Amphioxus adenosine-to-inosine tRNA-editing enzyme that can perform C-to-U and A-to-I deamination of DNA.

Adenosine-to-inosine tRNA-editing enzyme has been identified for more than two decades, but the study on its DNA editing activity is rather scarce. We show that amphioxus (Branchiostoma japonicum) ADAT2 (BjADAT2) contains the active site 'HxE-PCxxC' and the key residues for target-base-binding, and amphioxus ADAT3 (BjADAT3) harbors both the N-terminal positively charged region and the C-terminal pseudo-catalytic domain important for recognition of substrates. The sequencing of BjADAT2-transformed Escherichia coli genome suggests that BjADAT2 has the potential to target E. coli DNA and can deaminate at TCG and GAA sites in the E. coli genome. Biochemical analyses further demonstrate that BjADAT2, in complex with BjADAT3, can perform A-to-I editing of tRNA and convert C-to-U and A-to-I deamination of DNA. We also show that BjADAT2 preferentially deaminates adenosines and cytidines in the loop of DNA hairpin structures of substrates, and BjADAT3 also affects the type of DNA substrate targeted by BjADAT2. Finally, we find that C89, N113, C148 and Y156 play critical roles in the DNA editing activity of BjADAT2. Collectively, our study indicates that BjADAT2/3 is the sole naturally occurring deaminase with both tRNA and DNA editing capacity identified so far in Metazoa.

Posture Monitoring of Basketball Training Based on Intelligent Wearable Device.

The relevant content of China's sport work clearly pointed out that it is necessary to improve the physical and mental health of the citizens. Combining the current development hotspots of mobile smart terminals and smart wearable devices, smart wearable devices are analyzed from the function and development history in order to find an effective combination of smart wearable devices and sports work. This article describes the youth as the main body receiving grassroots physical education of nonsports special students and basketball enthusiasts. In the process of physical education teaching, the problem of pain points is more prominent because most teenagers in the basketball education at the grass-roots level do not have the opportunity to get one-to-one private counseling. When there are problems in the operation, students cannot be guided to conduct training or practice by themselves. This paper aims to meet the demand of real-time monitoring of dribbling posture in basketball dribbling training and proposes a low-cost product solution to help teenagers carry out basketball dribbling training by themselves: intelligent wearable product of head wrist and dribbling assistant DribbleAid, which is used to monitor common bad posture problems of users in dribbling training and give corresponding reminders to deal with users' pain points. In this work, various experiments have been carried out for the proposed method and system. A large number of experimental results show that the method designed in this paper can effectively monitor the basketball training posture.

Identification of amphioxus protein disulfide isomerase as both an enzyme and an immunocompotent factor.

Previous studies have shown that protein disulfide isomerase (PDI), a member of the thioredoxin (TRX) superfamily, are broadly associated with immune responses in a variety of animals. However, it remains largely unknown about the direct roles of PDIs during a bacterial infection. In this study, we identified the presence of a single pdi gene in the amphioxus Branchiostoma japonicum, Bjpdi. The deduced protein BjPDI is structurally characterized by the presence of four Trx-like domains in the order of a, b, b' and a' and a short acidic C-terminal tail, that are characteristic of PDIs. We demonstrated that rBjPDI displayed both thiol reductase and disulfide bond isomerase activities, indicating comparability of BjPDI with PDIs in term of enzymatic activities. We also showed that rBjPDI induced bacterial agglutination and exhibited a lectin-like activity capable of binding both bacteria (E. coli and S. aureus) and their signature molecules LPS and LTA. Furthermore, BjPDI could kill S. aureus via inducing membrane depolarization and intracellular ROS production in vitro, and treatment of amphioxus with a blocking anti-PDI antibody in vivo markedly reduced the survival rate of amphioxus following attack by S. aureus. Collectively, our study demonstrates that amphioxus protein disulfide isomerase acts as both an enzyme and an immunocompotent factor, and reports the specific function and mode of action of PDIs in immune responses.

Hepatic cecum: a key integrator of immunity in amphioxus.

The vertebrate liver is regarded as an organ essential to the regulation of immunity and inflammation as well as being central to the metabolism of nutrients. Here, we discuss the functions that the hepatic cecum of amphioxus plays in the regulation of immunity and inflammation, and the molecular basis of this. It is apparent that the hepatic cecum performs important roles in the immunity of amphioxus including immune surveillance, clearance of pathogens and acute phase response. Therefore, the hepatic cecum, like the vertebrate liver, is an organ functioning as a key integrator of immunity in amphioxus.

Preserved antibacterial activity of ribosomal protein S15 during evolution.

Conventional role of ribosomal proteins is ribosome assembly and protein translation, but some ribosomal proteins also show antimicrobial peptide (AMP) activity, though their mode of action remains ill-defined. Here we demonstrated for the first time that amphioxus RPS15, BjRPS15, was a previously uncharacterized AMP, which was not only capable of identifying Gram-negative and -positive bacteria via interaction with LPS and LTA but also capable of killing the bacteria. We also showed that both the sequence and 3D structure of RPS15 and its prokaryotic homologs were highly conserved, suggesting its antibacterial activity is universal across widely separated taxa. Actually this was supported by the facts that the residues positioned at 45-67 formed the core region for the antimicrobial activity of BjRPS15, and its prokaryotic counterparts, including Nitrospirae RPS1933-55, Aquificae RPS1933-55 and P. syringae RPS1950-72, similarly displayed antibacterial activities. BjRPS15 functioned by both interaction with bacterial surface via LPS and LTA and membrane depolarization as well as induction of intracellular ROS. Moreover, we showed that RPS15 existed extracellularly in amphioxus, shrimp, zebrafish and mice, hinting it may play a critical role in systematic immunity in different animals. In addition, we found that neither BjRPS15 nor its truncated form BjRPS1545-67 were toxic to mammalian cells, making them promising lead molecules for the design of novel AMPs against bacteria. Collectively, these indicate that RPS15 is a new member of AMP with ancient origin and high conservation throughout evolution.

Identification of ß tubulin IVb as a pattern recognition receptor with opsonic activity.

Previous studies have shown that tubulins play important role in immune responses of both plants and animals, but no experiments have been performed to study the mode of action of tubulins in immune defense. In addition, there is little convincing experimental evidence of functional commitment for specific tubulin isotypes in animals. In the present, we showed that expression of ß-tubulin IVb gene was affected by both LPS and LTA, hinting its involvement in anti-infectious response. We also showed that recombinant zebrafish ß-tubulin IVb not only interacted with LPS and LTA as well as Gram-negative and -positive bacteria but also agglutinated both Gram-negative and -positive bacteria in a Ca2+-dependent fashion. Interestingly, recombinant ß-tubulin IVb could enhance the phagocytosis of bacteria by macrophages. Moreover, we demonstrated that ß-tubulin IVb was present extracellularly in the serum of zebrafish and mouse. Collectively, these suggest that ß-tubulin IVb may be physiologically involved in the systematic immunity of host via acting as a pattern recognition receptor and an opsonin. This also provides a new angle to understand the roles of ß-tubulin IVb.

Identification and functional characterization of ribosomal protein S23 as a new member of antimicrobial protein.

Previous studies show that some ribosomal proteins possess antimicrobial peptide (AMP) activity. However, information as such remains rather fragmentary and rather limited. We showed here for the first time that amphioxus RPS23, BjRPS23, was a previously uncharacterized AMP. It not only acted as a pattern recognition receptor, capable of identifying LPS, LTA and PGN, but also an effector, capable of killing the Gram-negative and -positive bacteria. We also showed that the residues positioned at 67-84 formed the core region for the antimicrobial activity of BjRPS23, and its orthologues Verrucomicrobia RPS1268-85 and Thermotoga RPS1265-82 similarly displayed some antibacterial activities. BjRPS23 functioned by a combined action of membranolytic mechanisms including interaction with bacterial membrane via LPS, LTA and PGN, and membrane depolarization. BjRPS23 also stimulated production of intracellular ROS in bacteria. Moreover, we demonstrated that RPS23 existed across widely separated taxa, and might play a universal role in protection against bacterial infection in different animals. In addition, we found that neither BjRPS23 nor its truncated form BjRPS2367-84 were cytotoxic to mammalian cells, making them promising lead molecules for the design of novel peptide antibiotics against bacteria. Collectively, these indicate that RPS23 is a new member of AMP with ancient origin and high conservation.

Characterization of a novel protein identified by proteomics analysis as a modulator of inflammatory networks in amphioxus.

Inflammatory response is an innate host defense mechanism, and its regulation is essential for the host to get rid of harm by the excessive reactions. We first utilized proteomics approach to identify amphioxus humoral fluid proteins in response to LPS-induced inflammation. A total of 26 differentially expressed proteins, mainly involved in energy metabolism and cytoskeleton rearrangement processes, were identified between LPS-treated and control animals. Furthermore, we found a single uncharacterized protein (termed BjIM1) out of the most up-regulated ones, and examined its role in the regulation of immune and inflammatory responses. BjIM1 is predominantly expressed in the hepatic caecum, and its promoter sequence includes many binding sites for immune-relevant transcription factors. Importantly, recombinant BjIM1 (rBjIM1) is able to inhibit LPS-induced up-regulation of TLR pathway genes, such as MyD88, IKK, NF-κB1, Rel, p38, JNK and AP-1, indicating that BjIM1 may negatively regulate the TLR signaling pathway in amphioxus. Moreover, rBjIM1 also modulates the expression of genes involved in the interaction network of inflammation, energy metabolism and cytoskeleton rearrangement, including SIRT1, Rac1 and NOX2, in the LPS-induced inflammatory response in amphioxus. Collectively, our studies suggest that BjIM1 is an uncharacterized protein functioning as a modulator of inflammatory networks in amphioxus.

Lectin-like and bacterial-agglutinating activities of heat shock proteins Hsp5 and Hsp90α from amphioxus Branchiostoma japonicum.

Previous studies have shown that heat shock proteins (Hsps) are broadly associated in immune responses in a variety of animals. However, it remains largely unknown about the direct roles of Hsps during a bacterial infection. In this study, we have cloned and characterized the cDNAs of two Hsp genes in the amphioxus Branchiostoma japonicum, termed Bjhsp5 and Bjhsp90α, the first ones in this evolutionarily important animal. Both Bjhsp5 and Bjhsp90α showed distinct tissue expression patterns, and were inducible by challenge with lipopolysaccharide (LPS) and lipoteichoic acid (LTA), suggesting they may be involved in anti-infectious responses. We also showed that both BjHsp5 and BjHsp90α displayed lectin-like property with affinity to both the Gram-negative and -positive bacteria as well as their signature molecules LPS and LTA, hinting they may both act as a pattern recognition receptor, capable of identifying pathogens. In addition, we found that BjHsp5 and BjHsp90α were both able to agglutinate the Gram-negative and -positive bacteria in the presence of Ca2+, suggesting they may be able to trap the invading pathogens together in vivo, avoiding them moving around and thereby protecting the host from pathogenic attack. These data provide a new angle to the roles of Hsps in immune defense.

Subtle Difference Generates Big Dissimilarity: Comparison of Enzymatic Activity in KL1 and KL2 Domains of Lancelet Klotho.

Klotho, a putative aging suppressor, shares sequence similarity with members of the glycosidase family 1. It has been identified in several vertebrate species, but only mouse Klotho has so far been proven to exhibit ß-glucuronidase activity. Thus, the argument that Klotho from animals other than mouse has glycosidase activity remains open. Moreover, little information is available regarding the structure-activity relationship of Klotho. Here, we demonstrate the presence of a single klotho gene in the amphioxus Branchiostoma japonicum, Bjklotho, which possesses two tandem domains named BjKL1 and BjKL2, and each of them has two glutamic acid residues that have been shown to be involved in the catalytic activity of family 1 glycosidase. Enzymatic activity assays of the recombinant proteins BjKL1 and BjKL2 revealed that only BjKL2 displayed ß-glucosidase activity, but BjKL1 did not. Structural analysis showed that there existed nine consecutive but not conserved residues in the ß6α6 loop, which affects the conformational form in the entrance to the catalytic pocket of BjKL1 and BjKL2, thereby leading to a subtle difference in the enzyme-substrate binding and interaction. Furthermore, the substitution of the nine residues 354QNRVDPNDT362 in BjKL1 by the residues 884EDNVVVGAA892 in BjKL2 resulted in significant increase in ß-glucosidase activity in the BjKL1 mutant. Our results indicate that BjKL2 possesses ß-glucosidase, the first data as such in invertebrates. We also identify, for the first time, the residues 884EDNVVVGAA892 in BjKL2 a sequence critical and indispensable for glucosidase.

NEFLb impairs early nervous system development via regulation of neuron apoptosis in zebrafish.

Neurofilament light chain (NEFL), a subunit of neurofilament, has been shown to play an important role in pathogenic neurodegenerative disease and in radial axonal growth. However, information remains largely lacking regarding the function of NEFL in early development to date. In this study, we demonstrated the presence of two nefl genes, nefla and neflb, in zebrafish, generated by fish-specific third round genome duplication. These duplicated nefl genes were predominantly expressed in the nervous system with an overlapping and distinct expression pattern. Both gene knockdown and rescue experiments show that it was neflb rather than nefla that played an indispensable role in nervous system development. It was also found that neflb knockdown resulted in striking apoptosis of the neurons in the brain and spinal cord, leading to morphological defects such as brain structure disorder and trunk bending. Thus, we report a previously uncharacterized role of NEFL that NEFLb impairs the early development of zebrafish nervous system via regulation of the neuron apoptosis in the brain and spinal cord.

Identification and functional characterization of amphioxus Miple, ancestral type of vertebrate midkine/pleiotrophin homologues.

Midkine (MK) and pleiotrophin (PTN) are the only two members of heparin-binding growth factor family. MK/PTN homologues found from Drosophila to humans are shown to have antibacterial activities and their antibacterial domains are conserved during evolution. However, little is known about MK/PTN homologue in the basal chordate amphioxus, and overall, information regarding MK/PTN homologues is rather limited in invertebrates. In this study, we identified a single MK/PTN homologue in Branchiostoma japonicum, termed BjMiple, which has a novel domain structure of PTN-PTNr1-PTNr2, and represents the ancestral form of vertebrate MK/PTN family proteins. BjMiple was expressed mainly in the ovary in a tissue-dependent fashion, and its expression was remarkably up-regulated following challenge with bacteria or their signature molecules LPS and LTA, suggesting its involvement in antibacterial responses. Functional assays revealed that BjMiple had strong antimicrobial activity, capable of killing a panel of Gram-negative and Gram-positive bacteria via a membranolytic mechanism, including interaction with bacterial membrane via LPS and LTA, membrane depolarization and high intracellular levels of ROS. Importantly, strong antibacterial activity was localized in PTN42-61 and PTNr142-66. Additionally, BjMiple and its derived peptides PTN42-61 and PTNr142-66 were not cytotoxic to human RBCs and mammalian cells. Taken together, our study suggests that amphioxus Miple is the ancestral type of vertebrate MK/PTN family homologues, and can play important roles as innate peptide antibiotics, which renders it a promising template for the design of novel peptide antibiotics against multi-drug resistant bacteria.

Identification and characterization of properdin in amphioxus: Implications for a functional alternative complement pathway in the basal chordate.

A complement system operating via the alternative pathway similar to that of vertebrates has been demonstrated in the primitive chordate amphioxus. However, the factor P (fP), a positive regulator of the alternative pathway, remains elusive in amphioxus to date. In this study, we identified and characterized a properdin gene in the amphioxus B. japonicum, BjfP, which represents an archetype of vertebrate properdins. Real-time PCR analysis showed that the BjfP was ubiquitously expressed and its expression was significantly up-regulated following the challenge with bacteria or lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Recombinant BjfP (rBjfP) and its truncated proteins including rTSR1-3, rTSR4-6 and rTSR7-8, were all capable of interacting with both Gram-negative and positive bacteria as well as LPS and LTA. Moreover, rBjfP, rTSR1-3 and rTSR4-6 could also specifically bind to C3b. Importantly, both rTSR1-3 and rTSR4-6 could inhibit the binding of rBjfP to C3b, and thus suppress the activation of the alternative pathway of complement, suggesting the involvement of BjfP in the alternative pathway. This is the first report showing that a properdin protein in invertebrates plays similar roles to vertebrate properdins. Collectively, these data suggest that BjfP might represent the ancient molecule from which vertebrate properdins evolved.

Identification and functional characterization of a novel member of low-density lipoprotein receptor-related protein (LRP)-like family in amphioxus.

Low-density lipoprotein receptor-related protein (LRP) is a group of important endocytic receptors contributing to binding ligands and maintaining internal environment. In this study, we identified a soluble LRP-like molecule in the amphioxus B. japonicum, BjLRP, with an uncharacterized domain structure combination of LY-EGF-CRD-EGF-CRD. It was mainly expressed in the gill, muscle, notochord and testis, and was significantly up-regulated following the challenge with bacteria. Recombinant BjLRP was capable of interacting with both Gram-negative and positive bacteria as well as PAMPs including lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PGN). Interestingly, recombinant LY peptide was also able to bind to the Gram-negative and positive bacteria as well as the PAMPs LPS, LTA and PGN. By contrast, none of recombinant EGF1, EGF2, CRD1 and CRD2 had affinity to the bacteria and the PAMPs. In addition, BjLRPΔLY had no affinity to the PAMPs, although BjLRPΔLY showed slight affinity to the bacteria. These suggest that the interaction of BjLRP with the bacteria and PAMPs was primarily attributable to the LY domain. It is clear that BjLRP is a novel pattern recognition protein capable of identifying and interacting with invading bacteria in amphioxus.

A new LDLa domain-containing C-type lectin with bacterial agglutinating and binding activity in amphioxus.

Over 1200 C-type lectin gene models have been identified in amphioxus, but only a few of them have been functionally characterized. In this study, we identified a C-type lectin, BjCTL, with domain structure of LDLa-CTLD-EGF_Lam, the first such data in chordates. It was expressed mainly in the notochord and ovary in a tissue-dependent fashion. Recombinant BjCTL was characterized as a typical Ca2+-dependent carbohydrate-binding protein capable of agglutinating and binding to both Gram-negative and positive bacteria we tested. In addition, it specifically bound to insoluble lipopolysaccharide, lipoteichoic acid and peptidoglycan, which can be inhibited by galactose. We also showed that the interaction of BjCTL with the bacteria is primarily attributable to CTLD domain. Thus, BjCTL is a novel pattern recognition protein involved in lectin-mediated innate immunity.