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Organisms produce antimicrobial peptides (AMPs) either in response to infection (induced) or continuously (constitutively) to combat microbes encountered during normal trophic activities and/or through pathogenic infections. The expression of AMPs is tightly regulated, often with specificity to particular tissues or developmental stages. As a result, AMPs face varying selective pressures based on the microbes the organism's tissue or developmental stage encounters. Here, we analyzed the evolution and developmental-specific expression of Defensins, which are ancient AMPs in insects, in the stable fly (Stomoxys calcitrans). Stable fly larvae inhibit microbe-rich environments, whereas adult flies, as blood-feeders, experience comparatively fewer encounters with diverse microbial communities. Using existing RNA-seq datasets, we identified six Defensins that were only expressed in larvae (larval Defensins) and five that were not expressed in larvae (non-larval Defensins). Each of the non-larval Defensins was expressed in at least one adult tissue sample. Half of the larval Defensins were induced by mating or feeding in adults, and all three of the induced Defensins were located downstream of canonical binding sites for an Imd transcription factor involved in the highly conserved NF-κB signaling that regulates induction of AMPs. The larval and non-larval Defensins were located in distinct genomic regions, and the amino acid sequences of the larval Defensins formed a monophyletic clade. There were more amino acid substitutions across non-larval Defensins, with multiple genes losing a highly conserved furin cleavage site thought to be required for the removal of the amino terminus from the mature Defensin domain. However, larval Defensins had a higher proportion of radical amino acid substitutions, altering amino acid size and polarity. Our results reveal insights into the developmental stage-specific regulation of AMPs, and they suggest different regulatory regimes impose unique selection pressures on AMPs, possibly as a result of variation in exposure to microbial communities across development., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

The immune system of ticks, along with that of other invertebrates, is comparatively simpler than that of vertebrates, relying solely on innate immune responses. Direct antimicrobial defence is provided by the synthesis of antimicrobial peptides (AMPs), including defensins. The aim of this study was to investigate the differences in defensin genes expression between questing and engorged Ixodes ricinus (def1 and def2) and Dermacentor reticulatus (defDr) ticks, in the presence of selected pathogens: Borrelia spp., Rickettsia spp., Babesia spp., Anaplasma phagocytophilum, and Neoehrlichia mikurensis in the natural environment. After pathogen screening by PCR/qPCR, the expression of defensin genes in pathogen positive ticks and ticks without any of the tested pathogens, was analysed by reverse transcription qPCR. The results showed an increased expression of defensin genes in I. ricinus ticks after blood feeding and I. ricinus and D. reticulatus ticks during in cases of co-infection. In particular, the expression of defensins genes was higher in questing D. reticulatus than in questing and engorged I. ricinus ticks, when borreliae were detected. This study contributes to uncovering the expression patterns of defensin genes in the presence of several known tick pathogens, the occurrence of these pathogens and possible regulatory mechanisms of defensins in tick vector competence., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Plant Snakin/GASA and defensin peptides are cysteine-rich molecules with a wide range of biological functions. They are included within the large family of plant antimicrobial peptides (AMPs), characterized by their structural stability, broad spectrum of activity, and diverse mechanisms of action. The Dilatata group of Paspalum includes five allotetraploids that share an equivalent genomic formula IIJJ. From RNA-seq data of seedling tissues, we performed an in silico characterization of the defensin and Snakin/GASA genes in these species and diploids with a II and JJ genome formula and studied the evolutionary consequences of polyploidy on the expression of the two AMPs families. A total of 107 defensins (distributed in eight groups) and 145 Snakin/GASA (grouped in three subfamilies) genes were identified. Deletions, duplications and/or gene silencing seem to have mediated the evolution of these genes in the allotetraploid species. In defensin genes, the IIJJ allopolyploids retained the I subgenome defensin copies in some of the identified groups supporting the closeness of their nuclear genome with the I subgenome species. In both AMPs families, orthologous genes in tetraploids exhibit higher similarity to each other than with diploids. This data supports the theory of a single origin for the allotetraploids. Several copies of both defensin and Snakin/GASA genes were detected in the five polyploids which could have arisen due to duplication events occurring independently during the diploidization processes in the allotetraploid taxa., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Accumulation of cadmium (Cd) in rice is not only harmful to the growth of plants but also poses a threat to human health. Exposure to Cd triggers unfolded protein response (UPR) within cells, a process that is still not completely understood. The study demonstrated that the lack of OsbZIP39, an essential endoplasmic reticulum (ER)-resident regulator of the UPR, resulted in decreased Cd intake and reduced Cd levels in the roots, stems, and grains of rice. Upon exposure to Cd stress, GFP-OsbZIP39 translocated from ER to nucleus, initiating UPR. Further investigation revealed that Cd treatment caused changes in sphingolipid levels in the membrane, influencing the localization and activation of OsbZIP39. Yeast one-hybrid and dual-LUC assays were conducted to validate the interaction between activated OsbZIP39 and the promoter of the defensin-like gene OsCAL2, resulting in an increase in its expression. Different variations were identified in the coding region of OsbZIP39, which may explain the varying levels of Cd accumulation observed in the indica and japonica subspecies. Under Cd treatment, OsbZIP39 ind exhibited a more significant enhancement in the transcription of OsCAL2 compared to OsbZIP39 jap . Our data suggest that OsbZIP39 positively regulates Cd uptake in rice, offering an encouraging objective for the cultivation of low-Cd rice., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Pathogenesis-related proteins (PR), including osmotins, play a vital role in plant defense, being activated in response to diverse biotic and abiotic stresses. Despite their significance, the mechanistic insights into the role of osmotins in plant defense have not been extensively explored. The present study explores the cloning and characterization of the osmotin gene (WsOsm) from Withania somnifera, aiming to illuminate its role in plant defense mechanisms. Quantitative real-time PCR analysis revealed significant induction of WsOsm in response to various phytohormones e.g. abscisic acid, salicylic acid, methyl jasmonate, brassinosteroids, and ethrel, as well as biotic and abiotic stresses like heat, cold, salt, and drought. To further elucidate WsOsm's functional role, we overexpressed the gene in Nicotiana tabacum, resulting in heightened resistance against the Alternaria solani pathogen. Additionally, we observed enhancements in shoot length, root length, and root biomass in the transgenic tobacco plants compared to wild plants. Notably, the WsOsm- overexpressing seedlings demonstrated improved salt and drought stress tolerance, particularly at the seedling stage. Confocal histological analysis of H 2 O 2 and biochemical studies of antioxidant enzyme activities revealed higher levels in the WsOsm overexpressing lines, indicating enhanced antioxidant defense. Furthermore, a pull-down assay and mass spectrometry analysis revealed a potential interaction between WsOsm and defensin, a known antifungal PR protein (WsDF). This suggests a novel role of WsOsm in mediating plant defense responses by interacting with other PR proteins. Overall, these findings pave the way for potential future applications of WsOsm in developing stress-tolerant crops and improving plant defense strategies against pathogens., (© 2024 Scandinavian Plant Physiology Society.)

Aims/hypothesis: Defensins play a crucial role in the innate immune system's first defense against microbial threats. However, little is known about the defensin system in the pancreas, especially in relation to Type 1 diabetes. We explore the expression of defensins in different disease stages of Type 1 diabetes and correlated obtained findings to the degree of inflammation, providing new insights into the disease and the innate immune system., Material and Methods: Pancreases from non-diabetic human organ donors of different age groups and donors with Type 1 diabetes with different disease duration were examined. Sections from head, body and tail of the pancreas were stained for eight different defensins and for immune cells; CD3+, CD45+, CD68+ and NES+ (granulocytes)., Results: In non-diabetic adult controls the level of expression for defensins Beta-1,Alpha-1, Cathelicidin and REG3A correlated with the level of inflammation. In contrast, individuals with Type  1 diabetes exhibit a reduction or absence of several central defensins regardless of the level of inflammation in their pancreas. The expression of Cathelicidin is present in neutrophils and macrophages but not in T-cells in subjects with Type 1 diabetes., Conclusions: Obtained findings suggest a pancreatic dysfunction in the innate immune system and the bridging to the adaptive system in Type 1 diabetes. Further studies on the role of the local innate immune system in Type 1 diabetes is needed., (© 2024. The Author(s).)

Animals encounter diverse microbial communities throughout their lifetime, which exert varying selection pressures. Antimicrobial peptides (AMPs), which lyse or inhibit microbial growth, are a first line of defense against some of these microbes. Here we examine how developmental variation in microbial exposure has affected the evolution of expression and amino acid sequences of Defensins (an ancient class of AMPs) in the house fly ( Musca domestica ). The house fly is a well-suited model for this work because it trophically associates with varying microbial communities throughout its life history and its genome contains expanded families of AMPs, including Defensins. We identified two subsets of house fly Defensins: one expressed in larvae or pupae, and the other expressed in adults. The amino acid sequences of these two Defensin subsets form distinct monophyletic clades, and they are located in separate gene clusters in the genome. The adult-expressed Defensins evolve faster than larval/pupal Defensins, consistent with different selection pressures across developmental stages. Our results therefore suggest that varied microbial communities encountered across life history can shape the evolutionary trajectories of immune genes., Competing Interests: The authors declare there are no competing interests.

Defensins are antimicrobial peptides involved in innate immunity, and gene number differs amongst eutherian mammals. Few studies have investigated defensins in marsupials, despite their potential involvement in immunological protection of altricial young. Here we use recently sequenced marsupial genomes and transcriptomes to annotate defensins in nine species across the marsupial family tree. We characterised 35 alpha and 286 beta defensins; gene number differed between species, although Dasyuromorphs had the largest repertoire. Defensins were encoded in three gene clusters within the genome, syntenic to eutherians, and were expressed in the pouch and mammary gland. Marsupial beta defensins were closely related to eutherians, however marsupial alpha defensins were more divergent. We identified marsupial orthologs of human DEFB3 and 6, and several marsupial-specific beta defensin lineages which may have novel functions. Marsupial predicted mature peptides were highly variable in length and sequence composition. We propose candidate peptides for future testing to elucidate the function of marsupial defensins., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The activation of the vitamin D receptor (VDR) in the ileum has been shown to regulate Paneth cell-specific defensins, a large family of antimicrobial peptides; hence, this may serve as a potential mechanism to maintain intestinal homeostasis. Previously, we have demonstrated that a combination of vitamin D 3 (VD) and fructooligosaccharides (FOSs) upregulates colonic Vdr in mice. Here, we aim to examine the effect of VD, alone or in combination with FOSs, on intestinal barrier integrity and the secretion of antimicrobial peptides, as well as the gut microbial community. Male and female C57BL/6J mice at 6 weeks old were randomized into three groups to receive the following dietary regimens (n = 10/sex/group) for 8 weeks: (1) standard AIN-93G control diet (CTR), (2) CTR + 5000 IU vitamin D 3 (VD), and (3) VD + 5% fructooligosaccharides (VF). VD and VF differentially regulated the mRNA expressions of tight junction proteins in the colon and ileum. VF suppressed the upregulation of colonic ZO-1 and occludin , which was induced by VD supplementation alone. In the ileum, occludin but not ZO-1 was upregulated 20-fold in the VF-treated mice. While VD did not alter the mRNA expressions of Vdr and defensins in the ileum, these targets were downregulated by VF. Microbial analysis further reveals a shift of microbial beta diversity and a reduction in Romboutsia ilealis , a pathobiont, in VF-treated mice. Though the implications of these phenotypical and microbial changes remain to be determined, the administration of FOSs in the presence of VD may serve as an effective dietary intervention for maintaining intestinal homeostasis.

Trace amine-associated receptors (TAARs) are a class of G protein-coupled receptors, playing an immunomodulatory function in the neuroinflammatory responses. In the present study, a TAAR homologue with a 7tm&#95;classA&#95;rhodopsin-like domain (designated as CgTAAR1L) was identified in oyster Crassostrea gigas. The abundant CgTAAR1L transcripts were detected in visceral ganglia and haemocytes compared to other tissues, which were 55.35-fold and 32.95-fold (p < 0.01) of those in adductor muscle, respectively. The mRNA expression level of CgTAAR1L in haemocytes significantly increased and reached the peak level at 3 h after LPS or Poly (I:C) stimulation, which was 4.55-fold and 12.35-fold of that in control group, respectively (p < 0.01). After the expression of CgTAAR1L was inhibited by the injection of its targeted siRNA, the mRNA expression levels of interleukin17s (CgIL17-1, CgIL17-5 and CgIL17-6), and defensin (Cgdefh1) significantly decreased at 3 h after LPS stimulation, which was 0.51-fold (p < 0.001), 0.39-fold (p < 0.01), 0.48-fold (p < 0.05) and 0.41-fold (p < 0.05) of that in the control group, respectively. The nuclear translocation of Cgp65 protein was suppressed in the CgTAAR1L-RNAi oysters. Furthermore, the number of Vibrio splendidus in the haemolymph of CgTAAR1L-RNAi oysters significantly increased (4.11-fold, p < 0.001) compared with that in the control group. In contrast, there was no significant difference in phagocytic rate of haemocytes to V. splendidus in the CgTAAR1L-RNAi oysters. These results indicated that CgTAAR1L played an important role in the immune defense against bacterial infection by inducing the expressions of interleukin and defensin., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Nicotine, a naturally occurring alkaloid found in tobacco, is a potent neurotoxin extensively used to control Nilaparvata lugens (Stål), a destructive insect pest of rice crops. The insect gut harbors a wide array of resident microorganisms that profoundly influence several biological processes, including host immunity. Maintaining an optimal gut microbiota and immune homeostasis requires a complex network of reciprocal regulatory interactions. However, the underlying molecular mechanisms driving these symbiotic exchanges, particularly between specific gut microbe and immunity, remain largely unknown in insects. Our previous investigations identified and isolated a nicotine-degrading Burkholderia cepacia strain (BsNLG8) with antifungal properties. Building on those findings, we found that nicotine intake significantly increased the abundance of a symbiotic bacteria BsNLG8, induced a stronger bacteriostatic effect in hemolymph, and enhanced the nicotine tolerance of N. lugens. Additionally, nicotine-induced antimicrobial peptides (AMPs) exhibited significant antibacterial effects against Staphylococcus aureus. We adopted RNA-seq to explore the underlying immunological mechanisms in nicotine-stressed N. lugens. Bioinformatic analyses identified numerous differentially expressed immune genes, including recognition/immune activation (GRPs and Toll) and AMPs (i.e., Defensin, Lugensin, lysozyme). Temporal expression profiling (12, 24, and 48 hours) of immune genes revealed pattern recognition proteins and immune effectors as primary responders to nicotine-induced stress. Defensin A, a broad-spectrum immunomodulatory cationic peptide, exhibited significantly high expression. RNA interference-mediated silencing of Defensin A reduced the survival, enhanced nicotine sensitivity of N. lugens to nicotine, and decreased the abundance of BsNLG8. The reintroduction of BsNLG8 improved the expression of immune genes, aiding nicotine resistance of N. lugens. Our findings indicate a potential reciprocal immunomodulatory interaction between Defensin A and BsNLG8 under nicotine stress. Moreover, this study offers novel and valuable insights for future research into enhancing nicotine-based pest management programs and developing alternative biocontrol methods involving the implication of insect symbionts., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest, (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

A distinct family of plant-specific WRKY transcription factors plays a crucial role in modulating responses to biotic and abiotic stresses. In this investigation, we unveiled a signaling pathway activated in the desert shrub Ammopiptanthus nanus during feeding by the moth Spodoptera exigua. The process involves a Ca 2+ flux that facilitates interaction between the protein kinase AnCIPK12 and AnWRKY29. AnWRKY29 directly interacts with the promoters of two key genes encoding AnPDF1 and AnHsfB1, involved in the biosynthesis of plant defensins. Consequently, AnWRKY29 exerts its transcriptional regulatory function, influencing plant defensins biosynthesis. This discovery implies that A. nanus can bolster resistance against herbivorous insects like S. exigua by utilizing this signaling pathway, providing an effective natural defense mechanism that supports its survival and reproductive success., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Insect host defense comprises two complementary dimensions, microbial killing-mediated resistance and microbial toxin neutralization-mediated resilience, both jointly providing protection against pathogen infections. Insect defensins are a class of effectors of innate immunity primarily responsible for resistance to Gram-positive bacteria. Here, we report a newly originated gene from an ancestral defensin via genetic deletion following gene duplication in Drosophila virilis, which confers an enhanced resilience to Gram-positive bacterial infection. This gene encodes an 18-mer arginine-rich peptide (termed DvirARP) with differences from its parent gene in its pattern of expression, structure and function. DvirARP specifically expresses in D. virilis female adults with a constitutive manner. It adopts a novel fold with a 3 10 helix and a two CXC motif-containing loop stabilized by two disulfide bridges. DvirARP exhibits no activity on the majority of microorganisms tested and only a weak activity against two Gram-positive bacteria. DvirARP knockout flies are viable and have no obvious defect in reproductivity but they are more susceptible to the DvirARP-resistant Staphylococcus aureus infection than the wild type files, which can be attributable to its ability in neutralization of the S. aureus secreted toxins. Phylogenetic distribution analysis reveals that DvirARP is restrictedly present in the Drosophila subgenus, but independent deletion variations also occur in defensins from the Sophophora subgenus, in support of the evolvability of this class of immune effectors. Our work illustrates for the first time how a duplicate resistance-mediated gene evolves an ability to increase the resilience of a subset of Drosophila species against bacterial infection., (© 2024. The Author(s).)

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., (© 2024. The Author(s).)

The purpose of this study was to investigate whether a defensin-like antimicrobial peptide (C-13326 peptide) identified in Hermetia illucens could possess protective effect against multidrug-resistant Aeromonas schubertii in hybrid snakehead (Channa maculate ♀ × Channa argus ♂). The cDNA of C-13326 peptide comprised 243 nucleotides encoding 80 amino acids, with six conserved cysteine residues and the classical CSαβ structure. The recombinant expression plasmid pPIC9K-C-13326 was constructed and transformed into GS115 Pichia pastoris, and the C-13326 peptide was expressed by induction with 1% methanol. The crude extract of C-13326 peptide was precipitated by ammonium sulfate, assayed by Braford method, detected by tricine-SDS-PAGE, evaluated by BandScan software and identified by liquid chromatography-mass spectrometry. The C-13326 peptide was shown to have inhibitory activity against the growth of multidrug-resistant A. schubertii DM210910 by using the minimum growth inhibitory concentration and Oxford cup method. In addition, scanning electron microscopy analysis suggested that C-13326 peptide inhibited the growth of A. schubertii DM210910 by damaging the bacterial cell membrane. To explore the role of peptide C-13326 in vivo, hybrid snakehead was fed with peptide C-13326 as feed additives for 7 days. The results revealed that C-13326 peptide could significantly down-regulate the expression levels of IL-1β, IL-8, IL-12 and TNF-α (p < .05), and significantly improved the survival rate of hybrid snakehead after challenging with A. schubertii DM210910. Therefore, the C-13326 peptide is a promising antimicrobial agent for A. schubertii treatment in aquaculture., (© 2024 John Wiley & Sons Ltd.)

Due to rapidly emerging resistance to single-site fungicides in fungal pathogens of plants, there is a burgeoning need for safe and multisite fungicides. Plant antifungal peptides with multisite modes of action (MoA) have potential as bioinspired fungicides. Medicago truncatula defensin MtDef4 was previously reported to exhibit potent antifungal activity against fungal pathogens. Its MoA involves plasma membrane disruption and binding to intracellular targets. However, specific biochemical processes inhibited by this defensin and causing cell death have not been determined. Here, we show that MtDef4 exhibited potent antifungal activity against Botrytis cinerea. It induced severe plasma membrane and organelle irregularities in the germlings of this pathogen. It bound to fungal ribosomes and inhibited protein translation in vitro. A MtDef4 variant lacking antifungal activity exhibited greatly reduced protein translation inhibitory activity. A cation-tolerant MtDef4 variant was generated that bound to β-glucan of the fungal cell wall with higher affinity than MtDef4. It also conferred a greater reduction in the grey mould disease symptoms than MtDef4 when applied exogenously on Nicotiana benthamiana plants, tomato fruits and rose petals. Our findings revealed inhibition of protein synthesis as a likely target of MtDef4 and the potential of its cation-tolerant variant as a peptide-based fungicide., (© 2024 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Insect defensins are a large family of antimicrobial peptides primarily active against Gram-positive bacteria. Here, we explore their hidden anti-Gram-negative bacterial potential via a nature-guided strategy inspired by natural deletion variants of Drosophila defensins. Referring to these variants, we deleted the equivalent region of an insect defensin with the first cysteine-containing N-terminus, and the last three cysteine-containing C-terminal regions remained. This 15-mer peptide exhibits low solubility and specifically targets Gram-positive bacteria. Further deletion of alanine-9 remarkably improves its solubility, unmasks its hidden anti-Gram-negative bacterial activity, and alters its states in different environments. Intriguingly, compared with the oxidized form, the 14-mer reduced peptide shows increased activity on Gram-positive and Gram-negative bacteria through a membrane-disruptive mechanism. The broad-spectrum activity and tolerance to high-salt environments and human serum, together with no toxicity to mammalian or human cells, make it a promising candidate for the design of new peptide antibiotics against Gram-negative bacterial infections.

Chronic endometritis has a high incidence in infertile women, which is caused by endometrial microbiome infection. In response to microbial infection, the role of defensins during chronic endometritis need explored. Besides, the expression of estrogen and its receptors vary in different menstrual cycles, but their roles in chronic endometritis are still unclear. In this study, we used the human endometrial tissues to examine the expression of antimicrobial peptides (AMPs) α-defensin hNP-1 and β-defensins hBD-1, hBD-2, hBD-3, hBD-4 and LCN2. We found the expression of hBD-1 and LCN2 were downregulated in endometritis tissues, while the expressions of hBD-2, hBD-3, hBD-4, hNP-1, and estrogen and ERα were upregulated in chronic endometritis tissues compared to normal tissues. The expression and phosphorylation of STING, which is a crucial mediator of mammalian innate immunity in response to pathogens, was regulated with the treatment of ERα inhibitor raloxifene (Rx). Furthermore, using with the estrogen receptor inhibitor Rx and STING inhibitor H-151 significantly decreases the LCN2 expression. Taken together, these results suggested ERα was upregulated to modulate STING expression inducing LCN2 antimicrobial peptide expression to modulate the mucosal immunity during chronic endometritis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Subtilisin-like enzymes are recognized as key players in many infectious agents. In this context, its inhibitors are very valuable molecular lead compounds for structure based drug discovery and design. Marine invertebrates offer a great source of bioactive molecules, including protease inhibitors. In this work, we describe a new subtilisin inhibitor, from the sea anemone Condylactis gigantea (CogiTx1). CogiTx1 was purified using a combination of cation exchange chromatography, size exclusion chromatography and RP-HPLC chromatography. CogiTx1 it is a protein with 46 amino acid residues, with 4970.44 Da and three disulfide bridges. Is also able to inhibit subtilisin-like enzymes and pancreatic elastase. According to the amino acid sequence, it belongs to the defensin 4 family of proteins. The sequencing showed that CogiTx1 has an amidated C-terminal end, which was confirmed by the presence of the typical -XGR signal for amidation in the protein sequence deduced from the cDNA. This modification was described at protein level for the first time in this family of proteins. CogiTx1 is the first subtilisin inhibitor from the defensin 4 family and accordingly it has a folding consisting primarily in beta-strands in agreement with the analysis by CD and 3D modelling. Therefore, future in-depth functional studies may allow a more detailed characterization and will shed light on structure-function properties., Competing Interests: Declaration of competing interest The authors have no relevant financial or non-financial interests to disclose., (Copyright © 2023 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Ectopic expression of defensins in plants correlates with their increased capacity to withstand abiotic and biotic stresses. This applies to Arabidopsis thaliana, where some of the seven members of the PLANT DEFENSIN 1 family (AtPDF1) are recognised to improve plant responses to necrotrophic pathogens and increase seedling tolerance to excess zinc (Zn). However, few studies have explored the effects of decreased endogenous defensin expression on these stress responses. Here, we carried out an extensive physiological and biochemical comparative characterization of (i) novel artificial microRNA (amiRNA) lines silenced for the five most similar AtPDF1s, and (ii) a double null mutant for the two most distant AtPDF1s. Silencing of five AtPDF1 genes was specifically associated with increased aboveground dry mass production in mature plants under excess Zn conditions, and with increased plant tolerance to different pathogens - a fungus, an oomycete and a bacterium, while the double mutant behaved similarly to the wild type. These unexpected results challenge the current paradigm describing the role of PDFs in plant stress responses. Additional roles of endogenous plant defensins are discussed, opening new perspectives for their functions., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

As a family of cationic host defense peptides, defensins are mainly synthesized by Paneth cells, neutrophils, and epithelial cells, contributing to host defense. Their biological functions in innate immunity, as well as their structure and activity relationships, along with their mechanisms of action and therapeutic potential, have been of great interest in recent years. To highlight the key research into the role of defensins in human and animal health, we first describe their research history, structural features, evolution, and antimicrobial mechanisms. Next, we cover the role of defensins in immune homeostasis, chemotaxis, mucosal barrier function, gut microbiota regulation, intestinal development and regulation of cell death. Further, we discuss their clinical relevance and therapeutic potential in various diseases, including infectious disease, inflammatory bowel disease, diabetes and obesity, chronic inflammatory lung disease, periodontitis and cancer. Finally, we summarize the current knowledge regarding the nutrient-dependent regulation of defensins, including fatty acids, amino acids, microelements, plant extracts, and probiotics, while considering the clinical application of such regulation. Together, the review summarizes the various biological functions, mechanism of actions and potential clinical significance of defensins, along with the challenges in developing defensins-based therapy, thus providing crucial insights into their biology and potential clinical utility., (© 2023. West China Hospital, Sichuan University.)

The depletion of cellular zinc (Zn) adversely affects plant growth. Plants have adaptation mechanisms for Zn-deficient conditions, inhibiting growth through the action of transcription factors and metal transporters. We previously identified three defensin-like (DEFL) proteins (DEFL203, DEFL206 and DEFL208) that were induced in Arabidopsis thaliana roots under Zn-depleted conditions. DEFLs are small cysteine-rich peptides involved in defense responses, development and excess metal stress in plants. However, the functions of DEFLs in the Zn-deficiency response are largely unknown. Here, phylogenetic tree analysis revealed that seven DEFLs (DEFL202-DEFL208) were categorized into one subgroup. Among the seven DEFLs, the transcripts of five (not DEFL204 and DEFL205) were upregulated by Zn deficiency, consistent with the presence of cis-elements for basic-region leucine-zipper 19 (bZIP19) or bZIP23 in their promoter regions. Microscopic observation of GFP-tagged DEFL203 showed that DEFL203-sGFP was localized to the apoplast and plasma membrane. Whereas a single mutation of the DEFL202 or DEFL203 genes only slightly affected root growth, defl202 defl203 double mutants showed enhanced root growth under all growth conditions. We also showed that the size of the root meristem was increased in the double mutants compared with the wild type. Our results suggest that DEFL202 and DEFL203 are redundantly involved in the inhibition of root growth under Zn-deficient conditions through a reduction in root meristem length and cell number., (© 2023 Society for Experimental Biology and John Wiley & Sons Ltd.)

Thionins are important antibacterial peptides in plants. However, the roles of plant thionins, especially the defensin-dissimilar thionins, in alleviating heavy-metal toxicity and accumulation remain unclear. Here, cadmium (Cd)-related functions and mechanisms of the defensin-dissimilar rice thionin OsThi9 were investigated. OsThi9 was significantly upregulated in response to Cd exposure. OsThi9 was localized to the cell wall and was shown to bind Cd; these characters help to increase Cd tolerance. In Cd-exposed rice plants, OsThi9 overexpression significantly increased cell wall Cd binding, decreasing upward Cd translocation and subsequent Cd accumulation in shoots and straw, while OsThi9 knockout had inverse effects. Importantly, in rice plants grown in Cd-contaminated soils, OsThi9 overexpression significantly reduced Cd accumulation in brown rice (decrease of ≥ 51.8%) without negatively impairing the crop yield and essential elements. Thus, OsThi9 plays an important role in the alleviation of Cd toxicity and accumulation and has significant potential for developing low-Cd rice.

Background: The body and head lice of humans are conspecific, but only the body louse functions as a vector to transmit bacterial pathogens such as Bartonella quintana. Both louse subspecies have only two antimicrobial peptides, defensin 1 and defensin 2. Consequently, any differences in the molecular and functional properties of these two louse subspecies may be responsible for the differential vector competence between them., Methods: To elucidate the molecular basis of vector competence, we compared differences in the structural properties and transcription factor/microRNA binding sites of the two defensins in body and head lice. Antimicrobial activity spectra were also investigated using recombinant louse defensins expressed via baculovirus., Results: The full-length amino acid sequences of defensin 1 were identical in both subspecies, whereas the two amino acid residues in defensin 2 were different between the two subspecies. Recombinant louse defensins showed antimicrobial activities only against the representative Gram-positive Staphylococcus aureus but not against either Gram-negative Escherichia coli or the yeast Candida albicans. However, they did show considerable activity against B. quintana, with body louse defensin 2 being significantly less potent than head louse defensin 2. Regulatory sequence analysis revealed that the gene units of both defensin 1 and defensin 2 in body lice possess decreased numbers of transcription factor-binding sites but increased numbers of microRNA binding sites, suggesting relatively lower transcription activities of body louse defensins., Conclusions: The significantly lower antibacterial activities of defensin 2 along with the reduced probability of defensin expression in body lice likely contribute to the relaxed immune response to B. quintana proliferation and viability, resulting in higher vector competence of body lice compared to head lice., (© 2023. The Author(s).)

Plant defensins are a potential tool in crop improvement programs through biotechnology. Their antifungal action makes them attractive molecules for the production of transgenic plants. Information is currently lacking on what happens to the expression of defense genes in transgenic plants that overexpress a defensin. Here we show the relative expression of four defense-related genes: Mn-sod, PAL1, aos1 and HPL evaluated in two transgenic soybean events (Def1 and Def17) constitutively expressing the NmDef02 defensin gene from Nicotiana megalosiphon. The expression of these defense genes showed a differential profile in the transgenic events, with the increased expression of the aos1 gene and the repression of the Mn-sod gene in both events, when compared to the non-transgenic control. Furthermore, the expression of the PAL1 gene only increased in the Def17 event. The results indicate that although there were some changes in the expression of defense genes in transgenic plants overexpressing the defensin NmDef02; the morphoagronomic parameters evaluated were similar to the non-transgenic control. Understanding the molecular changes that occur in these transgenic plants could be of interest in the short, medium and long term., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Divergent evolution for more than 310 million years has resulted in an avian immune system that is complex and more compact than that of primates, sharing much of its structure and functions. Not surprisingly, well conserved ancient host defense molecules, such as defensins and cathelicidins, have diversified over time. In this review, we describe how evolution influenced the host defense peptides repertoire, its distribution, and the relationship between structure and biological functions. Marked features of primate and avian HDPs are linked to species-specific characteristics, biological requirements, and environmental challenge., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The emergence of multi-resistant pathogens have increased dramatically in recent years, becoming a major public-health concern. Among other promising antimicrobial molecules with potential to assist in this worldwide struggle, cysteine-stabilized αβ (CS-αβ) defensins are attracting attention due their efficacy, stability, and broad spectrum against viruses, bacteria, fungi, and protists, including many known human pathogens., Results: Here, 23 genomes of ciliated protists were screened and two CS-αβ defensins with a likely antifungal activity were identified and characterized, using bioinformatics, from a culturable freshwater species, Laurentiella sp. (LsAMP-1 and LsAMP-2). Although any potential cellular ligand could be predicted for LsAMP-2; evidences from structural, molecular dynamics, and docking analyses suggest that LsAMP-1 may form stably associations with phosphatidylinositol 4,5-bisphosphates (PIP2), a phospholipid found on many eukaryotic cells, which could, in turn, represent an anchorage mechanism within plasma membrane of targeted cells., Conclusion: These data stress that more biotechnology-oriented studies should be conducted on neglected protists, such ciliates, which could become valuable sources of novel bioactive molecules for therapeutic uses., (© 2023. The Author(s).)

Lepidopteran insect pest Helicoverpa armigera is one of the most destructive pests of crop plants and several biotechnological approaches are being developed for its control. Plant defensins are small cationic and cysteine-rich peptides that play a role in plant defense. Ingestion of a defensin from Capsicum annuum (CanDef-20) induced a dose-dependent reduction in larval and pupal mass, delayed metamorphosis and also severely reduced fecundity and fertility in H. armigera. To understand the molecular mechanisms of CanDef-20 ingestion-mediated antibiosis in H. armigera larvae, a comparative transcriptomics analysis was carried out. Predominant downregulation of GOs represents serine-type endopeptidases, structural constituents of ribosomes and integral membrane components and differential upregulation of ATP binding, nucleus and translation, while up-regulation of nucleic acid binding represented by transposable elements, were detected. Different isoforms of lipase, serine endopeptidase, glutathione S-transferase, cadherin, alkaline phosphatase and aminopeptidases were found to be upregulated as a compensatory response to CanDef-20 ingestion. In vitro enzyme assays and qPCR analysis of some representative genes associated with vital cellular processes like metamorphosis, food digestion and gut membrane indicated adaptive differential regulations in CanDef-20 fed H. armigera larvae. We conclude that CanDef-20 ingestion affects insect metabolism in a number of ways through its interaction with cell membrane, enzymes, cytoplasmic proteins and triggering transposon mobilization which are linked to growth retardation and adaptive strategies in H. armigera., (© 2023. The Author(s).)

Background: Reproductive tract infection is an important factor leading to male and female infertility. Among female infertility factors, microbial and viral infections are the main factors affecting female reproductive health and causing tubal infertility, ectopic tubal pregnancy and premature delivery. Among male infertility factors, 13-15% of male infertility is related to infection. Defensins are cationic antibacterial and antiviral peptides, classified into α-defensins, β-defensins and θ-defensins. Humans only have α-defensins and β-defensins. Apart from their direct antimicrobial functions, defensins have an immunomodulatory function and are involved in many physiological processes. Studies have shown that defensins are widely distributed in the female reproductive tract (FRT) and male reproductive tract (MRT), playing a dual role of host defence and fertility protection. However, to our knowledge, the distribution, regulation and function of defensins in the reproductive tract and their relation to reproduction have not been reviewed., Objective and Rationale: This review summarizes the expression, distribution and regulation of defensins in the reproductive tracts to reveal the updated research on the dual role of defensins in host defence and the protection of fertility., Search Methods: A systematic search was conducted in PubMed using the related keywords through April 2022. Related data from original researches and reviews were integrated to comprehensively review the current findings and understanding of defensins in the human reproductive system. Meanwhile, female and male transcriptome data in the GEO database were screened to analyze defensins in the human reproductive tracts., Outcomes: Two transcriptome databases from the GEO database (GSE7307 and GSE150852) combined with existing researches reveal the expression levels and role of the defensins in the reproductive tracts. In the FRT, a high expression level of α-defensin is found, and the expression levels of defensins in the vulva and vagina are higher than those in other organs. The expression of defensins in the endometrium varies with menstrual cycle stages and with microbial invasion. Defensins also participate in the local immune response to regulate the risk of spontaneous preterm birth. In the MRT, a high expression level of β-defensins is also found. It is mainly highly expressed in the epididymal caput and corpus, indicating that defensins play an important role in sperm maturation. The expression of defensins in the MRT varies with androgen levels, age and the status of microbial invasion. They protect the male reproductive system from bacterial infections by neutralizing lipopolysaccharide and downregulating pro-inflammatory cytokines. In addition, animal and clinical studies have shown that defensins play an important role in sperm maturation, motility and fertilization., Wider Implications: As a broad-spectrum antimicrobial peptide without drug resistance, defensin has great potential for developing new natural antimicrobial treatments for reproductive tract infections. However, increasing evidence has shown that defensins can not only inhibit microbial invasion but can also promote the invasion and adhesion of some microorganisms in certain biological environments, such as human immunodeficiency virus. Therefore, the safety of defensins as reproductive tract anti-infective drugs needs more in-depth research. In addition, the modulatory role of defensins in fertility requires more in-depth research since the current conclusions are based on small-size samples. At present, scientists have made many attempts at the clinical transformation of defensins. However, defensins have problems such as poor stability, low bioavailability and difficulties in their synthesis. Therefore, the production of safe, effective and low-cost drugs remains a challenge., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Grain cadmium (Cd) is translocated from source to sink tissues exclusively via phloem, though the phloem Cd unloading transporter has not been identified yet. Here, we isolated and functionally characterized a defensin-like gene DEFENSIN 8 (DEF8) highly expressed in rice (Oryza sativa) grains and induced by Cd exposure in seedling roots. Histochemical analysis and subcellular localization detected DEF8 expression preferentially in pericycle cells and phloem of seedling roots, as well as in phloem of grain vasculatures. Further analysis demonstrated that DEF8 is secreted into extracellular spaces possibly by vesicle trafficking. DEF8 bound to Cd in vitro, and Cd efflux from protoplasts as well as loading into xylem vessels decreased in the def8 mutant seedlings compared with the wild type. At maturity, significantly less Cd accumulation was observed in the mutant grains. These results suggest that DEF8 is a dual function protein that facilitates Cd loading into xylem and unloading from phloem, thus mediating Cd translocation from roots to shoots and further allocation to grains, representing a phloem Cd unloading regulator. Moreover, essential mineral nutrient accumulation as well as important agronomic traits were not affected in the def8 mutants, suggesting DEF8 is an ideal target for breeding low grain Cd rice., (© The Author(s) 2022. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Insects can assemble defensive microbiomes on their body surfaces to defend against fungal parasitic infections. The strategies employed by fungal pathogens to combat host cuticular microbiotas remains unclear. Here, we report the identification and functional characterization of the defensin-like antimicrobial gene BbAMP1 encoded by the entomopathogenic fungus Beauveria bassiana. The mature peptide of BbAMP1 can coat fungal spores and can be secreted by the fungus to target and damage Gram-positive bacterial cells. Significant differences in insect survival were observed between the wild-type and BbAMP1 mutant strains during topical infection but not during injection assays that bypassed insect cuticles. Thus, BbAMP1 deletion considerably reduced fungal virulence while gene overexpression accelerated the fungal colonization of insects compared with the wild-type strain in natural infections. Topical infection of axenic Drosophila adults evidenced no difference in fly survivals between strains. However, the gnotobiotic infections with the addition of Gram-positive but not Gram-negative bacterial cells in fungal spore suspensions substantially increased the survival of the flies treated with ∆BbAMP1 compared to those infected by the wild-type and gene-overexpression strains. Bacterial colony counts and microbiome analysis confirmed that BbAMP1 could assist the fungus to manipulate insect surface bacterial loads. This study reveals that fungal defensin can suppress the host surface defensive microbiomes, which underscores the importance to extend the research scope of fungus-host interactions., (© 2022. The Author(s), under exclusive licence to International Society for Microbial Ecology.)

Background: Chickpea (Cicer arietinum L.), a major nutritional source cultivated worldwide, is vulnerable to several abiotic and biotic stresses, including different types of soil-borne pathogens like Fusarium oxysporum f. sp. ciceri, which causes root rot disease and severely affects productivity., Methods and Results: In this study, putative transgenic plants were obtained with the Radish defensin (Rs-AFP2) gene through Agrobacterium tumefaciens mediated transformation using the embryo axis explants. Transgenes were confirmed in 18 putative transgenic plants with PCR-specific primers for nptII and Rs-AFP2 genes. Twelve transgenic plants were established successfully under greenhouse conditions. The T0 plants were allowed for self-pollination to obtain T1 seeds. The T1 plants, selected for Fusarium wilt assay using Fusarium oxysporum f. sp. Cicero, showed different resistance levels, from moderate to high levels in comparison to control plants (wild-type) which exhibited severe wilt symptoms., Conclusion: Our results suggest the application of Radish defensins (RsAFP1/RsAFP2 genes) for improving pathogen resistance in chickpea., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Recombinant promoters are of high value in translational research. Earlier, we developed two recombinant promoters, namely MUAS35SCP and FUAS35SCP, and their transcriptional activities were found to be stronger than that of the most widely used CaMV35S promoter in dicot plants. Presently, we are reporting constitutive expression of both GUS and GFP reporters under the control of these promoters in several monocots, including rice, wheat, and pearl millet. We observed that these promoters could express the reporter genes constitutively, and their expression abilities were almost equal to that of the CaMV35S 2 promoter. Plant-derived enriched PaDef (Persea americana var. drymifolia defensin) and NsDef2 (Nigella sativa L. defensin 2) antimicrobial peptides expressed under the control of these promoters arrest the growth of devastating phytopathogens like Pseudomonas syringae, Rhodococcus fascians, and Alternaria alternata. We observed that plant-derived NsDef2 and PaDef under control of these promoters showed approximately 80-90% inhibitory activity against Pseudomonas syringae. Hence, these promoters were constitutive and universal, as they can drive the expression of transgenes in both dicot and monocot plants. Alongside, these promoters could become a valuable tool for raising genetically modified plants with in-built resistance toward phytopathogens., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In this study, seven transcripts representing a novel antimicrobial peptide (AMP) family with structural features similar to those of arthropod defensins were identified from Mytilus coruscus. These novel defensins from the Mytilus AMP family were named myticofensins. To explore the possible immune-related functions of these myticofensins, we examined their expression profiles in different tissues and larval stages, as well as in three immune-related tissues under the threat of different microbes. Our data revealed that the seven myticofensins had relatively high expression levels in immune-related tissues. Most myticofensins were undetectable, or had low expression levels, in different larval mussel stages. Additionally, in vivo microbial challenges significantly increased the expression levels of myticofensins in M. coruscus hemocytes, gills, and digestive glands, showing different immune response patterns under challenges from different microbes. Our data indicates that different myticofensins may have different immune functions in different tissues. Furthermore, peptide sequences corresponding to the beta-hairpin, alpha-helix, and N-terminal loop of myticofensin were synthesized and the antimicrobial activities of these peptide fragments were tested. Our data confirms the diversity of defensins in Mytilus and reports the complex regulation of these defensins in the mussel immune response to different microbes in immune-related tissues. The immune system of Mytilus has been studied for years as they are a species with strong environmental adaptations. Our data can be regarded as a step forward in the study of the adaptation of Mytilus spp. to an evolving microbial world., Competing Interests: Declaration of competing interest The authors have declared that no conflict of interests exist., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Commiphora gileadensis L. is a medicinal plant, known as balsam, with pharmaceutical potential for its phytochemical activities and chemical constituents. Genetic diversity is a genetic tool used in medicinal plant evolution and conservation. Three accessions from C. gileadensis were collected from three localities in Saudi Arabia (Jeddah, Jizan and Riyadh). Genetic characterization was carried out using physio-biochemical parameters, molecular markers (inter-simple sequence repeat (ISSR) and start codon targeted (SCoT)), DNA barcoding (18 S rRNA and ITS rDNA regions), relative gene expressions (phenylalanine ammonia-lyase 1 (PAL1), defensin (PR-12)) and pathogenesis-related protein (AFPRT). The results of this study showed that C. gileadensis accession C3, collected from Riyadh, had the highest content from the physio-biochemical parameters perspective, with values of 92.54 mg/g and 77.13 mg/g for total phenolic content (TPC) and total flavonoid content (TFC), respectively. Furthermore, the highest content of antioxidant enzyme activity was present in accession C3 with values of 16.87, 60.87, 35.76 and 27.98 U mg -1 for superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) (mol/min/mg FW) and ascorbate peroxidase (APX) (U mg -1 protein), respectively. The highest total number of bands and number of unique bands were 138 and 59, respectively, for the SCoT marker. The SCoT marker was the most efficient for the genetic diversity of C. gileadensis by producing the highest polymorphism (75.63%). DNA barcoding using 18 S and ITS showed the nearby Commiphora genus and clustered C . gileadensis accessions from Jeddah and Jizan in one clade and the C. gileadensis accession from Ryiadh in a separate cluster. Moreover, relative gene expression of the PAL1, defensin (PR-12) and AFPRT (PR1) genes was upregulated in the C . gileadensis accession from Ryiadh. In conclusion, ecological and environmental conditions in each locality affect the genomic expression and genetic diversity, which can help the evolution of important medicinal plants and improve breeding and conservation systems.

Aphids (Hemiptera: Aphidoidea) are among the most detrimental insects for agricultural plants, and their management is a great challenge in agronomical research. A new class of proteins, called Bacteriocyte-specific Cysteine-Rich (BCR) peptides, provides an alternative to chemical insecticides for pest control. BCRs were initially identified in the pea aphid Acyrthosiphon pisum . They are small disulfide bond-rich proteins expressed exclusively in aphid bacteriocytes, the insect cells that host intracellular symbiotic bacteria. Here, we show that one of the A. pisum BCRs, BCR4, displays prominent insecticidal activity against the pea aphid, impairing insect survival and nymphal growth, providing evidence for its potential use as a new biopesticide. Our comparative genomics and phylogenetic analyses indicate that BCRs are restricted to the aphid lineage. The 3D structure of BCR4 reveals that this peptide belongs to an as-yet-unknown structural class of peptides and defines a new superfamily of defensins.

Background: Pulmonary embolism (PE) is a leading cause of death in stroke patients and a severe health burden worldwide. There is a pressing need to understand the mechanisms by which it occurs and to identify at-risk patients efficiently and accurately., Methods: First, based on data from GWAS in European populations, we performed a linkage disequilibrium score regression (LDSC) analysis of plasma proteins and PE in 3,283 individuals and additionally analyzed the genetic association between PE and fracture. Then, we performed a TWAS on PE GWAS data using skeletal muscle and blood for gene expression references. Finally, we validated the genetic correlation between PE and human plasma proteins by co-matching the genes encoding the identified proteins and those identified using TWAS with the differentially expressed genes obtained from mRNA expression profiling of PE., Results: We identified five plasma proteins associated with PE, including hydroxycarboxylic acid receptor 2, defensin 118, and bone morphogenetic protein (BMP) 7, as well as a relationship between PE and fracture. Comparison of genes encoding these proteins with genes obtained from TWAS and then with differentially expressed genes obtained from PE mRNA expression profiling revealed that PE was highly correlated with the BMP family of genes.

Studies on phylogeography and population structure of Ixodes ricinus have been carried out in Europe for decades, but the number of specimens from the Middle East included in these analyses is relatively small, despite the wide distribution of the species in this area. This study aimed to clarify the phylogenetic positions of I. ricinus from Turkey as well as to investigate the presence of Ixodes inopinatus in Anatolia. For this purpose, one mitochondrial (mt 16S rDNA) and one nuclear gene (defensin) were used to generate molecular data from I. ricinus samples, which were collected from 17 locations across the species' distributional range in Turkey. Bayesian inference was used to investigate phylogenetic relationships. Globally, the mt 16S rDNA lineages correspond to the lineages revealed by defensin; I. ricinus and I. inopinatus sequences clustered separately. However, a discordant genetic pattern was observed between the phylogenetic position of turkish I. ricinus revealed by nuclear versus mitochondrial genes. All Turkish haplotypes of mt 16SrDNA clustered with I. ricinus samples from Europe, which might be the result of extensive gene flow between populations of Europe and the Middle East. On the other hand, a sample from Thrace Region grouped within I. inopinatus clade. Thus, the occurrence of I. inopinatus in Turkey was demonstrated for the first time using molecular data. Moreover, four individuals were found to be heterozygous for the defensin. The potential evolutionary processes that underlie this observed discrepancy between the phylogenetic trees of two genes have been discussed., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Proline-rich peptide (CgPrp) and defensin (CgDef), oyster (Crassostrea gigas)-originated antimicrobial peptides (AMPs), were produced by the recombinant technique in Komagataella phaffii GS115 cells. For this purpose, the nucleotide sequences encoding the CgPrp and CgDef peptides were synthesized by the recursive PCR technique, and ligated in pPICZaA expression vector. Additionally, the expression cassettes of pPICZαA-CgDef and pPICZαA-CgPrp were combined using in vitro multimer ligation strategy to construct the coexpression vector pPICZaA-CgPrp-CgDef. The expression and coexpression vectors transformed into K. phaffii GS115 cells by electroporation. At the end of the 0.5% methanol-induced expression stage for 96 h, the recombinant peptides were purified from the culture medium. The concentrations of purified peptides were changed between 1.05 and 1.21 mg/L. The recombinant peptides successfully inhibited the growth of tested Gram-positive bacterial strains belonging to Staphylococcus aureus, methicillin-resistant S. aureus (MRSA), Listeria monocytogenes, and Bacillus cereus. The minimum inhibitory concentrations (MIC) of recombinant CgPrp, CgDef, and CgPrp-CgDef peptides against tested bacteria were in the range of 12.50-25.00, 18.75-75.00, and 5.80-11.60 pg/μl, respectively. The results of the study proved that the recombinant CgPrp, CgDef, and CgPrp-CgDef peptides expressed in K. phaffii might have good potential for the inhibition of common Gram-positive pathogenic bacteria, including drug-resistant MRSA., (© 2021 International Union of Biochemistry and Molecular Biology, Inc.)

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&#95;024085718.1), CL-defensin2 (XP&#95;014240919.1)), and two new defensin isoforms (CL-defensin3a (XP&#95;014240918.1), CL-defensin3b (XP&#95;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.

During infection, Bacillus anthracis bacilli encounter potent antimicrobial peptides (AMPs) such as defensins. We examined the role that B. anthracis capsule plays in protecting bacilli from defensins and other cationic AMPs by comparing their effects on a fully virulent encapsulated wild type (WT) strain and an isogenic capsule-deficient capA mutant strain. We identified several human defensins and non-human AMPs that were capable of killing B. anthracis. The human alpha defensins 1-6 (HNP-1-4, HD-5-6), the human beta defensins 1-4 (HBD-1-4), and the non-human AMPs, protegrin, gramicidin D, polymyxin B, nisin, and melittin were all capable of killing both encapsulated WT and non-encapsulated capA mutant B. anthracis. However, non-encapsulated capA mutant bacilli were significantly more susceptible than encapsulated WT bacilli to killing by nearly all of the AMPs tested. We demonstrated that purified capsule bound HBD-2, HBD-3, and HNP-1 in an electrophoretic mobility shift assay. Furthermore, we determined that the capsule layer enveloping WT bacilli bound and trapped HBD-3, substantially reducing the amount reaching the cell wall. To assess whether released capsule might also play a protective role, we pre-incubated HBD-2, HBD-3, or HNP-1 with purified capsule before their addition to non-encapsulated capA mutant bacilli. We found that free capsule completely rescued the capA mutant bacilli from killing by HBD-2 and -3 while killing by HNP-1 was reduced to the level observed with WT bacilli. Together, these results suggest an immune evasion mechanism by which the capsule, both that enveloping the bacilli and released fragments, contributes to virulence by binding to and inhibiting the antimicrobial activity of cationic AMPs., Competing Interests: The authors have declared that no competing interests exist.

Healthy skin maintains a diverse microbiome and a potent immune system to fight off infections. Here, we discovered that the epithelial-cell-derived antimicrobial peptides defensins activated orphan G-protein-coupled receptors (GPCRs) Mrgpra2a/b on neutrophils. This signaling axis was required for effective neutrophil-mediated skin immunity and microbiome homeostasis. We generated mutant mouse lines lacking the entire Defensin (Def) gene cluster in keratinocytes or Mrgpra2a/b. Def and Mrgpra2 mutant animals both exhibited skin dysbiosis, with reduced microbial diversity and expansion of Staphylococcus species. Defensins and Mrgpra2 were critical for combating S. aureus infections and the formation of neutrophil abscesses, a hallmark of antibacterial immunity. Activation of Mrgpra2 by defensin triggered neutrophil release of IL-1β and CXCL2 which are vital for proper amplification and propagation of the antibacterial immune response. This study demonstrated the importance of epithelial-neutrophil signaling via the defensin-Mrgpra2 axis in maintaining healthy skin ecology and promoting antibacterial host defense., Competing Interests: Declaration of interests Xinzhong Dong is a co-founder and a scientific advisory board member of Escient Pharmaceuticals, a company focused on developing small molecules targeting MRGPRs. N.K.A. has received previous grant support from Pfizer and Boehringer Ingelheim and was a paid consultant for Janssen Pharmaceuticals. L.S.M. is currently a full-time paid employee of Janssen Research & Development, the pharmaceutical companies of Johnson & Johnson, and owns Johnson & Johnson stock. L.S.M. performed all work at his prior affiliation at Johns Hopkins University School of Medicine and he has received prior grant support from AstraZeneca, Pfizer, Boehringer Ingelheim, Regeneron Pharmaceuticals, and Moderna Therapeutics; was a paid consultant for Armirall and Janssen Research and Development; was on the scientific advisory board of Integrated Biotherapeutics; and is a shareholder of Noveome Biotherapeutics, which are all developing therapeutics against infections (including S. aureus and other pathogens) and/or inflammatory conditions. B.S.K. has served as a consultant for AbbVie, ABRAX Japan, Almirall, Cara Therapeutics, Maruho, Menlo Therapeutics, Pfizer, and Third Rock Ventures. He has also participated on the advisory board for Almirall, Boehringer Ingelheim, Cara Therapeutics, Kiniksa Pharmaceuticals, Menlo Therapeutics, Regeneron Pharmaceuticals, Sanofi Genzyme, and Trevi Therapeutics. He is also Founder, Chief Scientific Officer, and stockholder of Nuogen Pharma, Inc. He is stockholder of Locus Biosciences. C.V. is a paid consultant and stockholder of AstraZeneca., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mesobuthus martensii , a famous and important Traditional Chinese Medicine has a long medical history and unique functions. It is the first scorpion species whose whole genome was sequenced worldwide. In addition, it is the most widespread and infamous poisonous animal in northern China with complex habitats. It possesses several kinds of toxins that can regulate different ion channels and serve as crucial natural drug resources. Extensive and in-depth studies have been performed on the structures and functions of toxins of M . martensii . In this research, we compared the morphology of M. martensii populations from different localities and calculated the COI genetic distance to determine intraspecific variations. Transcriptome sequencing by RNA-sequencing of the venom glands of M . martensii from ten localities and M. eupeus from one locality was analyzed. The results revealed intraspecific variation in the expression of sodium channel toxin genes, potassium channel toxin genes, calcium channel toxin genes, chloride channel toxin genes, and defensin genes that could be related to the habitats in which these populations are distributed, except the genetic relationships. However, it is not the same in different toxin families. M . martensii and M. eupeus exhibit sexual dimorphism under the expression of toxin genes, which also vary in different toxin families. The following order was recorded in the difference of expression of sodium channel toxin genes: interspecific difference; differences among different populations of the same species; differences between sexes in the same population, whereas the order in the difference of expression of potassium channel toxin genes was interspecific difference; differences between both sexes of same populations; differences among the same sex in different populations of the same species. In addition, there existed fewer expressed genes of calcium channel toxins, chloride channel toxins, and defensins (no more than four members in each family), and their expression differences were not distinct. Interestingly, the expression of two calcium channel toxin genes showed a preference for males and certain populations. We found a difference in the expression of sodium channel toxin genes, potassium channel toxin genes, and chloride channel toxin genes between M . martensii and M. eupeus . In most cases, the expression of one member of the toxin gene clusters distributed in series on the genome were close in different populations and genders, and the members of most clusters expressed in same population and gender tended to be the different. Twenty-one toxin genes were found with the MS/MS identification evidence of M . martensii venom. Since scorpions were not subjected to electrical stimulation or other special treatments before conducting the transcriptome extraction experiment, the results suggested the presence of intraspecific variation and sexual dimorphism of toxin components which revealed the expression characteristics of toxin and defensin genes in M . martensii . We believe this study will promote further in-depth research and use of scorpions and their toxin resources, which in turn will be helpful in standardizing the identification and medical applications of Quanxie in traditional Chinese medicine.

Salmonella, one of the major infectious diseases in poultry, causes considerable economic losses in terms of mortality and morbidity, especially in countries that lack effective vaccination programs. Besides being resistant to diseases, indigenous chicken breeds are also a potential source of animal protein in developing countries. For understanding the disease resistance, an indigenous chicken line Kashmir faverolla, and commercial broiler were selected. RNA-seq was performed after challenging the chicken with Salmonella Typhimurium. Comparative differential expression results showed that following infection, a total of 3153 genes and 1787 genes were differentially expressed in the liver and spleen, respectively. The genes that were differentially expressed included interleukins, cytokines, NOS2, Avβ-defensins, toll-like receptors, and other immune-related gene families. Most of the genes and signaling pathways involved in the innate and adaptive immune responses against bacterial infection were significantly enriched in the Kashmir faverolla. Pathway analysis revealed that most of the enriched pathways were MAPK signaling pathway, NOD-like receptor signaling pathway, TLR signaling pathway, PPAR signaling pathway, endocytosis, etc. Surprisingly some immune-related genes like TLRs were upregulated in the susceptible chicken breed. On postmortem examination, the resistant birds showed small lesions in the liver compared to large necrotic lesions in susceptible birds. The pathological manifestations and RNA sequencing results suggest a balancing link between resistance and infection tolerance in Kashmir faverolla. Here we also developed an online Poultry Infection Database (https://skuastk.org/pif/index.html), the first publicly available gene expression resource for disease resistance in chickens. The available database not only shows the data for gene expression in chicken tissues but also provides quick search, visualization and download capacity., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 μM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages., Competing Interests: The authors have declared that no competing interests exist.

Antimicrobial resistance requires urgent efforts towards the discovery of active antimicrobials, and the development of strategies to sustainably produce them. Defensin and defensin-like antimicrobial peptides (AMPs) are increasingly gaining pharmacological interest because of their potency against pathogens. In this study, we expressed two AMPs: defensin-d2 derived from spinach, and defensin-like actifensin from Actinomyces ruminicola . Recombinant pTXB1 plasmids carrying the target genes encoding defensin-d2 and actifensin were generated by the MEGAWHOP cloning strategy. Each AMP was first expressed as a fusion protein in Escherichia coli , purified by affinity chromatography, and was thereafter assayed for antimicrobial activity against multidrug-resistant (MDR) pathogens. Approximately 985 µg/mL and 2895 µg/mL of recombinant defensin-d2 and actifensin, respectively, were recovered with high purity. An analysis by MALDI-TOF MS showed distinct peaks corresponding to molecular weights of approximately 4.1 kDa for actifensin and 5.8 kDa for defensin-d2. An in vitro antimicrobial assay showed that MDR Pseudomonas aeruginosa and Candida albicans were inhibited at minimum concentrations of 7.5 µg/mL and 23 µg/mL for recombinant defensin-d2 and actifensin, respectively. The inhibitory kinetics of the peptides revealed cidal activity within 4 h of the contact time. Furthermore, both peptides exhibited an antagonistic interaction, which could be attributed to their affinities for similar ligands, as deduced by peptide-ligand profiling. Moreover, both peptides inhibited biofilm formation, and they exhibited no resistance potential and low hemolytic activity. The peptides also possess the ability to permeate and disrupt the cell membranes of MDR P. aeruginosa and C. albicans . Therefore, recombinant actifensin and defensin-d2 exhibit broad-spectrum antimicrobial activity and have the potential to be used as therapy against MDR pathogens.

Tuberculosis (TB) and human immunodeficiency virus (HIV) represent a significant burden of disease on a global scale. Despite improvements in the global epidemic status, largely facilitated by increased access to pharmacotherapeutic interventions, slow progress in the development of new clinical interventions coupled with growing antimicrobial resistance to existing therapies represents a global health crisis. There is an urgent need to expand the armamentarium of TB and HIV therapeutic strategies. Host mediated immune responses represent an untapped reservoir of novel approaches for TB and HIV. Antimicrobial peptides (AMPs) are an essential aspect of the immune system. Cathelicidins and defensins AMPs have been studied for their potential applications in TB and HIV therapeutic interventions. Genetic polymorphism across different population groups may affect endogenous expression or activity of AMPs, potentially influencing therapeutic outcomes. However, certain genetic polymorphisms in autophagy pathways may alter the downstream effects of nano-delivery of cathelicidin. On the other hand, certain genetic polymorphisms in beta-defensins may provide a protective role in reducing HIV-1 mother-to-child-transmission. Pharmaceutical development of cathelicidins and defensins is disadvantaged with complex challenges. Nanoparticle formulations improve pharmacokinetics and biocompatibility while facilitating targeted drug delivery, potentially minimising the risk of immunogenicity or non-specific haemolytic activity. This review aims to explore the potential viability of using cathelicidins and defensins as novel pharmacotherapy in the management of TB and HIV, highlight potential pharmacogenomic implications in host mediated immunity and AMP therapeutic applications, as well as propose novel drug delivery strategies represented by nanomedicine for AMPs., (Copyright © 2022 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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 β1-α1-α2-β2 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., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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 β-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-X 12 -C-X 3 -C-X 9 -C-X 5 -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., (Copyright © 2021. Published by Elsevier Inc.)

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., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)