A chimeric hairpin DNA aptamer-based biosensor for monitoring the therapeutic drug bevacizumab.

The accurate and rapid detection of specific antibodies in blood is very important for efficient diagnosis and precise treatment. Conventional methods often suffer from time-consuming operations and/or a narrow detection range. In this work, for the rapid determination of bevacizumab in plasma, a series of chimeric hairpin DNA aptamer-based probes were designed by the modification, labeling and theoretical computation of an original aptamer. Then, the dissociation constant of the modified hairpin DNA to bevacizumab was measured and screened using microscale thermophoresis. The best chimeric hairpin DNA aptamer-based probe was then selected, and a one-step platform for the rapid determination of bevacizumab was constructed. This strategy has the advantages of being simple, fast and label-free. Because of the design and screening of the hairpin DNA, as well as the optimization of the concentration and electrochemical parameters, a low detection limit of 0.37 pM (0.054 ng mL-1) with a wide linear range (1 pM-1 µM) was obtained. Finally, the rationally constructed biosensor was successfully applied to the determination of bevacizumab in spiked samples, and it showed good accuracy and precision. This method is expected to truly realize accurate and rapid detection of bevacizumab and provides a new idea for the precise treatment of diseases.

Plants of the Genus Mahonia as a Potential Traditional Chinese Medicine for the Prevention and Treatment of Alzheimer's Disease.

Alzheimer's disease (AD), a prevalent multiple neurodegenerative disease, has gained attention, particularly in the aging population. However, presently available therapies merely focus on alleviating the symptoms of AD and fail to slow disease progression significantly. Traditional Chinese medicine (TCM) has been used to ameliorate symptoms or interfere with the pathogenesis of aging-associated diseases for many years based on disease-modifying in multiple pathological roles with multi-targets, multi-systems and multi-aspects. Mahonia species as a TCM present potential for anti-inflammatory activity, antioxidant activity, anti-acetylcholinesterase activity, and antiamyloid- beta activity that was briefly discussed in this review. They are regarded as promising drug candidates for AD therapy. The findings in this review support the use of Mahonia species as an alternative therapy source for treating AD.

SUMO and PIAS repress NF-κB activation in a basal chordate.

Small ubiquitin-like modifier (SUMO) regulates various biological processes, including the MyD88/TICAMs-IRAKs-TRAF6-NF-κB pathway, one of the core immune pathways. However, its functions are inconsistent between invertebrates and vertebrates and have rarely been investigated in lower chordates, including amphioxus and fishes. Here, we investigated the SUMOylation gene system in the amphioxus, a living basal chordate. We found that amphioxus has a SUMOylation system that has a complete set of genes and preserves several ancestral traits. We proceeded to study their molecular functions using the mammal cell lines. Both amphioxus SUMO1 and SUMO2 were shown to be able to attach to NF-κB Rel and to inhibit NF-κB activation by 50-75% in a dose-dependent fashion. The inhibition by SUMO2 could be further enhanced by the addition of the SUMO E2 ligase UBC9. In comparison, while human SUMO2 inhibited RelA, human SUMO1 slightly activated RelA. We also showed that, similar to human PIAS1-4, amphioxus PIAS could serve as a SUMO E3 ligase and promote its self-SUMOylation. This suggests that amphioxus PIAS is functionally compatible in human cells. Moreover, we showed that amphioxus PIAS is not only able to inhibit NF-κB activation induced by MyD88, TICAM-like, TRAF6 and IRAK4 but also able to suppress NF-κB Rel completely in the presence of SUMO1/2 in a dose-insensitive manner. This suggests that PIAS could effectively block Rel by promoting Rel SUMOylation. In comparison, in humans, only PIAS3, but not PIAS1/2/4, has been reported to promote NF-κB SUMOylation. Taken together, the findings from amphioxus, together with those from mammals and other species, not only offer insights into the functional volatility of the animal SUMO system, but also shed light on its evolutionary transitions from amphioxus to fish, and ultimately to humans.

Differential roles of the fish chitinous membrane in gut barrier immunity and digestive compartments.

The chitin-based peritrophic matrix (PM) is a structure critical for both gut immunity and digestion in invertebrates. PM was traditionally considered lost in all vertebrates, but a PM-like chitinous membrane (CM) has recently been discovered in fishes, which may increase the knowledge on vertebrate gut physiology and structural evolution. Here, we show that in zebrafish, the CM affects ingestion behavior, microbial homeostasis, epithelial renewal, digestion, growth, and longevity. Young mutant fish without CM appear healthy and are able to complete their life cycle normally, but with increasing age they develop gut inflammation, resulting in gut atrophy. Unlike mammals, zebrafish have no visible gel-forming mucin layers to protect their gut epithelia, but at least in young fish, the CM is not a prerequisite for the antibacterial gut immunity. These findings provide new insights into the role of the CM in fish prosperity and its eventual loss in tetrapods. These findings may also help to improve fish health and conservation, as well as to advance the understanding of vertebrate gut physiology and human intestinal diseases.

Deep Learning to Reveal the Distribution and Diffusion of Water Molecules in Fuel Cell Catalyst Layers.

Water management in the catalyst layers (CLs) of proton-exchange membrane fuel cells is crucial for its commercialization and popularization. However, the high experimental or computational cost in obtaining water distribution and diffusion remains a bottleneck in the existing experimental methods and simulation algorithms, and further mechanistic exploration at the nanoscale is necessary. Herein, we integrate, for the first time, molecular dynamics simulation with our customized analysis framework based on a multiattribute point cloud dataset and an advanced deep learning network. This was achieved through our workflow that generates simulated transport data of water molecules in the CLs as the training and test dataset. Deep learning framework models the multibody solid-liquid system of CLs on a molecular scale and completes the mapping from the Pt/C substrate structure and Nafion aggregates to the density distribution and diffusion coefficient of water molecules. The prediction results are comprehensively analyzed and error evaluated, which reveals the highly anisotropic interaction landscape between 50,000 pairs of interacting nanoparticles and explains the structure and water transport property relationship in the hydrated Nafion film on the molecular scale. Compared to the conventional methods, the proposed deep learning framework shows computational cost efficiency, accuracy, and good visual display. Further, it has a generality potential to model macro- and microscopic mass transport in different components of fuel cells. Our framework is expected to make real-time predictions of the distribution and diffusion of water molecules in CLs as well as establish statistical significance in the structural optimization and design of CLs and other components of fuel cells.

Two novel mollusk short-form ApeC-containing proteins act as pattern recognition proteins for peptidoglycan.

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.

A Systematic Review and Meta-Analysis on the Efficacy of Puerarin Injection as Adjunctive Therapy for Unstable Angina Pectoris.

Background: As adjunctive therapy, puerarin injection has been widely applied for the treatment of unstable angina pectoris (UAP) in China during the past decades. However, the efficacy of puerarin injection as adjunctive therapy for UAP has not been well confirmed. The purpose of this meta-analysis was to summarize the available evidence to estimate the efficacy of puerarin injection in treating UAP. Objective: A systematic review and meta-analysis with subgroup analysis and sensitivity analysis according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) principle were carried out to evaluate the efficacy of puerarin injection as adjunctive therapy in treating UAP. Methods: To obtain the published randomized controlled trials (RCTs) on puerarin injection, databases, namely, China National Knowledge Infrastructure (CNKI), Wanfang Database, Chinese Biomedical Literature Database, Sino-Med, PubMed, China Science and Technology Journal Database (VIP), Medline, Google Scholar, Cochrane Library, Chinese Science Citation Database, and Embase were systematically searched until June 2021. In this meta-analysis, Review Manager version 5.3 software and Stata version 12.0 software were employed to analyze the collected data. Based on the methodological quality, years of publications, sample size and dosages, sensitivity analysis, and subgroup analysis were performed. The GRADE assessment was conducted by the software GRADEpro version 3.6 software. Results: A total of 17 RCTs involving 1,459 patients were included in this meta-analysis. Results indicated that puerarin injection as adjunctive therapy was more superior than conventional Western medicine alone in reducing angina symptoms [risk ratio (RR) = 1.22, 95% CI 1.16 to 1.28, Z = 8.11, p < 0.00001] and improving ECG (RR = 1.32, 95% CI 1.20 to 1.44, Z = 6.00, p < 0.00001), meanwhile reducing the frequency of angina attack [mean difference (MD) = -2.22, 95% CI -2.53 to -1.90, Z = 13.97, p < 0.00001] and the duration of angina attack (MD = -2.00, 95% CI -2.39 to -1.61, Z = 9.99, p < 0.00001) for the treatment of UAP. Results from the GRADE assessment suggested that the comprehensive quality of this evidence was low. Conclusion: This meta-analysis indicated that puerarin injection was more effective than using conventional Western medicine alone in the treatment of UAP. However, because of the low methodological quality of the included RCTs, more evidence was still needed to verify the efficacy of puerarin injection.

Application of deep learning for predicting the treatment performance of real municipal wastewater based on one-year operation of two anaerobic membrane bioreactors.

In this study, data-driven deep learning methods were applied in order to model and predict the treatment of real municipal wastewater using anaerobic membrane bioreactors (AnMBRs). Based on the one-year operating data of two AnMBRs, six parameters related to the experimental conditions (temperature of reactor, temperature of environment, temperature of influent, influent pH, influent COD, and flux) and eight parameters for wastewater treatment evaluation (effluent pH, effluent COD, COD removal efficiency, biogas composition (CH4, N2, and CO2), biogas production rate, and oxidation-reduction potential) were selected to establish the data sets. Three deep learning network structures were proposed to analyze and reproduce the relationship between the input parameters and output evaluation parameters. The statistical analysis showed that deep learning closely agrees with the AnMBR experimental results. The prediction accuracy rate of the proposed densely connected convolutional network (DenseNet) can reach up to 97.44%, and the single calculation time can be reduced to within 1 s, suggesting the high performance of AnMBR treatment prediction with deep learning methods.

Efficacy of Ligustrazine Injection as Adjunctive Therapy in Treating Acute Cerebral Infarction: A Systematic Review and Meta-Analysis.

Background: Ligustrazine injection has been widely used as adjunctive therapy in the treatment of acute cerebral infarction (ACI) during the past decades in China, but its clinical efficacy is not yet well confirmed. This study aims to evaluate the efficacy of ligustrazine injection as adjunctive therapy for ACI. Methods: Databases including China National Knowledge Infrastructure (CNKI), China Science and Technology Journal Database (VIP), PubMed, Medline, Google Scholar, Chinese Biomedical Literature Database, Cochrane Library, Embase, Sino-Med, Wanfang Database, and Chinese Science Citation Database were systematically searched for the published randomized controlled trials (RCTs) on ligustrazine injection in the treatment of ACI until November 2020. Meta-analysis was performed on the primary outcome measure (i.e., clinical effective rate) and the secondary outcome measure [i.e., neurological deficit score (NDS), fibrinogen, low shear blood viscosity (LBV), and high shear blood viscosity (HBV)]. The quality of the included RCTs was assessed according to the M scoring system (the refined Jadad scale). Sensitivity analysis and subgroup analysis were conducted according to the methodological quality, years of publication, and sample size. Results: Nineteen RCTs, containing 2022 patients, were included in this study. Meta-analysis indicated that ligustrazine injection combined with Western medicine could achieve a better effect in the treatment of ACI than using Western medicine alone in terms of clinical effective rate (RR = 1.24; 95% CI, 1.19-1.29), NDS (MD = -3.88; 95%CI, -4.51 to -3.61), fibrinogen (MD = -0.59; 95% CI, -0.76 to -0.42), LBV (MD = -2.11; 95% CI, -3.16 to -1.06), and HBV (MD = -0.88; 95% CI, -1.20 to -0.55). Conclusions: This research indicated that ligustrazine injection as adjunctive therapy seemed to be more effective than using western medicine alone in treating ACI. However, more evidence is required to confirm the efficacy of ligustrazine injection due to the low methodological quality of the included RCTs.

Two Amphioxus ApeC-Containing Proteins Bind to Microbes and Inhibit the TRAF6 Pathway.

The apextrin C-terminal (ApeC) domain is a class of newly discovered protein domains with an origin dating back to prokaryotes. ApeC-containing proteins (ACPs) have been found in various marine and aquatic invertebrates, but their functions and the underlying mechanisms are largely unknown. Early studies suggested that amphioxus ACP1 and ACP2 bind to bacterial cell walls and have a role in immunity. Here we identified another two amphioxus ACPs (ACP3 and ACP5), which belong to the same phylogenetic clade with ACP1/2, but show distinct expression patterns and sequence divergence (40-50% sequence identities). Both ACP3 and ACP5 were mainly expressed in the intestine and hepatic cecum, and could be up-regulated after bacterial challenge. Both prokaryotic-expressed recombinant ACP3 and ACP5 could bind with several species of bacteria and yeasts, showing agglutinating activity but no microbicidal activity. ELISA assays suggested that their ApeC domains could interact with peptidoglycan (PGN), but not with lipoteichoic acid (LTA), lipopolysaccharides (LPS) and zymosan A. Furthermore, they can only bind to Lys-type PGN from Staphylococcus aureus, but not to DAP-type PGN from Bacillus subtilis and not to moieties of PGN such as MDPs, NAMs and NAGs. This recognition spectrum is different from that of ACP1/2. We also found that when expressed in mammalian cells, ACP3 could interact with TRAF6 via a conserved non-ApeC region, which inhibited the ubiquitination of TRAF6 and hence suppressed downstream NF-κB activation. This work helped define a novel subfamily of ACPs, which have conserved structures, and have related yet diversified molecular functions. Its members have dual roles, with ApeC as a lectin and a conserved unknown region as a signal transduction regulator. These findings expand our understanding of the ACP functions and may guide future research on the role of ACPs in different animal clades.

Identification and Characterization of the Amphioxus Lck and Its Associated Tyrosine Phosphorylation-Dependent Inhibitory LRR Receptor.

Amphioxus (e.g., Branchiostoma belcheri, Bb) has recently emerged as a new model for studying the origin and evolution of vertebrate immunity. Mammalian lymphocyte-specific tyrosine kinase (Lck) plays crucial roles in T cell activation, differentiation and homeostasis, and is reported to phosphorylate both the ITIM and ITSM of PD-1 to induce the recruitment of phosphatases and thus the inhibitory function of PD-1. Here, we identified and cloned the amphioxus homolog of human Lck. By generating and using an antibody against BbLck, we found that BbLck is expressed in the amphioxus gut and gill. Through overexpression of BbLck in Jurkat T cells, we found that upon TCR stimulation, BbLck was subjected to tyrosine phosphorylation and could partially rescue Lck-dependent tyrosine phosphorylation in Lck-knockdown T cells. Mass spectrometric analysis of BbLck immunoprecipitates from immunostimulants-treated amphioxus, revealed a BbLck-associated membrane-bound receptor LRR (BbLcLRR). By overexpressing BbLcLRR in Jurkat T cells, we demonstrated that BbLcLRR was tyrosine phosphorylated upon TCR stimulation, which was inhibited by Lck knockdown and was rescued by overexpression of BbLck. By mutating single tyrosine to phenylalanine (Y-F), we identified three tyrosine residues (Y539, Y655, and Y690) (3Y) of BbLcLRR as the major Lck phosphorylation sites. Reporter gene assays showed that overexpression of BbLcLRR but not the BbLcLRR-3YF mutant inhibited TCR-induced NF-κB activation. In Lck-knockdown T cells, the decline of TCR-induced IL-2 production was reversed by overexpression of BbLck, and this reversion was inhibited by co-expression of BbLcLRR but not the BbLcLRR-3YF mutant. Sequence analysis showed that the three tyrosine-containing sequences were conserved with the tyrosine-based inhibition motifs (ITIMs) or ITIM-like motifs. And TCR stimulation induced the association of BbLcLRR with tyrosine phosphatases SHIP1 and to a lesser extent with SHP1/2. Moreover, overexpression of wild-type BbLcLRR but not its 3YF mutant inhibited TCR-induced tyrosine phosphorylation of multiple signaling proteins probably via recruiting SHIP1. Thus, we identified a novel immunoreceptor BbLcLRR, which is phosphorylated by Lck and then exerts a phosphorylation-dependent inhibitory role in TCR-mediated T-cell activation, implying a mechanism for the maintenance of self-tolerance and homeostasis of amphioxus immune system and the evolutionary conservatism of Lck-regulated inhibitory receptor pathway.

Broad distribution, high diversity and ancient origin of the ApeC-containing proteins.

Apextrin C-terminal (ApeC) is a novel protein domain with unknown functions, although early studies suggest that some ApeC-containing proteins (ACPs) bind to carbohydrates and have a role in development and immunity. Here we investigated the taxonomic distribution, sequence diversification and origin of ACPs in Metazoa. Most ACPs are present in invertebrates from aquatic or moist environments, including cnidarians, mollusks, echinoderms, cephalochordates, flatworms, water bears, nematodes and annelids. However, ACPs are absent in vertebrates and in most arthropod lineages (e.g. insects and crustaceans) except arachnids. ACPs apparently undergo rapid turnover and diversification, hence no orthologs could be found between (sub)phyla. ApeC can function either as a standalone domain or as a partner domain. It has been found to pair up with over ten different domain types in different ACPs. The partner domains are related to immunity, extracellular matrix, protein-protein and protein-carbohydrate interactions. Notably, the domain pair with the widest taxonomic distribution is MACPF/perforin-ApeC, which represent a classic group of ACPs called apextrins. ApeC also frequently pairs up with itself to form dual-ApeC modules in different phyla. Notably, in parasite flatworms, dual-ApeCs are present in 70% of ACPs and all inherited from a common ancestor. The broad distribution of MACPF-ApeC and dual-ApeC suggest their conserved yet unknown functions. We also discovered distant ApeC homologs in bacteria, hence tracing the origin of ApeC back to prokaryotes. Our findings show that ApeC has an ancient origin and is able to function alone or in complex domain architectures, though it is less prevalent than other versatile domains such as immunoglobulin domains and C-type lectin domains. This work provides a foundation for further functional study of this novel domain type.

Anti-fouling poly adenine coating combined with highly specific CD20 epitope mimetic peptide for rituximab detection in clinical patients' plasma.

In this study, a high-performance anti-fouling coating based on poly adenine (polyAn) as well as a highly specific cluster of differentiation 20 (CD20) epitope mimetic peptide (CN14) were employed to synergistically construct a facile biosensor for the rapid and sensitive determination of rituximab in lymphoma patients' plasma. The well-designed and optimized polyAn coating displayed excellent stability, hydrophilicity, thanks to its intrinsic affinity with gold surface and thoroughly exposed hydrophilic phosphate groups. Moreover, the proposed strategy avoids the necessity to modify binding groups (e.g. thiol), making it more facile, repeatable and efficient. When dealing with complex clinical plasma samples, the polyAn coating demonstrated better anti-fouling performance and lower background signal in comparison with mercaptan and bovine serum albumin coatings. The dissociation constant (~60 nM) between CN14 and rituximab was measured by microscale thermophoresis and their binding mechanism was further explained using computer simulation. The constructed GE/CN14/polyA20 biosensor displayed satisfactory performance with detection limit of 35.26 ng/mL. Finally, the proposed biosensor was successfully applied for rapidly determining rituximab in lymphoma patients' plasma, and exhibited comparable accuracy to the commercial ELISA, but has advantages including a shorter detection time, wider detection range and lower cost. It's worth noting that the anti-fouling polyAn coating can be tailored according to the surface property of sensing interface and can be easily expanded to other gold electrode related biosensors.

Development of histidine-tagged cyclic peptide functionalized monolithic material for the affinity purification of antibodies in biological matrices.

The rapidly increasing applications of monoclonal antibodies (mAbs) in therapy have necessitated the development of mAb production and purification technologies for both academic and industrial usage. Herein, a histidine-tagged cyclic peptide (HHHHHHGSGSGSDC*AWHLGELVWC*T, the disulfide-bonded cysteines of which are indicated by asterisks, named HT25-cyclopeptide) functionalized monolithic material was developed by the metal ion chelation-based approach. The resulting material possessed suitable affinity and peptide ligand density (13.8 mg peptide ligand per mL of material), good porosity (67.1 %), acceptable specific surface area (52.95 m2/g), and lots of macropores (4.13 µm). Moreover, excellent antibody-specific selectivity, comparable or even better binding capacity (for dried material, maximum static binding capacity and dynamic binding capacity are about 119.3 mg/g and 17.05 mg/g, respectively) for antibody compared to previously developed affinity materials, acceptable resistance to trypsin digestion, and negligible nonspecific protein adsorption, were also achieved on this novel monolithic material. Compared with the corresponding cyclic peptide-based sepharose material, milder elution conditions were employed for the HT25-cyclopeptide-based monolithic material, which could effectively prevent the aggregation and denaturation of the enriched antibodies. This novel material was then successfully applied to the affinity enrichment and purification of mAbs (including infliximab and rituximab) in different cell culture media or IgG in human serum.

Rapid preparation of 1-vinylimidazole based non-affinity polymers for the highly-selective purification of antibodies from multiple biological sources.

There is an urgent need for developing advanced purification techniques with the merits of low cost and satisfactory capacity in order to meet the challenges in the current downstream purification of monoclonal antibodies (mAbs). Herein, a simple and inexpensive nitrogen heterocycle molecule, 1-vinylimidazole (VIM), was proposed as the capture ligand of antibodies for the first time. The corresponding VIM-based non-affinity polymeric material (polyVIM) was then fabricated via a one-step polymerization for use in the highly selective purification of antibodies. Compared to the previously reported materials, this novel material exhibited many advantages without clearly sacrificing selectivity, such as a simpler and faster fabrication (within 1.5 h), comparable or even higher binding capacity (saturated static adsorption capacity > 190 mg/g polymer, dynamic binding capacity about 31.62 mg/g polymer), lower non-specific protein adsorption, and much lower cost. Notably, the polyVIM can effectively purify the antibodies from multiple biological sources with high purity (95.4% for mAbs in the cell culture medium, 93.3% for hIgG in the human serum), with an acceptable recovery (91.6% for mAbs, 77.0% for hIgG), and good reusability (> 10 times). Moreover, the target ELISA binding assay and NFAT-luc reporter gene assay demonstrated that the enriched antibodies can well maintain their binding activity and bioactivity during the whole purification process. The excellent performance of the polyVIM material may be attributed to the high recognition ability of VIM for antibodies, as well as the biocompatible and antifouling properties of the porous polymer. This study provides a promising alternative material for the purification of mAbs in downstream processes and the enrichment of hIgG in human serum.

Functional Variation of IL-1R-Associated Kinases in the Conserved MyD88-TRAF6 Pathway during Evolution.

IL-1R-associated kinases (IRAK) are important regulators in the TLR/IL-1R pathways, but their function appears inconsistent between Drosophila, bony fishes, and vertebrates. This causes a difficulty to understand the IRAK functions. As a step to reveal the evolution of IRAKs, in this study, we performed comparative and functional analysis of IRAKs by exploiting the amphioxus, a pivotal taxon connecting invertebrates and vertebrates. Sequence and phylogenetic analysis indicated three major IRAK lineages: IRAK1/2/3 is a vertebrate-specific lineage, IRAK4 is an ancient lineage conserved between invertebrate and vertebrates, and Pelle is another ancient lineage that is preserved in protostomes and invertebrate deuterostomes but lost in vertebrate deuterostomes. Pelle is closer neither to IRAK4 nor to IRAK1/2/3, hence suggesting no clear functional analogs to IRAK1/2/3 in nonvertebrates. Functional analysis showed that both amphioxus IRAK4 and Pelle could suppress NF-κB activation induced by MyD88 and TRAF6, which are unlike mammalian and Drosophila IRAKs, but, surprisingly, similar to bony fish IRAK4. Also unlike Drosophila IRAKs, no interaction was detected between amphioxus IRAK4 and Pelle, although both of them were shown capable of binding MyD88. These findings, together with previous reports, show that unlike other signal transducers in the TLR/IL-1R pathways, such as MyD88 and TRAF6, the functions of IRAKs are highly variable during evolution and very specialized in different major animal taxa. Indeed, we suggest that the functional variability of IRAKs might confer plasticity to the signal transduction of the TLR/IL-1R pathways, which in return helps the species to evolve against the pathogens.

Chordate PIAS proteins act as conserved repressors of the TRAF6 self-polyubiquitination.

In mammals, PIAS proteins are important SUMO E3 ligases and act as versatile regulators of over sixty different proteins, including components from the NF-κB pathways. But the PIAS functions are not well-understood due to complicated molecular mechanisms and multiple gene paralogs with overlapping roles, which is especially true in lower vertebrates where dedicated studies are scarce. As a basal chordate with a single PIAS gene, amphioxus is a convenient model to study PIAS from the evolutionary perspective. TRAF6 is a critical adaptor of the NF-κB pathways but it is not known whether TRAF6 is regulated by PIAS. Here we discover that in mammalian cells, amphioxus PIAS inhibited NF-κB activation by co-localizing and binding with TRAF6. The interaction relied on the N-terminal SAP and PINIT domains of PIAS. TRAF6 is an E3 ubiquitin ligase, which initiates downstream NF-κB signaling by promoting its self-ubiquitination. Both amphioxus SUMO1 and Ubc9 (SUMO E2 ligase) could suppress TRAF6 self-ubiquitination and NF-κB activation, suggesting that the SUMOylation activity competed away the ubiquitination activity of TRAF6. However, we show that the wild-type PIAS and the mutant PIAS without SUMO E3 ligase activity both could inhibit TRAF6-mediated NF-κB activation by reducing TRAF6 self-ubiquitination. This implies that SUMO ligase activity is not the only mechanism for PIAS to negatively regulate TRAF6. Finally, we tested the interactions between human PIAS1-4 and TRAF6. It reveals that human PIAS1, 3 and 4, but not 2, were able to repress NF-κB activation by reducing TRAF6 self-ubiquitination. Taken together, our study discovers a conserved regulatory interaction between chordate PIAS and TRAF6. It therefore sheds light on the complicated role of PIAS in immune regulation, and may help to understand the PIAS functions in other lower chordate taxa, such as jawless and jawed fishes.

The family of amphioxus chitin synthases offers insight into the evolution of chitin formation in chordates.

Chitin is a very important and widely-used biopolymer in fungi and lower metazoans, but mysteriously disappears in mammals. Recent studies reveal that at least lower vertebrates have chitin synthases (CS) and use them to synthesize endogenous chitin. Amphioxus, a basal chordate, therefore becomes critical to understand the evolution of CS, as it occupies the transitional position from invertebrates to vertebrates, and is considered as a good proxy to the chordate ancestor. Here, by exploiting multiple genome assemblies, high-depth RNA-seq data and synteny relations, we identify 11-12 CS genes for each amphioxus species. It represents the largest CS gene pool ever found in eukaryotes so far. As comparison, most metazoans have one or two CSs. Amphioxus is the only chordate that has both the very ancient type-I CS family and the more broadly distributed type-II CS family. Specifically, amphioxus has only one type-II CS but 10-11 type-I CSs, which means that amphioxus is the only metazoan with a greatly expanded type-I CS family. Further analysis suggests that the chordate ancestor have at least one type-II CS and an expanded of type-I CS family. We hypothesize that: these ancient CSs are mostly retained in amphioxus; but the whole type-I CS family was lost in urochordates and vertebrates; the type-II CS was later duplicated into two lineages in vertebrates and followed by stochastic losses, till all type-II CSs were eventually lost in birds and mammals. Finally, our expression profiling and preliminary gene knockout analysis suggest that amphioxus CSs could have highly diverse but mildly overlapping functions in various tissues and organs. Taken together, these findings not only provide insights into the evolution of chordate CSs, lay a foundation for further functional study of the chordate CSs. After all, it is mysterious that our chordate ancestor needed so many isoenzymes for chitin formation.

UPObase: an online database of unspecific peroxygenases.

There are many unspecific peroxygenases (UPOs) or UPO-like extracellular enzymes secreted by fungal species. These enzymes are considered special in their ways of catalyzing a wide variety of reactions such as epoxidation, peroxygenation and electron oxidations. This enzyme family exhibits diverse functions with thousands of UPOs and UPO-like sequences. These sequences are difficult to analyze without proper management tool and therefore desperately calls for a unified platform that can aide with annotation, classification, navigation and easy sequence retrieval. This prompted us to create an online database called Unspecific Peroxygenase Database (UPObase) (upobase.bioinformaticsreview.com) which currently includes 1948 peroxygenase-encoding protein sequences mined from more than 800 available fungal genomes. It provides information such as classification and motifs about each sequence and has functions such as homology search against UPObase sequence analyses such as multiple sequence alignments and phylogenetic trees. It also provides a new sequence submission facility. The database has been made user-friendly facilitating systematic search and filters. UPObase allows users to search for the sequences by organism name, cluster ID and accession number. Notably, in our previous study, 113 UPOs were classified into five subfamilies (I, II, III, IV and V) and an undetermined group (Pog) which remain established. In this study, using 1948 UPOs in our database, we were able to further identify six novel sub-superfamilies (Pog-a, Pog-b, Pog-c, Pog-d, Pog-e and Pog-f) with signature motifs and two distinct groups in Subfamily I and III, Ia and Ib, IIIa and IIIb, respectively. With the novel UPO-like sequences and classification, UPObase may serve for researchers working in the area of enzyme engineering and related fields.

LanceletDB: an integrated genome database for lancelet, comparing domain types and combination in orthologues among lancelet and other species.

Lancelet (amphioxus) represents the most basally divergent extant chordate (cephalochordates) that diverged from the other two chordate lineages (urochordates and vertebrates) more than half a billion years ago. As it occupies a key position in evolution, it is considered as one of the best proxies for understanding the chordate ancestral state. Thus, the construction of a database with multiple lancelet genomes and gene annotation data, including protein domains, is urgently needed to investigate the loss and gain of domains in orthologues among species, especially ancient domain types (non-vertebrate-specific domains) and novel domain combination, which is helpful for providing new insight into the chordate ancestral state and vertebrate evolution. Here, we present an integrated genome database for lancelet, LanceletDB, which provides reference haploid genome sequence and annotation data for lancelet (Branchiostoma belcheri), including gene models and annotation, protein domain types, gene expression pattern in embryogenesis, different expression sequence tag sets and alternative polyadenylation (APA) sites profiled by the sequencing APA sites method. Especially, LanceletDB allows comparison of domain types and combination in orthologues among type species so as to decode the ancient domain types and novel domain combination during evolution. We also integrated the released diploid lancelet genome annotation data (Branchiostoma floridae) to expand LanceletDB and extend its usefulness. These data are available through the search and analysis page, basic local alignment search tool page and genome browser to provide an integrated display.