A novel family of defensin-like peptides from Hermetia illucens with antibacterial properties.

BACKGROUND: The world faces a major infectious disease challenge. Interest in the discovery, design, or development of antimicrobial peptides (AMPs) as an alternative approach for the treatment of bacterial infections has increased. Insects are a good source of AMPs which are the main effector molecules of their innate immune system. Black Soldier Fly Larvae (BSFL) are being developed for large-scale rearing for food sustainability, waste reduction and as sustainable animal and fish feed. Bioinformatic studies have suggested that BSFL have the largest number of AMPs identified in insects. However, most AMPs identified in BSF have not yet undergone antimicrobial evaluation but are promising leads to treat critical infections. RESULTS: Jg7197.t1, Jg7902.t1 and Jg7904.t1 were expressed into the haemolymph of larvae following infection with Salmonella enterica serovar Typhimurium and were predicted to be AMPs using the computational tool ampir. The genes encoding these proteins were within 2 distinct clusters in chromosome 1 of the BSF genome. Following removal of signal peptides, predicted structures of the mature proteins were superimposed, highlighting a high degree of structural conservation. The 3 AMPs share primary sequences with proteins that contain a Kunitz-binding domain; characterised for inhibitory action against proteases, and antimicrobial activities. An in vitro antimicrobial screen indicated that heterologously expressed SUMO-Jg7197.t1 and SUMO-Jg7902.t1 did not show activity against 12 bacterial strains. While recombinant SUMO-Jg7904.t1 had antimicrobial activity against a range of Gram-negative and Gram-positive bacteria, including the serious pathogen Pseudomonas aeruginosa. CONCLUSIONS: We have cloned and purified putative AMPs from BSFL and performed initial in vitro experiments to evaluate their antimicrobial activity. In doing so, we have identified a putative novel defensin-like AMP, Jg7904.t1, encoded in a paralogous gene cluster, with antimicrobial activity against P. aeruginosa.

Membrane binding properties of plant defensins.

The membrane interaction characteristics of five antifungal plant defensin peptides: NaD1, and the related HXP4 and L5, as well as NaD2 and the related ZmD32 were studied. These peptides were chosen to cover a broad range of cationic charges with little structural variations, allowing for assessment of the role of charge in their membrane interactions. Membrane permeabilizing activity against C. albicans was confirmed and quantified for benchmarking purposes. Viscoelastic characteristics of the membrane interactions were studied in typical neutral and charged model membranes using quartz crystal microbalance with dissipation (QCM-D. Frequency-dissipation fingerprinting analysis of the QCM-D results revealed that all of the peptides were able to bind to all studied model membranes albeit with slightly different viscoelastic character for each membrane type. However, characteristic disruption patterns were not observed suggesting that the membrane disrupting activity of these defensins is mostly specific to fungal membranes, and that increasing the peptide charge does not enhance their action. The results also show that the presence of specific sterols has a profound effect on the ability of the peptides to disrupt the membrane.

Structural and functional studies of scorpine: A channel blocker and cytolytic peptide.

Scorpine is an antimicrobial and antimalarial peptide isolated from Pandinus imperator scorpion venom. As there are few functional and structural studies reported on scorpine-like peptides, we investigated the recombinant truncated N- and C-terminal domains as well as complete scorpine using biological assays and determined the N- and C-terminal structures using solution nuclear magnetic resonance. The study was conducted using recombinant N- and C-terminal peptides and complete scorpine expressed in Escherichia coli. The results showed that N-scorpine presented a random coil structure in water and adopted α-helical folding in the presence of 50% trifluoroethanol (TFE). C-scorpine contains three disulfide bonds with two structural domains: an unstructured N-terminal domain in water that can form a typical secondary alpha-helix structure in 50% TFE and a C-terminal domain with the CS-αß motif. Our findings demonstrate cytolytic activity associated with C-scorpine, N-scorpine, and scorpine, as well as channel blocking activity associated with the C-scorpine domain.

In silico analysis of the C-terminal domain of big defensin from the Pacific oyster.

Defensins are antimicrobial peptides consisting of intramolecular disulphide bonds in a complex folded arrangement of two or three antiparallel ß-sheets with or without an α-helical structure. They are produced by a vast range of organisms being constitutively expressed or induced in various tissues against different stimuli like infection, injury or other inflammatory factors. Two classes of invertebrate defensin exist, namely CS-αß and big defensin, the latter being predominantly present in molluscs. Intriguingly, an invertebrate big defensin gene has been hypothesized as the most probable ancestor of vertebrate ß-defensins. Here, conserved residues were identified for both big defensin and ß-defensin. In silico mutation on conserved amino acid positions of the ß-defensin-like domain of big defensin from Crassostrea gigas was carried out to understand the effects of mutation on the structure and function of the protein. R64A and E71A have been identified as deleterious as well as destabilizing for the protein. Changes in amino acid network and aggregation propensity were also observed upon mutating these two charged residues. 100 ns molecular dynamics simulations of wild-type, R64A and E71A structures revealed significant conformational changes in the case of mutants. Furthermore, molecular docking highlighted the significance of R64 in ligand interaction. In conclusion, these results provide the first in-depth understanding of the structural and functional importance imparted by two conserved charged residues in the C-terminal region of big defensin. It also enhances the existing knowledge about this antimicrobial peptide for application in therapeutics and other aspects of protein engineering.Communicated by Ramaswamy H. Sarma.

Characterization of New Defensin Antimicrobial Peptides and Their Expression in Bed Bugs in Response to Bacterial Ingestion and Injection.

Common bed bugs, Cimex lectularius, can carry, but do not transmit, pathogens to the vertebrate hosts on which they feed. Some components of the innate immune system of bed bugs, such as antimicrobial peptides (AMPs), eliminate the pathogens. Here, we determined the molecular characteristics, structural properties, and phylogenetic relatedness of two new defensins (CL-defensin1 (XP_024085718.1), CL-defensin2 (XP_014240919.1)), and two new defensin isoforms (CL-defensin3a (XP_014240918.1), CL-defensin3b (XP_024083729.1)). The complete amino acid sequences of CL-defensin1, CL-defensin2, CL-defensin3a, and CL-defensin3b are strongly conserved, with only minor differences in their signal and pro-peptide regions. We used a combination of comparative transcriptomics and real-time quantitative PCR to evaluate the expression of these defensins in the midguts and the rest of the body of insects that had been injected with bacteria or had ingested blood containing the Gram-positive (Gr+) bacterium Bacillus subtilis and the Gram-negative (Gr-) bacterium Escherichia coli. We demonstrate, for the first time, sex-specific and immunization mode-specific upregulation of bed bug defensins in response to injection or ingestion of Gr+ or Gr- bacteria. Understanding the components, such as these defensins, of the bed bugs' innate immune systems in response to pathogens may help unravel why bed bugs do not transmit pathogens to vertebrates.

Defensins as a promising class of tick antimicrobial peptides: a scoping review.

BACKGROUND: Ticks are hematophagous parasites that transmit an extensive range of pathogens to their vertebrate hosts. Ticks can destroy invading microorganisms or alleviate infection via their rudimentary but orchestrated innate immune system. Antimicrobial peptides (AMPs) are important components of tick innate immunity. Among these humoral effector molecules, defensins are well-studied and widely identified in various species of Ixodidae (hard ticks) and Argasidae (soft ticks). This review was aimed at presenting the characterization of tick defensins from structure-based taxonomic status to antimicrobial function. MAIN TEXT: All published papers written in English from 2001 to May 2022 were searched through PubMed and Web of Science databases with the combination of relevant terms on tick defensins. Reports on identification and characterization of tick defensins were included. Of the 329 entries retrieved, 57 articles were finally eligible for our scoping review. Tick defensins mainly belong to the antibacterial ancient invertebrate-type defensins of the cis-defensins superfamily. They are generally small, cationic, and amphipathic, with six cysteine residues forming three intra-molecular disulfide bonds. Tick defensins primarily target membranes of a variety of pathogens, including Gram-positive and Gram-negative bacteria, fungi, viruses, and protozoa. Since tick defensins have a high degree of variability, we summarize their common biological properties and enumerate representative peptides. Along with the various and potent antimicrobial activities, the role of tick defensins in determining vector competence is discussed. CONCLUSIONS: Due to their broad-spectrum antimicrobial activities, tick defensins are considered novel candidates or targets for controlling infectious diseases.

Comprehensive Structural and Functional Characterization of a Seed γ-thionin as a Potent Bioactive Molecule Against Fungal Pathogens and Insect Pests.

BACKGROUND: Fungi and insect pests ruin stored crop grain, which results in millions of dollars of damage, presenting an ongoing challenge for farmers in addition to diminishing the safety of stored food. A wide-range defensive system against pathogens is needed to reduce or even eliminate the dependence of the crop yield upon the use of pesticides. Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) that are a component of the host defense system. They are known to interact with cell membranes to exhibit antifungal and insecticidal activity. They exhibit a broad range of activities against fungi and insects and are effective at low concentrations. Thionins act on membranes, greatly reducing the development of pathogen resistance. OBJECTIVE: The aim of this study is to investigate a bioactive molecule that acts against fungal pathogens and stored grain insect pests. METHODS: γ-thionin protein was extracted from Brassica oleracea L. var. capitata f. alba (white cabbage) seed powder in phosphate buffer (100 mM, pH 7.0) and was identified by MALDI-TOF/TOF. The crude extract was subjected to 70% ammonium sulfate saturation followed by gel filtration chromatography. The disc diffusion assay along with a microtiter bioassay was used to determine the antifungal activity of the protein against phytopathogenic fungi. The insecticidal efficacy was evaluated by feeding insect pests with food contaminated with the purified protein. Additionally, an in silico molecular structure prediction study of the protein was performed using Auto Dock Vina for molecular docking of the protein with either fungal membrane moieties or α-amylase from Tenebrio molitor L. MD simulations of protein-ligand complexes were conducted using Schrodinger's Desmond module. RESULTS: γ-Thionin (BoT) was purified from white cabbage seeds and showed 100% homology with thionin (Brassica oleracea L. var. viridis) and 80% homology with defensin-like protein 1 (Raphanus sativus L.), respectively. BoT significantly inhibited the mycelial growth of Aspergillus niger van Tieghem and Aspergillus flavus Link at a concentration of 2 µM. Similarly, 0.12 µM BoT treatment resulted in significant mortality of Tribolium castaneum Herbst and Sitophilus oryzae L. Molecular docking and MD simulation of BoT confirmed the strong binding affinity with fungal membrane moieties (phosphatidylinositol 4,5-bisphosphate and phosphatidic acid), which causes disruption of the cell membrane and leakage of the cellular contents, leading to cell death. BoT blocked the active site of α-amylase, and as a result of the inactivation of this gut enzyme, the digestive systems of insects were disturbed, resulting in their deaths. CONCLUSION: This study revealed that γ-thionin is a good antifungal and insecticidal agent that could be used as an alternate to fungicides and insecticides.

Identification, characterization, and antimicrobial activity of a novel big defensin discovered in a commercial bivalve mollusc, Tegillarca granosa.

Molluscs, the second largest animal phylum on earth, primarily rely on cellular and humoral immune responses to fight against pathogen infection. Although antimicrobial peptides (AMPs) such as big defensin play crucial roles in the humoral immune response, it remains largely unknown in the ecological and economic important blood clam (Tegillarca granosa). In this study, a novel big defensin gene (TgBD) was identified in T. granosa through transcripts and whole genome searching. Bioinformatic analyses were conducted to explore the molecular characteristics of TgBD, and comparisons of TgBD with those reported in other molluscs were performed by multiple alignments and phylogenetic analysis. In addition, the expression patterns of TgBD in various tissues and upon bacterial challenge were investigated while the antimicrobial activity of synthetic N-terminal domain of TgBD was confirmed in vitro by radial diffusion experiment. Results obtained showed TgBD had an open reading frame (ORF) of 369 bp, encoding a prepropeptide containing a signal peptide and a propeptide. Similar to big defensins reported in other species, TgBD consists of a hydrophobic N-terminal domain containing ß1-α1-α2-ß2 folds and a cysteine-rich cationic C-terminal domain with three disulfide bonds between C1-C5, C2-C4, and C3-C6. Phylogenetic analysis showed that TgBD shared 76.80% similarity to its close relative ark shell (Scapharca broughtoni). In addition, TgBD expression was observed in all tissues investigated under normal conditions and was significantly induced by injection of Vibrio parahaemolyticus. Furthermore, synthetic N-terminal peptide of TgBD exhibited strong antimicrobial activity against Gram-positive bacteria tested. Our results indicated that TgBD is a constitutive and inducible acute phase AMP, which provides a universal and prompt protection for T. granosa.

Structure, dynamics, and function of PsDef2 defensin from Pinus sylvestris.

Plant defensins demonstrate high structural stability at extreme temperatures and pH values and, in general, are non-toxic to mammalian cells. These properties make them attractive candidates for use in biotechnology and biomedicine. Knowing the structure-function relationship is desirable to guide the design of plant defensin-based applications. Thus far, the broad range of biological activities was described only for one defensin from gymnosperms, the defensin PsDef1 from Scots pine. Here, we report that closely related defensin from the same taxonomy group, PsDef2, differing from PsDef1 by six amino acids, also possesses antimicrobial, antibacterial, and insect α-amylase inhibitory activities. We also report the solution structure and dynamics properties of PsDef2 assessed using a combination of experimental nuclear magnetic resonance (NMR) techniques. Lastly, we perform a comparative analysis of PsDef2 and PsDef1 gaining a molecular-level insight into their structure-dynamics-function relationship.

Identification and Functional Analysis of a Defensin CcDef2 from Coridius chinensis.

Coridius chinensis belongs to Dinidoridae, Hemiptera. Previous studies have indicated that C. chinensis contains abundant polypeptides with antibacterial and anticancer activities. Antimicrobial peptides (AMPs), as endogenous peptides with immune function, play an indispensable role in the process of biological development and immunity. AMPs have become one of the most potential substitutes for antibiotics due to their small molecular weight and broad-spectrum antimicrobial activity. In this study, a defensin CcDef2 from C. chinensis was characterized based on bioinformatics and functional analyses. The mature peptide of CcDef2 is a typical cationic peptide composed of 43 amino acid residues with five cations, and contains three intramolecular disulfide bonds and a typical cysteine-stabilized αß motif in defensins. Phylogenetic analysis showed that CcDef2 belongs to the insect defensin family. Analysis of gene expression patterns showed that CcDef2 was expressed throughout developmental stages of C. chinensis with high levels at the nymphal stage and in adult tissues tested with the highest level in the fat body. In addition, the CcDef2 expression was significantly upregulated in adults infected by bacteria. After expressed in Escherichia coli BL21(DE3) and renatured, the recombinant CcDef2 showed a significant antibacterial effect on three kinds of Gram-positive bacteria. These results indicate that CcDef2 is an excellent antibacterial peptide and a highly effective immune effector in the innate immunity of C. chinensis. This study provides a foundation for further understanding the function of CcDef2 and developing new antimicrobial drugs.

Human defensin-inspired discovery of peptidomimetic antibiotics.

SignificanceWe report the development of peptidomimetic antibiotics derived from a natural antimicrobial peptide, human α-defensin 5. By engaging multiple bacterial targets, the lead compound is efficacious in vitro and in vivo against bacteria with highly inducible antibiotic resistance, promising a useful therapeutic agent for the treatment of infections caused by antibiotic-resistant bacteria.

Divergence and conservation of defensins and lipid transfer proteins (LTPs) from sugarcane wild species and modern cultivar genomes.

Plant defensins and lipid transfer proteins (LTPs) constitute a large and evolutionarily diverse family of antimicrobial peptides. Defensins and LTPs are two pathogenesis-related proteins (PR proteins) whose characterization may help to uncover aspects about the sugarcane response to pathogens attack. LTPs have also been investigated for their participation in the response to different types of stress. Despite the important roles of defensins and LTPs in biotic and abiotic stresses, scarce knowledge is found about these proteins in sugarcane. By using bioinformatics approaches, we characterized defensins and LTPs in the sugarcane wild species and modern cultivar genomes. The identification of defensins and LTPs showed that all five defensins groups and eight of the nine LTPs have their respective genes loci, although some was only identified in the cultivar genome. Phylogenetic analysis showed that defensins appear to be more conserved among groups of plants than LTPs. Some defensins and LTPs showed opposite expression during pathogenic and benefic bacterial interactions. Interestingly, the expression of defensins and LTPs in shoots and roots was completely different in plants submitted to benefic bacteria or water depletion. Finally, the modeling and comparison of isoforms of LTPs and defensins in wild species and cultivars revealed a high conservation of tertiary structures, with variation of amino acids in different regions of proteins, which could impact their antimicrobial activity. Our data contributed to the characterization of defensins and LTPs in sugarcane and provided new elements for understanding the involvement of these proteins in sugarcane response to different types of stress.

Structural and functional characterizations and heterogenous expression of the antimicrobial peptides, Hidefensins, from black soldier fly, Hermetia illucens (L.).

Insect defensins are effector components of the innate defense system. Defensins, which are widely distributed among insects, are a type of small cysteine-rich plant antimicrobial peptides with broad-spectrum antimicrobial activity. Here, the cDNAs of the black soldier fly, Hermetia illucens (L.), encoding six defensins, designated herein as Hidefensin1-1, 2, 3, 4, 5, 6. Moreover, Hidefensin1-1, 2, and 5 were identified for the first time by genome-targeted analysis. These Hidefensins were found to mainly adopt α-helix and ß-sheet conformation homology as modeled by PRABI, Swiss-Model and ProFunc server. Six conserved cysteine residues that contribute to three disulfide bonds formed the spacing pattern "C-X12-C-X3-C-X9-C-X5-C-X-C", which play a vital role in the molecular stability of Hidefensins. Phylogenetic analysis revealed that the homology of five Hidefensins (except Hidefensin4) was about 59%-92% compared with other insect defensins, indicating that they are novel antimicrobial peptides genes in black soldier fly. Furthermore, the Hidefensin1-1 was expressed in the Escherichia coli strain BL21(DE3) as a fusion protein with thioredoxin. Results showed that the purified TRX-Hidefensin1-1 exerted strong inhibitory effects against the Gram-positive bacteria Staphylococcus aureus and the Gram-negative bacteria Escherichia coli. The inhibitory efficacy of TRX-Hidefensin1-1 against Gram-positive bacteria was better than that against Gram-negative bacteria. These results indicated that Hidefensin1-1 has potent antimicrobial activities against test pathogens.

Antifungal activity and mode of action of synthetic peptides derived from the tick OsDef2 defensin.

Candida albicans is the principal opportunistic fungal pathogen in nosocomial settings and resistance to antifungal drugs is on the rise. Antimicrobial peptides from natural sources are promising novel therapeutics against C. albicans. OsDef2 defensin was previously found to be active against only Gram-positive bacteria, whereas derived fragments Os and its cysteine-free analogue, Os-C, are active against Gram-positive and Gram-negative bacteria at low micromolar concentrations. In this study, OsDef2-derived analogues and fragments were screened for anticandidal activity with the aim to identify peptides with antifungal activity and in so doing obtain a better understanding of the structural requirements for activity and modes of action. Os, Os-C and Os(11-22)NH2 , a Os-truncated carboxy-terminal-amidated fragment, had the most significant antifungal activities, with minimum fungicidal concentrations (MFCs) in the micromolar range (6-28 µM). C. albicans killing was rapid and occurred within 30-60 min. Further investigations showed all three peptides interacted with cell wall derived polysaccharides while both Os and Os(11-22)NH2 permeabilized fungal liposomes. Confocal laser scanning microscopy confirmed that Os-C and Os(11-22)NH2 could enter the cytosol of live cells and subsequent findings suggest that the uptake of Os and Os-C, in contrast to Os(11-22)NH2 , is energy dependent. Although Os, Os-C and Os(11-22)NH2 induced the production of reactive oxygen species (ROS), co-incubation with ascorbic acid revealed that only ROS generated by Os-C and to a lesser extent Os(11-22)NH2 resulted in cell death. Overall, Os, Os-C and Os(11-22)NH2 are promising candidacidal agents.

Simplified Theta-defensin [Ser3,7,12,16] RTD-2 Analog Is Involved in Proteasomal Degradation Pathway in Breast Cancer.

BACKGROUND/AIM: Antimicrobial peptides are part of the innate immune response, regulate inflammation and initiate acquired immunity. This study focused on theta-defensins that have been shown to have anticancer properties. MATERIALS AND METHODS: RTD-2 analogs were synthesized on a peptide synthesizer. Cell viability was estimated using the MTT test. Immunoprecipitation assay was conducted to determine the molecular partner of the [Ser3,7,12,16]-RTD-2 analog. RESULTS: Here, we present the biologically active [Ser3,7,12,16]-RTD-2 analog that selectively targets various types of breast cancer cells. Immunoprecipitation protein-protein interaction studies showed eleven proteins common to MDA-MB-231 and T47D cell lines. Taking into account their cellular location, it can be concluded that the synthesized peptide interacts mainly with nuclear proteins, which correlates with the obtained microscopic image. CONCLUSION: Proteins that interact strongly with the [Ser3,7,12,16]-RTD-2 analog are closely related to the proteasomal protein degradation pathway. As the activity of the ubiquitin-proteasome system is markedly increased in patients with breast cancer, it is likely that selective modulation of this system may be a useful method for breast cancer treatment.

Defensins: The natural peptide antibiotic.

Defensins are a family of cationic antimicrobial peptides active against a broad range of infectious microbes including bacteria, viruses and fungi, playing important roles as innate effectors and immune modulators in immunological control of microbial infection. Their antibacterial properties and unique mechanisms of action have garnered considerable interest in developing defensins into a novel class of natural antibiotic peptides to fend off pathogenic infection by bacteria, particularly those resistant to conventional antibiotics. However, serious pharmacological and technical obstacles, some of which are unique to defensins and others are common to peptide drugs in general, have hindered the development and clinical translation of defensins as anti-infective therapeutics. To overcome them, several technologies have been developed, aiming for improved functionality, prolonged circulation time, enhanced proteolytic stability and bioavailability, and efficient and controlled delivery and release of defensins to the site of infection. Additional challenges include the alleviation of potential toxicity of defensins and their cost-effective manufacturing. In this review, we briefly introduce defensin biology, focus on various transforming strategies and practical techniques developed for defensins and their derivatives as antibacterial therapeutics, and conclude with a summation of future challenges and possible solutions.

Differential activity on trypanosomatid parasites of a novel recombinant defensin type 1 from the insect Triatoma (Meccus) pallidipennis.

Defensins are one of the major families of antimicrobial peptides (AMPs) that are widely distributed in insects. In Triatomines (Hemiptera: Reduviidae) vectors of Trypanosoma cruzi the causative agent of Chagas disease, two large groups of defensin isoforms have been described: type 1 and type 4. The aim of this study was to analyze the trypanocidal activity of a type 1 recombinant defensin (rDef1.3) identified in Triatoma (Meccus) pallidipennis, an endemic specie from México. The trypanocidal activity of this defensin was evaluated in vitro, against the parasites T. cruzi, T. rangeli, and two species of Leishmania (L. mexicana and L. major) both causative agents of cutaneous leishmaniasis. Our data demonstrated that the defensin was active against all the parasites although in different degrees. The defensin altered the morphology, reduced the viability and inhibited the growth of T.cruzi. When tested against T. rangeli (a parasite that infects a variety of mammalian species), stronger morphological effects where observed. Surprisingly the greatest effects were observed against the two Leishmania species, of which L. major was the parasite most affected with 50% of dead cells or with damaged membranes, in addition of a reduction in its proliferative capacity in culture. These results suggest that rDef1.3 has an important antimicrobial effect against trypanosomatids which cause some of the more important neglected tropical diseases transmitted by insect vectors.

Radiosynthesis and in silico bioevaluation of 131 I-Sulfasalazine as a highly selective radiotracer for imaging of ulcerative colitis.

This study demonstrated the tracking of ulcerative colitis, which is considered a stressful immune disease. Although there are many ways to test for this disease including dependence on gases, dyes, and painful anal endoscopy, these treatment modalities have many disadvantages. Hence, it is the utmost need of time to discover new methods to detect this chronic immune disease and to avoid the defects of traditional methodologies. Sulfasalazine (SSD) was labeled with iodine-131 (half-life: 8 days, Energy: 971 keV) under optimum reaction conditions including the amount of reducing agent, pH factor, chloramine-T (Ch-T) amount, and incubation period. Characterization was performed using 1 H/ 13 C-NMR, ESI-MS, and HPLC (UV/ Radio) techniques. The biodistribution study was performed in normal and ulcerative mice models, and in silico molecular docking study was performed to evaluate the possible mechanism of action to target peroxisome proliferator-activated receptor gamma (PPARγ). The high radiolabeling yield of [131 I]-sulfasalazine ([131 I]-SSD) was achieved ≥90% through the direct labeling method with radioactive iodine-131 in the presence of chloramine-T (100 µg). The radiotracer [131 I]-SSD was observed to be stable in normal saline and freshly eluted serum up to 12 hr at ambient temperature (37℃ ± 2℃). The radiotracer [131 I]-SSD showed the highest uptake in the targeted organ (i.e., ulcerative colon) which was observed to be ≥75% injected dose per gram (% ID/g) organ for 24 hr postinjection (p.i). Furthermore, in silico data collected from molecular modeling analysis of SSD and [131 I]-SSD with antimicrobial protein (PDB code: 3KEG) and peroxisome proliferator-activated receptor gamma (PPARγ) (PDB code: 4XTA) showed azoreductase activity and high binding potential for PPAR-γ site, respectively. The results of biological studies obtained in this study enlighten the usefulness of radiotracer [131 I]-SSD as a potential imaging agent for ulcerative colitis.

Mechanism through Which Retrocyclin Targets Flavivirus Multiplication.

Currently, there are no approved drugs for the treatment of flavivirus infection. Accordingly, we tested the inhibitory effects of the novel θ-defensin retrocyclin-101 (RC-101) against flavivirus infection and investigated the mechanism underlying the potential inhibitory effects. First, RC-101 robustly inhibited both Japanese encephalitis virus (JEV) and Zika virus (ZIKV) infections. RC-101 exerted inhibitory effects on the entry and replication stages. Results also indicated that the nonstructural protein NS2B-NS3 serine protease might serve as a potential viral target. Furthermore, RC-101 inhibited protease activity at the micromolar level. We also demonstrated that with respect to the glycoprotein E protein of flavivirus, the DE loop of domain III (DIII), which is the receptor-binding domain of the E protein, might serve as another viral target of RC-101. Moreover, a JEV DE mutant exhibited resistance to RC-101, which was associated with deceased binding affinity of RC-101 to DIII. These findings provide a basis for the development of RC-101 as a potential candidate for the treatment of flavivirus infection. IMPORTANCE Retrocyclin is an artificially humanized circular θ-defensin peptide, containing 18 residues, previously reported to possess broad antimicrobial activity. In this study, we found that retrocyclin-101 inhibited flavivirus (ZIKV and JEV) infections. Retrocyclin-101 inhibited NS2B-NS3 serine protease activity, suggesting that the catalytic triad of the protease is the target. Moreover, retrocyclin-101 bound to the DE loop of the E protein of flavivirus, which prevented its entry.

Cassava (Manihot esculenta) defensins: Prospection, structural analysis and tissue-specific expression under biotic/abiotic stresses.

Defensins are a prominent family of antimicrobial peptides. They play sophisticated roles in the defense against pathogens in all living organisms, but few works address their expression under different conditions and plant tissues. The present work prospected defensins of Manihot esculenta Crantz, popularly known as cassava. Five defensin candidates (MeDefs) were retrieved from the genome sequences and characterized. Considering chromosome distribution, only MeDef1 and 2 occupy adjacent positions in the same chromosome arm. All 3D structures had antiparallel ß-sheets, an α-helix, and amphipathic residues distributed throughout the peptides with a predominance of cationic surface charge. MeDefs expression was validated by RT-qPCR, including two stress types (biotic: fungus Macrophomina pseudophaseolina, and abiotic: mechanical injury) and a combination of both stresses (fungus+injury) in three different tissues (root, stem, and leaf). For this purpose, ten reference genes (RGs) were tested, and three were chosen to characterize MeDef expression. MeDef3 was up-regulated at roots in all stress situations tested. MeDef1 and MeDef5 were induced in leaves under biotic and abiotic stresses, but not in both stress types simultaneously. Only MeDef2 was down-regulated in the stem tissue also with biotic/abiotic combined stresses. These results indicate that although defensins are known to be responsive to pathogen infection, they may act as preformed defense or, still, have tissue or stress specificities. Aspects of their structure, stability and evolution are also discussed.