Analysis and Review of Downregulated Actin Cytoskeletal Proteins in Non-Small Cell Lung Cancer

Actin, a highly conserved protein, plays a dominant role in Non-small cell lung cancer (NSCLC). Late diagnosis and the aggressive nature of NSCLC pose a significant threat. Studying the clinic pathological properties of NSCLC proteins is a potential alternative for developing treatment strategies. Towards this, 35 downregulated actin cytoskeletal proteins on NSCLC prognosis and treatment were studied by examining their protein-protein interactions, gene ontology enrichment terms, and signaling pathways. Using PubMed, various proteins in NSCLC were identified. The protein-protein interactions and functional associations of these proteins were examined using the STRING database. The focal adhesion signaling pathway was selected from all available KEGG and Wiki pathways because of its role in regulating gene expression, facilitating cell movement and reproduction, and significantly impacting NSCLC. The protein-protein interaction network of the 35 downregulated actin cytoskeleton proteins revealed that ACTG1, ACTR2, ACTR3, ANXA2, ARPC4, FLNA, TLN1, CALD1, MYL6, MYH9, MYH10, TPM1, TPM3, TPM4, PFN1, IQGAP1, MSN, and ZXY exhibited the highest number of interactions. Whereas HSPB1, CTNNA1, KRT17, KRT7, FLNB, SEPT2, and TUBA1B displayed medium interactions, while UTRN, TUBA1B, and DUSP23 had relatively fewer interactions. It was discovered that focal adhesions are critical in connecting membrane receptors with the actin cytoskeleton. In addition, protein kinases, phosphatases, and adapter proteins were identified as key signaling molecules in this process, greatly influencing cell shape, motility, and gene expression. Our analysis shows that the focal adhesion pathway plays a crucial role in NSCLC and is essential for developing effective treatment strategies and improving patient outcomes.

Effect of glycosides of Cistanche on the expression of mitochondrial precursor protein and keratin type Ⅱ cytoskeletal 6A in a rat model of vascular dementia

Glycosides of Cistanche（GC）is a preparation used extensively for its neuroprotective effect against neurological diseases,but its mechanisms of action remains incompletely understood.Here,we established a bilateral common carotid artery occlusion model of vascular dementia in rats and injected the model rats with a suspension of GC（10 mg/kg/day,intraperitoneally）for 14 consecutive days.Immunohistochemistry showed that GC significantly reduced p-tau and amyloid beta（Aβ）immunoreactivity in the hippocampus of the model rats.Proteomic analysis demonstrated upregulation of mitochondrial precursor protein and downregulation of keratin type II cytoskeletal6A after GC treatment compared with model rats that had received saline.Western blot assay confirmed these findings.Our results suggest that the neuroprotective effect of GC in vascular dementia occurs via the promotion of neuronal cytoskeleton regeneration.

Effect of focal adhesion kinase on cytoskeletal arrangement of HepG2 cells induced by hypoxia

Objective： To study focal adhesion kinase （FAK） expression in hypoxic HepG2 cells and the effect of FAK siRNA on cytoskeletal arrangement of HepG2 cells induced by hypoxia. Methods： HepG2 cells were cultured in 21% O2 and 1% O2. Morphological changes were observed after hypoxia treatment. Western blot was used to measure FAK expression. The siRNA expression vector pshRNA-FAK targeting the mRNA of FAK and vector pGensil-2 （as a control） were constructed, and then transfected into HepG2 cells. Western blot was used to detect FAK. The cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by phalloidin. The migratory ability of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was analyzed by cell migration assay. Results： Hypoxia-treated cells displayed a more elongated shape with a large degree of cell detachment. FAK expression increased in hypoxic HepG2 cells. FAK protein level was decreased by 75.64% ± 3.12% （P 〈 0.01） after the pshRNA-FAK transfection. Hypoxia induced cytoskeletal arrangement of HepG2 cells. However, cytoskeletal arrangement of HepG2 cells transfected with pshRNA-FAK induced by hypoxia was inhibited in 1% O2. As cell migration assay showed, the migrating number of HepG cells transfected with pshRNA-FAK was significantly lower than that of control （P 〈 0.05）. Conclusion： The expression of FAK in hypoxic HCC might have a close relationship to the cytoskeletal arrangement of HepG2 cells induced by hypoxia. Up-regulation of FAK expression may be one of mechanisms of cytoskeletal arrangement and invasion of hepatocellular carcinoma induced by hypoxia.

Acrylamide Alters Cytoskeletal Protein Level in Rat Serum

To study biomarker of acrylamide （ACR） induced neuropathy, Wistar rats received 20 or 40 mg/kg of ACR by ip injection and the levels of light neurofilament （NF-L）, middle NF （NF-M）, heavy NF （NF-H）, β-actin, α-tubulin, and β-tubulin proteins in serum were evaluated using both SDS-PAGE and Western blotting. Compared to controls, NF-L and NF-M decreased,

Distinct Cytoskeletal Injuries Induced by As,Cd,Co,Cr,and Ni Compounds

The risks of metal compounds to human health are highlighted by the ubiquity of exposure and their persistence in the environment.Although compounds of As,Cd,Co,Cr,and Ni are known or“reasonably anticipated”to be carcinogenic to humans and/or experimental animals, the cellular targets of these health hazards and the underlying mechanisms of their carcinogenic- ity are still unclear.We show in this report that dramatic,time-and dose-dependent cytoskeletal perturbations,especially in the distribution and organization patterns of microtubules and mi- crofilaments,two of the principal components of the cytoskeleton,occurred in 3T3 cells upon exposure to these metal salts.Each metal salt appeared to induce a different,typical pattern of cytoskeletal injury,probably reflecting the specificity of action of each metal ion.These results suggest that the cytoskeleton can indeed act as a target for injury by epigenetic carcinogenic metal compounds in the environment.These findings should help our efforts to understand the mechanisms of action of metal compounds at the subcellular and molecular levels.1989 Academic Press,Inc.

Gene expression of 49 kDa apyrase,cytoskeletal proteins,ATPase,ADPase and amino acid contents of Pisum sativum(L.)cells germinated in Euryops arabicus(Steud.ex Jaub.&Spach)water extract

The present research reports of quick and marked changes induced by plant extract of Euryops arabicus in the gene expression of 49-kDa apyrases,cytoskeletal proteins,ATPases,ADPase and amount of amino acid of pea(Pisum sativum L.var.Alaska).Pellets of cytoskeletals proteins(27000 xg)were probed with anti-apyrase antibody,biotinylated anti-rat,actin and alpha and beta-tubulin for Western blotting.ATPase and ADPase activities were determined based on the hydrolytic efficacy of adenine triphosphate and adenine diphosphate.By 72 hours,the abundance of apyrases,cytoskeletal proteins and amount of amino acid in pellets of 27000 xg of germinated pea seeds in E.arabicus extracts were sharply increased than those sown in distilled water.All the samples exhibited that the stems had more amount from apyrases,cytoskeletal proteins,amino acids and ATPase and ADPase activities than primary leaves and primary roots that were germinated either on E.arabicus water extract or in distilled water.Based on the enzyme’s capability to hydrolyse nucleotide triphosphate and nucleotide diphosphate as well as the direct association between expression of 49-kDa apyrase and cytoskeletal proteins,E.arabicus water extract had an important effect on plant germinations.

Thermal Fluctuation Induced Piezoelectric Effect in Cytoskeletal Microtubules: Model for Energy Harvesting and Their Intracellular Communication

Cytoskeletal microtubules have long been conjectured to have piezoelectric properties. They have been shown to behave as nematic liquid crystals which oscillate along their director axis due to the prevalent thermal fluctuations. In this work, we develop a theoretical model of the mechanics of microtubules in the cytosolic space based on the buckling of its structure due to these thermal fluctuations. This cytosolic space has been considered as a viscoelastic medium in which microtubule oscillations have been considered. As a result of resilience of cytosol and neighbouring filaments from the axial force due to thermal fluctuations, the surface traction acting laterally on the microtubule structure has been further used to elucidate its piezoelectric behaviour in vivo. After the piezoelectric properties induced by thermal fluctuations (in addition to the buckling) of microtubules have been discussed, we propose a model discussing how microtubules behave as energy harvesters and communicate via electromagnetic radiation, with each other, in an intracellular environment.

IgA1 from HSP Patients Trigger Apoptosis and Inhibit Cytoskeletal Proteins in HUVEC

Background: Henoch-Schonlein purpura (HSP) is a kind of systemic small vessel vasculitis in children. Endothelium cells injury induced by IgA1 is considered important in the pathogenesis of HSP. Research found that the apoptosis of vein endothelial cells was related to the vasculitis in HSP patients. Purpose: To observe the effect of IgA1 from HSP patients on the apoptosis of HUVEC and firstly analyze the mechanism of the apoptosis of HUVEC induced by IgA1. Methods: HUVECs were cultured in 3 different conditional media with IgA1 from HSP patients, normal healthy children and simply medium (blank control). Serum IgA1 was purified by jacalin affinity chromatography. The rates of apoptosis in HUVEC incubated with IgA1 were determined by TUNEL method and flow cytometry, respectively. The expression of the cytoskeletal proteins, such as FAK, Vinculin and MLCK was detected with the methods of Real-time PCR and Westernblot, respectively. Results: The present study showed that the apoptosis rate of HUVEC by IgA1 isolated from HSP patients was higher than blank control (14.77% ± 2.23% vs 2.25% ± 0.77%) (P < 0.01) and the rate of HUVEC by IgA1 from normal healthy children was higher than blank control (9.97% ± 1.48% vs 2.25% ± 0.77%) (P < 0.01). The cytoskeletal proteins, such as FAK, Vinculin and MLCK expression were down-regulated in HUVEC co-cultured with IgA1 isolated from HSP patients for 24h. Conclusion: These findings firstly on IgA1 from HSP patients may induce apoptosis of vascular endothelial cells through inhibiting the cytoskeletal proteins expression. IgA1 may accelerate progression of HSP by inducing apoptosis of vascular endothelial cells.

Towards a better recording of microtubule cytoskeletal spatial organization and dynamics in plant cells

Numerous fluorescent marker lines are currently available to visualize microtubule(MT)architecture and dynamics in living plant cells, such as markers expressing p35S::GFP-MBD or p35S::GFP-TUB6.However, these MT marker lines display obvious defects that affect plant growth or produce unstable fluorescent signals. Here, a series of new marker lines were developed, including the pTUB6::VisGreen-TUB6-expressing line in which TUB6 is under the control of its endogenous regulatory elements and e GFP is replaced with VisGreen, a brighter fluorescent protein. Moreover, two different markers were combined into one expression vector and developed two dual-marker lines.These marker lines produce bright, stable fluorescent signals in various tissues, and greatly shorten the screening process for generating dual-marker lines.These new marker lines provide a novel resource for MT research.

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.

Reorganization of cytoskeletal proteins of mouse oocytes mediated by integrins

To study whether integrins on cell membrane ligate with intracellular cytoskeletal proteins and mediate their reorganization in egg activation, female mice were used for su- perovulation. The zona-free oocytes were incubated separately with specific ligand of integrins, an active RGD peptide, in vitro for certain period of time. RGE peptide and mouse capacitated sperm were used as controls. Freshly ovulated oocytes and those treated with different factors were immunostained with FITC-labeled anti-actin antibody, then detected with confocal micro- scope. The results demonstrated that freshly ovulated mouse oocytes, oocytes incubated for 2 h in vitro and those treated with control RGE peptide for 15 min showed hardly visible fluorescene or only thin fluorescence in plasma membrane region. Oocytes coincubated with sperms for 15 min and those treated with active RGD peptide for 10 min, 30 min and 2 hours respectively had strong and thick fluorescence in the plasma membrane and cortical region of oocytes, and some of them showed asymmetrically fluorescent distribution. It is proved that integrins on membrane are ligated directly with cytoskeletal protein. Integrins binding with their ligands regulate reor- ganization of cytoskelal protein, which may be involved in transmembrane signaling in egg acti- vation.

Use of artificial neural networks to identify and analyze polymerized actin-based cytoskeletal structures in 3D confocal images

Background:Living cells need to undergo subtle shape adaptations in response to the topography of their substrates.These shape changes are mainly determined by reorganization of their internal cytoskeleton,with a major contribution from filamentous(F)actin.Bundles of F-actin play a major role in determining cell shape and their interaction with substrates,either as“stress fibers,”or as our newly discovered“Concave Actin Bundles”(CABs),which mainly occur while endothelial cells wrap micro-fibers in culture.Methods:To better understand the morphology and functions of these CABs,it is necessary to recognize and analyze as many of them as possible in complex cellular ensembles,which is a demanding and time-consuming task.In this study,we present a novel algorithm to automatically recognize CABs without further human intervention.We developed and employed a multilayer perceptron artificial neural network(“the recognizer”),which was trained to identify CABs.Results:The recognizer demonstrated high overall recognition rate and reliability in both randomized training,and in subsequent testing experiments.Conclusion:It would be an effective replacement for validation by visual detection which is both tedious and inherently prone to errors.

Tetrandrine targeting SIRT5 exerts anti-melanoma properties via inducing ROS, ER stress, and blocked autophagy

Tetrandrine(TET),a natural bisbenzyl isoquinoline alkaloid extracted from Stephania tetrandra S.Moore,has diverse pharmacological effects.However,its effects on melanoma remain unclear.Cellular prolif-eration assays,multi-omics analyses,and xenograft models were used to determine the effect of TET on melanoma.The direct target of TET was identified using biotin-TET pull-down liquid chromatograph-mass spectrometry(LC-MS),cellular thermal shift assays,and isothermal titration calorimetry(ITC)analysis.Our findings revealed that TET treatment induced robust cellular autophagy depending on activating transcription factor 6(ATF6)-mediated endoplasmic reticulum(ER)stress.Simultaneously,it hindered autophagic flux by inducing cytoskeletal protein depolymerization in melanoma cells.TET treatment resulted in excessive accumulation of reactive oxygen species(Ros)and simultaneously triggered mitophagy.Sirtuin 5(SIRT5)was ultimately found to be a direct target of TET.Mechanistically,TET led to the degradation of SIRT5 via the ubiquitin(Ub)-26S proteasome system.SIRT5 knockdown induced ROS accumulation,whereas SIRT5 overexpression attenuated the TET-induced ROS accumula-tion and autophagy.Importantly,TET exhibited anti-cancer effects in xenograft models depending on SIRT5 expression.This study highlights the potential of TET as an antimelanoma agent that targets SIRT5.These findings provide a promising avenue for the use of TET in melanoma treatment and underscore its potential as a therapeutic candidate.

Establishment and characterization of a rat pancreatic stellate cell line by spontaneous immortalization

AIM: Activated pancreatic stellate cells (PSCs) have been implicated in the pathogenesis of pancreatic fibrosis and inflammation. Primary PSCs can be subcultured only several times because of their limited growth potential. A continuous cell line may therefore be valuable in studying molecular mechanisms of these pancreatic disorders. The aim of this study was to establish a cell line of rat PSCs by spontaneous immortalization.METHODS: PSCs were isolated from the pancreas of male Wistar rats, and conventional subcultivation was performed repeatedly. Telomerase activity was measured using the telomere repeat amplification protocol. Activation of transcription factors was assessed by electrophoretic mobility shift assay.Activation of mitogen-activated protein (MAP) kinases was examined by Western blotting using anti-phosphospecific antibodies. Expression of cytokine-induced neutrophil chemoattractant-1 was determined by enzyme immunoassay.RESULTS: Conventional subcultivation yielded actively growing cells. One clone was obtained after limiting dilution,and designated as SIPS. This cell line has been passaged repeatedly more than 2 years, and is thus likely immortalized.SIPS cells retained morphological characteristics of primary,culture-activated PSCs. SIPS expressed α-smooth muscle actin, glial acidic fibrillary protein, vimentin, desmin, type Ⅰ collagen, fibronectin, and prolyl hydroxylases. Telomerase activity and p53 expression were negative. Proliferation of SIPS cells was serum-dependent, and stimulated with platelet-derived growth factor-BB through the activation of extracellular signal-regulated kinase. Interleukin-1β activated nuclear factor-κB, activator protein-1, and MAP kinases.Interleukin-1β induced cytokine-induced neutrophil chemoattractant-1 expression through the activation of nuclear factor-κB and MAP kinases.CONCLUSION: SIPS cells can be useful for in vitro studies of cell biology and signal transduction of PSCs.

Muscularis mucosae in desmoplastic stroma formation of early invasive rectal adenocarcinoma

The origin of myofibroblasts or myofibroblastic cells in the desmoplastic stroma associated with carcinoma invasion has been controversial. In the early invasive area of a rectal adenocarcinoma reported here, an obvious transition between the muscularis mucosa and the bundles of eosinophilic stromal cells observed in the carcinomatous stroma was demonstrated both in morphology and in their cytoskeletal phenotype, which conceivably suggests that the smooth muscle cells of the muscularis mucosa could convert to the eosinophilic stromal cells, namely myofibroblasts. Moreover, type I procollagen was demonstrated in both protein and mRNA levels in the areas of eosinophilic stromal cells with a lesser degree of differentiated smooth muscle phenotype that showed a transition from the muscularis mucosa, implying that the myofibroblastic cells converted from smooth muscle cells of the muscularis mucosa could be responsible for type I collagen production. These findings suggest that the muscularis mucosae may not be a passive barrier through which colorectal carcinomas infiltrate into the submucosa, but may play an active role in the formation and remodeling of tumor stroma.

The expressions and significance of α-,β-catenins and cyclin D1 in breast cancers

Objective： To study the relationship between expressions of α-, β-catenins and cyclin D1 and the occurrence, infiltration and metastasis of breast cancer. Methods： High sensitive S-P immunohistochemical method was used to detect the protein expressions of α-, β-catenins and cyclin D1 in the 60 cases of breast cancer tissues. Results： Abnormal immunoreactivities of α- and β-catenins were observed in 37 （61.7%） and 42 （70%） cases of breast cancer tissues, respectively. There were 28 cases （46.7%） who showed cyclin D1 overexpression. The abnormal expression rates of α- and β-catenins in infiltrating Iobular carcinoma （ILC） were significantly higher than those in infiltrating ductal carcinoma （IDC） （P 〈 0.05）, but they had no relations to the extent of differentiation and lymphatic metastasis of breast cancer （P 〉 0.05）. The overexpression rate of cyclin D1 was correlated with tumor stage and lymphatic metastasis of breast cancer （P 〈 0.05）, but not with histological type and the extent of differentiation （P 〉 0.05）. Cyclin D1 overexpression was observed in 57.1% （24/42） of these cases that showed abnormal staining of β-catenin, but only observed in 22.2% （4/18） of these cases with normal membranous staining of β-catenin. There was a significantly positive correlation between the abnormal expression of β-catenin and overexpression of cyclin D1 （r, = 0.321, P 〈 0.05）. Conclusion： The abnormal expression of β-catenin may play an important role in the genesis of breast cancer by triggering cyclin D1 overexpression in breast cancer. The abnormal expressions of α- and β-catenins are not a key factor in malignant cell metastasis in breast cancer, but may also involve in the progress.

Synthesized flavanoid-derived ligand reduced dengue virus type-2 replication in vitro

Objective: To investigate the antiviral property of a lead ligand, YK51 that was synthesized based on the flavanoid of a natural product toward dengue virus type-2(DENV2)replication.Methods: c RNA was isolated from HepG2 cells inoculated with 1 000 median tissue culture infective dose of DENV2 and treated with different doses of the ligand followed by RT-PCR to quantify the virus gene copies. Confocal microscopy of actin and tubulin redistribution was also performed.Results: The quantitative RT-PCR result showed reduction of the DENV2 gene copies as the ligand concentration was increased. The confocal microscopy result showed increase in the tubulin intensity(79.6%) of infected BHK21 cells treated with the ligand,compared with the non-treated cells(54.8%). The 1.5-fold increase in the intensity of tubulin suggested that the ligand inhibitory effect stabilized the cellular microtubule structure.Conclusions: The synthesized ligand YK51 reduced DENV2 viral load by inhibiting virus replication thus is highly potential to be developed as antiviral agent.

Chemical Physics in Living Cells-using Light to Visualize and Control Intracellular Signal Transduction

Cells are crowded microenvironments filled with macromolecules undergoing constant phys- ical and chemical interactions. The physicochemical makeup of the cells aff）cts various cellular responses, determines cell-cell interactions and influences cell decisions. Chemical and physical properties diff）r between cells and within cells. Moreover, these properties are subject to dynamic changes in response to environmental signals, which often demand adjustments in the chemical or physical states of intracellular molecules. Indeed, cellular responses such as gene expression rely on the faithful relay of information from the outside to the inside of the cell, a process terrned signal transduction. The signal often traverses a complex path across subcellular spaces with variable physical chemistry, sometimes even influencing it. Understanding the molecular states of such signaling molecules and their intracellular environments is vital to our understanding of the cell. Exploring such intricate spaces is possible today largely because of experimental and theoretical tools. Here, we focus on one tool that is commonly used in chemical physics studies light. We summarize recent work which uses light to both visualize the cellular environment and also control intracel- lular processes along the axis of signal transduction. We highlight recent accomplishments in optical microscopy and optogenetics, an emerging experimental strategy which utilizes light to control the molecular processes in live cells. We believe that optogenetics lends un- precedented spatiotemporal precision to the manipulation of physicochemical properties in biological contexts. We hope to use this work to demonstrate new opportunities for chemical physicists who are interested in pursuing biological and biomedical questions.

Impact of chitosan-based nanocarriers on cytoskeleton dynamics:Current status and challenges

Chitosan-based nanocarriers(CS-NCs)show a promising role in improving drugs and bioactive compounds delivery for therapy.However,the effects exerted by CS-NCs at the cellular level,including their recognition and uptake,have not been fully investigated yet.Many factors,including size,shape,concentration,and surface chemistry of CS-NCs,play an important role in determining the types of intracellular signals triggered.The mechanism of uptake and the involvement of the cytoskeleton during the CS-NCs endocytosis variates among the different cell types as well as further effects observed inside cells.In the present work,we discuss the effects induced by CS-NCs per se on the cytoskeleton,a key component in cell architecture and physiology.The focus of this report is made on tumoral and normal biological models in which CS-NCs could differentially affect the cell cytoskeleton.The recent years reports regarding the impact of CS-NCs on cytoskeleton dynamics and the current techniques for its evaluation are summarized and discussed.Understanding mechanisms underlying cytoskeletal impact after cell exposure to CS-NCs is critical for the design of safest value-added formulations in the biomedical field.Furthermore,this revision points out some interesting aspects of cytoskeletal changes and cell death encompassing anti-tumoral effects.

Protective Effect of Space Ginseng Yeast on Human Primary Fibroblasts under Simulated Microgravity Condition

[Objectives] To study the protective effect of Space Ginseng Yeast on human primary fibroblasts. [Methods] Multiple indicators were used to simulate the microgravity effect,so as to study the protective effect of Space Ginseng Yeast on human primary fibroblasts under simulated microgravity condition. [Results]Space Ginseng Yeast could improve changes of cell morphology and decline of the survival rate caused by microgravity,restore the expression and effective arrangement of cytoskeletal protein Tubulin,extracellular matrix proteins Integrin and Fibronectin,and can inhibit the abnormal expression of MMP-1. The protective effect of Space Ginseng Yeast diluted 20-500 times is better,and the protective effect of Space Ginseng Yeast diluted 100 times is the most obvious. [Conclusions] Space Ginseng Yeast can protect damage of human primary fibroblasts caused by simulated microgravity.