Regeneration of Mechanically Enhanced Tissue-Engineered Cartilage Based on the Decalcified Bone Matrix Framework.

Human decalcified bone matrix (HDBM) is a framework with a porous structure and good biocompatibility. Nevertheless, its oversized pores lead to massive cell loss when seeding chondrocytes directly over it. Gelatin (GT) is a type of protein obtained by partial hydrolysis of collagen. The GT scaffold can be prepared from the GT solution through freeze-drying. More importantly, the pore size of the GT scaffold can be controlled by optimizing the concentration of the GT solution. Similarly, when different concentrations of gelatin are combined with HDBM and then freeze-dried, the pore size of the HDBM can be modified to different degrees. In this study, the HDBM framework was modified with 0.3, 0.6, and 0.9%GT, resulting in an improved pore size and adhesion rate. Results showed that the HDBM framework with 0.6%GT (HDBM-0.6%GT) had an average pore size of 200 µm, which was more suitable for chondrocyte seeding. Additionally, our study validated that porcine decalcified bone matrix (PDBM) had a proper pore structure. Chondrocytes were in vitro seeded on the three frameworks for 4 weeks and then implanted in nude mice and autologous goats, respectively. The in vivo cartilage regeneration results showed that HDBM-0.6%GT and PDBM frameworks compensated for the oversized pores of the HDBM framework. Moreover, they showed successfully regenerated more mature cartilage tissue with a certain shape in animals.

Tailoring a local acid-like microenvironment for efficient neutral hydrogen evolution.

Electrochemical hydrogen evolution reaction in neutral media is listed as the most difficult challenges of energy catalysis due to the sluggish kinetics. Herein, the Ir-HxWO3 catalyst is readily synthesized and exhibits enhanced performance for neutral hydrogen evolution reaction. HxWO3 support is functioned as proton sponge to create a local acid-like microenvironment around Ir metal sites by spontaneous injection of protons to WO3, as evidenced by spectroscopy and electrochemical analysis. Rationalize revitalized lattice-hydrogen species located in the interface are coupled with Had atoms on metallic Ir surfaces via thermodynamically favorable Volmer-Tafel steps, and thereby a fast kinetics. Elaborated Ir-HxWO3 demonstrates acid-like activity with a low overpotential of 20 mV at 10 mA cm-2 and low Tafel slope of 28 mV dec-1, which are even comparable to those in acidic environment. The concept exemplified in this work offer the possibilities for tailoring local reaction microenvironment to regulate catalytic activity and pathway.

High expression of GPR50 promotes the proliferation, migration and autophagy of hepatocellular carcinoma cells in vitro.

G protein-coupled receptors (GPCRs) play important roles in tumorigenesis and the development of hepatocellular carcinoma (HCC). GPR50 is an orphan GPCR. Previous studies have indicated that GPR50 could protect against breast cancer development and decrease tumor growth in a xenograft mouse model. However, its role in HCC remains indistinct. To detect the role and the regulation mechanism of GPR50 in HCC, GPR50 expression was analyzed in HCC patients (gene expression omnibus database (GEO) (GSE45436)) and detected in HCC cell line CBRH-7919, and the results showed that GPR50 was significantly up-regulated in HCC patients and CBRH-7919 cell line compared to the corresponding normal control. Gpr50 cDNA was transfected into HCC cell line CBRH-7919, and we found that Gpr50 promoted the proliferation, migration, and autophagy of CBRH-7919. The regulation mechanism of GPR50 in HCC was detected by isobaric tags for relative and absolute quantification (iTRAQ) analysis, and we found that GPR50 promoted HCC was closely related to CCT6A and PGK1. Taken together, GPR50 may promote HCC progression via CCT6A-induced proliferation and PGK1-induced migration and autophagy, and GPR50 could be an important target for HCC.

Regeneration of articular cartilage defects: Therapeutic strategies and perspectives.

Articular cartilage (AC), a bone-to-bone protective device made of up to 80% water and populated by only one cell type (i.e. chondrocyte), has limited capacity for regeneration and self-repair after being damaged because of its low cell density, alymphatic and avascular nature. Resulting repair of cartilage defects, such as osteoarthritis (OA), is highly challenging in clinical treatment. Fortunately, the development of tissue engineering provides a promising method for growing cells in cartilage regeneration and repair by using hydrogels or the porous scaffolds. In this paper, we review the therapeutic strategies for AC defects, including current treatment methods, engineering/regenerative strategies, recent advances in biomaterials, and present emphasize on the perspectives of gene regulation and therapy of noncoding RNAs (ncRNAs), such as circular RNA (circRNA) and microRNA (miRNA).

Surface-Sensitive Imaging Analysis of Cell-Microenvironment Interactions by Electrochemiluminescence Microscopy.

A complex and heterogeneous cell microenvironment offers not only structural support for cells but also myriad biochemical and biophysical cues. These outside-in signals transmit into cells primarily through integrins, which are the important components of cell-matrix adhesions to direct and maintain cell behaviors and fate. In this work, we report a surface-sensitive imaging methodology for evaluating the difference in cell-matrix adhesions at the single cell level to dissect the impact of the chemical microenvironment on cell behaviors. Cells were cultured on silica nanochannel membrane (SNM) modified indium tin oxide (ITO) electrodes (SNM/ITO) with different terminal surfaces and imaged by electrochemiluminescence microscopy (ECLM). The results show that the surface tethered with Arg-Gly-Asp (RGD) groups can mediate robust cell-microenvironment interaction and those coated with silanol and (3-aminopropyl)triethoxysilane (APTES) groups transmit an intermediate adhesion, while oligo(ethylene glycol) (OEG) coated surface conveys the weakest cell-matrix adhesion. Specific recognition of integrins to different surfaces was further explored in conjunction with selective immunoblocking of different subunits. α6, α5, and α1 integrin subunits were found to recognize SNM, RGD/OEG, and APTES surfaces, respectively. The work provides not only insights into cell-microenvironment interaction but also guideline in the design and development of functional and biomimetic surface materials.

The orphan GPR50 receptor interacting with TßRI induces G1/S-phase cell cycle arrest via Smad3-p27/p21 in BRL-3A cells.

The liver has the powerful capacity to regenerate after injury or resection. In one of our previous studies, GPR50 was observed to be significantly upregulated at 6 h, following a partial hepatectomy (PH) in rat liver regeneration (LR) via gene expression profile. However, little research has been done on the regulation and mechanism of GPR50 in the liver. Herein, we observed that the overexpression of GPR50 inhibited the proliferation of BRL-3A cells. To further explore the molecular mechanisms of GPR50 in the regulation of BRL-3A cell proliferation, interaction between GPR50 and transforming growth factor-beta I (TßRI) and iTRAQTM differential proteomic analysis were elucidated, which suggested that GPR50 may interact with TßRI to activate the TGF-ß signaling pathway and arrest BRL-3A cell cycle G1/S transition. Subsequently, the potential mechanism underlying the role of GPR50 in hepatocyte growth was also explored through the addition of a signaling pathway inhibitor. These data suggested that interaction between the orphan GPR50 receptor and TßRI induced the G1/S-phase cell cycle arrest of BRL-3A cells via the Smad3-p27/p21 pathway.

circRNA-14723 promotes hepatocytes proliferation in rat liver regeneration by sponging rno-miR-16-5p.

Circular RNA (circRNA) is a subclass of noncoding RNA (ncRNA) detected within mammalian tissues and cells. However, its regulatory role during the proliferation phase of rat liver regeneration (LR) remains unreported. This study was designed to explore their regulatory mechanisms in cell proliferation of LR. The circRNA expression profile was detected by high-throughput sequencing. It was indicated that 260 circRNAs were differentially expressed during the proliferation phase of rat LR. Among them, circ-14723 displayed a significantly differential expression. We further explored its regulatory mechanism in rat hepatocytes (BRL-3A cells). First, EdU, flow cytometry and western blot (WB) indicated that knocking down circ-14723 inhibited BRL-3A cells proliferation. Second, RNA-Pulldown and dual-luciferase report assay showed that circ-14723 could sponge rno-miR-16-5p. At last, WB showed that the reported target genes of rno-miR-16-5p, CCND1, and CCNE1 were downregulated after knocking down circ-14723. In conclusion, we found that circ-14723 exerted a critical role in G1/S arrest to promote cell proliferation via rno-miR-16-5p/CCND1 and CCNE1 axis in rat LR. This finding further revealed the regulatory mechanisms of circRNA on cell proliferation of LR, and might provide a potential target for clinical problems.

Serine peptidase inhibitor Kazal type III (SPINK3) promotes BRL-3A cell proliferation by targeting the PI3K-AKT signaling pathway.

The serine protease inhibitor, Kazal type III (SPINK3), is a trypsin inhibitor associated with liver disease, which highly overexpresses in a variety of cancers. In one of our previous studies of our laboratory, Spink3 was observed to be significantly upregulated in rat liver regeneration (LR) via a gene expression profile. For the current study, rat hepatocyte BRL-3A cells were treated by gene addition/interference, and the addition of the exogenous rat recombinant protein SPINK3. It was revealed that both the overexpression of endogenous Spink3 and addition of exogenous rat recombinant SPINK3 (rrSPINK3) significantly promoted the cell proliferation of BRL-3A cells, whereas cell proliferation was inhibited when Spink3 was interfered. Furthermore, quantitative reverse transcription polymerase chain reaction and western blot results revealed that three signaling pathways, including extracellular-signal-regulated kinase 1/2 (ERK1/2), Janus kinase (JAK)-signal transducer and activator of transcription (STAT), and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT), as well as their related genes, were altered following endogenous Spink3 addition/interference. Also, the PI3K-AKT and SRC-p38 pathways and their related genes were modified following exogenous SPINK3 treatment. Among them, the common signaling pathway was PI3K-AKT pathway. We concluded that SPINK3 could activate the PI3K-AKT pathway by enhancing the expression of AKT1 to regulate the proliferation of BRL-3A cells. This study may contribute to shedding light on the potential mechanisms of SPINK3 that regulate the proliferation of BRL-3A cells.

Pickering Interfacial Catalysts with CO2 and Magnetic Dual Response for Fast Recovering in Biphasic Reaction.

Pickering interfacial catalysis provides an excellent platform for biphasic reactions, but the separation and recycling of nanocatalysts is a challenge because of high adsorption energy of nanocatalysts at the liquid-liquid interface. In this work, we represent a new type of versatile Pickering emulsion based on magnetic and CO2-responsive nanohybrids Fe3O4@SiO2@P(TMA-DEA). The smart nanoparticles can stabilize the water-in-oil Pickering emulsion in the biphasic system and achieve the subsequent demulsification by bubbling CO2 ascribed to their reversible switching surface. In the absence of energy barrier, the nanohybrids can be easily captured in situ by magnetic field in 2 min and showed excellent recyclability. In the Anelli system for alcohol oxidation, the nanocatalyst exhibited threefold enhancement in catalytic efficiency in comparison with an unemulsified two-phase and little loss on activity after five cycles. The conceptually novel dual-responsive system offers a green and energy-saving strategy for effective recycling of the nanocatalyst and intensification of biphasic reaction.

FAM134B promotes adipogenesis by increasing vesicular activity in porcine and 3T3-L1 adipocytes.

Family with sequence similarity 134, Member B (FAM134B), is a cis-Golgi transmembrane protein that is known to be necessary for the long-term survival of nociceptive and autonomic ganglion neurons. Recent work has shown that FAM134B plays a pivotal role in autophagy-mediated turnover of endoplasmic reticulum (ER) membranes, tumor inhibition and lipid homeostasis. In this study, we provide mechanistic links between FAM134B and ARF-related protein 1 (ARFRP1) and further show that FAM134B resides in the Golgi apparatus. Here, we found that FAM134B increased lipid accumulation in adipocytes. Transport vehicle number and ADP-ribosylation factor (ARF) family gene expression were also increased by FAM134B overexpression, suggesting that vesicular transport activity enhanced lipid accumulation. ARF-related protein 1 (ARFRP1) is a GTPase that promotes protein trafficking. We show that FAM134B regulates the expression of ARFRP1, and the knockdown of ARFRP1 abolishes enhancement on lipid accumulation caused by FAM134B. In addition, FAM134B upregulates the PAT family protein (PAT), which associates with the lipid droplets (LDs) surface and promotes lipolysis by recruiting adipocyte triglyceride lipase (ATGL). These findings indicate that FAM134B promotes lipid accumulation and adipogenic differentiation by increasing vesicle transport activity in the Golgi apparatus and inhibiting the lipolysis of LDs.

Roles of Cyclin A, Myc, Jun and Ppm1l in tumourigenic transformation of NIH3T3 cell.

To analyse the mechanism of tumourigenic transformation of NIH3T3 cells at the transcriptional level, we used cancerogen 3-methylcholanthrene (3-MCA) and cancerogenic substance phorbol-12-myristate-13-acetate (PMA) to transform NIH3T3 cells and the assessment of transformation was performed using Giemsa staining and methylcellulose colony formation assay. Changes in gene expression profile were detected by Mouse Genome 430 2.0 microarray; and quantitative real-time polymerase chain reaction and Western blotting were used to verify the expression changes of mRNAs and proteins, respectively. With the aid of bioinformatics method, five signalling pathways were identified to participate in different stages of NIH3T3 cell transformation. Further, our study suggested that oncogenes Cyclin A, Myc, Jun and the tumour suppressor gene Ppm1l may play important roles in these pathways.

In situ fabrication of cobalt nanoflowers on sulfonated and fluorinated poly (arylene ether ketone-benzimidazole) template film for the electrocatalytic oxidation of glucose.

Using sulfonated and fluorinated poly (arylene ether ketone) comprising functional strong coordination group benzimidazole (SPAEK-F-BI) as a template film, a novel fabrication method of cobalt nanoflowers (CoNFs) and non-enzymatic glucose electrochemical sensor was developed in this work. After the precursors Co2+ ions were cooperatively bound by sulfonate and imidazole functionalities contained in SPAEK-F-BI film through ion exchange and strong coordination action, cobalt colloid nuclei were formed and grew to flower-like nanostructures by subsequent in-situ electrochemical reduction on SPAEK-F-BI film modified GCE. Characterization of SPAEK-F-BI film and CoNFs/SPAEK-F-BI film on GCE was performed in detail by FT-IR spectroscopy and scanning electron microscopy (SEM) attached with energy dispersive spectroscopy (EDS), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The results of SEM showed that beautiful CoNFs constructed by Co colloid nanosheets with just a few nanometers thickness were well dispersed on uniform SPAEK-F-BI film modified GCE, and the density of CoNFs was mainly influenced by the concentration of the precursor solution CoSO4. The CoNFs/SPAEK-F-BI composite modified electrode exhibited good electrocatalytic activity toward glucose oxidation in 0.1M NaOH solution, and the kinetic parameters of glucose oxidation were determined using chronoamperometry. When it was applied for the determination of glucose by amperometry at a potential of 0.6V versus Ag/AgCl, the linear range from 5µM to 1.14mM and the detection limit of 800nM (S/N = 3) were obtained. Finally, it was successfully employed to detect the glucose in human serum real samples, and the results were agreed closely with those measured in hospital.

PAM, OLA, and LNA are Differentially Taken Up and Trafficked Via Different Metabolic Pathways in Porcine Adipocytes.

Dietary fatty acids have different effects on fat deposition in pigs. To clarify the underlying mechanisms of this difference, we compared the metabolism of palmitic (PAM, saturated), oleic (OLA, monounsaturated) and linoleic acid (LNA, polyunsaturated) in porcine adipocytes treated with 100 µM PAM, OLA or LNA. We observed that the adipocytes incubated with LNA accumulated more lipids compared with those treated with PAM and OLA. We then probed the metabolism of these fatty acids in porcine adipocytes by using isotope-labelled fatty acids. The results showed that 42% of the [1-14C] LNA, 34% of the [1-14C] PAM and 28% of the [1-14C] OLA were recovered in the cellular lipids. The gene expression analyses showed that LNA significantly increased the expression of adipogenesis- and oxidation-related genes including PPARγ, C/EBPα, ap2 and NRF1. In addition, the cells incubated with LNA showed a decreased Ser112 phosphorylation in PPARγ compared to those incubated with PAM and OLA. Furthermore, when PPARγ Ser112 phosphorylation was inhibited, no significant difference in the triacylglycerol contents in the adipocytes was observed. These results showed the dietary fatty acids had different metabolism pathways in porcine adipocytes, and LNA significantly promoted lipid accumulation, probably by regulating PPARγ phosphorylation in adipocytes.

Serine peptidase inhibitor Kazal type I (SPINK1) promotes BRL-3A cell proliferation via p38, ERK, and JNK pathways.

Serine peptidase inhibitor Kazal type I (SPINK1) has the similar spatial structure as epidermal growth factor (EGF); EGF can interact with epidermal growth factor receptor (EGFR) to promote proliferation in different cell types. However, whether SPINK1 can interact with EGFR and further regulate the proliferation of hepatocytes in liver regeneration remains largely unknown. In this study, we investigated the role of SPINK1 in a rat liver hepatocyte line of BRL-3A in vitro. The results showed the upregulation of endogenous Spink1 (gene addition) significantly increased not only the cell viability, cell numbers in S and G2 /M phase, but also upregulated the genes/proteins expression related to cell proliferation and anti-apoptosis in BRL-3A. In contrast, the cell number in G1 phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased. The similar results were observed when the cells were treated with exogenous rat recombinant SPINK1. Immunoblotting suggested SPINK1 can interact with EGFR. By Ingenuity Pathway Analysis software, the SPINK1 signalling pathway was built; the predicted read outs were validated by qRT-PCR and western blot; and the results showed that p38, ERK, and JNK pathways-related genes/proteins were involved in the cell proliferation upon the treatment of endogenous Spink1 and exogenous SPINK1. Collectively, SPINK1 can associate with EGFR to promote the expression of cell proliferation-related and anti-apoptosis-related genes/proteins; inhibit the expression of pro-apoptosis-related genes/proteins via p38, ERK, and JNK pathways; and consequently promote the proliferation of BRL-3A cells. For the first time, we demonstrated that SPINK1 can associate with EGFR to promote the proliferation of BRL-3A cells via p38, ERK, and JNK pathways. This work has direct implications on the underlying mechanism of SPINK1 in regulating hepatocytes proliferation in vivo and liver regeneration after partial hepatectomy.

Ion-exchange chromatography combined with direct current amperometric detection at CuNPs/reduced graphene oxide-chitosan composite film modified electrode for determination of monosaccharide composition of polysaccharides from Phellinus igniarius.

A novel Cu nanoparticles/reduced graphene oxide-chitosan (CuNPs/r-GO-chitosan) composite film modified glassy carbon electrode (GCE) was fabricated by dispersing CuNPs uniformly on a stable r-GO-chitosan thin film through electrodeposition process. The modified electrode was characterized by cyclic voltammetry, scanning electron microscopy (SEM) and electrochemical impedance spectroscopy (EIS), and exhibited efficiently electrocatalytic oxidation toward monosaccharides with high stability. The good electrocatalytic activity of this modified electrode might be attributed to the synergistic effect of r-GO and CuNPs, and the stability might be attributed to the r-GO and chitosan thin matrix film. When the CuNPs/r-GO-chitosan/GCE was used as an electrochemical sensor in high performance anion exchange chromatography-direct current amperometric detection (HPAEC-DC) flowing system for the determination of monosaccharides under constant working potential of +0.55 V, the detection limits (S/N=3) ranged from 0.006 to 0.02 mg L(-1) for the analyzed sugars, and the dynamic linear ranges spanned from 0.02 to 500 mg L(-1). The proposed method has been applied for the determination of monosaccharide composition of crude polysaccharides from phellinus igniarius real samples, and the results were satisfactory.

Simultaneous determination of glucose, D-gluconic, 2-keto-D-gluconic and 5-keto-D-gluconic acids by ion chromatography-pulsed amperometric detection with column-switching technique.

A simple ion chromatographic (IC) method for simultaneous determination of glucose, D-gluconic acid (DGA), 2-keto-D-gluconic acid (2-KDG) and 5-keto D-gluconic acid (5-KDG) was proposed, with pulsed amperometric detection (PAD) and column-switching technique. Using this technique, the four compounds were detected simultaneously in a short time with strongly retained compounds (2-KDG and 5-KDG) eluted out prior to weakly retained compounds (glucose and DGA). Under the optimized conditions, the method showed good linearity in the range of 0.01-20 mg L(-1) with determination coefficients (R(2))≥ 99.84%. Low detection limits (LODs) in the range of 0.87-2.59 µg L(-1) and good repeatability (RSD<3%, n=6) were obtained. The proposed method has been successfully applied to the analysis of the four compounds in the fermentation broth, in which Gluconobacter oxydans was used to produce gluconic acids from glucose.

Determination of methotrexate and folic acid by ion chromatography with electrochemical detection on a functionalized multi-wall carbon nanotube modified electrode.

A simple and sensitive method was developed for simultaneous determination of methotrexate (MTX) and folic acid (FA) by ion chromatography with electrochemical detection (IC-ECD). Quaternary amine functionalized multi-wall carbon nanotubes (q-MWNTs) modified glassy carbon electrode (GCE) was used as an amperometric sensor to determine MTX and FA. The electrochemical behaviors of MTX and FA at the q-MWNTs/GCE were studied by cyclic voltammetry (CV). Results indicated that this modified electrode exhibited electrocatalytic oxidation toward MTX and FA with high sensitivity, stability and long life. Good separation of MTX and FA was demonstrated by IC on an anion-exchange column with phosphate buffer solution (PBS) as eluent. Under the optimal conditions, the linear ranges were from 0.01 to 20mg/L for both MTX and FA with correlation coefficients r ≥ 0.9994. The obtained detection limits (LODs) for MTX and FA were 0.2 and 0.4 µg/L (S/N=3), respectively. The method has been successfully applied to the determination of MTX and FA in plasma and urine of patients of rheumatoid arthritis.

Determination of phenols with ion chromatography-online electrochemical derivatization based on porous electrode-fluorescence detection.

The current study describes the determination of phenols using ion chromatography-online electrochemical derivatization-fluorescence detection (IC/ED/FD). Six model phenols including 4-methylphenol (pMP), 2, 4-dimethylphenol (DMP), 4-tert-butylphenol (TBP), 4-hydroxylphenolacetic acid (pHPA), 4-acetamidophenol (pAAP), and phenol (P) were well separated on an anion-exchange column under ion exchange mode using NaOH with small amount of acetonitrile added as eluent. Online electrochemical derivatization performed via a laboratory-made electrolytic cell (EC), consisting of porous titanium electrode and cation-exchange membrane (CEM), allows the oxidation products that are strongly fluorescent to be detected by the fluorescence detector. NaOH eluent used in the present method matches well with the maximal fluorescence intensity obtained at alkaline condition for oxidized phenols, thus the addition of specific buffer solution after oxidation encountered in previous report could be eliminated. This process leads to a simplified procedure. The proposed method was sensitive to the limits of detection in the range of 0.4 µg/L and 3.8 µg/L and the limits of quantification between 1.2 µg/L and 13 µg/L due to the strong electro-oxidation capacity of porous titanium electrode, as well as the implementation of time-programmed potential over EC. The linear ranges were 2.0-1.0 × 10(4) µg/L for pAAP and DMP, and 10-1.0 × 10(4) µg/L for P, pMP, pHPA, and TBP, respectively. The relative standard deviations range from 0.9% to 4.8%. The utilization of the method was demonstrated by the analysis of real samples. The average spiked recoveries of target analytes in pool water were 81.0-118%.

Analysis of etimicin sulfate by liquid chromatography with pulsed amperometric detection.

A new method for determination of etimicin's (ETM) purity and content is developed by liquid chromatography (LC) and pulsed amperometric detection (PAD). A reversed-phase ion-pair LC method with pulsed amperometric detection on a gold electrode after post-added NaOH is described. The mobile phase consisted of an aqueous solution containing 0.033 mol L(-1) oxalic acid, 0.012 mol L(-1) heptafluorobutyric acid, and 210 mL L(-1) acetonitrile with pH 3.40 adjusting by dilute NaOH solution. The total analysis time was not more than 30 min. The effects of the different chromatographic parameters on the separation were also investigated. A number of commercial samples of etimicin sulfate were analyzed using this method.