RGS2 attenuates alveolar macrophage damage by inhibiting the Gq/11-Ca2+ pathway during cowshed PM2.5 exposure, and aberrant RGS2 expression is associated with TLR2/4 activation.

Staff and animals in livestock buildings are constantly exposed to fine particulate matter (PM2.5), which affects their respiratory health. However, its exact pathogenic mechanism remains unclear. Regulator of G-protein signaling 2 (RGS2) has been reported to play a regulatory role in pneumonia. The aim of this study was to explore the therapeutic potential of RGS2 in cowshed PM2.5-induced respiratory damage. PM2.5 was collected from a cattle farm, and the alveolar macrophages (NR8383) of the model animal rat were stimulated with different treatment conditions of cowshed PM2.5. The RGS2 overexpression vector was constructed and transfected it into cells. Compared with the control group, cowshed PM2.5 significantly induced a decrease in cell viability and increased the levels of apoptosis and proinflammatory factor expression. Overexpression of RGS2 ameliorated the above-mentioned cellular changes induced by cowshed PM2.5. In addition, PM2.5 has significantly induced intracellular Ca2+ dysregulation. Affinity inhibition of Gq/11 by RGS2 attenuated the cytosolic calcium signaling pathway mediated by PLCß/IP3R. To further investigate the causes and mechanisms of action of differential RGS2 expression, the possible effects of oxidative stress and TLR2/4 activation were investigated. The results have shown that RGS2 expression was not only regulated by oxidative stress-induced nitric oxide during cowshed PM2.5 cells stimulation but the activation of TLR2/4 had also an important inhibitory effect on its protein expression. The present study demonstrates the intracellular Ca2+ regulatory role of RGS2 during cellular injury, which could be a potential target for the prevention and treatment of PM2.5-induced respiratory injury.

Taxonomic identification, phenol biodegradation and soil remediation of the strain Rhodococcus sacchari sp. nov. Z13T.

Phenols are highly toxic chemicals that are extensively used in industry and produce large amounts of emissions. Notably, phenols released into the soil are highly persistent, causing long-term harm to human health and the environment. In this study, a gram-positive, aerobic, and rod-shaped bacterial strain, Z13T, with efficient phenol degradation ability, was isolated from the soil of sugarcane fields. Based on the physiological properties and genomic features, strain Z13T is considered as a novel species of the genus Rhodococcus, for which the name Rhodococcus sacchari sp. nov. is proposed. The type strain is Z13T (= CCTCC AB 2022327T = JCM 35797T). This strain can use phenol as its sole carbon source. Z13T was able to completely degrade 1200 mg/L phenol within 20 h; the maximum specific growth rate was µmax = 0.93174 h-1, and the maximum specific degradation rate was qmax = 0.47405 h-1. Based on whole-genome sequencing and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, strain Z13T contains a series of phenol degradation genes, including dmpP, CatA, dmpB, pcaG, and pcaH, and can metabolize aromatic compounds. Moreover, the potential of strain Z13T for soil remediation was investigated by introducing Z13T into simulated phenol-contaminated soil, and the soil microbial diversity was analyzed. The results showed that 100% of the phenol in the soil was removed within 7.5 d. Furthermore, microbial diversity analysis revealed an increase in the relative species richness of Oceanobacillus, Chungangia, and Bacillus.

The effects of acute ammonia stress on liver antioxidant, immune and metabolic responses of juvenile yellowfin tuna (Thunnus albacares).

The impact of acute ammonia nitrogen (NH3-N) stress on the antioxidant, immune, and metabolic capabilities of the liver in juvenile yellowfin tuna (Thunnus albacares) is not yet fully understood. This study set NH3-N concentrations at 0 (natural seawater, control group), 5, and 10 mg/L, and sampled the liver at 6, 24, and 36 h for analysis. As time progresses, NH3-N exposure leads to an increase in malondialdehyde (MDA) concentrations. The activity of superoxide dismutase (SOD) and the relative expression levels of related genes, as well as the activity of immune enzymes and ATPase, decrease. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and interleukin-10 (IL-10) exhibit different fluctuation patterns. Low concentrations of NH3-N increase the activity of catalase (CAT) and glutathione peroxidase (GHS-PX) and the relative expression levels of the Na+K+-ATPase gene. The relative expression levels of the interleukin-6 receptor (IL-6r) gene show a decreasing trend. High concentrations of NH3-N decrease the activity of CAT, GSH-PX, and the relative expression levels of related genes. When the NH3-N concentration is below 5 mg/L, the stress duration should not exceed 36 h. When the NH3-N concentration is between 5 and 10 mg/L, the stress duration should not exceed 24 h, otherwise, it will have a negative impact on the liver of the juvenile yellowfin tuna. This study provides scientific data for the artificial breeding and recirculating aquaculture of juvenile yellowfin tuna.

Characteristics and health impacts of bioaerosols in animal barns: A comprehensive study.

Bioaerosols produced during animal production have potential adverse effects on the health of workers and animals. Our objective was to investigate characteristics, antibiotic-resistance genes (ARGs), and health risks of bioaerosols in various animal barns. Poultry and swine barns had high concentrations of airborne bacteria (11156 and 10917 CFU/m3, respectively). Acinetobacter, Clostridium sensu stricto, Corynebacterium, Pseudomonas, Psychrobacter, Streptococcus, and Staphylococcus were dominant pathogenic bacteria in animal barns, with Firmicutes being the most abundant bacterial phylum. Based on linear discriminant analysis effect size (LEfSe), there were more discriminative biomarkers in cattle barns than in poultry or swine barns, although the latter had the highest abundance of bacterial pathogens and high abundances of ARGs (including tetM, tetO, tetQ, tetW sul1, sul2, ermA, ermB) and intI1). Based on network analyses, there were higher co-occurrence patterns between bacteria and ARGs in bioaerosol from swine barns. Furthermore, in these barns, relative abundance of bacteria in bioaerosol samples was greatly affected by environmental factors, mainly temperature, relative humidity, and concentrations of CO2, NH3, and PM2.5. This study provided novel data regarding airborne bio-contaminants in animal enclosures and an impetus to improve management to reduce potential health impacts on humans and animals.

Poly (I:C)-Induced microRNA-30b-5p Negatively Regulates the JAK/STAT Signaling Pathway to Mediate the Antiviral Immune Response in Silver Carp (Hypophthalmichthys molitrix) via Targeting CRFB5.

In aquaculture, viral diseases pose a significant threat and can lead to substantial economic losses. The primary defense against viral invasion is the innate immune system, with interferons (IFNs) playing a crucial role in mediating the immune response. With advancements in molecular biology, the role of non-coding RNA (ncRNA), particularly microRNAs (miRNAs), in gene expression has gained increasing attention. While the function of miRNAs in regulating the host immune response has been extensively studied, research on their immunomodulatory effects in teleost fish, including silver carp (Hyphthalmichthys molitrix), is limited. Therefore, this research aimed to investigate the immunomodulatory role of microRNA-30b-5p (miR-30b-5p) in the antiviral immune response of silver carp (Hypophthalmichthys molitrix) by targeting cytokine receptor family B5 (CRFB5) via the JAK/STAT signaling pathway. In this study, silver carp were stimulated with polyinosinic-polycytidylic acid (poly (I:C)), resulting in the identification of an up-regulated miRNA (miR-30b-5p). Through a dual luciferase assay, it was demonstrated that CRFB5, a receptor shared by fish type I interferon, is a novel target of miR-30b-5p. Furthermore, it was found that miR-30b-5p can suppress post-transcriptional CRFB5 expression. Importantly, this study revealed for the first time that miR-30b-5p negatively regulates the JAK/STAT signaling pathway, thereby mediating the antiviral immune response in silver carp by targeting CRFB5 and maintaining immune system stability. These findings not only contribute to the understanding of how miRNAs act as negative feedback regulators in teleost fish antiviral immunity but also suggest their potential therapeutic measures to prevent an excessive immune response.

Cooperative Positioning Method of a Multi-UAV Based on an Adaptive Fault-Tolerant Federated Filter.

Aiming at the problem of the low cooperative positioning accuracy and robustness of multi-UAV formation, a cooperative positioning method of a multi-UAV based on an adaptive fault-tolerant federated filter is proposed. Combined with the position of the follower UAV and leader UAV, and the relative range between them, a cooperative positioning model of the follower UAV is established. On this basis, an adaptive fault-tolerant federated filter is designed. Fault detection and isolation technology are added to improve the positioning accuracy of the follower UAV and the fault tolerance performance of the filter. Meanwhile, the measurement noise matrix is adjusted by the adaptive information allocation coefficient to reduce the impact of undetected fault information on the sub-filter and global estimation accuracy. The simulation results show that the adaptive fault-tolerant federated algorithm can greatly improve the positioning accuracy, which is 83.4% higher than that of the absolute positioning accuracy of a single UAV. In the case of a gradual fault, the method has a stronger fault-tolerant performance and reconstruction performance.

Rhizoma curcumae Longae ameliorates high dietary carbohydrate-induced hepatic oxidative stress, inflammation in golden pompano Trachinotus ovatus.

In general, starch, as a complex carbohydrate, is the most economical energy source in aquaculture for its relatively low cost. However, excessive dietary levels of carbohydrate result in pathological conditions. An 8-week feeding trial with CT (control diet, containing 21% carbohydrate), HC (a high-carbohydrate diet, containing 50% carbohydrate) and HCR (a HC diet supplemented with 0.015% Rhizoma curcumae Longae) was performed to investigate the protective effect of curcumin on high-carbohydrate-induced hepatic oxidative stress and intestine lesion in juvenile Trachinotus ovatus. In the current study, HC group significantly decreased WGR, SGR, plasma CAT activity, intestinal C4 levels, hepatic Nrf2, Keap1, Bach1, HO1, CAT, and GPX mRNA expression as well as ZO-1, Occludin, and Claudin-3, TGF-ß mRNA transcription levels, while the opposite was true for plasma AST activity, hepatic MDA contents, intestinal Claudin-15, NF-κB, IL-1ß, IL-6, and TNF-α mRNA expression. In contrast with the HC group, the HCR group significantly increased the activities of hepatic CAT, SOD, intestinal C3, C4, IgG and LZM levels, hepatic Nrf2, Bach1, CAT, and GPX mRNA expression as well as intestinal ZO-1, Occludin, Claudin-3, TGF-ß and IL-10 mRNA expression levels, but the opposite trend was found in plasma triglyceride content, hepatic lipid deposition, hepatic Keap1 mRNA level as well as intestinal NF-κB, IL-6. In conclusion, high-carbohydrate diet can cause detrimental effect on physiological health status in Trachinotus ovatus, while adding Rhizoma curcumae Longae can improve hepatic and intestinal health status via attenuating the oxidative stress, inflammation, and reducing lipid deposition.

Acute acidification stress weakens the head kidney immune function of juvenile Lates calcarifer.

Acidized water environment can impact many physiological processes of aquatic animals. The response of the head kidney to acidification, especially the immune response, is of great significance to health. This study analyzed the histological and transcriptional changes under different acidification levels (C group, pH 8.1; P group, pH 7.4; E group, pH 3.5) in the short term (12 h, 36 h and 60 h) in the head kidney of juvenile L. calcarifer. The results showed that the acidification of the water environment caused tissue damage to the head kidney of L. calcarifer, and the damage appeared earlier and was stronger in the extreme pH group. The transcriptional response of L. calcarifer head kidney increased with the increase of acidification level. The two treatments transcriptional responses showed different trends in terms of time. After KEGG function enrichment, with the increase of stimulation time, the proportion of down-regulated pathways was increasing, and the types of pathway enrichment at different acidification levels were quite different at the initial stage. At 12 h, the first category in the P group with the most significant number of pathways was 'Metabolism', and the first category in the E group with the largest number of pathways was 'Human Diseases'. At 60 h, the enrichment pathways of the two groups were highly overlapping in immune-related pathways, which contained 26 common DEGs. They had a dominant expression pattern. In the P group, the expression level decreased with time. In the E group, the down-regulation degree of expression level at 12 h reached the level of the P group at 60 h, and the expression level remained low until 60 h. Through the correlation network, interferon regulatory factor 7 (IRF7), Tripartite motif containing-21 (TRIM21), Signal transducer and activator of transcription 1 (STAT1) and Signal transducer and activator of transcription 3 (STAT3) were found to have the most correlation with other genes. In this study, juvenile L. calcarifer showed different coping strategies to different levels of acute acidification stress, but all of them resulted in the extensive weakening of head kidney immune function.

Lignin degradation by water buffalo.

So far, few animals with the ability of lignin degradation have been reported except termite and longicorn. In this study, it was found that the crude fiber and acid detergent lignin (ADL) of rice straw can be degraded dramatically higher by buffalo than those by cattle. In order to further study this ability of buffalo, the digestion of roughages in buffalo rumen was studied using rumen nylon bag experiment, scanning electron microscopy (SEM), and Van Soest fiber analysis. The SEM results showed that the degradation degree of rice straw was dramatically higher in buffalo than that in cattle. The digestibility of crude fiber was significantly higher in buffalo than that in cattle (P < 0.01). The digestibility of ADL, cellulose, hemicellulose, acid detergent, fiber, and neutral detergent fiber of rice straw in buffalo rumen was significantly higher than that in cattle (P < 0.05). The ADL degradation rate of rice straw in buffalo rumen was significantly higher than that in cattle rumen, indicating that buffalo was capable of utilizing lignin and had superior utilizing capability than cattle. It was observed that various roughages can be dramatically digested by buffalo rumen with the ranking of ADL degradation rate: peanut vine (15.04%) > rice silage > maize silage > rice straw > corn stover > wheat stalk > bract leaf > potato vine (7.22%), verifying that buffalo rumen possessed the ability to digest universal roughages. In conclusion, this study revealed that buffalo was more efficient in ADL degradation compared with cattle.

Detection of miR-122 by fluorescence real-time PCR in blood from patients with chronic hepatitis B and C infections.

BACKGROUND: This study aims to determine whether relative miR-122 levels in peripheral blood are correlated with chronic hepatitis B (CHB) and chronic hepatitis C (CHC) virus infection and viral replication to determine whether miR-122 can be a new marker for liver injury. METHODS: MicroRNA (miRNA) was extracted from the peripheral blood of 20 CHB patients, 20 CHC patients, and 20 healthy controls. The levels of miR-122 were determined using fluorescence real-time reverse transcription PCR. Then, the associations of miR-122 with CHB and CHC were analyzed, and its correlation with other markers of liver function and viral replication were determined. RESULTS: The expression level of miR-122 in patients with CHB was significantly higher when compared to subjects in the control group (P = 0.007) or CHC patients (P = 0.005). Furthermore, the miR-122 level in patients with CHC was somewhat higher when compared to healthy controls (66% higher), but the difference was not statistically significant (P = 0.229). MiR-122 levels were significantly correlated with ALT (correlation coefficient [R] = 0.7, P < 0.001), AST (R = 0.71, P < 0.001), and HBV NA (R = 0.9, P < 0.001). The regression analysis indicated that the AUC of miR-122 levels in the diagnosis of CHB was 0.87, with a sensitivity of 0.8 and a specificity of 0.8. CONCLUSION: MiR-122 can be used to distinguish healthy persons and patients with CHB infection with high sensitivity and specificity. These present findings presented that the complex and context-specific associations of miR-122 with liver diseases, suggesting that this may be a promising marker for liver injury.

Highly Selective Binding and Inhibition of Pyr-His-Pro-NH2 (TRH) Function using a Polypyridinyl Macrocyclic Receptor with an Amphiphilic Cavity.

Macrocycle, cyclo[4] [(1,3-(4,6)-dimethylbezene)[4](2,6-(3,5)-dimethylpyridine (B4P4), shows highly selective binding affinity with protirelin (Pyr-His-Pro-NH2 ; TRH) among the tested 26 drug or drug adductive substrates. The stable complexation in a 1:1 manner was fully characterized in solution, gas phase, and solid state study. Furthermore, B4P4 acts as an efficient TRH inhibitor even at [macrocycle]:[drug] <1:300, both in membrane transport and cellar incubation. The current work provides an unprecedented strategy for macrocycles to be efficiently used in drug target therapy.

Identification and characterization of interferon regulatory factor 1 from Lateolabrax japonicus involved in antiviral immune response against grouper nervous necrosis virus infection.

Interferon regulatory factors (IRFs) play a key role in mediating the host response against pathogen infection and other important biological processes. In the present study, an interferon regulation factor 1 gene was identified from Lateolabrax japonicus (designated LjIRF-1), the cDNA sequence of LjIRF-1 was 1394 bp long, and with an open reading frame (ORF) of 945 bp that encodes a peptide of 314 amino acids. Bioinformatics data showed that LjIRF-1 possesses a DNA-binding domain (DBD) and two low complexity regions, which shared 56-81% identity to other fish IRF-1s. The LjIRF-1 transcripts were detectable in all examined tissues of healthy L. japonicus, with higher levels in the blood, head-kidney, intestine, gill and spleen. When challenged with grouper nervous necrosis virus (GNNV) and poly (I:C) infection, both the mRNA expression levels of LjIRF-1 and L. japonicus interferon-1 gene (designated LjIFN-1) were significantly up-regulated. Furthermore, like with poly (I:C), the active purified recombinant protein (rLjIRF-1) was also capable of increasing the expression level of LjIFN-1; controlling the copy number of GNNV under lethiferous titer (1011-1012 copies/µL) and promoting the survival rate of GNNV infected L. japonicas. Combine all the results, we deduced that LjIRF-1 is involved in defending GNNV infection by simulating LjIFN-1 signal pathway in L. japonicas.

Transcriptional analysis reveals physiological response to acute acidification stress of barramundi Lates calcarifer (Bloch) in coastal areas.

To understand the physiological response of estuarine fish to acidification, barramundi (Lates calcarifer) juveniles were exposed to acidified seawater in experimental conditions. The molecular response of barramundi to acidification stress was assessed by RNA-seq analysis. A total of 2188 genes were identified as differential expression genes. The gene ontology classification system and Kyoto Encyclopedia of Genes and Genomes database analysis showed that acidification caused differential expressions of genes and pathways in the gills of barramundi. Acidification had a great influence on the signal transduction pathway in cell process. Furthermore, we detected that numerous unigenes involved in the pathways associated with lipid metabolism, carbohydrate metabolism, amino acid metabolism, glycan biosynthesis and metabolism specific and non-specific immunity were changed. This study indicates that the physiological responses in barramundi especially the immune system and energy allocation correspond to the variation of environmental pH. This study reveals the necessity for assessment of the potential of estuarine fishes to cope with acidification of the environment and the need to develop strategies for fish conservation in coastal areas.

The gut microbiota community and antioxidant enzymes activity of barramundi reared at seawater and freshwater.

To understand the physiological responses of euryhaline fish to reared salinity, the gut microbiota composition and antioxidant enzymes activity of same batch barramundi Lates calcarifer reared in two extreme salinity condition (seawater and freshwater) were studied, under laboratory condition. The gut bacterial composition was analyzed using high-throughput Illumina sequencing. Acid phosphatase (ACP), catalase (CAT), glutathione peroxidase (GSH-Px), lipid peroxide (LPO), lysozyme (LZM), malonaldehyde (MDA), peroxidase (POD), and superoxide dismutase (SOD) were used as biochemical indicators. The reared salinity did not change the major composition of barramundi gut bacteria under circulating aquaculture system. Proteobacteria, Firmicutes were the most phylum in barramundi gut microbiota community, and Exiguobacterium, Citrobacter, Acinetobacter, Pseudomonas were the dominate genus. CAT and ACP activity in barramundi liver were found significantly different between freshwater and seawater group. GSH-Px, LZM, POD, SOD activity and MDA, LPO levels were not significantly affected by reared salinity. This study is the first high-throughput analyses of the gut microbiota diversity in barramundi from same batch of siblings reared under two extreme salinity condition. And the findings in the present study can be instructive to the management of animal health in barramundi circulating farming activities, and further euryhaline fish gut microecology research.

Dietary non-protein energy source regulates antioxidant status and immune response of barramundi (Lates calcarifer).

This study evaluates the effects of different dietary sources of non-protein energy on growth performance, histological structure, antioxidant status and immune response of barramundi Lates calcarifer. Fish were fed with isoenergetic diets (18 kJ/g) with two types of non-protein energy in the experimental groups and a regular diet was used as the control for 56 days. The specific growth rate and survival of fish were not significantly different between experimental diets. Hepatic histology did not reveal significant differences between dietary treatments at cellular level. The activity of most antioxidant enzymes in the lipid group significantly increased, and the antioxidant capacity in the carbohydrate group was significantly higher than that in other treatments. In the TOR pathway, LST8 homolog (mLST8) expression in the high lipid group was downregulated, and the mechanistic target of rapamycin (mTOR) expression in the high carbohydrate group was downregulated and eIF4E expression was upregulated. The C-reactive protein (CRP) expression in the high lipid and high carbohydrate groups was upregulated. The expression levels of heat shock protein genes in the high lipid group and the high carbohydrate group were significantly downregulated. This study indicates that the lipid diet have less effect in fish immunity but is more suitable as a non-protein ingredient for energy supply for barramundi.

Mineral and bone disorder in hemodialysis patients in the Tibetan Plateau: a multicenter cross-sectional study.

Background: Mineral and bone disorder (MBD) in hemodialysis patients is associated with increased morbidity and mortality. Studies on the MBD status of hemodialysis patients at high altitudes are extremely limited. Methods: A total of 146 hemodialysis patients from 5 local hospitals across all districts with hemodialysis centers in the Tibetan Plateau were enrolled in this cross-sectional study. Parameters related to MBD, including serum phosphorus (P), calcium (Ca), and intact parathyroid hormone (iPTH) levels, were measured. The achievement of MBD goals was compared with the achievement in the Dialysis Outcomes and Practice Study (DOPPS) 3, DOPPS 4 and a multicenter study of MBD in China. Factors associated with hyperphosphatemia were examined. Results: Altogether, 146 hemodialysis patients were recruited from the Tibetan Plateau. According to the K/DIGO guidelines, there were low achievement rates for serum Ca (40.4%), P (29.7%), and iPTH (47.1%). As for the (KDOQI) guidelines, the rates of achievement of defined targets were 38.4%, 33.7% and 16.4% for serum Ca, P and iPTH, respectively. The percentages of patients reaching the KDOQI targets for corrected Ca, P, and iPTH were significantly lower for Tibetan patients than the percentages found in DOPPS 3 (38.4% vs. 50.4%, 33.7% vs. 49.8%, and 16.4% vs. 31.4%, respectively, all p < .001) and DOPPS 4 (38.4% vs. 56.0%, 33.7% vs. 54.5%, and 16.4% vs. 35.3%, respectively, all p < .001). The percentage of patients reaching the KDOQI targets for iPTH was significantly lower in Tibet than in the plain areas of China (16.4% vs. 26.5%, p < .001). The proportion of patients with hypocalcemia was higher in Tibet than in the plain areas (44.5% vs. 19.4%, p < .001). The percentage of local patients with optimal P was significantly higher for patients with an activated vitamin D prescription than for patients without an activated vitamin D prescription (45.3% vs. 19.3%, p < .001). Age and the activated vitamin D prescription were independently associated with hyperphosphatemia. Conclusion: The MBD status of hemodialysis patients in Tibet is far from the ideal level. High altitude is one of the possible causes of the differences found, but not the principal one. It is necessary for medical staff in Tibet to improve the detection and treatment of MBD.

Nutrition and water temperature regulate the expression of heat-shock proteins in golden pompano larvae (Trachinotus ovata, Limmaeus 1758).

Understanding fish larval development is of a great interest for aquaculture production efficiency. Identifying possible indicators of fish larvae stress could improve the production and limit the mortality rate that larval stage is subjected to. Heat-shock proteins (HSPs) and heat-shock factors (HSFs) are well known as indicators of response to many kinds of stressor (e.g., environmental, morphological, or pathological changes). In this study, golden pompano larvae were raised at different temperatures (23 °C, 26 °C, and 29 °C), as well as three different diets (Artemia nauplii unenriched, Artemia nauplii enriched with Nannochloropsis sp., and Artemia nauplii enriched with Algamac 3080), and the expression of HSP60, HSP70, HSF1, HSP2, and GRP94 were monitored. While stress genes were widely expressed in the larval tissues, HSP60 and HSP70 were principally from the gills and heart; HSF1 principally from the muscle, brain, and heart; and GRP94 principally from the head kidney and spleen. Golden pompano larvae were found to be more sensitive to thermal changes at later larval stage, and 29 °C was showed to likely be the best condition for golden pompano larval development. Nannochloropsis sp.-enriched Artemia nauplii treatment was found to be the most appropriate feed type with moderate relative expressions of HSP60, HSP70, HSF1, HSF2, and GRP94.

Characterization, expression and silencing by RNAi of p53 from Penaeus monodon.

The tumor suppressor p53 is a sequence-specific transcription factor, whose target genes can regulate genomic stability, the cellular response to DNA damage and cell-cycle progression. In the present study, the full-length complementary DNA (cDNA) sequence of p53 gene from Penaeus monodon (Pmp53) was cloned by the technology of rapid amplification of cDNA ends (RACE). The cDNA of Pmp53 was 2239 bp, encoding a protein of 450 amino acids with calculated molecular weight of 50.62 kDa. The temporal expression of Pmp53 in different tissues (ovary, heart, intestine, brain, muscles, stomach and gills) and different developmental stages of ovary was investigated by real-time quantitative PCR (RT-qPCR). The lowest expression level of Pmp53 was observed in the stomach, while the highest expression level was detected in the brain. During the ovary development stages, the expression level of Pmp53 reached the peak at stage III. RNA interference (RNAi) and serotonin (5-hydroxytryptamine, 5-HT) injection experiments were conducted to study the expression profile of Pmp53 and PmCDK2 (cyclin-dependent kinase 2, CDK2). Knocked down of Pmp53 by dsRNA-p53 was sequence-specific and successful. Expression levels of Pmp53 and PmCDK2 in ovary of P. monodon were significantly increased at 12-96 h post 5-HT injection. These results indicate that Pmp53 may be involved in the regulation of ovarian development of P. monodon.

Thioredoxin of golden pompano involved in the immune response to Photobacterium damselae.

Thioredoxin (TRX) is one of the key systems responsible for keeping the intracellular environment in a highly reduced state. In this study, a full-length TRX cDNA sequence (ToTRX) from golden pompano Trachinotus ovatus was identified after pyrosequencing of golden pompano cDNA library. ToTRX cDNA is comprised of 786 bp, and contained a 324 bp open reading frame (ORF) encoding a 107 amino acid polypeptide, a 5' untranslated region (UTR) of 116 bp, and a long 3'- UTR of 346 bp. Multiple sequence alignment revealed that ToTRX contained the highly conserved redox active disulphide/dithiol site (CGPC) of the thioredoxin active family, and phylogenetic tree showed that ToTRX had a closer evolution relationship with TRX from Oplegnathus fasciatus and Anoplopoma fimbria. ToTRX mRNA is ubiquitously expressed in all detected tissues with the higher expression levels in the stomach, gill and fin tissues. The expression of ToTRX mRNA was significantly up-regulated in liver, kidney, intestine and spleen of golden pompano injected with Photobacterium damselae. The recombinant ToTRX protein (rToTRX) was expressed in Escherichia coli BL21 (DE3), and then purified and refolded. The insulin disulfides assay was performed to investigate the enzymatic oxidoreductase activity of rToTRX, and the results demonstrated that rToTRX exhibited a high reducing activity in presence of DTT, while no activity was observed in the regroup without DTT and blank control group. Over all, the study provided the useful information to help further understand the functional mechanism of TRX in marine fish immunity.