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Rice production worldwide is threatened by the disease Bacterial Panicle Blight (BPB) caused by Burkholderia glumae . Despite the threat, resources to control this disease such as completely resistant cultivars or effective chemical methods are still lacking. However, the need to control this disease has paved the way to explore biologically based approaches harnessing the antimicrobial activities of environmental bacteria. Previously, the bacterium Pseudomonas protegens PBL3 was identified as a potential biological control agent against B. glumae due to its antimicrobial activity against B. glumae . Such antimicrobial activity in vitro and in planta was associated with the P. protegens PBL3 bacteria-free secreted fraction (secretome), although the specific molecules responsible for this activity have remained elusive. In this work, we advance the characterization of the P. protegens PBL3 secretome, by evaluating the antimicrobial activity in vitro of selected secondary metabolites predicted by the P. protegens PBL3 genomic sequence against B. glumae . In addition, using Reversed Phase Liquid Chromatography Tandem Mass Spectrometry (RPLC-MS/MS), of the P. protegens PBL3 secretome, enabled us to successfully detect and quantify Pyoluteorin, 2,4-diacetylphloroglucinol (2,4-DAPG) and Pyochelin. Among those, Pyoluteorin and 2,4-DAPG reduced the growth of B. glumae in vitro along with reducing the symptoms of BPB and bacterial growth in planta, suggesting that these compounds could be effective as biopesticides to mitigate BPB.

Antimicrobial peptides (AMPs) are compounds with a variety of bioactive properties. Especially promising are their antibacterial activities, often toward drug-resistant pathogens. Across different AMP sources, AMPs expressed within plants are relatively underexplored with a limited number of plant AMP families identified. Recently, we identified the novel AMPs CC-AMP1 and CC-AMP2 in ghost pepper plants ( Capsicum chinense x frutescens ), exerting promising antibacterial activity and not classifying into any known plant AMP family. Herein, AMPs related to CC-AMP1 and CC-AMP2 were identified within both Capsicum annuum and Capsicum baccatum . In silico predictions throughout plants were utilized to illustrate that CC-AMP1-like and CC-AMP2-like peptides belong to two broader AMP families, with three-dimensional structural predictions indicating that CC-AMP1-like peptides comprise a novel subfamily of α-hairpinins. The antibacterial activities of several closely related CC-AMP1-like peptides were compared with a truncated version of CC-AMP1 possessing significantly more activity than the full peptide. This truncated peptide was further characterized to possess broad-spectrum antibacterial activity against clinically relevant ESKAPE pathogens. These findings illustrate the value in continued study of plant AMPs toward characterization of novel AMP families, with CC-AMP1-like peptides possessing promising bioactivity.

Tardigrades are renowned for their ability to survive a wide array of environmental stressors. In particular, tardigrades can curl in on themselves while losing a significant proportion of their internal water content to form a structure referred to as a tun. In surviving varying conditions, tardigrades undergo distinct morphological transformations that could indicate different mechanisms of stress sensing and tolerance specific to the stress condition. Methods to effectively distinguish between morphological transformations, including between tuns induced by different stress conditions, are lacking. Herein, an approach for discriminating between tardigrade morphological states is developed and utilized to compare sucrose- and CaCl 2 -induced tuns, using the model species Hypsibius exemplaris. A novel approach of shadow imaging with confocal laser scanning microscopy enabled production of three-dimensional renderings of Hys. exemplaris in various physiological states resulting in volume measurements. Combining these measurements with qualitative morphological analysis using scanning electron microscopy revealed that sucrose- and CaCl 2 -induced tuns have distinct morphologies, including differences in the amount of water expelled during tun formation. Further, varying the concentration of the applied stressor did not affect the amount of water lost, pointing towards water expulsion by Hys. exemplaris being a controlled process that is adapted to the specific stressors., (© 2024. The Author(s).)

Tardigrades can survive remarkable doses of ionizing radiation, up to about 1,000 times the lethal dose for humans. How they do so is incompletely understood. We found that the tardigrade Hypsibius exemplaris suffers DNA damage upon gamma irradiation, but the damage is repaired. We show that this species has a specific and robust response to ionizing radiation: irradiation induces a rapid upregulation of many DNA repair genes. This upregulation is unexpectedly extreme-making some DNA repair transcripts among the most abundant transcripts in the animal. By expressing tardigrade genes in bacteria, we validate that increased expression of some repair genes can suffice to increase radiation tolerance. We show that at least one such gene is important in vivo for tardigrade radiation tolerance. We hypothesize that the tardigrades' ability to sense ionizing radiation and massively upregulate specific DNA repair pathway genes may represent an evolved solution for maintaining DNA integrity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

There have been significant advances in the flexibility and power of in vitro cell-free translation systems. The increasing ability to incorporate noncanonical amino acids and complement translation with recombinant enzymes has enabled cell-free production of peptide-based natural products (NPs) and NP-like molecules. We anticipate that many more such compounds and analogs might be accessed in this way. To assess the peptide NP space that is directly accessible to current cell-free technologies, we developed a peptide parsing algorithm that breaks down peptide NPs into building blocks based on ribosomal translation logic. Using the resultant data set, we broadly analyze the biophysical properties of these privileged compounds and perform a retrobiosynthetic analysis to predict which peptide NPs could be directly synthesized in augmented cell-free translation reactions. We then tested these predictions by preparing a library of highly modified peptide NPs. Two macrocyclases, PatG and PCY1, were used to effect the head-to-tail macrocyclization of candidate NPs. This retrobiosynthetic analysis identified a collection of high-priority building blocks that are enriched throughout peptide NPs, yet they had not previously been tested in cell-free translation. To expand the cell-free toolbox into this space, we established, optimized, and characterized the flexizyme-enabled ribosomal incorporation of piperazic acids. Overall, these results demonstrate the feasibility of cell-free translation for peptide NP total synthesis while expanding the limits of the technology. This work provides a novel computational tool for exploration of peptide NP chemical space, that could be expanded in the future to allow design of ribosomal biosynthetic pathways for NPs and NP-like molecules.

Chelidonium majus , known as Greater Celandine, is a latex-bearing plant that has been leveraged for its anticancer and antimicrobial properties. Herein, C. majus aerial tissue is mined for the presence of antimicrobial peptides. A highly abundant cysteine-rich peptide with a length of 25 amino acids, deemed CM-AMP1, is characterized through multiple mass spectrometric approaches. Electron-activated dissociation is leveraged to differentiate between isoleucine and leucine residues and complement conventional collision-induced dissociation to gain full sequence coverage of the full-length peptide. CM-AMP1 shares little sequence similarity with any proteins in publicly available databases, highlighting the novelty of its cysteine landscape and core motif. The presence of three disulfide bonds in the native peptide confers proteolytic stability, and antimicrobial activity is greatly decreased upon the alkylation of the cysteine residues. Synthetic variants of CM-AMP1 are used to confirm the activity of the full-length sequence and the core motif. To assess the biological impact, E. coli was grown in a sublethal concentration of CM-AMP1 and quantitative proteomics was used to identify proteins produced by the bacteria under stress, ultimately suggesting a membrane lytic antimicrobial mechanism of action. This study integrates multiple analytical methods for molecular and biological characterization of a unique antimicrobial peptide identified from C. majus.

Tardigrades, commonly known as 'waterbears', are eight-legged microscopic invertebrates renowned for their ability to withstand extreme stressors, including high osmotic pressure, freezing temperatures, and complete desiccation. Limb retraction and substantial decreases to their internal water stores results in the tun state, greatly increasing their ability to survive. Emergence from the tun state and/or activity regain follows stress removal, where resumption of life cycle occurs as if stasis never occurred. However, the mechanism(s) through which tardigrades initiate tun formation is yet to be uncovered. Herein, we use chemobiosis to demonstrate that tardigrade tun formation is mediated by reactive oxygen species (ROS). We further reveal that tuns are dependent on reversible cysteine oxidation, and that this reversible cysteine oxidation is facilitated by the release of intracellular reactive oxygen species (ROS). We provide the first empirical evidence of chemobiosis and map the initiation and survival of tardigrades via osmobiosis, chemobiosis, and cryobiosis. In vivo electron paramagnetic spectrometry suggests an intracellular release of reactive oxygen species following stress induction; when this release is quenched through the application of exogenous antioxidants, the tardigrades can no longer survive osmotic stress. Together, this work suggests a conserved dependence of reversible cysteine oxidation across distinct tardigrade cryptobioses., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Smythers et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The post-translational modification of cysteine to diverse oxidative states is understood as a critical cellular mechanism to combat oxidative stress. To study the role of cysteine oxidation, cysteine enrichments and subsequent analysis via mass spectrometry are necessary. As such, technologies and methods are rapidly developing for sensitive and efficient enrichments of cysteines to further explore its role in signaling pathways. In this review, we analyze recent developments in methods to miniaturize cysteine enrichments, analyze the underexplored disulfide bound redoxome, and quantify site-specific cysteine oxidation. We predict that further development of these methods will improve cysteine coverage across more diverse organisms than those previously studied and elicit novel roles cysteines play in stress response., 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 article., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Introduction: Adverse Childhood Experiences (ACEs) are associated with a range of negative physical and mental health outcomes, yet there is limited research focused on the effect of ACEs on stress responses during pregnancy. Expectant mothers experience an increase in cortisol levels as pregnancy progresses, with this increase having important implications for fetal and early infant development. Little is known about the impact of ACEs on maternal cortisol levels. This study explored the relationship between maternal ACEs and cortisol response among expectant mothers nearing or in the third trimester of pregnancy., Methods: 39 expectant mothers were exposed to a Baby Cry Protocol via an infant simulator, with salivary cortisol collected at five points in time (N = 181). Stepwise, multilevel model creation resulted in a random intercept and random slope model with an interaction term for total number of ACEs and week of pregnancy., Results: The repeated measures data showed that cortisol levels decreased across collection times, from arrival at the lab, through the Baby Cry Protocol, to recovery. Predictive margins for the interaction term showed that while exposure to a greater number of ACEs was associated with higher cortisol levels early in the third trimester, the expected increase in cortisol late in pregnancy was blunted for expectant mothers who were exposed to a greater number of ACEs., Discussion: These findings findings suggest the importance of ACEs screening and intervention efforts as part of prenatal care., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Antenatal depression (AD) is the most common complication of pregnancy in developed countries and increases the risk of preterm birth (PTB). Many pregnant individuals with AD do not obtain treatment due in part to risks associated with antidepressant medications, the expense and wait times for psychological services, and perceived stigma. Accessible and timely treatment of antenatal depression is crucial to minimize foetal impacts and associated long-term child health outcomes. Previous studies show that behavioural activation and peer support are promising avenues of treatment for perinatal depression. Additionally, remote and paraprofessional counselling interventions show promise as more accessible, sustainable, and cost-effective treatment avenues than traditional psychological services. The primary aim of this trial is to test the effectiveness of a remote, behavioural activation and peer support intervention, administered by trained peer para-professionals, for increasing gestational age at delivery among those with antenatal depression. The secondary aims are to evaluate the effectiveness for treating AD prior to delivery, with persistence into the postpartum; improving anxiety symptoms; and improving parenting self-efficacy compared to controls., Methods: A two-arm, single-blinded, parallel groups randomized controlled trial (RCT) with repeated measures will be conducted. Participants scoring >10 on the Edinburgh Postnatal Depression Scale will be recruited from the larger P3 cohort and invited to enroll. Assessments will be conducted prior to 27 weeks' gestation at trial intake (T1), post-intervention, prior to delivery (T2), 5-6 months postpartum (T3), and 11-12 months postpartum (T4) and will include self-report questionnaires and linked medical records., Discussion: Our remote, peer paraprofessional-delivered behavioural activation plus peer support intervention has the potential to successfully reduce symptoms of AD, which may in turn decrease the risk of PTB and subsequent health impacts. The current trial builds on previous findings and uses a patient-oriented approach to address priorities for patient care and to provide a cost-effective, accessible, and evidence-based treatment to pregnant individuals with AD., Trial Registration: International Standard Randomised Controlled Trial Number (ISRCTN) registry (ISRCTN51098220) ISRCTN51098220. Registered on April 7, 2022., (© 2023. The Author(s).)

The plant hormone abscisic acid (ABA) plays crucial roles in regulation of stress responses and growth modulation. Heterotrimeric G-proteins are key mediators of ABA responses. Both ABA and G-proteins have also been implicated in intracellular redox regulation; however, the extent to which reversible protein oxidation manipulates ABA and/or G-protein signaling remains uncharacterized. To probe the role of reversible protein oxidation in plant stress response and its dependence on G-proteins, we determined the ABA-dependent reversible redoxome of wild-type and Gβ-protein null mutant agb1 of Arabidopsis. We quantified 6891 uniquely oxidized cysteine-containing peptides, 923 of which show significant changes in oxidation following ABA treatment. The majority of these changes required the presence of G-proteins. Divergent pathways including primary metabolism, reactive oxygen species response, translation and photosynthesis exhibited both ABA- and G-protein-dependent redox changes, many of which occurred on proteins not previously linked to them. We report the most comprehensive ABA-dependent plant redoxome and uncover a complex network of reversible oxidations that allow ABA and G-proteins to rapidly adjust cellular signaling to adapt to changing environments. Physiological validation of a subset of these observations suggests that functional G-proteins are required to maintain intracellular redox homeostasis and fully execute plant stress responses., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Purpose: Perinatal mental health disorders are common, and rates have increased during the COVID-19 pandemic. It is unclear where providers may improve perinatal mental health care, particularly in countries lacking national guidelines, such as Canada., Methods: A cross-sectional survey of perinatal health providers was conducted to describe the landscape of perinatal mental health knowledge, screening, and treatment practices across Canada. Providers were recruited through listservs, social media, and snowball sampling. Participants completed an online survey that assessed their perinatal mental health training, service provision types, their patient wait times, and treatment barriers, and COVID-19 pandemic-related impacts., Results: A total of 435 providers completed the survey, including physicians, midwives, psychologists, social workers, nurses, and allied non-mental health professionals. Most (87.0%) did not have workplace mandated screening for perinatal mental illness but a third (66%) use a validated screening tool. Many (42%) providers stated their patients needed to wait more than 2 months for services. More than half (57.3%) reported they did not receive or were unsure if they received specialized training in perinatal mental health. Most (87.0%) indicated there were cultural, linguistic, and financial barriers to accessing services. Over two-thirds (69.0%) reported the COVID-19 pandemic reduced access to services., Conclusion: Survey findings reveal significant gaps in training, screening tool use, and timely and culturally safe treatment of perinatal mental health concerns. There is critical need for coordinated and nationally mandated perinatal mental health services in Canada to improve care for pregnant and postpartum people., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hicks, Ou, Charlebois, Tarasoff, Pawluski, Roos, Hooykaas, Fairbrother, Carter and Tomfohr-Madsen.)

Amaranthus hypochondriacus is a nutritious alternative grain native to Central and South America. Increased interest in the impact of A. hypochondriacus on the human body has driven characterization of bioactive secondary metabolites. The seeds are known to contain bioactive small molecules but little is known regarding endogenous peptides. Cysteine-rich peptides (CRPs) in foodstuffs are particularly relevant because they are stabilized by disulfide bonds enhancing resistance to digestion. Here, in silico predictions, proteomics, and simulated gastrointestinal digestions are leveraged to identify digestion resistant CRPs within A. hypochondriacus seeds. Thirteen in silico predicted CRPs were detected in a seed extract providing evidence for the translation of five CRP families. Mature forms of six CRPs were characterized via top-down proteomics revealing multiple post-translational modifications. All six peptides demonstrated resistance to simulated gastrointestinal digestion, suggesting that A. hypochondriacus CRPs may exhibit bioactivity after consumption and should be prioritized for further characterization., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In humans, an adaptable internal biological system generates circadian rhythms that maintain synchronicity of behavior and physiology with the changing demands of the 24-h environment. Development of the circadian system begins in utero and continues throughout the first few years of life. Maturation of the clock can be measured through sleep/wake patterns and hormone secretion. Circadian rhythms, by definition, can persist in the absence of environmental input; however, their ability to adjust to external time cues is vital for adaptation and entrainment to the environment. The significance of these external factors that influence the emergence of a stable circadian clock in the first years of life remain poorly understood. Infants raised in our post-modern world face adverse external circadian signals, such as artificial light and mistimed hormonal cues via breast milk, which may increase interference with the physiological mechanisms that promote circadian synchronization. This review describes the very early developmental stages of the clock and common circadian misalignment scenarios that make the developing circadian system more susceptible to conflicting time cues and temporal disorder between the maternal, fetal, infant, and peripheral clocks., (© 2022. The Author(s).)

Antimicrobial peptides (AMPs) are promising compounds for the treatment of antibiotic-resistant bacteria and are found across all organisms, including plants. Unlike most antibiotics, AMPs tend to act on more generalized and multiple targets, making development of resistance more difficult. Conventional approaches toward AMP identification include bioactivity-guided fractionation and genome mining. Complementary methods leveraging bioactivity-guided fractionation, cysteine motif-guided in silico AMP prediction, and mass spectrometric approaches can be combined to expand botanical AMP discovery. Herein, we present an integrated workflow which serves to streamline implementation toward a robust botanical AMP discovery pipeline., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Antimicrobial resistance (AMR) is a significant public health issue that threatens our ability to treat common infections. AMR often emerges in bacteria through upregulation of proteins that allow a subpopulation of resistant bacteria to proliferate through natural selection. Identifying these proteins is crucial for understanding how AMR develops in bacteria and is essential in developing novel therapeutics to combat the threat of widespread AMR. Mass spectrometry-based proteomics is a powerful tool for understanding the biochemical pathways of biological systems, lending remarkable insight into AMR mechanisms in bacteria through measuring the changing protein abundances as a result of antibiotic treatment. Here, we describe a serial passaging method for evolving resistance in bacteria that implements quantitative proteomics to reveal the differential proteomes of resistant bacteria. The focus herein is on antimicrobial peptides (AMPs), but the approach can be generalized for any antimicrobial compound. Comparative proteomics of sensitive vs. resistance strains in response to AMP treatment reveals mechanisms to survive the bioactive compound and points to the mechanism of action for novel AMPs., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Antimicrobial peptides (AMPs) are host defense peptides with a range of functions/activities/modes of action that are ubiquitously expressed across all forms of life. Continued discovery of novel AMPs presents exciting opportunities to address evolving resistance to existing treatments in multiple fields, including agricultural pathogens/pests as well as antimicrobial and chemotherapeutics for human health. However, typical discovery methods including bioassay-guided fractionation and genome mining generally lack the capacity for robust AMP discovery in non-model/unsequenced organisms. PepSAVI-MS (Statistically guided bioactive peptides prioritized via mass spectrometry) was developed as an AMP discovery approach that addresses some of the limitations associated with these standard methods. PepSAVI-MS is a multi-pronged pipeline that includes peptide library creation, bioactivity screening, LC-MS analysis, and statistical modeling for putative AMP identification. The original implementation of PepSAVI-MS outlined strategies for the fractionation of plant extracts with strong cation exchange chromatography (SCX). Herein, we elaborate on recent improvements to peptide library creation through the use of orthogonal fractionation methods, specifically crude SCX chromatography and reversed-phase liquid chromatography (RPLC). This optimization of the "peptide library creation" step has demonstrated improvements for processing and AMP identifications via PepSAVI-MS., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Post-translational modifications (PTMs) regulate complex biological processes through the modulation of protein activity, stability, and localization. Insights into the specific modification type and localization within a protein sequence can help ascertain functional significance. Computational models are increasingly demonstrated to offer a low-cost, high-throughput method for comprehensive PTM predictions. Algorithms are optimized using existing experimental PTM data, thus accurate prediction performance relies on the creation of robust datasets. Herein, advancements in mass spectrometry-based proteomics technologies to maximize PTM coverage are reviewed. Further, requisite experimental validation approaches for PTM predictions are explored to ensure that follow-up mechanistic studies are focused on accurate modification sites., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Interactomics is an emerging field that seeks to identify both transient and complex-bound protein interactions that are essential for metabolic functions. Crosslinking mass spectrometry (XL-MS) has enabled untargeted global analysis of these protein networks, permitting largescale simultaneous analysis of protein structure and interactions. Increased commercial availability of highly specific, cell permeable crosslinkers has propelled the study of these critical interactions forward, with the development of MS-cleavable crosslinkers further increasing confidence in identifications. Herein, the global interactome of the unicellular alga Chlamydomonas reinhardtii was analyzed via XL-MS by implementing the MS-cleavable disuccinimidyl sulfoxide (DSSO) crosslinker and enriching for crosslinks using strong cation exchange chromatography. Gentle lysis via repeated freeze-thaw cycles facilitated in vitro analysis of 157 protein-protein crosslinks (interlinks) and 612 peptides linked to peptides of the same protein (intralinks) at 1% FDR throughout the C. reinhardtii proteome. The interlinks confirmed known protein relationships across the cytosol and chloroplast, including coverage on 42% and 38% of the small and large ribosomal subunits, respectively. Of the 157 identified interlinks, 92% represent the first empirical evidence of interaction observed in C. reinhardtii. Several of these crosslinks point to novel associations between proteins, including the identification of a previously uncharacterized Mg-chelatase associated protein (Cre11.g477733.t1.2) bound to seven distinct lysines on Mg-chelatase (Cre06.g306300.t1.2). Additionally, the observed intralinks facilitated characterization of novel protein structures across the C. reinhardtii proteome. Together, these data establish a framework of protein-protein interactions that can be further explored to facilitate understanding of the dynamic protein landscape in C. reinhardtii .

Stress and nutrient availability influence cell proliferation through complex intracellular signalling networks. In a previous study it was found that pyro-inositol polyphosphates (InsP 7 and InsP 8 ) produced by VIP1 kinase, and target of rapamycin (TOR) kinase signalling interacted synergistically to control cell growth and lipid metabolism in the green alga Chlamydomonas reinhardtii. However, the relationship between InsPs and TOR was not completely elucidated. We used an in vivo assay for TOR activity together with global proteomic and phosphoproteomic analyses to assess differences between wild-type and vip1-1 in the presence and absence of rapamycin. We found that TOR signalling is more severely affected by the inhibitor rapamycin in a vip1-1 mutant compared with wild-type, indicating that InsP 7 and InsP 8 produced by VIP1 act independently but also coordinately with TOR. Additionally, among hundreds of differentially phosphorylated peptides detected, an enrichment for photosynthesis-related proteins was observed, particularly photosystem II proteins. The significance of these results was underscored by the finding that vip1-1 strains show multiple defects in photosynthetic physiology that were exacerbated under high light conditions. These results suggest a novel role for inositol pyrophosphates and TOR signalling in coordinating photosystem phosphorylation patterns in Chlamydomonas cells in response to light stress and possibly other stresses., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Traditional medicinal plants contain a variety of bioactive natural products including cysteine-rich (Cys-rich) antimicrobial peptides (AMPs). Cys-rich AMPs are often crosslinked by multiple disulfide bonds which increase their resistance to chemical and enzymatic degradation. However, this class of molecules is relatively underexplored. Herein, in silico analysis predicted 80-100 Cys-rich AMPs per species from three edible traditional medicinal plants: Linum usitatissimum (flax), Trifolium pratense (red clover), and Sesamum indicum (sesame). Bottom-up proteomic analysis of seed peptide extracts revealed direct evidence for the translation of 3-10 Cys-rich AMPs per species, including lipid transfer proteins, defensins, α-hairpinins, and snakins. Negative activity revealed by antibacterial screening highlights the importance of employing a multi-pronged approach for AMP discovery. Further, this study demonstrates that flax, red clover, and sesame are promising sources for further AMP discovery and characterization.

Enterococcus faecalis is a Gram-positive bacterium that is a major cause of hospital-acquired infections due, in part, to its intrinsic resistance to cell wall-active antimicrobials. One critical determinant of this resistance is the transmembrane kinase IreK, which belongs to the penicillin-binding protein and serine/threonine kinase-associated kinase family of bacterial signaling proteins involved with the regulation of cell wall homeostasis. The activity of IreK is enhanced in response to cell wall stress, but direct substrates of IreK phosphorylation, leading to antimicrobial resistance, are largely unknown. To better understand stress-modulated phosphorylation events contributing to antimicrobial resistance, wild type E. faecalis cells treated with cell wall-active antimicrobials, chlorhexidine or ceftriaxone, were examined via phosphoproteomics. Among the most prominent changes was increased phosphorylation of divisome components after both treatments, suggesting that E. faecalis modulates cell division in response to cell wall stress. Phosphorylation mediated by IreK was then determined via a similar analysis with a E. faecalis Δ ireK mutant strain, revealing potential IreK substrates involved with the regulation of peptidoglycan biosynthesis and within the E. faecalis CroS/R two-component system, another signal transduction pathway that promotes antimicrobial resistance. These results reveal critical insights into the biological functions of IreK.

Capsicum spp. (hot peppers) demonstrate a range of interesting bioactive properties spanning anti-inflammatory, antioxidant, and antimicrobial activities. While several species within the genus are known to produce antimicrobial peptides (AMPs), AMP sequence mining of genomic data indicates this space remains largely unexplored. Herein, in silico AMP predictions were paired with peptidomics to identify novel AMPs from the interspecific hybrid ghost pepper ( Capsicum chinense × frutescens ). AMP prediction algorithms revealed 115 putative AMPs within the Capsicum chinense genome, of which 14 were identified in the aerial tissue peptidome. PepSAVI-MS, de novo sequencing, and complementary approaches were used to fully molecularly characterize two novel AMPs, CC-AMP1 and CC-AMP2, including elucidation of a pyroglutamic acid post-translational modification of CC-AMP1 and disulfide bond connectivity of both. Both CC-AMP1 and CC-AMP2 have little homology with known AMPs and exhibited low μM antimicrobial activity against Gram-negative bacteria, including Escherichia coli . These findings demonstrate the complementary nature of peptidomics, bioactivity-guided discovery, and bioinformatics-based investigations to characterize plant AMP profiles.

The complex reservoir of metabolite-producing bacteria in the gastrointestinal tract contributes tremendously to human health and disease. Bacterial composition, and by extension gut metabolomic composition, is undoubtably influenced by the use of modern antibiotics. Herein, we demonstrate that polymyxin B, a last resort antibiotic, influences the production of the genotoxic metabolite colibactin from adherent-invasive Escherichia coli (AIEC) NC101. Colibactin can promote colorectal cancer through DNA double stranded breaks and interstrand cross-links. While the structure and biosynthesis of colibactin have been elucidated, chemical-induced regulation of its biosynthetic gene cluster and subsequent production of the genotoxin by E . coli are largely unexplored. Using a multiomic approach, we identified that polymyxin B stress enhances the abundance of colibactin biosynthesis proteins (Clb's) in multiple pks+ E . coli strains, including pro-carcinogenic AIEC, NC101; the probiotic strain, Nissle 1917; and the antibiotic testing strain, ATCC 25922. Expression analysis via qPCR revealed that increased transcription of clb genes likely contributes to elevated Clb protein levels in NC101. Enhanced production of Clb's by NC101 under polymyxin stress matched an increased production of the colibactin prodrug motif, a proxy for the mature genotoxic metabolite. Furthermore, E . coli with a heightened tolerance for polymyxin induced greater mammalian DNA damage, assessed by quantification of γH2AX staining in cultured intestinal epithelial cells. This study establishes a key link between the polymyxin B stress response and colibactin production in pks+ E . coli . Ultimately, our findings will inform future studies investigating colibactin regulation and the ability of seemingly innocuous commensal microbes to induce host disease.

Protein phosphorylation, which is one of the most important post-translational modifications (PTMs), is involved in regulating myriad cellular processes. Herein, we present a novel deep learning based approach for organism-specific protein phosphorylation site prediction in Chlamydomonas reinhardtii, a model algal phototroph. An ensemble model combining convolutional neural networks and long short-term memory (LSTM) achieves the best performance in predicting phosphorylation sites in C. reinhardtii. Deemed Chlamy-EnPhosSite, the measured best AUC and MCC are 0.90 and 0.64 respectively for a combined dataset of serine (S) and threonine (T) in independent testing higher than those measures for other predictors. When applied to the entire C. reinhardtii proteome (totaling 1,809,304 S and T sites), Chlamy-EnPhosSite yielded 499,411 phosphorylated sites with a cut-off value of 0.5 and 237,949 phosphorylated sites with a cut-off value of 0.7. These predictions were compared to an experimental dataset of phosphosites identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) in a blinded study and approximately 89.69% of 2,663 C. reinhardtii S and T phosphorylation sites were successfully predicted by Chlamy-EnPhosSite at a probability cut-off of 0.5 and 76.83% of sites were successfully identified at a more stringent 0.7 cut-off. Interestingly, Chlamy-EnPhosSite also successfully predicted experimentally confirmed phosphorylation sites in a protein sequence (e.g., RPS6 S245) which did not appear in the training dataset, highlighting prediction accuracy and the power of leveraging predictions to identify biologically relevant PTM sites. These results demonstrate that our method represents a robust and complementary technique for high-throughput phosphorylation site prediction in C. reinhardtii. It has potential to serve as a useful tool to the community. Chlamy-EnPhosSite will contribute to the understanding of how protein phosphorylation influences various biological processes in this important model microalga.

Plants rapidly respond to environmental fluctuations through coordinated, multi-scalar regulation, enabling complex reactions despite their inherently sessile nature. In particular, protein post-translational signaling and protein-protein interactions combine to manipulate cellular responses and regulate plant homeostasis with precise temporal and spatial control. Understanding these proteomic networks are essential to addressing ongoing global crises, including those of food security, rising global temperatures, and the need for renewable materials and fuels. Technological advances in mass spectrometry-based proteomics are enabling investigations of unprecedented depth, and are increasingly being optimized for and applied to plant systems. This review highlights recent advances in plant proteomics, with an emphasis on spatially and temporally resolved analysis of post-translational modifications and protein interactions. It also details the necessity for generation of a comprehensive plant cell atlas while highlighting recent accomplishments within the field., (© 2021 The Author(s).)

Background: Plant defensins are a broadly distributed family of antimicrobial peptides which have been primarily studied for agriculturally relevant antifungal activity. Recent studies have probed defensins against Gram-negative bacteria revealing evidence for multiple mechanisms of action including membrane lysis and ribosomal inhibition. Herein, a truncated synthetic analog containing the γ-core motif of Amaranthus tricolor DEF2 (Atr-DEF2) reveals Gram-negative antibacterial activity and its mechanism of action is probed via proteomics, outer membrane permeability studies, and iron reduction/chelation assays., Results: Atr-DEF2(G39-C54) demonstrated activity against two Gram-negative human bacterial pathogens, Escherichia coli and Klebsiella pneumoniae. Quantitative proteomics revealed changes in the E. coli proteome in response to treatment of sub-lethal concentrations of the truncated defensin, including bacterial outer membrane (OM) and iron acquisition/processing related proteins. Modification of OM charge is a common response of Gram-negative bacteria to membrane lytic antimicrobial peptides (AMPs) to reduce electrostatic interactions, and this mechanism of action was confirmed for Atr-DEF2(G39-C54) via an N-phenylnaphthalen-1-amine uptake assay. Additionally, in vitro assays confirmed the capacity of Atr-DEF2(G39-C54) to reduce Fe 3+ and chelate Fe 2+ at cell culture relevant concentrations, thus limiting the availability of essential enzymatic cofactors., Conclusions: This study highlights the utility of plant defensin γ-core motif synthetic analogs for characterization of novel defensin activity. Proteomic changes in E. coli after treatment with Atr-DEF2(G39-C54) supported the hypothesis that membrane lysis is an important component of γ-core motif mediated antibacterial activity but also emphasized that other properties, such as metal sequestration, may contribute to a multifaceted mechanism of action.

Protein homeostasis (proteostasis) is crucial for proper cellular function, including the production of peptides with biological functions through controlled proteolysis. Proteostasis has roles in maintenance of cellular functions and plant interactions with the environment under physiological conditions. Plant stress continues to reduce agricultural yields causing substantial economic losses; thus, it is critical to understand how plants perceive stress signals to elicit responses for survival. As previously shown in Arabidopsis thaliana, thimet oligopeptidases (TOPs) TOP1 (also referred to as organellar oligopeptidase) and TOP2 (also referred to as cytosolic oligopeptidase) are essential components in plant response to pathogens, but further characterization of TOPs and their peptide substrates is required to understand their contributions to stress perception and defense signaling. Herein, label-free peptidomics via liquid chromatography-tandem mass spectrometry was used to differentially quantify 1111 peptides, originating from 369 proteins, between the Arabidopsis Col-0 wild type and top1top2 knock-out mutant. This revealed 350 peptides as significantly more abundant in the mutant, representing accumulation of these potential TOP substrates. Ten direct substrates were validated using in vitro enzyme assays with recombinant TOPs and synthetic candidate peptides. These TOP substrates are derived from proteins involved in photosynthesis, glycolysis, protein folding, biogenesis, and antioxidant defense, implicating TOP involvement in processes aside from defense signaling. Sequence motif analysis revealed TOP cleavage preference for non-polar residues in the positions surrounding the cleavage site. Identification of these substrates provides a framework for TOP signaling networks, through which the interplay between proteolytic pathways and defense signaling can be further characterized., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Covering: Up to July 2020Ribosomal antimicrobial peptide (AMP) natural products, also known as ribosomally synthesized and post-translationally modified peptides (RiPPs) or host defense peptides, demonstrate potent bioactivities and impressive complexity that complicate molecular and biological characterization. Tandem mass spectrometry (MS) has rapidly accelerated bioactive peptide sequencing efforts, yet standard workflows insufficiently address intrinsic AMP diversity. Herein, orthogonal approaches to accelerate comprehensive and accurate molecular characterization without the need for prior isolation are reviewed. Chemical derivatization, proteolysis (enzymatic and chemical cleavage), multistage MS fragmentation, and separation (liquid chromatography and ion mobility) strategies can provide complementary amino acid composition and post-translational modification data to constrain sequence solutions. Examination of two complex case studies, gomesin and styelin D, highlights the practical implementation of the proposed approaches. Finally, we emphasize the importance of heterogeneous AMP peptidoforms that confer varying biological function, an area that warrants significant further development.

One of the key proposed agents of fetal programming is exposure to maternal glucocorticoids. Experimental animal studies provide evidence that prenatal exposure to elevated maternal glucocorticoids has consequences for hypothalamic-pituitary-adrenal (HPA) axis functioning in the offspring. There are very few direct tests of maternal glucocorticoids, such as cortisol, during human pregnancy and associations with infant cortisol reactivity. The current study examined the link between maternal prenatal cortisol trajectories and infant cortisol reactivity to the pain of inoculation in a sample of 152 mother-infant (47.4% girls) pairs. The results from the current study provide insight into fetal programming of the infant HPA axis, demonstrating that elevated prenatal maternal cortisol is associated with a larger infant cortisol response to challenge at both 6 and 12 months of age., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Traditional medicinal plants are rich reservoirs of antimicrobial agents, including antimicrobial peptides (AMPs). Advances in genomic sequencing, in silico AMP predictions, and mass spectrometry-based peptidomics facilitate increasingly high-throughput bioactive peptide discovery. Herein, Amaranthus tricolor aerial tissue was profiled via MS-based proteomics/peptidomics, identifying AMPs predicted in silico. Bottom-up proteomics identified seven novel peptides spanning three AMP classes including lipid transfer proteins, snakins, and a defensin. Characterization via top-down peptidomic analysis of Atr-SN1, Atr-DEF1, and Atr-LTP1 revealed unexpected proteolytic processing and enumerated disulfide bonds. Bioactivity screening of isolated Atr-LTP1 showed activity against the high-risk ESKAPE bacterial pathogens ( Enterococcus faecium , Staphylococcus aureus , Klebsiella pneumoniae , Acinetobacter baumannii , and Enterobacter cloacae ). These results highlight the potential for integrating AMP prediction algorithms with complementary -omics approaches to accelerate characterization of biologically relevant AMP peptidoforms.

Upon pathogen infection, receptors in plants will activate a localized immune response, the effector-triggered immunity (ETI), and a systemic immune response, the systemic acquired response (SAR). Infection also induces oscillations in the redox environment of plant cells, triggering response mechanisms involving sensitive cysteine residues that subsequently alter protein function. Arabidopsis thaliana thimet oligopeptidases TOP1 and TOP2 are required for plant defense against pathogens and the oxidative stress response. Herein, we evaluated the biochemical attributes of TOP isoforms to determine their redox sensitivity using ex vivo Escherichia coli cultures and recombinant proteins. Moreover, we explored the link between their redox regulation and plant immunity in wild-type and mutant Arabidopsis lines. These analyses revealed that redox regulation of TOPs occurs through two mechanisms: (1) oxidative dimerization of full-length TOP1 via intermolecular disulfides engaging cysteines in the N-terminal signal peptide, and (2) oxidative activation of all TOPs via cysteines that are unique and conserved. Further, we detected increased TOP activity in wild-type plants undergoing ETI or SAR following inoculation with Pseudomonas syringae strains. Mutants unable to express the chloroplast NADPH-dependent thioredoxin reductase C (NTRC) showed elevated TOP activity under unstressed conditions and were SAR-incompetent. A top1top2 knockout mutant challenged with P. syringae exhibited misregulation of ROS-induced gene expression in pathogen-inoculated and distal tissues. Furthermore, TOP1 and TOP2 could cleave a peptide derived from the immune component ROC1 with distinct efficiencies at common and specific sites. We propose that Arabidopsis TOPs are thiol-regulated peptidases active in redox-mediated signaling of local and systemic immunity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Despite the continuous emergence of multi-drug resistant pathogens, the number of new antimicrobials reaching the market is critically low. Natural product peptides are a rich source of bioactive compounds, and advances in mass spectrometry have achieved unprecedented capabilities for the discovery and characterization of novel molecular species. However, traditional bioactivity assay formats hinder the discovery and biochemical characterization of natural product antimicrobial peptides (AMPs), necessitating large sample quantities and significant optimization of experimental parameters to achieve accurate/consistent activity measurements. Microfluidic devices offer a promising alternative to bulk assay systems. Herein, a microfluidics-based bioassay was compared to the traditional 96-well plate format in respective commercially-available hardware. Bioactivity in each assay type was compared using a Viola inconspicua peptide library screened against E. coli ATCC 25922. Brightfield microcopy was used to determine bioactivity in microfluidic channels while both common optical and fluorescence-based measurements of cell viability were critically assessed in plate-based assays. Exhibiting some variation in optical density and fluorescence-based measurements, all plate-based assays conferred bioactivity in late eluting V. inconspicua library fractions. However, significant differences in the bioactivity profiles of plate-based and microfluidic assays were found, and may be derived from the materials comprising each assay device or the growth/assay conditions utilized in each format. While new technologies are necessary to overcome the limitations of traditional bioactivity assays, we demonstrate that off-the-shelf implementation of microfluidic devices is non-trivial and significant method development/optimization is required before conventional use can be realized for sensitive and rapid detection of AMPs in natural product matrices., (Copyright © 2020 Parsley, Smythers and Hicks.)

Natural product extracts present inherently complex matrices in which the identification of novel bioactive peptide species is challenged by low-abundance masses and significant structural and sequence diversity. Additionally, discovery efforts often result in the re-identification of known compounds, where modifications derived in vivo or during sample handling may obscure true sequence identity. Herein, we identify mass spectral (MS 2 ) "fingerprint" ions characteristic of cyclotides, a diverse and biologically active family of botanical cysteine-rich peptides, based on regions of high sequence homology. We couple mass shift analysis with MS 2 spectral fingerprint ions cross referenced with CyBase-a cyclotide database-to discern unique mass species in Viola communis extracts from mass species that are likely already characterized and those with common modifications. The approach is extended to a related class of cysteine-rich peptides, the trypsin inhibitors, using the characterized botanical species Lagenaria siceraria . Coupling the observation of highly abundant MS 2 ions with mass shift analysis, we identify a new set of small, highly disulfide-bound cysteine-rich L. siceraria peptides.

Cysteine is the most intrinsically nucleophilic residue in proteins and serves as a mediator against increasing reactive oxygen species (ROS) via reversible thiol oxidation. Despite the importance of cysteine oxidation in understanding biological stress response, cysteine sites most reactive toward ROS remain largely unknown and are a major analytical challenge. Herein, a chemical proteomic method to quantify site-specific cysteine reactivity using a maleimide-activated, thiol-reactive probe ( N -propargylmaleimide, NPM) is described. Implementation of a gel-based approach via conjugation of rhodamine-azide to NPM-labeled cysteine residues by copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry allowed simple and highly sensitive fluorescence profiling. Relative quantification of >1500 unique cysteine sites from greater than 800 proteins was achieved by conjugating dialkoxydiphenylsilane (DADPS) biotin-azide by the CuAAC reaction and subsequently performing biotin-streptavidin affinity purification and mass-spectrometry-based proteomics. Taken together, this work defines a novel role for the NPM probe in chemical proteomics and presents a robust method for determination of cysteine reactivity during oxidative stress response.

As global temperatures climb to historic highs, the far-reaching effects of climate change have impacted agricultural nutrient availability. This has extended to low latitude oceans, where a deficit in both nitrogen and phosphorus stores has led to dramatic decreases in carbon sequestration in oceanic phytoplankton. Although Chlamydomonas reinhardtii , a freshwater model green alga, has shown drastic systems-level alterations following nitrogen deprivation, the mechanisms through which these alterations are triggered and regulated are not fully understood. This study examined the role of reversible oxidative signaling in the nitrogen stress response of C. reinhardtii . Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 7889 unique oxidized cysteine thiol identifiers were quantified, with 231 significantly changing peptides from 184 proteins following 2 h of nitrogen deprivation. These results demonstrate that the cellular response to nitrogen assimilation, photosynthesis, pigment biosynthesis, and lipid metabolism are regulated by reversible oxidation. An enhanced role of non-damaging oxidative pathways is observed throughout the photosynthetic apparatus that provides a framework for further analysis in phototrophs.

The unicellular alga Chlamydomonas reinhardtii is a model photosynthetic organism for the study of microalgal processes. Along with genomic and transcriptomic studies, proteomic analysis of Chlamydomonas has led to an increased understanding of its metabolic signaling as well as a growing interest in the elucidation of its phosphorylation networks. To this end, mass spectrometry-based proteomics has made great strides in large-scale protein quantitation as well as analysis of posttranslational modifications (PTMs) in a high-throughput manner. An accurate quantification of dynamic PTMs, such as phosphorylation, requires high reproducibility and sensitivity due to the substoichiometric levels of modified peptides, which can make depth of coverage challenging. Here we present a method using TiO 2 -based phosphopeptide enrichment paired with label-free LC-MS/MS for phosphoproteome quantification. Three technical replicate samples in Chlamydomonas were processed and analyzed using this approach, quantifying a total of 1775 phosphoproteins with a total of 3595 phosphosites. With a median CV of 21% across quantified phosphopeptides, implementation of this method for differential studies provides highly reproducible analysis of phosphorylation events. While the culturing and extraction methods used are specific to facilitate coverage in algal species, this approach is widely applicable and can easily extend beyond algae to other photosynthetic organisms with minor modifications.

Target of rapamycin complex 1 (TORC1) is a central regulator of cell growth. It balances anabolic and catabolic processes in response to nutrients, growth factors, and energy availability. Nitrogen- and carbon-containing metabolites have been shown to activate TORC1 in yeast, animals, and plants. Here, we show that phosphorus (P) regulates TORC1 signaling in the model green alga Chlamydomonas ( Chlamydomonas reinhardtii ) via LST8, a conserved TORC1 subunit that interacts with the kinase domain of TOR. P starvation results in a sharp decrease in LST8 abundance and downregulation of TORC1 activity. A hypomorphic lst8 mutation resulted in decreased LST8 abundance, and it both reduced TORC1 signaling and altered the cellular response to P starvation. Additionally, we found that LST8 levels and TORC1 activity were not properly regulated in a mutant defective in the transcription factor PSR1, which is the major mediator of P deprivation responses in Chlamydomonas. Unlike wild-type cells, the psr1 mutant failed to downregulate LST8 abundance and TORC1 activity when under P limitation. These results identify PSR1 as an upstream regulator of TORC1 and demonstrate that TORC1 is a key component in P signaling in Chlamydomonas., (© 2020 American Society of Plant Biologists. All rights reserved.)

The gas-phase linearization of cyclotides via site-selective ring opening at dehydroalanine residues and its application to cyclotide sequencing is presented. This strategy relies on the ability to incorporate dehydroalanine into macrocyclic peptide ions, which is easily accomplished through an ion/ion reaction. Triply protonated cyclotide cations are transformed into radical cations via ion/ion reaction with the sulfate radical anion. Subsequent activation of the cyclotide radical cation generates dehydroalanine at a single cysteine residue, which is easily identified by the odd-electron loss of ·SCH 2 CONH 2 . The presence of dehydroalanine in cyclotides provides a site-selective ring-opening pathway that, in turn, generates linear cyclotide analogues in the gas phase. Unlike cyclic variants, product ions derived from the linear peptides provide rich sequence information. The sequencing capability of this strategy is demonstrated with four known cyclotides found in Viola inconspicua , where, in each case, greater than 93% sequence coverage was observed. Furthermore, the utility of this method is highlighted by the partial de novo sequencing of an unknown cyclotide with much greater sequence coverage than that obtained with a conventional Glu-C digestion approach. This method is particularly well-suited for cyclotide species that are not abundant enough to characterize with traditional methods.

As molecular on-off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein-coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor-like kinase (RLK) BRI1-associated Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. Twenty-two trans-phosphorylation sites on AtGPA1 are mapped by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis-phosphorylation sites are also identified on these RLKs, some newly shown to be dual specific kinases. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and/or pThr53 of the conserved P-loop that directly binds nucleotide/phosphate, pThr164, and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 and/or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are potentially nucleotide-dependent phosphorylation patterns, such as Ser52/Thr53 in the P-loop and Thr193 and/or Thr194 in SwI., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Traditional medicinal plants are a rich source of antimicrobials; however, the bioactive peptide constituents of most ethnobotanical species remain largely unexplored. Herein, PepSAVI-MS, a mass spectrometry-based peptidomics pipeline, was implemented for antimicrobial peptide (AMP) discovery in the medicinal plant Amaranthus tricolor . This investigation revealed a novel 1.7 kDa AMP with strong activity against Escherichia coli ATCC 25922, deemed Atr-AMP1. Initial efforts to determine the sequence of Atr-AMP1 utilized chemical derivatization and enzymatic digestion to provide information about specific residues and post-translational modifications. EThcD (electron-transfer/higher-energy collision dissociation) produced extensive backbone fragmentation and facilitated de novo sequencing, the results of which were consistent with orthogonal characterization experiments. Additionally, multistage HCD (higher-energy collisional dissociation) facilitated discrimination between isobaric leucine and isoleucine. These results revealed a positively charged proline-rich peptide present in a heterogeneous population of multiple peptidoforms, possessing several post-translational modifications including a disulfide bond, methionine oxidation, and proline hydroxylation. Additional bioactivity screening of a simplified fraction containing Atr-AMP1 revealed activity against Staphylococcus aureus LAC, demonstrating activity against both a Gram-negative and a Gram-positive bacterial species unlike many known short chain proline-rich antimicrobial peptides.

The target of rapamycin (TOR) kinase is a master metabolic regulator with roles in nutritional sensing, protein translation, and autophagy. In Chlamydomonas reinhardtii , a unicellular green alga, TOR has been linked to the regulation of increased triacylglycerol (TAG) accumulation, suggesting that TOR or a downstream target(s) is responsible for the elusive "lipid switch" in control of increasing TAG accumulation under nutrient limitation. However, while TOR has been well characterized in mammalian systems, it is still poorly understood in photosynthetic systems, and little work has been done to show the role of oxidative signaling in TOR regulation. In this study, the TOR inhibitor AZD8055 was used to relate reversible thiol oxidation to the physiological changes seen under TOR inhibition, including increased TAG content. Using oxidized cysteine resin-assisted capture enrichment coupled with label-free quantitative proteomics, 401 proteins were determined to have significant changes in oxidation following TOR inhibition. These oxidative changes mirrored characterized physiological modifications, supporting the role of reversible thiol oxidation in TOR regulation of TAG production, protein translation, carbohydrate catabolism, and photosynthesis through the use of reversible thiol oxidation. The delineation of redox-controlled proteins under TOR inhibition provides a framework for further characterization of the TOR pathway in photosynthetic eukaryotes.

The emergence of rapidly evolving multidrug-resistant pathogens and a deficit of new compounds entering the clinical pipeline necessitate the exploration of alternative sources of antimicrobial therapeutics. Cyclotides revealed in Viola spp . are a class of highly stable, cyclic, and disulfide-bound peptides with diverse intrinsic bioactivities. Herein we have identified a novel complement of 42 putative cyclotide masses in the plant species Viola inconspicua . Cyclotide-containing fractions of a V. inconspicua peptide library revealed potent bioactivities against the Gram-negative bacteria Escherichia coli ATCC 25922 and the highly virulent and multidrug-resistant Klebsiella pneumoniae VK148. As such, six previously uncharacterized cyclotides, cycloviolacins I1-6 (cyI1-cyI6), were prioritized for molecular characterization. Cyclotides cyI3-cyI6 contain a novel "TLNGNPGA" motif in the highly variable loop six region, expanding the already substantial sequence diversity of this peptide class. Library fractions comprised of cyclotides cyI3-cyI6 exhibited MIC values of 18 and 35 μM against E. coli and K. pneumoniae , respectively, whereas isolated cyI3 killed ∼50% of E. coli at 60 μM and isolated cyI4 demonstrated no killing at concentrations >60 μM against both pathogens. This work expands the repertoire of bioactive cyclotides found in Viola spp . and highlights the potential of these antibacterial cyclic peptides.

The rice heterotrimeric G-protein complex, a guanine-nucleotide-dependent on-off switch, mediates vital cellular processes and responses to biotic and abiotic stress. Exchange of bound GDP (resting state) for GTP (active state) is spontaneous in plants including rice and thus there is no need for promoting guanine nucleotide exchange in vivo as a mechanism for regulating the active state of signaling as it is well known for animal G signaling. As such, a master regulator controlling the G-protein activation state is unknown in plants. Therefore, an ab initio approach is taken to discover candidate regulators. The rice Gα subunit (RGA1) is used as bait to screen for nucleotide-dependent protein partners. A total of 264 proteins are identified by tandem mass spectrometry of which 32 were specific to the GDP-bound inactive state and 22 specific to the transition state. Approximately, 10% are validated as previously identified G-protein interactors., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Despite the significant health benefits of breastfeeding for the mother and the infant, economic class and race disparities in breastfeeding rates persist. Support for breastfeeding from the father of the infant is associated with higher rates of breastfeeding initiation. However, little is known about the factors that may promote or deter father support of breastfeeding, especially in fathers exposed to contextual adversity such as poverty and violence. Using a mixed methods approach, the primary aims of the current work were to (1) elicit, using qualitative methodology, the worries, barriers and promotive factors for breastfeeding that expectant mothers and fathers identify as they prepare to parent a new infant, and (2) to examine factors that influence the parental breastfeeding intentions of both mothers and fathers using quantitative methodology. A sample (N=95) of expectant, third trimester mothers and fathers living in a low-income, urban environment in Midwestern USA, were interviewed from October 2013 to February 2015 about their infant feeding intentions. Compared with fathers, mothers more often identified the benefits of breastfeeding for the infant's health and the economic advantage of breastfeeding. Mothers also identified more personal and community breastfeeding support resources. Fathers viewed their own support of breastfeeding as important but expressed a lack of knowledge about the breastfeeding process and often excluded themselves from discussions about infant feeding. The results point to important targets for interventions that aim to increase breastfeeding initiation rates in vulnerable populations in the US by increasing father support for breastfeeding.

A surge in the accumulation of oxidants generates shifts in the cellular redox potential during early stages of plant infection with pathogens and activation of effector-triggered immunity (ETI). The redoxome, defined as the proteome-wide oxidative modifications of proteins caused by oxidants, has a well-known impact on stress responses in metazoans. However, the identity of proteins and the residues sensitive to oxidation during the plant immune response remain largely unknown. Previous studies of the thimet oligopeptidases TOP1 and TOP2 placed them in the salicylic acid dependent branch of ETI, with a current model wherein TOPs sustain interconnected organellar and cytosolic pathways that modulate the oxidative burst and development of cell death. Herein, we characterized the ETI redoxomes in Arabidopsis ( Arabidopsis thaliana ) wild-type Col-0 and top1top2 mutant plants using a differential alkylation-based enrichment technique coupled with label-free mass spectrometry-based quantification. We identified cysteines sensitive to oxidation in a wide range of protein families at multiple time points after pathogen infection. Differences were detected between Col-0 and top1top2 redoxomes regarding the identity and number of oxidized cysteines, and the amplitude of time-dependent fluctuations in protein oxidation. Our results support a determining role for TOPs in maintaining the proper level and dynamics of proteome oxidation during ETI. This study significantly expands the repertoire of oxidation-sensitive plant proteins and can guide future mechanistic studies., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Background: Nearly a third of adults report childhood trauma in their youth and approximately 700,000 cases of child maltreatment were reported in 2016. Both history of childhood trauma and current trauma symptoms in adults are linked to child maltreatment, although many trauma-exposed individuals are warm and nurturing parents. Identifying resiliency factors in adults with risk factors for harsh parenting may illuminate new pathways to sensitive parenting. Mindfulness is reported to improve trauma and mental health symptoms but the relationship between mindfulness, trauma, and child abuse potential is not yet understood., Objective: This cross-sectional study investigated the relationship between mindfulness, childhood trauma experiences, trauma symptoms and child abuse potential., Participants and Setting: Our participants were 102 expectant parents recruiting from obstetric clinics and agencies Detroit, MI (58.8% African American, 27.5% Caucasian)., Method: Bivariate correlations were examined using validated, self-report questionnaires. Significant variables were included in a hierarchical linear regression to identify predicting factors that contribute to child abuse potential scores., Results: Significant correlations between child abuse potential with current trauma symptoms (r = .53, p < .01) and mindfulness (r = -.32, p < .01) were found, but no link with past childhood trauma experiences and child abuse potential were identified. The model significantly predicts child abuse potential (ΔR 2  = .10, F(5, 96), = 12.48, p < .001). Trauma symptoms (B = .09, p <  .001, 95% confidence interval [CI][-.40, -.07]) and mindfulness nonreactivity (B = -.24, p <  .01, 95% CI[.05, .14]) predicted higher potential for child abuse scores., Conclusion: Findings suggest increased mindfulness, especially nonreactivity to one's own thoughts, may be an important factor to protect against child abuse potential. Interventions to increase parental mindfulness may reduce child abuse potential and improve child well-being, but further mechanistic research is needed to determine this., (Copyright © 2019 Elsevier Ltd. All rights reserved.)