Accumulation and toxicity of biologically produced gold nanoparticles in different types of specialized mammalian cells.

The biologically produced gold nanoparticles (AuNPs) are novel carriers with promising use in targeted tumor therapy. Still, there are no studies regarding the efficacy of nanoparticle internalization by cancer and noncancer cells. In this study, AuNPs were produced by Fusarium oxysporum and analyzed by spectrophotometry, transmission electron microscopy (TEM), energy dispersive x-ray spectroscopy (EDS), and Zetasizer. Obtained AuNPs were about 15 nm in size with a zeta potential of -35.8 mV. The AuNPs were added to cancer cells (4T1), noncancer cells (NIH/3T3), and macrophages (RAW264.7). The viability decreased in 4T1 (77 ± 3.74%) in contrast to NIH/3T3 and RAW264.7 cells (89 ± 4.9% and 90 ± 3.5%, respectively). The 4T1 cancer cells also showed the highest uptake and accumulation of Au (∼80% of AuNPs was internalized) as determined by graphite furnace atomic absorption spectroscopy. The lowest amount of AuNPs was internalized by the NIH/3T3 cells (∼30%). The NIH/3T3 cells exhibited prominent reorganization of F-actin filaments as examined by confocal microscopy. In RAW264.7, we analyzed the release of proinflammatory cytokines by flow cytometry and we found the AuNP interaction triggered transient secretion of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ). In summary, we proved the biologically produced AuNPs entered all the tested cell types and triggered cell-specific responses. High AuNP uptake by tumor cells was related to decreased cell viability, while low nanoparticle uptake by fibroblasts triggered F-actin reorganization without remarkable toxicity. Thus, the biologically produced AuNPs hold promising potential as cancer drug carriers and likely require proper surface functionalization to shield phagocytizing cells.

The anti-neoplastic effects of metformin modulate the acquired phenotype of fibroblast cells in the breast cancer-normal fibroblast co-culture system.

Intracellular communications between breast cancer and fibroblast cells were reported to be involved in cancer proliferation, growth, and therapy resistance. The hallmarks of cancer-fibroblast interactions, consisting of caveolin 1 (Cav1) and mono-carboxylate transporter 4 (MCT4) (metabolic coupling markers), along with IL-6, TGFß, and lactate secretion, are considered robust biomarkers predicting recurrence and metastasis. In order to promote a novel phenotype in normal fibroblasts, we predicted that breast cancer cells could be able to cause loss of Cav1 and increase of MCT4, as well as elevate IL-6 and TGFß in nearby normal fibroblasts. We created a co-culture model using breast cancer (4T1) and normal fibroblast (NIH3T3) cell lines cultured under specific experimental conditions in order to directly test our theory. Moreover, we show that long-term co-culture of breast cancer cells and normal fibroblasts promotes loss of Cav1 and gain of MCT4 in adjacent fibroblasts and increase lactate secretion. These results were validated using the monoculture of each group separately as a control. In this system, we show that metformin inhibits IL-6 and TGFß secretion and re-expresses Cav1 in both cells. However, MCT4 and lactate stayed high after treatment with metformin. In conclusion, our work shows that co-culture with breast cancer cells may cause significant alterations in the phenotype and secretion of normal fibroblasts. Metformin, however, may change this state and affect fibroblasts' acquired phenotypes. Moreover, mitochondrial inhibition by metformin after 8 days of treatment, significantly hinders tumor growth in mouse model of breast cancer.

The flower of Abelmoschus manihot (L.) medik exerts antioxidant effects by regulating the Nrf2 signalling pathway in scald injury.

Scald is a common skin injury in daily life. It is well known that skin burns are associated with inflammation and oxidative stress. In our previous study, we found that Abelmoschus manihot (L.) medik had excellent therapeutic effects on scald-induced inflammation, but its effect on scald-induced oxidative stress was not reported. In this study, a deep second-degree scald model in mice was established, and the wound healing rate, healing time, malondialdehyde (MDA) and total superoxide dismutase (T-SOD) levels, and nuclear factor erythroid 2-related Factor 2 (Nrf2) expression in wound tissue were measured to evaluate the scald wound healing performance of extraction from A. manihot (L.) medik (EAM). Scalding activity in mice was examined in vivo by hot water-induced finger swelling. The treatment scald activities were also examined in vivo by subjecting mice to thermal water-induced digit swelling. Additionally, the antioxidant effect of EAM on fibroblasts was also used to determine the mechanism in vitro. The results showed that EAM not only decreased the wound healing time but also effectively regulated the levels of oxidising, MDA and T-SOD in wound tissue. Concurrently, EAM suppressed digit swelling and hyperalgesia. Furthermore, EAM had a significant protective effect on NIH-3T3 cells after H2 O2 injury by regulating the Nrf2 signalling pathway against oxidative injury. Therefore, EAM is a promising drug for the treatment of scald-induced inflammation.

Chitin Nanofibrils Enabled Core-Shell Microcapsules of Alginate Hydrogel.

An engineered 3D architectural network of the biopolymeric hydrogel can mimic the native cell environment that promotes cell infiltration and growth. Among several bio-fabricated hydrogel structures, core-shell microcapsules inherit the potential of cell encapsulation to ensure the growth and transport of cells and cell metabolites. Herein, a co-axial electrostatic encapsulation strategy is used to create and encapsulate the cells into chitin nanofibrils integrated alginate hydrogel microcapsules. Three parameters that are critical in the electrostatic encapsulation process, hydrogel composition, flow rate, and voltage were optimized. The physicochemical characterization including structure, size, and stability of the core-shell microcapsules was analyzed by scanning electron microscope (SEM), FTIR, and mechanical tests. The cellular responses of the core-shell microcapsules were evaluated through in vitro cell studies by encapsulating NIH/3T3 fibroblast cells. Notably, the bioactive microcapsule showed that the cell viability was found excellent for more than 2 weeks. Thus, the results of this core-shell microcapsule showed a promising approach to creating 3D hydrogel networks suitable for different biomedical applications such as in vitro tissue models for toxicity studies, wound healing, and tissue repair.

The Expression Pattern of tRNA-Derived Small RNAs in Adult Drosophila and the Function of tRF-Trp-CCA-014-H3C4 Network Analysis.

tRNA-derived small RNAs (tsRNAs) are derived from tRNA and include tRNA halves (tiRNAs) and tRNA fragments (tRFs). tsRNAs have been implicated in a variety of important biological functions, such as cell growth, transcriptional regulation, and apoptosis. Emerging evidence has shown that Ago1-guided and Ago2-guided tsRNAs are expressed at 3 and 30 days in Drosophila and that tRF biogenesis in fruit flies affects tRNA processing and tRNA methylation. However, a wide analysis of tsRNA patterns in different ages of Drosophila have not been reported via the small RNA sequencing method. In the present study, tsRNAs of young (7 days) and old (42 days) Drosophila were sequenced and their expression characteristics were analysed. Then, a specific tRF (named tRF-Trp-CCA-014) was determined and was found to be conserved in fruit flies, mice, and humans. The expression patterns of tRF-Trp-CCA-014 in different tissues and stages of fruit flies and mice, and mouse NIH/3T3 cells were detected. Furthermore, mouse embryonic fibroblast NIH/3T3 cells were used as a model to analyse the function and targets of tRF-Trp-CCA-014. The RNA-seq data of six groups (Mimics, Mimic NC, Inhibitors, Inhibitor NC, Aging (adriamycin), and Control (Normal)) in mouse NIH3T3 cells were analysed. The results showed that the number of tsRNAs at 42 days (417) was more than at 7 days (288); thus, it was enriched with age. tRFs-1 were the most enriched, followed by 5'-tRFs and 3'-tRFs. Twenty-one differentially expressed tsRNAs were identified between 7 days and 42 days. Then, the conserved tRF tRF-Trp-CCA-014 was identified and found to accumulate in aged fruit flies and aged mouse NIH3T3 cells. RNA-seq data showed that most differentially expressed genes were involved in the immune system, cancer: overview, and signal translation. Furthermore, tRF-Trp-CCA-014 was found to bind to the 3'UTR of H3C4 in a dual-luciferase reporter gene assay. tRF-Trp-CCA-014 and H3C4 were detected in the cytoplasm of aged NIH3T3 cells by RNA in situ hybridization. These results suggest that the H3C4 gene is the target of tRF-Trp-CCA-014. This study will advance the current understanding of tRF roles and their implication in Drosophila and mouse studies.

Protective effects of Cu/Zn-SOD and Mn-SOD on UVC radiation-induced damage in NIH/3T3 cells and murine skin.

Superoxide dismutase (SOD) is an antioxidant enzyme with multiple metal cofactors that can specifically clear reactive oxygen species (ROS), which plays an important role in a variety of ultraviolet-induced lesions. Therefore, SOD has the anti-ultraviolet radiation effect. The objective of this study was to compare the differences in the anti-ultraviolet radiation effect of SOD with distinct metal cofactors: Cu/Zn-SOD and Mn-SOD. SOD was first purified using hydrophobic interaction chromatography and ion-exchange chromatography. Second, the Methylthiazolyldiphenyl-tetrazolium bromide method and cell senescence kits were used to study the protective effect of SOD on ultraviolet-induced cell damage. Finally, the protective effect of SOD on ultraviolet -induced skin damage was histopathologically evaluated, and the expression levels of malondialdehyde (MDA) and matrix metalloproteinases (MMPs) in tissues were detected. The results showed that Cu/Zn-SOD was superior to Mn-SOD in promoting cell proliferation, alleviating cell damage, protecting skin structure, and regulating the expression levels of MDA and MMPs, and it has no side effects. In conclusion, Cu/Zn-SOD had a better anti-ultraviolet radiation effect than Mn-SOD, and it can be used in anti-aging and anti-ultraviolet skin-care products.

Mesoporous silica nanoparticles adsorb aflatoxin B1 and reduce mycotoxin-induced cell damage.

The present study examined the effects of mesoporous silica nanoparticles (MSNs) on its adsorption capacity of aflatoxin B1 (AFB1). Moreover, the study evaluated the toxicity of MSNs with AFB1 using NIH3T3 cells and hemolysis test. The obtained MSNs were spherical, irregular-like in shape, having a mean size of 39.97 ± 7.85 nm and a BET surface area of 1195 m2/g. At 0.1 mg mL-1 concentration of MSN, the AFB1 adsorption capacity was 30%, which reached 70% when the MSN concentration increased to 2.0 mg mL-1. Our findings showed that AFB1 was adsorbed (∼67%) in the first few minutes on being in contact with MSNs, reaching an adsorption capacity of ∼70% after 15 min. Thereafter, the adsorption capacity remained constant in solution, demonstrating that the MSNs adsorbed toxins even beyond overnight. MSN treatment (0.5-2.0 mg mL-1) using NIH3T3 cells did not result in any reduction in cell viability. In addition, MSN treatment completely reversed the cytotoxic effect of AFB1 at all concentrations. Hemolysis test also revealed no hemolysis in MSNs evaluated alone and in those combined with AFB1. To the best of our knowledge, this study is the first to demonstrate that MSN can reduce cell toxicity produced by AFB1 due to its potential to adsorb mycotoxins.

Cytotoxicity evaluation of haloperidol, clozapine and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells

Abstract Schizophrenia is an illness that affects 26 million people worldwide. However, conventional antipsychotics present side effects and toxicity, highlighting the need for new antipsychotics. We aimed to evaluate the cytotoxicity of haloperidol (HAL), clozapine (CLO), and a new molecule with antipsychotic potential, PT-31, in NIH-3T3 cells. The neutral red uptake assay and the MTT assay were performed to evaluate cell viability and mitochondrial activity, morphological changes were assessed, and intracellular reactive oxygen species (ROS) detection was performed. HAL and CLO (0.1 µM) showed a decrease in cell viability in the neutral red uptake assay and in the MTT assay. In addition, cell detachment, content decrease, rounding and cell death were also observed at 0.1 µM for both antipsychotics. An increase in ROS was observed for HAL (0.001, 0.01 and 1 µM) and CLO (0.01 and 1 µM). PT-31 did not alter cell viability in any of the assays, although it increased ROS at 0.01 and 1 µM. HAL and CLO present cytotoxicity at 0.1 µM, possibly through apoptosis and necrosis. In contrast, PT-31 does not present cytotoxicity to NIH-3T3 cells. Further studies must be performed for a better understanding of these mechanisms and the potential risk of conventional antipsychotics

Uptake of nanoparticles from sunscreen physical filters into cells arising from increased environmental microwave radiation: increased potential risk of the use of sunscreens to human health.

This study examines the microwave chemical risks posed by photocatalysts present in sunscreens (physical filters) against the increasing use of microwaves (radio waves) in the environment, sometimes referred to as electronic smog. Specifically, the study assesses the damage caused by silica-coated physical filters (photocatalysts, TiO2⋅ and/or ZnO) contained in commercially available sunscreens and fresh silica-coated ZnO for sunscreens to mouse skin fibroblasts cells (NIH/3T3) evaluated in vitro by the life/death of cells using two types of electromagnetic waves: UV light and microwave radiation, and under simultaneous irradiation with both UV light and microwaves. Conditions of the electromagnetic waves were such as to be of lower light irradiance than that of UVA/UVB radiation from incident sunlight, and with microwaves near the threshold power levels that affect human health. The photocatalytic activity of the physical filters was investigated by examining the degradation of the rhodamine B (RhB) dye in aqueous media and by the damage caused to DNA plasmids from E. coli. Compared to the photocatalytic activity of ZnO and TiO2 when irradiated with UV light alone, a clear enhanced photocatalytic activity was confirmed upon irradiating these physical filters concurrently with UV and microwaves. Moreover, the uptake of these metal oxides into the NIH/3T3 cells led to the death of these cells as a result of the enhanced photocatalytic activity of the metal oxides on exposure to microwave radiation.

Proteomic analysis of chemically transformed NIH-3T3 cells reveals novel mechanisms of action of amaranth lunasin-like peptide.

Food-derived biopeptides can interact with genes and proteins to preserve health and prevent the development of diseases. Lunasin is a soybean cancer-preventive peptide that has been well characterized; however, few studies have been carried out to characterize the function of amaranth lunasin-like peptide (AhLun). The aim of this work was to analyze the proteomic profile changes in NIH-3T3 cells when they are chemically transformed with the carcinogen 3-methylcholanthrene (3MC) in the absence or presence of AhLun. The addition of AhLun into the culture medium did not affect the cell morphology; however, as a chemopreventive agent, it significantly reduced anisokaryosis formation when cells were treated with 3MC. Changes in protein accumulation in NIH-3T3 cells were evaluated by gel-based proteomics analysis. Differentially accumulated protein spots that exhibited at least a twofold change in spot intensity (p < 0.05), when compared with control cells, were analyzed by LC-MS/MS. Successfully identified proteins were grouped into six main categories according to their localization and function (nuclear, ribosomal, mitochondrial, metabolism, cytoskeletal, and miscellaneous). The gel-based proteomic approach for the evaluation of the chemopreventive potential of AhLun reveals novel pathways of action and provides new clues about the possible mechanisms of action of this bioactive peptide present in amaranth seeds.

Facile Green Synthesis of Titanium Dioxide Nanoparticles by Upcycling Mangosteen (Garcinia mangostana) Pericarp Extract.

In the present report, green synthesis of titanium dioxide nanoparticles (TiO2 NPs) was performed by upcycling mangosteen (Garcinia mangostana) pericarp extract (methanol and ethyl acetate extracts). Field emission scanning electron microscopy images revealed an aggregated structure with a highly porous network of TiO2 NPs. TiO2 NPs synthesized with ethyl acetate extract (EtOAc-TiO2 NPs) exhibited more monodispersity and possessed smoother surfaces than the control TiO2 NPs (Con-TiO2 NPs) and TiO2 NPs synthesized with methanol extract (MeOH-TiO2 NPs). High-resolution X-ray diffraction patterns clearly confirmed that TiO2 NPs had a crystalline nature. A mixture of anatase and rutile was observed in Con-TiO2 NPs and MeOH-TiO2 NPs, while EtOAc-TiO2 NPs had only anatase with the smallest size (12.50 ± 1.81 nm). Ethyl acetate extract contained the highest amount of α-mangostin; thus, the surface of TiO2 NPs was functionalized with ethyl acetate extract. The functionalized TiO2 NPs synthesized with ethyl acetate extract (EtOAc-TiO2-αm) showed the highest 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazyl (DPPH) radical scavenging activity. In vitro cell viability on mouse fibroblast cells (NIH3T3) indicated that the newly synthesized TiO2 NPs did not show any significant cytotoxicity. Therefore, the TiO2 NPs in the present report have the potential to be used in cosmetic applications such as sunscreens.

Functions of key genes involved in TGF-β/Smad signaling pathway in progression of pulmonary fibrosis / 环境与职业医学

Background Although transforming growth factor-β (TGF-β)/Smad signaling pathway is important in regulating the occurrence and development of pulmonary fibrosis, the pathogenesis of pulmonary fibrosis remains elusive. Objective To explore the functions of genes associated with TGF-β/Smad signaling pathway in the progression of pulmonary fibrosis. Methods A NIH-3T3 fibroblast model induced by TGF-β1 was established. The experiment samples were divided into a control group and a TGF-β1 treatment group. The control group was exposed to normal saline, while the TGF-β1 treatment group was exposed to 10 ng·mL−1 TGF-β1 for 12 h. The RNAs of the two groups were extracted, sequenced, and analyzed by bioinformatics methods to identify seven key genes in TGF-β pathway, including Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3. The gene expression levels of five markers [Collagen1α1, Collagen1α2, α-smooth muscle actin (α-SMA), TGF-β1, and TGF-β3] and the seven key genes were detected by quantitative real-time PCR (qRT-PCR). The proteins of the two groups were extracted. The important marker protein expression levels of Smad3, the phosphorylation of Smad3 (P-Smad3), and α-SMA were detected by Western blotting. At the same time, 30 healthy SPF-grade C57BL/6 mice were randomly divided into three groups, with 10 mice in each group: a control group, a SiO2 inhalation exposure group for 28 d (10 mice), and a SiO2 inhalation exposure group for 56 d (10 mice). The mice in the two treatment groups were exposed to a natural SiO2 environment for 4 h per day with a 10-min pause for breathing fresh air at 2 h intervals. The lung tissues of the mice were taken after execution. The changes of pulmonary fibrosis were detected by Masson staining, and mRNAs and proteins were extracted to detect the expression of the above key genes and proteins. Results The expression levels of the five marker genes Collagen1α1, Collagen1α2, α-SMA, TGF-β1, and TGF-β3 were significantly increased in the TGF-β1-induced NIH-3T3 fibroblasts than those in the control group (P < 0.01); the expression levels of P-Smad3 and α-SMA proteins increased significantly (P < 0.01); the expression results of the seven key genes screened in the TGF pathway were that Dcn and Smad3 were obviously down-regulated (P < 0.01), and Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 were obviously up-regulated (P < 0.01). The changes in gene expression levels of the transcriptome sequencing showed the same trend. The results of Masson staining showed that the content of collagen fibers in the lung tissues also increased in the SiO2 inhalation exposure groups over time. In the mouse experiment, five marker genes were obviously up-regulated compared with the control group (P < 0.01); no obvious change was found in the expression of Smad3 protein, and the expression levels of P-Smad3 and α-SMA were obviously higher in the SiO2 exposure groups than those in the control group (P < 0.01); the expression levels of Dcn and Smad3 showed a down-regulated trend, while the expression levels of Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3 showed an up-regulated trend with the increase of SiO2 inhalation exposure days (P < 0.01). The expression levels of the above five marker genes, three important marker proteins, and seven key genes were consistent with the expression trends of TGF-β1-induced NIH-3T3 fibroblasts. Conclusion The expression levels of pulmonary fibrosis-related marker genes and proteins change significantly in TGF-β1-induced fibroblast cells, and the lung tissues of mice under natural SiO2 inhalation exposure has obvious fibrosis characteristics. Seven genes (Dcn, Smad3, Smad7, Fbn1, Thbs1, TGF-β1, and TGF-β3) may be involved in the regulation of pulmonary fibrosis by the TGF-β/Smad signaling pathway.

Human Hair Keratin Composite Scaffold: Characterisation and Biocompatibility Study on NIH 3T3 Fibroblast Cells.

The aim of this study was to transform human hair keratin waste into a scaffold for soft tissue engineering to heal wounds. The keratin was extracted using the Shindai method. Keratin and polyvinyl alcohol (PVA) was cross-linked with alginate dialdehyde and converted into a scaffold by the freeze-drying method using gentamycin sulphate (GS) as a model drug. The scaffold was subjected to Fourier transform infrared spectra (FTIR), swelling index, porosity, water absorption, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), X-ray diffraction (XRD), drug release, and cell viability (MTT) analysis. The scaffold was tested for keratinocyte growth using the murine fibroblast cell line (NIH 3T3 cells). The outcome from the keratin had a molecular weight band between 52-38 kDa in SDS-PAGE (Sodium dodecylsulfate-Polyacrylamide gel electrophoresis). A porous scaffold was capable of water absorption (73.64 ± 14.29%), swelling ability (68.93 ± 1.33%), and the release of GS shown as 97.45 ± 4.57 and 93.86 ± 5.22 of 1:4 and 1:3 scaffolds at 16 h. The physicochemical evaluation revealed that the prepared scaffold exhibits the proper structural integrity: partially crystalline with a strong thermal property. The scaffold demonstrated better cell viability against the murine fibroblast cell line (NIH 3T3 cells). In conclusion, we found that the prepared composite scaffold (1:4) can be used for wound healing applications.

Steroid sulfatase in the mouse NIH-3T3 fibroblast cell line: Characterization, and downregulation by glucocorticoids.

Steroid hormones often circulate in the blood as inactive sulfated forms, such as estrone sulfate and dehydroepiandrosterone sulfate. The enzyme steroid sulfatase (STS) converts these steroids into active forms, mainly estrogens, in peripheral tissues. We have previously characterized STS activity in human and mouse breast and bone tissues, and we have shown that STS can provide estrogens to these tissues from circulating sulfated precursors. This study was designed to characterize STS activity in a mouse fibroblast cell line (NIH-3T3). Using a radioactive estrone sulfate (E1S) conversion assay, we detected STS activity in cultured NIH-3T3 cells. This activity was blocked by the STS inhibitors EMATE and STX-64, indicating authentic STS activity. We also found that microsomes prepared from NIH-3T3 cells had relatively high STS activity and that cytosols had low activity, consistent with the known distribution of this enzyme to the endoplasmic reticulum. Michaelis-Menten analysis of the NIH-3T3 microsomes indicated a Km of 10.9 µM using E1S as substrate. Primary fibroblasts prepared from mouse ears and tails also had measurable STS activity, as indicated by 3H-E1S conversion assay, further supporting the conclusion that fibroblasts possess STS. Furthermore, Western blotting confirmed the presence of immunoreactive STS in NIH-3T3 microsomes. With regard to regulation, treatments of cultured NIH-3T3 cells revealed that cortisol and the synthetic glucocorticoids dexamethasone and prednisolone decreased STS activity, as we have found for cell lines from other tissues. The effect of cortisol was seen at both 10 µM and 1.0 µM but not at 0.1 µM. Western blotting also indicated a decrease in STS immunoreactivity in cortisol-treated microsomes. The reduction in STS activity by dexamethasone in whole cells was reversed by the glucocorticoid receptor antagonist RU-486, indicating that glucocorticoid downregulation of STS activity is receptor mediated. An inhibition assay on NIH-3T3 microsomes revealed that STS activity was inhibited significantly by 10 µM estradiol-17ß, a known substrate inhibitor of E1S for STS, but not by 10 µM cortisol. This is consistent with the idea that cortisol inhibits STS in NIH-3T3 cells through a regulatory mechanism rather than by substrate inhibition. Our results could have important implications regarding local estrogen production by STS in fibroblasts, which are the most common connective tissue cells in the body, and on possible regulation of local estrogen levels by cortisol.

Glycosylation at Asn254 Is Required for the Activation of the PDGF-C Protein.

Platelet-derived growth factor C (PDGF-C) is a member of the PDGF/VEGF (vascular endothelial growth factor) family, which includes proteins that are well known for their mitogenic effects on multiple cell types. Glycosylation is one of the most important forms of posttranslational modification that has a significant impact on secreted and membrane proteins. Glycosylation has many well-characterized roles in facilitating protein processing and contributes to appropriate folding, conformation, distribution, and stability of proteins that are synthesized intracellularly in the endoplasmic reticulum (ER) and Golgi apparatus. Although the general process and functions of glycosylation are well documented, there are most likely others yet to be discovered, as the glycosylation of many potential substrates has not been characterized. In this study, we report that the PDGF-C protein is glycosylated at three sites, including Asn25, Asn55, and Asn254. However, we found that mutations at any of these sites do not affect the protein expression or secretion. Similarly, disruption of PDGF-C glycosylation had no impact on its progression through the ER and Golgi apparatus. However, the introduction of a mutation at Asn254 (N254 A) prevents the activation of full-length PDGF-C and its capacity for signaling via the PDGF receptor. Our findings reveal that glycosylation affects PDGF-C activation rather than the protein synthesis or processing. This study characterizes a crucial modification of the PDGF-C protein, and may shed new light on the process and function of glycosylation.

Study on the biocompatibility of Ga-based and Al-assisted self-driven liquid metals in cell and animal experiments for drug delivery.

BACKGROUND: With inherent flexibility, high electroconductivity, excellent thermal conductivity, easy printability and biosafety, Ga-based functional liquid metals (LMs) have been extensively evaluated for biomedical applications. When implanted in the biological environment, the safety of the LMs is a major concern for future application. METHODS: In this study, we conducted several biocompatibility assessments through immersion experiments, in vitro cytotoxicity experiments and in vivo embedding experiments. RESULTS: The results showed that both the Al-assisted self-driven LM and the LM per se own good biocompatibility and retrievable properties when contacted with living organisms for a relatively long period of time. CONCLUSION: This study provides preliminary evidence about the biocompatibility of the functional LM materials, such as LM-based soft machine, which would promote and inspire other research to address other tough biomedical issues.

Suppression of systemic inflammation and signs of acute and chronic cholangitis by multi-kinase inhibitor 1-(4-Cl-benzyl)-3-chloro-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione.

An aberrant activity of growth factor receptors followed by excessive cell proliferation plays a significant role in pathogenesis of cholangitis. Therefore, inhibition of these processes could be a fruitful therapeutic strategy. The effects of multi-kinase inhibitor 1-(4-Cl-benzyl)-3-chloro-4-(CF3-phenylamino)-1H-pyrrole-2,5-dione (MI-1) on the hepatic and systemic manifestations of acute and chronic cholangitis in rats were addressed. MI-1 (2.7 mg/kg per day) was applied to male rats that experienced α-naphthylisothiocyanate-induced acute (3 days) or chronic (28 days) cholangitis. Liver autopsy samples, blood serum markers, and leukograms were studied. MI-1 localization in liver cells and its impact on viability of HepG2 (human hepatoma), HL60 (human leukemia), and NIH3T3 (normal murine fibroblasts) cell lines and lymphocytes of human peripheral blood (MTT, DNA fragmentation, DNA comet assays, Propidium Iodide staining) were assessed. Under both acute and chronic cholangitis, MI-1 substantially reduced liver injury, fibrosis, and inflammatory scores (by 46-86%) and normalized blood serum markers and leukograms. Moreover, these effects were preserved after a 28-day recovery period (without any treatment). MI-1 inhibited the HL60, HepG2 cells, and human lymphocytes viability (IC50 0.6, 9.5 and 8.3 µg/ml, respectively), while NIH3T3 cells were resistant to that. Additionally, HepG2 cells and lymphocytes being incubated with MI-1 demonstrated insignificant pro-apoptotic and pro-necrotic changes and DNA single-strand breaks, suggesting that MI-1 effects in liver might be partly caused by its cytotoxic action towards liver cells and lymphocytes. In conclusion, MI-1 attenuated the systemic inflammation and signs of acute and chronic cholangitis partly through cytotoxicity towards cells of hepatic and leukocytic origin.

Characterization of Betulinic Acid-Multiwalled Carbon Nanotubes Modified with Hydrophilic Biopolymer for Improved Biocompatibility on NIH/3T3 Cell Line.

The biocompatibility of carbon nanotubes (CNT) is fairly a challenging task for their applications in nanomedicine. Therefore, the objective of this research was to formulate four types of highly biocompatible betulinic acid-loaded biopolymer nanocomposites, namely chitosan-multiwalled carbon nanotubes (MWBA-CS), polyethylene glycol-multiwalled carbon nanotubes (MWBA-PG), Tween 20-multiwalled carbon nanotubes (MWBA-T2) and Tween 80-multiwalled carbon nanotubes (MWBA-T8). The physico-chemical properties of the modified nanocomposites were determined by Fourier transform infrared spectroscopy (FTIR), thermal analysis (TGA) and Raman spectroscopy, while the surface morphology of the resulting nanocomposites was studied using field emission scanning electron microscopy (FESEM). All data revealed that the external surface of MWBA nanocomposites was successfully coated with the respective polymer molecules through hydrophobic and electrostatic interactions with improved thermal profiles. The cell viability assay, which was performed on cultured normal embryonic mouse fibroblast cells, confirmed their excellent biocompatibility in phosphate-buffered saline aqueous media. Overall, our findings herein suggest that the synthesized biopolymer-coated MWBA nanocomposites are promising nanomaterials for drug delivery applications as they enhance the solubility and dispersibility of CNT with significantly reduced cytotoxic effect, especially in normal cells.

Proteomic Analysis Reveals the Effect of Trichostatin A and Bone Marrow-Derived Dendritic Cells on the Fatty Acid Metabolism of NIH3T3 Cells under Oxygen-Glucose Deprivation Conditions.

Fibroblasts mediate acute wound healing and long-term tissue remodeling with scarring after tissue injury. Following myocardial infarction (MI), necrotized cardiomyocytes become replaced by secreted extracellular matrix proteins produced by fibroblasts. Dendritic cells (DCs) can migrate from the bone marrow to the infarct areas and infarct border areas to mediate collagen accumulation after MI. Trichostatin A (TSA) is known to regulate apoptosis and proliferation in fibroblasts and affect the functions of DCs under oxygen-glucose deprivation (OGD) conditions. In this study, we used label-free quantitative proteomics to investigate the effects of TSA and bone marrow-derived dendritic cells (BMDCs) on NIH3T3 fibroblasts under OGD conditions. The results showed that the fatty acid degradation pathway was significantly upregulated in NIH3T3 cells under OGD conditions and that the fatty acid synthesis pathway was significantly downregulated in NIH3T3 cells treated with conditioned media (CM) from BMDCs treated with TSA under OGD conditions [BMDCs-CM(TSA)]. In addition, BMDCs-CM(TSA) significantly decreased the levels of triglycerides and free fatty acids and mediated fatty acid metabolism-related proteins in NIH3T3 cells under OGD conditions. In summary, this proteomics analysis showed that TSA and BMDCs affect fatty acid metabolism in NIH3T3 cells under OGD conditions.

Tagging and Deleting of Endogenous Caveolar Components Using CRISPR/Cas9 Technology.

Here, we describe how to utilize CRISPR/Cas9 technology in the generation of tissue culture cells with fluorescently tagged caveolar components as well as cells deleted of endogenous caveolar components. As one example, we will describe tagging of EHD2, caveolar neck protein, with Green Fluorescent protein (eGFP) from endogenous loci (knock-in, KI). As another example, we will describe deletion (knock-out, KO) of Caveolin1 (Cav1), an essential caveolar component in NIH/3T3 cells. In both instances, the modifications were achieved by using Cas9 delivery on plasmid DNA by electroporation and by utilizing FACS cell sorting for selection or enrichment of edited population of cells. We also provide a list with tested gRNA sequences to successfully produce KI and KO of other caveolar components.