Evaluation of the safety, immunogenicity and protective effect of an attenuated Pseudomonas plecoglossicida strain ΔgacS as the live vaccine for the large yellow croaker (Larimichthys crocea).

Pseudomonas plecoglossicida is one of most important pathogenic bacterial species in large yellow croaker and several other commercially valuable fish species. In our previous study, a GacS deficient mutant (ΔgacS) was constructed and its virulence showed substantially attenuated. In present study, the safety, immunogenicity and protective effect of the ΔgacS were evaluated in large yellow croaker as a live-attenuated vaccine candidate. It was shown that the ΔgacS strain exhibited good safety to large yellow croaker and there was no mortality or clinical symptoms observed in all fish that infected by ΔgacS strain with the doses range from 2 × 105～107 CFU per fish via intraperitoneal injection (IP) or immersion (IM), and almost all bacteria were cleaned up in the spleen of the fish at 14-day post infection. Specific antibodies could be detected at 7-day and 14-day post infection by direct agglutination method, and the valences of antibodies and bactericidal activities of the serum were significant increased with vaccination doses and vaccination time. Moreover, the expressions of some molecules and cytokines involved in specific immune responses were detected in the ΔgacS strain immunization group and control group. After challenged by the wild-type (WT) strain XSDHY-P, the relative percentage survival (RPS) showed highly correlated with the immunized dosage regardless of vaccination methods. It showed that the RPS of the IP groups were 39.47 %, 57.89 %, 71.05 % with the immune dosage in a descending order, respectively, and the RPS of the IM groups were 26.31 %, 36.84 %, 76.31 % with the immune dosage in a descending order, respectively. In summary, the ΔgacS strain exhibited safety and good protective effect to large yellow croaker and was a potential live vaccine candidate.

Swelling of Multilayered Calcium Alginate Microspheres for Drug-Loaded Dressing Induced Rapid Lidocaine Release for Better Pain Control.

The development of effective drug-loaded dressings has been considered a hot research topic for biomedical therapeutics, including the use of botanical compounds. For wound healing, adequate dressings can provide a good microenvironment for drug release, such as lidocaine. Biological macromolecular materials such as alginate show excellent properties in wound management. This study involves the preparation and evaluation of biocompatible multilayered-structure microspheres composed of chitosan, porous gelatin, and calcium alginate microspheres. The multilayered structure microspheres were named chitosan@ porous gelatin@ calcium alginate microspheres (CPAMs) and the drugs were rapidly released by the volume expansion of the calcium alginate microspheres. The in vitro release curve revealed that the peak release of lidocaine from CPAMs was reached within 18[Formula: see text]min. After 21[Formula: see text]min, the remaining lidocaine was then slowly released, and the active drug release was converted to a passive drug release phase. The initial release effect of lidocaine was much better than that reported in the published studies. Additionally, blood coagulation experiments showed that CPAMs coagulated blood in 60[Formula: see text]s, and the blood liquidity of the CPAMs group was worse than that of the woundplast group. Therefore, the coagulation characteristics of CPAMs were superior to the commonly used woundplast containing lidocaine healing gel. These study outcomes indicated that the CPAMs acted as fast-release dressings for faster pain control and better coagulation properties.

Morphological Characterization and Molecular Phylogenetic Analysis of Kudoa iwatai from Large Yellow Croaker (Larimichthys crocea) as a New Host in China.

Kudoa (Myxosporea: Multivalvulida) parasites are critical pathogens in marine and freshwater fish associated with significant economic losses and reduced market prices caused by post-mortem myoliquefaction or numerous cysts on muscles. In the present study, large yellow croakers infected by Kudoa were found during fish disease surveillance in China in November 2020 and used for morphological observation and characterization using light DIC microscopy and electron microscopy. Numerous creamy-white oval plasmodia were observed in muscles and on the surface of brain cartilage, gill arches, and serosal surfaces. The spores were considerably longer and thicker than previously reported Kudoa, with protruding polar filaments (PFs) in the mature spores, fingertip-shaped apical projections (APs), and polar capsules. Phylogenetic analyses with SSU rDNA, LSU rDNA, and mitochondrial DNA showed that the Kudoa-infected sample (LcK-2020) had the highest similarity to Kudoa iwatai reported in Japan. Based on the morphological characterization and phylogenetic analysis, it could be concluded that the sample LcK-2020 was infected by Kudoa iwatai, which would be the first report of Kudoa iwatai infection in large yellow croaker in China.

An edible molecularly imprinted material prepared by a new environmentally friendly deep eutectic solvent for removing oxalic acid from vegetables and human blood.

A novel deep eutectic solvent-magnetic molecularly imprinted polymer (DES-MMIP) for the specific removal of oxalic acid (OA) was prepared by an environmentally friendly deep eutectic solvent, consisting of betaine, citric acid, and glycerol, which acted as the functional monomer for polymerization. The structure and morphology of DES-MMIPs were studied by X-ray diffraction, scanning and transmission electron microscopy, thermal gravimetric analysis, Fourier transform infrared spectroscopy, and vibrating sample magnetometer. DES-MMIPs had a core-shell structure, with magnetic iron oxide as the core, and showed good thermal stability and high adsorption capacity (18.73 mg/g) for OA. The adsorption process of OA by DES-MMIPs followed the pseudo-second-order kinetic model and Langmuir isotherm model. DES-MMIPs had significant selectivity for OA and their imprinting factor was 3.26. When applied to real samples, high performance liquid chromatography analysis showed that DES-MMIPs could remove OA from both spinach and blood serum. These findings provide potential methods for removal of OA from vegetables and for specific removal of OA in renal dialysis.

Synergistic recognition of transferrin by using performance dual epitope imprinted polymers.

Transferrin (Trf) is a new type of active drug targeting carrier and disease biomarker that regulates the balance of iron ions in human body. The recognition and isolation of Trf is of great significance for disease diagnosis and treatment. Thus, a new type of magnetic dual affinity epitope molecularly imprinted polymer coated on Fe3O4 nanoparticles (Fe3O4@DEMIP) was successfully prepared for specific recognition of Trf. C-terminal nonapeptide and Trf glycan were selected as bi-epitope templates for metal chelation and boron affinity immobilization, respectively. 4-vinylphenylboric acid (4-VP), N-isopropyl acrylamide (NIPAM) and zinc acrylic were used as functional monomers. Results showed that Fe3O4@DEMIP exhibited excellent specific recognition ability adsorption capacity toward Trf, with an adsorption of 43.96 mg g-1 (RSD = 3.28%) and a more satisfactory imprinting factor (about 6.60) than that of other reported imprinting methods. In addition, Fe3O4@DEMIP displayed pH, temperature and magnetic sensitivity properties to realize temperature and pH-controlled recognition and release of target proteins and magnetic rapid separation. Furthermore, the Fe3O4@DEMIP coupled with high-performance liquid chromatography (HPLC) analysis was successfully used for specific recognition of Trf in biosamples. This study provides a reliable protocol for preparing metal chelation and boron affinity dual affinity bi-epitope molecularly imprinted polymers for synergistic and efficient recognition of biomacromolecules in the complex biological systems.

Specific adsorption and determination of aspartame in soft drinks with a zein magnetic molecularly imprinted modified MGCE sensor.

In this work, an efficient and sensitive magnetic molecularly imprinted polymer with zein and deep eutectic solvents (ZDM-MIPs) was designed and synthesized to exclusively adsorb and detect aspartame (ASP). We used zein, together with deep eutectic solvents (DESs) and Fe3O4 as the cross-linker, functional monomer and support material, respectively. A magnetic glassy carbon electrode (MGCE) modified with ZDM-MIPs was used for selective recognition of ASP. The electrochemical response of the ZDM-MIPs-MGCE for quantification of ASP was evaluated with a portable electrochemical detection station with differential pulse voltammetry and cyclic voltammetry. The responses of ZDM-MIPs-MGCE signified a good linear relationship with ASP concentrations in the range of 0.1-50 µg mL-1. The sensor systems showed good accuracy and precision, with recovery percentages between 84% and 107%. These results suggested that the obtained ZDM-MIPs exhibited good adsorption performance for ASP in soft drinks, and this method could be used to determine ASP content in actual food samples.

Selective separation and inexpensive purification of paclitaxel based on molecularly imprinted polymers modified with ternary deep eutectic solvents.

Paclitaxel (PTX) is a powerful anticancer natural product, with its separation and purification having been widely studied. In this work, new molecular imprinted polymers (MIPs) using deep eutectic solvents (DESs) with different molar ratios were prepared as functional monomers. These were then used as adsorbents in solid phase extraction (SPE) for the separation of PTX from its structural analogs. The polymers were characterized by energy disperive X-rays (EDX), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and fourier transform infrared spectroscopy (FT-IR). The results suggested that the formative regular DES-MIPs had an even pore-size distribution and a large specific surface area. The dynamic adsorption and static adsorption showed that the DES-MIPs had excellent adsorption performance, with a maximum adsorption capacity and optimum adsorption time of 87.08 mg/g and 180 min, respectively. The selective adsorption experiments showed that the material had outstanding selectivity, and the maximum selectivity factor was 6.20. For stability, after six consecutive adsorption and desorption cycles, the DES-MIPs maintained the perfect stability and reusability. Furthermore, the fabricated SPE column was successfully utilized for extracting and eluting PTX. This study provides a reliable protocol for the separation and purification PTX from its structural analogs and the DES-MIPs materials have excellent potential application value in pharmaceutical industry.

Specific adsorption of tetracycline from milk by using biocompatible magnetic molecular imprinting material and evaluation by ECD.

Biocompatible magnetic molecularly imprinted polymers (BMMIPs) were prepared with Zein for the first time, and were used to enrich tetracycline compounds selectively. Innovative combination of BMMIPs and electrochemistry to obtain lower detection line to satisfy industrial detection demands. Using Zein as the crosslinking agent, the polymers were synthesized on the surface of Fe3O4 particles. The scanning electron microscope, transmission electron microscope and X-ray diffraction technologies were used to characterize BMMIPs. Through optimization, BMMIPs attained large adsorption capacity (236.40 mg/g) with fast kinetics (40 min) and followed the Langmuir isotherm and pseudo-second-order kinetic models. BMMIPs had good recognition ability, the selective factors of oxytetracycline, chlortetracycline, doxycycline were 4.78, 4.23, and 3.39, respectively. Excellent linearity was attained in the range of 0.025-500 µg/mL, with low detection limits and low quantitation limits of 0.025 and 0.083 µg/mL. According to our exploring, BMMIPs was ideal materials for enrichment of tetracycline in complex biological samples.

Development and validation of a more accurate estimating equation for glomerular filtration rate in a Chinese population.

Previously published equations to estimate glomerular filtration rate (GFR) have limited accuracy in Asian populations. We aimed to develop and validate a more accurate equation for estimated GFR (eGFR) in the Chinese population, using data from 8571 adults who were referred for direct measurement of GFR by renal dynamic imaging (mGFR) at 3 representative hospitals in China. Patients from the Third Xiangya Hospital were included in our development (n=1730) and internal validation sets (n=1042) and patients from the other hospitals comprised the external validation set (n=5799). We excluded patients who were prescribed medications known to influence the tubular secretion of creatinine, patients on dialysis, kidney transplant recipients, and patients with missing creatinine values or with creatinine >700 µmol/l. We derived a novel eGFR equation by linear regression analysis and compared the performance to 12 creatinine-based eGFR equations, including previously published equations for use in Chinese or Asian populations. In the development and internal validation sets, the novel Xiangya equation had high accuracy (accuracy within 30% [P30], 79.21% and 84.33%, respectively), low bias (mean difference between mGFR and eGFR, -1.97 and -1.85 ml/min per 1.73 m2, respectively), and high precision (interquartile range of the differences, 21.13 and 18.88 ml/min per 1.73 m2, respectively). In external validation, the Xiangya equation had the highest P30 among all eGFR equations, with P30 ≤ 75% for the other 12 equations. This novel equation provides more accurate GFR estimates in Chinese adults and could replace existing eGFR equations for use in the Chinese population.

Identification of Carassius auratus gibelio liver cell proteins interacting with the GABAA receptor γ2 subunit using a yeast two-hybrid system.

The γ-aminobutyric acid type A (GABAA) receptor is an important pentameric inhibitory neurotransmitter receptor, and the γ2 subunit of this receptor plays a key role in potentiation of the GABAA response. We previously detected that the expression of GABAA receptor in the livers of Carassius auratus gibelio significantly increased after medication (avermectin and difloxacin treatment). In order to better understand the mechanism of action of the GABAA receptor γ2 subunit in the livers of C. auratus gibelio, we constructed a C. auratus gibelio liver cDNA library (the titer value of 1.2 × 106 cfu/mL) and identified the proteins that interact with the GABAA receptor γ2 subunit by using a yeast two-hybrid assay. The yeast two-hybrid screening yielded seven positive clones, namely, prelid3b, cdc42, sgk1, spg21, proteasome, chia.5, and AP-3 complex subunit beta-1, all of which have been annotated by the NCBI database. The functions of these proteins are complex; therefore, additional studies are required to determine the specific interactions of these proteins with the GABAA receptor γ2 subunit in the liver of C. auratus gibelio. Although the interactions identified by the yeast two-hybrid system should be considered as preliminary results, the findings of this study may provide further direction and a foundation for future research focusing on the mechanisms of the GABAA receptor γ2 subunit in C. auratus gibelio livers.

Evaluating tacrolimus pharmacokinetic models in adult renal transplant recipients with different CYP3A5 genotypes.

PURPOSE: Numerous studies have been conducted on the population pharmacokinetics of tacrolimus in adult renal transplant recipients. It has been reported that the cytochrome P450 (CYP) 3A5 genotype is an important cause of variability in tacrolimus pharmacokinetics. However, the predictive performance of population pharmacokinetic (PK) models of tacrolimus should be evaluated prior to their implementation in clinical practice. The aim of the study reported here was to test the predictive performance of these published PK models of tacrolimus. METHODS: A literature search of the PubMed and Web of Science databases ultimately led to the inclusion of eight one-compartment models in our analysis. We collected a total of 1715 trough concentrations from 174 patients. Predictive performance was assessed based on visual and numerical comparison bias and imprecision and by the use of simulation-based diagnostics and Bayesian forecasting. RESULTS: Of the eight one-compartment models assessed, seven showed better predictive performance in CYP3A5 extensive metabolizers in terms of bias and imprecision. Results of the simulation-based diagnostics also supported the findings. The model based on a Chinese population in 2013 (model 3) showed the best and most stable predictive performance in all the tests and was more informative in CYP3A5 extensive metabolizers. As expected, Bayesian forecasting improved model predictability. Diversity among models and between different CYP3A5 genotypes of the same model was also narrowed by Bayesian forecasting. CONCLUSIONS: Based on our results, we recommend using model 3 in CYP3A5 extensive metabolizers in clinical practice. All models had a poor predictive performance in CYP3A5 poor metabolizers, and they should be used with caution in this patient population. However, Bayesian forecasting improved the predictability and reduced differences, and thus the models could be applied in this latter patient population for the design of maintenance dose.

Utility of cystatin C-based equations in patients undergoing dialysis.

Serum cystatin C (CysC) measurement is regarded as a simple and practical alternative to measure residual renal function for dialysis patients. Recent studies have shown that CysC has better diagnostic accuracy or at least equivalence to creatinine in predicting the early stages of renal damage, and is closely related to clinical outcomes of dialysis patients. Thus, the applicability of CysC-derived equations in patients undergoing dialysis should be paid attention. Here, we review the role of CysC in diagnosis, renal function evaluation, and prognosis outcomes for dialysis patients, so as to provide them with useful suggestions on evaluating renal function and predicting adverse outcomes in clinical practice.

[Sagittal otolith morphology and the relationship between its mass and the age of Liza haematocheila in the Yangtze Estuary, China]. / é•¿æ±Ÿå£é®»çŸ¢è€³çŸ³å½¢æ€ç‰¹å¾åŠè´¨é‡ä¸Žå¹´é¾„çš„å ³ç³».

To examine the relationship of morphological characters of sagittal otolith and the age of Liza haematocheila in the Yangtze Estuary, we analyzed the morphological parameters of 324 pairs of otoliths extracted from 358 L. haematocheila specimens from the Yangtze Estuary in February to June of 2017. The results showed that sagittal otolith had rostrum, antirostrum and obvious central notch. The size and shape of sagittal otolith significantly changed with their growth, from regular melon seeds shape outline to long narrow leaf shape and increasing irregular wavy outline. The average density of sagittal otolith was 1.52 mg·mm-2. The average rectangularity was 0.68. The length of sagittal otolith was 0.021%-0.047% of entire body length (BL), the width was 0.009%-0.021% of entire BL, and the mass was 0.045‰-0.731‰ of the entire body mass (BM). Otolith length (OL), otolith width (OW) and otolith mass (OM) were all significantly related to the BL, with the determination coefficient for OW and OM model being the highest (R2=0.928). The relationship between OM and BL was described best by exponential regression: OM=0.0009BL1.8737(R2=0.967). The relationships between OM and age (A), BL and A were well fitted by multinomial regressions, respectively: OM=2.9262A2+4.8437A+2.1894 (R2=0.847), BL=-3.2248A2+102.54A+38.373 (R2=0.858). In addition, OM was linearly correlated with A. The estimated otolith's ages from the model did not significantly variate from the real ages counting from annulus counts. Therefore, OM could be an effective parameter for the age estimation of L. haematocheila.

A Simple Clinical Pre-procedure Risk Model for Predicting Thrombocytopenia Associated With Periprocedural Use of Tirofiban in Patients Undergoing Percutaneous Coronary Intervention.

Background: No risk model for predicting thrombocytopenia associated with periprocedural tirofiban exposure is available. The purpose of this study was to develop a simple clinical pre-procedure risk model based on pre-procedural characteristics for early prediction of thrombocytopenia before patients were exposed to tirofiban. Methods: The series included 1862 patients who underwent percutaneous coronary intervention with tirofiban exposure. Baseline demographic and clinical characteristics were collected from the hospital information system on admission. The earliest pro-procedural platelets within 72 h were used to evaluate the thrombocytopenia incidence. Risk factors associated with thrombocytopenia in patients with tirofiban exposure were investigated by univariable and multivariable analyses. Locally weighted scatterplot smoothing procedure was used to identify the cut points for the numeric variables. The discriminatory power of the scoring system was assessed with the receiver operating characteristic (ROC) curve analysis. Results: The occurrence of thrombocytopenia was 4.02% (75 of 1862), 4.01% (56 of 1396), and 4.08% (19 of 466) in the overall, developmental, and validation data sets, respectively. The risk score was developed based on five independent predictors: age ≥65y, white blood cell ≥12 × 109/L, diabetes mellitus, congestive heart failure, and chronic kidney disease. This tool was well calibrated (Hosmer Lemeshow χ2 = 6.914; P = 0.546) and good discrimination was well obtained in validation data set (C-statistic, 0.82). Conclusion: The clinical pre-procedure risk model is a simple and accurate tool for early identification of high-risk patients of thrombocytopenia before tirofiban exposure, allowing for timely and appropriate intervention.

Population Pharmacokinetic Modeling of Diltiazem in Chinese Renal Transplant Recipients.

BACKGROUND AND OBJECTIVES: Diltiazem is a benzothiazepine calcium blocker and widely used in renal transplant patients since it improves the level of tacrolimus or cyclosporine A concentration. Several population pharmacokinetic (PopPK) models had been established for cyclosporine A and tacrolimus but no specific PopPK model was established for diltiazem. The aim of the study is to develop a PopPK model for diltiazem in renal transplant recipients and provide relevant pharmacokinetic parameters of diltiazem for further pharmacokinetic interaction study. METHODS: Patients received tacrolimus as primary immunosuppressant agent after renal transplant and started administration of diltiazem 90 mg twice daily on 5th day. The concentration of diltiazem at 0, 0.5, 1, 2, 8, and 12 h was measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Genotyping for CYP3A4*1G, CYP3A5*3, and MDR1 3435 was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). 25 covariates were considered in the stepwise covariate model (SCM) building procedure. RESULTS: One-compartment structural pharmacokinetic model with first-order absorption and elimination was used to describe the pharmacokinetic characteristics of diltiazem. Total bilirubin (TBIL) influenced apparent volume of distribution (V/F) of diltiazem in the forward selection. The absorption rate constant (K a), V/F, and apparent oral clearance (CL/F) of the final population pharmacokinetic (PopPK) model of diltiazem were 1.96/h, 3550 L, and 92.4 L/h, respectively. CONCLUSION: A PopPK model of diltiazem is established in Chinese renal transplant recipients and it will provide relevant pharmacokinetic parameters of diltiazem for further pharmacokinetic interaction study.

Analysis of Saprolegnia parasitica Transcriptome following Treatment with Copper Sulfate.

BACKGROUND: Massive infection caused by oomycete fungus Saprolegnia parasitica is detrimental to freshwater fish. Recently, we showed that copper sulfate demonstrated good efficacy for controlling S. parasitica infection in grass carp. In this study, we investigated the mechanism of inhibition of S. parasitica growth by copper sulfate by analyzing the transcriptome of copper sulfate-treated S. parasitica. To examine the mechanism of copper sulfate inhibiting S. parasitica, we utilized RNA-seq technology to compare differential gene expression in S. parasitica treated with or without copper sulfate. RESULTS: The total mapped rates of the reads with the reference genome were 90.50% in the control group and 73.50% in the experimental group. In the control group, annotated splice junctions, partial novel splice junctions and complete novel splice junctions were about 83%, 3% and 14%, respectively. In the treatment group, the corresponding values were about 75%, 6% and 19%. Following copper sulfate treatment, a total 310 genes were markedly upregulated and 556 genes were markedly downregulated in S. parasitica. Material metabolism related GO terms including cofactor binding (33 genes), 1,3-beta-D-glucan synthase complex (4 genes), carboxylic acid metabolic process (40 genes) were the most significantly enriched. KEGG pathway analysis also determined that the metabolism-related biological pathways were significantly enriched, including the metabolic pathways (98 genes), biosynthesis of secondary metabolites pathways (42 genes), fatty acid metabolism (13 genes), phenylalanine metabolism (7 genes), starch and sucrose metabolism pathway (12 genes). The qRT-PCR results were largely consistent with the RNA-Seq results. CONCLUSION: Our results indicate that copper sulfate inhibits S. parasitica growth by affecting multiple biological functions, including protein synthesis, energy biogenesis, and metabolism.

Determination of calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine by HPLC.

A reversed-phase high-performance liquid chromatographic (HPLC) assay for calycosin-7-O-beta-D-glucopyranoside in rat plasma and urine with solid-phase extraction (SPE) was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) at a flow rate of 1.0 mL/min. Detection was set at 280 nm. The limit of quantitation of calycosin-7-O-beta-D-glucopyranoside was 0.2 microg/mL in both plasma and urine. The standard curve was linear from 0.2 to 10.0 microg/mL in plasma, and 0.2 to 5.0 microg/mL in urine. Both intra- and inter-day precision of the calycosin-7-O-beta-d-glucopyranoside were determined and their RSD did not exceed 10%. The method was successfully applied to the analysis of samples obtained from a basic pharmacokinetic study, in which calycosin-7-O-beta-d-glucopyranoside was administered orally to rats.

High-performance liquid chromatographic method for the determination of mangiferin in rat plasma and urine.

A reversed-phase high-performance liquid chromatography assay for mangiferin in rat plasma and urine was developed. Rutin was employed as an internal standard. The mobile phase consisted of acetonitrile-water (16:84, v/v) containing 3% acetic acid at a flow rate of 1 mL/min. Detection was at 257 and 365 nm for mangiferin in plasma and urine, respectively. The limit of quantitation (LOQ) of mangiferin was 0.6 microg/mL in plasma, and 0.48 microg/mL in urine. The standard curve was linear from 0.6 to 24 microg/mL in plasma, and 0.48 to 24 microg/mL in urine, both intra- and inter-day precision of the mangiferin were determined and their RSD did not exceed 10%. The method provides a technique for rapid analysis of mangiferin in rat plasma and urine, which can be used in pharmacokinetic studies.