LPS-induced endothelial cytoskeleton remodeling in human lung vessels and related miRNAs-profiling / 细胞与分子免疫学杂志

Objective To investigate the effects of lipopolysaccharide (LPS) on human pulmonary vascular endothelial cells (HPVECs) cytoskeleton and perform biological analysis of the microRNA (miRNA) spectrum. Methods The morphology of HPVECs was observed by microscope, the cytoskeleton by FITC-phalloidin staining, and the expression of VE-cadherin was detected by immunofluorescence cytochemical staining; the tube formation assay was conducted to examine the angiogenesis, along with cell migration test to detect the migration, and JC-1 mitochondrial membrane potential to detect the apoptosis. Illumina small-RNA sequencing was used to identify differentially expressed miRNAs in NC and LPS group. The target genes of differentially expressed miRNAs were predicted by miRanda and TargetScan, and the functional and pathway enrichment analysis was performed on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG). Further biological analysis of related miRNAs was carried out. Results After the LPS got induced, the cells became round and the integrity of cytoskeleton was destroyed. The decreased expression of VE-cadherin was also observed, along with the decreased ability of angiogenesis and migration, and increased apoptosis. Sequencing results showed a total of 229 differential miRNAs, of which 84 miRNA were up-regulated and 145 miRNA were down-regulated. The target gene prediction and functional enrichment analysis of these differential miRNA showed that they were mainly concentrated in pathways related to cell connection and cytoskeleton regulation, cell adhesion process and inflammation. Conclusion In vitro model of lung injury, multiple miRNAs are involved in the process of HPVECs cytoskeleton remodeling, the reduction of barrier function, angiogenesis, migration and apoptosis.

The p21-activated kinases in neural cytoskeletal remodeling and related neurological disorders

The serine/threonine p21-activated kinases (PAKs), as main effectors of the Rho GTPases Cdc42 and Rac, represent a group of important molecular switches linking the complex cytoskeletal networks to broad neural activity. PAKs show wide expression in the brain, but they differ in specific cell types, brain regions, and developmental stages. PAKs play an essential and differential role in controlling neural cytoskeletal remodeling and are related to the development and fate of neurons as well as the structural and functional plasticity of dendritic spines. PAK-mediated actin signaling and interacting functional networks represent a common pathway frequently affected in multiple neurodevelopmental and neurodegenerative disorders. Considering specific small-molecule agonists and inhibitors for PAKs have been developed in cancer treatment, comprehensive knowledge about the role of PAKs in neural cytoskeletal remodeling will promote our understanding of the complex mechanisms underlying neurological diseases, which may also represent potential therapeutic targets of these diseases.

Microenvironmental stiffness mediates cytoskeleton re-organization in chondrocytes through laminin-FAK mechanotransduction / 国际口腔科学杂志·英文版

Microenvironmental biophysical factors play a fundamental role in controlling cell behaviors including cell morphology, proliferation, adhesion and differentiation, and even determining the cell fate. Cells are able to actively sense the surrounding mechanical microenvironment and change their cellular morphology to adapt to it. Although cell morphological changes have been considered to be the first and most important step in the interaction between cells and their mechanical microenvironment, their regulatory network is not completely clear. In the current study, we generated silicon-based elastomer polydimethylsiloxane (PDMS) substrates with stiff (15:1, PDMS elastomer vs. curing agent) and soft (45:1) stiffnesses, which showed the Young's moduli of ~450 kPa and 46 kPa, respectively, and elucidated a new path in cytoskeleton re-organization in chondrocytes in response to changed substrate stiffnesses by characterizing the axis shift from the secreted extracellular protein laminin β1, focal adhesion complex protein FAK to microfilament bundling. We first showed the cellular cytoskeleton changes in chondrocytes by characterizing the cell spreading area and cellular synapses. We then found the changes of secreted extracellular linkage protein, laminin β1, and focal adhesion complex protein, FAK, in chondrocytes in response to different substrate stiffnesses. These two proteins were shown to be directly interacted by Co-IP and colocalization. We next showed that impact of FAK on the cytoskeleton organization by showing the changes of microfilament bundles and found the potential intermediate regulators. Taking together, this modulation axis of laminin β1-FAK-microfilament could enlarge our understanding about the interdependence among mechanosensing, mechanotransduction, and cytoskeleton re-organization.

Nuclear elongation correlates with neurite induced cellular elongation during differentiation of SH-SY5Y neural cells / El alargamiento del núcleo se correlaciona con el alargamiento celular inducido por neuritas durante la diferenciación de las células neuronales Sh-Sy5Y

SUMMARY: Cellular differentiation is a highly regulated process that has vast implications for the mechanics of the cell. The interplay between differentiation induced cytoskeletal mechanical changes and strain on the nucleus is a potential cause of gene level changes. This study explores mechanical changes in SH-SY5Y neural cells during differentiation mediated by Retinoic Acid (RA) across Days 0 through 9. Findings suggest that cellular elongation increases 1.92-fold over a 10-day differentiation period, from 48.97 ±16.85µm to 93.96 ± 31.20 µm over 3 repeated trials and across multiple cells analyzed on ImageJ. Nuclear elongation increases less substantially from 17.51 ± 2.71 µm to 23.26 ± 3.10 µm over 3 repeated trials and across multiple cells. Results are statistically significant at a significance level of α = 0.05. This study is one of the first studies to show that during the process of RA mediated neural differentiation in SH-SY5Y neural cells, nuclear elongation is initially not significantly correlated with cellular elongation, but it becomes correlated during the differentiation process with an overall correlation coefficient of 0.4498 at a significance level of α = 0.05. Given the time course of the mechanical changes and the known coupling between the cytoskeleton and nuclear lamina, this study suggests a causative and correlative relationship between neurite-driven cellular elongation and nuclear elongation during neural differentiation.

RESUMEN: La diferenciación celular es un proceso altamente regulado que tiene vastas implicaciones para la mecánica de la célula. La interacción entre los cambios mecánicos citoesqueléticos inducidos por la diferenciación y la tensión en el núcleo es una causa potencial de cambios a nivel genético. Este estudio explora los cambios mecánicos en las células neurales SH-SY5Y durante la diferenciación mediada por el ácido retinoico (RA) durante los días 0 a 9. Los resultados sugieren que el alargamiento celular aumenta 1,92 veces durante un período de diferenciación de 10 días, de 48,97 ± 16,85 µm a 93,96 ± 31,20 µm en 3 ensayos repetidos y en múltiples células analizadas en Image J. El alargamiento nuclear aumenta menos sustancialmente de 17,51 ± 2,71 µm a 23,26 ± 3,10 µm durante 3 ensayos repetidos y en múltiples células. Los resultados son estadísticamente significativos a un nivel de significancia de α = 0,05. Esta investigación es uno de los primeros estudios en demostrar que durante el proceso de diferenciación neural mediada por RA en las células neurales SH-SY5Y, el alargamiento nuclear inicialmente no se correlaciona significativamente con el alargamiento celular, pero se correlaciona durante el proceso de diferenciación con un coeficiente de correlación global de 0,4498 a un nivel de significancia de α = 0,05. Dado el curso temporal de los cambios mecánicos y el acoplamiento conocido entre el citoesqueleto y la lámina nuclear, este estudio sugiere una relación causal y correlativa entre el alargamiento celular impulsado por neuritas y el alargamiento nuclear durante la diferenciación neural.

The Role of Zyxin in Regulating Platelet Cytoskeleton Distribution / 中国实验血液学杂志

OBJECTIVE@#To investigate the regulatory effect of zyxin on the distribution of platelet cytoskeleton.@*METHODS@#Platelets were isolated from zyxin-knockout (Zyx@*RESULTS@#After zyxin gene was knockout, the expressions of cytoskeleton proteins β-actin, α-actinin, filamin A, and myosin Ⅱ A in resting and Jas-induced platelets were significantly increased. In the platelet spreading on fibrinogen surface, F-actin was increased in Zyx@*CONCLUSION@#Zyxin significantly regulates the distribution of platelet cytoskeleton, which plays an important role in maintaining platelet cytoskeleton homeostasis.

Relationship between PI3K/mTOR/RhoA pathway-regulated cytoskeletal rearrangements and phagocytic capacity of macrophages

The objective of this study was to investigate the relationship between PI3K/mTOR/RhoA signaling regulated cytoskeletal rearrangements and phagocytic capacity of macrophages. RAW264.7 macrophages were divided into four groups; blank control, negative control, PI3K-RNAi, and mTOR-RNAi. The cytoskeletal changes in the macrophages were observed. Furthermore, the phagocytic capacity of macrophages against Escherichia coli is reported as mean fluorescence intensity (MFI) and percent phagocytosis. Transfection yielded 82.1 and 81.5% gene-silencing efficiencies against PI3K and mTOR, respectively. The PI3K-RNAi group had lower mRNA and protein expression levels of PI3K, mTOR, and RhoA than the blank and negative control groups (Р<0.01). The mTOR-RNAi group had lower mRNA and protein levels of mTOR and RhoA than the blank and the negative control groups (Р<0.01). Macrophages in the PI3K-RNAi group exhibited stiff and inflexible morphology with short, disorganized filopodia and reduced number of stress fibers. Macrophages in the mTOR-RNAi group displayed pronounced cellular deformations with long, dense filopodia and an increased number of stress fibers. The PI3K-RNAi group exhibited lower MFI and percent phagocytosis than blank and negative control groups, whereas the mTOR-RNAi group displayed higher MFI and percent phagocytosis than the blank and negative controls (Р<0.01). Before and after transfection, the mRNA and protein levels of PI3K were both positively correlated with mTOR and RhoA (Р<0.05), but the mRNA and protein levels of mTOR were negatively correlated with those of RhoA (Р<0.05). Changes in the phagocytic capacity of macrophages were associated with cytoskeletal rearrangements and were regulated by the PI3K/mTOR/RhoA signaling pathway.

Silence of cytoskeleton-associated protein 2 represses cell proliferation and migration and promotes apoptosis in liver cancer cell lines / 中南大学学报(医学版)

OBJECTIVES@#To investigate the roles of cytoskeleton-associated protein 2 (CKAP2) in proliferation, apoptosis, and migration in liver cancer cells and the potential mechanisms.@*METHODS@#Human normal hepatocyte L02 and liver cancer cell lines HepG2, Huh7, and SMMC-7721 were cultured. The CKAP2 expression was detected by real-time PCR and Western blotting. HepG2 cells were randomly divided into a control group, a negative control (NC) group, and a CKAP2 silencing (siCKAP2) group. CCK-8 and BrdU assays were used to evaluate cell viability and proliferation, respectively. Transwell assay was employed to determine cell migration and invasion. The protein levels of cleaved-caspase 3, Bax, E-cadherin, N-cadherin, Vimentin, phosphorylated Janus kinase 2 (p-JAK2), and phosphorylated signal transducer and activator of transcription 3 (p-STAT3) were determined by Western blotting.@*RESULTS@#Compared with normal hepatocyte L02, CKAP2 was highly expressed in liver cancer cell lines HepG2, Huh7, and SMMC-7721 (all <0.05). Compared with the NC group, cell viability and proliferation rate of the siCKAP2 group were decreased (both <0.05). The apoptotic rate, protein expression of cleaved-caspase 3 and Bax in the siCKAP2 group were significantly higher than those in the NC group (all <0.05). Compared with the NC group, cell migration and invasion rates of the siCKAP2 group were significantly attenuated (both <0.05). Compared with the NC group, E-cadherin protein expression in siCKAP2 group was increased, while protein expression levels of Vimentin, N-cadherin, p-JAK2, and p-STAT3 were decreased (all <0.05).@*CONCLUSIONS@#CKAP2 gene silence inhibits proliferation, migration, and invasion, and promotes apoptosis in liver cancer cells, while JAK2/STAT3 signaling pathway may be involved in these processes.

Microtubular Dysfunction and Male Infertility

Dact1 involvement in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation by regulating cx43

Abstract Objective: To determine the role of the dishevelled binding antagonist of beta catenin 1 (DACT1) in the cytoskeletal arrangement of cardiomyocytes in atrial fibrillation (AF). Methods: The DACT1 expression and its associations with the degree of fibrosis and β-catenin in valvular disease patients were analyzed by immunohistochemistry and Masson's staining. DACT1 was overexpressed in the atrial myocyte cell line (HL-1) and the cardiac cell line (H9C2) by adenoviral vectors. Alterations in the fibrous actin (F-actin) content and organization and the expression of β-catenin were detected by flow cytometry, immunofluorescence, and Western blotting. Additionally, the association of DACT1 with gap junctions connexin 43 (Cx43) was detected by immunohistochemistry, immunofluorescence, and Western blotting. Results: Decreased cytoplasmic DACT1 expression in the myocardium was associated with AF (P=0.037) and a high degree of fibrosis (weak vs. strong, P=0.028; weak vs. very strong, P=0.029). A positive association was observed between DACT1 and β-catenin expression in clinical samples (P=0.028, Spearman's rho=0.408). Furthermore, overexpression of DACT1 in HL-1 and H9C2 cells induced an increase in β-catenin and subsequent partial colocalization of DACT1 and β-catenin. In addition, F-actin content and organization were enhanced. Interestingly, DACT1 was positively correlated with the Cx43 expression in clinical samples (P=0.048, Spearman's rho=0.370) and changed the Cx43 distribution in cardiac cell lines. Conclusion: DACT1 proved to be a novel AF-related gene by regulating Cx43 via cytoskeletal organization induced by β-catenin accumulation in cardiomyocytes. DACT1 could thus serve as a potential therapeutic marker for AF.

Antitumoral effects of Amblyomma sculptum Berlese saliva in neuroblastoma cell lines involve cytoskeletal deconstruction and cell cycle arrest / Efeito antitumoral da saliva do carrapato Amblyomma sculptum Berlese em células de neuroblastoma envolve desconstrução do citoesqueleto e parada do ciclo celular

Abstract The antitumor properties of ticks salivary gland extracts or recombinant proteins have been reported recently, but little is known about the antitumor properties of the secreted components of saliva. The goal of this study was to investigate the in vitro effect of the saliva of the hard tick Amblyomma sculptum on neuroblastoma cell lines. SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32, and CHLA-20 cells were susceptible to saliva, with 80% reduction in their viability compared to untreated controls, as demonstrated by the methylene blue assay. Further investigation using CHLA-20 revealed apoptosis, with approximately 30% of annexin-V positive cells, and G0/G1-phase accumulation (>60%) after treatment with saliva. Mitochondrial membrane potential (Δψm) was slightly, but significantly (p < 0.05), reduced and the actin cytoskeleton was disarranged, as indicated by fluorescent microscopy. The viability of human fibroblast (HFF-1 cells) used as a non-tumoral control decreased by approximately 40%. However, no alterations in cell cycle progression, morphology, and Δψm were observed in these cells. The present work provides new perspectives for the characterization of the molecules present in saliva and their antitumor properties.

Resumo As propriedades antitumorais de extratos de glândulas salivares de carrapatos ou proteínas recombinantes foram relatadas recentemente, mas pouco se sabe sobre as propriedades antitumorais dos componentes secretados da saliva. O objetivo deste estudo foi investigar o efeito in vitro da saliva bruta do carrapato duro Amblyomma sculptum sobre as linhagens celulares de neuroblastoma. Células SK-N-SK, SH-SY5Y, Be(2)-M17, IMR-32 e CHLA-20 foram suscetíveis à saliva, com redução de 80% na sua viabilidade em comparação com controles não tratados, como demonstrado pelo ensaio de Azul de Metileno. Investigações posteriores utilizando CHLA-20 revelaram apoptose, com aproximadamente 30% de células positivas para anexina-V, e G0/G1 (> 60%) após tratamento com saliva. O potencial de membrana mitocondrial (Δψm) foi reduzido significativamente (p <0,05), e o citoesqueleto de actina foi desestruturado, como indicado pela microscopia de fluorescência. A viabilidade do fibroblasto humano (células HFF-1), usado como controle não tumoral, diminuiu em aproximadamente 40%. No entanto, não foram observadas alterações na progressão do ciclo celular, morfologia e Δψm nestas células. O presente trabalho fornece novas perspectivas para a caracterização das moléculas presentes na saliva e suas propriedades antitumorais.

“Hairiness” is a Facsimile of Reorganized Cytoskeletons: A Cytopathic Effect of Coxiella burnetii

In 1993, I reported that Coxiella burnetii transforms human B cells into hairy cells (cbHCs), the first hairy cell reported outside of hairy cell leukemia (HCL). Over last few decades, advances in molecular biology have provided evidence supporting that C. burnetii induces hairiness and inhibits the apoptosis of host cells. The present review summarizes new information in support of cbHC. C. burnetii was shown to induce reorganization of the cytoskeleton and to inhibit apoptosis in host cells. Peritoneal B1a cells were found to be permissive for virulent C. burnetii Nine Mile phase I (NMI) strains in mice. C. burnetii severely impaired E-cad expression in circulating cells of Q fever patients. B-cell non-Hodgkin lymphoma was linked to C. burnetii. Mutation of BRAF V600E was pronounced in HCL, but “hairiness” was not linked to the mutation. Risk factors shared among coxiellosis and HCL in humans and animals were reported in patients with Q-fever. Accordingly, I propose that C. burnetii induces reorganization of the cytoskeleton and inhibits apoptosis as cytopathic effects that are not target cell specific. The observed hairiness in cbHC appears to be a fixed image of dynamic nature, and hairy cells in HCL are distinct among lymphoid cells in circulation. As the cytoskeleton plays key roles in maintaining cell structural integrity in health and disease, the pathophysiology of similar cytopathic effects should be addressed in other diseases, such as myopathies, B-cell dyscrasias, and autoimmune syndromes.

JNK/c-Jun signaling pathway mediates arginine vasopressin neuron regeneration by promoting cytoskeleton reconstruction in rats with electrical lesions of the pituitary stalk / 南方医科大学学报

OBJECTIVE@#To investigate the mechanism by which doublecortin promotes the recovery of cytoskeleton in arginine vasopressin (AVP) neurons in rats with electrical lesions of the pituitary stalk (PEL).@*METHODS@#Thirty-two SD rats were randomized into PEL group with electrical lesions of the pituitary stalk through the floor of the skull base (=25) and sham operation group (=7), and the daily water consumption (DWC), daily urine volume (DUV) and urine specific gravity (USG) of the rats were recorded. Four rats on day 1 and 7 rats on each of days 3, 7 and 14 after PEL as well as the sham-operated rats were sacrificed for detection of the expressions of β-Tubulin (Tuj1), doublecortin and caspase- 3 in the AVP neurons of the supraoptic nucleus using immunofluorescence assay and Western blotting.@*RESULTS@#After PEL, the rats exhibited a typical triphasic pattern of diabetes insipidus, with the postoperative days 1-2 as the phase one, days 3-5 as the phase two, and days 6-14 as the phase three. Immunofluorescent results indicated the repair of the AVP neurons evidenced by significantly increased doublecortin expressions in the AVP neurons following PEL; similarly, the expression of Tuj1 also increased progressively after PEL, reaching the peak level on day 7 after PEL. The apoptotic rates of the AVP neurons exhibited a reverse pattern of variation, peaking on postoperative day 3 followed by progressive reduction till day 14. Western blotting showed that the expressions of c-Jun and p-c-Jun were up-regulated significantly on day 3 ( < 0.05) and 7 ( < 0.01) after PEL, while an upregulated p-JNK expression was detected only on day 3 ( < 0.05), as was consistent with the time-courses of neuronal recovery and apoptosis after PEL.@*CONCLUSIONS@#JNK/c-Jun pathway is activated after PEL to induce apoptosis of AVP neurons in the acute phase and to promote the repair of neuronal cytoskeleton by up-regulation of doublecortin and Tuj1 expressions.

Memantine Improves Cognitive Function and Alters Hippocampal and Cortical Proteome in Triple Transgenic Mouse Model of Alzheimer's Disease

Memantine is a non-competitive N-methyl-D-aspartate receptor (NMDAR) antagonist clinically approved for moderate-to-severe Alzheimer's disease (AD) to improve cognitive functions. There is no report about the proteomic alterations induced by memantine in AD mouse model yet. In this study, we investigated the protein profiles in the hippocampus and the cerebral cortex of AD-related transgenic mouse model (3×Tg-AD) treated with memantine. Mice (8-month) were treated with memantine (5 mg/kg/bid) for 4 months followed by behavioral and molecular evaluation. Using step-down passive avoidance (SDA) test, novel object recognition (NOR) test and Morris water maze (MWM) test, it was observed that memantine significantly improved learning and memory retention in 3xTg-AD mice. By using quantitative proteomic analysis, 3301 and 3140 proteins in the hippocampus and the cerebral cortex respectively were identified to be associated with AD abnormalities. In the hippocampus, memantine significantly altered the expression levels of 233 proteins, among which PCNT, ATAXIN2, TNIK, and NOL3 were up-regulated, and FLNA, MARK 2 and BRAF were down-regulated. In the cerebral cortex, memantine significantly altered the expression levels of 342 proteins, among which PCNT, PMPCB, CRK, and MBP were up-regulated, and DNM2, BRAF, TAGLN 2 and FRY1 were down-regulated. Further analysis with bioinformatics showed that memantine modulated biological pathways associated with cytoskeleton and ErbB signaling in the hippocampus, and modulated biological pathways associated with axon guidance, ribosome, cytoskeleton, calcium and MAPK signaling in the cerebral cortex. Our data indicate that memantine induces higher levels of proteomic alterations in the cerebral cortex than in the hippocampus, suggesting memantine affects various brain regions in different manners. Our study provides a novel view on the complexity of protein responses induced by memantine in the brain of AD.

A case of FLNA gene mutation with respiratory insufficiency and periventricular heterotopia

Filamin A is an actin-binding protein and, in humans, is encoded by FLNA gene in the long arm of X chromosome. Filamin A plays a role in the formation of cytoskeleton by crosslinking actin filaments in cytoplasm. FLNA mutations affect cytoskeletal regulatory processes and cellular migrating abnormalities that result in periventricular heterotopia. A 5-month-old girl was hospitalized because of breathing difficulty and was diagnosed as having periventricular heterotopia with laryngomalacia, cricopharyngeal incoordination, pulmonary hypertension, and chronic lung disease. A genetic test was performed to find the cause of periventricular heterotopia, and FLNA gene mutation (c.5998+1G>A) was confirmed for the first time in Korea. After discharge, she developed respiratory failure due to a viral infection at 8 months of her age. In spite of management with mechanical ventilation, she died of pneumothorax and pulmonary hemorrhage. Herein, we report a case of FLNA gene mutation who presented with periventricular nodular heterotopia with respiratory insufficiency.

L-plastin: Structure, Regulation, and Roles in Cancer Invasion and in Macrophages

The cytoskeleton consists of 3 filamentous components: intermediate filaments, microtubules, and actin filaments. Actin filaments continuously assemble and disassemble far out of equilibrium to adapt cells in response to external stimuli. Actin filaments organization and dynamic are controlled by a multitude of actin-binding proteins including actin-bundling proteins. L-plastin, expressed abundantly in lymphocytes and monocytes, is an actin-bundling protein that roles in immune defense and in metastatic invasion of cancer cells. The actin-bundling activity of L-plastin is regulated not only by intracellular calcium concentration, but by phosphorylation of Ser5. The actin-bundling activity of L-pastin decreases by increased calcium concentration but is promoted by phosphorylation of Ser5. The morphology changes and motility of cells requires continuous remodeling of actin filaments which demands the sensitive nature of L-plastin to Ca2+-signal, phosphorylation of Ser5, and probably additional regulation. This review briefly describes the structure and regulation of L-plastin, and roles for L-plastin in cancer invasion and in macrophages.

Mesenchymal Stem Cells Isolated from the Anterior Cruciate Ligament: Characterization and Comparison of Cells from Young and Old Donors

PURPOSE: Mesenchymal stem cells (MSCs) isolated from the anterior cruciate ligament (ACL) share multiple characteristics of bone marrow-derived mesenchymal stem cells (BMSCs), allowing their use for regenerative therapies. Injuries to the ACL can affect people of all ages. This study assesses whether the regenerative potential of ACL-derived MSCs (ACL-MSCs) from old donors is as high as the potential of ACL-MSCs from young donors. MATERIALS AND METHODS: ACL-MSCs were isolated from ACL tissues obtained from young and old donors at the time of ACL reconstruction or arthroplasty. Proliferative capacity, multilineage differentiation potential (chondrogenic, osteogenic, and adipogenic lineages), and transcriptome-wide gene expression were assessed and compared between young and old donors. BMSCs of middle-aged donors served as an additional comparator. RESULTS: No substantial differences between ACL-MSCs from young and old donors were observed in their proliferative capacity and multilineage differentiation potential. The latter did not substantially differ between both ACL-MSC groups and BMSCs. Differential expression of genes related to the cytoskeleton and to protein dephosphorylation amongst other pathways was detected between ACL-MSCs from young and old donors. CONCLUSIONS: Regenerative potential of ACL-MSCs from old donors was not substantially lower than that from young donors, suggesting that regenerative therapies of ACL tears are feasible in both age groups. In vivo studies of the effect of age on the efficacy of such therapies are needed.

Relevant coexpression of STMN1, MELK and FOXM1 in glioblastoma and review of the impact of STMN1 in cancer biology / Coexpressão relevante de STMN1, MELK e FOXM1 em glioblastoma e revisão do impacto de stmn1 na biologia do câncer

OBJECTIVE: To analyze the associated expression of STMN1, MELK and FOXM1 in search of alternative drugable target in glioblastoma (GBM) and to review relevant functional roles of STMN1 in cancer biology. METHOD: STMN1, MELK and FOXM1 expressions were studied by quantitative PCR and their coexpressions were analyzed in two independent glioblastoma cohorts. A review of articles in indexed journals that addressed the multiple functional aspects of STMN1 was conducted, focusing on the most recent reports discussing its role in cancer, in chemoresistance and in upstream pathways involving MELK and FOXM1. RESULTS: Significant associated expressions of MELK and FOXM1 were observed with STMN1 in GBM. Additionally, the literature review highlighted the relevance of STMN1 in cancer progression. CONCLUSION: STMN1 is very important to induce events in cancer development and progression, as cellular proliferation, migration, and drug resistance. Therefore, STMN1 can be an important therapeutic target for a large number of human cancers. In glioblastoma, the most aggressive brain tumor, the MELK/FOXM1/STMN1 presented significant associated expressions, thus pointing MELK and FOXM1 as alternative targets for therapy instead of STMN1, which is highly expressed in normal brain tissue. Continuous functional research to understand the STMN1 signaling pathway is worthwhile to improve the therapeutic approaches in cancer.

OBJETIVO: Analisar as expressões associadas de STMN1, MELK e FOXM1 na procura de alvos alternativos de tratamento em glioblastoma (GBM) e revisar os papeis funcionais relevantes de STMN1 na biologia do câncer. MÉTODO: As expressões de STMN1, MELK e FOXM1 foram estudadas por PCR quantitativo e suas coexpressões foram analisadas em dois coortes independentes de GBM. A revisão dos artigos publicados em revistas indexadas na procura dos aspectos funcionais múltiplos de STMN1 foi conduzida focando-se nos estudos mais recentes discutindo o seu papel em câncer, quimiorresistência e vias de sinalização envolvendo MELK e FOXM1. RESULTADOS: Observou-se expressões associadas significantes de MELK e FOXM1 com STMN1. Adicionalmente, a revisão da literatura salientou a relevância do STMN1 na progressão do câncer. CONCLUSÃO: STMN1 é muito importante nos eventos relacionados ao desenvolvimento e progressão do câncer, como proliferação celular, migração e resistência ao tratamento. Desta forma, STMN1 pode ser um forte alvo terapêutico em um grande número de cânceres humanos. Em GBM, o tumor cerebral mais agressivo, MELK/FOXM1/STMN1 apresentaram significativa associação em suas expressões gênicas, indicando, portanto, MELK e FOXM1 como alvos alternativos para terapia em substituição ao STMN1, que apresenta alta expressão no tecido cerebral normal. Perseverar nos estudos funcionais para o entendimento da via de sinalização do STMN1 é relevante para melhorar os esquemas terapêuticos para câncer.

Avaliação do citoesqueleto e da barreira endotelial pulmonar na malária experimental / Evaluation of the cytoskeleton and pulmonary endothelial barrier in experimental malaria

Infecções por Plasmodium sp. podem levar a um quadro respiratório grave, com complicações pulmonares denominadas lesão pulmonar aguda e síndrome do desconforto respiratório agudo (LPA/SDRA). Inflamação aguda, lesão do endotélio alveolar e do parênquima pulmonar, disfunção e aumento da permeabilidade da barreira alvéolo-capilar e, consequente, formação de edema, caracterizam esta síndrome. O modelo experimental, que utiliza o parasita murino Plasmodium berghei ANKA e camundongos da linhagem DBA/2, é empregado no estudo de mediadores imunológicos e fatores que propiciam o estabelecimento das lesões pulmonares associados à LPA/SDRA. Diversos estímulos podem atuar diretamente no aumento da permeabilidade endotelial por meio da desestabilização dos microtúbulos, rearranjo dos microfilamentos de actina e contração das células endoteliais, via sinalização de Rho-GTPases, causando disfunção da barreira endotelial. Desta forma, este trabalho tem como objetivo avaliar as alterações do citoesqueleto em células endoteliais primárias pulmonares de camundongos DBA/2 (CEPP-DBA/2), as vias de sinalização das principais Rho-GTPases e o estresse oxidativo, causados pela presença de eritrócitos parasitados com esquizontes de P. berghei ANKA (EP-PbA). As CEPP-DBA/2 foram estimuladas com TNF, VEGF ou IFNγ, em diferentes tempos de exposição, seguido da incubação com EP-PbA. Assim, foram realizados ensaios de imunofluorescência para análise do rearranjo de microfilamentos de actina e da desestabilização de microtúbulos. As vias de sinalização das Rho-GTPases foram avaliadas por Western blot, para as expressões proteicas de RhoA, Cdc42 e MLC. Além disso, ensaio fluorométrico foi realizado para detectar a produção de espécies reativas de oxigênio, resultantes do estímulo com eritrócitos parasitados. CEPP-DBA/2 estimuladas por EP-PbA, VEGF, TNF ou IFNγ, em associação ou não, apresentaram alterações morfológicas nos microfilamentos de actina e aumento dos espaços interendoteliais. Imagens de imunofluorescência também mostram desestabilização de microtúbulos e desfosforilação de FAK, causadas por EP-PbA. Os ensaios de permeabilidade validam que os eritrócitos parasitados com formas maduras de P. berghei induziram aumento da permeabilidade microvascular nas CEPP-DBA/2. Além disso, estas células, estimuladas com EP-PbA, demonstraram elevada produção de espécies reativas de oxigênio (EROs), o que pode estar contribuindo com o desenvolvimento de estresse oxidativo e com a injúria endotelial, assim como, com o aumento da permeabilidade vascular. O mais interessante é que estas alterações endoteliais podem estar relacionadas ao aumento da razão RhoA/Cdc42, da expressão proteica de MLC fosforilada e do sinal de ativação de RhoA. Em conjunto, estes resultados mostram envolvimento dos eritrócitos parasitados com esquizontes de Plasmodium berghei ANKA na desorganização do citoesqueleto e na disfunção da barreira alvéolo-capilar, via RhoA/Rho-kinase, o que pode estar contribuindo com a patogênese da LPA/SDRA associada à malária

Infections by Plasmodium sp. can lead to a serious respiratory condition with pulmonary complications, named acute lung injury and acute respiratory distress syndrome (ALI/ARDS). Acute inflammation, alveolar endothelium and lung parenchyma injuries, dysfunction and increased permeability of the pulmonary alveolar-capillary barrier and consequent formation of edema characterize this syndrome. Several stimuli can directly increase endothelial permeability through actin microfilaments rearrangement, via Rho- GTPases signaling, leading to endothelial barrier dysfunction. DBA/2 mice infected with Plasmodium berghei ANKA develop ALI/ARDS similar to that observed in humans. The purpose of this research was to assess cytoskeletal changes in DBA/2 mice primary microvascular lung endothelial cells (PMLEC), verify the signaling pathways of the Rho- GTPases and analyze the oxidative stress on these cells in the presence of P. berghei ANKA-infected red blood cells (PbA-iRBC). PMLEC were stimulated by TNF, VEGF or IFNγ followed by incubation with PbA-iRBC. Immunofluorescence assays were performed to analyze actin microfilaments rearrangement and microtubules destabilization. Western blot for RhoA, Cdc42 and MLC proteins were conducted to assess alterations in signaling pathways of Rho-GTPases. In addition, a fluorimetric assay was performed to detect the production of reactive oxygen species resulting from PbA-iRBC stimulus. P. berghei ANKA, VEGF, TNF and IFNγ stimuli, in association or not, caused morphological disturbances in actin microfilaments of PMLEC and an increase of intercellular spaces. Moreover, immunofluorescence images showed microtubules destabilization and FAK dephosphorylation in these cells, caused by PbA-iRBC. The permeability assay showed that PbA-iRBC induced an increase of microvascular permeability in PMLEC. In addition, PMLEC stimulated by PbA-iRBC, showed elevated production of ROS, which may be contributing to oxidative stress and increasing the damage of endothelial cells, as well as an increase of vascular permeability. Interestingly, these endothelial changes may be related to the increased RhoA/Cdc42 protein expressions ratio, augmented protein expression of phosphorylated MLC and RhoA activation signal. Taken together, these data demonstrate the involvement of P. berghei ANKA-infected red blood cells in cytoskeleton disorganization and alveolar-capillary barrier dysfunction, through of RhoA / Rho-kinase signaling pathway, which may contribute to ALI/ARDS pathogenesis

Outer dense fiber 2 and sperm function: Progress in studies / 中华男科学杂志

Oligoasthenozoospermia, teratozoospermia or low sperm motility is the main cause of male infertility. Low sperm motility can be induced by abnormalities of the sperm tail structure and sperm function. The outer dense fiber protein 2 (ODF2) is a protein fiber maintaining cytoskeleton, as a major component of the mammalian sperm tail and centrosome, and its abnormality is closely related to asthenospermia. Recent studies indicate that ODF2 includes many proteins of the same name and homologous splices located in the sperm centrosomes and spindles of cleaved-embryos, necessary for animal ciliogenesis and associated with sperm capacitation. The features of ODF2 indicate that it is not a single-structural protein. This paper reviews the known functions of ODF2, paving a ground for further studies of the relationship between the ODF2 protein and fertilization.

Cyclic Stretch Effects on Adipose-Derived Stem Cell Stiffness, Morphology and Smooth Muscle Cell Gene Expression

Recent investigations consider adipose-derived stemcells (ASCs) as a promising source of stemcells for clinical therapies. To obtain functional cells with enhanced cytoskeleton and aligned structure, mechanical stimuli are utilized during differentiation of stem cells to the target cells. Since function of muscle cells is associated with cytoskeleton, enhanced structure is especially essential for these cells when employed in tissue engineering. In this study by utilizing a custom-made device, effects of uniaxial tension (1Hz, 10% stretch) on cytoskeleton, cell alignment, cell elastic properties, and expression of smooth muscle cell (SMC) genes in ASCs are investigated.Due to proper availability ofASCs, results can be employed in cardiovascular engineeringwhen production of functional SMCs in arterial reconstruction is required. Results demonstrated that cells were oriented after 24 hours of cyclic stretch with aligned pseudo-podia. Staining of actin filaments confirmed enhanced polymerization and alignment of stress fibers. Such phenomenon resulted in stiffening of cell body which was quantified by atomic force microscopy (AFM). Expression of SM α-actin and SM22 α-actin as SMC associated genes were increased after cyclic stretch while GAPDH was considered as internal control gene. Finally, it was concluded that application of cyclic stretch on ASCs assists differentiation to SMC and enhances functionality of cells.