Effects of Different Salinity Stress on the Transcriptomic Responses of Freshwater Crayfish (Procambarus clarkii, Girard, 1852).

Salinization of freshwater ecosystems is a pressing global issue. Changes in salinity can exert severe pressure on aquatic animals and jeopardize their survival. Procambarus clarkii is a valuable freshwater aquaculture species that exhibits some degree of salinity tolerance, making it an excellent research model for freshwater aquaculture species facing salinity stress. In the present study, crayfish were exposed to acute low salt (6 ppt) and high salt (18 ppt) conditions. The organisms were continuously monitored at 6, 24, and 72 h using RNA-Seq to investigate the mechanisms of salt stress resistance. Transcriptome analysis revealed that the crayfish responded to salinity stress with numerous differentially expressed genes, and most of different expression genes was observed in high salinity group for 24h. GO and KEGG enrichment analyses indicated that metabolic pathways were the primary response pathways in crayfish under salinity stress. This suggests that crayfish may use metabolic pathways to compensate for energy loss caused by osmotic stress. Furthermore, gene expression analysis revealed the differential expression of immune and antioxidant-related pathway genes under salinity stress, implying that salinity stress induces immune disorders in crayfish. More genes related to cell proliferation, differentiation, and apoptosis, such as the Foxo, Wnt, Hippo, and Notch signaling pathways, responded to high-salinity stress. This suggests that regulating the cellular replication cycle and accelerating apoptosis may be necessary for crayfish to cope with high-salinity stress. Additionally, we identified 36 solute carrier family (SLC) genes related to ion transport, depicting possible ion exchange mechanisms in crayfish under salinity stress. These findings aimed to establish a foundation for understanding crustacean responses to salinity stress and their osmoregulatory mechanisms.

Evaluation of Genetic Parameters and Comparison of Stress Tolerance Traits in Different Strains of Litopenaeus vannamei.

Litopenaeus vannamei stands out globally in aquaculture for its fast growth, broad salt tolerance, disease resistance, and high protein levels. Selective breeding requires the precise estimation of the variance components and genetic parameters for important traits. This study formed lineages from 20 full sibling families of L. vannamei, with progenitors from Thailand and the USA. We then assessed the genetic resilience traits of juvenile shrimp from these families to high ammonia-N, high pH, and low salinity by performing a 96 h acute toxicity test. Mortality rates for the families under 96 h exposure to high ammonia-N, high pH, and low salinity were 19.52-92.22%, 23.33-92.22%, and 19.33-80.00%, respectively, showing significant variance in stress tolerance among families (p < 0.05). Survival heritability estimates, using threshold male and female models, were 0.44 ± 0.12 in high ammonia-N, 0.41 ± 0.12 in high pH, and 0.27 ± 0.08 in low salinity, respectively. Genetic correlations between growth and stress resistance traits varied from 0.0137 ± 0.2406 to 0.8327 ± 0.0781, and phenotypic correlations ranged from 0.0019 ± 0.0590 to 0.6959 ± 0.0107, indicating a low-to-high positive correlation significant at (p < 0.05). It was found that the survival rate of families No. 2 and No. 9 was higher under high ammonia-N and high pH stresses, while the survival rate of family No. 10 was higher under low salinity stress after comparing two selection criteria, the breeding values and phenotypic values. Thus, these three families are identified as potential breeding program candidates. Through the creation of a genetic parameter estimation model, the genetic variances across mating combinations for stress resistance traits were obtained and families with heightened stress resistance were identified, laying the groundwork for enhanced genetic selection of L. vannamei.

Effect of Fly Maggot Protein as Dietary on Growth and Intestinal Microbial Community of Pacific White Shrimp Litopenaeus vannamei.

As the intensive development of aquaculture persists, the demand for fishmeal continues to grow; however, since fishery resources are limited, the price of fishmeal remains high. Therefore, there is an urgent need to develop new sources of protein. They are rich in proteins, fatty acids, amino acids, chitin, vitamins, minerals, and antibacterial substances. Maggot meal-based diet is an ideal source of high-quality animal protein and a new type of protein-based immune enhancer with good application prospects in animal husbandry and aquaculture. In the present study, we investigated the effects of three different diets containing maggot protein on the growth and intestinal microflora of Litopenaeus vannamei. The shrimp were fed either a control feed (no fly maggot protein added), FM feed (compound feed with 30% fresh fly maggot protein added), FF feed (fermented fly maggot protein), or HT feed (high-temperature pelleted fly maggot protein) for eight weeks. The results showed that fresh fly maggot protein in the feed was detrimental to shrimp growth, whereas fermented and high-temperature-pelleted fly maggot protein improved shrimp growth and survival. The effects of different fly maggot protein treatments on the intestinal microbiota of L. vannamei also varied. Fermented fly maggot protein feed and high-temperature-pelleted fly maggot protein feed increased the relative abundance of Ruegeria and Pseudomonas, which increased the abundance of beneficial bacteria and thus inhibited the growth of harmful bacteria. In contrast, fresh fly maggot proteins alter the intestinal microbiome, disrupting symbiotic relationships between bacteria, and causing invasion by Vibrio and antibiotic-resistant bacteria. These results suggest that fresh fly maggot proteins affect the composition of intestinal microorganisms, which is detrimental to the intestinal tract of L. vannamei, whereas fermented fly maggot protein feed affected the growth of L. vannamei positively by improving the composition of intestinal microorganisms.

First transcriptome profiling in gill and hepatopancrease tissues of Metapenaeus ensis in response to acute ammonia-N stress.

The greasyback shrimp, Metapenaeus ensis, suffers from ammonia-N stress during intensive factory aquaculture. Optimizing ammonia-N stress tolerance has become an important issue in M. ensis breeding. The metabolic and adaptive mechanisms of ammonia-N toxicity in M. ensis have not been comprehensively understood yet. In this study, a large number of potential simple sequence repeats (SSRs) in the transcriptome of M. ensis were identified. Differentially expressed genes (DEGs) in the gill and hepatopancreas at 24 h post-challenges under high concentrations of ammonia-N treatment were detected. We obtained 20,108,851-27,681,918 clean reads from the control and high groups, assembled and clustered a total of 103,174 unigenes with an average of 876 bp and an N50 of 1189 bp. Comparative transcriptome analyses identified 2000 different expressed genes in the gill and 2010 different expressed genes in the hepatopancreas, a large number of which were related to immune function, oxidative stress, metabolic regulation, and apoptosis. The results suggest that M. ensis may counteract ammonia-N toxicity at the transcriptome level by increasing the expression of genes related to immune stress and detoxification metabolism, and that selected genes may serve as molecular indicators of ammonia-N. By exploring the genetic basis of M. ensis' ammonia-N stress adaptation, we constructed the genetic networks for ammonia-N adaptation. These findings will accelerate the understanding of M. ensis' ammonia-N adaptation, contribute to the research of future breeding, and promote the level of factory aquaculture of M. ensis.

Transcriptome and molecular regulatory mechanisms analysis of gills in the black tiger shrimp Penaeus monodon under chronic low-salinity stress.

Background: Salinity is one of the main influencing factors in the culture environment and is extremely important for the survival, growth, development and reproduction of aquatic animals. Methods: In this study, a comparative transcriptome analysis (maintained for 45 days in three different salinities, 30 psu (HC group), 18 psu (MC group) and 3 psu (LC group)) was performed by high-throughput sequencing of economically cultured Penaeus monodon. P. monodon gill tissues from each treatment were collected for RNA-seq analysis to identify potential genes and pathways in response to low salinity stress. Results: A total of 64,475 unigenes were annotated in this study. There were 1,140 upregulated genes and 1,531 downregulated genes observed in the LC vs. HC group and 1,000 upregulated genes and 1,062 downregulated genes observed in the MC vs. HC group. In the LC vs. HC group, 583 DEGs significantly mapped to 37 signaling pathways, such as the NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, and PI3K-Akt signaling pathway; in the MC vs. HC group, 444 DEGs significantly mapped to 28 signaling pathways, such as the MAPK signaling pathway, Hippo signaling pathway and calcium signaling pathway. These pathways were significantly associated mainly with signal transduction, immunity and metabolism. Conclusions: These results suggest that low salinity stress may affect regulatory mechanisms such as metabolism, immunity, and signal transduction in addition to osmolarity in P. monodon. The greater the difference in salinity, the more significant the difference in genes. This study provides some guidance for understanding the low-salt domestication culture of P. monodon.

Effect of Air Exposure and Re-Submersion on the Histological Structure, Antioxidant Response, and Gene Expression of Procambarus Clarkii.

Air exposure is an important environmental stressor during the transportation and cultivation of Procambarus clarkii. We evaluated the effect of re-submersion for 24 h after dry transportation for 24 h on the histological structure, antioxidant activity, and gene expression of crayfish. The antioxidant parameters of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactate dehydrogenase (LDH), and the relative expression of CAT, SOD, HSP70, and ferritin genes were subsequently measured in the hepatopancreas and gills at both stages. Histopathology found that air exposure led to vacuolation of the hepatopancreas and disorderly arrangement of respiratory epithelial cells (REC) in the gills. The activities of catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA), and lactic dehydrogenase (LDH) in the hepatopancreas and gills increased with short-term air exposure. The relative expression of genes (CAT, SOD, HSP70, and Ferritin) were induced after short-term air exposure. During re-submersion, MDA content and CAT and SOD activities in the hepatopancreas and gills were restored after 24 h, however, LDH activity and hepatopancreatic tissue damage were not repaired. Our results indicate that air exposure can cause oxidative damage to P. clarkii, and CAT and SOD can be used to determine the response of crayfish exposed to air, in addition to some damage that can be eliminated after re-submersion to a limited degree. This study provides foundational data that re-submersion can improve crayfish performance under hypoxic stress to a certain extent and will lead to the development of more effective transportation strategies and decrease economic losses in the future.

Application of heart-cutting two-dimensional liquid chromatography-mass spectrometry to the characterization of highly polar impurities in calcium gluconate injection.

The separation and characterization of small polar impurities in polar drugs such as calcium gluconate products are always challenging, due to their poor retention on traditional reversed phase (RP) columns. Although ion-pair reversed-phase liquid chromatography (IPRP-LC) and hydrophilic interaction liquid chromatography (HILIC) are commonly used methods for polar compound analysis, both methods have some drawbacks. For example, IPRP-LC is incompatible with mass spectrometry (MS) due to the presence of non-volatile salts in its mobile phase and HILIC has limited sensitivity due to the poor solubility of polar drugs in the organic-rich sample diluents used in HILIC separations. In order to characterize the highly polar impurities in calcium gluconate injections, a heart-cutting two-dimensional liquid chromatography (2D-LC) method coupled with quadrupole time-of-flight mass spectrometry (Q-TOF/MS) was developed in this study. An IPRP-LC method in the first dimension (1D) provided the selectivity for the separation of polar analytes, using a 100% aqueous mobile phase containing phosphate buffer and ion-pair reagent. Heart cuts of target peaks were collected with sample loops and transferred to the second dimension (2D) HILIC column using an organic-rich mobile phase. In order to solve the mobile phase mismatch problem between the two dimensions, a make-up flow module was introduced in the 2D-LC system to dilute the 1D-water-rich fractions with acetonitrile before entering the sample loops. By optimizing the loop size and dilution factor, good retention and peak shape of the highly polar impurities were obtained on the 2D-HILIC column, and the ion suppression effect for MS detection from the ion-pair reagent and non-volatile salt in the 1D-effluent was minimized. A total of five impurities were identified through fragmentation studies by Q-TOF/MS analysis and their fragmentation pathways were proposed. Four of them were further confirmed by reference substances. This study not only provided useful information for quality control of calcium gluconate injections, but also provided an alternative method for polar impurity characterization in pharmaceuticals.

Transcriptome analysis of hepatopancreas in penaeus monodon under acute low pH stress.

The decrease of seawater pH can affect the metabolism, acid-base balance, immune response and immunoprotease activity of aquatic animals, leading to aquatic animal stress, impairing the immune system of aquatic animals and weakening disease resistance, etc. In this study, we performed high-throughput sequencing analysis of the hepatopancreas transcriptome library of low pH stress penaeus monodon, and after sequencing quality control, a total of 43488612-56271828 Clean Reads were obtained, and GO annotation and KEGG pathway enrichment analysis were performed on the obtained Clean Reads, and a total of 395 DEGs were identified. we mined 10 differentially expressed and found that they were significantly enriched in the Metabolic pathways (ko01100), Biosynthesis of secondary metabolites (ko01110), Nitrogen metabolism (ko00910) pathways, such as PIGA, DGAT1, DGAT2, UBE2E on Metabolic pathways; UGT, GLT1, TIM genes on Biosynthesis of secondary metabolites; CA, CA2, CA4 genes on Nitrogen metabolism, are involved in lipid metabolism, induction of oxidative stress and inflammation in the muscular body of spot prawns. These genes play an important role in lipid metabolism, induction of oxidative stress and inflammatory response in the muscle of the shrimp. In summary, these genes provide valuable reference information for future breeding of low pH-tolerant shrimp.

Expression Analysis of a Novel Oxidoreductase Glutaredoxin 2 in Black Tiger Shrimp, Penaeus monodon.

Glutaredoxin (Grx) is a glutathione-dependent oxidoreductase that is an important component of the redox system in organisms. However, there is a serious lack of sequence information and functional validation related to Grx in crustaceans. In this study, a novel Grx was identified in Penaeus monodon (PmGrx2). The full-length cDNA of PmGrx2 is 998 bp, with an open reading frame (ORF) of 441 bp, encoding 119 amino acids. Sequence alignment showed that PmGrx2 had the highest identity with Grx2 of Penaeus vannamei at 96.64% and clustered with Grx2 of other crustaceans. Quantitative real-time PCR (qRT-PCR) analysis showed that PmGrx2 was expressed in all examined tissues, with higher expression levels in the stomach and testis. PmGrx2 was continuously expressed during development and had the highest expression level in the zygote stage. Both ammonia-N stress and bacterial infection could differentially induce the expression of PmGrx2 in hepatopancreas and gills. When PmGrx2 was inhibited, the expression of antioxidant enzymes was suppressed, the degree of apoptosis increased, and the GSH content decreased with the prolongation of ammonia-N stress. Inhibition of PmGrx2 resulted in shrimp being exposed to a greater risk of oxidative damage. In addition, an SNP locus was screened on the exons of PmGrx2 that was significantly associated with an ammonia-N-stress-tolerance trait. This study suggests that PmGrx2 is involved in redox regulation and plays an important role in shrimps' resistance to marine environmental stresses.

Identification and Expression Analysis of Dsx and Its Positive Transcriptional Regulation of IAG in Black Tiger Shrimp (Penaeus monodon).

Doublesex (Dsx) is a polymorphic transcription factor of the DMRTs family, which is involved in male sex trait development and controls sexual dimorphism at different developmental stages in arthropods. However, the transcriptional regulation of the Dsx gene is largely unknown in decapods. In this study, we reported the cDNA sequence of PmDsx in Penaeus monodon, which encodes a 257 amino acid polypeptide. It shared many similarities with Dsx homologs and has a close relationship in the phylogeny of different species. We demonstrated that the expression of the male sex differentiation gene Dsx was predominantly expressed in the P. monodon testis, and that PmDsx dsRNA injection significantly decreased the expression of the insulin-like androgenic gland hormone (IAG) and male sex-determining gene while increasing the expression of the female sex-determining gene. We also identified a 5'-flanking region of PmIAG that had two potential cis-regulatory elements (CREs) for the PmDsx transcription. Further, the dual-luciferase reporter analysis and truncated mutagenesis revealed that PmDsx overexpression significantly promoted the transcriptional activity of the PmIAG promoter via a specific CRE. These results suggest that PmDsx is engaged in male reproductive development and positively regulates the transcription of the PmIAG by specifically binding upstream of the promoter of the PmIAG. It provides a theoretical basis for exploring the sexual regulation pathway and evolutionary dynamics of Dmrt family genes in P. monodon.

Identification of multifunctionality of the PmE74 gene and development of SNPs associated with low salt tolerance in Penaeus monodon.

Members of the E74-like factor (ELF) subfamily are involved in the immune stress process of organisms by regulating immune responses and the development of immune-related cells. PmE74 of Penaeus monodon was characterized and functionally analyzed in this study. The full length of PmE74 was 3106 bp, with a 5'-UTR of 297 bp, and a 3'-UTR of 460 bp. The ORF (Open reading frame) was 2349 bp and encoded 782 amino acids. Domain analysis showed that PmE74 contains a typical Ets domain. Multiple sequence alignment and phylogenetic tree analysis showed that PmE74 clustered with Litopenaeus vannamei E74 and displayed significant similarity (98.98%). PmE74 was expressed in all tissues tested in P. monodon, with the highest levels of expression observed in the testis, intestine, and epidermis. Different pathogen stimulation studies have revealed that PmE74 expression varies in response to different pathogen stimuli. A 96-h acute low salt stress study revealed that PmE74 in the hepatopancreas was upregulated and downregulated in the salinity 17 group and considerably downregulated in the salinity 3 group, whereas PmE74 in gill tissue was considerably downregulated in both groups. Further, by knocking down PmE74 and learning the trends of its linkage genes PmAQP1, PmNKA, PmE75, PmFtz-f1, PmEcR, and PmRXR in response to low salt stress, it was further indicated that PmE74 could have a vital role in the regulation of low salt stress. The SNP test revealed that PmE74-In1-53 was significantly associated with low salt tolerance traits in P. monodon (P < 0.05). The findings of this study can aid in the advancement of molecular marker-assisted breeding in P. monodon, as well as provide fundamental data and methodologies for further investigation of its low salt tolerance strains in P. monodon.

Comparative transcriptome analysis of differentially expressed genes and pathways in Procambarus clarkii (Louisiana crawfish) at different acute temperature stress.

Procambarus clarkii is an important economic species in China, and exhibit heat and cold tolerance in the main culture regions. To understand the mechanisms, we analyzed the hepatopancreas transcriptome of P. clarkii treated at 10 °C, 25 °C, and 30 °C, then 2092 DEGs and 6929 DEGs were found in 30 °C stress group and 10 °C stress group, respectively. KEGG pathway enrichment results showed that immune pathway is the main stress pathway for 10 °C treatment and metabolic pathway is the main response pathway for 30 °C treatment, which implies low temperature stress induces the damage of the immune system and increases the susceptibility of bacteria while the body response to high temperature stress through metabolic adjustment. In addition, flow cytometry proved that both high and low temperature stress caused different degrees of apoptosis of hemocytes, and dynamic transcription heat map analysis also identified the differential expression of HSPs family genes and apoptosis pathway genes under different heat stresses. This indicates that preventing damaged protein misfolding and accelerating cell apoptosis are necessary mechanisms for P. clarkii to cope with high and low temperature stress. Our research has deepened our understanding of the complex molecular mechanisms of P. clarkii in response to acute temperature stress, and provided a potential strategy for aquatic animals to relieve environmental duress.

Single-Cell Sequencing Reveals Types of Hepatopancreatic Cells and Haemocytes in Black Tiger Shrimp (Penaeus monodon) and Their Molecular Responses to Ammonia Stress.

The cell types and developmental trajectories of shrimp cells based on the transcriptional level have not been established, and gene expression profile and function at the single-cell level is unclear. We aimed to use scRNA-seq to construct a single-cell resolution transcriptional map of hepatopancreas and haemocytes in shrimp to analyse the molecular mechanisms of the immune response to ammonia nitrogen stress. In the present study, seven cell clusters were successfully identified in each of the two tissues (haemocytes, Hem1-7; hepatopancreas, Hep1-7) based on specifically-expressed marker genes. The developmental starting points of haemocytes and hepatopancreatic cells were Hem2 and Hep1, respectively. We propose that Hem2 has oligopotent potential as the initiation site for haemocyte development and that Hem4 and Hem5, located at the end of development, are the most mature immune cell types in haemocytes. Hep5 and Hep6 were the developing terminal cells of hepatopancreas. The antioxidant system and proPO system of shrimp were activated under ammonia nitrogen stress. A large number of DEGs were involved in oxidative stress, detoxification metabolism, and immune defence. In particular, important response genes such as AMPs, proPO, and GST were not only marker genes for identifying cell groups but also played an important role in shrimp cell differentiation and functional plasticity. By successfully applying 10× Genomics based scRNA-seq to the study of shrimp, the single-cell transcriptional profiles of hepatopancreatic cells and haemocytes of shrimp innate immune responses under ammonia stress were constructed for the first time. This atlas of invertebrate hepatopancreatic cells and haemocytes at single-cell resolution identifies molecular events that underpin shrimp innate immune system responses to stress.

Development of FGF-2-loaded electrospun waterborne polyurethane fibrous membranes for bone regeneration.

Guided bone regeneration (GBR) membrane has been used to improve functional outcomes for periodontal regeneration. However, few studies have focused on the biomimetic membrane mimicking the vascularization of the periodontal membrane. This study aimed to fabricate waterborne polyurethane (WPU) fibrous membranes loaded fibroblast growth factor-2 (FGF-2) via emulsion electrospinning, which can promote regeneration of periodontal tissue via the vascularization of the biomimetic GBR membrane. A biodegradable WPU was synthesized by using lysine and dimethylpropionic acid as chain extenders according to the rule of green chemical synthesis technology. The WPU fibers with FGF-2 was fabricated via emulsion electrospinning. The results confirmed that controlled properties of the fibrous membrane had been achieved with controlled degradation, suitable mechanical properties and sustained release of the factor. The immunohistochemical expression of angiogenic-related factors was positive, meaning that FGF-2 loaded in fibers can significantly promote cell vascularization. The fiber scaffold loaded FGF-2 has the potential to be used as a functional GBR membrane to promote the formation of extraosseous blood vessels during periodontal repairing.

Anti-tumor effect of local injectable hydrogel-loaded endostatin alone and in combination with radiotherapy for lung cancer.

Endostatin (ES) can effectively inhibit neovascularization in most solid tumors and has the potential to make oxygen delivery more efficient and increase the efficacy of radiotherapy (RT). With a short half-life, ES is mainly administered systemically, which leads to low intake in tumor tissue and often toxic systemic side effects. In this study, we used hyaluronic acid-tyramine as a carrier to synthesize an ES-loaded hydrogel drug (ES/HA-Tyr) that can be injected locally. ES/HA-Tyr has a longer half-life and fewer systemic toxic side effects, and it exerts a better anti-angiogenic effect and anti-tumor effect with RT. In vitro, ES/HA-Tyr showed sustained release in the release assay and a stronger ability to inhibit the proliferation of human umbilical vascular endothelial cells (HUVECs) in the MTT assay; it exhibited a more potent effect against HUVEC invasion and a stronger anti-angiogenic effect on HUVECs in tube formation. In vivo, ES/HA-Tyr increased local drug concentration, decreased blood drug concentration, and caused less systemic toxicity. Further, ES/HA-Tyr effectively reduced tumor microvessel density, increased tumor pericyte coverage, decreased tumor hypoxia, and increased RT response. ES/HA-Tyr + RT also had increased anti-tumor and anti-angiogenic effects in Lewis lung cancer (LLC) xenograft models. In conclusion, ES/HA-Tyr showed sustained release, lower systemic toxicity, and better anti-tumor effects than ES. In addition, ES/HA-Tyr + RT enhanced anti-angiogenic effects, reduced tumor hypoxia, and increased the efficacy of RT in LLC-bearing mice.

Transcriptome reveals the important role of metabolic imbalances, immune disorders and apoptosis in the treatment of Procambarus clarkii at super high temperature.

Temperature is an important environmental factor in the living environment of crustaceans. Changes in temperature can affect their normal growth and metabolism and even cause bacterial disease. Currently, the potential anti-reverse molecular reaction mechanism of crustaceans during high-temperature conditions has not yet been fully understood. Therefore, in this study, we characterised the transcriptome of Procambarus clarkii using RNA sequencing and performed a comparison between super-high-temperature treated samples and controls. After assembly and annotation, 81,097 unigenes with an average length of 069 bp and 358 differentially expressed genes (DEGs) were identified. Among these DEGs, 264 were differentially upregulated and 94 were differentially downregulated. To obtain comprehensive gene function information, we queried seven databases, namely, Nr, Nt, Pfam, KOG, Swiss-Prot, KEGG, and GO to annotate gene functions. Transcriptome analysis revealed that the identified DEGs have significant effects on immune-related pathways, including lysosomal and phagosomal pathways, and that super-high-temperature conditions can cause disease in P. clarkii. Some significantly downregulated genes are involved in oxidative phosphorylation and the PPAR signalling pathway; this suggests a metabolic imbalance in P. clarkia during extreme temperature conditions. In addition, elevated temperature changed the expression patterns of key apoptosis genes XIAP, CASP2, CASP2, CASP8, and CYTC, thereby confirming that high-temperature conditions caused immune disorders, metabolic imbalance, and, finally, triggered apoptosis. Our results provide a useful foundation for understanding the molecular mechanisms underlying the responses of P. clarkii during high-temperature conditions.

Comprehensive expression analysis of the beta integrin from Penaeus monodon indicating its participation in innate immunity and ammonia nitrogen stress response.

The aim of the present study was to investigate the function of the beta integrin (PmItgb) in Penaeus monodon. The 3011 bp cDNA sequence of PmItgb was cloned from P. monodon using rapid amplification of cDNA ends (RACE) PCR. Phylogenetic tree analyses indicated that the amino acid sequence of PmItgb should be merged into Fenneropenaeus chinensis (93%). Quantitative real-time PCR (q RT-PCR) revealed that PmItgb mRNA was highly expressed in the hemocytes. In addition, with regard to developmental stages, PmItgb showed significantly higher expression in oosperm, nauplius IV, zoea I and III, and post larval stages than that in other development stages. PmItgb expression in the shrimp epidermis was higher in the postmolt (B) stage, and lower in other molting stages. We also found that Vibrio harveyi and V. anguillarum challenge enhanced PmItgb expression in the hepatopancreas and gills. When PmItgb was inhibited, innate immunity-related genes such as ALF, crustin 1, crustin 7, penaeidin 3, and penaeidin 5 were significantly down-regulated. Furthermore, we demonstrated that PmItgb knock-down by specific dsRNA reduced bacterial clearance. In high ammonia nitrogen concentrations, PmItgb was significantly up-regulated in the hepatopancreas and gills. After PmItgb was silenced, the rate of mortality owing to high ammonia nitrogen concentrations decreased; the expression of related anti-apoptotic genes was up-regulated, and that of the apoptotic genes was slightly down-regulated. These results suggested that PmItgb may be involved in shrimp innate immunity and mediate apoptosis of hepatopancreatic cells induced by high ammonia nitrogen environments.

Up-regulation of Insulin-like Growth Factor Binding Protein-3 Is Associated with Brain Metastasis in Lung Adenocarcinoma.

The brain is the most common metastatic site of lung adenocarcinoma; however, the mechanism of this selective metastasis remains unclear. We aimed to verify the hypothesis that exposure of tumor cells to the brain microenvironment leads to changes in their gene expression, which promotes their oriented transfer to the brain. A549 and H1299 lung adenocarcinoma cells were exposed to human astrocyte-conditioned medium to simulate the brain microenvironment. Microarray analysis was used to identify differentially expressed genes, which were confirmed by quantitative real-time PCR and western blotting. Knockdown experiments using microRNAs and the overexpression of genes by cell transfection were performed in addition to migration and invasion assays. In vitro findings were confirmed in clinical specimens using immunohistochemistry. We found and confirmed a significant increase in insulin-like growth factor binding protein-3 (IGFBP3) levels. Our results also showed that the up-regulation of IGFBP3 promoted A549 cell epithelial-mesenchymal transition, migration, and invasion, while the knockdown of IGFBP3 resulted in decreased cell motility. We also found that Transforming growth factor-ß (TGF-ß)/Mothers against decapentaplegic homolog 4 (Smad4)-induced epithelial-mesenchymal transition was likely IGFBP3-dependent in A549 cells. Finally, expression of IGFBP3 was significantly elevated in pulmonary cancer tissues and intracranial metastatic tissues. Our data indicate that up-regulation of IGFBP3 might mediate brain metastasis in lung adenocarcinoma, which makes it a potential therapeutic target.

Characterization of a group D anti-lipopolysaccharide factor (ALF) involved in anti-Vibrio response in Penaeus monodon.

Antimicrobial peptides (AMPs) are an essential component of innate immunity of invertebrates. Anti-lipopolysaccharide factor (ALF), as a main type of AMPs in crustaceans, attends in the disease prevention in general. In this research, a novel Group D ALF was identified and characterized from Penaeus monodon, named PenmonALF8. It was an anionic peptide, with both the full-length peptide and lipopolysaccharide binding domain (LBD) a low isoelectric point. PenmonALF8, composed of a signal peptide of 26 amino acids and a mature peptide of 98 amino acids, probably contained three alpha helixes and four beta sheets. Moreover, PenmonALF8 was detected in all tested tissues of P. monodon, and the expression level in hemocyte and intestine was relatively high. When challenged by Vibrio parahaemolyticus, PenmonALF8 showed 30-100 times higher expression level in all the tissues except in hemocyte and intestine, indicating that PenmonALF8 played a very important role in the immune response of P. monodon. By fusing to a SUMO protein, PenmonALF8 was successfully over-expressed in E. coli and purified by affinity chromatography. Additionally, the reconstituted PenmonALF8 and its LBD region displayed modest antimicrobial activity. This is the first research about the Group D ALF in P. monodon, which provides more information for humoral immunity study of shrimps.

C-type lectin response to bacterial infection and ammonia nitrogen stress in tiger shrimp (Penaeus monodon).

C-type lectins (CTLs) are pattern recognition receptors (PRRs) that are important in invertebrate innate immunity for the recognition and elimination of pathogens. Although they were reported in many shrimp, C-type lectins subfamily contain a large number of members with different functions that need to research in deep. In this present study, a new type of CTL, PmCL1 with 861 bp long full-length cDNA, that encodes a protein with 164-amino acid from a 495-bp open reading frame, was isolated and characterized from tiger shrimp (Penaeus monodon). The mRNA transcript of PmCL1 showed the highest expression in the hepatopancreas, whereas it was barely detected in the ovary. After the shrimp were stimulated by Vibrio harveyi and Vibrio anguillarum, PmCL1 expression in the hepatopancreas and gill was significantly upregulated. A carbohydrate-binding assay revealed the specificity of PmCL1 for pathogen-associated molecular patterns (PAMPs) that included peptidoglycan (PGN) and lipopolysaccharide (LPS), and saccharides that included d-glucose, galactosamine, α-lactose, treholose, and d-mannose. Recombinant PmCL1 agglutinated gram-positive (Staphylococcus aureus) and gram-negative bacteria (V. harveyi, V. anguillarum, Vibrio alginolyticus, Vibrio parahemolyticus, Vibrio vulnificus, and Aeromonas hydrophila) in the presence of calcium ions and enhanced the efficiency of clearing the invading bacteria. Collectively, our results suggested that PmCL1 might play an important role as a pattern recognition receptor (PRR) in the immune response towards pathogen infections, as well as the response towards ammonia nitrogen stress.