RESUMO
Plants deploy versatile scaffold proteins to intricately modulate complex cell signaling. Among these, RACK1A (Receptors for Activated C Kinase 1A) stands out as a multifaceted scaffold protein functioning as a central integrative hub for diverse signaling pathways. However, the precise mechanisms by which RACK1A orchestrates signal transduction to optimize seedling development remain largely unclear. Here, we demonstrate that RACK1A facilitates hypocotyl elongation by functioning as a flexible platform that connects multiple key components of light signaling pathways. RACK1A interacts with PHYTOCHROME INTERACTING FACTOR (PIF)3, enhances PIF3 binding to the promoter of BBX11 and down-regulates its transcription. Furthermore, RACK1A associates with ELONGATED HYPOCOTYL 5 (HY5) to repress HY5 biochemical activity toward target genes, ultimately contributing to hypocotyl elongation. In darkness, RACK1A is targeted by CONSTITUTIVELY PHOTOMORPHOGENIC (COP)1 upon phosphorylation and subjected to COP1-mediated degradation via the 26 S proteasome system. Our findings provide new insights into how plants utilize scaffold proteins to regulate hypocotyl elongation, ensuring proper skoto- and photo-morphogenic development.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Hipocótilo , Receptores de Quinase C Ativada , Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Receptores de Quinase C Ativada/metabolismo , Receptores de Quinase C Ativada/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Transdução de Sinais , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Transdução de Sinal Luminoso , FosforilaçãoRESUMO
Nocardia seriolae is the primary pathogen causing nocardiosis in various fish species, leads to significant economic losses in the aquaculture industry. In this study, 10 bacterial strains isolated from Micropterus salmoides and Channa argus infected with nocardiosis, were identified as N. seriolae by physiological and biochemical identification, as well as 16S rDNA sequencing. Moreover, the key virulence-related genes such as ESX-1, T7SS-2, T7SS-3, EspG1, sodC, sod2 and ESAT6 were all positive, and showing high homology among different strains. Pathogenicity testing revealed mortality rates ranging from 70 to 100%, accompanied by the presence of white nodules in the viscera of deceased fish. The drug sensitivity test demonstrated that LY21811, the most lethal strain, exhibited high sensitivity to nine types of antibiotics, including azithromycin, doxycycline, florfenicol and compound sulfamethoxazole, yet showed complete resistance to ß-lactam antibiotics. Additionally, the tannic acid also demonstrated potent inhibitory effects against LY21811, with a minimum inhibitory concentration of 0.0625 mg/mL. These results showed that N. seriolae originated from M. salmoides and C. argus in Zhejiang Province were highly conserved, demonstrating a high homogeneity in genetic characteristics, pathogenicity and antimicrobial susceptibilities. These results provide a foundation for further research on the pathogenic characteristics and disease prevention of N. seriolae infections.
RESUMO
Objective@#To investigate the prevalence of major chronic diseases among residents in Xiamen City, Fujian Province, so as to provide insights into enhancing the prevention and control of chronic diseases.@*Methods@#The residents aged 18 years and older and lived for 6 months and more were selected from 6 districts in Xiamen City from 2021 to 2022 using the stratified random sampling method. Demographic information, disease history, blood pressure, fasting blood glucose and blood cholesterol were collected through questionnaire surveys, physical examination and laboratory tests. The prevalence rates of hypertension, diabetes and hypercholesterolemia were calculated and standardized using the 2020 National Population Census data, and were analyzed by age and gender.@*Results@#Totally 22 289 residents were investigated, including 10 444 males (46.86%) and 11 845 females (53.14%). The prevalence rates of hypertension, diabetes and hypercholesterolemia were 15.35%, 4.30% and 10.45%, and the standardized prevalence rates were 16.25%, 4.81% and 10.21%, respectively. The standardized prevalence rates of hypertension, diabetes and hypercholesterolemia in males were 19.20%, 5.71% and 9.97%, respectively, and were higher than those in females, which were 13.62%, 4.03% and 10.29%, respectively (all P<0.05). The crude prevalence rates of hypertension and diabetes in males aged 18 to 59 years were higher than those in females, the crude prevalence rate of hypercholesterolemia in males aged 18 to 49 years was higher than that in females, and the crude prevalence rate of hypercholesterolemia aged 50 years and over in males was lower than that in females (all P<0.05).@*Conclusion@#From 2017 to 2022, the prevalence rates of hypertension, diabetes, and hypercholesterolemia in males were higher than those in females in Xiamen City, while the prevalence rate of hypercholesterolemia in females was higher than that in males after the age of 50 years.
RESUMO
Macrobrachium rosenbergii Taihu virus (MrTV) is a virulent pathogen that mainly threatens M. rosenbergii larvae. Rab proteins, which are essential for controlling intracellular membrane trafficking, are hijacked by multiple viruses to complete their life cycle. In this paper, we studied the function of M. rosenbergii Rab1A (MrRab1A) in the MrTV infection. Upon MrTV infection, the transcription level of MrRab1A was significantly up-regulated, indicating MrRab1A was a MrTV responsive gene and might be important for MrTV infection. Co-IP and co-localization assays revealed that MrRab1A could directly bind with MrTV and its capsid protein VP3. Moreover, the in vivo neutralization assay demonstrated that pre-incubation of MrTV with recombinant MrRab1A could partially block MrTV infection. These findings indicated that MrRab1A functioned as a virus-binding protein involved in MrTV infection, which shed new light on the mechanism of MrTV infection and provided a potential target for developing anti-MrTV therapies.
Assuntos
Palaemonidae , Viroses , Animais , Palaemonidae/genética , Proteínas de Transporte , Proteínas do Capsídeo/genética , Proteínas ViraisRESUMO
Nucleic acid sequence-based amplification (NASBA) provides a fast and convenient approach for nucleic acid amplification under isothermal conditions, and its combination with an immunoassay-based lateral flow dipstick (LFD) could produce a higher detection efficiency for M. rosenbergii nodavirus isolated from China (MrNV-chin). In this study, two specific primers and a labelled probe of the capsid protein gene of MrNV-chin were constructed. The process of this assay mainly included a single-step amplification at a temperature of 41 â for 90 min, and hybridization with an FITC-labeled probe for 5 min, with the hybridization been required for visual identification during LFD assay. The test results indicated that, the NASBA-LFD assay showed sensitivity for 1.0 fg M. rosenbergii total RNA with MrNV-chin infection, which was 104 times that of the present RT-PCR approach for the detection of MrNV. In addition, no products were created for shrimps with infection of other kinds of either DNA or RNA virus, which indicated that the NASBA-LFD was specific for MrNV. Therefore, the combination of NASBA and LFD is a new alternative detection method for MrNV which is rapid, accurate, sensitive and specific without expensive equipment and specialised personnel. Early detection of this infectious disease among aquatic organisms will help implement efficient therapeutic strategy to prevent its spread, enhance animal health and limit loss of aquatic breeds in the event of an outbreak.
Assuntos
Nodaviridae , Palaemonidae , Vírus de RNA , Animais , Replicação de Sequência Autossustentável , Nodaviridae/genética , Vírus de RNA/genética , Técnicas de Amplificação de Ácido Nucleico/métodosRESUMO
Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/µl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.
Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Ranavirus , Animais , Ranavirus/genética , Reprodutibilidade dos Testes , Infecções por Vírus de DNA/diagnóstico , Infecções por Vírus de DNA/veterinária , Reação em Cadeia da Polimerase/veterinária , Reação em Cadeia da Polimerase/métodos , Proteínas do Capsídeo/genéticaRESUMO
Thiourea derivatives are expected to be potential monomers of As(Ш) molecular imprinted polymers (MIPs) which are used to specifically recognize As(Ш). However, the specific recognition and binding mechanisms between template and monomers are unclear, which limits the practical applications of MIPs in As(Ш)detection. In this work, density functional theory (DFT) calculations, molecular dynamics (MD) simulations and experimental methods were jointly applied to explore the binding interactions between H3AsO3 and thiourea derivatives and environmental factors influences, aiming to find out the best monomer and optimal preparation conditions for H3AsO3 MIPs. Among five monomer candidates, (2, 6-difluorophenyl) thiourea (FT) was calculated to be the most potential one, while allyl thiourea (AT) was the second choice. Configurations of the most stable binding complexes were found out. The optimal solvent was found to be toluene and the bindings were more favorable at pH 7.5 in aqueous solution. Besides, EGDMA was proved as the best cross-linker with the optimal ratio of template: monomer: cross-linker= 2:3:20. Moreover, the binding interactions were identified to be hydrogen bonds, and the non-covalent nature was revealed. These findings provide references for efficient design and preparation of good-performance H3AsO3 MIPs, which can be used to detect and remove As(Ш) from environment.
Assuntos
Impressão Molecular , Polímeros , Ligação de Hidrogênio , Impressão Molecular/métodos , Polímeros/química , Solventes , Tioureia , ÁguaRESUMO
In this study, we established and characterized a continuous cell line from the spinal cord tissue of mandarin fish, Siniperca chuatsi and assessed its susceptibility to infectious spleen and kidney necrosis virus (ISKNV), Siniperca chuatsi ranavirus (SCRaV) and Siniperca chuatsi rhabdovirus (SCRV). The cell line, named SCC, has been successively cultured up to 40 passages. The optimal growing conditions of SCC cells were in Leibovitz's L-15 medium supplemented with 20% foetal bovine serum (FBS) at 28°C. Karyotype analysis demonstrated 48 normal diploid chromosomes in the cells. The identity of S. chuatsi origin of SCC cells was confirmed by partial sequencing of the 16S rRNA and cytochrome oxidase I (COI) genes. Infection susceptibility assessment showed that ISKNV, SCRIV and SCRV and can be stably produced and transmitted in SCC cells, and the replication efficiency of ISKNV, SCRaV and SCRV ranged from 107.4 to 109.6 TCID50 /ml. In addition, transmission electron microscopy analysis of ISKNV, SCRAV and SCRV infected SCC cells showed numerous viral particles. In conclusion, the newly established SCC cells provide an important tool for isolation and production of viruses, as well as for molecular and cell biology studies.
Assuntos
Infecções por Vírus de DNA , Doenças dos Peixes , Iridoviridae , Perciformes , Rhabdoviridae , Animais , Linhagem Celular , Peixes/genética , Iridoviridae/genética , Perciformes/genética , RNA Ribossômico 16S , Rhabdoviridae/genética , Medula EspinalRESUMO
Largemouth bass iridovirus (LMBV) can cause high mortality and lead to heavy economic loss in the cultivation of largemouth bass, but there was no effective treatment. Here, the present study constructed a recombinant Pichia pastoris expressing LMBV major capsid protein (MCPD). The recombinant GS115-pW317-MCPD was then used to immunize largemouth bass via oral administration, and mucosal immune response mediated by immunoglobulins (Igs) was measured after oral immunization. Serum antibody levels were measured by ELISA, neutralizing antibody titers were determined by serum neutralization test (SNT), antigen presentation-related gene expressions were detected by RT-PCR, and the histopathological characteristics of immunized fish were assessed after challenging with 0.1 ml 107.19 TCID50/ml LMBV. The relative percentage survival (RPS) was also determined. Our results showed that the serum antibody titers of immunized fish were significantly higher than that of control groups (P < 0.05). IgT and IgM expressions in gut were increased significantly after vaccination with GS115-pW317-MCPD; however, much stronger response in gut was observed as compared with gill. The expression levels of major histocompatibility complex (MHC) II, CD8, and T-cell receptor (TCR) were significantly elevated in GS115-pW317-MCPD group (P < 0.05), while CD4 and MHC I transcription levels remained unchanged after oral immunization (P > 0.05). The RPS of fish orally immunized with 1.0 × 108 CFU/g GS115-pW317-MCPD was reached up to 41.6% after challenge with 0.1 ml 109.46 TCID50/ml LMBV. Moreover, orally immunizing with GS115-pW317-MCPD can relieve the pathological damage caused by LMBV. Therefore, GS115-pW317-MCPD showed a promising potential against LMBV.
Assuntos
Bass , Infecções por Vírus de DNA , Doenças dos Peixes , Iridovirus , Animais , Proteínas do Capsídeo/genética , Doenças dos Peixes/prevenção & controle , Pichia/genética , Saccharomycetales , VacinaçãoRESUMO
Climate change disproportionately affects natural resource-dependent communities in the ecologically vulnerable regions of western China. This study used the household livelihood vulnerability index under the Intergovernmental Panel on Climate Change (HLV-IPCC) to assess vulnerability. Data were collected from 823 households in Ningxia, Gansu, Guangxi, and Yunnan provinces, these being ecologically vulnerable regions in China. With a composite HLVI-IPCC and multiple regression model, the factors that affect households' adaptive capability to HLVI-IPCC was estimated. Results indicate that Ningxia is the most vulnerable community, while Guangxi is the least vulnerable community across all indices. Moreover, Gansu has the heaviest sensitivity and exposure to climate change, whereas Ningxia has the highest adaptive capability to climate change. In addition, the age of household head and distance of the home to the town center had significant negative impacts on households' adaptive capacity to HLVI-IPCC. The results also suggest that the HLVI assessment can provide an effective tool for local authorities to formulate prioritizing strategies with promoting climate-resilient development and increasing long-term adaptive capacity.
Assuntos
Mudança Climática , Características da Família , China , CidadesRESUMO
With no lysine (K) (WNK) kinases comprise a family of serine/threonine kinases belonging to an evolutionary branch of the eukaryotic kinome. These special kinases contain a unique active site and are found in a wide range of eukaryotes. The model plant Arabidopsis has been reported to have 11 WNK members, of which WNK8 functions as a negative regulator of abscisic acid (ABA) signaling. Here, we found that the expression of WNK8 is post-transcriptionally regulated through an upstream open reading frame (uORF) found in its 5' untranslated region (5'-UTR). This uORF has been predicted to encode a conserved peptide named CPuORF58 in both monocotyledons and dicotyledons. The analysis of the published ribosome footprinting studies and the study of the frameshift CPuORF58 peptide with altered repression capability suggested that this uORF causes ribosome stalling. Plants transformed with the native WNK8 promoter driving WNK8 expression were comparable with wild-type plants, whereas the plants transformed with a similar construct with mutated CPuORF58 start codon were less sensitive to ABA. In addition, WNK8 and its downstream target RACK1 were found to synergistically coordinate ABA signaling rather than antagonistically modulating glucose response and flowering in plants. Collectively, these results suggest that the WNK8 expression must be tightly regulated to fulfill the demands of ABA response in plants.
Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fases de Leitura Aberta , Biossíntese de Proteínas , Proteínas Serina-Treonina Quinases/genética , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Germinação/genética , Desenvolvimento Vegetal/genética , Proteínas Serina-Treonina Quinases/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Transdução de Sinais , SinteniaRESUMO
Macrobrachium rosenbergii Taihu virus (MrTV) is a fierce pathogen that causes high mortality in M. rosenbergii larvae. Little is known about the pathogenesis of MrTV and host-virus interactions. In this study, a virus overlay protein binding assay (VOPBA), followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, was carried out to search for novel host molecules that bind with VP3, one of the main capsid proteins of MrTV. Macrobrachium rosenbergii 14-3-3 protein (Mr14-3-3) was identified as the binding protein of VP3, which was further confirmed by co-immunoprecipitation (Co-IP) and co-localization assay. A preincubation assay was developed, which indicated that preincubation with recombinant Mr14-3-3 (rMr14-3-3) could significantly decrease the expression level of VP3 in MrTV-infected M. rosenbergii larvae, suggesting that preincubation with rMr14-3-3 could partially block MrTV infection. This study revealed that Mr14-3-3 acts as a binding protein for MrTV-VP3 and plays an important role in MrTV infection, offering a potential target for the development of anti-MrTV therapies.
Assuntos
Proteínas 14-3-3/metabolismo , Proteínas do Capsídeo/metabolismo , Dicistroviridae/imunologia , Palaemonidae/imunologia , Viroses/imunologia , Proteínas 14-3-3/genética , Animais , Cromatografia Líquida , Interações entre Hospedeiro e Microrganismos/imunologia , Larva/virologia , Palaemonidae/virologia , Espectrometria de Massas em Tandem , Viroses/mortalidadeRESUMO
Carbonic anhydrases (CAs) are ubiquitous zinc metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. Higher plants mainly contain the three evolutionarily distinct CA families αCA, ßCA, and γCA, with each represented by multiple isoforms. Alternative splicing (AS) of the CA transcripts is common. However, there is little information on the spliced variants of individual CA isoforms. In this study, we focused on the characterization of spliced variants of ßCA1 from Arabidopsis. The expression patterns and subcellular localization of the individual spliced variants of ßCA1 were examined. The results showed that the spliced variants of ßCA1 possessed different subcellular and tissue distributions and responded differently to environmental stimuli. Additionally, we addressed the physiological role of ßCA1 in heat stress response and its protein-protein interaction (PPI) network. Our results showed that ßCA1 was regulated by heat stresses, and ßca1 mutant was hypersensitive to heat stress, indicating a role for ßCA1 in heat stress response. Furthermore, PPI network analysis revealed that ßCA1 interacts with multiple proteins involved in several processes, including photosynthesis, metabolism, and the stress response, and these will provide new avenues for future investigations of ßCA1.
RESUMO
The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.
Assuntos
Adesinas Bacterianas/imunologia , Vacinas Bacterianas/imunologia , Doenças dos Peixes/imunologia , Imunogenicidade da Vacina , Vibrioses/veterinária , Vibrio mimicus/imunologia , Animais , Peixes-Gato , Vacinas de Subunidades Antigênicas/imunologia , Vibrioses/imunologiaRESUMO
Citrobacter freundii refers to a fish pathogen extensively reported to be able to cause injury and high mortality. Phage therapy is considered a process to alternatively control bacterial infections and contaminations. In the present study, the isolation of a virulent bacteriophage IME-JL8 isolated from sewage was presented, and such bacteriophage was characterized to be able to infect Citrobacter freundii specifically. Phage IME-JL8 has been classified as the member of the Siphoviridae family, which exhibits the latent period of 30-40 min. The pH and thermal stability of phage IME-JL8 demonstrated that this bacteriophage achieved a pH range of 4-10 as well as a temperature range of 4, 25, and 37°C. As revealed from the results of whole genomic sequence analysis, IME-JL8 covers a double-stranded genome of 49,838 bp (exhibiting 47.96% G+C content), with 80 putative coding sequences contained. No bacterial virulence- or lysogenesis-related ORF was identified in the IME-JL8 genome, so it could be applicable to phage therapy. As indicated by the in vitro experiments, phage IME-JL8 is capable of effectively removing bacteria (the colony count decreased by 6.8 log units at 20 min), and biofilm can be formed in 24 h. According to the in vivo experiments, administrating IME-JL8 (1 × 107 PFU) was demonstrated to effectively protect the fish exhibiting a double median lethal dose (2 × 109 CFU/carp). Moreover, the phage treatment led to the decline of pro-inflammatory cytokines in carp with lethal infections. IME-JL8 was reported to induce efficient lysis of Citrobacter freundii both in vitro and in vivo, thereby demonstrating its potential as an alternative treatment strategy for infections attributed to Citrobacter freundii.
RESUMO
At present, there is an abundance of quality domestic rabbit breeds in China. However, due to the lack of technical standards for the genetic evaluation of rabbit germplasm resources, there have been a number of problems, such as poor breed conservation. By studying the genetic diversity of 130 New Zealand white rabbits (regardless of generation), we obtained the best simple sequence repeat (SSR) marker combination. We found that, when using microsatellite markers for the effective genetic evaluation of domestic rabbits, the number of records should be greater than 60 and the marker number more than 22. Through the comparative analysis of 30 combinations of 22 markers, the optimal combination of 22 markers was determined, and the 22 SSR polymorphic loci were distributed on different chromosomes. We performed a genetic analysis of 200 New Zealand white rabbits corresponding to two generations, using the best SSR polymorphic loci combination. There were no significant differences in the genetic diversity parameters between the two generations of rabbits (p > 0.05), indicating that the characteristics of this excellent rabbit germplasm have been effectively preserved. At the same time, we verified that the established method can be used to evaluate the breed conservation of rabbit germplasm resources.
RESUMO
BACKGROUND: Melanocytes play an extremely important role in the process of skin and coat colors in mammals which is regulated by melanin-related genes. Previous studies have demonstrated that KIT is implicated in the process of determining the color of the coat in Rex rabbits. However, the effect of KIT on the proliferation and apoptosis of melanocytes and melanogenesis has not been clarified. METHODS: The mRNA and protein expression levels of KIT were quantified in different coat colored rabbits by qRT-PCR and a Wes assay. To identify whether KIT functions by regulating of melanogenesis, KIT overexpression and knockdown was conducted in melanocytes, and KIT mRNA expression and melanin-related genes TYR, MITF, PMEL and DCT were quantified by qRT-PCR. To further confirm whether KIT influences melanogenesis in melanocytes, melanin content was quantified using NaOH lysis after overexpression and knockdown of KIT. Melanocyte proliferation was estimated using a CCK-8 assay at 0, 24, 48 and 72 h after transfection, and the rate of apoptosis of melanocytes was measured by fluorescence-activated cell sorting. RESULTS: KITmRNA and protein expression levels were significantly different in the skin of Rex rabbits with different color coats (P < 0.05), the greatest levels observed in those with black skin. The mRNA expression levels of KIT significantly affected the mRNA expression of the pigmentation-related genes TYR, MITF, PMEL and DCT (P < 0.01). Melanin content was evidently regulated by the change in expression patterns of KIT (P < 0.01). In addition, KIT clearly promoted melanocyte proliferation, but inhibited apoptosis. CONCLUSIONS: Our results reveal that KIT is a critical gene in the regulation of melanogenesis, controlling proliferation and apoptosis in melanocytes, providing additional evidence for the mechanism of pigmentation of animal fur.
RESUMO
BACKGROUND: In recent years, crucian carp hematopoietic necrosis caused by Cyprinid herpesvirus 2 (CyHV-2) infection has caused an enormous economic loss to the aquaculture industry. METHODS: In this study antigenic epitope analysis was performed on the membrane proteins of CyHV-2, and 8 antigen-rich peptide fragments were selected for prokaryotic expression. Then, the immunogenicity of the recombinant proteins was analyzed. On this basis, DNA vaccines were constructed for immunization of hybridized Prussian carps. The protective effect of DNA vaccines against challenge in hybridized Prussian carps was evaluated. RESULTS: The results showed that all 8 recombinant proteins were successfully expressed. Among the recombinant proteins, ORF16, tORF25, tORF64, and ORF146, gave a positive serum reaction with CyHV-2. Of the four proteins used for the immunization of silver crucian carps, the antibody titer induced by tORF25 was the highest. The DNA vaccine, pEGFP-N1-ORF25, was constructed based on ORF25 and able to induce production of specific antibodies in carps, while up-regulating the expression of MHCâ , IL-1ß, C3, and TF-A in the kidneys of carps. Moreover, the immunoprotective rate was increased to 70% in hybridized Prussian carps. CONCLUSION: The results showed that the DNA vaccine constructed based on the ORF25 gene had a greater immune protective effect and can be used as a candidate vaccine for immunoprotection against CyHV-2.
Assuntos
Antígenos Virais/imunologia , Doenças dos Peixes/prevenção & controle , Infecções por Herpesviridae/veterinária , Herpesviridae/imunologia , Vacinas Virais/imunologia , Animais , Anticorpos Antivirais/sangue , Anticorpos Antivirais/imunologia , Antígenos Virais/genética , Aquicultura , Carpas , Citocinas/genética , Herpesviridae/genética , Infecções por Herpesviridae/prevenção & controle , Imunização/veterinária , Rim/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Vacinas de DNA/administração & dosagem , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/administração & dosagem , Vacinas Virais/genéticaRESUMO
The present study was conducted to evaluate the influence of Glycyrrhiza uralensis (G. uralensis) extracts on the growth performance, histological structure, immune response and disease resistance against Flavobacterium columnare (F. columnare) of yellow catfish. Fish were fed with two different diets, i.e., basal diet as control group (CG) and diet containing G. uralensis extracts as experimental group (GG). After 60 days feeding, growth performance of GG fish was significantly improved, with increased WG and SGR but decreased FCR compared to CG fish. Fish were then challenged with F. columnare for two times, as fish showed rare mortality after the first infection. GG fish showed significantly lower cumulative mortality during F. cloumnare infection than CG fish after 21 days infection (dpi). Epithelial cell exfoliation and obvious cellular vacuolization in the skin and congestion of gill lamellae were detected in CG fish, while GG fish showed increased width of epidermis and mucous cells number in skin, and increased length of secondary lamina in gill. GG fish also exhibited higher enzyme activity of lysozyme in serum and mRNA expression of lysozyme in head kidney than CG fish at most time points post infection. G. uralensis extracts supplementation also induced earlier serum anti-oxidative responses, with increased superoxide dismutase activity and total antioxidant capacity in GG fish at 1 dpi. Compared to CG fish, GG fish showed increased expression level of genes involved in TLRs-NFκB signaling (TLR2, TLR3, TLR5, TLR9, Myd88, and p65NFκB), resulting in higher expression levels of pro-inflammatory cytokines (IL-1ß and IL-8) in the head kidney post infection. However, these genes showed deviation in the gill of GG fish, which increased at some time points but decreased at other time points. Moreover, G. uralensis extracts supplementation also significantly unregulated the expression levels of IgM and IgD in head kidney, and the expression levels of IgM in the gill of yellow catfish, suggesting the elevated humoral immune response during F. columnare infection. All these results contributed to the elevated disease resistance ability against F. cloumnare infection of yellow catfish after dietary G. uralensis extracts supplementation.
Assuntos
Peixes-Gato/crescimento & desenvolvimento , Peixes-Gato/imunologia , Resistência à Doença , Doenças dos Peixes/prevenção & controle , Infecções por Flavobacteriaceae/veterinária , Glycyrrhiza uralensis/química , Extratos Vegetais/administração & dosagem , Ração Animal/análise , Animais , Peixes-Gato/microbiologia , Suplementos Nutricionais , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/prevenção & controle , Flavobacterium , Extratos Vegetais/imunologia , Transdução de SinaisRESUMO
Aeromonas veronii is an emerging aquatic pathogen causing hemorrhagic septicemia in humans and animals. Probiotic is an effective strategy for controlling enteric infections through reducing intestinal colonization by pathogens. Here we report that the consumption of Bacillus velezensis regulated the intestinal innate immune response and decreased the degree of intestinal inflammation damage caused by the A. veronii in Crucian carp. In this study, we isolated four strains of B. velezensis, named C-11, S-22, L-17 and S-14 from apparently healthy Crucian carp, which exerted a broad-spectrum antimicrobial activity inhibiting both Gram-positive and Gram-negative bacteria especially the fish pathogens. B. velezensis isolates showed typical Bacillus characteristics by endospore staining, physiological and biochemical test, enzyme activity analysis (amylase, protease, and lipase), and molecular identification. Here, Bacillus-containing dietary was orally administrated to Crucian carp for 8 weeks before A. veronii challenge. Immunological parameters and the expression of immune-related genes were measured at 2, 4, 6, 8, and 10 weeks post-administration. The results showed that B. velezensis was found to promote the increase in the phagocytic activities of peripheral blood leukocytes (PBLs) and head kidney leukocytes (HKLs), as well as the increase in interleukin 1ß (IL-1ß), IL-10 and tumor necrosis factor α (TNF-α) concentration of serum. Lysozyme levels (113.76 U/mL), ACP activity (25.32 U/mL), AKP activity (130.08 U/mL), and SOD activity (240.63 U/mL) were maximum (P < 0.05) in the B. velezensis C-11 treated group at 8 week. Our results showed that Crucian carp fed with the diet containing B. velezensis C-11 and S-22 developed a strong immune response with significantly higher (P < 0.05) levels of IgM in samples of serum, mucus of skin and intestine compared to B. velezensis L-17 and S-14 groups. Moreover, B. velezensis spores appeared to show no toxicity and damage in fish, which could inhabit the gut of Crucian carp. B. velezensis restrained up-regulation of pro-inflammation cytokines (IL-1ß, IFN-γ, and TNF-α) mRNA levels in the intestine and head kidney at final stage of administration, and the expression of IL-10 was increased throughout the 10-week trial. A. veronii infection increased the population of inflammatory cells in the intestinal villi in the controls. In contrast, numerous goblet cells and few inflammatory cells infiltrated the mucosa in the B. velezensis groups after challenge with A. veronii. Compared with A. veronii group, B. velezensis could safeguard the integrity of intestinal villi. The highest post-challenge survival rate (75.0%) was recorded in B. velezensis C-11 group. The present data suggest that probiotic B. velezensis act as a potential gut-targeted therapy regimens to protecting fish from pathogenic bacteria infection. IMPORTANCE: In this work, four Bacillus velezensis strains isolated from apparently healthy Crucian carp, which exhibited a broad-spectrum antibacterial activity especially the fish pathogens. Administration of B. velezensis induced the enhancement of the intestinal innate immune response through reducing intestinal colonization by pathogens. The isolation and characterization would help better understand probiotic can be recognized as an alternative of antimicrobial drugs protecting human and animal health.