Easy recognition and high autoimmune hepatitis specificity of smooth muscle antibodies giving an actin microfilament immunofluorescent pattern on embryonal vascular smooth muscle cells.

Smooth muscle antibodies (SMA) with anti-microfilament actin (MF-SMA) specificity are regarded as highly specific markers of type 1 autoimmune hepatitis (AIH-1) but their recognition relying on immunofluorescence of vessel, glomeruli, and tubules (SMA-VGT pattern) in rodent kidney-tissue, is restricted by operator-dependent interpretation.A gold standard method for their identification is not available. We assessed and compared the diagnostic accuracy for AIH-1 of an embryonal-aorta vascular-smooth-muscle(VSM) cell line-based assay with those of the rodent-tissue based assay for the detection of MF-SMA pattern in AIH-1 patients and controls. Sera from 138 AIH-1 patients and 295 controls (105 primary biliary cholangitis,40 primary sclerosing cholangitis,50 chronic viral hepatitis,20 alcohol-related liver disease,40 steatotic liver disease,and 40 healthy controls) were assayed for MF-SMA and for SMA-VGT using VSM-based and rodent tissue-based assays, respectively. MF-SMA and SMA-VGT were found in 96(70%) and 87(63%) AIH-1 patients, and 2 controls (p<0.0001).Compared with SMA-VGT, MF-SMA showed similar specificity (99%), higher sensitivity (70% vs 63%,p=ns) and likelihood ratio for a positive test (70 vs 65). Nine (7%) AIH-1 patients were MF-SMA positive despite being SMA-VGT negative. Overall agreement between SMA-VGT and MF-SMA was 87% (kappa coefficient 0.870,[0.789-0.952]). MF-SMA were associated with higher serum γ-globulin [26(12-55) vs 20 g/l(13-34),p<0.005] and immunoglobulin G (IgG) levels [3155(1296-7344) vs 2050 mg/dl(1377-3357), p<0.002]. The easily recognizable IFL MF-SMA pattern on VSM cells strongly correlated with SMA-VGT and has an equally high specificity for AIH-1. Confirmation of these results in other laboratories would support the clinical application of the VSM cell-based assay for reliable detection of AIH-specific SMA.

Role of Cytoskeletal Elements in Regulation of Synaptic Functions: Implications Toward Alzheimer's Disease and Phytochemicals-Based Interventions.

Alzheimer's disease (AD), a multifactorial disease, is characterized by the accumulation of neurofibrillary tangles (NFTs) and amyloid beta (Aß) plaques. AD is triggered via several factors like alteration in cytoskeletal proteins, a mutation in presenilin 1 (PSEN1), presenilin 2 (PSEN2), amyloid precursor protein (APP), and post-translational modifications (PTMs) in the cytoskeletal elements. Owing to the major structural and functional role of cytoskeletal elements, like the organization of axon initial segmentation, dendritic spines, synaptic regulation, and delivery of cargo at the synapse; modulation of these elements plays an important role in AD pathogenesis; like Tau is a microtubule-associated protein that stabilizes the microtubules, and it also causes inhibition of nucleo-cytoplasmic transportation by disrupting the integrity of nuclear pore complex. One of the major cytoskeletal elements, actin and its dynamics, regulate the dendritic spine structure and functions; impairments have been documented towards learning and memory defects. The second major constituent of these cytoskeletal elements, microtubules, are necessary for the delivery of the cargo, like ion channels and receptors at the synaptic membranes, whereas actin-binding protein, i.e., Cofilin's activation form rod-like structures, is involved in the formation of paired helical filaments (PHFs) observed in AD. Also, the glial cells rely on their cytoskeleton to maintain synaptic functionality. Thus, making cytoskeletal elements and their regulation in synaptic structure and function as an important aspect to be focused for better management and targeting AD pathology. This review advocates exploring phytochemicals and Ayurvedic plant extracts against AD by elucidating their neuroprotective mechanisms involving cytoskeletal modulation and enhancing synaptic plasticity. However, challenges include their limited bioavailability due to the poor solubility and the limited potential to cross the blood-brain barrier (BBB), emphasizing the need for targeted strategies to improve therapeutic efficacy.

Characterization of Intrinsically Disordered Proteins in Healthy and Diseased States by Nuclear Magnetic Resonance.

Intrinsically Disordered Proteins (IDPs) are active in different cellular procedures like ordered assembly of chromatin and ribosomes, interaction with membrane, protein, and ligand binding, molecular recognition, binding, and transportation via nuclear pores, microfilaments and microtubules process and disassembly, protein functions, RNA chaperone, and nucleic acid binding, modulation of the central dogma, cell cycle, and other cellular activities, post-translational qualification and substitute splicing, and flexible entropic linker and management of signaling pathways. The intrinsic disorder is a precise structural characteristic that permits IDPs/IDPRs to be involved in both one-to-many and many-to-one signaling. IDPs/IDPRs also exert some dynamical and structural ordering, being much less constrained in their activities than folded proteins. Nuclear magnetic resonance (NMR) spectroscopy is a major technique for the characterization of IDPs, and it can be used for dynamic and structural studies of IDPs. This review was carried out to discuss intrinsically disordered proteins and their different goals, as well as the importance and effectiveness of NMR in characterizing intrinsically disordered proteins in healthy and diseased states.

Plasmonic cellulose microfilament assisted SERS detection in microfluidics.

Limited by the narrow enhanced area of nanoscale on the metal surface, the sensitivity of surface-enhanced Raman spectroscopy (SERS) detection in solution is usually much lower than the detection in a solid substrate, which is dramatic in microfluidics for online detection. In this work, a cellulose microfilament embraced by Ag nanoparticles, called plasmonic cellulose microfilament, is located in a microchannel for SERS detection in microfluidics. Benefiting from the congestion caused by the plasmonic cellulose microfilament in a microchannel, the trace molecule in the solution is much easier to gather in Ag nanoparticles for Raman enhancement. To obtain high sensitivity, the structure of plasmonic cellulose microfilament is optimized. The SERS spectra collected in this novel microfluidics demonstrate that the plasmonic cellulose microfilament presents a high sensitivity at 10-13 M and good reproducibility in SERS detection. In addition, automatic identification of urea presence or absence was achieved based on deep learning (DL) here. The results show excellent diagnostic accuracy (99 %), which suggests that a fast, label-free urea screening tool can be developed. These results point out this SERS microfluidics with plasmonic cellulose microfilament has a great application prospective in online SERS detection with high sensitivity.

Research progress on the roles of actin-depolymerizing factor in plant stress responses.

Actin-depolymerizing factors (ADFs) are highly conserved small-molecule actin-binding proteins found throughout eukaryotic cells. In land plants, ADFs form a small gene family that displays functional redundancy despite variations among its individual members. ADF can bind to actin monomers or polymerized microfilaments and regulate dynamic changes in the cytoskeletal framework through specialized biochemical activities, such as severing, depolymerizing, and bundling. The involvement of ADFs in modulating the microfilaments' dynamic changes has significant implications for various physiological processes, including plant growth, development, and stress response. The current body of research has greatly advanced our comprehension of the involvement of ADFs in the regulation of plant responses to both biotic and abiotic stresses, particularly with respect to the molecular regulatory mechanisms that govern ADF activity during the transmission of stress signals. Stress has the capacity to directly modify the transcription levels of ADF genes, as well as indirectly regulate their expression through transcription factors such as MYB, C-repeat binding factors, ABF, and 14-3-3 proteins. Furthermore, apart from their role in regulating actin dynamics, ADFs possess the ability to modulate the stress response by influencing downstream genes associated with pathogen resistance and abiotic stress response. This paper provides a comprehensive overview of the current advancements in plant ADF gene research and suggests that the identification of plant ADF family genes across a broader spectrum, thorough analysis of ADF gene regulation in stress resistance of plants, and manipulation of ADF genes through genome-editing techniques to enhance plant stress resistance are crucial avenues for future investigation in this field.

Chorein sensitive microtubule organization in tumor cells.

Background: The purpose of this study is to analyzed the involvement of chorein in microtubules organization of three types of malignant; rhabdomyosarcoma tumor cells (ZF), rhabdomyosarcoma cells (RH30), and rhabdomyosarcoma cells (RD). ZF are expressing high chorein levels. Previous studies revealed that chorein protein silencing in ZF tumor cells persuaded apoptotic response followed by cell death. In addition, in numerous malignant and non-malignant cells this protein regulates actin cytoskeleton structure and cellular signaling. However, the function of chorein protein in microtubular organization is yet to be established. Methods: In a current research study, we analyzed the involvement of chorein in microtubules organization by using three types of malignant rhabdomyosarcoma cells. We have applied confocal laser-scanning microscopy to analyze microtubules structure and RT-PCR to examine cytoskeletal gene transcription. Results: We report here that in rhabdomyosarcoma cells (RH30), chorein silencing induced disarrangement of microtubular network. This was documented by laser scanning microscopy and further quantified by FACS analysis. Interestingly and in agreement with previous reports, tubulin gene transcription in RH cells was unchanged upon silencing of chorein protein. Equally, confocal analysis showed minor disordered microtubules organization with evidently weakened staining in rhabdomyosarcoma cells (RD and ZF) after silencing of chorein protein. Conclusion: These results disclose that chorein silencing induces considerable structural disorganization of tubulin network in RH30 human rhabdomyosarcoma tumor cells. Additional studies are now needed to establish the role of chorein in regulating cytoskeleton architecture in tumor cells.

Visualization of Cell Membrane Tension Regulated by the Microfilaments as a "Shock Absorber" in Micropatterned Cells.

The extracellular stress signal transmits along the cell membrane-cytoskeleton-focal adhesions (FAs) complex, regulating the cell function through membrane tension. However, the mechanism of the complex regulating membrane tension is still unclear. This study designed polydimethylsiloxane stamps with specific shapes to change the actin filaments' arrangement and FAs' distribution artificially in live cells, visualized the membrane tension in real time, and introduced the concept of information entropy to describe the order degree of the actin filaments and plasma membrane tension. The results showed that the actin filaments' arrangement and FAs' distribution in the patterned cells were changed significantly. The hypertonic solution resulted in the plasma membrane tension of the pattern cell changing more evenly and slowly in the zone rich in cytoskeletal filaments than in the zone lacking filaments. In addition, the membrane tension changed less in the adhesive area than in the non-adhesive area when destroying the cytoskeletal microfilaments. This suggested that patterned cells accumulated more actin filaments in the zone where FAs were difficult to generate to maintain the stability of the overall membrane tension. The actin filaments act as shock absorbers to cushion the alternation in membrane tension without changing the final value of membrane tension.

The novel role of etoposide in inhibiting the migration and proliferation of small cell lung cancer and breast cancer via targeting Daam1.

OBJECTIVES: Daam1 (Dishevelled-associated activator of morphogenesis 1) is a Wnt/PCP signaling protein that engages in cytoskeleton reorganization and is abnormally activated in certain tumors. Daam1 is closely related to cancer metastasis, which is expected to become a target for cancer treatment. However, the natural small molecules targeting Daam1 have not been identified. MATERIALS AND METHODS: We screened several natural small molecules that may bind to Daam1 by Sybyl molecular simulation docking technique. As a first-line drug for the treatment of small cell lung cancer, etoposide was chosen for further investigation. Next, we used Micro Scale Thermophoresis (MST) to verify the interaction of etoposide and Daam1. Small cell lung cancer H446 cells and breast cancer MCF-7 cells were treated with etoposide and subjected to Western blotting to measure the Daam1 expression. The effect of etoposide on cell proliferation was determined by CCK-8 assay in vitro and by a tumor-bearing mouse model in vivo. Wound healing assay and Boyden chamber assay were used to evaluate the role of etoposide in the migration and invasion ability of tumor cells. The effect of etoposide on the microfilament assembly was visualized by immunofluorescence staining with phalloidine. Finally, the possible mechanism of down-regulation of Daam1 expression after etoposide-induced small cell lung cancer cells was detected by a half-life experiment and immunofluorescence staining with lysosomal marker LAMP1. RESULTS: Sybyl molecular modeling docking technique was performed to screen a natural chemical library for molecules that bound to the FH2 domain of Daam1 and found etoposide was virtually interacted with Daam1. MST validated etoposide directly bound to the FH2 domain of Daam1. Etoposide significantly down-regulated the expression of Daam1 in small cell lung cancer H446 cells and breast cancer MCF-7 cells. Moreover, 270 µmol/L etoposide largely inhibited the proliferation, migration, and invasion of H446 cells and MCF-7 cells. Immunofluorescence staining experiments revealed that etoposide induced the disassembly of microfilaments in H446 cells and MCF-7 cells, which were rescued by Daam1 overexpression. In nude mice transplanted with H446 cells, 5, 10, 20 mg/kg etoposide (drug/weight) injected via tail vein largely retarded the proliferation of subcutaneous tumors. Etoposide induced Daam1 to shorten its half-life and enter the lysosome degradation pathway, and eventually leading to the downregulation of Daam1 expression. CONCLUSIONS: Etoposide is a novel natural small molecule targeting Daam1. Etoposide inhibits the proliferation, migration and invasion of small cell lung cancer cells and breast cancer cells, and also suppresses tumor proliferation of small cell lung cancer in vivo.

Actin: Static and Dynamic Studies.

The actin cytoskeleton is a highly dynamic network in plant cells, which is precisely regulated by numerous actin-binding proteins. Hence, characterizing the biochemical activities of actin-binding proteins is of great importance. Here we describe methods for determining the binding and bundling of microfilaments as well as methods for visualizing microfilaments using fluorescent phalloidin and single-molecule TIRF imaging.

Assessment of plaque control efficacy using two types of toothbrushes in young adults: An interventional crossover study.

OBJECTIVE: Evaluation of plaque removal efficacy of short-headed toothbrush as compared to conventional/traditional toothbrush. METHOD: A total of 20 subjects meeting the inclusion criteria were considered. Selected subjects were divided into test and control groups. Initially, subjects were advised to refrain from brushing teeth for 24 h, to determine the plaque index (PI) and gingival index (GI) at the baseline. Further, crossover observation between the groups was recorded based on short-headed toothbrush (Curaprox CS 5460) and traditional brushes (ultrasoft). Finally, a questionnaire survey was conducted to gather preferences and experiences of each subject. RESULTS: In terms of plaque removal, the short-headed toothbrush and the conventional toothbrush demonstrated comparable results. In terms of PI and GI, the intergroup comparison revealed no significant differences (p = 0.878). Individual acceptability of the short-headed toothbrush was shown to be higher in the questionnaire survey. CONCLUSION: It was observed that both toothbrushes showed similar efficacy. However, the subjects preferred short-headed toothbrush. CLINICAL RELEVANCE: Short-headed toothbrushes comprising a higher number of bristles can be recommended in terms of better oral hygiene.

miR-196a enhances polymerization of neuronal microfilaments through suppressing IMP3 and upregulating IGF2 in Huntington's disease.

Huntington's disease (HD) is one of the inheritable neurodegenerative diseases, and these diseases share several similar pathological characteristics, such as abnormal neuronal morphology. miR-196a is a potential target to provide neuroprotective functions, and has been reported to enhance polymerization of neuronal microtubules in HD. While microtubules and microfilaments are two important components of the neuronal cytoskeleton, whether miR-196a improves neuronal microfilaments is still unknown. Here, we identify insulin-like growth factor 2 mRNA binding protein 3 (IMP3), and show that miR-196a directly suppresses IMP3 to increase neurite outgrowth in neurons. In addition, IMP3 disturbs neurite outgrowth in vitro and in vivo, and worsens the microfilament polymerization. Moreover, insulin-like growth factor-II (IGF2) is identified as the downstream target of IMP3, and miR-196a downregulates IMP3 to upregulate IGF2, which increases microfilamental filopodia numbers and activates Cdc42 to increase neurite outgrowth. Besides, miR-196a increases neurite outgrowth through IGF2 in different HD models. Finally, higher expression of IMP3 and lower expression IGF2 are observed in HD transgenic mice and patients, and increase the formation of aggregates in the HD cell model. Taken together, miR-196a enhances polymerization of neuronal microfilaments through suppressing IMP3 and upregulating IGF2 in HD, supporting the neuroprotective functions of miR-196a through neuronal cytoskeleton in HD.

mTORC1/C2 regulate spermatogenesis in Eriocheir sinensis via alterations in the actin filament network and cell junctions.

Spermatogenesis is a finely regulated process of germ cell proliferation and differentiation that leads to the production of sperm in seminiferous tubules. Although the mammalian target of rapamycin (mTOR) signaling pathway is crucial for spermatogenesis in mammals, its functions and molecular mechanisms in spermatogenesis remain largely unknown in nonmammalian species, particularly in Crustacea. In this study, we first identified es-Raptor (the core component of mTOR complex 1) and es-Rictor (the core component of mTOR complex 2) from the testis of Eriocheir sinensis. Dynamic localization of es-Raptor and es-Rictor implied that these proteins were indispensable for the spermatogenesis of E. sinensis. Furthermore, es-Raptor and es-Rictor knockdown results showed that the mature sperm failed to be released, causing almost empty lumens in the testis. We investigated the reasons for these effects and found that the actin-based cytoskeleton was disrupted in the knockdown groups. In addition, the integrity of the testis barrier (similar to the blood-testis barrier in mammals) was impaired and affected the expression of cell junction proteins. Further study revealed that es-Raptor and es-Rictor may regulate spermatogenesis via both mTORC1- and mTORC2-dependent mechanisms that involve es-rpS6 and es-Akt/es-PKC, respectively. Moreover, to explore the testis barrier in E. sinensis, we established a cadmium chloride (CdCl2)-induced testis barrier damage model as a positive control. Morphological and immunofluorescence results were similar to those of the es-Raptor and es-Rictor knockdown groups. Altogether, es-Raptor and es-Rictor were important for spermatogenesis through maintenance of the actin filament network and cell junctions in E. sinensis.

Facile Preparation of PET/PA6 Bicomponent Microfilament Fabrics with Tunable Porosity for Comfortable Medical Protective Clothing.

Medical protective materials have broadly drawn attention due to their ability to stop the spread of infectious diseases and protect the safety of medical staff. However, creating medical protective materials that combine excellent liquid shielding performance and outstanding mechanical properties with high breathability is still a challenging task. Herein, a polyester/polyamide 6 (PET/PA6) bicomponent microfilament fabric with tunable porosity for comfortable medical protective clothing was prepared via dip-coating technology and an easy and effective thermal-belt bonding process. The dip coating of the C6-based fluorocarbon polymer endowed the samples with excellent hydrophobicity (alcohol contact angles, 130-128°); meanwhile, by adjusting the temperature and pressure of the thermal-belt bonding process, the porosity of the samples was adapted in the range of 64.19-88.64%. Furthermore, benefitting tunable porosity and surface hydrophobicity, the samples also demonstrated an excellent softness score (24.3-34.5), agreeable air permeability (46.3-27.8 mm/s), and high hydrostatic pressure (1176-4130 Pa). Significantly, the created textiles successfully filter aerosol from the air and display highly tensile strength. These excellent comprehensive performances indicate that the prepared PET/PA6 bicomponent microfilament fabrics would be an attractive choice for medical protective apparel.

Membrane-organizing extension spike protein and its role as an emerging biomarker in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is the most malignant tumor worldwide with a relatively poor prognosis. This can be due to lack of using new specific biomarkers as a mode of pristine interventional therapy for detecting the lesions at an early stage, thereby not allowing it to proceed to a severe advanced stage. Biomarkers, being the products of malignant cells, can prove to be promising prognostic factors in understanding the molecular pathogenesis of oral cancer. One such biomarker is membrane-organizing extension spike protein (MOESIN). Belonging to the family of ezrin/radixin/MOESIN proteins, MOESIN acts as a structural linker between plasma membrane and actin filament of the cell moiety and is involved in regulating many fundamental cellular processes such as cell morphology, adhesion and motility. This narrative review is a systematic compilation on MOESIN and its role as an emerging biomarker in OSCC.

Microcystin-LR induced microfilament rearrangement and cell invasion by activating ERK/VASP/ezrin pathway in DU145 cells.

Microcystin-LR (MC-LR) is an environmental toxin that is synthesized by cyanobacteria and considered a potential human carcinogen. However, the role of MC-LR in prostate cancer progression has not been elucidated. The purpose of this study was to investigate the effect of MC-LR on prostate cancer cell invasion and its underlying mechanisms. Transwell assay was performed, and the result showed that MC-LR increased DU145 cell invasion in a concentration-dependent manner. The result of Western blot showed that MC-LR promoted ERK phosphorylation, while enhancing VASP and ezrin phosphorylation. Moreover, PD0325901 was used to verify the role of the ERK/VASP/ezrin axis in MC-LR-promoted cell invasion. The results revealed that MC-LR promoted microfilament rearrangement and cell invasion by activating the ERK/VASP/ezrin pathway in DU145 cells. Finally, in vivo assay was performed, and the result suggested that MC-LR promoted p-ERK, p-VASP and p-ezrin expression and local invasion in nude mice model. Taken together, our data proved that MC-LR induced microfilament rearrangement and cell invasion by activating the ERK/VASP/ezrin pathway in DU145 cells.

Cytochalasin Q exerts anti-melanoma effect by inhibiting creatine kinase B.

Due to the pivotal role of microfilament in cancer cells, targeting microfilaments with cytochalasins is considered a promising anticancer strategy. Here, we obtained cytochalasin Q (CQ) from Xylaria sp. DO1801, the endophytic fungi from the root of plant Damnacanthus officinarum, and discovered its anti-melanoma activity in vivo and in vitro attributing to microfilament depolymerization. Mechanistically, CQ directly bound to and inactivated creatine kinase B (CKB), an enzyme phosphorylating creatine to phosphocreatine (PCr) and regenerating ATP to cope with high energy demand, and then inhibited the creatine metabolism as well as cytosolic glycolysis in melanoma cells. Preloading PCr recovered ATP generation, reversed microfilament depolymerization and blunted anti-melanoma efficacy of CQ. Knockdown of CKB resulted in reduced ATP level, perturbed microfilament, inhibited proliferation and induced apoptosis, and manifested lower sensitivity to CQ. Further, we found that either CQ or CKB depletion suppressed the PI3K/AKT/FoxO1 pathway, whereas 740Y-P, a PI3K agonist, elevated protein expression of CKB suppressed by CQ. Taken together, our study highlights the significant anti-melanoma effect and proposes a PI3K/AKT/FoxO1/ CKB feedback circuit for the activity of CQ, opening new opportunities for current chemotherapy.

[Effects of adenovirus-mediated shRNA down-regulates PTEN expression on fibril-binding proteins vinculin, filamin A and cortactin in activated hepatic stellate cells].

Objective: To investigate the effect of adenovirus-mediated shRNA down-regulating phosphatase and tensin homolog deleted on chromosome 10 (PTEN) expression on vinculin, filamin A, and cortactin in activated hepatic stellate cells (HSCs). Methods: Activated rats hepatic stellate cell line (HSC-T6) was cultured in vitro. Recombinant adenovirus Ad-shRNA/PTEN carrying PTEN targeted RNA interference sequence [short hairpin RNA (shRNA)] and empty control virus Ad-GFP were transfected into HSCs. The PTEN mRNA and protein expression of HSCs in each group were detected by real-time fluorescence quantitative PCR and Western blot. The expressional change of vinculin, filamin A and cortactin in HSCs of each group were detected by confocal laser scanning immunofluorescence microscope. Image-pro plus 6.0 software was used for image analysis and processing. The integrated optical density (IOD) of the fluorescence protein expression was measured. The experiment was divided into three groups: control group (DMEM instead of adenovirus solution in the adenovirus transfection step), Ad-GFP group (transfected with empty virus Ad-GFP only expressing green fluorescent protein), and Ad-shRNA/PTEN group (recombinant adenovirus Ad-shRNA/PTEN carrying shRNA targeting PTEN and expressing green fluorescent protein). One-way analysis of variance was used for comparison of mean value among the three groups, and LSD-test was used for comparison between the groups. Results: shRNA targeted PTEN was successfully transfected and the expression of PTEN mRNA and protein in HSC (P < 0.05) was significantly down-regulated. HSCs vinculin was mainly expressed in the cytoplasm. HSCs vinculin fluorescence IOD in the Ad-shRNA/PTEN group (19 758.83 ± 1 520.60) was higher than control (7 737.16 ± 279.93) and Ad-GFP group (7 725.50 ± 373.03) (P < 0.05), but there was no statistically significant difference between control group and Ad-GFP group (P > 0.05). There was no statistically significant difference in the fluorescence IOD of Filamin A among the three groups (P > 0.05), but the subcellular distribution of Filamin A among the three groups were changed. Filamin A in the Ad-shrNA /PTEN HSC group was mainly distributed in the cytoplasm. Filamin A HSC was mainly located in the nucleus.The filamin A HSC in the control group and Ad-GFP group was mainly located in the nucleus. The nucleocytoplasmic ratio of Filamin A in the AD-shrNA /PTEN group (0.60 ± 0.15) was significantly lower than control group (1.20 ± 0.15) and Ad-GFP group (1.08 ± 0.23), P < 0.05. but there was no statistically significant difference in filamin A nucleocytoplasmic ratio of HSC between the control group and the Ad-GFP group (P > 0.05). Cortactin HSCs in the three groups was mainly distributed in the cytoplasm. The cortactin fluorescence IOD of HSCs in the Ad-shRNA/PTEN group was significantly higher than control group (22 959.94 ± 1 710.42) and the Ad-GFP group (22 547.11 ± 1 588.72 ) (P < 0.05), while there was no statistically significant difference in the IOD of cortactin fluorescence in HSCs between the control group and the Ad-GFP group (P > 0.05). Conclusion: The down-regulation of PTEN expression raises the expression of microfilament-binding protein vinculin and cortactin, and changes the subcellular distribution of another microfilament binding protein filamin A, that is, translocation from nucleus to the cytoplasm in activated HSC in vitro.

[Mechanism study of the protective effects of selective cyclooxygenase-2 enzyme inhibitors on the liver of rats with type 2 diabetes mellitus combined with nonalcoholic steatohepatitis via Rho/ROCK pathway].

Objective: To investigate whether the selective cyclooxygenase-2 enzyme inhibitors celecoxib has protective effect on the liver of rats with type 2 diabetes mellitus (T2DM) combined with nonalcoholic steatohepatitis (NASH) via inhibiting the expression of Rho/ROCK pathway. Methods: Forty male SD rats were randomly divided into four groups: type 2 diabetes mellitus combined with nonalcoholic steatohepatitis (T2DM-NASH) group, T2DM-NASH + celecoxib group, control group, and control+celecoxib group. The T2DM-NASH and T2DM-NASH + celecoxib groups were fed with high-sugar and fat diet, and the control group and control + celecoxib group were fed with basal diet (25 kJ/kg). Four weeks later, streptozotocin (STZ, 30 mg/kg) was intraperitoneally injected into the NASH group and T2DM-NASH + celecoxib group to induce T2DM model, and the control group and control + celecoxib group were intraperitoneally injected with isovolumic citric acid-sodium citrate buffer. Four weeks after STZ injection, the T2DM-NASH + celecoxib group and the control + celecoxib group were gavaged with celecoxib (10 mg·kg·d) dissolved in normal saline for 4 weeks, and the remaining two groups of rats were gavaged with isovolumic normal saline for 4 weeks. Animals were sacrificed at the end of the 12- weeks, and the liver tissue was collected. Liver pathological changes were observed by HE staining. The expressions of RhoA, RhoA, ROCK1 and ROCK2 proteins in liver were detected by immunohistochemistry and western blot. The expressional condition of RhoA, ROCK1 and ROCK2 mRNA in liver were detected by real-time quantitative PCR. The differences were compared between protein and mRNA expression among the groups by analysis of variance and t-test. Results: Compared with the control group and the control + celecoxib group, the liver tissue of the T2DM-NASH group and the T2DM-NASH + celecoxib group had severe steatosis, and there was partial inflammatory cell infiltration under the light microscope. The expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were significantly increased (P < 0.05) in each liver tissue, while liver steatosis was reduced to certain extent in T2DM-NASH + celecoxib group than T2DM-NASH group, and the expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were decreased in each liver tissue of T2DM-NASH group (P < 0.05). Conclusion: The selective cyclooxygenase-2 enzyme inhibitors celecoxib has a protective effect on the liver of rats with T2DM-NASH, and its effect may be achieved by inhibiting the expression of Rho/ROCK pathway.

XB130 Plays an Essential Role in Folliculogenesis Through Mediating Interactions Between Microfilament and Microtubule Systems in Thyrocytes.

Background: XB130 (actin filament-associated protein 1-like 2, AFAP1L2) is a thyroid-abundant adaptor/scaffold protein. Xb130-/- mice exhibit transient growth retardation postnatally due to congenital hypothyroidism with diminished thyroglobulin iodination and release at both embryonic and early postnatal stages due to disorganized thyroid apical membrane structure and function. We hypothesized that XB130 is crucial for polarity and folliculogenesis by mediating proper cytoskeletal structure and function in thyrocytes. Methods: Primary thyrocytes isolated from thyroid glands of Xb130-/- mice and their wild-type littermates at postnatal week 2 were cultured in 10% Matrigel for different time periods. Folliculogenesis was studied with immunofluorescence staining, followed by confocal microscopy. Cells were also transfected to express human XB130 fused Green Fluorescent Protein (XB130-GFP) or Green Fluorescent Protein (GFP) only before morphological analysis. Cytoskeletal structures from embryo and postnatal thyroid glands were also studied. Results: In three-dimensional cultures of thyrocytes, XB130, aligned with actin filaments, participated in defining the site of apical membrane formation and coalescence to form a thyroid follicle lumen. Xb130-/- thyrocytes displayed delayed folliculogenesis, reduced recruitment of a microtubule (MT)-associated proteins, and disorganized acetylated tubulin under the apical membrane, resulting in delayed folliculogenesis with reduced efficiency in formation of the thyroid follicle lumen. Conclusions: XB130 critically regulates thyrocyte polarization by functioning as a link between the actin filament cortex and MT network at the apical membrane of thyrocytes. Defects of adaptor scaffold proteins may affect cellular polarity and cytoskeletal structure and function and result in disorders of epithelial function, such as congenital hypothyroidism.

Mechanism study of the protective effects of selective cyclooxygenase-2 enzyme inhibitors on the liver of rats with type 2 diabetes mellitus combined with nonalcoholic steatohepatitis via Rho/ROCK pathway / 中华肝脏病杂志

Objective: To investigate whether the selective cyclooxygenase-2 enzyme inhibitors celecoxib has protective effect on the liver of rats with type 2 diabetes mellitus (T2DM) combined with nonalcoholic steatohepatitis (NASH) via inhibiting the expression of Rho/ROCK pathway. Methods: Forty male SD rats were randomly divided into four groups: type 2 diabetes mellitus combined with nonalcoholic steatohepatitis (T2DM-NASH) group, T2DM-NASH + celecoxib group, control group, and control+celecoxib group. The T2DM-NASH and T2DM-NASH + celecoxib groups were fed with high-sugar and fat diet, and the control group and control + celecoxib group were fed with basal diet (25 kJ/kg). Four weeks later, streptozotocin (STZ, 30 mg/kg) was intraperitoneally injected into the NASH group and T2DM-NASH + celecoxib group to induce T2DM model, and the control group and control + celecoxib group were intraperitoneally injected with isovolumic citric acid-sodium citrate buffer. Four weeks after STZ injection, the T2DM-NASH + celecoxib group and the control + celecoxib group were gavaged with celecoxib (10 mg·kg·d) dissolved in normal saline for 4 weeks, and the remaining two groups of rats were gavaged with isovolumic normal saline for 4 weeks. Animals were sacrificed at the end of the 12- weeks, and the liver tissue was collected. Liver pathological changes were observed by HE staining. The expressions of RhoA, RhoA, ROCK1 and ROCK2 proteins in liver were detected by immunohistochemistry and western blot. The expressional condition of RhoA, ROCK1 and ROCK2 mRNA in liver were detected by real-time quantitative PCR. The differences were compared between protein and mRNA expression among the groups by analysis of variance and t-test. Results: Compared with the control group and the control + celecoxib group, the liver tissue of the T2DM-NASH group and the T2DM-NASH + celecoxib group had severe steatosis, and there was partial inflammatory cell infiltration under the light microscope. The expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were significantly increased (P < 0.05) in each liver tissue, while liver steatosis was reduced to certain extent in T2DM-NASH + celecoxib group than T2DM-NASH group, and the expression levels of RhoA, ROCK1 and ROCK2 protein and mRNA were decreased in each liver tissue of T2DM-NASH group (P < 0.05). Conclusion: The selective cyclooxygenase-2 enzyme inhibitors celecoxib has a protective effect on the liver of rats with T2DM-NASH, and its effect may be achieved by inhibiting the expression of Rho/ROCK pathway.