An Aerolysin-like Pore-Forming Protein Complex Targets Viral Envelope to Inactivate Herpes Simplex Virus Type 1.

Because most of animal viruses are enveloped, cytoplasmic entry of these viruses via fusion with cellular membrane initiates their invasion. However, the strategies in which host cells counteract cytoplasmic entry of such viruses are incompletely understood. Pore-forming toxin aerolysin-like proteins (ALPs) exist throughout the animal kingdom, but their functions are mostly unknown. In this study, we report that ßγ-crystallin fused aerolysin-like protein and trefoil factor complex (ßγ-CAT), an ALP and trefoil factor complex from the frog Bombina maxima, directly blocks enveloped virus invasion by interfering with cytoplasmic entry. ßγ-CAT targeted acidic glycosphingolipids on the HSV type 1 (HSV-1) envelope to induce pore formation, as indicated by the oligomer formation of protein and potassium and calcium ion efflux. Meanwhile, ßγ-CAT formed ring-like oligomers of ∼10 nm in diameter on the liposomes and induced dye release from liposomes that mimic viral envelope. Unexpectedly, transmission electron microscopy analysis showed that the ßγ-CAT-treated HSV-1 was visibly as intact as the vehicle-treated HSV-1, indicating that ßγ-CAT did not lyse the viral envelope. However, the cytoplasmic entry of the ßγ-CAT-treated HSV-1 into HeLa cells was totally hindered. In vivo, topical application of ßγ-CAT attenuated the HSV-1 corneal infection in mice. Collectively, these results uncovered that ßγ-CAT possesses the capacity to counteract enveloped virus invasion with its featured antiviral-acting manner. Our findings will also largely help to illustrate the putative antiviral activity of animal ALPs.

A secreted pore-forming protein modulates cellular endolysosomes to augment antigen presentation.

Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that ßγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of ßγ-CAT. The cellular action of ßγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of ßγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.

Isolation and characterization of the major centipede allergen Sco m 5 from Scolopendra subspinipes mutilans.

BACKGROUND: Allergic reactions have been observed following both direct centipede bites and the clinical use of centipede-containing medicines, such as traditional Chinese medicines utilizing Scolopendra subspinipes mutilans; however, no natural centipede allergen has yet been characterized. METHODS: An allergen was purified from S. s. mutilans venom using Superdex 75 gel filtration and RESOURCE S ion chromatography, and its primary structure was determined via a combination of LC-MS-MS, MALDI-TOF/TOF and protein sequencing techniques. Its potential allergenicity was evaluated by immunoblotting, ELISAs, skin prick tests (SPTs) and mast cell activation assays. RESULTS: A novel allergen Sco m 5 (210 amino acids long) was successfully purified from crude S. s. mutilans venom. Sco m 5 could promote the degranulation of a human mast cell line, HMC-1. Among centipede-allergic patients, Sco m 5 showed an 83.3% IgE-binding frequency and a 66.7% positive reaction frequency, as detected by immunoblotting and SPTs, respectively. Sco m 5 IgE-binding frequencies of common Chinese population was found to be 9%-16%. Sera positive for Sco m 5 IgE-binding was cross-reactive against venom from the wasp Vespa mandaeinia. CONCLUSIONS: The present study isolated and characterized a novel allergen termed as Sco m 5 from the centipede S. s. mutilans. The use of Sco m 5 to identify centipede-allergic individuals could be important, given the high potential allergenicity of Sco m 5 among the general Chinese population, along with the likely possibility of cross-reactivity against wasp venom among centipede-allergic patients.

Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.

Tissue repair is a highly dynamic process, and the immediate onset of acute inflammation has been considered necessary for repair. Pore-forming proteins are important, both in pathogen invasion and host immunity. However, their roles in wound healing and tissue repair are unclear. ßγ-crystallin fused aerolysin-like protein (α-subunit) and trefoil factor (ß-subunit) complex (ßγ-CAT) is a complex of a bacterial pore-forming toxin aerolysin-like protein and trefoil factor identified in the frog Bombina maxima. In this study, we established mouse cutaneous wound models to explore the effects of ßγ-CAT on skin wound healing. ßγ-CAT accelerated the healing of full-thickness wounds by improving re-epithelialization. This complex relieved dermal edema and promoted scarless healing. ßγ-CAT treatment resulted in a rapid release of IL-1ß, which initiated an acute inflammation response in the early stage of healing. Meanwhile, the expression levels of TGF-ß1, VEGF, and bFGF and the recruitment of M2 macrophages around the wound significantly increased after ßγ-CAT treatment. ßγ-CAT protected skin wounds against methicillin-resistant Staphylococcus aureus by improving neutrophil recruitment at the site of the wound. Overall, our results suggest that ßγ-CAT can promote tissue repair and protect skin wounds against antibiotic-resistant bacterial infection by triggering the acute inflammatory response. This is the first example that aerolysin-like pore-forming proteins widely existing in plants and animals may act in wound healing and tissue repair.-Gao, Z.-H., Deng, C.-J., Xie, Y.-Y., Guo, X.-L., Wang, Q.-Q., Liu, L.-Z., Lee, W.-H., Li, S.-A., Zhang, Y. Pore-forming toxin-like protein complex expressed by frog promotes tissue repair.

Proteotranscriptomic Analysis and Discovery of the Profile and Diversity of Toxin-like Proteins in Centipede.

Centipedes are one of the oldest venomous animals and use their venoms as weapons to attack prey or protect themselves. Their venoms contain various components with different biomedical and pharmacological properties. However, little attention has been paid to the profiles and diversity of their toxin-like proteins/peptides. In this study, we used a proteotranscriptomic approach to uncover the diversity of centipede toxin-like proteins in Scolopendra subspinipes mutilans Nine hundred twenty-three and 6,736 peptides, which were separately isolated from venom and torso tissues, respectively, were identified by ESI-MS/MS and deduced from their transcriptomes. Finally, 1369 unique proteins were identified in the proteome, including 100 proteins that exhibited overlapping expression in venom and torso tissues. Of these proteins, at least 40 proteins were identified as venom toxin-like proteins. Meanwhile, transcriptome mining identified ∼10-fold more toxin-like proteins and enabled the characterization of the precursor architecture of mature toxin-like peptides. Importantly, combined with proteomic and transcriptomic analyses, 25 toxin-like proteins/peptides (neurotoxins accounted for 50%) were expressed outside the venom gland and involved in gene recruitment processes. These findings highlight the extensive diversity of centipede toxin-like proteins and provide a new foundation for the medical-pharmaceutical use of centipede toxin-like proteins. Moreover, we are the first group to report the gene recruitment activity of venom toxin-like proteins in centipede, similar to snakes.

Generation of dual specific bivalent BiTEs (dbBIspecific T-cell engaging antibodies) for cellular immunotherapy.

BACKGROUND: Bispecific T-cell engaging antibodies (BiTES), comprising dual anti-CD3 and anti-tumor antigen scFv fragments, are important therapeutic agents for the treatment of cancer. The dual scFv construct for BiTES requires proper protein folding while their small molecular size leads to rapid kidney clearance. METHODS: An intact (150 kDa) anti-tumor antigen antibody to CEA was joined in high yield (ca. 30%) to intact (150 kDa) anti-murine and anti-human CD3 antibodies using hinge region specific Click chemistry to form dual-specific, bivalent BiTES (dbBiTES, 300 kDa). dbBiTEs were tested in vitro by EM, flow cytometry and cell cytoxicity and in vivo by PET tumor imaging and redirected T-cell therapy. RESULTS: The interlocked hinge regions are compatible with a structural model that fits the electron micrographs of 300 kDa particles. Compared to intact anti-CEA antibody, dbBiTES exhibit high in vitro cytotoxicity, high in vivo tumor targeting as demonstrated by PET imaging, and redirected dbBiTE coated T-cells (1 microgram/10 million cells) that kill CEA+ target cells in vivo in CEA transgenic mice. CONCLUSION: dbBiTE redirected T-cell therapy is a promising, efficient approach for targeting and killing cancer cells.

Host Pore-Forming Protein Complex Neutralizes the Acidification of Endocytic Organelles to Counteract Intracellular Pathogens.

Many intracellular pathogens invade cells via endocytic organelles and have adapted to the drop in pH along the endocytic pathway. However, the strategy by which the host cell counteracts this pathogen adaptation remains unclear. ßγ-CAT is an aerolysin-like pore-forming protein and trefoil factor complex in the frog Bombina maxima. We report here that ßγ-CAT, as a host-secreted factor with an intrinsic channel-forming property, is the first example of a molecule that actively neutralizes the acidification of endocytic organelles to counteract Listeria monocytogenes infection. Immunodepletion of endogenous ßγ-CAT largely impaired the control of L. monocytogenes by frog cells. ßγ-CAT elevates the pH of L. monocytogenes-containing vacuoles to limit the vacuole escape of L. monocytogenes to cytosol. Furthermore, ßγ-CAT promotes intracellular L. monocytogenes clearance via autophagy and by that the nonlytic expulsion of the bacteria from host cells. Finally, ßγ-CAT attenuated the dissemination of L. monocytogenes in vivo. These findings reveal a novel host strategy and effectors that combat pathogen adaptation to acidic conditions along the endocytic pathway.

Characterization of an insulinotropic peptide from skin secretions of Odorrana andersonii.

Insulinotropic peptide agents are regarded as potential candidates for anti-diabetic treatment. In the present study, a novel insulinotropic peptide, termed OA-A1, was purified from frog skin secretions of Odorrana andersonii. Mature OA-A1 was determined to be a 1965.049 Da peptide with an amino acid sequence of LVGKLLKGAVGDVCGLLPIC, in which an intramolecular disulfide bridge was formed by two cysteine residues. At the cellular level, OA-A1 exhibited potent proliferation promoting effects on mouse-derived pancreatic ß-TC-6 cells and significantly stimulated insulin release in ß-TC-6 cells at a minimum concentration of 1 nM. In the animal model, OA-A1 also showed a dose-dependent insulin-releasing role in mice. At concentrations ranging from 1 nmol/kg to 1 µmol/kg, OA-A1 had a significant acute hypoglycemic effect on streptozotocin (STZ)-induced diabetic mice. The pancreatic islet areas of diabetic mice increased dose-dependently after 21 days of OA-A1 treatment (1-100 nmol/kg) compared with those of the saline control group. Moreover, OA-A1 significantly improved the oral glucose tolerance of STZ-induced diabetic mice. Taken together, these results suggest that OA-A1 provides an excellent template for the development of novel anti-diabetic therapeutic agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Host-derived, pore-forming toxin-like protein and trefoil factor complex protects the host against microbial infection.

Aerolysins are virulence factors belonging to the bacterial ß-pore-forming toxin superfamily. Surprisingly, numerous aerolysin-like proteins exist in vertebrates, but their biological functions are unknown. ßγ-CAT, a complex of an aerolysin-like protein subunit (two ßγ-crystallin domains followed by an aerolysin pore-forming domain) and two trefoil factor subunits, has been identified in frogs (Bombina maxima) skin secretions. Here, we report the rich expression of this protein, in the frog blood and immune-related tissues, and the induction of its presence in peritoneal lavage by bacterial challenge. This phenomena raises the possibility of its involvement in antimicrobial infection. When ßγ-CAT was administrated in a peritoneal infection model, it greatly accelerated bacterial clearance and increased the survival rate of both frogs and mice. Meanwhile, accelerated Interleukin-1ß release and enhanced local leukocyte recruitments were determined, which may partially explain the robust and effective antimicrobial responses observed. The release of interleukin-1ß was potently triggered by ßγ-CAT from the frog peritoneal cells and murine macrophages in vitro. ßγ-CAT was rapidly endocytosed and translocated to lysosomes, where it formed high molecular mass SDS-stable oligomers (>170 kDa). Lysosomal destabilization and cathepsin B release were detected, which may explain the activation of caspase-1 inflammasome and subsequent interleukin-1ß maturation and release. To our knowledge, these results provide the first functional evidence of the ability of a host-derived aerolysin-like protein to counter microbial infection by eliciting rapid and effective host innate immune responses. The findings will also largely help to elucidate the possible involvement and action mechanisms of aerolysin-like proteins and/or trefoil factors widely existing in vertebrates in the host defense against pathogens.

[Identification and bactericidal activity of a novel Cathelicidin family member from skin of Bufu bufo gargarizans].

The skin transcriptome of Bufu bufo gargarizans was determined by conventional methods. A novel full length cDNA coding for a Cathelicidin precursor was identified by transcriptomic data assembling, annotation and blast search of corresponding data banks. According to the known processing methods of Cathelicidin family members, present reported novel Cathelicidin precursor of B. bufo gargarizans might be cleaved at 2 possible sites of the same precursor and generate both BG-CATH25 and BG-CATH29 as mature molecules. The deduced BG-CATH25 and BG-CATH29 were synthesized with purity>95% to evaluate the properties and bactericidal activities. The secondary structural characteristics of both BG-CATH25 and BG-CATH29 in different solutions were determined by Circular Dichroism (CD) Analysis. CD results indicated that random coil conformation were the main structural elements for both BG-CATH25 and BG-CATH29 in different buffer systems. Antimicrobial activities against tested bacterial strains were carried out by plating method. Both BG-CATH25 and BG-CATH29 showed strong antibacterial activities against Aeromonas hydrophila, with MIC values of 1.25, 10 mg•L⁻¹, respectively. However, both of them showed weak bactericidal activities against human pathogenic bacteria, like Escherichia coli (ATCC25922）,Staphylococcus aureus (ATCC25923）and Pseudomonas aeruginosa (ATCC 27853).

Prohibitin is involved in the activated internalization and degradation of protease-activated receptor 1.

The protease-activated receptor 1 (PAR1) is a G-protein-coupled receptor that is irreversibly activated by either thrombin or metalloprotease 1. Due this irrevocable activation, activated internalization and degradation are critical for PAR1 signaling termination. Prohibitin (PHB) is an evolutionarily conserved, ubiquitously expressed, pleiotropic protein and belongs to the stomatin/prohibitin/flotillin/HflK/C (SPFH) domain family. In a previous study, we found that PHB localized on the platelet membrane and participated in PAR1-mediated human platelet aggregation, suggesting that PHB likely regulates the signaling of PAR1. Unfortunately, PHB's exact function in PAR1 internalization and degradation is unclear. In the current study, flow cytometry revealed that PHB expressed on the surface of endothelial cells (HUVECs) but not cancer cells (MDA-MB-231). Further confocal microscopy revealed that PHB dynamically associates with PAR1 in a time-dependent manner following induction with PAR1-activated peptide (PAR1-AP), though differently between HUVECs and MDA-MB-231 cells. Depletion of PHB by RNA interference significantly inhibited PAR1 activated internalization and led to sustained Erk1/2 phosphorylation in the HUVECs; however, a similar effect was not observed in MDA-MB-231 cells. For both the endothelial and cancel cells, PHB repressed PAR1 degradation, while knockdown of PHB led to increased PAR1 degradation, and PHB overexpression inhibited PAR1 degradation. These results suggest that persistent PAR1 signaling due to the absence of membrane PHB and decreased PAR1 degradation caused by the upregulation of intracellular PHB in cancer cells (such as MDA-MB-231 cells) may render cells highly invasive. As such, PHB may be a novel target in future anti-cancer therapeutics, or in more refined cancer malignancy diagnostics.

Inhibition of S-phase kinase-associated protein 2 (Skp2) reprograms and converts diabetogenic T cells to Foxp3+ regulatory T cells.

Autoreactive pathogenic T cells (Tpaths) and regulatory T cells (Tregs) express a distinct gene profiles; however, the genes and associated genetic/signaling pathways responsible for the functional determination of Tpaths vs. Tregs remain unknown. Here we show that Skp2, an E3 ubiquitin ligase that affects cell cycle control and death, plays a critical role in the function of diabetogenic Tpaths and Tregs. Down-regulation of Skp2 in diabetogenic Tpaths converts them into Foxp3-expressing Tregs. The suppressive function of the Tpath-converted Tregs is dependent on increased production of TGF-ß/IL-10, and these Tregs are able to inhibit spontaneous diabetes in NOD mice. Like naturally arising Foxp3(+) nTregs, the converted Tregs are anergic cells with decreased proliferation and activation-induced cell death. Skp2 down-regulation leads to Tpath-Treg conversion due at least in part to up-regulation of several genes involved in cell cycle control and genes in the Foxo family. Down-regulation of the cyclin-dependent kinase inhibitor p27 alone significantly attenuates the effect of Skp2 on Tpaths and reduces the suppressive function of converted Tregs; its effect is further improved with concomitant down-regulation of p21, Foxo1, and Foxo3. In comparison, Skp2 overexpression does not change Tpath function, but significantly decreases Foxp3 expression and abrogates the suppressive function of nTregs. These findings support the critical role of Skp2 in functional specification of Tpaths and Tregs, and demonstrate an important molecular mechanism mediating Skp2 function in balancing immune tolerance during autoimmune disease development.

Therapeutic potential of the antimicrobial peptide OH-CATH30 for antibiotic-resistant Pseudomonas aeruginosa keratitis.

The therapeutic potential of antimicrobial peptides (AMPs) has been evaluated in many infectious diseases. However, the topical application of AMPs for ocular bacterial infection has not been well investigated. The AMP OH-CATH30, which was identified in the king cobra, exhibits potent antimicrobial activity. In this study, we investigated the therapeutic potential of OH-CATH30 for Pseudomonas aeruginosa keratitis. Ten isolates of P. aeruginosa from individuals with keratitis were susceptible to OH-CATH30 but not to cefoperazone, ciprofloxacin, gentamicin, and levofloxacin. The microdilution checkerboard assay showed that OH-CATH30 exhibited synergistic activity with ciprofloxacin and levofloxacin against antibiotic-resistant P. aeruginosa. Meanwhile, P. aeruginosa did not develop resistance to OH-CATH30, even after exposure at 0.5× the MIC for up to 25 subcultures. Furthermore, treatment with OH-CATH30, alone or in combination with levofloxacin, significantly improved the clinical outcomes of rabbit keratitis induced by antibiotic-resistant P. aeruginosa. Taken together, our data indicate that the topical application of OH-CATH30 is efficacious against drug-resistant P. aeruginosa keratitis. In addition, our study highlights the potential application of AMPs in treating ocular bacterial infections.

Killer cell Ig-like receptor (KIR) 3DL1 down-regulation enhances inhibition of type 1 diabetes by autoantigen-specific regulatory T cells.

Both Foxp3(+) regulatory T cells (Tregs) and antigen-expanded Foxp3(-) Tregs play an important role in regulating immune responses as well as in preventing autoimmune diseases and graft rejection. Molecular mechanisms modulating Treg function remain largely unclear, however. We report here on the expression and function of an inhibitory killer cell Ig-like receptor, KIR3DL1, in a nonobese diabetic (NOD) mouse-derived autoantigen-specific Treg (2D2), which protects from type 1 diabetes (T1D) in adoptive transfer experiments. This gene is not expressed in T1D pathogenic T cells (Tpaths) or non-Tpath T cells. KIR genes are known to play an important role in regulating natural killer (NK) cell function, but their role in Tregs and T1D is unknown. To examine whether KIR3DL1 expression may modulate Treg function, we used shRNA to down-regulate KIR3DL1 expression (2D2-shKIR). We find that KIR3DL1 down-regulation enhances in vitro function, as measured by improved suppression of target cell proliferation. Antibody blockade of IL-10 but not IL-4 partially abrogated suppressive function. In vivo function is also improved. Adoptive transfer of 2D2-shKIR into 10-wk-old NOD mice prevented spontaneous insulitis and T1D, and the inhibitory effect was further improved if the cells were transferred earlier into 6-wk-old NOD mice. These studies indicate that KIR3DL1 expression may negatively regulate Treg function and suggest a previously undescribed target for improving immune tolerance for potential treatment of autoimmune diseases like T1D.

Controlled Cascade-Release and High Selective Sterilization by Core-Shell Nanogels for Microenvironment Regulation of Aerobic Vaginitis.

Aerobic vaginitis (AV) is a gynecological disease associated with vaginal flora imbalance. The nonselective bactericidal nature of antibiotics and low customization rate of probiotic supplementation in existing treatments lead to AV recurrence. Here, a drug delivery strategy is proposed that works with the changing dynamics of the bacterial flora. In particular, a core-shell nanogel (CSNG) is designed to encapsulate prebiotic inulin and antimicrobial peptide Cath 30. The proposed strategy allows for the sequential release of both drugs using gelatinase produced by AV pathogenic bacteria, initially selectively killing pathogenic bacteria and subsequently promoting the proliferation of beneficial bacteria in the vagina. In a simulated infection environment in vitro, the outer layer of CSNGs, Cath 30 is rapidly degraded and potently killed the pathogenic bacterium Staphylococcus aureus at 2-6 h. CSNGs enhances proliferation of the beneficial bacterium Lactobacillus crispatus by more than 50% at 24 h. In a rat AV model, the drug delivery strategy precisely regulated the bacterial microenvironment while controlling the inflammatory response of the vaginal microenvironment. This new treatment approach, configured on demand and precisely controlled, offers a new strategy for the treatment of vaginal diseases.

Extremely abundant antimicrobial peptides existed in the skins of nine kinds of Chinese odorous frogs.

Peptide agents are regarded as hopeful candidates to solve life-threatening resistance of pathogenic microorganisms to classic antibiotics due to their unique action mechanisms. Peptidomic and genomic investigation of natural antimicrobial peptides (AMPs) from amphibian skin secretions can provide a large amount of structure-functional information to design peptide antibiotics with therapeutic potential. In the present study, we identified a large number of AMPs from the skins of nine kinds of Chinese odorous frogs. Eighty AMPs were purified from three different odorous frogs and confirmed by peptidomic analysis. Our results indicated that post-translational modification of AMPs rarely happened in odorous frogs. cDNAs encoding precursors of 728 AMPs, including all the precursors of the confirmed 80 native peptides, were cloned from the constructed AMP cDNA libraries of nine Chinese odorous frogs. On the basis of the sequence similarity of deduced mature peptides, these 728 AMPs were grouped into 97 different families in which 71 novel families were identified. Out of these 728 AMPs, 662 AMPs were novel and 28 AMPs were reported previously in other frog species. Our results revealed that identical AMPs were widely distributed in odorous frogs; 49 presently identified AMPs could find their identical molecules in different amphibian species. Purified peptides showed strong antimicrobial activities against 4 tested microbe strains. Twenty-three deduced peptides were synthesized and their bioactivities, including antimicrobial, antioxidant, hemolytic, immunomodulatory and insulin-releasing activities, were evaluated. Our findings demonstrate the extreme diversity of AMPs in amphibian skins and provide plenty of templates to develop novel peptide antibiotics.

Venomic and transcriptomic analysis of centipede Scolopendra subspinipes dehaani.

Centipedes have venom glands in their first pair of limbs, and their venoms contain a large number of components with different biochemical and pharmacological properties. However, information about the compositions and functions of their venoms is largely unknown. In this study, Scolopendra subspinipes dehaani venoms were systematically investigated by transcriptomic and proteomic analysis coupled with biological function assays. After random screening approximately 1500 independent clones, 1122 full length cDNA sequences, which encode 543 different proteins, were cloned from a constructed cDNA library using a pair of venom glands from a single centipede species. Neurotoxins, ion channel acting components and venom allergens were the main fractions of the crude venom as revealed by transcriptomic analysis. Meanwhile, 40 proteins/peptides were purified and characterized from crude venom of S. subspinipes dehaani. The N-terminal amino acid sequencing and mass spectrum results of 29 out of these 40 proteins or peptides matched well with their corresponding cDNAs. The purified proteins/peptides showed different pharmacological properties, including the following: (1) platelet aggregating activity; (2) anticoagulant activity; (3) phospholipase A(2) activity; (4) trypsin inhibiting activity; (5) voltage-gated potassium channel activities; (6) voltage-gated sodium channel activities; (7) voltage-gated calcium channel activities. Most of them showed no significant similarity to other protein sequences deposited in the known public database. This work provides the largest number of protein or peptide candidates with medical-pharmaceutical significance and reveals the toxin nature of centipede S. subspinipes dehaani venom.

Efficacy of OH-CATH30 and its analogs against drug-resistant bacteria in vitro and in mouse models.

Antimicrobial peptides (AMPs) have been considered alternatives to conventional antibiotics for drug-resistant bacterial infections. However, their comparatively high toxicity toward eukaryotic cells and poor efficacy in vivo hamper their clinical application. OH-CATH30, a novel cathelicidin peptide deduced from the king cobra, possesses potent antibacterial activity in vitro. The objective of this study is to evaluate the efficacy of OH-CATH30 and its analog OH-CM6 against drug-resistant bacteria in vitro and in vivo. The MICs of OH-CATH30 and OH-CM6 ranged from 1.56 to 12.5 µg/ml against drug-resistant clinical isolates of several pathogenic species, including Escherichia coli, Pseudomonas aeruginosa, and methicillin-resistant Staphylococcus aureus. The MICs of OH-CATH30 and OH-CM6 were slightly altered in the presence of 25% human serum. OH-CATH30 and OH-CM6 killed E. coli quickly (within 60 min) by disrupting the bacterial cytoplasmic membrane. Importantly, the 50% lethal doses (LD(50)) of OH-CATH30 and OH-CM6 in mice following intraperitoneal (i.p.) injection were 120 mg/kg of body weight and 100 mg/kg, respectively, and no death was observed at any dose up to 160 mg/kg following subcutaneous (s.c.) injection. Moreover, 10 mg/kg OH-CATH30 or OH-CM6 significantly decreased the bacterial counts as well as the inflammatory response in a mouse thigh infection model and rescued infected mice in a bacteremia model induced by drug-resistant E. coli. Taken together, our findings demonstrate that the natural cathelicidin peptide OH-CATH30 and its analogs exhibit relatively low toxicity and potent efficacy in mouse models, indicating that they may have therapeutic potential against the systemic infections caused by drug-resistant bacteria.

CD4+ CD25+ regulatory T cells prevent type 1 diabetes preceded by dendritic cell-dominant invasive insulitis by affecting chemotaxis and local invasiveness of dendritic cells.

Development of type 1 diabetes (T1D) is preceded by invasive insulitis. Although CD4(+)CD25(+) regulatory T cells (nTregs) induce tolerance that inhibits insulitis and T1D, the in vivo cellular mechanisms underlying this process remain largely unclear. Using an adoptive transfer model and noninvasive imaging-guided longitudinal analyses, we found nTreg depletion did not affect systemic trafficking and tissue localization of diabetogenic CD4(+) BDC2.5 T (BDC) cells in recipient mice prior to development of T1D. In addition, neither the initial expansion/activation of BDC cells nor the number of CD11c(+) or NK cells in islets and pancreatic lymph nodes were altered. Unexpectedly, our results showed nTreg depletion led to accelerated invasive insulitis dominated by CD11c(+) dendritic cells (ISL-DCs), not BDC cells, which stayed in the islet periphery. Compared with control mice, the phenotype of ISL-DCs and their ability to stimulate BDC cells did not change during invasive insulitis development. However, ISL-DCs from nTreg-deficient recipient mice showed increased in vitro migration toward CCL19 and CCL21. These results demonstrated invasive insulitis dominated by DCs, not CD4(+) T cells, preceded T1D onset in the absence of nTregs, and suggested a novel in vivo function of nTregs in T1D prevention by regulating local invasiveness of DCs into islets, at least partly, through regulation of DC chemotaxis toward CCL19/CCL21 produced by the islets.

16S rDNA analysis of osteoporotic rats treated with osteoking.

Introduction. Osteoporosis (OP) is characterized by microstructural degeneration of bone tissue, low bone mass, bone fragility and even brittle fracture (osteoporotic fracture, OPF). OP and OPF are common and there are many disadvantages to the current medications for OP/OPF. Osteoking is a traditional Chinese medicine (TCM) originating from the Yi nationality (Yunnan, China) that has been used to treat bone diseases for decades.Hypothesis/Gap Statement. This study will reveal the changes in the intestinal microbiota of OP rats after 70 days of osteoking treatment.Method. With duplication of sham and OP rats, eight groups were established, with six rats in each group. The intestinal microbiotas were analysed by 16S rDNA sequencing.Results. The results showed that osteoking changed the intestinal microbiota of sham rats and OP rats. The mechanism by which osteoking improves OP is related to the functions of the intestinal microbiota. After 70 days of treatment with osteoking, the contents of Pseudonocardia, Pedomicrobium, Variovorax, Niastella and Actinosynnema were decreased in OP rats. The functions of the above intestinal microbiota related to iron metabolism affected calcifediol and 25(OH)D, and measuring these bone metabolic indicators is required for further study.Conclusion. Osteoking changes the intestinal microbiota to improve OP, and further study which reveals these intestinal microbiota and mechanism is needed.