Functional characterization of NOD1 from golden pompano Trachinotus ovatus.

Fish rely on innate immune system for immunity, and nucleotide-binding oligomerization domain-like receptors (NLRs) are a vital group of receptor for recognition. In the present study, NOD1 gene was cloned and characterized from golden pompano Trachinotus ovatus, a commercially important aquaculture fish species. The ORF of T. ovatus NOD1 was 2820 bp long, encoding 939 amino acid residues with a highly conserved domains containing CARD-NACHT-LRRs. Phylogenetic analysis revealed that the T. ovatus NOD1 clustered with those of fish and separated from those of birds and mammals. T. ovatus NOD1 has wide tissue distribution with the highest expression in gills. Bacterial challenges (Streptococcus agalactiae and Vibrio alginolyticus) significantly up-regulated the expression of NOD1 with different response time. The results of T. ovatus NOD1 ligand recognition and signaling pathway analysis revealed that T. ovatus NOD1 could recognize iE-DAP at the concentration of ≧ 100 ng/mL and able to activate NF-κB signaling pathway. This study confirmed that NOD1 play a crucial role in the innate immunity of T. ovatus. The findings of this study improve our understanding on the immune function of NOD1 in teleost, especially T. ovatus.

Effect of frozen storage conditions on antithrombin protein C and protein S activity assay stability.

BACKGROUND: Inherited antithrombin, protein C, and protein S deficiency increase the risk of venous thromboembolism. The presence of defects can be identified by clinical laboratory assays. In most Chinese clinical laboratories, the screening tests for antithrombin, protein C, and protein S deficiency are their activity assays. Ensuring appropriate pre-analytical storage conditions for activity tests is essential. This study aimed to assess the effects of storage conditions on antithrombin, protein C, and protein S activity in frozen plasma. METHODS: We collected the remaining plasma of 29 patients. The baseline of antithrombin, protein C, and protein S activity values were tested within 4 h. Then, each sample was sub-packaged into 4 EP tubes, and was stored at -20 °C for 3 days, -20 °C for 7 days, -80 °C for 3 days, and - 80 °C for 7 days, respectively. After thawing, samples were tested by two systems. RESULTS: The percentage deviation of antithrombin and protein C activity assay was<10% compared with the initial values. Protein S activity showed a significant reduction in frozen plasma, with a deviation > 10%. Some samples, initially within the normal range, were classified as abnormal after freezing storage. CONCLUSIONS: Our study indicated that antithrombin and protein C remain stable when stored at -20 °C or -80 °C in a week. We argued that Protein S activity is not stable in frozen plasma. The use of frozen-thawed plasma for PS activity assay may result in overdiagnosis of protein S deficiency.

Effects of Sophora flavescens dietary supplementation on the growth performance, haematological indices, and intestinal bacterial community of Oreochromis niloticus.

Sophora flavescens is considered to be of various medicinal and economic importance. Previous studies have shown that it has antibacterial, antioxidant, and immune-enhancing functions. To explore the full potential of S. flavescens in fish feed development, we investigated the effects of different levels of S. flavescens added to the diet on the growth performance, haematological indices, and the intestinal bacterial community of Nile tilapia (Oreochromis niloticus). The feeding trial lasted for 56 days, and the initial weight of the fish was 40.00 ± 5.00 g. Nile tilapia were randomly divided into six groups: a control group (CN) and five S. flavescens supplementation groups (0.02%, 0.04%, 0.08%, 0.16%, and 0.3% [w/w] of S. flavescens in diet). The growth performance of fish increased first and then decreased with the increase of S. flavescens supplemental level. Compared with other experimental groups, the growth performance of fish supplemented with 0.08% S. flavescens was significantly improved (p < 0.05). Haematological indices exhibited that erythrocyte (RBC), along with leucocyte (WBC) indices, exhibited a secondary trend of increasing and then decreasing with the increase of S. flavescens, reaching the highest level at 0.08% (p < 0.05). However, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), and mean corpuscular haemoglobin concentration (MCHC) exhibited a secondary trend of first decreasing and then increasing with the increase of S. flavescens and reached the lowest value at 0.08% (p < 0.05). Compared with the CN, alkaline phosphatase (AKP) significantly decreased and changed when the additional amount of S. flavescens was 0.04% in each treatment group (p < 0.05). In another way, the gut microbial profiles revealed that Bacteroides dominated the gut communities, and compared with the control group, two uncultured bacteria were suppressed. In addition, when the supplemental level of S. flavescens was more significant than 0.04%, the proliferation of Arcobacter cryaerophilus, Solibacillus silvestris, and Escherichia sp. in the gut of Nile tilapia was promoted. The results revealed that S. flavescens, as an additive to the Nile tilapia diet with levels ranging from 0.04% to 0.08%, can enhance the growth performance and immunity and promote the proliferation of intestinal beneficial bacteria of Nile tilapia.

Molecular characterization of complement regulatory factor CD46 in Trachinotus ovatus and its role in the antimicrobial immune responses and complement regulation.

CD46, as a cofactor of complement I factor, not only regulates the complement system but also functions as a pathogen receptor and is involved in controlling early pathogen infection through autophagy. In this study, a new CD46 gene (ToCD46) was identified from golden pompano (Trachinotus ovatus), which showed higher sequence homology with other teleosts CD46. Homology comparison showed that ToCD46 had higher sequence homology (46.95-52.85%) with other teleosts CD46 and lower homology with mammal. Tissue expression profile analysis showed that ToCD46 was generally expressed in all tissues with the highest expression level in liver, followed by head kidney, and showed different patterns of up-regulation in immune-related tissues after stimulation by Streptococcus agalactiae and Vibrio alginolyticus. The hemolytic activity analysis and apoptosis assay showed that rToCD46 decreased the hemolytic activity of serum of golden pompano and effectively inhibited the damage of A549 cells, suggesting that ToCD46 might be involved in the regulation of complement activation of golden pompano. In vitro antibacterial experiments showed that rToCD46 had antibacterial activity against gram negative bacteria V. alginolyticus but no effect on positive bacteria S. agalactiae. These results suggest that ToCD46 may be involved in the immune response of golden pompano to pathogens, which will provide important basic information for elucidating the evolutionary history of the complement system of golden pompano.

Hemostatic alterations during extracorporeal membrane oxygenation in ovine veno-venous and veno-arterial models.

BACKGROUND: Extracorporeal membrane oxygenation (ECMO) has salvaged many people's life during global pandemics. However, ECMO is associated with a high incidence of hemostatic complications. This study aims to explore the effects of the ECMO system on the coagulation system in the healthy ovine ECMO model. METHODS: Ten healthy male sheep were included. Five received the veno-arterial ECMO and five received the veno-venous ECMO. Heparin was infused for systemic anticoagulation and was adjusted according to the activated clotting time. Blood routine tests, coagulation factors, anticoagulation proteins, and fibrinolysis markers were tested at the baseline and every 24 h. After weaning, the pump heads were dissected to explore thrombosis. RESULTS: Platelets decreased in the first 72 h and returned to the baseline at the 120th hour. The neutrophils increased in the first 24 h and returned to the baseline at the 48th hour. Factors II, VII, and X decreased in the first 24 h and gradually increased, while factors VIII, IX, XI, and XII decreased in the first 24 h and remained at a low level. The baseline antithrombin was 73.2 ± 14.4% and reduced to 42.6 ± 9.9% at the 168th hour. Pathology showed seven sheep developed thrombus, but no clinically relevant bleeding or thrombosis events occurred. CONCLUSIONS: The study explored hemostatic alterations during ECMO in healthy animal models, which eliminated the confounding under critically ill conditions. The study may provide insights into ECMO hemostatic disorders and aid the design of optimal therapeutic strategies.

Identification and functional characterization of complement regulatory protein CD59 in golden pompano (Trachinotus ovatus).

CD59, one of the essential inhibitors of the complement membrane attack complex (MAC), plays a crucial role in regulation of complement activation. In this study, we cloned and identified the CD59 gene (named ToCD59) of golden pompano (Trachinotus ovatus). The ORF sequence of ToCD59 is 357 bp long encoding 118 amino acids with a molecular weight of 13.09 kDa. Prediction of protein domains showed that ToCD59 contained an Lu domain and a C-terminal glycosylphosphatidylinositol (GPI) partial anchor. Homology comparisons indicated that ToCD59 shared the high sequence similarity with other fish CD59. RT-qPCR analysis showed that ToCD59 was expressed in all tested healthy tissues of golden pompano, with the highest level of expression in the brain. After stimulation with bacteria, ToCD59 expression levels were significantly up-regulated in head kidney, liver, gill and brain, but down-regulated in spleen. Subcellular localization results showed that ToCD59 localized to the cytoplasm of A549 cells. The hemolytic activity analysis showed that rToCD59 might have complement inhibitory activity through the alternative complement pathway. In addition, antibacterial test showed that rToCD59 had antibacterial ability against S. agalactiae and V. alginolyticus in vitro. These results suggest that ToCD59 might play an important role in the immune response against pathogens, which would provide basic information for elucidating the functional evolutionary history of complement system in teleost.

Adult Acute Myeloid Leukemia with the KMT2A-Mixed Lineage Leukemia T10 Fusion: An Analysis of 10 Cases Showed Common Features and Frequent Mutations in the RAS Signaling Pathway.

Mixed lineage leukemia (MLL) T10 is a relatively rare partner for the KMT2A lysine (K)-specific methyltransferase 2A gene. The common features and coexisting mutations of acute myeloid leukemia (AML) patients with KMT2A-MLLT10 remain unknown. In this study, 10 adult AML patients with KMT2A-MLLT10 fusions were picked up from 496 AML patients by using RT-polymerase chain reaction (PCR) and/or fluorescence in situ hybridization, and then screened for mutations in the 49 genes panel with next-generation sequencing and PCR, followed by direct Sanger sequencing. Of the 10 unique individuals identified, 6 were male and 4 were female (M:F ratio, 1.5:1) with ages ranging from 19 to 52 years (median 39.5 years). Most (90%, 9/10) patients with KMT2A-MLLT10 were accompanied by additional mutations. Twelve mutated genes were detected, averaging 2.1 mutations per patient (range, 0-4). The most frequently mutated gene was NRAS (n = 5). Clinical and laboratory data pointed to common features: French American British-M5 subtype (n = 7), a high rate of relapse, and biomarkers CD33 (n = 10), CD117 (n = 9), CD13 (n = 8), and CD64 (n = 8). Overall, most patients harbored at least one mutation. A high incidence of mutations affecting the RAS signaling pathway or RAS regulating components was found in 50% (5/10) patients. The overall survival is about 12.0 months. Allogeneic-hematopoietic stem cell transplantation trends to improve survival in selected patients.

A novel C-type lectin from Crassostrea gigas involved in the innate defense against Vibrio alginolyticus.

C-type lectins (CTLs) are important immune molecules that participate in invertebrate defense response. In the present work, a novel structural CTL (CgLec-4E) was identified from Crassostrea gigas, which encodes 237 amino acids (aa) with an extra long chain of aa and in the C-type CRD domain with EPA, QPG and WHD mutated motifs respectively. rCgLec-4E could agglutinate and inhibit the growth of Vibrio alginolyticus, except Chlorella, which might be relevant to three mutated motifs. CgLec-4E was mainly expressed in digestive gland, and its expression level was significantly up-regulated post V. alginolyticus challenge, indicating that the high expression of CgLec-4E could provide necessary mucosal immune protections and might involve in food particle recognition for C. gigas. Moreover, the subcellular locations indicated that CgLec-4E might play different roles in the immune response. Taken together, our results enrich our understanding of the structures and function of CTLs in invertebrates.

Human Acellular Amniotic Matrix with Previously Seeded Umbilical Cord Mesenchymal Stem Cells Restores Endometrial Function in a Rat Model of Injury.

BACKGROUND: Abnormal endometrial repair after injury results in the formation of intrauterine adhesions (IUA) and a thin endometrium, which are key causes for implantation failure and infertility. Stem cell transplantation offers a potential alternative for some cases of severe Asherman's syndrome that cannot be treated with surgery or hormonal therapy. Umbilical cord-derived mesenchymal stem cells (UCMSCs) have been reported to repair the damaged endometrium. However, there is no report on the effects of UCMSCs previously seeded on human acellular amniotic matrix (AAM) on endometrial injury. METHODS: Absolute ethanol was injected into rat uteri to damage the endometrium. UCMSCs previously seeded on AAM were surgically transplanted. Using a variety of methods, the treatment response was assessed by endometrial thickness, endometrial biomarker expression, endometrial receptivity, cell proliferation, and inflammatory factors. RESULTS: Endometrial thickness was markedly improved after UCMSC-AAM transplantation. The expression of endometrial biomarkers, namely, vimentin, cytokeratin, and integrin ß3, in treated rats increased compared with untreated rats. In the UCMSC-AAM group, the VEGF expression decreased, whereas that of MMP9 increased compared with the injury group. Moreover, in the AAM group, the MMP9 expression increased. The expression of proinflammatory factors (IL-2, TNFα, and IFN-γ) in the UCMSC-AAM group decreased compared with the untreated group, whereas the expression of anti-inflammatory factors (IL-4, IL-10) increased significantly. CONCLUSIONS: UCMSC transplantation using AAM as the carrier can be applied to treat endometrial injury in rats. The successful preparation of lyophilized AAM provides the possibility of secondary infectious disease screening and amniotic matrix quality detection, followed by retrospective analysis. The UCMSC-AAM complex may promote the better application of UCMSCs on the treatment of injured endometrium.

Preparation and anti-Raji lymphoma efficacy of a novel pH sensitive and magnetic targeting nanoparticles drug delivery system.

BACKGROUND: Non-Hodgkin's lymphoma (NHL) is a heterogeneous class of cancers that arises in lymph nodes or other lymphatic tissues, which causes many deaths worldwide and its incidence is increasing. METHODS: In this study, a pH-responsive DMSA-Fe3O4 magnetic nanoparticles (MNPs) covalently connect with ADM and As2O3 as a drug delivery system was invented to discuss the anticancer efficacy in non-Hodgkin's lymphoma (NHL) cell line--Raji. RESULTS: Detailedly, according to the chelation of ADM and Fe2+, the release rate of ADM was accelerated in acidic environment, and slowed down/blocked in neutral environment. The inhibitory effect to induce apoptosis of Fe3O4/As2O3+Doxil on Raji cells was obvious compared with that of single-drug group. Furthermore, the expression of Bcl-2 gene in Raji cells was suppressed under the action of MNPs. CONCLUSION: Taken together, the novel pH-responsive MNPs was proven to be a promising synergistic form of magnetic targeted drugs for clinical treatment of human Raji lymphoma.

Preparation, characterization, and pharmacodynamic study on deep second degree burns of total flavonoids composite phospholipids liposome gel of Oxytropis falcata Bunge.

Wound healing is the treatment problem after deep second degree (II°) burns. The p38 mitogen-activated protein kinase (p38 MAPK) and nuclear factor-κB/inhibitory factor-κB (NF-κB/IκB) signal pathways play significant role in angiogenesis and wound repair after burns.This study aimed to investigate the preparation, characterization and pharmacodynamics of the total flavonoids composite phospholipids liposome of Oxytropis falcata Bunge (TFOFB-CPL) on deep II° burns to research its biological activity and underlying mechanism. The TFOFB-CPL was prepared by thin-film dispersion method and the preparation process was optimized via central composite design. The TFOFB-CPL was then characterized by using particle size, polydispersity indexes (PDIs), zeta potential, encapsulation efficiency (EE) and morphology. Moerover, in vitro transdermal test and in vivo pharmacodynamic study included wound healing rate, hematoxylin-eosin (HE) staining, masson staning, western blotting and RT-PCR. The results showed that the therapeutic effects of TFOFB-CPL gel on deep II° burns, especially during wound healing were significant. TFOFB-CPL gel has a sustained-release effect during the treatment of deep II° burns with forming drug depot in the dermis layer. The wound healing rate of TFOFB-CPL gel group was near positive group and better than the other groups. TFOFB-CPL gel could promote the growth of epidermis, skin appendages, fibrovascular and collagen fibers, and had obvious anti-inflammatory effects. Moreover, TFOFB-CPL gel inhibited the activation of p38MAPK and the degradation of IκBα, and promoted the neonatal wounds during the early stage. Therefore, TFOFB-CPL gel could be considered as a novel preparation for treating deep II° burns.

[Analysis of ASXL1 gene variant in patients with myelodysplastic syndrome].

OBJECTIVE: To detect ASXL1 gene variants among patients with myelodysplastic syndrome (MDS) and explore their correlation with variants of other genes and clinical features of patients. METHODS: For 149 patients with MDS, genomic DNA was amplified by PCR and subject to direct sequencing to identify variants of ASXL1, U2AF1, SF3B1, DNMT3A, TET2, IDH1/2, NPM1, FLT3-ITD and C-KIT genes. RESULTS: ASXL1 variants were found among 37 patients (24.8%). Other commonly mutated genes included U2AF1 (22.8%), TET2 (11.4%), DNMT3A (9.4%), NPM1 (8.1%) and SF3B1 (6.0%). The frequency of concurrent U2AF1 and TET2 variants among patients with ASXL1 variants was slightly higher than that of wild-type patients. No significant difference was found in median age, MDS subtype, karyotype, peripheral leukocytes, hemoglobin, platelet levels, and bone marrow blast counts between the ASXL1-variant and the wild-type groups (P> 0.05). Twenty-nine patients harboring ASXL1 variants were followed up, 37.9% progressed to acute myeloid leukemia (AML). The rate of transformation in ASXL1-variant group was significantly higher than the wild-type group (37.9% vs. 14.1%, P< 0.01). CONCLUSION: ASXL1 showed a high frequency of variant among MDS patients, which was frequently accompanied with U2AF1 and TET2 variants. Compared with the wild type group, patients with ASXL1 variants were more likely to progress to AML.

In Situ Growth of Metal-Organic Frameworks in Three-Dimensional Aligned Lumen Arrays of Wood for Rapid and Highly Efficient Organic Pollutant Removal.

Organic contaminants in water have become one of the most serious environmental problems worldwide. Adsorption is one of the most promising approaches to remove organic pollutants from water. However, the existing adsorbents have relatively low removal efficiency, complex preparation processes, and high cost, which limit their practical applications. Here, we developed three-dimensional (3D) zirconium metal-organic frameworks (MOFs) encapsulated in a natural wood membrane (UiO-66/wood membrane) for highly efficient organic pollutant removal from water. UiO-66 MOFs were in situ grown in the 3D low-tortuosity wood lumens by a facile solvothermal strategy. The resulting UiO-66/wood membrane contains the highly mesoporous UiO-66 MOF structure as well as many elongated and open lumens along the direction of the wood growth. Such a unique structural feature improves the mass transfer of organic pollutants and increases the contact probability of organic contaminants with UiO-66 MOFs as the water flows through the membrane, thereby improving the removal efficiency. Furthermore, the integrated multilayer filter consisting of three pieces of UiO-66/wood membranes exhibits a high removal efficiency (96.0%) for organic pollutants such as rhodamine 6G, propranolol, and bisphenol A at the flux of 1.0 × 103 L·m-2·h-1. The adsorbed capacity of UiO-66/wood for Rh6G (based on the content of UiO-66 MOFs) is calculated to be 690 mg·g-1. We believe that such low-cost and scalable production of the UiO-66/wood membrane has broad applications for wastewater treatment and other related pollutant removal.

[Mutation analysis of 77 patients with normal-karyotype myelodysplastic syndrome].

OBJECTIVE: To carry out mutation analysis for patients with myelodysplastic syndromes (MDS) and a normal karyotype. METHODS: Targeted capture and next-generation sequencing (NGS) was carried out using a customized 49-gene panel. FLT3 internal tandem duplication (FLT3-ITD), CALR, NPM1 and CEBPA mutations were detected by PCR and Sanger sequencing. RESULTS: Sixty-two patients (80.5%) were found to harbor at least one mutation. Each patient has carried 2.21 mutations in average. Coexistence of ≥ 3 mutations was common (43.7%). The most commonly mutated genes were RUNX1 (23.4%, 18/77), ASXL1 (18.2%, 14/77), NPM1 (15.6%, 12/77), U2AF1 (15.6%, 12/77), DNMT3A (11.7%, 9/77). Patients with SF3B1 mutations were significantly older than those with ASXL1 mutations (P=0.023). Mutations of the DNMT3A gene were significantly associated with the blood platelet level compared with BCOR mutations (P=0.02). No significant difference was found in the number and rate of mutations between those under or above 60-year-old. Among 67 patients with clinical follow-up, 20 (29.8%) has transformed to acute myeloid leukemia, and the time of transformation has ranged from 1 to 44 months, with a average of 5.3 months. RUNX1, U2AF1 and FLT3 mutations are associated with leukemic transformation. CONCLUSION: Coexistence of ≥ 3 mutations are frequent among patients with normal-karyotype MDS. Certain mutations are associated with age and leukemic transformation.

Gene expression profiles of HTR8-S/Vneo cells after changes in ABCA1 expression.

ABCA1 is expressed in placental trophoblasts, such that when the expression level of ABCA1 changes, the function of trophoblasts dramatically changes. However, the mechanism by which ABCA1 affects the function of trophoblast cells remains unclear. Here, we used biochemical and transcriptomic to uncover the potential mechanism of the effect of ABCA1 on trophoblast function. HTR8/SVneo cells were either treated with the agonist T0901317 or transfected with siRNA to regulate ABCA1 expression levels. A human gene expression microarray was used to analyze the expression spectrum of ABCA1. Microarray results were confirmed by Western blotting and RT-PCR. Immunofluorescence allowed detection of the cellular localization of ABCA1, CCL8, CXCL10, CXCL11, and S1PR1 in HTR8/SVneo cells. Co-immunoprecipitation was used to test interactions among these proteins. Concomitant with an increase in ABCA1 expression, S1PR1 expression increased, whereas expression of CCL8, CXCL10, and CXCL11 decreased significantly; opposite effects were observed with a decrease in ABCA1 expression. Thus, changes in ABCA1 expression may lead to changes in downstream gene expression. Whereas the interaction between ABCA1 and S1PR1 was direct, interactions among ABCA1 and CCL8, CXCL10, and CXCL11 were indirect. We propose that, in conjunction with S1PR1, ABCA1 regulates expression levels of CCL8, CXCL10, and CXCL11; this may lead to changes in the immune function of trophoblastic cells. Thus, we suspect that the effect of ABCA1 on trophoblast function may involve many biological processes, molecular function changes, and the activation of multiple signaling pathways.

Psychological Capital and Perceived Professional Benefits: Testing the Mediating Role of Perceived Nursing Work Environment Among Chinese Nurses.

The current descriptive cross-sectional study aimed to explore the mediating role of perceived nursing work environment (PNWE) in the relationship between psychological capital (PsyCap) and perceived professional benefits among Chinese nurses. Participants (N = 351) working in two large general hospitals in Guangdong, China completed self-report questionnaires from March to May 2017. Linear regression analyses and structural equation modeling were performed to explore the mediating effect. PsyCap (particularly for hope and optimism) had a positive effect on perceived professional benefits, and PNWE was a mediator in this relationship among Chinese nurses. A good working environment can be regarded as a mediator variable, increasing staff's competence and sense of belonging to a team. For successful implementation, nurse managers should use effective strategies to increase nurses' confidence and hope while providing a comfortable work environment. [Journal of Psychosocial Nursing and Mental Health Services, 56(4), 38-47.].

Construction of a Streptococcus agalactiae phoB mutant and evaluation of its potential as an attenuated modified live vaccine in golden pompano, Trachinotus ovatus.

Streptococcus agalactiae is a Gram-positive pathogen that can survive inside professional phagocytes and nonphagocytic cells to cause septicemia and meningoencephalitis in freshwater and marine fish. However, vaccines based on extracellular products (ECP) and formalin-killed whole S. agalactiae cells, as well as subunit vaccine are unable to protect fish from infection by variant serotypes S. agalactiae. The search for live attenuated vaccine with highly conserved and virulent-related genes is essential for producing a vaccine to help understand and control streptococcosis In this study, the phoB gene was cloned from pathogenic S. agalactiae TOS01 strain and the mutant strain SAΔphoB was constructed via allelic exchange mutagenesis. The results showed that the deduced amino acid of S. agalactiae TOS01 shares high similarities with other Streptococcus spp. and has high conserved response regulator receiver domain (REC) and DNA-binding effector domain of two-component system response regulators (Trans_reg_C). Cell adherence and invasion assays, challenge experiments and histopathological changes post-vaccination were performed and observed, the results showed that the mutant strain SAΔphoB has a lower adherence and invasion rate and less virulent than the wild type strain in golden pompano, and it doesn't induce clinical symptoms and obvious pathological changes in golden pompano, thereby indicating that the deletion of phoB affects the virulence and infectious capacity of S. agalactiae. Golden pompano vaccinated via intraperitoneal injection SAΔphoB had the relative percent survival value of 93.1% after challenge with TOS01, demonstrating its high potential as an effective attenuated live vaccine candidate. Real-time PCR assays showed that the SAΔphoB was able to enhance the expression of immune-related genes, including MHC-I, MyD88, IL-22 and IL-10 after vaccination, indicating that the SAΔphoB is able to induce humoral and cell-mediated immune response in golden pompano over a long period of time.

Molecular characterization and expression of interleukin-10 and interleukin-22 in golden pompano (Trachinotus ovatus) in response to Streptococcus agalactiae stimulus.

In the present study, members of the interleukin (IL)-10 family of cytokines, including IL-10 (TOIL-10) and IL-22 (TOIL-22) of golden pompano (Trachinotus ovatus), were cloned for the first time, and their expression patterns and 3D structures analyzed. The full-length cDNA sequences of TOIL-10 and TOIL-22 contained open reading frames of 564 and 567 bp, respectively. TOIL-10 and TOIL-22 shared higher homology (78%-89%) with the corresponding genes from various fish relative to other species (25%-34%) and contained the IL-10 family signature and four cysteine residues that are well conserved in other vertebrate IL-10 members. Phylogenetic tree analysis of our sequences alongside other IL-10 family proteins revealed that TOIL-10 and TOIL-22 cluster together with other teleost IL-10 and IL-22 molecules. Expression of TOIL-10 and TOIL-22 genes was ubiquitous in all tissues examined. The TOIL-10 gene was also highly expressed in skin, heart, gill, spleen, kidney, brain and liver, and lower levels were detected in intestine and muscle. High expression of the TOIL-22 gene was observed in gill, intestine, kidney, spleen, with the lowest levels in liver. TOIL-10 and TOIL-22 were rapidly activated after SAΔphoB immunization and significantly increased to peak levels at 12 h and 4 d in golden pompano kidney and spleen respectively following challenge. Expression in the brain reached peak levels at 4 d and 3 d respectively after post-immunization. Our results collectively indicate that TOIL-10 and TOIL-22 participate in the host immune response to bacterial infection. Moreover, TOIL-22 plays a potentially important role in mucosal immunity.

Characterization of Full-Length Genomes of Hepatitis B Virus Quasispecies in Sera of Patients at Different Phases of Infection.

Hepatitis B virus (HBV) infection results in different clinical presentation due to different levels of immune response. Our study aimed to characterize HBV full-length genome quasispecies (QS) in patients with different phases of infection to better understand its pathogenesis. Forty treatment-naive HBV-infected patients were enrolled, including 10 cases of acute hepatitis B (AHB), 9 cases of immunotolerant (IT) HBV carriers, 11 cases of chronic hepatitis B (CHB), and 10 cases of acute-on-chronic liver failure (ACLF). The present study was conducted by clone-based sequencing. QS heterogeneity within each open reading frame was calculated. The mutation frequency index (MFI) and amino acid variations within the large HBsAg, HBcAg, and HBxAg regions were analyzed based on the different infection phases. In total, 606 HBV full-length sequences were obtained. HBV QS had higher heterogeneity in ACLF and CHB than that in IT among chronically infected individuals. AHB patients had the lower QS heterogeneity at onset than those with chronic infection. ACLF patients had the highest frequency of mutations in the core promoter and precore region. A triple mutation (A1762T/G1764A/G1896A) was observed more frequently in genotype C than in genotype B. The MFI indicated that specific peptides of the studied regions had more frequent mutations in ACLF. Furthermore, several amino acid variations, known as T- and B-cell epitopes, were potentially associated with the immunoactive phase of infection. More HBV genome mutations and deletions were observed in patients with more severe diseases, particularly in specific regions of the core and preS regions, the clinical significance and mechanism of which need to be further investigated.

Silica-based 2-(N,N-dimethylamino)-1,3-propanediol hydrophilic interaction liquid chromatography stationary phase for separating cephalosporins and carbapenems.

A silica-based stationary phase bearing both hydrophilic hydroxyl and amino groups was developed by covalently bonding a small molecular N,N-dimethylamino 1,3-propanediol moiety onto silica beads via copper(I)-catalyzed Huisgen azide-alkyne 1,3-dipolar cycloaddition (CuAAC). This new stationary phase showed good HILIC characteristics and high column efficiency (the theoretical plate number is up to 37000 plates m(-1) in the case of inosine) in the separation of polar compounds, such as nucleosides and bases, organic acids, cephalosporins, and carbapenems.