Tumor Targeted Cuprous-Based Nanocomposite as Responsive Cascade Nanocatalyst for Efficient Tumor Synergistic Therapy.

The single-functionality of traditional chemodynamic therapy (CDT) reagents usually limits the therapeutic efficacy of cancer treatment. Synergistic nanocomposites that involve cascade reaction provide a promising strategy to achieve satisfactory anticancer effects. Herein, a cuprous-based nanocomposite (CCS@GOx@HA) is fabricated, which owns the tumor targeting ability and can undergo tumor microenvironment responsive cascade reaction to enhance the tumor therapeutic efficiency significantly. Surface modification of nanocomposite with hyaluronic acid enables the targeted delivery of the nanocomposite to cancer cells. Acid-triggered decomposition of nanocomposite in cancer cell results in the release of Cu+ , Se2- and GOx. The Cu+ improves the Fenton-like reaction with endogenous H2 O2 to generate highly toxic • OH for CDT. While GOx can not only catalyze the in situ generation of endogenous H2 O2 , but also accelerate the consumption of intratumoral glucose to reduce nutrient supply in tumor site. In addition, Se2- further improves the therapeutic effects of CDT by upregulating the reactive oxygen species (ROS) in tumor cells. Meanwhile, the surface modification endows the nanocomposite the good water dispersibility and biocompatibility. Moreover, in vitro and in vivo experiments demonstrate satisfactory anti-cancer therapeutic performance by the synergistic cascade function of CCS@GOx@HA than CDT alone.

Mechanism of Chinese sturgeon IFN-γ inhibition on Mycobacterium marinum (Acipenser sinensis).

IFN-γ plays a crucial role in both innate and adaptive immune responses and is a typical Th1 cytokine that promotes Th1 response and activates macrophages. When macrophages were incubated with IFN-γ, their phagocytosis ratio against Mycobacterium marinum increased significantly, as observed under fluorescence microscopy. The macrophages engulfed a large number of M. marinum. The proliferative ability of macrophages treated with IFN-γ was significantly weaker on the 4th and 7th day after phagocytosis and subsequent re-infection with marine chlamydia (P < 0.001). This suggests that IFN-γ enhances the phagocytosis and killing ability of macrophages against M. marinum. IFN-γ protein also significantly increased the production of reactive oxygen species (H2O2) and nitric oxide (NO) by macrophages. Additionally, the expression levels of toll-like receptor 2 (tlr2) and caspase 8 (casp8) were significantly higher in macrophages after IFN-γ incubation compared to direct infection after 12 h of M. marinum stimulation. Apoptosis was also observed to a higher degree in IFN-γ incubated macrophage. Moreover, mRNA expression of major histocompatibility complex (MHC) molecules produced by macrophages after IFN-γ incubation was significantly higher than direct infection. This indicates that IFN-γ enhances antigen presentation by upregulating MHC expression. It also upregulates tlr2 and casp8 expression through the TLR2 signaling pathway to induce apoptosis in macrophages. The pro-inflammatory cytokine showed an initial increase followed by a decline, suggesting that IFN-γ enhances the immune response of macrophages against M. marinum infection. On the other hand, the anti-inflammatory cytokine showed a delayed increase, significantly reducing the expression of pro-inflammatory cytokines. The expression of both cytokines balanced each other and together regulated the inflammatory reaction against M. marinum infection.

Role of creatine supplementation on the myofibre characteristics and muscle protein synthesis of grass carp (Ctenopharyngodon idellus).

To assess the role of dietary creatine on myofibre characteristics and protein synthesis in muscle, we fed grass carp (Ctenopharyngodon idellus, initial body weight: 88·47 ± 1·44 g) creatine-supplemented diets (1·84, 5·91, 8·48 and 15·44 g/kg diet) for 8 weeks. Creatine supplementation did not affect growth performance, but significantly increased creatine contents in muscle and liver. At 8·48 g/kg, creatine decreased the activities of alanine transaminase and aspartate aminotransferase in serum and improved hardness and chewiness of muscle due to shorter myofibre mean diameter, higher myofibre density and the frequencies of the diameters of classes I and III and collagen content, longer sarcomere length and upregulated mRNA levels of slow myosin heavy chains. Creatine supplementation upregulated the mRNA expressions of myogenic regulatory factors. The 8·48 g/kg creatine-supplemented diet significantly increased the contents of protein, total amino acids (AA), essential AA and free flavour AAs in muscle, the protein levels of insulin-like growth factor I, myogenic differentiation antigen and PPAR-γ coactlvator-1α in muscle and stimulated the phosphorylation of target of rapamycin (TOR) pathway in muscle. In summary, 8·48 mg/kg creatine improved fish health and skeletal muscle growth and increased hardness and protein synthesis in muscle of grass carp by affecting myofibre characteristics and the TOR signalling pathway. A second-order regression model revealed that the optimal dietary creatine supplementation of grass carp ranges between 8·48 and 12·04 g/kg.

Effects of dietary nucleotide and yeast cell wall on growth performance, feed utilization, anti-oxidative and immune response of grass carp (Ctenopharyngodon idella).

A 70-day feeding trial was conducted to study the effects of dietary nucleotide, yeast cell wall (containing 20% ß -glucan) and their combination on growth performance, feed utilization and immune response of grass carp (Ctenopharyngodon idella) with 69.97 ± 0.05 g of initial body weight. Four isonitrogenous (about 38% crude protein) and isolipidic (about 5% crude lipid) diets were established. Based on the control diet (CD), the other three experimental diets were prepared by adding 0.01% of nucleotide (NT), 0.1% of yeast cell wall (YCW) and NT (0.01%) +YCW (0.1%), respectively. Results showed that no significant difference was found in survival of grass carp ranging from 94.44% to 97.78% among all the groups (P > 0.05). Compared with the control group, weight gain rate, muscle crude protein content, serum protein, trypsin and chymotrypsin activities in midgut, lysozyme and immunoglobulin M in serum significantly increased in fish fed the YCW diet (P < 0.05). The significantly highest weight gain rate, villus height and digestive enzyme activities in midgut and innate immune parameters in serum were found in fish fed the NT + YCW diet (P < 0.05). The gene expressions of ß-defensin, hepcidin, il-10 and tgf-ß1 in the midgut, and tor and s6k1 in liver significantly increased in fish fed the NT + YCW diet. Meanwhile, the gene expressions of il-1ß and tnf-α in the midgut decreased significantly (P < 0.05). The liver histology showed the better development in dietary NT and/or YCW supplemented groups than those in the control group. In conclusion, combination of dietary NT and YCW had significantly synergetic improvements on the growth, feed utilization, digestive enzymes, innate immunity and histology of midgut and liver of grass carp.

Dietary Cholesterol Requirements of Large Red Swamp Crayfish (Procambarus clarkii).

To investigate the dietary cholesterol requirements of large red swamp crayfish (Procambarus clarkii), crayfish (initial body weight: 13.49 ± 0.22 g) were hand-fed six diets containing 2.47 (C0), 4.27 (C1), 6.80 (C2), 8.77 (C3), 11.74 (C4), and 14.24 (C5) g/kg cholesterol. After 8 weeks of feeding, the maximum weight gain rate and specific growth rate occurred in group C4. The lowest feed conversion ratio was observed in group C3. Total flesh percentage increased significantly by 15.33% in group C2 compared to group C0. The increase in dietary cholesterol resulted in significant quadratic trends in concentrations of crude protein and lipid in muscle and whole body; cholesterol and free fatty acid in hemolymph, hepatopancreas, and muscle; activities of lipase and amylase in hepatopancreas and intestine; and total antioxidant capacity and catalase activity in hepatopancreas. Group C3 experienced a noteworthy increase in hemolymph glucose and total protein content compared to group C0. Additionally, malondialdehyde content and superoxide dismutase activity in hepatopancreas displayed significant linear and quadratic trends. The optimal dietary cholesterol level for large P. clarkii is between 7.42 and 10.93 g/kg, as revealed by the quadratic regression analysis.

Reassortment with dominant chicken H9N2 influenza virus contributed to the fifth H7N9 virus human epidemic.

H9N2 Avian influenza virus (AIV) is regarded as a principal donor of viral genes through reassortment to co-circulating influenza viruses that can result in zoonotic reassortants. Whether H9N2 virus can maintain sustained evolutionary impact on such reassortants is unclear. Since 2013, avian H7N9 virus had caused five sequential human epidemics in China; the fifth wave in 2016-2017 was by far the largest but the mechanistic explanation behind the scale of infection is not clear. Here, we found that, just prior to the fifth H7N9 virus epidemic, H9N2 viruses had phylogenetically mutated into new sub-clades, changed antigenicity and increased its prevalence in chickens vaccinated with existing H9N2 vaccines. In turn, the new H9N2 virus sub-clades of PB2 and PA genes, housing mammalian adaptive mutations, were reassorted into co-circulating H7N9 virus to create a novel dominant H7N9 virus genotype that was responsible for the fifth H7N9 virus epidemic. H9N2-derived PB2 and PA genes in H7N9 virus conferred enhanced polymerase activity in human cells at 33°C and 37°C, and increased viral replication in the upper and lower respiratory tracts of infected mice which could account for the sharp increase in human cases of H7N9 virus infection in the 2016-2017 epidemic. The role of H9N2 virus in the continual mutation of H7N9 virus highlights the public health significance of H9N2 virus in the generation of variant reassortants of increasing zoonotic potential.IMPORTANCEAvian H9N2 influenza virus, although primarily restricted to chicken populations, is a major threat to human public health by acting as a donor of variant viral genes through reassortment to co-circulating influenza viruses. We established that the high prevalence of evolving H9N2 virus in vaccinated flocks played a key role, as donor of new sub-clade PB2 and PA genes in the generation of a dominant H7N9 virus genotype (G72) with enhanced infectivity in humans during the 2016-2017 N7N9 virus epidemic. Our findings emphasize that the ongoing evolution of prevalent H9N2 virus in chickens is an important source, via reassortment, of mammalian adaptive genes for other influenza virus subtypes. Thus, close monitoring of prevalence and variants of H9N2 virus in chicken flocks is necessary in the detection of zoonotic mutations.

Development and initial validation of the HS-IGA: a novel hidradenitis suppurativa-specific investigator global assessment for use in interventional trials.

BACKGROUND: Few validated instruments exist for use in hidradenitis suppurativa (HS) trials. OBJECTIVES: To develop a novel HS Investigator Global Assessment (HS-IGA) and to validate its psychometric properties. METHODS: Development of HS-IGA involved discussion among stakeholders, including patients, within HISTORIC. Data from replicate phase III randomized controlled trials evaluating HS treatment were utilized. Multivariate models identified lesion type and body region as variables of importance. Classification and regression trees for ordinal responses were built. Validation included assessment of test-retest reliability, predictive validity, responsiveness and clinical meaningfulness. RESULTS: There were 3024 unique measurements available in PIONEER I. Mean and median lesion counts by region were largely <10 and were highest in axillary and inguinal regions. The mean and median number of regions involved were ≤ 3 for individual lesions and combinations. Regardless of lesion type, axillary and inguinal regions most influenced the HS-IGA score. Accordingly, regions were combined into a six-point IGA based on the maximum lesion number in either upper or lower body regions with a score of 0 (0-1 lesions), 1 (2-5), 2 (6-10), 3 (11-15), 4 (16-20) and 5 (≥ 20 lesions). The intraclass correlation coefficient for test-retest reliability was 0·91 (95% confidence interval 0·87-0·94). Spearman's rank order correlations (SROCs) with HS-PGA and Hidradenitis Suppurativa Clinical Response (HiSCR) were 0·73 and 0·51, respectively (P < 0·001 for both comparisons). SROCs with Dermatology Life Quality Index (DLQI), pain numerical rating scale and HS-QoL were 0·42, 0·34 and -0·25, respectively (P < 0·001 for all comparisons). HS-IGA was responsive at weeks 12 and 36. Predictive convergent validity was very good with HS-PGA (area under the curve = 0·89) and with HiSCR (area under the curve = 0·82). Predictive divergent validity was low with DLQI and HS-QoL. CONCLUSIONS: HS-IGA has moderate-to-strong psychometric properties and is simple to calculate.

Effects of phytase supplementation of high-plant-protein diets on growth, phosphorus utilization, antioxidant, and digestion in red swamp crayfish (Procambarus clarkii).

Fish meal is increasingly being replaced by plant protein raw materials, meanwhile, it brings phytic acid, which combines with phosphorus to form phytate phosphorus and leads to a low utilization rate of phosphorus in shrimp. To solve this problem, this study investigated the effects of phytase supplementation on growth performance, phosphorus utilization, antioxidants, and digestion in red swamp crayfish (Procambarus clarkii). Crayfish (initial mean weight: 8.69 ± 0.15 g, N = 324) were randomly divided into six groups each with three replicates of 18 individuals each, and hand-fed for 8 weeks with one of six experimental diets (50 and 490 g kg-1 animal and plant protein raw material, respectively): negative control (NC; 11.0 g kg-1 phosphorus), positive control (PC; 15 g kg-1 NaH2PO4 added to NC; 14.7 g kg-1 phosphorus), and phytase supplementation diets (P1-P4: 0.1, 0.2, 0.4, and 0.6 g kg-1 phytase added to NC, respectively). The feeding trial was performed in a micro-flow water culture system. P2 showed a significantly higher weight gain rate (WGR), specific growth rate, protein efficiency ratio, and protein retention efficiency (PRE) but showed the lowest feed conversion ratio (FCR) than other groups. Broken-line regression analyses using WGR, FCR, and PRE as evaluation indices showed that the optimal dietary phytase supplementation level was 0.233, 0.244, and 0.303 g kg-1, respectively. P2 showed the highest crude protein content of whole crayfish and abdominal muscle, and phosphorus deposition rate, which was significantly higher than that in NC and PC. P3 showed the highest calcium and phosphorus contents in whole crayfish and phosphorus content in abdominal muscle, and calcium and inorganic phosphorus content in serum, which were significantly higher than those in NC. P3 showed significantly lowest serum alkaline phosphatase, alanine aminotransferase, aspartate transaminase activities, malondialdehyde content in hepatopancreas, and highest catalase activity, which were significantly lower and higher, respectively, than those in NC and PC. In summary, the addition of 0.2-0.4 g kg-1 phytase significantly improves the growth performance, feed utilization, digestive enzyme activity, and antioxidant of P. clarkii, which has a similar effect to the direct addition of NaH2PO4 at 15 g kg-1 to the feed.

Effects of recombinant anti-lipopolysaccharide factor expressed by Pichia pastoris on the growth performance, immune response and disease resistance of Litopenaeusvannamei.

The codon-optimized anti-lipopolysaccharide factor (ALF) sequence was introduced into pPICZαA vector and transformed into Pichia pastoris GS115. The recombinant ALF yeast supernatant (rALF-mix) was freeze-dried and evaluated as a feed additive for Litopenaeus vannamei. It was found by antibacterial activity test in vitro that the rALF-mix had antibacterial activity under different pH and temperature conditions. The 0, 0.00375%, 0.0075%, 0.015%, 0.03% and 0.06% of rALF-mix were added respectively to make the six experimental diets. After a 10-week feeding trial with shrimps (2.36 ± 0.02 g), it was found that the weight gain rate (WGR) and protein efficiency ratio (PER) of shrimp in the groups with 0.0075%, 0.015% and 0.03% of dietary rALF-mix supplementation were significantly higher than those in the control group (P < 0.05). Dietary rALF-mix supplementation significantly increased the total haemocyte count, respiratory burst, phagocytic activity, total anti-oxidative capacity (T-AOC), phenol oxidase activity, nitric oxide synthase activity, lysozyme (LYZ) activity, serum antibacterial capacity in the hemolymph and the T-AOC, LYZ in the hepatopancreas of shrimps (P < 0.05). The malondialdehyde contents in hemolymph and hepatopancreas were significantly decreased (P < 0.05). Meanwhile, the expression levels of toll, immune deficiency, heat shock protein 70, crustin and lipopolysaccharide-ß-glucan binding protein in the gill of shrimps were significantly increased (P < 0.05). After the challenge test, it was showed that dietary rALF-mix supplementation significantly improved the resistance of L. vannamei to Vibrio parahaemolyticus (P < 0.05). In conclusion, the rALF-mix can be used as a functional feed additive to improve the growth, immunity and disease resistance of shrimp. Based on the quadratic regression analysis for WGR, the optimal supplemental level of rALF-mix in diet for shrimp was estimated to be 0.02813%.

Impact of nitrite exposure on oxidative stress and antioxidative-related genes responses in the gills of Procambarus clarkii.

Nitrite is the major environmental pollutant in the freshwater aquaculture environment, which has a negative impact on aquatic species growth. Currently, we know that the main way nitrite enters crustaceans is through their gills. In this study, a total of 96 h acute nitrite stress (60 mg/L) experiments were conducted, and the impact of the serum biochemical parameters, gill oxidase activity and oxidative-related gene expression of red swamp crayfish were evaluated. After exposure to nitrite for 0, 6, 12, 24, 48, and 96 h, hemolymph and gills samples were taken at each time point. In the serum, acute nitrite stress significantly increased glutamic-oxaloacetic transaminase (GOT) and alanine aminotransferase (ALT) activities after 6 h of exposure, decreased total protein (TP) and albumin (ALB) levels after 24 h and 48 h of exposure, respectively. In the gills, the activities of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were enhanced to the maximum level at 12 h, 24 h and 24 h, respectively. The contents of malondialdehyde (MDA) and lipid peroxide (LPO) were increased significantly after 12 h and 24 h exposure, respectively. In addition, the expression levels of antioxidative-related genes, including hsp70, fer and mt, were significantly upregulated in the gills after 6 h of exposure. The results indicated that acute nitrite stress changed the serum physiological status, induced oxidative stress and caused damage to gill cells in P. clarkii.

Swine MicroRNAs ssc-miR-221-3p and ssc-miR-222 Restrict the Cross-Species Infection of Avian Influenza Virus.

Avian influenza virus (AIV) can cross species barriers to infect humans and other mammals. However, these species-cross transmissions are most often dead-end infections due to host restriction. Current research about host restriction focuses mainly on the barriers of cell membrane, nuclear envelope, and host proteins; whether microRNAs (miRNAs) play a role in host restriction is largely unknown. In this study, we used porcine alveolar macrophage (PAM) cells as a model to elucidate the role of miRNAs in host range restriction. During AIV infection, 40 dysregulation expressed miRNAs were selected in PAM cells. Among them, two Sus scrofa (ssc; swine) miRNAs, ssc-miR-221-3p and ssc-miR-222, could inhibit the infection and replication of AIV in PAM cells by directly targeting viral genome and inducing cell apoptosis via inhibiting the expression of anti-apoptotic protein HMBOX1. Avian but not swine influenza virus caused upregulated expressions of ssc-miR-221-3p and ssc-miR-222 in PAM cells. We further found that NF-κB P65 was more effectively phosphorylated upon AIV infection and that P65 functioned as a transcription activator to regulate the AIV-induced expression of miR-221-3p/222 Importantly, we found that ssc-miR-221-3p and ssc-miR-222 could also be specifically upregulated upon AIV infection in newborn pig tracheal epithelial (NPTr) cells and also exerted anti-AIV function. In summary, our study indicated that miRNAs act as a host barrier during cross-species infection of influenza A virus.IMPORTANCE The host range of an influenza A virus is determined by species-specific interactions between virus and host cell factors. Host miRNAs can regulate influenza A virus replication; however, the role of miRNAs in host species specificity is unclear. Here, we show that the induced expression of ssc-miR-221-3p and ssc-miR-222 in swine cells is modulated by NF-κB P65 phosphorylation in response to AIV infection but not swine influenza virus infection. ssc-miR-221-3p and ssc-miR-222 exerted antiviral function via targeting viral RNAs and causing apoptosis by inhibiting the expression of HMBOX1 in host cells. These findings uncover miRNAs as a host range restriction factor that limits cross-species infection of influenza A virus.

Effects of Yeast Autolysate in the Practical Diet on the Growth Performance, Immune Response, and Disease Resistance of Pacific White Shrimp.

The present study was conducted to investigate the effects of supplementing the practical diet with yeast autolysate (YA) on the growth performance, immunity, and disease resistance of Pacific white shrimp Litopenaeus vannamei. Four isonitrogenous and isolipidic practical diets were formulated. The relatively high-fish-meal control diet contained 25% fish meal without YA supplementation (E1). The other control diet contained 20% fish meal without YA (E2). With the E2 diet as the basis, two additional experimental diets were created by further supplementation with 1% YA (E3) and 2% YA (E4). The shrimp (initial weight: 0.30 ± 0.02 g) were fed with the four experimental diets for 8 weeks and then challenged with Vibrio parahaemolyticus. The results indicated that there were no significant differences in survival rate (SR) or feed intake (FI) among these groups. The weight gain rate (WGR) of group E1 was not significantly different from that of groups E3 and E4. The feed conversion ratio (FCR) in group E4 was lower than that of group E2, and group E4 had the highest protein efficiency ratio (PER). The total hemocyte counts (THC) and lysozyme activities in group E3 and group E4 were significantly higher than those of the other groups. Group E3 had the highest respiratory burst (RB). After V. parahaemolyticus administration, group E3 and group E4 had significantly lower cumulative mortalities than group E1 did. In conclusion, the 20% fish meal diet without YA supplementation (E2) yielded a significantly lower growth rate than the 25% fish meal diet without YA supplementation (E1) did. Furthermore, the Pacific white shrimp that received dietary supplementation with 1% YA demonstrated improved growth rate, immune response, and resistance to the V. parahaemolyticus challenge compared with those that were fed the 20% fish meal diet without YA supplementation (E2).

Prevailing I292V PB2 mutation in avian influenza H9N2 virus increases viral polymerase function and attenuates IFN-ß induction in human cells.

Adaptation of PB2 protein is important for the establishment of avian influenza viruses in mammalian hosts. Here, we identify I292V as the prevalent mutation in PB2 of circulating avian H9N2 and pandemic H1N1 viruses. The same dominant PB2 mutation is also found in most human isolates of emergent avian H7N9 and H10N8 viruses. In human cells, PB2-292V in H9N2 virus has the combined ability of conferring higher viral polymerase activity and stronger attenuation of IFN-ß induction than that of its predecessor PB2-292I. IFN-ß attenuation is accompanied by higher binding affinity of PB2-292V for host mitochondrial antiviral signalling protein, an important intermediary protein in the induction of IFN-ß. In the mouse in vivo model, PB2-292V mutation increases H9N2 virus replication with ensuing increase in disease severity. Collectively, PB2-292V is a new mammalian adaptive marker that promotes H9N2 virus replication in mammalian hosts with the potential to improve transmission from birds to humans.

Sequence analysis and characterization of type I interferon and type II interferon from the critically endangered sturgeon species, A. dabryanus and A. sinensis.

In the present study, we identify three type I interferon (IFN) genes (Ad/AsIFNe1-3) and a type II IFN gene (Ad/AsIFNγ) from the Dabry's sturgeon (Acipenser dabryanus) and the Chinese sturgeon (Acipenser sinensis). Sequence analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ contain several conserved characteristics, including signal peptides, interferon alpha, beta, and delta (IFabd) domains, and N-glycosylation sites. Ad/AsIFNe1-3 belongs to the type I IFN group I subgroup, possessing two conserved cysteines residues (C1 and C3), and Ad/AsIFNγ contained a conserved nuclear localization sequence (NLS) motif. Ad/AsIFNe1-3 and Ad/AsIFNγ contain signature motifs indicative of their corresponding IFN group. The Ad/AsIFNe1-3 and Ad/AsIFNγ genes were found to consist of 5 exons/4 introns and 4 exons/3 introns, respectively. These IFNs were separated by four phase 0 introns (type I IFN) and three phase 0 introns (type II IFN). The sequences of IFNe1-3 and IFNγ from the Dabry's sturgeon and the Chinese sturgeon were closely aligned, suggested that these two species are closely related. Phylogenetic analysis revealed that Ad/AsIFNe1-3 and Ad/AsIFNγ clustered together with the corresponding homologous proteins from other fish species. AdIFNe1-3 were found to be high expressed in early embryonic development, suggesting that AdIFNe1-3 might indicate maternal transmission, while AdIFNγ may not mediate embryonic development. Tissue distribution analysis revealed that AdIFNe1-3 and AdIFNγ carry out biological functions in immune and non-immune tissues compartments. AdIFNe1-3 and AdIFNγ can be stimulated by polyinosinic-polycytidylic acid (poly I:C) and lipopolysaccharides (LPS). AdIFNe1-3 have stronger antiviral activity than AdIFNγ, and AdIFNγ has a stronger antibacterial activity than AdIFNe1-3. The differential responses of these genes to poly I:C and LPS suggest differences in the mechanisms of defense against viruses and bacteria.

Molecular characterization and expression analysis of Asian swamp eel (Monopterus albus) CXC chemokine receptor (CXCR) 1a, CXCR1b, CXCR2, CXCR3a, CXCR3b, and CXCR4 after bacteria and poly I:C challenge.

The CXC chemokine receptors (CXCRs) play critical roles in innate and adaptive immune systems. In this study, six Asian swamp eel (Monopterus albus) CXCRs (MaCXCR1-4) were identified and their molecular characterization and expression patterns were analyzed. The open reading frames (ORFs) of MaCXCR1a, MaCXCR1b, MaCXCR2, MaCXCR3a, MaCXCR3b, and MaCXCR4 were 1074 bp (base pairs), 1080 bp, 1125 bp, 1146 bp, 1083 bp, and 1140 bp, and encoded proteins of 357 aa (amino acids), 359 aa, 374 aa, 381 aa, 360 aa, and 379 aa, respectively. All these CXCRs have seven conserved transmembrane domains and four cysteines (with the exception of MaCXCR3b). Multiple sequence alignment revealed that the MaCXCRs possess a typical G-protein receptor family 1 signature and a DRY motif. There are also one to four potential N-glycosylation sites in the extracellular regions of the MaCXCRs, mainly distributed in the N-terminus and extracellular hydrophilic loop (ECL) 2 region. Phylogenetic analysis demonstrated that the MaCXCRs were clustered together with homologous proteins from other fish. Taken together with the amino acid identity and similarity analysis, these results suggested that the MaCXCRs are conserved with other homologous genes, in which CXCR4 is more conserved than CXCR1-3. The MaCXCRs loci showed conserved synteny among teleost fish, and we found that human CXCR1 shares a common ancestor with fish CXCR1a. MaCXCRs were constitutively expressed in a wide range of tissues (especially in immune-related tissues) with different expression levels, suggesting that the MaCXCRs have different roles in un-stimulated tissues, and may play vital roles under normal conditions. MaCXCRs showed different fold changes in the spleen after Aeromonas veronii and polyinosinic-polycytidylic acid (poly I:C) challenge, which suggested that MaCXCR1a and MaCXCR3a have longer antiviral activities compared with their antibacterial functions, and that MaCXCR1b possesses stronger antiviral than antibacterial activity. MaCXCR4 may play vital roles during bacterial and viral infection; however, MaCXCR2 has relatively small effect in antibacterial and antiviral responses. The differential responses of these genes to bacteria and poly I:C implied the differences in the mechanisms of defense against viruses and bacteria.

Sequence characterization and expression pattern analysis of six kinds of IL-17 family genes in the Asian swamp eel (Monopterus albus).

Interleukin-17 (IL-17) is an important cytokine that plays a critical role in the inflammatory response and host defense against extracellular pathogens. In the present study, six novel IL-17 family genes (MaIL-17) were identified by analyzing Asian swamp eel (Monopterus albus) genome. Sequence analysis revealed that the MaIL-17 family genes shared similar features, comprising a signal peptide, an IL-17 superfamily region, and four conserved cysteines. Phylogenetic analysis showed that the MaIL-17 genes were clustered together with their corresponding IL-17 genes from other species. The similarity and identity of all IL-17 family genes indicated that the MaIL-17 genes are conserved among teleosts, while Ma-IL-17D is more conserved than the other Ma-IL-17s. Except for MaIL-17A/F3 and MaIL-17D, all MaIL-17s shared the same genomic structure as the genes from other fish, namely three exons and two introns. The MaIL-17s showed conserved synteny among fish, and we found that the MaIL-17D locus has a more conserved syntenic relationship with the loci from other fish and humans. These results demonstrated that MaIL-17D and human IL-17D might have evolved from a common ancestral gene and subsequently diverged. The analysis of swamp eel reference genes revealed that EEF1A1 (encoding eukaryotic translation elongation factor 1 alpha 1) was an ideal reference gene for accurate real-time qRT-PCR normalization in the swamp eel. The MaIL-17 genes are widely distributed throughout tissues, suggesting that MaIL-17s carry out their biological functions in immune and non-immune tissues compartments. The transcript of Ma-IL17s exhibited different fold changes in head kidney cells in response to Aeromonas veronii phorbol 12-myristate 13-acetate (PMA) and polyinosinic:polycytidylic acid (poly I:C) challenge, showing that MaIL-17A/F1 has stronger antiviral activities compared with other MaIL-17 family genes, and that MaIL-17A/F3 and MaIL-17A/F2 possess stronger effects against extracellular pathogens compared with the others; however, MaIL-17C2 and MaIL-17D may play vital roles during pathogen infection. The differential immune responses of these genes to Aeromonas veronii, PMA and poly I:C implied distinct mechanisms of host defense against extracellular pathogens.

The Impact of Fetal Anomalies on Contemporary Labor Patterns.

OBJECTIVE: To compare labor patterns in pregnancies affected by fetal anomalies to low-risk singletons. STUDY DESIGN: Labor data from the Consortium on Safe Labor, a multicenter retrospective study from 19 U.S. hospitals, including 98,674 low-risk singletons compared with 6,343 pregnancies with fetal anomalies were analyzed. Repeated-measures analysis constructed mean labor curves by parity, gestational age, and presence of fetal anomaly in women who reached full dilation. Interval-censored regression analysis adjusted for covariables was used to determine the median traverse times for labor progression. RESULTS: Labor curves for all groups indicated slower labor progress for patients with fetal anomalies. The most significant trends in median traverse times were observed in the preterm nulliparous and term multiparous groups. The median traverse times from 4 cm to complete dilation in the preterm nulliparous control versus anomaly groups were 5.0 and 5.4 hours (p < 0.0001). CONCLUSION: Labor proceeds at a slower rate in pregnancies affected by anomalies.

M Gene Reassortment in H9N2 Influenza Virus Promotes Early Infection and Replication: Contribution to Rising Virus Prevalence in Chickens in China.

Segment reassortment and base mutagenesis of influenza A viruses are the primary routes to the rapid evolution of high-fitness virus genotypes. We recently described a predominant G57 genotype of avian H9N2 viruses that caused countrywide outbreaks in chickens in China during 2010 to 2013, which led to the zoonotic emergence of H7N9 viruses. One of the key features of the G57 genotype is the replacement of the earlier A/chicken/Beijing/1/1994 (BJ/94)-like M gene with the A/quail/Hong Kong/G1/1997 (G1)-like M gene of quail origin. We report here the functional significance of the G1-like M gene in H9N2 viruses in conferring increased infection severity and infectivity in primary chicken embryonic fibroblasts and chickens. H9N2 virus housing the G1-like M gene, in place of the BJ/94-like M gene, showed an early surge in viral mRNA and viral RNA (vRNA) transcription that was associated with enhanced viral protein production and with an early elevated release of progeny virus comprising largely spherical rather than filamentous virions. Importantly, H9N2 virus with the G1-like M gene conferred extrapulmonary virus spread in chickens. Five highly represented signature amino acid residues (37A, 95K, 224N, and 242N in the M1 protein and 21G in the M2 protein) encoded by the prevalent G1-like M gene were demonstrated to be prime contributors to enhanced infectivity. Therefore, the genetic evolution of the M gene in H9N2 virus increases reproductive virus fitness, indicating its contribution to the rising virus prevalence in chickens in China.IMPORTANCE We recently described the circulation of a dominant genotype (genotype G57) of H9N2 viruses in countrywide outbreaks in chickens in China, which was responsible, through reassortment, for the emergence of H7N9 viruses that cause severe human infections. A key feature of the genotype G57 H9N2 virus is the presence of the quail-origin G1-like M gene, which had replaced the earlier BJ/94-like M gene. We found that H9N2 virus with the G1-like M gene, but not the BJ/94-like M gene, showed an early surge in progeny virus production and more severe pathology and extrapulmonary virus spread in chickens. Five highly represented amino acid residues in the M1 and M2 proteins derived from the G1-like M gene were shown to mediate enhanced virus infectivity. These observations enhance what we currently know about the roles of reassortment and mutations in virus fitness and have implications for assessing the potential of variant influenza viruses that can cause a rising prevalence in chickens.

Reduced glutathione supplementation in practical diet improves the growth, anti-oxidative capacity, disease resistance and gut morphology of shrimp Litopenaeus vannamei.

The present study was conducted to investigate the effects of reduced glutathione (GSH) supplementation in practical diet on growth performance, anti-oxidative response, disease resistance and intestine morphology of shrimp Litopenaeus vannamei. Two control diets based on the commercial formulation were designed with high level (27%) and low level (22%) of fish meal, respectively. Based on the control diet with low level of fish meal, 75, 150 and 225 mg/kg of GSH were added, respectively, to make the other three experimental diets. The five formulated diets were named as C1, C2, G1, G2 and G3, respectively. The shrimp (initial body weight: 0.30 ± 0.02 g) were fed with the five experimental diets for 8 weeks followed by a challenge test with Vibrio parahaemolyticus. The results showed that the specific growth rate (SGR) of shrimp in the C2 group was significantly lower than that in C1. The SGRs in G1 and G2 had no significant difference with those in C1 and C2. However, the SGR in G3 was significantly lower than that in C1. The serum GSH concentration in C2 was significantly lower than the other groups, but the malondialdehyde concentration was significantly higher. The supplementation of dietary GSH significantly improved the total anti-oxidative capacity and activities of glutathione S-transferase and glutathione peroxidase in serum. The villus height of intestine in the GSH supplemented groups had no significant difference with C1, but was significantly higher than C2. The jejunum wall thickness of intestine in G2 and G3 was significantly higher than those in the other groups. After the challenge test, the cumulative mortalities in G1 and G2 were significantly lower than C1 and C2. However, there was no significant difference in cumulative mortalities among G3, C1 and C2. In conclusion, based on the present experimental conditions, 75-150 mg/kg of GSH was suggested to be supplemented into the practical diet to improve the growth, anti-oxidative capacity, disease resistance and gut health of shrimp L. vannamei.

Functional characterization of interferon regulatory factor 5 and its role in the innate antiviral immune response.

In mammals, type I interferons (IFNs) are primarily regulated by transcription factors of the IFN regulatory (IRF) family. Interferon regulatory factor 5 (IRF-5) plays pivotal roles in antiviral and inflammatory responses. In the present study, we found that zebrafish (Danio rerio) IRF5 is a key player in the regulation of the expression of type I IFN and its antiviral immune response. IRF5 was upregulated in zebrafish embryonic fibroblast cells (ZF4) when challenged with grass carp reovirus (GCRV). Moreover, the expression profiles of Mx, IFN, Viperin, and IRF7, but not IRF3, were upregulated by overexpression of IRF5 in Epithelioma papulosum cyprinid cells (EPCs). Luciferase assays revealed that the activation of the IFNϕ1 promoter was stimulated by overexpression of IRF5 and IRF5-△IAD (IRF5 lacking the IRF-associated domain), respectively. However, overexpression of IRF5 or IRF5-△IAD inhibited the activity of the IFNϕ3 promoter. IRF5-△DBD (lacking the DNA-binding domain) had no influence in the activation of the IFNϕ1 and IFNϕ3 promoters. Furthermore, the determination of the cytopathic effect (CPE) numbers and viral titers revealed that the viral concentration was reduced by ectopic expression of IRF5 in EPC cells. Ectopic expression of IRF5 in EPC cells could protect cells from GCRV and significantly inhibited GCRV virus replication. These data indicated that IRF5 could limit viral replication through an IFN-dependent pathway.