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1. Chickpea NCR13 disulfide cross-linking variants exhibit profound differences in antifungal activity and modes of action

   https://doi.org/10.1371/journal.ppat.1012745  

   Godwin, James; Djami-Tchatchou, Arnaud Thierry; Velivelli, Siva_L S; Tetorya, Meenakshi; Kalunke, Raviraj; Pokhrel, Ambika; Zhou, Mowei; Buchko, Garry W; Czymmek, Kirk J; Shah, Dilip M (December 2024, PLOS Pathogens)

   Dinesh-Kumar, Savithramma P (Ed.)

   Small cysteine-rich antifungal peptides with multi-site modes of action (MoA) have potential for development as biofungicides. In particular, legumes of the inverted repeat-lacking clade express a large family of nodule-specific cysteine-rich (NCR) peptides that orchestrate differentiation of nitrogen-fixing bacteria into bacteroids. These NCRs can form two or three intramolecular disulfide bonds and a subset of these peptides with high cationicity exhibits antifungal activity. However, the importance of intramolecular disulfide pairing and MoA against fungal pathogens for most of these plant peptides remains to be elucidated. Our study focused on a highly cationic chickpea NCR13, which has a net charge of +8 and contains six cysteines capable of forming three disulfide bonds. NCR13 expression inPichia pastorisresulted in formation of two peptide folding variants, NCR13_PFV1 and NCR13_PFV2, that differed in the pairing of two out of three disulfide bonds despite having an identical amino acid sequence. The NMR structure of each PFV revealed a unique three-dimensional fold with the PFV1 structure being more compact but less dynamic. Surprisingly, PFV1 and PFV2 differed profoundly in the potency of antifungal activity against several fungal plant pathogens and their multi-faceted MoA. PFV1 showed significantly faster fungal cell-permeabilizing and cell entry capabilities as well as greater stability once inside the fungal cells. Additionally, PFV1 was more effective in binding fungal ribosomal RNA and inhibiting protein translationin vitro. Furthermore, when sprayed on pepper and tomato plants, PFV1 was more effective in reducing disease symptoms caused byBotrytis cinerea, causal agent of gray mold disease in fruits, vegetables, and flowers. In conclusion, our work highlights the significant impact of disulfide pairing on the antifungal activity and MoA of NCR13 and provides a structural framework for design of novel, potent antifungal peptides for agricultural use. 

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2. Gambierone and Sodium Channel Specific Bioactivity Are Associated with the Extracellular Metabolite Pool of the Marine Dinoflagellate Coolia palmyrensis

   https://doi.org/10.3390/md21040244  

   Leynse, Alexander K.; Mudge, Elizabeth M.; Turner, Andrew D.; Maskrey, Benjamin H.; Robertson, Alison (April 2023, Marine Drugs)

   Tropical epibenthic dinoflagellate communities produce a plethora of bioactive secondary metabolites, including the toxins ciguatoxins (CTXs) and potentially gambierones, that can contaminate fishes, leading to ciguatera poisoning (CP) when consumed by humans. Many studies have assessed the cellular toxicity of causative dinoflagellate species to better understand the dynamics of CP outbreaks. However, few studies have explored extracellular toxin pools which may also enter the food web, including through alternative and unanticipated routes of exposure. Additionally, the extracellular exhibition of toxins would suggest an ecological function and may prove important to the ecology of the CP-associated dinoflagellate species. In this study, semi-purified extracts obtained from the media of a Coolia palmyrensis strain (DISL57) isolated from the U.S. Virgin Islands were assessed for bioactivity via a sodium channel specific mouse neuroblastoma cell viability assay and associated metabolites evaluated by targeted and non-targeted liquid chromatography tandem and high-resolution mass spectrometry. We found that extracts of C. palmyrensis media exhibit both veratrine enhancing bioactivity and non-specific bioactivity. LC-HR-MS analysis of the same extract fractions identified gambierone and multiple undescribed peaks with mass spectral characteristics suggestive of structural similarities to polyether compounds. These findings implicate C. palmyrensis as a potential contributor to CP and highlight extracellular toxin pools as a potentially significant source of toxins that may enter the food web through multiple exposure pathways. 

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3. Comparative Molecular Immunological Activity of Physiological Metal Oxide Nanoparticle and its Anticancer Peptide and RNA Complexes

   https://doi.org/10.3390/nano9121670  

   DeLong, Robert K.; Comer, Jeffrey; Mathew, Elza Neelima; Jaberi-Douraki, Majid (December 2019, Nanomaterials)

   null (Ed.)

   Currently, there is a great interest in nanoparticle-based vaccine delivery. Recent studies suggest that nanoparticles when introduced into the biological milieu are not simply passive carriers but may also contribute immunological activity themselves or of their own accord. For example there is considerable interest in the biomedical applications of one of the physiologically-based inorganic metal oxide nanoparticle, zinc oxide (ZnO). Indeed zinc oxide (ZnO) NP are now recognized as a nanoscale chemotherapeutic or anticancer nanoparticle (ANP) and several recent reports suggest ZnO NP and/or its complexes with drug and RNA induce a potent antitumor response in immuno-competent mouse models. A variety of cell culture studies have shown that ZnO NP can induce cytokines such as IFN-γ, TNF-α, IL-2, and IL-12 which are known to regulate the tumor microenvironment. Much less work has been done on magnesium oxide (MgO), cobalt oxide (Co3O4), or nickel oxide (NiO); however, despite the fact that these physiologically-based metal oxide NP are reported to functionally load and assemble RNA and protein onto their surface and may thus also be of potential interest as nanovaccine platform. Here we initially compared in vitro immunogenicity of ZnO and Co3O4 NP and their effects on cancer-associated or tolerogenic cytokines. Based on these data we moved ZnO NP forward to testing in the ex vivo splenocyte assay relative to MgO and NiO NP and these data showed significant difference for flow cytometry sorted population for ZnO-NP, relative to NiO and MgO. These data suggesting both molecular and cellular immunogenic activity, a double-stranded anticancer RNA (ACR), polyinosinic:poly cytidylic acid (poly I:C) known to bind ZnO NP; when ZnO-poly I:C was injected into B16F10-BALB/C tumor significantly induced, IL-2 and IL-12 as shown by Cohen’s d test. LL37 is an anticancer peptide (ACP) currently in clinical trials as an intratumoral immuno-therapeutic agent against metastatic melanoma. LL37 is known to bind poly I:C where it is thought to compete for receptor binding on the surface of some immune cells, metastatic melanoma and lung cells. Molecular dynamic simulations revealed association of LL37 onto ZnO NP confirmed by gel shift assay. Thus using the well-characterized model human lung cancer model cell line (BEAS-2B), poly I:C RNA, LL37 peptide, or LL37-poly I:C complexes were loaded onto ZnO NP and delivered to BEAS-2B lung cells, and the effect on the main cancer regulating cytokine, IL-6 determined by ELISA. Surprisingly ZnO-LL37, but not ZnO-poly I:C or the more novel tricomplex (ZnO-LL37-poly I:C) significantly suppressed IL-6 by >98–99%. These data support the further evaluation of physiological metal oxide compositions, so-called physiometacomposite (PMC) materials and their formulation with anticancer peptide (ACP) and/or anticancer RNA (ACR) as a potential new class of immuno-therapeutic against melanoma and potentially lung carcinoma or other cancers. 

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4. Entomopathogenic Nematode Species Vary in Their Behavior and Virulence in Response to Cardiac Glycosides Within and Around Insect Hosts

   https://doi.org/10.1007/s10886-025-01563-9  

   Achi, Perla; Christensen, Preston; Iglesias, Victoria; McCarthy, Cullen; Pena, Robert; Bavier, Lanie; Goldy, Connor; Agrawal, Anurag A; Groen, Simon C; Dillman, Adler R (February 2025, Journal of Chemical Ecology)

   Abstract Plants produce defensive toxins to deter herbivores. In response, some specialized herbivores evolved resistance and even the capacity to sequester toxins, affecting interactions at higher trophic levels. Here, we test the hypothesis that potential natural enemies of specialized herbivores are differentially affected by plant toxins depending on their level of adaptation to the plant-herbivore system. We focus on toxic cardiac glycosides (CGs) from milkweeds (Asclepiasspp.), which inhibit animal Na⁺/K⁺-ATPases, and two CG-resistant insects, the large milkweed bugOncopeltus fasciatusand a CRISPR-editedDrosophila melanogaster. Both have CG-resistant Na⁺/K⁺-ATPases through a set of key amino acid substitutions, which facilitate CG sequestration. We conducted infection experiments with entomopathogenic nematodes (Steinernema carpocapsae,S. feltiae, andS. hermaphroditum) as natural enemies on host insects containing mixtures of milkweed-derived CGs or purified CGs (ouabain, digoxin, and digitoxin) that vary in toxicity. The nematodeS. carpocapsaeis known to occur in soil near milkweed plants and naturally has several of the same Na⁺/K⁺-ATPase substitutions as the milkweed bugO. fasciatusand ourDrosophilamutant. This nematode not only exhibited higher fecundity in hosts that carried CGs relative to the other nematode species (which have sensitive Na⁺/K⁺-ATPases), but also showed attraction to mixtures of CGs in milkweed root extracts and to purified ouabain when tested on agar plates. A coiling phenotype, which is a symptom of neurotoxicity, was observed more frequently inS. feltiaeandS. hermaphroditumupon exposure to milkweed root extracts than inS. carpocapsae. Nematode behavior was further tested in sand, and while attraction to CGs was found forS. carpocapsae, nematodes of the other species tended to migrate away from milkweed root chemicals. Thus,S. carpocapsaecan tolerate CGs and may use these as chemical cues to locate insect hosts that live on or around milkweed plants. 

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5. Phytochemical Screening and Antioxidant Activity Evaluation of Selected Philippine Fruit Peels and Pulps

   Pates, Mildred D; Walag, Angelo MP; Rosario, Romeo RM (January 2024, Asian journal of biological and life sciences)

   This research aimed to explore the potential of underutilized plant waste products from Philippine fruits as sustainable sources of phytochemicals, assessing the viability of green extraction methods. It focused on the antioxidant activities of extracts from fruit peels and pulps, comparing these to the benchmark antioxidant, L-Ascorbic acid (Vitamin C). Materials and Methods: Peels and pulps from selected local fruits, including Annona squamosa (sugar apple; atis), Musa acuminata (banana; lakatan), Sandoricum koetjape (cotton fruit; santol), Mangifera altissima (mango; paho) and Ananas comosus (pineapple; piña), were utilized. Standard phytochemical screening methods were employed to identify the presence of secondary metabolites in aqueous extracts. Quantification of antioxidant activities was conducted against DPPH. Results: Antioxidant activities of S. koetjape (cotton fruit; santol) and M. altissima (mango; paho) fruit extracts demonstrated better or comparable efficacy to L-Ascorbic acid (Vitamin C). Evidence was established that water-based extraction of secondary metabolites, which are polar, is both feasible and environmentally sustainable. Conclusion: Extracts from Philippine fruit waste products are viable sources of phytochemicals with significant antioxidant activities. Furthermore, water-based, green extraction methods are beneficial for environmental sustainability and the promotion of green waste management. 

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