Atomically dispersed Cu coordinated Rh metallene arrays for simultaneously electrochemical aniline synthesis and biomass upgrading.

Organic electrocatalytic conversion is an essential pathway for the green conversion of low-cost organic compounds to high-value chemicals, which urgently demands the development of efficient electrocatalysts. Here, we report a Cu single-atom dispersed Rh metallene arrays on Cu foam for cathodic nitrobenzene electroreduction reaction and anodic methanol oxidation reaction. In the coupled electrocatalytic system, the Cusingle-atom-Rh metallene arrays on Cu foam requires only the low voltages of 1.18 V to reach current densities of 100 mA cm-2 for generating aniline and formate, with up to ~100% of nitrobenzene conversion/ aniline selectivity and over ~90% of formate Faraday efficiency, achieving synthesis of high-value chemicals. Density functional theory calculations reveal the electron effect between Cu single-atom and Rh host and catalytic reaction mechanism. The synergistic catalytic effect and H*-spillover effect can improve catalytic reaction process and reduce energy barrier for reaction process, thus enhancing electrocatalytic reaction activity and target product selectivity.

Metabolic degradation of polysaccharides from Lentinus edodes by Kupffer cells via the Dectin-1/Syk signaling pathway.

It had been shown that lentinan (LNT) was mainly distributed in the liver after intravenous administration. The study aimed to investigate the integrated metabolic processes and mechanisms of LNT in the liver, as these have not been thoroughly explored. In current work, 5-([4,6-dichlorotriazin-2-yl] amino) fluorescein and cyanine 7 were used to label LNT for tracking its metabolic behavior and mechanisms. Near-infrared imaging demonstrated that LNT was captured mainly by the liver. Kupffer cell (KC) depletion reduced LNT liver localization and degradation in BALB/c mice. Moreover, experiments with Dectin-1 siRNA and Dectin-1/Syk signaling pathway inhibitors showed that LNT was mainly taken up by KCs via the Dectin-1/Syk pathway and promoted lysosomal maturation in KCs via this same pathway, which in turn promoted LNT degradation. These empirical findings offer novel insights into the metabolism of LNT in vivo and in vitro, which will facilitate the further application of LNT and other ß-glucans.

Comparison and the lipid-lowering ability evaluation method discussion of Dendrobium officinale polysaccharides from different origins based on principal component analysis.

As typical acetylated glucomannans, Dendrobium officinale polysaccharides (DOPs) from different origins differ in their structural characteristics and some of their physicochemical properties. To rapidly select D. officinale plants, we systematically investigate the differences among DOPs from different origins and analyzed the structural characteristics, such as the degree of acetylation and monosaccharide composition; the physicochemical properties, such as solubility, water absorption and apparent viscosity; and the lipid-lowering activity of the obtained DOPs. Principal component analysis (PCA), a method for analyzing multiple variables, was used to analyze the relationship between the physicochemical and structural properties, and lipid-lowering activity. It was found that the structural and physicochemical characteristics had significant effects on lipid-lowering activity, and DOPs with a high degree of acetylation, high apparent viscosity and large D-mannose-to-d-glucose ratio were associated with greater lipid-lowering activity. Therefore, this study provides a reference for the selection and application of D. officinale.

Interfacial engineering of hydrophobic octadecanethiol/Pd metallene toward electrocatalytic nitrogen reduction.

In this work, we propose the modification of ultrathin and wrinkled Pd metallene by hydrophobic octadecanethiol (Pdene@C18) via Pd-S bonds for the nitrogen reduction reaction. The hydrophobic self-assembled monolayer C18 can effectively capture more N2 and inhibit the hydrogen evolution reaction. As a result, a high NH3 yield and Faraday efficiency of 27.97 µg h-1 mgcat.-1 and 14.29% are achieved for Pdene@C18 under neutral conditions, respectively, highlighting the modification of hydrophobic monolayers for efficient nitrogen electro-reduction to ammonia.

Sulfur Vacancy-Rich Amorphous Rh Metallene Sulfide for Electrocatalytic Selective Synthesis of Aniline Coupled with Efficient Sulfion Degradation.

The construction of efficient and stable electrocatalysts is of widespread research significance for electrocatalytic coupling reactions. Herein, an amorphous Rh metallene sulfide with sulfur-rich vacancies (a-RhS2-x metallene) is synthesized for the cathodic nitrobenzene (Ph-NO2) electroreduction reaction (ERR) to aniline (Ph-NH2) coupled with the anodic sulfur ion (S2-) oxidation reaction (SOR) in a coelectrolysis system. On the one hand, the amorphous Rh metallene structure can provide enough of a reactive site. On the other hand, the amorphization and the introduced S vacancies can generate rich defects and ligand unsaturated sites to improve the intrinsic activity of the active sites. Due to these advantages, the a-RhS2-x metallene exhibits superior electrocatalytic performance for Ph-NO2 ERR and SOR. Inspiringly, in the assembled electrocatalytic coupling system, the required overpotential is only 0.442 V at 10 mA cm-2 to drive the cathodic Ph-NO2 ERR and anodic SOR, which allows for promising energy-efficient electrolysis to generate high value-added chemicals.

Metabolism and Biodegradation of ß-Glucan in vivo.

The ß-Glucans widely exist in plants and edible fungi, and their diverse bioactivities and good physicochemical properties have been widely reported. In addition, ß-glucan intravenous injections (such as lentinan and schizophyllan) have been clinically used as immunomodulators and antitumor polysaccharides. However, the pharmacokinetic studies of ß-glucans only stay on the level of plasma concentration and biodistribution in vivo, and little is known about their metabolism and degradation in vivo, which severely limits the further application of ß-glucans in the field of medicine and biomaterials. The aim of this paper is to explore the metabolism and degradation process of lentinan (as a representative of ß-glucans) in vivo by labeling it with water-soluble fluorescein 5-([4, 6-Dichlorotriazin-2-yl]amino)fluorescein (DTAF). Fluorescently labeled lentinan (FLNT) was intravenously administered to rats at a single dose of 8 mg/kg. The degradation of LNT in blood, liver, kidney, and urine was evaluated by the gel permeation chromatography. Our results showed that although LNT could be degraded in blood, liver, kidney, and urine, there were still some prototypes until excreted in urine due to the incomplete degradation of LNT in each step. To the best of our knowledge, this is the first report to comprehensively study LNT metabolic degradation in rats. These results provide an important reference for further exploration and application of LNT and other ß-glucans.

Damage to the Blood Brain Barrier Structure and Function from Nano Titanium Dioxide Exposure Involves the Destruction of Key Tight Junction Proteins in the Mouse Brain.

Numerous studies have proven that nano titanium dioxide (nano TiO2) can accumulate in animal brains, where it damages the blood brain barrier (BBB); however, whether this process involves destruction of tight junction proteins in the mouse brain has not been adequately investigated. In this study, mice were exposed to nano TiO2 for 30 consecutive days, and then we used transmission electron microscopy to observe the BBB ultrastructure and the Evans blue assay to evaluate the permeability of the BBB. Our data suggested that nano TiO2 damaged the BBB ultrastructure and increased BBB permeability. Furthermore, we used immunofluorescence and Western blotting to examine the expression of key tight junction proteins, including Occludin, ZO-1, and Claudin-5 in the mouse brain. Our data showed that nano TiO2 reduced Occludin, ZO-1 and Claudin-5 expression. Taken together, nano TiO2-induced damage to the BBB structure and function may involve the destruction of key tight junction proteins.

PAF enhances cancer stem cell properties via ß-catenin signaling in hepatocellular carcinoma.

Increasing proofs have declared that liver cancer stem cells (CSCs) are the main contributors to tumor initiation, metastasis, therapy resistance, and recurrence of hepatocellular carcinoma (HCC). However, the molecular mechanisms underlying CSCs regulation remain largely unclear. Recently, PCNA-associated factor (PAF) was identified to play a key role in maintaining breast cancer cell stemness, but its role in liver cancer stem cells has not been declared yet. Herein, we found that both mRNA and protein expression levels of PAF were significantly higher in HCC tissues and cell lines than normal controls. CSC-enriched hepatoma spheres displayed an increase in PAF expression compared to monolayer-cultured cells. Both loss-of-function and gain-of-function experiments revealed that PAF enhanced sphere formation and the percentage of CD133+ or EpCAM+ cells in HCCLM3 and Huh7 cells. In the xenograft HCC tumor model, tumor initiation rates and tumor growth were suppressed by knockdown of PAF. Mechanistically, PAF can amplify the self-renewal of liver CSCs by activating ß-catenin signaling. Taken together, our results demonstrate that PAF plays a crucial role in maintaining the hepatoma cell stemness by ß-catenin signaling.Abbreviations: CSCs: cancer stem cells; HCC: hepatocellular carcinoma; PAF: pCNA-associated factor.

Titanium Dioxide Inhibits Hippocampal Neuronal Synapse Growth Through the Brain-Derived Neurotrophic Factor-Tyrosine Kinase Receptor B Signaling Pathway.

Nanoparticulate titanium dioxide (nano-TiO2) is a commonly used nanoparticle material and has been widely used in the fields of medicine, cosmetics, construction, and environmental protection. Numerous studies have demonstrated that nano-TiO2 has toxic effects on neuronal development, which lead to defects in learning and memory functions. However, it is still unclear whether nano-TiO2 inhibits the development of synapse and the underlying molecular mechanism is still unknown. In this study, nano-TiO2 was administered to rat primary hippocampal neurons for 24 h to investigate the underlying molecular mechanisms behind the inhibition of neuronal synaptic development by nano-TiO2. We used hippocampal neurons as a model to study the effect of nano-TiO2 on synaptic development. Our results demonstrated that dendritic development that represented synaptic plasticity in hippocampal neurons was significantly inhibited in a concentration-dependent manner after exposure to nano-TiO2 for 24 h. Experiments with varying concentrations of nano-TiO2 (5, 15, and 30 g/mL) indicated that the apoptotic rate of hippocampal neurons increased, development of neuronal synapses were inhibited, and synaptic densities decreased by 24.29%, 54.29%, and 72.86%, respectively, in post-treatment with nano-TiO2. Furthermore, the results indicated that the expressions of Synapsin I (SYN I) and postsynaptic density 95 (PSD95) in neuron synapse were also significantly inhibited, particularly SYN I decreased by 18.43%, 37.2%, and 51.6%, and PSD95 decreased by 16.02%, 24.06%, and 38.74% after treatment with varying concentrations of nano-TiO2, respectively. In addition, experiments to assess the BDNF-TrkB signaling pathway indicated that nano-TiO2 inhibited the expressions of key proteins in the downstream MEK/ERK and PI3K/Akt signaling pathways by inhibiting the expression of BDNF. With concentrations of nano-TiO2 at 5, 15, and 30 µg/mL, the expression of BDNF decreased by 22.64%, 33.3%, and 53.58% compared with the control group. Further, the expression ratios of downstream key proteins p-CREB/CREB decreased by 3.03%, 18.11%, and 30.57%; p-ERK1/2/ERK1/2 ratios decreased by 19.11%, 28.82%, and 58.09%, and p-Akt1/Akt1 ratios decreased by 1.92%, 27.79%, and 41.33%, respectively. These results demonstrated that nano-TiO2 inhibited the normal function of the BDNF-TrkB signaling pathway, which is closely related to neuronal synapse. Thus, it can be hypothesized that the inhibition of neuronal synaptic growth by nano-TiO2 may be related to the inhibition of BDNF-TrkB signaling pathway.

Genome analysis of Salmonella phage vB_SalM_8-19 (genus Rosemountvirus).

This work describes the characterization and genome annotation of Salmonella phage vB_SalM_8-19 (referred to as 8-19) isolated from sewage samples collected in a pig farm in Jilin, China. This phage was capable of infecting 60% Salmonella strains in our lab stock. The genome of phage 8-19 is composed of linear double-stranded DNA that is 52,648 bp in length with a G + C content of 46.02%; containing 74 ORFs and no tRNA genes. In October 2019, phylogenetic analyses indicated that phage 8-19 might belong to a novel cluster among the other similar phages which have not been specifically classified within some new genus in family Myoviridae. Recently, the International Committee on Taxonomy of Viruses (ICTV) defined phage 8-19 and its related phages as genus Rosemountvirus, family Myoviridae. This new genus, known as Rosemountvirus, is rarely reported in the literature.

Synthesis of 2-ethoxycarbonylthieno[2,3-b]quinolines in biomass-derived solvent γ-valerolactone and their biological evaluation against protein tyrosine phosphatase 1B.

A series of 2-ethoxycarbonylthieno[2,3-b]quinolines were synthesized in the bio-derived "green" solvent γ-valerolactone (GVL) and evaluated for their inhibitory activities against PTP1B, the representative compound 6a displayed an IC50 value of 8.04 ± 0.71 µM with 4.34-fold preference over TCPTP. These results provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.

Molecular mechanism of nanoparticulate TiO2 induction of axonal development inhibition in rat primary cultured hippocampal neurons.

Numerous studies have demonstrated the in vitro and in vivo neurotoxicity of nanoparticulate titanium dioxide (nano-TiO2 ), a mass-produced material for a large number of commercial and industrial applications. The mechanism of nano-TiO2 -induced inhibition of axonal development, however, is still unclear. In our study, primary cultured hippocampal neurons of 24-hour-old fetal Sprague-Dawley rats were exposed to 5, 15, or 30 µg/mL nano-TiO2 for 6, 12, and 24 hours, and the toxic effects of nano-TiO2 exposure on the axons development were detected and its molecular mechanism investigated. Nano-TiO2 accumulated in hippocampal neurons and inhibited the development of axons as nano-TiO2 concentrations increased. Increasing time in culture resulted in decreasing axon length by 32.5%, 36.6%, and 53.8% at 6 hours, by 49.4%, 53.8%, and 69.5% at 12 hours, and by 44.5%, 58.2%, and 63.6% at 24 hours, for 5, 15, and 30 µg/mL nano-TiO2 , respectively. Furthermore, nano-TiO2 downregulated expression of Netrin-1, growth-associated protein-43, and Neuropilin-1, and promoted an increase of semaphorin type 3A and Nogo-A. These studies suggest that nano-TiO2 inhibited axonal development in rat primary cultured hippocampal neurons and this phenomenon is related to changes in the expression of axon growth-related factors.

Suppression of testosterone production by nanoparticulate TiO2 is associated with ERK1/2-PKA-PKC signaling pathways in rat primary cultured Leydig cells.

BACKGROUND: Nanoparticulate titanium dioxide (nano-TiO2) enters the body through various routes and causes organ damage. Exposure to nano-TiO2 is reported to cause testicular injury in mice or rats and decrease testosterone synthesis, sperm number, and motility. Importantly, nano-TiO2 suppresses testosterone production by Leydig cells (LCs) and impairs the reproductive capacity of animals. METHODS: In an attempt to establish the molecular mechanisms underlying the inhibitory effect of nano-TiO2 on testosterone synthesis, primary cultured rat LCs were exposed to varying concentrations of nano-TiO2 (0, 10, 20, and 40 µg/mL) for 24 hours, and alterations in cell viability, cell injury, testosterone production, testosterone-related factors (StAR, 3ßHSD, P450scc, SR-BI, and DAX1), and signaling molecules (ERK1/2, PKA, and PKC) were investigated. RESULTS: The data show that nano-TiO2 crosses the membrane into the cytoplasm or nucleus, triggering cellular vacuolization and nuclear condensation. LC viability decreased in a time-dependent manner at the same nano-TiO2 concentration, nano-TiO2 treatment (10, 20, and 40 µg/mL) decreased MMP (36.13%, 45.26%, and 79.63%), testosterone levels (11.40% and 44.93%), StAR (14.7%, 44.11%, and 72.05%), 3ßHSD (26.56%, 50%, and 79.69%), pERK1/2 (27.83%, 63.61%, and 78.89%), PKA (47.26%, 70.54%, and 85.61%), PKC (30%, 50%, and 71%), SR-BI (16.41%, 41.79%, and 67.16%), and P450scc (39.41%, 55.26%, and 86.84%), and upregulated DAX1 (1.31-, 1.63-, and 3.18-fold) in primary cultured rat LCs. CONCLUSION: Our collective findings indicated that nano-TiO2-mediated suppression of testosterone in LCs was associated with regulation of ERK1/2-PKA-PKC signaling pathways.

Co-delivery nanoparticles with characteristics of intracellular precision release drugs for overcoming multidrug resistance.

Combination chemotherapy in clinical practice has been generally accepted as a feasible strategy for overcoming multidrug resistance (MDR). Here, we designed and successfully prepared a co-delivery system named S-D1@L-D2 NPs, where denoted some smaller nanoparticles (NPs) carrying a drug doxorubicin (DOX) were loaded into a larger NP containing another drug (vincristine [VCR]) via water-in-oil-in-water double-emulsion solvent diffusion-evaporation method. Chitosan-alginate nanoparticles carrying DOX (CS-ALG-DOX NPs) with a smaller diameter of about 20 nm formed S-D1 NPs; vitamin E D-α-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles carrying VCR (TPGS-PLGA-VCR NPs) with a larger diameter of about 200 nm constituted L-D2 NPs. Some CS-ALG-DOX NPs loaded into TPGS-PLGA-VCR NPs formed CS-ALG-DOX@TPGS-PLGA-VCR NPs. Under the acidic environment of cytosol and endosome or lysosome in MDR cell, CS-ALG-DOX@TPGS-PLGA-VCR NPs released VCR and CS-ALG-DOX NPs. VCR could arrest cell cycles at metaphase by inhibiting microtubule polymerization in the cytoplasm. After CS-ALG-DOX NPs escaped from endosome, they entered the nucleus through the nuclear pore and released DOX in the intra-nuclear alkaline environment, which interacted with DNA to stop the replication of MDR cells. These results indicated that S-D1@L-D2 NPs was a co-delivery system of intracellular precision release loaded drugs with pH-sensitive characteristics. S-D1@L-D2 NPs could obviously enhance the in vitro cytotoxicity and the in vivo anticancer efficiency of co-delivery drugs, while reducing their adverse effects. Overall, S-D1@L-D2 NPs can be considered an innovative platform for the co-delivery drugs of clinical combination chemotherapy for the treatment of MDR tumor.

Enhanced differentiation of human amniotic fluid-derived stem cells into insulin-producing cells in vitro.

AIMS/INTRODUCTION: To investigate the ability of human amniotic fluid stem cells (hAFSCs) to differentiate into insulin-producing cells. MATERIALS AND METHODS: hAFSCs were induced to differentiate into pancreatic cells by a multistep protocol. The expressions of pancreas-related genes and proteins, including pancreatic and duodenal homeobox-1, insulin, and glucose transporter 2, were detected by polymerase chain reaction and immunofluorescence. Insulin secreted from differentiated cells was tested by enzyme-linked immunosorbent assay. RESULTS: hAFSCs were successfully isolated from amniotic fluid that expressed the pluripotent markers of embryonic stem cells, such as Oct3/4, and mesenchymal stem cells, such as integrin ß-1 and ecto-5'-nucleotidase. Here, we first obtained the hAFSCs that expressed pluripotent marker stage-specific embryonic antigen 1. Real-time polymerase chain reaction analysis showed that pancreatic and duodenal homeobox-1, paired box gene 4 and paired box gene 6 were expressed in the early phase of induction, and then stably expressed in the differentiated cells. The pancreas-related genes, such as insulin, glucokinase, glucose transporter 2 and Nkx6.1, were expressed in the differentiated cells. Immunofluorescence showed that these differentiated cells co-expressed insulin, C-peptide, and pancreatic and duodenal homeobox-1. Insulin was released in response to glucose stimulation in a manner similar to that of adult human islets. CONCLUSIONS: The present study showed that hAFSCs, under selective culture conditions, could differentiate into islet-like insulin-producing cells, which might be used as a potential source for transplantation in patients with type 1 diabetes mellitus.

CS/PAA@TPGS/PLGA nanoparticles with intracellular pH-sensitive sequential release for delivering drug to the nucleus of MDR cells.

Development of novel nano-drug delivery systems (NDDS) that can transport anticancer drugs into cell nuclei is still a highly desirable strategy for reversing multi-drug resistance (MDR) in cancer therapy. Herein, we designed and prepared a novel NDDS, designated S@L NPs, in which several smaller nanoparticles are contained within a larger nanoparticle. Our S@L NPs (CS/PAA/VP-16@TPGS/PLGA NPs) possess a structure in which smaller nanoparticles (Chitosan-Poly(acrylic acid) nanoparticles, CS/PAA NPs) containing the drug etoposide (VP-16) are loaded within a larger nanoparticle (Vitamin E d-a-tocopheryl polyethylene glycol 1000 succinate-modified poly(lactic-co-glycolic acid) nanoparticles, TPGS/PLGA NPs). The system utilizes intracellular pH gradients to achieve pH-sensitive sequential release within different intracellular domains of MDR cells. S@L NPs could be triggered to degrade and release CS/PAA/VP-16 NPs in the acid environment of the cytosol, endosomes or lysosomes, and CS/PAA/VP-16 NPs were capable of entering the nucleus through nucleopores. It is significant that CS/PAA/VP-16 NPs exhibit disaggregation in the alkaline environment of the nucleus and thereby release the contained anticancer drug. Further mechanistic studies showed that CS/PAA/VP-16 NPs escaped retention and degradation within lysosomes and protected the drug from P-glycoprotein-induced efflux. Simultaneously, S@L NPs enhanced the anticancer effect of the loaded drug by inducing autophagy and apoptosis of MDR cells. This novel NDDS may provide a promising platform for nuclear drug delivery for reversing MDR.

Hyperbranched polymers with thermoresponsive property highly sensitive to ions.

The salt effects on the water solubility of thermoresponsive hyperbranched polyethylenimine and polyamidoamine possessing large amounts of isobutyramide terminal groups (HPEI-IBAm and HPAMAM-IBAm) were studied systematically. Eight anions with sodium as the counterion and ten cations with chloride as the counterion were used to measure the anion and cation effects on the cloud point temperature (T(cp)) of these dendritic polymers in water. It was found that the T(cp) of these dendritic polymers was much more sensitive to the addition of salts than that of the traditional thermoresponsive linear polymers. At low anion concentration, the electrostatic interaction between anions and the positively charged groups of these polymers was dominant, resulting in the unusual anion effect on the T(cp) of these polymers in water, including (1) T(cp) of these dendritic polymers decreasing nonlinearly with the increase of kosmotropic anion concentration; (2) the chaotropic anions showing abnormal salting-out property at low salt concentration and the stronger chaotropes having much pronounced salting-out ability; (3) anti-Hofmeister ordering at low salt concentration. At moderate to high salt concentration, the specific ranking of these anions in reducing the T(cp) of HPEI-IBAm and HPAMAM-IBAm polymers was PO(4)(3-) > CO(3)(2-) > SO(4)(2-) > S(2)O(3)(2-) > F(-) > Cl(-) > Br(-) > I(-), in accordance with the well-known Hofmeister series. At moderate to high salt concentration, the specific ranking order of inorganic cations in reducing the T(cp) of HPEI-IBAm polymer was Sr(2+) ≈ Ba(2+) > Na(+) ≈ K(+) ≈ Rb(+) > Cs(+) > NH(4)(+) ≈ Ca(2+) > Li(+) ≈ Mg(2+). This sequence was only partially similar to the typical Hofmeister cation series, whereas at low salt concentration the cation effect on T(cp) of the dendritic polymer was insignificant and no obvious specific ranking order could be found.

[Crop geometry identification based on inversion of semiempirical BRDF models].

With the rapid development of remote sensing technology, the application of remote sensing has extended from single view angle to multi-view angles. It was studied for the qualitative and quantitative effect of average leaf angle (ALA) on crop canopy reflected spectrum. Effect of ALA on canopy reflected spectrum can not be ignored with inversion of leaf area index (LAI) and monitoring of crop growth condition by remote sensing technology. Investigations of the effect of erective and horizontal varieties were conducted by bidirectional canopy reflected spectrum and semiempirical bidirectional reflectance distribution function (BRDF) models. The sensitive analysis was done based on the weight for the volumetric kernel (fvol), the weight for the geometric kernel (fgeo), and the weight for constant corresponding to isotropic reflectance (fiso) at red band (680 nm) and near infrared band (800 nm). By combining the weights of the red and near-infrared bands, the semiempirical models can obtain structural information by retrieving biophysical parameters from the physical BRDF model and a number of bidirectional observations. So, it will allow an on-site and non-sampling mode of crop ALA identification, which is useful for using remote sensing for crop growth monitoring and for improving the LAI inversion accuracy, and it will help the farmers in guiding the fertilizer and irrigation management in the farmland without a priori knowledge.

[Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells].

OBJECTIVE: To construct two types of Wnt-inducible secreted protein 3(WISP3) gene's mutants(1000T/C,840delT) found in spondyloepiphyseal dysplasia tarda with progressive anthopathy (SEDT-PA) patients, and to observe their expression in COS-7 cells. METHODS: Full-length cDNA of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT1000T/C and MUT840delT). The recombined plasmids WT-WISP3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+) and MUT840delT/pcDNA3.1(+) were transfected transiently into COS-7 cells by liposome-mediated method, and pcDNA3.1(+) vector was used as a control. The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot. RESULTS: By restriction endonuclease analysis and sequencing, the sequence of MUT1000T/C and MUT840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells. CONCLUSION: WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 cells, which lays the foundation for the further study on its molecular functions in SEDT-PA.

Construction of WISP3 gene's mutants in SEDT-PA and their expression in COS-7 cells / 中南大学学报(医学版)

OBJECTIVE@#To construct two types of Wnt-inducible secreted protein 3(WISP3) gene's mutants(1000T/C,840delT) found in spondyloepiphyseal dysplasia tarda with progressive anthopathy (SEDT-PA) patients, and to observe their expression in COS-7 cells.@*METHODS@#Full-length cDNA of wild type WISP3 gene(WT-WISP3) was amplified from human chondrocytes by RT-PCR, and site-directed mutagenesis was used to obtain full-length cDNAs of the mutated WISP3 genes(MUT1000T/C and MUT840delT). The recombined plasmids WT-WISP3/pcDNA3.1(+), MUT1000T/C/pcDNA3.1(+) and MUT840delT/pcDNA3.1(+) were transfected transiently into COS-7 cells by liposome-mediated method, and pcDNA3.1(+) vector was used as a control. The total RNA and protein of the transfected COS-7 cells were extracted after 48 hours of transfection. The expression of WISP3 gene in the transfected COS-7 cells was detected by semi-quantitative RT-PCR and Western blot.@*RESULTS@#By restriction endonuclease analysis and sequencing, the sequence of MUT1000T/C and MUT840delT were consistent with that mutated in SEDT-PA, and the open reading frames matched with the vector sequence. Semi-quantitative RT-PCR and Western blot showed that the recombined plasmids were highly expressed in COS-7 cells.@*CONCLUSION@#WISP3 gene's mutants of SEDT-PA are successfully constructed by genetic recombination, and expressed in COS-7 cells, which lays the foundation for the further study on its molecular functions in SEDT-PA.