[Screening and activity analysis of nanobodies against lymphocyte activation gene-3(LAG-3)].

Objective To generate the phage display nanobody library immunized by lymphocyte-activation gene 3 (LAG-3) and to validate the functional activity of obtained anti-LAG-3 nanobodies. Methods The peripheral blood cDNA library was isolated from the adult llama which was immunized by human LAG-3 protein. The nanobodies sequences were obtained by nested PCR and cloned into the phagemid vector pComb3XSS, then transformed into Escherichia coli XL1-Blue cells for library generation and quality analysis. Anti-LAG-3 specific nanobodies were screened by phage display and sequenced by next-generation sequencing. Nanobodies were cloned into pET-22b (+) vector and Escherichia coli BL21 (DE3) cells were used for protein expression. The proteins were purified by using the Prism A column, then HPLC-MS, ELISA, Western blot, and surface plasmon resonance technology (SPR) were performed to characterize the nanobodies. Results The library capacity of the nanobody phage immune library with great diversity was 7.20×108 CFU/mL. After four rounds of biopanning, three individual nanobodies with distinct amino acid sequences VHH-L1-3, VHH-L3-2 and VHH-L13-2 were picked. The purity of the purified nanobodies was more than 95%. All of these three nanobodies exhibited high binding affinities with recombinant human LAG-3 specifically, among which the KD value of VHH-L13-2 was 3.971×10-9 mol/L. VHH-L13-2 exhibited the inhibitory effects on the association of LAG-3 and its ligand FGL-1, and the half maximal inhibitory concentration (IC50) value was 15.58 nmol/L. Conclusion The anti-LAG-3 phage display nanobody library is generated successfully. The anti-LAG-3 nanobodies possess high specificity and binding affinity and exhibit the inhibitory effects on the association of LAG-3 and its ligand.

Molecular Characterization of U6 Promoters from Orange-Spotted Grouper (Epinephelus coioides) and Its Application in DNA Vector-Based RNAi Technology.

The U6 promoter, a typical RNA polymerase III promoter, is widely used to transcribe small RNAs in vector-based siRNA systems. The RNAi efficiency is mainly dependent on the transcriptional activity of the U6 promoter. However, studies have found that U6 promoters isolated from some fishes do not work well in distantly related species. To isolate a U6 promoter with high transcriptional efficiency from fish, in this study, we cloned five U6 promoters in orange-spotted grouper, of which only the grouper U6-1 (GU6-1) promoter contains the OCT element in the distant region. Functional studies revealed that the GU6-1 promoter has high transcriptional ability, which could efficiently transcribe shRNA and result in target gene knockdown in vitro and in vivo. Subsequently, the deletion or mutation of the OCT motif resulted in a significant decrease in promoter transcriptional activity, demonstrating that the OCT element plays an important role in enhancing the grouper U6 promoter transcription. Moreover, the transcriptional activity of the GU6-1 promoter showed little species specificity. It not only works in the grouper but also possesses high transcriptional activity in the zebrafish. Knockdown of the mstn gene in zebrafish and grouper through shRNA driven by the GU6-1 promoter could promote fish growth, suggesting that the GU6-1 promoter can be used as a potential molecular tool in aquaculture practice.

Inhalable GSH-Triggered Nanoparticles to Treat Commensal Bacterial Infection in In Situ Lung Tumors.

Bacterial infection has been considered one of the primary reasons for low survival rate of lung cancer patients. Herein, we demonstrated that a kind of mesoporous silica nanoparticles loaded with anticancer drug doxorubicin (DOX) and antimicrobial peptide HHC36 (AMP) (MSN@DOX-AMP) can kill both commensal bacteria and tumor cells under GSH-triggering, modulating the immunosuppressive tumor microenvironment, significantly treating commensal bacterial infection, and eliminating in situ lung tumors in a commensal model. Meanwhile, MSN@DOX-AMP encapsulated DOX and AMP highly efficiently via a combined strategy of physical adsorption and click chemistry and exhibited excellent hemocompatibility and biocompatibility. Importantly, MSN@DOX-AMP could be inhaled and accumulate in lung by a needle-free nebulization, achieving a better therapeutic effect. This system is expected to serve as a straightforward platform to treat commensal bacterial infections in tumors and promote the translation of such inhaled GSH-triggered MSN@DOX-AMP to clinical treatments of lung cancer.

Effects of different concentrations of mesenchymal stem cells treatment on LPS-induced acute respiratory distress syndrome rat model.

PURPOSE: This study was prospectively designed to investigate the effects of different concentrations of mesenchymal stem cells treatment on respiratory mechanics, oxygenation, hemodynamics and inflammatory response in LPS-induced acute respiratory distress syndrome (ARDS) rat model. Methods: One hundred and twenty six LPS-induced ARDS model rats (weighted 200-220 g) were randomly divided into three groups: 1) Control group (N = 42); 2) low-dose hUC-MSC treatment group (MSC group 1, 1x107 cell/kg, N = 42); 3) high-dose hUC-MSC treatment group (MSC group 2, 2x107 cell/kg, N = 42), sham operation group as healthy group (N = 15). The rats were observed closely for 24 hours after hUC-MSC treatment, and the survival rate was calculated. At 24 hours, all rats were tested for hemodynamics, blood gas analysis, heart, lung, liver and kidney functions, inflammatory factors detection in blood samples and broncho-alveolar lavage fluid (BALF). The lung tissue of the rats was collected for HE staining analysis. Results: After LPS injection, ARDS was obvious in all LPS-infused rat groups, consistent with severe acute lung injury and high death rate. However, compared with the control group, a single intravenous injection hUC-MSC at dose of 1 × 107 cells/kg (low dose group) and 2 × 107 cells/kg (high dose group) reduced the mortality of rats with LPS-induced ARDS, as well as improving the lung function, increased the arterial oxygen pressure, improved the heart function, and reduced the levels of inflammatory factors including IL-1ß, IL-6, and TNF-α. In addition, the high dose MSC group showed better lung injury therapeutic effects than the low dose MSC group. Data from this study demonstrated that injection of hUC-MSC had a significant therapeutic effect in treating the rat model of LPS-induced ARDS and multiple organ function injury.

Pharmacokinetics, tissue distribution and excretion of a novel long-acting human insulin analogue - recombinant insulin LysArg in rats.

As a novel long-acting recombinant human insulin analogue, it is necessary to carry out the preclinical research for insulin LysArg. The purpose of this study was to characterise the pharmacokinetic, tissue distribution and excretion of insulin LysArg and provide a reference for its development. Three methods were used to measure the content of insulin LysArg in biological samples after a single subcutaneous administration in rats, including radioassay, radioassay after precipitation with TCA and separation by HPLC. After Subcutaneous administration of recombinant insulin LysArg 1, 2, 4 U/kg in rats, it showed both Cmax and AUC0-t were positively correlated with the dose. In the meanwhile, after a single subcutaneous administration of recombinant insulin LysArg at 2 U/kg in rats, the amount of radioactivity in most organs was highest at 1.5 h and then decreased gradually, no accumulation was found. The highest level of insulin LysArg was observed in the kidney. Like other macromolecules, insulin LysArg was mainly excreted from urine. The study fully illustrated the pharmacokinetic pattern of insulin LysArg, provided valuable informations to support its further development about safety and toxicology.

Transporters (OATs and OATPs) contribute to illustrate the mechanism of medicinal compatibility of ingredients with different properties in yuanhuzhitong prescription.

Various medicinal ingredients with different tastes are combined according to the theory of compatibility in Chinese materia medica to achieve a better efficacy, while the mechanism was not very clear. Here, the authors studied the interaction between ingredients and human transporters such as the kidney transporters OAT1 and OAT3, the liver transporters OATP1B1 and OATP1B3, and the intestine transporter OATP2B1 to discern the compatibility mechanism of ingredients with different tastes in the Yuanhuzhitong preparation (YHP) comprising Corydalis yanhusuo (CYH) and Angelica dahurica (AD), which could relieve pain by restraining the central system. The results show that tetrahydropalmatine (TDE), the major component of CYH, could be transported by OAT3 into kidney, OATP1B1 and OATP1B3 into liver, while imperatorin (IPT) and isoimperatorin (ISP), the two key components of AD, and AD extract showed strong inhibition to OAT1 and OAT3. What's more, AD extract also exerted strongly inhibition to human transporters OATP1B1 and OATP1B3. It was also detected that IPT, ISP, and AD extract significantly downregulated the expression of Oatp1a1, Oatp1a4, and Oatp1b2 of liver in mice. The in vivo results show that the concentration of TDE in liver and kidney significantly decreased, while the TDE concentration in blood and brain were both significantly enhanced in the presence of IPT, ISP, and AD extract. These results suggest that the ingredients in AD with pungent taste could enhance the exposure of TDE in blood and brain by inhibiting the uptake of TDE in liver and kidney. That is to say, TDE with bitter taste could "flood up" into the central nervous system to play its therapeutic effect by the cut-off of that into liver and kidney in the presence of ingredients within AD. This paper not only proves the meridian distribution of CYH in liver and kidney with the role of OAT3, OATP1B1, and OATP1B3, but also illustrates how to improve the efficacy of CYH by reasonable compatibility with AD. This study may offer a valuable clue to illustrate the mechanism of compatibility theory.

Responses of Manila Grass (Zoysia matrella) to chilling stress: From transcriptomics to physiology.

Manila grass (Zoysia matrella), a warm-season turfgrass, usually wilts and browns by late autumn because of low temperature. To elucidate the molecular mechanisms regarding Manila grass responses to cold stress, we performed transcriptome sequencing of leaves exposed to 4°C for 0 (CK), 2h (2h_CT) and 72h (72h_CT) by Illumina technology. Approximately 250 million paired-end reads were obtained and de novo assembled into 82,605 unigenes. A total of 34,879 unigenes were annotated by comparing their sequence to public protein databases. At the 2h- and 72h-cold time points, 324 and 5,851 differentially expressed genes (DEGs) were identified, respectively. Gene ontology (GO) and metabolism pathway (KEGG) enrichment analyses of DEGs indicated that auxin, gibberellins, ethylene and calcium took part in the cold signal transduction in the early period. And in the late cold period, electron transport activities, photosynthetic machinery and activity, carbohydrate and nitrogen metabolism, redox equilibrium and hormone metabolism were disturbed. Low temperature stress triggered high light, drought and oxidative stress. At the physiological level, cold stress induced a decrease in water content, an increase in levels of total soluble sugar, free proline and MDA, and changes in bioactive gibberellins levels, which supported the changes in gene expression. The results provided a large set of sequence data of Manila grass as well as molecular mechanisms of the grass in response to cold stress. This information will be helpful for future study of molecular breeding and turf management.

Sugar transporter genes in grass carp (Ctenopharyngodon idellus): molecular cloning, characterization, and expression in response to different stocking densities.

Glucose and fructose play a central role in the metabolism and cellular homeostasis of organisms. Their absorption is co-mediated by two families of glucose transporters, Na+-coupled glucose co-transporters (SGLTs) and facilitative Na+-independent sugar carriers (GLUTs), in the intestine. However, limited information has been available on these transporters in fish. Therefore, we studied glut2, sglt1, and sglt4 genes in grass carp (Ctenopharyngodon idellus). The full-length cDNAs of glut2 was 2308 bp, with an open reading frame (ORF) of 503 amino acids (AAs). The full-length cDNAs of sglt1 was 2890 bp, with an ORF of 658 AAs. Additionally, the full-length cDNAs of sglt4 was 2090 bp, with an ORF encoding 659 AAs. The three deduced AA sequences showed high homology between grass carp and other cyprinid fish species. Based on homology modeling, three-dimensional models of GLUT2, SGLT1, and SGLT4 proteins were created and transmembrane domains were noted. glut2, sglt1, and sglt4 were abundantly expressed in the anterior and mid intestine. In particular, glut2 was markedly expressed in liver (P < 0.05). Additionally, the results indicated that different stocking densities (0.9 or 5.9 kg m-2) did not alter intestinal section-dependent expression patterns of the three transporter genes. However, high stocking density impacted segmental mRNA expression levels. This work demonstrated that mRNA expression of sugar transporter genes in the fish intestine was segment specific, and crowding stress may affect the activity of intestinal sugar transporters. These results provided new insights into the relationship between crowding stress and intestinal sugar transporters in fish.

The Identification of the Deformation Stage of a Metal Specimen Based on Acoustic Emission Data Analysis.

The acoustic emission (AE) signals of metal materials have been widely used to identify the deformation stage of a pressure vessel. In this work, Q235 steel samples with different propagation distances and geometrical structures are stretched to get the corresponding acoustic emission signals. Then the obtained acoustic emission signals are de-noised by empirical mode decomposition (EMD), and then decomposed into two different frequency ranges, i.e., one mainly corresponding to metal deformation and the other mainly corresponding to friction signals. The ratio of signal energy between two frequency ranges is defined as a new acoustic emission characteristic parameter. Differences can be observed at different deformation stages in both magnitude and data distribution range. Compared with other acoustic emission parameters, the proposed parameter is valid in different setups of the propagation medium and the coupled stiffness.

Characterization of ß-glucosidase from Aspergillus terreus and its application in the hydrolysis of soybean isoflavones.

An extracellular ß-glucosidase produced by Aspergillus terreus was identified, purified, characterized and was tested for the hydrolysis of soybean isoflavone. Matrix-assisted laser desorption/ionization with tandem time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) revealed the protein to be a member of the glycosyl hydrolase family 3 with an apparent molecular mass of about 120 kDa. The purified ß-glucosidase showed optimal activity at pH 5.0 and 65 °C and was very stable at 50 °C. Moreover, the enzyme exhibited good stability over pH 3.0-8.0 and possessed high tolerance towards pepsin and trypsin. The kinetic parameters Km (apparent Michaelis-Menten constant) and Vmax (maximal reaction velocity) for p-nitrophenyl-ß-D-glucopyranoside (pNPG) were 1.73 mmol/L and 42.37 U/mg, respectively. The Km and Vmax for cellobiose were 4.11 mmol/L and 5.7 U/mg, respectively. The enzyme efficiently converted isoflavone glycosides to aglycones, with a hydrolysis rate of 95.8% for daidzin, 86.7% for genistin, and 72.1% for glycitin. Meanwhile, the productivities were 1.14 mmol/(L·h) for daidzein, 0.72 mmol/(L·h) for genistein, and 0.19 mmol/(L·h) for glycitein. This is the first report on the application of A. terreus ß-glucosidase for converting isoflavone glycosides to their aglycones in soybean products.

[Attachment and spreading of fibroblasts on titanium coatings by different micro-arc oxidation time].

PURPOSE: To investigate the attachment and spreading of fibroblasts on titanium coatings by different micro-arc oxidation(MAO) time. METHODS: Micro-arc oxidation film was formed on the titanium surface by using micro-arc oxidation. The film morphology and the cell shape on the samples were observed by scanning electron microscopy (SEM) and the surface roughness of film with different time was analyzed by surface coarseness profiling instrument. SPSS11.5 software package was used to analyze the data. RESULTS: The experiment result showed, the coatings were rough and porous, the longer oxidation time, the larger diameter of porous but the less porous numbers. As the treatment time increased, the surface roughness of micro-arc oxidation specimens increased (P<0.05). Compared with titanium, fibroblasts on titanium MAO coatings had more abundant spreading and adhesion. At 24 h, 48 h and 72 h, the cell number on coatings adhesion increased, but with different oxidation time, the cell shape, number and adhesion weren't obviously changed. CONCLUSIONS: The results indicate that microarc oxidation increases the surface roughness, have a good influence on cell adhesion and spreading.

[Preparation of hydroxyapatite-containing titania film on the surface of pure titanium by micro-arc oxidation technique].

A porous hydroxylapatite-containing titania film was prepared by an electrochemical oxidation method, i.e. micro-arc oxidation (MAO). During the oxidation treatment, the titanium sample was immersed in electrolytic solution containing calcium acetate monohydrate and sodium biphosphate dihydrate by using a pulse power supply. The thickness, phase, composition and morphology of the oxide coating were monitored with X-ray diffraction (XRD) and scanning electron microscopy (SEM) with energy dispersive X-ray spectrometer (EDS). The thickness of the MAO film is about 20 microm and the coating where each porous size is no more than 5 microm was porous and uneven, without apparent interface to the titanium substrates. The coating formed in the Ca- and P-containing solution with MAO contained Ca and P along with Ti and O. The Ca/P ratio on the surface is 1.63, while that in the interface is 0.51. XRD showed that the porous coating was made up of anatase, rutile and hydroxyapatite. Such MAO films are expected to have significant medical applications as dental implants and artificial bone joints.