Dermaseptin B2 bioactive gene's potential for anticancer and anti-proliferative effect is linked to the regulation of the BAX/BBC3/AKT pathway.

Dermaseptin B2 (DrsB2) is an antimicrobial peptide with anticancer and angiostatic properties. We aimed to assess the in vitro inhibitory effect of pDNA/DrsB2 on the growth of breast cancer cells and its impact on the expression of genes involved in the BAX/BBC3/AKT pathway. The nucleic acid sequence of DrsB2 was artificially synthesized and inserted into the pcDNA3.1( +) Mammalian Expression Plasmid. PCR testing and enzyme digesting procedures evaluated the accuracy of cloning. The vectors were introduced into cells using LipofectamineTM2000 transfection reagent. The breast cancer cells were assessed by flow cytometry, MTT assessment, soft agar colony method, and wound healing investigation. The gene's transcription was evaluated using real-time PCR with a significance level of P < 0.05. The recombinant plasmid harboring the pDNA/DrsB2 vector was effectively produced, and the gene sequence showed absolute homogeneity (100% similarity) with the DrsB2 gene. The transfection effectiveness of MCF-7 and MCF-10A cells was 79% and 68%, respectively. The findings are measured using the growth inhibition 50% (GI50) metric, which indicates the concentration of pDNA/DrsB2 that stops 50% of cell growth. The proportions of early apoptosis, late apoptosis, necrosis, and viable MCF-7 cells in the pDNA/DrsB2 group were 40.50%, 2.31%, 1.69%, and 55.50%, respectively. The results showed a 100% increase in gene expression in programmed cell death following treatment with pDNA/DrsB2 (**P < 0.01). To summarize, the results described in this work offer new possibilities for treating cancer by targeting malignancies via pDNA/DrsB2 and activating the BAX/BBC3/AKT signaling pathways.

Characterization, expression pattern and antiviral activities of oligoadenylate synthetase in Chinese Giant Salamander, Andrias davidianus.

The enzyme 2'-5'-oligoadenylate synthetase (OAS) is an antiviral protein induced by interferons (IFNs), which plays an important role in IFN-mediated antiviral signaling pathway. In this study, the OAS of Chinese Giant Salamander, Andrias davidianus (AdOAS) was identified for the first time, and the expression profiles in vivo and the antiviral activities in vitro were investigated. The open reading frame (ORF) of AdOAS gene is 1185 bp in length, encoding a putative protein of 394 amino acids, in which a Nucleotidyltransferase (NTase) domain (40-143 aa) and a conserved OAS1 C superfamily domain (165-341 aa) are included. qRT-PCR analysis revealed a broad expression of AdOAS in vivo, with the highest expression level in intestine and heart. After infection with Chinese giant salamander iridovirus (GSIV), the mRNA level of AdOAS in liver increased significantly at 24 h and 48 h post infection and reached the peak at 72 h compared with the control group. The AdOAS mRNA level in kidney increased slightly at 6 h and 12 h post infection, declined to the initial level at 24 h and peaked at 48 h post infection, while in spleen it was slightly up-regulated at 6 h, inhibited at 12 h, 24 h and 48 h, and then significantly increased to the peak at 72 h post infection. In vitro, AdOAS mRNA level in Chinese giant salamander muscle (GSM) cells was not noticeably up-regulated until 24 h and then peaked at 48 h post GSIV infection. In antiviral activity test, the mRNA transcription and protein level of virus major capsid protein (MCP) in AdOAS over-expressed cells was significantly reduced compared with that in control cells by qRT-PCR and western blot analysis. In addition, ddPCR results showed that lower MCP gene copy was found in AdOAS over-expressed cells compared with the control group. These results collectively suggest that AdOAS plays a crucial role against GSIV infection in Chinese giant salamander, and provide a solid base for the further studies on the mechanism of immune defense and the control of the disease in this animal.

Drivers and suppressors of triple-negative breast cancer.

To identify regulators of triple-negative breast cancer (TNBC), gene expression profiles of malignant parts of TNBC (mTNBC) and normal adjacent (nadj) parts of the same breasts have been compared. We are interested in the roles of estrogen receptor ß (ERß) and the cytochrome P450 family (CYPs) as drivers of TNBC. We examined by RNA sequencing the mTNBC and nadj parts of five women. We found more than a fivefold elevation in mTNBC of genes already known to be expressed in TNBC: BIRC5/survivin, Wnt-10A and -7B, matrix metalloproteinases (MMPs), chemokines, anterior gradient proteins, and lysophosphatidic acid receptor and the known basal characteristics of TNBC, sox10, ROPN1B, and Col9a3. There were two unexpected findings: 1) a strong induction of CYPs involved in activation of fatty acids (CYP4), and in inactivation of calcitriol (CYP24A1) and retinoic acid (CYP26A1); and 2) a marked down-regulation of FOS, FRA1, and JUN, known tethering partners of ERß. ERß is expressed in 20 to 30% of TNBCs and is being evaluated as a target for treating TNBC. We used ERß+ TNBC patient-derived xenografts in mice and found that the ERß agonist LY500703 had no effect on growth or proliferation. Expression of CYPs was confirmed by immunohistochemistry in formalin-fixed and paraffin-embedded (FFPE) TNBC. In TNBC cell lines, the CYP4Z1-catalyzed fatty acid metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) increased proliferation, while calcitriol decreased proliferation but only after inhibition of CYP24A1. We conclude that CYP-mediated pathways can be drivers of TNBC but that ERß is unlikely to be a tumor suppressor because the absence of its main tethering partners renders ERß functionless on genes involved in proliferation and inflammation.

Frog Skin Derived Peptides With Potential Protective Effects on Ultraviolet B-Induced Cutaneous Photodamage.

Hyla annectans is a tree frog living in the southwestern plateau area of China where there is strong ultraviolet radiation and long duration of sunshine. So their naked skin may possess chemical defense components that protect it from acute photo-damage. However, no such peptide or components has been identified till to date. In the current work, two novel peptides (FW-1, FWPLI-NH2 and FW-2, FWPMI-NH2) were identified from the skin of the tree frog. Five copies of FW-1 and four copies of FW-2 are encoded by an identical gene and released from the same protein precursor, which possess 167 amino acid residues. FW-1 and -2 can exert significant anti-inflammatory functions by directly inhibiting Ultraviolet B irradiation (UVB)-induced secretion of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). They may achieve this function by modulating the UV-induced stress signaling pathways such as Mitogen-activated protein kinases (MAPK) and Nuclear Factor Kappa B (NF-κB). Besides, FW-1 and -2 showed potential antioxidant effects on epidermis by attenuating the UVB-induced reactive oxygen species (ROS) production through an unknown mechanism. Considering small peptides' easy production, storage, and potential photo-protective activity, FW-1/2 might be exciting leading compounds or templates for the development of novel pharmacological agents for the suppression of UVB-induced skin inflammation. Moreover, this study might expand our knowledge on skin defensive mechanism of tree frog upon UVB irradiation.

Thyroid Hormone Receptor Is Essential for Larval Epithelial Apoptosis and Adult Epithelial Stem Cell Development but Not Adult Intestinal Morphogenesis during Xenopus tropicalis Metamorphosis.

Vertebrate postembryonic development is regulated by thyroid hormone (T3). Of particular interest is anuran metamorphosis, which offers several unique advantages for studying the role of T3 and its two nuclear receptor genes, TRα and TRß, during postembryonic development. We have recently generated TR double knockout (TRDKO) Xenopus tropicalis animals and reported that TR is essential for the completion of metamorphosis. Furthermore, TRDKO tadpoles are stalled at the climax of metamorphosis before eventual death. Here we show that TRDKO intestine lacked larval epithelial cell death and adult stem cell formation/proliferation during natural metamorphosis. Interestingly, TRDKO tadpole intestine had premature formation of adult-like epithelial folds and muscle development. In addition, T3 treatment of premetamorphic TRDKO tadpoles failed to induce any metamorphic changes in the intestine. Furthermore, RNA-seq analysis revealed that TRDKO altered the expression of many genes in biological pathways such as Wnt signaling and the cell cycle that likely underlay the inhibition of larval epithelial cell death and adult stem cell development caused by removing both TR genes. Our data suggest that liganded TR is required for larval epithelial cell degeneration and adult stem cell formation, whereas unliganded TR prevents precocious adult tissue morphogenesis such as smooth-muscle development and epithelial folding.

A novel strategy for engineering of a smart generation of immune ribonucleases against EGFR+ cells.

The overexpression of epidermal growth factor receptor (EGFR) could result in the development of solid tumors of prostate, breast, gastric, colorectal, ovarian, and head and neck, leading to carcinoma. Antibody therapies are ideal methods to overcome malignant diseases. However, immunoribonucleases are a new generation of antibodies in which an RNase binds to a specific antibody and shows a stronger ability to terminate cancer cells. In this study, we engineered Rana pipiens RNase to bind to the scFv of human antiepidermal growth factor receptor antibody. The molecular dynamic simulations confirmed protein stability and the ability of scFv-ranpirnase (rantoxin) to bind to epidermal growth factor receptor protein. Then, the rantoxin construct was synthesized in a pCDNA 3.1 Neo vector. CHO-K1 cells were used as expression hosts and the construct was transfected. Cells were selected by antibiotic therapies using neomycin, 120 mg/ml, and the high-yield colony was screened by real-time polymerase chain reaction (PCR) methods. Then, the recombinant protein production was confirmed using the sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot analyses. The molecular dynamic simulation (MDS) confirmed that the I467, S468, Q408, and H409 amino acids of EGFR bonded well to rantoxin. As revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analyses, the rantoxin production and PCR analysis showed that the T3 colony can produce rantoxin messenger RNA fourfold higher than the GAPDH gene. The immunotoxin function was assessed in A431 cancer cells and EGFR-negative HEK293 cells, and IC50  values were estimated to be 22.4 ± 3 and >620.4 ± 5 nM, respectively. The results indicated that the immunotoxins produced in this study had the potential for use as anticancer drugs.

Identification and characterization of novel bi-functional cathelicidins from the black-spotted frog (Pelophylax nigromaculata) with both anti-infective and antioxidant activities.

Cathelicidins are an important family of antimicrobial peptides (AMPs), which play pivotal roles in vertebrate immune responses against microbial infections. They are regarded as potential drug leads for the development of novel antimicrobial agents and three related drugs have been developed into clinical trials. Thus, it is meaningful to identify more cathelicidins from vertebrate species. Cathelicidins from ranid frogs possess special structural characteristics and activities, but to date only 12 ranid frog cathelicidins have been identified. In the present study, two novel cathelicidins (PN-CATH1 and 2) were identified from the black-spotted frog, Pelophylax nigromaculata. PN-CATHs possess low sequence similarity with the known cathelicidins. They exhibited moderate, but broad-spectrum and rapid antimicrobial activities against the tested bacteria. They kill bacteria by mainly inducing bacterial membrane disruption and possibly generating intracellular ROS formation. They also possess potent anti-biofilm and persister cell killing activity, indicating their potential in combating infections induced by biofilms-forming bacteria. Besides direct antimicrobial activity, they exhibited potent anti-inflammatory activity by effectively inhibiting the LPS-induced production of pro-inflammatory cytokines in mouse macrophages, which could be partly ascribed to their direct LPS-neutralizing ability. Furthermore, PN-CATHs demonstrated powerful in vitro free radical scavenging activities. Ultraviolet radiation significantly increased their in vivo gene expression in frog skin. Meanwhile, they possess weak cytotoxic activity and extremely low hemolytic activity. PN-CATHs represent the first discovery of cathelicidins family AMPs with both potent anti-infective and antioxidant activities. The discovery of PN-CATHs provides potential peptide leads for the development of novel anti-infective and antioxidant drugs.

Characterisation of a novel peptide, Brevinin-1H, from the skin secretion of Amolops hainanensis and rational design of several analogues.

As drug-resistant bacteria have become a serious health problem and have caused thousands of deaths, finding new antibiotics has become an urgent research priority. A novel antimicrobial peptide, named Brevinin-1H, was identified in the skin secretion of Amolops hainanensis through 'shotgun' cloning. It has broad-spectrum antimicrobial activity against tested micro-organisms and has anticancer cell activity. To improve its bioactivity and decrease its cytotoxicity, two structural analogues-Brevinin-1Ha and Brevinin-1HY-were designed based on the secondary structure of the natural peptide. Brevinin-1HY, in which tyrosine substituted Pro11 , had similar activity to the natural peptide against Gram-negative bacteria and cancer cells, but showed a dramatic increase in haemolytic activity and cytotoxicity at its minimum inhibitory concentration. Brevinin-1Ha, which transferred the Rana-box from the C-terminal to a central position, had significantly decreased haemolytic activity, but also in antimicrobial and anticancer activity. The present data suggest that increasing the proportion of α-helix structure in an AMP can increase its target micro-organism bioactivity to some extent.

Exploring the relationships between amphibian (Xenopus laevis) myeloid cell subsets.

The differentiation of distinct leukocyte subsets is governed by lineage-specific growth factors that elicit disparate expression of transcription factors and markers by the developing cell populations. For example, macrophages (Mφs) and granulocytes (Grns) arise from common granulocyte-macrophage progenitors in response to distinct myeloid growth factors. In turn, myelopoiesis of the Xenopus laevis anuran amphibian appears to be unique to other studied vertebrates in several respects while the functional differentiation of amphibian Mφs and Grns from their progenitor cells remains poorly understood. Notably, the expression of colony stimulating factor-1 receptor (CSF-1R) or CSF-3R on granulocyte-macrophage progenitors marks their commitment to Mφ- or Grn-lineages, respectively. CSF-1R is activated by the colony stimulating factor-1 (CSF-1) and interleukin (IL-34) cytokines, resulting in morphologically and functionally distinct Mφ cell types. Conversely, CSF-3R is ligated by CSF-3 in a process indispensable for granulopoiesis. Presently, we explore the relationships between X. laevis CSF-1-Mφs, IL-34-Mφs and CSF-3-Grns by examining their expression of key lineage-specific transcription factor and myeloid marker genes as well as their enzymology. Our findings suggest that while the CSF-1- and IL-34-Mφs share some commonalities, the IL-34-Mφs possess transcriptional patterns more akin to the CSF-3-Grns. IL-34-Mφs also possess robust expression of dendritic cell-associated transcription factors and surface marker genes, further underlining the difference between this cell type and the CSF-1-derived frog Mφ subset. Moreover, the three myeloid populations differ in their respective tartrate-resistant acid phosphatase, specific- and non-specific esterase activity. Together, this work grants new insights into the developmental relatedness of these three frog myeloid subsets.

Cloning of Hynobius lichenatus (Tohoku hynobiid salamander) p53 and analysis of its expression in response to radiation.

BACKGROUND: Caudata species such as salamanders are easily affected by environmental changes, which can drastically reduce their population. The effects of acute X-rays and chronic γ-irradiation on Hynobius lichenatus, the Japanese Tohoku hynobiid salamander, are known. However, the expression of radiation-inducible genes, such as the DNA-damage checkpoint response gene p53, has not been analyzed in H. lichenatus. This has not occurred because there is no established method for mRNA quantification in H. lichenatus due to a lack of information on available nucleotide sequences corresponding to both radiation-inducible genes and endogenous control genes such as ACTB (ß-actin). RESULTS: In this study, we aimed to evaluate the effects of radiation on gene expression in H. lichenatus. Using RNA extracted from irradiated salamanders, we performed rapid amplification of cDNA ends (RACE) and cloned H. lichenatus ß-actin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and p53. We confirmed that the cloned cDNAs were able to synthesize salamander proteins by western blotting after transfection into cultured HEK293 cells. Proliferation assays using HEK293 cells stably expressing H. lichenatus p53 protein showed that this protein has antiproliferative effects, similar to that of mammalian p53. Furthermore, RT-qPCR analysis using gene-specific primers revealed that p53 mRNA expression in H. lichenatus was upregulated upon exposure to radiation. CONCLUSION: Our results suggest that H. lichenatus p53 protein take an important role in regulating the cellular responses to various stimuli as mammalian p53 does. Furthermore, our study provides novel data to select appropriate primers to analyze internal control mRNA expression in H. lichenatus and to evaluate p53 expression as a marker of radiation and environmental stimuli.

Purification of Soluble Membrane-Bound Ambystoma mexicanum Epidermal Lipoxygenase from E. coli and Its Growth Effect on Human Fetal Foreskin Fibroblast.

Lipoxygenases are non-heme iron-containing lipid dioxygenases enzymes that catalyze the hydroperoxidation of lipids. The Mexican axolotl (Ambystoma mexicanum) is a prominent source of the enzyme with a regeneration capacity in limbs. It has been shown that transfected human osteosarcoma and keratinocyte cells with epidermal lipoxygenase (LOXe) have an increased rate of cell migration. In the present study, LOXe, a peripheral membrane protein, was produced in Escherichia coli. The enzyme was purified using different detergents, anionic solutions, and gel filtration chromatography. Kinetic assay of the enzyme activity was carried out by the spectroscopy method using arachidonic acid as a substrate. Finally, the enzyme was characterized and its growth effect on human fibroblast cells was examined by MTT viability assay. Enzyme kinetic parameters including Km of 90.4 µM and Vmax of 2.63 IU were determined for LOXe. The enzyme with 0.1 nM end concentration promoted the growth of 5000 cells/well human fibroblast cells up to 11% (P < 0.01). In the present study, we introduce an E. coli expression system to produce an excessive amount of soluble LOXe and the efficient purification method to provide a soluble and active form of LOXe that is effective in stimulating human fibroblast cell proliferation.

Brevinin-1GHd: a novel Hylarana guentheri skin secretion-derived Brevinin-1 type peptide with antimicrobial and anticancer therapeutic potential.

Host-defense antimicrobial peptides (AMPs) from amphibians are usually considered as one of the most promising next-generation antibiotics because of their excellent antimicrobial properties and low cytotoxicity. In the present study, one novel Brevinin-1 type peptide, Brevinin-1GHd, was isolated and characterized from the skin secretion of the frog, Hylarana guentheri. Brevinin-1GHd was found to possess a wide range of antimicrobial activity through penetrating the bacterial membrane within a short time while showing low hemolysis at bactericidal concentrations, even against the resistant strains. It also inhibited and eradicated biofilms that are thought to be closely related to the rise in resistance. Meanwhile, Brevinin-1GHd exhibited wide-spectrum anti-proliferation activity toward human cancer lines. Taken together, these results indicate that Brevinin-1GHd with its excellent antimicrobial and anticancer activities is a promising candidate for a novel antibiotic agent, and study of its structure-activity relationships also provided a rational template for further research and peptide analog design.

Neutralization of Bombina variegata peptide 8 suppresses retinal neovascularization in two different murine models: The oxygen-induced retinopathy model and the rhodopsin promoter/VEGF transgenic mouse model.

Bombina variegata 8 (Bv8), also known as prokineticin-2 (PK-2), is a potent pro-angiogenic factor. However, its role in retinal neovascularization (RNV) remains unknown. In this study, we explored the role of Bv8 in the pathogenesis of RNV. We found that the expression of Bv8 was significantly increased in two different models of retinal neovascularization: the oxygen-induced retinopathy (OIR) mouse model and the rhodopsin promoter (rho)/VEGF transgenic mouse model. Neutralization of Bv8 by intravitreal injections of its antibody, not only inhibited retinal and subretinal neovascularization but also decreased the mRNA and protein levels of several pro-angiogenic factors. Our in vitro assay showed that recombinant human Bv8 (RhBv8) protein promoted human retinal microvascular endothelial cells (HRECs) tube-formation, cell proliferation and vascular endothelial growth factor receptor 1 (VEGFR1) and receptor 2 (VEGFR2) expression. Our findings suggest that Bv8 could be used as a novel target for the treatment of RNV-related ocular diseases.

Bioevaluation of Ranatuerin-2Pb from the Frog Skin Secretion of Rana pipiens and its Truncated Analogues.

Antimicrobial peptides (AMPs) are considered as a promising agent to overcome the drug-resistance of bacteria. Large numbers of AMPs have been identified from the skin secretion of Rana pipiens, including brevinins, ranatuerins, temporins and esculentins. In this study, the cDNA precursor of a broad-spectrum antimicrobial peptide, ranatuerin-2Pb, was cloned and identified. Additionally, two truncated analogues, RPa and RPb, were synthesised to investigate the structure-activity relationship of ranatuerin-2Pb. RPa lost antimicrobial activity against Candida albicans, MRSA, Enterococcus faecalis and Pseudomonas aeruginosa, while RPb retained its broad-spectrum antimicrobial activity. Additionally, ranatuerin-2Pb, RPa and RPb demonstrated inhibition and eradication effects against Staphylococcusaureus biofilm. RPb showed a rapid bacterial killing manner via membrane permeabilization without damaging the cell membrane of erythrocytes. Moreover, RPb decreased the mortality of S. aureus infected Galleria mellonella larvae. Collectively, our results suggested that RPb may pave a novel way for natural antimicrobial drug design.

What lies beneath? Molecular evolution during the radiation of caecilian amphibians.

BACKGROUND: Evolution leaves an imprint in species through genetic change. At the molecular level, evolutionary changes can be explored by studying ratios of nucleotide substitutions. The interplay among molecular evolution, derived phenotypes, and ecological ranges can provide insights into adaptive radiations. Caecilians (order Gymnophiona), probably the least known of the major lineages of vertebrates, are limbless tropical amphibians, with adults of most species burrowing in soils (fossoriality). This enigmatic order of amphibians are very distinct phenotypically from other extant amphibians and likely from the ancestor of Lissamphibia, but little to nothing is known about the molecular changes underpinning their radiation. We hypothesised that colonization of various depths of tropical soils and of freshwater habitats presented new ecological opportunities to caecilians. RESULTS: A total of 8540 candidate groups of orthologous genes from transcriptomic data of five species of caecilian amphibians and the genome of the frog Xenopus tropicalis were analysed in order to investigate the genetic machinery behind caecilian diversification. We found a total of 168 protein-coding genes with signatures of positive selection at different evolutionary times during the radiation of caecilians. The majority of these genes were related to functional elements of the cell membrane and extracellular matrix with expression in several different tissues. The first colonization of the tropical soils was connected to the largest number of protein-coding genes under positive selection in our analysis. From the results of our study, we highlighted molecular changes in genes involved in perception, reduction-oxidation processes, and aging that likely were involved in the adaptation to different soil strata. CONCLUSIONS: The genes inferred to have been under positive selection provide valuable insights into caecilian evolution, potentially underpin adaptations of caecilians to their extreme environments, and contribute to a better understanding of fossorial adaptations and molecular evolution in vertebrates.

An annual cycle of gene regulation in the red-legged salamander mental gland: from hypertrophy to expression of rapidly evolving pheromones.

BACKGROUND: Cell differentiation is mediated by synchronized waves of coordinated expression for hundreds to thousands of genes, and must be regulated to produce complex tissues and phenotypes. For many animal species, sexual selection has driven the development of elaborate male ornaments, requiring sex-specific differentiation pathways. One such male ornament is the pheromone-producing mental gland of the red-legged salamander (Plethodon shermani). Mental gland development follows an annual cycle of extreme hypertrophy, production of pheromones for the ~ 2 month mating season, and then complete resorption before repeating the process in the following year. At the peak of the mating season, the transcriptional and translational machinery of the mental gland are almost exclusively redirected to the synthesis of rapidly evolving pheromones. Of these pheromones, Plethodontid Modulating Factor (PMF) has experienced an unusual history: following gene duplication, the protein coding sequence diversified from positive sexual selection while the untranslated regions have been conserved by purifying selection. The molecular underpinnings that bridge the processes of gland hypertrophy, pheromone synthesis, and conservation of the untranslated regions remain to be determined. RESULTS: Using Illumina sequencing, we prepared a de novo transcriptome of the mental gland at six stages of development. Differential expression analysis and immunohistochemistry revealed that the mental gland initially adopts a highly proliferative, almost tumor-like phenotype, followed by a rapid increase in pheromone mRNA and protein. One likely player in this transition is Cold Inducible RNA Binding Protein (CIRBP), which selectively and cooperatively binds the highly conserved PMF 3' UTR. CIRBP, along with other proteins associated with stress response, have seemingly been co-opted to aid in mental gland development by helping to regulate pheromone synthesis. CONCLUSIONS: The P. shermani mental gland utilizes a complex system of transcriptional and post-transcriptional gene regulation to facilitate its hypertrophication and pheromone synthesis. The data support the evolutionary interplay of coding and noncoding segments in rapid gene evolution, and necessitate the study of co-evolution between pheromone gene products and their transcriptional/translational regulators. Additionally, the mental gland could be a powerful emerging model of regulated tissue proliferation and subsequent resorption within the dermis and share molecular links to skin cancer biology.

Targeting microbial pathogens by expression of new recombinant dermaseptin peptides in tobacco.

Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin-binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD-DrsB1 and pGSA/DrsB1-CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi-quantitative RT-PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD-DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1-CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.

The Amphibian Diacylglycerol O-acyltransferase 2 (DGAT2): a 'paleo-protein' with Conserved Function but Unique Folding.

Amphibians are, currently, considered the first vertebrates that had performed the aquatic to terrestrial transition during evolution; therefore, water balance and dehydration control were prerequisites for such environment conquering. Among anurans, Phyllomedusa is a well-studied genus, due to its peptide-rich skin secretion. Here, we have analyzed the skin secretion of Phyllomedusa distincta targeting the proteins present in the skin secretion. The major soluble protein was chromatographically isolated and utilized to immunize rabbits. Through proteomics approaches, we were able to identify such protein as being the diacylglycerol O-acyltransferase 2 (DGAT2), a crucial enzyme involved in lipid synthesis and in the skin water balance. Immunohistochemistry assays revealed the protein tissular distribution for different animal species, belonging to different branches of the phylogenetic tree. Specifically, there was positivity to the anti-DGAT2 on Amphibians' skin, and no antibody recognition on fish and mammals' skins. The DGAT2 multiple sequence alignment reveals some degree of conservation throughout the genera; however, there is a different cysteine pattern among them. Molecular modeling analyses corroborate that the different cysteine pattern leads to distinct 3D structures, explaining the different antibody recognition. Moreover, the protein phylogenetic analyses place the Xenopus DGAT2 (the available amphibian representative) next to the Coelacanthus enzyme, which have led the authors to term this a 'paleo-protein'. DGAT2 would be, therefore, an ancient protein, crucial to the terrestrial environment conquest, with a unique folding-as indicated by the molecular models and immunohistochemistry analyses-a consequence of the different cysteine pattern but with conserved biological function.

Production of a Recombinant Dermaseptin Peptide in Nicotiana tabacum Hairy Roots with Enhanced Antimicrobial Activity.

Expression of strong antimicrobial peptides in plants is of great interest to combat a wide range of plant pathogens. To bring the Dermaseptin B1 (DrsB1) peptide to the intimate contact of the plant pathogens cell wall surface, the DrsB1 encoding sequence was fused to the C-terminal part of the two copies of the chitin-binding domain (CBD) of the Avr4 effector protein and used for Agrobacterium rhizogenes-mediated transformation. The expression of the recombinant protein in the tobacco hairy roots (HRs) was confirmed by molecular analysis. Antimicrobial activity analysis of the recombinant protein purified from the transgenic HRs showed that the (CBD)2-DrsB1 recombinant protein had a significant (p < 0.01) antimicrobial effect on the growth of different fungal and bacterial pathogens. The results of this study indicated that the recombinant protein had a higher antifungal activity against chitin-producing Alternaria alternata than Pythium spp. Scanning electron microscopy images demonstrated that the recombinant protein led to fungal hypha deformation, fragmentation, and agglutination of growing hypha, possibly by dissociating fungal cell wall components. In vitro evidences suggest that the expression of the (CBD)2-DrsB1 recombinant protein in plants by generating transgenic lines is a promising approach to produce disease-resistant plants, resistance to chitin-producing pathogenic fungi.

The defensive system of tree frog skin identified by peptidomics and RNA sequencing analysis.

The diversity of defensive peptides from skin of amphibians has been demonstrated. These peptides may have resulted from the diversity of microorganisms encountered by amphibians. In this study, peptidomics and RNA sequencing analyses were used to study deeply the defensive peptides of the skin secretions from Polypedates megacephalus. A total of 99 defensive peptides have been identified from the skin secretions. Among these peptides, 3 peptides were myotropical peptides and 34 peptides classified as protease inhibitor peptides. 5 lectins, 8 antimicrobial peptides, 26 immunomodulatory peptides, 10 wound-healing peptides and 13 other bioactive peptides were identified as belonging to the innate immune system. One antimicrobial peptide Pm-amp1 showed high similarity to antimicrobial peptide marcin-18. This peptide was successfully expressed and showed moderate activity against four tested strains. These identified peptides highlight the extensive diversity of defensive peptides and provide powerful tools to understand the defense weapon of frog.