NIH 3T3 Mouse Embryonic Fibroblast Research Articles

Improved penetration of wild ginseng extracts into the skin using low-temperature atmospheric pressure plasma

Wild ginseng (WG) is a well-known traditional medicinal plant that grows in natural environments in deep mountains. WG has been thought to exert potent physiological and medicinal effects, and, recently, its use in skin care has attracted much interest. This study investigated the efficient penetration of WG extracts into the skin by means of low-temperature atmospheric pressure plasma (LTAPP), and its effects on the skin at the cellular and tissue levels. NIH3T3 mouse embryonic fibroblasts and HRM-2 hairless mice were used to confirm the improved absorption of WG extracts into the skin using LTAPP. The gene expression levels in NIH3T3 cells and morphological changes in skin tissues after WG treatment were monitored using both in vitro and in vivo experiments. Although WG extracts did not show any significant effects on proliferative activity and cytotoxicity, at a concentration of 1:800, it significantly increased the expression of fibronectin and vascular endothelial growth factor. In the in vivo study, the combinational treatment of LTAPP and WG markedly induced the expression of fibronectin and integrin α6, and it thickened. Our results showed that LTAPP treatment safely and effectively accelerated the penetration of the WG extracts into the skin, thereby increasing the effects of WG on the skin.

Broad-Spectrum Antibacterial Activity of Proteolytically Stable Self-Assembled αγ-Hybrid Peptide Gels.

Bacterial infections pose a serious threat to mankind, and there is immense interest in the design and development of self-assembled peptide gels using ultrashort peptides for antibacterial applications. The peptide gels containing natural amino acids suffer from poor stability against proteolytic enzymes. Therefore, there is a need to design and develop peptide gels with improved stability against proteolytic enzymes. In the present work, we report the synthesis and characterization of α/γ hybrid peptides Boc-D-Phe-γ4-L-Phe-PEA (NH007) and Boc-L-Phe-γ4-L-Phe-PEA (NH009) to improve the proteolytic stability. Both of the dipeptides were found to self-assemble into gels in aqueous DMSO (3-5% w/v), and the self-assembly process was studied using FTIR and CD, which indicated antiparallel β-sheet formation with random coils in NH007 gels and random or unordered conformation in NH009. The rheological studies indicated viscoelastic characteristics for both gels; the storage modulus ( G') for NH007 and NH009 gels (3% w/v) was estimated as 0.2 and 0.5 MPa, higher than the loss modulus ( G''). Also, both gels demonstrated self-healing characteristics for six consecutive cycles when subjected to varying strains of 0.1 and 30% (200 s each). The peptide gels were incubated with a mocktail of proteolytic enzymes, proteinase K, pepsin, and chymotrypsin, and stability was monitored using RP HPLC. Up to 23 and 40% degradation was observed for NH007 (3%, w/v) in 24 and 36 h, and 77 and 94% degradation was observed for NH009 (3%, w/v), within the same period. Thus α/γ hybrid peptide gels containing D-Phe exhibited higher stability than gels fabricated using L-Phe. The use of D-residue in α/γ hybrid peptide significantly enhanced the stability of peptides against proteolytic enzymes, as the stability data reported in this work are possibly the best in class. Both peptide gels exhibited broad-spectrum antibacterial activity against Gram-negative and Gram-positive bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. The Pseudomonas aeruginosa and Staphylococcus aureus, in particular, are known to develop resistance. The NH007 (3%, w/v) demonstrated 65% inhibition, whereas NH009 (3%, w/v) showed 78% inhibition, with potent activity against Pseudomonas aeruginosa. Mechanistic studies, using SEM, HR-TEM, and bacterial live-dead assay, indicated entrapment of bacteria in gel networks, followed by interaction with cell membrane components and lysis. Cell viability (MTT assay) and toxicity (LDH assay) studies showed that both gels are not toxic to NIH 3T3 mouse embryonic fibroblast cells (mammalian). MTT assay showed >85% cell viability, and LDH assay exhibited not more than 15% cytotoxicity, even at higher concentrations (5%, w/v) and prolonged exposures (48 h). Overall, studies indicate the potential application of gels developed from the α/γ hybrid peptides in preventing biomaterial-related infections.

Retracted: MicroRNA‐29b‐3p prevents Schistosoma japonicum‐induced liver fibrosis by targeting COL1A1 and COL3A1

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, causing granulomatous inflammation and cumulative fibrosis. This study explored in vivo and vitro effects of miR-29b-3p in granulomatous liver fibrosis by targeting COL1A1 and COL3A1 in Schistosoma japonicum infection. Thirty male Balb/c mice were assigned to normal control and model (percutaneous infection of cercariae of S. japonicum) groups. NIH-3T3 mouse embryonic fibroblasts were designated into blank, NC, miR-29b-3p mimic, TGF-β1, TGF-β1 + NC, and TGF-β1 + miR-29b-3p mimic groups. HE and Masson staining were employed to observe the pathological changes and collagenous fibrosis. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was determined by immunohistochemistry. The RT-qPCR, Western blotting and immunofluorescence staining were conducted to determine expression of miR-29b-3p, COL1A1, and COL3A1. CCK-8 assay and flow cytometry were performed to evaluate viability and apoptosis. The relative expression of miR-29b-3p decreased in the model group. The model group showed marked fibrosis in liver tissues. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was higher in the model group than that in the normal control group. Dual luciferase reporter gene assay revealed that miR-29b-3p directly targeted COL1A1 and COL3A1. Compared with the blank, NC, TGF-β1 and TGF-β1 + NC groups, the miR-29b-3p mimic group exhibited up-regulated expression of miR-29b-3p and MMP-9 but down-regulated expression of TIMP-1, HSP47, α-SMA, COL1A1, and COL3A1; while lower cell viability but higher apoptosis rate showed. It indicated that miR-29b-3p prevents S. japonicum-induced liver fibrosis by inhibiting COL1A1 and COL3A1.

Molecular mechanisms of Ellis‑van Creveld gene variations in ventricular septal defect.

The Ellis-van Creveld (EVC) gene is associated with various congenital heart diseases. However, studies on EVC gene variations in ventricular septal defect (VSD) and the underlying molecular mechanisms are sparse. The present study detected 11 single-nucleotide polymorphisms (SNPs) in 65 patients with VSD and 210 control patients from the Chinese Han population. Of the identified SNPs only the c.1727G>A SNP site was positively associated with the development of VSD (P<0.007). A known mutation, c.343C>G, was also identified, which causes a leucine to valine substitution at amino acid 115 of the EVC protein (p.L115V). The results of functional prediction indicated that c.343C>G may be a pathogenic mutation. In addition, in NIH3T3 mouse embryonic fibroblast cells, the EVC c.343C>G mutation significantly decreased cell proliferation and increased apoptosis. Further investigation demonstrated that in NIH3T3 cells, overexpression of EVC c.343C>G mutation reduced the binding between EVC and smoothened, which further downregulated the activity of the hedgehog (Hh) signaling pathway and the expression of downstream cyclin D1 and B-cell lymphoma 2 proteins with SAG. The c.1727G>A SNP of the EVC gene increased VSD susceptibility in patients from the Chinese Han population. The molecular mechanism underlying the development of VSD induced by the EVC c.343C>G mutation may be due to a reduction in the anti-apoptotic and proliferative abilities of cardiomyocytes via downregulation of Hh pathway activity. The results of the present study may provide novel targets for the diagnosis and treatment of patients with VSD.

In vitro mesenchymal-epithelial transition in NIH3T3 fibroblasts results in onset of low-dose radiation hypersensitivity coupled with attenuated connexin-43 response

BackgroundMesenchymal-to-epithelial transition (MET) is associated with altered cell adhesion patterns. Independent studies showed that cellular adhesion regulates low-dose hyper-radiosensitivity (HRS), a phenomenon reported widely in tumour cells. Therefore, present study aimed to investigate whether MET and associated cellular adhesion alterations affect cellular radiosensitivity. MethodsWe established multiple stages of MET by in vitro transformation of NIH3T3 mouse embryonic fibroblasts. Nutritional deprivation followed by repetitive treatment cycles of 3-methylcholanthrene and phorbol-12-myristate-13-acetate with frequent isolation of foci established three progressive strains (NIH3T3.1, NIH3T3x3, NIH3T3x8x3) depicting MET, and one strain (NIH3T3x12) with partial reversion. Alterations in morphology, cell adhesion properties, expression/intracellular localization of cell adhesion proteins, microRNA expression and cellular radiosensitivity were studied in these stably transformed cell strains. ResultsAll four transformants had increased proliferation rate, saturation density, bipolarity, E-cadherin expression; coupled with reduced cell size/spreading, pseudopodia/migration, and fibroblast marker protein and vimentin. The most aggressive trans-differentiated (phenotypically epithelial) cell strain, NIH3T3x8x3 acquired ~30% higher growth potential associated with more than two-fold reduction in cell size and migration. These phenotypic changes accompanied ~40% reduction in endogenous or radiation-induced connexin-43 expression/mitochondrial translocation. Incidentally, all three progressive strains displayed prominent HRS (αs/αr: 7.95–37.29) whereas parental (NIH3T3) and reverting (NIH3T3x12) strains lacked HRS and had distinct radiation-induced Cx43 translocation into mitochondria. ConclusionOur study shows that trans-differentiating fibroblasts progressively acquiring epithelial features during MET process, display low-dose hyper-radiosensitivity associated with altered Cx43 behaviour. General significanceThis study demonstrates that MET progression triggers low-dose hyper-radiosensitivity in trans-differentiating cells, which has significant therapeutic implications.

Urea dimethacrylates functionalized with bisphosphonate/bisphosphonic acid for improved dental materials

ABSTRACTIncorporation of bisphosphonate/bisphosphonic acid groups in dental monomer structures should increase interaction of these monomers with dental tissue as these groups have strong affinity for hydroxyapatite. Therefore, new urea dimethacrylates functionalized with bisphosphonate (1a, 1b) and bisphosphonic acid (2a, 2b) groups are synthesized and evaluated for dental applications. Monomers 1a and 1b are synthesized from 2‐isocyanatoethyl methacrylate (IEM) and two bisphosphonated amines (BPA1 and BPA2), prepared as reported elsewhere. Selective dealkylation of the bisphosphonate ester groups of 1a and 1b using trimethylsilyl bromide (TMSBr) gives monomers (2a and 2b) with bisphosphonic acid functionality. X‐ray diffractometer (XRD), Raman spectroscopy, and X‐ray photoelectron spectroscopy (XPS) analyses of monomer‐treated HAP particles show that 2a induces formation of stable monomer‐calcium salts, similar to 10‐methacryloyloxydecyl dihydrogen phosphate (MDP), with higher chemical interaction than 2b. The photopolymerization studies indicate good copolymerizability with commercial dental monomers. In vitro studies on NIH 3T3 mouse embryonic fibroblast cells have clearly shown that the tested monomers (1b and 2b) are not toxic according to the MTT standards. All these properties make these monomers suitable as biocompatible cross‐linkers/adhesives for dental applications. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 3195–3204

Effect of processing parameters on the deposition of SiOx-like coatings on the surface of polypropylene films using glow discharge plasma assisted polymerization for tissue engineering applications

In this investigation hexamethyldisiloxane (HMDSO) was used to deposit SiOx-like coatings on the surface of polypropylene (PP) films using an Ar + O2 glow discharge. Discharge potential and deposition time were varied systematically to optimize various SiOx coating properties such as hydrophilicity, oxidation state and surface homogeneity for possible tissue engineering applications. X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and contact angle were employed to analyze the said changes. Cell compatibility study of the SiOx films was done by in-vitro analysis, using NIH-3T3 (mouse embryonic fibroblast) cells to study the influence of the surface characteristics on cell adhesion and proliferation. The SiOx films were found to be highly hydrophilic, based on the contact angle and surface energy results. XPS confirmed the efficient incorporation of various oxidation states of Si and oxygen containing polar functional groups on the surface of PP film. Overall, a relatively smooth surface topography was observed, with distinct differences, depending on the applied plasma parameters. The in-vitro analysis characterized SiOx films as cyto-compatible, i.e improved non-toxicity, adhesion and proliferation of NIH-3T3 fibroblast cells. Overall, the developed films were found to exhibit a high potential for possible tissue engineering applications.

Influence of extremely low-frequency magnetic field on circadian rhythm of cryptochrome in mouse embryonic fibroblasts

Objective: To investigate the influence of extremely low-frequency magnetic field on periodical expression of cryptochrome (Cry) gene in mouse embryonic fibroblast NIH3T3 cells. Methods: The NIH3T3 cells were divided into magnetic field group and sham-exposure group. The NIH3T3 cells in the magnetic field group were stimulated by horse serum and then exposed to an extremely low-frequency magnetic field (50 Hz and 0.3 mT) for 48 hours, and those in the sham-exposure group were also stimulated by horse serum and then exposed to a coil for 48 hours. The NIH3T3 cells were collected, total RNA was extracted, and cDNA was obtained via reverse transcription. Real-time fluorescent quantitative RT-PCR was used to measure the changes in transcription cycles of Cry and Period genes in both groups. Results: There was no significant difference in the proliferation rate at 0, 12, 24, and 48 hours of exposure between the two groups (P>0.05) . Both sham-exposure group and magnetic field group showed a rhythmic change in the expression of Cry gene, and compared with the sham-exposure group, the magnetic field group had a significantly shortened circadian rhythm of Cry gene in NIH3T3 cells (t=2.57, P<0.05) . Both groups had rhythmic and periodical expression of Period gene and there was no significant difference between the two groups (t=0.70, P>0.05) . Conclusion: Extremely low-frequency magnetic field can significantly shorten the circadian rhythm of Cry gene in mouse embryonic fibroblasts, while there is no significant change in the circadian rhythm of Period gene.

Far-infrared suppresses skin photoaging in ultraviolet B-exposed fibroblasts and hairless mice.

Ultraviolet (UV) induces skin photoaging, which is characterized by thickening, wrinkling, pigmentation, and dryness. Collagen, which is one of the main building blocks of human skin, is regulated by collagen synthesis and collagen breakdown. Autophagy was found to block the epidermal hyperproliferative response to UVB and may play a crucial role in preventing skin photoaging. In the present study, we investigated whether far-infrared (FIR) therapy can inhibit skin photoaging via UVB irradiation in NIH 3T3 mouse embryonic fibroblasts and SKH-1 hairless mice. We found that FIR treatment significantly increased procollagen type I through the induction of the TGF-β/Smad axis. Furthermore, UVB significantly enhanced the expression of matrix metalloproteinase-1 (MMP-1) and MMP-9. FIR inhibited UVB-induced MMP-1 and MMP-9. Treatment with FIR reversed UVB-decreased type I collagen. In addition, FIR induced autophagy by inhibiting the Akt/mTOR signaling pathway. In UVB-induced skin photoaging in a hairless mouse model, FIR treatment resulted in decreased skin thickness in UVB irradiated mice and inhibited the degradation of collagen fibers. Moreover, FIR can increase procollagen type I via the inhibition of MMP-9 and induction of TGF-β in skin tissues. Therefore, our study provides evidence for the beneficial effects of FIR exposure in a model of skin photoaging.

Bisphosphonate‐functionalized poly(β‐amino ester) network polymers

Three novel bisphosphonate-functionalized secondary diamines are synthesized and incorporated into poly(β-amino ester)s (PBAEs) to investigate the effects of bisphosphonates on biodegradation and toxicity of PBAE polymer networks. These three novel amines, BPA1, BPA2, and BPA3, were prepared from the reactions of 1,4-butanediamine, 1,6-hexanediamine, or 4,9-dioxa-1,12-dodecanediamine with tetraethyl vinylidene bisphosphonate, respectively. The PBAE macromers were obtained from the aza-Michael addition reaction of these amines to 1,6-hexane diol diacrylate (HDDA) and poly(ethylene glycol) diacrylate (PEGDA, Mn = 575) and photopolymerized to produce biodegradable gels. These gels with different chemistries exhibited similar degradation behavior with mass loss of 53-73% within 24 h, indicating that degradation is mostly governed by the bisphosphonate group. Based on the in vitro cytotoxicity evaluation against NIH 3T3 mouse embryonic fibroblast cells, the degradation products do not exhibit significant toxicity in most cases. It was also shown that PBAE macromers can be used as cross-linkers for the synthesis of 2-hydroxyethyl methacrylate hydrogels, conferring small and customizable degradation rates upon them. The materials reported have potential to be used as nontoxic degradable biomaterials. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1412-1421, 2017.

Effect of Surface Potential on the Adhesion Behavior of NIH3T3 Cells Revealed by Quartz Crystal Microbalance with Dissipation Monitoring (QCM-D)

Cell adhesion is crucial to cell behaviors including survival, growth, and differentiation. In recent years, quartz crystal microbalance with dissipation monitoring (QCM-D) has exhibited advantages in examining real-time viscoelastic changes of surface interactions. Self-assembled monolayers (SAMs) are known for their convenience and versatility in modifying surfaces. A series of ζ-potentials can be obtained by introducing two functional groups of opposite charge to gold surfaces, namely, 6-amino-1-hexanethiol and 6-mercaptohexanoic acid. In this work, NIH3T3 mouse embryonic fibroblasts were chosen for examining the cell–surface, extracellular matrix (ECM)–surface, and cell–ECM interactions of these binary SAM-modified surfaces of serial surface potentials. The effect of surface potential on focal adhesion was also characterized by immunofluorescence staining. Combining an optical microscope with the QCM-D system, in-situ and real-time cell morphology and corresponding viscoelastic changes were obtained i...

Hsd11β2 Is Enriched in Medulloblastoma and Generates Ciliary Oxysterols to Stimulate Hedgehog Signaling

Vertebrate hedgehog (Hh) signals are transduced through the primary cilium, an antenna that projects from the surface of most cells including the progenitors of Hh-associated medulloblastoma within the cerebellar external granule layer (EGL). Hh ligands relieve Patched1 (Ptch1) repression of Smoothened (Smo), which moves Smo into cilia to activate the Hh transcriptional program. Synthetic oxysterols bind the Smo extracellular domain (ECD) to activate Hh signaling in vitro, but how Smo is activated in vivo remains unknown. To understand how ciliary Hh signaling is misactivated to cause cancer, we identify the enzyme Hsd11β2 as a novel Hh pathway activator that (i) is enriched in Hh-associated medulloblastoma and (ii) synthesizes endogenous ciliary oxysterols (ECOs) that bind the Smo ECD to accumulate Smo in cilia and stimulate Hh signaling. We created a mouse genetic model of Hh-associated medulloblastoma by conditionally expressing the oncogenic SmoM2 allele in the EGL using Cre under the control of Math1 regulatory sequences. RNA sequencing of mouse tumors revealed 250±41-fold enrichment of Hsd11β2. Genomic and protein expression comparisons to human tumors confirmed Hsd11β2 as a promising target for further investigation. NIH3T3 cells and mouse embryonic fibroblasts (MEFs) derived from knockout animals were used to study Hsd11β2-mediated Hh signaling by qRT-PCR and luciferase assays. Smo localization to cilia was assessed by immunofluorescence microscopy (IF). Lipidomic high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) was used to measure oxysterols from purified cilia. Small molecule and shRNA inhibition of Hsd11β2 have no effect in Smo-/- MEFs, but inhibit Hh signaling in Ptch1-/- MEFs which have constitutive pathway activation due to Smo de-repression. Re-expression of Smo in Smo-/- MEFs restores sensitivity to Hsd11β2 inhibition in a manner dependent on the Smo ECD. Consistently, Hsd11β2 inhibition in NIH3T3 cells blocks Smo accumulation in cilia through competitive inhibition of the Smo ECD. Lipidomic HPLC-MS/MS identifies a family of ECOs that bind the Smo ECD and activate Hh signaling. ECOs epistatically restore Hh signaling after inhibition of Hsd11β2 and related oxysterol synthases, leading to a model of Hh-activating ciliary oxysterol biosynthesis. A GFP-tagged construct containing the Smo ECD and a single TMD was sufficient for ciliary localization in response to ECOs and Hh signals in a manner that is modulated by Hsd11β2 activity. Hsd11β2 generates endogenous ciliary oxysterols to mediate Smo accumulation in cilia and activate the Hh pathway. These findings illuminate how the Hh pathway and lipids functions in malignancy, and also shed light on the fundamental mechanisms of Hh signaling relevant to normal development. Experiments are underway to determine whether genetic or pharmacological inhibition of Hsd11β2 blocks Hh-associated medulloblastoma growth in mice.

Frequently rearranged in advanced T‑cell lymphomas‑1 demonstrates oncogenic properties in prostate cancer.

Prostate cancer is the fifth most common cause of cancer-associated mortality for males worldwide. Although dysregulation of the β-catenin/T-cell factor (TCF) pathway has been previously reported in prostate cancer, the mechanisms underlying this process remain unknown. Frequently rearranged in advanced T-cell lymphomas-1 (FRAT1) functions as a positive regulator of the β-catenin/TCF signaling pathway. However, to the best of our knowledge, the molecular association between FRAT1 and the β-catenin/TCF pathway in prostate cancer has not been investigated. In the present study, FRAT1 expression was analyzed in normal prostate tissues and prostate adenocarcinoma samples using publicly available databases, a commercial tissue microarray and immunohistochemistry techniques. In addition, FRAT1 expression levels were altered by overexpression or RNA interference-mediated depletion in prostate cancer cells. The effects of FRAT1 expression on tumor growth were determined using cell growth curves in vitro and xenografts in nude mice in vivo. The effects of FRAT1 on β-catenin/TCF activity were measured using the TOPFLASH reporter assay. FRAT1 was expressed exclusively in the nuclei of normal prostate basal cells, and nuclear FRAT1 was detected in 68% (40/59) of prostate adenocarcinoma samples. In addition, FRAT1 activated the TCF luciferase reporter gene promoter in prostate cancer cells, and was observed to promote the growth of prostate cancer cells in vitro. Furthermore, FRAT1 expression was sufficient to transform NIH3T3 mouse embryonic fibroblast cells and lead to tumor formation in vivo. These results suggest that FRAT1 demonstrates oncogenic properties in prostate cancer, potentially by suppressing the inhibitory effect of nuclear glycogen synthase 3β against β-catenin/TCF activity, thus activating the Wnt/β-catenin signaling pathway and promoting cell growth.

Accumulation and Toxicity of Superparamagnetic Iron Oxide Nanoparticles in Cells and Experimental Animals.

The uptake and distribution of negatively charged superparamagnetic iron oxide (Fe3O4) nanoparticles (SPIONs) in mouse embryonic fibroblasts NIH3T3, and magnetic resonance imaging (MRI) signal influenced by SPIONs injected into experimental animals, were visualized and investigated. Cellular uptake and distribution of the SPIONs in NIH3T3 after staining with Prussian Blue were investigated by a bright-field microscope equipped with digital color camera. SPIONs were localized in vesicles, mostly placed near the nucleus. Toxicity of SPION nanoparticles tested with cell viability assay (XTT) was estimated. The viability of NIH3T3 cells remains approximately 95% within 3–24 h of incubation, and only a slight decrease of viability was observed after 48 h of incubation. MRI studies on Wistar rats using a clinical 1.5 T MRI scanner were showing that SPIONs give a negative contrast in the MRI. The dynamic MRI measurements of the SPION clearance from the injection site shows that SPIONs slowly disappear from injection sites and only a low concentration of nanoparticles was completely eliminated within three weeks. No functionalized SPIONs accumulate in cells by endocytic mechanism, none accumulate in the nucleus, and none are toxic at a desirable concentration. Therefore, they could be used as a dual imaging agent: as contrast agents for MRI and for traditional optical biopsy by using Prussian Blue staining.

Manipulation of Cell Cycle and Chromatin Configuration by Means of Cell-Penetrating Geminin

Geminin regulates chromatin remodeling and DNA replication licensing which play an important role in regulating cellular proliferation and differentiation. Transcription of the Geminin gene is regulated via an E2F-responsive region, while the protein is being closely regulated by the ubiquitin-proteasome system. Our objective was to directly transduce Geminin protein into cells. Recombinant cell-penetrating Geminin (CP-Geminin) was generated by fusing Geminin with a membrane translocating motif from FGF4 and was efficiently incorporated into NIH 3T3 cells and mouse embryonic fibroblasts. The withdrawal study indicated that incorporated CP-Geminin was quickly reduced after removal from medium. We confirmed CP-Geminin was imported into the nucleus after incorporation and also that the incorporated CP-Geminin directly interacted with Cdt1 or Brahma/Brg1 as the same manner as Geminin. We further demonstrated that incorporated CP-Geminin suppressed S-phase progression of the cell cycle and reduced nuclease accessibility in the chromatin, probably through suppression of chromatin remodeling, indicating that CP-Geminin constitutes a novel tool for controlling chromatin configuration and the cell cycle. Since Geminin has been shown to be involved in regulation of stem cells and cancer cells, CP-Geminin is expected to be useful for elucidating the role of Geminin in stem cells and cancer cells, and for manipulating their activity.

Sonochemically-Produced Metal-Containing Polydopamine Nanoparticles and Their Antibacterial and Antibiofilm Activity.

A facile one-pot sonochemical synthesis of Cu-, Ag-, and hybrid Cu/Ag-based polydopamine nanoparticles (Cu-, Ag-, and Cu/Ag-PDA-NPs) and the mechanisms by which they exert antibacterial and antibiofilm activities are reported. We showed that the nanoparticles are spherical with a core-shell structure. Whereas Cu is chelated to the shell of Cu-PDA-NPs in oxidation states of +1/+2, the core of Ag-PDA-NPs is filled with elemental Ag°. Sonochemical irradiation of dopamine in the presence of both Cu(2+) and Ag(+) generates hybrid Cu/Ag-PDA-NPs, whose shells are composed of Cu-chelated PDA with Ag° in the core. The redox potential of the metals was found to be the main determinant of the location and oxidation state of the metals. Leaching studies under physiological conditions reveal a relatively fast release of Cu ions from the shell, whereas Ag leaches very slowly from the core. The metal-containing PDA-NPs are highly microbicidal and exhibit potent antibiofilm activity. The combination of both metals in Cu/Ag-PDA-NPs is especially effective against bacteria and robust biofilms, owing to the dual bactericidal mechanisms of the metals. Most importantly, both Ag- and Cu/Ag-PDA-NPs proved to be significantly more antibacterial than commercial Ag-NPs while exhibiting lower toxicity toward NIH 3T3 mouse embryonic fibroblasts. Mechanistically, the metal-containing PDA-NPs generate stable PDA-semiquinone and reactive oxygen species under physiological conditions, which contribute at least partly to the antimicrobial activity. We also demonstrated that simple treatment of surfaces with Ag-PDA-NPs converts them to antibacterial, the activity of which was preserved even after prolonged storage under ambient conditions.

Cold atmospheric pressure (CAP) plasma assisted tailoring of LDPE film surfaces for enhancement of adhesive and cytocompatible properties: Influence of operating parameters

Low density polyethylene (LDPE) plays a vital role in tissue engineering application because of its superior bulk properties. However, the cell affinity is one of the significant factors in determining the cell compatibility of polymers which depends on the surface properties such as hydrophilicity, surface chemistry and morphology. Due to intrinsic poor surface properties, the cell affinity is one of the major deterrents of LDPE films in biomedical applications. Hence, in the present investigation we discuss the influence of operating parameters such as applied potential, exposure time and type of plasma processing gases on the improvement of surface and cell compatible properties of the LDPE film using cold atmospheric pressure (CAP) plasma. The surface chemistry and hydrophilicity of the LDPE films were examined using X-ray photoelectron spectroscopy (XPS), contact angle (CA) respectively. Further, surface energy (SE) of the LDPE films were estimated from CA data by Fowke's approximation method. The quantification of topographical changes on the surface of LDPE films was carried out by atomic force microscopy (AFM). Adhesive strength of the surface modified films was analyzed by T-peel and lap shear tests. Finally, in vitro cytocompatibility studies of the surface modified LDPE films were carried out using NIH3T3 (mouse embryonic fibroblast) cells. The results obtained from various characterization techniques evidently revealed that CAP plasma treatment enhanced the surface properties (hydrophilicity, surface morphology and surface chemistry) of the LDPE films. These physico-chemical changes induced by the CAP plasma treatment facilitates the improvement in adhesive strength as well as adhesion and proliferation of cells on the surface of LDPE films.

Influence of non-thermal TiCl4/Ar + O2 plasma-assisted TiOx based coatings on the surface of polypropylene (PP) films for the tailoring of surface properties and cytocompatibility

The superior bulk properties (corrosion resistance, high strength to weight ratio, relatively low cost and easy processing) of hydrocarbon based polymers such as polypropylene (PP) have contributed significantly to the development of new biomedical applications such as artificial organs and cell scaffolds. However, low cell affinity is one of the main draw backs for PP due to its poor surface properties. In tissue engineering, physico-chemical surface properties such as hydrophilicity, polar functional groups, surface charge and morphology play a crucial role to enrich the cell proliferation and adhesion. In this present investigation TiOx based biocompatible coatings were developed on the surface of PP films via DC excited glow discharge plasma, using TiCl4/Ar+O2 gas mixture as a precursor. Various TiOx-based coatings are deposited on the surface of PP films as a function of discharge power. The changes in hydrophilicity of the TiOx/PP film surfaces were studied using contact angle analysis and surface energy calculations by Fowke's approximation. X-ray photo-electron spectroscopy (XPS) was used to investigate the surface chemical composition of TiOx/PP films. The surface morphology of the obtained TiOx/PP films was investigated by scanning electron and transmission electron microscopy (SEM &TEM). Moreover, the surface topography of the material was analyzed by atomic force microscopy (AFM). The cytocompatibility of the TiOx/PP films was investigated via in vitro analysis (cell viability, adhesion and cytotoxicity) using NIH3T3 (mouse embryonic fibroblast) cells. Furthermore the antibacterial activities of TiOx/PP films were also evaluated against two distinct bacterial models namely Gram positive Staphylococcus aureus (S.aureus) and Gram negative Escherichia coli DH5α. (E.coli) bacteria. XPS results clearly indicate the successful incorporation of TiOx and oxygen containing polar functional groups on the surface of plasma treated PP films. Moreover the surface of modified PP films exhibited nano structured morphology, as confirmed by SEM, TEM and AFM. The physico-chemical changes have improved the hydrophilicity of the PP films. The in-vitro analysis clearly confirms that the TiOx coated PP films performs as good as the standard tissue culture plates and also are unlikely to impact the bacterial cell viability.

Study of smart antibacterial PCL-xFe3 O4 thin films using mouse NIH-3T3 fibroblast cells in vitro.

Surface energy plays a major role in prokaryotic and eukaryotic cell interactions with biomedical devices. In the present study, poly(ε-caprolactone)-xFe3 O4 nanoparticles (PCL-xFO NPs; x = 0, 10, 20, 30, 40, 60 wt% FO concentration in PCL) composite thin films were developed for skin tissue regeneration. The surface properties in terms of roughness, surface energy, wettability of the thin films were altered with the incorporation of Fe3 O4 NPs. These thin films show antimicrobial properties and cyto-compatibility with NIH 3T3 mouse embryonic fibroblast cells. The porosity and thickness of the films were controlled by varying RPM of the spin coater. Interestingly, at 1000 RPM the roughness of the film decreased with increasing concentrations of FO NPs in PCL, whereas the surface energy increased with increasing FO NPs concentrations. Furthermore, the spreading of NIH-3T3 cells grown on PCL-xFO thin films was less as compared to control (TCPS), however cells overcame this effect after 48 h of seeding and cells spread similarly to those grown on TCPS after 48 h. Also, the incorporation of FO NPs in thin films induced inner membrane permeabilization in E. coli bacteria leading to bacterial cell death. The viability of E. coli bacteria decreased with increasing concentration of FO NPs in PCL. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 795-804, 2017.

Di-Sulfide Linked Polyethylenimine Coated Gold Nanoparticles as a Non-Viral Gene Delivery Agent in NIH-3T3 Mouse Embryonic Fibroblast.

Di-sulfide linked polyethylenimine coated gold nanoparticles (ssPEI-GNPs) of 20 nm size was prepared in order to deliver the genes to target site. DLS and TEM analysis demonstrated that the GNPs have average size of 13 nm in diameter. Upon coating the GNPs with ssPEI in the weight ratio of 1:3, the average hydrodynamic diameter of the ssPEI-GNPs was found to 19±1.14 nm and a zeta potential value 41±1.23 mV was observed. TEM analysis of ssPEI-GNPs demonstrated that the nanoparticles have spherical morphology. Thermogravemetric analysis of the prepared ssPEI-GNPs showed that the estimated composition of the ssPEI coated over the GNPs was approximately 5% (w/w). Gene expression capabilities of the nanoparticles were confirmed by fluorescent microscopy and luciferase assay, which demonstrated the transgene delivery capability of the ssPEI-GNPs. These results demonstrate that ssPEI-GNPs could be used as gene delivery agent.