Alterations In Cytoskeleton Research Articles

Content of GFAP in the Brain of BALB/C Mice with the Antiphospholipid Syndrome: Effects of L-Arginine and Aminoguanidine

We investigated the influence of the nitric oxide precursor L-arginine and selective inhibitor of inducible NOS aminoguanidine on the content of glial fibrillary acidic protein (GFAP) in the cerebellum and cerebral hemispheres of BALB/c mice with the experimental antiphospholipid syndrome (APS). In the APS, the total amount of GFAP and amount of its component GFAP 49–37 kDa in the cerebellum were 280 and 640%, as compared with the control, while the respective figures in the cerebral cortex were 118% and 155%. Injections of L-arginine led to a further increase in the GFAP content in the cerebellum of animals with the APS. Administration of aminoguanidine alone and of aminoguanidine together with L-arginine provided decreases in the GFAP content in the cerebellum of APS mice. Aminoguanidine also reduced the GFAP content in the cerebral hemispheres in APS animals. The increased GFAP content in the cerebellum and cerebral hemispheres in mice with the APS is indicative of the development of reactive astrogliosis. Changes in the NO synthesis due to introduction of its modulators significantly affect the GFAP content in the above brain regions in animals with the APS. These facts indirectly confirm the role of NO in the regulation of the GFAP expression in astrocytes under conditions of this pathology. The presence of GFAP 49–37 kDa (mainly in the cerebellum) in BALB/c mice with APS appears to be a sign of proteolytic degradation of GFAP, suggesting alterations in the astrocyte cytoskeleton (intermediate filaments) in the respective brain regions.

Klebsiella pneumoniae Redistributes Katanin Severing Proteins and Alters Astral Microtubules during Mitosis.

Klebsiella pneumoniae has become a growing concern within hospitals due to multidrug resistant strains and increasing mortality rates. Recently, we showed that at the subcellular level, K. pneumoniae compromises the integrity of the epithelia by disassembling the microtubule networks of cells through the actions of katanin microtubule severing proteins. In this study, we report on the observation that mitotic cells are targeted by K. pneumoniae and that during infections, the katanin proteins are excluded from the microtubule organizing centers of dividing cells, resulting in the alteration of the microtubule cytoskeleton. Anat Rec, 2019. © 2019 American Association for Anatomy Anat Rec, 303:1859-1864, 2020. © 2019 American Association for Anatomy.

Molecular Characterization of Human Lymph Node Stromal Cells During the Earliest Phases of Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a progressive, destructive autoimmune arthritis. Break of tolerance and formation of autoantibodies occur years before arthritis. Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSCs) play a crucial role in shaping the immune response and maintaining peripheral tolerance. Here we performed the first epigenomic characterization of LNSCs during health and early RA, by analyzing their transcriptome and DNA methylome in LNSCs isolated from lymph node needle biopsies obtained from healthy controls (HC), autoantibody positive RA-risk individuals and patients with established RA. Of interest, LNSCs from RA-risk individuals and RA patients revealed a common significantly differential expressed gene signature compared with HC LNSCs. Pathway analysis of this common signature showed, among others, significant enrichment of pathways affecting the extracellular matrix (ECM), cholesterol biosynthesis and immune system. In a gel contraction assay LNSCs from RA-risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSCs. In RA LNSCs a significant enrichment was observed for genes involved in cytokine signaling, hemostasis and packaging of telomere ends. In contrast, in RA-risk LNSCs pathways in cancer (cell cycle related genes) were differentially expressed compared with HC, which could be validated in vitro using a proliferation assay, which indicated a slower proliferation rate. DNA methylation analyses revealed common and specific differentially methylated CpG sites (DMS) in LNSC from RA patients and RA-risk individuals compared with HC. Intriguingly, shared DMS were all associated with antigen processing and presentation. This data point toward alterations in cytoskeleton and antigen-processing and presentation in LNSC from RA-risk individuals and RA patients. Further studies are required to investigate the consequence of this LNSC abnormality on LNSC-mediated immunomodulation.

TRPC6 Binds to and Activates Calpain, Independent of Its Channel Activity, and Regulates Podocyte Cytoskeleton, Cell Adhesion, and Motility.

Mutations in the transient receptor potential channel 6 (TRPC6) gene are associated with an inherited form of FSGS. Despite widespread expression, patients with TRPC6 mutations do not present with any other pathologic phenotype, suggesting that this protein has a unique yet unidentified role within the target cell for FSGS, the kidney podocyte. We generated a stable TRPC6 knockout podocyte cell line from TRPC6 knockout mice. These cells were engineered to express wild-type TRPC6, a dominant negative TRPC6 mutation, or either of two disease-causing mutations of TRPC6, G109S or K874*. We extensively characterized these cells using motility, detachment, and calpain activity assays; immunofluorescence; confocal or total internal reflection fluorescence microscopy; and western blotting. Compared with wild-type cells, TRPC6-/- podocytes are less motile and more adhesive, with an altered actin cytoskeleton. We found that TRPC6 binds to ERK1/2 and the actin regulatory proteins, caldesmon (a calmodulin- and actin-binding protein) and calpain 1 and 2 (calcium-dependent cysteine proteases that control the podocyte cytoskeleton, cell adhesion, and motility via cleavage of paxillin, focal adhesion kinase, and talin). Knockdown or expression of the truncated K874* mutation (but not expression of the gain-of-function G019S mutation or dominant negative mutant of TRPC6) results in the mislocalization of calpain 1 and 2 and significant downregulation of calpain activity; this leads to altered podocyte cytoskeleton, motility, and adhesion-characteristics of TRPC6-/- podocytes. Our data demonstrate that independent of TRPC6 channel activity, the physical interaction between TRPC6 and calpain in the podocyte is important for cell motility and detachment and demonstrates a scaffolding role of the TRPC6 protein in disease.

Transcriptomic analysis of clam extrapallial fluids reveals immunity and cytoskeleton alterations in the first week of Brown Ring Disease development

The Brown Ring Disease is an infection caused by the bacterium Vibrio tapetis on the Manila clam Ruditapes philippinarum. The process of infection, in the extrapallial fluids (EPFs) of clams, involves alteration of immune functions, in particular on hemocytes which are the cells responsible of phagocytosis. Disorganization of the actin-cytoskeleton in infected clams is a part of what leads to this alteration. This study is the first transcriptomic approach based on collection of extrapallial fluids on living animals experimentally infected by V. tapetis. We performed differential gene expression analysis of EPFs in two experimental treatments (healthy-against infected-clams by V. tapetis), and showed the deregulation of 135 genes. In infected clams, a downregulation of transcripts implied in immune functions (lysosomal activity and complement- and lectin-dependent PRR pathways) was observed during infection. We also showed a deregulation of transcripts encoding proteins involved in the actin cytoskeleton organization such as an overexpression of β12-Thymosin (which is an actin sequestration protein) or a downregulation of proteins that closely interact with capping proteins such as Coactosin, that counteract action of capping proteins, or Profilin. We validated these transcriptomic results by cellular physiological analyses that showed a decrease of the lysosome amounts and the disorganization of actin cytoskeleton in infected hemocytes.

Tubulin in Platelets: When the Shape Matters.

Platelets are anuclear cells with a short lifespan that play an essential role in many pathophysiological processes, including haemostasis, inflammation, infection, vascular integrity, and metastasis. Billions of platelets are produced daily from megakaryocytes (platelet precursors). Despite this high production, the number of circulating platelets is stable and, under resting conditions, they maintain their typical discoid shape thanks to cytoskeleton proteins. The activation of platelets is associated with dynamic and rapid changes in the cytoskeleton. Two cytoskeletal polymer systems exist in megakaryocytes and platelets: actin filaments and microtubules, based on actin, and α- and β-tubulin heterodimers, respectively. Herein, we will focus on platelet-specific tubulins and their alterations and role of the microtubules skeleton in platelet formation (thrombopoiesis). During this process, microtubules mediate elongation of the megakaryocyte extensions (proplatelet) and granule trafficking from megakaryocytes to nascent platelets. In platelets, microtubules form a subcortical ring, the so-called marginal band, which confers the typical platelet discoid shape and is also responsible for changes in platelet morphology upon activation. Molecular alterations in the gene encoding β1 tubulin and microtubules post-translational modifications may result in quantitative or qualitative changes in tubulin, leading to altered cytoskeleton reorganization that may induce changes in the platelet number (thrombocytopenia), morphology or function. Consequently, β1-tubulin modifications may participate in pathological and physiological processes, such as development.

A Proteomic View of Cellular Responses to Anticancer Quinoline-Copper Complexes.

Metal-containing drugs have long been used in anticancer therapies. The mechansims of action of platinum-based drugs are now well-understood, which cannot be said of drugs containing other metals, such as gold or copper. To gain further insights into such mechanisms, we used a classical proteomic approach based on two-dimensional elelctrophoresis to investigate the mechanisms of action of a hydroxyquinoline-copper complex, which shows promising anticancer activities, using the leukemic cell line RAW264.7 as the biological target. Pathway analysis of the modulated proteins highlighted changes in the ubiquitin/proteasome pathway, the mitochondrion, the cell adhesion-cytoskeleton pathway, and carbon metabolism or oxido-reduction. In line with these prteomic-derived hypotheses, targeted validation experiments showed that the hydroxyquinoline-copper complex induces a massive reduction in free glutathione and a strong alteration in the actin cytoskeleton, suggesting a multi-target action of the hydroxyquinoline-copper complex on cancer cells.

Transcriptomic analysis of clam extra pallial fluids reveals immunity and cytoskeleton alterations in the first week of brown ring disease development

Transcriptomic analysis of clam extra pallial fluids reveals immunity and cytoskeleton alterations in the first week of brown ring disease development

Inflammatory cytokines associated with cancer growth induce mitochondria and cytoskeleton alterations in cardiomyocytes.

Cardiac dysfunction is often observed in patients with cancer also representing a serious problem limiting chemotherapeutic intervention and even patient survival. In view of the recently established role of the immune system in the control of cancer growth, the present work has been undertaken to investigate the effects of a panel of the most important inflammatory cytokines on the integrity and function of mitochondria, as well as of the cytoskeleton, two key elements in the functioning of cardiomyocytes. Either mitochondria features or actomyosin cytoskeleton organization of in vitro‐cultured cardiomyocytes treated with different inflammatory cytokines were analyzed. In addition, to investigate the interplay between tumor growth and cardiac function in an in vivo system, immunocompetent female mice were inoculated with cancer cells and treated with the chemotherapeutic drug doxorubicin at a dosing schedule able to suppress tumor growth without inducing cardiac alterations. Analyses carried out in cardiomyocytes treated with the inflammatory cytokines, such as tumor necrosis factor α (TNF‐α), interferon γ (IFN‐γ), interleukin 6 (IL‐6), IL‐8, and IL‐1β revealed severe phenotypic changes, for example, of contractile cytoskeletal elements, mitochondrial membrane potential, mitochondrial reactive oxygen species production and mitochondria network organization. Accordingly, in immunocompetent mice, the tumor growth was accompanied by increased levels of the inflammatory cytokines TNF‐α, IFN‐γ, IL‐6, and IL‐8, either in serum or in the heart tissue, together with a significant reduction of ventricular systolic function. The alterations of mitochondria and of microfilament system of cardiomyocytes, due to the systemic inflammation associated with cancer growth, could be responsible for remote cardiac injury and impairment of systolic function observed in vivo.

Cell Type‐Specific Ephrin expression in the Diabetic Coronary Circulation

Ephrin signaling between vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) is crucial during normal vascular development and homeostasis, primarily by influencing cell behavior, resulting in alterations of the cell cytoskeleton and cell adhesion. Ephrins are re‐expressed in vascular injury and disease states such as cancer and chronic inflammation; therefore the goal of this study was to determine the impact of type 2 diabetes on ephrin expression in coronary VSMCs and ECs. Low‐passage primary coronary VMSCs were isolated from normal (n=6) and type 2 diabetic db/db mice (n=4) and were either cultured alone or co‐cultured with normal or type 2 diabetic human coronary microvascular endothelial cells (hCMECs), respectively, and RNA was collected for PCR analysis. Normal (n=5) and type 2 diabetic (T2DM) (n=4) human coronary vascular smooth muscle cells (CVSMCs) were also cultured and protein lysates collected for Western Blot analysis. Ephrin B2 ligand gene expression was significantly increased in diabetic CRM VSMCs in both mono‐ and co‐culture conditions (1.00 ± 0.13 vs. 1.57 ± 0.24, p=0.049 and 1.00 ± 0.14 vs. 1.60 ± 0.22, p=0.044, respectively). There were no significant differences in ephrin B4 receptor levels in normal vs. diabetic coronary VMSCs. In human coronary VSMCs, ephrin B2 ligand protein expression was also significantly increased in diabetic coronary VSMCs over control VSMCs (normal 1.00±0.05 vs. T2DM 2.91±0.29, p=0.019). Interestingly, there was a different trend in the diabetic hCMECs. Ephrin B2 gene expression was significantly reduced in diabetic hCMECs in both mono‐ and co‐culture conditions (1.00 ± 0.05 vs. 0.75 ± 0.07, p=0.02 and 1.00 ± 0.10 vs. 0.50 ± 0.07, p=0.007, respectively). Ephrin B4 receptor gene expression was significantly reduced in diabetic hCMECs in both mono‐ and co‐culture conditions (1.00 ± 0.07 vs. 0.55 ± 0.05, p=0.002 and 1.00 ± 0.03 vs. 0.77 ± 0.03, p=0.0008, respectively). Collectively, these data demonstrate that, in both mice and humans, diabetes alters ephrin expression in VSMCs and ECs in a cell‐specific manner. Further studies will address how alterations in ephrin expression influence coronary cell behavior in the setting of diabetes.Support or Funding InformationNIH R00 HL116769, NIH R21 EB026518, and Nationwide Children's Hospital to AJTThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Endothelial Differentiation G Protein-Coupled Receptor 5 Plays an Important Role in Induction and Maintenance of Pluripotency.

Direct reprogramming of human somatic cells toward induced pluripotent stem cells holds great promise for regenerative medicine and basic biology. We used a high‐throughput small interfering RNA screening assay in the initiation phase of reprogramming for 784 genes belonging to kinase and phosphatase families and identified 68 repressors and 22 effectors. Six new candidates belonging to the family of the G protein‐coupled receptors (GPCRs) were identified, suggesting an important role for this key signaling pathway during somatic cell‐induced reprogramming. Downregulation of one of the key GPCR effectors, endothelial differentiation GPCR5 (EDG5), impacted the maintenance of pluripotency, actin cytoskeleton organization, colony integrity, and focal adhesions in human embryonic stem cells, which were associated with the alteration in the RhoA‐ROCK‐Cofilin‐PAXILLIN‐actin signaling pathway. Similarly, downregulation of EDG5 during the initiation stage of somatic cell‐induced reprogramming resulted in alteration of cytoskeleton, loss of human‐induced pluripotent stem cell colony integrity, and a significant reduction in partially and fully reprogrammed cells as well as the number of alkaline phosphatase positive colonies at the end of the reprogramming process. Together, these data point to an important role of EDG5 in the maintenance and acquisition of pluripotency. Stem Cells 2019;37:318–331

Combination therapy targeting the elevated interleukin-6 level reduces invasive migration of BRAF inhibitor-resistant melanoma cells.

The identification of novel antimetastatic therapeutic targets is necessary for improved treatment of patients with acquired BRAF inhibitor‐resistant (BRAFi‐R) melanoma, in whom metastasis is a major concern. Our present study focused on the identification of such targets to explore novel antimetastatic therapeutic options for BRAFi‐R melanoma patients. We confirmed the development of BRAFi resistance in our BRAFi‐treated melanoma cell lines by demonstrating reduced sensitivity to BRAF inhibitors, increased ERK1/2 activity and increased WNT5A expression. Here, we demonstrated for the first time that high secretion of interleukin‐6 (IL‐6) was associated with increased invasive migration of BRAFi‐R melanoma cells. This finding could be readily explained by the increased expression of WNT5A in BRAFi‐R melanoma cells and the presence of an IL‐6/WNT5A positive feedback loop in parental melanoma cells. Surprisingly, however, we found that the IL‐6/WNT5A positive feedback loop present in parental melanoma cells was lost during the development of acquired BRAFi resistance, meaning that IL‐6 and WNT5A signalling were independent events in BRAFi‐R melanoma cells. Despite the absence of an IL‐6/WNT5A loop, we found that both an IL‐6 blocking antibody and the WNT5A antagonist Box5 alone impaired the elevated invasive migration of BRAFi‐R melanoma cells, but combined use of the two was more effective. This impaired invasive migration of BRAFi‐R melanoma cells correlated well with the reduction in Cdc42‐GTPase activity and alterations of the actin cytoskeleton in these cells. In summary, our novel identification of IL‐6 as a key independent promoter of the invasive migration of BRAFi‐R melanoma cells stresses that a combination of a blocking IL‐6 antibody and administration of the WNT5A antagonist Box5 might be an attractive antimetastatic approach for future treatment of BRAFi‐R melanoma patients.

Cadmium induces actin cytoskeleton alterations and dysfunction in Neuro-2a cells.

Cadmium (Cd) is considered a possible etiological factor in neurodegenerative diseases. However, the exact mechanism by which Cd induces neurotoxicity is not well elucidated. In this study, Neuro-2a cells were treated with 0, 10, 20, and 40 μM cadmium chloride for 24 hours to investigate the effects of Cd on the cytoskeleton of nerve cells. MTT assay and ELISA assay were used to examine cell viability and release of lactate dehydrogenase (LDH) from cells, respectively. Results showed that Cd reduced cell viability and increased the release of LDH in a dose-dependent manner (P < 0.05). The morphology of treated cell was damaged as indicated by cell collapse and dimensionality reduction. Moreover, the axonal spines and normal features of Cd-treated neurons disappeared. We checked the ultrastructure of Neuro-2a cells and found that Cd-induced swelling, membrane damage, overflow of cytoplasm contents, and cell fragmentation. Damaged mitochondria, expanded endoplasmic reticulum, and abnormal microfilaments were found in Cd-treated cells rather than in untreated cells. Compared with the control group, the relative release of glutamate in the supernatant after Cd treatment was reduced, indicating that Cd exposure could reduce the release of glutamate by inhibiting the function of nerve-2a cells. Cd decreased the mRNA and protein expression levels of cytoskeletal proteins including DBN, SYP, and TAU, which might promote cytoskeleton alterations in Cd-treated cells. In conclusion, Cd-induced actin cytoskeleton alterations and dysfunction of cultured neurons. The results of the present study provide new insights for the investigation of Cd-induced neurotoxicity.

Prevalence of infections with Clostridium difficile on potential pathology groups

Abstract Enterotoxins produced by Clostridium difficile cause a series of biochemical and immunological manifestations in the cascade leading to alteration of the enterocitus cytoskeleton, intestinal inflammation and diarrhea that can greatly impair the patient’s biological status. The genome of the Clostridium difficile bacterium shows a series of evolutionary adaptations that can give it a high degree of resistance or adaptability to many known pharmacological classes. Changing the diversity of intestinal microbiota induced by the use of antibiotics creates a favorable environment from all points of view for Clostridium difficile spore activity. The theme addresses in an original way but related to the epidemiological studies presented in the literature a correlative aspect between the pathological group and the infection with Clostridium difficile. From the data presented, there is a direct correlation between Clostridium difficile infection and the use of antibiotic therapy as a curative or preventive treatment. Gastrointestinal and neurological pathologies, due to the use of curative but also preventive antibiotic therapy, are at increased risk for the installation of Clostridium difficile infection. The study presented may be a first step in raising awareness of the rational use of antibiotics and avoiding non-assisted community antibiotic therapy.

Stimuli-responsive polymer-doxorubicin conjugate: Antitumor mechanism and potential as nano-prodrug

Polymer-drug conjugates has significantly improved the anti-tumor efficacy of chemotherapeutic drugs and alleviated their side effects. N-(1,3-dihydroxypropan-2-yl) methacrylamide (DHPMA) copolymer was synthesized via RAFT polymerization and polymer-doxorubicin (DOX) (diblock pDHPMA-DOX) were formed by conjugation, resulting in a self-aggregation-induced nanoprodrug with a favorable size of 21 nm and great stability. The nanoprodrug with a molecular weight (MW) of 95 kDa released drugs in response to tumor microenvironmental pH variations and they were enzymatically hydrolyzed into low MW segments (45 kDa). The nanoprodrug was transported through the endolysosomal pathway, released the drug into the cytoplasm and some was localized in the mitochondria, resulting in disruption of the cellular actin cytoskeleton. Cellular apoptosis was also associated with reduction in the mitochondrial potential caused by the nanoprodrug. Notably, the nanoprodrug had a significantly prolonged blood circulation time with an elimination half time of 9.8 h, displayed high accumulation within tumors, and improved the in vivo therapeutic efficacy against 4T1 xenograft tumors compared to free DOX. The tumor xenograft immunohistochemistry study clearly indicated tumor inhibition was through the inhibition of cell proliferation and antiangiogenic effects. Our studies demonstrated that the diblock pDHPMA-DOX nanoprodrug with a controlled molecular structure is promising to alleviate adverse effects of free DOX and have a great potential as an efficient anticancer agent. Statement of SignificanceIn this work, we prepared a biodegradable diblock DHPMA polymer-doxorubicin conjugate via one-pot of RAFT polymerization and conjugate chemistry. The conjugate-based nanoprodrug was internalized by endocytosis to intracellularly release DOX and further induce disruption of mitochondrial functions, actin cytoskeleton alterations and cellular apoptosis. The nanoprodrug with a high molecular weight (MW) (95 kDa) showed a long blood circulation time and achieved high accumulation into tumors. The nanoprodrug was degraded into low MW (∼45 kDa) products below the renal threshold, which ensured its biosafety. Additionally, the multi-stimuli-responsive nanoprodrug demonstrated an enhanced antitumor efficacy against 4T1 breast tumors and alleviated side effects, showing a great potential as an efficient and safe anticancer agent.

An Introduction to Actin and Actin-Rich Structures.

The actin cytoskeleton has long been recognized as a crucial sub-cellular filament system that is responsible for governing fundamental events ranging from cell division and muscle contraction to whole cell motility and the maintenance of tissue integrity. Consequently, it is not surprising that this network is the focus of over 100,000 different manuscripts. Alterations in the actin cytoskeleton lead to an assortment of diseases and serve as a target for a variety of pathogens. Here we have brought together a collection of primary research articles and reviews that underscore the broad influence this filament system has on organisms. Anat Rec, 301:1986-1990, 2018. © 2018 Wiley Periodicals, Inc.

SFRP1 exerts effects on gastric cancer cells through GSK3β/Rac1‑mediated restraint of TGFβ/Smad3 signaling.

Secreted frizzled-related protein 1 (sFRP1) is an inhibitor of canonical Wnt signaling; however, previous studies have determined a tumor-promoting function of sFRP1 in a number of different cancer types. A previous study demonstrated that sFRP1 overexpression was associated with an aggressive phenotype and the activation of transforming growth factor β (TGFβ) signaling. sFRP1 overexpression and sFRP1 knockdown cell models were established. Immunoblotting was conducted to examine the protein levels of the associated molecules. Immunofluorescence staining followed by confocal microscopy was performed to visualize the cytoskeleton alterations and subcellular localization of key proteins. sFRP1 overexpression restored glycogen synthase kinase 3β (GSK3β) activity, which activated Rac family small GTPase 1 (Rac1). GSK3β and Rac1 mediated the effect of sFRP1 on the positive regulation of cell growth and migration/invasion. Inhibition of GSK3β or Rac1 abolished the regulation of sFRP1 on TGFβ/SMAD family member 3 (Smad3) signaling and the aggressive phenotype; however, GSK3β or Rac1 overexpression increased cell migration/invasion and restrained Smad3 activity by preventing its nuclear translocation and limiting its transcriptional activity. The present study demonstrated a tumor-promoting function of sFRP1-overexpression by selectively activating TGFβ signaling in gastric cancer cells. GSK3β and Rac1 serve an important function in mediating the sFRP1-induced malignant alterations and signaling changes.

Elevated Autoantibodies in Subacute Human Spinal Cord Injury Are Naturally Occurring Antibodies.

Spinal cord injury (SCI) results in long-term neurological and systemic consequences, including antibody-mediated autoimmunity, which has been related to impaired functional recovery. Here we show that autoantibodies that increase at the subacute phase of human SCI, 1 month after lesion, are already present in healthy subjects and directed against non-native proteins rarely present in the normal spinal cord. The increase of these autoantibodies is a fast phenomenon–their levels are already elevated before 5 days after lesion–characteristic of secondary immune responses, further supporting their origin as natural antibodies. By proteomics studies we have identified that the increased autoantibodies are directed against 16 different nervous system and systemic self-antigens related to changes known to occur after SCI, including alterations in neural cell cytoskeleton, metabolism and bone remodeling. Overall, in the context of previous studies, our results offer an explanation to why autoimmunity develops after SCI and identify novel targets involved in SCI pathology that warrant further investigation.

Effects of RCCS simulated microgravity on proliferation and cell cytoskeleton of human HaCaT keratinocyte

Objective To investigate the effects of simulated microgravity by RCCS on proliferation and cell cytoskeleton of human HaCaT keratinocyte. Methods The rotary cell culture system (RCCS) was used to simulate the microgravity environment, and human HaCaT keratinocytes were divided randomly(random number) into the simulated microgravity group (SMG) and normal gravity group (NG). HaCaT cells in the two groups were harvested respectively after 32, 36 and 42 h culture. The HaCaT cells proliferation and cycles were detected by flow cytometry, the concentration of hb-EGF in supernatant was detected by ELISA, and the cell cytoskeleton was observed after 42 hours’ culture under laser confocal microscope with FITC-labeled technique. SPSS 23.0 statistical software was used for statistical analysis, and P <0.05 was considered statistically significant. Results The flow cytometry showed that the proportions of human HaCaT keratinocytes in G1 and G2/M phases were increased while the proportion of HaCaT cells in S stage was decreased significantly after 32, 36 and 42 h RCCS culture compared with those in the normal gravity group. The HaCaT cells in G1 stage were declined along with incubation time. ELISA results showed that the hb-EGF concentration in HaCaT supernatant under simulated microgravity culture for 24 and 36 h was lower than that in the normal control group (P<0.01). The laser confocal microscope revealed that the HaCaT fluorescence intensity was decreased, and there were disordered microfilaments, structural ambiguity, pseudopodia reduction and irregular shape among FITC-labeled HaCaT cells cultured 42 h in RSSC compared with the normal gravity group. Conclusions RCCS simulated microgravity environment could inhibit the cell cycle transformation and proliferation of human HaCaT keratinocyte, affect the keratinocyte-secreting function, and induce alterations of the cell cytoskeleton. Key words: Simulated microgravity; RCCS; Keratinocyte; HaCaT cell line; Proliferation; Cell cycle; hb-EGF; Cytoskeleton

Application of laser scanning cytometry in vascular smooth muscle remodeling.

Pulmonary artery hyperplasia is the result of proliferation of the pulmonary arterial smooth muscles (PASM). Hypoxia-induced PASM proliferation in the fetus and the newborn is the primary cause of persistent pulmonary hypertension of the newborn (PPHN). This study was performed to characterize the utility of the Laser Scanning Cytometry (LSC) method in elucidating arterial cytoskeletal remodeling in an in vitro model of PPHN. The aim was to demonstrate the following: (a) LSC is a valid method for the analysis of nuclear and cytosolic fluorescence and (b) the cumulative effects of mechanical stretch together with hypoxia promote reactive oxygen species (ROS) formation. The molecular events in response to hypoxia and the mechanical overload of the pulmonary circuit were demonstrated in vitro by subjecting hypoxic cultured primary PASM or human airway smooth muscles (hASM) to repetitive stretch-relaxation cycles at rates comparable to dynamic stretch in vivo. The altered cytoskeleton in the form of filamentous to globular actin (F:G actin) ratio was imaged and quantified at the cellular level by LSC as an endpoint. LSC can remove the nuclear G-actin fluorescence from the total G-actin fluorescence. Pulsatile stretch was found to significantly increase the total endogenous ROS and superoxide anion release in normoxic and hypoxic conditions in primary PASM fibers. The effect of stretch was predominant in increasing superoxide anion release, only under hypoxic conditions. These findings, obtained by LSC in vitro are amenable to validation in any in vivo model of interest. The in vitro model is clinically relevant to human pulmonary vascular remodeling.