NIH 3T3 Mouse Fibroblast Cells Research Articles

Sustainable bioactive food packaging based on electrospun zein-polycaprolactone nanofibers integrated with aster yomena extract loaded halloysite nanotubes

Compostable zein-polycaprolactone (PZ) electrospun nanofiber integrated with different concentrations of Aster yomena extract loaded halloysite nanotubes (A. yomena-HNT) as bioactive nanofibrous food packaging is reported. SEM micrographs reveal heterogeneous nanofibers. A. yomena extract used in the study showed weak antioxidant activity with AAI and TEAC values of 0.229 and 0.346. In vitro, release profile over 7 days of A. yomena indicates a controlled, sustained, and prolonged release. The prepared nanofibers were effective against both gram-positive and gram-negative bacteria. The prepared composite nanofibers were rendered biocompatible and nontoxic when subjected to WST-1 and LDH assay after incubating with NIH 3T3 mouse fibroblast cell line. PZ-15 nanofiber packaging showed the best postharvest quality preservation in Black mulberry fruits after 4 days of storage at 25 °C and 85 % Rh. Moreover, the in vitro decomposition test reveals that the fabricated nanofibers decompose in the soil and do not pose as a threat to the environment.

Surface modification of a commercial bone plate (Ti6Al4V) implant for improved antibacterial and cytocompatibility via thermal dewetting of a silver thin film

The high demand for bone grafts has motivated the development of implants with excellent osteogenic activity, whereas the risk of implant-associated infection, particularly given the rise of antimicrobial resistance, has compelled the development of implants with innovative antimicrobial strategies in which a small amount of bactericidal agent can effectively kill a wide range of bacteria. To induce antibacterial property, the surface of Grade-5 bone plate titanium implants used in clinical applications was modified using direct current (DC) sputter coating followed by thermal annealing. The 15 nm silver film-coated implants were thermally annealed in the furnace for 15 min at 750 °C. The modified implant surface’s antibacterial efficacy against Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Salmonella typhi, and Methicillin-resistant staphylococcus aureus bacteria has been assessed using a colony-forming assay. On the modified implant surface, the growth of E. coli and S. aureus bacteria is reduced by 99.72%, while highly drug-resistant bacteria are inhibited by 96.59%. The MTT assay was used to assess the cytotoxicity of the modified bone-implant surface against NIH3T3 mouse fibroblast cells. The modified bone-implant surface promoted fibroblast growth and demonstrated good cytocompatibility. Furthermore, the mechanical properties of the implant were not harmed by this novel surface modification method. This method is simple and provides new insight into surface modification of commercial metallic implants to have effective antibacterial properties against various classes of bacteria.

Polymer-lipid hybrid nanomedicines to deliver siRNA in and against glioblastoma cells

Small interfering RNA (siRNA) holds great potential to treat many difficult-to-treat diseases, but its delivery remains the central challenge. This study aimed at investigating the suitability of polymer-lipid hybrid nanomedicines (HNMeds) as novel siRNA delivery platforms for locoregional therapy of glioblastoma. Two HNMed formulations were developed from poly(lactic-co-glycolic acid) polymer and a cationic lipid: 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or 3ß-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol). After characterization of the HNMeds, a model siRNA was complexed onto their surface to form HNMed/siRNA complexes. The physicochemical properties and siRNA binding ability of complexes were assessed over a range of nitrogen-to-phosphate (N/P) ratios to optimize the formulations. At the optimal N/P ratio of 10, complexes effectively bound siRNA and improved its protection from enzymatic degradation. Using the NIH3T3 mouse fibroblast cell line, DOTAP-based HNMeds were shown to possess higher cytocompatibility in vitro over the DC-Chol-based ones. As proof-of-concept, uptake and bioefficacy of formulations were also assessed in vitro on U87MG human glioblastoma cell line expressing luciferase gene. Complexes were able to deliver anti-luciferase siRNA and induce a remarkable suppression of gene expression. Noteworthy, the effect of DOTAP-based formulation was not only about three-times higher than DC-Chol-based one, but also comparable to lipofectamine model transfection reagent. These findings set the basis to exploit this nanosystem for silencing relevant GB-related genes in further in vitro and in vivo studies.

Bioinspired synthesis and characterization of zinc oxide nanoparticles and assessment of their cytotoxicity and antimicrobial efficacy

Zinc oxide nanoparticles (ZnO NPs) are versatile and promising, with diverse applications in environmental remediation, nanomedicine, cancer treatment, and drug delivery. In this study, ZnO NPs were synthesized utilizing extracts derived from Acacia catechu, Artemisia vulgaris, and Cynodon dactylon. The synthesized ZnO NPs showed an Ultraviolet–visible spectrum at 370 nm, and X-ray diffraction analysis indicated the hexagonal wurtzite framework with the average crystallite size of 15.07 nm, 16.98 nm, and 18.97 nm for nanoparticles synthesized utilizing A. catechu, A. vulgaris, and C. dactylon respectively. Scanning electron microscopy (SEM) demonstrated spherical surface morphology with average diameters of 18.5 nm, 17.82 nm, and 17.83 nm for ZnO NPs prepared from A. catechu, A. vulgaris, and C. dactylon, respectively. Furthermore, ZnO NPs tested against Staphylococcus aureus, Kocuria rhizophila, Klebsiella pneumonia, and Shigella sonnei demonstrated a zone of inhibition of 8 to 14 mm. The cell viability and cytotoxicity effects of ZnO NPs were studied on NIH-3T3 mouse fibroblast cells treated with different concentrations (5 μg/mL, 10 μg/mL, and 50 μg/mL). The results showed biocompatibility of all samples, except with higher doses causing cell death. In conclusion, the ZnO NPs synthesized through plant-mediated technique showed promise for potential utilization in various biomedical applications in the future.

Cell-free chromatin particles released from dying cells inflict mitochondrial damage and ROS production in living cells

Mitochondrial damage and the resultant oxidative stress are associated with neurodegenerative diseases, ageing, and cancer. However, the triggers of mitochondrial damage remain unclear. We previously reported that cell-free chromatin particles (cfChPs) released from the billions of cells that die in the body every day can readily enter healthy cells and damage their DNA. Here, we show that cfChPs isolated from the sera of healthy individuals, when applied to NIH3T3 mouse fibroblast cells, cause physical damage to mitochondrial DNA (mtDNA). cfChPs also induce ultrastructural changes, increase mitochondrial mass, alter mitochondrial shape, upregulate mitochondrial outer membrane protein translocase of the outer membrane 20, and change mitochondrial membrane potential. Furthermore, a marked increase was observed in mitochondrial superoxide (ROS) production, as detected by MitoSOX Red, and intracellular superoxide dismutase-1 activation. ROS production was also activated when a conditioned medium containing cfChPs released from hypoxia-induced dying NIH3T3 cells was applied to healthy NIH3T3 cells. ROS activation was significantly reduced when the conditioned medium was pre-treated with three different cfChP-deactivating agents: anti-histone antibody-complexed nanoparticles, DNase I, and the novel pro-oxidant combination of the nutraceuticals resveratrol and copper. Given that 1 × 109–1 × 1012 cells die in the body every day, we hypothesise that cfChPs from dying cells are the major physiological triggers for mtDNA damage and ROS production. Deactivation of cfChPs may provide a novel therapeutic approach to retard ageing and associated degenerative conditions linked to oxidative stress.

Evaluation of the anti-androgenic and cytotoxic effects of benzophenone-3 in male Sprague-Dawley rats

ABSTRACT Benzophenone-3 (BP-3, 2-hydroxy-4-methoxybenzophenone, oxybenzone) is one of the most widely used types of benzophenone organic sunscreen. However, this compound is a potentially harmful toxicant. The aim of this study was 2-fold to: (1) utilize a Hershberger bioassay in vivo in castrated male Sprague-Dawley rats to investigate the anti-androgenic activities of BP-3, and (2) use in vitro a methyl tetrazolium assay to compare the toxicity between Leydig cells (TM3 cells) and mouse fibroblast (NIH-3T3) cell lines. In the Hershberger assay, rats were divided into 6 groups (each of n = 7): a vehicle control, negative control, positive control, PB-3 low (40 mg/kg), BP-3 intermediate (200 mg/kg), and BP-3 high (1000 mg/kg)-dose. The weight of the ventral prostate was significantly decreased at BP-3 doses of 200 or 1,000 mg/kg/day. In addition, the levator anibulbocavernosus muscle weights were also significantly reduced at BP-3 doses of 40, 200, or 1,000 mg/kg/day. In the MTT assay, the viability of NIH-3T3 mouse fibroblast cells was within the normal range. However, the TM3 mouse testis Leydig cell viability was significantly lowered in a concentration-dependent manner. Therefore, data indicate that BP-3 might exert in vivo anti-androgenic and in vitro cytotoxic effects in cells associated with the male reproductive system compared to normal non-reproductive cells. Abbreviation: BP-3: benzophenone-3; CG: Cowper’s gland; DMEM: Dulbecco’s modified Eagle’s medium; DMSO: dimethyl sulfoxide; GP: glans penis; LABC: levator anibulbocavernosus muscle; MTT: methyl tetrazolium; NC: negative control; PC: positive control; SV: seminal vesicle; TP: testosterone propionate; VC: vehicle control; VP: ventral prostate

Coordinately unsaturated single Fe-atoms with N vacancies and enhanced sp3 carbon defects in Fe-N(sp2)-C structural units for suppression of cancer cell metabolism and electrochemical oxygen evolution.

Installing coordinately unsaturated Fe-N-C structural units on polymer-composite-derived N-doped carbon offers highly active Fe-Nx sites for the electrochemical oxygen evolution reaction (OER) and reactive oxygen species (ROS) generation in tumor cells. An NH4Cl-driven high-temperature etching method was employed for the formation of FeSA950NC with coordinately unsaturated single Fe-atoms in an Fe-N(sp2)-C structural unit together with N vacancies (VN) and sp3 defects. The carbonization of Fe-phen@ZIF-8 at 800 °C for 30 min under argon, followed by grinding Fe-ZIF-8@RF-urea with NH4Cl at 950 °C for 2 hours, resulted in sp3 carbon defects and VN sites with coordination unsaturation in Fe-Nx due to NH4Cl decomposition to NH3 and HCl, which produced substantial internal stress for etching the carbon matrix. FeSA950NC was used to treat both A549 lung cancer cells and NIH3T3 mouse fibroblast cells to determine its potential as an efficient tumor therapeutic strategy using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and ROS assays. Additionally, FeSA950NC provided high stability and excellent OER activity through the Fe-N(sp2)-C structural unit on pyridinic nitrogen by delivering at a minimum overpotential of 300 mV, which is much lower than that of structurally similar Fe-atom sites. The significantly stronger ROS and OER activities of FeSA950NC suggested the role of VN and sp3-carbon defects with coordinately unsaturated Fe-N2 sites in improving its catalytic performance.

Assessment of the photocatalytic performance and cytotoxic effects of barium sulfate nanoparticles synthesized with a one-step hydrothermal method

The article introduces a novel method for the production of barium sulfate nanoparticles (BaSO4-NPs), which was executed utilizing a particular methodology that entailed the reaction between barium chloride and sodium sulfate. FT-IR, PXRD, UV–Vis, FESEM/EDX, TEM, and PSA analyses were employed to approve the fabrication of BaSO4-NPs. The XRD pattern showed that the NPs had a higher occurrence of the orthorhombic phase with the Pnma space group. Furthermore, the synthesis of BaSO4-NPs was confirmed utilizing FTIR analysis, which displayed their strong bands. The FESEM pictures of BaSO4-NPs exhibited spherical morphology and uniform distribution. The proficiency of photocatalysis by BaSO4-NPs was evaluated in the degradation of Methylene Blue (MB) dye. After 150 min, the observed degradation percentage was 89%. In the current research, the cytotoxicity of the BaSO4-NPs was studied on normal mouse fibroblast NIH3T3 cell and cancer mouse melanoma B16F0 cell lines and the IC50 value was 823.8 µg/mL for cancer B16F0 cells and the results revealed a slight reduction in the viability of cancer cells compared to normal cells.

Co-electrospun PCL-collagen nanofibers containing saffron-synthesized gold nanoparticles as an antioxidant wound dressing.

Oxidant environment and inflammation are the leading cause of chronic wounds such as diabetic ulcers. A dressing containing antioxidants would ensure accelerated wound healing. In this study, electrospun gold nanoparticle (GNP)-embedded nanofibers were developed. GNPs (about 7 nm) were synthesized using saffron extract as a reducing and capping agent (GNP-EXT). For comparison, nanoparticles of the same size were also synthesized using citrate (GNP-CIT). Nanoparticle colloids showed a zeta potential of -27 mV. FTIR confirmed the presence of the extract molecules on the nanoparticles. DPPH assay demonstrated the significant radical scavenging properties of the GNP-EXT. The effect of nanoparticles on the viability of NIH3T3 mouse fibroblast cells was evaluated with an MTT assay that showed no significant toxicity of nanoparticles even in the highest concentration of 250 ppm. Then poly (ɛ-caprolactone) (PCL)- Collagen nanofibers containing GNPs were electrospun. By using SEM, TEM, ATR-FTIR, and contact angle measurement, the nanofibers were characterized. Proper cell adhesion and spreading was observed on nanofibers by SEM and Alamar blue assay illustrated appropriate cyto-compatibility on the obtained nanofibers after 5 days of cell seeding. Wound healing assay also confirmed the cell supporting properties and biocompatibility. The results suggest that saffron-synthesized GNP-loaded nanofibers would be considered as potential wound dressings.

Alleviation of acetaminophen-induced liver failure using silibinin nanoliposomes: An in vivo study

BackgroundAcetaminophen (Act) overdose is a known inducer of liver failure in both children and adults. Cell annihilation ensues following acetaminophen overdose and its toxic metabolites by depleting cellular GSH storage and increasing ROS levels. Silymarin extract and its major compound silibinin (SLB) possess robust antioxidant properties by inducing ROS elimination; however, low bioavailability and rapid metabolism limit their applications. Herein, we aimed at using SLB liposomes to combat acetaminophen-induced acute liver toxicity. MethodsWe have developed a SLB-lipid complex to improve SLB loading efficiency within nanoliposome by using the lipid film method. Liposomes were characterized by using DLS and TEM analysis, and the release pattern, and toxicity profile on the normal cells as well as histopathological and serum analysis were investigated to reveal relevant enzyme activities in an animal model. ResultsData demonstrated that negatively-charged SLB liposomes of 115 nm had homogeneous spherical morphology, and entrapped a considerable quantity of SLB of almost 40%. Liposomes shows a favorable release pattern and were not toxic against NIH3T3 mouse fibroblast cells. The animal study revealed that treatment of mice with SLB nanoliposomes could significantly preserve liver function as revealed by the reduced levels of ALT and AST hepatic enzymes as well as ALP in the serum. Our data indicated that intraperitoneal administration of SLB Lip could significantly reduce ALT enzyme levels (p < 0.05) compared to N-acetylcysteine, while i.v administration resulted in no significant difference compared to control animals with no treatment. ConclusionThe results of this study support the significant hepatoprotective effect of SLB nanoliposomes against acetaminophen-induced toxicity depending on the route of administration.

Construction and characterization of ribonuclease H2 C subunit-knockout NIH3T3 cells.

Mammalian ribonuclease (RNase) H2 is a trimer consisting of catalytic A and accessory B and C subunits. RNase H2 is involved in the removal of misincorporated ribonucleotides from genomic DNA. In humans, mutations in RNase H2 gene cause a severe neuroinflammatory disorder, Aicardi-Goutières syndrome (AGS). Here, we constructed RNase H2 C subunit (RH2C)-knockout mouse fibroblast NIH3T3 cells. Compared with the wild-type NIH3T3 cells, the knockout cells exhibited a decreased single ribonucleotide-hydrolyzing activity and an increased accumulation of ribonucleotides in genomic DNA. Transient expression of wild-type RH2C in the knockout cells increased this activity and decreased this ribonucleotide accumulation. Same events were observed when RH2C variants with an AGS-causing mutation, R69W or K145I, were expressed. These results corresponded with our previous results on the RNase H2 A subunit (RH2A)-knockout NIH3T3 cells and the expression of wild-type RH2A or RH2A variants with an AGS-causing mutation, N213I and R293H, in the RH2A-knockout cells.

1-Tetracosanol isolated from the leaves of Eupatorium glandulosum, accelerates wound healing by expressing inflammatory cytokines and matrix metalloproteinase

Ethnopharmacological relevanceThe leave paste of the plant, Eupatorium glandulosum H. B & K, has been traditionally used to treat cuts and wounds by the tribal community of the Nilgiris district of Tamilnadu, India. Aim of the studyThe present study was carried out to investigate the wound healing potential of this plant extract and the compound, 1-Tetracosanol, isolated from the ethyl acetate fraction. Materials and methodsAn in vitro study was designed to compare the viability, migration and apoptosis of the fresh methanolic extract fractions and 1-Tetracosanol using mouse fibroblast NIH3T3 cell lines and human keratinocytes HaCaT cell lines, respectively. 1-Tetracosanol was evaluated for its viability, migration, qPCR analysis, in silico, in vitro and in vivo. Results1-Tetracosanol at the concentration of 800, 1600, 3200 μM has significant wound closure of 99% at 24 h. The compound when screened in silico against various wound healing markers, TNF-α, IL-12, IL-18, GM-CSF and MMP-9, revealed high binding energy of −5, 4.9 and −6.4 kcal/mol for TNF-α, IL-18 and MMP-9, respectively. Gene expression and the release of cytokines increased at an early stage of the wound repair. 1-Tetracosanol, at 2% gel showed 97.35 ± 2.06% wound closure at 21st day. Conclusion1-Tetracosanol is a good lead for drug development targeted towards wound healing activity and work in this direction is in progress.

Purification and Biological Properties of Raniseptins-3 and -6, Two Antimicrobial Peptides from Boana raniceps (Cope, 1862) Skin Secretion.

The number of multidrug-resistant pathogenic microorganisms has been growing in recent years, most of which is due to the inappropriate use of the commercial antibiotics that are currently available. The dissemination of antimicrobial resistance represents a serious global public health problem. Thus, it is necessary to search for and develop new drugs that can act as antimicrobial agents. Antimicrobial peptides are a promising alternative for the development of new therapeutic drugs. Anurans' skin glands are a rich source of broad-spectrum antimicrobial compounds and hylids, a large and diverse family of tree frogs, are known as an important source of antimicrobial peptides. In the present study, two novel antimicrobial peptides, named Raniseptins-3 and -6, were isolated from Boana raniceps skin secretion and their structural and biological properties were evaluated. Raniseptins-3 and -6 are cationic, rich in hydrophobic residues, and adopt an α-helix conformation in the presence of SDS (35 mM). Both peptides are active against Gram-negative bacteria and Gram-positive pathogens, with low hemolytic activity at therapeutic concentrations. No activity was observed for yeasts, but the peptides are highly cytotoxic against B16F10 murine melanoma cells and NIH3T3 mouse fibroblast cells. None of the tested compounds showed improvement trends in the MTT and LDH parameters of MHV-3 infected cells at the concentrations tested.

Design of novel polyurethane-based ionene nanocarriers for cancer therapy: Synthesis, in-vitro, and in-vivo studies

New strategies for constructing versatile nanocarriers are needed for cancer therapy to overcome the multiple challenges of targeted delivery. This work explores the advantages of polyurethane with main-chain quaternary ammonium salt moieties (ionene) as a novel carrier for targeted drug delivery. We have developed a novel cationic soybean oil-based polyurethane ionene nanocarrier (CPUI) that can act as an effective anticancer agent and efficiently deliver the anticancer drug 5-fluorouracil (5FU). We also report a potential anticancer drug delivery system targeting the folate receptor. In vitro experiments with blank CPUI carriers on the 4T1 (mouse breast cancer cell line) and the NIH-3T3 (mouse fibroblast cell line) revealed high cytotoxicity for the cancer cells but only low cytotoxicity for the normal fibroblast cells. The CPUI nanoparticles were readily loaded with 5FU (5FU-CPUI) in water using electrostatic interactions between the cationic quaternary ammonium groups of ionene and the anionic 5FU. The in vivo study in mice with tumors showed that the blank CPUI carriers significantly inhibited tumor growth, even more than the free drug (5FU). The inhibitory effect on tumor growth was slightly enhanced when the carriers were loaded with 5FU. The prepared nanoparticles had a high loading capacity of 41.8 %. Further enhancement of the inhibitory effect was observed when folic acid (FA) was added as a targeting moiety to the system via ion exchange with the bromine counterion of the quaternary ammonium moieties. The results suggest that the efficacy of FA-CPUI-5FU nanoparticles as vehicles for drug delivery can be enhanced via folate receptor (FR) mediated endocytosis in 4T1 cells and these novel nanocarriers may provide a potential platform for effective targeted drug delivery to tumor tissue and breast cancer therapy in the clinic.

Study of the Effect of Cell Prestress on the Cell Membrane Penetration Behavior by Atomic Force Microscopy

In recent years, atomic force microscopes have been used for cell transfection because of their high-precision micro-indentation mode; however, the insertion efficiency of the tip of AFM into cells is extremely low. In this study, NIH3T3 mouse fibroblast cells cultured on a flexible dish with micro-groove patterns were subjected to various substrate strains at 5%, 10%, 15%, and 20%. It was found that the cell stiffness depends on the prestress of the cell membrane, and that the insertion rate of AFM tips into the cell membrane is proportional to the stiffness through the AFM indentation experiment. The finite element analysis proves that prestress increases the bending stiffness of the cytoskeleton, allowing it to better support the cell membrane, which realizes the stress concentration in the contact area between the AFM tip and the cell membrane. The results indicate that the prestress contributes to the mechanical properties of the cell and suggest that the insertion efficiency could be greatly improved with an increase of the prestress of the cell membrane.

The phytochemical curcumin inhibits steroid sulfatase activity in rat liver tissue and NIH-3T3 mouse fibroblast cells

Curcumin is a phytochemical derived from the spice turmeric that is reported to have therapeutic effects. We are studying the enzyme steroid sulfatase (STS), which removes the sulfate group from inactive steroid hormones in peripheral tissues and we were interested in the effect of curcumin on STS activity due to its structural composition (polyphenolic). We sought to determine if curcumin affects STS activity in two model systems, rat liver and NIH-3T3 mouse fibroblast cells. STS assays were performed on tissue extracts of rat liver, and on NIH-3T3 microsomes and cells, with and without curcumin. Male and female rat liver extracts contained substantial amounts of STS activity, with males averaging higher (4–11 %) levels. Estradiol inhibited STS activity in livers of both sexes at 20 and 10 µM. Curcumin acted as a competitive inhibitor of STS activity in rat liver extracts, with a Ki of 19.8 µM in males and 9.3 µM in females. Curcumin also inhibited STS activity in NIH-3T3 microsomes at both 20 µM and 10 µM, and in whole NIH-3T3 cells at 20 µM. These data are the first to demonstrate STS inhibition by curcumin. Inhibition of STS results in lower active steroid hormone (estrogens and androgens) levels in tissues, possibly altering modulation of immune responses by these steroids.

PVA-Based Nanofibers Containing Chitosan Modified with Graphene Oxide and Carbon Quantum Dot-Doped TiO2 Enhance Wound Healing in a Rat Model.

Electrospun nanofibrous constructs based on nanoparticles and biopolymers have recently been used in tissue engineering because of their similarity to the extracellular matrix in nature. In this study, electrospun chitosan-carbon quantum dot-titanium dioxide-graphene oxide (CS-CQD-TiO2-GO) nanofibrous mats were synthesized for use as wound dressings by the electrospinning method. To increase the biodegradation rate and water resistance, the fabricated nanofibrous mats were cross-linked. SEM images showed a uniform and coherent structure of CS-CQD-TiO2-GO nanocomposites and CS-CQD-TiO2-GO electrospun nanofibers mats. FTIR analysis, XRD pattern, SEM mapping, and EDS spectrum demonstrate the accuracy of the synthesis as well as the elemental and chemical structure of the nanofibrous mat. The water contact angle indicated that the nanofibrous mat had a hydrophilic property, which is essential for controlling wound exudates. The tensile strength and elongation tests showed that the nanofibrous mat has suitable mechanical properties for wound dressing, including significant flexibility and strength. Interestingly, antimicrobial testing illustrated that the fabricated nanofibrous mat had antibacterial activity against Gram-negative and Gram-positive bacteria. Appropriate cell viability and cytocompatibility of treated mouse fibroblast NIH3T3 cells with the nanofibrous mat were determined using an MTT assay. The animal study results confirmed the proper potential of the nanofibrous mat in wound dressing applications.

Analysis of ribonucleotide content in the genomic DNA of ribonuclease H2 A subunit (RH2A)-knockout NIH3T3 cells after transient expression of wild-type RH2A or RH2A variants with an Aicardi-Goutières syndrome-causing mutation.

Ribonuclease (RNase) H2 is involved in the removal of ribonucleotides embedded in genomic DNA. Eukaryotic RNase H2 is a heterotrimer consisting of the catalytic A subunit (RH2A) and the accessory B and C subunits. This study aimed to compare the cellular activities of wild-type ribonuclease (RNase) H2 and its variants with a mutation causing neuroinflammatory autoimmune disease, Aicardi-Goutières syndrome (AGS). We first analyzed cellular RNase H2 activity and ribonucleotide content in the genomic DNA of RH2A-knockout (KO) mouse fibroblast NIH3T3 cells after transfection with a transient expression plasmid encoding mouse wild-type RH2A. From 4h after transfection, the RNase H2 activity increased and the amount of ribonucleotides decreased, as compared with the corresponding non-transfected RH2A-KO cells. This demonstrated the rapidness of ribonucleotide turnover in mammalian genomic DNA and the importance of continuous expression of RNase H2 to maintain the ribonucleotide amount low. Next, we expressed mouse RH2A variants with a mutation corresponding to a human AGS-causing mutation in RH2A-KO NIH3T3 cells. Neither increase in RNase H2 activity nor decrease in ribonucleotide amount was observed for G37S; however, both conditions were observed for N213I and R293H. This corresponded with our previous results on the activity of recombinant human RNase H2 variants.

Long-Term Storage Stability and Nitric Oxide Release Behavior of (N-Acetyl-S-nitrosopenicillaminyl)-S-nitrosopenicillamine-Incorporated Silicone Rubber Coatings.

Physical incorporation of nitric oxide (NO) releasing materials in biomedical grade polymer matrices to fabricate antimicrobial coatings and devices is an economically viable process. However, achieving long-term NO release with a minimum or no leaching of the NO donor from the polymer matrix is still a challenging task. Herein, (N-acetyl-S-nitrosopenicillaminyl)-S-nitrosopenicillamine (SNAP-SNAP), a penicillamine dipeptide NO-releasing molecule, is incorporated into a commercially available biomedical grade silicone rubber (SR) to fabricate a NO-releasing coating (SNAP-SNAP/SR). The storage stabilities of the SNAP-SNAP powder and SNAP-SNAP/SR coating were analyzed at different temperatures. The SNAP-SNAP/SR coatings with varying wt % of SNAP-SNAP showed a tunable and sustained NO release for up to 6 weeks. Further, S-nitroso-N-acetylpenicillamine (SNAP), a well-explored NO-releasing molecule, was incorporated into a biomedical grade silicone polymer to fabricate a NO-releasing coating (SNAP/SR) and a comparative analysis of the NO release and S-nitrosothiol (RSNO) leaching behavior of 10 wt % SNAP-SNAP/SR and 10 wt % SNAP/SR was studied. Interestingly, the 10 wt % SNAP-SNAP/SR coatings exhibited ∼36% higher NO release and 4 times less leaching of NO donors than the 10 wt % SNAP/SR coatings. Further, the 10 wt % SNAP-SNAP/SR coatings exhibited promising antibacterial properties against Staphylococcus aureus and Escherichia coli due to the persistent release of NO. The 10 wt % SNAP-SNAP/SR coatings were also found to be biocompatible against NIH 3T3 mouse fibroblast cells. These results corroborate the sustained stability and NO-releasing properties of the SNAP-SNAP in a silicone polymer matrix and demonstrate the potential for the SNAP-SNAP/SR polymer in the fabrication of long-term indwelling biomedical devices and implants to enhance biocompatibility and resist device-related infections.

Fluorescent half-sandwich iridium picolinamidate complexes for in-cell visualization

In this work, we report on the development of fluorescent half-sandwich iridium complexes using a fluorophore attachment strategy. These constructs consist of pentamethylcyclopentadienyl (Cp*) iridium units ligated by picolinamidate donors conjugated to green-emitting boron-dipyrromethene (bodipy) dyes. Reaction studies in H2O/THF mixtures showed that the fluorescent Ir complexes were active as catalysts for transfer hydrogenation, with activities similar to that of their non-fluorescent counterparts. The iridium complexes were taken up by NIH-3T3 mouse fibroblast cells, with 50% inhibition concentrations ranging from ~20–70 μM after exposure for 3 h. Visualization of the bodipy-functionalized Ir complexes in cells using fluorescence microscopy revealed that they were localized in the mitochondria and lysosome but not the nucleus. These results indicate that our fluorescent iridium complexes could be useful for future biological studies requiring intracellular catalyst tracking.