Purification, conformational analysis and cytotoxic activities of host-defense peptides from the Tungara frog Engystomops pustulosus (Leptodactylidae; Leiuperinae).

The amphibian family Leptodactylidae is divided into three sub-families: Leiuperinae, Leptodactylinae, and Paratelmatobiinae. Host-defense peptides (HDPs) present in the skins of frogs belonging to the Leptodactylinae have been studied extensively, but information is limited  regarding peptides from Leiuperinae species. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Tungara frog Engystomops pustulosus (Leiuperinae) collected in Trinidad led to the isolation and structural characterization of previously undescribed pustulosin-1 (FWKADVKEIG KKLAAKLAEELAKKLGEQ), [Q28E] pustulosin-1 (pustulosin-2), and pustulosin-3 (DWKETAKELLKKIGAKVAQVISDKLNPAPQ). The primary structures of these peptides do not resemble those of previously described frog skin HDPs. In addition, the secretions contained tigerinin-1EP (GCKTYLIEPPVCT) with structural similarity to the tigerinins previously identified in skin secretions from frogs from the family Dicroglossidae. Pustulosin-1 and -3 adopted extended α-helical conformations in 25% trifluoroethanol-water and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). Pustulosin-1 and -3 displayed cytotoxic activity against a range of human tumor-derived cell lines (A549, MDA-MB-231, and HT29), but their therapeutic potential for development into anti-cancer agents is limited by their comparable cytotoxic activity against non-neoplastic human umbilical vein endothelial cells. The peptides also displayed weak antimicrobial activity against Escherichia coli (MIC = 125 µM) but were inactive against Staphylococcus aureus. Tigerinin-1EP was inactive against both the tumor-derived cells and bacteria.

Impact of Carbon Source Supplementations on Pseudomonas aeruginosa Physiology.

Pseudomonas aeruginosa is an opportunistic pathogen highly resistant to a wide range of antimicrobial agents, making its infections very difficult to treat. Since microorganisms need to perpetually adapt to their surrounding environment, understanding the effect of carbon sources on P. aeruginosa physiology is therefore essential to avoid increasing drug-resistance and better fight this pathogen. By a global proteomic approach and phenotypic assays, we investigated the impact of various carbon source supplementations (glucose, glutamate, succinate, and citrate) on the physiology of the P. aeruginosa PA14 strain. A total of 581 proteins were identified as differentially expressed in the 4 conditions. Most of them were more abundant in citrate supplementation and were involved in virulence, motility, biofilm development, and antibiotic resistance. Phenotypic assays were performed to check these hypotheses. By coupling all this data, we highlight the importance of the environment in which the bacterium evolves on its metabolism, and thus the necessity to better understand the metabolic pathways implied in its adaptative response according to the nutrient availability.

Venom Peptide Repertoire of the European Myrmicine Ant Manica rubida: Identification of Insecticidal Toxins.

Using an integrated transcriptomic and proteomic approach, we characterized the venom peptidome of the European red ant, Manica rubida. We identified 13 "myrmicitoxins" that share sequence similarities with previously identified ant venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O-fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC venom fractions on the blowfly Lucilia caesar, permitting us to identify six myrmicitoxins (i.e., U3-, U10-, U13-, U20-MYRTX-Mri1a, U10-MYRTX-Mri1b, and U10-MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U10-MYRTX-Mri1a, -Mri1b, -Mri1c, and U20-MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g-1). U13-MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g-1). Finally, U3-MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g-1. Thus, M. rubida employs a paralytic venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.

MIB-MIP is a mycoplasma system that captures and cleaves immunoglobulin G.

Mycoplasmas are "minimal" bacteria able to infect humans, wildlife, and a large number of economically important livestock species. Mycoplasma infections include a spectrum of clinical manifestations ranging from simple fever to fulminant inflammatory diseases with high mortality rates. These infections are mostly chronic, suggesting that mycoplasmas have developed means to evade the host immune response. Here we present and functionally characterize a two-protein system from Mycoplasma mycoides subspecies capri that is involved in the capture and cleavage of IgG. The first component, Mycoplasma Ig binding protein (MIB), is an 83-kDa protein that is able to tightly bind to the Fv region of a wide range of IgG. The second component, Mycoplasma Ig protease (MIP), is a 97-kDa serine protease that is able to cleave off the VH domain of IgG. We demonstrate that MIB is necessary for the proteolytic activity of MIP. Cleavage of IgG requires a sequential interaction of the different partners of the system: first MIB captures the IgG, and then MIP is recruited to the MIB-IgG complex, enabling protease activity. MIB and MIP are encoded by two genes organized in tandem, with homologs found in the majority of pathogenic mycoplasmas and often in multiple copies. Phylogenetic studies suggest that genes encoding the MIB-MIP system are specific to mycoplasmas and have been disseminated by horizontal gene transfer. These results highlight an original and complex system targeting the host immunoglobulins, playing a potentially key role in the immunity evasion by mycoplasmas.

Processing and Maturation of Cathepsin C Zymogen: A Biochemical and Molecular Modeling Analysis.

Cysteine cathepsin C (CatC) is a ubiquitously expressed, lysosomal aminopeptidase involved in the activation of zymogens of immune-cell-associated serine proteinases (elastase, cathepsin G, proteinase 3, neutrophil serine proteinase 4, lymphocyte granzymes, and mast cell chymases). CatC is first synthetized as an inactive zymogen containing an intramolecular chain propeptide, the dimeric form of which is processed into the mature tetrameric form by proteolytic cleavages. A molecular modeling analysis of proCatC indicated that its propeptide displayed a similar fold to those of other lysosomal cysteine cathepsins, and could be involved in dimer formation. Our in vitro experiments revealed that human proCatC was processed and activated by CatF, CatK, and CatV in two consecutive steps of maturation, as reported for CatL and CatS previously. The unique positioning of the propeptide domains in the proCatC dimer complex allows this order of cleavages to be understood. The missense mutation Leu172Pro within the propeptide region associated with the Papillon-Lefèvre and Haim-Munk syndrome altered the proform stability as well as the maturation of the recombinant Leu172Pro proform.

SAG12, a Major Cysteine Protease Involved in Nitrogen Allocation during Senescence for Seed Production in Arabidopsis thaliana.

SAG12 is the most widely used senescence-associated reference gene for characterizing leaf senescence, and the increase in SAG12 protein during leaf senescence is remarkable. However, the role of this cysteine protease in N remobilization and the leaf senescence process remains unclear. The role of SAG12 has been poorly investigated and the few reports dealing with this are somewhat controversial. Indeed, sag12 Arabidopsis mutants have not shown any phenotype, while OsSAG12-1 and OsSAG12-2 overexpression in rice moderates senescence progression. Therefore, this study aims at clarifying the role of the SAG12 cysteine protease during the entire plant life span and during leaf senescence. Arabidopsis thaliana plants knocked-out for the SAG12 gene (sag12) did not exhibit any special phenotypic traits when grown under optimal nitrogen supply (HN), suggesting that other cysteine proteases could provide compensatory effects. Moreover, for the first time, this study shows that aspartate protease activity is significantly increased in sag12. Among the putative aspartate proteases involved, a CND41-like aspartate protease has been identified. Under low nitrogen (LN) availability, when inducible proteolytic systems are not sufficient to cope with SAG12 depletion, a decrease in yield is observed. Altogether, these results show that SAG12 (and perhaps also aspartate proteases) could be involved in RuBisCO degradation during the leaf senescence associated with seed filling.

Cytotoxic peptides with insulin-releasing activities from skin secretions of the Italian stream frog Rana italica (Ranidae).

Peptidomic analysis of norepinephrine-stimulated skin secretions from Italian stream frog Rana italica led to the purification and characterization of two host-defense peptides differing by a single amino acid residue belonging to the brevinin-1 family (brevinin-1ITa and -1ITb), a peptide belonging to the temporin family (temporin-ITa) and a component identified as prokineticin Bv8. The secretions contained relatively high concentrations of the methionine-sulphoxide forms of brevinin-1ITa and -1ITb suggesting that these peptides may have a role as antioxidants in the skin of this montane frog. Brevinin-1ITa (IVPFLLGMVPKLVCLITKKC) displayed potent cytotoxicity against non-small cell lung adenocarcinoma A549 cells (LC50  = 18 µM), breast adenocarcinoma MDA-MB-231 cells (LC50  = 8 µM) and colorectal adenocarcinoma HT-29 cells (LC50  = 18 µM), but the peptide was also strongly hemolytic against mouse erythrocytes (LC50  = 7 µM). Temporin-ITa (VFLGAIAQALTSLLGKL.NH2 ) was between three and fivefold less potent against these cells. Brevinin-1ITa inhibited growth of both Gram-positive Staphylococcus epidermidis and Gram-negative Escherichia coli as well as a strain of the opportunist yeast pathogen Candida parapsilosis, whereas temporin-ITa was active only against S. epidermidis and C. parapsilosis. Both peptides stimulated the release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥1 nM, but brevinin-1ITa was cytotoxic to the cells at concentrations ≥3 µM. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Purification, Conformational Analysis, and Properties of a Family of Tigerinin Peptides from Skin Secretions of the Crowned Bullfrog Hoplobatrachus occipitalis.

Four host-defense peptides belonging to the tigerinin family (tigerinin-1O: RICTPIPFPMCY; tigerinin-2O: RTCIPIPLVMC; tigerinin-3O: RICTAIPLPMCL; and tigerinin-4O: RTCIPIPPVCF) were isolated from skin secretions of the African crowned bullfrog Hoplobatrachus occipitalis. In aqueous solution at pH 4.8, the cyclic domain of tigerinin-2O adopts a rigid amphipathic conformation that incorporates a flexible N-terminal tail. The tigerinins lacked antimicrobial (MIC > 100 µM) and hemolytic (LC50 > 500 µM) activities but, at a concentration of 20 µg/mL, significantly (P < 0.05) inhibited production of interferon-γ (IFN-γ) by peritoneal cells from C57BL/6 mice without affecting production of IL-10 and IL-17. Tigerinin-2O and -4O inhibited IFN-γ production at concentrations as low as 1 µg/mL. The tigerinins significantly (P ≤ 0.05) stimulated the rate of insulin release from BRIN-BD11 clonal ß-cells without compromising the integrity of the plasma membrane. Tigerinin-1O was the most potent (threshold concentration 1 nM) and the most effective (395% increase over basal rate at a concentration of 1 µM). Tigerinin-4O was the most potent and effective peptide in stimulating the rate of glucagon-like peptide-1 release from GLUTag enteroendocrine cells (threshold concentration 10 nM; 289% increase over basal rate at 1 µM). Tigerinin peptides have potential for development into agents for the treatment of patients with type 2 diabetes.

Unraveling the effects of static magnetic field stress on cytosolic proteins of Salmonella by using a proteomic approach.

The present study investigated the adaptation of Salmonella enterica subsp. enterica serovar Hadar to static magnetic field (SMF) exposure (200 mT, 9 h). The proteomic analysis provides an overview of potentially important cytosolic proteins that Salmonella needs to regulate to survive and adapt to magnetic stress. Via 2-dimensional electrophoresis and liquid chromatography tandem mass spectrometry, we compared cytosolic proteomes before and after exposure to magnetic field. A total of 35 proteins displaying more than a 2-fold change were differentially expressed in exposed cells, among which 25 were upregulated and 10 were downregulated. These proteins can be classified mainly into 6 categories: (i) proteins involved in metabolic pathways of carbohydrates, (ii) chaperones and proteins produced in response to oxidative stress, (iii) proteins involved in energy homeostasis, (iv) elongation factors (EF-Tu and EF-Ts), (v) proteins involved in motility, and (vi) proteins involved in molecules transport. Many of the presented observations could be explained, while some represent still-unknown mechanisms. In addition, this study reveals 5 hypothetical proteins. It seems that the stress response to SMF (200 mT) is essentially set up to avoid oxidative damages, with the overexpression of proteins directly involved in oxidative stress response and metabolic switches to counteract oxidative stress. Interestingly, several proteins induced under SMF exposure are found to overlap with those induced by other stresses, such as heat shock and starvation.

Antimicrobial Peptide LL-37 Is Both a Substrate of Cathepsins S and K and a Selective Inhibitor of Cathepsin L.

Lung cysteine cathepsins B, K, L, and S contribute to physiological and pathological processes including degradation of antimicrobial peptides/proteins (AMPs) such as surfactant protein SP-A, lactoferrin, secretory leukocyte peptidase inhibitor, and beta-defensins-2 and -3. Substantial amounts of uncleaved LL-37, a 37-mer cationic AMP, were observed in the sputum of patients with cystic fibrosis (CF). Nevertheless LL-37 was degraded after prolonged incubation in CF sputum, and the hydrolysis was blocked by E-64, a selective inhibitor of cysteine proteases. Cathepsins K and S, expressed in human alveolar macrophages, thoroughly hydrolyzed LL-37 in vitro, whereas it competitively inhibited cathepsin L (Ki = 150 nM). Cleavage of LL-37 by cathepsins S and K impaired its antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus, in a time- and concentration-dependent manner. The exchange of residues 67 and 205 in the S2 pockets of cathepsins L (Leu67Tyr/Ala205Leu) and K (Tyr67Leu/Leu205Ala) switched the specificity of these mutants toward LL-37. Molecular modeling suggested that LL-37 interacted with the active site of cathepsin L in both forward (i.e., substrate-like) and reverse orientations with similar binding energies. Our data support the hypothesis that cysteine cathepsins modulate the innate immunity response by degrading distinct and representative members of the AMP family.

Determination of Multimodal Isotopic Distributions: The Case of a (15)N Labeled Protein Produced into Hairy Roots.

Isotopic labeling is widely used in various fields like proteomics, metabolomics, fluxomics, as well as in NMR structural studies, but it requires an efficient determination of the isotopic enrichment. Mass spectrometry is the method of choice for such analysis. However, when complex expression systems like hairy roots are used for production, multiple populations of labeled proteins may be obtained. If the isotopic incorporation determination is actually well-known for unimodal distributions, the multimodal distributions have scarcely been investigated. Actually, only a few approaches allow the determination of the different labeled population proportions from multimodal distributions. Furthermore, they cannot be used when the number of the populations and their respective isotope ratios are unknown. The present study implements a new strategy to measure the (15)N labeled populations inside a multimodal distribution knowing only the peptide sequence and peak intensities from mass spectrometry analyses. Noteworthy, it could be applied to other elements, like carbon and hydrogen, and extended to a larger range of biomolecules.

Proteomic profile of pre - B2 lymphoblasts from children with acute lymphoblastic leukemia (ALL) in relation with the translocation (12; 21).

BACKGROUND: Until now, the major prognostic factors for pediatric acute lymphoblastic leukemia (ALL), age, white blood cell count and chromosomal alterations are initially taken into account for the risk stratification of patients. In the light of protein marker studies to classify subtypes of Acute Myeloblastic Leukemia efficiently, we have compared the lymphoblastes proteome in Childhood ALL in accordance with the presence of t(12;21), indicator of good prognosis, usually. METHODS: Protein expression in pre-B2 lymphoblastic cells, collected from residual bone marrow cells after diagnostic procedures, was analyzed using two dimensional gel electrophoresis protocol. Protein spots whose average normalized volumes were statistically different in the two patients groups (n = 13; student t test p < 0.01), were excised. Tryptic peptides were then analyzed using a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a HPLC-chip cube interface. The tandem mass spectrometry peak lists extracted using the DataAnalysis program, were compared with the protein database Mascot Daemon. RESULTS: We focused on twelve spots corresponding to sixteen identified candidate proteins among the 26 found differentially expressed (p ≤ 0.05) regarding the presence of t(12;21). Among over expressed proteins, two proteins were implicated in cellular growth arrest (i.e. calponine 2, p ≤ 0.001 and phosphatidylinositol transfer protein beta, p ≤ 0.001) in accordance with good prognosis, while two other proteins favored cell cycle proliferation (i.e. methionine adenosyl transferase 2ß, p ≤ 0.005 and heterogeneous nuclear ribonucleo-proteins A2 p ≤ 0.01) and could therefore be good marker candidates of aggressiveness. Level of expression of proteasome subunit beta type-2 (p ≤ 0.01) and protein casein kinase 2α (p ≤ 0.01) which both favored apoptosis, deubiquitinating enzyme OTUB1 (p ≤ 0.05) and MLL septin-like fusion protein MSF-B, septin 9 i4 (p ≤ 0.01) were in accord with a good prognosis related to t(12;21) lymphoblasts. CONCLUSION: By drawing up the protein map of leukemic cells, these new data identified marker candidates of leukemic aggressiveness and new t(12;21) patients subgroups. These preliminary results will be in the near future confirmed by using a larger sample of pre-B2 childhood ALLs from national lymphoblastic cell collections.

Multifunctional host-defense peptides isolated from skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae).

Five host-defense peptides (figainin 2PL, hylin PL, raniseptin PL, plasticin PL, and peptide YL) were isolated from norepinephrine-stimulated skin secretions of the banana tree dwelling frog Boana platanera (Hylidae; Hylinae) collected in Trinidad. Raniseptin PL (GVFDTVKKIGKAVGKFALGVAKNYLNS.NH2) and figainin 2PL (FLGTVLKLGKAIAKTVVPMLTNAMQPKQ. NH2) showed potent and rapid bactericidal activity against a range of clinically relevant Gram-positive and Gram-negative ESKAPE + pathogens and Clostridioides difficile. The peptides also showed potent cytotoxic activity (LC50 values < 30 µM) against A549, MDA-MB-231 and HT29 human tumor-derived cell lines but appreciably lower hemolytic activity against mouse erythrocytes (LC50 = 262 ± 14 µM for raniseptin PL and 157 ± 16 µM for figainin 2PL). Hylin PL (FLGLIPALAGAIGNLIK.NH2) showed relatively weak activity against microorganisms but was more hemolytic. The glycine-leucine-rich peptide with structural similarity to the plasticins (GLLSTVGGLVGGLLNNLGL.NH2) and the non-cytotoxic peptide YL (YVPGVIESLL.NH2) lacked antimicrobial and cytotoxic activities. Hylin PL, raniseptinPL and peptide YL stimulated the rate of release of insulin from BRIN-BD11 clonal ß-cells at concentrations ≥100 nM. Peptide YL was the most effective (2.3-fold increase compared with basal rate at 1 µM concentration) and may represent a template for the design of a new class of incretin-based anti-diabetic drugs.

BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.

Evaluation of the skin peptide defenses of the Oregon spotted frog Rana pretiosa against infection by the chytrid fungus Batrachochytrium dendrobatidis.

Population declines due to amphibian chytridiomycosis among selected species of ranid frogs from western North America have been severe, but there is evidence that the Oregon spotted frog, Rana pretiosa Baird and Girard, 1853, displays resistance to the disease. Norepinephrine-stimulated skin secretions were collected from a non-declining population of R. pretiosa that had been exposed to the causative agent Batrachochytrium dendrobatidis. Peptidomic analysis led to identification and isolation, in pure form, of a total of 18 host-defense peptides that were characterized structurally. Brevinin-1PRa, -1PRb, -1PRc, and -1PRd, esculentin-2PRa and -PRb, ranatuerin-2PRa, -2PRb, -2PRc, and -2PRe, temporin-PRb and -PRc were identified in an earlier study of skin secretions of frogs from a different population of R. pretiosa known to be declining. Ranatuerin-2PRf, -2PRg, -2PRh, temporin-PRd, -PRe, and -PRf were not identified in skin secretions from frogs from the declining population, whereas temporin-PRa and ranatuerin-2PRd, present in skin secretions from the declining population, were not detected in the current study. All purified peptides inhibited the growth of B. dendrobatidis zoospores. Peptides of the brevinin-1 and esculentin-2 families displayed the highest potency (minimum inhibitory concentration = 6.25-12.5 µM). The study provides support for the hypothesis that the multiplicity and diversity of the antimicrobial peptide repertoire in R. pretiosa and the high growth-inhibitory potency of certain peptides against B. dendrobatidis are important in conferring a measure of resistance to fatal chytridiomycosis.

Purification, Conformational Analysis and Cytotoxic Activities of Host-Defense Peptides from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae).

Frogs from the extensive amphibian family Hylidae are a rich source of peptides with therapeutic potential. Peptidomic analysis of norepinephrine-stimulated skin secretions from the Giant Gladiator Treefrog Boana boans (Hylidae: Hylinae) collected in Trinidad led to the isolation and structural characterization of five host-defense peptides with limited structural similarity to figainin 2 and picturin peptides from other frog species belonging to the genus Boana. In addition, the skin secretions contained high concentrations of tryptophyllin-BN (WRPFPFL) in both C-terminally α-amidated and non-amidated forms. Figainin 2BN (FLGVALKLGKVLG KALLPLASSLLHSQ) and picturin 1BN (GIFKDTLKKVVAAVLTTVADNIHPK) adopt α-helical conformations in trifluroethanol-water mixtures and in the presence of cell membrane models (sodium dodecylsulfate and dodecylphosphocholine micelles). The CD data also indicate contributions from turn structures. Both peptides and picturin 2BN (GLMDMLKKVGKVALT VAKSALLP) inhibited the growth of clinically relevant Gram-negative and Gram-positive bacteria with MIC values in the range 7.8-62.5 µM. Figainin 2BN was potently cytotoxic to A549, MDA-MB-231 and HT-29 human tumor-derived cells (LC50 = 7-14 µM) but displayed comparable potency against non-neoplastic HUVEC cells (LC50 = 15 µM) indicative of lack of selectivity for cancer cells.

Peptidomic analysis of the host-defense peptides in skin secretions of the Amazon River frog Lithobates palmipes (Ranidae).

Skin secretions of certain frog species represent a source of host-defense peptides (HDPs) with therapeutic potential and their primary structures provide insight into taxonomic and phylogenetic relationships. Peptidomic analysis was used to characterize the HDPs in norepinephrine-stimulated skin secretions from the Amazon River frog Lithobates palmipes (Ranidae) collected in Trinidad. A total of ten peptides were purified and identified on the basis of amino acid similarity as belonging to the ranatuerin-2 family (ranatuerin-2PMa, -2PMb, -2PMc, and-2PMd), the brevinin-1 family (brevinin-1PMa, -1PMb, -1PMc and des(8-14)brevinin-1PMa) and the temporin family (temporin-PMa in C-terminally amidated and non-amidated forms). Deletion of the sequence VAAKVLP from brevinin-1PMa (FLPLIAGVAAKVLPKIFCAISKKC) in des[(8-14)brevinin-1PMa resulted in a 10-fold decrease in potency against Staphylococcus aureus (MIC = 31 µM compared with 3 µM) and a > 50-fold decrease in hemolytic activity but potency against Echerichia coli was maintained (MIC = 62.5 µM compared with 50 µM). Temporin-PMa (FLPFLGKLLSGIF.NH2) inhibited growth of S. aureus (MIC = 16 µM) but the non-amidated form of the peptide lacked antimicrobial activity. Cladistic analysis based upon the primary structures of ranaturerin-2 peptides supports the division of New World frogs of the family Ranidae into the genera Lithobates and Rana. A sister-group relationship between L. palmipes and Warszewitsch's frog Lithobates warszewitschii is indicated within a clade that includes the Tarahumara frog Lithobates tarahumarae. The study has provided further evidence that peptidomic analysis of HDPs in frog skin secretions is a valuable approach to elucidation of the evolutionary history of species within a particular genus.

Identification of an Antimicrobial Peptide from the Venom of the Trinidad Thick-Tailed Scorpion Tityus trinitatis with Potent Activity against ESKAPE Pathogens and Clostridioides difficile.

Envenomation by the Trinidad thick-tailed scorpion Tityus trinitatis may result in fatal myocarditis and there is a high incidence of acute pancreatitis among survivors. Peptidomic analysis (reversed-phase HPLC followed by MALDI-TOF mass spectrometry and automated Edman degradation) of T. trinitatis venom led to the isolation and characterization of three peptides with antimicrobial activity. Their primary structures were established asTtAP-1 (FLGSLFSIGSKLLPGVFKLFSRKKQ.NH2), TtAP-2 (IFGMIPGLIGGLISAFK.NH2) and TtAP-3 (FFSLIPSLIGGLVSAIK.NH2). In addition, potassium channel and sodium channel toxins, present in the venom in high abundance, were identified by CID-MS/MS sequence analysis. TtAP-1 was the most potent against a range of clinically relevant Gram-positive and Gram-negative aerobes and against the anaerobe Clostridioides difficile (MIC = 3.1-12.5 µg/mL). At a concentration of 1× MIC, TtAP-1 produced rapid cell death (<15 min against Acinetobacter baumannii and Staphylococcus aureus). The therapeutic potential of TtAP-1 as an anti-infective agent is limited by its high hemolytic activity (LC50 = 18 µg/mL against mouse erythrocytes) but the peptide constitutes a template for the design of analogs that maintain the high bactericidal activity against ESKAPE pathogens but are less toxic to human cells. It is suggested that the antimicrobial peptides in the scorpion venom facilitate the action of the neurotoxins by increasing the membrane permeability of cells from either prey or predator.

Transportation of patients under extracorporeal membrane oxygenation support on an airliner: Flying bridge to transplantation.

BACKGROUND: A retrieval programme was developed in Martinique (French West Indies) to provide extracorporeal membrane oxygenation for patients in the Caribbean, where heart transplantation and ventricular assist devices are not available. In 2011, the Department of Cardiac Surgery at the University Hospital of Fort-de-France (Martinique) developed a transfer programme to Paris (France) on an airliner, to refer patients for whom extracorporeal membrane oxygenation was not weanable to heart transplantation or a ventricular assist device. AIM: To report this unique experience of transportation of patients under extracorporeal membrane oxygenation support on an airliner from the French West Indies to Paris. METHODS: This was an observational and retrospective study of all patients under extracorporeal membrane oxygenation support who were transferred from Martinique to the Pitié-Salpêtrière Hospital/Sorbonne University in Paris between September 2011 and September 2019. Transport characteristics, complications during repatriation, cost and clinical outcomes at 30days and 1year were reported. RESULTS: Twenty-six patients were transferred on an airliner; the retrieval distance was 7260km, and the mean duration was 14hours. Only two patients developed complications (pulmonary oedema and leg ischaemia), and no patient died during the flight. Nine patients had a ventricular assist device implanted, and six patients were transplanted. Thirty-day survival was 65.4%, and 1-year survival was 38.5%. CONCLUSIONS: Transport under extracorporeal membrane oxygenation support on an airliner is safe and efficient, with an acceptable cost. This programme allowed patients under extracorporeal membrane oxygenation support in a remote centre, without access to transplantation or a ventricular assist device, to be referred for these techniques in specialized centres. This experience strengthens the strategy of developing regional networks around specialized extracorporeal membrane oxygenation centres.

Structure-function analysis of grass clip serine protease involved in Drosophila Toll pathway activation.

Grass is a clip domain serine protease (SP) involved in a proteolytic cascade triggering the Toll pathway activation of Drosophila during an immune response. Epistasic studies position it downstream of the apical protease ModSP and upstream of the terminal protease Spaetzle-processing enzyme. Here, we report the crystal structure of Grass zymogen. We found that Grass displays a rather deep active site cleft comparable with that of proteases of coagulation and complement cascades. A key distinctive feature is the presence of an additional loop (75-loop) in the proximity of the activation site localized on a protruding loop. All biochemical attempts to hydrolyze the activation site of Grass failed, strongly suggesting restricted access to this region. The 75-loop is thus proposed to constitute an original mechanism to prevent spontaneous activation. A comparison of Grass with clip serine proteases of known function involved in analogous proteolytic cascades allowed us to define two groups, according to the presence of the 75-loop and the conformation of the clip domain. One group (devoid of the 75-loop) contains penultimate proteases whereas the other contains terminal proteases. Using this classification, Grass appears to be a terminal protease. This result is evaluated according to the genetic data documenting Grass function.