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Mesenchymal-epithelial transition factor (MET) is a receptor tyrosine kinase that serves a critical function in numerous developmental, morphogenic, and proliferative signaling pathways. If dysregulated, MET has been shown to be involved in the development and survival of several cancers, including non-small cell lung cancer (NSCLC), renal cancer, and other epithelial tumors. Currently, the clinical efficacy of FDA approved MET inhibitors is limited by on-target acquired resistance, dose-limiting toxicities, and less than optimal efficacy against brain metastasis. Therefore, there is still an unmet medical need for the development of MET inhibitors to address these issues. Herein we report the application of structure-based design for the discovery and development of a novel class of brain-penetrant MET inhibitors with enhanced activity against clinically relevant mutations and improved selectivity. Compound 13 with a MET D1228N cell line IC 50 value of 23 nM showed good efficacy in an intracranial tumor model and increased the median overall survival of the animals to 100% when dosed orally at 100 mg/kg daily for 21 days.

Abscisic acid (ABA) is a phytohormone that promotes leaf senescence in response to environmental stress. We previously identified methyl CpG-binding domain 10 (MBD10) as a phosphoprotein that becomes differentially phosphorylated after ABA treatment in Arabidopsis. ABA-induced leaf senescence was delayed in mbd10 knockout plants but accelerated in MBD10-overexpressing plants, suggesting that MBD10 positively regulates ABA-induced leaf senescence. ABA-induced phosphorylation of MBD10 occurs in planta on Thr-89, and our results demonstrated that Thr-89 phosphorylation is essential for MBD10's function in leaf senescence. The in vivo phosphorylation of Thr-89 in MBD10 was significantly downregulated in a quadruple mutant of group C MAPKs (mpk1/2/7/14), and group C MAPKs directly phosphorylated MBD10 in vitro. Furthermore, mpk1/2/7/14 showed a similar phenotype as seen in mbd10 for ABA-induced leaf senescence, suggesting that group C MAPKs are the cognate kinases of MBD10 for Thr-89. Because group C MAPKs have been reported to function downstream of SnRK2s, our results indicate that group C MAPKs and MBD10 constitute a regulatory pathway for ABA-induced leaf senescence., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Many plant pathogenic bacteria suppress host defenses by secreting small molecule toxins or immune-suppressing proteins into host cells, processes that likely require close physical contact between pathogen and host. Yet, in most cases, little is known about whether phytopathogenic bacteria physically attach to host surfaces during infection. Here we report that Pseudomonas syringae pv. tomato strain DC3000, a Gram-negative bacterial pathogen of tomato and Arabidopsis, attaches to polystyrene and glass surfaces in response to chemical signals exuded from Arabidopsis seedlings and tomato leaves. We characterized the molecular nature of these attachment-inducing signals and discovered that multiple hydrophilic metabolites found in plant exudates, including citric acid, glutamic acid, and aspartic acid, are potent inducers of surface attachment. These same compounds were previously identified as inducers of P. syringae genes encoding a type III secretion system (T3SS), indicating that both attachment and T3SS deployment are induced by the same plant signals. To test if surface attachment and T3SS are regulated by the same signaling pathways, we assessed the attachment phenotypes of several previously characterized DC3000 mutants, and found that the T3SS master regulator HrpL was partially required for maximal levels of surface attachment, whereas the response regulator GacA, a negative regulator of T3SS, negatively regulated DC3000 surface attachment. Together, our data indicate that T3SS deployment and surface attachment by P. syringae may be co-regulated by the same host signals during infection, possibly to ensure close contact necessary to facilitate delivery of T3SS effectors into host cells., Competing Interests: The authors declare that they have no competing interests., (© 2023 O’Malley et al.)

Certain cultivars of maize show increased tolerance to water deficit conditions by maintenance of root growth. To better understand the molecular mechanisms related to this adaptation, nodal root growth zone samples were collected from the reference inbred line B73 and inbred line FR697, which exhibits a relatively greater ability to maintain root elongation under water deficits. Plants were grown under various water stress levels in both field and controlled environment settings. FR697-specific RNA-Seq datasets were generated and used for a de novo transcriptome assembly to characterize any genotype-specific genetic features. The assembly was aided by an Iso-Seq library of transcripts generated from various FR697 plant tissue samples. The Necklace pipeline was used to combine a Trinity de novo assembly along with a reference guided assembly and the Viridiplantae proteome to generate an annotated consensus "SuperTranscriptome" assembly of 47,915 transcripts with a N50 of 3152 bp in length. The results were compared by Blastn to maize reference genes, a Benchmarking Universal Single-Copy Orthologs (BUSCO) genome completeness report and compared with three maize reference genomes. The resultant 'SuperTranscriptome' was demonstrated to be of high-quality and will serve as an important reference for analysis of the maize nodal root transcriptomic response to environmental perturbations., (© 2023. The Author(s).)

Anthracnose is a fungal disease caused by members of Colletotrichum that affect a wide range of crop plants. Strategies to improve crop resistance are needed to reduce the yield losses; and one strategy is to manipulate protein kinases that catalyze reversible phosphorylation of proteins regulating both plant immune responses and fungal pathogenesis. Hence, in this review, we present a summary of the current knowledge of protein kinase signaling pathways in plant- Colletotrichum interaction as well as the relation to a more general understanding of protein kinases that contribute to plant immunity and pathogen virulence. We highlight the potential of combining genomic resources and phosphoproteomics research to unravel the key molecular components of plant- Colletotrichum interactions. Understanding the molecular interactions between plants and Colletotrichum would not only facilitate molecular breeding of resistant cultivars but also help the development of novel strategies for controlling the anthracnose disease., 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 Jiang, Zhang, Su, Peck and Luo.)

The phytohormone abscisic acid (ABA) plays a major role in abiotic stress responses in plants, and subclass III SNF1-related protein kinase 2 (SnRK2) kinases mediate ABA signaling. In this study, we identified Raf36, a group C Raf-like protein kinase in Arabidopsis , as a protein that interacts with multiple SnRK2s. A series of reverse genetic and biochemical analyses revealed that 1) Raf36 negatively regulates ABA responses during postgermination growth, 2) the N terminus of Raf36 is directly phosphorylated by SnRK2s, and 3) Raf36 degradation is enhanced in response to ABA. In addition, Raf22, another C-type Raf-like kinase, functions partially redundantly with Raf36 to regulate ABA responses. A comparative phosphoproteomic analysis of ABA-induced responses of wild-type and raf22raf36-1 plants identified proteins that are phosphorylated downstream of Raf36 and Raf22 in planta. Together, these results support a model in which Raf36/Raf22 function mainly under optimal conditions to suppress ABA responses, whereas in response to ABA, the SnRK2 module promotes Raf36 degradation as a means of alleviating Raf36-dependent inhibition and allowing for heightened ABA signaling to occur., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Introduction: Hemiarthroplasty is increasingly used for the treatment of geriatric femoral neck fractures in an effort to optimize value-based care. The current American Association of Orthopaedic Surgeons (AAOS) guidelines released in 2014 for the treatment of geriatric hip fractures recommend the utilization of monopolar cemented constructs. The purpose of this study was to evaluate hip hemiarthroplasty implant cost variability and implant selection trends from 2006 to 2018., Materials and Methods: A retrospective review of 940 geriatric hip fractures treated with hemiarthroplasty was conducted across 3 institutions from 2006 to 2018. Variables examined were construct type, surgeon, operative time, patient mortality, and implant cost. Statistical analysis consisted of multigroup comparative tests and multiple linear regression analyses to evaluate correlative measures., Results: The study population was 85.0 ± 7.9 years of age with a body mass index of 24.0 ± 5.5. A total of 33 (3.5%) patients were deceased at the 90-day postoperative mark and 45 (4.8%) patients at the 1-year mark. There was no statistical difference in terms of mortality between the 4 implant cohorts at the 90-day mark ( P = .56) and 1-year mark ( P = .24). The bipolar press-fit construct was the most expensive, US$3900.61 ± US$2607.54, and the monopolar cemented construct was the least expensive, US$2618.68 ± US$1834.16. The mean operative time was 6 minutes greater for press-fit implants, 93.6 ± 32.0, than cemented implants, 87.1 ± 33.6 ( P = .02). The use of monopolar cemented implants increased from 12.1% to 83.3%, while bipolar press-fit decreased from 57.6% to 4.6% from 2013 to 2018., Discussion: The use of a bipolar and/or press-fit implant significantly increases construct cost despite little evidence in the literature of improved outcomes. Contrary to previous research, cemented implants do not increase the operative time., Conclusions: Encouragingly, selection of the most cost-conscience implant, monopolar cemented, has been increasing since 2014, which may reflect the influence of current AAOS guidelines., Level of Evidence: Diagnostic Level III., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2020.)

The plasma membrane (PM) provides a critical interface between plant cells and their environment to control cellular responses. To perceive the bacterial flagellin peptide flg22 for effective defense signaling, the immune receptor FLAGELLIN SENSING2 (FLS2) needs to be at its site of function, the PM, in the correct abundance. However, the intracellular machinery that controls PM accumulation of FLS2 remains largely undefined. The Arabidopsis ( Arabidopsis thaliana ) clathrin adaptor EPSIN1 (EPS1) is implicated in clathrin-coated vesicle formation at the trans-Golgi network (TGN), likely aiding the transport of cargo proteins from the TGN for proper location; but EPS1's impact on physiological responses remains elusive. Here, we identify EPS1 as a positive regulator of flg22 signaling and pattern-triggered immunity against Pseudomonas syringae pv tomato DC3000. We provide evidence that EPS1 contributes to modulating the PM abundance of defense proteins for effective immune signaling because in eps1 , impaired flg22 signaling correlated with reduced PM accumulation of FLS2 and its coreceptor BRASSINOSTEROID INSENSITIVE1-ASSOCIATED RECEPTOR KINASE1 (BAK1). The eps1 mutant also exhibited reduced responses to the pathogen/damage-associated molecular patterns elf26 and AtPep1, which are perceived by the coreceptor BAK1 and cognate PM receptors. Furthermore, quantitative proteomics of enriched PM fractions revealed that EPS1 was required for proper PM abundance of a discrete subset of proteins with different cellular functions. In conclusion, our study expands the limited understanding of the physiological roles of EPSIN family members in plants and provides novel insight into the TGN-associated clathrin-coated vesicle trafficking machinery that impacts plant PM-derived defense processes., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

GacS/GacA is a conserved two-component system that functions as a master regulator of virulence-associated traits in many bacterial pathogens, including Pseudomonas spp., that collectively infect both plant and animal hosts. Among many GacS/GacA-regulated traits, type III secretion of effector proteins into host cells plays a critical role in bacterial virulence. In the opportunistic plant and animal pathogen Pseudomonas aeruginosa, GacS/GacA negatively regulates the expression of type III secretion system (T3SS)-encoding genes. However, in the plant pathogenic bacterium Pseudomonas syringae, strain-to-strain variation exists in the requirement of GacS/GacA for T3SS deployment, and this variability has limited the development of predictive models of how GacS/GacA functions in this species. In this work we re-evaluated the function of GacA in P. syringae pv. tomato DC3000. Contrary to previous reports, we discovered that GacA negatively regulates the expression of T3SS genes in DC3000, and that GacA is not required for DC3000 virulence inside Arabidopsis leaf tissue. However, our results show that GacA is required for full virulence of leaf surface-inoculated bacteria. These data significantly revise current understanding of GacS/GacA in regulating P. syringae virulence., (© 2019 The Authors. Molecular Plant Pathology published by British Society for Plant Pathology and John Wiley & Sons Ltd.)

Abscisic acid (ABA) is a phytohormone and a major determinant of seed dormancy in plants. Seed dormancy is gradually lost during dry storage, a process known as 'after-ripening', and this dormancy decay is related to a decline in ABA content and sensitivity in seeds after imbibition. In this study, we aimed at investigating the effect of after-ripening on ABA signaling in barley, our cereal model species. Phosphosignaling networks in barley grains were investigated by a large-scale analysis of phosphopeptides to examine potential changes in response pathways to after-ripening. We used freshly harvested (FH) and after-ripened (AR) barley grains which showed different ABA sensitivity. A total of 1,730 phosphopeptides were identified in barley embryos isolated from half-cut grains. A comparative analysis showed that 329 and 235 phosphopeptides were upregulated or downregulated, respectively after ABA treatment, and phosphopeptides profiles were quite different between FH and AR embryos. These results were supported by peptide motif analysis which suggested that different sets of protein kinases are active in FH and AR grains. Furthermore, in vitro phosphorylation assays confirmed that some phosphopeptides were phosphorylated by SnRK2s, which are major protein kinases involved in ABA signaling. Taken together, our results revealed very distinctive phosphosignaling networks in FH and AR embryos of barley, and suggested that the after-ripening of barley grains is associated with differential regulation of phosphosignaling pathways leading to a decay of ABA signaling., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

A key remit of the NSF-funded "Arabidopsis Research and Training for the 21 st Century" (ART-21) Research Coordination Network has been to convene a series of workshops with community members to explore issues concerning research and training in plant biology, including the role that research using Arabidopsis thaliana can play in addressing those issues. A first workshop focused on training needs for bioinformatic and computational approaches in plant biology was held in 2016, and recommendations from that workshop have been published (Friesner et al., Plant Physiology , 175, 2017, 1499). In this white paper, we provide a summary of the discussions and insights arising from the second ART-21 workshop. The second workshop focused on experimental aspects of omics data acquisition and analysis and involved a broad spectrum of participants from academics and industry, ranging from graduate students through post-doctorates, early career and established investigators. Our hope is that this article will inspire beginning and established scientists, corporations, and funding agencies to pursue directions in research and training identified by this workshop, capitalizing on the reference species Arabidopsis thaliana and other valuable plant systems.

Hydrogen sulfide (H 2 S) production in the intestinal microbiota has many contributions to human health and disease. An important source of H 2 S in the human gut is anaerobic respiration of sulfite released from the abundant dietary and host-derived organic sulfonate substrate in the gut, taurine (2-aminoethanesulfonate). However, the enzymes that allow intestinal bacteria to access sulfite from taurine have not yet been identified. Here we decipher the complete taurine desulfonation pathway in Bilophila wadsworthia 3.1.6 using differential proteomics, in vitro reconstruction with heterologously produced enzymes, and identification of critical intermediates. An initial deamination of taurine to sulfoacetaldehyde by a known taurine:pyruvate aminotransferase is followed, unexpectedly, by reduction of sulfoacetaldehyde to isethionate (2-hydroxyethanesulfonate) by an NADH-dependent reductase. Isethionate is then cleaved to sulfite and acetaldehyde by a previously uncharacterized glycyl radical enzyme (GRE), isethionate sulfite-lyase (IslA). The acetaldehyde produced is oxidized to acetyl-CoA by a dehydrogenase, and the sulfite is reduced to H 2 S by dissimilatory sulfite reductase. This unique GRE is also found in Desulfovibrio desulfuricans DSM642 and Desulfovibrio alaskensis G20, which use isethionate but not taurine; corresponding knockout mutants of D. alaskensis G20 did not grow with isethionate as the terminal electron acceptor. In conclusion, the novel radical-based C-S bond-cleavage reaction catalyzed by IslA diversifies the known repertoire of GRE superfamily enzymes and enables the energy metabolism of B. wadsworthia This GRE is widely distributed in gut bacterial genomes and may represent a novel target for control of intestinal H 2 S production., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The C-terminal domain of the dehydrophos biosynthetic enzyme DhpH (DhpH-C) catalyzes the condensation of Leu-tRNALeu with (R)-1-aminoethylphosphonate, the aminophosphonate analog of alanine called Ala(P). The product of this reaction, Leu-Ala(P), is a phosphonodipeptide, a class of compounds that have previously been investigated for use as clinical antibiotics. In this study, we show that DhpH-C is highly substrate tolerant and can condense various aminophosphonates (Gly(P), Ser(P), Val(P), 1-amino-propylphosphonate, and phenylglycine(P)) to Leu. Moreover, the enzyme is also tolerant with respect to the amino acid attached to tRNALeu. Using a mutant of leucyl tRNA synthetase that is deficient in its proofreading ability allowed the preparation of a series of aminoacyl-tRNALeu derivatives (Ile, Ala, Val, Met, norvaline, and norleucine). DhpH-C accepted these aminoacyl-tRNA derivatives and condensed the amino acid with l-Ala(P) to form the corresponding phosphonodipeptides. A subset of these peptides displayed antimicrobial activities demonstrating that the enzyme is a versatile biocatalyst for the preparation of antimicrobial peptides. We also investigated another enzyme from the dehydrophos biosynthetic pathway, the 2-oxoglutarate dependent enzyme DhpA. This enzyme oxidizes 2-hydroxyethylphosphonate to 1,2-dihydroxyethylphosphonate en route to l-Ala(P), but longer incubation results in overoxidation to 1-oxo-2-hydroxyethylphosphonate. This α-ketophosphonate was converted by the pyridoxal phosphate dependent enzyme DhpD into l-Ser(P). Thus, the dehydrophos biosynthetic enzymes can generate not only l-Ala(P) but also l-Ser(P).

Mitogen-Activated Protein Kinase (MAPK) cascades are conserved signaling modules that integrate multiple signaling pathways. One level of control on the activity of MAPKs is through their negative regulators, MAPK phosphatases (MKPs). Therefore, MKPs also play an integrative role for plants responding to diverse environmental stimulus; but the mechanism(s) by which these phosphatases contribute to specific signals remains largely unknown. In this review, we summarize recent advances in characterizing the biological functions of a sub-class of MKPs, dual-specificity phosphatases (DSPs), ranging from controlling plant growth and development to modulating stress adaptation. We also discuss putative regulatory mechanisms of DSP-type MKPs, which plants may use to control the correct level of responses at the right place and time. We highlight insights into potential regulation of cross-talk between different signaling pathways, facilitating the development of strategies for targeting such cross-talk and to help improve plant resistance against adverse environmental conditions without affecting the growth and development.

Methylphosphonate synthase (MPnS) produces methylphosphonate, a metabolic precursor to methane in the upper ocean. Here, we determine a 2.35-angstrom resolution structure of MPnS and discover that it has an unusual 2-histidine-1-glutamine iron-coordinating triad. We further solve the structure of a related enzyme, hydroxyethylphosphonate dioxygenase from Streptomyces albus ( Sa HEPD), and find that it displays the same motif. Sa HEPD can be converted into an MPnS by mutation of glutamine-adjacent residues, identifying the molecular requirements for methylphosphonate synthesis. Using these sequence markers, we find numerous putative MPnSs in marine microbiomes and confirm that MPnS is present in the abundant Pelagibacter ubique. The ubiquity of MPnS-containing microbes supports the proposal that methylphosphonate is a source of methane in the upper, aerobic ocean, where phosphorus-starved microbes catabolize methylphosphonate for its phosphorus., (Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Plants perceive potential pathogens via the recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors, which initiates a series of intracellular responses that ultimately limit bacterial growth. PAMP responses include changes in intracellular protein phosphorylation, including the activation of mitogen-activated protein kinase (MAPK) cascades. MAP kinase phosphatases (MKPs), such as Arabidopsis ( Arabidopsis thaliana ) MKP1, are important negative regulators of MAPKs and play a crucial role in controlling the intensity and duration of MAPK activation during innate immune signaling. As such, the mkp1 mutant lacking MKP1 displays enhanced PAMP responses and resistance against the virulent bacterium Pseudomonas syringae pv tomato DC3000. Previous in vitro studies showed that MKP1 can be phosphorylated and activated by MPK6, suggesting that phosphorylation may be an important mechanism for regulating MKP1. We found that MKP1 was phosphorylated during PAMP elicitation and that phosphorylation stabilized the protein, resulting in protein accumulation after elicitation. MKP1 also can be stabilized by the proteasome inhibitor MG132, suggesting that MKP1 is constitutively degraded through the proteasome in the resting state. In addition, we investigated the role of MKP1 posttranslational regulation in plant defense by testing whether phenotypes of the mkp1 Arabidopsis mutant could be complemented by expressing phosphorylation site mutations of MKP1. The phosphorylation of MKP1 was found to be required for some, but not all, of MKP1's functions in PAMP responses and defense against bacteria. Together, our results provide insight into the roles of phosphorylation in the regulation of MKP1 during PAMP signaling and resistance to bacteria., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

In this monograph, we investigate the developmental trajectories of a predominantly middle-class, community-based sample of European American and African American adolescents growing up in urban, suburban, and rural areas in Maryland, United States. Within risk-protection and positive youth development frameworks, we selected developmental measures based on the normative tasks of adolescence and the most widely studied indicators in the three major contexts of development: families, peer groups, and schools. Using hierarchical linear growth models, we estimated adolescents' growth trajectories from ages 12 to 20 with variation accounted for by socioeconomic status (SES), gender, race/ethnicity, and the gender by race/ethnicity interaction. In general, the results indicate that: (a) periods of greatest risk and positive development depended on the time frame and outcome being examined and (b) on average, these adolescents demonstrated much stronger evidence of positive than problematic development, even at their most vulnerable times. Absolute levels of their engagement in healthy behaviors, supportive relationships with parents and friends, and positive self-perceptions and psychological well-being were much higher than their reported angry and depressive feelings, engagement in risky behaviors, and negative relationships with parents and peers. We did not find evidence to support the idea that adolescence is a time of heightened risk. Rather, on average, these adolescents experienced relatively stable and developmentally healthy trajectories for a wide range of characteristics, behaviors, and relationships, with slight increases or decreases at different points in development that varied according to domain. Developmental trajectories differed minimally by SES but in some expected ways by gender and race/ethnicity, although these latter differences were not very marked. Overall, most of the young people navigated through their adolescence and arrived at young adulthood with good mental and physical health, positive relationships with their parents and peers, and high aspirations and expectations for what their future lives might hold., (© 2017 The Society for Research in Child Development, Inc.)

Within the field of relationship science there is increasing interest in the connections between close relationships and physical health. In the present study, we examined whether adolescents' (~12 years old) and young adults' (~20 years old) perceptions of their parents as a secure base prospectively predict C-reactive protein (CRP), a commonly used marker of inflammatory activity, at age 32 in a well-characterized sample of African Americans. We utilized existing data collected as part of the Maryland Adolescent Development in Context Study (MADICS) to construct measures of perceptions of parental secure base support (SBS), general parental support, and peer support in early adolescence and early adulthood. In the present study, SBS was operationalized as the perceived ability to depend on parents in times of need. Fifty-nine African American MADICS participants who reported on perceived support in early adolescence and early adulthood participated in a follow-up home visit at age 32 during which serum CRP was measured via a blood draw. After controlling for inflammation-related confounds (e.g., tobacco use, body mass index), adolescents' perceptions of parental SBS, but not peer support or general parental support, predicted lower CRP values at age 32 ( b = -.92, SE = .34, p < .05). None of the support variables in early adulthood predicted CRP at 32 years. This study adds to a growing literature on relationships and health-related outcomes and provides the first evidence for a link between parental SBS in adolescence and a marker of inflammatory activity in adulthood.

Plant immunity is initiated by extracellular detection of pathogen-associated molecular patterns (PAMPs) through surface-localized pattern recognition receptors (PRRs). PRR activation induces many responses including the activation of mitogen-activated protein kinases (MAPKs) that ultimately limit bacterial growth. Previous work identified Arabidopsis MAP kinase phosphatase 1 (MKP1) as a negative regulator of signaling pathways required for some, but not all, of PAMP-initiated responses. Specifically, loss of MAPK MPK6 in an mkp1 background suppressed a subset of the mkp1-dependent biological phenotypes, indicating the requirement for MPK6 in MKP1-dependent signaling. To further genetically separate the outputs of PAMP-responsive signaling pathways, we performed a transcriptome analysis in Arabidopsis wild type, mkp1 and mkp1 mpk6 seedlings treated with the bacterially derived PAMP elf26 for 0, 30, and 90 min. Using differential genetic and temporal clustering analyses between and within genotypes, we identified and separated 6963 elf26-responsive transcripts based on both genetic requirements of MKP1 (with or without a requirement for MPK6) and temporal transcriptional accumulation patterns, and some of these novel response markers were validated by qRT-PCR over a more extended time course. Taken together, our transcriptome analysis provides novel information for delineating PAMP signaling pathways., (© Society for Experimental Biology 2017.)

Vocal learning from social partners is crucial for the successful development of communication in a wide range of species. Social interactions organize attention and enhance motivation to learn species-typical behaviour. However, the neurobiological mechanisms connecting social motivation and vocal learning are unknown. Using zebra finches ( Taeniopygia guttata ), a ubiquitous model for vocal learning, we show that manipulations of nonapeptide hormones in the vasopressin family (arginine vasotocin, AVT) early in development can promote or disrupt both song and social motivation. Young male zebra finches, like human infants, are socially gregarious and require interactive feedback from adult tutors to learn mature vocal forms. To investigate the role of social motivational mechanisms in song learning, in two studies, we injected hatchling males with AVT or Manning compound (MC, a nonapeptide receptor antagonist) on days 2-8 post-hatching and recorded song at maturity. In both studies, MC males produced a worse match to tutor song than controls. In study 2, which experimentally controlled for tutor and genetic factors, AVT males also learned song significantly better compared with controls. Furthermore, song similarity correlated with several measures of social motivation throughout development. These findings provide the first evidence that nonapeptides are critical to the development of vocal learning., (© 2017 The Author(s).)

This review discusses the current mechanistic understanding of a group of mononuclear non-heme iron-dependent enzymes that catalyze four-electron oxidation of their organic substrates without the use of any cofactors or cosubstrates. One set of enzymes acts on α-ketoacid-containing substrates, coupling decarboxylation to oxygen activation. This group includes 4-hydroxyphenylpyruvate dioxygenase, 4-hydroxymandelate synthase, and CloR involved in clorobiocin biosynthesis. A second set of enzymes acts on substrates containing a thiol group that coordinates to the iron. This group is comprised of isopenicillin N synthase, thiol dioxygenases, and enzymes involved in the biosynthesis of ergothioneine and ovothiol. The final group of enzymes includes HEPD and MPnS that both carry out the oxidative cleavage of the carbon-carbon bond of 2-hydroxyethylphosphonate but generate different products. Commonalities amongst many of these enzymes are discussed and include the initial substrate oxidation by a ferric-superoxo-intermediate and a second oxidation by a ferryl species.

The human microbiome encodes vast numbers of uncharacterized enzymes, limiting our functional understanding of this community and its effects on host health and disease. By incorporating information about enzymatic chemistry into quantitative metagenomics, we determined the abundance and distribution of individual members of the glycyl radical enzyme superfamily among the microbiomes of healthy humans. We identified many uncharacterized family members, including a universally distributed enzyme that enables commensal gut microbes and human pathogens to dehydrate trans -4-hydroxy-l-proline, the product of the most abundant human posttranslational modification. This "chemically guided functional profiling" workflow can therefore use ecological context to facilitate the discovery of enzymes in microbial communities., (Copyright © 2017, American Association for the Advancement of Science.)

Activation of O-H bonds by inorganic metal-oxo complexes has been documented, but no cognate enzymatic process is known. Our mechanistic analysis of 2-hydroxyethylphosphonate dioxygenase (HEPD), which cleaves the C1-C2 bond of its substrate to afford hydroxymethylphosphonate on the biosynthetic pathway to the commercial herbicide phosphinothricin, uncovered an example of such an O-H-bond-cleavage event. Stopped-flow UV-visible absorption and freeze-quench Mössbauer experiments identified a transient iron(IV)-oxo (ferryl) complex. Maximal accumulation of the intermediate required both the presence of deuterium in the substrate and, importantly, the use of 2 H 2 O as solvent. The ferryl complex forms and decays rapidly enough to be on the catalytic pathway. To account for these unanticipated results, a new mechanism that involves activation of an O-H bond by the ferryl complex is proposed. This mechanism accommodates all available data on the HEPD reaction., Competing Interests: The authors declare no competing financial interest.

The plasma membrane (PM) forms a barrier between a plant cell and its environment. Proteins at this subcellular location play diverse and complex roles, including perception of extracellular signals to coordinate cellular changes. Analyses of PM proteins, however, are often limited by the relatively low abundance of these proteins in the total cellular protein pool. Techniques traditionally used for enrichment of PM proteins are time consuming, tedious, and require extensive optimization. Here, we provide a simple and reproducible enrichment procedure for PM proteins from Arabidopsis thaliana seedlings starting from total microsomal membranes isolated by differential centrifugation. To enrich for PM proteins, total microsomes are treated with the nonionic detergent Brij-58 to decrease the abundance of contaminating organellar proteins. This protocol combined with the genetic resources available in Arabidopsis provides a powerful tool that will enhance our understanding of proteins at the PM.

Previous work on maize (Zea mays L.) primary root growth under water stress showed that cell elongation is maintained in the apical region of the growth zone but progressively inhibited further from the apex. These responses involve spatially differential and coordinated regulation of osmotic adjustment, modification of cell wall extensibility, and other cellular growth processes that are required for root growth under water-stressed conditions. As the interface between the cytoplasm and the apoplast (including the cell wall), the plasma membrane likely plays critical roles in these responses. Using a simplified method for enrichment of plasma membrane proteins, the developmental distribution of plasma membrane proteins was analysed in the growth zone of well-watered and water-stressed maize primary roots. The results identified 432 proteins with differential abundances in well-watered and water-stressed roots. The majority of changes involved region-specific patterns of response, and the identities of the water stress-responsive proteins suggest involvement in diverse biological processes including modification of sugar and nutrient transport, ion homeostasis, lipid metabolism, and cell wall composition. Integration of the distinct, region-specific plasma membrane protein abundance patterns with results from previous physiological, transcriptomic and cell wall proteomic studies reveals novel insights into root growth adaptation to water stress., (© 2016 John Wiley & Sons Ltd.)

Cellular membranes define the boundaries between organelles and the cytosol or the extracellular environment, thus providing functional separation between subcellular compartments. In addition, membranes assist in a diverse range of cellular functions, including serving as signaling platforms, mediating transport of molecules, and facilitating trafficking of cargo between cellular compartments. Because membrane functionality is largely defined by protein composition, exploring the roles of membrane proteins is of interest to many researchers. This article focuses on the subcellular fractionation of microsomes, which are membrane-derived vesicles formed during cell lysis. In plants, microsomes mainly consist of the plasma membrane and membranes derived from the endoplasmic reticulum, Golgi apparatus, trans-Golgi network, and tonoplast. The article describes the different steps involved in enriching for and solubilizing microsomal membrane proteins from Arabidopsis thaliana seedlings and cultured cells by differential centrifugation. Solubilized microsomal proteins can be used for subsequent immunoblot analysis, co-immunoprecipitation, or proteomic studies. © 2016 by John Wiley & Sons, Inc., (Copyright © 2016 John Wiley & Sons, Inc.)

Perceived racial discrimination (PRD) has been associated with altered diurnal cortisol rhythms in past cross-sectional research. We investigate whether developmental histories of PRD, assessed prospectively, are associated with adult diurnal cortisol profiles. One-hundred and twelve (N=50 Black, N=62 White) adults from the Maryland Adolescent Development in Context Study provided saliva samples in adulthood (at approximately age 32 years) at waking, 30min after waking, and at bedtime for 7 days. Diurnal cortisol measures were calculated, including waking cortisol levels, diurnal cortisol slopes, the cortisol awakening response (CAR), and average daily cortisol (AUC). These cortisol outcomes were predicted from measures of PRD obtained over a 20-year period beginning when individuals were in 7th grade (approximately age 12). Greater average PRD measured across the 20-year period predicted flatter adult diurnal cortisol slopes for both Black and White adults, and a lower CAR. Greater average PRD also predicted lower waking cortisol for Black, but not White adults. PRD experiences in adolescence accounted for many of these effects. When adolescent and young adult PRD are entered together predicting cortisol outcomes, PRD experiences in adolescence (but not young adulthood) significantly predicted flatter diurnal cortisol slopes for both Black and White adults. Adolescent, but not young adult PRD, also significantly predicted lower waking and lower average cortisol for Black adults. Young adult PRD was, however, a stronger predictor of the CAR, predicting a marginally lower CAR for Whites, and a significantly larger CAR for Blacks. Effects were robust to controlling for covariates including health behaviors, depression, income and parent education levels. PRD experiences interacted with parent education and income to predict aspects of the diurnal cortisol rhythm. Although these results suggest PRD influences on cortisol for both Blacks and Whites, the key findings suggest that the effects are more pervasive for Blacks, affecting multiple aspects of the cortisol diurnal rhythm. In addition, adolescence is a more sensitive developmental period than adulthood for the impacts of PRD on adult stress biology., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Nonheme iron oxygenases that carry out four-electron oxidations of substrate have been proposed to employ iron(III) superoxide species to initiate this reaction [Paria, S.; Que, L.; Paine, T. K. Angew. Chem. Int. Ed. 2011, 50, 11129]. Here we report experimental evidence in support of this proposal. (18)O KIEs were measured for two recently discovered mononuclear nonheme iron oxygenases: hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS). Competitive (18)O KIEs measured with deuterated substrates are larger than those measured with unlabeled substrates, which indicates that C-H cleavage must occur before an irreversible reductive step at molecular oxygen. A similar observation was previously used to implicate copper(II) superoxide in the H-abstraction reactions catalyzed by dopamine β-monooxygenase [Tian, G. C.; Klinman, J. P. J. Am. Chem. Soc. 1993, 115, 8891] and peptidylglycine α-hydroxylating monooxygenase [Francisco, W. A.; Blackburn, N. J.; Klinman, J. P. Biochemistry 2003, 42, 1813].

2-Hydroxyethylphosphonate dioxygenase (HEPD) and methylphosphonate synthase (MPnS) are nonheme iron oxygenases that both catalyze the carbon-carbon bond cleavage of 2-hydroxyethylphosphonate but generate different products. Substrate labeling experiments led to a mechanistic hypothesis in which the fate of a common intermediate determined product identity. We report here the generation of a bifunctional mutant of HEPD (E176H) that exhibits the activity of both HEPD and MPnS. The product distribution of the mutant is sensitive to a substrate isotope effect, consistent with an isotope-sensitive branching mechanism involving a common intermediate. The X-ray structure of the mutant was determined and suggested that the introduced histidine does not coordinate the active site metal, unlike the iron-binding glutamate it replaced.

This chapter discusses efforts to define and improve the quality of afterschool services, highlighting areas of agreement and identifying leading-edge issues. We conclude that the afterschool field is especially well positioned to deliver high-quality services and demonstrate effectiveness at scale because a strong foundation has been built for continuous improvement of service quality., (© 2014 WILEY PERIODICALS, INC.)

Environmental stimuli, including elevated carbon dioxide levels, regulate stomatal development; however, the key mechanisms mediating the perception and relay of the CO2 signal to the stomatal development machinery remain elusive. To adapt CO2 intake to water loss, plants regulate the development of stomatal gas exchange pores in the aerial epidermis. A diverse range of plant species show a decrease in stomatal density in response to the continuing rise in atmospheric CO2 (ref. 4). To date, one mutant that exhibits deregulation of this CO2-controlled stomatal development response, hic (which is defective in cell-wall wax biosynthesis, ref. 5), has been identified. Here we show that recently isolated Arabidopsis thaliana β-carbonic anhydrase double mutants (ca1 ca4) exhibit an inversion in their response to elevated CO2, showing increased stomatal development at elevated CO2 levels. We characterized the mechanisms mediating this response and identified an extracellular signalling pathway involved in the regulation of CO2-controlled stomatal development by carbonic anhydrases. RNA-seq analyses of transcripts show that the extracellular pro-peptide-encoding gene EPIDERMAL PATTERNING FACTOR 2 (EPF2), but not EPF1 (ref. 9), is induced in wild-type leaves but not in ca1 ca4 mutant leaves at elevated CO2 levels. Moreover, EPF2 is essential for CO2 control of stomatal development. Using cell-wall proteomic analyses and CO2-dependent transcriptomic analyses, we identified a novel CO2-induced extracellular protease, CRSP (CO2 RESPONSE SECRETED PROTEASE), as a mediator of CO2-controlled stomatal development. Our results identify mechanisms and genes that function in the repression of stomatal development in leaves during atmospheric CO2 elevation, including the carbonic-anhydrase-encoding genes CA1 and CA4 and the secreted protease CRSP, which cleaves the pro-peptide EPF2, in turn repressing stomatal development. Elucidation of these mechanisms advances the understanding of how plants perceive and relay the elevated CO2 signal and provides a framework to guide future research into how environmental challenges can modulate gas exchange in plants.

Free zinc is required for proper lipopolysaccharide (LPS)-stimulated signaling, but potential sites of action in the pathway have not been defined. In this work, we provide in vitro and ex vivo evidence that zinc is not required for phosphorylation or ubiquitylation of IRAK1, a kinase functioning early in the TLR4 pathway. However, degradation of ubiquitylated IRAK1 occurred via a zinc-dependent, proteasome-independent pathway. These results provide evidence of a novel site of action for zinc during TLR4-mediated inflammatory responses., (Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Racial/ethnic (R/E) socialization is widely practiced in R/E minority families. However, only recently have models been developed to understand how parents' R/E socialization messages influence adolescent development. The primary goal of the present study was to clarify and extend existing work on R/E socialization in African American (Black) families by distinguishing between parent and youth reports of parents' R/E socialization messages and examining the extent to which adolescents and their parents agree about these socialization messages. In addition, we tested a theoretical model in which parent-reported R/E socialization messages have an indirect effect on the development of youth R/E identity through youth reports of their parents' R/E socialization messages. Using a combination of open- and close-ended data from a longitudinal study of self-identified Black adolescents and their parents, we found statistically significant parent-youth agreement about whether parents send both general R/E socialization messages and, for daughters, specific R/E socialization messages. R/E socialization messages focused on promoting cultural pride and history were associated positively with R/E identity development, whereas messages focused on preparing youth for discrimination tended to be unrelated to R/E identity development. The results largely supported the hypothesis that parent reports of parents' R/E socialization messages are related indirectly to the development of adolescent R/E identity via youth reports of parents' R/E socialization messages., ((PsycINFO Database Record (c) 2014 APA, all rights reserved).)

Genes encoding the virulence-promoting type III secretion system (T3SS) in phytopathogenic bacteria are induced at the start of infection, indicating that recognition of signals from the host plant initiates this response. However, the precise nature of these signals and whether their concentrations can be altered to affect the biological outcome of host-pathogen interactions remain speculative. Here we use a metabolomic comparison of resistant and susceptible genotypes to identify plant-derived metabolites that induce T3SS genes in Pseudomonas syringae pv tomato DC3000 and report that mapk phosphatase 1 (mkp1), an Arabidopsis mutant that is more resistant to bacterial infection, produces decreased levels of these bioactive compounds. Consistent with these observations, T3SS effector expression and delivery by DC3000 was impaired when infecting the mkp1 mutant. The addition of bioactive metabolites fully restored T3SS effector delivery and suppressed the enhanced resistance in the mkp1 mutant. Pretreatment of plants with pathogen-associated molecular patterns (PAMPs) to induce PAMP-triggered immunity (PTI) also restricts T3SS effector delivery and enhances resistance by unknown mechanisms, and the addition of the bioactive metabolites similarly suppressed both aspects of PTI. Together, these results demonstrate that DC3000 perceives multiple signals derived from plants to initiate its T3SS and that the level of these host-derived signals impacts bacterial pathogenesis.

Phosphonates (C-PO₃²⁻) have applications as antibiotics, herbicides, and detergents. In some environments, these molecules represent the predominant source of phosphorus, and several microbes have evolved dedicated enzymatic machineries for phosphonate degradation. For example, most common naturally occurring phosphonates can be catabolized to either phosphonoacetaldehyde or phosphonoacetate, which can then be hydrolyzed to generate inorganic phosphate and acetaldehyde or acetate, respectively. The phosphonoacetaldehyde oxidase gene (phnY) links these two hydrolytic processes and provides a previously unknown catabolic mechanism for phosphonoacetate production in the microbial metabolome. Here, we present biochemical characterization of PhnY and high-resolution crystal structures of the apo state, as well as complexes with substrate, cofactor, and product. Kinetic analysis of active site mutants demonstrates how a highly conserved aldehyde dehydrogenase active site has been modified in nature to generate activity with a phosphonate substrate., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

During many biological responses, changes in protein modifications (e.g., phosphorylation) are often more critical than changes in protein abundance in determining the outcome of cellular responses. These important regulatory changes can alter a protein's location, activity, or binding partners. Monitoring modifications such as phosphorylation is often impeded, or even prevented, because of the need for specialized reagents and equipment that are expensive and/or time-consuming to produce. However, many protein modifications alter the isoelectric point (pI) of a protein. Therefore, we developed a denaturing, one-dimensional isoelectric focusing (IEF) procedure that separates proteins based on their pI to resolve different isoforms, allowing a relatively simple strategy for detecting changes in protein modifications. Although similar results can be achieved by two-dimensional gel electrophoresis, the method described here uses a multi-well SDS-PAGE format that allows many more samples to be assayed within a single gel, thereby greatly decreasing both the time and cost needed to assess modifications of a single protein in response many different treatment conditions. To increase the sensitivity of detection, we also optimized a procedure to transfer proteins from these gels to membranes for subsequent immunodetection. This combination of techniques provides the means of interrogating the number and stoichiometry of isoforms from total protein extracts without a priori knowledge of which modification may occur.

The iron-dependent epoxidase HppE converts (S)-2-hydroxypropyl-1-phosphonate (S-HPP) to the antibiotic fosfomycin [(1R,2S)-epoxypropylphosphonate] in an unusual 1,3-dehydrogenation of a secondary alcohol to an epoxide. HppE has been classified as an oxidase, with proposed mechanisms differing primarily in the identity of the O2-derived iron complex that abstracts hydrogen (H•) from C1 of S-HPP to initiate epoxide ring closure. We show here that the preferred cosubstrate is actually H2O2 and that HppE therefore almost certainly uses an iron(IV)-oxo complex as the H• abstractor. Reaction with H2O2 is accelerated by bound substrate and produces fosfomycin catalytically with a stoichiometry of unity. The ability of catalase to suppress the HppE activity previously attributed to its direct utilization of O2 implies that reduction of O2 and utilization of the resultant H2O2 were actually operant.