Hierarchically patterned protein scaffolds with nano-fibrillar and micro-lamellar structures modulate neural stem cell homing and promote neuronal differentiation.

Biophysical factors are essential in cell survival and behaviors, but constructing a suitable 3D microenvironment for the recruitment of stem cells and exerting their physiological functions remain a daunting challenge. Here, we present a novel silk fibroin (SF)-based fabrication strategy to develop hierarchical microchannel scaffolds for biomimetic nerve microenvironments in vitro. We first modulated the formation of SF nanofibers (SFNFs) that mimic the nanostructures of the native extracellular matrix (ECM) by using graphene oxide (GO) nanosheets as templates. Then, SFNF-GO systems were shaped into 3D porous scaffolds with aligned micro-lamellar structures by freeze-casting. The interconnected microchannels successfully induced cell infiltration and migration to the SFNF-GO scaffolds' interior. Meanwhile, the nano-fibrillar structures and the GO component significantly induced neural stem cells (NSCs) to differentiate into neurons within a short timeframe of 14 d. Importantly, these 3D hierarchical scaffolds induced a mild inflammatory response, extensive cell recruitment, and effective stimulation of NSC neuronal differentiation when implanted in vivo. Therefore, these SFNF-GO lamellar scaffolds with distinctive nano-/micro-topographies hold promise in the fields of nerve injury repair and regenerative medicine.

Graphene Oxide-Coated Patterned Silk Fibroin Films Promote Cell Adhesion and Induce Cardiomyogenic Differentiation of Human Mesenchymal Stem Cells.

Cardiac tissue engineering is a promising strategy for the treatment of myocardial damage. Mesenchymal stem cells (MSCs) are extensively used in tissue engineering. However, transformation of MSCs into cardiac myocytes is still a challenge. Furthermore, weak adhesion of MSCs to substrates often results in poor cell viability. Here, we designed a composite matrix based on silk fibroin (SF) and graphene oxide (GO) for improving the cell adhesion and directing the differentiation of MSCs into cardiac myocytes. Specifically, patterned SF films were first produced by soft lithographic. After being treated by air plasma, GO nanosheets could be successfully coated on the patterned SF films to construct the desired matrix (P-GSF). The resultant P-GSF films presented a nano-topographic surface characterized by linear grooves interlaced with GO ridges. The P-GSF films exhibited high protein absorption and suitable mechanical strength. Furthermore, the P-GSF films accelerated the early cell adhesion and directed the growth orientation of MSCs. RT-PCR results and immunofluorescence imaging demonstrated that the P-GSF films significantly improved the cardiomyogenic differentiation of MSCs. This work indicates that patterned SF films coated with GO are promising matrix in the field of myocardial repair tissue engineering.

Identification and function analysis of two fibroblast growth factor receptor (FGFR) from Scylla paramamosain: The evidence of FGFR involved in innate immunity in crustacean.

The fibroblast growth factor receptor (FGFR) belongs to the tyrosine kinase family consisting of four members (FGFR1-4). This study involved identification and characterization of FGFR1 and FGFR3 from mud crab Scylla paramamosain for the first time. The obtained cDNAs of SpFGFR1 and SpFGFR3 were 2,380 bp and 2,982 bp in length with a 1,503 bp and 2,310 bp open reading frame, respectively. The predicted SpFGFR1 protein included three immunoglobulin domains and a transmembrane region, while SpFGFR3 protein possessed a typical TyrKc (Tyrosine kinase, catalytic) domain. Real-time PCR analysis showed that SpFGFR1 and SpFGFR3 were highly expressed in the hepatopancreas. Furthermore, the expression levels of SpFGFR1 and SpFGFR3 in the hepatopancreas were enhanced following challenges with Vibro alginolyticus, Staphylococcus aureus, Poly (I:C) and White spot syndrome virus, which shows the involvement of SpFGFR1 and SpFGFR3 in innate immune response to infections from bacteria and virus. There was significant suppression of six antimicrobial peptide genes (SpALF1-5 and SpCrustin) and three NF-κB members (SpDorsal, SpIKK and SpRelish) when SpFGFR1 and SpFGFR3 was interfered in vivo. Also, treatment of the hemocytes with specific inhibitor of SpFGFR for 24 h consistently down-regulated SpDorsal, SpRelish and AMPs. These results suggested that SpFGFR1 and SpFGFR3 played important roles in regulating the Toll signaling pathway and immune deficiency (IMD) pathway through NF-κB signaling pathway. These findings may provide new insights into the role of FGFRs in the innate immune function of crustaceans.

Biomimetic Nucleation of Metal-Organic Frameworks on Silk Fibroin Nanoparticles for Designing Core-Shell-Structured pH-Responsive Anticancer Drug Carriers.

Silk fibroin (SF) is a biomacromolecule that can be assembled into nanostructures and induce biomimetic nucleation of inorganic materials. Zeolitic imidazolate framework-8 (ZIF-8), a metal-organic framework (MOF), can be dissolved selectively under acidic pH. Here, we integrated SF and ZIF-8 to develop novel drug carriers that selectively release drug in the acidic intracellular environment of cancer cells. Specifically, SF was assembled into nanoparticles (SF-NPs), which were then loaded with an antitumor drug, doxorubicin (DOX), to form DSF-NPs. Due to the SF-mediated organization of ZIF-8 precursors such as zinc ions, the DSF-NPs further templated the nucleation of ZIF-8 onto their surface to generate core-shell-structured NPs (termed DSF@Z-NPs) with ZIF-8 as a shell and DSF-NP as a core. We found that the DSF@Z-NPs, highly stable under neutral conditions, could be uptaken by breast cancer cells, release DOX selectively owing to dissolution of ZIF-8 shells in the acidic intracellular environment in a controlled manner, and induce cell apoptosis. We also confirmed that the DSF@Z-NPs could inhibit tumor growth more efficiently to reach a higher survival rate than their controls by inducing cell apoptosis in vivo. Our study suggests that SF and MOF could be combined to design a new type of cancer therapeutics.

Polydopamine modification of silk fibroin membranes significantly promotes their wound healing effect.

Natural polymer-based wound dressings have gained great attention in skin tissue engineering. Silk fibroin (SF) spun from Bombyx mori (B. mori) is a potential wound dressing material due to its outstanding biocompatibility and biodegradability, however, its wound healing effect is still limited. To maximize the wound healing effect of SF-based wound dressing, we first fabricated fibrous electrospun SF (ESF) membranes with large porosity and specific surface area, and then formed polydopamine (PDA) coating on the ESF fibers to form PESF membranes. We found that PDA coating enabled the PESF membranes to outperform the ESF membranes in enhancing the hydrophilicity and protein adsorption ability of the membranes as well as the attachment, spreading and proliferation of fibroblasts on the membranes in vitro. Our further in vivo histological analysis confirmed that the PESF membranes accelerated wound healing in a rat skin wound model more effectively within 2 weeks than both the ESF membrane and a commercial dressing (3M™ Tegaderm™). The enhanced wound healing effect of the PESF membranes was further proved by the increase in the content of hydroxyproline (a constituent in collagen) in the wound treated by the PESF membranes. Therefore, the PESF membranes could be used as a promising wound dressing for wound healing and skin regeneration.

Polydopamine-Coated Antheraea pernyi (A. pernyi) Silk Fibroin Films Promote Cell Adhesion and Wound Healing in Skin Tissue Repair.

Wound dressings are important materials for the successful recovery of skin trauma. Traditional wound dressings such as gauzes are not efficient in wound healing. Here we show that silk fibroin, spun from a wild silkworm Antheraea pernyi (A. pernyi) and rich in Arg-Gly-Asp (RGD) sequences, can be developed into a wound dressing after proper modification for improving the cell adhesion to accelerate the skin repair. Specifically, polydopamine (PDA) was coated on an A. pernyi silk fibroin (AF) film to form the PAF film to achieve enhanced cell adhesion and would healing. The PDA coating significantly increased the roughness and hydrophilicity of the AF film and thus its protein absorption capability. Furthermore, the PAF films promoted the adhesion and migration of mesenchymal stem cells (MSCs) in the in vitro wound healing assay. In vivo testing confirmed that wound covered with the PAF film was completely healed with the formation of the new skin and hair within 14 days post trauma. Histological examination indicated that, compared to the AF film and gauze control, the PAF film did not cause significant inflammation in the wound but promoted the epithelialization and well-organized collagen deposition in the dermis. This work indicates that AF films coated with PDA are promising wound dressings for skin tissue repair.

The nuclear factor interleukin 3-regulated (NFIL3) transcription factor involved in innate immunity by activating NF-κB pathway in mud crab Scylla paramamosain.

NFIL3 is a transcriptional activator of the IL-3 promoter in T cells. In vertebrates, it has been characterized as an essential regulator of several cellular processes such as immunity response, apoptosis and NK cells maturation. However, the identification and functional characterization of NFIL3 still remains unclear in arthropods. In this study, the NFIL3 homologue was firstly cloned and characterized in mud crab Scylla paramamosain. The full-length of SpNFIL3 was 2, 041 bp in length with an open reading frame of 1, 509 bp, containing a conserved basic region of leucin zipper domain. The qRT-PCR analysis indicated that SpNFIL3 was significantly highly expressed in hepatopancreas and in hemocytes. Moreover, the SpNFIL3 transcription could be up-regulated after the challenge of Vibrio alginolyticus or virus-analog Poly (I:C). The dual-luciferase reporter assays revealed that SpNFIL3 could activate NF-κB pathway. The immunofluorescence assay indicated SpNFIL3 was located in nucleus. After NFIL3 was interfered in vivo and in vitro, the expressions of two NF-κB members (SpRelish and SpDorsal), six antimicrobial peptide genes (SpCrustin and SpALF2-6) and pro-inflammatory cytokine SpIL-16 were suppressed, and the bacteria clearance capacity of crabs was also markedly impaired in NFIL3 silenced crabs. These results indicated that SpNFIL3 played crucial role in the innate immunity of S. paramamosain and it also brought new insight into the origin and evolution of NFIL3 in arthropods and even in invertebrates.

Transforming growth factor-ß-activating kinase 1 and its binding protein 1 participate in the innate immune responses via modulating the IMDNFκB signaling in mud crab (Scylla paramamosain).

Transforming growth factor-ß-activating kinase 1 (TAK1) is essential for diverse important biological functions, such as innate immunity, development and cell survival. In the present study, the homologs of TAK1 and TAK1-binding protein 1 (TAB1) were identified and characterized from mud crab Scylla paramamosain for the first time. The full-length cDNAs of SpTAK1 and SpTAB1 were 2, 226 bp and 2, 433 bp with 1, 782 bp and 1, 533 bp open reading frame (ORF), respectively. The deduced SpTAK1 protein contained a conserved S_TKc (Serine/threonine protein kinases, catalytic) domain, and the putative SpTAB1 protein possessed a typical PP2Cc (Serine/threonine phosphatases, family 2C, catalytic) domain and a potential TAK1 docking motif. Real-time PCR analysis showed that SpTAK1 and SpTAB1 were highly expressed at early development stages, suggesting their participation in crab's development process. Moreover, the expression levels of SpTAK1 and SpTAB1 in hepatopancreas were positively stimulated after challenge with Vibro alginolyticus and Poly (I:C), implying the involvement of SpTAK1 and SpTAB1 in innate immune responses against both bacterial and viral infections. When SpTAK1 or SpTAB1 were silenced in vivo, the expression levels of two IMDNFκB signaling components (SpIKKß and SpRelish) and six antimicrobial peptide (AMP) genes (SpALF1-5 and SpCrustin) were significantly reduced, and the bacteria clearance capacity of crabs was also markedly impaired in SpTAK1 or SpTAB1 silenced crabs. Additionally, overexpression of SpTAK1 and SpTAB1 in HEK293T cells could markedly activate the mammalian NF-κB signaling. Collectively, our results suggested that TAK1 and TAB1 regulated crab's innate immunity via modulating the IMDNFκB signaling. These findings may provide new insights into the TAK1/TAB1-mediated signaling cascades in crustaceans and pave the way for a better understanding of crustacean innate immune system.

Identification and functional analysis of immune deficiency (IMD) from Scylla paramamosain: The first evidence of IMD signaling pathway involved in immune defense against bacterial infection in crab species.

Immune deficiency (IMD) pathway, one of the most essential pattern recognition receptor signaling pathways, plays vital roles in innate immune responses to eliminate pathogen infection in invertebrates. In the present study, an immune deficiency (IMD) gene and two NF-κB family members, Relish and Dorsal, were identified and characterized in mud crab Scylla paramamosain for the first time. The deduced SpIMD, SpRelish and SpDorsal protein contained conserved death domain and classical NF-κB domains, respectively. Phylogenetic analysis suggested that SpIMD was classified into the invertebrate IMD branch, and SpRelish could be classified into the type I NF-κB class while SpDorsal could be grouped into the type II NF-κB class. Tissue distribution results showed these three genes were ubiquitously expressed in all tested tissues. The expression patterns of IMD signaling pathway and NF-κB genes, including SpIMD, SpIKKß, SpIKKε, SpRelish and SpDorsal, were distinct when crabs were stimulated with Vibro alginolyticus, indicating that they might be involved in responding to bacterial infection. When SpIMD was silenced by in vivo RNA interference assay, the expression levels of IMD pathway and antimicrobial peptides (AMPs) genes, including SpIKKß, SpRelish, SpALF1-6 and SpCrustin, were significantly down-regulated (p < 0.05). Correspondingly, the bacteria clearance ability of hemolymph was extremely impaired in IMD silenced crabs. Overall, the IMD played vital roles in innate immune response by regulating the expressions of its down-stream signaling genes and AMPs in S. paramamosain. These findings might pave the way for a better understanding of innate immune system and establish a fundamental network for the IMD signaling pathway in crustaceans.

Identification and functional analysis of transforming growth factor-ß type I receptor (TßR1) from Scylla paramamosain: The first evidence of TßR1 involved in development and innate immunity in crustaceans.

The transforming growth factor-ß (TGF-ß) receptor-mediated TGF-ß signaling cascade plays important roles in diverse cellular processes, including cell proliferation, differentiation, growth, apoptosis and inflammation in vertebrates. In the present study, the type I TGF-ß receptor (TßR1) was firstly identified and characterized in mud crab Scylla paramamosain. The full-length cDNA of SpTßR1 was 1, 986 bp with a 1, 608 bp open reading frame, which encoded a putative protein of 535 amino acids including a typical transmembrane region, a conserved glycine-serine (GS) motif and a S_TKc domain (Serine/Threonine protein kinases, catalytic domain). Real-time PCR analysis showed that SpTßR1 was predominantly expressed at early embryonic development stage and was highly expressed at postmolt stages during molt cycle, suggesting its participation in development and growth. Moreover, the expression levels of SpTßR1 in hepatopancreas and hemocytes were positively induced after the challenges of Vibro alginolyticus and Poly (I:C), indicating the involvement of SpTßR1 in responding to both bacterial and viral infections. The in vivo RNA interference assays demonstrated that the expression levels of two NF-κB members (SpRelish and SpDorsal) and six antimicrobial peptide (AMP) genes (SpCrustin and SpALF2-6) were significantly suppressed when the SpTßR1 was silenced. Additionally, the expression levels of SpTßR1, SpRelish, SpDorsal and AMPs were consistently down-regulated or up-regulated when the primary cultured hemocytes were treated with TßR1 antagonist or agonist for 24 h. These results indicated that TßR1 not only contributed to the crabs' development and growth but also played vital role in the innate immunity of S. paramamosain, and it also provided new insights into the origin or evolution of TGF-ß receptors in crustacean species and even in invertebrates.

Expression plasticity and evolutionary changes extensively shape the sugar-mimic alkaloid adaptation of nondigestive glucosidase in lepidopteran mulberry-specialist insects.

During the co-evolutionary arms race between plants and herbivores, insects evolved systematic adaptive plasticity to minimize the chemical defence effects of their host plants. Previous studies mainly focused on the expressional plasticity of enzymes in detoxification and digestion. However, the expressional response and adaptive evolution of other fundamental regulators against host phytochemicals are largely unknown. Glucosidase II (GII), which is composed of a catalytic GIIα subunit and a regulatory GIIß subunit, is an evolutionarily conserved enzyme that regulates glycoprotein folding. In this study, we found that GIIα expression of the mulberry-specialist insect was significantly induced by mulberry leaf extract, 1-deoxynojirimycin (1-DNJ), whereas GIIß transcripts were not significantly changed. Moreover, positive selection was detected in GIIα when the mulberry-specialist insects diverged from the lepidopteran order, whereas GIIß was mainly subjected to purifying selection, thus indicating an asymmetrically selective pressure of GII subunits. In addition, positively selected sites were enriched in the GIIα of mulberry-specialist insects and located around the 1-DNJ-binding sites and in the C-terminal region, which could result in conformational changes that affect catalytic activity and substrate-binding efficiency. These results show that expression plasticity and evolutionary changes extensively shape sugar-mimic alkaloids adaptation of nondigestive glucosidase in lepidopteran mulberry-specialist insects. Our study provides novel insights into a deep understanding of the sequestration and adaptation of phytophagous specialists to host defensive compounds.

Silkworm Pupa Protein Hydrolysate Induces Mitochondria-Dependent Apoptosis and S Phase Cell Cycle Arrest in Human Gastric Cancer SGC-7901 Cells.

Silkworm pupae (Bombyx mori) are a high-protein nutrition source consumed in China since more than 2 thousand years ago. Recent studies revealed that silkworm pupae have therapeutic benefits to treat many diseases. However, the ability of the compounds of silkworm pupae to inhibit tumourigenesis remains to be elucidated. Here, we separated the protein of silkworm pupae and performed alcalase hydrolysis. Silkworm pupa protein hydrolysate (SPPH) can specifically inhibit the proliferation and provoke abnormal morphologic features of human gastric cancer cells SGC-7901 in a dose- and time-dependent manner. Moreover, flow cytometry indicated that SPPH can induce apoptosis and arrest the cell-cycle in S phase. Furthermore, SPPH was shown to provoke accumulation of reactive oxygen species (ROS) and depolarization of mitochondrial membrane potential. Western blotting analysis indicated that SPPH inhibited Bcl-2 expression and promoted Bax expression, and subsequently induced apoptosis-inducing factor and cytochrome C release, which led to the activation of initiator caspase-9 and executioner caspase-3, cleavage of poly (ADP-ribose) polymerase (PARP), eventually caused cell apoptosis. Moreover, SPPH-induced S-phase arrest was mediated by up-regulating the expression of E2F1 and down-regulating those of cyclin E, CDK2 and cyclin A2. Transcriptome sequencing and gene set enrichment analysis (GSEA) also revealed that SPPH treatment could affect gene expression and pathway regulation related to tumourigenesis, apoptosis and cell cycle. In summary, our results suggest that SPPH could specifically suppress cell growth of SGC-7901 through an intrinsic apoptotic pathway, ROS accumulation and cell cycle arrest, and silkworm pupae have a potential to become a source of anticancer agents in the future.

Effect of cadmium exposure on hepatopancreas and gills of the estuary mud crab (Scylla paramamosain): Histopathological changes and expression characterization of stress response genes.

Cadmium (Cd) is a heavy metal that accumulates easily in organisms and causes several detrimental effects, including tissue damage. Cd contamination from anthropogenic terrestrial sources flows into rivers, and through estuaries to the ocean. To evaluate the toxic effects of Cd on estuary crustaceans, we exposed the mud crab Scylla paramamosain to various Cd concentrations (0, 10.0, 20.0, and 40.0mg/L) for 24h. We also exposed mud crabs to a fixed Cd concentration (20.0mg/L) for various periods of time (0, 6, 12, 24, 48, and 72h). We observed that after exposure to Cd, the surfaces of the gill lamellae were wrinkled, and the morphologies of the nuclei and mitochondria in the hepatopancreas were altered. We analyzed the expression profiles of 36 stress-related genes after Cd exposure, including those encoding metallothioneins, heat shock proteins, apoptosis-related proteins, and antioxidant proteins, with quantitative reverse transcription PCR. We found that exposure to Cd altered gene expression, and that some genes might be suitable bioindicators of Cd stress. Gene expression profiles were organ-, duration-, and concentration-dependent, suggesting that stress-response genes might be involved in an innate defense system for handling heavy metal exposure. To the best of our knowledge, this study is the first one of histopathology and stress-response gene expression pattern of Scylla paramamosain after Cd exposure. Our work could increase our understanding of the effect of environmental toxins on estuary crustaceans.

Hemocytes of the mud crab Scylla paramamosain: Cytometric, morphological characterization and involvement in immune responses.

Hemocytes play essential roles in the innate immune system of crustaceans. Characterization of hemocytes from estuary mud crab Scylla paramamosain was performed by flow cytometry and morphological studies such as cytochemical staining and electron microscopy. The hemocyte subsets were further separated using a modified Percoll density gradient centrifugation method. Based on the morphological characteristics of the cells, three distinct categories of hemocytes were identified: granulocytes with abundant large granularity representing 5.27 ± 0.42%, semigranulocytes with small or less granularity representing 76.03 ± 3.34%, and hyalinocytes (18.70 ± 3.92%) which were almost no granularity. The total hemocyte cell count and the percentage of hemocyte subsets varied after pathogen infection, including Vibrio alginolyticus and the viral double-stranded RNA analog Poly (I:C). The phagocytic process is of fundamental importance for crustaceans' cellular immune response as well as development and survival. The results of the in vitro phagocytosis assays analyzed by flow cytometry demonstrated that granulocytes and semigranulocytes had significantly higher phagocytic ability than hyalinocytes. A primary culture system, L-15 medium supplemented with 5-10% fetal bovine serum, was developed to further investigate the immune function of hemocytes. Furthermore, adenovirus can be utilized to effectively transfer GFP gene into hemocytes. Overall, three hemocyte sub-populations of S. paramamosain were successfully discriminated, moreover, their response to pathogen infections, phagocytic activity and adenovirus mediated transfection were also investigated for the first time. This study may contribute to a better understanding of the innate immune system of estuary crabs.

Identification and characterization of six peroxiredoxin transcripts from mud crab Scylla paramamosain: The first evidence of peroxiredoxin gene family in crustacean and their expression profiles under biotic and abiotic stresses.

The peroxiredoxins (Prxs) define a novel and evolutionarily conserved superfamily of peroxidases able to protect cells from oxidative damage by catalyzing the reduction of a wide range of cellular peroxides. Prxs have been identified in prokaryotes as well as in eukaryotes, however, the composition and number of Prxs family members vary in different species. In this study, six Prxs were firstly identified from the mud crab Scylla paramamosain by RT-PCR and RACE methods. Six SpPrxs can be subdivided into three classes: (a) three typical 2-Cys enzymes denominated as Prx1/2, 3, 4, (b) two atypical 2-Cys enzymes known as Prx5-1 and Prx5-2, and (c) a 1-Cys isoform named Prx6. The evolutionarily conserved signatures of peroxiredoxin catalytic center were identified in all six SpPrxs. Phylogenetic analysis revealed that SpPrx3, SpPrx4, SpPrx5s and SpPrx6 were clearly classified into Prx3-6 subclasses, respectively. Although SpPrx1/2 could not be grouped into any known Prx subclasses, SpPrx1/2 clustered together with other arthropods Prx1 or unclassified Prx and could be classified into the typical 2-Cys class. The comparative and evolutionary analysis of the Prx gene family in invertebrates and vertebrates were also conducted for the first time. Tissue-specific expression analysis revealed that these six SpPrxs were expressed in different transcription patterns while the highest expression levels were almost all in the hepatopancreas. Quantitative RT-PCR analysis exhibited that the gene expression profiles of six SpPrxs were distinct when crabs suffered biotic and abiotic stresses including the exposures of Vibrio alginolyticus, poly (I:C), cadmium and hypoosmotic salinity, suggesting that the SpPrxs might play different roles in response to various stresses. The recombinant proteins including the SpPrx1/2, SpPrx4, SpPrx5-1 and SpPrx6 were purified and the peroxidase activity assays indicated that all these proteins can reduce H2O2 in a typical DTT-dependent manner. To our knowledge, this is the first study about the comprehensive characterization of Prx gene family in Scylla paramamosain and even in crustaceans. These results would broaden the current knowledge of the whole Prx family as well as be helpful to understand and clarify the evolutionary pattern of Prx family in invertebrate and vertebrate taxa.

Identification and characterization of pro-interleukin-16 from mud crab Scylla paramamosain: The first evidence of proinflammatory cytokine in crab species.

IL-16 is a pro-inflammatory cytokine originally designated as a lymphocyte chemoattractant factor. In mammal and avian, it has been characterized as an essential regulator of various cellular processes including cell recruitment and activation against pathogen invasion. So far, neither of the full-length of IL-16 homologue nor the response mechanism against pathogen was reported in crab species. In the present study, the pro-IL-16 homologue was firstly cloned and characterized from mud crab Scylla paramamosain. The full-length Sp-pro-IL-16 consisted of 4107 bp with an opening reading frame encoding 1369 amino acids. Multiple alignment analysis showed the putative amino acid sequence of Sp-pro-IL-16 had about 73.86% identity with Litopenaeus vannamei pro-IL-16. Additionally, two conserved PDZ domains and protein binding sites were found in Sp-pro-IL-16 and showed high similarities about 94.19% and 51.14% with their Litopenaeus vannamei and Mus musculus counterparts. RT-PCR analysis indicated that Sp-pro-IL-16 transcripts were constitutively expressed in all tissues examined with an extreme high level in hepatopancreas. Moreover, Sp-pro-IL-16 transcripts in hepatopancreas were significantly up-regulated 15-fold at 72 h after Vibrio alginolyticus challenge and 3.5-fold at 12 h after virus-analog Poly (I:C) challenge. The Western blot analysis revealed that Sp-pro-IL-16 can be cleaved to its bioactive form, an approximately 35 kDa mature IL-16, and the protein levels of both pro-IL-16 and mature IL-16 increased after Vibrio alginolyticus challenge. It is the first experimental identification of pro-inflammatory cytokine IL-16 in arthropods. This study could shed new light on further understanding of the response mechanism of pro-inflammatory cytokine IL-16 in Scylla paramamosain against pathogens. Meanwhile, it brought new insight into the origin and evolution of IL-16 in crab species.

Identification and characterization of atypical 2-cysteine peroxiredoxins from mud crab Scylla paramamosain: The first evidence of two peroxiredoxin 5 genes in non-primate species and their involvement in immune defense against pathogen infection.

Peroxiredoxin 5 (Prx5) belongs to a novel family of evolutionarily conserved antioxidant proteins that protect cells against various oxidative stresses. Generally, no more than one Prx5 transcript had been reported in non-primate species. In this study, two Prx5 genes (coined as SpPrx5-1 and SpPrx5-2) were firstly isolated from the mud crab, Scylla paramamosain, through RT-PCR and RACE methods. The open reading frame of SpPrx5-1 and SpPrx5-2 were 561 bp and 429 bp in length, encoding 186 and 142 amino acids polypeptide, respectively. Both the conserved signatures of peroxiredoxin catalytic center and Prx5-specific domain were identified in SpPrx5-1 and SpPrx5-2. Phylogenetic analysis indicated that both SpPrx5 clustered together with other animal Prx proteins and were classified into Prx5 subfamily. Tissue-specific expression analysis revealed that both SpPrx5-1 and SpPrx5-2 were ubiquitously expressed, highest in hepatopancreas, and showed remarkably similar transcription patterns. Quantitative RT-PCR analysis exhibited that both SpPrx5 genes changed dramatically in hepatopancreas, although showing different expression profiles, after virus-analog poly (I:C) or Vibrio alginolyticus challenge. The expression levels of both SpPrx5s were significantly enhanced in hepatopancreas after poly (I:C) stimulation, while SpPrx5-2 exhibited a more prompt response than SpPrx5-1. Nevertheless, the expression levels of both SpPrx5s were significantly reduced in hepatopancreas after Vibrio alginolyticus challenge in which SpPrx5-1 showed a more prompt response than SpPrx5-2. These results suggested the involvement of SpPrx5s in responses against viral and bacterial infections and further highlighted their functional importance in the immune system of Scylla paramamosain.

Molecular evolutionary mechanisms driving functional diversification of α-glucosidase in Lepidoptera.

The digestive tract of lepidopteran insects is unique given its highly alkaline pH. The adaptive plasticity of digestive enzymes in this environment is crucial to the highly-efficient nutritional absorption in Lepidoptera. However, little is known about the molecular adaptation of digestive enzymes to this environment. Here, we show that lepidopteran α-glucosidase, a pivotal digestive enzyme, diverged into sucrose hydrolase (SUH) and other maltase subfamilies. SUH, which is specific for sucrose, was only detected in Lepidoptera. It suggests that lepidopteran insects have evolved an enhanced ability to hydrolyse sucrose, their major energy source. Gene duplications and exon-shuffling produced multiple copies of α-glucosidase in different microsyntenic regions. Furthermore, SUH showed significant functional divergence (FD) compared with maltase, which was affected by positive selection at specific lineages and codons. Nine sites, which were involved in both FD and positive selection, were located around the ligand-binding groove of SUH. These sites could be responsible for the ligand-binding preference and hydrolytic specificity of SUH for sucrose, and contribute to its conformational stability. Overall, our study demonstrated that positive selection is an important evolutionary force for the adaptive diversification of α-glucosidase, and for the exclusive presence of membrane-associated SUHs in the unique lepidopteran digestive tract.

Evaluation of differentially expressed immune-related genes in intestine of Pelodiscus sinensis after intragastric challenge with lipopolysaccharide based on transcriptome analysis.

Pelodiscus sinensis is the most common turtle species that has been raised in East and Southeast Asia. However, there are still limited studies about the immune defense mechanisms in its small intestine until now. In the present research, histological analysis and transcriptome analysis was performed on the small intestine of P. sinensis after intragastric challenge with LPS to explore its mechanisms of immune responses to pathogens. The result showed the number of intraepithelial lymphocytes (IELs) and goblet cells (GCs) in its intestine increased significantly at 48 h post-challenge with LPS by intragastrical route, indicating clearly the intestinal immune response was induced. Compared with the control, a total of 748 differentially expressed genes (DEGs) were identified, including 361 up-regulated genes and 387 down-regulated genes. Based on the Gene Ontology (GO) annotation and the Kyoto Encyclopedia of Genes and Genomes (KEGG), 48 immune-related DEGs were identified, which were classified into 82 GO terms and 14 pathways. Finally, 18 DEGs, which were randomly selected, were confirmed by quantitative real-time PCR (qRT-PCR). Our results provide valuable information for further analysis of the immune defense mechanisms against pathogens in the small intestine of P. sinensis.

Characterization of Transgenic Silkworm Yielded Biomaterials with Calcium-Binding Activity.

Silk fibers have many inherent properties that are suitable for their use in biomaterials. In this study, the silk fibroin was genetically modified by including a Ca-binding sequence, [(AGSGAG)6ASEYDYDDDSDDDDEWD]2 from shell nacreous matrix protein. It can be produced as fibers by transgenic silkworm. The Ca-binding activity and mineralization of the transgenic silk fibroin were examined in vitro. The results showed that this transgenic silk fibroin had relatively higher Ca-binding activity than unmodified silk fibroin. The increased Ca-binding activity could promote the usage of silk fibroin as a biomaterial in the pharmaceutical industry. This study shows the possibility of using silk fibroin as a mineralization accelerating medical material by generating genetically modified transgenic silkworm.