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Lampreys are blood-sucking vampires in marine environments. From a survival perspective, it is expected that the lamprey buccal gland exhibits a repository of pharmacologically active components to modulate the host's homeostasis, inflammatory and immune responses. By analyzing the metabolic profiles of 14 different lamprey tissues, we show that two groups of metabolites in the buccal gland of lampreys, prostaglandins and the kynurenine pathway metabolites, can be injected into the host fish to assist lamprey blood feeding. Prostaglandins are well-known blood-sucking-associated metabolites that act as vasodilators and anticoagulants to maintain vascular homeostasis and are involved in inflammatory responses. The vasomotor reactivity test on catfish aortic ring showed that kynurenine can also relax the blood vessels of the host fish, thus improving the blood flow of the host fish at the bite site. Finally, a lamprey spatial metabolomics database (https://www.lampreydb.com) was constructed to assist studies using lampreys as animal model. A spatial metabolomics study of the lampreys leads to the identification of the kynurenine pathway metabolites and prostaglandins in their buccal gland secretions (saliva), which are likely involved in promoting blood flow when feeding on host fishes. [ABSTRACT FROM AUTHOR]

As a critical evolutionary pivot between invertebrates and vertebrates, lampreys provide rich genetic information. Lamprey immune protein (LIP) is a key immune regulator. MicroRNAs, well-conserved in the response to immunological stress, remain understudied in lamprey immunity. We generated a lamprey microRNA expression atlas, using deep sequencing, upon Vibrio anguillarum infection. Using comparative methods, we found that miR-4561 potentially regulates innate immunity via interaction with lip. We found a sequence in the 3′-UTR region of LIP mRNA complementary to the miR-4561 seed region; miR-4561 expression was negatively correlated with LIP. During V. anguillarum infection, miR-4561 inhibited LIP expression and bacterial clearance. Notably, LIP expression in supraneural body cells was necessary for the Gram-negative immune response. Additionally, we observed that overexpression of miR-4561 induced apoptosis in embryonic cells, suggesting a role in embryonic development. Collectively, we show lamprey microRNAs may significantly affect gene regulation and provide new insights on LIP-mediated immune regulation. Ma et al characterise differentially expressed miRNAs in Lamprey upon bacterial infection. They identify miR-4561 as being decreased after infection and show that it targets the 3′UTR of the mRNA encoding lamprey immune protein (LIP) involved in lamprey innate immunity and regulates survival in lamprey embryos. [ABSTRACT FROM AUTHOR]

Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are well-known viral RNA sensors in the cytoplasm. RIG-I-mediated antiviral signals are activated by interacting with the adapter protein mitochondrial antiviral signaling (MAVS), which triggers interferon (IFN) responses via a signaling cascade. Although the complete RIG-I receptor signaling pathway has been traced back to teleosts, definitive evidence of its presence in lampreys is lacking. Here, we identified 13 pivotal molecules in the RIG-I signaling pathway in lamprey, and demonstrated that the original RIG-I/MAVS signaling pathway was activated and mediated the expression of unique immunity factors such as RRP4, to inhibit viral proliferation after viral infection in vivo and in vitro. This study confirmed the conservation of the RIG-I pathway, and the uniqueness of the RRP4 effector molecule in lamprey, and further clarified the evolutionary process of the RIG-I antiviral signaling pathway, providing evidence on the origins of innate antiviral immunity in vertebrates. • Identification of 13 pivotal molecules and demonstrated the original RIG-I/MAVS signaling pathway was activated in lamprey. • High expression of RRP4 to inhibit viral proliferation after viral infection in vivo and in vitro. • Constructed a putative model of RIG-I mediated antiviral pathways in lampreys. [ABSTRACT FROM AUTHOR]

• This study used transcriptome sequencing technology to successfully obtain transcriptome data of Vibro splendidus , Aeromonas hydrophila and Saprolegnia parasitica infected lamprey larvae. • In this study, we found differentially expressed genes caused by lamprey larvae infected with three pathogens, and the signaling pathways were queried by GO and KEGG enrichment. • This study provides useful information for further understanding mechanisms of antibacterial immunity in lamprey larvae and the artificial propagation of lamprey. Based on the large number of deaths of lamprey larvae infected by pathogens in the culture process, the infection response of lamprey larvae to different pathogens was studied. Thus, high-throughput sequencing is firstly used to analyze the lamprey larvae infected by Aeromonas hydrophila , Vibrosplendidus , and Saprolegnia parasitica. A high quality of 338,628,426 Reads were obtained and 203,829 transcripts were assembled. A total of 158,001 Unigenes were annotated by Blast comparison, and a relatively comprehensive transcriptome database of lamprey larvae was established. Based on the transcriptome of lamprey larvae infected by three aquatic pathogens, classification results of COG and GO show that the genes classified into signal transduction mechanism accounted for the largest proportion, and 2316 differentially expressed genes were identified and screened out. After infection by the three pathogens, 16 genes and 19 genes expression were up-regulated and down-regulated, respectively. KEGG pathway analysis of differentially expressed genes showed that lamprey larvae were mainly affected their metabolism and oxidative phosphorylation pathways after infection by two gram-negative bacteria, Vibrosplendidus and Aeromonas hydrophila. As a fungus, Saprolegnia parasitica mainly affected ribosome synthesis in lamprey larvae. The results were validated by detecting the expression levels of 13 DEGs at 24 h after pathogens treatment using Q-PCR. The results provide valuable information for further research into the development of immunity and the innate immune response against pathogen invasion during the artificial propagation of lamprey larvae. [ABSTRACT FROM AUTHOR]

Serine protease inhibitors (serpins) are a large protein family that is involved in various physiological processes and is known to regulate innate immunity pathways. However, research for the functional study of serpins in lamprey is limited. In the present study, a serpin gene was cloned and characterized from Lampetra japonica at molecular, protein and cellular levels, named L-serpin which belongs to family F serine protease inhibitors (serpin family). The L-serpin includes a serpin domain in the N-terminus. The mRNA transcript of L-serpin was extensively expressed in kidney, supraneural body, intestine, liver, heart, gill and the highest expression in leukocytes. The mRNA expression level of L-serpin increased significantly after Vibrio anguillarum, Staphylocccus aureus and Poly I:C stimulation and dramatically peak at 8 h. It is demonstrated that the L-serpin protected cells from lethal Gram-negative endotoxemia through associating with inhibition of lipopolysaccharide (LPS)-triggered cell death and inflammatory factors expression. Surface plasmon resonance (SPR) and the microbe binding assay were used to determine that L-serpin interacts directly with LPS (KD = 6.14 × 10−7 M). Furthermore, we confirmed L-serpin is a major inhibitor of complement activation by inactivating lamprey-C1q protein (KD = 2.06 × 10−6 M). Taken together, these findings suggest that L-serpin is a endogenous anti-inflammatory factor to defend against Gram-negative bacterial challenge and involved in lamprey innate immunity. • A serpin gene named L-serpin belongs to clade F and group V4 serpin family was cloned and characterized from lamprey. • Bacterial multi-stimulation induces L-serpin expression in leukocytes and serum. • L-serpin protects cells against LPS-mediated cell death and displays LPS binding activities. • L-serpin inhibits the complement-dependent cytotoxicity of lamprey serum via binding to L-C1qDC-1. [ABSTRACT FROM AUTHOR]

Two endo-functionalized organic cages as oxyanion hole mimics were achieved via dynamic covalent chemistry, which exhibit good size selectivity, catalytic activity and broad substrate scopes for Friedel–Crafts reactions. The modular character of the synthesis provides an easy way to modify the size, shape and inner function of the molecular cavity. [ABSTRACT FROM AUTHOR]

We have identified several important features in the neutron scattering data of cuprates that are difficult lo explain in terms of d-wave and isotropic s-wave order parameters. Alternatively, we show that the neutron data are in quantitative agreement with an order parameter that has an extended s-wave (AIg) symmetry Lind opposite sign in the bonding and antibonding electron bands formed within the Cu2O4 bilayers. The extended s-wave has eight line nodes and changes sign when a node is crossed. This AIg pairing symmetry may be compatible with a charge-fluctuation-mediated pairing mechanism. [ABSTRACT FROM AUTHOR]

The mammalian testis and ovary possess special immunocompetence, which is central to provide protection against pathogens. However, the innate immune responses to immune challenges in lamprey gonads are poorly understood. In this study, we extracted RNA from testis and ovary tissues of lampreys at 0 hour, 8 hours and 17 days after lipopolysaccharides (LPS) stimulation and performed transcriptome sequencing. While the transcriptome profiles of the two tissues were different for the most part, genes LIP, LECT2, LAL2, GRN, ITLN, and C1q were found to be the most significantly up-regulated genes in both. Quantitative Real-time PCR (qRT-PCR) analysis confirmed that these genes were upregulated after stimulation. Furthermore, immunohistochemical staining showed that these genes in lamprey gonads are expressed in high quantities and have a specific distribution. Taken together, our results suggest that these genes could play an essential role in response of the gonads to LPS induction. This research establishes a basis for investigating the immune mechanism of vertebrate gonads and presents a fresh concept for gaining insight into the evolutionary development of jawless vertebrates. [ABSTRACT FROM AUTHOR]

Background: In previous research, we found that lamprey immune protein (LIP) possessed cytocidal activity against tumor cells, but the mechanism of the selective recognition and killing of tumor cells by LIP was not identified. Methods: Superresolution microscopy, crystallographic structural analysis, glycan chip assay, SPR experiments, FACS assays, computational studies and mass spectrometric analysis firmly establish the mode of action of LIP, which involves dual selective recognition and efficient binding. Results: We determined the overall crystallographic structure of LIP at a resolution of 2.25 Å. LIP exhibits an elongated structure with dimensions of 105 Å × 30 Å × 30 Å containing an N-terminal lectin module and a C-terminal aerolysin module. Moreover, the Phe209-Gly232 region is predicted to insert into the lipid bilayer to form a transmembrane β-barrel, in which the hydrophobic residues face the lipid bilayer, and the polar residues constitute the hydrophilic lumen of the pore. We found that LIP is able to kill various human cancer cells with minimal effects on normal cells. Notably, by coupling biochemical and computational studies, we propose a hypothetical mechanism that involves dual selective recognition and efficient binding dependent on both N-linked glycans on GPI-anchored proteins (GPI-APs) and sphingomyelin (SM) in lipid rafts. Furthermore, specific binding of the lectin module with biantennary bisialylated nonfucosylated N-glycan or sialyl Lewis X-containing glycan structures on GPI-APs triggers substantial conformational changes in the aerolysin module, which interacts with SM, ultimately resulting in the formation of a membrane-bound oligomer in lipid rafts. Conclusions: LIP holds great potential for the application of a marine protein towards targeted cancer therapy and early diagnosis in humans. [ABSTRACT FROM AUTHOR]

Motivation Robust and reproducible data is essential to ensure high-quality analytical results and is particularly important for large-scale metabolomics studies where detector sensitivity drifts, retention time and mass accuracy shifts frequently occur. Therefore, raw data need to be inspected before data processing to detect measurement bias and verify system consistency. Results Here, we present RawHummus, an R Shiny app for an automated raw data quality control (QC) in metabolomics studies. It produces a comprehensive QC report, which contains interactive plots and tables, summary statistics and detailed explanations. The versatility and limitations of RawHummus are tested with 13 metabolomics/lipidomics datasets and 1 proteomics dataset obtained from 5 different liquid chromatography mass spectrometry platforms. Availability and implementation RawHummus is released on CRAN repository (https://cran.r-project.org/web/packages/RawHummus), with source code being available on GitHub (https://github.com/YonghuiDong/RawHummus). The web application can be executed locally from the R console using the command 'runGui()'. Alternatively, it can be freely accessed at https://bcdd.shinyapps.io/RawHummus/. Supplementary information Supplementary data are available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

Background: Previous studies have shown that lamprey buccal glands contain some regulators related to anticoagulation, nociception, and immune responses due to the blood sucking habit. Regrettably, the protein expression profile in the buccal glands of feeding lampreys has never been reported yet. The present study was performed in order to further identify more proteins which are closely associated with lamprey feeding process. Methods: 2D-PAGE, NanoLC–MS/MS with higher resolution, Ensembl lamprey and NCBI protein databases, as well as western blot was used to compare the proteomics of buccal gland secretion from China northeast lampreys (Lampetra morii) which had been fed for 0, 10, and 60 min, respectively. Results: In the present study, the number of identified protein species in the buccal glands of feeding groups (60 min) was increased significantly, nearly ten times of that in the fasting group. During the feeding stage, novel proteins emerged in the buccal gland secretion of lampreys. According to gene ontology (GO) analysis and function predictions, these proteins were summarized and discussed based on their potential roles during feeding process. Furthermore, some of the identified proteins were confirmed to express during the feeding time of lampreys. Conclusion: When lampreys attack host fishes to suck blood and flesh, their buccal glands could secrete enough proteins to suppress blood coagulation, nociception, oxidative stress, immune response, as well as other adverse effects encountered during their parasitic lives. The present study would provide clues to clarify the feeding mechanism of the bloodsucking lampreys. [ABSTRACT FROM AUTHOR]

Cold-inducible RNA-binding protein (CIRBP) responds to a wide array of cellular stresses such as cold shock, hypoxia, and inflammatory responses. However, functional studies of CIRBP in jawless vertebrates are limited. In this study, a CIRBP homolog from the jawless vertebrate lamprey (Lethenteron reissneri) was cloned and characterized (named Lr-CIRBP). The cDNA fragment of Lr-CIRBP has a 516 bp open reading frame (ORF) that encodes 171 amino acids, comprising a glycine-rich region at the C-terminal, similar to higher vertebrates but slightly shorter, and an RNA recognition motif (RRM) domain at the N-terminus. The predicted Lr-CIRBP sequence had 51.4 ~ 70.6% similarity with CIRBPs from other vertebrates. Further phylogenetic analysis revealed that Lr-CIRBP is located in the outgroup of vertebrates and is the ancestor of vertebrates. Based on real-time quantitative PCR experimental analysis, Lr-CIRBP expression was highest in leukocytes and increased significantly after multi-stimulation, peaking at 12 h. RNA interference showed that Lr-CIRBP knockdown can down-regulate the expression of inflammatory factors in Lethenteron reissneri. In conclusion, our study successfully clarifies the ancestral features and functions of CIRBP, while revealing valuable insight into how the protein is involved in the immune responses of a jawless vertebrate. [ABSTRACT FROM AUTHOR]

Simple Summary: Takifugu obscurus is an economically important fish because of its fast growth rate, tender meat, and high nutritional value. The rapid development of the aquaculture industry in recent years has led to the increasing persistence of hypoxia in aquaculture water systems. Dissolved oxygen is an important factor for fish survival, and low oxygen stress can cause slow growth and reduced immunity of fish. Gills are the main respiratory organs of fish and are greatly affected by changes in dissolved oxygen levels. In this study, the transcriptomic responses of T. obscurus gill tissues were compared under acute hypoxic stress relative to normoxic and reoxygenated conditions to identify differentially expressed genes associated with hypoxia and understand the adaptive changes of these genes in fish responses to hypoxia. This study provides new insights into the molecular mechanisms underlying the responses of T. obscurus to hypoxic stress. Takifugu obscurus has relatively small gills and gill pores, leading to a relatively low respiratory capacity and increased vulnerability to low dissolved oxygen (DO) levels compared to other fish. To investigate the responses of T. obscurus to acute hypoxic stress, high-throughput-sequencing-based transcriptomic analyses were conducted here to assess the responses of T. obscurus gills to acute hypoxic stress. Three environmental conditions were compared including normoxia (DO: 7.0 ± 0.2 mg/L), hypoxic stress (DO: 0.9 ± 0.2 mg/L), and reoxygenation (4, 8, 12, and 24 h after return to normoxia) conditions to identify differentially expressed genes (DEGs) responsive to hypoxia. A total of 992, 877, 1561, 1412, and 679 DEGs were identified in the normoxia and reoxygenation for 4, 8, 12, and 24 h groups in comparison to the hypoxia groups, respectively. The DEGs were primarily associated with oxidative stress, growth and development, and immune responses. Further functional annotation enrichment analysis of the DEGs revealed that they were primarily related to cytokine–cytokine interactions, transforming growth factor β receptor (TGF-β), cell adhesion molecules (CAMs), the vascular endothelial growth factor (VEGF) signaling pathway, and the mitogen-activated protein kinase (MAPK) signaling pathway. These results provide new insights into the physiological and biochemical mechanisms of T. obscurus adaptations to hypoxic stress. Furthermore, these results provide a framework for future studies into the molecular mechanisms of hypoxia tolerance and the healthy culture of T. obscurus and other fish. [ABSTRACT FROM AUTHOR]

In this study, metagenomics was used to analyze microbial succession and predict changes in raw milk during 6 days of storage at 4°C, aimed at determining how microorganisms drive the deterioration of refrigerated raw milk. The microbial community in raw milk changed significantly with an extension of refrigeration time (p <.01). The dominant bacterial genera gradually evolved from Acinetobacter, Streptococcus, and Anaplasma to Flavobacterium, Pseudomonas, and Lactococcus. KEGG annotation results indicated that the main role of the bacterial community included amino acid biosynthesis, carbon metabolism, and functioning as an ABC transporter. Additionally, lipid, amino acid, and carbohydrate metabolism pathways were significantly expressed at the beginning, middle, and end of refrigeration, respectively. Refrigeration time is an important factor affecting the composition of the microbial flora in raw milk. The results of this study illustrate the role of microorganisms in the deterioration of refrigerated raw milk. [ABSTRACT FROM AUTHOR]

The clinical management of bladder cancer (BCa) is hindered by the lack of reliable biomarkers. We aimed to investigate the potential of lamprey immunity protein (LIP), a lectin that specifically binds to multi-antennary sialylated N -glycolylneuraminic acid (Neu5Gc) structures on UMOD glycoproteins in the urine of BCa patients. Primary BCa patients had higher levels of LIP-bound Neu5Gc in urine than healthy participants and patients receiving postoperative treatment did. In addition, lectin chip assay and mass spectrometry were used to analyze the glycan chain structure, which can recognize the UMOD glycoprotein decorated with multi-antennary sialylated Neu5Gc structures. Furthermore, compared with urine samples from healthy patients (N = 2821, T/C = 0.12 ± 0.09) or benign patients (N = 360, T/C = 0.11 ± 0.08), the range of the urine T/C ratio detected using LIP test paper was 1.97 ± 0.32 in patients with bladder cancer (N = 518) with significant difference (P < 0.0001). Our results indicate that LIP may be a tool for early BCa identification, diagnosis, and monitoring. Neu5Gc-modified UMOD glycoproteins in urine and Neu5Gc-modified N -glycochains and sialyltransferases may function as potential markers in clinical trials. • To establish a new ELISA method for LIP protein for diagnosis of bladder cancer • Identify LIP protein to identify sialic acid modified sugar chain in urine • Based on the principle of TRFIA, the LIP protein strip kit is established [ABSTRACT FROM AUTHOR]

Human B cell adaptor for phosphoinositide 3-kinase (BCAP) is identified as an adaptor protein expressed in B cells and plays a critical immunomodulatory role in B cell receptor signaling and humoral immune response. In the current study, a homolog of BCAP (Lja-BCAP) was identified in Lampetra japonica. The open reading frame of Lja-BCAP contains 2181bp nucleotides and encodes a protein of 726 amino acids. After being stimulated by mixed bacteria, the mRNA and protein expression levels of Lja-BCAP and the activation levels of tyrosine kinases increased significantly in peripheral blood lymphocytes, gills and supraneural myeloid bodies, respectively. However, after the knockdown of Lja-BCAP by RNAi in vivo, the activation of tyrosine kinases was inhibited in the above tissues, which indicated that Lja-BCAP participated in the anti-bacterial immune response of lampreys. After lipopolysaccharide (LPS) stimulation, the expression of Lja-BCAP in peripheral blood lymphocytes, gills and supraneural myeloid bodies were significantly up-regulated 2.5, 2.2, and 11.1 times (p < 0.05) compared to the control group, respectively; while after phytohemagglutinin (PHA) stimulation, the up-regulation of Lja-BCAP was only detected in peripheral blood lymphocytes. The above results show that Lja-BCAP mainly participates in the LPS-mediated immune response of lampreys. [ABSTRACT FROM AUTHOR]

Liver-expressed antimicrobial peptide 2 (LEAP2) is a member of the antimicrobial peptides family and plays a key role in the innate immune system of organisms. LEAP2 orthologs have been identified from a variety of fish species, however, its function in primitive vertebrates has not been clarified. In this study, we cloned and identified Lc-LEAP2 from the primitive jawless vertebrate lamprey (Lethenteron camtschaticum) which includes a 25 amino acids signal peptide and a mature peptide of 47 amino acids. Although sequence similarity was low compared to other species, the mature Lc-LEAP2 possesses four conserved cysteine residues, forming a core structure with two disulfide bonds between the cysteine residues in the relative 1–3 (Cys 58 and Cys 69) and 2–4 (Cys 64 and Cys 74) positions. Lc-LEAP2 was most abundantly expressed in the muscle, supraneural body and buccal gland of lamprey, and was significantly upregulated during LPS and Poly I:C stimulations. The mature peptide was synthesized and characterized for its antibacterial activity against different bacteria. Lc-LEAP2 possessed inhibition of a wide range of bacteria with a dose-dependence, disrupting the integrity of bacterial cell membranes and binding to bacterial genomic DNA, although its inhibitory function is weak compared to that of higher vertebrates. These data suggest that Lc-LEAP2 plays an important role in the innate immunity of lamprey and is of great value in improving resistance to pathogens. In addition, the antimicrobial mechanism of LEAP2 has been highly conserved since its emergence in primitive vertebrates. • Lc-LEAP2 was cloned from the Lethenteron camtschaticum and was significantly overexpressed after multi-stimulation. • Lc-LEAP2 was abundantly expressed in the muscle, supraneural body and buccal gland. • Lc-LEAP2 possessed inhibition of bacteria with a dose dependence while its inhibitory function was weak. • Ancestral characteristics and functions of the LEAP2 were clarified. [ABSTRACT FROM AUTHOR]

• Four RHR genes identified and cloned from Lethenteron reissneri. • The evolutionary relationship of NFAT and RBPJ genes among different species is indicated. • RHR gene family plays a significant role in the immune defense of lamprey. • The study provides a new understanding for origin and evolution of the RHR gene family. Nuclear factor of activated T cells (NFAT) and recombination signal binding protein (RBP) belong to the family of Rel homology region (RHR) transcription factors which regulate the expression of genes involved in different aspects of the immune response. To gain insights into the evolution and characterisation of RHR genes in lampreys, a jawless vertebrate, four RHR genes, including nuclear factor of activated T cells (NFAT) and recombination signal binding protein for immunoglobulin kappa J region (RBPJ), have been identified and cloned from the lamprey (Lethenteron reissneri) database. Evolutionary relationships of NFAT and RBPJ genes among different species were determined through molecular phylogenetic analysis. Motif, genetic structure, and tertiary structure analyses showed that NFATs and RBPJ are conserved and contain RHD and IPT domains. Moreover, synteny analysis showed that the neighbourhood genes of Lr-NFATs and Lr-RBPJ have undergone significant changes compared to jawed vertebrates. Real-time quantitative results demonstrated that the RHR gene family plays a significant role in immune defence. This study provides a new understanding of the origin and evolution of the RHR gene family in different species. [ABSTRACT FROM AUTHOR]

The insecticide dimethoate, an organophosphate, has been used on crops, soybeans, fruits, and vegetables since the 1960s and is considered one of the most widely used pesticides. However, the understanding of the molecular mechanisms of dimethoate in crops, especially crop seedlings, is still limited. The green vegetable soya bean (Glycine max merr) is usually used as a vegetable-like fruit of soybean in many Asian countries. This study aimed to analyze the effect of dimethoate on the growth of green vegetable soya bean seedlings at the metabolic and transcriptional levels. An integrated analysis of the transcriptome and metabolome was performed to determine the responses of green vegetable soya bean seedlings to different concentrations (D1 for low dose, D2 for high dose and C for control) of dimethoate. In omics analyses, 4156 differentially expressed genes (DEGs) and 1935 differentially abundant metabolites (DAMs) were identified in the D1/C comparison, and 11,162 DEGs and 819 DAMs were identified in D2/C. Correlation analyses revealed dimethoate affected the metabolic pathways of green vegetable soya beans such as the biosynthesis of secondary metabolites and microbial metabolism in diverse environmental pathways, demonstrating that even small doses of dimethoate can affect green vegetable soya bean seedlings in a short period of time. Our study further enriches our understanding of the molecular mechanisms by which green vegetable soya beans are treated with dimethoate and provides a deeper understanding of the effects of dimethoate on crops. [ABSTRACT FROM AUTHOR]

Information on the prostaglandin pathway in lampreys is limited. Here, five genes related to the prostaglandin pathway from synthesis to inactivation, namely, phospholipase A 2 , cyclooxygenase-2 , prostaglandin E synthase 3 , prostaglandin D synthase , and 15-hydroxyprostaglandin dehydrogenase [NAD(+)] , were screened and cloned from the lamprey, Lethenteron camtschaticum. Bioinformatic analysis showed that these lamprey genes are relatively conserved with teleost genes in domains, motifs, gene structure and 3D structure. Analysis of expression distribution of the genes in lamprey tissues revealed that a complete prostaglandin pathway from synthesis to inactivation exists in the oral gland of lamprey, especially the key gene of prostaglandin synthesis cyclooxygenase-2 , which was highly expressed in the oral gland. Furthermore, cyclooxygenase-2 expression increased after LPS and Poly I:C stimulations. Using our established spatial metabolite database LampreyDB, six prostaglandin-related metabolites were screened from the oral gland of lamprey, four of which were highly expressed in the oral gland. This study provides new insights into prostaglandin synthesis and inactivation pathways in lamprey, thereby improving our understanding of the origin and evolution of the prostaglandin pathway and contributing to the recognition of lamprey regulatory mechanisms in development and immunity. • Prostaglandins play a crucial function in aquatic animal immunity. • A complete prostaglandin pathway is conserved in lamprey (Lethenteron camtschaticum). • Lc-COX-2 was highly expressed in the oral gland of Lethenteron camtschaticum , and responded to immune stimulation. [ABSTRACT FROM AUTHOR]

The complement component 3 of the lamprey, a jawless vertebrate, functions as an opsonin during the phagocytosis of rabbit red cells. Furthermore, lamprey C3 may be activated and cleaved into C3b, which is attached to the surface of target cells in the cytolytic process. However, the mechanism mediating the biological function of C3 in the lamprey is unknown. To our knowledge, this study is the first to show that variable lymphocyte receptors (VLRs) expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. We identified the C3 gene in the lamprey genome based on its orthologs, conserved synteny, functional domains, phylogenetic tree, and conserved motifs. Additionally, we determined the optimal infection concentration of Aeromonas hydrophila to perform immune stimulation experiments in the lamprey larvae. The quantitative real-time polymerase chain reaction and immunofluorescence analyses revealed that the expression of Lampetra morii C3 (lmC3) was significantly upregulated in the larvae infected with 107 CFU/mL of A. hydrophila. The lmC3 morphants (lmC3 MO) of lamprey larvae were generated by morpholino-mediated knockdown. The lmC3 MO larvae were highly susceptible to A. hydrophila infection, which indicated that lmC3 is critical in lamprey immune response. The expression of a selected panel of orthologous genes was comparatively analyzed in the infected wild type, infected lmC3 MO, infected control MO, uninfected wild type and uninfected lmC3 MO one-month-old ammocoete larvae. The knockdown of lmC3 strongly affected the expression of VLRA+/VLRB+/VLRC+-associated genes, which was also confirmed by immunohistochemical analysis. Thus, VLR expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. • We conducted an analysis of multiple C3 gene family in vertebrates. • We report that A. hydrophila has been characterized as a rapidly lethal pathogen for L.morii. • VLRs expression were significantly affected by complement C3 knockdown morphants in Lampetra morii. [ABSTRACT FROM AUTHOR]

In jawed vertebrates, B cell receptors (BCR) are primary pathogen detectors that activate downstream signaling pathways to express adaptive immune effectors. In jawless vertebrates, the variable lymphocyte receptors (VLR) B positive lymphocytes can express and secrete specific VLRB molecules in an analogous manner to that of immunoglobulins by B cells in jawed vertebrates. Our study is the first to demonstrate the possibility of incubation of fertilized eggs and artificial breeding of Lampetra morii larvae throughout their life cycle under laboratory condition. We also found that VLRB, lamprey B-cell linker (L-BLNK), and lamprey nuclear factor-kappa B (L-NF-κB) play key roles in early larval development. Aeromonas hydrophila was found to be a lethal pathogen of L. morii larvae causing rapid infection at a concentration of 107 cfu/mL qRT-PCR results revealed that gene expression levels of VLRB, L-BLNK, and L-NF-κB were up-regulated significantly. Ten-day infection trials showed that VLRB, L-BLNK, and L-NF-κB are crucial for lamprey immune response. Furthermore, the expression levels of L-BLNK and L-NF-κB were down-regulated drastically both at mRNA and protein levels after bacterial infection than in the naive group of VLRB morphants. A similar expression pattern of VLRB and L-BLNK was found in L-NF-κB morphants post bacterial infection. The results were strikingly different in the other two morphants. The VLRB and L-NF-κB expression levels were found to be down-regulated at mRNA and protein levels by less than 30% and 45%, respectively, in L-BLNK morphants compared to those in the naive group. These results indicate that L-BLNK and L-NF-κB might participate in VLRB-mediated immune response. Additionally, in VLRB morphants, the mRNA expression levels of some genes, especially the ones expressed in VLRB+ lymphocytes but not in VLRA+ lymphocytes, were found to be affected. Therefore, these findings of B-like lymphocytes in lamprey offer key evidence with regard to the evolution of adaptive immunity. • We report that L. morii has been cultured in laboratory through the complete life cycle, and VLRB, L-BLNK and L-NF-κB have been proven to be critical for its early development. • We provide evidences that L-BLNK and L-NF-κB seem to be key signaling molecules in VLRB signaling pathway in immune responses of VLRB+ lymphocytes. • These findings of B-like lymphocytes in lampreys offers key evidences into the evolution of adaptive immunity. [ABSTRACT FROM AUTHOR]

The tryptophan–kynurenine (TRP–KYN) pathway is involved in several biological functions, including immunosuppression, inflammatory response, and tumor suppression. Six TRP–KYN pathway-related genes, tryptophan 2,3-dioxygenase (TDO), indoleamine 2,3-dioxygenase 2 (IDO2), aminoadipate aminotransferase (AADAT), glutamate oxaloacetate transaminase 2 (GOT2), kynurenine monooxygenase (KMO), and kynureninase (KYNU) have been identified and cloned from the jawless vertebrate lamprey (Lampetra japonica) to gain insights into their evolution and characterization. Expression distribution showed that the key gene Lj-TDO was highly expressed in the oral gland. Real-time quantitative PCR showed that TRP–KYN pathway-related genes were significantly overexpressed after multi-stimulation. RNA interference showed that Lj-IDO2 knockdown regulated the expression of inflammatory factors. In conclusion, our study successfully clarified the ancestral features and functions of the TRP–KYN pathway, while providing valuable insights into the involvement of this pathway in the immune responses of a jawless vertebrate. • Six TRP-KYN pathway-related genes cloned from Lampetra japonica were significantly overexpressed after multiple stimuli. • Lj-TDO was highly expressed in the oral gland of L. japonica. • RNA interference showed that Lj-IDO2 knockdown regulated the expression of inflammatory factors. • Ancestral characteristics and functions of the TRP-KYN pathway were clarified. [ABSTRACT FROM AUTHOR]

Peperomin E is a natural secolignan existing distributed in the plants of the genus Peperomia. Previous investigations demonstrated that peperomin E showed potential antitumor activity in some cancer lines, but it is unclear whether peperomin E has an effect on prostate cancer cell lines. The aim of the present study is to investigate its effects on proliferation inhibition, apoptosis-inducing and cell-cycle arrest activity using a prostate cancer PC-3 cell line. The proliferation inhibition was evaluated by MTT assay, apoptosis was detected by Annexin V/propidium iodide (PI) staining and Hoechst 33258 staining, cell cycle distributions were measured by flow cytometry, and western blot analysis was used to determine specific cellular apoptotic protein expressions of Bcl-2, Bax, caspase-3 and cleaved-caspase-3. According to the results of this study, peperomin E exhibited significant anti-proliferation activity on PC-3 cell lines in vitro in a dose-dependent manner. Peperomin E treatments lead to marked morphological changes. Apoptotic cell count and cell-cycle distribution at G2/M phase significantly increased with increasing concentrations of peperomin E. The down-regulated expression level of Bcl-2 and up-regulated expression level of Bax and cleaved-caspase-3 compared with the controls were also observed after peperomin E treatment. These data suggest that peperomin E exhibited proliferation inhabitation, apoptosis-inducing and cell-cycle arrest activity on PC-3 cell lines. The anti-proliferation effect of peperomin E on PC-3 cells should result partly from its cell-cycle arrest and apoptosis-inducing activity, whereas the increasing of the Bax/Bcl-2 ratio and activation of caspases-3 play an important role in the development of apoptosis. [ABSTRACT FROM AUTHOR]

Ovarian carcinoma is a tumor derived from ovary, which brings relatively higher mortality rate among the fatal gynecological cancers. Recently, lots of studies have concentrated on the anti-tumor effects of Arg-Gly-Asp (RGD) motif containing peptides due to their integrin binding properties. In order to meet the criterion of genetic engineering drugs, a recombinant RGD toxin protein (rLj-RGD3) without a His-tag was cloned from the buccal glands of Lampetra japonica in the present study. After endotoxin removal, the His-tag removed rLj-RGD3 was shown to inhibit the proliferation of HeyA8 cells. According to the confocal microscope, flow cytometry and western blot analysis, rLj-RGD3 could trigger HeyA8 cells apoptosis by changing mitochondrial membrane potential, arrangement of F-actin, protein level of BCL2, BAX, caspase 3, and cleaved caspase 3, concentration of cytoplasmic calcium, as well as phosphorylation level of ERK/JNK/p38. Furthermore, rLj-RGD3 was also able to suppress the adhesion, migration, and invasion processes of HeyA8 cells by disturbing the organization of F-actin and reducing the level of p -FAK. In addition, rLj-RGD3 could inhibit the adhesion of HeyA8 cells to extracellular matrix proteins by competitively binding to integrins, indicated that rLj-RGD3 might act as an anti-tumor drug to treat ovarian carcinoma patients in the future. [ABSTRACT FROM AUTHOR]

Tryptophan is mainly degraded through kynurenine pathway (KP) in vertebrates which is closely related to the nerve and depression, while the studies on immunity is still limited. This study aims to explore the functions of tryptophan in the innate immunity of primitive vertebrate lamprey. MTT (3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide) assay showed that tryptophan had no obvious effect on cell viability. Tryptophan was transported into leukocytes and degraded via the KP after tryptophan supplement. Tryptophan treatment (T1x and T2x) failed to alter the total antioxidant capacity regardless of stimulation and exposure time. Real-time quantitative PCR and western blotting results revealed that tryptophan was not only able to reduce the expression of pro-inflammatory factors Lj-TNF-α, Lj-IL1β and Lj–NF–κB, but also to upregulate the expression of anti-inflammatory factor Lj-TGF-β independent of stimulation and time. In addition, tryptophan can exert immune tolerance function by inhibiting TLR-MyD88 and promoting (Indoleamine 2, 3-Dioxygenase) IDO-kynurenine-AHR (aryl hydrocarbon receptor) pathways. This study provides a new understanding for tryptophan-kynurenine metabolism and mechanism of immune tolerance function in primitive vertebrate lamprey. • Tryptophan can be transported into leukocytes and degraded through the kynurenine pathway. • Tryptophan-kynurenine metabolism can exert immune tolerance function. • Provides further insights for the origin of immunological tolerance function of tryptophan metabolism in lower vertebrates. [ABSTRACT FROM AUTHOR]

Cysteine-rich secretory proteins (CRISPs), characterized by 16 conserved cysteines, are distributed in a wide range of organisms, such as secernenteas, amphibians, reptiles and mammals. In the previous studies, a novel CRISP family member (cysteine-rich buccal gland protein, CRBGP) was separated from the buccal gland of lampreys ( Lampetra japonica , L. japonica ). Lamprey CRBGP could not only suppress depolarization-induced contraction of rat tail arterial smooth muscle, but also block voltage-gated sodium channels (VGSCs). In the present study, the anti-angiogenic activities of lamprey CRBGP were investigated using endothelial cells and chick chorioallantoic membrane (CAM) models. In vitro assays, lamprey CRBGP is able to induce human umbilical vein endothelial cells (HUVECs) apoptosis by disturbing the calcium homeostasis and mitochondria functions. In addition, lamprey CRBGP could inhibit proliferation, adhesion, migration, invasion and tube formation of HUVECs by affecting the organization of F-actin and expression level of matrix metallo-proteinase 2 (MMP-2), matrix metallo-proteinase 9 (MMP-9) and vascular endothelial growth factor A (VEGFA) which are related to angiogenesis. In vivo assays, lamprey CRBGP could suppress the blood vessel formation in CAM models. Therefore, lamprey CRBGP is an important protein present in the buccal gland of lampreys and might help lampreys suppress the contraction of blood vessels, nociceptive responses and wound healing of host fishes during their feeding time. In addition, lamprey CRBGP might have the potential to act as an effective anti-angiogenic factor for the treatment of abnormal angiogenesis induced diseases. [ABSTRACT FROM AUTHOR]

The non-receptor protein tyrosine kinase (nrPTK) Fyn, a member of the avian sarcoma virus transforming gene (Src) kinase family, plays a very significant role in cell growth, survival, apoptosis, tumor formation and immune response. In this study, a homolog of nrPTK Fyn was identified for the first time in the lamprey, Lampetra japonica and was named “Lja-Fyn”. The cDNA fragment of lamprey lja-fyn contains a 1611-bp open reading frame, which encodes a protein of 537 amino acids. Multiple sequence alignment analysis showed that it shares four conserved domains (Src homology (SH) 4, SH3, SH2 and protein kinases catalytic domains) and a variable unique domain with vertebrates Fyn molecules. Though Lja-Fyn has high sequence similarity with typical Fyn and Yes molecules of jawed vertebrates, the identities among Lja-Fyn and typical Fyn molecules in unique domain are relatively higher than that among Lja-Fyn and typical Yes molecules. The result indicates that Lja-Fyn is a homolog of Fyn rather than Yes. The phylogenetic analysis showed that Fyn, Yes and Src molecules are grouped into three distinct phylogenetic clusters, and Lja-Fyn is grouped as a single branch in Fyn cluster. The real-time quantitative PCR assay revealed the wide distribution of the lja-fyn mRNA in lamprey immune related tissues. After stimulation with mixed antigens, the levels of lja-fyn mRNA were obviously up-regulated in the gill and lymphocyte-like cells, and the similar results were got by western blot analysis of Lja-Fyn protein expression. These results indicated that nrPTK Lja-Fyn was likely to be involved in immune response. Furthermore, our present findings also provide the necessary information for understanding the distinction between lamprey Lja-Fyn and other members of jawed vertebrates in Src family. [ABSTRACT FROM AUTHOR]

CD82, a member of the tetraspanins, is originally identified as an accessory molecule in T cell activation, and it participates in the formation of immune synapse both in T cells and antigen-presenting cells of jawed vertebrates. In the present study, a CD82 homologous complementary DNA (cDNA) sequence is identified in the lamprey Lampetra japonica. The open reading frame of this sequence is 801 bp long and encodes a 266-amino acid protein. The multialignment of this sequence with several typical CD82s and CD37s of jawed vertebrates shows that it also possesses their conserved four transmembrane domains and a six-cysteine motif Cys-Cys-Gly...Cys-Ser-Cys...Cys...Cys, which is a characteristic motif of CD82 and CD37 vertebrate tetraspanin sequences. Since it is close to CD82s in sequence similarity, we name it as Lja-CD82-like. From the distribution profile of the conserved motifs of CD82-like, CD82, and CD37 molecules from molluscas to mammals, it seems that the CD82s and CD37s evolved from a common ancestral gene through a gene duplication event to their modern forms by a short insertion or substitution approaches. The phylogenetic analysis indicated that CD82 and CD37 molecules of jawed vertebrates originated from a common ancestral gene which is close to agnathan CD82-like and evolved into two distinct paralogous groups maybe after the divergence of jawed and jawless vertebrates. An expression vector with trigger factor (TF) was constructed to ensure that Lja-CD82-like express in prokaryotic expression host. The expressions of Lja- CD82- like messenger RNA (mRNA) and protein in immune-related tissues of lamprey were detected by real-time quantitative polymerase chain reaction and western blotting. Results showed that the mRNA and the protein levels of Lja- CD82- like were significantly upregulated in lymphocyte-like cells, gills, and supraneural myeloid bodies after stimulation with mixed antigens, respectively. Our data provided a foundation for the further study of Lja-CD82-like and its role in immune response process of jawless vertebrates. [ABSTRACT FROM AUTHOR]

Aldehyde dehydrogenases (ALDHs), which oxidize aldehyde to corresponding acids, play a major role in the detoxification of various endogenous and exogenous aldehydes. In this study, we cloned and characterized ALDH9 (designated LjALDH9) from Arctic lamprey Lampetra japonica . The open reading frame of LjALDH9 was 1566 bp, encoding 521 amino acids with a predicted molecular mass of 55.68 kDa. LjALDH9 protein had a signal peptide and Aldedh domain with the active site Cys315. In addition, LjALDH9 shares high sequence homology with ALDH9 of jawed vertebrates. Real-time quantitative PCR revealed that LjALDH9 was highly expressed in the buccal gland. A reactive LjALDH9 protein was obtained by prokaryotic expression, two-step-denaturing and refolding and affinity purification. During enzyme activity analysis of recombinant LjALDH9, we found that the most suitable reaction conditions were pH 7.0, 16–23 °C and Mn 2 + as the activator. Our study provides theoretical proof that LjALDH9 plays an important role in the parasitic life phase of lamprey. [ABSTRACT FROM AUTHOR]

Intelectins play an important role in innate immune response. In a previous study, lamprey inteletins purified by galactose-Sepharose were inactive and insoluble. Herein, we provided a simple and effective method to purify natural intelectins from the serum of lamprey (Lethenteron japonicum) using proteinG agarose. SDS-PAGE, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), and mass spectrometry (MS) were used to analyze the purified proteins. The purified proteins were identified to be lamprey serum lectin and intelectinB. The activity analysis results indicated that the proteins had certain extent agglutination activity. The effective method will be useful to study their immune functions and molecular mechanisms. [ABSTRACT FROM PUBLISHER]

Cathepsin D (EC 3.4.23.5) is a lysosomal aspartic proteinase of the pepsin superfamily which participates in various digestive processes within the cell. In the present study, the full length cDNA of a novel cathepsin D homologue was cloned from the buccal glands of lampreys ( Lampetra japonica ) for the first time, including a 124-bp 5′ terminal untranslated region (5′-UTR), a 1194-bp open reading frame encoding 397 amino acids, and a 472-bp 3′-UTR. Lamprey cathepsin D is composed of a signal peptide (Met 1-Ala 20), a propeptide domain (Leu 21-Ala 48) and a mature domain (Glu 76-Val 397), and has a conserved bilobal structure. Cathepsin D was widely distributed in the buccal glands, immune bodies, hearts, intestines, kidneys, livers, and gills of lampreys. After challenging with Escherichia coli or Staphylococcus aureus , the expression level of lamprey cathepsin D in the buccal gland was 8.5-fold or 6.5-fold higher than that in the PBS group. In addition, lamprey cathepsin D stimulated with Escherichia coli was also up-regulated in the hearts, kidneys, and intestines. As for the Staphylococcus aureus challenged group, the expression level of lamprey cathepsin D was found increased in the intestines. The above results revealed that lamprey cathepsin D may play key roles in immune response to exogenous pathogen and could serve as a potential antibacterial agent in the near future. In addition, lamprey cathepsin D was subcloned into pcDNA 3.1 vector and expressed in the human embryonic kidney 293 cells. The recombinant lamprey cathepsin D could degrade hemoglobin, fibrinogen, and serum albumin which are the major components in the blood, suggested that lamprey cathepsin D may also act as a digestive enzyme during the adaptation to a blood-feeding lifestyle. [ABSTRACT FROM AUTHOR]

Temporin-1CEb shows antimicrobial activity against Gram-positive bacteria, but its therapeutic potential is limited by its haemolysis. In this study, eight temporin-1CEb analogues with altered cationicities and hydrophobicities were synthesized. Increasing cationicity and amphipathicity by substituting neutral and non-polar amino acid residues on the hydrophilic face of the α-helix by five or six lysines increased antimicrobial potency approximately 10-fold to 40-fold, although when the number of positive charges was increased from +6 to +7, the antimicrobial potency was not additionally enhanced. The substitution of an l-lysine with a d-lysine, meanwhile maintaining the net charge and the mean hydrophobicity values, had only a minor effect on its antimicrobial activity, whereas significantly led a decrease in its haemolytic activity. Of all the peptides, l-K6 has the best potential as an antimicrobial agent because its antimicrobial activity against both Gram-positive and Gram-negative bacteria is substantial, and its haemolytic activity is negligible. l-K6 adopts an α-helix in 50% trifluoroethanol/water and 30 m m SDS solutions. l-K6 killed 99.9% of E. coli and S. aureus at 4× MIC in 60 min, and its postantibiotic effect was >5 h. l-K6 affects the integrity of E. coli and S. aureus plasma membranes by rapidly inducing membrane depolarization. [ABSTRACT FROM AUTHOR]

This study was designed to investigate proteome changes in Japanese puffer fish (Takifugu rubripes) during short- and long-term frozen storage. In total, 1484 proteins were quantified, and 164 proteins were identified as differential abundance proteins (DAPs) in Japanese puffer fish from two frozen storage treatment groups (14 days and 60 days) compared with the fresh control group. Correlation analysis between the DAPs and quality traits of the puffer fish muscle showed that 106 proteins were correlated closely with colour and texture (hardness, elasticity, and chewiness). Bioinformatics analysis revealed and Western blot analysis verified that Putative prothymosin alpha species, Bridging integrator 3, NADH: the ubiquinone oxidoreductase subunit and Mx species are candidate biomarkers for puffer fish properties. This study offers valuable evidence to improve the quality control and monitoring of Japanese puffer fish during transportation and storage. [ABSTRACT FROM AUTHOR]

In jawless vertebrates, the lamprey complement component C1q (LC1q) acts as a lectin and activates lamprey complement component C3 (LC3) in association with mannose-binding lectin (MBL)-associated serine protease (MASP) via the lectin pathway. Furthermore, LC1q may interact with variable lymphocyte receptor B (VLRB) in a complex with antigens and mediate the activation of LC3, leading to cytolysis. In the present study, we found, for the first time, that LC1q plays a critical role in VLRA/VLRC-mediated immune response. Escherichia coli , Shigella flexneri , Aeromonas hydrophila , Pseudomonas plecoglossicida , Aeromonas allosaccharophila , P. luteola , Brevundimonas diminuta , and Bacillus cereus were isolated from infected Lampetra morii in our laboratory and identified using the 16s rRNA method. A. hydrophila was confirmed as a rapidly spreading lethal pathogen in the larvae of L. morii and was used in subsequent immune stimulation experiments. The results of real-time quantitative polymerase chain reaction (Q-PCR) and immunofluorescence analyses indicated that the RNA and protein expression levels of LC1q were upregulated following exposure to 107 cfu/mL of A. hydrophila, compared to the levels of the naïve group. We obtained LC1q morphants (LC1q MO) of lamprey larvae by morpholino-mediated knockdowns. We found that LC1q played key roles in the embryonic development of lamprey. The median lethal time (LT50) of LC1q MO larvae was 2 d after being exposed to the pathogens, whereas the LT50 of control MO was 5 d. The drastic decrease in LT50 values after LC1q knockdown implies that LC1q plays a critical role in lamprey immune response. Gene expression profiles of LC1q-deficient A. hydrophila , control MO A. hydrophila, wild type A. hydrophila , and naive 1-month-old ammocoetes larvae were compared by examining the expression levels of a selected panel of orthologous genes. It is worth mentioning that LC1q MO affected the VLRA+/VLRC + population genes but did not affect the VLRB + populations. Immunohistochemical data indicated that LC1q deficiency also affected VLRA and VLRC but not VLRB. Thus, LC1q plays a critical role in VLRA/VLRC-mediated immune response in lamprey. • We report that LC1q have been proven to be critical for L.morii early development. • LC1q play important roles in resisting the A. hydrophila invasion in its proammocoete larvae stage. • LC1q plays a critical role in VLRA/VLRC mediated immune response in the lamprey. [ABSTRACT FROM AUTHOR]