Bacillus subtilis partially inhibits African swine fever virus infection in vivo and in vitro based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II.

IMPORTANCE: African swine fever virus (ASFV) is a highly fatal swine disease that severely affects the pig industry. Although ASFV has been prevalent for more than 100 years, effective vaccines or antiviral strategies are still lacking. In this study, we identified four Bacillus subtilis strains that inhibited ASFV proliferation in vitro. Pigs fed with liquid biologics or powders derived from four B. subtilis strains mixed with pellet feed showed reduced morbidity and mortality when challenged with ASFV. Further analysis showed that the antiviral activity of B. subtilis was based on its metabolites arctiin and genistein interfering with the function of viral topoisomerase II. Our findings offer a promising new strategy for the prevention and control of ASFV that may significantly alleviate the economic losses in the pig industry.

Meta-Analysis of the Prevalence of Giardia duodenalis in Cattle in China.

Giardia duodenum (G. duodenalis) can cause giardiasis and infect a variety of hosts. So far, there have been no detailed data regarding the positive rate of G. duodenalis in cattle in China. Here, a systematic literature review was carried out to investigate the epidemiology of bovine G. duodenalis in China. To perform the meta-analysis, the databases China National Knowledge Infrastructure, VIP Chinese Journal Databases, WanFang Databases, PubMed, and ScienceDirect were employed for screening studies related to the prevalence of G. duodenalis in cattle in China. The total prevalence of G. duodenalis in cattle was estimated to be 8.00% (95% confidence interval [CI]: 5.51-11.62). In the age subgroup, the prevalence of G. duodenalis in calves (11.72%; 95% CI: 7.75-17.73) was significantly higher than that in cattle of other age groups. An analysis based on seasons showed that the prevalence of G. duodenalis in cattle was higher in summer (9.69%; 95% CI: 2.66-35.30) than that in other seasons. The prevalence of G. duodenalis in cattle in 2016 or later was 11.62% (95% CI: 6.49-20.79), which was significantly higher than that before 2016 (3.65%; 95% CI: 2.17-6.12). The highest prevalence of G. duodenalis in cattle was 74.23% (95% CI: 69.76-78.45) recorded in South China. The NOAA's National Center for Environmental Information (https://gis.ncdc.noaa.gov/maps/ncei/cdo/monthly) was used to extract relevant geoclimatic data (latitude, longitude, elevation, temperature, precipitation, humidity, and climate). By analyzing the data of each subgroup, it was shown that age of cattle, sampling year, province, region, temperature, and climate were potential risk factors for giardiasis prevalence in cattle. Based on the analysis of common factors and geographical factors, it is recommended to strengthen effective management measures (e.g., ventilation and disinfection in warm and humid areas) and formulate relevant policies according to local conditions. Breeders should pay more attention to the detection of G. duodenalis in calves, to prevent giardiasis prevalence in cattle of different ages, thereby reducing the economic losses of animal husbandry in China.

Genome-Wide Identification and Expression Analysis of NCED Gene Family in Pear and Its Response to Exogenous Gibberellin and Paclobutrazol.

The 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme for the process of ABA synthesis that plays key roles in a variety of biological processes. In the current investigation, genome-wide identification and comprehensive analysis of the NCED gene family in 'Kuerle Xiangli' (Pyrus sinkiangensis Yu) were conducted using the pear genomic sequence. In total, nineteen members of PbNCED genes were identified from the whole genome of pear, which are not evenly distributed over the scaffolds, and most of which were focussed in the chloroplasts. Sequence analysis of promoters showed many cis-regulatory elements, which presumably responded to phytohormones such as abscisic acid, auxin, etc. Synteny block indicated that the PbNCED genes have experienced strong purifying selection. Multiple sequence alignment demonstrated that these members are highly similar and conserved. In addition, we found that PbNCED genes were differentially expressed in various tissues, and three PbNCED genes (PbNCED1, PbNCED2, and PbNCED13) were differentially expressed in response to exogenous Gibberellin (GA3) and Paclobutrazol (PP333). PbNCED1 and PbNCED13 positively promote ABA synthesis in sepals after GA3 and PP333 treatment, whereas PbNCED2 positively regulated ABA synthesis in ovaries after GA3 treatment, and PbNCED13 positively regulated ABA synthesis in the ovaries after PP333 treatment. This study was the first genome-wide report of the pear NCED gene family, which could improve our understanding of pear NCED proteins and provide a solid foundation for future cloning and functional analyses of this gene family. Meanwhile, our results also give a better understanding of the important genes and regulation pathways related to calyx abscission in 'Kuerle Xiangli'.

Genome-Wide Identification, Classification, Expression and Duplication Analysis of bZIP Family Genes in Juglans regia L.

Basic leucine zipper (bZIP), a conserved transcription factor widely found in eukaryotes, has important regulatory roles in plant growth. To understand the information related to the bZIP gene family in walnut, 88 JrbZIP genes were identified at the genome-wide level and classified into 13 subfamilies (A, B, C, D, E, F, G, H, I, J, K, M, and S) using a bioinformatic approach. The number of exons in JrbZIPs ranged from 1 to 12, the number of amino acids in JrbZIP proteins ranged from 145 to 783, and the isoelectric point ranged from 4.85 to 10.05. The majority of JrbZIP genes were localized in the nucleus. The promoter prediction results indicated that the walnut bZIP gene contains a large number of light-responsive and jasmonate-responsive action elements. The 88 JrbZIP genes were involved in DNA binding and nucleus and RNA biosynthetic processes of three ontological categories, molecular functions, cellular components and biological processes. The codon preference analysis showed that the bZIP gene family has a stronger bias for AGA, AGG, UUG, GCU, GUU, and UCU than other codons. Moreover, the transcriptomic data showed that JrbZIP genes might play an important role in floral bud differentiation. The results of a protein interaction network map and kegg enrichment analysis indicated that bZIP genes were mainly involved in phytohormone signaling, anthocyanin synthesis and flowering regulation. qRT-PCR demonstrated the role of the bZIP gene family in floral bud differentiation. Co-expression network maps were constructed for 29 walnut bZIP genes and 6 flowering genes, and JrCO (a homolog of AtCO) was significantly correlated (p < 0.05) with 13 JrbZIP genes in the level of floral bud differentiation expression, including JrbZIP31 (homolog of AtFD), and JrLFY was significantly and positively correlated with JrbZIP10,11,51,59,67 (p < 0.05), and the above results suggest that bZIP family genes may act together with flowering genes to regulate flower bud differentiation in walnut. This study was the first genome-wide report of the walnut bZIP gene family, which could improve our understanding of walnut bZIP proteins and provide a solid foundation for future cloning and functional analyses of this gene family.

Comprehensive Identification and Analyses of the GRF Gene Family in the Whole-Genome of Four Juglandaceae Species.

The GRF gene family plays an important role in plant growth and development as regulators involved in plant hormone signaling and metabolism. However, the Juglandaceae GRF gene family remains to be studied. Here, we identified 15, 15, 19, and 20 GRF genes in J. regia, C. illinoinensis, J. sigillata, and J. mandshurica, respectively. The phylogeny shows that the Juglandaceae family GRF is divided into two subfamilies, the ε-group and the non-ε-group, and that selection pressure analysis did not detect amino acid loci subject to positive selection pressure. In addition, we found that the duplications of the Juglandaceae family GRF genes were all segmental duplication events, and a total of 79 orthologous gene pairs and one paralogous homologous gene pair were identified in four Juglandaceae families. The Ka/KS ratios between these homologous gene pairs were further analyzed, and the Ka/KS values were all less than 1, indicating that purifying selection plays an important role in the evolution of the Juglandaceae family GRF genes. The codon bias of genes in the GRF family of Juglandaceae species is weak, and is affected by both natural selection pressure and base mutation, and translation selection plays a dominant role in the mutation pressure in codon usage. Finally, expression analysis showed that GRF genes play important roles in pecan embryo development and walnut male and female flower bud development, but with different expression patterns. In conclusion, this study will serve as a rich genetic resource for exploring the molecular mechanisms of flower bud differentiation and embryo development in Juglandaceae. In addition, this is the first study to report the GRF gene family in the Juglandaceae family; therefore, our study will provide guidance for future comparative and functional genomic studies of the GRF gene family in the Juglandaceae specie.

Decreased Tissue Omega-6/Omega-3 Fatty Acid Ratio Prevents Chemotherapy-Induced Gastrointestinal Toxicity Associated with Alterations of Gut Microbiome.

Gastrointestinal toxicity (GIT) is a debilitating side effect of Irinotecan (CPT-11) and limits its clinical utility. Gut dysbiosis has been shown to mediate this side effect of CPT-11 by increasing gut bacterial ß-glucuronidase (GUSB) activity and impairing the intestinal mucosal barrier (IMB). We have recently shown the opposing effects of omega-6 (n-6) and omega-3 (n-3) polyunsaturated fatty acids (PUFA) on the gut microbiome. We hypothesized that elevated levels of tissue n-3 PUFA with a decreased n-6/n-3 PUFA ratio would reduce CPT-11-induced GIT and associated changes in the gut microbiome. Using a unique transgenic mouse (FAT-1) model combined with dietary supplementation experiments, we demonstrate that an elevated tissue n-3 PUFA status with a decreased n-6/n-3 PUFA ratio significantly reduces CPT-11-induced weight loss, bloody diarrhea, gut pathological changes, and mortality. Gut microbiome analysis by 16S rRNA gene sequencing and QIIME2 revealed that improvements in GIT were associated with the reduction in the CPT-11-induced increase in both GUSB-producing bacteria (e.g., Enterobacteriaceae) and GUSB enzyme activity, decrease in IMB-maintaining bacteria (e.g., Bifidobacterium), IMB dysfunction and systemic endotoxemia. These results uncover a host-microbiome interaction approach to the management of drug-induced gut toxicity. The prevention of CPT-11-induced gut microbiome changes by decreasing the tissue n-6/n-3 PUFA ratio could be a novel strategy to prevent chemotherapy-induced GIT.

Enhancing the selectivity of liquid chromatography-mass spectrometry by using trilinear decomposition on LC-MS data: An application to three-way calibration of coeluting analytes in human plasma.

The high selectivities of liquid chromatography and mass spectrometry make liquid chromatography-mass spectrometry one of the most popular tools for quantitative analysis in complex chemical, biological, and environmental systems, while the potential mathematical selectivity of liquid chromatography-mass spectrometry is rarely investigated. This work discussed the mathematical selectivity of liquid chromatography-mass spectrometry by three-way calibration based on the trilinear model, with an application to quantitative analysis of coeluting aromatic amino acids in human plasma. By the trilinear decomposition of the constructed liquid chromatography-mass spectrometry-sample trilinear model and individual regression of the decomposed relative intensity versus concentration, the proposed three-way calibration method successfully achieved quantitative analysis of coeluting aromatic amino acids in human plasma, even in the presence of uncalibrated interferent(s) and a varying background. This analytical method can ease the requirements for sample preparation and complete chromatographic separation of components, reduce the use of organic solvents, decrease the time of chromatographic separation, and increase the peak capacity of liquid chromatography-mass spectrometry. As a "green analytical method", the liquid chromatography-mass spectrometry three-way calibration method can provide a promising tool for direct and fast quantitative analysis in complex systems containing uncalibrated spectral interferents, especially for the situation where the coelution problem is difficult to overcome.

Deep Learning Correction Algorithm for The Active Optics System.

The correction of wavefront aberration plays a vital role in active optics. The traditional correction algorithms based on the deformation of the mirror cannot effectively deal with disturbances in the real system. In this study, a new algorithm called deep learning correction algorithm (DLCA) is proposed to compensate for wavefront aberrations and improve the correction capability. The DLCA consists of an actor network and a strategy unit. The actor network is utilized to establish the mapping of active optics systems with disturbances and provide a search basis for the strategy unit, which can increase the search speed; The strategy unit is used to optimize the correction force, which can improve the accuracy of the DLCA. Notably, a heuristic search algorithm is applied to reduce the search time in the strategy unit. The simulation results show that the DLCA can effectively improve correction capability and has good adaptability. Compared with the least square algorithm (LSA), the algorithm we proposed has better performance, indicating that the DLCA is more accurate and can be used in active optics. Moreover, the proposed approach can provide a new idea for further research of active optics.

[Discussion on research thought of dry granulation of traditional Chinese medicine: research progress at home and abroad based on dry granulation technology].

Dry granulation technology is a great innovation in granulation technology,which saves many intermediate links and reduces many intermediate costs. It is closely related to the characteristics of materials,dry granulation equipment and process. Dry granulation technology is a systematic engineering science covering many technical fields. The process of dry granulation involves complex mathematical model mechanisms of temperature field,pressure field and velocity field,closely related to the characteristics of materials and drying equipment. However,due to the late start of research on dry granulation technology of traditional Chinese medicine,basic research is still weak. The research on dry granulation technology has achieved great results in the fields of food,chemical industry,agriculture and forestry,showing great reference significance. The advantage of dry granulation of traditional Chinese medicine is that it can be directly granulated by adding an appropriate amount of auxiliary materials in the extract powder of traditional Chinese medicine,without the need of wetting,mixing,drying and other processes. The process is simple and can effectively guarantee the quality of traditional Chinese medicine. The granules obtained by the dry granulation technique are important intermediates for preparing the solid preparations of traditional Chinese medicines,which would directly affect the subsequent molding process and the quality of the preparation products. Therefore,based on the characteristics of dry granulation method in traditional Chinese medicine and by referring to the advanced research results of dry granulation technology in other fields,we would discuss the research ideas of dry granulation in traditional Chinese medicine in terms of the mechanism of dry granulation equipment,technology,on-line detection technology and mathematical model of dry granulation process,hoping to provide reference for the research of dry granulation method in traditional Chinese medicine.

Characterization of roles of SpaA in Erysipelothrix rhusiopathiae adhesion to porcine endothelial cells.

Erysipelothrix rhusiopathiae is the causative agent of animal erysipelas and human erysipeloid. The major protective antigen SpaA was suggested to play important roles in E. rhusiopathiae adhesion to host cells, but there is no specific study on SpaA pathogenic roles in adhesion. In this study we characterized direct and indirect roles of SpaA in E. rhusiopathiae adhesion to porcine endothelial cells. Recombinant E. rhusiopathiae SpaA (rSpaA) successfully binded to porcine iliac arterial endothelial cells. rSpaA protein pre-incubating endothelial cells or rSpaA antiserum pre-incubating E. rhusiopathiae significantly decreased E. rhusiopathiae adhesion to endothelial cells. rSpaA successfully binded host plasminogen and fibronectin, and rSpaA antiserum significantly decreased plasminogen-recruitment activity but not fibronectin-recruitment activity of E. rhusiopathiae. In conclusion, SpaA acts as adhesin in E. rhusiopathiae adhesion to host cells, and SpaA binding activity to host plasminogen highly likely play roles in this adhesion.

The expression of VILL protein is hypoosmotic-dependent in the lamellar gill ionocytes of Otocephala teleost fish, Chanos chanos.

Milkfish, a species within the primitive teleost lineage Otocephala, can survive in water conditions ranging from hypo- to hyper-saline. This study explored the effects of environmental salinity on apical morphologies of ionocytes and the expression of villin homologs in the gills of milkfish acclimated to either seawater (SW) or fresh water (FW). Scanning electron microscopy revealed that the ionocytes in the gill filaments of SW and FW milkfish, respectively, cellular apical morphologies were hole-type and squint-type. The flat-type ionocytes were observed in the gill lamellae of FW milkfish. Furthermore, apical surfaces of some lamellar ionocytes exhibited microvilli. Villin 1 is a microvilli marker expressed in the epithelial cells of various vertebrates. In the phylogenetic tree of villin 1 homologs, primitive teleosts exhibit villin 1-like (VILL) and villin 1 proteins. Two mRNA sequences, villin 1 and VILL, were identified from the milkfish transcriptome by next generation sequencing. Low but constant expression of villin 1 (gene and protein) was observed in the gills for both SW and FW fish. VILL gene and protein expression levels in the gills were higher in FW fish, compared to SW fish. Double immunofluorescence staining demonstrated that VILL protein was present in some lamellar ionocytes of FW milkfish, but not in the filament ionocytes of either FW or SW milkfish. Taken together, these findings indicated that the VILL expression of ionocytes is hypoosmotic-dependent. The VILL might be involved in the formation of microvilli in the lamellar ionocytes for hyperosmoregulation of the milkfish.

Effects of Noggin-Transfected Neural Stem Cells on Neural Functional Recovery and Underlying Mechanism in Rats with Cerebral Ischemia Reperfusion Injury.

OBJECTIVE: To investigate neuroprotection of noggin-transfected neural stem cells (NSCs) against focal cerebral ischemia reperfusion injury (IRI) in rats. METHODS: Eighty Wistar rats were randomly divided into the sham, IRI, NSCs, and noggin + NSCs groups. Noggin containing adenoviral vectors was transfected into rat NSCs. Rats were subjected to 2.0 hours middle cerebral artery occlusion and reperfusion 1.0 hour, followed by infusion into the lateral ventricles of NSCs alone, noggin-transfected NSCs, and saline at 3 days in the NSCs, noggin + NSCs, and sham groups, respectively. All rats were sacrificed on 1, 3, 7, and 28 days after transplantation; the colorimetric method was used to detect the levels of superoxide dismutase (SOD) and the malondialdehyde (MDA) content after the behavior capability determined. Western blot was performed for detecting the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) proteins. The TUNEL-positive and BrdU/nestin double-positive cells were observed under a light microscope and quantitative analysis was performed by morphometric technique. RESULTS: Noggin-transfected NSCs significantly decreased the infarct volume and improved the neurological scores. Noggin-transfected NSCs also reduced the percentage of apoptotic neurons and relieved neuronal morphological damage. Noggin-transfected NSC transplantation markedly decreased the MDA levels and increased the SOD activity, and simultaneously downregulated the BMP4 (bone morphogenesis protein), VEGF, and bFGF proteins. CONCLUSIONS: The present study demonstrates that grafting NSCs modified by noggin gene provides better neuroprotection for cerebrovascular disease.

A Stenohaline Medaka, Oryzias woworae, Increases Expression of Gill Na(+), K(+)-ATPase and Na(+), K(+), 2Cl(-) Cotransporter 1 to Tolerate Osmotic Stress.

The present study aimed to evaluate the osmoregulatory mechanism of Daisy's medaka, O. woworae,as well as demonstrate the major factors affecting the hypo-osmoregulatory characteristics of euryhaline and stenohaline medaka. The medaka phylogenetic tree indicates that Daisy's medaka belongs to the celebensis species group. The salinity tolerance of Daisy's medaka was assessed. Our findings revealed that 20‰ (hypertonic) saltwater (SW) was lethal to Daisy's medaka. However, 62.5% of individuals survived 10‰ (isotonic) SW with pre-acclimation to 5‰ SW for one week. This transfer regime, "Experimental (Exp.) 10‰ SW", was used in the following experiments. After 10‰ SW-transfer, the plasma osmolality of Daisy's medaka significantly increased. The protein abundance and distribution of branchial Na(+), K(+)-ATPase (NKA) and Na(+), K(+), 2Cl(-) cotransporter 1 (NKCC1) were also examined after transfer to 10‰ SW for one week. Gill NKA activity increased significantly after transfer to 10‰ SW. Meanwhile, elevation of gill NKA αα-subunit protein-abundance was found in the 10‰ SW-acclimated fish. In gill cross-sections, more and larger NKA-immunoreactive (NKA-IR) cells were observed in the Exp. 10‰ SW medaka. The relative abundance of branchial NKCC1 protein increased significantly after transfer to 10‰ SW. NKCC1 was distributed in the basolateral membrane of NKA-IR cells of the Exp. 10‰ SW group. Furthermore, a higher abundance of NKCC1 protein was found in the gill homogenates of the euryhaline medaka, O. dancena, than in that of the stenohaline medaka, O. woworae.

Towards Acid-Tolerated Ethanol Dehydration: Chitosan-Based Mixed Matrix Membranes Containing Cyano-Bridged Coordination Polymer Nanoparticles.

Prussian blue (PB) nanoparticles, one of many cyano-bridged coordination polymers, are successfully incorporated into chitosan (CS) polymer to prepare PB/CS mixed matrix membranes (MMMs). The PB nanoparticles are uniformly distributed in the MMMs without the collapse of the original PB structure. As-prepared PB/CS MMMs are used for ethanol dehydration at 25 °C in the pervaporation process. The effect of loading PB in CS matrix on pervaporation performance is carefully investigated. The PB/CS membrane with 30 wt% PB loading shows the best performance with a permeate flux of 614 g. m-2 . h-1 and a separation factor of 1472. The pervaporation using our PB/CS membranes exhibits outstanding performance in comparison with the previously reported CS-based membranes and MMMs. Furthermore, the addition of PB allows PB/CS MMMs to be tolerant of acidic environment. The present work demonstrates good pervaporation performance of PB/CS MMMs for the separation of an ethanol/water (90:10 in wt%) solution. Our new system provides an opportunity for dehydration of bioethanol in the future.

FXYD11 mediated modulation of Na(+)/K(+)-ATPase activity in gills of the brackish medaka (Oryzias dancena) when transferred to hypoosmotic or hyperosmotic environments.

FXYD proteins regulate Na(+)/K(+)-ATPase (NKA), which is a primary active pump that provides the driving force that triggers osmoregulatory systems in teleosts. To explore the regulatory mechanisms between FXYD and NKA in euryhaline teleosts, the expression of NKA (mRNA, protein, and activity) and FXYD11 and their interaction were examined in the gills of brackish medaka (Oryzias dancena) when transferred from brackish water (BW; 15‰) to fresh water (FW) or seawater (SW; 35‰). The mRNA expression of Odfxyd11 and Odnka-α was elevated 48h post-hypoosmotic transfer. Moreover, FXYD11 protein and NKA activity were upregulated 12h after transfer to FW. When transferred to SW, the protein abundance of FXYD11 and the NKA α-subunit did not show apparent changes, while Odfxyd11 and Odnka-α mRNA expression and NKA activity increased significantly 12h and 1h post-transfer, respectively. To clarify the FXYD11 mechanisms involved in modulating NKA activity via their interaction, co-immunoprecipitation was further applied to O. dancena gills. The results revealed that the levels of protein-protein interaction between branchial NKA and FXYD11 increased acutely 12h after the transfer from BW to FW. However, immediate upregulation of NKA activity 1h following post-exposure to SW, without the elevation of protein-protein interaction levels, was found. Hence, branchial NKA activity of O. dancena was suggested to be rapidly regulated by FXYD11 interaction with NKA when acclimated to hypoosmotic environments. To the best of our knowledge, this is the first study that focuses on the efficacy of interactions between FXYD11 and NKA in the gills of euryhaline teleosts.

[Research on M Dwarf Sub-Classification Based on the Measurement of Residual Distribution].

The classification of stellar spectra is an important job in data processing of astronomy, which is mainly used for searching celestial spectra with known types in massive data survey. This paper focuses on LAMOST M dwarfs fine classification based on measurement of residual distribution. Residual distribution measurement is a measurement method used to measure the distance between two spectra. In the process of calculating the distance between two spectra, normalized processing should come first. Then the residuals of the sampling points of corresponding wavelength are calculated. Eventually the standard deviation of the residual distribution as the distance between the spectra is calculated. In this paper, the M star of LAMOST DR2 is used as the experimental data of classification. The experimental results show that the spectra data can be classified more accurately with the measurement method of residual distribution than the use of other traditional classification methods. The effect of spectral classification is affected by signal to noise ratio, outliers, residual standardized coefficient and other factors.

The inner opercular membrane of the euryhaline teleost: a useful surrogate model for comparisons of different characteristics of ionocytes between seawater- and freshwater-acclimated medaka.

The inner opercular membranes of the brackish medaka, Oryzias dancena, have numerous ionocytes, similar to the gill epithelia. By histological observation, this study demonstrated that it is possible to investigate the cellular morphology and function of ionocytes in the opercular membrane. The mitochondria-rich ionocytes in the opercular membranes were traced using rhodamine 123 and a cytochrome c oxidase IV antibody in vital and fixed situations, respectively. To validate different morphologies of seawater (SW)-type and freshwater (FW)-type ionocytes of the opercular membrane of euryhaline brackish medaka, a method of dual observation including immunofluorescence staining and subsequent scanning electron microscopy was used. The apical morphologies of SW- and FW-type ionocytes were hole and flat opening, respectively. In addition, the microvilli were found on the apical surface of the FW-type ionocytes. The SW-type ionocytes exhibited basolateral Na(+), K(+), 2Cl(-) cotransporter and the apical cystic fibrosis transmembrane conductance regulator. In contrast, in the apical region of FW-type ionocytes, Na(+), Cl(-) cotransporter and villin 1-like protein were expressed. In addition, histochemical staining of AgCl precipitation counterstained with a Na(+), K(+)-ATPase α-subunit antibody on the opercular membrane illustrated the role of Cl(-) secretion in the SW-type ionocytes of the brackish medaka. A combination of different observations in this study indicated that the opercular membrane could be a useful surrogate model for histological and functional studies on the epithelial ionocytes of fish gills.

Establishment of a mouse model to express bovine CD14 short hairpin RNA.

BACKGROUND: Cluster of differentiation 14 (CD14) functions as a co-receptor for Toll-like receptor (TLR)-4 and myeloid differentiation factor (MD)-2 in detecting bacterial lipopolysaccharide. Together, these complexes promote the phagocytosis and digestion of Gram-negative bacteria, and initiate immune responses. To date, much of our understanding of CD14 function during Gram-negative bacterial inflammation comes from studies on mouse knockout models and cell transfection. To identify the effect of CD14 knockdown in this process in large livestock animals, we established a mouse model expressing bovine CD14 short hairpin (sh) RNA. shRNA fragments targeting bovine CD14 were screened by co-transfection in HEK 293 cells, and the most effective CD14 shRNA fragment was cloned into the eukaryotic expression vector pSilencer4.1-CD14 shRNA-IRES (internal ribosome entry site) and transferred into mouse zygotes by pronuclear microinjection to obtain transgenic mice. Expression of the enhanced green fluorescent protein (EGFP) reporter and genes related to the TLR4 signaling pathway was detected by immunohistochemistry (IHC) and quantitative polymerase chain reaction (PCR), respectively. RESULTS: One effective shRNA fragment (shRNA-674) targeting bovine CD14 was obtained, the sequence of which was shown to be conserved between cows, buffalos, sheep, and humans. Thirty-seven founder pups were obtained by pronuclear microinjection, of which three were positive for the transgene. In the F(1) generation, 11 of 33 mice (33%) were positive for the transgene as detected by PCR. IHC analysis detected exogenous EGFP expression in the liver, kidney, and spleen of transgenic F(1) mice, indicating that they were chimeric. The expression of endogenous CD14 mRNA in the heart, liver, spleen, lung, and kidney of transgenic F(1) mice was decreased 8-, 3-, 19.5-, 6-, and 11-fold, respectively. The expression patterns of endogenous MD-2, TLR4, interleukin-6 and tumor necrosis factor-α genes in transgenic mice also varied. CONCLUSIONS: This study confirms that transgenic mice expressing bovine CD14 shRNA can be generated by pronuclear microinjection, and demonstrates inhibited endogenous mouse CD14 expression that alters gene expression related to the TLR4 signaling pathway.

Fast quantitative analysis of four tyrosine kinase inhibitors in different human plasma samples using three-way calibration-assisted liquid chromatography with diode array detection.

A simple method has been developed by combining high-performance liquid chromatography with diode array detection with the alternating trilinear decomposition method for simultaneous determination of four tyrosine kinase inhibitors in different human plasma samples. Chromatographic separation of the analytes was performed on a reversed-phase column with methanol (65%, v/v, A) and 0.1% aqueous solution of formic acid (35%, v/v, B). Analysis time was 5.0 min per run and analytes could be completely eluted within 2.8--3.8 min. The calibration concentration ranges of vandetanib, pazopanib, afatinib and dasatinib were designed as 0.50-6.10, 0.50-6.10, 0.70-7.00 and 0.70-7.00 µg·mL(-1), respectively. The intra- and inter-day RSDs ranged between 0.1 and 8.9%. Quantitative information could be extracted from the unsegregated interferences of different human plasma samples with the aid of the "second-order advantage" of three-way (second-order) calibration methods. All results demonstrated that the proposed method for direct quantitative analysis of four tyrosine kinase inhibitors in different complex systems possessed good characteristics of rapidity, sensitivity and efficiency, and it is expected to be an attractive choice in the fast analysis of clinical samples.

Cloning and expression of a novel prolyl endopeptidase from Aspergillus oryzae and its application in beer stabilization.

A novel prolyl endopeptidase gene from Aspergillus oryzae was cloned and expressed in Pichia pastoris. Amino acid sequence analysis of the prolyl endopeptidase from Aspergillus oryzae (AO-PEP) showed that this enzyme belongs to a class serine peptide S28 family. Expression, purification and characterization of AO-PEP were analyzed. The optimum pH and temperature were pH 5.0 and 40 °C, respectively. The enzyme was activated and stabilized by metal ion Ca(2+) and inhibited by Zn(2+), Mn(2+), Al(3+), and Cu(2+). The K m and k cat values of the purified enzyme for different substrates were evaluated. The results implied that the recombinant AO-PEP possessed higher affinity for the larger substrate. A fed-batch strategy was developed for the high-cell-density fermentation and the enzyme activity reached 1,130 U/l after cultivation in 7 l fermentor. After addition of AO-PEP during the fermentation phase of beer brewing, demonstrated the potential application of AO-PEP in the non-biological stability of beer, which favor further industrial development of this new enzyme in beer stabilization, due to its reducing operational costs, as well as no beer losses unlike regeneration process and beer lost with regenerated polyvinylpolypyrrolidone system.