The Impact of ADP Inhibition on Traumatic Brain Injury Outcomes.

Platelet inhibition correlates with severity of traumatic brain injury and may be associated with mortality. Adenosine diphosphate participates in platelet aggregation via the activation of the ADP receptors, P2Y1 and P2Y12. Prior work suggests this ADP pathway is significant in managing patients with head injuries. This study aimed to measure the influence of ADP inhibition on outcomes after a traumatic brain injury (TBI), as measured by thromboelastography with platelet mapping (TEG-PM). Outcomes were defined as (a) hospital length of stay; (b) ICU length of stay, (c) mortality, and (d) progression of hemorrhage on CT. The resulting cohort was split into quartiles to compare the effect of increasingly inhibited ADP values on the identified outcomes. Comparisons of 2 groups of patients were also conducted, one defined by ADP inhibition less than or equal to 60% and the other group by ADP inhibition of greater than 60%. 98 patients were included in final analysis, with 72.4% having ADP inhibition less than 60%. These patients were significantly older and had lower global injury severity scores (ISSs), although their head-specific ISS was equivalent. Compared to the group with ADP inhibition over 60%, there was no significant difference in mortality, hospital or ICU length of stay, or progression of lesion on CT. Patients with ADP less than 60% inhibited had smaller ISS and higher GCS, indicating they were less injured than those with greater ADP inhibition, consistent with prior literature. The equivalent ICU and hospital length of stay and mortality suggests that ADP inhibition plays a smaller role in outcomes. Additional study with a larger sample size and guideline-based assessments is necessary to further define the impact of ADP inhibition and to determine the role of platelet transfusion in this population.

Suppression of pinoid mutant phenotypes by mutations in PIN-FORMED 1 and PIN1-GFP fusion.

Disruption of either the auxin transporter PIN-FORMED 1 (PIN1) or the protein kinase PINOID (PID) leads to the development of pin-like inflorescences. Previous studies have shown that phosphoregulation of PIN1 by AGC kinases including PID directs auxin flux to drive organ initiation. Here, we report unexpected findings on the genetic interactions between these two genes. We deleted the first 2/3 of the PIN1 coding sequence using CRISPR/Cas9, and the resulting pin1 mutant (pin1-27) was a strong allele. Surprisingly, heterozygous pin1-27 suppressed two independent pid null mutants, whereas homozygous pin1-27 enhanced the phenotypes of the pid mutants during embryogenesis. Furthermore, we show that deletion of either the hydrophilic loop or the second half of PIN1 also abolished PIN1 function, yet those heterozygous pin1 mutants were also capable of rescuing pid nulls. Moreover, we inserted green fluorescent protein (GFP) into the hydrophilic loop of PIN1 through CRISPR-mediated homology-directed repair (HDR). The GFP signal and pattern in the PIN1-GFPHDR line are similar to those in the previously reported PIN1-GFP transgenic lines. Interestingly, the PIN1-GFPHDR line also rescued various pid null mutant alleles in a semidominant fashion. We conclude that decreasing the number of functional PIN1 copies is sufficient to suppress the pid mutant phenotype, suggesting that PIN1 is likely part of a larger protein complex required for organogenesis.

Nuclear Pyruvate Dehydrogenase Complex Regulates Histone Acetylation and Transcriptional Regulation in the Ethylene Response.

Ethylene plays its essential roles in plant development, growth, and defense responses by controlling the transcriptional reprograming, in which EIN2-C-directed regulation of histone acetylation is the first key-step for chromatin to perceive ethylene signaling. But how the nuclear acetyl coenzyme A (acetyl CoA) is produced to ensure the ethylene-mediated histone acetylation is unknown. Here we report that ethylene triggers the accumulation of the pyruvate dehydrogenase complex (PDC) in the nucleus to synthesize nuclear acetyl CoA to regulate ethylene response. PDC is identified as an EIN2-C nuclear partner, and ethylene triggers its nuclear accumulation. Mutations in PDC lead to an ethylene-hyposensitivity that results from the reduction of histone acetylation and transcription activation. Enzymatically active nuclear PDC synthesize nuclear acetyl CoA for EIN2-C-directed histone acetylation and transcription regulation. These findings uncover a mechanism by which PDC-EIN2 converges the mitochondrial enzyme mediated nuclear acetyl CoA synthesis with epigenetic and transcriptional regulation for plant hormone response.

Plant height heterosis is quantitatively associated with expression levels of plastid ribosomal proteins.

The use of hybrids is widespread in agriculture, yet the molecular basis for hybrid vigor (heterosis) remains obscure. To identify molecular components that may contribute to trait heterosis, we analyzed paired proteomic and transcriptomic data from seedling leaf and mature leaf blade tissues of maize hybrids and their inbred parents. Nuclear- and plastid-encoded subunits of complexes required for protein synthesis in the chloroplast and for the light reactions of photosynthesis were expressed above midparent and high-parent levels, respectively. Consistent with previous reports in Arabidopsis, ethylene biosynthetic enzymes were expressed below midparent levels in the hybrids, suggesting a conserved mechanism for heterosis between monocots and dicots. The ethylene biosynthesis mutant, acs2/acs6, largely phenocopied the hybrid proteome, indicating that a reduction in ethylene biosynthesis may mediate the differences between inbreds and their hybrids. To rank the relevance of expression differences to trait heterosis, we compared seedling leaf protein levels to the adult plant height of 15 hybrids. Hybrid/midparent expression ratios were most positively correlated with hybrid/midparent plant height ratios for the chloroplast ribosomal proteins. Our results show that increased expression of chloroplast ribosomal proteins in hybrid seedling leaves is mediated by reduced expression of ethylene biosynthetic enzymes and that the degree of their overexpression in seedlings can quantitatively predict adult trait heterosis.

Vision, challenges and opportunities for a Plant Cell Atlas.

With growing populations and pressing environmental problems, future economies will be increasingly plant-based. Now is the time to reimagine plant science as a critical component of fundamental science, agriculture, environmental stewardship, energy, technology and healthcare. This effort requires a conceptual and technological framework to identify and map all cell types, and to comprehensively annotate the localization and organization of molecules at cellular and tissue levels. This framework, called the Plant Cell Atlas (PCA), will be critical for understanding and engineering plant development, physiology and environmental responses. A workshop was convened to discuss the purpose and utility of such an initiative, resulting in a roadmap that acknowledges the current knowledge gaps and technical challenges, and underscores how the PCA initiative can help to overcome them.

Nitrate triggered phosphoproteome changes and a PIN2 phosphosite modulating root system architecture.

Nitrate commands genome-wide gene expression changes that impact metabolism, physiology, plant growth, and development. In an effort to identify new components involved in nitrate responses in plants, we analyze the Arabidopsis thaliana root phosphoproteome in response to nitrate treatments via liquid chromatography coupled to tandem mass spectrometry. 176 phosphoproteins show significant changes at 5 or 20 min after nitrate treatments. Proteins identified by 5 min include signaling components such as kinases or transcription factors. In contrast, by 20 min, proteins identified were associated with transporter activity or hormone metabolism functions, among others. The phosphorylation profile of NITRATE TRANSPORTER 1.1 (NRT1.1) mutant plants was significantly altered as compared to wild-type plants, confirming its key role in nitrate signaling pathways that involves phosphorylation changes. Integrative bioinformatics analysis highlights auxin transport as an important mechanism modulated by nitrate signaling at the post-translational level. We validated a new phosphorylation site in PIN2 and provide evidence that it functions in primary and lateral root growth responses to nitrate.

Genetic elucidation of interconnected antibiotic pathways mediating maize innate immunity.

Specialized metabolites constitute key layers of immunity that underlie disease resistance in crops; however, challenges in resolving pathways limit our understanding of the functions and applications of these metabolites. In maize (Zea mays), the inducible accumulation of acidic terpenoids is increasingly considered to be a defence mechanism that contributes to disease resistance. Here, to understand maize antibiotic biosynthesis, we integrated association mapping, pan-genome multi-omic correlations, enzyme structure-function studies and targeted mutagenesis. We define ten genes in three zealexin (Zx) gene clusters that encode four sesquiterpene synthases and six cytochrome P450 proteins that collectively drive the production of diverse antibiotic cocktails. Quadruple mutants in which the ability to produce zealexins (ZXs) is blocked exhibit a broad-spectrum loss of disease resistance. Genetic redundancies ensuring pathway resiliency to single null mutations are combined with enzyme substrate promiscuity, creating a biosynthetic hourglass pathway that uses diverse substrates and in vivo combinatorial chemistry to yield complex antibiotic blends. The elucidated genetic basis of biochemical phenotypes that underlie disease resistance demonstrates a predominant maize defence pathway and informs innovative strategies for transferring chemical immunity between crops.

Dynamic regulation of Pep-induced immunity through post-translational control of defence transcript splicing.

The survival of all living organisms requires the ability to detect attacks and swiftly counter them with protective immune responses. Despite considerable mechanistic advances, the interconnectivity of signalling modules often remains unclear. A newly characterized protein, IMMUNOREGULATORY RNA-BINDING PROTEIN (IRR), negatively regulates immune responses in both maize and Arabidopsis, with disrupted function resulting in enhanced disease resistance. IRR associates with and promotes canonical splicing of transcripts encoding defence signalling proteins, including the key negative regulator of pattern-recognition receptor signalling complexes, CALCIUM-DEPENDENT PROTEIN KINASE 28 (CPK28). On immune activation by Plant Elicitor Peptides (Peps), IRR is dephosphorylated, disrupting interaction with CPK28 transcripts and resulting in the accumulation of an alternative splice variant encoding a truncated CPK28 protein with impaired kinase activity and diminished function as a negative regulator. We demonstrate a new mechanism linking Pep-induced post-translational modification of IRR with post-transcriptionally mediated attenuation of CPK28 function to dynamically amplify Pep signalling and immune output.

Meta Gene Regulatory Networks in Maize Highlight Functionally Relevant Regulatory Interactions.

The regulation of gene expression is central to many biological processes. Gene regulatory networks (GRNs) link transcription factors (TFs) to their target genes and represent maps of potential transcriptional regulation. Here, we analyzed a large number of publically available maize (Zea mays) transcriptome data sets including >6000 RNA sequencing samples to generate 45 coexpression-based GRNs that represent potential regulatory relationships between TFs and other genes in different populations of samples (cross-tissue, cross-genotype, and tissue-and-genotype samples). While these networks are all enriched for biologically relevant interactions, different networks capture distinct TF-target associations and biological processes. By examining the power of our coexpression-based GRNs to accurately predict covarying TF-target relationships in natural variation data sets, we found that presence/absence changes rather than quantitative changes in TF gene expression are more likely associated with changes in target gene expression. Integrating information from our TF-target predictions and previous expression quantitative trait loci (eQTL) mapping results provided support for 68 TFs underlying 74 previously identified trans-eQTL hotspots spanning a variety of metabolic pathways. This study highlights the utility of developing multiple GRNs within a species to detect putative regulators of important plant pathways and provides potential targets for breeding or biotechnological applications.

Wnt5a induces ROR1 to recruit cortactin to promote breast-cancer migration and metastasis.

ROR1 is a conserved oncoembryonic surface protein expressed in breast cancer. Here we report that ROR1 associates with cortactin in primary breast-cancer cells or in MCF7 transfected to express ROR1. Wnt5a also induced ROR1-dependent tyrosine phosphorylation of cortactin (Y421), which recruited ARHGEF1 to activate RhoA and promote breast-cancer-cell migration; such effects could be inhibited by cirmtuzumab, a humanized mAb specific for ROR1. Furthermore, treatment of mice bearing breast-cancer xenograft with cirmtuzumab inhibited cortactin phosphorylation in vivo and impaired metastatic development. We established that the proline at 841 of ROR1 was required for it to recruit cortactin and ARHGEF1, activate RhoA, and enhance breast-cancer-cell migration in vitro or development of metastases in vivo. Collectively, these studies demonstrate that the interaction of ROR1 with cortactin plays an important role in breast-cancer-cell migration and metastasis.

Multiple genes recruited from hormone pathways partition maize diterpenoid defences.

Duplication and divergence of primary pathway genes underlie the evolution of plant specialized metabolism; however, mechanisms partitioning parallel hormone and defence pathways are often speculative. For example, the primary pathway intermediate ent-kaurene is essential for gibberellin biosynthesis and is also a proposed precursor for maize antibiotics. By integrating transcriptional coregulation patterns, genome-wide association studies, combinatorial enzyme assays, proteomics and targeted mutant analyses, we show that maize kauralexin biosynthesis proceeds via the positional isomer ent-isokaurene formed by a diterpene synthase pair recruited from gibberellin metabolism. The oxygenation and subsequent desaturation of ent-isokaurene by three promiscuous cytochrome P450s and a new steroid 5α reductase indirectly yields predominant ent-kaurene-associated antibiotics required for Fusarium stalk rot resistance. The divergence and differential expression of pathway branches derived from multiple duplicated hormone-metabolic genes minimizes dysregulation of primary metabolism via the circuitous biosynthesis of ent-kaurene-related antibiotics without the production of growth hormone precursors during defence.

Auxin Induces Widespread Proteome Remodeling in Arabidopsis Seedlings.

It is known that auxin induces rapid gene expression changes throughout plant development, but how these transcriptional responses relate to changes in protein abundance is not well characterized. This report identifies early auxin responsive proteins in whole Arabidopsis seedlings using an isobaric tags for relative and absolute quantification-based quantitative proteomics approach. Approximately 25% of the detected proteins (1045 out of 4257 proteins) are auxin responsive, which is in line with the central role of auxin in the regulation of plant growth and development. Several well-known auxin pathway proteins are identified as differentially expressed, validating this quantitative proteomics approach. Additionally, functional categorization of these auxin responsive proteins indicates that rapid and complex metabolic changes occur in seedlings in response to auxin, including lipid metabolism. Altogether, these data describe novel auxin-regulated proteins and are an excellent resource for identifying new downstream signaling components related to auxin-mediated plant growth and development.

Identification of the expressome by machine learning on omics data.

Accurate annotation of plant genomes remains complex due to the presence of many pseudogenes arising from whole-genome duplication-generated redundancy or the capture and movement of gene fragments by transposable elements. Machine learning on genome-wide epigenetic marks, informed by transcriptomic and proteomic training data, could be used to improve annotations through classification of all putative protein-coding genes as either constitutively silent or able to be expressed. Expressed genes were subclassified as able to express both mRNAs and proteins or only RNAs, and CG gene body methylation was associated only with the former subclass. More than 60,000 protein-coding genes have been annotated in the reference genome of maize inbred B73. About two-thirds of these genes are transcribed and are designated the filtered gene set (FGS). Classification of genes by our trained random forest algorithm was accurate and relied only on histone modifications or DNA methylation patterns within the gene body; promoter methylation was unimportant. Other inbred lines are known to transcribe significantly different sets of genes, indicating that the FGS is specific to B73. We accurately classified the sets of transcribed genes in additional inbred lines, arising from inbred-specific DNA methylation patterns. This approach highlights the potential of using chromatin information to improve annotations of functional genes.

LIKE SEX4 1 Acts as a ß-Amylase-Binding Scaffold on Starch Granules during Starch Degradation.

In Arabidopsis (Arabidopsis thaliana) leaves, starch is synthesized during the day and degraded at night to fuel growth and metabolism. Starch is degraded primarily by ß-amylases, liberating maltose, but this activity is preceded by glucan phosphorylation and is accompanied by dephosphorylation. A glucan phosphatase family member, LIKE SEX4 1 (LSF1), binds starch and is required for normal starch degradation, but its exact role is unclear. Here, we show that LSF1 does not dephosphorylate glucans. The recombinant dual specificity phosphatase (DSP) domain of LSF1 had no detectable phosphatase activity. Furthermore, a variant of LSF1 mutated in the catalytic cysteine of the DSP domain complemented the starch-excess phenotype of the lsf1 mutant. By contrast, a variant of LSF1 with mutations in the carbohydrate binding module did not complement lsf1 Thus, glucan binding, but not phosphatase activity, is required for the function of LSF1 in starch degradation. LSF1 interacts with the ß-amylases BAM1 and BAM3, and the BAM1-LSF1 complex shows amylolytic but not glucan phosphatase activity. Nighttime maltose levels are reduced in lsf1, and genetic analysis indicated that the starch-excess phenotype of lsf1 is dependent on bam1 and bam3 We propose that LSF1 binds ß-amylases at the starch granule surface, thereby promoting starch degradation.

Magnesium Flux Modulates Ribosomes to Increase Bacterial Survival.

Bacteria exhibit cell-to-cell variability in their resilience to stress, for example, following antibiotic exposure. Higher resilience is typically ascribed to "dormant" non-growing cellular states. Here, by measuring membrane potential dynamics of Bacillus subtilis cells, we show that actively growing bacteria can cope with ribosome-targeting antibiotics through an alternative mechanism based on ion flux modulation. Specifically, we observed two types of cellular behavior: growth-defective cells exhibited a mathematically predicted transient increase in membrane potential (hyperpolarization), followed by cell death, whereas growing cells lacked hyperpolarization events and showed elevated survival. Using structural perturbations of the ribosome and proteomic analysis, we uncovered that stress resilience arises from magnesium influx, which prevents hyperpolarization. Thus, ion flux modulation provides a distinct mechanism to cope with ribosomal stress. These results suggest new approaches to increase the effectiveness of ribosome-targeting antibiotics and reveal an intriguing connection between ribosomes and the membrane potential, two fundamental properties of cells.

Quantitative Early Auxin Root Proteomics Identifies GAUT10, a Galacturonosyltransferase, as a Novel Regulator of Root Meristem Maintenance.

Auxin induces rapid gene expression changes throughout root development. How auxin-induced transcriptional responses relate to changes in protein abundance is not well characterized. This report identifies early auxin responsive proteins in roots at 30 min and 2 h after hormone treatment using a quantitative proteomics approach in which 3,514 proteins were reliably quantified. A comparison of the >100 differentially expressed proteins at each the time point showed limited overlap, suggesting a dynamic and transient response to exogenous auxin. Several proteins with established roles in auxin-mediated root development exhibited altered abundance, providing support for this approach. While novel targeted proteomics assays demonstrate that all six auxin receptors remain stable in response to hormone. Additionally, 15 of the top responsive proteins display root and/or auxin response phenotypes, demonstrating the validity of these differentially expressed proteins. Auxin signaling in roots dictates proteome reprogramming of proteins enriched for several gene ontology terms, including transcription, translation, protein localization, thigmatropism, and cell wall modification. In addition, we identified auxin-regulated proteins that had not previously been implicated in auxin response. For example, genetic studies of the auxin responsive protein galacturonosyltransferase 10 demonstrate that this enzyme plays a key role in root development. Altogether these data complement and extend our understanding of auxin response beyond that provided by transcriptome studies and can be used to uncover novel proteins that may mediate root developmental programs.

The Promises and Pitfalls of Engaging Male Juvenile Offenders in Gender Violence Prevention and Bystander Education.

Drawing on interviews with and observations of boys enrolled in a bystander violence prevention program at a juvenile detention center, this article provides a sociological case study on how the boys' biographies and violent lived experiences shaped their engagement with the program. Previous research on bystander prevention programs has typically focused on men enrolled in college who do not have the same kinds of violent histories as the boys in this study do. This article builds upon prior research on prevention programs by demonstrating how at-risk youth participants understand and access the program. We offer suggestions for tailoring bystander prevention programs to more adequately address the specific needs of these populations.

Dynamic Patterns of Gene Expression Additivity and Regulatory Variation throughout Maize Development.

Gene expression variation is a key component underlying phenotypic variation and heterosis. Transcriptome profiling was performed on 23 different tissues or developmental stages of two maize inbreds, B73 and Mo17, as well as their F1 hybrid. The obtained large-scale datasets provided opportunities to monitor the developmental dynamics of differential expression, additivity for gene expression, and regulatory variation. The transcriptome can be divided into ∼30 000 genes that are expressed in at least one tissue of one inbred and an additional ∼10 000 ″silent" genes that are not expressed in any tissue of any genotype, 90% of which are non-syntenic relative to other grasses. Many (∼74%) of the expressed genes exhibit differential expression in at least one tissue. However, the majority of genes with differential expression do not exhibit consistent differential expression in different tissues. These genes often exhibit tissue-specific differential expression with equivalent expression in other tissues, and in many cases they switch the directionality of differential expression in different tissues. This suggests widespread variation for tissue-specific regulation of gene expression between the two maize inbreds B73 and Mo17. Nearly 5000 genes are expressed in only one parent in at least one tissue (single parent expression) and 97% of these genes are expressed at mid-parent levels or higher in the hybrid, providing extensive opportunities for hybrid complementation in heterosis. In general, additive expression patterns are much more common than non-additive patterns, and this trend is more pronounced for genes with strong differential expression or single parent expression. There is relatively little evidence for non-additive expression patterns that are maintained in multiple tissues. The analysis of allele-specific expression allowed classification of cis- and trans-regulatory variation. Genes with cis-regulatory variation often exhibit additive expression and tend to have more consistent regulatory variation throughout development. In contrast, genes with trans-regulatory variation are enriched for non-additive patterns and often show tissue-specific differential expression. Taken together, this study provides a deeper understanding of regulatory variation and the degree of additive gene expression throughout maize development. The dynamic nature of differential expression, additivity, and regulatory variation imply abundant variability for tissue-specific regulatory mechanisms and suggest that connections between transcriptome and phenome will require expression data from multiple tissues.

Wnt5a induces ROR1 to recruit DOCK2 to activate Rac1/2 in chronic lymphocytic leukemia.

Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncoembryonic protein expressed on chronic lymphocytic leukemia (CLL) that can serve as a receptor for Wnt5a, which can promote leukemia cell migration, proliferation, and survival. We found Wnt5a could induce ROR1 to complex with DOCK2 (dedicator of cytokinesis 2) and induce activation of Rac1/2; these effects could be blocked by cirmtuzumab, a humanized anti-ROR1 monoclonal antibody. We find that silencing DOCK2 specifically impaired the capacity of Wnt5a to induce activation of Rac1/2 or enhance CLL cell proliferation. We generated truncated forms of ROR1 and found the cytoplasmic proline-rich domain (PRD) of ROR1 was required for Wnt5a to induce ROR1 to complex with DOCK2 and activate Rac1/2 in the CLL cell-line MEC1. We introduced single amino acid substitutions of proline (P) to alanine (A) in the ROR1-PRD at potential binding sites for the Src-homology 3 domain of DOCK2. In contrast to wild-type ROR1, or other ROR1 P→A variants, ROR1P808A was unable to recruit DOCK2 in response to Wnt5a. Moreover, unlike MEC1 cells transfected with wild-type ROR1 or ROR1 with P→A substitutions at positions 784, 826, or 841, MEC1 cells transfected to express ROR1P808A did not have a growth advantage over MEC1 cells that do not express ROR1. This study reveals that the recruitment of DOCK2 may be critical for the capacity of Wnt5a to enhance CLL proliferation, which may contribute to the observed increased tendency for disease progression in patients who have CLL cells that express high levels of ROR1.