Some in-field experiences of non-synchronous vibrations in large rotating machinery

Some problems associated with non-synchronous vibrations are analyzed by describing three cases experienced with fairly large rotating machines in operating conditions. In each case, a brief description is first given of the machine and of the instrumentation used. The experimental results are then presented, with reference to time or frequency domain recordings. The lines followed in diagnosis are then discussed and, lastly, the corrective action undertaken is presented

Response to 'The evolving mystery of why skeletal muscle is spared in seropositive neuromyelitis optica'

Dear Editor, We would like to thank you for the opportunity to respond to the questions raised in Dr Verkman's letter and to elucidate related aspects. We also thank Dr Verkman and colleagues for their attention to our study. The use of g‐STED super‐resolution microscopy versus freeze‐fracture electron microscopy (FFEM) to analyse skeletal muscle and brain AQP4 supramolecular assemblies (OAPs) used in our study 1 has been disputed by Verkman et al. While we agree that FFEM is the gold standard to visualise OAPs and also measure their size, we also are aware that the very small amount of the plasma membrane that can be suitable for analysis represents a major limit to obtaining statistically significant data (such as the OAP dimension) representative of the entire tissue. In contrast, STED microscopy has the enormous advantage of analysing, in real time, very large portions of the plasma membrane, with a resolution that in our setup can reach approximately 30 nm, providing the possibility to have a more complete vision of the entire tissue and handle a large amount of data. Considering the ‘contradiction of available data’, Verkman et al. refer to an FFEM study on OAP structure and organisation performed before the identification of AQP4 (or MIWC) as the molecular determinant of OAPs 2. Moreover, the same study did not directly compare muscle and brain OAP size 2. It was Verkman's group that later performed the first study 3 in which the role of AQP4 in OAP formation and in different tissues was directly analysed by FFEM in AQP4‐WT and null mice. This study literally reported: ‘The density of OAPs in brain was similar to that of OAPs in muscle, however, the patch sizes were somewhat bigger than in muscle…’ 3. Therefore, our interpretation is that our results are rather ‘in line with available data’ with a step forward in which g‐STED has helped to quantify the Verkman group's observation that OAPs in brain are ‘somewhat bigger that in muscle’. Anyhow, it is not of secondary importance that, independently of the size of skeletal muscle OAPs, super‐resolution microscopy revealed that AQP4 sarcolemma organisation in fast‐twitch skeletal muscle fibres is different compared to brain perivascular astrocyte endfeet. A second issue raised by Verkman et al. refers to their own studies in which they have demonstrated that small changes in isoform ratio should not substantially affect NMO‐IgG binding 4. In this case, we have to take into account that those studies have two major limits: (1) they were obtained in heterologous systems in which only two isoforms were over‐expressed; and (2) they were obtained mainly using a recombinant monoclonal antibody, very far from the complexity of real human polyclonal autoantibodies, as demonstrated by epitope mapping studies 5. We believe that the unquestionable advantage of our study is that it has been performed on tissues expressing endogenous AQP4 with all the players (known and unknown) for AQP4 clustering. One of these players is the recently identified AQP4ex isoform 6, which is strongly expressed in skeletal muscle. As AQP4ex modulates AQP4 cluster size, 6 it may, for example, have a role in the different supramolecular organisation. With regard to the concern about the use of non‐fixed frozen tissues in our study, it is well‐established that the use of unfixed tissue for immunofluorescence is crucial to preserve the conformational epitopes necessary for AQP4‐IgG binding 5. While the fascinating mystery of why skeletal muscle is spared in seropositive neuromyelitis optica will certainly benefit from further studies, we believe that a very small piece has been added in this direction here. We would like to thank all involved for the opportunity to continue this fascinating discussion and look forward to hearing from you. Yours sincerely

Low cost vacuum web coating system

A low cost solution for a mini roll to roll web coating system is presented. The design is very simple and involves only three active rolls, two winding/unwinding rolls and a cooling drum. No extra load cells are used to control the web winding mechanism operation. To reach such result it has been necessary to develop an adequate control solution which acts on the two winding roll torques to make the web moving properly. The effect of the control mechanism is to increase electronically the total mechanical inertia of the roll to roll system. In such manner the stick-slip motion of the web, induced by the dry friction affecting the rotation of the rolls, is avoided. The effectiveness of this strategy has been corroborated: a first test showed that the web moves continuously while it is kept tense; in a second experiment a-Si material has been deposited by hot-wire chemical vapor deposition technique. For that material the optical transmission measurements at several points over the deposited area indicate a satisfactory uniformity. The presented tests validate the goodness of the new control method

Technological solution for the automatic replacement of the catalytic filaments in HWCVD

The degradation of the catalytic filaments is the main factor limiting the industrial implementation of the hot wire chemical vapor deposition (HWCVD) technique. Up to now, no solution has been found to protect the catalytic filaments used in HWCVD without compromising their catalytic activity. Probably, the definitive solution relies on the automatic replacement of the catalytic filaments. In this work, the results of the validation tests of a new apparatus for the automatic replacement of the catalytic filaments are reported. The functionalities of the different parts have been validated using a 0.2 mm diameter tungsten filament under uc-Si:H deposition conditions

Degradation of thin tungsten filaments at high temperature in HWCVD

The degradation of the filaments is usually studied by checking the silicidation or carbonization status of the refractory metal used as catalysts, and their effects on the structural stability of the filaments. In this paper, it will be shown that the catalytic stability of a filament heated at high temperature is much shorter than its structural lifetime. The electrical resistance of a thin tungsten filament and the deposition rate of the deposited thin film have been monitored during the filament aging. It has been found that the deposition rate drops drastically once the quantity of dissolved silicon in the tungsten reaches the solubility limit and the silicides start precipitating. This manuscript concludes that the catalytic stability is only guaranteed for a short time and that for sufficiently thick filaments it does not depend on the filament radius

UM ESTUDO SOBRE O USO DAS TÉCNICAS DE SIMULAÇÃO NO PROCESSO DE ELABORAÇÃO E EXECUÇÃO DOS PLANEJAMENTOS ESTRATÉGICO E OPERACIONAL

This research wants to demonstrate how simulation is related to the elaboration of both the strategic planning and the operational planning, and how it can be used to improve manager´s analytical and decision making abilities. Supported by a bibliographic research and using a deductive method, we tried to verify the usage of this tool in association with decision trees. The study´s scope ranges from trend analysis in the establishment of strategies to their quantification in operational planning, allowing an optimal usage of resources. On reflection, we concluded that the simulation technique can give us good results in terms of economy of values and corrective action anticipation, as it predicts possible future results. We verify that the usage of decision tree diagrams gives us a simple and objective view in relation to the way that the simulation can be used in the strategic and operational planning. Besides, through the analysis of alternatives with different final results, these tools lead to the optimization in the use of company´s resources.Esta pesquisa buscou demonstrar como a simulação está associada à elaboração do planejamento estratégico e do planejamento operacional e como pode ser utilizada para melhorar a capacidade de análise e decisão do gestor. Através do método dedutivo e apoiado em uma pesquisa bibliográfica, procurou-se verificar a utilidade dessa ferramenta associada a árvores de decisão. Abordou-se desde a análise de tendências na definição de estratégias, até sua quantificação no planejamento operacional, proporcionando a utilização de recursos de forma otimizada. Após as reflexões, concluiu-se que a técnica da simulação pode proporcionar bons resultados em relação à economia de valores e à antecipação da correção de rumos ao permitir a análise de resultados futuros. Constatou-se que a utilização dos diagramas de árvores de decisão proporciona uma visão simples e objetiva em relação à forma como a simulação pode ser usada no planejamento estratégico e operacional. Ainda, a análise das alternativas de ações pode contribuir para a otimização do uso dos recursos da empresa

Glio-vascular modifications caused by Aquaporin-4 deletion in the mouse retina

Aquaporin-4 (AQP4) is the Central Nervous System water channel highly expressed at the perivascular glial domain. In the retina, two types of AQP4 expressing glial cells take part in the blood-retinal barrier (BRB), astrocytes and Müller cells. The aim of the present study is to investigate the effect of AQP4 deletion on the retinal vasculature by looking at typical pathological hallmark such as BRB dysfunction and gliotic condition.AQP4 dependent BRB properties were evaluated by measuring the number of extravasations in WT and AQP4 KO retinas by Evans blue injection assay. AQP4 deletion did not affect the retinal vasculature, as assessed by Isolectin B4 staining, but caused BRB impairment to the deep plexus capillaries while the superficial and intermediate capillaries were not compromised. To investigate for gliotic responses caused by AQP4 deletion, Müller cells and astrocytes were analysed by immunofluorescence and western blot, using the Müller cell marker Glutamine Synthetase (GS) and the astrocyte marker GFAP. While GS expression was not altered in AQP4 KO retinas, a strong GFAP upregulation was found at the level of AQP4 KO astrocytes at the superficial plexus and not at Müller cells at the intermediate and deep plexi. These data, together with the upregulation of inflammatory markers (TNF-α, IL-6, IL-1β and ICAM-1) in AQP4 KO retinas indicated AQP4 deletion as responsible for a gliotic phenotype. Interestingly, no GFAP altered expression was found in AQP4 siRNA treated astrocyte primary cultures. All together these results indicate that AQP4 deletion is directly responsible for BRB dysfunction and gliotic condition in the mouse retina. The selective activation of glial cells at the primary plexus suggests that different regulatory elements control the reaction of astrocytes and Müller cells. Finally, GFAP upregulation is strictly linked to gliovascular crosstalk, as it is absent in astrocytes in culture. This study is useful to understand the role of AQP4 in the perivascular domain in the retina and its possible implications in the pathogenesis of retinal vascular diseases and of Neuromyelitis Optica, a human disease characterized by anti-AQP4 auto-antibodies

AQP4-independent TRPV4 modulation of plasma membrane water permeability

: Despite of the major role of aquaporin (AQP) water channels in controlling transmembrane water fluxes, alternative ways for modulating water permeation have been proposed. In the Central Nervous System (CNS), Aquaporin-4 (AQP4) is reported to be functionally coupled with the calcium-channel Transient-Receptor Potential Vanilloid member-4 (TRPV4), which is controversially involved in cell volume regulation mechanisms and water transport dynamics. The present work aims to investigate the selective role of TRPV4 in regulating plasma membrane water permeability in an AQP4-independent way. Fluorescence-quenching water transport experiments in Aqp4-/- astrocytes revealed that cell swelling rate is significantly increased upon TRPV4 activation and in the absence of AQP4. The biophysical properties of TRPV4-dependent water transport were therefore assessed using the HEK-293 cell model. Calcein quenching experiments showed that chemical and thermal activation of TRPV4 overexpressed in HEK-293 cells leads to faster swelling kinetics. Stopped-flow light scattering water transport assay was used to measure the osmotic permeability coefficient (Pf, cm/s) and activation energy (Ea, kcal/mol) conferred by TRPV4. Results provided evidence that although the Pf measured upon TRPV4 activation is lower than the one obtained in AQP4-overexpressing cells (Pf of AQP4 = 0.01667 ± 0.0007; Pf of TRPV4 = 0.002261 ± 0.0004; Pf of TRPV4 + 4αPDD = 0.007985 ± 0.0006; Pf of WT = 0.002249 ± 0.0002), along with activation energy values (Ea of AQP4 = 0.86 ± 0.0006; Ea of TRPV4 + 4αPDD = 2.73 ± 1.9; Ea of WT = 8.532 ± 0.4), these parameters were compatible with a facilitated pathway for water movement rather than simple diffusion. The possibility to tune plasma membrane water permeability more finely through TRPV4 might represent a protective mechanism in cells constantly facing severe osmotic challenges to avoid the potential deleterious effects of the rapid cell swelling occurring via AQP channels

Role of the H-bond between L53 and T56 for Aquaporin-4 epitope in Neuromyelitis Optica

Aquaporin-4 (AQP4) is the CNS water channel organized into well-ordered protein aggregates called Orthogonal Arrays of Particles (OAPs). Neuromyelitis Optica (NMO) is an autoimmune disease caused by anti-OAP autoantibodies (AQP4-IgG). Molecular Dynamics (MD) simulations have identified an H-bond between L53 and T56 as the key for AQP4 epitope and therefore of potential interest for drug design in NMO field. In the present study, we have experimentally tested this MD-prediction using the classic mutagenesis approach. We substituted T56 with V56 and tested this mutant for AQP4 aggregates and AQP4-IgG binding. gSTED super-resolution microscopy showed that the mutation does not affect AQP4 aggregate dimension; immunofluorescence and cytofluorimetric analysis demonstrated its unaltered AQP4-IgG binding, therefore invalidating the MD-prediction. We later investigated whether AQP4, expressed in Sf9 insect and HEK-293F cells, is able to correctly aggregate before and after the purification steps usually applied to obtain AQP4 crystal. The results demonstrated that AQP4-IgG recognizes AQP4 expressed in Sf9 and HEK-293F cells by immunofluorescence even though BN-PAGE analysis showed that AQP4 forms smaller aggregates when expressed in insect cells compared to mammalian cell lines. Notably, after AQP4 purification, from both insect and HEK-293F cells, no aggregates are detectable by BN-PAGE and AQP4-IgG binding is impaired in sandwich ELISA assays. All together these results indicate that 1) the MD prediction under analysis is not supported by experimental data and 2) the procedure to obtain AQP4 crystals might affect its native architecture and, as a consequence, MD simulations. In conclusion, given the complex nature of the AQP4 epitope, MD might not be the suitable for molecular medicine advances in NMO

Potential role of the methylation of VEGF gene promoter in response to hypoxia in oxygen-induced retinopathy: beneficial effect of the absence of AQP4

Hypoxia-dependent accumulation of vascular endothelial growth factor (VEGF) plays a major role in retinal diseases characterized by neovessel formation. In this study, we investigated whether the glial water channel Aquaporin-4 (AQP4) is involved in the hypoxia-dependent VEGF upregulation in the retina of a mouse model of oxygen-induced retinopathy (OIR). The expression levels of VEGF, the hypoxia-inducible factor-1a (HIF-1a) and the inducible form of nitric oxide synthase (iNOS), the production of nitric oxide (NO), the methylation status of the HIF-1 binding site (HBS) in the VEGF gene promoter, the binding of HIF-1a to the HBS, the retinal vascularization and function have been determined in the retina of wild-type (WT) and AQP4 knock out (KO) mice under hypoxic (OIR) or normoxic conditions. In response to 5 days of hypoxia, WT mice were characterized by (i) AQP4 upregulation, (ii) increased levels of VEGF, HIF-1a, iNOS and NO, (iii) pathological angiogenesis as determined by engorged retinal tufts and (iv) dysfunctional electroretinogram (ERG). AQP4 deletion prevents VEGF, iNOS and NO upregulation in response to hypoxia thus leading to reduced retinal damage although in the presence of high levels of HIF-1a. In AQP4 KO mice, HBS demethylation in response to the beginning of hypoxia is lower than in WT mice reducing the binding of HIF-1a to the VEGF gene promoter. We conclude that in the absence of AQP4, an impaired HBS demethylation prevents HIF-1 binding to the VEGF gene promoter and the relative VEGF transactivation, reducing the VEGF-induced retinal damage in response to hypoxia