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.

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.

A novel gene encoding 55 amino-acid residues has been identified from the brooding pouch cDNA library of Hippocampus kuda Bleeker. The deduced amino-acid sequence is highly homologous to several pleurocidin-like peptides from the winter flounder and comprises a signal peptide, a pro-peptide and a mature peptide. The glycine-rich mature peptide, designated HKPLP, contains 24 amino-acid residues and has been synthesized by solid-phase peptide synthesis. The purified HKPLP exhibits antimicrobial activity against several Gram-positive and Gram-negative bacterial strains at low concentrations (MIC 1.5–7.5 μM). Thermal stability assay data show good heat stability. CD spectroscopy experiments indicate that the dominant contents are anti-parallel and parallel sheets, which may have β-sheet or β-strand motif. It is inferred that HKPLP participates in the host defense during egg fertilization and embryo development as an antimicrobial peptide in brooding pouch.

The MyD88-independent pathway, one of the two crucial TLR signaling routes, is thought to be a vertebrate innovation. However, a novel Toll/interleukin-1 receptor (TIR) adaptor, designated bbtTICAM, which was identified in the basal chordate amphioxus, links this pathway to invertebrates. The protein architecture of bbtTICAM is similar to that of vertebrate TICAM1 (TIR-containing adaptor molecule-1, also known as TRIF), while phylogenetic analysis based on the TIR domain indicated that bbtTICAM is the oldest ortholog of vertebrate TICAM1 and TICAM2 (TIR-containing adaptor molecule-2, also known as TRAM). Similar to human TICAM1, bbtTICAM activates NF-κB in a MyD88-independent manner by interacting with receptor interacting protein (RIP) via its RHIM motif. Such activation requires bbtTICAM to form homodimers in endosomes, and it may be negatively regulated by amphioxus SARM (sterile α and armadillo motif-containing protein) and TRAF2. However, bbtTICAM did not induce the production of type I interferon. Thus, our study not only presents the ancestral features of vertebrate TICAM1 and TICAM2, but also reveals the evolutionary origin of the MyD88-independent pathway from basal chordates, which will aid in understanding the development of the vertebrate TLR network.

Three novel Ru(II) complexes of the general formula [Ru(N–N)2(Norharman)2](SO3CF3)2, where N–N = 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), 4,7-diphenyl-1,10-phenanthroline (DIP, 3) and Norharman (9H-pyrido[3,4-b]indole) is a naturally occurring β-carboline alkaloid, have been synthesized and characterized. The molecular structures of 1 and 2 have been determined by X-ray diffraction analysis. The cellular uptake efficiencies, in vitro cytotoxicities and apoptosis-inducing properties of these complexes have been extensively explored. Notably, 1–3 exhibit potent antiproliferative activities against a panel of human cancer cell lines with IC50 values lower than those of cisplatin. Further studies show that 1–3 can cause cell cycle arrest in the G0/G1 phase and induce apoptosis through mitochondrial dysfunction and reactive oxygen species (ROS) generation. In vitro DNA binding studies have also been conducted to provide information about the possible mechanism of action.

Proteins fused to the elastin-like polypeptide (ELP) tag can be selectively separated from crude cell extract without chromatography. To avoid the interference of the ELP tag on properties of the target protein, it is necessary to remove the ELP tag from target protein by protease digestion. Therefore, an additional chromatographic purification step is required to remove the proteases, and this is time- and labor-consuming. Here we demonstrate the utility of the ELP-tagged proteases for cleavage of ELP fusion proteins, allowing one-step removal of the cleaved ELP tag and ELP-tagged proteases without chromatography.

Smad family proteins are identified as intracellular signal mediators of the TGF-β superfamily. In this study, we identified two novel members of the Smad family, termed as AmphiSmad1/5/8 and AmphiSmad4, from Chinese amphioxus. Both AmphiSmad1/5/8 and AmphiSmad4 showed a typical domain structure of Smad proteins consisting of conserved MH1 and MH2 domains. Phylogenetic analysis placed AmphiSmad1/5/8 in the Smad1, 5 and 8 subgroup of the R-Smad subfamily, and AmphiSmad4 in the Co-Smad subfamily. The spatial and temporal gene expression patterns of AmphiSmad1/5/8 and AmphiSmad4 showed that they may be involved in the embryonic development of notochord, myotome and alimentary canal, and may help to establish the specification of dorsal-ventral axis of amphioxus. Moreover, AmphiSmad1/5/8 and AmphiSmad4 showed extensive distribution in all adult tissues examined, suggesting that these two genes may play important roles in the morphogenesis of a variety of tissues especially notochord and gonad.

The role of autophagy in cancer development and response to cancer therapy has been a subject of debate. Here we demonstrate that a series of ruthenium(II) complexes containing a β-carboline alkaloid as ligand can simultaneously induce autophagy and apoptosis in tumor cells. These Ru(II) complexes are nuclear permeable and highly active against a panel of human cancer cell lines, with complex 3 displaying activities greater than those of cisplatin. The antiproliferative potentialities of 1−3 are in accordance with their relative lipophilicities, cell membrane penetration abilities, and in vitro DNA binding affinities. Complexes 1−3 trigger release of reactive oxygen species (ROS) and attenuation of ROS by scavengers reduced the sub-G1 population, suggesting ROS-dependent apoptosis. Inhibition of ROS generation also reduces autophagy, indicating that ROS triggers autophagy. Further studies show that suppression of autophagy using pharmacological inhibitors (3-methyladenine and chloroquine) enhances apoptotic cell death.

Death domain–dependent signaling in the basal chordate amphioxus suggests that extrinsic apoptotic signaling is not restricted to vertebrates.

In this work, a novel M-superfamily conotoxin, designated lt3a, was purified from the crude venom of Conus litteratus. Combined with peptide sequencing, MALDI-TOF mass spectrometry and cDNA cloning techniques, the amino acid sequence of lt3a was supposed to be DγCCγ OQWCDGACDCCS, where O is hydroxyproline and γ is carboxyglutamate. The Cys framework of lt3a (–CC–C–C–CC–) is similar to that of ψ-, μ-, κM-conotoxins, which are representatives of M-conotoxins. Peptide lt3a is categorized into M1 branch based on the number of residues in the last Cys loop. Whole cell patch-clamp study on adult rat dorsal root ganglion neurons indicated that lt3a could enhance tetrodotoxin-sensitive sodium currents. This is a previously unknown function of M-superfamily conotoxins.

•We identified a vermivorous conotoxin lt16a with a cysteine framework XVI.•lt16a was successfully expressed as a fusion protein with TRX in Escherichia coli.•lt16a can inhibit both the TTX-sensitive and TTX-resistant sodium currents.A peptide toxin, lt16a, from the venom of the worm-hunting Conus litteratus, shares the typical signal peptide sequences of M-superfamily conotoxins, which usually contain six cysteine residues that are arranged in a CC-C-C-CC pattern. Interestingly, lt16a comprises 21 amino acid residues in its mature region and has a cysteine framework XVI, which is arranged in a C-C-CC pattern. The coding region of lt16a was cloned into the pTRX vector and the fusion protein was overexpressed in Escherichia coli. After cleaving the fusion protein and purifying the protein lt16a using chromatography, the mass of lt16a was found by mass spectrometry to be consistent with the expected mass of 2357.7 Da. Whole-cell patch clamp experiments demonstrated that lt16a could inhibit both the TTX-sensitive and TTX-resistant sodium currents in adult rat dorsal root ganglion neurons. The inhibition of lt16a on TTX-resistant sodium currents was stronger than on TTX-sensitive sodium currents. To our knowledge, this is the first report of a framework XVI conotoxin that can inhibit voltage-gated sodium channel currents in mammalian sensory neurons. This report helps facilitates an understanding of the sequence diversity of conotoxins.

•We identified a vermivorous conotoxin lt16a with a cysteine framework XVI.•lt16a was successfully expressed as a fusion protein with TRX in Escherichia coli.•lt16a can inhibit both the TTX-sensitive and TTX-resistant sodium currents.A peptide toxin, lt16a, from the venom of the worm-hunting Conus litteratus, shares the typical signal peptide sequences of M-superfamily conotoxins, which usually contain six cysteine residues that are arranged in a CC-C-C-CC pattern. Interestingly, lt16a comprises 21 amino acid residues in its mature region and has a cysteine framework XVI, which is arranged in a C-C-CC pattern. The coding region of lt16a was cloned into the pTRX vector and the fusion protein was overexpressed in Escherichia coli. After cleaving the fusion protein and purifying the protein lt16a using chromatography, the mass of lt16a was found by mass spectrometry to be consistent with the expected mass of 2357.7 Da. Whole-cell patch clamp experiments demonstrated that lt16a could inhibit both the TTX-sensitive and TTX-resistant sodium currents in adult rat dorsal root ganglion neurons. The inhibition of lt16a on TTX-resistant sodium currents was stronger than on TTX-sensitive sodium currents. To our knowledge, this is the first report of a framework XVI conotoxin that can inhibit voltage-gated sodium channel currents in mammalian sensory neurons. This report helps facilitates an understanding of the sequence diversity of conotoxins.

The Drung ethnic minority is one of the smallest ethnic groups of China, geographically isolated by mountains and rivers. Before 1949, Drung society maintained many vestiges of the primitive commune system. The origin and migration of the Drung and their genetic background are still unknown because of limited records about this population. Here, we for the first time demonstrated the unique distribution of HLA alleles in the Drung by high-resolution sequence-based typing (SBT) method. Number of alleles detected is obviously less than expected and only a few alleles with a high homozygosity in each locus are predominant in this minority. The characteristics of HLA allele distribution in the Drung could reflect founder effects, suggesting the Drung probably descended from very few ancestors. The statistical analysis based on allele frequencies indicated that the Drung was an isolated ethnic group, but it also provided the clue that the Drung was genetically related to Chinese southwestern ethnic groups. Significant reduced allelic diversity and genetic isolate in the Drung make it an ideal homogeneous population and very useful model to study the evolution of HLA and the origin and migration of Chinese ethnic groups. The research paved a way to elucidate the genetic background of this mysterious minority and disease predisposition.

•A novel conotoxin cysteine scaffold, framework XV (-C-C-CC-C-C-C-C-), was first identified from Conus litteratus.•By fused with thioredoxin and 6 × His tag, six XV-conotoxins were successfully expressed in E. coli.•Different framework XV conotoxins have distinct biological activities on mice and frogs.•Because of the indel and point mutations occurred in their mature peptides, all the framework XV conotoxins were converged into two branches.•Position-specific codon conservation is not only restricted to cysteines, but also to other conserved residues.The conotoxin cysteine framework XV (-C-C-CC-C-C-C-C-), which was named Lt15a, was firstly identified from the cDNA library of Conus litteratus. After that, 18 new framework XV conotoxin sequences were cloned from nine Conus species. Like other conopeptides, the XV-conotoxins have the conserved signal peptide and propeptide, and there are also some conserved residues in their mature peptide. All the framework XV conotoxins were apparently converged into two branches, because of the indel and point mutations occurred in their mature peptides. By fused with thioredoxin and 6 × His tag, six XV-conotoxins were successfully expressed in Escherichia coli and purified. Different framework XV conotoxins have distinct biological activities on mice and frogs, and that may be related to the diversity of the toxin sequences. All the six XV-conotoxins had no obvious effects on the sodium currents of DRG neuron cells of Sprague–Dawley (SD) rats. The identification of this framework of conotoxins enriches our understanding of the structural and functional diversity of conotoxin.

A big bang expansion of the Vertebrate-type (V-type) TLRs was reported in amphioxus. To shed lights on its implications, a unique TLR which is reversely inserted into an intron of amphioxus PSMB7–10 by retrotransposition in the highly polymorphic proto-MHC region was cloned from Chinese amphioxus (Branchiostoma belcheri tsingtauense) and named as bbtTLR1. In situ assays showed that bbtTLR1 was predominantly expressed in pharynx and gut from larva to adult stages, which are considered as the first frontlines of amphioxus defense system. Acute immune challenges revealed that the expression of bbtTLR1 was stimulated by bacteria and their cell wall components, while suppressed by Glucan and Poly I:C in the digestive system. Amphioxus also had dozens of TIR adaptors from which we cloned bbtMyD88. BbtMyD88 expressed in 293T cells led to the activation of NF-κB pathway through its DEATH and middle domains. Moreover, this activation could be enhanced by bbtTLR1 through the direct association with bbtMyD88. In summary, this study provides evidence for the immune-relation of amphioxus V-type TLRs, and suggests that amphioxus TLR1 and MyD88 represent a basic evolutionary pathway.

Zebrafish has emerged as a valuable model for immunological studies. However, little is known about the overall picture of its immune response to infectious pathogens. Here we present the first systematic study of its immune response to Aeromonas salmonicida and Staphylococcus aureus, a Gram-negative and a Gram-positive bacteria, respectively. Genes induced upon infection were identified with suppression subtractive hybridization, with many of them encoding acute phase proteins (APPs). When compared with mammals, striking similarities and obvious differences have been observed. Both similar APPs (SAA, hepcidin and haptoglobin, etc.) and a similar system for the induction of APPs (which involves the TLRs, pro-inflammatory cytokines and C/EBPs) were identified, implying evolutionary conserved mechanisms among fish and mammals. Some novel APPs were also discovered, suggesting different immune strategies adopted by fish species. Among which, LECT2 was induced by up to 1000-fold upon infection, shedding new lights on the function of this gene. Our results constitute the first demonstration of a similar while different immune response in zebrafish and open new avenues for the investigation of evolutionary conserved and fish specific mechanisms of innate immunity.

•The digestive system is the major defense line of cephalochordate amphioxus.•Amphioxus has lymphocyte-likes cells, macrophage-like cells and monocyte-like cells.•Amphioxus has some basic components of vertebrate adaptive immunity (VAI).•Amphioxus has an extraordinarily complex innate immunity, created by huge expansions of many innate gene families.•Amphioxus has unique features to become an important model for understanding the phylogeny of immunity.As the most basal chordate, the cephalochordate amphioxus has unique features that make it a valuable model for understanding the phylogeny of immunity. Vertebrate adaptive immunity (VAI) mediated by lymphocytes bearing variable receptors has been well-studied in mammals but not observed in invertebrates. However, the identification of lymphocyte-like cells in the gill along with genes related with lymphoid proliferation and differentiation indicates the presence of some basic components of VAI in amphioxus. Without VAI, amphioxus utilizes about 10% of its gene repertoires, and an ongoing domain reshuffling mechanism among these genes, for innate immunity, suggesting extraordinary innate complexity and diversity not observed in other species. Innate diversity may not be comparable to the somatic diversity of the VAI, but there is no doubt of the success of this immune system, since amphioxus has existed for over 500 million years. Studies of amphioxus immunity may provide information on the reduction of innate immune complexity and the conflict between microbiota and host shaped the evolution of adaptive immune systems (AIS) during chordate evolution.

A novel conotoxin lt14a containing 13 amino acid residues with an amidated C-terminus derived from Conus litteratus, belongs to C–C–C–C cysteine pattern. As the smallest peptide of conotoxin framework 14, lt14a could inhibit nicotinic acetylcholine receptor and suppress pain. To elucidate structure–function relationship, we determine the solution structure by NMR and find that lt14a comprises a short duple β-strand region and β-turn motif. An analog [K7A]-lt14a of Ala substitution for Lys in position 7 is designed. Interestingly, [K7A]-lt14a exhibits higher activity than lt14a as long-lasting analgesic in the hotplate pain model in mice. Additionally, MTT assay reveals that the two peptides have low toxicity to human cells. The studies suggest that positively charged residue may not be involved in the blocking mechanism. However, due to the Ala substitution, hydrophobic residues’ patch expansion strengthens the binding ability. A hypothesis is given that in conotoxin lt14a, hydrophobic residues rather than charged residues play a key role during target binding.Research highlights▶ Determined the solution structure of conotoxin lt14a by NMR. ▶ An analog [K7A]-lt14a exhibited higher activity than lt14a as long-lasting analgesic. ▶ Both lt14a and [K7A]-lt14a had low toxicity to human cells. ▶ It is assumed that hydrophobic residues play a key role in binding processing of lt14a.

Two novel tumor necrosis factor receptors, Bbt-TNFR1 and Bbt-TNFR2, were isolated from Chinese amphioxus, the closest relative to vertebrate. The mRNA of Bbt-TNFR1 encoded a type I membrane protein of 452 amino acids, including four cysteine-rich domains in the extracellular region and a putative TRAF6-binding site at its 154aa long cytoplasmic tail. Bbt-TNFR2 was a 304aa long type I membrane protein, featuring three cysteine-rich domains and a short cytoplasmic tail of just 13 amino acids. Southern blot revealed that Bbt-TNFR1 was a single copy gene, while Bbt-TNFR2 was presented in multiple copies. Sequence comparison indicated that both Bbt-TNFR1 and Bbt-TNFR2 were weakly similar to LT-bR, HVEM, TNFR2, CD40, OX40 and DcR3. Real-time PCR showed that Bbt-TNFR1 and Bbt-TNFR2 were regulated during development and finally had high expression in mucosa-rich tissues in adult stage. Furthermore, up-regulated expression of both genes was also observed in guts after Gram-positive bacteria challenge. However, not like Bbt-TNFR2's slowly and gradually augmentation in the following 48 h, expression of Bbt-TNFR1 dramatically surged up within 4 h and then subsided rapidly. Taking together, Bbt-TNFR1 and Bbt-TNFR2 may involve in the host defense of Chinese amphioxus via distinct fashions.

Amphioxus is considered to be the basal chordate. However, the structural and anatomical features of the amphioxus immune system are still elusive. Here we report a profile of structural studies of the amphioxus gill and gut, the first line of defending against microbes, through optical and electron microscopy. The amphioxus gut and gill are characterized by the following morphological criteria compared with vertebrates: primary and secondary lymphoid-like tissue clustered in the gill, a thicker basement membrane with a large villus channel and lack of muscular layer in the gut, along with blood vessels that fill with phagocytes following microbial challenge. The phenomena of tissue repair after microbial invasion was observed, though no phagocytes were observed in the region of tissue necrosis. The epithelium cells of amphioxus gut showed active phagocytosis after the microbial challenge. A small number of free and fixed macrophage-like cells were also found in the amphioxus gut. The current results described the structure of the immune system and cellular defense against infection in a protochordate, which may help us in understanding the structural origin of the vertebrate immune system.

Conotoxins are a diverse array of small peptides mostly with multiple disulfide bridges. These peptides become an increasing significant source of neuro-pharmacological probes and drugs as a result of the high selectivity for ion channels and receptors. Usually, the analogue of natural conotoxins is produced by means of chemical synthesis. Here, we present a simple and fast strategy of producing disulfide-rich conotoxins via recombinant expression. By fused with thioredoxin and His tag, a novel O-superfamily conotoxin lt7a was successfully expressed in Escherichia coli and purified, resulting in a high yield of recombinant lt7a about 6 mg/l. The purity of target protein is up to 95% as identified by HPLC results. Whole cell patch-clamp recording revealed that the new conotoxin blocked voltage-sensitive sodium channels in rat dorsal root ganglion neurons, indicating it might be a novel μO-conotoxin.

Amphioxus is traditionally considered as the living invertebrate most closely related to vertebrate. However, no systematic study was performed about how the amphioxus defends against the microbial invasion. Here we reported a profile of gene transcription after Staphylococcus aureus (S.c) and Vibrio parahaemolyticus (V.p) challenged by suppression subtractive hybridization (SSH). When compared with mammals, amphioxus has the same acute immune defense genes (lectins, metalloproteinase, lysozymes and antimicrobial peptide, etc.) as well as a similar pattern and level of temporal gene expression. In contrast, amphioxus was demonstrated to have some novel acute immune response genes in response to the microbial challenge, such as apextrin and dermatopontin, which have a 3500-fold and 900-fold induction after the V.p infection, respectively, suggesting new functions in early immune system for these two genes. Our results reported for the first time a profile of primitive immune system defense against infection in protochordate.

•Lt14a show analgesic activity in two animal pain model.•Lt14a could suppressed the mRNA expressions of c-fos and nos of rats.•Lt14a could suppress (ERK1/2) phosphorylation aroused by Ach in PC12 cells.•Lt14a did not induce jumping number increasing and body weight loss of mice.Conotoxin lt14a is a small peptide consisting of 13 amino acids. It was originally identified from the cDNA of Conus litteratus in the South China Sea. Previous reports showed lt14a exhibited antinociceptive activity using a hot plate-induced pain mouse model and acted as an antagonist of neuronal nicotinic acetylcholine receptors. We confirmed that conotoxin lt14a administration resulted in antinociception activity using a mouse inflammatory pain model and a rat model of mechanically-induced pain. The mRNA expression of c-fos and NOS in the spinal cord of rats was suppressed by lt14a. Labeling of lt14a with an Alexa Fluor 488 ester showed that lt14a was bound to the surface of PC12 cells and that this binding was inhibited by pre-application of the nicotinic acetylcholine receptor (nAChR) antagonist tubocurarine chloride (TUB) and the nAChR blocker hexamethonium bromide (HB). These data confirm previous reports that showed lt14a binds to the surface of PC12 cells via nAChRs with patch clamp whole-cell recordings. Additional results showed that lt14a suppressed extracellular signal-regulated kinase (ERK1/2) phosphorylation in PC12 cells activated by Ach. Our results showed that lt14a did not induce drug dependence but rather suppressed morphine withdrawal symptoms. Our work suggests that lt14a is a novel antinociceptive agent that targets the nAChR receptor without inducing drug dependence.

•Identical M-superfamily conotoxins can be found in different Conus species.•Ten of the 18 cysteine frameworks may be generated from framework III.•Position-specific codon conservation is not restricted to cysteines, but also to other conserved residues.•M-superfamily conotoxins may not evolve in a concerted manner but were subject to birth-and-death evolution.•Indels are involved in the creation of new functions and structures of the M-superfamily conotoxins.Conotoxins from cone snails are valuable in physiology research and therapeutic applications. Evolutionary mechanisms of conotoxins have been investigated in several superfamilies, but there is no phylogenetic analysis on M-superfamily conotoxins. In this study, we characterized identical sequences, gene structure, novel cysteine frameworks, functions and evolutionary mechanisms of M-superfamily conotoxins. Identical M-superfamily conotoxins can be found in different Conus species from the analysis of novel 467 M-superfamily conotoxin sequences and other published M-superfamily conotoxins sequences. M-superfamily conotoxin genes consist of two introns and three exons from the results of genome walking. Eighteen cysteine frameworks were identified from the M-superfamily conotoxins, and 10 of the 18 may be generated from framework III. An analysis between diet types and phylogeny of the M-superfamily conotoxins indicate that M-superfamily conotoxins might not evolve in a concerted manner but were subject to birth-and-death evolution. Codon usage analysis shows that position-specific codon conservation is not restricted to cysteines, but also to other conserved residues. By analysing primary structures and physiological functions of M-superfamily conotoxins, we proposed a hypothesis that insertions and deletions, especially insertions in the third cysteine loop, are involved in the creation of new functions and structures of the M-superfamily conotoxins.

Three novel Ru(II) complexes of the general formula [Ru(N–N)2(Norharman)2](SO3CF3)2, where N–N = 2,2′-bipyridine (bpy, 1), 1,10-phenanthroline (phen, 2), 4,7-diphenyl-1,10-phenanthroline (DIP, 3) and Norharman (9H-pyrido[3,4-b]indole) is a naturally occurring β-carboline alkaloid, have been synthesized and characterized. The molecular structures of 1 and 2 have been determined by X-ray diffraction analysis. The cellular uptake efficiencies, in vitro cytotoxicities and apoptosis-inducing properties of these complexes have been extensively explored. Notably, 1–3 exhibit potent antiproliferative activities against a panel of human cancer cell lines with IC50 values lower than those of cisplatin. Further studies show that 1–3 can cause cell cycle arrest in the G0/G1 phase and induce apoptosis through mitochondrial dysfunction and reactive oxygen species (ROS) generation. In vitro DNA binding studies have also been conducted to provide information about the possible mechanism of action.