Immune-enhancing effects of Astragalus polysaccharides and Ganoderma lucidum polysaccharides on Vibrio harveyi flgJ DNA vaccine in grouper.

Astragalus polysaccharides (APS) and Ganoderma lucidum polysaccharides (GLP) have been shown to possess strong immunoregulatory properties in aquatic animals. In this study, the fragment containing Vibrio harveyi flgJ gene was ligated into pcDNA3.1(+) vector and pcDNA3.1(+)-flgJ was constructed as DNA vaccine. APS and GLP were used as DNA vaccine adjuvants to evaluate the immunoregulatory effect by intramuscular injection to pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚E. lanceolatus). The results showed that pcDNA3.1(+)-flgJ combined with APS or GLP could significantly up-regulate the innate and adaptive immune response in fish, including serum-specific antibody titres, catalase and lysozyme activities. At the same time, DNA vaccine combined with APS or GLP significantly up-regulated the expression levels of CD8α, IgM, IL-1ß, MHC-Iα, MyD88 and TLR3 genes in thymus, head kidney, spleen and liver of pearl gentian grouper in comparison with those of the pFlgJ group. After 42 days post-vaccination, V. harveyi was used to challenge pearl gentian grouper by intraperitoneal injection. The relative percentage of survival (RPS) of pFlgJ, pFlgJ +APS, pFlgJ +GLP and pFlgJ+APS+GLP groups were 69%, 81%, 77% and 88%, respectively. These results suggested APS and GLP were potential adjuvants for DNA vaccine against V. harveyi infection in pearl gentian grouper.

Construction of a phosphodiesterase mutant and evaluation of its potential as an effective live attenuated vaccine in pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus).

Vibrio alginolyticus is a dominant pathogen that causes vibriosis of fish and shellfish. VAGM003125 is a specific phosphodiesterase bearing HD-GYP domain, which extensively regulates multicellular behavior and physiological processes in bacteria. In this study, an in-frame deleted ΔVAGM003125 mutant was constructed and changes of ΔVAGM003125 mutant in physiology and pathogenicity were examined. The potential application of ΔVAGM003125 mutant as a live attenuated vaccine was also assessed. The ΔVAGM003125 mutant displayed no significant differences in the growth rate and morphology in comparison to the wild type strain. However, the ΔVAGM003125 mutant significantly enhanced biofilm formation compared to the wild type strain. Also, the ΔVAGM003125 mutant was noted as being able to attenuate swarming motility, ECPase, and adherence compared to the wild type strain. Moreover, the ΔVAGM003125 mutant induced high antibody titers and provided effective immune protection, which was evidenced with a relative survival rate of 81% without histopathological abnormality. Following ΔVAGM003125 mutant vaccination, immune-related genes of pearl gentian grouper (♀Epinephelus fuscoguttatus × â™‚Epinephelus lanceolatus) including IgM, MHC-Iα, IL-16, IL-1, and TNF-α was up-regulated. Taken together, the present data suggested that the ΔVAGM003125 mutant might be applied as an attenuated live vaccination against V. alginolyticus during fish culture.

Ketamine Induces Lasting Antidepressant Effects by Modulating the NMDAR/CaMKII-Mediated Synaptic Plasticity of the Hippocampal Dentate Gyrus in Depressive Stroke Model.

Background: Ketamine has been shown to possess lasting antidepressant properties. However, studies of the mechanisms involved in its effects on poststroke depression are nonexistent. Methods: To investigate these mechanisms, Sprague-Dawley rats were treated with a single local dose of ketamine after middle cerebral artery occlusion and chronic unpredicted mild stress. The effects on the hippocampal dentate gyrus were analyzed through assessment of the N-methyl-D-aspartate receptor/calcium/calmodulin-dependent protein kinase II (NMDAR/CaMKII) pathway, synaptic plasticity, and behavioral tests. Results: Ketamine administration rapidly exerted significant and lasting improvements of depressive symptoms. The biochemical analysis showed rapid, selective upregulation and downregulation of the NMDAR2-ß and NMDAR2-α subtypes as well as their downstream signaling proteins ß-CaMKII and α-phosphorylation in the dentate gyrus, respectively. Furthermore, the colocalization analysis indicated a significant and selectively increased conjunction of ß-CaMKII and postsynaptic density protein 95 (PSD95) coupled with a notable decrease in NMDAR2-ß association with PSD95 after ketamine treatment. These changes translated into significant and extended synaptic plasticity in the dentate gyrus. Conclusions: These findings not only suggest that ketamine represents a viable candidate for the treatment of poststroke depression but also that ketamine's lasting antidepressant effects might be achieved through modulation of NMDAR/CaMKII-induced synaptic plasticity in key brain regions.

[Impacts of Erythropoietin on Vascular Endothelial Growth Factor Receptor 2 by the Extracellular Signal-regulated Kinase Signaling Pathway in a Neonatal Rat Model of Periventricular White Matter Damage].

OBJECTIVE: To explore the impacts of erythropoietin on vascular endothelial growth factor receptor 2 (VEGFR2) by the extracellular signal-regulated kinase (ERK) signaling pathway in a neonatal rat model of periventricular white matter damage. METHODS: All of postnatal day 4 rats were randomized into three groups: the sham group [without hypoxia-ischemia (HI)], the HI group (HI with saline administration), and the erythropoietin (EPO) group [HI with recombinant human erythropoietin (rh-EPO) administration]. Rat pups underwent permanent ligation of the right common carotid artery, followed by 6% O2 for 2 hours or sham operation and normoxic exposure. Immediately after the HI, rats received a single intraventricular injection of rh-EPO (0.6 IU/g body mass) or saline. ERK and phosphorylation-ERK were examined at 60 minutes and 90 minutes after operation, and VEGFR2 were detected at 2 and 4 days after operation by using Western blot. RESULTS: At 60 minutes and 90 minutes after operation, the proteins of phosphorylation-ERK were significantly higher in HI rats than in the sham rats and significantly higher in HI+EPO rats than in the HI rats (P<0.05). Two days after operation, VEGFR2 was not significantly different between sham and HI rats. However, the proteins of VEGFR2 were increased after administration of rh-EPO (P<0.05). Four days after operation, the proteins of VEGFR2 were significantly higher in HI rats than in the sham rats and significantly higher in HI+EPO rats than in the HI rats (P<0.05). CONCLUSION: EPO may regulate VEGFR2 expression by affecting the intracranial ERK signaling pathways.

Design and characterization of Fe(II) complexes with tetradentate ligands exhibiting spin-crossover near room temperature.

Construction of spin-crossover (SCO) materials is very appealing for applications such as molecular switches and information storage. This study focuses on the design of Fe(II) complexes using N,N'-bis(2-pyridinylmethyl)-1,2-ethanediamine-based ligands with an N4 structure for SCO material development. By incorporating para-substituted benzene groups into the ligand's pyridine moiety, two polymorphs, α and ß, were obtained, both exhibiting SCO activity. Notably, the ß polymorph displayed a spin crossover temperature of 270 K, which is approaching room temperature. Structural analyses were conducted to compare the differences between the polymorphs, along with a literature review of related complexes, providing insights into the characteristics of SCO behavior.

Characterization of fliR-deletion mutant ΔfliR from Vibrio alginolyticus and the evaluation as a live attenuated vaccine.

Vibrio alginolyticus is the common pathogen affecting various species of marine organisms. It has been demonstrated that fliR is a necessary virulence factor to adhere and infect their hosts for pathogenic bacteria. Frequent disease outbreaks in aquaculture have highlighted the necessity of developing effective vaccines. In the present study, in order to investigate the function of fliR in V.alginolyticus, the fliR deletion mutant ΔfliR was constructed and its biological properties were evaluated, additionally, the differences in gene expression levels between wild-type and ΔfliR were analyzed by transcriptomics. Finally, ΔfliR was used as a live attenuated vaccine to immunize grouper via the intraperitoneal route to evaluate its protective effect. Results show that fliR gene of V. alginolyticus was identified as being 783 bp in length, encoding 260 amino acids, and showing significant similarity to homologs of other Vibrio species. The fliR-deletion mutant ΔfliR of V. alginolyticus was successfully constructed, and its biological phenotype analysis showed no significant differences in growth capacity and extracellular enzyme activity compared to the wild-type. However, a substantial reduction of motility ability was detected in ΔfliR. Transcriptomic analysis revealed that the absence of fliR gene is responsible for a significantly decreased expression of flagellar genes, including flaA, flaB, fliS, flhB and fliM. The fliR-deletion mainly affects the related pathways involved in cell motility, membrane transport, signal transduction, carbohydrate metabolism, and amino acid metabolism in V. alginolyticus. The efficacy of ΔfliR as a candidate of live attenuated vaccine were evaluated by intraperitoneal injection in grouper. The ΔfliR provided the RPS (Relative protection rate) of 67.2% against V. alginolyticus in groupers. The ΔfliR efficiently stimulated antibody production with specific IgM still detected at 42 d post-vaccination, and significantly elevated the activity of antioxidant enzymes like Catalase (CAT), Superoxide dismutase (SOD), and lactate dehydrogenase (LDH) in the serum. The higher expression levels of immune-related genes were observed in the immune tissues of inoculated grouper compared to the control. In conclusion, ΔfliR effectively improved the immunity of inoculated fish. The results suggest that ΔfliR is an effective live attenuated vaccine against vibriosis in in grouper.

Complete mitochondrial genome of broadbanded cardinalfish (Ostorhinchus fasciatus) and phylogenetic analysis.

In this study, the complete mitochondrial genome (mitogenome) of Ostorhinchus fasciatus was first determined and its phylogenetic position was investigated. The mitogenome was 16568 bp long and showed a typical teleost orders, containing 13 protein-coding genes (PCGs), 2 ribosome RNA genes (rRNAs), 22 transfer RNA genes (tRNAs), and a D-loop region. The overall nucleotide composition included A, 25.89%; C, 30.40%; G, 17.46%; and T, 26.26%. Except for nad6 was located on the light strand, the other PCGs were encoded on the heavy strand. Phylogenetic analysis suggested that O. fasciatus shared a close relationship with Sphaeramia orbicularis and Pterapogon kauderni.

Leukemia Inhibitory Factor Is Essential for the Self-Renewal of Embryonic Stem Cells from Nile Tilapia (Oreochromis niloticus) Through Stat3 Signaling.

To date, little is known about the mechanisms underlying the self-renewal of embryonic stem (ES) cells from fish species. In this study, we report that the leukemia inhibitory factor (LIF; named as OnLif) from a teleost fish, Nile tilapia (Oreochromis niloticus), is essential for the proliferation, survival, and pluripotency maintenance of Nile tilapia ES cells (TES1) by activating the signal transducer and activator of transcription 3 (Stat3). This protein has 221 amino acid residues with similar sequence features to mammalian LIF. By fusing to a small ubiquitin-related modifier and inducing expression at 16°C, the soluble tag-free protein had been successfully obtained. Further investigation indicates that OnLif could significantly enhance the proliferation and survival of TES1. Moreover, it contributed to the pluripotency maintenance of TES1 characteristic of high expression of pluripotency genes, no or low expression of differentiation genes, and strong alkaline phosphatase activity. Notably, it mediated Stat3 phosphorylation, whose inhibitor treatment could lead to apoptosis. In addition, OnLif significantly enhanced the proliferation of ES cells from medaka (Oryzias latipes), suggesting its potential role in other fish ES cells. These data first suggest that Lif/Stat3 signaling has an essential role in the self-renewal of ES cells from fish, just like that in the ground state pluripotency maintenance of mouse and human ES cells. Our study would not only be helpful for the understanding of molecular mechanisms underlying self-renewal of ES cells from the perspective of evolution but also facilitate ES-based biotechnology application in fishery.

Both Gfrα1a and Gfrα1b Are Involved in the Self-renewal and Maintenance of Spermatogonial Stem Cells in Medaka.

Glial cell-derived neurotrophic factor family receptor alpha-1 (GFRα1) plays a crucial role in the self-renewal and maintenance of spermatogonial stem cells (SSCs) from mammals. However, to date, our knowledge about its role in fish SSCs is limited. In the present study, the medaka (Oryzias latipes) gfrα1 duplicate genes, Olgfrα1a and Olgfrα1b, were cloned and characterized. Furthermore, their expression profile and biological activity were investigated. OlGfrα1a and OlGfrα1b predict 524 and 466 amino acid residues, respectively. Both are orthologous to mammalian Gfrα1 by sequence analyses and appear high in spermatogonia by in situ hybridization assay. The knockdown of OlGfrα1a and/or OlGfrα1b via Vivo-Morpholino oligos significantly inhibited the self-renewal and maintenance of SSCs, as evidenced by the decreased proliferation activity of SG3 cells (a spermatogonial stem cell line derived from adult medaka testis) as well as spermatogonia in the testicular organ culture and by the decreased survival rate and expression levels of pluripotency-related genes (klf4, lin28b, bcl6b, and etv5) in SG3 cells. Additionally, our study indicates that OlGfrα1a might function by binding either Gdnfa or Gdnfb (the two medaka Gdnf homologs), whereas OlGfrα1b function by binding Gdnfa not Gdnfb. Taken together, our study indicates that both OlGfrα1a and OlGfrα1b are involved in the self-renewal and maintenance of SSCs by binding Gdnfa and/or Gdnfb, respectively. These findings suggest that the GDNF/GFRα1 signaling pathway might be conserved from mammals to fish species.

Recombinant Human Erythropoietin Augments Neovascularization Responses in a Neonatal Rat Model of Premature Brain Damage by Phosphatidylinositol 3 Kinase/Akt Pathway.

BACKGROUND: Recombinant human-erythropoietin (rh-EPO) has therapeutic efficacy for premature infants with brain damage during the active rehabilitation and anti-inflammation. In the present study, we found that the rh-EPO was related to the promotion of neovascularization. Our aim was to investigate whether rh-EPO augments neovascularization in the neonatal rat model of premature brain damage through the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) signaling pathway. METHODS: Postnatal day 5 (PD5), rats underwent permanent ligation of the right common carotid artery and were exposed to hypoxia for 2 h. All the rat pups were randomized into five groups as follows: (1) control group; (2) hypoxia-ischemic (HI) group; (3) HI + LY294002 group; (4) HI + rh-EPO group; and (5) HI + rh-EPO + LY294002 group. The phospho-Akt protein was tested 90 min after the whole operation, and CD34, vascular endothelial growth factor receptor 2 (VEGFR2), and vascular endothelial growth factor (VEGF) were also tested 2 days after the whole operation. RESULTS: In the hypoxic and ischemic zone of the premature rat brain, the rh-EPO induced CD34+ cells to immigrate to the HI brain zone (P < 0.05) and also upregulated the VEGFR2 protein expression (P < 0.05) and VEGF mRNA level (P < 0.05) through the PI3K/Akt (P < 0.05) signaling pathway when compared with other groups. CONCLUSIONS: The rh-EPO treatment augments neovascularization responses in the neonatal rat model of premature brain damage through the PI3K/Akt signaling pathway. Besides, the endogenous EPO may exist in the HI zone of rat brain and also has neovascularization function through the PI3K/Akt signaling pathway.

Corticosteroid therapy in ulcerative colitis: Clinical response and predictors.

AIM: To evaluate clinical response to initial corticosteroid (CS) treatment in Chinese ulcerative colitis patients (UC) and identify predictors of clinical response. METHODS: Four hundred and twenty-three UC patients who were initially treated with oral or intravenous CS from 2007 to 2011 were retrospectively reviewed at eight inflammatory bowel disease centers in China, and 101 consecutive cases with one-year follow-up were analyzed further for clinical response and predictors. Short-term outcomes within one month were classified as primary response and primary non-response. Long-term outcomes within one year were classified as prolonged CS response, CS dependence and secondary non-response. CS refractoriness included primary and secondary non-response. Multivariate analyses were performed to identify predictors associated with clinical response. RESULTS: Within one month, 95.0% and 5.0% of the cases were classified into primary response and non-response, respectively. Within one year, 41.6% of cases were assessed as prolonged CS response, while 49.5% as CS dependence and 4.0% as secondary non-response. The rate of CS refractoriness was 8.9%, while the cumulative rate of surgery was 6.9% within one year. After multivariate analysis of all the variables, tenesmus was found to be a negative predictor of CS dependence (OR = 0.336; 95%CI: 0.147-0.768; P = 0.013) and weight loss as a predictor of CS refractoriness (OR = 5.662; 95%CI: 1.111-28.857; P = 0.040). After one-month treatment, sustained high Sutherland score (≥ 6) also predicted CS dependence (OR = 2.347; 95%CI: 0.935-5.890; P = 0.014). CONCLUSION: Tenesmus was a negative predictor of CS dependence, while weight loss and sustained high Sutherland score were strongly associated with poor CS response.

MicroRNA-185 suppresses growth and invasion of colon cancer cells through inhibition of the hypoxia­inducible factor-2α pathway in vitro and in vivo.

MicroRNAs (miRs) are small non­coding RNAs with regulatory roles, which are involved in a broad spectrum of physiological and pathological processes, including cancer development and progression. However, the function of miR­185 in the development of human colon cancer has not yet been investigated. In this study, the association between miR­185 expression and the clinicopathological characteristics of patients with colon cancer was analyzed using quantitative polymerase chain reaction (qPCR). Using a gain­of­function approach, the effects of miR­185 overexpression on the expression of hypoxia­inducible factor­2α (HIF­2α), proliferating cell nuclear antigen (PCNA) and matrix metallopeptidase­2 (MMP­2) were investigated in SW620 colon cancer cells using qPCR and western blotting. Functional analysis of cellular proliferative activities, by MTT assay, and invasive potential, by Transwell assay, was conducted on SW620 cells expressing low levels of miR­185. miR­185 was found to be significantly downregulated in cancer tissues compared with adjacent non­cancerous tissues, and was negatively correlated with lymph node metastasis of colon cancer (P<0.001). miR­185 overexpression in vitro impeded cellular proliferation and invasive potential with reduced expression of HIF­2α, PCNA and MMP­2 in SW620 cells transfected with an miR­185 mimic. In addition, the tumor volumes in SW620 subcutaneous nude mouse models treated with miR­185 were significantly smaller than those of the control group. In conclusion, these findings indicate that miR­185 as a tumor suppressor may affect the development of colon cancer cells via inhibition of HIF­2α signaling, suggesting that miR­185 may serve as a potential therapeutic target in cancer treatment.

Recovery from TNBS-induced colitis leads to the resistance to recurrent colitis and an increased ratio of FOXP3 to CD3 mRNA.

OBJECTIVE: The aim of our study was to investigate the effects of the recovery from acute colitis on recurrent colitis with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice. METHODS: Acute colitis was induced via an intrarectal injection of TNBS. For recurrent colitis, mice were intrarectally treated with a repeated TNBS 14 days after the first TNBS administration. And another two groups (control and recovery groups) were added to the analysis. Disease activity index (DAI), macroscopic and histological assessments, mRNA expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor (TNF), IL-10, forkhead box P3 (FOXP3) and the ratio of FOXP3 to CD3 in the colonic tissues were evaluated. RESULTS: Mice developed colitis after treated with TNBS. After the last TNBS administration, DAI in the recurrent colitis group was lower than that in the acute colitis group. In the recurrent colitis group, the mice exhibited longer colon length, reduced histological damage and lower IL-1ß, IL-6, TNF, IL-10 mRNA expression in the colon compared with the acute colitis group. The ratio of FOXP3 to CD3 mRNA expression in the colon of recurrent colitis was higher than that in the acute colitis. There was a significant negative correlation between the ratio of FOXP3 to CD3 mRNA expression and DAI in the acute and recurrent colitis groups (r= -0.808, P<0.05). CONCLUSIONS: Mice that recovered from TNBS-induced acute colitis with intestinal epithelial disruption are resistant to recurrent colitis, which is associated with an increased ratio of FOXP3 to CD3 mRNA expression.

BMP2 induces PANC-1 cell invasion by MMP-2 overexpression through ROS and ERK.

The emerging roles of bone morphogenetic proteins (BMPs) in the initiation and progression of multiple cancers have drawn great attention in cancer research. We hypothesized that BMP2 promotes cancer metastasis by modulating MMP-2 secretion and activity through intracellular ROS regulation and ERK activation in human pancreatic cancer. Our data show that stimulation of PANC-1 cells with BMP2 induced MMP-2 secretion and activation, associated with decreased E-cadherin expression, resulting in epithelial-to-mesenchymal transformation (EMT) and cell invasion. Blockade of ROS by the ROS scavenger, 2-MPG, abolished cell invasion, inhibited the EMT process and decreased MMP-2 expression, suggesting ROS accumulation caused an increase in MMP-2 expression in BMP2-stimulated PANC-1 cell invasion. Furthermore, treatment of PANC-1 cells with 2-MPG or ERK inhibitor PD98059 reduced the phosphorylation of ERK, resulting in attenuation of BMP2-induced cell invasion and MMP-2 activation. Taken together, these results suggest that BMP2 induces the cell invasion of PANC-1 cells by enhancing MMP-2 secretion and acting through ROS accumulation and ERK activation.

The origins of vacularization in tumors.

Vascularization is crucial for tumor growth and metastasis. Angiogenesis and vasculogenesis are widely accepted processes of tumor vascularization, particularly for endothelium-dependent vessels. In both these processes, the tumor vascular endothelial cells are derived from the host cells, including cells in normal tissues around the tumor or endothelial progenitor cells. In addition, the mosaic vessels occur as a transitional pattern between endothelium-dependent vessels and vasculogenic mimicry (VM), wherein both host endothelium and tumor cells participate in tumor vascularization. VM provides a special passage not involving endothelial cells and is conspicuously different from angiogenesis and vasculogenesis. The biological features of the tumor cells that form VM remain unknown. Tumor stem cells may participate in VM. In this review, we discuss the patterns involved in the origin of vascularization in tumors.

MicroRNA155 is induced in activated CD4(+) T cells of TNBS-induced colitis in mice.

AIM: To investigate the expression of microRNA155 (miRNA155) in trinitrobenzene sulphonic acid (TNBS)-induced colitis and the relationship between miRNA155 and tumor necrosis factor (TNF) expressions. METHODS: In TNBS colitis mice, miRNA155 and TNF mRNA expressions were measured in colons and CD4(+) T cells of draining lymph nodes (LNs). CD4(+) T cells were cultured in vitro with or without anti-CD3/CD28 antibody, and the expressions of miRNA155 and TNF mRNA in cells and TNF concentration in culture media were examined. RESULTS: miRNA155 and TNF mRNA expressions in colons and in cells of LNs were significantly increased in TNBS colitis compared with controls. In TNBS colitis, miRNA155 and TNF mRNA expressions in CD4(+) T cells of LNs and TNF concentration in CD4(+) T cells culture media increased compared with controls. When cultured with anti-CD3/CD28 antibody, miRNA155 and TNF mRNA expressions in CD4(+) T cells and TNF concentration in the CD4(+) T cells culture media were significantly higher than those cultured without anti-CD3/CD28 antibody. Following analysis using the Pearson's correlation coefficient, miRNA155 expression had a significant positive correlation with either TNF mRNA expression in CD4(+) T cells (r = 0.860, P < 0.05) or TNF concentration in CD4(+) T cells culture media (r = 0.892, P < 0.05). CONCLUSION: miRNA155 is induced in colons and activated CD4(+) T cells in TNBS colitis, and the levels of miRNA155 and TNF expressions have a significant positive correlation.