Yuzhu Song

Toward an Understanding of the Molecular Mechanism for Successful Blood Feeding by Coupling Proteomics Analysis with Pharmacological Testing of Horsefly Salivary Glands

Horseflies are economically important blood-feeding arthropods and also a nuisance for humans and vectors for filariasis. They rely heavily on the pharmacological properties of their saliva to get a blood meal and suppress immune reactions of hosts. Little information is available on antihemostatic substances in horsefly salivary glands; especially no horsefly immune suppressants have been reported. By proteomics or peptidomics and coupling transcriptome analysis with pharmacological testing, several families of proteins or peptides, which act mainly on the hemostatic system or immune system of the host, were identified and characterized from 30,000 pairs salivary glands of the horsefly Tabanus yao (Diptera, Tabanidae). They are: (i) a novel family of inhibitors of platelet aggregation including two members, which possibly inhibit platelet aggregation by a novel mechanism and act on platelet membrane, (ii) a novel family of immunosuppressant peptides including 12 members, which can inhibit interferon-γ production and increase interleukin-10 secretion, (iii) a serine protease inhibitor with 56 amino acid residues containing anticoagulant activity, (iv) a serine protease with anticoagulant activity, (v) a protease with fibrinogenolytic activity, (vi) three families of antimicrobial peptides including six members, (vii) a hyaluronidase, (viii) a vasodilator peptide, which is an isoform of vasotab identified from Hybomitra bimaculata, and interestingly (ix) two metallothioneins, which are the first metallothioneins reported from invertebrate salivary glands. The current work will facilitate the understanding of the molecular mechanisms of the ectoparasite-host relationship and help in identifying novel vaccine targets and novel leading pharmacological compounds.

A new family of antimicrobial peptides from skin secretions of Rana pleuraden

While conducting experiments to investigate antimicrobial peptides of amphibians living in the Yunnan-Guizhou region of southwest China, a new family of antimicrobial peptides was identified from skin secretions of the Yunnan frog, Rana pleuraden. Members of the new peptide family named pleurain-As are composed of 26 amino acids with a unique N-terminal sequence (SIIT) and a disulfide-bridged heptapeptide sequence (CRLYNTC). By BLAST search, pleurain-As had no significant similarity to any known peptides. Native and synthetic peptides showed antimicrobial activities against tested microorganisms including Gram-negative and Gram-positive bacteria and fungi. Twenty different cDNAs encoding pleurain-As were cloned from the skin cDNA library of R. pleuraden. The precursors of pleurain-As are composed of 69 amino acid residues including predicted signal peptides, acidic propieces, and cationic mature antimicrobial peptides. The preproregion of pleurain-A precursor comprises a hydrophobic signal peptide of 22 residues followed by an 18 residue acidic propiece which terminates by a typical prohormone processing signal Lys-Arg. The preproregions of precursors are very similar to other amphibian antimicrobial peptide precursors but the mature pleurain-As are different from other antimicrobial peptide families. The remarkable similarity of preproregions of precursors that give rise to very different antimicrobial peptides in distantly related frog species suggests that the corresponding genes form a multigene family originating from a common ancestor. Furthermore, pleurain-As could exert antimicrobial capability against Helicobacter pylori. This is the first report of naturally occurring peptides with anti-H. pylori activity from Rana amphibians.

Isolation and cDNA cloning of cholecystokinin from the skin of Rana nigrovittata

Many neuroendocrine peptides that are distributed in amphibian gastrointestinal tract and central nervous system are also found in amphibian skins, and these peptides are classified into skin-gut-brain triangle peptides, such as bombesins, gastrin-releasing peptides. Cholecystokinins (CCKs) are neuroendocrine peptides known for their production in the gastrointestinal tract and central nervous system of mammalians. Several CCKs have been identified from two amphibians, Rana catesbeiana and Xenopus laevis. These amphibian CCKs are found to be express in brain and in the gastrointestinal tract, but not in skin. In the current report, a cholecystokinin (CCK) isoform was identified from skin secretions of the frog, Rana nigrovittata. Its amino acid sequence is RVDGNSDQKAVIGAMLAKDLQTRKAGSSTGRYAVLPNR PVIDPTHRINDRDYMGWMDF, which is the same with that of CCK from R. catesbeiana. Four different cDNAs (GenBank accession nos. EF608063-6) encoding CCK precursors were cloned from the cDNA library of the skin of R. nigrovittata. The present data demonstrated that amphibian CCK could also be expressed in gastrointestinal tract, central nervous system and skin as other amphibian skin-gut-brain triangle peptides.

Targeted next-generation sequencing of candidate genes reveals novel mutations in patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) is a major cause of sudden cardiac death and heart failure, and it is characterized by genetic and clinical heterogeneity, even for some patients with a very poor clinical prognosis; in the majority of cases, DCM necessitates a heart transplant. Genetic mutations have long been considered to be associated with this disease. At present, mutations in over 50 genes related to DCM have been documented. This study was carried out to elucidate the characteristics of gene mutations in patients with DCM. The candidate genes that may cause DCM include MYBPC3, MYH6, MYH7, LMNA, TNNT2, TNNI3, MYPN, MYL3, TPM1, SCN5A, DES, ACTC1 and RBM20. Using next-generation sequencing (NGS) and subsequent mutation confirmation with traditional capillary Sanger sequencing analysis, possible causative non-synonymous mutations were identified in ~57% (12/21) of patients with DCM. As a result, 7 novel mutations (MYPN, p.E630K; TNNT2, p.G180A; MYH6, p.R1047C; TNNC1, p.D3V; DES, p.R386H; MYBPC3, p.C1124F; and MYL3, p.D126G), 3 variants of uncertain significance (RBM20, p.R1182H; MYH6, p.T1253M; and VCL, p.M209L), and 2 known mutations (MYH7, p.A26V and MYBPC3, p.R160W) were revealed to be associated with DCM. The mutations were most frequently found in the sarcomere (MYH6, MYBPC3, MYH7, TNNC1, TNNT2 and MYL3) and cytoskeletal (MYPN, DES and VCL) genes. As genetic testing is a useful tool in the clinical management of disease, testing for pathogenic mutations is beneficial to the treatment of patients with DCM and may assist in predicting disease risk for their family members before the onset of symptoms.

Study on electroless Ni-P-ZrO2 composite coatings on AZ91D magnesium alloys

Abstract Electroless Ni–P–ZrO2 composite coatings on AZ91D magnesium alloys was developed and their characteristics were investigated. The experimental results showed that the composite coatings were uniform and compact. The hardness of Ni–P–ZrO2 composite coatings reached the maximum value at 350°C heat treatment, and then decreased at 400°C. The nickel phosphor alloys provided solution strengthening and precipitation strengthening, while the ZrO2 nanoparticles provided dispersion hardening, making the composite coatings harder than that of Ni–P coatings. The wear resistance of Ni–P–ZrO2 composite coatings was obviously superior to Ni–P coatings, especially with longer wear time, which can be attributed to the dispersion strengthening and load support effect of ZrO2 in the coatings. The Ni–P–ZrO2 coatings exhibited better corrosion resistance than that of Ni–P coatings owing to codeposition of ZrO2 together with NiP leading to a more compact composite coating.

Purification, characterization and cloning of two novel tigerinin-like peptides from skin secretions of Fejervarya cancrivora

While investigating the innate defense of brackish water-living amphibian and its comparison with freshwater-living amphibians, two novel 12-residue antimicrobial peptides were purified from the skin secretions of the crab-eating frog, Fejervarya cancrivora which typically inhabits brackish water of mangrove forests of Southeast Asia. These two antimicrobial peptides, tigerinin-RC1 and -RC2 share significant structural similarity with tigerinins found in the skin of Indian frog, Hoplobatrachus tigerinus. cDNAs encoding tigerinin-RC1 and -RC2 were also cloned from the skin cDNA library of F. cancrivora. Tigerinin-RC precursors are composed of 71 amino acid residues including a signal peptide, acidic spacer peptide, which are very similar to other amphibian antimicrobial peptide precursors and mature tigerinin-RC. The current results confirmed that both amphibians inhabiting freshwater and brackish water share the same antimicrobial peptide family to exert innate defense. Furthermore, the current work was also the first report of precursor and cDNA cloning of the tigerinin antimicrobial peptide family.

Five novel antimicrobial peptides from skin secretions of the frog, Amolops loloensis

While investigating antimicrobial peptide diversity of Amolops loloensis, five novel antimicrobial peptides belonging to two families were identified from skin secretions of this frog. The first family including two members is esculentin-2-AL (esculentin-2-ALa and -ALb); the second family including three members is temporin-AL (temporin-ALd to -ALf). The family of esculentin-2-AL is composed of 37 amino acid residues (aa); the family of temporin-AL is composed of 16, 13 and 10 aa, respectively. All of these antimicrobial peptides showed antimicrobial activities against tested microorganisms. cDNAs encoding precursors of esculentin-2-ALs and temporin-ALs were cloned from the skin cDNA library of A. loloensis. All the precursors share similar overall structures. There is a typical prohormone processing signal (Lys-Arg) located between the acidic propiece and the mature peptide. The antimicrobial peptide family of esculentin-2 is firstly reported in the genus of Amolops. Combined with previous reports, a total of four antimicrobial peptide families have been identified from the genus of Amolops; three of them are also found in the genus of Rana. These results suggest the possible evolutionary connection between the genera Amolops and Rana.

A horsefly saliva antigen 5-like protein containing RTS motif is an angiogenesis inhibitor

The Ag5 proteins are the most abundant and immunogenic proteins in the venom secretory ducts of stinging insects. An antigen 5-like protein (named tabRTS) composed of 221 amino acid residues was purified and characterized from the salivary glands of the horsefly, Tabanus yao (Diptera, Tabanidae). Its cDNA was cloned from the cDNA library of the horseflys salivary gland. TabRTS containing the SCP domain (Sc7 family of extracellular protein domain) was found in insect antigen 5 proteins. More interestingly, there is an Arg-Thr-Ser (RTS) disintegrin motif at the C-terminus of tabRTS. The RTS motif is positioned in a loop bracketed by cysteine residues as those found in RTS-disintegrins of Crotalidae and Viperidae snake venoms, which act as angiogenesis inhibitors. Endothelial Cell Tube formation assay in vitro and chicken chorioallantoic membrane (CAM) angiogenesis assay in vivo were performed as to investigate the effect of tabRTS on angiogenesis. It was found that tabRTS could significantly inhibit angiogenesis in vitro and in vivo. Anti-alpha(1)beta(1) monoclonal antibody could dose-dependently inhibit the anti-angiogenic activity of tabRTS. This result indicated that tabRTS possibly targets the alpha(1)beta(1) integrin to exert the anti-angiogenic activity as snake venom RTS-/KTS-disintegrins do. The current work revealed the first angiogenesis inhibitor protein containing RTS motif from invertebrates, a possible novel type of RTS-disintegrin.

Scorpion Toxin, BmP01, Induces Pain by Targeting TRPV1 Channel

The intense pain induced by scorpion sting is a frequent clinical manifestation. To date, there is no established protocol with significant efficacy to alleviate the pain induced by scorpion envenomation. One of the important reasons is that, little information on pain-inducing compound from scorpion venoms is available. Here, a pain-inducing peptide (BmP01) has been identified and characterized from the venoms of scorpion (Mesobuthus martensii). In an animal model, intraplantar injection of BmP01 in mouse hind paw showed significant acute pain in wild type (WT) mice but not in TRPV1 knock-out (TRPV1 KO) mice during 30 min recording. BmP01 evoked currents in WT dorsal root ganglion (DRG) neurons but had no effect on DRG neurons of TRPV1 KO mice. Furthermore, BmP01 evoked currents on TRPV1-expressed HEK293T cells, but not on HEK293T cells without TRPV1. These results suggest that (1) BmP01 is one of the pain-inducing agents in scorpion venoms; and (2) BmP01 induces pain by acting on TRPV1. To our knowledge, this is the first report about a scorpion toxin that produces pain by targeting TRPV1. Identification of a pain-inducing compound may facilitate treating pain induced by scorpion envenomation.

High corrosion resistance multilayer nickel coatings on AZ91D magnesium alloys

Abstract The multilayer coatings consisting of electroless Ni–P, Ni–P–ZrO2 and electroplating Ni coatings were investigated. The surface and cross-section morphologies of coatings were observed with SEM. The microstructures were analysed by XRD. The corrosion resistance was evaluated by potentiodynamic polarisation curves and salt spray tests. The results showed that the electroplating Ni coating was crystal structure, whereas the electroless Ni–P and Ni–P–ZrO2 coatings displayed amorphous characteristic. The electroplating Ni coating exhibited more positive corrosion potential Ecorr and lower corrosion current density Icorr than that of electroless Ni–P and Ni–P–ZrO2 coatings. The samples covered with multilayer coatings of Ni–P–ZrO2/Ni/Ni–P (from substrate to surface) indicated the best corrosion resistance among six samples, and the salt spray tests were carried out for more than 1000 h. According to the analysis of corrosion mechanism, the upper Ni–P layer served as sacrificial protection anode due to its more negative corrosion potential Ecorr in comparison with the middle electroplating Ni layer. When the upper Ni–P layer was consumed, the middle Ni layer went to prevent from corroding. Until the corrosion pits penetrated all of the protective coatings, the substrate of magnesium alloys was destroyed completely.