Ficus hirta Vahl. alleviate LPS induced apoptosis via down-regulating of miR-411 in orange-spotted grouper (Epinephelus coioides) spleen cell.

Ficus hirta Vahl. (FhV) has been shown to have antimicrobial and antiviral efficacy. To further ascertain the pharmacological properties of FhV., and to search for alternatives to antibiotics. An in vitro experiment was carried out to evaluate what influence FhV. would have on LPS-induced apoptosis. In this study, Fas, an apoptosis receptor, was cloned, which included a 5'-UTR of 39 bp, an ORF of 951 bp, a protein of 316 amino acids, and a 3'-UTR of 845 bp. EcFas was most strongly expressed in the spleen tissue of orange-spotted groupers. In addition, the apoptosis of fish spleen cells induced by LPS was concentration-dependent. Interestingly, appropriate concentrations of FhV. alleviated LPS-induced apoptosis. Inhibition of miR-411 further decreased the inhibitory effect of Fas on apoptosis, which reduced Bcl-2 expression and mitochondrial membrane potential, enhanced the protein expression of Bax and Fas. More importantly, the FhV. could activate miR-411 to improve this effect. In addition, luciferase reporter assays showed that miR-411 binds to Fas 3'-UTR to inhibit Fas expression. These findings provide evidence that FhV. alleviates LPS-induced apoptosis by activating miR-411 to inhibit Fas expression and, therefore, provided possible strategies for bacterial infections in fish.

miR-2765 involved in ammonia nitrogen stress via negative regulation of autophagy in shrimp.

MicroRNA (miRNA) is a highly conserved non-coding tiny endogenous RNA molecule that regulates various cellular functions by inhibiting mRNA translation or promoting the degradation of proteins. In this study, we identified a specific miRNA (designed as Pva-miR-2765) from Penaeus vannamei, which widely distributed in different tissues of shrimp, with the highest concentration found in the intestine. Through fluorescence in situ hybridization (FISH), we observed that Pva-miR-2765 is primarily located in the cytoplasm. Interestingly, we found that the expression of Pva-miR-2765 significantly decreased in hemocytes, hepatopancreas and gill under ammonia nitrogen stress. Furthermore, when Pva-miR-2765 was silenced, the autophagy level in shrimp significantly increased. Additionally, Pva-miR-2765 was found to promote pathological damage in the hepatopancreas of shrimp. Subsequently, correlation analysis revealed a negative relationship between the expression of Pva-miR-2765 and PvTBC1D7. To confirm this interaction, we conducted a dual luciferase reporter gene assay, which demonstrated that Pva-miR-2765 inhibit the expression of PvTBC1D7 by interacting with its 3'UTR. And the expression level of PvTBC1D7 in shrimp decreased significantly under ammonia nitrogen stress in Pva-miR-2765 overexpressed. Our findings suggest that Pva-miR-2765 can reduce autophagy in P. vannamei by inhibiting the regulation of PvTBC1D7, thereby participating in the oxidative stress of shrimp caused by ammonia nitrogen stress.

c-Jun N-terminal kinase activation contributes to improving low temperature tolerance via regulating apoptosis in the Pacific white shrimp Penaeus vannamei.

Temperature is an essential environmental factor for the survival of aquatic animals. Low temperature stress can induce mitochondria to produce excessive ROS and free radicals, and destroy homeostasis. c-Jun N-terminal kinase (JNK) is involved in regulating various physiological processes, including inflammatory responses, cell cycle, reproduction, and apoptosis. Here, we investigated the mechanism of ROS/JNK pathway under low temperature stress both in vitro and in vivo. In this study, transcriptome analysis revealed that apoptosis, autophagy, calcium channel, and antioxidant were involved in the mediation of low temperature tolerance in Pacific white shrimp (penaeus vannamei). PvJNK was activated in response to low temperature stress. Treatments with different temperature caused oxidative stress as demonstrated by increased intensity of the ROS indicator H2DCF-DA, and induced apoptosis as confirmed by indicator FITC. Pretreatment with N-acetylcysteine, an ROS scavenger, attenuated low temperature induced apoptosis, and inhibited the expression of PvJNK. In addition, we demonstrate that mediator PvJNK translocated to nuclear through interacting with PvRheb. By using flow cytometry, inhibiting PvJNK can increase the expression of apoptosis related genes, accelerate tissue damage, and induce ROS and cell apoptosis. The ultimate inhibition of PvJNK accelerates the mortality of shrimp under low temperature stress. Overall, these findings suggest that during low temperature stress, PvJNK was activated by ROS to regulates apoptosis via interacting with PvRheb to promote PvJNK into the nucleus and to improve low temperature tolerance of shrimp.

Transcriptome profiling reveals characteristics of hairy root and the role of AhGLK1 in response to drought stress and post-drought recovery in peanut.

BACKGROUND: HR (hairy root) has emerged as a valuable tissue for the rapid characterization of plant gene function and enzyme activity in vivo. AhGLK1 (Arachis hypogaea L. golden2-like 1) is known to play a role in post-drought recovery. However, it is unclear (a) whether HR has properties that are distinct from those of PR (primary root); and (b) which gene networks are regulated by AhGLK1 in response to drought stress and recovery in peanut. RESULTS: We found that cells of the root tip cortex were larger in HR than in PR, while a total of 850 differentially expressed genes (DEGs) were identified in HR compared to PR. Eighty-eight of these DEGs, relating to chlorophyll and photosynthesis, were upregulated in HR. In addition, AhGLK1-OX (AhGLK1-overexpressing) HR showed a green phenotype, and had a higher relative water content than 35 S::eGFP (control) HR during drought stress. RNA-seq analysis showed that 74 DEGs involved both in the drought response and the post-drought recovery process were significantly enriched in the galactose metabolism pathway. GO terms enrichment analysis revealed that 59.19%, 29.79% and 17.02% of the DEGs mapped to the 'biological process' (BP), 'molecular function' (MF) and 'cellular component' (CC) domains, respectively. Furthermore, 20 DEGs involved in post-drought recovery were uniquely expressed in AhGLK1-OX HR and were significantly enriched in the porphyrin metabolism pathway. GO analysis showed that 42.42%, 30.30% and 27.28% of DEGs could be assigned to the BP, MF and CC domains, respectively. Transcription factors including bHLH and MYB family members may play a key role during drought stress and recovery. CONCLUSION: Our data reveal that HR has some of the characteristics of leaves, indicating that HR is suitable for studying genes that are mainly expressed in leaves. The RNA-seq results are consistent with previous studies that show chlorophyll synthesis and photosynthesis to be critical for the role of AhGLK1 in improving post-drought recovery growth in peanut. These findings provide in-depth insights that will be of great utility for the exploration of candidate gene functions in relation to drought tolerance and/or post-drought recovery ability in peanut.

scRNA-seq analysis reveals toxicity mechanisms in shrimp hemocytes subjected to nitrite stress.

In shrimp, hemocytes play an important role in detoxification and immune defense, and are where nitrite accumulates during exposure to this toxic environmental pollutant. However, the heterogeneity mechanisms of toxicity have not been reported under nitrite expose in shrimp. Here, we used single-cell RNA-seq to resolve 24,000 cells, which the responses of different cell populations of hemocytes under nitrite exposure in Penaeus vannamei. We identified 394 specific nitrite-responsive genes in 9 clusters of hemocytes, and found heterogeneity in the nitrite response of the three subpopulations of hemocytes (hyaline, semi-granular and granular cells). In hyaline, the response appeared modest, whereas nitrite-related dysregulation of metabolic processes in granular and semi-granular was pronounced. Ammonia nitrogen will rapidly accumulate in hemocytes of shrimp under nitrite stress. In semi-granular, excessive ammonia will interfere with oxidative phosphorylation and antioxidant system, thus inducing the production of reactive oxygen species. In granular, the abnormality of urea cycle caused by ammonia accumulation is the main toxic factor, which by inhibits arginase and arginine kinase. Collectively, our data provide a single-cell atlas for the dissection of shrimp hemocyte complexity, and reveal the toxicity mechanisms associated with nitrite exposure.

miR-144 and DJ-1/NF-κB regulates UCP4 maintain mitochondrial homeostasis in Penaeus vannamei.

UCP4, as an uncoupling protein in mitochondrial intima, is closely related to the resistance to oxidative stress and the function of mitochondria. However, whether and how its antioxidant capacity also works in crustaceans has not been reported in detail. This study showed that the expression of PvUCP4 was negatively correlated with the expression of pva-miR-144. The content of reactive oxygen species (ROS), ATP, and apoptosis was significantly increased, while the mitochondrial membrane potential (MMP) was seriously depolarized, Edema, vacuolation, and ambiguity of cristae and membrane were observed clearly in mitochondria after the knockdown of PvUCP4 induced by V. alginolyticus. The sharp drop in THC and severe damage in the hepatopancreas were all due to the knockout of PvUCP4 under the stress of V. alginolyticus. The co-transfection of pva-miR-144 and PvUCP4 could partially recover MMP compared with the abnormal expression of pva-miR-144. Similarly, co-transfection of pva-miR-144 and PvUCP4 could partially eliminate apoptosis compared with the abnormal expression of pva-miR-144. In addition, PvUCP4 3'-UTR has a pva-miR-144 predicted binding site in 1417-1428, which also was confirmed by the dual luciferase reporter assay. By the way, the results of ROS, MMP, and apoptosis showed that PvDJ-1 regulated the expression of PvUCP4 through PvNF-κB. Altogether, these results indicated that PvUCP4 has the antioxidant function of resisting oxidation reaction and weakening oxidative damage, to protect the normal operation of mitochondrial function and maintaining the cell homeostasis in shrimp.

MYC drives autophagy to adapt to stress in Penaeus vannamei.

MYC proto-oncogene (MYC), a first oncogenic nuclear transcription factor isolated from the human genome, belongs to the helix loop helix/leucine zipper protein family (bHLHzip). MYC plays an important part in the process of various physiological and biochemical of vertebrate, such as cell growth, proliferation, cycle, and autophagy. However, its molecular regulation mechanism and function in invertebrates are still unclear. In this study, a novel transcription factor MYC gene was screened, cloned, and characterized from Penaeus vannamei. The open reading frame of PvMYC was 1593bp, encode a polypeptide of 530 amino acids with molecular weight of 58.5 kDa, and a theoretical PI of 5.75. The results of tissue distribution showed that PvMYC was constitutively expressed in all detected tissues, and highest expression in hepatopancreas. The expression level of PvMYC up-regulated significantly and responded to low temperature stress by nuclear ectopic after low temperature stress. Overexpression of PvMYC in shrimp hemocytes negatively regulated the expression of Beclin-1 and reduced the conversion from LC3I to LC3II, yet p62 was decreased significantly. Meanwhile, RAPA eliminated the inhibition of autophagy caused by overexpression of PvMYC. ROS levels and autophagy flux showed the similar trend under low temperature stress after silencing PvMYC. The expression levels of Beclin-1, key ATG gene and LC3II increased significantly, while p62 decreased significantly under the same conditions. In addition, the Total hemocyte count (THC) decreased sharply, and accelerated the injury of hepatopancreas under low temperature stress after silencing PvMYC. Collectively, these results suggest that PvMYC has vital role in the cold adaptation mechanism of P. vannamei by negatively regulating autophagy.

Integrative analysis of the miRNA-mRNA regulation network in hemocytes of Penaeus vannamei following Vibrio alginolyticus infection.

Penaeus vannamei is an important cultured shrimp that has high commercial value in the worldwide. However, the industry suffers heavy economic losses each year due to disease outbreaks caused by pathogenic bacteria. In the present study, after Vibrio alginolyticus infection, DNA damage in the hemocytes of the shrimp markedly increased, and autophagy and apoptosis increased significantly. Subsequently, hemocytes were sampled from the control and infected shrimp and sequenced for mRNA and microRNA (miRNA) 24 h after V. alginolyticus infection to better understand the response mechanism to bacterial infection in P. vannamei. We identified 1,874 and 263 differentially expressed mRNAs (DEGs) and miRNAs (DEMs) respectively, and predicted that 997 DEGs were targeted by DEMs. These DEGs were involved in the regulation of multiple signalling pathways, such as Toll and IMD signalling, TGF-beta signalling, MAPK signalling, and cell apoptosis, during Vibrio alginolyticus infection of the shrimp. We identified numerous mRNA-miRNA interactions, which provide insight into the defense mechanism that occur during the antimicrobial process of P. vannamei.

TBC domain family 7-like enhances the tolerance of Penaeus vannamei to ammonia nitrogen by the up-regulation of autophagy.

TBC domain family 7 (TBC1D7) is one of the subunits of tuberous sclerosis complex (TSC) and an important regulator of autophagosome biogenesis. However, the function of TBC1D7 is not fully understood in crustaceans. In the present study, TBC1D7 was identified from Penaeus vannamei. The complete coding sequence of PvTBC1D7 was of 960 bp encoding a predicted polypeptide of 319 amino acids with one conserved TBC domain, which shared high similarity with TBC1D7 of that other species. The mRNA of PvTBC1D7 was highly expressed in hemocyte and hepatopancreas, and the PvTBC1D7 protein was localized specifically in the cytoplasm of hemocyte of shrimp. Besides, PvTBC1D7 was co-localized with PvTSC1 in the cytoplasm of shrimp, indicating that there might existed a binding relationship between PvTBC1D7 and PvTSC1. During the ammonia nitrogen stress, the mRNA transcripts of PvTBC1D7 were significantly upregulated in hemocyte, hepatopancreas, and gill. Functionally, overexpression of PvTBC1D7 in vitro restored the inhibition to autophagy caused by chloroquine (CLQ) and increased the autophagy level, while the silencing of PvTBC1D7 could inhibit the autophagy. More importantly, after interfering with PvTBC1D7, the autophagy level decreased significantly both in hepatopancreas and hemocyte of P. vannamei, the mRNA expression of PvmTOR was increased remarkably with the significantly decrease of autophagy-related genes (PvATG12 and PvATG14). And the reduction of PvTBC1D7 remarkably exacerbated the damage of hepatopancreas, increased the accumulation of ROS, and reduced the survival proportion of shrimp under ammonia nitrogen stress. Altogether, these results indicated that PvTBC1D7 might positively regulate the autophagy by stabilizing the negative regulation of mTOR by TSC complex, reduce the oxidative stress damage and improve shrimp ammonia nitrogen tolerance.

Ficus hirta Vahl. promotes antioxidant enzyme activity under ammonia stress by inhibiting miR-2765 expression in Penaeus vannamei.

Ficus hirta Vahl. has been reported to have hepatoprotective, antitumor, antibacterial functions, and is used to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Ammonia nitrogen is one of the most common environmental stress factors in aquaculture. Long-term exposure to high concentrations of ammonia nitrogen can induce oxidative stress and increase the risk of infections. However, whether Ficus hirta Vahl. has effect on ammonia nitrogen stress is unclear. In present study we report that Ficus hirta Vahl. improves the activity of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) of shrimp and decreases shrimp mortality caused by ammonia nitrogen stress. It is demonstrated that miR-2765 is negatively regulate the antioxidant capacity. We find that SOD was a direct target gene of miR-2765. MiR-2765 can bind to 3'-untranslated region (3'-UTR) of SOD to inhibit its transcription. Furthermore, Ficus hirta Vahl. down-regulates miR-2765 to activate the antioxidant capacity to alleviate the damage caused by ammonia nitrogen stress. Interestingly, overexpression of miR-2765 could attenuate the protective effect of Ficus hirta Vahl. on shrimp under ammonia nitrogen stress. These data indicate that Ficus hirta Vahl. alleviates the damage of ammonia nitrogen stress in shrimp by repressing miR-2765 and activating the antioxidant enzyme system. This study will provide a theoretical basis and a new perspective for assessing the toxicity mechanism of ammonia nitrogen in the process of farming on shrimp.

Pva-miR-252 participates in ammonia nitrogen-induced oxidative stress by modulating autophagy in Penaeus vannamei.

MicroRNAs (miRNAs) are critical post-transcriptional regulators, which play a crucial role in resistance to adverse environmental stress by regulating autophagy. However, the mechanism of miRNA involved in the autophagy regulation of shrimp under ammonia nitrogen stress is still limited. In the present study, ammonia nitrogen could induce hepatopancreas injury and oxidative stress of P. vannamei, and significantly increase the content of ROS in hemocytes by flow cytometry. Simultaneously, it is accompanied by autophagy occurred in the hemocytes and hepatopancreas. Furthermore, the qRT-PCR analysis revealed that the expression of pva-miR-252 in P. vannamei decreased significantly after ammonia nitrogen stress, and pva-miR-252 negatively regulated PvPI3K by binding to 3'UTR of PvPI3K by double-luciferase assay. Pva-miR-252 overexpression could significantly increase the level of autophagy, and restore the autophagy inhibition caused by Chloroquine in vitro , whereas silencing of pva-miR-252 resulted in the opposite effect. More importantly, overexpression of pva-miR-252 could enhance the activity of antioxidant enzymes and reduced the production of ROS of shrimp under ammonia nitrogen stress. In conclusion, pva-miR-252 could positively regulate autophagy through PvPI3K and improve the antioxidant enzyme activity of P. vannamei under ammonia nitrogen stress, and our study provides a novel theoretical molecular mechanism for further understanding the shrimp cope with a high ammonia nitrogen environment.

Potential roles for microRNAs in facilitating physiological adaptation to low-temperature stress in Penaeus vannamei.

Water temperature is one of the most common physiological stressors in aquaculture. Previous studies demonstrate that organisms require miRNA activity for survival in various unfavourable environmental conditions. However, the detailed role of miRNA in response to low-temperature stress is still unclear. This study was conducted to construct a comprehensive miRNA dataset for the Penaeus vannamei after low-temperature stress. A total of 329 known miRNAs and 60 putative novel miRNAs were identified. Among them, 17 miRNAs were identified with the most significant differences, and they were found to be involved in stimulation or stress processes. The main enriched target pathways of the 17 miRNAs were the Hippo signalling pathway, autophagy, apoptosis and MAPK signalling. In addition, all the 17 miRNAs identified were up-regulated, suggesting that miRNA by inhibiting the expression of target genes constitutes an effective strategy for Penaeus vannamei to cope with low-temperature stress. The 35-putative target of the 17 miRNAs was related to apoptosis and autophagy-related proteins, such as Pxt, DRAM2, cytochrome c, ATG2B, JNK, ATG4 and API5. The analysis of miRNA expression profiles contributes to the understanding of the molecular mechanisms of low-temperature tolerance in Penaeus vannamei. This study's findings enrich current miRNA resources and offer the possibility to validate the involvement of 17 miRNAs in the response of shrimp to low-temperature stress.

miR-151 Affects Low-Temperature Tolerance of Penaeus vannamei by Modulating Autophagy Under Low-Temperature Stress.

MicroRNAs (miRNAs) play key roles in many physiologic and pathologic processes, including autophagy. Autophagy is cellular in an emergency response mechanism of environment stress, but their complex molecular regulatory mechanism under low-temperature stress is largely unknown in shrimp, especially miRNA-mediated regulation of autophagy in low-temperature tolerance. In this article, a shrimp PvTOR and miRNA pva-miR-151 cooperation in response to low-temperature stress has been reported. Pva-miR-151 showed expression patterns opposite to target PvTOR under low-temperature stress. The pva-miR-151 targets the 3'-UTR region of PvTOR, regulate the formation of autophagosome, which contribute to the degradation and recycling of damaged organelles. In addition, the low-temperature tolerance was correlated positively with autophagy in shrimp. Silenced pva-miR-151 increased sensitivity to low-temperature stress, whereas overexpression pva-miR-151 decreased the expression of PvTOR and p-TOR and increased tolerance to low-temperature stress by improving the formation of autophagosome and total hemocyte count. In addition, the TOR activator 3BDO can partially rescue autophagy induced by overexpression of pva-miR-151; these results indicate that miR-151 was necessary for the low-temperature tolerance in shrimp. Taken together, we provide a novel strategy and mechanism for shrimp breeding to improve shrimp low-temperature tolerance.

A Novel Kelch-Like-1 Is Involved in Antioxidant Response by Regulating Antioxidant Enzyme System in Penaeus vannamei.

Heavy metals are typical cumulative pollutants that can enter and poison the human body through the food chain. However, the molecular mechanism of heavy metal-induced oxidative stress is unclear. In this study, we characterize PvKelch-like-1 from P. vannamei and explore its antioxidant roles in immune regulation of crustaceans. PvKelch-like-1 full length contains 2107 nucleotides, consists of a 5' untranslated region (UTR) of 79 bp, a 3' UTR of 180 bp, and a ORF of 1848 encoded 615 amino acids, which contain a BTB, BACK and Kelch motif, putative molecular mass and isoelectric point were 69 KDa and 6.54. PvKelch-like-1 mRNA was ubiquitously expressed in all detected tissue of P. vannamei, and mRNA expression levels were significantly up-regulated from 6 to 24 h after cadmium stress and reached the highest level (3.2-fold) at 12 h in the hepatopancreas. Subcellular localization analysis revealed that PvKelch-like-1 was localized in the nucleus. Silencing PvKelch-like-1 significantly increased reactive oxygen species (ROS) (1.61-fold) production and DNA damage (1.32-fold) in the shrimp hemolymph, and significantly decreased total hemocyte counts (THC) (0.64-fold) at 6 h in hemolymph. Additionally, the antioxidant genes PvCAT (0.43-fold), PvMnSOD (0.72-fold), PvGST (0.31-fold) and PvGPx (0.59-fold) at 6 h were decreased significantly in PvKelch-like-1 silenced shrimp after cadmium stress. Overexpression of PvKelch-like-1 has the opposite results in enzyme activity. The SOD (2.44-fold) and CAT (2.19-fold) activities were significantly increased after overexpressing PvKelch-like-1. These results suggest that PvKelch-like-1 plays a vital role in shrimp innate immune defense by positively regulating the expression of antioxidant enzyme genes to respond to cadmium stress.

A Litopenaeus vannamei p70S6K gene is involved in the antioxidative and apoptosis under low temperature.

p70S6K is involved in cellular response, such as tumor metastases, the immune response and tissue repair in vertebrates. The role of p70S6K in these physiological processes in crustaceans remains, however, unknown. In this study, the Lvp70S6K was identified, containing a 5' UTR of 294 bp, an ORF of 1494 bp ad a 3' UTR of 211 bp, encoding 497 amino acids with a theoretical molecular weight of 70 kDa and an estimated isoelectric point of (pI) of 5.16. The multiple alignment found that Lvp70S6K was highly homologous with other invertebrates. Lvp70S6K mRNA was detected in all the tested tissues and the Lvp70S6K expression levels was significantly down-regulated and reached the lowest level (0.44-fold, p < 0.01) at 1.5 h after low temperature stress. The subcellular localization of Lvp70S6K could be detected in cytoplasm. ROS production was significantly up-regulation (1.19-fold, p < 0.01), total hemocyte count (THC) was significantly down-regulation (0.22-fold, p < 0.01), apoptosis rate was markedly increased (1.09-fold, p < 0.01), apoptosis-related genes of LvPDCD4 (1.61-fold, p < 0.01) and LvCyt.C (1.23-fold, p < 0.01) were up-regulated, and anti-apoptotic gene of LvBcl-2 (0.69-fold, p < 0.01), LvIAP1 (0.68-fold, p < 0.01) and LvIAP2 (0.45-fold, p < 0.01) were decreased after low temperature stress in hemolymph of Lvp70S6K-silenced shrimp at 1.5 h. Silencing of LvPTEN significantly increased Lvp70S6K, LvPI3K, LvAKT and LvmTOR expression. In summary, these results indicated that Lvp70S6K play a crucial role in oxidative and apoptosis, which was able to negatively regulate by PTEN.

MiR-122 is involved in immune response by regulating Interleukin-15 in the orange-spotted grouper (Epinephelus coioides).

Epinephelus coioides is an important economic culture marine fish and is susceptible to various pathogenic diseases. Increasingly evidences showed that miRNAs participated in the regulation of the cell proliferation, differentiation and immune response. MiR-122 has been reported to play an essential role in immune response by triggering an inflammatory reaction. However, the function of miR-122 in response to bacterial infection is unclear in Epinephelus coioides. Herein, we report that miR-122 is involved in response to Aeromonas hydrophila infection of grouper spleen cells (GS). IL-15, IL-6 and IL-1ß are inhibited in overexpression miR-122 GS cells, while induced in silence miR-122 GS cells. In addition, IL-15 is predicted to be the target gene of miR-122, which is further confirmed by LUC. Taken together, we propose that miR-122 regulates the immune response to bacterial infection by triggering IL-15.

A sensitive pH fluorescent probe based on triethylenetetramine bearing double dansyl groups in aqueous solutions and its application in cells.

pH fluorescent probes possess many advantages, including intracellular detection, rapid response time and nondestructive testing. In this paper, a highly selective and sensitive fluorescent pH probe based on triethylenetetramine bearing double dansyl groups (1) was synthesized. This probe offers fluorescent measurement of pH value in the range of 5.81-7.21 in aqueous solution, with an 8.64-fold enhancement of fluorescent emission intensity over the unmodified probe. Probe 1 shows a fluorescent color change from a pale yellow to bright green when the pH is increased from 5.81 to 7.21. In addition, probe 1 shows good potential as a fluorescent visualizing sensor for pH values in living GS cells of epinepheluscoioides. The mechanism of the fluorescent response of probe 1 to solution pHs was further clarified by NMR, fluorescent spectra, and UV-vis absorption spectra. The results indicate that the fluorescent emission will shift with an increase in solution pHs, due to increasing deprotonation of the nitrogen atom on the sulfonamides. Deprotonation of the sulfonamide group will inhibits the intramolecular charge transfer process between the imino group and the naphthalene ring, resulting in the recognition phenomenon of blue shift and enhancement of fluorescent emission intensity.

Functional analysis target of rapamycin (TOR) on the Penaeus vannamei in response to acute low temperature stress.

Target of rapamycin (TOR) is an atypical of Ser/Thr protein kinase that plays an important role in many aspects such as cell growth, reproduction, differentiation, cell cycle regulation, autophagy and apoptosis. However, little information is known about the enzyme in crustaceans. Here, a novel TOR was identified from shrimp Penaeus vannamei (PvTOR) and its biological functions were investigated in response low temperature stress. The PvTOR gene encoded a polypeptide of 2464 amino acids with an estimated molecular mass of 279.4 kD and a predicted isoelectronic point (pI) of 7.30. Phylogenetic analysis revealed that PvTOR shared high similarity with other known species. PvTOR mRNA was detected in all the tested tissues and highest transcription in muscle and hepatopancreas. PvTOR transcriptional level was up-regulated significantly at 1.5 h and 3 h, and down-regulated at 12 h and 24 h after low temperature stress. TEM and autophagy indicator system GFP-PvLC3 suggested that low temperature induced autophagy generation. ROS, Ca2+ concentration and apoptosis rate were increased significantly in TOR-knockdown shrimp after low temperature stress. The autophagy associated gene ATG8II/I, PvBeclin-1, PvATG14, apoptosis gene PvPARP, Pvcasp-3, PvBAX and Pvp53 transcripts, and casp-3/8 activity in hemocyte were increased significantly in TOR-knockdown group shrimp at 3 h after low temperature stress. Additionally, THC counts of TOR-knockdown group were significantly higher than the dsGFP group. In summary, these results suggested that PvTOR plays an important role in the adaptation mechanisms of shrimp at low temperature by regulating autophagy and apoptosis.

The key differentially expressed genes and proteins related to immune response in the spleen of pufferfish (Takifugu obscurus) infected by Aeromonas hydrophila.

The immune mechanism elicited in pufferfish (Takifugu obscurus) against the invasion of Aeromonas hydrophila is still poorly understood. We examined the spleen of pufferfish at the transcriptome and proteome levels by using Illumina-seq and TMT coupled mass spectrometry after 12 h infection by A. hydrophila, respectively. A total of 2,339 genes (1,512 up-regulated and 827 down-regulated) and 537 (237 up-regulated and 300 down-regulated) proteins were identified. GO and KEGG analyses revealed that the responses to stimulus were the main biological processes, intestinal immune network for IgT production and calcium signaling pathway. Fourteen genes (8 up-regulated and 6 down-regulated) and proteins (5 up-regulated and 9 down-regulated) involved immune responses or signal transduction were validated by qRT-PCR and parallel reaction monitoring to confirm the reliability of the transcriptomic and proteomic analyses, respectively. Moreover, qRT-PCR and flow cytometry were used to detect dynamics of the genes in calcium signaling pathway and changes of concentration of cytoplasm Ca2+ in spleen cells within a 72 h challenge. This study provides the findings regarding immune response, especially intestinal immune network for IgT production pathway and calcium signaling pathway at the molecular, protein and cellular in pufferfish after infection by A. hydrophila. These results would provide a new insight and molecular targets into the response to pathogenic infection in pufferfish.

Integrative mRNA-miRNA interaction analysis associated with the immune response of Epinephelus coioddes to Vibrio alginolyticus infection.

MicroRNAs (miRNAs) are a kind of small non-coding RNAs that have been reported to play a vital role in mediating host-pathogen interactions. High-throughput sequencing technology was applied to identify and illuminate mRNAs and miRNAs from grouper infected with Vibrio alginolyticus. The KEGG pathway enrichment analysis showed that the most significate DEGs are associated with Toll-like receptor signaling pathway and NOD-like receptor signaling pathway. We obtained 374 known miRNAs and 116 novel miRNAs. During them, there are 31 up-regulated miRNAs and 93 down-regulated miRNAs. miRNA-mRNA GO and KEGG analysis show that there are 90 miRNAs associated with the immune system. The target genes of immune-related miRNAs (miR-142, miR-146, miR-150, miR-155, miR-203, miR-205, miR-24, miR-31) and genes (CD80, IL-2, AMPK, PI3K) in Epinephelus coioddes were predicted and validated. This study provides an opportunity to further understanding the molecular mechanisms especially the immune system of miRNA regulation in Epinephelus coioddes host-pathogen interactions.