Search

Orchestrated apoptosis in planarian Dugesia japonica is very important for its degrowth and regeneration. Apoptosis Inhibitor-5 (API5) is an anti-apoptotic factor that negatively regulates cell apoptosis. We characterized the conserved structure of DjApi5, however, the biological function of DjApi5 in planarians needs further investigation. In this study, we found that DjApi5 and its interacting molecular DjAcinus are required for planarian homeostasis, which may be correlated with their specific localization in neoblasts in addition to their anti-apoptosis functions. We further demonstrated the increased expression of DjApi5 during planarian regeneration, and DjApi5 deficiency affects normal regeneration processes. These results indicated new functions of DjApi5 in development.

As a typical organism of platyhelminth, Dugesia japonica attracts more and more attention for its strong regenerative ability. Protein arginine methyltransferase (PRMT) family is composed of a class of enzymes with methyltransferase activities, which play fundamental roles in vivo in many important physiological processes. PRMT1 is a predominant type Ⅰ PRMT, which has been reported to be expressed in Schmidtea mediterranea. Nevertheless, the existence and the specific biological functions of PRMT1 in Dugesia japonica need further investigation. In this study, we acquired the full-length sequence of DjPRMT1 and confirmed it was a conserved protein. Thereafter, whole-mount in situ hybridization results showed DjPRMT1 was mainly expressed in neoblasts of adult worms, and obvious aggregation of DjPRMT1 was observed at the wound site in early stages of regeneration. Silencing of the DjPRMT1 gene retarded the movement of planarians with decreased DjPIWI-A expression, and DjPRMT1 knockdown also affected planarian regeneration with slightly attenuated proliferation around the blastema of posterior-facing wounds regeneration. In summary, these preliminary results demonstrated DjPRMT1 was involved in the regeneration of planarian.

Pharmaceutical cocrystal as an alternative modification strategy presents opportunities for the formulation development of drugs with undesirable pharmacokinetic properties. Metronidazole (MNZ) is a water-soluble antimicrobial drug that can cause high plasma concentration peaks after administration, which may relate to serious encephalopathy. In the present study, the properties of MNZ were optimized through a cocrystallization strategy. A cocrystal of MNZ is prepared using gallic acid (GA) as cocrystal former. The structure of the prepared MNZ–GA cocrystal is studied by means of molecular dynamics and density functional theory calculations, powder X-ray diffraction, infrared spectroscopy, and thermal analysis. Comparative studies on the dissolution and absorption behaviors of raw MNZ and MNZ–GA cocrystal are completed. The results show that the MNZ–GA cocrystal exhibits lower Cmax and longer Tmax than pure metronidazole, suggesting that the prepared MNZ–GA cocrystal may find application in the formulation of MNZ.

Retinoic acid-inducible gene I (RIG-I), an RNA sensor with a conserved structure, activates the host interferon (IFN) system to produce IFNs and cytokines for eliminating pathogens upon recognizing PAMPs. However, the biological functions and the mechanism by which RIG-I regulates the innate immunity response in invertebrates are still unknown at present. Here we identified an atypical RIG-I in planarian Dugesia japonica. Sequence analysis, 3D structure modeling and phylogenetic analysis showed that this atypical protein was clustered into a single clade at the base of the tree in invertebrates, suggesting that DjRIG-I is an ancient and unique protein of the RIG-I-like receptors (RLRs). In situ hybridization analysis revealed that the DjRIG-I mRNAs were predominantly expressed in the pharynx and head of the adult and regenerative planarians. Stimulation with PAMPs induced the over-expression of DjRIG-I in planarians. The molecular simulation demonstrated that DjRIG-I formed a large hole-structure for the docking of dsRNAs, and the pull-down assay confirmed the interaction between DjRIG-I and viral analog poly(I:C). Importantly, some representative antiviral/antibacterial genes in the RIG-I-mediated IFN and P38 signaling pathway, TBK1, IRF-3, Mx, and P38, were significantly upregulated in planarians stimulated with PAMPs. Interference of the DjRIG-I expression by RNAi, inhibited the PAMPs-induced over-expression, suggesting that DjRIG-I is a key player for downstream signaling events. These results indicate that DjRIG-I triggered the intracellular signaling cascades independent of the classical CARD domains and played an essential role in the virus/bacteria-induced innate immunity of planarian.

The T7 phage is a virulent phage hosted by Escherichia coli, which poses no threat to animals and plants. Due to the advantages of small genome, well elucidated functional genomics, fast life cycle, and high stability, T7 phage has been widely used in many fields, including biology and medicine. In this review, we focus on the research of T7 phages in biological sciences and medicine, including the application of T7 phages and T7 phage products, T7 phage display systems, and recombinant T7 phages in the treatment and diagnosis of infectious diseases (bacteria, viruses, parasites) and tumor diseases. In addition, we also introduce the therapeutic application of T7 phage in various diseases such as allergic reaction, Alzheimer's disease, inflammatory reaction, and other diseases, and finally discuss the future direction of T7 phage application in the biomedical field.

The tumor necrosis factor (TNF) superfamily consists of a wide variety of inflammatory cytokine, including cell-bound and secreted proteins. These TNFs function through binding and activation of the TNF receptors for modulating TNF-associated intracellular signals. A set of mammalian TNF receptor-associated factors (TRAFs) that have emerged as the major signal transducers for the TNF receptor superfamily, play an important role in both adaptive and innate immunity. However, the existence of TRAFs and their biological functions in planarian are still unknown. In this study, a new member of TRAFs, DjTRAF2, was identified in planarian Dugesia japonica. Phylogenetic analysis revealed that DjTRAF2 could be a new member of the invertebrate TRAF2 family. Sequence analysis showed that the open reading frame of DjTRAF2 had 1353 bp in length and encoded a putative protein of 450 amino acids with a predicted molecular mass of ~51.8 kDa and an isoelectric point of 7.052. Whole-mount in situ hybridization showed that DjTRAF2 was predominantly expressed in adult and regenerative pharynx, which is an important immune organ of planarian. Quantitative real-time PCR revealed that the transcriptional level of DjTRAF2 was significantly up-regulated after induced by pathogen-associated molecular patterns (polyinosinic-polycytidylic acid, lipopolysaccharide, peptidoglycan and β-glucan), suggesting that DjTRAF2 is involved in the immune response against pathogen invasion. Collectively, these results demonstrated that DjTRAF2 might play important roles in the innate immunity of planarian.

The La(III) and Ce(III) complexes with mixed ligands of salicylic acid (Hsal) and 1,10-phenanthroline (phen), namely [La(sal)3(Phen)2] and [Ce(sal)3(phen)2], were synthesized and characterized by elemental analysis, thermal analysis, infrared, UV–visible and fluorescence spectroscopy, and single-crystal X-ray diffraction. The crystal structure analyses reveal that the Ce(III) complex is isostructural with the La(III) complex, where the Ce(III) center is chelated by two phen and three sal ligands. The comprehensive investigation on antibacterial activities and underlying mechanisms of the La(III) and Ce(III) complexes were done in vitro, in silico, and in vivo. In vitro experiments show that the coordination of the metal ions increased the antibacterial activity of the ligands. The scanning electron microscopy (SEM) images show that the Ce(III) complex may destroy the cell envelope. The molecular dynamics simulations provide complementary results that the Ce(III) complex is membrane-active against bacteria. In vivo assessments conducted using the infection wound model in mice, together with the preliminary safety evaluation, suggest that the La(III) and Ce(III) complexes may be developed as effective antimicrobial candidates. The research may contribute to the development of novel antimicrobial agents by providing new ideas.

In this study, the mechanism that lithium (Li) promotes the production of reactive oxygen species (ROS) via the glycogen synthase kinase-3β (GSK-3β)/tuberous sclerosis complex 2 (TSC2)/target of rapamycin (TOR) signaling was investigated in the gill of zebrafish (Danio rerio). After the zebrafish were treated by 25 and 50 mg/L Li+, the mRNA expression of GSK-3β and TSC2 was inhibited, but the expression of TOR was induced in the gill of zebrafish. The levels of hydrogen peroxide (H2O2), superoxide anion (O2·-), and hydroxy radical (·OH) as well as the activity of superoxide dismutase (SOD) were increased, while the activities of catalase (CAT), glutathione peroxidase (GSH-PX), and peroxidase (POD) were decreased by 25 and 50 mg/L Li+ treatments. In the ZF4 cells, the mRNA expression of GSK-3β and TSC2 was inhibited, but TOR expression was induced by 1, 5, and 10 mmol/L Li+ treatments. To further confirm that lithium promoted ROS production via GSK-3β inhibition, GSK-3β RNA was interfered. It was found that the interference of GSK-3β RNA induced the TSC2/TOR signaling. The levels of H2O2, O2·-, and ·OH were increased, but the activities of CAT, GSH-PX, and POD were decreased by GSK-3β RNA interference. In addition, lithium decreased the mitochondrial membrane potential (MMP) with Rhodamine-123 assay, but increased the levels of ROS by 2′,7′-dichlorofluorescein diacetate (DCFH-DA) assay. The present results indicated that lithium promoted the ROS production through the GSK-3β/TSC2/TOR signaling in the gill of zebrafish.

Betaine has three active methyl groups, which determine its unique function in the metabolism of animals. In this study, the mechanism that betaine regulates the synthesis of fatty acids via mTOR signaling was investigated in zebrafish. It was found that betaine induced the protein expression of mTOR, P-4EBP1, and P-S6, but inhibited the gene expression of GSK-3β, ACC, ACL, and FAS in the muscle of zebrafish. In addition, betaine decreased the content of NEFA and TG as well as the activities of ACC, ACL, and FAS. The total content of PUFA was elevated, but the total content of SFA was decreased by betaine treatments. Moreover, the interference of GSK-3β gene induced mTOR signaling but decreased the gene expression of ACL, ACC, and FAS in muscle. In all, betaine could induce mTOR signaling, which may further participate in regulating the synthesis of fatty acids in the muscle of zebrafish.

β-Thymosin is a multifunctional peptide ubiquitously expressed in vertebrates and invertebrates. Many studies have found β-thymosin is critical for wound healing, angiogenesis, cardiac repair, hair regrowth, and anti-fibrosis in vertebrates, and plays an important role in antimicrobial immunity in invertebrates. However, whether β-thymosin participates in the regeneration of organisms is still poorly understood. In this study, we identified a β-thymosin gene in Dugesia japonica which played an important role in stem cell proliferation and neuron regeneration during the tissue repair process in D. japonica. Sequencing analysis showed that β-thymosin contained two conserved β-thymosin domains and two actin-binding motifs, and had a high similarity with other β-thymosins of invertebrates. In situ or fluorescence in situ hybridization analysis revealed that Djβ-thymosin was co-localized with DjPiWi in the neoblast cells of intact adult planarians and the blastema of regenerating planarians, suggesting Djβ-thymosin has a potential function of regeneration. Disruption Djβ-thymosin by RNA interference results in a slightly curled up head of planarian and stem cell proliferation defects. Additionally, we found that, upon amputation, Djβ-thymosin RNAi-treated animals had impaired regeneration ability, including impaired blastema formation, delayed eyespot formation, decreased brain area, and disrupted central CNS formation, implying Djβ-thymosin is an essential regulator of stem cell proliferation and neuron regeneration.

14-3-3 proteins are a family of highly conserved acidic proteins that regulate cellular processes. They act as a kind of important signaling molecules taking part in many crucial decisions throughout the development process. We have isolated and characterized two members of the 14-3-3 family, namely, Dj14-3-3 α and Dj14-3-3 ζ in the planarian Dugesia japonica. The Dj14-3-3 α and ζ genes encode polypeptides of 260 and 255 amino acids respectively. We have proved that the Dj14-3-3 α and ζ genes were especially expressed in the pharynx in adult and regenerating planarians by in situ hybridization and they were not involved in regeneration process. Besides, Dj14-3-3 α and ζ genes can compensate each other in planarians by RNA interference. The Dj14-3-3 α and ζ were significantly up-regulated expression when planarians were stimulated with the pathogen-associated molecular patterns including lipopolysaccharide (LPS), peptidoglycan (PGN), β-Glu and Poly (I:C), indicating that the Dj14-3-3 α and ζ may be involved in the immune responses.

The powerful regenerative ability of planarians has long been a concern of scientists, but recently, their efficient immune system has attracted more and more attention from researchers. Gamma-interferon-inducible lysosomal thiol reductase (GILT) is related not only to antigen presentation but also to bacteria invasions. But the systematic studies are not yet to be conducted on the relationship between bacterial infection. Our study reveals for the first time that GILT of planarian (DjGILT) plays an essential role in the clearance of Gram-negative bacteria by conducting H2O2 concentration in planarians. In animals that DjGILT was silenced, it persisted for up to 9 days before all bacteria were cleared, compared with 6 days of the control group. When infected with E. coli and V. anguillarum, the level of H2O2 was significantly increased in DjGILT-silenced planarians, and concomitantly, mRNA level of C-type lectin DjCTL, which modulates agglutination and clearance efficiency of invading bacteria, was decreased. Further study showed that the decrease of H2O2 level led to a significant increase in DjCTL transcripts. Collectively, we proposed a mechanism model for the involvement of GILT gene in bacterial elimination. We have for the first time revealed the specific mechanism of GILT in innate immune response against bacterial infection.

A new mononuclear ternary La(III) complex with mixed ligands of salicylic acid (Hsal) and 1,10-phenanthroline (phen), namely [La(sal)3(phen)2], has been synthesized and characterized. The structure of the La(III) complex was studied by elemental analysis, thermal analysis, infrared, UV–visible, and fluorescence spectroscopy, single-crystal X-ray diffraction, and tight-binding quantum chemical calculation. The crystal structure analysis reveals the La center was chelated by two phen and three sal ligands. Comparative studies on the bacteriostatic and bactericidal activities of the La(III) complex, ligands, and the lanthanum salt were completed against gram-positive and gram-negative bacteria. These studies demonstrate that the La(III) complex has efficient antibacterial activities toward the selected bacteria. The safety profile of the complex was studied using mice as animal models. The results show that the La(III) complex has low toxicity. The morphology of bacteria was observed by field emission scanning electron microscopy, showing that the La(III) complex may act on the cell envelope of bacteria to achieve good bactericidal effects. The results suggest that the La(III) complex may be developed as effective antibacterial drug candidates.

Previous studies have shown that the toxicity of graphene nanomaterials (GNMs) to bacteria are related to the surface functionalization, however, the involved mechanisms are not fully understood. The present study aims to explore the toxic mechanisms of differentially functionalized GNMs to bacteria from the aspects of physical interaction, oxidative damage and cell autolysis. Three basic functionalization of GNMs including carboxylation (G-COOH), hydroxylation (G-OH) and amination (G-NH2) were studied. G-COOH (66% viability vs CT group) and G-OH (54%) graphene showed higher toxicity to E. coli than G-NH2 (96%) within 3 h at a concentration of 50 mg/L. The three materials showed distinct physical interaction modes with bacterial cells. G-COOH and G-OH contact with cell membrane via their sharp edges thus causing more damage than G-NH2 which covered the bacteria attaching along the basal plane. The three GNMs showed similar radical generation capacities, thus the direct generation of radicals is not the mechanism causing the toxicity. Instead, the GNMs can oxidize the cellular antioxidant glutathione (GSH) thereby causing oxidative damage. The oxidative capacity follows the order: G-COOH > G-OH > G-NH2, which correlated with the antibacterial activity. Cell autolysis, the degradation of cell wall component peptidoglycan, was found to be a new mechanism inducing the death of bacteria. G-COOH and G-OH caused more cell autolysis than G-NH2, which accounts partially for the different toxicity of the three GNMs. The findings provide significant insights into the mechanism of GNMs toxicity to bacteria for not only the risk assessment of GNMs but also the design of graphene based antibacterial materials.

Vinculin is a cytoskeletal protein associated with cell-cell and cell-matrix junctions, playing an important role in linkage of integrin adhesion molecules to the actin cytoskeleton. The planarian nervous system is a fascinating system for studying the organogenesis during regeneration. In this paper, a homolog gene of Vinculin, DjVinculin, was identified and characterized in Dugesia japonica. The DjVinculin sequence analysis revealed that it contains an opening reading frame encoding a putative protein of 975 amino acids with functionally domains that are highly conserved, including eight anti-parallel α-helical bundles organized into five distinct domains. Whole mount in situ hybridization showed that DjVinculin was predominantly expressed in the brain of intact and regenerating planarians. RNA interference of DjVinculin caused distinct defects in brain morphogenesis and influences the regeneration of planarian GABAergic neurons. The expression level of DjGAD protein was decreased in the DjVinculin-knockdown planarians. These findings suggest that DjVinculin is required for GABAergic neurons regeneration.

Prophenoloxidase (tyrosinase) widely distributed in invertebrates and vertebrates, and plays a crucial role in the innate immune. In the present study, the full-length cDNA of a tyrosinase-like (designated AmphiTYR) was cloned from amphioxus Branchiostoma japonicum by PCR techniques. The full-length cDNA of AmphiTYR is 2314 bp, and its predicted open-reading frame codes for a protein of 544 amino acids with a predicted molecular mass of approximately 60.9 kDa and an isoelectric point of 5.65. It has a conserved putative copper-binding domain with six histidines in tyrosinase proteins. Six potential N-linked glycosylation sites and 14 conserved cysteine residues were also predicted to be present in B. japonicum tyrosinase. Homology analysis revealed that AmphiTYR was higher similar to vertebrates tyrosinases (32.5-40.5%) than to invertebrates phenoloxidase (6.4-25.4%). In the adult, AmphiTYR mRNA was expressed in the muscle, epidermis, notochord, ovary, hepatic caecum, pharynx and gill, but not in the neural tube, intestines and testis. During the different development stages from unfertilized egg to larvae of amphioxus, AmphiTYR expressed during all the amphioxus development. These results indicated that AmphiTYR gene not only play a pivotal role in innate immune but also play an important role during embryonic development of cephalochordate amphioxus.

Freshwater planarians have traditionally been favored animal models in regenerative and developmental biology. Little is known about the immunological research in these animals. Here, we provide spectrophotometric, electrophoretic and histochemical evidence demonstrating the presence and characterization of phenoloxidase (EC 1.14.18.1; PO) activity in the planaria Dugenia japonica. PO activity was optimal at pH 7.5 and at a temperature of 45 degrees C. ProPO in the humoral fluid was readily activated by trypsin and zymosan, and was susceptible to phenylthiourea. Two bands of PO activity in the humoral fluid with 150 and 300 kDa by native PAGE and a single band with 150 kDa after SDS-PAGE were resolved. Enzyme histochemical staining demonstrated the presence of PO in the epidermal cell and pharyngeal epithelium, which indicate that PO could serve as a protecting agent against environmental pathogens in D. japonica.

The functional properties of complement in invertebrate deuterostomes are rather ill-defined. Here we showed that the humoral fluids from amphioxus Branchiostoma belcheri were capable of causing lysis of some Vibrio species including Vibrio alginolyticus HW284, Vibrio parahaemolyticus HW458 and Vibrio harvey SF-1, the first such data in the invertebrate deuterostomes. The fluid bacteriolytic activity was abolished by pre-incubation with heat-inactivated rabbit anti-human C3 serum, heating at 45 degrees C for 30 min, and repeated thawing and freezing. Additionally, the bacteriolytic activity was Mg(2+)-dependent and Ca(2+)-independent, and selective activation of the alternative pathway by zymosan A induced a loss of bacteriolytic activity. This strongly suggests that activation of the alternative complement pathway is responsible for the fluid bacteriolytic activity. It was also shown that some Vibrio species like Vibrio cincinnatiensis HW287 appeared resistant to the complement-mediated lysis. The LPS profiling revealed that the fluid-resistant V. cincinnatiensis HW287 had an LPS profile with a ladder of both high-molecular-weight (HMW) and low-molecular-weight (LMW) O-antigen bands, whereas the fluid-sensitive V. alginolyticus HW284 had few HMW O-antigen bands, suggesting a positive correlation between O-antigen size and humoral fluid resistance. Moreover, complement consumption assays demonstrated that both V. alginolyticus HW284 and V. cincinnatiensis HW287 consumed complement, with the former consuming significantly higher complement than the latter. Overall, it is suggested that HMW O-antigens may protect the fluid-resistant Vibrio species by a dual act of avoiding initiating complement activation as well as sterically hindering complement from gaining access to and damaging the cell membrane.

The significance of the presence of vitellogenin (Vg), a female-specific protein, in male and juvenile fish is uncertain. Here we show that the Vg purified from the rosy barb Puntius conchonius possesses both antibacterial and hemagglutinating activities in vitro, and the male fish challenged with Escherichia coli synthesize Vg. These suggest that in addition to being involved in yolk protein formation, Vg is also involved in defense reactions, a novel function assigned to Vg.

Phenoloxidase (PO) from the humoral fluid of amphioxus B. belcheri tsingtauense was purified using a sequential combination of ammonium sulphate precipitation, Sephadex G-200 chromatography and DEAE Sepharose Fast Flow chromatography. In PAGE, the purified enzyme exhibited a single band of 150 kDa under non-reducing conditions, and was resolved to three bands with molecular masses of 72, 46 and 44 kDa, respectively, under reducing conditions, suggesting that the PO in amphioxus humoral fluid seems to be a heterotrimer of three polypeptides held together by disulphide bonds. The substrate specificity and inhibition characteristics both indicate that the PO isolated from amphioxus humoral fluid is a tyrosinase-type enzyme. In addition, mouse antisera against the purified PO were prepared, and their specificity was confirmed by Western blotting, facilitating the future determination of the origin of PO in the humoral fluid and the distribution of PO-synthesising tissues in amphioxus.

The knowledge concerning the humoral immunity is scarce in amphioxus Branchiostoma belcheri. This study measured the humoral parameters including lysozyme, antimicrobial activity, microbial agglutinin and haemagglutinin in amphioxus humoral fluids before and after Vibrio alginolyticus challenge. After challenged with V. alginolyticus, the lysozyme activity, growth inhibiting activities against Escherichia coli and V. alginolyticus and microbial agglutinating activities against Micrococcus lysodeikticus, Bacillus subtilis and Staphylococcus aureus were all increased significantly and haemagglutinating activities against rabbit and human A and O erythrocytes in the humoral fluids were all increased earlier. In contrast, the agglutinating activities against Vibrio harvey and E. coli in the humoral fluids were reduced in response to V. alginolyticus challenge and the haemagglutinating activity against human B erythrocytes increased later.

The humoral immune responses of amphioxus Branchiostoma belcheri to microbial challenge remain open to date. Here we examined the changes in PO activity in the humoral fluids in amphioxus before and after challenge with Escherichia coli and Vibrio alginolyticus. It was found that PO activity in the humoral fluids is markedly increased by challenge with E. coli and V. alginolyticus; and the microbial challenge results in a significant rise in subunit 2 of the three PO subunits, making PO subunit 2 a marker enzyme responsive to microbial challenge. This is the first report on microbial induction of the immune-related molecules like PO in B. belcheri.