F-actin Staining Research Articles

3D bioprinting of conductive hydrogel for enhanced myogenic differentiation.

Recently, hydrogels have gained enormous interest in three-dimensional (3D) bioprinting toward developing functional substitutes for tissue remolding. However, it is highly challenging to transmit electrical signals to cells due to the limited electrical conductivity of the bioprinted hydrogels. Herein, we demonstrate the 3D bioprinting-assisted fabrication of a conductive hydrogel scaffold based on poly-3,4-ethylene dioxythiophene (PEDOT) nanoparticles (NPs) deposited in gelatin methacryloyl (GelMA) for enhanced myogenic differentiation of mouse myoblasts (C2C12 cells). Initially, PEDOT NPs are dispersed in the hydrogel uniformly to enhance the conductive property of the hydrogel scaffold. Notably, the incorporated PEDOT NPs showed minimal influence on the printing ability of GelMA. Then, C2C12 cells are successfully encapsulated within GelMA/PEDOT conductive hydrogels using 3D extrusion bioprinting. Furthermore, the proliferation, migration and differentiation efficacies of C2C12 cells in the highly conductive GelMA/PEDOT composite scaffolds are demonstrated using various in vitro investigations of live/dead staining, F-actin staining, desmin and myogenin immunofluorescence staining. Finally, the effects of electrical signals on the stimulation of the scaffolds are investigated toward the myogenic differentiation of C2C12 cells and the formation of myotubes in vitro. Collectively, our findings demonstrate that the fabrication of the conductive hydrogels provides a feasible approach for the encapsulation of cells and the regeneration of the muscle tissue.

Degradation-Dependent Controlled Delivery of Doxorubicin by Glyoxal Cross-Linked Magnetic and Porous Chitosan Microspheres.

Glyoxal cross-linked porous magnetic chitosan microspheres, GMS (∼170 μm size), with a tunable degradation profile were synthesized by a water-in-oil emulsion technique to accomplish controlled delivery of doxorubicin (DOX), a chemotherapeutic drug, to ensure prolonged chemotherapeutic effects. The GMS exhibit superparamagnetism with saturation magnetization, Ms = 7.2 emu g–1. The in vitro swelling and degradation results demonstrate that a swelling plateau of GMS is reached at 24 h, while degradation can be modulated to begin at 96–120 h by formulating the cross-linked network using glyoxal. MTT assay, live/dead staining, and F-actin staining (actin/DAPI) validated the cytocompatibility of GMS, which further assured good drug loading capacity (35.8%). The release mechanism has two stages, initiated by diffusion-inspired release of DOX through the swollen polymer network (72 h), which is followed by a disintegration-tuned release profile (>96 h) conferring GMS a potential candidate for DOX delivery.

Long non-coding RNA ARHGAP5-AS1 inhibits migration of breast cancer cell via stabilizing SMAD7 protein

PurposeTumor metastasis is the main cause of death from breast cancer patients and cell migration plays a critical role in cancer metastasis. Recent studies have shown long non-coding RNAs (lncRNAs) play an essential role in the initiation and progression of cancer. In the present study, the role of an LncRNA, Rho GTPase Activating Protein 5- Antisense 1 (ARHGAP5-AS1) in breast cancer was investigated.MethodsRNA sequencing was performed to find out dysregulated LncRNAs in MDA-MB-231-LM2 cells. Transwell migration assays and F-actin staining were utilized to estimate cell migration ability. RNA pulldown assays and RNA immunoprecipitation were used to prove the interaction between ARHGAP5-AS1 and SMAD7. Western blot and immunofluorescence imaging were used to examine the protein levels. Dual luciferase reporter assays were performed to evaluate the activation of TGF-β signaling.ResultsWe analyzed the RNA-seq data of MDA-MB-231 and its highly metastatic derivative MDA-MB-231-LM2 cell lines (referred to as LM2) and identified a novel lncRNA (NR_027263) named as ARHGAP5-AS1, which expression was significantly downregulated in LM2 cells. Further functional investigation showed ARHGAP5-AS1 could inhibit cell migration via suppression of stress fibers in breast cancer cell lines. Afterwards, SMAD7 was further identified to interact with ARHGAP5-AS1 by its PY motif and thus its ubiquitination and degradation was blocked due to reduced interaction with E3 ligase SMURF1 and SMURF2. Moreover, ARHGAP5-AS1 could inhibit TGF-β signaling pathway due to its inhibitory role on SMAD7.ConclusionARHGAP5-AS1 inhibits breast cancer cell migration via stabilization of SMAD7 protein and could serve as a novel biomarker and a potential target for breast cancer in the future.

3D-Printed Ginsenoside Rb1-Loaded Mesoporous Calcium Silicate/Calcium Sulfate Scaffolds for Inflammation Inhibition and Bone Regeneration.

Bone defects are commonly found in the elderly and athletic population due to systemic diseases such as osteoporosis and trauma. Bone scaffolds have since been developed to enhance bone regeneration by acting as a biological extracellular scaffold for cells. The main advantage of a bone scaffold lies in its ability to provide various degrees of structural support and growth factors for cellular activities. Therefore, we designed a 3D porous scaffold that can not only provide sufficient mechanical properties but also carry drugs and promote cell viability. Ginsenoside Rb1 (GR) is an extract from panax ginseng, which has been used for bone regeneration and repair since ancient Chinese history. In this study, we fabricated scaffolds using various concentrations of GR with mesoporous calcium silicate/calcium sulfate (MSCS) and investigated the scaffold’s physical and chemical characteristic properties. PrestoBlue, F-actin staining, and ELISA were used to demonstrate the effect of the GR-contained MSCS scaffold on cell proliferation, morphology, and expression of the specific osteogenic-related protein of human dental pulp stem cells (hDPSCs). According to our data, hDPSCs cultivated in GR-contained MSCS scaffold had preferable abilities of proliferation and higher expression of the osteogenic-related protein and could effectively inhibit inflammation. Finally, in vivo performance was assessed using histological results that revealed the GR-contained MSCS scaffolds were able to further achieve more effective hard tissue regeneration than has been the case in the past. Taken together, this study demonstrated that a GR-containing MSCS 3D scaffold could be used as a potential alternative for future bone tissue engineering studies and has good potential for clinical use.

Dental follicle cell differentiation towards periodontal ligament-like tissue in a self-assembly three-dimensional organoid model.

Periodontitis remains an unsolved oral disease, prevalent worldwide and resulting in tooth loss due to dysfunction of the periodontal ligament (PDL), a tissue connecting the tooth root with the alveolar bone. A scaffold-free three-dimensional (3D) organoid model for in vitro tenogenesis/ligamentogeneis has already been described. As PDL tissue naturally arises from the dental follicle, the aim of this study was to investigate the ligamentogenic differentiation potential of dental follicle cells (DFCs) in vitro by employing this 3D model. Human primary DFCs were compared, in both two- and three-dimensions, to a previously published PDL- hTERT cell line. The 3D organoids were evaluated by haematoxylin and eosin, 4',6-diamidino-2-phenylindole and F-actin staining combined with detailed histomorphometric analyses of cell-row structure, angular deviation and cell density. Furthermore, the expression of 48 tendon/ligament- and multilineage-related genes was evaluated using quantitative polymerase chain reaction, followed by immunofluorescent analyses of collagen 1 and 3. The results showed that both cell types were successful in the formation of scaffold-free 3D organoids. DFC organoids were comparable to PDL-hTERT in terms of cell density; however, DFCs exhibited superior organoid morphology, cell-row organisation (p < 0.0001) and angular deviation (p < 0.0001). Interestingly, in 2 dimensions as well as in 3D, DFCs showed significantly higher levels of several ligament- related genes compared to the PDL-hTERT cell line. In conclusion, DFCs exhibited great potential to form PDL-like 3D organoids in vitro suggesting that this strategy can be further developed for functional PDL engineering.

Antidiabetic activities of Bolanthus spergulifolius (Caryophyllaceae) extracts on insulin-resistant 3T3-L1 adipocytes.

Diabetes mellitus (DM) is a metabolic disorder with chronic hyperglycemia featured by metabolic outcomes owing to insufficient insulin secretion and/or insulin effect defect. It is critical to investigate new therapeutic approaches for T2DM and alternative, natural agents that target molecules in potential signal pathways. Medicinal plants are significant resources in the research of alternative new drug active ingredients. Bolanthus spergulifolius (B. spergulifolius) is one of the genera of the family Caryophyllaceae. In this study, it was explored the potential anti-diabetic effects in vitro of B. spergulifolius extracts on 3T3-L1 adipocytes. The total phenolic contents (TPC) of methanolic (MeOH), ethyl acettate (EA) and aqueous extracts of B. spergulifolius were evaluated via Folin-Ciocateau. B. spergulifolius extracts showing highly TPC (Aqueous< MeOH< EA) and their different concentrations were carried out on preadipocytes differentiated in to mature 3T3-L1 adipocytes to investigate their half-maximal (50%) inhibitory concentration (IC50) value by using Thiazolyl blue tetrazolium bromide (MTT) assay. The IC50 of MeOH, EA and Aqueous extracts were observed as 305.7 ± 5.583 μg/mL, 567.4 ± 3.008 μg/mL, and 418.3 ± 4.390 μg/mL and used for further experiments. A live/dead assay further confirmed the cytotoxic effects of MeOH, EA and Aqueous extracts (respectively, 69.75 ± 1.70%, 61.75 ± 1.70%, 70 ± 4.24%, and for all p< 0.05). Also, effects of extracts on lipid accumulation in mature 3T3-L1 adipocytes were evaluated by Oil-Red O staining assay. The extracts effectively decreased lipid-accumulation compared to untreated adipocytes (for all p< 0.05). Moreover, effect of extracts on apoptosis regulated by the Bax and Bcl-2 was investigated by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The extracts significantly induced apoptosis by up-regulating pro-apoptotic Bax expression but down-regulated anti-apoptotic Bcl-2 gene expression compared to untreated adipocytes (for all p< 0.05). The Glut-4 expression linked with insulin resistance was determined by qRT-PCR, Western-blot analysis, and immunofluorescence staining. In parallel, the expression of Glut-4 in adipocytes treated with extracts was significantly higher compared to untreated adipocytes (for all p< 0.05). Extracts significantly suppressed cell migration after 30 h of wounding in a scratch-assay (for all p< 0.05). Cell morphology and diameter were further evaluated by phase-contrast microscopy, scanning electron microscopy, Immunofluorescence with F-Actin and Giemsa staining. The adipocytes treated with extracts partially lost spherical morphology and showed smaller cell-diameter compared to untreated adipocytes (for all p< 0.05). In conclusion, these results suggest that extracts of B. spergulifolius cause to an induce apoptosis, decrease lipid-accumulation, wound healing, up-regulating Glut-4 level and might contribute to reducing of insulin-resistance in DM.

Radiopacity endowed magnetic nanocomposite with hyperthermia and in vitro mineralization potential: a combinatorial therapeutic system for osteosarcoma

The development of clinically advanced multifaceted therapeutic materials for osteosarcoma is at the forefront of cancer research. Accordingly, this work presents the design of a multifunctional magnetic nanocomposite composed of maghemite, strontium doped hydroxyapatite and silica nanoparticles prospectively holding indispensable therapeutic features such as magnetic hyperthermia, in vitro biomineralization, sustained drug release and intrinsic radiopacity for the treatment of osteosarcoma. The optimal composition has been identified by sequentially modulating the ratio of precursors of the magnetic nanocomposite synthesized by sol–gel technique. Structural and morphological characterization by x-ray diffraction, fourier transform infrared spectrum, Brunauer–Emmet–Teller and transmission electron microscopy analyses followed by VSM, hyperthermia and micro-CT analyses essentially assisted in the selective configuration of biofunctional properties. Results exemplify that MSHSr1 has a saturation magnetization of 47.4 emu g−1 and attained hyperthermia temperature (42 °C) at a very low exposure time of 4 min. MSHSr1 is further unique with respect to its exceptional x-ray attenuation ability (contrast enhancement 154.5% in digital radiography; CT number 3100 HU), early biomimetic mineralization (in vitro) evident by the formation of spheroidal apatite layer (Ca/P ratio 1.33) harvested from FESEM–EDX analysis and controlled release of Doxorubicin, the clinically used chemotherapeutic drug: 87.7% at 120 h in tumour analogous pH (6.5) when compared to physiological pH (71.3% at 7.4). MTT assay complemented with cytoskeleton (F-actin) staining of human osteosarcoma (HOS) cells affirm biocompatibility of MSHSr1. In vitro biomineralization authenticated by Alizarin red S and von Kossa staining has been further corroborated by semi-quantitative calcium estimation of HOS cells cultured with MSHSr1 for two weeks. The results therefore validate the multifunctionality of MSHSr1, and hence could be proposed as a combinatorial therapeutic nanocomposite for osteosarcoma treatment.

Punicalagin Inhibited Inflammation and Migration of Fibroblast-Like Synoviocytes Through NF-κB Pathway in the Experimental Study of Rheumatoid Arthritis.

BackgroundThe aggressive phenotype of fibroblast-like synoviocytes (FLSs) is essential in the synovitis and bone destruction in rheumatoid arthritis (RA). Punicalagin is a natural polyphenol extracted in pomegranate juice, which possesses antioxidant, anti-inflammatory and anti-tumor properties suggesting it may be a potent drug for RA therapy. However, there is paucity of information on its effect in RA.ObjectiveTo investigate the effects of punicalagin on synovial inflammation and bone destruction in RA.MethodsFLSs were isolated from synovial tissue of RA patients. The mRNA levels were evaluated by quantitative real-time PCR. Western blot was used for protein level measurements. The secretion of pro-inflammatory cytokines and metalloproteinases (MMPs) was detected by ELISA assays. Edu staining assays were carried out to investigate the proliferation of FLSs. Cell migration was assessed by Boyden chambers, wound scratch assays and F-actin staining in vitro. The intracellular translocation of nuclear factor kappa B (NF-κB) was investigated using immunofluorescence. The effects of punicalagin in vivo were measured by using collagen-induced arthritis (CIA) mice.ResultsPunicalagin treatments significantly reduced the TNF-α induced expressions of pro-inflammatory cytokines (IL-1β, IL-6, IL-8 and IL-17A) and MMPs (MMP-1 and MMP-13) of RA FLSs. Punicalagin also suppressed the proliferation and migration of RA FLSs. Moreover, punicalagin (50mg/kg/d) alleviated arthritis severity and bone destruction, and decreased serum IL-6 and TNF-α in CIA mice. Further mechanism studies indicated that punicalagin blocked NF-κB activation via suppressing phosphorylation of IKK and IkBα, and preventing the translocation of 65.ConclusionOur findings suggested that punicalagin might be one of natural therapeutic compounds for relieving RA progress via suppressing FLSs inflammation and migration through modulating NF-κB pathways.

Glaucocalyxin A suppresses osteoclastogenesis induced by RANKL and osteoporosis induced by ovariectomy by inhibiting the NF-κB and Akt pathways

Ethnopharmacological relevanceGlaucocalyxin A (GLA), the most abundant active component of the aboveground sections of Rabdosia japonica (Burm. f.) Hara var. glaucocalyx (Maxim.) Hara, possesses various pharmacological activities, such as antioxidant, antithrombosis, anticoagulation, antibacterial, antitumor, anti-inflammatory activities. According to previous studies, inflammation is closely associated with osteoclast differentiation and activity. Although GLA has demonstrated effective anti-inflammatory properties, its effects on osteoclast differentiation remain unclear. Aim of the studyTo examine the possible inhibitory effects of GLA and its molecular mechanisms in osteogenesis induced by RANKL as well as ovariectomy (OVX)-induced osteoporosis (OP) in mice. Materials and methodsTartrate-resistant acid phosphatase (TRAP) staining, F-actin staining, and a bone resorption pit assay were applied for identifying the effects of GLA on the differentiation of osteoclasts and the function of bone resorption. The mRNA expression of the genes related to osteoclast differentiation was measured by quantitative PCR. Protein expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), c-fos and phosphorylation of inhibitor of nuclear factor kappa B (IκBα), protein kinase B (AKT), c–Jun N–terminal kinase (JNK), extracellular signal–regulated kinase (ERK), and p38 in RANKL-induced osteoclasts was determined using western blotting. The effect of GLA on OP was studied using a mouse model of OVX. ResultsAt nontoxic concentrations ≤0.5 μM in vitro, GLA suppressed the formation of osteoclasts induced by RANKL with the decreased number and area size of TRAP-positive multinuclear osteoclasts, and the resorption of bone function by reducing F-actin ring number and bone resorption pit areas. It also reduced the expression of the genes specific for osteoclasts, which included genes encoding NFATc1, cathepsin K, c-fos, TRAP, vacuolar-type ATPase d2, and dendritic cell-specific transmembrane protein. Moreover, GLA repressed NF-κB and Akt pathway activation induced by RANKL. Micro-CT analysis of femur samples indicated decreased bone loss and greater trabecular bone density after GLA treatment, which showed that GLA played a protective role by inhibiting bone loss in OVX-induced OP mice in vivo. ConclusionsOur study is the first to show that GLA has significant therapeutic potential in OP, which is the disease of osteoclast increase caused by estrogen deficiency.

Topical fluoride varnishes promote several biological responses on human gingival cells

ObjectiveTo compare the in vitro cytotoxicity of four commercial topical fluoride varnishes widely used in daily dental practice for the prevention of caries on human fibroblasts: Cervitec F, Fixofluor, Fluor Protector S and Duraphat. Material and methodsHuman gingival fibroblasts (hGF) were exposed to different concentrations of fluoride varnishes extracts. Biological assays, including MTT and IC50 value determination, annexin-V/7-AAD staining, cell migration and F-actin staining with phalloidin were carried out. Statistical analyses were performed using one-way ANOVA and Tukey’s post hoc test. ResultsAt 4% concentration, all of the fluoride varnishes extracts affected fibroblasts metabolic activity, exhibiting a high degree of cytotoxicity at all measured time points. At 0.1% and 1%, Duraphat and Fixofluor or Fluor Protector S and Cervitec F exerted the lowest or highest cytotoxic effects, respectively. Similar effects were evidenced when induction of apoptosis/necrosis and cell migration assays were analyzed. Immunocytochemical assays revealed a similar number of fibroblasts, without changes in the morphology and F-actin content at 0.1% concentration of all tested materials, while at 1% concentration, Fluor Protector S and Cervitec F showed few cells with aberrant morphology or non-adhered cells, respectively. ConclusionsDifferent commercial topical fluoride varnishes with the same therapeutic indication may exhibit different biological effects and cytotoxicity on fibroblasts.

Spatiotemporal and functional characterisation of transient receptor potential vanilloid 4 (TRPV4) in the murine intervertebral disc.

The molecular regulators of mechano-transduction in intervertebral disc (IVD) cells are not well understood. The aim of the present study was to characterise the expression and function of the mechano-sensitive ion channel TRPV4 in the IVD. A novel transgenic reporter mouse, in which the endogenous Trpv4 locus drove the expression of LacZ, was used to localise Trpv4 expression at specific stages of spine development [embryonic day (E) 8.5, 12.5, 17.5, postnatal day 1] and time points following skeletal maturity (2.5, 6, 9 and 12 months). The TRPV4-specific agonist GSK1016790A and antagonist GSK2193874 were used to assess the functional response of annulus fibrosus (AF) cells using epifluorescence imaging with Ca2+-sensitive Fura-2 dye and F-actin staining. The effects of TRPV4 agonism and antagonism in mechanically stimulated AF cells were quantified by gene expression analysis. Trpv4 expression was specific to the developing notochord and intervertebral mesenchyme at E12.5. At 2.5, 6 and 9 months, Trpv4 expression was detected in the nucleus pulposus, inner AF, cartilage endplate and vertebral growth plate. AF cells treated with GSK1016790A demonstrated heterogeneity in TRPV4-dependent Ca2+ responses (no response, calcium oscillation or sustained response). TRPV4-induced Ca2+ signalling was associated with Rho/ROCK-dependent actin cytoskeleton remodelling and stress-fibre formation. In AF cells, cyclic-tensile-strain-induced changes in Acan and Prg4 expression were mediated by TRPV4 channel activation. These data establish TRPV4 as an important mechano- sensor regulating IVD mechano-biology.

Nanodiamonds Inhibit Cancer Cell Migration by Strengthening Cell Adhesion: Implications for Cancer Treatment.

Nanodiamonds (NDs) are a type of biocompatible nanomaterial with easily modified surfaces and are considered as promising candidates in biomedicine. In this work, the inhibition of tumor cell migration by carboxylated nanodiamonds (cNDs) was investigated. AFM-based single cell adhesion and F-actin staining experiments demonstrated that cNDs treatment could enhance cell adhesion and impair assembly of the cytoskeleton. The mechanism analysis of the regulatory protein expression level also proved that cNDs could inhibit the migration of Hela cells by preventing the epithelial-mesenchymal transition (EMT) process through the transforming growth factor β (TGF-β) signaling pathway. The in vivo pulmonary metastasis model also showed that cNDs effectively reduced the metastasis of murine B16 melanoma cells. In summary, cNDs have been demonstrated to inhibit cancer cell migration in vitro and decrease tumor metastasis in vivo. Therefore, cNDs might have potential utility for specific cancer treatment.

Abstract PS19-06: Role of the calcium-sensing receptor (CaSR) in invasive lobular breast carcinoma metastasis to the ovary

Abstract Invasive lobular carcinoma (ILC) is the second most common histological subtype of breast cancer, after the more common invasive ductal carcinoma (IDC). ILC accounts for 10 to 15% of all invasive breast carcinomas, affecting approx. 26,000-40,000 women in 2020 in the US alone. Despite 95% of ILC being Luminal A conferring patients a favorable prognosis, patients with ILC have poorer long-term outcomes when compared to patients with Luminal A IDC. ILC spread to common sites of ER+ breast cancer metastasis such as the bones, but are also three times more likely to spread to the ovaries, peritoneum, and gastrointestinal tract compared to IDC, and these unique aspects of metastases remain poorly understood. To better understand metastasis to the ovary we performed DNA and RNA sequencing of 11 pairs of primary breast tumor and ovarian metastasis as well as 13 orphan breast cancer ovarian metastases. Our cohort was enriched for lobular histology, with 13 samples originating from ILC, 6 from IDC and 6 from mixed ILC/IDC. We found mutations in CDH1 (43%), PIK3CA (40%), and FOXA1 (29%) highlighting the enrichment in ILC cases present in our cohort. Gene expression analysis lead to identification of 874 differentially expressed between primary tumors and ovarian metastases. We identified the calcium-sensing receptor CaSR as one of the top upregulated in ovarian metastases compared to primary tumor in 10 out of the 11 paired samples. Other genes highly expressed in ovarian metastases included TAC3, GLRA2, ALLC, MUC19, CHRNA2, PIP, CST4, and TEX15. Pathways analyses showed an enrichment of signaling through glutamate receptor and glycine receptor families. To assess the contribution of CaSR to breast cancer cell proliferation and metastatic properties, and due to the absence of breast cancer cell lines expressing CaSR, we generated CaSR overexpression models using lentiviral infection in MDA-MB-134, MDA-MB-330, BCK4 and SUM44PE ILC cell lines. While overexpression of CaSR alone did not confer a cell growth advantage or migratory ability to the cell lines, stimulation with calcium or a calcium mimetic resulted in enhanced migration in transwell assays in 3 of the 4 cell lines. Scratch assays further confirmed the stimulation of cell migration in cells with CaSR overexpression in the presence of calcium. Cell migration in CaSR overexpression models could be stimulated with the calcimimetic R568, and inhibited by the calcilytics NPS2143, and F-actin staining confirmed the need to activate the receptor to enhance migratory properties. Our studies further revealed that the induced migratory properties of CaSR overexpressing cells required estradiol and ER signaling, and that migration could be blocked with ER inhibitors such as ICI 182,780 and tamoxifen. Western blotting data revealed that the enhanced cell migratory properties of CaSR overexpressing cells was via activation of the MEK/ERK pathway and migration could be inhibited using specific small molecule pathway inhibitors.Altogether, our study provides insight on the potential mechanism by which upregulation of the CaSR supports breast cancer ovarian metastasis. We hope that these studies will not only deepen our understanding of ILC ovarian metastasis but will eventually lead to the development of more effective therapies and improve the outcome of patients with this understudied type of breast cancer. Citation Format: Ye Qin, Laura Savariau, Ahmed Mohammed Basudan, Osama Shah, Zheqi Li, Tiantong Liu, Nilgun Tasdemir, Lan Coffman, Esther Elishaev, Jennifer M Atkinson, Peter Lucas, Adrian V Lee, Steffi Oesterreich. Role of the calcium-sensing receptor (CaSR) in invasive lobular breast carcinoma metastasis to the ovary [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-06.

Cobalt-containing calcium phosphate induces resorption of biomineralized collagen by human osteoclasts

BackgroundBiomineralized collagen, consisting of fibrillar type-I collagen with embedded hydroxyapatite mineral, is a bone-mimicking material with potential application as a bone graft substitute. Despite the chemical and structural similarity with bone extracellular matrix, no evidence exists so far that biomineralized collagen can be resorbed by osteoclasts. The aim of the current study was to induce resorption of biomineralized collagen by osteoclasts by a two-fold modification: increasing the calcium phosphate content and introducing cobalt ions (Co2+), which have been previously shown to stimulate resorptive activity of osteoclasts.MethodsTo this end, we produced biomineralized collagen membranes and coated them with a cobalt-containing calcium phosphate (CoCaP). Human osteoclasts, derived from CD14+ monocytes from peripheral blood, were differentiated directly on the membranes. Upon fluorescent staining of nuclei, F-actin and tartrate-resistant alkaline phosphatase, the cells were analyzed by laser confocal microscopy. Their resorption capacity was assessed by scanning electron microscopy (SEM), as well as indirectly quantified by measuring the release of calcium ions into cell culture medium.ResultsThe CoCaP coating increased the mineral content of the membranes by 4 wt.% and their elastic modulus from 1 to 10 MPa. The coated membranes showed a sustained Co2+ release in water of about 7 nM per 2 days. In contrast to uncoated membranes, on CoCaP-coated biomineralized collagen membranes, osteoclasts sporadically formed actin rings, and induced formation of resorption lacunae, as observed by SEM and confirmed by increase in Ca2+ concentration in cell culture medium. The effect of the CoCaP layer on osteoclast function is thought to be mainly caused by the increase of membrane stiffness, although the effect of Co2+, which was released in very low amounts, cannot be fully excluded.ConclusionsThis work shows the potential of this relatively simple approach to induce osteoclast resorption of biomineralized collagen, although the extent of osteoclast resorption was limited, and the method needs further optimization. Moreover, the coating method is suitable for incorporating bioactive ions of interest into biomineralized collagen, which is typically not possible using the common biomineralization methods, such as polymer-induced liquid precursor method.

Effect of HIF1α on the TRPC6 channel of glomerular podocytes under chronic hypoxia

BackgroundChronic hypoxia plays an important role in the initiation and progression of chronic renal disease. The pathogenic role of chronic hypoxia in tubulointerstitial injury has been investigated widely, but little is known about acute hypoxia implications in glomerular damage. In this study, we investigated the effect of chronic hypoxia on transient receptor potential cation channel 6 (TRPC6) and the underlying mechanism in cultured human podocytes. MethodsFluo-3 was used as a calcium indicator of the OAG-induced receptor operated calcium entry (ROCE) and basal [Ca2+]i levels were monitored using laser scanning confocal microscope after exposure of cells to chronic hypoxia. 2-aminoethoxydiphenylborane (2-APB), a pharmacological blocker of TRPCs channels, was used to determine the role of TRPC6 in podocytes under chronic hypoxia. The mRNA expression and protein levels of TRPC6 were determined using Real-time RT-PCR and Western Blotting under normoxic and chronic hypoxic conditions. Actin arrangement was analyzed by confocal microscopy using phalloidin staining of F-actin in podocytes. ResultsCytosolic free Ca2+ was increased by hypoxia or the treatment of TRPC6 agonist OAG under normoxic conditions. The increase of intracellular Ca2+ induced by hypoxia was time- and dose-dependent, which can be inhibited by 2-APB, demonstrating that the changes of intracellular Ca2+ induced by OAG depend on the activation of TRPC6. Further study showed that the TRPC6 expression levels were significantly increased by hypoxia, which were inhibited by the HIF1α inhibitor in podocytes. Similarly, the increase of intracellular Ca2+ induced by hypoxia was decreased when the podocytes were incubated with HIF1α inhibitor. We also found that F-actin was ruptured by hypoxia in podocytes, showing cytoskeleton reorganization. ConclusionsTRPC6 mRNA and protein expression levels were significantly increased in podocytes under hypoxia, which may result in the increase of intracellular Ca2+. This alternation of TRPC6 may be relevant to the modulation of HIF1α. Hypoxia in podocytes can result in cytoskeleton reorganization, which further leads to podocytes injury and disfunction.

PolyIC Induces Retinoic Acid-inducible Gene-I and Melanoma Differentiation-associated Gene 5 and Modulates Inflammation in Podocytes.

Viral infection often exacerbates proteinuria, which has been suggested to be due to antiviral responses of podocytes. We examined the effect of polyinosinic-polycytidylic acid (polyIC) on the expression of retinoic acid-inducible gene-I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) in differentiated human podocytes in culture. The podocytes were treated with 2 ng/ml to 500 μg/ml of polyIC for 3 to 36 h, and also transfected with siRNA against RIG-I and MDA5. F-actin staining was performed to assess actin reorganization. PolyIC induced the expression of RIG-I and MDA5 in dose- and time-dependent manner, accompanied with interferon-β (IFN-β) and interleukin-6 (IL-6) up-regulation and actin reorganization. Temporal knockdown of RIG-I by siRNA decreased IFN-β expression, while MDA5 siRNA inhibited IFN-β and IL-6 expression. Actin reorganization was attenuated by RIG-I and MDA5 knockdown. RIG-I and MDA5 may play a role in the antiviral responses of podocytes.

Intrinsically radiopaque and antimicrobial cellulose based surgical sutures from mechanically powerful Agave sisalana plant leaf fibers.

The judicious configuration of a flexible radiopaque suture would be exemplary to facilitate effortless tracking and precise diagnosis of the sutured surgical site by various X-ray assisted imaging modalities and simultaneously serve as a complementary tool for monitoring the fate of the suture material during the post-operative course. A unique radiopaque cellulose based surgical suture (RF) with good mechanical properties was developed by strategically controlled mercerization and bleaching of mechanically strong natural cellulosic fibers extracted from Agave sisalana plant leaves followed by the facile dip-coating of SrO integrated polylactic acid (PLA). RF exhibited admirable straight-pull tensile strength (184 MPa) and commendable contrast enhancement (277.4%) under digital X-ray radiographic imaging which was further validated by micro-CT analysis. Further, RF has a controlled hydrolytic degradation profile favorable for surgical suturing (mass loss ∼22% in 28 days). The microporous surface architecture of RF (pore size < 10 μm) as a result of SrO-PLA coating enabled the loading of antibiotic (ciprofloxacin) deep inside the pores with a cumulative release of 24% at 28 days under physiological conditions substantiating its feasibility to be used as an efficient antimicrobial suture (CRF) that prevents possible bacterial infections at the surgical site. This has been demonstrated by antibacterial disc diffusion assay performed against two Gram-positive and two Gram-negative bacterial strains. Significantly, both RF and CRF are highly biocompatible as confirmed by MTT assay and F-actin staining. Hence, CRF would be a good biocompatible suture candidate holding good tensile properties, exceptional antimicrobial property and intrinsic radiopacity retention for a period >28 days.

An L314Q mutation in Map3k1 gene results in failure of eyelid fusion in the N-ethyl-N-nitrosourea-induced mutant line.

In this study, we describe an N-ethyl-N-nitrosourea-induced mouse model with a corneal opacity phenotype that was associated with “eye open at birth” (EOB). Histological and immunohistochemistry staining analysis showed abnormal differentiation of the corneal epithelial cells in the mutant mice. The EOB phenotype was dominantly inherited on a C57BL/6 (B6) background. This allele carries a T941A substitution in exon 4 that leads to an L314Q amino acid change in the open reading frame of MAP3K1 (MEEK1). We named this novel Map3k1 allele Map3k1L314Q. Phalloidin staining of F-actin was reduced in the mutant epithelial leading edge cells, which is indicative of abnormality in epithelial cell migration. Interestingly enough, not only p-c-Jun and p-JNK but also c-Jun levels were decreased in the mutant epithelial leading edge cells. This study identifies a novel mouse Map3k1 allele causing EOB phenotype and the EOB phenotype in Map3k1L314Q mouse may be associated with the reduced level of p-JNK and c-Jun.

Beyond Microcystins: Cyanobacterial Extracts Induce Cytoskeletal Alterations in Rice Root Cells.

Microcystins (MCs) are cyanobacterial toxins and potent inhibitors of protein phosphatases 1 (PP1) and 2A (PP2A), which are involved in plant cytoskeleton (microtubules and F-actin) organization. Therefore, studies on the toxicity of cyanobacterial products on plant cells have so far been focused on MCs. In this study, we investigated the effects of extracts from 16 (4 MC-producing and 12 non-MC-producing) cyanobacterial strains from several habitats, on various enzymes (PP1, trypsin, elastase), on the plant cytoskeleton and H2O2 levels in Oryza sativa (rice) root cells. Seedling roots were treated for various time periods (1, 12, and 24 h) with aqueous cyanobacterial extracts and underwent either immunostaining for α-tubulin or staining of F-actin with fluorescent phalloidin. 2,7-dichlorofluorescein diacetate (DCF-DA) staining was performed for H2O2 imaging. The enzyme assays confirmed the bioactivity of the extracts of not only MC-rich (MC+), but also MC-devoid (MC−) extracts, which induced major time-dependent alterations on both components of the plant cytoskeleton. These findings suggest that a broad spectrum of bioactive cyanobacterial compounds, apart from MCs or other known cyanotoxins (such as cylindrospermopsin), can affect plants by disrupting the cytoskeleton.

Nuclear F-actin Cytology in Oral Epithelial Dysplasia and Oral Squamous Cell Carcinoma

Oral cavity cancer has a low 5-y survival rate, but outcomes improve when the disease is detected early. Cytology is a less invasive method to assess oral potentially malignant disorders relative to the gold-standard scalpel biopsy and histopathology. In this report, we aimed to determine the utility of cytological signatures, including nuclear F-actin cell phenotypes, for classifying the entire spectrum of oral epithelial dysplasia and oral squamous cell carcinoma. We enrolled subjects with oral potentially malignant disorders, subjects with previously diagnosed malignant lesions, and healthy volunteers without lesions and obtained brush cytology specimens and matched scalpel biopsies from 486 subjects. Histopathological assessment of the scalpel biopsy specimens classified lesions into 6 categories. Brush cytology specimens were analyzed by machine learning classifiers trained to identify relevant cytological features. Multimodal diagnostic models were developed using cytology results, lesion characteristics, and risk factors. Squamous cells with nuclear F-actin staining were associated with early disease (i.e., lower proportions in benign lesions than in more severe lesions), whereas small round parabasal-like cells and leukocytes were associated with late disease (i.e., higher proportions in severe dysplasia and carcinoma than in less severe lesions). Lesions with the impression of oral lichen planus were unlikely to be either dysplastic or malignant. Cytological features substantially improved upon lesion appearance and risk factors in predicting squamous cell carcinoma. Diagnostic models accurately discriminated early and late disease with AUCs (95% CI) of 0.82 (0.77 to 0.87) and 0.93 (0.88 to 0.97), respectively. The cytological features identified here have the potential to improve screening and surveillance of the entire spectrum of oral potentially malignant disorders in multiple care settings.