Discovery of an Active RAG Transposon Illuminates the Origins of V(D)J Recombination.

Co-option of RAG1 and RAG2 for antigen receptor gene assembly by V(D)J recombination was a crucial event in the evolution of jawed vertebrate adaptive immunity. RAG1/2 are proposed to have arisen from a transposable element, but definitive evidence for this is lacking. Here, we report the discovery of ProtoRAG, a DNA transposon family from lancelets, the most basal extant chordates. A typical ProtoRAG is flanked by 5-bp target site duplications and a pair of terminal inverted repeats (TIRs) resembling V(D)J recombination signal sequences. Between the TIRs reside tail-to-tail-oriented, intron-containing RAG1-like and RAG2-like genes. We demonstrate that ProtoRAG was recently active in the lancelet germline and that the lancelet RAG1/2-like proteins can mediate TIR-dependent transposon excision, host DNA recombination, transposition, and low-efficiency TIR rejoining using reaction mechanisms similar to those used by vertebrate RAGs. We propose that ProtoRAG represents a molecular "living fossil" of the long-sought RAG transposon.

Downregulation of CPSF6 leads to global mRNA 3' UTR shortening and enhanced antiviral immune responses.

Alternative polyadenylation (APA) is a widespread mechanism of gene regulation that generates mRNA isoforms with alternative 3' untranslated regions (3' UTRs). Our previous study has revealed the global 3' UTR shortening of host mRNAs through APA upon viral infection. However, how the dynamic changes in the APA landscape occur upon viral infection remains largely unknown. Here we further found that, the reduced protein abundance of CPSF6, one of the core 3' processing factors, promotes the usage of proximal poly(A) sites (pPASs) of many immune related genes in macrophages and fibroblasts upon viral infection. Shortening of the 3' UTR of these transcripts may improve their mRNA stability and translation efficiency, leading to the promotion of type I IFN (IFN-I) signalling-based antiviral immune responses. In addition, dysregulated expression of CPSF6 is also observed in many immune related physiological and pathological conditions, especially in various infections and cancers. Thus, the global APA dynamics of immune genes regulated by CPSF6, can fine-tune the antiviral response as well as the responses to other cellular stresses to maintain the tissue homeostasis, which may represent a novel regulatory mechanism for antiviral immunity.

Characterization of GSDME in amphioxus provides insights into the functional evolution of GSDM-mediated pyroptosis.

Members of the gasdermin (GSDM) family are pore-forming effectors that cause membrane permeabilization and pyroptosis, a lytic proinflammatory type of cell death. To reveal the functional evolution of GSDM-mediated pyroptosis at the transition from invertebrates to vertebrates, we conducted functional characterization of amphioxus GSDME (BbGSDME) and found that it can be cleaved by distinct caspase homologs, yielding the N253 and N304 termini with distinct functions. The N253 fragment binds to cell membrane, triggers pyroptosis, and inhibits bacterial growth, while the N304 performs negative regulation of N253-mediated cell death. Moreover, BbGSDME is associated with bacteria-induced tissue necrosis and transcriptionally regulated by BbIRF1/8 in amphioxus. Interestingly, several amino acids that are evolutionarily conserved were found to be important for the function of both BbGSDME and HsGSDME, shedding new lights on the functional regulation of GSDM-mediated inflammation.

Insights into RAG Evolution from the Identification of "Missing Link" Family A RAGL Transposons.

A series of "molecular domestication" events are thought to have converted an invertebrate RAG-like (RAGL) transposase into the RAG1-RAG2 (RAG) recombinase, a critical enzyme for adaptive immunity in jawed vertebrates. The timing and order of these events are not well understood, in part because of a dearth of information regarding the invertebrate RAGL-A transposon family. In contrast to the abundant and divergent RAGL-B transposon family, RAGL-A most closely resembles RAG and is represented by a single orphan RAG1-like (RAG1L) gene in the genome of the hemichordate Ptychodera flava (PflRAG1L-A). Here, we provide evidence for the existence of complete RAGL-A transposons in the genomes of P. flava and several echinoderms. The predicted RAG1L-A and RAG2L-A proteins encoded by these transposons intermingle sequence features of jawed vertebrate RAG and RAGL-B transposases, leading to a prediction of DNA binding, catalytic, and transposition activities that are a hybrid of RAG and RAGL-B. Similarly, the terminal inverted repeats (TIRs) of the RAGL-A transposons combine features of both RAGL-B transposon TIRs and RAG recombination signal sequences. Unlike all previously described RAG2L proteins, RAG2L-A proteins contain an acidic hinge region, which we demonstrate is capable of efficiently inhibiting RAG-mediated transposition. Our findings provide evidence for a critical intermediate in RAG evolution and argue that certain adaptations thought to be specific to jawed vertebrates (e.g. the RAG2 acidic hinge) actually arose in invertebrates, thereby focusing attention on other adaptations as the pivotal steps in the completion of RAG domestication in jawed vertebrates.

Effect of tire wear particle accumulation on nitrogen removal and greenhouse gases abatement in bioretention systems: Soil characteristics, microbial community, and functional genes.

Tire wear particles (TWPs), as predominant microplastics (MPs) in road runoff, can be captured and retained by bioretention systems (BRS). This study aimed to investigate the effect of TWPs accumulation on nitrogen processes, focusing on soil characteristics, microbial community, and functional genes. Two groups of lab-scale bioretention columns containing TWPs (0 and 100 mg g-1) were established. The removal efficiencies of NH4+-N and TN in BRS significantly decreased by 7.60%-24.79% and 1.98%-11.09%, respectively, during the 101 days of TWPs exposure. Interestingly, the emission fluxes of N2O and CO2 were significantly decreased, while the emission flux of CH4 was substantially increased. Furthermore, prolonged TWPs exposure significantly influenced the contents of soil organic matter (increased by 27.07%) and NH4+-N (decreased by 42.15%) in the planting layer. TWPs exposure also significantly increased dehydrogenase activity and substrate-induced respiration rate, thereby promoting microbial metabolism. Microbial sequencing results revealed that TWPs decreased the relative abundance of nitrifying bacteria (Nitrospira and Nitrosomonas) and denitrifying bacteria (Dechloromonas and Thauera), reducing the nitrification rate by 42.24%. PICRUSt2 analysis further indicated that TWPs changed the relative abundance of functional genes related to nitrogen and enzyme-coding genes.

ZNFX1 antisense RNA1 promotes antiviral innate immune responses via modulating ZNFX1 function.

Increasing evidence suggests that natural antisense transcriptional lncRNAs regulate their adjacent coding genes to mediate diverse aspects of biology. Bioinformatics analysis of the previously identified antiviral gene ZNFX1 revealed neighboring lncRNA ZFAS1 transcribed on the opposite strand from ZNFX1. Whether ZFAS1 exerts antiviral function via regulating the dsRNA sensor ZNFX1 is unknown. Here we found that ZFAS1 was upregulated by RNA and DNA viruses and type I IFNs (IFN-I) dependent on Jak-STAT signaling, similar to the transcription regulation of ZNFX1. Knockdown of endogenous ZFAS1 partially facilitated viral infection, while ZFAS1 overexpression showed opposite effects. In addition, mice were more resistant to VSV infection with the delivery of human ZFAS1. We further observed that ZFAS1 knockdown significantly inhibited IFNB1 expression and IFR3 dimerization, whereas ZFAS1 overexpression positively regulated antiviral innate immune pathways. Mechanistically, ZFAS1 positively regulated ZNFX1 expression and antiviral function by enhancing the protein stability of ZNFX1, thereby establishing a positive feedback loop to enhance antiviral immune activation status. In short, ZFAS1 is a positive regulator of antiviral innate immune response via regulating its neighbor gene ZNFX1, adding new mechanistic insight into lncRNA-mediated regulation of signaling in innate immunity.

Degradation of WTAP blocks antiviral responses by reducing the m6 A levels of IRF3 and IFNAR1 mRNA.

N6 -methyladenosine (m6 A) is a chemical modification present in multiple RNA species and is most abundant in mRNAs. Studies on m6 A reveal its comprehensive roles in almost every aspect of mRNA metabolism, as well as in a variety of physiological processes. Although some recent discoveries indicate that m6 A can affect the life cycles of numerous viruses as well as the cellular antiviral immune response, the roles of m6 A modification in type I interferon (IFN-I) signaling are still largely unknown. Here, we reveal that WT1-associated protein (WTAP), one of the m6 A "writers", is degraded via the ubiquitination-proteasome pathway upon activation of IFN-I signaling. With the degradation of WTAP, the m6 A levels of IFN-regulatory factor 3 (IRF3) and interferon alpha/beta receptor subunit 1 (IFNAR1) mRNAs are reduced, leading to translational suppression of IRF3 and instability of IFNAR1 mRNA. Thus, the WTAP-IRF3/IFNAR1 axis may serve as negative feedback pathway to fine-tune the activation of IFN-I signaling, which highlights the roles of m6 A in the antiviral response by dictating the fate of mRNAs associated with IFN-I signaling.

Obesity-induced inflammatory miR-133a mediates apoptosis of granulosa cells and causes abnormal folliculogenesis.

Obesity has been reported to promote disordered folliculogenesis, but the exact molecular mechanisms are still not fully understood. In this study, we find that miR-133a is involved in obesity-induced follicular development disorder. After feeding with a high-fat diet (HFD) and fructose water for nine weeks, the mouse body weight is significantly increased, accompanied by an inflammatory state and increased expression of miR-133a in the adipose tissues and ovaries as well as accelerated follicle depletion. Although miR-133a is increased in the fat and ovaries of HFD mice, the increased miR-133a in the HFD ovaries is not derived from exosome transferred from obese adipose tissues but is synthesized by ovarian follicular cells in response to HFD-induced inflammation. In vivo experiments show that intrabursal injection of miR-133a agomir induces a decrease in primordial follicles and an increase in antral follicles and atretic follicles, which is similar to HFD-induced abnormal folliculogenesis. Overexpression of miR-133a modestly promotes granulosa cell apoptosis by balancing the expression of anti-apoptotic proteins such as C1QL1 and XIAP and pro-apoptotic proteins such as PTEN. Overall, this study reveals the function of miR-133a in obesity-induced ovarian folliculogenesis dysfunction and sheds light on the etiology of female reproductive disorders.

Application of biochar as an innovative soil ameliorant in bioretention system for stormwater treatment: A review of performance and its influencing factors.

As an emerging environment functional material, biochar has become a research hotspot in environmental fields because of its excellent ecological and environmental benefits. Recently, biochar has been used as an innovative soil ameliorant in bioretention systems (BRS) to effectively enhance pollutant removal efficiency for BRS. This paper summarizes and evaluates the performance and involved mechanisms of biochar amendment in BRS with respect to the removal of nutrients (TN (34-47.55%) and PO43--P (47-99.8%)), heavy metals (25-100%), pathogenic microorganisms (Escherichia coli (30-98%)), and organic contaminants (77.2-100%). For biochar adsorption, the pseudo-second-order and Langmuir models are the most suitable kinetic and isothermal adsorption models, respectively. Furthermore, we analyzed and elucidated some factors that influence the pollutant removal performance of biochar-amended BRS, such as the types of biochar, the preparation process and physicochemical properties of biochar, the aging of biochar, the chemical modification of biochar, and the hydraulic loading, inflow concentration and drying-rewetting alternation of biochar-amended BRS. The high potential for recycling spent biochar in BRS as a soil ameliorant is proposed. Collectively, biochar can be used as an improved medium in BRS. This review provides a foundation for biochar selection in biochar-amended BRS. Future research and practical applications of biochar-amended BRS should focus on the long-term stability of treatment performances under field conditions, chemical modification with co-impregnated nanomaterials in biochar surface, and the durability, aging, and possible negative effects of biochar.

Recent advances of SBA-15-based composites as the heterogeneous catalysts in water decontamination: A mini-review.

As an emerging class of silica-based mesoporous materials with incorporation of active components (e.g., transition metals/metal oxides and nanocarbons), SBA-15-based composites (X@SBA-15) have been attracting increasing attention in the field of water treatment owing to their unique characteristics and excellent remediation performance. This paper reviews recent advances in catalytic applications of X@SBA-15 to remove organic contaminants from water. Emphasis is made on the use of X@SBA-15 in four advanced oxidation processes (AOPs) (i.e., photocatalysis, Fenton-like oxidation, catalytic ozonation, and sulfate radical-based oxidation). Impregnation and hydrothermal methods are two most widely used synthetic approaches to combine the active composites with SBA-15, obtaining a synergistic effect with significant improvement in their individual catalytic activity for pollution remediation. The enhanced generation of highly reactive hydroxyl radicals from the surface of X@SBA-15 was widely recognized as being responsible for water decontamination using these AOPs, while sulfate radicals were also involved during activation of persulfate or peroxymonosulfate. Especially, X@SBA-15 could significantly enhance the light harvest and reduce the recombination of photo-induced electrons and holes during photocatalytic treatment, which also played the critical role in oxidizing the organics. The superior catalytic performance of X@SBA-15 without leaching metal ions during successive runs demonstrated the excellent reusability and structural stability. Together with the reduced toxicity of the treated solutions and the cost-effective characteristics of X@SBA-15 nanohybrids reported in the published literature, their great potential as the efficient and environmentally friendly heterogeneous catalysts in a real use scenario is suggested. Finally, the future perspectives on the development and practical utilization of X@SBA-15 are addressed.

Peri-ovarian adipose tissue contributes to intraovarian control during folliculogenesis in mice.

Peri-ovarian adipose tissue (POAT) is a kind of intra-abdominal white adipose tissue that is present surrounding the ovaries in rodents. Recent studies demonstrated that POAT-deficient mice displayed a phenotype of delayed antral follicular development, for which decreases in serum estrogen, serum FSH and FSHR levels were responsible. However, folliculogenesis is regulated by endocrine signals and also modulated by a number of locally produced intraovarian factors whose acts are both autocrine and paracrine. Here, we used a model of surgical removal of POAT unilaterally and contralateral ovaries as controls, as both were under the same endocrine control, to assess the paracrine effect of the POAT on folliculogenesis. Surgical removal of unilateral POAT resulted in delayed antral follicular development and the increased number of atretic follicles, accompanied by decreased levels of intraovarian adipokines and growth factors, lipid accumulation and steroidogenic enzyme expression. POAT-deficient ovaries displayed compensatory increased expressions of intraovarian genes, such as Vegf and Adpn for angiogenesis, Acc, Fasn, and Gapdh involved in lipogenesis and Fshr in response to FSH stimulation. Furthermore, we demonstrated that removal of POAT promoted follicular apoptosis, caused retention of cytoplasmic YAP and inhibited PTEN-AKT-mTOR activation. These alterations were observed only in the POAT-deficient ovaries but not in the contralateral ovaries (with POAT), which suggests that a paracrine interaction between POAT and ovaries is important for normal folliculogenesis.

Autophagy participates in innate immune defense in lamprey.

Autophagy is a homeostatic process which degrades cytoplasmic constituents to maintain the balance of organs when they were challenged with nutrient stress. It also participates in cancer, neurodegenerative disorders, aging and innate immune defense. In order to reveal how autophagy participates in innate immune response when invertebrates evolved into vertebrates. Firstly, we performed a systematic analysis of Atg genes and found that they are highly conserved among lancelet, lamprey and zebrafish. Then, we observed autophagosomes upon starvation by TEM in lancelet, lamprey and zebrafish and found that the morphology of autophagosome is similar to that was observed in yeast and mammals. In addition, rapamycin can induce autophagy in lamprey leukocytes and the deficiency of human Beclin1 protein can be rescued by lancelet and lamprey Beclin1 proteins. When lamprey leukocytes were treated with polyI:C and LPS, autophagy was induced. Moreover, when lamprey leukocytes were challenged with live E. coli, phagocytosis along with autophagy was triggered to degrade pathogenic bacteria. In all, our study here indicated that autophagy is highly conserved during evolution and plays a key role in innate defense when invertebrates evolved into vertebrates.

Characterization of Amphioxus IFN Regulatory Factor Family Reveals an Archaic Signaling Framework for Innate Immune Response.

The IFN regulatory factor (IRF) family encodes transcription factors that play important roles in immune defense, stress response, reproduction, development, and carcinogenesis. Although the origin of the IRF family has been dated back to multicellular organisms, invertebrate IRFs differ from vertebrate IRFs in genomic structure and gene synteny, and little is known about their functions. Through comparison of multiple amphioxus genomes, in this study we suggested that amphioxus contains nine IRF members, whose orthologs are supposed to be shared among three amphioxus species. As the orthologs to the vertebrate IRF1 and IRF4 subgroups, Branchiostoma belcheri tsingtauense (bbt)IRF1 and bbtIRF8 bind the IFN-stimulated response element (ISRE) and were upregulated when amphioxus intestinal cells were stimulated with poly(I:C). As amphioxus-specific IRFs, both bbtIRF3 and bbtIRF7 bind ISRE. When activated, they can be phosphorylated by bbtTBK1 and then translocate into nucleus for target gene transcription. As transcriptional repressors, bbtIRF2 and bbtIRF4 can inhibit the transcriptional activities of bbtIRF1, 3, 7, and 8 by competing for the binding of ISRE. Interestingly, amphioxus IRF2, IRF8, and Rel were identified as target genes of bbtIRF1, bbtIRF7, and bbtIRF3, respectively, suggesting a dynamic feedback regulation among amphioxus IRF and NF-κB. Collectively, to our knowledge we present for the first time an archaic IRF signaling framework in a basal chordate, shedding new insights into the origin and evolution of vertebrate IFN-based antiviral networks.

Novel Toll/IL-1 Receptor Homologous Region Adaptors Act as Negative Regulators in Amphioxus TLR Signaling.

Studies have shown that the basal chordate amphioxus possesses an extraordinarily complex TLR system, including 39 TLRs and at least 40 Toll/IL-1R homologous region (TIR) adaptors. Besides homologs to MyD88 and TIR domain-containing adaptor molecule (TICAM), most amphioxus TIR adaptors exhibit domain architectures that are not observed in other species. To reveal how these novel TIR adaptors function in amphioxus Branchiostoma belcheri tsingtauense (bbt), four representatives, bbtTIRA, bbtTIRB, bbtTIRC, and bbtTIRD, were selected for functional analyses. We found bbtTIRA to show a unique inhibitory role in amphioxus TICAM-mediated pathway by interacting with bbtTICAM and bbt receptor interacting protein 1b, whereas bbtTIRC specifically inhibits the amphioxus MyD88-dependent pathway by interacting with bbtMyD88 and depressing the polyubiquitination of bbt TNFR-associated factor 6. Although both bbtTIRB and bbtTIRD are located on endosomes, the TIR domain of bbtTIRB can interact with bbtMyD88 in the cytosol, whereas the TIR domain of bbtTIRD is enclosed in endosome, suggesting that bbtTIRD may be a redundant gene in amphioxus. This study indicated that most expanded TIR adaptors play nonredundant regulatory roles in amphioxus TLR signaling, adding a new layer to understanding the diversity and complexity of innate immunity at basal chordate.

Emergence of the A20/ABIN-mediated inhibition of NF-κB signaling via modifying the ubiquitinated proteins in a basal chordate.

In the past decade, ubiquitination has been well documented to have multifaceted roles in regulating NF-κB activation in mammals. However, its function, especially how deubiquitinating enzymes balance the NF-κB activation, remains largely elusive in invertebrates. Investigating bbtA20 and its binding proteins, bbt A20-binding inhibitor of NF-κB (bbtABIN1) and bbtABIN2, in Chinese amphioxus Branchiostoma belcheri tsingtauense, we found that bbtABIN2 can colocalize and compete with bbt TNF receptor-associated factor 6 to connect the K63-linked polyubiquitin chains, whereas bbtABIN1 physically links bbtA20 to bbt NF-κB essential modulator (bbtNEMO) to facilitate the K48-linked ubiquitination of bbtNEMO. Similar to human A20, bbtA20 is a dual enzyme that removes the K63-linked polyubiquitin chains and builds the K48-linked polyubiquitin chains on bbt receptor-interacting serine/threonine protein kinase 1b, leading to the inhibition of NF-κB signaling. Our study not only suggests that ubiquitination is an ancient strategy in regulating NF-κB activation but also provides the first evidence, to our knowledge, for ABINs/A20-mediated inhibition of NF-κB via modifying the ubiquitinated proteins in a basal chordate, adding information on the stepwise development of vertebrate innate immune signaling.

Two apextrin-like proteins mediate extracellular and intracellular bacterial recognition in amphioxus.

Animals exploit different germ-line-encoded proteins with various domain structures to detect the signature molecules of pathogenic microbes. These molecules are known as pathogen-associated molecular patterns (PAMPs), and the host proteins that react with PAMPs are called pattern recognition proteins (PRPs). Here, we present a novel type of protein domain structure capable of binding to bacterial peptidoglycan (PGN) and the minimal PGN motif muramyl dipeptide (MDP). This domain is designated as apextrin C-terminal domain (ApeC), and its presence was confirmed in several invertebrate phyla and subphyla. Two apextrin-like proteins (ALP1 and ALP2) were identified in a basal chordate, the Japanese amphioxus Branchiostoma japonicum (bj). bjALP1 is a mucosal effector secreted into the gut lumen to agglutinate the Gram-positive bacterium Staphylococcus aureus via PGN binding. Neutralization of secreted bjALP1 by anti-bjALP1 monoclonal antibodies caused serious damage to the gut epithelium and rapid death of the animals after bacterial infection. bjALP2 is an intracellular PGN sensor that binds to TNF receptor-associated factor 6 (TRAF6) and prevents TRAF6 from self-ubiquitination and hence from NF-κB activation. MDP was found to compete with TRAF6 for bjALP2, which released TRAF6 to activate the NF-κB pathway. BjALP1 and bjALP2 therefore play distinct and complementary functions in amphioxus gut mucosal immunity. In conclusion, discovery of the ApeC domain and the functional analyses of amphioxus ALP1 and ALP2 allowed us to define a previously undocumented type of PRP that is represented across different animal phyla.

Dynamic landscape of tandem 3' UTRs during zebrafish development.

Tandem 3' untranslated regions (UTRs), produced by alternative polyadenylation (APA) in the terminal exon of a gene, could have critical roles in regulating gene networks. Here we profiled tandem poly(A) events on a genome-wide scale during the embryonic development of zebrafish (Danio rerio) using a recently developed SAPAS method. We showed that 43% of the expressed protein-coding genes have tandem 3' UTRs. The average 3' UTR length follows a V-shaped dynamic pattern during early embryogenesis, in which the 3' UTRs are first shortened at zygotic genome activation, and then quickly lengthened during gastrulation. Over 4000 genes are found to switch tandem APA sites, and the distinct functional roles of these genes are indicated by Gene Ontology analysis. Three families of cis-elements, including miR-430 seed, U-rich element, and canonical poly(A) signal, are enriched in 3' UTR-shortened/lengthened genes in a stage-specific manner, suggesting temporal regulation coordinated by APA and trans-acting factors. Our results highlight the regulatory role of tandem 3' UTR control in early embryogenesis and suggest that APA may represent a new epigenetic paradigm of physiological regulations.

HaploMerger: reconstructing allelic relationships for polymorphic diploid genome assemblies.

Whole-genome shotgun assembly has been a long-standing issue for highly polymorphic genomes, and the advent of next-generation sequencing technologies has made the issue more challenging than ever. Here we present an automated pipeline, HaploMerger, for reconstructing allelic relationships in a diploid assembly. HaploMerger combines a LASTZ-ChainNet alignment approach with a novel graph-based structure, which helps to untangle allelic relationships between two haplotypes and guides the subsequent creation of reference haploid assemblies. The pipeline provides flexible parameters and schemes to improve the contiguity, continuity, and completeness of the reference assemblies. We show that HaploMerger produces efficient and accurate results in simulations and has advantages over manual curation when applied to real polymorphic assemblies (e.g., 4%-5% heterozygosity). We also used HaploMerger to analyze the diploid assembly of a single Chinese amphioxus (Branchiostoma belcheri) and compared the resulting haploid assemblies with EST sequences, which revealed that the two haplotypes are not only divergent but also highly complementary to each other. Taken together, we have demonstrated that HaploMerger is an effective tool for analyzing and exploiting polymorphic genome assemblies.

The archaic roles of the amphioxus NF-κB/IκB complex in innate immune responses.

NF-κB transcription factors play important roles in immune responses and the development of the immune system. Many aspects of NF-κB signaling differ significantly among distinct species, although many similarities in signaling exist in flies and humans. Thus, to understand the functional refinement of the NF-κB cascade from invertebrates to vertebrates, the Rel and NF-κB proteins, identified as bbtRel and bbtp105, were characterized in a basal chordate amphioxus. Consistent with the sequence similarities, bbtRel was found to interact with a mammalian κB response element, to move into the nucleus when activated, and to be inhibited by the NF-κB-specific inhibitor helenalin. Similar to the other class I members, bbtp105 could be cleaved into the mature form p58. Such endoproteolysis depends on the GRR sequence and requires both protease degradation and caspase 8 cleavage. Furthermore, we found that bbtIκB and the unprocessed bbtp105 can inhibit the transcriptional activity of bbtRel, whereas bbtp58 forms homodimers or heterodimers with bbtRel to create a mature NF-κB complex. Finally, we found that the survival rate and the expression of bbtIκB and TNF-α-like genes were decreased when adult amphioxus were treated with helanalin before immune challenge, suggesting the archaic roles for NF-κB signaling in innate immune responses in a basal chordate. The presence of the NF-κB-IκB cascade in amphioxus indicates that it is a significant feature linking invertebrates to vertebrates and is refined in vertebrates through the expansion and divergence of genes involved in the cascade.

The evolution and regulation of the mucosal immune complexity in the basal chordate amphioxus.

Both amphioxus and the sea urchin encode a complex innate immune gene repertoire in their genomes, but the composition and mechanisms of their innate immune systems, as well as the fundamental differences between two systems, remain largely unexplored. In this study, we dissect the mucosal immune complexity of amphioxus into different evolutionary-functional modes and regulatory patterns by integrating information from phylogenetic inferences, genome-wide digital expression profiles, time course expression dynamics, and functional analyses. With these rich data, we reconstruct several major immune subsystems in amphioxus and analyze their regulation during mucosal infection. These include the TNF/IL-1R network, TLR and NLR networks, complement system, apoptosis network, oxidative pathways, and other effector genes (e.g., peptidoglycan recognition proteins, Gram-negative binding proteins, and chitin-binding proteins). We show that beneath the superficial similarity to that of the sea urchin, the amphioxus innate system, despite preserving critical invertebrate components, is more similar to that of the vertebrates in terms of composition, expression regulation, and functional strategies. For example, major effectors in amphioxus gut mucous tissue are the well-developed complement and oxidative-burst systems, and the signaling network in amphioxus seems to emphasize signal transduction/modulation more than initiation. In conclusion, we suggest that the innate immune systems of amphioxus and the sea urchin are strategically different, possibly representing two successful cases among many expanded immune systems that arose at the age of the Cambrian explosion. We further suggest that the vertebrate innate immune system should be derived from one of these expanded systems, most likely from the same one that was shared by amphioxus.