Crystal structure of bacterial ubiquitin ADP-ribosyltransferase CteC reveals a substrate-recruiting insertion.

ADP-ribosylation is a post-translational modification involved in regulation of diverse cellular pathways. Interestingly, many pathogens have been identified to utilize ADP-ribosylation as a way for host manipulation. A recent study found that CteC, an effector from the bacterial pathogen Chromobacterium violaceum, hinders host ubiquitin (Ub) signaling pathways via installing mono-ADP-ribosylation on threonine 66 of Ub. However, the molecular basis of substrate recognition by CteC is not well understood. In this article, we probed the substrate specificity of this effector at protein and residue levels. We also determined the crystal structure of CteC in complex with NAD+, which revealed a canonical mono-ADP-ribosyltransferase fold with an additional insertion domain. The AlphaFold-predicted model differed significantly from the experimentally determined structure, even in regions not used in crystal packing. Biochemical and biophysical studies indicated unique features of the NAD+ binding pocket, while showing selectivity distinction between Ub and structurally close Ub-like modifiers and the role of the insertion domain in substrate recognition. Together, this study provides insights into the enzymatic specificities and the key structural features of a novel bacterial ADP-ribosyltransferase involved in host-pathogen interaction.

Identification of the principal neuropeptide MIP and its action pathway in larval settlement of the echiuran worm Urechis unicinctus.

BACKGROUND: Larval settlement and metamorphosis represent critical events in the life history of marine benthic animals. Myoinhibitory peptide (MIP) plays a pivotal role in larval settlement of marine invertebrates. However, the molecular mechanisms of MIP involved in this process are not well understood. RESULTS: In this study, we evaluated the effects of thirteen MIP mature peptides on triggering the larval settlement of Urechis unicinctus (Xenopneusta, Urechidae), and determined that MIP2 was the principal neuropeptide. Transcriptomic analysis was employed to identify differentially expressed genes (DEGs) between the MIP2-treated larvae and normal early-segmentation larvae. Both cAMP and calcium signaling pathways were enriched in the DEGs of the MIP2-treated larvae, and two neuropeptide receptor genes (Spr, Fmrfar) were up-regulated in the MIP2-treated larvae. The activation of the SPR-cAMP pathway by MIP2 was experimentally validated in HEK293T cells. Furthermore, fourteen cilia-related genes, including Tctex1d2, Cfap45, Ift43, Ift74, Ift22, Cav1 and Mns1, etc. exhibited down-regulated expression in the MIP2-treated larvae. Whole-mount in situ hybridization identified two selected ciliary genes, Tctex1d2 and Cfap45, were specially expressed in circumoral ciliary cells of the early-segmentation larvae. Knocking down Tctex1d2 mRNA levels by in vivo RNA interference significantly increased the larval settlement rate. CONCLUSION: Our findings suggest that MIP2 inhibits the function of the cilia-related genes, such as Tctex1d2, through the SPR-cAMP-PKA pathway, thereby inducing larval settlement in U. unicinctus. The study contributes important data to the understanding of neuropeptide regulation in larval settlement.

The regulatory function of GATA3 on immune response in Japanese flounder (Paralichthys olivaceus).

GATA3 belongs to the GATA family, and it could interact with the target gene promoter. It has been reported to play a central role in regulating lymphocyte differentiation. In this study, the GATA3 cDNA sequence was identified by a homologous clone and the RACE technology from Japanese flounder (Paralichthys olivaceus). The full-length of the GATA3 cDNA sequence was 2904 bp, including 1332 bp open reading frame (ORF), 265 bp 5 '-untranslated region (5' UTR), and 1308 bp 3 '-UTR, encoding 443 amino acids. GATA3 protein sequence was conserved in vertebrates and invertebrates, including two zinc finger domains. qRT-PCR showed that the expression of GATA3 was high in the gill, kidney, and spleen. Expression of GATA3 slowly increased at the earlier stages and culminated at the late gastrula and somatic stages. Immunohistochemistry (IHC) results showed that the GATA3 protein was expressed in lymphocyte cells, undifferentiated basal and pillar cells of the gills, as well as lymphocyte cells and melanin macrophages of the kidney. The expression of GATA3 was significantly regulated in tissues and different types of lymphocytes after stimulation with Edwardsiella tarda. Dual-luciferase reporter assay indicated that the GATA3 protein could directly interact with promoters of target genes involved in the immune response. These findings suggested that GATA3 plays a major role in regulating the immune response. This study provided a theoretical basis for the immune response mechanism of teleost and a useful reference for later research on fish immunology.

Identification, Characterization and Functional Analysis of Fibroblast Growth Factors in Black Rockfish (Sebastes schlegelii).

Fibroblast growth factors (FGFs) are short polypeptides that play essential roles in various cellular biological processes, including cell migration, proliferation, and differentiation, as well as tissue regeneration, immune response, and organogenesis. However, studies focusing on the characterization and function of FGF genes in teleost fishes are still limited. In this study, we identified and characterized expression patterns of 24 FGF genes in various tissues of embryonic and adult specimens of the black rockfish (Sebates schlegelii). Nine FGF genes were found to play essential roles in myoblast differentiation, as well as muscle development and recovery in juvelines of S. schlegelii. Moreover, sex-biased expression pattern of multiple FGF genes was recorded in the species' gonads during its development. Among them, expression of the FGF1 gene was recorded in interstitial and sertoli cells of testes, promoting germ-cell proliferation and differentiation. In sum, the obtained results enabled systematic and functional characterization of FGF genes in S. schlegelii, laying a foundation for further studies on FGF genes in other large teleost fishes.

Morphological Observation and Transcriptome Analysis of Ciliogenesis in Urechis unicinctus (Annelida, Echiura).

During the early development of marine invertebrates, planktic larvae usually occur, and their body surfaces often form specific types of cilia that are involved in locomotion and feeding. The echiuran worm Urechis unicinctus sequentially undergoes the formation and disappearance of different types of body surface cilia during embryonic and larval development. The morphological characteristics and molecular mechanisms involved in the process remain unclear. In this study, we found that body surface cilia in U. unicinctus embryos and larvae can be distinguished into four types: body surface short cilia, apical tufts, circumoral cilia and telotrochs. Further, distribution and genesis of the body surface cilia were characterized using light microscope and electron microscope. To better understand the molecular mechanism during ciliogenesis, we revealed the embryonic and larval transcriptome profile of the key stages of ciliogenesis in U. unicinctus using RNA-Seq technology. A total of 29,158 differentially expressed genes (DEGs) were obtained from 24 cDNA libraries by RNA-Seq. KEGG pathway enrichment results showed that Notch, Wnt and Ca2+ signaling pathways were significantly enriched during the occurrence of apical tufts and circumoral cilia. Furthermore, all DEGs were classified according to their expression pattern, and DEGs with similar expression pattern were grouped into a module. All DEG co-expression modules were correlated with traits (body surface short cilia, apical tufts, circumoral cilia and telotrochs) by WGCNA, the results showed DEGs were divided into 13 modules by gene expression patterns and that the genes in No. 7, No. 8 and No. 10 modules were to be highly correlated with the occurrence of apical tufts, circumoral cilia and telotrochs. The top 10 hub genes in the above three modules were identified to be highly correlated with ciliogenesis, including the reported cilium-related gene Cnbd2 and unreported cilium-related candidate genes FAM181B, Capsl, Chst3, TMIE and Innexin. Notably, Innexin was included in the top10 hub genes of the two modules (No. 7 and No. 8), suggesting that Innexin may play an important role in U. unicinctus apical tufts, circumoral cilia and telotrochs genesis. This study revealed the characteristics of ciliogenesis on the body surface of U. unicinctus embryos and larvae, providing basic data for exploring the molecular mechanism of ciliogenesis on the body surface.

Characterization of channel catfish (Ictalurus punctatus) melanocortin-3 receptor reveals a potential network in regulation of energy homeostasis.

The melanocortin-3 receptor (MC3R) is known to be involved in regulation of energy homeostasis, regulating feed efficiency and nutrient partitioning in mammals. Its physiological roles in non-mammalian vertebrates, especially economically important aquaculture species, are not well understood. Channel catfish (Ictalurus punctatus) is the main freshwater aquaculture species in North America. In this study, we characterized the channel catfish MC3R. The mc3r of channel catfish encoded a putative protein (ipMC3R) of 367 amino acids. We transfected HEK293T cells with ipMC3R plasmid for functional studies. Five agonists, including adrenocorticotropin, α-melanocyte stimulating hormone (α-MSH), ß-MSH, [Nle4, D-Phe7]-α-MSH, and D-Trp8-γ-MSH, were used in the pharmacological studies. Our results showed that ipMC3R bound ß-MSH with higher affinity and D-Trp8-γ-MSH with lower affinity compared with human MC3R. All agonists could stimulate ipMC3R and increase intracellular cAMP production with sub-nanomolar potencies. The extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation could also be triggered by ipMC3R. The ipMC3R exhibited constitutive activities in both cAMP and ERK1/2 pathways, and Agouti-related protein served as an inverse agonist at ipMC3R, potently inhibiting the high basal cAMP level. Moreover, we showed that melanocortin receptor accessory protein 2 (MRAP2) preferentially modulated ipMC3R in cAMP production rather than ERK1/2 activation. Our study will assist further investigation of the physiological roles of the ipMC3R, especially in energy homeostasis, in channel catfish.

Genome-wide identification, characterization and expression analyses of TLRs in Yesso scallop (Patinopecten yessoensis) provide insight into the disparity of responses to acidifying exposure in bivalves.

Toll-like receptors (TLRs) play a crucial role in innate immunity by recognizing specific pathogen-associated molecular patterns, including lipoproteins, lipopeptides, lipopolysaccharide, flagellin, dsRNA, ssRNA and CpG DNA motifs. Although significant effects of TLRs on immunity have been reported in most vertebrates and some invertebrates, the complete TLR superfamily has not been systematically characterized in scallops. In this study, 18 TLR genes were identified from Yesso scallop (Patinopecten yessoensis) using whole-genome scanning. Phylogenetic and protein structural analyses were performed to determine the identities and evolutionary relationships of the 18 genes. Extensive expansion of TLR genes from the Yesso scallop genome indicated gene duplication events. In addition, expression profiling of PyTLRs was performed at different acidifying exposure levels (pH = 6.50, 7.50) with different challenge durations (3, 6, 12 and 24 h) via in silico analysis using transcriptome and genome databases. Our results confirmed the inducible expression patterns of PyTLRs under acidifying exposure, and the responses to immune stress may have arisen through adaptive recruitment of tandem duplications of TLR genes. Collectively, this study provides novel insight into PyTLRs as well as the specific role and response of TLR signaling pathways in host immune responses against acidifying exposure in bivalves.

Surface structure feature matching algorithm for cardiac motion estimation.

BACKGROUND: Cardiac diseases represent the leading cause of sudden death worldwide. During the development of cardiac diseases, the left ventricle (LV) changes obviously in structure and function. LV motion estimation plays an important role for diagnosis and treatment of cardiac diseases. To estimate LV motion accurately for cine magnetic resonance (MR) cardiac images, we develop an algorithm by combining point set matching with surface structure features of myocardium. METHODS: The structure features of myocardial wall are described by estimating the normal directions of points locating on the myocardium contours using an approximation approach. The Gaussian mixture model (GMM) of structure features is used to represent LV structure feature distribution. A new cost function is defined to represent the differences between two Gaussian mixture models, which are the GMM of structure features and the GMM of positions of two point sets. To optimize the cost function, its gradient is derived to use the Quasi-Newton (QN). Furthermore, to resolve the dis-convergence issue of Quasi-Newton for high-dimensional parameter space, Stochastic Gradient Descent (SGD) is used and SGD gradient is derived. Finally, the new cost function is solved by optimization combining SGD with QN. With the closed form expression of gradient, this paper provided a computationally efficient registration algorithm. RESULTS: Three public datasets are employed to verify the performance of our algorithm, including cardiac MR image sequences acquired from 33 subjects, 14 inter-subject heart cases, and the data obtained in MICCAI 2009s 3D Segmentation Challenge for Clinical Applications. We compare our results with those of the other point set registration methods for LV motion estimation. The obtained results demonstrate that our algorithm shows inherent statistical robustness, due to the combination of SGD and Quasi-Newton optimization. Furthermore, our method is shown to outperform other point set matching methods in the registration accuracy. CONCLUSIONS: We provide a novel effective algorithm for cardiac motion estimation by introducing LV surface structure feature to point set matching. A new cost function is defined to measure the discrepancy between GMMs of two point sets. The GMM of point positions and the GMM of surface structure descriptor are defined at the same time. Optimization by combining SGD and Quasi-Newton is performed to solve the cost function. We experimentally demonstrate that our algorithm shows improved registration accuracy, and is convergent when used in high-dimensional parameter space.

Polyketides from the Mangrove-Derived Endophytic Fungus Nectria sp. HN001 and Their α-Glucosidase Inhibitory Activity.

Four new polyketides: nectriacids A-C (1-3) and 12-epicitreoisocoumarinol (4), together with three known compounds: citreoisocoumarinol (5), citreoisocoumarin (6), and macrocarpon C (7) were isolated from the culture of the endophytic fungus Nectria sp. HN001, which was isolated from a fresh branch of the mangrove plant Sonneratia ovata collected from the South China Sea. Their structures were determined by the detailed analysis of NMR and mass spectroscopic data. The absolute configuration of the stereogenic carbons for compound 4 was further assigned by Mosher's ester method. All of the isolated compounds were tested for their α-glucosidase inhibitory activity by UV absorbance at 405 nm, and new compounds 2 and 3 exhibited potent inhibitory activity with IC50 values of 23.5 and 42.3 µM, respectively, which were more potent than positive control (acarbose, IC50, 815.3 µM).

The Preparation of Crumpled Graphene Oxide Balls and Research in Tribological Properties.

In this study, crumpled graphene oxide balls (CGBs) were prepared via capillary compression using a rapidly evaporating aerosol droplet method. The CGBs were observed using scanning electron microscopy (SEM), high-resolution transmission electron microscopy (HRTEM), and Raman spectroscopy. The size distributions of crumpled particles were obtained using a laser nanometer particle size analyzer (DLS). The dispersibility of the water and the ionic liquid (IL) was tested by ultrasonic dispersion. The tribological properties of water or ionic liquids containing crumpled graphene oxide ball additives (W/IL-CGB) were tested by a reciprocating friction tester and compared with water/ionic liquids with graphene oxide. The morphology of the wear scar was observed by a three-dimensional optical microscope and its lubrication mechanism was analyzed. The results show that the CGBs were successfully prepared by rapid evaporation of aerosol droplets, and the obtained CGBs were crumpled paper spheres. The CGBs had good water dispersion and ionic liquid dispersion, and IL-CGB has excellent anti-friction and anti-wear effects on steel-steel friction pairs. During the friction process, the CGB was adsorbed at the interface of the steel-steel friction pair to form a protective layer, which avoids the direct contact of the friction pair, thereby reducing friction and wear.

Legionella metaeffector MavL reverses ubiquitin ADP-ribosylation via a conserved arginine-specific macrodomain.

ADP-ribosylation is a reversible post-translational modification involved in various cellular activities. Removal of ADP-ribosylation requires (ADP-ribosyl)hydrolases, with macrodomain enzymes being a major family in this category. The pathogen Legionella pneumophila mediates atypical ubiquitination of host targets using the SidE effector family in a process that involves ubiquitin ADP-ribosylation on arginine 42 as an obligatory step. Here, we show that the Legionella macrodomain effector MavL regulates this pathway by reversing the arginine ADP-ribosylation, likely to minimize potential detrimental effects caused by the modified ubiquitin. We determine the crystal structure of ADP-ribose-bound MavL, providing structural insights into recognition of the ADP-ribosyl group and catalytic mechanism of its removal. Further analyses reveal DUF4804 as a class of MavL-like macrodomain enzymes whose representative members show unique selectivity for mono-ADP-ribosylated arginine residue in synthetic substrates. We find such enzymes are also present in eukaryotes, as exemplified by two previously uncharacterized (ADP-ribosyl)hydrolases in Drosophila melanogaster. Crystal structures of several proteins in this class provide insights into arginine specificity and a shared mode of ADP-ribose interaction distinct from previously characterized macrodomains. Collectively, our study reveals a new regulatory layer of SidE-catalyzed ubiquitination and expands the current understanding of macrodomain enzymes.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The intracellular bacterial pathogen Legionella pneumophila modulates host cell functions by secreting multiple effectors with diverse biochemical activities. In particular, effectors of the SidE family interfere with host protein ubiquitination in a process that involves production of phosphoribosyl ubiquitin (PR-Ub). Here, we show that effector LnaB converts PR-Ub into ADP-ribosylated ubiquitin, which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (SHxxxE) present in a large family of toxins from diverse bacterial pathogens. Thus, our study sheds light on the mechanisms by which a pathogen maintains ubiquitin homeostasis and identifies a family of enzymes capable of protein AMPylation.

Legionella maintains host cell ubiquitin homeostasis by effectors with unique catalytic mechanisms.

The reversal of ubiquitination induced by members of the SidE effector family of Legionella pneumophila produces phosphoribosyl ubiquitin (PR-Ub) that is potentially detrimental to host cells. Here we show that the effector LnaB functions to transfer the AMP moiety from ATP to the phosphoryl moiety of PR-Ub to convert it into ADP-ribosylated ubiquitin (ADPR-Ub), which is further processed to ADP-ribose and functional ubiquitin by the (ADP-ribosyl)hydrolase MavL, thus maintaining ubiquitin homeostasis in infected cells. Upon being activated by Actin, LnaB also undergoes self-AMPylation on tyrosine residues. The activity of LnaB requires a motif consisting of Ser, His and Glu (S-HxxxE) present in a large family of toxins from diverse bacterial pathogens. Our study not only reveals intricate mechanisms for a pathogen to maintain ubiquitin homeostasis but also identifies a new family of enzymes capable of protein AMPylation, suggesting that this posttranslational modification is widely used in signaling during host-pathogen interactions.

Characterization of Deubiquitinase Catalytic State Using a Structure-Based Approach.

The activity of deubiquitinases (DUBs) is tightly regulated in eukaryotes via various mechanisms. One of the regulatory strategies is substrate-induced catalytic triad rearrangement, where ubiquitin-binding helps the DUB adopt an active conformation for catalysis. The crystal structure of the apo form of such a DUB, when not bound to ubiquitin, reveals an inactive conformation of the catalytic residues, necessitating the structure of the ubiquitin-bound form to visualize the active state of the DUB. Comparing the apo and ubiquitin-bound structures reveals conformational changes leading to catalytic activation. To capture the deubiquitinase in its ubiquitin-bound form, a series of activity-based ubiquitin probes (Ub-ABPs) harboring C-terminal electrophiles were designed to react with the catalytic nucleophile of cysteine protease DUBs. The resulting covalently linked DUB-ubiquitin complex is amendable for structural studies to probe the DUB-ubiquitin interface and the potential conformational change of the DUB. Here, we present a detailed protocol for the generation and purification of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) in complex with a Ub-ABP, ubiquitin-vinyl methyl ester (UbVME), and the subsequent structural analysis to characterize the catalytic state of the DUB.

Pathogen-defending deubiquitinase possesses distinct specificity towards K6-polyubiquitination.

The bacterial pathogen Legionella pneumophila encodes numerous effectors to manipulate host ubiquitin signaling. Recently, Warren et al. revealed the structural basis of K6-polyubiquitination recognition by Legionella deubiquitinase LotA, while validating its potential as an enzymatic tool to study linkage-specific ubiquitination. During Legionella infection, LotA counteracts valosin-containing protein (VCP) recruitment to the Legionella-containing vacuole.

Research on skull trauma biomechanical stress distribution in case of dental implants existence.

BACKGROUND: When the jaw bone is subjected to an external force, the stress is transmitted from the force point along the alveolar bone to the skull and skull base. In the case of a dental implant, the stress distribution is mainly dependent on the implant position, type, and mechanical properties. OBJECTIVE: To investigate the dental implant position influence on the stress distribution and transmission in case of facial frontal trauma. Furthermore, the correlation between facial trauma and skull trauma in the case of a dental implant exists. METHODS: In this study, a Finite Element Method (FEM) model was constructed based on a real skull shape, size, and anatomy. Dental implants were modeled based on imported CAD Data. Five cases were investigated including no dental implant and the replacement of teeth no. 18, 19 20 and 21. Facial trauma was mimicked by applying an external load on the lower frontal jaw. Finally, the stress distribution based on the bone geometry and implant position were evaluated and compared. RESULTS: Results suggested that a dental implant could significantly influence the stress distribution in the skull in case of facial trauma. In addition, the dental position greatly affects stress transmission from the mandible to the skull bones through the zygomatic arch. CONCLUSION: The position of the dental implant could have a significant role in stress transmission and distribution in case of facial or even brain trauma. Thus, increasing the possibility of a correlation between facial and brain trauma.

The Preparations of Fluorographene Nanosheets and Research in Tribological Properties in High Vacuum.

In this study, fluorographene nanosheets (FG nanosheets) were prepared via the solvent-ultrasonic exfoliation method. The fluorographene sheets were observed using field-emission scanning electron microscopy (FE-SEM). The microstructure of the as-prepared FG nanosheets was characterized by X-ray diffraction (XRD) and a thermal analyzer (TG). The tribological properties of FG nanosheets as an additive in ionic liquids in high vacuum were compared to that of ionic liquid (IL) with graphene (IL-G). The wear surfaces and transfer films were analyzed via an optical microscope, Raman spectroscopy, scanning electron microscopy (SEM), and X-ray photoelectron spectroscopy (XPS). The results show that FG nanosheets can be obtained from the simple solvent-ultrasonic exfoliation method. The prepared G nanosheets are a sheet, and the longer the ultrasonic time is, the thinner the sheet is. Ionic liquids with FG nanosheets had low friction and a low wear rate under high vacuum conditions. The improved frictional properties were attributed to the transfer film of FG nanosheets and more formation film of Fe-F.

Omics insights into spermatozoa activation induced by Fetal bovine serum in viviparous black rockfish (Sebastes schlegelii).

Black rockfish (Sebastes schlegelii) is an economically important marine species with the characteristics of viviparity. The spermatozoa were transferred into the ovary by mating and stored for several months until fertilization. Little is known about spermatozoa activation and its mechanism in black rockfish. In this study, the suitable medium for spermatozoa activation in vitro was explored, and the underlying mechanism was studied by omics analysis. Fetal bovine serum (FBS) could significantly enhance spermatozoa motility in vitro. Omics analysis showed 559 differentially expressed genes (DEGs) and 1311 differentially methylated genes (DMGs) were identified after FBS treatment. Transcriptome analysis revealed that FBS-induced spermatozoa motility activation is associated with spermatozoa capacitation regulated by the cAMP-SRC-PKA, cGMP-PKG and phospholipase D signaling pathway. Spermatozoa capacitation-related gene hsp90aa1 and chemotaxis-related gene cxcr4 were two of the important DMGs. Methylome analysis further revealed that FBS-induced epigenetic modifications are involved in spermatozoa capacitation and chemotaxis. 36 overlaps were identified between DMGs and DEGs, of which five genes were demonstrated to play a role in spermatozoa physiology, required for flagellum stability and spermatozoa motility. The results could provide new clues for understanding spermatozoa activation's molecular mechanism and help establish activation and/or immobilizing media for improving either artificial fertilization or cryopreservation in black rockfish.

Study on the relationship between permeability coefficient and porosity, the confining and osmotic pressure of attapulgite-modified loess.

This study investigated attapulgite-modified loess as an efficient and cost-effective method for creating an impermeable liner for landfills in regions with scarce clay resources. Laboratory permeability tests were conducted using a flexible wall permeameter to determine the permeability of compacted loess and attapulgite mixtures under varying osmotic conditions. The relationship between the permeability coefficient, attapulgite dosage, radial pressure, and osmotic pressure was analyzed. Nuclear magnetic resonance and scanning electron microscopy were also used to observe the microstructure of the modified loess. The results showed that attapulgite dosage significantly reduced the permeability coefficient, but the effect became limited when the content surpassed 10%. The decrease of the permeability coefficient of the modified loess is mainly due to the filling of pores between the loess by attapulgite, which makes the pore size and throat size of the modified loess smaller. The modified loess displayed a sheet structure that contributed to an increased permeability coefficient due to increased radial pressure. This study provides valuable insights into using attapulgite-modified loess as a material for landfill lining in regions with scarce clay resources.

Identification and expression analysis of receptors that mediate MIP regulating larval settlement in Urechis unicinctus.

Larval attachment and metamorphosis are important processes during the development of some marine invertebrates. Myoinhibitory peptides (MIPs), a class of small molecular neuropeptides, have been revealed to be involved in regulating the larval settlement. In this paper, we identified two types of MIP membrane receptors, G-protein coupled receptor SPR and MIP-gated ion channel receptors MGIC1 and MGIC2 based on sequence homology with other species in the transcriptome database of Echiuroidea Urechis unicinctus (Xenopneusta, Urechidae). The results of in situ hybridization showed that positive signals of these receptors were obviously located in the apex of the segmentation larvae, a critical stage of U. unicinctus larval settlement. Further, these receptors were determined on the membrane of HEK293 cells by immunohistochemistry. Also, we verified that U. unicinctus MIP can activate its SPR receptor based on the results of the significantly decreased cAMP concentration in HEK293 cells. Our data will provide scientific reference for elucidating mechanism of neuropeptide regulating the larval attachment and metamorphosis in marine invertebrates.