Establishment of a microtubule-fluorescent fusion protein mosaically labeled zebrafish motor neuron system / 生理学报

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.

Sex determining region Y-box 9 induced microtubule formation and epithelial⁃mesenchymal transition in human oral squamous cell carcinoma CAL27 cells / 华西口腔医学杂志

OBJECTIVES@#This study aimed to explore the effect of sex determining region Y-box 9 (SOX9) on the microtubule formation and epithelial-mesenchymal transition (EMT) of human oral squamous cell carcinoma (OSCC) CAL27 and the underlying mechanism.@*METHODS@#SOX9-shRNA1 and SOX9-shRNA2 were designed and synthesized and then transfected into CAL27 cells. The expression of SOX9 was detected by quantitative real-time polymerase chain reaction. Microtubule formation assay was used to detect the change in the number of microtubule nodules after interfering with SOX9. Immunofluorescence was used to detect the Vimentin content. Western blot was used to detect the protein expression of EMT marker molecules and Wnt/β-catenin pathway-related proteins, such as E-cadherin, N-cadherin, Fibronectin, Wnt, β-catenin, T-cell factor-4 (TCF-4).@*RESULTS@#The expression level of SOX9 significantly decreased after transfection with SOX9-shRNA1 and SOX9-shRNA2 in CAL27 cells (@*CONCLUSIONS@#Interference with SOX9 decreased Vimentin content and inhibited the microtubule formation and protein expression of EMT marker molecules, as well as the expression of proteins related to the Wnt/β-catenin pathway. Thus, SOX9 can induce microtubule formation and EMT in CAL27, which was related to the inhibition of the Wnt/β-catenin pathway activation.

Comparison of interscalene brachial plexus block and intra-articular local anesthetic administration on postoperative pain management in arthroscopic shoulder surgery / Comparação de bloqueio do plexo braquial por via interescalênica e administração de anestésico local intra-articular no manejo da dor no pós-operatório de cirurgia artroscópica do ombro / Comparación de bloqueo del plexo braquial por vía interescalénica y administración de anestésico local intraarticular en el manejo del dolor en el postoperatorio de cirugía artroscópica del hombro

BACKGROUND AND OBJECTIVES: In this study, the aim was to compare postoperative analgesia effects of the administration of ultrasound-guided interscalene brachial plexus block and intra-articular bupivacaine carried out with bupivacaine. METHODS: In the first group of patients 20 mL 0.25% bupivacaine and ultrasound-guided interscalene brachial plexus block (ISPB) were applied, while 20 mL 0.25% bupivacaine was given via intra-articular (IA) administration to the second group patients after surgery. Patients in the third group were considered the control group and no block was performed. Patient-controlled analgesia (PCA) with morphine was used in all three groups for postoperative analgesia. RESULTS: In the ISPB group, morphine consumption in the periods between 0-4, 6-12 and 12-24 postoperative hours and total consumption within 24 h was lower than in the other two groups. Morphine consumption in the IA group was lower than in the control group in the period from 0 to 6 h and the same was true for total morphine consumption in 24 h. Postoperative VASr scores in the ISPB group were lower than both of the other groups in the first 2 h and lower than the control group in the 4th and 6th hours (p < 0.05). In the IA group, VASr and VASm scores in the 2nd, 4th and 6th hours were lower than in the control group (p < 0.05). CONCLUSION: Interscalene brachial plexus block was found to be more effective than intra-articular local anesthetic injection for postoperative analgesia. .

JUSTIFICATIVA E OBJETIVOS: Comparar os efeitos na analgesia no pós-operatório da administração de bloqueio do plexo braquial por via interescalênica guiado por ultrassom e bupivacaína intra-articular, feito com bupivacaína. MÉTODOS: No primeiro grupo de pacientes, 20 mL de bupivacaína a 0,25% e bloqueio do plexo braquial por via interescalênica guiado por ultrassom (BPBI) foram administrados, enquanto 20 mL de bupivacaína a 0,25% foram administrados por via intra-articular (IA) ao segundo grupo de pacientes após a cirurgia. Os pacientes do terceiro grupo foram considerados grupo controle e nenhum bloqueio foi feito. Analgesia controlada pelo paciente (ACP) com morfina foi usada nos três grupos para analgesia pós-operatória. RESULTADOS: No grupo BPBI, o consumo de morfina nos períodos entre 0-4, 6-12 e 12-24 horas após a cirurgia e o consumo total em 24 horas foram mais baixos do que nos outros dois grupos. O consumo de morfina no grupo IA foi menor do que no grupo controle no período de 0-6 horas, como também foi menor o consumo total de morfina em 24 horas. Os escores EVAr no pós-operatório do grupo BPBI foram menores do que os escores dos dois outros grupos nas primeiras duas horas e menores do que os do grupo controle nos períodos de 4 e 6 horas (p < 0,05). No grupo IA, os escores EVAr e EVAm nos períodos de 2, 4 e 6 horas foram menores do que no grupo controle (p < 0,05). CONCLUSÃO: O bloqueio do plexo braquial por via interescalênica mostrou ser mais eficaz do que a injeção intra-articular de anestésico local para analgesia pós-operatória. .

JUSTIFICACIÓN Y OBJETIVOS: En este estudio, nuestro objetivo fue comparar en el período postoperatorio los efectos analgésicos de la administración de la bupivacaína en el bloqueo del plexo braquial por vía interescalénica guiado por ecografía y bupivacaína intraarticular. MÉTODOS: En el primer grupo de pacientes se administraron 20 mL de bupivacaína al 0,25% y se llevó a cabo el bloqueo del plexo braquial por vía interescalénica (BPBI) guiado por ecografía, mientras que al segundo grupo de pacientes se le administraron 20 mL de bupivacaína al 0,25% por vía intraarticular (IA) tras la cirugía. Los pacientes del tercer grupo fueron considerados como grupo control y en ellos no se realizó ningún bloqueo. La analgesia controlada por el paciente con morfina se usó en los 3 grupos para la analgesia postoperatoria. RESULTADOS: En el grupo BPBI, el consumo de morfina en los períodos entre 0-4, 6-12 y 12-24 h del postoperatorio y el consumo total en 24 h fueron más bajos que en los otros 2 grupos. El consumo de morfina en el grupo IA fue menor que en el grupo control en el período de 0-6 h, como también fue menor el consumo total de morfina en 24 h. Las puntuaciones EVAr en el postoperatorio del grupo BPBI fueron menores que las de los otros 2 grupos en las primeras 2 h y menores que los del grupo control en los períodos de 4 y 6 h (p < 0,05). En el grupo IA, las puntuaciones EVAr y EVAm en los períodos de 2, 4 y 6 h fueron menores que en el grupo control (p < 0,05). CONCLUSIÓN: El BPBI mostró ser más eficaz que la inyección intraarticular de anestésico local para analgesia postoperatoria. .

Fatores psicossociais e socioeconômicos relacionados à insônia e menopausa: Estudo Pró-Saúde / Psychosocial and socioeconomic factors related to insomnia and menopause: Pró-Saúde Study / Factores psicosociales y socioeconómicos relacionados con el insomnio y la menopausia: Estudio Pro-Salud

Foi avaliada a associação entre menopausa e insônia e a influência de variáveis socioeconômicas e psicossociais, em estudo transversal com 2.190 funcionárias de uma universidade (Estudo Pró-Saúde), a partir de um questionário autopreenchível com variáveis sobre menopausa, insônia, transtorno mental comum, eventos de vida estressantes, apoio social e variáveis socioeconômicas. Odds ratios foram calculados por meio de regressão logística multivariada, com desfecho politômico. Após ajuste para potenciais confundidoras sociodemográficas, mulheres na menopausa há mais de 60 meses apresentaram maior chance de reportar queixas de sono frequentes (OR entre 1,53 e 1,86) do que as que estavam na menopausa há menos de 60 meses. Após os ajustes, no primeiro grupo, para as variáveis psicossociais, a magnitude dos ORs reduziu para 1,53 (IC95%: 0,92-2,52) para dificuldade em iniciar o sono, 1,81 (IC95%: 1,09-2,98) para dificuldade em manter o sono e 1,71 (IC95%: 1,08-2,73) para queixa geral de insônia. Fatores psicossociais podem mediar a manifestação da insônia em mulheres na menopausa.

This study evaluated the association between insomnia and menopausal status and the influence of socioeconomic and psychosocial variables on this association in a cross-sectional analysis of 2,190 university employees (the Pró-Saúde Study). A self-administered questionnaire was used, covering menopausal status, complaints of insomnia, common mental disorders, stressful life events, social support, and socioeconomic variables. Odds ratios were calculated using logistic regression with a polytomous outcome. After adjusting for potential socio-demographic confounders, women who had entered menopause more than 60 months previously were more likely to report complaints with sleep (OR 1.53-1.86) as compared to women in menopause for less than 60 months. After adjusting for psychosocial variables, in the first group the ORs decreased to 1.53 (95%CI: 0.92-2.52) for difficulty initiating sleep, 1.81 (95%CI: 1.09-2.98) for difficulty maintaining sleep, and 1.71 (95%CI: 1.08-2.73) for general complaints of insomnia. Psychosocial factors can mediate the manifestation of insomnia among menopausal women.

En este estudio se evaluó la asociación entre insomnio y menopausia y la influencia de las variables socioeconómicas y psicosociales, en un estudio transversal con 2.190 mujeres de una universidad (Estudio Pro-Salud), a partir de un cuestionario autoadministrado con variables de la menopausia, insomnio, trastornos mentales, situaciones de estrés vital, apoyo social y variables socioeconómicas. Se calcularon los odds ratio mediante regresión logística multivariante con desenlace politómico. Después de ajustar por factores de confusión sociodemográficos potenciales, las mujeres menopáusicas desde hace más de 60 meses fueron más propensas a reportar quejas frecuentes de sueño (OR entre 1,53 y 1,86) que las menopáusicas hace menos de 60 meses. Después de los ajustes, en el primer grupo, para las variables psicosociales la magnitud de los OR se redujo a 1,53 (IC95%: 0,92-2,52) para la dificultad para iniciar el sueño, un 1,81 (IC95%: 1,09-2,98) para mantener el sueño y un 1,71 (IC95%: 1,08-2,73) para las quejas de insomnio en general. Los factores psicosociales pueden mediar en la manifestación del insomnio en las mujeres menopáusicas.

Predictors of Success in the Treatment of Obstructive Sleep Apnea Syndrome with Mandibular Repositioning Appliance: A Systematic Review

Introduction Obstructive sleep apnea syndrome affects up to 4% of middle-aged men and 2% of adult women. It is associated with obesity. Objective The objective of this article is to review the literature to determine which factors best correlate with treatment success in patients with obstructive sleep apnea syndrome treated with a mandibular repositioning appliance. Data Synthesis A search was performed of the PubMed, Cochrane, Lilacs, Scielo, and Web of Science databases of articles published from January 1988 to January 2012. Two review authors independently collected data and assessed trial quality. Sixty-nine articles were selected from PubMed and 1 from Cochrane library. Of these, 42 were excluded based on the title and abstract, and 27 were retrieved for complete reading. A total of 13 articles and 1 systematic review were considered eligible for further review and inclusion in this study: 6 studies evaluated anthropomorphic and physiologic factors, 3 articles addressed cephalometric and anatomic factors, and 4 studies evaluated variables related to mandibular repositioning appliance design and activation. All the studies evaluated had low to moderate methodologic quality and were not able to support evidence on prediction of treatment success. Conclusion Based on this systematic review on obstructive sleep apnea syndrome treatment, it remains unclear which predictive factors can be used with confidence to select patients suitable for treatment with a mandibular repositioning appliance. .

Natural transovarial transmission of dengue virus 4 in Aedes aegypti from Cuiabá, State of Mato Grosso, Brazil

INTRODUCTION: Dengue is the most prevalent arboviral disease in tropical areas. In Mato Grosso, outbreaks are reported every year, but studies on dengue in this state are scarce. METHODS: Natural transovarial infection of Aedes aegypti by a flavivirus was investigated in the Jardim Industriário neighborhood of Cuiabá, Mato Grosso. Eggs were collected with ovitraps during the dry, intermediate, and rainy seasons of 2012. After the eggs hatched and the larvae developed to adulthood, mosquitoes (n = 758) were identified and allocated to pools of 1-10 specimens according to the collection location, sex, and climatic period. After RNA extraction, multiplex semi-nested RT-PCR was performed to detect the four dengue virus (DENV) serotypes, yellow fever virus, West Nile virus and Saint Louis encephalitis virus. RESULTS: DENV-4 was the only flavivirus detected, and it was found in 8/50 pools (16.0%). Three of the positive pools contained females, and five contained males. Their nucleotide sequences presented 96-100% similarity with DENV-4 genotype II strains from Manaus, Amazonas. The minimum infection rate was 10.5 per 1000 specimens, and the maximum likelihood estimator of the infection rate was 11.6 (95% confidence interval: 4.8; 23.3). CONCLUSIONS: This study provides the first evidence of natural transovarial infection by DENV-4 in Ae. Aegypti in Mato Grosso, suggesting that this type of infection might serve as a mechanism of virus maintenance during interepidemic periods in Cuiabá, a city where dengue epidemics are reported every year. These results emphasize the need for efficient vector population control measures to prevent arbovirus outbreaks in the state. .

The colchicine derivative CT20126 shows a novel microtubule-modulating activity with apoptosis

New colchicine analogs have been synthesized with the aim of developing stronger potential anticancer activities. Among the analogs, CT20126 has been previously reported to show immunosuppressive activities. Here, we report that CT20126 also shows potential anticancer effects via an unusual mechanism: the modulation of microtubule integrity and cell cycle arrest at the G2/M phase before apoptosis. When we treated COS-7 cells with CT20126 (5 muM), the normal thread-like microtubules were disrupted into tubulin dimers within 10 min and thereafter repolymerized into short, thick filaments. In contrast, cells treated with the same concentration of colchicine exhibited microtubule depolymerization after 20 min and never underwent repolymerization. Furthermore, optical density (OD) analysis (350 nm) with purified tubulin showed that CT20126 had a higher repolymerizing activity than that of Taxol, a potent microtubule-polymerizing agent. These results suggest that the effects of CT20126 on microtubule integrity differ from those of colchicine: the analog first destabilizes microtubules and then stabilizes the disrupted tubulins into short, thick polymers. Furthermore, CT20126 induced a greater level of apoptotic activity in Jurkat T cells than colchicine (assessed by G2/M arrest, caspase-3 activation and cell sorting). At 20 nM, CT20126 induced 47% apoptosis among Jurkat T cells, whereas colchicine induced only 33% apoptosis. Our results suggest that the colchicine analog CT20126 can potently induce apoptosis by disrupting microtubule integrity in a manner that differs from that of colchicine or Taxol.

A likely role for a novel PH-domain containing protein, PEPP2, in connecting membrane and cytoskeleton

PH domains (pleckstrin homology) are well known to bind membrane phosphoinositides with different specificities and direct PH domain-containing proteins to discrete subcellular apartments with assistances of alternative binding partners. PH domain-containing proteins are found to be involved in a wide range of cellular events, including signalling, cytoskeleton rearrangement and vesicular trafficking. Here we showed that a novel PH domain-containing protein, PEPP2, displayed moderate phosphoinositide binding specificity. Full length PEPP2 associated with both plasma membrane and microtubules. The membrane-associated PEPP2 nucleated at cell-cell contacts and the leading edge of migrating cells. Overexpression of PEPP2 increased membrane microviscosity, indicating a potential role of PEPP2 in regulating function of membrane and microtubules.

Biophysical aspects of cancer--electromagnetic mechanism.

Hypothesis of coherent vibration states in biological systems based on nonlinear interaction between longitudinal elastic and electric polarization fields with metabolic energy supply was formulated by Frohlich. Conditions for excitation of coherent states and generation of electromagnetic fields are satisfied in microtubules which form electrical polar structures. Numerical models are used for analysis of Frohlich's vibration states in cells. Reduction of activity and of energy production in mitochondria, and disintegration of cytoskeleton structures by phosphorylation on the pathway of cancer trasformation can diminish excitation of the Frohlich's vibration states and of the generated electromagnetic field, which results in disturbances of the interaction forces between cells. Interaction forces between cancer cells may be smaller than interaction forces between healthy cells and cancer cells as follows from numerical models. Mechanism of malignity, i.e. local invasion, detachment of cancer cells, and metastasis, is assumed to depend on the electromagnetic field.

Transferrin uptake may occur through detergent-resistant membrane domains at the cytopharynx of Trypanosoma cruzi epimastigote forms

Uptake of transferrin by epimastigote forms of the protozoan Trypanosoma cruzi occurs mainly through a cytostome/ cytopharynx, via uncoated endocytic vesicles that bud off from the bottom of the cytopharynx. We have here examined whether detergent-resistant membrane (DRM) domains might be involved in this process. Purified whole cell membrane fractions were assayed for cholesterol levels and used in dot blot analyses. Detergent-resistant membrane markers (cholera B toxin and anti-flotillin-1 antibody) presented positive reaction by dot blots in cholesterol-rich/ protein-poor membrane sub-fractions. The positive dot blot fraction was submitted to lipid composition analysis, showing composition similar to that of raft fractions described for other eukaryotic cells. Immunofluorescence assays allowed the localization of punctual positive signal for flotillin-1, matching the precise cytostome/ cytopharynx location. These data were confirmed by immunofluorescence assays with the co-localization of flotillin-1 and the transferrin uptake site. Our data suggest that DRM domains occur and are integrated at the cytostome/ cytopharynx of T. cruzi epimastigotes, being the main route for transferrin uptake.

Association of rotavirus viroplasms with microtubules through NSP2 and NSP5

Rotavirus replication and virus assembly take place in electrodense spherical structures known as viroplasms whose main components are the viral proteins NSP2 and NSP5. The viroplasms are produced since early times after infection and seem to grow by stepwise addition of viral proteins and by fusion, however, the mechanism of viropIasms formation is unknown. In this study we found that the viroplasms surface colocalized with microtubules, and seem to be caged by a microtubule network. Moreover inhibition of microtubule assembly with nocodazole interfered with viroplasms growth in rotavirus infected cells. We searched for a physical link between viroplasms and microtubules by co-immunoprecipitation assays, and we found that the proteins NSP2 and NSP5 were co-immunoprecipitated with anti-tubulin in rotavirus infected cells and also when they were transiently co-expressed or individually expressed. These results indicate that a functional microtubule network is needed for viroplasm growth presumably due to the association of viroplasms with microtubules via NSP2 and NSP5.

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P2] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.

Molecular Motor Proteins of the Kinesin Superfamily Proteins (KIFs): Structure, Cargo and Disease

Intracellular organelle transport is essential for morphogenesis and functioning of the cell. Kinesins and kinesin-related proteins make up a large superfamily of molecular motors that transport cargoes such as vesicles, organelles (e.g. mitochondria, peroxisomes, lysosomes), protein complexes (e.g. elements of the cytoskeleton, virus particles), and mRNAs in a microtubule- and ATP-dependent manner in neuronal and non-neuronal cells. Until now, more than 45 kinesin superfamily proteins (KIFs) have been identified in the mouse and human genomes. Elucidating the transport pathways mediated by kinesins, the identities of the cargoes moved, and the nature of the proteins that link kinesin motors to cargoes are areas of intense investigation. This review focuses on the structure, the binding partners of kinesins and kinesin-based human diseases.

The anthelmintic albendazole affects in vivo the dynamics and the detyrosination-tyrosination cycle of rat brain microtubules

Albendazole (ABZ) is an anthelmintic benzimidazole drug widely used in human and veterinary medicine. ABZ has binding affinity to both mammalian and helminth parasite tubulin. In the current work, we have performed in vitro assays and in vivo experiments in which rats were given ABZ orally to better characterize the action of the drug on the polymerization of rat brain microtubules and on the detyrosination/tyrosination cycle that occurs on the COOH-terminal end of alpha-tubulin. The results showed that ABZ inhibits brain microtubule polymerization in vitro, and significantly delayed microtubule assembly in vivo. The tyrosination reaction cycle was not affected in vitro; however, in rats to which the drug was administered orally, the levels of in vitro tyrosination were reduced when compared to the controls with mock treatment. These results suggest that this apparent inhibition would be due to a decrease in the amount of substrate caused by the depolymerizing effect of ABZ and the subsequent tyrosination in the intact brain with endogenous tyrosine. In conclusion, ABZ strongly affects tubulin dynamics both in vivo and in vitro. The outcome of these experiments is a contribution to the understanding of the molecular mechanisms involved in the antimicrotubular action of benzimidazole compounds.

Mouse brain tau proteins: microheterogeneity and phosphorylation

Tau proteins are involved in polymerization of tubulin into microtubules. They comprise a heterogeneous group of proteins that can be resolved by two-dimensional gel electrophoresis using a non-equilibrium pH gradient in the first dimension. Developmental studies show that mouse brain Tau proteins are more heterogeneous in 15-day old mice than in newborn pups or adults. Tau phosphorylation is also more heterogeneous at this stage