C-type lectins (CTLs) play crucial roles in innate immune responses in invertebrates by recognizing and eliminating microinvaders. In this study, a CTL from pacific white shrimp Litopenaeus vannamei (LvCTL3) was identified. LvCTL3 contains a single C-type lectin-like domain (CTLD), which shows similarities to those of other shrimp CTLs and has a mutated 'EPD' motif in Ca(2+)-binding site 2. LvCTL3 mRNA can be detected in all tested tissues and expression of LvCTL3 in gills was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges, suggesting activation responses of LvCTL3 to bacterial, virus and immune stimulant challenges. The 5'flanking regulatory region of LvCTL3 was cloned and we identified a NF-κB binding motif in the LvCTL3 promoter region. Dual-luciferase reporter assays indicated that over-expression of L. vannamei dorsal can dramatically up regulate the promoter activity of LvCTL3, suggesting that LvCTL3 expression could be regulated through NF-κB signaling pathway. As far as we know, this is the first report on signaling pathway involve in shrimp CTLs expression. The recombinant LvCTL3 protein was expressed in Escherichia coli and purified by Ni-affinity chromatography. The purified LvCTL3 can agglutinate Gram-negative microbe Vibrio alginolyticus and V. parahaemolyticus and Gram-positive bacteria Bacillus subtilis in the presence of calcium ions, but cannot agglutinate Gram-positive bacteria Streptococcus agalactiae. The agglutination activity of LvCTL3 was abolished when Ca(2+) was chelated with EDTA, suggesting the function of LvCTL3 is Ca(2+)-dependent. In vivo challenge experiments showed that the recombinant LvCTL3 protein can significantly reduce the mortalities of V. parahemolyticus and WSSV infection, indicating LvCTL3 might play significant roles in shrimp innate immunity defense against bacterial and viral infection.

The nuclear factor-kappa B (NF-κB) pathways play important roles in innate immune responses. IκB is the main cytoplasmic inhibitor of NF-κB. In this study, we identified the LvCactus gene from Litopenaeus vannamei, which is the first cloned IκB homologue in subphylum Crustacea. LvCactus contains six predicted ankyrin repeats, which show similarities to those of Cactus proteins from insects. LvCactus localizes in cytoplasm and interacts with LvDorsal, an L. vannamei homologue to Drosophila melanogaster Dorsal belonging to class II NF-κB family, to prevent its nuclear translocation. Contrary to that of LvDorsal, over-expression of LvCactus down-regulates the activities of shrimp antimicrobial peptides promoters, suggesting LvCactus is an inhibitor of LvDorsal. The promoter of LvCactus was predicted to contain five putative NF-κB binding motifs, among which four were proved to be bound by LvDorsal by chromatin immunoprecipitation assays. Dual-luciferase reporter assays also showed that transcription of LvCactus was promoted by LvDorsal but inhibited by LvCactus itself, indicating a feedback regulatory pathway between LvCactus and LvDorsal. Expression of LvCactus was up-regulated after Lipopolysaccharides, poly (I:C), Vibrio parahaemolyticus, and Staphylococcus aureus injections, suggesting an activation response of LvCactus to bacterial and immune stimulant challenges. Differently, the LvCactus expression levels obviously decreased during white spot syndrome virus (WSSV) infection, indicating the feedback regulatory pathway of LvCactus/LvDorsal could be modified by WSSV.

Our previous study showed that visceral adipose tissue-derived serpin (vaspin) was an independent predictor of coronary artery disease (CAD). Further, plasma vaspin levels in patients with unstable angina pectoris were lower than those in patients with stable angina pectoris. In this study, we investigated the prognostic relevance of plasma vaspin levels in patients with CAD and non-CAD.

The simultaneous improvement of grain quality and yield of cereal crops is a major challenge for modern agriculture. Here we show that a rice grain yield quantitative trait locus qLGY3 encodes a MADS-domain transcription factor OsMADS1, which acts as a key downstream effector of G-protein βγ dimers. The presence of an alternatively spliced protein OsMADS1lgy3 is shown to be associated with formation of long and slender grains, resulting in increases in both grain quality and yield potential of rice. The Gγ subunits GS3 and DEP1 interact directly with the conserved keratin-like domain of MADS transcription factors, function as cofactors to enhance OsMADS1 transcriptional activity and promote the co-operative transactivation of common target genes, thereby regulating grain size and shape. We also demonstrate that combining OsMADS1 lgy3 allele with high-yield-associated dep1-1 and gs3 alleles represents an effective strategy for simultaneously improving both the productivity and end-use quality of rice.

Doxorubicin (DOX) is a commonly studied chemotherapeutic agent for the treatment of solid tumors, but the severe side effects limit its clinical application. It is shown that DOX-metal chelate has lower in vitro cytotoxicity compared with DOX, as the anthracyclines of DOX can form coordinative interaction with transition metal ions. In addition, the transition metal ions could catalyze the production of hydroxyl radicals (·OH) via Fenton/Fenton-like reactions to achieve antitumor chemodynamic therapy (CDT). In this study, copper ions (Cu2+) were applied to obtain DOX/Cu(II) prodrug, and a liposomal formulation was used to avoid the rapid blood clearance and optimize the biodistribution of this prodrug. In vitro and in vivo antitumor results demonstrated that this pH sensitive Cu-chelating prodrug can reduce adverse effects of DOX but improve the antitumor efficiency due to the combination of chemotherapy and chemodynamic therapy. Our study provided a facile and effective approach of metal-chelating prodrug strategy for combination cancer therapy strategy.

c-Jun NH2-terminal kinase/stress-activated kinase (JNK/SAPK) and the p38 mitogen-activated protein kinase (p38 MAPK) are important components of cellular signal transduction pathways, which have been reported to be involved in viral replication. However, little is known about JNK1/2 and p38 MAPK signaling pathways in enterovirus 71 (EV71)-infected immature dendritic cells (iDCs). Thus, iDCs were induced from peripheral blood mononuclear cells (PBMC) and performed to explore the expressions and phosphorylation of molecules in the two signaling pathways as well as secretions of inflammatory cytokines and interferons during EV71 replication.

Interleukins are a group of cytokines that play essential roles in immune regulation. Almost all interleukin genes are only found in vertebrates. In this study, an interleukin-16-like gene (LvIL-16L) was identified from Pacific white shrimp, Litopenaeus vannamei. LvIL-16L was predicted to encode a precursor (pro-LvIL-16L) with 1378 amino acids, sharing similarities with predicted pro-IL-16-like proteins from insects. The C-terminus of pro-LvIL-16L protein contained two PDZ domains homologous to the mature IL-16 cytokine of vertebrates. In tissues, LvIL-16L could be processed into a ∼36 kDa mature peptide through a caspase-3 cleavage site, which was verified by in vitro site mutation analysis and in vivo RNA interference (RNAi) experiments. The LvIL-16L mRNA could be detected in all the analyzed tissues and the expression of LvIL-16L was significantly up-regulated after immune stimulation. Using RNAi strategy, the role of LvIL-16L in immune responses was initially investigated. Interestingly, knockdown of LvIL-16L could significantly increase the mortality of the Vibro parahaemolyticus infected shrimps but reduce that of the WSSV infected shrimps, suggesting that LvIL-16L could have opposite effects on the antiviral and antibacterial immune responses in shrimp. To our knowledge, this is the first study of an IL-16-like gene in invertebrates, which could help to elucidate interleukin evolution and regulatory mechanisms of shrimp immune responses.

Deep learning (DL) is currently revolutionizing peptide drug development due to both computational advances and the substantial recent expansion of digitized biological data. However, progress in oligopeptide drug development has been limited, likely due to the lack of suitable datasets and difficulty in identifying informative features to use as inputs for DL models. Here, we utilized an unsupervised deep learning model to learn a semantic pattern based on the intrinsically disordered regions of ~171 known osteogenic proteins. Subsequently, oligopeptides were generated from this semantic pattern based on Monte Carlo simulation, followed by in vivo functional characterization. A five amino acid oligopeptide (AIB5P) had strong bone-formation-promoting effects, as determined in multiple mouse models (e.g., osteoporosis, fracture, and osseointegration of implants). Mechanistically, we showed that AIB5P promotes osteogenesis by binding to the integrin α5 subunit and thereby activating FAK signaling. In summary, we successfully established an oligopeptide discovery strategy based on a DL model and demonstrated its utility from cytological screening to animal experimental verification.

A Dicer2 gene from Scylla paramamosain, named SpDicer2, was cloned and characterized. The full length of SpDicer2 mRNA contains a 121 bp 5'untranslated region (UTR), an open reading frame (ORF) of 4518 bp and a 3' UTR of 850 bp. The SpDicer2 protein contains seven characteristic Dicer domains and showed 34%-65% identity and 54%-79% similarity to other Dicer protein domains, respectively. The mRNA of SpDicer2 was high expressed in hemocytes, intestine and gill and low expressed in the eyestalk and muscle. Moreover, expression of SpDicer2 was significantly responsive to challenges by mud crab reovirus (MCRV), Poly(I:C), LPS, Staphylococcus aureus and Vibrio parahaemolyticus. SpDicer2 was dispersedly presented in the cytoplasm except for a small amount in the nucleus. SpDicer2 could activate SpSTAT to translocate from the cytoplasm to the nucleus, and significantly increase the transcription activity of the wsv069 promoter, suggesting that SpDicer2 activated the JAK/STAT pathway. Furthermore, silencing of SpDicer2 in vivo increased the mortality of MCRV infected mud crab and the viral load in tissues and down-regulated the expression of multiple components of Toll, IMD and JAK-STAT pathways and almost all the examined immune effector genes. These results suggested that SpDicer2 could play an important role in defense against MCRV via activating the JAK/STAT signaling pathways in mud crab.

The inverse design method based on a generative adversarial network (GAN) combined with a simulation neural network (sim-NN) and the self-attention mechanism is proposed in order to improve the efficiency of GAN for designing nanophotonic devices. The sim-NN can guide the model to produce more accurate device designs via the spectrum comparison, whereas the self-attention mechanism can help to extract detailed features of the spectrum by exploring their global interconnections. The nanopatterned power splitter with a 2 μm × 2 μm interference region is designed as an example to obtain the average high transmission (>94%) and low back-reflection (<0.5%) over the broad wavelength range of 1200~1650 nm. As compared to other models, this method can produce larger proportions of high figure-of-merit devices with various desired power-splitting ratios.

The systemic RNA interference defective protein (SID)-1 plays an important role in dsRNA uptake in cells. We identified the ScSidT2 gene from the mandarin fish (Siniperca chuatsi), which is the first-studied SID-1 homolog in fish. ScSidT2 mRNA is 3380 bp long and contains a 2568-bp open reading frame that encodes an 855-amino-acid protein with an N-terminal signal peptide and ten putative transmembrane domains. Tissue distribution profile in healthy fish and expression profiles of ScSidT2 in infectious spleen and kidney necrosis virus (ISKNV)-infected fish were analyzed. Overexpression of the ScSidT2 protein in fathead minnow (FHM) epithelial cells could remarkably increase the uptake of exogenous dsRNA. In tiger frog virus (TFV)-infected FHM cells, overexpression of ScSidT2 could suppress virus production, and this mechanism could be significantly enhanced by adding virus-specific dsRNA. In mandarin fish fry cells, ISKNV infection and reproduction could be promoted when the ScSidT2 protein was neutralized with specific antisera. These results suggest that ScSidT2 could be associated with the host's antiviral defense mechanism.

In the Hippo pathway, activation of Hippo and Warts (Wts) kinases results in the phosphorylation of Yorkie (Yki), to prevent its nuclear translocation. Shrimp aquaculture is threatened by Vibrio genus bacteria. In this study, we examine the role of the Hippo pathway in immune defense against Vibrio parahaemolyticus in Pacific white shrimp Penaeus vannamei. We show that V. parahaemolyticus infection promotes the expression of Yki and facilitates the dephosphorylation and nuclear translocation of Yki, indicating the inhibition of Hippo signaling upon bacterial infection. There is a complex regulatory relationship between the Hippo pathway components Hippo, Wts, and Yki and the immune-related transcription factors Dorsal, Relish, and STAT. Silencing of Hippo and Wts weakened hemocyte phagocytosis, while the silencing of Yki enhanced it, suggesting a positive regulation of shrimp cellular immunity by Hippo signaling activation. In vivo silencing of Hippo and Wts decreased the survival rates of V. parahaemolyticus-infected shrimp and elevated the bacterial content in tissues, while the silencing of Yki showed the opposite results. This suggests that the activation of Hippo signaling and the inhibition of Yki enhance antibacterial immunity in shrimp.

The emergence of multi-drug resistance (MDR) in esophageal carcinoma has severely affected the effect of chemotherapy and shortened the survival of patients. To this end, we intend to develop a biomimetic nano-targeting drug modified by cancer cell membrane, and investigate its therapeutic effect.

The NF-κB family transcription factors regulate a wide spectrum of biological processes, in particular immune responses. The studies in human suggest that the NF-κB repressing factor (NKRF) negatively regulates the activity of NF-κB through a direct protein-protein interaction. However, the function of NKRF has not been studied outside mammals up to now. The current study identified a NKRF gene (LvNKRF) from the Pacific white shrimp, Litopenaeus vannamei, which showed homology with NKRFs from insects, fishes and mammals. LvNKRF was high expressed in intestine, stomach and muscle tissues and was localized in the nucleus. LvNKRF could interact with both Dorsal and Relish, the two members of the shrimp NF-κB family. Interestingly, although sharing a similar protein structure with that of human NKRF, LvNKRF showed no inhibitory but instead enhancing effects on activities of Dorsal and Relish, which was contrary to those of mammalian NKRFs. The expression of LvNKRF could not be induced by Gram-positive and -negative bacteria and immunostimulants lipopolysaccharide (LPS) and poly (I:C) but was significantly up-regulated after white spot syndrome virus (WSSV) infection. Silencing of LvNKRF significantly decreased the mortalities of shrimp caused by WSSV infection and down-regulated the WSSV copies and the expression of WSSV structural gene in tissues. These suggested that LvNKRF could facilitate the infection of shrimp by WSSV, which may be an additional strategy for WSSV to hijack the host NF-κB pathway to favor its own replication. The current study could provide a valuable context for further investigating the evolutionary derivation of NKRFs and facilitate the study of regulatory mechanisms of invertebrate NF-κB pathways.

The forkhead box protein P (FoxP) family members have been known to be important for regulation of immune responses in vertebrates, but their roles in invertebrate immunity remain unclear. In this study, a novel FoxP gene (LvFoxP) was identified from Pacific white shrimp Litopenaeus vannamei and functionally studied in the context of immune response. Possessing a conserved FoxP coiled-coil domain and a forkhead domain, LvFoxP shared homology to vertebrate FoxP family members, in particular FoxP1. Expression of LvFoxP was detectable in all the examined tissues and could be up-regulated by immune challenge in gill and hemocytes. The LvFoxP protein was present in both the cytoplasm and nucleus of hemocytes and could be nuclear-translocated upon immune stimulation. Silencing of LvFoxP increased the susceptibility of shrimp to infections by Vibrio parahaemolyticus and white spot syndrome virus (WSSV) and down-regulated the expression of multiple components of NF-κB and JAK-STAT pathways and almost all the examined immune effector genes. Moreover, the phagocytic activity of hemocytes from LvFoxP-silenced shrimp against V. parahaemolyticus was decreased. These suggested that LvFoxP could play a positive role in immune response. The current study may provide novel insights into the immunity of invertebrates and the functional evolution of the FoxP family.

Growing evidence indicates that activator protein-1 (AP-1) plays a major role in stimulating the transcription of immune effector molecules in cellular response to an incredible array of stimuli, including growth factors, cytokines, cellular stresses and bacterial and viral infection. Here, we reported the isolation and characterization of a cDNA from Litopenaeus vannamei encoding the full-length c-Fos protein (named as Lvc-Fos). The predicted amino acid sequences of Lvc-Fos contained a basic-leucine zipper (bZIP) domain, which was characteristic of members of the AP-1 family. Immunoprecipitation and native-PAGE assays determined that Lvc-Fos could interact with the Lvc-Jun, a homolog of c-Jun family in L. vannamei, in a heterodimer manner. Further investigation demonstrated that Lvc-Fos and Lvc-Jun were expressed in all tested tissues and located in the nucleus. Real-time RT-PCR analysis showed both Lvc-Fos and Lvc-Jun in gills were up-regulated during Vibrio parahaemolyticus and white spot syndrome virus (WSSV) challenges. In addition, reporter gene assays indicated Lvc-Fos and Lvc-Jun could activate the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp, as well as WSSV immediate early (IE) genes wsv069 and wsv249, in a different manner. Knockdown of Lvc-Fos or Lvc-Jun by RNA interference (RNAi) resulted in higher mortalities of L. vannamei after infection with V. parahaemolyticus, suggesting that Lvc-Fos and Lvc-Jun might play protective roles in bacterial infection. However, silencing of Lvc-Fos or Lvc-Jun in shrimp caused lower mortalities and virus loads under WSSV infection, suggesting that Lvc-Fos and Lvc-Jun could be engaged for WSSV replication and pathogenesis. In conclusion, our results provided experimental evidence and novel insight into the roles of L. vannamei AP-1 in bacterial and viral infection.

Pacific white shrimp (Litopenaeus vannamei), the major species of farmed shrimps in the world, has been attracting extensive studies, which require more and more genome background knowledge. The now available transcriptome data of L. vannamei are insufficient for research requirements, and have not been adequately assembled and annotated.

Skin injury affects millions of people via the uncontrolled inflammation and infection. Many cellular components including fibroblasts and signaling pathways such as transforming growth factor-β (TGF-β) were activated to facilitate the wound healing to repair injured tissues. C57BL/6 female mice were divided into control and ozone oil treated groups. Excisional wounds were made on the dorsal skin and the fibroblasts were isolated from granulation tissues. The skin injured mouse model revealed that ozone oil could significantly decrease the wound area and accelerate wound healing compared with control group. QPCR and Western blotting assays showed that ozone oil up-regulated collagen I, α-SMA, and TGF-β1 mRNA and protein levels in fibroblasts. Wound healing assay demonstrated that ozone oil could increase the migration of fibroblasts. Western blotting assay demonstrated that ozone oil increased the epithelial-mesenchymal transition (EMT) process in fibroblasts via up-regulating fibronectin, vimentin, N-cadherin, MMP-2, MMP-9, insulin-like growth factor binding protein (IGFBP)-3, IGFBP5, and IGFBP6, and decreasing epithelial protein E-cadherin and cellular senescence marker p16 expression. Mechanistically, Western blotting assay revealed that ozone oil increased the phosphorylation of PI3K, Akt, and mTOR to regulate the EMT process, while inhibition of PI3K reversed this effect of ozone oil. At last, the results from Cytometric Bead Array (CBA) demonstrated ozone oil significantly decreased the inflammation in fibroblasts. Our results demonstrated that ozone oil facilitated the wound healing via increasing fibroblast migration and EMT process via PI3K/Akt/mTOR signaling pathway in vivo and in vitro The cellular and molecular mechanisms we found here may provide new therapeutic targets for the treatment of skin injury.

The toll-like receptor (TLR)/NF-κB signaling pathways play critical roles in the innate immune system. The intracellular signal transduction of most TLR pathways in invertebrate cells is triggered by formation of a heterotrimeric complex composed of MyD88, Tube and Pelle. In this study, we identified a Litopenaeus vannamei Pelle (LvPelle) and an isoform of L. vannamei Tube (LvTube) designated as LvTube-1. The interactions among LvPelle, LvTube/LvTube-1 and LvMyD88/LvMyD88-1 were elucidated and their functions during pathogen infections were investigated. Knockdowns of LvPelle and LvTube/LvTube-1 using RNAi strategy led to higher mortalities of shrimps during Vibrio parahemolyticus infection, and could reduce the genome copy number of white spot syndrome virus (WSSV) in the infected muscle tissue but did not affect the mortality caused by WSSV infection. The effects of LvPelle and LvTube/LvTube-1 on promoters containing NF-κB binding motifs were analyzed by dual-luciferase reporter assays and the results demonstrated that LvTube-1 could activate the NF-κB activity to significantly higher level than LvTube did. Moreover, tissue distributions of LvTube and LvTube-1 mRNAs and their expression profiles during pathogen and immune stimulant challenges were different, indicating that they could play different roles in immune responses. This is the first report of Tube isoforms in invertebrates. Together with our previous study on LvMyD88 isoforms, our results suggest that various isoforms of adaptor components may be involved in various regulatory patterns of signal transduction in invertebrate TLR/NF-κB pathway and this could be a strategy adopted by invertebrates to modulate immune responses.

Regulation of immune responses in animals is largely governed by the JAK-STAT and NF-κB pathways, which are conserved across vertebrates and invertebrates. At present, the relationship between these two pathways in invertebrates remains unclear. In the current study, a novel antimicrobial peptide termed LvSWD5 belonging to the Crustin family was identified from Pacific white shrimp Litopenaeus vannamei. The mature LvSWD5 peptide containing a single WAP domain (SWD) could directly bind bacteria and fungi and inhibit the growth of both Gram-positive and -negative bacteria in vitro. The LvSWD5 promoter was predicted to contain binding sites for STAT and NF-κB and could be regulted by the JAK-STAT and Relish pathways. The expression of LvSWD5 was up-regulated during bacterial, viral and fungal infections and silencing of LvSWD5 in vivo affected the expression of a series of immune related genes and decreased the phagocytic activity of hemocytes against V. Parahaemolyticus. Moreover, the susceptibility of shrimp to V. parahaemolyticus and white spot syndrome virus (WSSV) was significantly increased after silencing of LvSWD5, indicating that LvSWD5 could be involved in antibacterial and antiviral responses. These suggested that the JAK-STAT and NF-κB pathways could converge at the promoter level of a common target gene to regulate the immunity in shrimp.