A molecularly imprinted photoelectrochemical sensor based on an rGO/MoSSe heterojunction for the detection of chlortetracycline.

A reduced graphene oxide/molybdenum selenosulfide (rGO/MoSSe) heterojunction was synthesized, and a molecularly imprinted photoelectrochemical sensor for the detection of chlortetracycline was prepared. MoSSe was grown in situ on rGO by a hydrothermal method to form an rGO/MoSSe heterojunction, which acts as the sensitive film of the sensor. Since rGO can promote electron transfer and effectively inhibit electron-hole recombination, it effectively reduces the recombination probability of electrons and holes and improves the photoelectric efficiency, thus enhancing the detection sensitivity of the PEC sensor. The rGO/MoSSe was immobilized on an FTO electrode, and molecularly imprinted polymers (MIPs) were prepared by electropolymerization on the rGO/MoSSe-modified FTO electrode with chlortetracycline as the template molecule and o-phenylenediamine as the functional monomer, so as to construct a molecularly imprinted photoelectrochemical (MIP-PEC) sensor. The determination of chlortetracycline was realized by the strategy of a "gate-controlled effect", and the detection range of the chlortetracycline concentration was 5.0 × 10-13-5 × 10-9 mol L-1 with a detection limit of 1.57 × 10-13 mol L-1. The sensor has been applied to the determination of chlortetracycline in animal-derived food samples.

Optimization of Factor Combinations for Stem Cell Differentiations on a Design-of-Experiment Microfluidic Chip.

Directed differentiation of stem cells plays a vital role in cell replacement therapy. Many activators and inhibitors targeting different signaling pathways have been identified to contribute to each step of differentiation. Most studies relied on empirically optimizing the combinations of the aforementioned factors for each step to optimize the efficiency of differentiation, which are time-consuming and nonsystematic. Design-of-experiment (DOE) is a powerful strategy to identify the critical combinations from multiple factors systematically. However, it is prohibitively complicated for typical laboratories, given a large number of potential combinations. Here, we develop a multilayer polymethyl methacrylate-based, reusable microfluidic chip to directly facilitate the DOE in the differentiation of stem cells. The chip consists of an inlet layer and multiple disperse layers. Different solutions are injected simultaneously to the chip through the inlet layer. Subsequently, the channels in the disperse layers split and recombine the flow streams to generate solution combinations based on hard-wired DOE designs. We demonstrated that it is in quantitative agreement with the designs using fluorescent dyes. Moreover, we constructed a human-induced pluripotent stem reporter cell line to improve the consistency of the cellular state measurements and use the chip to identify critical factors for cell differentiation to definitive endoderm (DE). We found that the differentiation efficiencies under various factor combinations are significantly different, and CHIR99201 and GDF8 are the most critical factors for differentiation to DE. Our method is potentially applicable to the optimization of factor combinations for multi-step stem cell differentiation and combinatorial drug screening.

A boronate-modified molecularly imprinted polymer labeled with a SERS-tag for use in an antibody-free immunoassay for the carcinoembryonic antigen.

An antibody-free immunoassay that makes use of a boronate affinity molecularly imprinted polymer (MIP) and surface enhanced Raman scattering (SERS) tags is described. It was applied to the specific determination of the carcinoembryonic antigen (CEA) in human serum. For the preparation of the boronate affinity array, a polymer capable of adsorbing glycoproteins was first synthesized on the surface of a glass slide with four spots using 4-vinylbenzeneboronic acid (VPBA) as the functional monomer, ethylene glycol dimethacrylate (EGDMA) as the crosslinking agent, and ethylene glycol and cyclohexanol as porogens. The surface of the VPBA-Co-EGDMA can bind target glycoproteins. After specific capture of the glycoprotein, a "MIP-target glycoprotein-SERS tag" sandwich structure was formed by covalent interaction between the SERS nanotag (consisting of gold nanoparticles and 4-mercaptophenylboronic acid [MPBA]). CEA can be quantified in spiked serum with a detection limit of 0.1 ng·mL-1 via the specific Raman band at 1098 cm-1. Graphical abstractSchematic representation of the boronate affinity molecularly imprinted polymer (MIPs) array-based SERS sensor for rapid and sensitive detection of the carcinoembryonic antigen (CEA) from human serum. The boronate affinity MIPs array are used as capture probes, and MPBA@AuNPs are used as SERS tags.

Regulating protein corona on nanovesicles by glycosylated polyhydroxy polymer modification for efficient drug delivery.

The dynamic protein corona formed on nanocarriers has been revealed to strongly affect their in vivo behaviors. Precisely manipulating the formation of protein corona on nanocarriers may provide an alternative impetus for specific drug delivery. Herein, we explore the role of glycosylated polyhydroxy polymer-modified nanovesicles (CP-LVs) with different amino/hydroxyl ratios in protein corona formation and evolution. CP-LVs with an amino/hydroxyl ratio of approximately 0.4 (CP1-LVs) are found to efficiently suppress immunoglobulin adsorption in blood and livers, resulting in prolonged circulation. Moreover, CP1-LVs adsorb abundant tumor distinctive proteins, such as CD44 and osteopontin in tumor interstitial fluids, mediating selective tumor cell internalization. The proteins corona transformation specific to the environment appears to be affected by the electrostatic interaction between CP-LVs and proteins with diverse isoelectric points. Benefiting from surface modification-mediated protein corona regulation, paclitaxel-loaded CP1-LVs demonstrate superior antitumor efficacy to PEGylated liposomes. Our work offers a perspective on rational surface-design of nanocarriers to modulate the protein corona formation for efficient drug delivery.

Determination of Aminophylline in Human Serum Using Hydrogel Microspheres for Coupled Surface-Enhanced Raman Spectroscopy (SERS) and Solid-Phase Extraction.

Aminophylline (AMP) is a bronchodilator. The therapeutic and toxic doses are very close. Therefore, therapeutic drug monitoring (TDM) of AMP is essential in clinical practice. Microgels were synthesized by free radical precipitation polymerization. Silver@poly(N-isopropyl acrylamide) (Ag@PNIPAM) hybrid microgels were obtained by loading silver (Ag) nanoparticles into the three-dimensional network of the microgels by in situ reduction. The microgel is a three-dimensional reticular structure with tunable pore size, large specific surface area, and good biocompatibility, which can be used as a sorbent for solid-phase extraction (SPE) of target molecules in complex matrices and as a surface-enhanced Raman spectroscopy (SERS) substrate. We optimized the conditions affecting SERS enhancement, such as silver nitrate (AgNO3) concentration and SPE time, according to the SERS strategy of Ag@PNIPAM hybrid microgels to achieve label-free TDM for trace AMP in human serum. The results showed good linearity between the logarithmic concentration of AMP and its SERS intensity in the range of 1-1.1 × 102 µg/mL, with a correlation coefficient (R2) of 0.9947 and a low detection limit of 0.61 µg/mL. The assay accuracy was demonstrated by spiking experiments, with recoveries ranging from 93.0 to 101.8%. The method is rapid, sensitive, reproducible, requires simple sample pretreatment, and has good potential for use in clinical treatment drug monitoring.

[Expression of ß-glucosidase An-bgl3 from Aspergillus niger for conversion of scopolin].

Scopoletin is a coumarin compound with various biological activities including detumescence and analgesic, insecticidal, antibacterial and acaricidal effects. However, interference with scopolin and other components often leads to difficulties in purification of scopoletin with low extraction rates from plant resource. In this paper, heterologous expression of the gene encoding ß-glucosidase An-bgl3 derived from Aspergillus niger were carried out. The expression product was purified and characterized with further structure-activity relationship between it and ß-glucosidase analyzed. Subsequently, its ability for transforming scopolin from plant extract was studied. The results showed that the specific activity of the purified ß-glucosidase An-bgl3 was 15.22 IU/mg, the apparent molecular weight was about 120 kDa. The optimum reaction temperature and pH were 55 ℃ and 4.0, respectively. Moreover, 10 mmol/L metal ions Fe2+ and Mn2+ increased the enzyme activity by 1.74-fold and 1.20-fold, respectively. A 10 mmol/L solution containing Tween-20, Tween-80 and Triton X-100 all inhibited the enzyme activity by 30%. The enzyme showed affinity towards scopolin and tolerated 10% methanol and 10% ethanol solution, respectively. The enzyme specifically hydrolyzed scopolin into scopoletin from the extract of Erycibe obtusifolia Benth with a 47.8% increase of scopoletin. This demonstrated that the ß-glucosidase An-bgl3 from A. niger shows specificity on scopolin with good activities, thus providing an alternative method for increasing the extraction efficiency of scopoletin from plant material.

Construction of Cu-N coordination into natural biopolymer lignin backbone for highly efficient and selective removal of cationic dyes.

Here, a Cu2+-doped lignin-based adsorbent (Cu-AL) was fabricated via the amination and Cu2+-doping of industrial alkali lignin for massive and selective adsorption of cationic dyes azure B (AB) and saffron T (ST). The Cu-N coordination structures endowed Cu-AL with stronger electronegativity and higher dispersity. Through the electrostatic attraction, π-π interaction, H-bonding, and Cu2+ coordination, the adsorption capacities of AB and ST reached up to 1168 and 1420 mg g-1, respectively. The pseudo-second-order model and Langmuir isotherm model were more relevant to the AB and ST adsorption on Cu-AL. Based on the thermodynamic study, the adsorption progresses were endothermic, spontaneous, and feasible. The Cu-AL maintained high removal efficiency to dyes after 4 reuses (>80%). Importantly, the Cu-AL could efficiently remove and separate AB and ST from dye mixtures even in real time. All the above characteristics demonstrated that Cu-AL was an excellent adsorbent for fast wastewater treatment.

Deciphering the Heterogeneity Landscape of Mesenchymal Stem/Stromal Cell-Derived Extracellular Vesicles for Precise Selection in Translational Medicine.

Mesenchymal stem/stromal cell-derived extracellular vesicles (MSC-EVs) have been considered promising therapeutics for disease treatments. However, MSC-EVs harvested from different tissues present unique biological features reflective of their origins. The heterogeneity of MSC-EVs constitutes an important barrier to their precise application in clinical translation that may probably lead to uncertain therapeutic effects. To give hints for future clinical translation, five MSCs are employed, whose derived EVs are most intensively utilized, namely bone marrow mesenchymal stem/stromal cells (BMMSCs), umbilical cord stem/stromal cells (UCSCs), adipose-derived stem/stromal cells (ASCs), dermal stem/stromal cells (DSCs) and dental pulp stem/stromal cells (DPSCs) and the heterogeneity landscape of the corresponding MSC-EVs are documented. Overall, the basic parameters, stability, and biosafety of different MSC-EVs are indiscriminate. Strikingly, UCSC-EVs exhibit distinguishing productivity. UCSC-EVs as well as DPSC-EVs present better drug loading/delivery capacity. In addition, the heterogeneity of different MSC-EVs in cargo diversity, cellular affinity, organ biodistribution, and therapeutic effects may cue the rational selection in different disease treatments. Through a combined assessment, a rational strategy is combined for selecting MSC-EVs in future clinics. Offering a panoramic view of MSC-EVs harvested from different tissues, the current study may provide guidelines for the precise selection of MSC-EVs in next-generation therapeutics.

Targeting soluble epoxide hydrolase promotes osteogenic-angiogenic coupling via activating SLIT3/HIF-1α signalling pathway.

Type H vessels have recently been identified to modulate osteogenesis. Epoxyeicostrioleic acids (EETs) have an essential contribution to vascular homeostasis. However, whether increased EETs with soluble epoxide hydrolase (sEH) inhibitor TPPU enhance the coupling of angiogenesis and osteogenesis remains largely unknown. The effects of TPPU on cross-talk between co-cultured human umbilical vein endothelial cells (HUVECs) and human dental pulp stem cells (hDPSCs), and on long bone growth and calvarial defect repair in mice were investigated in vitro and in vivo. TPPU enhanced osteogenic differentiation of co-cultured HUVECs and hDPSCs in vitro and increased type H vessels, and long bone growth and bone repair of calvarial defect. Mechanistically, TPPU promoted cell proliferation and angiogenesis, reclined cell apoptosis, and significantly increased CD31hi EMCNhi endothelial cells (ECs) and SLIT3 and HIF-1α expression levels in co-cultured HUVECs and hDPSCs. Knockdown of Slit3 in hDPSCs or Hif-1α in HUVECs impaired the formation of CD31hi EMCNhi ECs and reversed TPPU-induced osteogenesis. We defined a previously unidentified effect of TPPU coupling angiogenesis and osteogenesis. TPPU induced type H vessels by upregulating the expression of hDPSCs-derived SLIT3, which resulted in the activation of ROBO1/YAP1/HIF-1α signalling pathway in ECs. Targeting metabolic pathways of EETs represents a new strategy to couple osteogenesis and angiogenesis, sEH is a promising therapeutic target for bone regeneration and repair.

Knockdown of FOXA1 enhances the osteogenic differentiation of human bone marrow mesenchymal stem cells partly via activation of the ERK1/2 signalling pathway.

BACKGROUND: The available therapeutic options for large bone defects remain extremely limited, requiring new strategies to accelerate bone healing. Genetically modified bone mesenchymal stem cells (BMSCs) with enhanced osteogenic capacity are recognised as one of the most promising treatments for bone defects. METHODS: We performed differential expression analysis of miRNAs between human BMSCs (hBMSCs) and human dental pulp stem cells (hDPSCs) to identify osteogenic differentiation-related microRNAs (miRNAs). Furthermore, we identified shared osteogenic differentiation-related miRNAs and constructed an miRNA-transcription network. The Forkhead box protein A1 (FOXA1) knockdown strategy with a lentiviral vector was used to explore the role of FOXA1 in the osteogenic differentiation of MSCs. Cell Counting Kit-8 was used to determine the effect of the knockdown of FOXA1 on hBMSC proliferation; real-time quantitative reverse transcription PCR (qRT-PCR) and western blotting were used to investigate target genes and proteins; and alkaline phosphatase (ALP) staining and Alizarin Red staining (ARS) were used to assess ALP activity and mineral deposition, respectively. Finally, a mouse model of femoral defects was established in vivo, and histological evaluation and radiographic analysis were performed to verify the therapeutic effects of FOXA1 knockdown on bone healing. RESULTS: We identified 22 shared and differentially expressed miRNAs between hDPSC and hBMSC, 19 of which were downregulated in osteogenically induced samples. The miRNA-transcription factor interaction network showed that FOXA1 is the most significant and novel osteogenic differentiation biomarker among more than 300 transcription factors that is directly targeted by 12 miRNAs. FOXA1 knockdown significantly promoted hBMSC osteo-specific genes and increased mineral deposits in vitro. In addition, p-ERK1/2 levels were upregulated by FOXA1 silencing. Moreover, the increased osteogenic differentiation of FOXA1 knockdown hBMSCs was partially rescued by the addition of ERK1/2 signalling inhibitors. In a mouse model of femoral defects, a sheet of FOXA1-silencing BMSCs improved bone healing, as detected by microcomputed tomography and histological evaluation. CONCLUSION: These findings collectively demonstrate that FOXA1 silencing promotes the osteogenic differentiation of BMSCs via the ERK1/2 signalling pathway, and silencing FOXA1 in vivo effectively promotes bone healing, suggesting that FOXA1 may be a novel target for bone healing.

Mitochondria-Targeting Polymer Micelle of Dichloroacetate Induced Pyroptosis to Enhance Osteosarcoma Immunotherapy.

Pyroptosis has been reported to improve the immunosuppressive tumor microenvironment and may be a strategy to enhance osteosarcoma treatment. The extent to which modulation of mitochondria could induce tumor pyroptosis to enhance immunotherapy has not been characterized. We synthesized a mitochondria-targeting polymer micelle (OPDEA-PDCA), in which poly[2-(N-oxide-N,N-diethylamino)ethyl methacrylate] (OPDEA) was used to target mitochondria and the conjugated dichloroacetate (DCA) was used to inhibit pyruvate dehydrogenase kinase 1 (PDHK1). This conjugate induced pyroptosis through initiation of mitochondrial oxidative stress. We found that OPDEA-PDCA targeted mitochondria and induced mitochondrial oxidative stress through the inhibition of PDHK1, resulting in immunogenic pyroptosis in osteosarcoma cell lines. Moreover, we showed that OPDEA-PDCA could induce secretion of soluble programmed cell death-ligand 1 (PD-L1) molecule. Therefore, combined therapy with OPDEA-PDCA and an anti-PD-L1 monoclonal antibody significantly suppressed proliferation of osteosarcoma with prolonged T cell activation. This study provided a strategy to initiate pyroptosis through targeted modulation of mitochondria, which may promote enhanced antitumor efficacy in combination with immunotherapy.

Delayed neurological deficits caused by cement extravasation following vertebroplasty: a case report.

Delayed neurological deficits secondary to percutaneous vertebroplasty caused by cement leakage is a rare condition. Although cement extravasation during percutaneous vertebroplasty is not uncommon, most cases are clinically asymptomatic, and symptomatic cement extravasation that requires surgical excision is rarely reported. Herein, a case of L4 radiculopathy secondary to cement leakage is reported that involved the delayed onset of neurological symptoms. The patient was treated using a minimally invasive transforaminal endoscopic approach. The clinical and imaging findings and treatment methods are discussed.

Preparation of a Sandwich-like Complex "MIPs-Target Molecule-Magnetic SERS Probe" and SERS Determination of Immunoglobulin G.

Based on the reversible covalent binding between borates and glycoproteins, we proposed a scheme that can specifically recognize and detect immunoglobulin G (IgG). The application of magnetic materials to enrich SERS probes can provide an enhanced signal and repeatability. The results showed that the prepared sandwich-like complex displayed high sensitivity and excellent selectivity for IgG. The Raman peak intensity showed a good linear relationship in the concentration range of 1 mg/mL - 1 ng/mL. The linear equation was y = 657.93x + 2963.9, R2 = 0.996. Parallel testing of this sandwich-like complex proved to have excellent repeatability. This method provided a new possibility for clinical non-immunity and the quantitative detection of IgG.

Development of a Remodeled Caspar Retractor and Its Application in the Measurement of Distractive Resistance in an In Vitro Anterior Cervical Distraction Model.

STUDY DESIGN: In vitro biomechanical study of the cervical intervertebral distraction using a remodeled Caspar retractor. OBJECTIVE: To investigate the torques required for distraction to different heights in an in vitro C3-C4 anterior cervical distraction model using a remodeled Caspar retractor, focusing on the influence of the intervertebral disk, posterior longitudinal ligament (PLL), and ligamentum flavum (LF). SUMMARY OF BACKGROUND DATA: No previous studies have reported on the torques required for distraction to various heights or the factors resisting distraction in anterior cervical discectomy and fusion. METHODS: Anterior cervical distractions at C3-C4 was performed in 6 cadaveric specimens using a remodeled Caspar retractor, under 4 conditions: A, before disk removal; B, after disk removal; C, after disk and PLL removal; and D, after disk and PLL removal and cutting of the LF. Distraction was performed for 5 teeth, and distractive torque of each tooth was recorded. RESULTS: The torque increased with distraction height under all conditions. There was a sudden increase in torque at the fourth tooth under conditions B and C, but not D. Under condition A, distraction to the third tooth required 84.8±13.3 cN m. Under conditions B and C, distraction to the third tooth required <13 cN m, and further distraction required dramatically increased torque. Under condition D, no marked increase in torque was recorded. CONCLUSIONS: Distraction of the intervertebral space was much easier after disk removal. An intact LF caused a sudden marked increase in the force required for distraction, possibly indicating the point at which the LF was fully stretched. This increase in resistance may help to determine the optimal distraction height to avoid excessive stress to the endplate spacer. The remodeled Caspar retractor in the present study may provide a feasible and convenient method for intraoperative measurement of distractive resistance.

[Finite element analysis of first maxillary molars restored with different post and core materials].

PURPOSE: To analyze the stress magnitude and distribution of residual dentin in maxillary first molar restored with post and crown using three-dimension finite element methods. METHODS: An intact maxillary first molar was scanned using a 3DX multi-image micro-CT. Three-dimensional finite element models simulated an endodontically treated first maxillary molar restored with post and crown, which were varied in different number and material of post. A load of 480 N, simulating intercuspal occlusion, was applied vertically to the occlusal surface and a load of 240 N simulating mastication was applied to the occlusal surface with a 45° angle to the long axis of the tooth. Von Mises stresses were calculated by MSC.Marc software. RESULTS: The maximum stresses among the post in the radicular portion increased as elastic modulus of the material increased. The stress values of remaining dentin observed in two-post group were lower than those in one post group and three-post group for cast metal post systems. In the test simulating mastication, the peak value of Von Mises stress was higher on remaining dentin among the post in the radicular portion and lower on the outer surface of residual tooth tissue than that in the test simulating intercuspal occlusion. CONCLUSIONS: The number, material of post and occlusal loading have influence on magnitude and distributionn of stress. Supported by Natural Science Foundation of China (81271175) and National Science and Technology Supporting Plan (2012BAI07B01).

Polyethylenimine-based amphiphilic core-shell nanoparticles: study of gene delivery and intracellular trafficking.

Amphiphilic core-shell nanoparticle, which is composed of a hydrophobic core and a branched polyethylenimine (PEI) shell, has been designed and synthesized as a novel gene delivery nanocarrier. In our previous study, we demonstrated that the core-shell nanoparticle was not only able to efficiently complex with plasmid DNA (pDNA) and protect it against enzymatic degradation, but also three times less cytotoxic, and threefold more efficient in gene transfection than branched 25 kDa PEI. This paper reports our further studies in the following three aspects: (1) the ability of the PEI-based nanoparticles to deliver gene in various mammalian cell lines; (2) intracellular distributions of the nanoparticles and their pDNA complexes in HeLa cells; and (3) incorporation of nuclear targeting agent into the nanoparticle/pDNA complexes to enhance the nuclear targeting ability. The PEI-based nanoparticles were able to transfect both human and non-human cell lines and their transfection efficiencies were cell-dependent. Within our four tested cell lines (MCF-7, BEL 7404, C6 and CHO-K1), gene transfer using PEI-based core-shell nanoparticles displayed gene expression levels comparable to, or even better than, the commercial Lipofectamine™ 2000. Confocal laser scanning microscopy showed that the nanoparticles and their pDNA complexes were effectively internalized into the HeLa cells. The in vitro time series experiments illustrated that both the nanoparticle/pDNA complexes and PEI-based nanoparticles were distributed in the cytoplasmic region after transfection for 10 and 60 min, respectively. Nuclear localization was also observed in both samples after transfection for 20 and 60 min, respectively. Incorporation of the high mobility group box 1 (HMGB1) protein for nuclear targeting has also been demonstrated with a simple approach: electrostatic complexation between the PEI-based nanoparticles and HMGB1. In the in vitro transfection study in MCF-7 cells, the expression level of the firefly luciferase gene encoded by the pDNA increased remarkably by up to eightfold when the HMGB1 protein was incorporated into the nanoparticle/pDNA complexes. Our results demonstrate that the PEI-based core-shell nanoparticles are promising nanocarriers for gene delivery.