso combinado de los agentes de suspensión fue más apropiado que usarlos por separado, debido al producto de látex y la baja conversión de los monómeros para AG y PVA, respectivamente. El aumento del % DVB de 18,8 a 50% mejoró la resistencia mecánica de la cubierta polimérica, aumentando el rendimiento de reacción hasta un 84,7% y manteniendo la encapsulación TOMAC (36,0%) y la esfericidad de las microcápsulas. El tamaño de partícula obtenido (40 μm) indicó que la principal aplicación de este material será en lechos fluidizados o en reactores perfectamente mezclados. En base a los resultados experimentales, una relación de masa AG:PVA de 1:1, una relación de volumen TOMAC:tolueno de 1:3 y un 50% de DVB se establecieron como las mejores condiciones para producir este tipo de microcápsulas, permitiendo su reproducción en una planta a escala piloto. Además, los altos valores del coeficiente de distribución para la remoción de mercurio confirman el éxito de este proceso de optimización

Comparison of clinical baseline characteristics between Asian and Western COPD patients in a prospective, international, multicenter study

The study was designed and coordinated by the Respiratory Effectiveness Group (REG; www.effectivenessevaluation.org; Cambridge, UK) and delivered by Optimum Patient Care (OPC; www.optimumpatientcare.org). The following investigators participated in the study: Spain: Marc Miravitlles, Cristina Esquinas, Miriam Barrecheguren, Alexa Nuñez, Hospital Universitari Vall d’Hebron, Barcelona. Bernardino Alcazar, Hospital de Alta Resolución de Loja. Juan Luis García-Rivero, Karina Hueso, Hospital Comarcal de Laredo, Cantabria. Miguel Roman-Rodríguez, Primary Health-care Center Son Pisà. IB-Salut. Palma de Mallorca. Poland: Pawel Sliwinsk Sliwinski, Katarzyna Iwan, Jacek Kolakowski, Institute of Tuberculosis and Lung Diseases, Warsaw. Korea: Chin Kook Rhee, Esther Ahn, St Mary’s Hospital. Seoul. Singapore: Jessica Tan, Therese Lapperre, Karen Tan Li Leng, Nicole Chia, Ong Thun How, Syifa Binte Shamsuddin, Sherine Lim Shu Gim, Yap Chwee Bee, Soh Rui Ya, Singapore General Hospital. Augustine Tee, Jun Jie Yan, Samuel Hong, William Tan, Jessica Tan, Changi General Hospital. UK: Victoria Carter, Latife Hardaker, Andrew McLaughlin, Optimum Patient Care, Cambridge. Malta: Caroline Gouder, Mater Dei Hospital. Ireland: Richard W Costello, Royal College of Surgeons. Dublin. The study was funded by an unrestricted grant from Novartis AG.Peer reviewedPublisher PD

Evaluation of criteria for clinical control in a prospective, international, multicenter study of patients with COPD

BACKGROUND: The concept of clinical control in COPD has been developed to help in treatment decisions, but it requires validation in prospective studies. METHOD: This international, multicenter, prospective study aimed to validate the concept of control in COPD [control = stability (no exacerbations or impairment in CAT scores) + low impact (low level of symptoms)]. Data from the screening visit was used to: investigate the level of control, compare characteristics of patients according to the control status, and perform a sensitivity analysis of the levels of control using either clinical criteria or questionnaires (COPD Assessment Test -CAT- or Clinical COPD Questionnaire -CCQ-). RESULTS: A total of 314 patients were analysed, mean age was 68.5 years and mean FEV1 was 52.6% of predicted. According to the prespecified criteria 21% of patients were classified as controlled, all of them with mild/moderate COPD (Body mass index, Obstruction, Dyspnea and Exacerbations, -BODEx-index <5). A high level of dyspnea, a high CAT score or an exacerbation in the previous 3 months were found, using univariate analysis, to be the main reasons for patients not being classified as controlled. Multivariate analysis showed that female sex, chronic bronchitis and having exacerbations in the previous year were associated with uncontrolled COPD. Changing the severity cut off of BODEx from 5 to 3 did not change significantly the percentage of patients fulfilling the criteria of control. CONCLUSIONS: The proposed criteria of control were only fulfilled by 21% of patients. The suggested cut offs and their predictive value for poor outcomes need to be refined in prospective studies

Changes in control status of COPD over time and their consequences : A prospective international, study

ACKNOWLEDGMENTS The study was designed and coordinated by the Respiratory Effectiveness Group (REG; www.effectivenessevaluation.org; Cambridge, UK) and delivered by Optimum Patient Care (OPC; www.optimumpatientcare.org). Funding: The study was funded by an unrestricted grant from Novartis AG.Peer reviewedPostprin

Predictive value of control of COPD for risk of exacerbations : An international, prospective study

Acknowledgements Collaborators/REG Investigators: Spain: Marc Miravitlles, Cristina Esquinas, Miriam Barrecheguren, Alexa Nuñez, Hospital Universitari Vall d'Hebron, Barcelona. Bernardino Alcazar, Hospital de Alta Resolución de Loja. Juan Luis García‐Rivero, Karina Hueso, Hospital Comarcal de Laredo, Cantabria. Miguel Roman‐Rodríguez, Primary Health‐Care Center Son Pisà, IB‐Salut, Palma de Mallorca. Poland: Pawel Sliwinsk, Katarzyna Iwan, Jacek Kolakowski, Institute of Tuberculosis and Lung Diseases, Warsaw. Korea: Chin Kook Rhee, Esther Ahn, St Mary's Hospital, Seoul. Singapore: Jessica Tan, Therese Laperre, Karen Tan Li Leng, Nicole Chia, Ong Thun How, Syifa Binte Shamsuddin, Sherine Lim Shu Gim, Yap Chwee Bee, Soh Rui Ya, Singapore General Hospital. Augustine Tee, Jun Jie Yan, Samuel Hong, William Tan, Changi General Hospital. UK: Victoria Carter, Latife Hardaker, Andrew McLaughlin, Optimum Patient Care, Cambridge. Malta: Caroline Gouder, Mater Dei Hospital. Ireland: Richard W Costello, Royal College of Surgeons, Dublin. The study was funded by an unrestricted grant from Novartis AG. The study was designed and coordinated by the Respiratory Effectiveness Group (REG; www.effectivenessevaluation.org; Cambridge, UK) and delivered by Optimum Patient Care (OPC; www.optimumpatientcare.org).Peer reviewedPostprin

Deficiency in the mRNA export mediator Gle1 impairs Schwann cell development in the zebrafish embryo

GLE1 mutations cause lethal congenital contracture syndrome 1 (LCCS1), a severe autosomal recessive fetal motor neuron disease, and more recently have been associated with amyotrophic lateral sclerosis (ALS). The gene encodes a highly conserved protein with an essential role in mRNA export. The mechanism linking Gle1 function to motor neuron degeneration in humans has not been elucidated, but increasing evidence implicates abnormal RNA processing as a key event in the pathogenesis of several motor neuron diseases. Homozygous gle1−/− mutant zebrafish display various aspects of LCCS, showing severe developmental abnormalities including motor neuron arborization defects and embryonic lethality. A previous gene expression study on spinal cord from LCCS fetuses indicated that oligodendrocyte dysfunction may be an important factor in LCCS. We therefore set out to investigate the development of myelinating glia in gle1−/− mutant zebrafish embryos. While expression of myelin basic protein (mbp) in hindbrain oligodendrocytes appeared relatively normal, our studies revealed a prominent defect in Schwann cell precursor proliferation and differentiation in the posterior lateral line nerve. Other genes mutated in LCCS have important roles in Schwann cell development, thereby suggesting that Schwann cell deficits may be a common factor in LCCS pathogenesis. These findings illustrate the potential importance of glial cells such as myelinating Schwann cells in motor neuron diseases linked to RNA processing defects

Arginine methylation of REF/ALY promotes efficient handover of mRNA to TAP/NXF1

The REF/ALY mRNA export adaptor binds TAP/NXF1 via an arginine-rich region, which overlaps with its RNA-binding domain. When TAP binds a REF:RNA complex, it triggers transfer of the RNA from REF to TAP. Here, we have examined the effects of arginine methylation on the activities of the REF protein in mRNA export. We have mapped the arginine methylation sites of REF using mass spectrometry and find that several arginines within the TAP and RNA binding domains are methylated in vivo. However, arginine methylation has no effect on the REF:TAP interaction. Instead, arginine methylation reduces the RNA-binding activity of REF in vitro and in vivo. The reduced RNA-binding activity of REF in its methylated state is essential for efficient displacement of RNA from REF by TAP in vivo. Therefore, arginine methylation fine-tunes the RNA-binding activity of REF such that the RNA–protein interaction can be readily disrupted by export factors further down the pathway